>>WE WOULD LIKE TO WELCOME
YOU AND THANK YOU FOR JOINING US
TO THE YET TO BE CHARTED
WORKSHOP WITH THE EMPHASIS ON
LYMPHATIC SYSTEM IN HEALTH AND
DISEASE.
LAST YEAR'S SUCCESSFUL WORKSHOP
IS AVAILABLE ON DEMAND IN NIH
VIDEO WEBSITE.
WE WOULD LIKE TO WELCOME
SPEAKERS, MODERATORS AND OVER
700 REGISTRANTS WITH A GREAT
COVERAGE OF ALL STAKEHOLDERS
INCLUDING ACADEMIA, GOVERNMENT
AND INDUSTRY RESEARCHERS,
CLINICIANS, AND PATIENT AND
PATIENT CAREGIVERS WHO REPRESENT
10% OF OUR REGISTRANTS.
WE ARE OVERWHELMED BY THE GREAT
RESPONSE WE RECEIVED AND HOPE TO
LIVE UP TO THE EXPECTATIONS OF
THE LYMPHATIC COMMUNITY AND HAVE
A SUCCESSFUL TWO-DAY WORKSHOP.
I'LL START BY BRIEF HOUSEKEEPING
ANNOUNCEMENTS.
THIS WORKSHOP IS RECORDED AND
WILL BE AVAILABLE FOR EVERYONE
IN PERPETUITY IN NIH VIDEOCAST
COLLECTION.
THE LINKS FOR THE VIDEOCAST
RECORDING ALONG WITH THE
DETAILED INFORMATION OF ALL
SPEAKERS CAN BE FOUND ON THE
REGISTRATION PAGE.
DURING THE WORKSHOP, THE
SPEAKERS WILL PRESENT THEIR
TALKS AND THEY WILL ANSWER THE
QUESTIONS AS A PANEL AT THE END
OF EVERY SESSION THAT WILL BE
LED BY THE SESSION MODERATOR.
PLEASE SUBMIT YOUR QUESTIONS
BOTH ZOOM AND VIDEOCAST
ATTENDEES, PUT YOUR QUESTIONS IN
THE Q & A AND THE MODERATOR WILL
READ AND DIRECT YOUR QUESTIONS
TO THE PANELISTS.
AND WE REQUEST THAT OUR SPEAKERS
SHOULD RAISE THEIR HANDS AND
WHEN YOU ARE CALLED, UNMUTE AND
ASK YOUR QUESTION LIVE.
NEXT I WOULD LIKE TO INTRODUCE
AND THANK TO ALL OUR ORGANIZING
COMMITTEE WHO WORKED TIRELESSLY
TO PUT TOGETHER THIS EXCITING
AGENDA OF THE WORKSHOP.
DR. RANDOLPH FROM WASHINGTON
UNIVERSITY, NIDDK, NATIONAL
HEART, LUNG AND BLOOD INSTITUTE
AND OUR WONDERFUL CHAIRS.
WITHOUT FURTHER ADO, I WOULD
LIKE TO PASS THE MIC TO OUR
CHAIRS.
>> THANK YOU.
GOOD MORNING AND WELCOME,
EVERYONE.
MY NAME IS BABAK MEHRARA, I'M A
SURGEON SCIENTIST AT MEMORIAL
SLOAN KETTERING SCIENCE CENTER.
>> GOOD MORNING AND WELCOME.
I'M ANDREA RADTKE.
MY RESEARCH FOCUSES AT THE
DEVELOPMENT AND APPLICATION OF
TECHNOLOGIES THAT ENABLE
DETAILED SPATIAL MAPPING OF
CELLS AND STRUCTURES WITHIN
HUMAN LYMPHOID ORGANS.
>> WE ARE HONORED THAT YOU CAN
JOIN US FOR THE YET TO BE
CHARTED LYMPHATIC SYSTEM IN
HEALTH AND DISEASE SEMINAR.
THE PRIMARY OBJECTIVE OF THIS
MEETING IS TO ADVANCE OUR
UNDERSTANDING OF HOW THE
LYMPHATIC SYSTEM DEVELOPS,
FUNCTIONS IN HEALTH AND DISEASE
AND MOST IMPORTANTLY HOW THE
DISSEMINATION OF THIS KNOWLEDGE
MAY IMPROVE HOW WE TREAT
LYMPHATIC DISORDERS.
A SPECIAL FOCUS HAS BEEN PLACED
ON LYMPHEDEMA AND THE ROLE OF
LIX IN
LYMPHATICS IN OBESITY.
>> WE ARE HAVE A POSTURE OF
GRATITUDE, WE ARE SO GRATEFUL
FOR THE DEDICATION IN CREATIVITY
AND PLANNING FOR THIS WORKSHOP.
WE WANT TO THANK NHLBI AND
NIDDK.
WE WANT TO NOTE THAT GWEN IS
UNABLE TO ATTEND DUE TO A
SCHEDULING CONFLICT BUT WE ARE
GRATEFUL FOR HER EXCELLENT
CONTRIBUTIONS.
WE'D ALSO LIKE TO THANK THE
TRANS-NIH LYMPHATIC COORDINATING
COMMITTEE AND, OF COURSE,
DR. ZORINA GALIS.
ZORINA IS ONE OF THE VISIONARIES
BEHIND MANY TRANS-NIH
INITIATIVES AND INVOLVED IN
ADVANCING OUR UNDERSTANDING OF
THE HUMAN BODY.
>> WE WANT TO THANK THE SPEAKERS
FOR THEIR COMMITMENT TO THE
FIELD AND PRESENTING THEIR WORK.
WE KNOW THIS IS A LOT OF WORK
AND PUTTING TOGETHER ANOTHER
TALK IS JUST ONE MORE THING BUT
WE WANT TO THANK THEM FOR DOING
SO.
ALSO THE LYMPHATIC EDUCATION AND
RESEARCH NETWORK AND ALL THE
ADVOCACY GROUPS INVOLVED FOR
THEIR TIEFERLESS TIRELESS.
LASTLY THANKS TO CAMERON,
CASSIE, CYNTHIA AND TAYLOR WHO
WILL SHARE THEIR PERSONAL
STORIES WITH ALL OF YOU
TOMORROW.
THANK YOU ALL FOR YOUR
COMMITMENT AND COMPASSION FOR
LYMPHATIC RESEARCH.
WE'RE HUMBLED TO WELCOME OVER
700 REGISTRANTS
>> THE SELECTION OF TOPICS AS A
MULTIDISCIPLINARY EFFORT IS
REQUIRED TO ADVANCE THE FIELD.
FURTHERMORE, IT IS IMPERATIVE
THAT WE HEAR FROM PATIENTS,
CLINICIANS, PROGRAM DIRECTORS,
JUNIOR AND SENIOR INVESTIGATORS,
AND ALL 700 OF YOU.
THE NAME OF THE SYMPOSIUM, YET
TO BE CHARTED, IS DELIBERATE AS
THE LYMPHATIC SYSTEM REPRESENTS
ONE OF THE LAST FRONTIERS OF
ANATOMICAL MEDICINE.
FURTHERMORE, A MAJOR THEME OF
THIS MEETING IS CREATING A
USABLE MAP FOR RESEARCHERS AND
CLINICIANS, A MAP, IT IS SAID,
ORGANIZES WONDER, AND THE
LYMPHATIC SYSTEM IS TRULY
WONDER-FILLED.
FIRST FUNCTIONS AND MOST ORGANS
AND TISSUES OF THE HUMAN BODY,
TRANSPORTS A THOUSAND-PLUS
PROTEINS IN LYMPH FLUID ALONE
AND MOST IMPORTANTLY, NEGATIVELY
IMPACTS THE LIVES OF HUNDREDS OF
MILLIONS OF PEOPLE SUFFERING
FROM LYMPHATIC DISEASES AROUND
THE WORLD.
TODAY WE WILL HEAR FROM JUST
FOUR OF THESE PATIENTS.
>> WE CLOSE BY WELCOMING YOU TO
THIS EXCITING MEETING AND THANK
YOU FOR YOUR TIME.
PLEASE JOIN US IN WELCOMING BACK
DR. PATRICIA GREENWEL FROM THE
DIVISION OF DIGESTIVE DISEASE
AND NUTRITION, THE NIDDK.
THANK YOU SO MUCH FOR HAVING US.
>> HELLO, GOOD MORNING.
I WOULD LIKE TO INTRODUCE
DR. STEVE JAMES, DIRECTOR OF THE
DIVISION OF DIGESTIVE DISEASES
AND NUTRITION, NIDDK.
STEVE?
>> CAN YOU HEAR ME?
I JUST LOGGED IN.
THANK YOU.
WELCOME, ALL OF YOU, WHEREVER
YOU MAY BE TO THIS VERY EXCITING
MEETING.
THE NIDDK AND NHLBI HAVE
CO-SPONSORED THIS WORKSHOP, AND
IT'S REALLY OUR PLEASURE TO
WELCOME YOU.
THE LYMPHATIC SYSTEM IS A
COMPLEX AND INTRICATE NETWORK OF
CAPILLARES
KAIB LARRYS AND VESSELS THAT
PERMEATE NEARLY EVERY ORGAN OF
THE BODY.
THEY'RE DYNAMIC AND PLAY
CRITICAL AND ACTIVE ROLES IN
MAINTENANCE OF FLUID
HOMEOSTASIS, TRAFFICKING AND
MATURATION OF THE IMMUNE CELLS,
ABSORPTION OF KEY NUTRIENTS,
SELECTIVE TRANSPORT OF SOME
HORMONES, AND MODULATION OF
INFLAMMATORY RESPONSES.
ALTERATIONS IN THE LYMPHATIC
SYSTEM INCLUDING ENHANCED
PROLIFERATION AND IN SOME
INSTANCES MORPHOLOGICAL CHANGES
HAVE BEEN OBSERVED IN MANY
DISEASE STATES.
SOME OF THESE CHANGES ARE
CONGENITAL AND OTHERS DEVELOP AS
A RESULT OF DISEASE OR INJURY.
IN THE UNITED STATES, SEVERAL
MILLION AMERICANS SUFFER FROM
LYMPHEDEMA AND OTHER DISEASES.
THERE'S AN ENORMOUS RENEWAL OF
INTEREST IN AND THE IMPORTANCF
LYMPHATICS -- TO NOW INCLUDE
ACUTE AND CHRONIC INFLAMMATION.
MORE GENERALLY.
THE DIGESTIVE SYSTEM CRITICALLY
DEPENDS ON ADEQUATE LYMPHATIC
FUNCTION.
ABOUT 70% OF THE TOTAL LYMPH
FORMED IN THE BODY ORIGINATES IN
THE SMALL INTESTINE AND LIVER.
DEFECTS IN GASTROINTESTINAL
LYMPHATIC DEVELOPMENT AND
STRUCTURE, LYMPH PUMP FUNCTION
OR REMODELING PRODUCE SEVERAL
FORMS OF GI PATHOLOGY, AND
EXACERBATE G.I. INJURY.
MALFUNCTIONS IN THE LYMPHATIC
SYSTEM HAVE BEEN IMPLICATED IN
SEVERAL DISEASE STATES, SUCH AS
OBESITY, METABOLIC SYNDROME,
INFLAMMATORY BOWEL DISEASES,
LIVER FIBROSIS AND CIRRHOSIS.
A BETTER UNDERSTANDING OF THE
LYMPHATIC SYSTEM IN HEALTH AND
DISEASE STATES COULD LEAD TO THE
DEVELOPMENT OF NOVEL THERAPIES
FOR MANY OF THESE CONDITIONS.
LYMPHATICS RESEARCH HAS BEEN A
PRIORITY AREA FOR NIDDK FOR
SEVERAL YEARS.
NIDDK HAS PARTICIPATED IN
SEVERAL NIH-WIDE ACTIVITIES TO
ENCOURAGE THE STUDY OF
LYMPHATICS IN HEALTH AND DISEASE
INCLUDE WORKSHOPS AND SEVERAL
FUNDING OPPORTUNITY
ANNOUNCEMENTS.
OVERALL, THESE INITIATIVES HAVE
BEEN QUITE SUCCESSFUL AND OUR
GRANT PORTFOLIO IN THIS AREA HAS
MORE THAN DOUBLED IN THE LAST
FIVE YEARS.
OUR INITIATIVES HAVE RESULTED IN
NEW INTERESTS FROM THE PART OF
INVESTIGATORS AT NIDDK-RELATED
TOPICS AND HAVE BEEN SPARCED NEW
COLLABORATIONS AND GRANT
APPLICATIONS IN AREAS SUCH AS
LYMPHATICS DEVELOPMENT,
REMODELING AND FUNCTION IN
HEALTH AND DISEASE OF THE
DIGESTIVE SYSTEM, THE ROLE OF
LYMPHATICS IN OBESITY AND
METABOLIC SYNDROME, AND THE ROLE
OF LYMPHATICS IN FAT AND OTHER
NUTRIENT ABSORPTION.
THE PURPOSE OF THIS TWO-DAY
WORKSHOP IS TO BRING TOGETHER
BASIC TRANSLATIONAL AND CLINICAL
LYMPHATICS EXPERTS TO PRESENT
THE LATEST ADVANCES IN THE
FIELD, ENGAGE INTERDISCIPLINARY
DISCUSSIONS, AND TO IDENTIFY
KNOWLEDGE GAPS AND OPPORTUNITIES
IN THE FIELD.
WE HOPE THIS EEFNTD EVENT WD
COLLABORATIVE PARTNERSHIPS AMONG
MANY.
I PARTICULARLY WANT TO THANK THE
COORGANIZERS OF THE WORKSHOP,
AND THE PROGRAM STAFF FROM
NHLBI, AND PATRICIA GREENWEL FRP
NIDDK.
I ALSO WANT TO THANK THE MANY
SPEAKERS WHO WILL INFORM US
ABOUT THE LATEST ADVANCES AND
IMPORTANTLY REPRESENTATIVES WHO
WILL TELL US ABOUT THE
PERSPECTIVE OF PATIENTS AND
THEIR FAMILIES.
I'LL NOW TURN THE MEETING BACK
TO DR. ROVIRA.
THANK YOU.
>> TODAY I HAVE THE PLEASURE TO
INTRODUCE DR. ZORINA GALIS,
CHIEF OF VASCULAR BIOLOGY AND
HYPERTENSION BRANCH AT NHLBI.
DR. GA LIST HAS EXTENSIVE
EXPERIENCE IN ACADEMIA INDUSTRY
AND GOVERNMENT, AND HER CAREER
CONTRIBUTIONS REFLECT HER SPIRIT
OF INNOVATION AND DEVOTION TO
SERVICE.
IT IS MY HONOR TO INTRODUCE
DR. ZORINA GALIS.
THANK YOU.
>> THANK YOU SO MUCH, ILSA, AND
THANK YOU AGAIN, EVERYONE.
I DON'T WANT TO REPEAT ALL THE
THANKS, I THINK IT'S VERY
OBVIOUS THAT IT TAKES A HUGE
TEAM AND HUGE EFFORT TO ORGANIZE
SUCH MEETINGS AND I AM SO
GRATEFUL THAT WE WERE ABLE TO BE
INTERESTING TO SO MANY PEOPLE
WHO ARE GOING TO HELP US FORWARD
RESEARCHING THIS FIELD.
I WOULD LIKE TO SHOW YOU SOME
SLIDES, SO HOPEFULLY I AM ABLE
TO SHARE MY SCREEN.
SO I WANTED TO JUST BRIEFLY
RE-INTRODUCE THE IDEA THAT NIH
IS REALLY DEDICATED TO
SUPPORTING THE LYMPHATIC
RESEARCH, AS WELL AS MANY OTHER
TOPICS, BUT WE ARE HERE TO
CELEBRATE AND TO FIND OUT WHAT
ELSE WE CAN DO IN ADVANCING THE
CURRENT KNOWLEDGE ABOUT THE
LYMPHATIC SYSTEM.
SO MANY OF YOU ALREADY KNOW THAT
NIH IS HART OF PART OF HHS .
GOVERNMENT AND THEREFORE WE ARE
RESPONSIBLE TO THE TAXPAYERS FOR
WHAT WE DO WITH THE INVESTMENTS.
THIS SLIDE REPRESENTS THE
BUDGETS THAT ARE ALLOCATED TO
NIH, AND IT'S THE 2021 BUDGET
BECAUSE THAT'S ALREADY CLOSED
THE BOOKS.
THE HEART, LUNG AND BLOOD
INSTITUTE IN TERMS OF FUNDING IS
RANKING CURRENTLY AS THE FOURTH
OF THE 27 DIFFERENT INSTITUTES
AND CENTERS.
WE RECEIVE A LOT OF QUESTIONS
AND FEEDBACK FROM POTENTIAL
APPLICANTS, GRANTEES AND SO
FORTH, SO I THOUGHT I WILL TRY
TO BRIEFLY RESPOND TO SOME OF
THESE QUESTIONS DURING MY TALK
AND SOME OF YOU ARE VERY
FAMILIAR WITH THE ANSWERS, FOR
SOME OF YOU, THIS MAY BE AN EYE
OPENER AND IT WILL HELP
HOPEFULLY PROVIDE MORE
INFORMATION ABOUT WHAT YOU NEED
TO KNOW.
THE MOST COMMON QUESTIONS WITH
OUR INSTITUTE OR NIDDK OR MAYBE
NIAID OR WHICH INSTITUTE IS
INTERESTED IN THE PROPOSAL
SOMEBODY IS THINKING ABOUT
APPLYING, WHAT PEER REVIEW
SECTION MIGHT REVIEW IT, WHO
COULD THEY TALK TO AT NIH IF
ANYBODY TO FIND OUT HOW TO MOVE
FORWARD WITH THEIR APPLICATIONS.
AND OF COURSE A LOT OF PEOPLE
ARE VERY INTERESTED IN HOW MUCH
MONEY IS NIH OR ANY SPECIFIC
INSTITUTE INVESTING IN LYMPHATIC
RESEARCH AND WHETHER WE HAVE
SPECIFIC INITIATIVES TO HELP
WITH THAT.
THAT IS A TOOL THAT IN THIS DAY
AND AGE, I'M SURE EVERYBODY WILL
BE VERY HAPPY TO KNOW IS AT
THEIR FINGERTIPS.
IT'S CALLED THE NIH MATCH MAKER,
AND YOU CAN JUST PUT IN YOUR
PROPOSAL, YOUR SPECIFIC AIMS, OR
SUMMARY, A POSTER, ANY TYPE OF
TEXT THAT DEVIBES WHAT YOU S
WHAT YOU INTEND TO DO AND BY
PRESSING THE BUTTON YOU CAN
IMMEDIATELY SEE WHICH INSTITUTE
MIGHT BE MOST IMMEDIATELY
INTERESTED IN YOUR RESEARCH,
WHAT MECHANISM YOU MIGHT WANT TO
USE, BUT AT THE SAME TIME, YOU
CAN ALSO FIND OUT WHAT IS THE
SIMILAR RESEARCH AND WHO IS
PERFORMING IT AT VARIOUS
INSTITUTES ACROSS THE U.S. OF A
THAT CAN HELP YOU FIND
COLLABORATORS YOU MAY NEED IN
ORDER TO PUT IN YOUR OWN
APPLICATION.
ANOTHER IMPORTANT QUESTION IS,
WHO IS GOING TO REVIEW, WHICH
STUDY SECTION IS GOING TO REVIEW
YOUR APPLICATION AND YOU'LL FIND
OUT THAT THERE IS A REVIEW ART
WHICH IS ASSISTIVE REFERRAL TOOL
THAT YOU CAN USE AND YOU CAN BE
MATCHED WITH VARIOUS SCIENTIFIC
APPLICATIONS AND YOU CAN ALSO
FIND OUT WHO IS RUNNING THAT
PEER REVIEW SECTION AND CALL
THEM FOR HELP.
MANY OF YOU MAY KNOW THIS
ALREADY, BUT I'VE BEEN A GRANTEE
MYSELF AND SOMETIMES THE NIH
LOOKS LIKE A BLACK BOX.
YOU SEND IN YOUR APPLICATION,
YOU HAVE NO IDEA WHAT'S
HAPPENING TO IT, WHERE IS IT, AT
WHICH POINT.
SO ONCE YOU SUBMIT YOUR
APPLICATION, IT GOES TO A CENTER
FOR SCIENTIFIC REVIEW, WHICH IS
SEPARATE FROM ALL THE OTHER
INSTITUTES, AND THAT WILL ASSIGN
TO THE REVIEW SECTION AS WELL TO
INSTITUTE THAT SEEMS MOST LIKELY
TO MATCH THE INTENT OF THE
APPLICATION.
SO YOU AS AN APPLICANT GOING TO
FIND OUT WHAT THE SCORE IS, THE
SCORE IS GOING TO BE REVIEWED BY
THE SCIENTIFIC COUNCIL OR THE
SPECIFIC INSTITUTE I EXEMPLIFY
HERE IN NHLBI.
AND YOU'LL FIND OUT AND LUCKILY
YOU TURNED INTO POTENTIALLY A
P.I. WHO HAS BEEN FUNDED, AND SO
AT THIS POINT IN TIME, YOU FIND
OUT ALSO HOW MUCH MONEY WAS
APPROVED FOR YOUR RESEARCH.
DURING THIS PROCESS, YOU
COMMUNICATE HOPEFULLY WITH THE
PERSON WHO LEADS THE REVIEW,
WHICH IS THE SCIENTIFIC REVIEW
OFFICER, WITH THE ASSIGNED
PROGRAM OFFICER AND WITH THE
GRANTS MANAGER AND ACTUALLY THEY
HAVE EXPERTISE TO ADVISE YOU ON
WHAT'S NEEDED TO BE APPROVED AND
INITIATE YOUR WORK, ANY MONEY OR
TIMELINE OR APPROVAL QUESTIONS,
THEY ARE THE BEST TO ANSWER.
SO THIS PROCESS IS ACTUALLY A
PRETTY LONG PROCESS.
IT TAKES IN THE BEST CASE
SCENARIO 10 MONTHS.
IN THE BEGINNING, YOU ARE IN
WHAT THEY CALL REVIEWLAND AND
YOU ARE IN PROGRAMLAND.
AT ANY TIME, THOUGH, THERE IS
STILL THE HUMAN TOUCH AND THERE
IS THIS MYSTERIOUS RED PHONE WE
HAVE AT NIH, WE NEVER KNEW
EXACTLY WHAT'S THE SIGNIFICANCE
OF THE RED COLOR, BUT IT'S A
TOUCH PHONE.
SO YOU CAN, ANY TIME, CALL THESE
PEOPLE, YOU CAN ALSO SEND AN
EMAIL AND MY FAVORITE EXAMPLE IS
THIS EMAIL THAT I RECEIVED BASED
ON SOMEBODY MATCHING THEIR IDEA
WITH ME, I APPEARED AS THE FIRST
MATCH, ALTHOUGH APPARENTLY NCI
WOULD HAVE BEEN THE BEST
INSTITUTE, BUT AS A PERSON
HAVING SO MANY LYMPHATIC
RESEARCH PROPOSALS, I GOT AN
EMAIL.
AS YOU CAN SEE, YOU CAN
IMMEDIATELY GET MY EMAIL OR
ANYBODY ELSE'S WHO MATCHES WITH
YOU FROM DR. DHRUV SINGHAL
APPLYING FOR HIS FIRST RSM R01 D
WE HAD A VERY NICE EXCHANGE OF
QUESTIONS AND ANSWERS AND HE
APPLIED AND HE IS NOW OUR
GRANTEE.
WE DO HAVE A COMMITTEE THAT
UNDERSTANDS THE FACT THAT SINCE
LYMPHATIC RESEARCH TOUCHES SO
MANY BODY ORGANS AND FUNCTIONS,
AS WELL AS DISEASE, MAY BE
SOMETIMES HARD TO PLACE THEM
WITHIN THE NIH, SO OUR TRANS-NIH
LYMPHATIC COORDINATING COMMITTEE
HAS A LOT OF MEMBERS,
INSTITUTES, AS WELL AS VERY
IMPORTANTLY RECEIVES FEEDBACK
FROM OUR GUEST SOCIETIES AND YOU
CAN SEE HERE THE CURRENT MEMBERS
AND GUESTS.
YOU CAN IMAGINE THAT WE'RE
MAINLY AN INFORMATION EXCHANGE,
WE TRY TO MAKE SURE THAT
EVERYBODY IS AWARE OF ALL THE
OPPORTUNITIES AND THE LATEST
INFORMATION IN TERMS OF SCIENCE,
OF DATABASES AND OTHER THINGS,
BUT WE ALSO WORK TOGETHER ON
SPECIFIC INITIATIVES SUCH AS A
COUPLE THAT I'M GOING TO
EXEMPLIFY NOW.
IN ORDER TO ANSWER THIS
QUESTION, IT'S A VERY COMPLEX
QUESTION ACTUALLY, HOW MUCH DOES
NIH OR IN OUR CASE NHLBI INVEST
IN LYMPHATIC RESEARCH, WE HAVE
TO DRAW FROM A LOT OF
INFORMATION.
THIS INFORMATION AGAIN IS AT
YOUR FINGERTIPS THROUGH THE NIH
REPORT, OPEN ACCESS DATA AND
TOOLS, AND THERE IS A SPECIFIC
SECTION THAT IS CALLED RESEARCH
CONDITION AND DISEASE
CATEGORIZATION OR RCDC.
IT'S IMPORTANT TO KNOW THAT
THESE ARE NOT REPRESENTING
STATIC NUMBERS.
THEY REALLY EVOLVE WITH SCIENCE,
BUDGET AND SO FORTH, AND THEY'RE
ALSO NOT MUTUALLY EXCLUSIVE, AS
IT'S VERY DIFFICULT TO PUT ANY
RESEARCH PROPOSAL, ESPECIALLY
ABOUT TOPICS SUCH AS LYMPHATIC
RESEARCH, IN JUST ONE CATEGORY.
SO WHAT I'M GOING TO SHOW YOU
REPRESENTS OUR BEST EFFORT TO DO
THIS THAT IS ASSIST BID MACHINE
LEARNING ASSISTED BY
TECHNOLOGY AND OTHER
TECHNOLOGIES NOW AVAILABLE.
AS OF 2022, WE LAUNCHED TWO NEW
RCD CCTT GRIS, D CCTT .
THE CATEGORY ALREADY HAD ABOUT
LYMPHOMA, SO AT ANY TIME
LYMPHATIC RESEARCH SHOULD BE
CONTAINING ONE OF THESE
CATEGORIES, THIS IS JUST SHOWING
SOME NUMBERS THAT ARE RELATED
ONLY TO THE TYPE OF R01s, TYPE
1 AND 2, THAT MEANS FIRST IN THE
RENEWAL, AND THEY WILL COVER
LYMPHATIC RESEARCH, INCLUDING
LYMPHOMA OR NOT INCLUDING
LYMPHOMA, AS WELL AS WE HAVE A
SPECIAL CATEGORY ON LYMPHEDEMA
NOW.
I'M GOING TO SHOW YOU YOU CAN
FIND MUCH MORE ABOUT THESE
THINGS, NOT ONLY THE TOTAL
AMOUNT, BUT WHAT TYPE OF
MECHANISM IS USED, WHAT'S THE
TITLE, WHO ARE THE
INVESTIGATORS, WHAT'S THE AMOUNT
OF FUNDING AND SO FORTH.
SO I INVITE YOU TO PLEASE LOOK
AT THESE.
YOU CAN ALSO BREAK DOWN THE
NUMBERS BY INSTITUTES, AND IF WE
LOOK AT RESEARCH THAT INCLUDES
LYMPHOMA, NCI IS ACTUALLY THE
NUMBER ONE FUNDER OF LYMPHATIC
RESEARCH.
IF LYMPHOMA IS EXCLUDED, NHLBI,
THE HEART, LUNG AND BLOOD
INSTITUTE, IS THE NUMBER ONE
FUNDER.
BARS HERE REPRESENT -- THE DARK
BAR REPRESENTS LYMPHATIC
RESEARCH NOT INCLUDING LYMPHOMA
AND THE ONE ABOVE INCLUDING
LYMPHOMA.
AS YOU CAN SEE OVERALL, THE
TRENDS ARE UP.
THE OTHER IMPORTANT QUESTION ON
EVERYBODY'S MIND, AM I GOING TO
BE SUCCESSFUL?
AND WHAT'S SHOWN HERE IS THE
SUCCESS OF LYMPHATIC RESEARCH --
I'M SORRY, MY COMPUTER IS TOO
JUMPY.
ON THE LEFT SIDE, YOU CAN SEE
THAT IT'S BEEN VARIABLE, BUT ON
AVERAGE, IS ABOUT 30%, WHICH
MEANS FOR THE FIRST APPLICATION
AND RENEWAL, WHILE THE OVERALL
SUCCESS OF R01s AT NIH HOVERS
AROUND 20%.
SO THE LYMPHATIC RESEARCH IS
PRETTY WELL RECEIVED, AND
FUNDED, BUT OF COURSE IT DEPENDS
ON HOW MANY APPLICATIONS ARE
RECEIVED.
ONE BRIGHT SPOT I THINK WE WANT
TO HIGHLIGHT IS THE FACT THAT
THERE ARE A LOT OF YOUNG
INVESTIGATORS WHO ARE INTERESTED
IN LYMPHATIC RESEARCH.
IF YOU LOOK HERE ON THE LEFT
SIDE, I'LL TRY NOT TO ADVANCE
SLIDE BY TOUCHING THE SCREEN,
THE ESI REPRESENT ABOUT A THIRD
OF ALL THE APPLICANTS AND THEIR
RATE OF SUCCESS IS EVEN BETTER
THAN THE NORMAL RATE OF SUCCESS,
SO I'M INVITING ALL YOU NEW
INVESTIGATORS TO APPLY.
NHLBI HAS A SPECIAL PROGRAM BY
WHICH YOU GET A 10-POINT BONUS
IF YOU APPLY.
ONE OF THESE APPLICANTS WAS
DR. SINGHAL.
HE DIDN'T NEED IT BUT IT'S OUT
THERE TO INVITE ALL OF YOU AND
ENHANCE THE NEW GENERATION OF
RESEARCHERS.
WE ALSO ARE VERY PROUD OF THE
OUTPUTS.
WE HAVE SUPPORTED A NUMBER OF
CLINICAL TRIALS, PATENTS,
DATASETS AND SO FORTH IN A LARGE
NUMBER OF HIGHLY CITED
PUBLICATIONS.
THE OTHER QUESTION THAT'S VERY
FREQUENTLY ON PEOPLE'S MIND IS
WHETHER WE SUPPORT SPECIFIC
INITIATIVES THAT ARE FOCUSED ON
THE LYMPHATIC SYSTEM.
ONE OF THESE INITIATIVES WHICH
IS FOCUSED ACTUALLY ON MAPPING
THE ENTIRE HUMAN BODY BUT NOW
CONTAINS A LOT OF THE
INFORMATION AND FUNDING TO MAP
THE LYMPHATIC SYSTEM IS THE
HUMAN BIOMOLECULAR ATLAS PROGRAM
THAT IS DEVELOPING A FRAMEWORK
ALLOWING US TO MAN THE MAP N
BODY FROM THE CLINICAL LEVEL ALL
THE WAY TO THE SINGLE CELL
RESOLUTION AND ADVANCE SCIENCE
ACROSS THE HUMAN BODY.
WE'RE DEVELOPING TECHNOLOGIES
AND MOST IMPORTANTLY WE'RE
DEVELOPING ALSO COLLABORATION
ACROSS EXPERTISE DOMAIN AND
ORGANS AND SO FORTH.
SO THIS PROGRAM IS ONE OF THE
INITIATIVES I'M VERY FAMILIAR
WITH, I'VE BEEN INVOLVED IN
LAUNCHING IT, AND I'M JUST GOING
TO SHOW VERY QUICKLY THAT IT
TAKES MANY, MANY YEARS TO
DEVELOP SUCH INITIATIVES.
WE INTENTIONALLY STARTED IN 2014
WITH THE REQUEST TO MAP THE
ENTIRE HUMAN VASCULATURE,
INCLUDING THE LYMPHATICS, AND WE
WENT THROUGH MANY DIFFERENT
ITERATIONS AND FINALLY WE WERE
APPROVED AS A PROGRAM THAT
ACTUALLY DEVELOPS A MAP OF THE
ENTIRE HUMAN BODY, AND THE FIRST
CALL FOR APPLICATION WAS
LAUNCHED IN 2018, SO IT TOOK
FOUR YEARS TO GET THERE.
INTERESTINGLY ENOUGH, WE JUST
HAD THE LAST ROUND OF CALL TO
APPLICATIONS AND WE RECEIVED
AWARDS THAT WERE MADE IN
SEPTEMBER OF THIS YEAR, SO VERY
RECENTLY.
WE BRING TOGETHER AS I SAID
PEOPLE FROM ACROSS THE UNITED
STATES AND SOMETIMES ACROSS THE
WORLD IF SUCH CAPABILITIES ARE
WARRANTED.
WE MAP A LOT OF ORGANS.
WE SPECIFICALLY -- I
SPECIFICALLY BROUGHT HERE A
COUPLE OF EXAMPLES THROUGH MANY
TECHNOLOGIES AND I SPECIFICALLY
BROUGHT A COUPLE OF EXAMPLES
THAT COME FROM OUR DISTINGUISHED
CHAIR, DR. ANGELA RADTKE WHO IS
ACTUALLY INSIDE THE CLINICAL
CENTER AT THE NIH BUT SHE IS
DOING FANTASTIC WORK IN
ADVANCING THE MAPPING OF THE
LYMPHATIC ORGANS.
AND WE ARE HERE, THIS LINE SHOWS
WE MADE NEW AWARDS THAT ARE
LOOKING AT MAPPING DIFFERENT
TISSUES AND ORGANS, AND WE MADE
ALSO AWARDS TO MAP -- TO DEVELOP
DEMONSTRATION PROJECT THAT
SHOWED THE BIOLOGICAL AND
MEDICAL UTILITY OF WHAT WE ARE
DOING.
I DO WANT TO SAY THAT WE
SPECIFICALLY NAMED LYMPHATIC
RESEARCH WHEN WE WROTE THE CALL
FOR APPLICATIONS, AND I WAS
INVOLVED IN MANY EFFORTS TO
ENGAGE THE LYMPHATIC RESEARCH
COMMUNITY.
WE GAVE AND ORGANIZED MANY
SEMINARS, INCLUDING WITH THE
HELP OF THE BOSTON LYMPHATIC,
WHERE WE BROADCASTED THIS
RESEARCH FUNDING OPPORTUNITY.
EVEN MORE IMPORTANTLY, WE ARE
VERY HAPPY TO ANNOUNCE THAT WE
HAVE THE NEWEST ADDITION TO THIS
PROGRAM IS THE MULTI-SCALE
MAPPING OF THE HUMAN LYMPHATIC
VESSELS, WHICH IS LED BY AN
OUTSTANDING GROUP OF RESEARCHERS
AND PHYSICIANS FROM ACROSS THE
U.S. OF A, DR. SINGHAL,
DR. VLACHOS AND DR. HONG.
AND WHAT THE BASIS OF THIS
PROPOSAL IS, IS DR. SINGHAL IS
INDICATING THAT HAVING A MAP OF
THE LYMPHATIC VESSEL IS ACTUALLY
THE FOUNDATION FOR THE CLINICAL
APPROACH ESPECIALLY IN
LYMPHEDEMA, KNOWING WHICH
PATIENTS WILL HAVE POTENTIAL
VARIATIONS OF THE LYMPHATIC
VESSELS AND HOW TO USE SUCH
KNOWLEDGE TO ACTUALLY TRY TO
PREVENT LYMPHEDEMA.
SO ONE OF THE MAJOR THINGS
THEY'RE GOING TO DO, THEY'RE
GOING TO ACTUALLY BRING BACK TO
THIS TIME A BOOK FROM 1938,
WHICH I UNDERSTAND IS THE MAIN
SOURCE OF INFORMATION STILL,
ROUVIERE, MAPPING THE LYMPHATIC
VESSELS TO SOMETHING MUCH MORE
MODERN.
THERE'S AN OPEN SOURCE PLATFORM
BEING DEVELOPED THAT'S GOING TO
BE AVAILABLE TO INVESTIGATE ALL
THE DETAILS OF THE LYMPHATIC
VASCULATURE.
I KNOW YOU CANNOT SEE REALLY
HERE IN THE SMALL IMAGES, BUT
THIS IS A QUITE SOPHISTICATED
APPROACH IN DEVELOPING KNOWLEDGE
OF THE MAP THAT'S GOING TO BE
USED BY PHYSICIAN RESEARCHERS
AND CAN BE TRAVELED JUST LIKE IN
THAT MOVIE THAT WE ALL REMEMBER,
TRAVELING THE VASCULATURE.
THE REASON IS THEY ARE ALSO
GOING TO APPLY VERY INTERESTING
NEW TECHNOLOGIES THAT GO FROM
THE 3D MAPPING TO SINGLE CELL
ANALYSIS, SO WE'RE GOING TO KNOW
HOW THE LYMPHATIC SYSTEM IS
ORGANIZED AND SPECIFICALLY THE
VASCULATURE IN THIS APPLICATION
FROM THE LEVEL -- THE CLINICAL
LEVEL WHERE THE PHYSICIANS AND
MOST OF THE PATIENTS OPERATE ALL
THE WAY DOWN TO THE SINGLE CELL
OF GENE EXPRESSION AND
FUNCTIONAL ANALYSIS, WHERE MOST
OF THE RESEARCHERS OPERATE, BUT
THAT'S NEEDED IN ORDER TO CREATE
THE MEDICAL KNOWLEDGE TO ADVANCE
THE FIELD.
SO THIS IS ALL I HAD TO SAY, BUT
I DO WANT TO LEAVE YOU WITH THE
MESSAGE THAT THIS IS A TEAM
SPORT, RESEARCH IS A TEAM SPORT.
WE ARE ON YOUR TEAM.
I WANT TO TELL YOU THAT A LOT OF
THIS INFORMATION IS AT YOUR
FINGERTIPS.
MY FAVORITE SEARCH ENGINE,
GOOGLE, WILL FIND THE QUESTIONS
AND PEOPLE WHO CAN HELP YOU WITH
THE QUESTIONS YOU HAVE ABOUT
NIH.
WE ARE HAPPY TO BE HERE.
WE CAME HERE AND I CAN VOUCH FOR
EVERYBODY I WORK WITH, THEY CAME
TO THE NIH NOT BECAUSE THEY
DIDN'T HAVE ANYTHING ELSE BETTER
TO DO, BUT BECAUSE WE ARE
DEDICATED TO SERVICE AND TO
SCIENCE AND HELPING YOU ADVANCE
SCIENCE.
I WANT TO GIVE SPECIAL THANKS IN
CONNECTION TO THIS TALK TO OUR
PORTFOLIO ANALYTICS BRANCH WHO
HELP WITH THE PORTFOLIO
ANALYSIS, AND MY FRIENDS AND
COLLEAGUES, ILSA ROVIRA AND
SELEN CATANIA FOR ALL THE
ENORMOUS WORK THEY DID TO PUT
TOGETHER THE WORKSHOP BUT ALSO
SPECIFICALLY FOR BEING SO KIND
AND HELPING WITH THIS TALK.
I BELIEVE THAT THERE IS TIME TO
ASK QUESTIONS AT THE END OF THE
SESSION OR SOMEBODY CAN TELL ME,
BUT I'M LOOKING FORWARD TO YOUR
QUESTIONS, BOTH DURING THIS
WORKSHOP AS WELL AS AFTER.
NOW YOU KNOW HOW TO FIND US.
THANK YOU, EVERYONE.
>> THANK YOU, ZORINA.
NEXT IS GOING TO BE DR. MICHAEL
DAVIS.
ANDREA, WILL YOU BE --
>> YES, HAPPY TO INTRODUCE HIM.
IT'S MY PLEASURE TO INTRODUCE
OUR FIRST KEYNOTE SPEAKER,
DR. MICHAEL DAVIS, A
DISTINGUISHED PROFESSOR AT THE
UNIVERSITY OF MISSOURI COLUMBIA
SCHOOL OF MEDICINE, AND LEADER
IN THE DEVELOPMENT OF TOOLS,
TECHNOLOGIES AND ANIMAL MODELS
TO STUDY LYMPHATIC FUNCTION.
THE TITLE OF HIS TALK IS
MECHANISMS OF LYMPHATIC
CONTRACTILE AND VALVE
DYSFUNCTION IN METABOLIC
SYNDROME.
WELCOME, DR. DAVIS.
>> THANK YOU.
I WILL START BY INTRODUCING THE
LYMPHATIC SYSTEM TO A GENERAL
AUDIENCE.
IT'S A ONE-WAY VASCULAR NETWORK
THAT RUNS IN PARALLEL TO THE
BLOOD VASCULAR SYSTEM.
IN ALMOST EVERY TISSUE, THERE
ARE NETWORKS OF LYMPHATIC
CAPILLARIES THAT ABSORB LYMPH,
COALESCE INTO COLLECTING
LYMPHATIC VESSELS WHICH HAVE AT
LEAST ONE CELL LAYER OF MUSCLE,
SMOOTH MUSCLE, AND A SERIES OF
ONE-WAY VALVES.
THESE SPONTANEOUSLY CONTRACT AND
PROPEL LYMPH CENTRALLY BUT MOST
OF THE LYMPH IS DRAINED INTO THE
CONFLUENCE OF THE GREAT VEINS IN
THE NECK.
HERE YOU SEE THESE LYMPHOVENOUS
VALVES AT THIS CONFLUENCE IN
PURPLE HERE.
THE GREEN STRUCTURES ARE VENOUS
VALVES.
THERE ARE THREE CLASSIC ROLES
FOR THE LYMPHATIC SYSTEM IN
HOMEOSTASIS, INTERSTITIAL FLUID
AND PROTEIN BALANCE, FAT
ABSORPTION FROM THE INS TESSAL
TRACT, IMMUNE CELL TRAFFICKING.
OVER THE PAST 10 YEARS, THERE
ARE AT LEAST 10 NEWLY
APPRECIATED ROLES, AND I'VE JUST
LISTED A FEW OF THEM.
I WON'T GO INTO DETAILS BECAUSE
THEY WILL BE COVERED BY SPEAKERS
IN THIS SYMPOSIUM.
I WILL JUST MENTION THAT
LYMPHEDEMA IS A SIGNIFICANT
HUMAN HEALTH PROBLEM.
PRIMARY LYMPHEDEMA, ALTHOUGH
RELATIVELY RARE, INVOLVES
GENETIC MUTATIONS OF LYMPHATIC
VESSEL DEVELOPMENT AND VALVE
DEVELOPMENT THAT PRODUCE PRETTY
CATASTROPHIC RESULTS IN TERMS OF
FAILURE TO ABSORB INTERSTITIAL
FLUID.
SECONDARY LYMPHEDEMA IS MUCH
MORE COMMON.
THERE ARE ABOUT 10 MILLION CASES
PER YEAR IN THE U.S.
MOSTLY A CONSEQUENCE OF
CONGESTIVE HEART FAILURE,
CHRONIC VENOUS DISEASE, AND
CANCER SURGERY AND RADIOTHERAPY.
BUT AROUND THE WORLD, THERE ARE
MANY MORE CASES, OVER
130 MILLION PREDICTED OR
FILARIA, PARASITES THAT PLUG UP
THE LYMPHATIC SYSTEM.
THE CONSEQUENCES OF UNTREATED
LYMPHEDEMA ARE PAIN, IMMOBILITY,
INCREASED RISK OF INFECTION,
FIBROSIS AND TISSUE DAMAGE.
COLLECTING LYMPHATICS ARE
CRITICAL FOR ACTIVE LYMPH
TRANSPORT.
IF YOU LOOK, A LOT OF TEXTBOOKS
STILL GIVE ONE THE IMPRESSION
THAT LYMPH PROPULSION IS PURELY
PASSIVE, FROM CONTRACTIONS OF
SKELETAL MUSCLES THAT COMPRESS
LYMPH VESSELS BUT THERE'S
ACTUALLY VERY GOOD STUDIES THAT
SHOW THAT TWO THIRDS OF PUMPING
IN DEPENDENT EXTREMITIES UNDER
QUIET STANDING ARE DUE TO ACTIVE
CONTRACTIONS.
SO THIS IS A RAT MESENTERIC
LYMPHATIC VESSEL.
WE'VE ISOLATED AND CANNULATED IN
MICRO PIPETTES.
I PUT IN RED CELLS AS FLOW
MARKERS.
YOU CAN SEE THAT WHEN THE VESSEL
EXPANDS IN DIASTOLE, IT DRAWS IN
FLUID AND CELLS FROM THE INFLOW
PIPETTE, AND WHEN IT CONTRACTS,
IT EXPELS A SMALL AMOUNT OF
FLUID AND THE OUTFLOW VALVE
SNAPS BACK TO PREVENT BACKFLOW.
THESE LYMPHATIC CONTRACTIONS ARE
INITIATED BY ACTION POTENTIALS
IN LYMPHATIC MUSCLE.
ACTION POTENTIALS LOOK SOMETHING
LIKE THIS.
THESE ARE GENERATED BY A CYCLE
OF IONIC INTERACTIONS THAT
PRODUCE DIASTOLIC
DEPOLARIZATION, EVENTUALLY
RESULT IN OPENING OF L-TYPE
CALCIUM CHANNELS WHICH ALLOW
CALCIUM INFLUX TO DRIVE THESE
TWITCH CONTRACTIONS.
SO EACH ACTION POTENTIAL WILL
DRIVE A SINGLE TWITCH
CONTRACTION, AND THE ONE-WAY
VALVES BASICALLY TRANSFORM THE
PULSATILE MOVEMENT INTO A
STEP-WISE INCREASE IN LYMPH
FLOW.
MY LAB HAS DEVELOPED MULTIPLE
METHODS FOR STUDYING
PARTICULARLY MOUSE LYMPHATIC
VESSELS, BUT WE ALSO STUDY
VESSELS FROM RATS AND HUMANS.
FROM 1 TO 4 SEGMENTS OF VALVES,
2-VALVE SEGMENTS FOR PUMP TESTS,
1-VALVE SEGMENTS FOR
QUANTITATIVE ANALYSIS OF
LYMPHATIC VALVE FUNCTION.
WE'VE DEVELOPED METHODS FOR
CALCIUM IMAGING,
ELECTROPHYSIOLOGY, AND WE
ACTUALLY DO SOME IN VIVO
STUDIES, WHICH A LOT OF PEOPLE
DON'T APPRECIATE THAT WE DO.
AND THIS SLIDE JUST SHOWS THE
TYPICAL ANALYSIS.
SO THIS IS A POPLITEAL LYMPHATIC
FROM THE LOWER LEG OF THE MOUSE
TAKEN OUT, CANNULATED ON MICRO
PIPETTES, CLEANED OF MOST FAT
AND CONNECTIVE TISSUE.
SO WE TYPICALLY USE THIS TYPE OF
ANALYSIS BECAUSE IT HAS DEFINED
PRESSURES AND FLOWS.
WE CAN CHANGE THE SOLUTIONS
INSIDE AND OUTSIDE THE VESSEL,
AND WE CAN USE DEFINED
CONCENTRATIONS OF AGONISTS AND
ANTAGONISTS.
THIS TRACE HERE, PRESSURE AND
DIAMETER OVER TIME, SHOW THE
EXQUISITE SENSITIVITY OF
LYMPHATIC CONTRACTIONS TO
PRESSURE.
SO EACH ONE OF THESE DOWNWARD
DEFLECTIONS IN DIAMETER IS A
3 TO 52ND CONTRACTION, AS WAS
SHOWN IN THE VIDEO.
YOU CAN SEE THAT THE PRESSURE
CHANGES FIVE FOLD BETWEEN
PRESSURE OF 3 CENTIMETERS WATER
AND A HALF COUNCILMEMBER OF 16-D
IT INCREASES WHEN YOU GO TO
THE -- PRESSURES.
WE PUT IT IN CALCIUM-FREE
SOLUTION, GET PASSIVE -- WE CAN
CALCULATE TONE, AMPLITUDE,
FREQUENCY, EJECTION FRACTION AND
INDEX OF FLOW WHICH WE CALL
FRACTIONAL PUMP FLOW BECAUSE THE
ABSOLUTE FLOWS IN MOUSE ARE IN
RANGE OF NANOLITERS PER MINUTE,
WHICH ARE TOO SMALL TO DIRECTLY
MEASURE WITH FLOW PROBES MPLE
THE FREQUENCY PRESSURE
RELATIONSHIP IN THE MOUSE
INCREASES FIVE FOLD ON AVERAGE
FOR VESSELS OVER THIS PRESSURE
RANGE.
THE AMPLITUDE OF CONTRACTIONS IS
OPTIMAL, SOMEWHERE AROUND 1 TO E
FRACTIONAL PUMP FLOW IS OPTIMAL
BETWEEN 3 AND 5 CENTIMETERS OF
WATER AND IT HAS SORT OF A BROAD
PEAK THAT LOOKS KIND OF LIKE A
CARDIAC FUNCTION CURVE.
A LOT OF PEOPLE USE THESE TYPE
OF ANALYSIS, BUT WE ALL MAKE
SOME ASSUMPTIONS WHEN WE USE
FRACTIONAL PUMP FLOW AS AN INDEX
OF PUMP OUTPUT.
THAT S WE ASSUME THAT THE VALVES
ARE 100% COMPETENT WITH NO
BACKLEAK, THAT THE CONTRACTION
WAVES ARE WELL ENTRAINED, SO
THAT THE CONTRACTION IS
EFFECTIVE, AND THAT THE VESSEL
WALL HAS LOW OR MINIMAL
PERMEABILITY.
MY LAB HAS NOTICED STUDYING
VARIOUS GENETIC MOUSE MODELS
OVER THE PAST 10 YEARS THAT A
LYMPHATIC DYSFUNCTION IN ALMOST
ALL OF THESE VARIABLES OCCURS.
SO THIS IS ONE EXAMPLE.
THESE ARE POPLITEAL VESSELS FROM
APOE KNOCKOUT MICE FED A HIGH
FAT DIET.
YOU CAN SEE -- THIS IS IN
COLLABORATION WITH ANGELI IN
SINGAPORE.
THE FREQUENCY VERSUS PRESSURE IS
NOT ALTERED BY THE APOE KNOCKOUT
BUT CONTRACTILE FUNCTION,
AMPLITUDE IS DEPRESSED BY 50% AT
ALL PRESSURES THAT WE STUDIED.
IF WE TAKE OUT SEGMENTS WITH
SINGLE VALVES AND DO
QUANTITATIVE MEASUREMENTS OF
VALVE BACKLEAK IN CLOSED VALVES,
I'M NOT GOING TO EXPLAIN THE
DETAILS BECAUSE JORGE WILL IN A
TALK LATER.
BUT THERE'S A SLIGHT BACKLEAK IN
VALVES FROM THE CONTROL GROUP,
BUT IT'S INCREASED FOUR FOLD IN
APOE KNOCKOUT VALVES.
THE NEXT TWO GRAPHS OVER HERE
SHOW THE BACK PRESSURE REQUIRED
TO CLOSE THE VALVE GIVES YOU AN
INDICATION OF THE STIFFNESS OF
THE VALVES.
NORMAL VALVES SHOW THIS KIND OF
CURVILINEAR RELATIONSHIP WITH
ADVERSE PRESSURE REQUIRED TO
CLOSE IT, AND THE DIAMETER.
SO AS THE VESSEL APPROACHES ITS
MAXIMAL DIAMETER, NORMALIZED TO
1 HERE, IT REQUIRES EVEN JUST A
FEW CENTIMETERS OF WATER TO
CLOSE THE VALVE, BUT ABOUT 30%
OF THE APOE KNOCKOUT VALVES
NEVER CLOSE UNDER ANYWHERE CLOSE
TO NORMAL PHYSIOLOGICAL
PRESSURES.
WHEN JOSH SCALLAN WAS IN MY LAB,
HE TOOK VESSELS OUT OF THESE
MICE AND DID QUANTITATIVE
MEASUREMENTS OF PERMEABILITY, OF
COLLECTING VESSEL PERMEABILITY.
THE NORMAL MICE ARE SOMEWHERE
CLOSE TO VENEAL LEVELS.
SO MY POSTDOC JINNY KIM HAS DONE
RECENT STUDIES IN DB/DB MICE.
SO THIS IS ONE MODEL, METABOLIC
SYNDROME.
MICE ARE EXTREMELY OWE OBESE,E
VERY HIGH GLUCOSE LEVELS.
THIS IS DB/DB MOUSE TRACE.
YOU CAN SEE THEY'RE VERY BLUNTED
CONTRACTIONS, THEY'RE IRREGULAR
IN FREQUENCY, THERE ARE NO
CONTRACTIONS AT THE LOWER
PRESSURES AND INTERESTINGLY, WE
FIND THAT GLABECTOMIDE RESCUES
THE FREQUENCY HERE.
SO YOU CAN SEE THE FREQUENCY
PATTERN LOOKS MORE LIKE THE WILD
TYPE.
IT DOESN'T RESCUE THE AMPLITUDE
UNLESS YOU ALSO ADD AN ACTIVATOR
OF L-TYPE CALCIUM CHANNELS.
THIS SHOWS THE AVERAGE DATA
HERE, SHOWING DEPRESSION BY --
IN THE DB/DB MICE.
JOSH SCALLAN HAD DONE
PERMEABILITY MEASUREMENTS ON
THESE VESSELS AND IN DB/DB MICE
FROM BOTH MALES AND FEMALES, THE
TWO DIFFERENT COLORS HERE, THERE
IS A 50 FOLD INCREASE IN
PERMEABILITY, THE LOG SCALE ON
THE Y AXIS.
SO JINNY JUST LOOKED AT THE
EFFECTS OF GLEUS GLUCOSE, PER
SE, USED OFTEN IN DIABETIC
STUDIES IN VITRO, AND SO YOU CAE
AFTER A FEW MINUTES, THE
FREQUENCY HAS DECREASED IN THIS
VESSEL AT THE CONTROL PRESSURE,
AND THEN WHEN WE LOWER TO
0.5 CENTIMETERS OF WATER,
THERE'S NO CONTRACTIONS OF THAT
PRESSURE, AND THIS IS PRETTY
MUCH RESCUED HERE.
THIS IS THE CONCENTRATION
RESPONSE CURVE TO GLUCOSE, AND
THE CONTROL IS FOR OSMOTIC
EFFECTS.
SO THIS IS SUMMARY DATA SHOWING
DEPRESSION OF FREQUENCY AT EVERY
PRESSURE IN THE PRESENCE OF HIGH
GLUCOSE RESCUED BY GLABEKLOMIDE.
INTERESTINGLY WHEN WE LOOK THE A
VESSELS FROM AN ANIMAL MODEL OF
KETP CHANNEL MODEL KNOCKOUT,
KIR6.1 -- AND THOSE MICE ARE --
THE VESSELS ARE RESISTANT TO THE
EFFECTS OF HIGH GLUCOSE.
SUGGESTING IT IS WORKING THROUGH
KATP CHANNELS.
IF YOU COMPARE THE PRESSURE
DIAMETER RELATIONSHIPS OVER TIME
IN WILD TYPE AND IN KNOCKOUT
MICE, THEY LOOK VERY SIMILAR.
VESSELS STILL SHOW FREQUENCY
MODULATION, THEY SHOW GOOD
ALMOST PLI TEUD AT ALL
PRESSURES.
NOW, IF WE TRY TO INDUCE
METABOLIC STRESS BY INHIBITING
SPECIFIC PARTS OF THE
MITOCHONDRIAL ELECTRON TRANSPORT
SYSTEM, IN THIS CASE INHIBITING
COMPLEX 3, IF YOU USE 30
NANOMOLAR ANTIMYCIN A -- IF YOU
ADD BACK GLIBENCLAMIDE, THIS CAN
BE REVERSED.
THE KNOCKOUT MICE ARE COMPLETELY
RESISTANT TO THE EFFECTS OF
ANTIMYCIN A.
SO WE THINK THAT THE KATP
SENSITIVE POTASSIUM CHANNELS ARE
NORMALLY NOT ACTIVATED AT HIGH
LEVELS IN LYMPHATIC MUSCLE, BUT
ONCE THEY'RE ACTIVATED, SAY BY
METABOLIC STRESS, THEY APPLY A
VERY STRONG BREAK TO THE
PACEMAKING MECHANISM, SLOWING
DOWN THE FREQUENCY AND COMPARING
CONTRACTILE ACTIVITY.
THIS ISN'T THE ONLY WAY THAT
THESE CHANNELS ARE REGULATED.
IT'S SHOWN THAT EXCESS CIV PRO
DUCKS OF NITRIC OXIDE THROUGH
INOS LEADS TO -- GMP LEVELS AND
THAT CAN CROSS ACTIVATE PKA AND
BOTH OF THESE ENZYMES ARE KNOWN
TO PHOSPHORYLATE THE CHANNEL TO
INCREASE ITS ACTIVITY.
AND THEN THERE IS A GENETIC
CONDITION CALLED CANTU SYNDROME
IN WHICH PATIENTS HAVE
MUTATIONS, GAIN OF FUNCTION
MUTATIONS IN EITHER OF THE TWO
KATP CHANNEL SUBUNITS, AND 50%
OF THESE PATIENTS HAVE
LYMPHEDEMA.
SO WE THINK THAT THESE
OBSERVATIONS ARE BASICALLY
CONSISTENT WITH A LOT OF THE
LITERATURE SHOWING IN METABOLIC
DISORDERS
DISORDERS, VARIOUS TYPES,
THERE'S LYMPHATIC CONTRACTILE
DYSFUNCTION, SECONDARY VALVE
DYSFUNCTION AND COLLECTING
VESTIBLE HYPERPERMEABILITY.
NOW I WANT TO TALK ABOUT THE
LAST TOPIC, WHICH IS ENTRAINMENT
OF CONTRACTION WAVES.
SO THIS MOVIE ON THE LEFT SHOWS
A LYMPHATIC VESSEL, A POPLITEAL
VESSEL ISOLATED FROM THE MOUSE
AND IT'S SHOWING SPONTANEOUS
CONTRACTIONS.
YOU NOTICE THAT IT'S ALMOST A
SYNCHRONOUS CONTRACTION OVER THE
WHOLE LENGTH OF THIS VESSEL.
SO THE WAY THAT WE QUANTIFY THIS
IS TO MEASURE DIAMETER AT EACH
POINT INSTEAD OF A SINGLE POINT,
EACH POINT ALONG THE VESSEL, AND
MAP IT AS A FUNCTION OF TIME.
SO EACH VERTICAL LINE ON THIS
SPATIO-TEMPORAL MAP IS A VIDEO
FRAME.
WHEN YOU COMPILE 10,000 VIDEO
FRAMES HERE, YOU GET THESE DARK
BANDS THAT REPRESENT SINGLE
CONTRACTIONS, SO FOUR SINGLE
CONTRACTIONS IN THAT MOVIE.
AND THE INTENSITY OF THE BAND IS
PROPORTIONAL TO THE DIAMETER
CHANGE.
SO THE STRONGER CONSTRICTION,
THE DARKER THE BAND.
THE LEFT EDGE REPRESENTS THE
SPEED OF CONDUCTION.
YOU CAN GET THE SLOPE THERE.
AND IF YOU LOOK AT COMPARISON TO
A VESSEL THAT COMES FROM A KATP
GAIN OF FUNCTION MOUSE, YOU CAN
SEE THAT THE CONTRACTION WAVES
ARE SLOWER CONDUCTING, THE
CONTRACTIONS ARE WEAKER, THERE
ARE MULTIPLE PACEMAKER SITES,
THE ST MAP LOOKS SOMETHING LIKE
THIS, YOU CAN SEE THE WAVES
CHANGING DIRECTIONS.
SO WHEN THE EXCITABILITY OF
LYMPHATICS MUSCLE IS REDUCED
THERE ARE CONTINUOUS CONTRACTION
WAVES, MULTIPLE PACEMAKER SITES,
CHANGES IN DIRECTION OF THE
WAVES AND REDUCED CONDUCTION
SPEED.
AND SO THIS LAST TEST IS THE
TEST THAT WE DEVELOPED THAT KIND
OF UNMASKS WHAT WE'VE BEEN
MISSING IN STANDARD CONTRACTION
ASSAYS.
SO THIS IS A TWO-VALVE SEGMENT,
INFLOW AND OUTFLOW VALVES, AND
WE'VE MAPPED OUT ALL THE
CONTRACTIONS TO SHOW IN THE WILD
TYPE VESSEL THEY'RE ALL
ENTRAINED, THEY GET WEAKER AS
YOU GO TO ELEVATED PRESSURES.
WE'VE GONE THROUGH THE VIDEOS
AND MEASURED THE POSITIONS OF
BOTH VALVES AT EACH POINT IN
TIME.
SO WHAT WE DO, WE HOILD THE
INFLOW PRESSURE CONSTANT AND --
TO MAKE THE VESSEL PUMP AGAINST
AN INCREASING OUTPUT LOAD.
SO AS OUTFLOW PLEASURE
INCREASES, THE VALVE GOES FROM
NORMALLY OPEN TO NORMALLY
CLOSED, AND IT ONLY OPENS
TRANSIENTLY DURING EACH
CONTRACTION AS IT EJECTS A SMALL
AMOUNT OF LYMPH.
WHEN THE PUMP REACHES A CERTAIN
POINT, IN THIS CASE ABOUT
9 CENTIMETERS OF WATER, IT NO
LONGER OPENS THE OUTPUT VALVE.
THAT'S AN INDICATOR THAT WE'VE
REACHED THE PUM LIMIT.
IF WE DO THIS SAME EXPERIMENT IN
A KATP GAIN OF FUNCTION MOUSE,
WE CAN SEE THAT THE PUMP LIMIT
HERE IS LESS THAN 1.
SO IT'S A REALLY PATHETIC PUMPER
EVEN THOUGH THE M PLI TEUD AND
FREQUENCY IS HIGHER, THE
AMPLITUDE IS NOT THAT MUCH
DIFFERENT THAN THE CONTROL.
SO IF WE COMPARE THE ANALYSIS OF
CONTRACTILE ACTIVITY WITH A
CONVENTIONAL ANALYSIS WITH THE
PUMP TEST, IT LOOKS LIKE THIS.
THERE'S BARELY ANY IMPAIRMENT IN
AMPLITUDE.
THE FREQUENCY IN THE
KNOCKOUTS -- OR KNOCK-IN MICE IS
ACTUALLY HIGHER, AND THERE'S NO
DIFFERENCE IN THE PUMP FLOW
CURVES.
BUT IF YOU LOOK AT THE VESSELS
WHEN THEY'RE FORCED TO PUMP
AGAINST A LOAD, IT'S VERY, VERY
CLEAR THAT THERE'S A HUGE
IMPAIRMENT IN THE SUR2 GAIN OF
FUNCTION MICE.
SO IN CONCLUSION, LYMPHATIC
DYSFUNCTION UNDER CONDITIONS OF
METABOLIC STRESS APPEARS TO
INCLUDE IMPAIRED CONTRACTILE
STRENGTH AND PACEMAKER
FREQUENCY, VALVE BACKLEAK AND
INCOMPETENCY, LACK OF
CONTRACTION WAVE ENTRAINMENT,
HYPERPERMEABILITY OF COLLECTING
VESSELS, AND THE UNDERLYING
MECHANISMS INCLUDE ACTIVATION OF
CHANNELS.
IN TERMS OF GAPS AND
OPPORTUNITIES, RELATED TO THIS
TALK, WE NEED TO UNDERSTAND THE
PRESSURES AND FLOWS IN HEALTHY
AND DYSFUNCTIONAL LYMPHATIC
NETWORKS, VIRTUALLY NOTHING IS
KNOWN IN MICE ABOUT THIS.
WE NEED TO UNDERSTAND THE
DEVELOP MEANT ORIGIN OF
LYMPHATIC MUSCLE CELLS, WHAT
CONTRACTILE PROTEINS THEY
EXPRESS, WHAT ION CHANNELS THEY
EXPRESS.
THESE TELL TELEUS HOW THE MUSCLE
FUNCTION IS REGULATED, AND
WHETHER THERE ARE REGIONAL
DIFFERENCES IN THESE THINGS.
AND THEN THE EXTENT TO WHICH
CONTRACTILE AND VALVE
DYSFUNCTION RESULT FROM, OTHER
PATHOLOGIES, OR CONTRIBUTE TO
THOSE PATHOLOGIES.
FINALLY, WE NEED TO DEVELOP MORE
RELEVANT ANIMAL MODELS OF THE
LYMPHATIC SYSTEM.
OUR CURRENT MOUSE MODELS OF
METABOLIC SYNDROME AND SECONDARY
LYMPHEDEMA ARE PRETTY
INADEQUATE.
AND SOME ASPECTS OF MOUSE
LYMPHATIC PHYSIOLOGY MAY NOT
APPLY TO HUMANS.
FOR EXAMPLE, VISCERAL AND
THORACIC CLEBTING LYMPHATICS DO
NOT PUMP AT ALL IN A MOUSE.
THEY DO IN EVERY OTHER SPECIES.
MICE ARE NOT EXPOSED TO CHRONIC
GRAVITATIONAL LOADS, WHICH IN
HUMANS IS ALMOST ALWAYS WHEN
LYMPHEDEMA DEVELOPS.
AND FINALLY, THERE ARE VERY FEW
FUNCTIONAL STUDIES OF HUMAN
VESSELS THAT HAVE BEEN
PERFORMED, ESPECIALLY HEALTHY
VESSELS.
SO I THANK MY COLLABORATORS, MY
CURRENT POST POSTDOC AND FORB
MEMBERS.
THANK YOU VERY MUCH.
>> THANK YOU VERY MUCH,
DR. DAVIS.
NOW IT'S MY PLEASURE TO WELCOME
OUR MODERATOR, DR. NATASZA
KURPIOS, CORNELL UNIVERSITY.
SHE'S CONTRIBUTED SIGNIFICANTLY
TO OUR UNDERSTANDING OF
LYMPHATIC DEVELOPMENT AS WELL AS
THE MECHANISMS IN ORGAN
ACEMETERY AND SHE'LL MODERATE
SESSION TWO.
SO TAKE IT AWAY, YOU CAN
INTRODUCE THE NEXT SPEAKER.
>> ANDREA, THANKS SO MUCH.
CAN YOU HEAR ME?
>> YES, WE CAN.
>> OKAY, FANTASTIC.
SO HELLO, EVERYBODY, AND WELCOME
TO SESSION TWO FROM WHEREVER YOU
ARE IN THE WORLD.
WE HAVE AN OUTSTANDING SESSION
ENTITLED LYMPHATIC DEVELOPMENT
AND FUNCTION IN HOMEOSTATIC
DEVELOPMENT, AND OUR FIRST
SPEAKER OF THE SESSION IS
DR. OLIVER STONE.
WELCOME, OLI.
AND THE TITLE OF OLIVER'S TALK
WILL BE LYMPHATIC DEVELOPMENT,
DEVELOPMENTAL ORIGINS OF
LYMPHATIC ENDOTHELIAL CELLS.
TAKE IT AWAY.
>> THANK YOU VERY MUCH.
SO I'M A DEVELOPMENTAL BIOLOGIST
WORKING AT THE UNIVERSITY OF
OXFORD, AND WE'RE BROADLY
INTERESTED IN UNDERSTANDING HOW
AND WHEN ENDOTHELIAL CELLS ARE
SPECIALIZED DURING EMBRYONIC
DEVELOPMENT TO ESTABLISH
HETEROGENEITY IN THE VASCULAR
SYSTEM.
THIS IS HOW WE SERENDIPITOUSLY
STUMBLED INTO THE LYMPHATIC
FIELD.
DURING EMBRYONIC DEVELOPMENT,
ENDOTHELIAL SPELLS ARE SPECIFIED
INTO ANGIOBLASTS THAT IMOA ON TO
PROLIFERATE, FURTHER
DIFFERENTIATE AND MIGRATE TO
PERFORM PRIMITIVE VASCULAR
NETWORKS.
WE'RE INTERESTED IN
UNDERSTANDING IF EVENTS AT THESE
VERY EARLY STAGES OF DEVELOPMENT
IMPACT THE EVENTUAL
DIVERSIFICATION OF ENDOTHELIAL
CELLS INTO THE KIND OF
ENDOTHELIAL CELLS WE WOULD FIND
IN CORONARY CELLS, IN THE LIVER,
FOR EXAMPLE, IMPACTED BY EARLY
EVENT, AND IMPORTANTLY AS I'M
SPEAKING TO YOU TODAY, THE
LYMPHATIC VASCULATURE.
SO TO GIVE YOU A BRIEF OVERVIEW
OF THE CURRENT KIND OF MODELS
WHICH EXIST ON LYMPHATIC
DEVELOPMENT, AT LEAST THIS IS
SPECIFICALLY REFERRING TO MICE.
SO THE KIND OF EXISTING MODELS
SUGGEST THAT AROUND EMBRYONIC
DAY 9.5, ENDOTHELIAL CELLS
WITHIN VENOUS ENDOTHELIUM, SO
THIS WOULD BE NOT ONLY THE
CARDINAL VEIN BUT ALSO
INTERSEGMENTAL VESSELS OF THE
MICE EMBRYO, SWITCH ON IN
RESPONSE TO SOX18, A
TRANSCRIPTION FACTOR KNOWN AS
PROX1.
IT'S BELIEVED THAT FROM THIS
STAGE, INDUCTION OF PROX1 LEADS
TO MIGRATION OF LYMPHATIC
ENDOTHELIAL CELLS FROM THE VEIN
AND THEN THESE LYMPHATIC
PROGENITORS SUBSEQUENTLY GO ON
TO FORM LYMPHATIC VESSELS.
NOW, OVER MORE RECENT YEARS,
THERE HAVE ACTUALLY BEEN A
NUMBER OF DESCRIPTIONS OF
NON-VENOUS SOURCES FOR LYMPHATIC
ENDOTHELIAL CELLS, SO THESE HAVE
BEEN DESCRIBED IN THE MESENTERY
IN MICE, THE CARDIAC LYMPHATICS
AND MORE BROADLY CONTRIBUTING TO
LYMPHATICS WITHIN THE SKIN.
BUT THESE ARE SOMEWHAT -- DEEMED
SOMEWHAT CONTROVERSIAL WITHIN
THE FIELD.
SO TO M COME BACK TO OUR
ORIGINAL INTEREST, SO WE WERE
INTERESTED IN UNDERSTANDING IF
CELLS FROM DIFFERENT SUBSETS OF
MESODERM CONTRIBUTES TO THE
VASCULATURE, IF THEY MAY HAVE
DIFFERENT FUNCTIONS THAT COME
FROM AND ARE LINKED TO THEIR
ORIGIN.
SO A NUMBER OF YEARS AGO NOW, WE
BEGAN BY LINEAGE TRACING CELLS
FROM MESODERM, WE FOUND THEY DO
CONTRIBUTE TO SOME EXTENT TO
BLOOD VESSELS, THEY VERY MUCH
PREFERENTIALLY CONTRIBUTE TO --
THIS IS EMBRYONIC DAY 9.5.
WITHIN THIS DORSAL PORTION OF
THE VEIN, YOU SEE ENDOTHELIAL
CELLS DERIVED FROM THIS LINEAGE.
A DAY LATER, THESE CELLS SWITCH
ON PROX1 AND IF WE LOOK USING
FLOW CYTOMETRY AT E13.5, WHAT WE
SEE IS SOME DEGREE OF
CONTRIBUTION, THE VAST MAJORITY
OF LYMPHATIC ENDOTHELIAL CELLS
ARE DERIVED FROM THIS LINEAGE.
SO THIS LED US TO PROPOSE A
REVISED MODEL, WHEREBY -- IT'S
THOSE CELLS WHICH GO ON TO
BECOME LYMPHATICS.
AND ACTUALLY TO FURTHER THIS, WE
HAVE DELETED PROX 1 WITHIN THE
PAX3 LINEAGE AND WHERE WE
ACHIEVED RECOMBINATION OF PROX1,
LYMPHATIC VESSELS DON'T FORM,
INDICATING THERE'S A
PREREQUIREMENT FOR CELLS TO PASS
THROUGH THIS LINEAGE AND THE
OTHER LINEAGES DON'T APPEAR TO
HAVE THE CAPACITY TO DO SO.
TO TRY AND UNDERSTAND WHAT MIGHT
BE DIFFERENT DURING EMBRYONIC
DEVELOPMENT, WHEREAS THE REST OF
THE VEIN IS NOT DOING SO, WE
CHOSE TO PERFORM SEQUENCING
ACROSS THESE KEY STAGES OF
DEVELOPMENT FOR LYMPHATIC
FORMATION.
SO WE ISOLATED -- FACS SORTED
CELLS, AND A STRIKING
OBSERVATION THAT WE MADE
INITIALLY LOOKING AT EMBRYONIC
DAY 9.5 WAS THE PRESENCE OF A
NUMBER OF DIFFERENT POPULATIONS
OF ETV 2 EXPRESSING ANGIOBLASTS.
FOR ME, AT LEAST, I THOUGHT THAT
THE PROCESS OF ANGIOBLAST
DIFFERENTIATION WAS OVER AT THIS
STAGE.
SO IT WAS QUITE AN INTRIGUING
FINDING.
SO THESE ETV 2 EXPRESSING
ANGIOBLASTS EMERGED FROM
MESENCHYMAL MESODERM, AND THEY
DID NOT EXPRESS MATURITY
ENDOTHELIAL MARKERS SUCH AS
PECAM.
IF YOU LOOK AT THE EMBRYO, THIS
IS ETV 2 EXPRESSIONS TOGETHER
WITH A MATURE MARKER OF
ENDOTHELIUM, WE ACTUALLY SEE A
NUMBER OF DISTINCT POPULATIONS
OF ANGIOBLASTS SPREAD THROUGHOUT
THE EMBRYO.
AND INTRIGUINGLY, WE FOUND ONE
OF THESE POPULATIONS APPEARS TO
BE GIVING RISE TO LYMPHATICS.
SO IF WE LOOK WITHIN OUR UMAP AT
THE EXPRESSION OF ETV 2 AND THIS
MASTER REGULATOR PROX1, WE SEE
AN OVERLAP OF EXPRESSION WITHIN
THESE LESS MATURE ENDOTHELIAL
CELLS AS THEY'RE DIFFERENTIATING
FROM MESODERM, AND WE ACTUALLY
SEE CELLS WHICH COEXPRESS PROX1
AND ETV 2 WHICH WE WOULD TERM
LYMPH ANGIOBLASTS, EXPRESSING
ENDOTHELIAL DIFFERENTIATION AND
LYMPHATIC FATE.
SO IF WE LOOK ACTUALLY WITHIN AN
EMBRYO, THIS IS EMBRYONIC DAY
9.5, WE SEE -- SO THIS IS A
CLOSE-UP HERE OF THIS REGION
HERE.
WE SEE CELLS WHICH SIT OUTSIDE
OF THE VENOUS ENDOTHELIUM IN
BETWEEN SEGMENTAL VESSELS WHICH
COEXPRESS PROX1 AND ETV 2.
THIS IS IN SITU, WE SEE THE SAME
WITH MRNA -- SORRY -- WITH
IMMUNE STAINING, AND ACTUALLY IN
OTHER REGIONS OF THE EMBRYO, SO
THESE IMAGES ARE TAKEN FROM THE
PHARYNGEAL ARTERIES SHOWN TO BE
A SOURCE OF LYMPHATIC
ENDOTHELIAL CELLS BY A COUPLE OF
OTHER GROUPS, AGAIN, WE FIND ETV
2 AND PROX1 COEXPRESSING CELLS
WHICH DO NOT EXPRESS CELL
MARKERS.
SO DE NOVO SYNTHSIFS LYMPHATIC
CELLS FROM A MESODERMAL
PROGENITOR, WE BELIEVE.
WE THEN TURNED TO TRY AND
UNDERSTAND WHERE THESE CELLS
MIGHT BE ENDING UP SO WE'VE USED
CONDITIONAL LINEAGE APPROACH,
INS DUED WITH TAMOXIFEN AT
VARIOUS STAGES DURING EMBRYONIC
DEVELOPMENT.
WHEN WE LOOK AT BLOOD
ENDOTHELIAL CELL
DIFFERENTIATION, IT'S
EFFECTIVELY LOST BY EMBRYONIC
DAY 9, WHEREAS IF WE LOOK AT
LYMPHATIC ENDOTHELIAL CELLS WE
STILL SEE A POPULATION OF CELLS
EMERGING WHICH ARE LABELED BY
TAMOXIFEN AT THIS STAGE,
INDICATING THAT THERE APPEARS TO
BE, DURING DEVELOPMENT, THE
ACQUISITION OF KIND OF A
RESTRICTED FATE OF CELLS
SPECIFICALLY GIVING RISE TO
LYMPHATICS, PROVIDING FURTHER
SUPPORT FOR LYMPH ANGIOBLAST.
IF WE LOOK WITHIN CROSS-SECTION
OF AN EMBRYO, EMBRYONIC DAY
10.5, AND THIS IS WITH TAMOXIFEN
ADMINISTERED EMBRYONIC DAY 9, WE
SEE CLOSE TO 70 TO 80% OF CELLS
OUTSIDE OF THE VEIN ARE LABELED
WITH TAMOXIFEN AT THIS STAGE,
WHEREAS THE VAST MAJORITY OF
VENOUS ENDOTHELIAL CELLS ARE NOT
LABELED.
ALTHOUGH WE DO BELIEVE THAT THE
VEIN ITSELF IS STILL GROWING AT
THESE STAGES, SO THERE IS
SOME DEGREE OF CONTRIBUTION.
IF WE LOOK AT SLIGHTLY LATER
STAGES, TO LOOK AT THE KIND OF
MORE INITIAL LYMPHATIC
STRUCTURE, WITH TAMOXIFEN AT
THESE EARLY STAGES WE SEE BROAD
LABELING OF ALL LYMPHATIC
ENDOTHELIAL CELLS AS WELL AS
SOME BLOOD ENDO THELIUM.
AT EMBRYONIC DAY 9, WE SEE MOST
LYMPHATIC ENDOTHELIAL CELLS ARE
LABELED AND THERE IS STILL SO
THE MINOR LABELING OF THE
CARDINAL VEIN, WHEREAS THIS
LABELING IS COMPLETELY LOST BY
EMBRYONIC DAY 10.
SO TO KIND OF COME BACK TO THE
EXISTING MODEL, IF THIS MODEL
WERE TO, IN FACT, BE PRECISELY
HOW THINGS HAPPEN, ONE WOULD
IMAGINE THAT PROLIFERATION OF
CELLS WITHIN THE EXISTING VENOUS
ENDOTHELIUM WOULD HAVE TO BE
VERY RAPID IN ORDER TO FULFILL
THE RAPID EXPANSION OF LYMPHATIC
ENDOTHELIAL CELLS THAT OCCURS
OVER DEVELOPMENT.
IN COLLABORATION WITH THE LAB IN
MUNSTER, WE LOOKED INTO THE
ABSOLUTE NUMBERS OF END ENDOTL
CELLS WHICH EMERGE DURING THESE
EARLY STAGES OF DEVELOPMENT AS
WELL AS THE VEIN ITSELF.
YOU CAN SEE THAT THERE IS VERY
RAPID EXPANSION OF -- SO IN
GREEN WE HAVE PROX1 POSITIVE
CELLS OUTSIDE OF THE VEIN, THEY
RAPIDLY INCREASE WITHIN THE
SPACE OF A DAY AND A HALF.
SO THIS WOULD SUGGEST THAT THE
VENOUS ENDOTHELIUM WOULD BE
EXTREMELY PROLIFERATIVE.
WHEN WE WENT BACK TO OUR SINGLE
CELL ANALYSIS, LOOKING
SPECIFICALLY AT CLUSTERS DERIVED
FROM VENOUS AND ANGIOBLAST
PROGENITOR, WE ACTUALLY SEE THIS
IS NOT THE CASE.
AND FURTHER WORK BY THE LAB OF
FREEDOM AND KIEFER HAS
DEMONSTRATED THAT, IN FACT, CELL
CYCLE DURATION IN CELLS WITHIN
THE VEIN IS ACTUALLY MUCH LOWER,
PROVIDING FURTHER EVIDENCE THAT
THE VEIN ITSELF IS UNLIKELY TO
PROVIDE A MASSIVE SOURCE OF
LYMPHATIC ENDOTHELIAL CELLS
DURING DEVELOPMENT.
SO TO COME BACK TO KIND OF THIS
MODEL, THERE'S A LOT OF DATA
THAT I HAVEN'T SHOWN WHICH IS IN
A PRE-PRINT THAT WE RECENTLY PUT
ON TO BIOARCHIVE, BUT WE NOW
BELIEVE THAT ACTUALLY A
SIGNIFICANT PROPORTION OF
LYMPHATIC ENDOTHELIAL CELLS
NEVER ENTER VENOUS ENDOTHELIUM
AND DIRECTLY ARE SPECIFIED TO
LYMPHATIC VESSELS, AND THAT
ACTUALLY THE PROPORTION OF CELLS
THAT DO LEAVE THE VEIN IS LIKELY
VERY LIMITED, AND PERHAPS
CONTRIBUTES MOSTLY TOWARDS
LYMPHOVENOUS VALVES.
IN TERMS OF WHAT I PERCEIVE IN
THE CONTEXT OF THIS TALK TO BE
GAPS, NEEDS AND OPPORTUNITIES, I
THINK THIS KIND OF NEW MODEL OF
HOW LYMPHATIC ENDOTHELIAL CELLS
ARE SPECIFIED EARLY IN
DEVELOPMENT GIVES US THE
OPPORTUNITY TO POTENTIALLY
IDENTIFY NEW GENES, CONTROLLING
THE EARLY STEPS OF LYMPHATIC
ENDOTHELIAL CELL SPECIFICATION.
AT THE SAME TIME, IDENTIFY
CAUSATIVE MUTATIONS IN NOVEL
GENES WHICH MIGHT UNDERPIN
PRIMARY LYMPHEDEMA IN HUMANS,
AND A GOAL OF MINE WOULD BE TO
DEVELOP IN VITRO SYSTEMS TO
DERIVE LYMPHATIC ENDOTHELIAL
CELLS FROM HUMAN EMBRYONIC STEM
CELLS ON THE BASIS OF THIS KIND
OF NEW PATHWAY OF
DIFFERENTIATION, WHICH WE
BELIEVE IS DOMINANT.
AND THAT JUST LEADS ME TO THANK
SOME OF THE PEOPLE THAT DID THE
WORK AT THE UNIVERSITY OF
OXFORD, AND ALSO OUR
COLLABORATORS, FRIEDEMANN AND
KIEFER'S LAB AS WELL AS SOPHIE
ASTROF.
>> THANK YOU VERY MUCH, THAT WAS
FANTASTIC.
WE'RE GOING TO HOLD THE
QUESTIONS FOR THE END OF THE
DISCUSSION, OKAY?
>> SOUNDS GOOD.
>> SO THANKS VERY MUCH.
SO OUR NEXT SPEAKER IS NATASZA
KURPIOS, SO THIS IS ME.
HI, EVERYBODY, AGAIN, AND I WILL
JUST SHARE MY SLIDES.
SO OLIVER, IF YOU CAN UNSHARE
YOUR SLIDES.
>> I THINK I HAVE.
>> OKAY.
GREAT.
AND SO AGAIN, HELLO, EVERYBODY,
AND WELCOME.
THANKS AGAIN FOR THE ORGANIZERS
TO PUT TOGETHER THIS REALLY
WONDERFUL AND ALSO REALLY
IMPORTANT MEETING.
SO THE TITLE OF MY TALK TODAY IS
THE ASYMMETRIC PITX2 GENE
REGULATES INTESTINAL MUSCULAR
LYMPHATIC MORPHOGENESIS AND
PROTECTS AGAINST FATTY LIVER
DISEASE.
TO SPECIFY LEFT VERSUS RIGHT
IDENTITY OF ORGANS.
ONE OF OUR FAVORITE MODELS TO
STUDY THIS IN A LAB IS THIS
BEAUTIFUL LOOPING MORPHOGENESIS
OF THE GI TRACT, AND THE REASON
IS BECAUSE THE LOOPING
MORPHOGENESIS IS ESSENTIAL TO
GUT FORM AND FUNCTION, BUT ALSO
THAT DIRECTION IS GOVERNED BY
RULES OF ASYMMETRY.
SO ONE OF THE MOST IMPORTANT
GENES TO SPECIFY THIS IS A
TRANSCRIPTION FACTOR KNOWN AS
PI.
PIT X2.
IT HAS THIS SHARP EXPRESSION
PATTERN RESTRICTED TO THE LEFT
HALF OF THE EMBRYO, AND THAT'S
WHERE PITX2 SPECIFIES THAT LEFT
SIDE IDENTITY AND THAT'S WHERE
PITX2'S ROLE IN LATERALITY HAS
BEEN EXTENSIVELY STUDIED.
HOWEVER, SEPARATE TO ITS ROLE IN
ORGAN LATERALITY, PITX2 HAS AN
ADDITIONAL ROLE IN ORGAN
MORPHOGENESIS.
BUT THIS FUNCTION OF PITX2 HAS
BEEN CHALLENGING TO STUDY
BECAUSE OF THE EARLY EMBRYONIC
LETHALITY THAT'S ASSOCIATED WITH
LOSS OF ALL OF PITX2 BUT HAS
BECOME A KEY FOCUS IN OUR LAB.
SO TO STUDY THIS ROLE IN
MORPHOGENESIS, OUR LAB SEVERAL
YEARS AGO HAS SHOWN THAT PITX2
EXPRESSION IN THE INTESTINE IS
SIMILARLY RESTRICTED TO THE LEFT
HALF OF THE DORSAL MESENTERY
WHERE IT FORMS THE MESENTERIC
TISSUE AND INITIATES THIS VERY
IMPORTANT ASYMMETRIC LOOPING
MORPHOGENESIS.
IN ADDITION, WE ALSO SHOWED
PITX2 PATTERNS THE VASCULATURE
LINKING THAT DEVELOPMENT TO
VASCULAR PA NERNING.
SO PATTERNING.
WE WANTED TO EXPAND THESE
FINDINGS AND ASK THE QUESTION
WHETHER PITX2 ALSO PLAYS A ROLE
IN VILLUS VASCULATURE AND
PERHAPS IN GUT ABSORPTIVE ACTION
POSTNATALLY.
MY LAB USES THIS HYPOMORPHIC
MOUSE MODEL WE CALL PITX2-ASE.
THIS MODEL IS VERY IMPORTANT TO
US BECAUSE LOSS OF ALL OF PITX2
RESULTS IN VERY EARLY EMBRYONIC
LETHALITY AND PRECLUDES LATER
ANALYSIS.
WITH ASE MODEL, WE CAN GENERATE
VIABLE PITX2 IN MUTANT MICE THAT
CAN VIEF BEYOND BIRTH.
THE FIRST THING WE NOTED IS
DESPITE COMPARABLE BODY SIZES
AND WEIGHTS AT BIRTH, THESE MICE
FAILED TO GAIN WAIT.
ONE HYPOTHESIS IS MAYBE PITX2
REGULATES AB SORPG.
SO OF COURSE WE LOOKED AT
LACTEALS.
THERE WERE FEWER IN THE
POSTNATAL INTENSATIVE, THIS IS
DAY 1.5 AFTER BIRTH, THEY HAD
REDUCED NUMBER OF LACTEAL --
LACK TEALS PROLIFERATE AND THESE
EXTENSIONS ARE VERY IMPORTANT
FOR EXTENSION AND HOMEOSTASIS.
THIS HAS BEEN SHOWN IN 20 BEN.
SO THIS WAS EXCITE BUG ALSO
SURPRISING BECAUSE PITX2
EXPRESSION IS ABSENT FROM
LYMPHATIC AND ENDOTHELIAL CELLS
SO THIS BRINGS ME TO THIS
BEAUTIFUL PICTURE OF AXIAL
SMOOTH MUSCLE FIBERS THAT ARE
SPECIFICALLY FORMED IN THE
VILLUS.
AND THIS PICTURE IS TAKEN FROM
THE PAPER FROM 2015, AND IT'S
SHOWING US THAT LACTEALS HERE IN
RED ARE ACTUALLY SURROUNDED BY
THIS NICHE OF MUSCLE FIBERS.
IT'S BEEN HYPOTHESIZED THAT
THESE FIBERS ARE IMPORTANT FOR
LACTEAL HOMEOSTASIS AND LATER IN
THE ADULT.
VERY RECENTLY IT'S BEEN SHOWN
THAT THESE MUSCLE FIBERS ARE
CONTRACTILE AND THEY SQUEEZE THE
ADJACENT RACK LACTEALS TO PRO
FACILITATE LIPID TRANSPORT.
WE ASKED THE QUESTION WHETHER
OUR PUPS ALREADY HAVE THESE
DIFFERENTIATED FIBERS.
INDEED WHEN WE LOOKED AT POST
DAY TALL
POSTNATAL DAY 9, THESE FIBERS IN
RED -- WE WERE ALSO EXCITED TO
KNOW THAT ABOUT HALF OF THESE
SMOOTH MUSCLE FIBERS IN THE
VILLUS WERE POSITIVE FOR PITX2
LINEAGE, SO WE HYPOTHESIZE THAT
PERHAPS PITX2 REGULATES THIS
MUSCULAR LACTEAL CO-DEVELOPMENT
THROUGH THE SMOOTH MUSCLE
LINEAGE.
SO INDEED WHAT WE SHOW THAT IN
THE MUTANTS, THESE FIBERS FAIL
TO ALIGN WITH LACTEAL, SO HERE
IN A WILD TYPE, AGAIN, THESE ARE
NEONATES, I APOLOGIZE FOR THE
JUMPY
JUMPY COMPUTER, THIS IS
POSTNATAL DAY 9, THIS IS
CONSISTENT WITH THE MUSCLE
FIBERS SQUEEZING THE ADJACENT
LACTEAL.
HOWEVER, IN PITX2 MUTANTS, WHAT
WE SAW IS SPACES BETWEEN THE
FIBERS, AND SPACES BETWEEN THE
LACTEAL.
SO AGAIN, A NUMBER OF QUESTIONS
AROSE LIKE, HOW DO THESE AXIAL
SMOOTH MUSCLE CELLS DEVELOP
ALONGSIDE LACTEALS AND DO THESE
LESIONS THAT WE SEE IN PITX2
MUTANTS HAVE ANYTHING TO DO WITH
DIETARY LIPID TRANSPORT?
SO BECAUSE WE ESSENTIALLY KNOW
NOTHING ABOUT THE ORIGIN OF
THESE VILLUS FIBERS, WE WANTED
TO DO TIME KINETICS TO SEE HOW
DO THEY FORM AND WHEN DO THEY
FORM.
SO WE FIRST NOTED CELLS THAT
HAVE LOW EXPRESSION OF SMOOTH
MUSCLE ACTIN AT EMBRYONIC DAY
16.5, RIGHT HERE.
AND THEY LOOKED LIKE STARFISH,
SO WE CALL THEM STAR CELLS.
THEN SHORTLY AFTER WITH THE
CO-APPEARANCE OF LACTEALS, WE
NOTED THAT THESE STAR CELLS
TRANSITION TO THESE ELONGATED
AXIAL SMOOTH MUSCLE-LIKE CELL
TYPES THAT ARE NOW VERY CLOSE TO
LACTEALS.
AND THEN BY POSTNATAL DAY 9, YOU
CAN SEE THESE AXIAL SMOOTH
MUSCLE CELLS WERE PREDOMINANT AT
THE EXPANSE OF LACTEALS.
SO WE HIGH HYPOTHESIZE -- JUE
THE SMOOTH MUSCLE WE SAW TO
POSITIVE LINEAGE.
WE ALSO SHOW THIS TRANSITION
FROM STAR CELLS TO
DIFFERENTIATED -- IS DEPENDENT
ON PITX2 2.
SO HOW ABOUT LIPID TRANSPORT?
SO WHAT DO WE KNOW?
WE KNOW THAT LACTEALS OF COURSE
ARE THE MAJOR ROUTE THROUGH
WHICH LIPIDS ARE ABSORBED INTO
THE HOST CIRCULATION.
IT'S THE MAJOR BUT NOT THE ONLY
ONE.
WE ALSO KNOW THAT SHORTER CHAIN
SAIDZ AND NON-FATTY NUTRIENTS
CAN ALSO DIRECTLY ENTER THE
PORTAL VEIN, MATE JUROR TD
SUPPLY TO THE LIVER.
LYMPHATIC VERSUS PORTAL
TRANSPORT HAVE VERY DIFFERENT
METABOLIC CONSEQUENCES.
LIPIDS ARE TAKEN UP DIRECTLY BY
ALL ORGANS IN THE BODY.
HOWEVER, IF THE LIPIDS ENTER THE
PORTAL VEIN, LIPIDS ARE TAKEN BY
THE LIVER PRIOR TO BE TAKEN UP
BY OTHER ORGANS, SO THIS REDUCES
THE AK ACCESSIBILITY TO OTHER
TISSUES.
THE REASON WHY WE BECAME VERY
INTERESTED IN THESE TWO
INDEPENDENT PATHWAYS IS SHOWN ON
THIS SLIDE, BECAUSE WHENEVER WE
ISOLATED LIVERS FROM OUR MUTANT
PUPS, IT NEVER SUNK TO THE
BOTTOM OF THE TUBE, SUGGESTING
THAT THEY ARE FATTY.
SO WE CONFIRMED THIS WITH
STAINING SHOWN HERE AND ALSO
WITH HISTOPATHOLOGY, CONCLUDING
THAT OUR MU TABT PUPS MUD
EARLY ONSET OF FAT EE TY LIVER
DISEASE SO WHY IS THAT?
THIS DELETION IMPAIRS THE NORMAL
FATE OF DIETARY LIPID TRANSPORT
AND INDEED WE SHOWED THIS WITH
USING A FLUORESCENT LIPID
TRASER, THIS IS A LONG CHAIN
FATTY ACID LIPID TRASER.
SO AS EXPECTED IN WILD TYPE,
BODIPY ACCUMULATED IN THE
MESENTERY COLLECTORS, AND
VERY -- SIGNAL INTO THE PORTAL
VEIN.
BOTH IN THE PORTAL VEIN AND
LIVER PARENCHYMA SUGGESTING THAT
INDEED THIS LIPID TRANSPORT
GROUP IS ALTERED IN PITX2 MUTANT
MICE.
SO WHAT WE CAN SUMMARIZE IS THAT
WE THINK PITX2 IS REQUIRED FOR
THE NORMAL ROUTE OF DIETARY
LIPID TRANSPORT AND PROTECTS
AGAINST FATTY LIVER DISEASE.
AND SO OUR DATA SUPPORTS THE
IMPORTANCE OF THIS MUSCULAR
LACTEAL COMPLEX IN LIPID
TRANSPORT FUNCTION.
AND WHAT WE NEED TO UNDERSTAND
THOUGH AND WHAT WE REALLY WANT
TO DO IN THE FUTURE IS
UNDERSTAND TO WHAT EXTENT THIS
MUSCULAR LYMPHATIC DYSFUNCTION
ACTUALLY CONTRIBUTES TO ABNORMAL
LIPID TRANSPORT AND LIVER
PATHOLOGIES, AND JUST TO REMIND
YOU, THIS IS A LIVER PATHOLOGY
OF THE ENTERIC ORIGIN, NOT THE
LIVER ORIGIN.
AND THE MAJOR GAPS FOR US HAS
BEEN THE LACK OF MOLECULAR
INFORMATION, THE LACK OF
MOLECULAR MARKERS TO
SPECIFICALLY PERTURB THE
RELEVANT CELL POPULATIONS
BECAUSE WE DON'T KNOW ANYTHING
ABOUT HOW THESE VILLUS -- FORM.
SO TO DO THAT WE RECENTLY BECAME
INTERESTED IN SINGLE CELL
APPROACHES TO SEQUENCE THE SMALL
INTESTINE AT VARIOUS STAGES OF
DEVELOPMENT, AND WE HAVE DONE
THIS IN COLLABORATION WITH
DEVLAMINCK'S LAB HERE AT CORNELL
UNIVERSITY.
BECAUSE OF THE SAKE OF TIME, I
AM ONLY GOING TO FOCUS ON THE
MUST
MUSCULARIS CLUSTER.
JUST WANT TO REMIND YOU THAT
EACH DOT ON THE MAPS REPRESENT A
SINGLE CELL AND EACH COLOR
REPRESENTS A DIFFERENT DAY.
SO FOR US, OUR LINEAGE TRACING
SPANS FROM EMBRYONIC DAY 12.5
ALL THE WAY TO POSTNATAL DAY 1.R
YOUNGEST CELLS ARE HERE IN BLUE
AND ORANGE AND OUR OLDER CELLS
ARE HERE ON THE PERIPHERY.
IMPORTANTLY FOR US, THE STAR
CELL TO ACCESS SMOOTH MUSCLE
TRANSITION, THIS PERINATAL
PERIOD IS NOTED RIGHT HERE.
SO WHERE ARE THE AXIAL SMOOTH
MUSCLE CLUSTER AND OUR STAR
CELLS?
SO WHAT WE DID IS WE FIRST USED
CANONICAL CELL TYPE SPECIFIC
MARKERS, SMOOTH MUSCLE ACTIN,
AND WE HAVE IDENTIFIED THREE
POTENTIAL CLUSTERS SMOOTH MUSCLE
ACTIN POSITIVE DEPICTED HERE IN
THE ASTERISKS.
ALSO THE NEXT MARKER WE USED WAS
A DIFFERENTIATED SMOOTH MUSCLE
MARKER, HEAVY CHAIN 11.
YOU CAN SEE WE HAVE IDENTIFIED
TWO OUT OF THE THREE CLUSTERS
THAT ARE DIFFERENTIATED SMOOTH
MUSCLE.
SO WE HIGH POTASSIUM SEISE E
CLUSTER HERE, THAT SMOOTH MUSCLE
ACTIN LOW COULD BE OUR STAR
CELLS.
NEXT WE WANTED TO DIFFERENTIATE
THE SMOOT MUSCLE OF THE VILLUS
FROM THE SMOOTH MUSCLE OF THE
GUT WALL.
WE USED C-KIT, SPECIFIC TO THE
GUT WALL, AND THAT WAS VERY
TELLING, BECAUSE IT ONLY MARKED
THIS ARM OF OUR CLUSTER,
SUGGESTING THAT THESE ARE OUR
GUT WALL SMOOTH MUSCLE AND THESE
ARE OUR AXIAL SMOOTH MUSCLE.
AND SO WE THINK THAT THIS LITTLE
CLUSTER HERE IS OUR
DIFFERENTIATED SMOOTH MUSCLE --
AXIAL SMOOTH MUSCLE POPULATION.
WE ALSO USE ANOTHER MARKER KNOWN
AS PERIOSTIN.
THIS IS LIKE OUR FIBROBLAST
CLUSTER.
YOU CAN SEE IN GENERAL, THIS
FIBROBLAST-LIKE CLUSTER WAS
NEGATIVE FOR SMOOTH MUSCLE ACTIN
EXPRESSION WITH THE EXCEPTION OF
THIS SMALL LITTLE CLUSTER WE
THINK ARE STAR CELLS.
SO HERE'S OUR ROAD MAP.
WE BELIEVE THAT THIS CLUSTER
HERE IS OUR DIFFERENTIATED AXIAL
SMOOTH MUSCLE CELLS OF THE
VILLUS AND THIS CLUSTER RIGHT
HERE IS OUR STAR CELL-LIKE
CLUSTER WITHIN THIS FIBROBLAST
POPULATION.
SO NOW OF COURSE WE HAVE TO
VALIDATE THIS, SO HOW DO WE DO
THIS?
SO HELP US VALIDATE THIS, WE
HAVE IDENTIFIED ANOTHER GENE
THAT I HOPE YOU CAN APPRECIATE
WAS EXCLUSIVELY EXPRESSED AND
WHAT WE THINK ARE STAR CELL
POPULATION BUT WAS NOT EXPRESSED
IN THE DIFFERENTIATED SMOOTH
MUSCLE.
THIS WAS EXCITING BECAUSE WE
KNOW THAT TENASCIN C PLAYS A
REALLY IMPORTANT ROLE FROM
FIBROBLAST EXPRESSION.
OF COURSE PDGFRA IS VERY
IMPORTANT DURING VILIFICATION.
WE FOUND IN OUR FIBROBLAST-LIKE
CLUSTER, I HOPE YOU CAN
APPRECIATE EXPRESSION WAS DEVOID
IN THE STAR CELL-LIKE CLUSTER.
THIS IS CON CONSISTENT WITH TE
FINDING THAT SUBEPITHELIAL ALPHA
POSITIVE CELLS ARE DISTINCT FROM
SMOOTH MUSCLE ACTIN POSITIVE
MYOFIBROBLASTS.
THIS IS WORK FROM KEN SAN SAN'
LAB.
WE HYPOTHESIZED THAT THEY GIVE
RISE TO TENASCIN POSITIVE
MYOFIBROBLAST STAR CELLS AND
SUBSEQUENTLY THESE DIFFERENTIATE
INTO AXIAL SMOOTH MUSCLE FIBERS
OF THE VILLUS.
SO NOW WE CAN GENETICALLY TEST
IT WITH CRE-SPECIFIC DRIVERS.
SO WE DID THAT.
SO INITIALLY, THIS IS JUST A
WILD TYPE INTESTINE, AND AGAIN I
HOPE YOU CAN APPRECIATE THAT
PDGFRA POSITIVE CELLS HERE IN
PURPLE ARE DISTINCT FROM THE
DIFFERENTIATED SMOOTH MUSCLE
ACTIN POSITIVE CELLS HERE SHOWN
IN BLUE.
AND THIS IS BOTH IN THE
EMBRYONIC INTESTINE AT 14.5 AND
ALSO IN THE DIFFERENTIATED
VILLUS SMOOTH MUSCLE CELL.
SO YOU CAN SEE THAT THEY ARE
DEVOID OF PDGFRA EXPRESSION
HERE.
HOWEVER, NOW IF WE INDUCE THE
PDGFRA LINEAGE WITH TAMOXIFEN AT
POSTNATAL DAY ZERO SO JUST IN
THAT NEONATAL TRANSITION, WE CAN
SEE ROBUST LABELING OF PDGFRA
LINEAGE IN THESE SMOOTH MUSCLE
BUNDLES AND ABOUT 80 TO 90% OF
DIFFERENTIATED BUNDLES ARE
LINEAGE POSITIVE.
IF WE REPEAT THESE INDUCTIONS AT
LATER TIME POINTS THAT NUMBER
DRAMATICALLY INCREASES.
HOW ABOUT DENA SIN C?
WE SHOW THAT TENASCIN C IS
INDEED COEXPRESSED WITH STAR LN
LIKE SHAPED CELLS, SMOOTH MUSCLE
ACTIVE LOW, BUT IT IS NOT
EXPRESSED IN THE DIFFERENTIATED
AXIAL SMOOTH MUSCLE CELLS.
HOWEVER, IF WE INDUCE TENASCIN C
LINEAGE WE SEE ABOUT 80% OF OUR
AXIAL SMOOTH MUSCLE FIBERS ARE
DERIVED FROM TENASCIN POSITIVE
LINEAGE.
IF WE REPEATED THE INDUCTIONS AT
LATER TIME POINTS, THAT NUMBER
STAYED RELATIVELY HIGH,
CERTAINLY BY COMPARISON TO
PDGFRA LINEAGE -- SUGGESTING
STAR CELLS REMAIN IN THE ADULT
AND POTENTIALLY COULD HAVE A
ROLE IN AXIAL SMOOTH MUSCLE CELL
HOMEOSTASIS AND MAYBE A DISEASE.
I'M OUT OF TIME, PLEASE ASK ME
QUESTIONS DURING THE QUESTION
AND ANSWER PERIOD.
WE COULDN'T HELP TO NOTICE THAT
OUR AXIAL SMOOTH MUSCLE CELLS
WERE POSITIVE FOR NOTCH 3 AND
ALSO A TRANSCRIPTIONAL ACTIVATOR
OF NOTCH SIGNALING SMG THE
REASON WHY WE BECAME REALLY
EXCITED ABOUT HAVING NOTCH
3 SPECIFICALLY IN OUR AXIAL
SMOOTH MUSCLE CELL CLUSTER IS
BECAUSE IT WAS PREVIOUSLY SHOWN
THAT DELTA 4, A LIGAND FOR NOTCH
3 RECEPTOR SIGNALING, IS
ABSOLUTELY CRITICAL.
SO WE COLLABORATED AND SHOWED
THAT INDEED IF WE DELETE NOTCH A
CRE THESE SMOOTH MUSCLE FIBERS
ARE MUCH, MUCH SHORTER THAN
LACTEALS AND NOW THE LACTEALS
LACK -- JUST LIKE IN THE DELTA .
SO WE ARE NOW WORKING WITH THIS
MODEL THAT PERHAPS NOTCH 3 DELTA
4 SIGNALING CASCADE COULD BE
IMPORTANT FOR THE PLEE OF THESE
AXIAL SMOOTH MUSCLE FIBERS ALONG
THEIR LACTEALS.
SO TO QUICKLY SUMMARIZE, WE
THINK STAR CELLS ARE TENASCIN
POSITIVE MYOFIBROBLAST-LIKE
POPULATION THAT GIVE RISE TO
VILLUS AXIAL CELLS, AND GIVES
RISE TO MYOFIBROBLAST-LIKE STAR
CELLS, AND WE THINK THAT
POPULATION COULD BE IMPORTANT
FOR THAT ADULT MAINTENANCE OF
THIS VILLUS MUSCLE.
SO TO ACKNOWLEDGE AND CONCLUDE,
WE HAVE A COMPUTATIONAL
COLLABORATOR WHO HAS DONE A LOT
OF DATA MINING FOR OUR SINGLE
CELLS AND THEN ALSO COLLEAGUES
AT CORNEL OUTSIDE ESPECIALLY I
WANT TO ACKNOWLEDGE -- AND OUR
FUNDING AND THANKS SO MUCH FOR
YOUR ATTENTION.
OUR NEXT SPEAK SER RACHEL NIEC,
LYMPHATIC CAPILLARIES ACT AS A
SIGNALING HUB IN THE INTESTINAL
STEM CELL NICHE.
ELWELL COME, RACHEL.
NICE TO HAVE YOU.
>> THANKS.
I REALLY COME TO THE LYMPHATICS
FROM A STEM CELL PERSPECTIVE AND
IMMUNOLOGY PERSPECTIVE, AND IT'S
JUST INSPIRING TO BE HERE AMONG
SUCH DIVERSE COLLEAGUES.
ARE YOU SEEING THE RIGHT SCREEN?
>> LOOKS GOOD.
>> PERFECT.
SO WE'RE ON THE TITLE SLIDE
AGAIN?
>> YES.
>> ALL RIGHT.
SO THE WORK I'LL TALK TO YOU
ABOUT TODAY HAS REALLY BEEN A
TEAM EFFORT BETWEEN MYSELF,
SHIRISGUR-COHEN, MARINA, AND
TINYI CHU, A COMPUTATIONAL
COLLABORATOR AND POSTDOC.
SO ACROSS THE BODY, TISSUE STEM
CELLS SUPPORT TISSUE-SPECIFIC
DEMANDS.
HAIR FOLLICLE STEM CELLS SHOWN
HERE ARE CYCLICALLY ACTIVE TO
GIVE RISE TO THE HAIR CYCLE AND
THE HAIR ON OUR BODIES AND HEADS
WHEREAS IN THE INTESTINE,
CRYPT-BASED INTESTINAL STEM
CELLS ARE CONSTANTLY RENEWING
AND DIFFERENTIATING TO GENERATE
THE DIVERSE EPITHELIAL CELL
TYPES.
HOW ARE THESE DIFFERENT STEM
CELL ACTIVITIES COORDINATED?
ABUNDANT EVIDENCE HAS SHOWN THAT
STEM CELL ACTIVITY IS LARGELY
INFLUENCED BY INTERACTIONS WITH
THEIR LOCAL MICROENVIRONMENT,
THEIR NICHE.
AND IN RECENT YEARS, THE
VASCULATURE HAS EMERGED AS A
NICHE COMPONENT IN MULTIPLE
TISSUES, AND IN THE LAB FOCUSING
ON LYMPHATICS, SHIRI IDENTIFIED
LYMPHATICS VASCULATURE AS A STEM
CELL NICHE FOR HAIR FOLLOW KEAL
STEM CELLS WHEREBY THEY
COORDINATE THE CYCLICAL ACTIVITY
OF THE HAIR FOLLICLE STEM CELLS
IN THE HAIR CYCLE.
AS A GASTROENTEROLOGIST,
PARTICULARLY SOMEONE STUDYING
INFLAMMATORY BOWEL DISEASE, THIS
WAS INCREDIBLY INTERESTING TO
ME, BECAUSE OF COURSE THE
INTESTINAL LYMPHATICS PLAY A KEY
ROLE IN NUTRIENT ABSORPTION AND
IMMUNITY, BUT MOREOVER,
DISRUPTION OF LYMPHATICS HAS
LONG BEEN ASSOCIATED WITH
INFLAMMATORY BOWEL DISEASE, BUT
THE FUNCTION OF THIS HAS BEEN
REALLY UNKNOWN.
SO THINKING ABOUT THE INTESTINE,
WE DIVIDE IT INTO TWO MAIN
ANATOMIC UNITS.
THE SMALL INTESTINE COMPRISED OF
BOTH CRYPT AND VILLI AND THE
VILLI MAKE UP THE LARGE
ABSORPTIVE SURFACE AIR OF THIS
TISSUE AND CONTAIN THE LACTEALS,
LYMPHATIC PROTRUSIONS JUST
DISCUSSED AND ARE IMPORTANT FOR
NUTRIENT ABSORPTION AND IMMUNE
SURVEILLANCE.
THE LARGE INTESTINE DOES NOT
CONTAIN VILLI OR LACTEALS AND
INSTEAD IS JUST COMPRISED OF
CRYPTS ALONE DENSELY COLONIZED
BY MICROBES.
LYMPHATICS IN THIS TISSUE ARE
LESS WELL CHARACTERIZED YET THEY
ALSO PLAY AN IMPORTANT ROLE IN
IMMUNE SURVEILLANCE.
IN BOTH TISSUES, LYMPHATICS ARE
REALLY EXPOSED TO THE METABOLIC
AND IMMUNE MILIEUS OF THE
TISSUE, MAKING THEM AN
INTRIGUING CANDIDATE TO
TRANSLATE THESE SORTS OF SIGNALS
INTO THE STEM CELL NICHE TO
COORDINATE APPROPRIATE STEM CELL
ACTIVITY.
SO WE'VE SET OUT TO DESCRIBE THE
VASCULATURE AND PARTICULARLY
LYMPHATICS IN THE INTESTINAL
STEM CELL NICHE USING FIRST
TISSUE CLEARING AND WHOLE MOUNT
THREE DIMENSIONAL IMAGING.
SO WHAT I'M SHOWING FIRST IS A
PICTURE OF THE SMALL INTESTINE
WITH THE SCA SCHEMATIC ON THE.
WE SEE THE LACTEALS AND WHAT WE
NOTICE RIGHT OFF WHEN WE CLEARED
THE TISSUE TO LOOK AT THE WHOLE
NETWORK WAS THAT THE LYMPHATIC
CAB
CAPILLARIES COLLECT AND FORM
THIS NETWORK THAT SIT JUST BELOW
THE CRYPTS WHERE THE STEM CELLS
ARE HOUSED.
THIS IS A SIDE VIEW AND A
TOP-DOWN VIEW AND THEN LOOKING
AT PSEUDOCOLORRED EM IMAGES, YOU
CAN SEE THESE LYMPHATIC
CAPILLARIES ARE FORMING THIS
NETWORK SITTING JUST BELOW THE
INTESTINAL STEM CELLS WITHIN THE
CRYPT.
AND OF COURSE ONE COULD THINK
THAT IN THE SMALL INTESTINE, THE
LACTEALS NEED TO COLLECT
SOMEWHERE, AND SO PERHAPS THEY
JUST COLLECT BELOW THE BASE OF
THE CRYPT SO THAT THEY CAN DRAIN
OUT INTO THE REST OF THE BODY,
BUT THE LARGE INTESTINE LACKS
LACTEAL, AND SO WE WANTED TO
KNOW IF THIS WAS ALSO TRUE IN
THE LARGE INTESTINE, AND INDEED
WE FOUND THAT THE LYMPHATIC
CAPILLARY NETWORK SITS JUST
BELOW THE BASE OF THE CRYPTS
WHERE THE STEM CELLS ARE HOUSED
IN THE LARGE INTESTINE.
SO TO GIVE YOU A BETTER VIEW OF
THIS, I'VE PUT TOGETHER THIS
VIDEO WHERE WE HAVE LYMPHATICS
IN WHITE, BLOOD VESSELS IN RED,
CRYPTS IN GREEN AND OF COURSE
THERE'S GAFFE.
WE CONSTRUCT THE CRYPTS IN
PURPLE, YOU CAN SEE THIS
LYMPHATIC NETWORK IS NESTING AND
CUPPING INDIVIDUAL CRYPTS AND
THEIR STEM CELLS.
AND THIS ASSOCIATION IS ALSO
CONSERVED IN HUMANS.
HERE YOU CAN SEE THE HUMAN SMALL
INTESTINE, THIS IS A BIOPSY
SPECIMEN TAKEN FROM A PATIENT,
SUBJECTED TO TISSUE CLEARING.
YOU CAN SEE CRYPTS IN GREEN AND
THE LYMPHATIC CAPILLARY NETWORK
SITTING ALONG THE BASE OF THE
CRYPTS.
AND ALSO IN THE COLON, THIS IS
AGAIN THE SIDE VIEW, YOU CAN SEE
THIS INDIVIDUAL CRYPT ALMOST HAS
ITS OWN LYMPHATIC NEST FORMING
AROUND ITS BASE.
I LIKE TO THINK THAT BUY BIOLC
FORM FOLLOWS FUNCTION, SO OF
COURSE WE WONDERED, DOES THE
PRESENCE OF LYMPHATIC
CAPILLARIES IN THE STEM CELL
NICHE HAVE IMPLICATIONS FOR
THESE STEM CELLS?
SO TO TEST THIS, WE TURN TODAY A
RATHER CRUDE LEVEL OF LYMPHATIC
ABLATION IN MICE, SO THAT WE
COULD TREAT MICE WITH TAMOXIFEN
TO INDUCE EXPRESSION OF
DIPHTHERIA TOXIN RECEPTOR ON THE
LYMPHATICS AND TREAT WITH DIP
TIER
DIPHTHERIA -- THE LYMPHATICS ARE
GONE.
JUST AFTER ABLATION WE SEE A
PRETTY PRONOUNCED DECREASE.
THIS IS CORRELATED WITH A
CORRESPONDING ACCELERATED
DIFFERENTIATION AND MATURATION
OF THE EPITHELIAL PROGENY OF
THESE STEM CELLS AS SHOWN HERE
IN THIS PULSE CHASE EXPERIMENT,
WHERE YOU CAN SEE THAT IN THE DT
TREATED MICE, EDU INCORPORATION
IS MUCH HIGHER IN THE CRYPT AT
30 MINTS AND THESE EDU-POSITIVE
CELLS HAVE MOVED UP OR
TRANSITIONED UP THE CRYPT VILLUS
ACCESS MUCH MORE QUICKLY SUCH
THAT THEY REPRESENT A HIGHER
POSITION AT 48 HOURS LATER.
SO THIS SUGGESTS ACCELERATED
DIFFERENTIATION AND MATURATION
OF STEM CELLS INTO
DIFFERENTIATED EPITHELIAL CELLS
UPON LYMPHATIC ABLATION.
AND OF COURSE THIS IS AN
EXPERIMENT FULL OF CAVEATS.
WE'VE KILLED A MASSIVE
COMPARTMENT OF CELLS JUST BELOW
THE BASE OF THE STEM CELL NICHE,
WE'VE GOT LYMPH DRAINAGE SO WE
NEEDED TO KNOW IF THIS WAS A
DIRECT EFFECT.
SO TO TEST THIS, WE USED A
CULTURE OF 3D ORGANOIDS REMOVING
CELL DEATH AND DRAINAGE FROM THE
EQUATION SO WE COULD EXCLUSIVELY
STUDY CELL-CELL COMMUNICATION
BETWEEN THESE TWO CELL TYPES.
SO WE FOUND THAT LYMPHATIC
ENDOTHELIAL CELL CONDITIONED
MEDIUM BUT NOT BLOOD OR
FIBROBLASTED CONDITIONED MEDIUM
INCREASED ORGANOID PRODUCTION
FROM SINGLE CELLS, A SURROGATE
OF STEMNESS.
AND ALSO, THAT LYMPHATIC
ENDOTHELIAL CONDITION MEDIUM WAS
ABLE TO MAINTAIN ORGANOIDS IN A
LEFT DIFFERENTIATED LESS MATURE
PROGENITOR-LIKE STATE AS SHOWN
BY INCREASED EXPRESSION OF SOX
9, A STEM CELL MARKER, AND
DECREASED EXPRESSION OF MATURE
DIFFERENTIATION MARKERS SHOWN IN
YELLOW.
SO THESE RESULTS ARE OPPOSITE
AND LESS COMPLEMENTARY TO ACUTE
LYMPHATIC ABLATION IN MICE, AND
TOGETHER THIS SUGGESTS THAT
LYMPHATIC SIGNALS ARE PROMOTING
STEM CELL MAINTENANCE, SO WE
WANTED TO KNOW WHICH SIGNALS AND
WHAT'S DRIVING THESE MOLECULAR
INTERACTIONS.
AND SO TO GET AT THIS, WE WANTED
TO MAP LYMPHATICS AT THE
TRANSCRIPTIONAL LEVEL TO REALLY
IDENTIFY LYMPHATIC FACTORS THAT
ARE BEING EXPRESSED AT THE CRYPT
BASE THAT HAD INTESTINAL STEM
CELL RECEPTORS AND SO WE USED A
SPATIAL TRANSCRIPTOMICS APPROACH
AND HERE'S A PICTURE OF THE
SLIDE WHERE YOU CAN SEE A ZOOMED
IN AREA OF A COUPLE OF
CRYPTOVILLUS UNITS, CAPILLARIES
IN RED, STEM CELLS IN GREEN.
EACH SPOT HERE REPRESENTS A
CAPTURE AREA THAT'S LABELED WITH
PROBES, SO WE QUICKLY
APPRECIATED THE LIMITATION OF
RESOLUTION HERE.
EACH SPOT IS CONTAINING UP TO 10
CELLS OR MORE, AND SO WE ENGAGED
TO HELP US OVERCOME THIS BY
DEVELOPING AN ALGORITHM USING
NON-SPATIAL DATA AS PRIOR
INFORMATION TO DEACON DE COE
EXPRESSION OF EACH SPOT ALONG
THE AXIS AND THEN FURTHER
IMPROVE RESOLUTION BY TAKING
ADVANTAGE OF THE STEREOTYPE
STRUCTURE OF THE INTESTINE,
THESE REPEATING CRYPT VILLUS
UNITS, TO THEN COMPRESS THE DATA
ON TO A SINGLE PSEUDOAXIS.
SO ONE DIMENSIONAL PSEUDOAXIS
REPRESENTING THE CRYPT-VILLUS
AXIS.
THIS IS WHAT THE DATA LOOKS
LIKE.
SO WHEN WE'VE REDUCED ALL THE
CRYPTS AND ALL THE VILLI FROM
THE TRANSCRIPT OMIC EXPERIMENT,
WE GENERATE THIS BEAUTIFUL CAR
CARTOGRAPH.
HERE WE HAVE LYMPHATICS SITTING
AROUND THE BASE AREA WITH THE
LACTEAL EXTEND EXTENDING UPN
THE NICHE PANETH STEM CELLS
SITTING BELOW AND OTHER IMMUNE
AND CELL TYPES WE'RE INTERESTED
TO EXPLORE.
BUT WITH THIS IN HAND, WE WANTED
TO LOOK FOR A LIST OF LYMPHATIC
VERY CREATED
SECRETED FACTORS.
THEN PLOT EXPRESSION OF THESE
LIGANDS ALONG THE CRYPT-VILLUS
AXIS WHICH IS NOW ON THE X AXIS
WITH THE CRYPT INDICATED IN THE
TRIANGLE.
SO EXCITINGLY, WE FOUND
LYMPHATICS WERE MAIN PRODUCERS
OF KEY ESTABLISHED STEM CELL
NICHE FACTORS SUCH AS WNTs.
TWO PAPERS DEMONSTRATED THE
IMPORTANT FUNCTION OF LYMPHATIC
DERIVED RSPONDIN.
WE WERE PARTICULARLY INTERESTED
IN -- BUT HAS A ROLE IN CARDIAC
PRO GENERATION FOLLOWING INJURY.
I THINK MORE WILL BE PRESENTED
ON THAT LATER.
WE WONDERED IF THIS ROLE IN
REGENERATION, A FEATURE OF STEM
CELLS, MIGHT BE CONSERVED.
SO THE LAB OLIVER LAB KINDLY S
TISSUE, MOREOVER PROVISION OF
EXOGENOUS -- TO OUR ORGANOID
COCULTURE SYSTEM MAINTAINED
ORGANOIDS IN THIS LESS MATURE,
LESS DIFFERENTIATED PHENOTYPE
THAT WE SAW WITH LYMPHATIC
CO-CULTURE OR LYMPHATIC
CONDITIONED MEDIUM.
AND WE SEE DIRECTLY THAT
ADDITION OF RELN CONTRIBUTES TO
PHOSPHORYLATION OF DOWN
SIGNALING -- INDICATING DIRECT
SIGNALING.
SO I'LL CONCLUDE BY JUST SAYING
THAT WHAT WE FOUND IS THAT
LYMPHATIC CAPILLARIES ARE
PHYSICALLY PROXIMAL TO THE SMALL
INTESTINE IN BOTH THE SMALL AND
LARGE INTESTINE IN BOTH MICE AND
HUMANS, AND THAT LYMPHATIC
ENDOTHELIAL CELL NETWORK IS A
SOURCE OF NICHE FACTORS OR A
SIGNALING HUB FOR THE INTESTINAL
STEM CELL NICHE, AND IN LOOKING
AT THIS DATA, WE WERE ABLE TO
IDENTIFY A NOVEL
LYMPHATIC-DERIVED FACTOR,
REELIN, IMPORTANT FOR STEM
CONTROL MAINTENANCE.
I'LL LEAVE YOU WITH SOME
KNOWLEDGE GAPS AND
OPPORTUNITIES.
I THINK ONE QUESTION THAT THIS
BEGS, OF COURSE, THINKING ABOUT
LYMPHATICS AND THE HEART AND
SKIN AND GUT LYMPHATICS IS A
CONSERVED FEATURE OF STEM CELL
NICHES OR REGENERATIVE NICHES IN
GENERAL.
DOES LYMPHATIC COMMUNICATION
WITH STEM CELLS RESPOND
DYNAMICALLY TO CHANGES IN THE
TISSUE?
OF COURSE LYMPHATICS ARE SEEING
EVERYTHING THAT'S DRAINED AND SO
ARE THEY RESPONDING AND
INSTRUCTING APPROPRIATE STEM
CELL OR OTHER CELLULAR BEHAVIOR?
AND THEN WITH THIS LITTLE
TEASER, ONE INTERESTING QUESTION
THAT I HAVE IS, HOW ARE
LYMPHATICS REGULATING IMMUNE
CELL AK ES TO ACCESS TO TH?
HERE YOU SEE IT GOING IN AND OUT
OF THE LYMPHATICS.
IF WE WERE TO WATCH THIS IN
TIME, I IMAGINE.
SO HOW MIGHT STEM CELLS BE
REGULATING THEIR LYMPHATIC NICHE
TO CHANGE THE IMMUNE MILIEU AND
OTHER SIGNALING MOLECULES IN THE
NICHE.
WITH THAT, I'LL THANK EVERYONE
INVOLVED IN THIS PROJECT.
I THINK I THANKED MY TEAM
ALREADY, THE PEER LAB HAS BEEN
AMAZING, OTHER COLLABORATORS ARE
SHOWN HERE AND I'LL THANK OUR
FUNDING SOURCES.
AND I'D LOVE TO HEAR YOUR
QUESTIONS AT THE END.
>> RACHEL, THANK YOU SO MUCH.
I'M ENVIOUS OF YOUR SPATIAL
TRANSCRIPTOMICS.
WE'RE DYING TO DO IT IN OUR
EMBRYONIC INTESTINES.
EXPWR WE'LL BE HAPPY TO SHARE TO
COMPARE.
IT WILL BE FUN.
>> THANK YOU SO MUCH.
OKAY.
SO LET'S MOVE ON TO OUR NEXT
SPEAKER, SO OUR NEXT SPEAKER IS
DR. YING YANG FROM UNIVERSITY OF
SOUTH FLORIDA.
AND THE TITLE OF HER TALK IS
NOVEL MECHANISMS TO ENHANCE
LYMPHATIC VALVE FUNCTION.
>> HOPEFULLY EVERYONE CAN SEE MY
SLIDES HERE.
SO VERY HAPPY TO BE HERE TO
PRESENT OUR WORK.
SO FIRST I WOULD LIKE TO THANK
THE ORGANIZERS FOR THIS GREAT
OPPORTUNITY TO SHARE OUR WORK.
UNDERSTANDING A NOVEL MECHANISM
THAT ENHANCES LYMPHATIC VALVE
FORMATION.
THIS IMMUNOSTAINING IMAGE
SHOWS -- IN LYMPHATIC VESSEL.
THE LYMPHATIC VASCULATURE IS THE
MAJOR TRANSPORTATION PATHWAY OF
TISSUE FLUIDS.
LYMPHATIC CAPILLARIES UPTAKE
FLUID -- TRANSPORT LINK.
AS DR. DAVIS MENTIONED EARLIER,
SMOOTH MUSCLE CELLS OUTSIDE OF
THE COLLECTING LYMPHATIC VESSELS
SPONTANEOUSLY CONTRIBUTE TO MOVE
LYMPH FORWARD AND -- ACT AS THE
GATE KEEPER TO PREVENT BACKFLOW
OF LYMPH.
THE FORMATION OF THE LYMPHATIC
VALVE IS A STEP WISE PROCESS
THAT STARTS AROUND EMBRYONIC DAY
15 TO 16.
THE INITIATION STEP OF VALVE
FORMATION IS ATTRIBUTED BY --
STRESS, WHICH INDUCED THE
UPREGULATION OF THREE
TRANSCRIPTION FACTORS, PROX1,
FOXC2 AND GATA2 IN VALVE FORMING
CELLS.
AFTER THE CONDENSATION AND
ELONGATION STEPS, IT HAS TWO
VALVE DEFECTS SHOWN HERE IN THE
IMAGE AT THE MATURATION STEP.
THEREFORE -- FLOW PLAYS AN
ESSENTIAL ROLE IN INDUCING VALVE
FORMATION.
THIS IS THE SHEAR STRESS
SIGNALING PATHWAY IN BLOOD
ENDOTHELIAL CELLS.
AS YOU CAN SEE HERE, AKT IS A
MAJOR DOWNSTREAM -- IN THE
PATHWAY AFTER SHEAR STRESS IS --
BY MORE MOLECULES ON THE CELL
SURFACE.
ALSO OUR PREVIOUS STUDY, WE
SHOWED THAT IN HUMAN ENDOTHELIAL
CELLS PHOSPHORYLATE AKT THE
ACTIVATED FORM OF AKT, WAS
UPREGULATED IN RESPONSE TO --
FLOW.
SO OUR QUESTION WAS, WHAT IS THE
DOWNSTREAM TARGET OF AKT THAT
CONTROLS LYMPHATIC VALVE GROWTH?
THE TRANSCRIPTION FACTOR
FOX01 AS DOWNSTREAM TARGET OF
AKT BECAME A CANDIDATE OF OUR
STUDY.
AS A TRANSCRIPTION FACTOR
FOX01 BINDS TO DNA TO REGULATE
GENE EXPRESSION.
HOWEVER, PHOSPHORYLATED AKT
CAN -- FOX01 AND -- OUT OF THE
NUCLEUS TO LOSE ITS NUCLEAR
ACTIVITY.
ALL THOAL FOX01 HAS THINGS
STUDIED IN MANY OTHER CELL
TYPES, HOWEVER, ITS ROLE IN
LYMPHATIC ENDOTHELIAL CELLS
REMAINS UNKNOWN.
SO THEREFORE WE DECIDED TO
DELETE FOX01 FROM THE LYMPHATIC
VASCULATURE USING PROX1 MOUSE
MODEL.
TO VISUALIZE THE LYMPHATIC
VASCULATURE, WE COMBINED THE
PROX1 GFP STRAIN TOGETHER, THE
ENTIRE LYMPHATIC VASCULATURE
WILL BE IN GFP COLOR.
WE DELETED FOX 01 FROM THE
EMBRYOS, THE POSTNATAL PUPS AND
THE ADULT MICE.
AND SINCE THE EMBRYONIC AND THE
ADULT DATA IS CONSISTENT WITH
THE POSTNATAL DATA, I WILL ONLY
SHOW THE POSTNATAL DATA HERE.
THAT MOX 15 TAMOXIFEN WAS T
POSTNATAL 1 AND 3.
AS YOU CAN SEE HERE, COMPARED TO
THE CONTROL VESSELS AT ALL THREE
STAGES, FOX01 DIVISION INDUCED
NEW VALVES TO GROW.
THESE VALVE-FORMING AREAS WERE
RARELY SEEN IN THE CONTROL
VESSELS.
IN OUR QUANTIFICATION DATA
CONFIRMED THAT, THAT NUMBER --
IN THE PUPS WITH FOX 01 DELETION
WAS SIGNIFICANTLY HIGHER THAN
THE CONTROL ANIMAL FLEURCHLT
MOREOVER, DELETION OF FOX 01 DID
NOT AFFECT THE DIAMETER OF THE
VESSELS, OR THE NUMBER OF
BRANCHES IN THESE ANIMALS.
SO FROM THESE DATA, WE CAN
CONCLUDE THAT FOX 01 IS A
REPRESSOR FOR DEFECTIVE VALVE
FORMATION.
DELETION INDUCED NEW VALVES TO
GROW.
SO YOU MIGHT WONDER HERE, SINCE
WE DON'T NEED TO ADD MORE VALVES
IN THE HEALTHY VESSELS, WHY IS
GROWING NEW VALVES IMPORTANT?
BECAUSE VALVE DEFECTS ARE
ASSOCIATED WITH SEVERAL TYPES OF
CONGENITAL LIAM LYMPHEDEMA AX
01 -- IN LYMPHATIC DISEASE.
HETEROZYGOSITY OF FOX C2 GENE
CAUSES -- IN HUMAN COMPARED TO
THE HEALTHY DRAINAGE OF THE
TRASER IN CONTROL PATIENTS,
THERMAL BACKFLOW HERE INDICATED
THAT LYMPHATIC VALVES ARE
DEFECTIVE IN THESE PATIENTS.
SO WE TESTED WHETHER FOX
01 DELETION CAN RESTORE THE
VALVE DEFECTS IN FOX
C2 HETEROZYGOUS MICE.
TO DO THIS, WE DELETED FOX 01 IN
THE FOX C2 HETEROZYGOUS
BACKGROUND.
WE USED FOX C2 CRE 2 MOUSE
MODEL.
IN THIS PARTICULAR MOUSE MODEL,
ONE ALLELE OF FOX C2 WAS
REPLACED WITH CRE ER T2.
THEREFORE, THIS MOUSE MODEL IS
BOTH HETEROZYGOSITY FOR FOX
C2 AND HAS CRE ER T2 ACTIVITY.
WE INCREASED -- AFTER BIRTH AND
WAS ANALYZED THREE WEEKS LATER.
OUR DATA SHOW HERE THAT FOX
C2 HETEROZYGOUS MICE HAD ABOUT
50% -- VALVES AT POSTNATAL DAY
51 COMPARED TO THE CONTROL AND
DELETION OF FOX 01 COMPLETELY
RESTORE THE VALVE NUMBER TO THE
Y
WILD TYPE LEVEL IN BOTH SHOWING
IN THE TOTAL NUMBER OF VALVE AND
THE VALVES PER MILLIMETER.
ADDITIONALLY, FOX
C2 HETEROZYGOUS MICE ALSO HAD A
SIGNIFICANT REDUCTION IN THE
BRANCH NUMBER.
AND THIS REDUCTION WAS ALSO
COMPLETELY RESTORED BY DELETION
OF FOX 01.
SO IN ADDITION TO RESCUING THE
MORPHOLOGY OF VALVES, WE ALSO
WANT TO TEST WHETHER FOX
01 DELETION CAN RESTORE THE
FUNCTION OF LYMPHATIC VALVES IN
FOX C2 HETEROZYGOUS MICE.
TO TEST VALVE FUNCTION, WE
COLLABORATED WITH DR. MICHAEL
DAVIS AT UNIVERSITY OF MISSOURI.
AFTER A SINGLE VESSEL CASS
CALCULATED ON TWO GLASS PIPETTES
THE OUTPUT PRESSURE WAS SLOWLY
INCREASED WHILE INPUT PRESSURE
REMAINS CONSTANT.
VALVES -- FROM A HEALTHY VALVE
CAN SEAL TIGHTLY TOGETHER TO
PREVENT BACKLEAK OF FLOAL.
AND ANY BACKLEAK HERE CAN BE
QUANTIFIED BY THE -- PIPETTE
RIGHT BEHIND THE VALVE LEAFLETS.
HERE OUR DATA SHOW SUCCESSFULLY
THAT THE LEAKY VALVES IN FOX
C2 HETEROZYGOUS MICE BECAME
SEALED TIGHTLY AGAINST TWO
DIFFERENT -- PRESSURES WHEN WE
DELETE EITHER ONE OR TWO ALEGALS
OF FOX 01.
THEREFORE, DELETION OF FOX
01 INDUCED THE NEW VALVES TO
GROW IN FOX C2 HETEROZYGOUS
MICE, AND MORE IMPORTANT -- ARE
FUNCTIONAL.
SO HOW DOES FOX 01 DELETION
INDUCE NEW VALVES TO GROW?
TO INVESTIGATE THIS, WE KNOCKED
DOWN FOX 01 IN HUMAN LYMPHATIC
ENDOTHELIAL CELLS AND TREATED
THESE WITH -- FLOW.
AS WE EXPECTED, THE EXPRESSION
LEVELS OF FLOW RESPONSIVE GENE
AND VALVE FORMING GENES
INCLUDING FOX C2, GATA2,
KLF4 AND KLF2 WAS -- MORE
IMPORTANTLY, THE EXPRESSION OF
ALL THESE GENES WAS
SIGNIFICANTLY INCREASED IN FOX
01 KNOCKDOWN CELLS ON BOTH MRNA
AND PROTEIN LEVELS.
IN THE PRESENCE OF -- FLOW, FOX
01 WAS EXCRETED OUT OF THE
NUCLEUS, SHOWN HERE IN
IMMUNOSTAINING, AND UNDER THEE
FLOW CONDITION ON POTASSIUM THE
LEVEL OF PHOSPHOAKT AND -- FOX
01 WERE UPREGULATED.
WHEN WE TREATED CELLS WITH AN
AKT ACTIVATOR SC-79, BOTH WERE
REGULATED.
SO ALL THESE DATA LEAD US TO
THIS MODEL THAT IN RESPONSE TO
SHEAR STRESS, ACTIVATED AKT AT10
WAITS THE NUCLEAR ACTIVITY OF
FOX 01 AND AS A VALVE GENE REPRE
SER IN
REPRESSOR IN THE NUCLEUS --
WHICH ALLOWS NEW VALVES TO GROW.
TO FURTHER SUPPORT OUR MODEL,
THE IMMUNO -- THE ASSAY SHOWED
THAT FOX 01 CAN DIRECTLY BIND TO
THE PROMOTER AREA OF FOX C2 AND
REG LAYING LATHE
REGULATE EXPRESSION.
RECENTLY WE TESTED -- IN
ADDITION TO GENETIC METHODS.
AS YOU CAN SEE HERE, AFTER 48
HOURS OF TREATMENT, FOX
01 INHIBITOR IS SIGNIFICANTLY
INCREASING THE LEVELS OF VALVE
FORMING GENES AND FLOW
RESPONSIVE GENES ON BOTH MRNA
AND THE PROTEIN LEVELS.
IN ADDITION TO THE INDIVIDUAL
DATA, IN VIVO INJECTION OF THIS
FOX 01 INHIBITOR SIGNIFICANTLY
INCREASED THE VALVE NUMBER IN
FOX C2 HETEROZYGOUS MICE.
ALTHOUGH THE VALVE NUMBER WAS
NOT RESTORED COMPLETELY TO WILD
TYPE LEVEL, THE FOX
01 INHIBITORS DO SHOW A PARTIAL
RESCUE OF THE VALVE NUMBER IN
FOX C2 HETEROSIGH DWUS. ZYGOUS.
SO TON CLEUD MY THOUGHT TODAY,
TRANSCRIPTION FACTOR THAT
REPRESSES LYMPHATIC VALVE
FORMATION, WE WERE ABLE TO SHOW
THAT AKT-FOX 01 SIGNALING
PATHWAY IS CRITICAL FOR
LYMPHATIC VALVE FORMATION, AND
FOX 01 DIRECTLY REPRESSES THE
EXPRESS OF THE KEY VALVE-FORMING
GENE FOX C2 TO REGULATE VALVE
FORMATION.
LASTLY, WE SHOWED LOSS OF FOX 0,
MORPHOLOGY AND FUNCTION OF
VALVES IN A LYMPHEDEMA MOUSE
MODEL.
SO THERE IS STILL MANY GAPS THAT
NEED MORE RESEARCH.
FOR EXAMPLE, WHICH TYPES OF
CONGENITAL LYMPHEDEMA CAN BE
RESTORED BY DELETION OF FOX 01?
ARE THERE ANY OTHER GENES ALSO
REPRESSING LYMPHATIC VALVE
FORMATION OTHER THAN FOX 01?
ARE THERE ANY OTHER LYMPHATIC
DISEASES BESIDES LYMPHEDEMA IN
WHICH INHIBITION OF FOX 01 CAN
IMPROVE LYMPHATIC FUNCTION?
NEVERTHELESS, OUR RESEARCH HAS
PROVIDED A POTENTIAL TARGET FOR
CONGENITAL LYMPHEDEMA.
WE THINK THE ABILITY TO
STIMULATE THE GROWTH OF
LYMPHATIC VALVES DE NOVO IN A
PHYSIOLOGICAL MANNER PROVIDES
OPPORTUNITIES TO FIND BE
POTENTIAL TARGETS FOR LYMPHATIC
DISEASES WHERE VALVES REGRESS OR
DISINTEGRATE, INCLUDING SEVERAL
TYPES OF PRIMARY LYMPHEDEMA AND
THE LYMPHATIC MALFORMATIONS.
SO LASTLY, I WOULD LIKE TO THANK
ALL OF MOUNTAIN BERMS FROM MY
LAB AND ALL MY COLLABORATORS FOR
SHARING THEIR MOUSE MODEL AND
FOR THEIR AMAZING TECHNICAL
SUPPORT.
I'D ALSO WANT TO THANK THE
SUPPORTERS FROM MY DEPARTMENT,
THE IMAGING CO-FACILITY AND THE
FACILITY AT USF, AND I ALSO WANT
TO THANK NIH FOR FUNDING
SUPPORT.
THANK YOU VERY MUCH FOR YOUR
ATTENTION.
AND I'M HAPPY TO TAKE QUESTIONS
LATER.
>> THANK YOU VERY MUCH, YANG.
>> THANK YOU.
AND OUR NEXT SPEAKER IS XIAOLEI
LIU, FROM TEMPLE UNIVERSITY.
TALKING ABOUT TARGETING CARDIAC
LYMPHATICS FOR TREATMENT OF
ISCHEMIC HEART DISEASE.
>> THANK YOU SO MUCH FOR THE
INTRODUCTION AND THANK YOU SO
MUCH FOR INVITING ME TO THIS
WORKSHOP.
IT HAS BEEN FANTASTIC.
SO WE HAVE JUST HEARD SO MANY
AWESOME TALKS ABOUT THE
DEVELOPMENT, ANATOMY AND
PHYSIOLOGIC CONDITION OF THE
LYMPH SYSTEM, AND WE KNOW THAT
IN GENERAL, LYMPHATICS PLAY A
MAJOR ROLE FOR TISSUE FLUID
BALANCE FOR IMMUNE SURVEILLANCE
AND LIPID ABSORPTION AT THE GUT
AREA.
INCREASING STUDIES SHOW THERE'S
NOVEL LYMPHATIC FUNCTIONS
IDENTIFIED.
MAINLY, I HAVE TO MENTION THAT
THESE FUNCTIONS ARE IN
ORGAN-SPECIFIC MANNER,
ESPECIALLY FINDING IN THE EYES,
IN THE CARDIOVASCULAR, IN THE
NEURAL AREA AND MORE TO BE
COMING IN THE FUTURE.
SO IN THE CASE OF THE HEART,
WHERE I AMPHOCUSSING ON, I HOPE
YOU CAN APPRECIATE THAT, THE
HEART IS ALSO ENRICHED BY A
COMPLEX NETWORK OF THE LYMPHATIC
VESSEL, HERE HIGHLIGHTED IN RED.
A STUDY IN THE PAST SEVERAL
YEARS HAS SHOWED THAT LYMPHATIC
VESSEL PLAY A FUNDAMENTAL ROLE
IN CARDIOVASCULAR DISEASE IN
TERMS OF ATHEROSCLEROSIS AND
MYOCARDIAL INFARCTION.
AS WE KNOW, THOSE ARE THE TWO
MAJOR DISEASE CONDITIONS THAT
LEAD TO HEART FAILURE, AND IT'S
STILL THE NUMBER ONE KILLER
WORLDWIDE.
SO UNDERSTANDING THE FUNCTIONAL
ROLE IN CARDIOVASCULAR DISEASE
IS REALLY A KEY AND RISING IN
THIS FIELD.
SO I HAVE TO MENTION PIONEER
WORK SEVERAL YEARS AGO FROM
DR. PAUL REILLY'S GROUP HAS
SHOWN THAT IN THE MOUSE MODEL,
AFTER MYOCARDIAL INFARCTION, YOU
CAN SEE THERE'S INCREASED
LYMPHOANGIOGENESIS IN THE
INFARCTED AREA.
AND IMPORTANTLY, WHEN YOU GIVE
VEGF C, A MAJOR FACTOR TO INDUCE
LYMPHOANGIOGENESIS, YOU CAN
CLEARLY SEE THAT COMPARED TO
CONTROL, THERE'S INCREASE IN --
GENESIS AND ALSO THERE'S
IMPROVED CARDIAC FUNCTION.
SO INDICATING THERAPEUTIC
POTENTIAL OF LYMPHATIC VESSEL IN
CARDIAC DISEASE.
I'M WORKING ON THIS FIELD.
I ALWAYS WONDER WHAT IS THE
MECHANISM, WHAT IS THE MECHANISM
TO HELP WITH THIS RECOVERY?
SO MANY OF THE STUDY CONCLUDE IT
LIKELY BY TRANSPORT OR BY REMOVE
THE DEBRIS OR IMMUNE CELLS FROM
THE INJURY SITE.
SO WHEN WE PER NORM MYOCARDIAL
INFARCTION TO THE HEART AND YOU
CAN SEE THE LYVE1 STAIN TO THE
VESSEL IS CLEARLY INCREASED AND
INFARCT THE AREA.
SO WE WONDER WHAT ARE THE
FUNCTIONS RELEVANT TO THIS
DISEASE RECOVERY?
SINCE I'M A DEVELOPMENTAL
BIOLOGIST, I WONDER WHETHER WE
CAN SORT OUT THIS MECHANISM FROM
DEVELOPMENTAL POINT OF VIEW, AND
TO ASK, DO WE KNOW THE
FUNCTIONAL ROLE OF THE
LYMPHATICS DURING HEART
DEVELOPMENT?
SO TO DO THIS, WE USE SEVERAL
GENETIC MOUSE MODEL LIKE IN
LYMPHATIC DEVELOPMENT AND WE ARE
USING CONDITIONAL DELETION TRAN
SCRIP TORE FACTOR, PROX1, AND
YOU CAN APPRECIATE HERE THAT
THIS EMBRYO SHOWS SEVERE EDEMA,
IN THE EMBRYO, AND IN THE HEART,
THERE'S -- TO OUR SURPRISE IS,
WE FOUND THAT THE HEART SIZE IS
DRAMATICALLY REDUCED COMPARED TO
THE CONTROL.
SO SUGGESTING THERE IS A ROLE OF
THE LYMPHATIC -- REGULATING
CARDIAC GROWTH, HOWEVER, BY
WHAT?
WHAT IS THE MECHANISM?
AND UNDERSTANDING THE HEART
ANATOMY, WE KNOW THAT ALTHOUGH
CARDIO -- IS CRITICAL, BUT IS
ONLY 30% OF THE MASS OF THE
HEART, AS AN INTERACTION BETWEEN
OTHER CELL TYPES WITH
CARDIOMYOCYTE HAS BEEN NO
CRITICAL IN THE
PHYSIOPATHOLOGICAL CONDITION OF
THE HEART DEVELOPMENT.
AND ONE OF THE MAJOR FOCUS IN
THE FIELD WE HAVE KNOWN IS
THAT -- FACTOR FROM ENDOTHELIAL
CELLS HAS BEEN TREMENDOUSLY
CRITICAL FOR THE CARDIAC
DEVELOPMENT AND DISEASE.
HOWEVER IN THIS PICTURE,
SOMETHING THAT'S LACKING IS
WHETHER THERE'S SUCH A SIGNAL
FROM LYMPHATICS THAT COULD ALSO
REGULATE CARDIO -- SO WE SET OUT
TO UNDERSTANDING WHETHER THERE
IS SUCH A ROLE BY USING IN VITRO
CELL CULTURE SYSTEM AND HAS
ALREADY MENTIONED A LITTLE BIT
ABOUT THAT.
WHAT WE DID IS COLLECT
COMMISSION MEDIUM AND USING
COCULTURE SYSTEM TO CULTURE THE
PRIMARY CARDIOMYOCYTE IN VITRO,
AND TO OUR SURPRISES, BY
CULTURING THE CARDIOMYOCYTE WITH
CONDITIONED MEDIUM, IT'S
SIGNIFICANTLY INCREASED THE
CARDIOMYOCYTE PROLIFERATION, AND
IT'S REDUCED THE CARDIOMYOCYTE
APOPTOSIS UNDER THE HYPOXIA
CONDITION.
SO SUGGESTING THERE IS INDEED
THERE IS A SIGNAL FROM THE
LYMPHATIC ENDOTHELIAL CELLS THAT
CAN REGULATE CARDIOMYOCYTE
PROLIFERATION AND SURVIVAL.
SO WHAT IS INSIDE OF THE
CONDITION MEDIUM?
WE USE MASS SPEC TO SCREEN THIS
CANDIDATE, AND WE FOUND LIKE
OVER 300 CANDIDATES, AND ONE OF
THE TOP ENRICHED FACTOR IS
REELIN.
YOU SEE WE DID IMMUNOSTAINING IN
THE HEART, YOU CAN CLEARLY SEE
THAT REELIN IS ENRICHED OR
EXPRESSED BY LYMPHATIC
ENDOTHELIAL CELLS IN THE HEART.
MOST IMPORTANTLY, WHEN WE DELETE
REELINUSING LYMPHATIC CRE AND
WHAT WE FOUND, IT'S KIND OF
RECAPTURED WHAT WE'VE SEEN IN
THE LYMPHATIC DEPLETION MUTANT
MICE SHOWN EARLIER AS THE HEART
SIZE IS DRAMATICALLY REDUCED
COMPARING TO THE CONTROL, AND IN
CONSISTENCY WITH THE SMALLER
HEART, WE ALSO SEE THE
CARDIOMYOCYTE PROLIFERATION IS
REDUCED, AND INSTEAD APOPTOSIS
IS INCREASED.
SO WITH THIS DEVELOPMENTAL
FINDING, WE WONDER WHETHER THE
SIGNAL ARE SIGNIFICANT IN TERMS
OF CARDIAC REPAIR.
SO WHAT WE DID IS WE USED THIS
IDENTIFIED FACTOR AND WE PUT
INTO THE HEART IMMEDIATELY AFTER
MYOCARDIAL INFARCTION AND TO SEE
WHETHER THIS FACTOR IS
BENEFICIAL.
AND IT'S SURPRISINGLY, YOU CAN
TELL THAT COMPARED TO THE
CONTROL PATCH, WHICH IS A
COLLAGEN PATCH INCORPORATED WITH
THIS FACTOR REELIN AND WE'VE
FOUND WITH REELIN THAT GREATLY
IMPROVED THE CARDIAC FUNCTION
THE DAYS AFTER M.I. AND WE ALSO
SEE THAT IS CLEARLY -- IT'S
RESORBED THE CARDIAC FIBROSIS AS
REELIN-TREATED GROUP SHOWS
REDUCED CARDIAC FIBROSIS
COMPARED TO THE CONTROL GROUP.
SO IT'S SUGGESTING THAT THIS
FACTOR WE FOUND FROM THE
DEVELOPMENTAL POINT OF VIEW IS
BENEFICIAL.
NOW THE NEXT QUESTION IS, DO WE
KNOW THAT LYMPHATIC DERIVED
RELIN IS CRITICAL DURING CARDIAC
REPAIR.
SO WE FIRST PERFORMED
IMMUNOSTAINING TO CHECK THE
REELIN EXPRESSION.
SURPRISINGLY IN A NON-INFARCTED
AREA REALLY EXPRESSION IS VERY
LOW, IT'S BARELY DETECTED.
HOWEVER, AFTER INFARCTION,
THERE'S NEWLY FORMED LYMPHATIC
VESSEL, THERE'S UPREGULATED OF
REELIN EXPRESSION IN THE HEART.
SO IT'S VERY INTERESTING, NOW WE
WANT TO ASK WHETHER THOSE REELIN
IN THIS -- ARE FUNCTIONAL.
WE PERFORMED -- -- WE PERFORM
M.I. AND WE CHECK THE CARDIAC
FUNCTION FOUR WEEKS AFTER, WHAT
WE HAVE FOUND IS THAT INDEED,
CARDIAC FUNCTION IS DRAMATICALLY
REDUCED BY LACKING OF THE REAL
ENIN THE LYMPHATIC AND WE SEE
CARDIAC FIBROSIS IS DRAMATICALLY
INCREASED IN THIS HEART POST
M.I. TOO.
SO WE FOUND REELIN LYMPHATIC
DERIVED FACTOR IS REQUIRED FOR
CARDIAC GROWTH BY IMPROVING
CARDIAC PROLIFERATION AND
SURVIVAL, AND WE ALSO SHOWED YOU
THAT -- IS REQUIRED FOR ADULT
CARDIAC REPAIR.
SO NOW WE PUT THE WHOLE PICTURE
TOGETHER AS WHEN WE CONSIDER
ABOUT LYMPHOANGIOGENESIS IN
CARDIAC REPAIR, WE ALWAYS THINK
LYMPHATIC HAS A ROLE TO CLEAR
THE IMMUNE CELLS.
NOW, WE IDENTIFY A NEW FUNCTION
THAT ACTIVELY SECRETES THIS
SIGNAL TO IMPROVE CARDIAC
SURVIVAL.
SO THE FUTURE DIRECTION ON TOP
OF THIS IS KIND OF TO DISSECT
THE CONTRIBUTION OF THE TWO
FUNCTIONS GIVEN THE CARDIAC
REPAIR, WHETHER THERE'S TEMPO
SPATIAL REGULATION, AND WHETHER
WE CAN OPTIMIZE BOTH OF THE
FUNCTION IN THE FUTURE CARDIAC
THERAPEUTIC STRATEGY FOR THE
CARDIAC REPAIR.
SO I THINK ANOTHER KEY POINT
ABOUT THIS FINDING IS NOT JUST
ONE FACTOR IDENTIFIED.
I THINK IT'S PROVIDE MOTIVATE --
TO THINK LYMPHATIC FUNCTION AS A
WHOLE IS NOT JUST A DRAINAGE BUT
ALSO TAKE INTO CONSIDERATION OF
THE LYMPHATIC SIGNAL.
AND I'M VERY EXCITED TO SAY THAT
AFTER THE PAPER CAME OUT, THERE
IS MANY, MANY -- THERE'S TWO
MAJOR FINDINGS THAT HAS BEEN
SHOWN IN ADIPOSE TISSUE, AND WE
HAVE JUST HEARD FROM RACHEL FROM
THE INTESTINE, AND WE ARE MORE
CONFIDENT ABOUT THAT THE
SIGNALING ARE IN AN
ORGAN-SPECIFIC MANNER BECAUSE A
DIFFERENT SIGNAL HAS BEEN
IDENTIFIED FROM DIFFERENT
LYMPHATIC NICHE.
BASED ON THIS, I THINK THERE IS
STILL GAPS AND A NEED AND
OPPORTUNITIES IN THIS FIELD IS
TO KIND OF DISSECT THIS
LYMPHATIC SIGNALING VERSUS
LYMPH -- FUNCTION DURING CARDIAC
REPAIR AND THE SECOND IS BL
THERE'S OTHER LYMPHATIC
SIGNALING AFFECT OTHER CELL
TYPES STILL IN THE HEART THAT IS
GOING TO BE CONTRIBUTED TO
DEVELOPMENT AND DISEASE.
WE'VE HEARD FROM OLIVER STONE
AND OTHER STUDIES IN THE FIELD
ABOUT THE ORIGIN ABOUT LYMPHATIC
VESSEL, AND WE WONDER WHETHER
THERE'S OTHER CONTRIBUTION TO
THIS DISEASE MODEL.
SO I'M GOING TO -- BASED ON
THOSE OPTION -- THIS
OPPORTUNITIES, I WANT TO
INTRODUCE TWO PROJECTS THAT IS
GOING ON IN MY LAB AT TEMPLE
UNIVERSITY.
THE FIRST ONE IS WE HAVE
IDENTIFIED THIS PAIR ALCRINE
SIGNALS TO CARDIOMYOCYTES NOW WE
ASK WHETHER THERE'S OTHER
SIGNALING FROM LYMPHATIC
ENDOTHELIAL CELLS TO OTHER CELL
TYPES DURING THE PATHOLOGIC PRO
GRETION OF THE
PROGRESSION OF THE CARDIAC
REPAIR AND USING IN VIVO AND IN
VITRO MODEL TO DISSECT THIS.
THE SECOND PROJECT IS, AS A
DEVELOPMENTAL BIOLOGIST, I
ALWAYS VALUE THE IMPORTANT AND
THE CRITICAL OF THE EARLY STEPS
OF THE LYMPHATIC DEVELOPMENT.
WE KNOW THAT THERE'S MANY KEY
GENES, NOW IT'S ALSO IMPORTANT
MARKERS IN THIS FIELD, HAS BEEN
IDENTIFIED TO CONTROL THE EARLY
SPROUTING MIGRATION AT
DEVELOPMENT OF THIS VESSEL.
COMPARED TO THE BLOOD VESSEL OR
OTHER SYSTEMS, THE UNDERSTANDING
IS FAR LESS UNDERSTOOD.
SO IDENTIFYING NOVEL PLAYER IS
STILL A NEED, AND IS A PART OF
MY PASSION TO IDENTIFY.
SO ONE OF THE CANDIDATES I
WANTED TO SHOW YOU HERE IS WE
IDENTIFIED ANOTHER GENE.
THAT IS VERY INTERESTINGLY
HIGHLY EXPRESSED IN THE
SPROUTING PART OF THE LYMPHATIC
VESSEL, AND IN THE LAB WE'RE
USING TRANSGENIC -- MOUSE MODEL
TO FURTHER IDENTIFY THE
FUNCTIONAL ROLE DURING THE
DEVELOPMENT.
AND WITH THE ULTIMATE GOAL IS TO
MODULATE THIS SIGNALING FOR THE
TREATMENT OF LYMPHATIC-RELATED
DISEASE.
SO WITH ALL OF THAT, I WOULD
LIKE TO ANNOUNCE THAT MY LAB IS
OFFICIALLY ESTABLISHED EARLY
THIS YEAR IN THE LEMOLE CENTER
FOR TEMPLE UNIVERSITY.
RIGHT NOW I HAVE A POSTDOC AND
PH.D. CANDIDATE IN THE LAB.
WE ARE STILL HIRING, SO ANYBODY
WHO ARE INTERESTED IN THIS PART
OF THE WORK, PLEASE FEEL FREE TO
CONTACT US, AND SO AT THE FIRST
PART OF THE WORK I HAVE
PRESENTED HAS BEEN DONE WHEN I
WAS A POSTDOC IN DR. OLIVER'S
LAB, RIGHT NOW WE STILL HAVE
ACTING COLLABORATION ON SOME OF
THE ONGOING PROJECT.
SINCE I JOINED TEMPLE, I HAVE
ESTABLISHED MY OWN COLLABORATION
WITH A NUMBER OF WONDERFUL
SCIENTISTS LISTED HERE, AND A
COLLABORATION OUTSIDE OF THE
TEMPLE.
THANK YOU SO MUCH FOR YOUR
ATTENTION.
I'M LOOKING FORWARD TO ANSWER
QUESTIONS AT THE END.
THANK YOU.
>> THANK YOU, THAT WAS VERY
EXCITING.
SO OUR NEXT AND FINAL SPEAKER OF
SESSION TWO IS JOSH SCALLAN FROM
UNIVERSITY OF SOUTH FLORIDA.
SO WELL WELCOME, JOSH.
JOSH IS GOING TO TALK TO US
ABOUT THE REGULATION OF BUTTON
JUNCTIONS IN THE LYMPHATIC
VASCULATURE.
>> THANK YOU FOR THE
INTRODUCTION AND THANK YOU TO
THE ORGANIZERS FOR INVITING US
TODAY.
THIS IS A NEW PROJECT IN OUR
LAB, WHICH IS FOCUSED ON BUTTON
JUNCTIONS IN THE LYMPHATIC
VASCULATURE AND JUST REAL
QUICKLY TO INTRODUCE WHAT A
BUTTON JUNCTION IS, IF YOU
IMMUNOSTAIN LYMPHATIC
VASCULATURE LIKE SHOWN HERE FOR
A JUNCTION PROTEIN IN BLOOD
VESSELS, YOU WILL SEE THE ENTIRE
CELL PERIPHERY OF EACH
INDIVIDUAL CELL.
AS YOU CAN NOTE HERE IN
LYMPHATIC VESSELS, THE JUNCTION
PATTERN LOOKS A LITTLE
DISCONTINUE WITH US AND BROKEN
AND WE REFER TO THESE AS
BUTTONS.
BEFORE I CONTINUE TALKING ABOUT
BUTTONS, I'D LIKE TO INTRODUCE
HOW WE GOT INTERESTED IN
MOLECULAR REGULATION OF BUTTONS.
SO THIS WAS A SLIDE THAT WAS
PRESENTED IN DR. YING YANG'S
TALK BUT REAL QUICK JUST TO GO
OVER IT, THIS IS A LIN PITOCIN G
ROW FEE, WHEN YOU INJECT RADIO
TRASER INTO FEET -- THAT TRAVELS
UP TO THE GROIN WHERE IT
ACCUMULATES IN LYMPH NODES.
IN FOX C2 PATIENTS, IT DOES
BACKFLOW INTO THE DERMAL CAB
LARRYS WHICH CAN ONLY HAPPEN
WHEN YOU HAVE EITHER MISSING OR
DYSFUNCTIONAL VALVES.
BUT WE WERE ACTUALLY REALLY
INTRIGUED BY ANOTHER PANEL IN
THIS PAPER WHICH SHOWED A
DIFFERENT LYMPHEDEMA FLOW
PATTERN WHICH OCCURS IN PATIENTS
WITH MISS ANS MUTATIONS IN THE
GENE VEGFR3, THE MOST
COMMONLY -- RADIO TRASER IS
SIMPLY NOT ABSORBED AT ALL BY
LYMPHATICS.
SO WE WANTED TO KNOW A LITTLE
BIT MORE ABOUT WHAT COULD BE
CAUSING THAT AND WHY IT'S
OCCURRING.
THERE ARE THREE VEGF RECEPTORS,
1, 2 AND 3, AND THESE RECEPTORS
ARE RECEPTOR TYROSINE KINASES
THAT FORM DIMERS -- EITHER HOMO
OR HETERODIMERS AND THE END
EFFECT OF BINDING THEIR LIGANDS
SUCH AS VEGF A OR VEGF C IS TO
NICHE YAIF LYMPH ANGIOGENESIS OR
ANGIOGENESIS, THE GROWTH OF
LYMPHATIC OR BLOOD VESSELS.
SO THE WAY WILD TYPE -- WHEN
VEGF C BINDS IT, THEY'LL AUTO
PHOSPHORYLATE ONE ANOTHER AND
INITIATE A BUNCH OF DIFFERENT
SIGNALING PATHWAYS WHICH WILL
ALL ACT TO INITIATE LYMPH
ANGIOGENESIS.
HOWEVER, IN MILL MILROY DISEE
THIS INACTIVATES THE TAIL SO
EVEN IF YOU HAVE A MUTATED
PROTEIN THAT PAIRS UP WITH A
WILD TYPE PROTEIN, YOU WILL NOT
GET ANY AUTO PHOSPHORYLATION
CYTOPLASMIC TAILS AND YOU WILL
HAVE ABLATED SIGNALING WHICH
WILL IN THIS DIAGRAM LEAD TO
DISRUPTED LYMPH ANGIOGENESIS.
SO TO EXPLAIN THE FLOW PATTERN I
SHOWED IN THE BEGINNING OF MY
TALK YOU MAY THINK MILROY
PATIENTS DON'T -- THEY DON'T
HAVE ANY LYMPHATIC VESSELS IN
THE FEET AT ALL.
THIS WAS ACTUALLY THE CONCLUSION
THAT MANY CAME TO, YOU KNOW, IN
THE LITERATURE.
JUST REAL QUICK, I WANT TO
HIGHLIGHT THAT VEG RECEPTOR 3 IS
THE KEY REGULATOR IN -- ANGIO16
CYST.
IF YOU DO A PUBMED SEARCH FOR
VEG RECEPTOR 3 OR VEG RECEPTOR A
STRONG DEGREE OF OVERLAP HERE,
INDICATING IT'S REALLY STUDIED
PRIMARILY IN THE CONTEXT OF
ANGIOGENESIS AND NOT ANYTHING
ELSE.
HOWEVER A MORE RECENT PAPER
SHOWED IT WASN'T ANY LACK OF
LYMPHATIC VESSELS BUT VESSELS
WITH DYSFUNCTION THAT CAUSES
MILROY DISEASE AND THEY TOOK
STUDIES FROM MILROY PATIENTS AND
IMMUNOSTAIN FOR BLOOD VESSELS
SHOWN IN RED, LYMPHATIC VESSELS
SHOWN IN GREE FROM CONTROL AND
MILROY PATIENTS.
FROM BOTH CONTROL IN MILROY
PATIENTS IN THE ARM AND IN THE
FEET, AND WHILE THERE IS A
SIGNIFICANT REDUCTION OF ALMOST
LYMPHATIC CAPILLARY DENSITY IN
MILROY PATIENTS AS YOU GO DEEPER
INTO THE SKIN, THIS DIFFERENCE
TENDS TO DISAPPEAR AND SO YOU
CAN APPRECIATE THAT MILROY
PATIENTS DEFINITELY HAVE A
NON-ZERO NUMBER OF LYMPHATIC
VESSELS THAT ARE PRESENT BUT
MUST BE DYSFUNCTIONAL IN SOME
WAY.
SO TO TRY TO GET AT THIS -- FOR
THE POSSIBLE CAUSE OF MISS
FUNCTION WE TURN TO
HYPOTHESIZING THAT MAYBE THE
LYMPHATIC CAPILLARIES ARE
PRESENT BUT THEY HAVE SOME KIND
OF A PROBLEM WITH THEIR BUTTON
JUNCTIONS.
THIS IS A TIME COURSE OF BUTTON
JUNCTION DEVELOPMENT FROM --
16 1/2, SO TWO MONTHS
POSTNATALLY IN THE TRACHEA.
IMMUNOSTAIN FOR -- YOU CAN SEE
THE CELL JUNCTIONS IN THE EMBRYO
ARE LARGELY CONTINUOUS BUT RIGHT
AROUND BIRTH, AND FOR SEVERAL
WEEKS AFTER BIRTH, YOU ARE
DEVELOPING MORE BUTTONS OVER
TIME AND THESE BUTTONS ARE
BECOMING MORE MATURE AND MORE
DISCRETE.
THIS IS A CARTOON SCHEMATIC
SHOWING THAT BUTTONS OCCUR IN --
THE PURPOSE OF THE BUTTON
JUNCTIONS IS TO FORM LARGE FLAPS
THAT ARE HIGHLY PERMEABLE WHICH
ALLOW THE LYMPHATIC CAPILLARIES
TO ABSORB LARGE AMOUNTS OF
FLUIDS, MACRO MOLECULES AND
LIPIDS.
SO WHAT DO WE KNOW ABOUT THE
MICRO REGULATION OF LYMPHATIC
BUTTON JUNCTIONS?
WE NODELL TA WE KNOW THAT D
4 PROMOTES CONTINUOUS LACTEAL
REGENERATION, NEVER FORM BUTTONS
IN THE FIRST PLACE DOWN HERE,
AND THIS IS QUANTIFIED HERE, YOU
CAN SEE THAT THERE ARE VERY FEW
CONTINUOUS ZIPPER-LIKE JUNCTIONS
IN HEALTHY WILD TYPE LACTEALS
BUT YOU HAVE EXPANSION OF
ZIPPERED UP JUNCTIONS IN DELTA
LIGAND 4 KNOCKOUTS.
IN THE SAME PAPER, IF YOU LOOK
AT SUBMUCOSAL LYMPHATIC
CAPILLARIES AND YOU DELETE DELTA
LIGHT LIGAND 4 THERE REALLY IS
NO DIFFERENCE IN THE NUMBER OF
BUTTON JUNCTIONS -- REGULATION
OF BUTTON JUNCTION DEVELOPMENT.
WE ALSO KNOW ANGIOPOIETIN
2 REGULATES THE TRANSFORMATION
AND INTEGRITY OF LYMPHATIC
ENDOTHELIAL CELL JUNCTIONS.
MORE RECENTLY, THEY SHOWED THAT
MICE ARE PROTECTED FROM A HIGH
FAT DIET FOR 16 WEEKS DUE TO
LACTEAL ZIPPERING, SO IF YOU
DELETE BOTH WHICH BIND VEGF A
THEN THE LACTEALS GO TO BEING
LARGELY ZIPPERED UP.
AND THE MECHANISM FOR HOW THIS
WORKS IS THAT RECEPTORS ON BLOOD
VESSELS THAT SEQUESTER VEGF A
AND PREVENT IT FROM ACCUMULATING
THE TISSUE.
WHEN YOU KNOCK THEM OUT YOU HAVE
A HIGH LEVEL IN THE TISSUE THAT
THEN HYPERACTIVATES -- R2,
ACTING AS A PRO ZIPPERING
PATHWAY.
THIS WAS CONFIRMED THIS YEAR IN
A PAPER WHERE MICE WERE INFECTED
WITH VACCINIA VIRUS WHICH TURNS
BUTTON JUNCTIONS INTO ZIPPER
JUNCTIONS, AND THAT COULD BE
RESCUED BY ANTIBODIES AGAINST
VEGF R2 OR VEGF A OR KNOCKING
OUT -- SHOWING THAT VEGF
R2 INDUCES ZIPPERING.
WE ON AT THE SAME TIME WITH PP
REPORTER SO WE CAN SEE ALL
LYMPHATICS.
WE INDUCED A LESION AT P1, RIGHT
WHEN BUTTONS ARE STARTING TO
BECOME FORMED, AND CONTINUED
DELETION UNTIL P21 BY INJECTING
TAMOXIFEN EVERY OTHER DAY.
SHOWN HERE, WE EFFECTIVELY
DELETED VEGF RECEPTOR 3, IF YOU
LOOK AT A HIGHER MAGNIFICATION,
YOU CAN SEE THERE ARE BUTTON
JUNCTIONS IN THE CONTROLS.
HOWEVER, KNOCKOUTS, THEY WERE
ALL ZIPPERED UP, AND THAT IS
QUANTIFIED HERE.
SO ZIPPERS IN BLUE AND BUTTONS
IN GREY.
SO WE HAD AN ALMOST COMPLETE
DISAPPEARANCE OF BUTTONS AND A
VERY BIG INCREASE IN NUMBER OF
ZIPPERS, JUST BY DELETING ZIPPER
3, INDICATING IT IS IMPORTANT
FOR BUTTER JUNCTION BUT.
WE ENSURED THIS WAS NOT SPECIFIC
TO THE DIAPHRAGM, THIS IS
MULTIPLE TISSUES WHERE WE HAVE
ZIPPERING.
I JUST WANTED TO POINT OUT THAT
WE DO NOT HAVE IMPAIRED LYMPH
ANGIOGENESIS BY -- POSE NATALLY
IN EITHER THE DIAPHRAGM OR THE
EAR.
THIS IS NOT THAT SURPRISING, BUT
IT MIGHT BE SURPRISING TO SOME
PEOPLE
PEOPLE.
IF YOU DELETE -- AFTER BUTTON
JUNCTIONS HAVE ALL BEEN BEEN
ESTABLISHED WE WANTED TO KNOW IF
IT WAS REQUIRED FOR MAINTENANCE
OF BUTTONS SORT OF LIKE HOW
OTHER GENES ARE REQUIRED FOR
MAINTENANCE OF OTHER LYMPHATIC
VALVES.
IF YOU LOOK AT AT THE BUTTONSW
THERE'S NO DIFFERENCE IN THE
NUMBER OF BUTTONS INDICATING
THAT VEGF RECEPTOR 3 IS
DISPENSABLE AFTER A SHORT
CRITICAL POSTNATAL WIN DOLE, WE
HAVE BUTTONS DEVELOPING SOMETIME
IN THE FIRST THREE WEEKS OF
LIFE.
AT LEAST IN MICE.
OKAY.
SO I ALREADY TOLD YOU THAT VEGF
R2 CAN INDUCE ZIPPERING.
SO IS IT POSSIBLE THAT SHOWM
WHEN WE DELETE VEG RECEPTOR 3,
DO WE HYPERACTIVATE VEGF R2 --
IF YOU ADDRESS WHETHER VEGF
RESPECT TORE 2 CAN REDUCE
ZIPPERING -- THIS IS SHOWING YOU
VEG RECEPTOR 2 WHICH IS
EXPRESSED IN MANY CELL TYPES AND
DIAPHRAGMS -- EPITHELIAL CELLS
AND AGAIN WE GET THE SAME
RESULTS, WE HAVE BUTTONS IN THE
CONTROLS AND ZIPPERS IN THE
DOUBLE KNOCKOUTS NOW, WHICH
REALLY LOOKS NO DIFFERENT THAN
OUR SINGLE VEG RECEPTOR
3 KNOCKOUTS, INDICATING THAT
DELETION OF VEG RECEPTOR 2 IS
NOT SUFFICIENT TO RESCUE LOSS OF
VEG RECEPTOR 3.
IT'S PROBABLY NOT PLAYING A
ROLE.
SO IT FIGURE OUT WHAT THE
MECHANISM WAS BEHIND WHY WE'RE
GETTING ZIPPERING, WE CULTURE
HD -- VEG RECEPTOR 2 DOES NOT
CHANGE WHEN WE DO THIS BUT NOTCH
SIGNALING IS STRONGLY -- AGAIN
THE PROTEIN LEVEL TOTAL VEGF
R2 AND PHOSPHORYLATED LEVELS
WE'RE NOT SEEING SIGNIFICANTLY
ALTERED BY KNOCKING DOWN VEG
RECEPTOR 3 AND PHOSPHORYLATED IS
VERY LOW IN LEZs, HOWEVER,
WHEN WE USE ANTIBODIES AGAINST
CLEAVES, NOTCH 1 OR NOTCH 4,
THEN NOTCH 1 WAS FOUND TO BE
HIGHLY EXPRESSED AND STRONGLY
DOWN REGULATED WHEN WE KNOCKED
DOWN VEG RECEPTOR 3 AND
NICD4 WAS REALLY NOT EXPRESSED
VERY HIGHLY AT ALL AND DIDN'T
CHANGE.
SO WHAT IS NOTCH SIGNALING?
NOTCH SIGNALING IS A SIGNALING
PATHWAY ACTIVATED BY CELL
MEMBRANE -- LIGANDS.
SO WHEN ONE CELL COMES INTO
CLOSE PROXIMITY TO ANOTHER CELL
EXPRESSING THE NOTCH RECEPTORS,
THE LIGAND COMBINES THE RECEPTOR
WHICH ACTIVATES CLEAVAGE OF THE
INTERCELLULAR DOMAIN OF NOTCH
WHICH THEN ALLOWS TO FREELY
TRANS LOCATE TO ACTIVATE GENE
TRANS KRIPTION AND IN THE
ABSENCE OF NICD, YOU USUALLY GET
GENE REPRESSION.
SO TO TEST WHETHER OR NOT WE
COULD OVEREXPRESS A
CONSTITUTIVELY ACTIVELY
NICD1 FRAG MANAGEMENT TO BETTER
RES KEUL
RESCUE KNOCKOUTS -- AND AGAIN,
I'M SHOWING YOU WE HAVE BUTTONS
IN THE CONTROLS, WE HAVE ZIPPERS
IN THE VEG RECEPTOR 3 KNOCK
YOWLTS BUT SURPRISINGLY IN THE
ABSENCE OF VEG RECEPTOR 3 BY
ACTIVATING NICD1, WE'RE ABLE TO
ACTIVATE -- BACK TO CONTROL
LEVELS INDICATING NOTCH 1 IS
SIGNIFICANT TO RESCUE THE LOGS
OF VEG RECEPTOR 3.
SO WE WANTED TO ALSO ADDRESS
FUNCTION.
WE INJECTED EVANS BLUE DYE.
IN THE R3 KNOCKOUTS IT MOVES
INTERSTISHLY BUT DOES NOT FILL
UP VASCULAR STRUCTURES.
HOWEVER IN NICD REGS RESCUESE
IS NOT ABLE TO FILL -- BY
RESTORING BUTTONS.
SO TO GO OVER MY CONCLUSIONS.
WHAT I SHOWED YOU TODAY IS THAT
VEG REACCEPT PORE 3 IS REQUIRED
FOR POSTNATAL BUTTON JUNCTION
FORMATION DURING A CRITICAL
WINDOW.
NOTCH 1 SIGNALING CAN RESCUE
BUTTON JUNCTION FORMATION AND
FUNCTION IN THE ABSENCE OF VEG
RECEPTOR 3.
WE PROPOSE THERE'S A VEG
RECEPTOR 3/DLL4 NOTCH SIGNALING
AXIS RESPONSIBLE FOR BUTTON
JUNCTION FORMATION AND BUTTON
JUNCTIONS FACILITATE
INTERSTITIAL FLUID ABSORPTION
AND FILE FAILURE TO FORM -- SO
FOR GAPS, NEEDS AND
OPPORTUNITIES, HOW DO WE
CLINICALLY TARGET AND INDUCE
BUTTON JUNCTION DEVELOPMENT IN
MILLROY PATIENTS AND OTHER
PATIENTS THAT MAYBE HAVE
DEFECTIVE BUTTON JUNCTION
FORMATION?
AND CAN WE TARGET NOTCH
SIGNALING SOMEHOW IN PATIENTS?
ARE THERE OTHER CONGENITAL GENE
MUTATIONS THAT CAUSE LYMPHEDEMA
BY INHIBITING BUTTON JUNCTION
DEVELOPMENT AND CAN THESE BE
RESCUED BY TARGETING THE NOTCH
PATHWAY?
IT'S WORTH NOTING THAT IN
ACQUIRED LYMPHEDEMA PATIENTS,
THERE IS A STRONG DOWNREGULATION
OF VEG RECEPTOR 3 AND NOTCH
1 ACTIVITY, SO MAYBE THERE COULD
BE A POTENTIAL ROLE FOR
DEFECTIVE BUTTON JUNCTIONS IN
SECONDARY LYMPHEDEMA.
AND FINALLY WHAT PROTEINS DOES
NOTCH COOPERATE WITH TO INDUCE
BUTTON DEVELOPMENT IN VIC
CAPILLARIES BUT NOT IN
COLLECTING VIC VESSELS?
WE COULD ACCELERATE BUTTON
FUNCTION EVEN EARLIER BUT
COLLECTING LYMPHATICS NEVER
DEVELOPED BUTTONS.
SO I'D LIKE TO ACKNOWLEDGE THE
PEOPLE IN MY LAB.
MY POSTDOC, MELANIE JANNAWAY WHO
DID A LOT OF WORK ON THIS
PROJECT, OTHER COLLABORATORS AND
NSF.
I'D LIKE TO THANK NIH FOR
FUNDING AS WELL AS DHA AND
OTHERS KIND ENOUGH TO SHARE
MOUSE MODELS, WITH THAT, I'D
LIKE TO TAKE ANY QUESTIONS.
THANK YOU.
>> JOSH, THANK YOU SO MUCH.
I WANT TO TAKE A SECOND TO THANK
ALL OF THE SPEAKERS FOR REALLY
ENERGETIC AND IMPORTANT TALKS.
SO I THINK WE WILL OFFICIALLY
START OUR Q & A DISCUSSION.
SO I ENCOURAGE THAT PANELISTS
AND THE SPEAKERS TO JUST RAISE
YOUR HAND OR JUST TO SHOUT IF
YOU HAVE -- OR NOT TOO LOUD --
IF YOU HAVE A QUESTION TO ANY OF
THE SPEAKERS, THIS INCLUDES
QUESTIONS TO MIKE DAVIS AS WELL
AND ALSO WHAT I'M GOING TO START
IS FOR ALL OF OUR PARTICIPANTS,
JUST PLACE YOUR QUESTIONS ON THE
CHAT AND I WILL TRY TO DO MY
BEST TO GO THROUGH ALL OF THEM.
SO WHAT I AM GOING TO DO IS I AM
GOING TO START WITH JUST THE
ORDER OF THE QUESTIONS THAT THEY
WERE POSTED.
SO WE WILL START WITH OLIVER
STONE, FROM SATESH.
THE QUESTION IS, HAVE YOU,
OLIVER, LOOKED AT ORGAN-SPECIFIC
LYMPHATIC VESSELS?
>> SO WE'VE LOOKED AT THIS WITH
A CONSTITUTIVE CRE AND ALSO WITH
CONDITIONAL LINEAGE LABELING AT
DIFFERENT STAGES.
WITH CONSTITUTIVE CRE, WE SEE
LABELING WITH A SIGNIFICANT
PROPORTION, THE MAJORITY OF
ENDOTHELIAL CELLS IN MOST
ORGANS.
WE DON'T SEE LABELING WITHIN THE
HEAD REGION.
THIS IS PURELY A CONSEQUENCE OF
THE FACT THAT PACKS 3 IS NOT
EXPRESSED IN MEES YOE DETERMINE
IN THE HEAD NOR IN THE
PHARYNGEAL MESOYOE MESODERM.
WE'RE ATTEMPTING TO UNDERSTAND
WHAT THIS MEANS AT THIS POINT IN
TIME.
SO WE WERE INITIALLY FOCUSED ON
THE EARLY STEPS OF
DIFFERENTIATION BUT WE ARE
CERTAINLY GOING BACK AND LOOKING
MORE INTO HOW CELLS END UP IN
DIFFERENT ORGANS.
>> OKAY.
OLIVER, THANKS SO MUCH.
SATESH, FEEL FREE TO EMAIL
OLIVER IF YOU WANT TO UNCOUNTER
THAT.
FOR RACHEL I'M WONDERING HOW YOU
THINK THAT IMPAIRED LYMPHATIC
FLOW WITH INTACT LYMPHATIC
ENDOTHELIUM MAY AFFECT THESE
INTERACTIONS WITH THE STEM CELL
NICHE?
THANKS VERY MUCH.
>> THANK YOU.
YOU'RE RIGHT ACROSS THE STREET,
WE SHOULD BE WAVING AT EACH
OTHER, THAT'S SO FUNNY.
I THINK IT'S A REALLY
FASCINATING QUESTION AND I THINK
A QUESTION THAT I'D REALLY LOVE
TO POSE TO SOME OF MY COLLEAGUES
HERE STUDYING LYMPHATIC FLOW,
WHAT WE KNOW ABOUT THE
TRANSCRIPTOME AND THE SECRETEOME
DURING FLOW OR STATIC STATES.
PERHAPS IT CHANGES, PERHAPS
THEY'RE SENSITIVE TO PRESSURE
AND HOW MUCH THEY'RE SECRETING
OR THE GEEBS GENES THEY'RE
TRANSCRIBING OR CHANGING AND
THAT WAY THEY CAN AFFECT CELLS
IN THEIR MICROENVIRONMENT.
ALSO OF COURSE ONE THING WE
THINK ABOUT IS IF THE LYMPHATICS
ARE FLOWING, IF THEY'RE DRAINING
FACTOR, THEY CAN BE DRAINING
IMPORTANT SIGNALING QUEUES OF
INFLAMMATION -- FROM THE STEM
CELL NICHE, EITHER LEAVING THEM
THERE TO PROMOTE APPROPRIATE
BEHAVIOR AND DRAINING THEM TO
CHANGE BEHAVIOR, SO THAT'S
SOMETHING THAT I'M REALLY
FASCINATED TO LOOK AT IN
INFLAMMATORY DISEASE
PARTICULARLY INFLAMMATORY BOWEL
DISEASE.
>> OKAY, RACHEL.
THANK YOU SO MUCH.
THERE'S A QUESTION FOR DR. YANG.
DO YOU THINK VAIR YATION IN N
VARIABLE PHENOTYPE IN LYMPHEDEMA
AND HAVE YOU LOOKED FOR EVIDENCE
OF THIS?
>> SO WE DID ACTUALLY.
FOX 02 IS IDENTICAL TO FOX 03.
SO WE ACTUALLY KNOCKED OUT FOX
03 FROM THE LYMPHATIC
ENDOTHELIAL CELLS USING THE SAME
CRE -- AND THEY LOOKED AT
WHETHER FOX 03 WOULD HAVE A
SIMILAR FUNCTION AS FOX
01 DELETION.
AND WHEN WE LOOK AT THE VALVE
NUMBER, WE CANNOT SEE AN
INCREASE OR DECREASE IN VALVE
NUMBER WHEN WE DELETED FOX 03.
SO IN THIS CASE, I THINK FOX
01 IS THE MAJOR FOX
0 TRANSCRIPTION FACTORS THAT
REGULATES LYMPHATIC VALVE
FORMATION.
AND ALSO FOX 03 EXPRESSION LEVEL
IS VERY LOW IN THE LYMPHATIC
ENDOTHELIAL CELLS THAT COULD
ALSO EXPLAIN THE REASON.
>> OKAY.
THERE'S ONE MORE QUESTION
WHETHER YOU THINK -- I THINK
IT'S FROM JIM MOORE.
I'M JUST WONDERING WHETHER THERE
ARE ANY SIMILARITIES BETWEEN
LYMPH ANGIOGENESIS, PARTICULARLY
VALVE FORMATION AND THE VALVE
FORMATION IN NEOANGIOGENESIS.
CAN YOU COMMENT ON THAT?
>> THANK YOU FOR THE QUESTION.
SO YES, THERE ARE SIM LAIRMTS.
SIMILARITIES.
FOX 01 IS A REPRESSOR FOR ALL
THREE PROCESS, INCLUDING
ANGIOGENESIS -- AND -- VALVE
FORMATION.
AND -- BUT THAT BECAUSE FOX
01 DELETION CAN CAUSE INCREASED
PROLIFERATION IN BOTH BLOOD
ENDOTHELIAL CELLS SHOWN BY
MICHAEL --'S LAB AND
INCREASED -- IN -- GENESIS SHOWN
BY MY LAB AND ALSO BY -- LAB,
BUT THE DIFFERENCE IS THAT WHEN
WE LOOKED AT PROLIFERATION
STATUS PARTICULARLY DURING
LYMPHATIC VALVE FORMATION, WE
DID NOT SEE A CHANGE IN
PROLIFERATION OF PARTICULARLY IN
POSTNATAL VESSELS.
SO WE RULED OUT ROW LIVE RANGE
OF MOTION FUNCTION IN
INDUCING -- GROWTH.
>> THANK YOU SO MUCH.
THERE IS A QUESTION FROM THE
AUDIENCE.
ASK YOUR QUESTION.
>> YES.
I WOULD LIKE TO ASK -- AND ALSO
REMIND EVERYONE THAT FOR MIKE
DAVIS AND -- THIS WOULD BE THE
TIME TO ASK QUESTIONS AND I HAVE
A QUESTION TO MIKE.
I WAS WONDERING IF YOU SEE ANY
EXTRACELLULAR MATRIX CHANGES IN
THOSE ANIMALS OF THE OBESE MICE,
ESPECIALLY, CAUSING THE
FUNCTIONAL CHANGES LIKE IN
ARTERIAL CONDITIONS, THEY BECOME
STIFF AND ARE NOT CONTRACTILE.
SO I WAS WONDERING IF YOU SEE
ANYTHING IN THE LYMPHATIC
VESSELS.
>> THAT'S A GOOD QUESTION.
WE HAVEN'T SPECIFICALLY LOOKED
AT THAT, BUT IF YOU DO PASSING
PRESSURE DIAMETER CURVES IN SOME
OF THOSE ANIMAL MODELS, THE
LYMPHATIC VESSELS DO SHOW
DIFFERENCES.
I THINK JORGE MAY BE ABLE TO
ADDRESS THAT LATER ON IN HIS
TALK.
>> IF I CAN FOLLOW UP JUST A
LITTLE BIT MORE THAT IF ALL
THOSE SMOOTH MUSCLE CELLS IN THE
LYMPHATIC VASCULATURE, BECAUSE
WE KNOW THAT IN ARTERIAL SMOOTH
MUSCLES, THEY CHANGE THEIR
RECEPTORS AND RECEPTOR
SIGNALING, AND THOSE CHANGES TO
REFLECT INTO THE -- FOR EXAMPLE,
CALCIUM SIGNALING CHANNELS AND
HOW THEY'RE EXPRESSED, SO I
WONDER IF -- I KNOW I'M ASKING A
LOT OF -- TO THE VERY BEGINNING
OF THIS WORK THAT YOU ARE
INITIATING, BUT MAYBE THOSE
MIGHT BE THE CHANGES THAT ARE
AFFECTING INITIALLY THE
LYMPHATIC VESSELS AND IN TURN
LYMPHATIC VESSELS INITIATE THE
CHANGES IN THE ARTERIAL VESSELS.
I WAS WONDERING WHAT YOU THINK
ABOUT THAT.
>> IT'S POSSIBLE.
I DON'T KNOW THE ANSWER TO THAT.
>> OKAY.
THANK YOU.
>> THANKS VERY MUCH, MIKE.
OKAY.
SO THERE IS A QUESTION FROM
JAMES MOORE TO YING.
HI, YING.
THANKS FOR THE NICE TALK.
DO YOU HAVE ANY ESTIMATE OF THE
OSCILLATORY SHEAR STRESS BEING
APPLIED IN THE TEST TUBE ROCKER
EXPERIMENT?
>> YES, THANK YOU FOR THE
QUESTION.
SO THE ESTIMATED OSCILLATORY
STRESS ON THE TEST TUBE ROCKER
WAS ABOUT 0.3 CENTIMETERS --
SQUARE CENTIMETER.
ALTHOUGH IT IS LOWER THAN THE
SHEAR STRESS IN THE LYMPHATIC
VESSELS IN VIVO EXPERIENCING
WHICH IS BETWEEN 1 TO 12
SQUARE CENTIMETER, BUT WE WERE
ABLE TO SEE THE UPREGULATION OF
VALVE GENES AND FOR RESPONSIVE
GENES, AND WE ALSO WERE ABLE TO
SEE THE NUCLEAR TRANSLOCATION OF
FOX 01 UNDER THAT CONDITION.
THANK YOU.
>> THANK YOU.
OKAY.
AND SO THERE'S A QUESTION FROM
ESTA STERNECK, FOR YOU AND OTHER
INVESTIGATORS USING LYMPHATIC
ENDOTHELIAL CONDITIONED MEDIA.
DO YOU USE OR ORGAN-SPECIFIC
PRIMARY ISOLATES AND IF NOT WHAT
IS THE STATUS OF YOUR
ENDOTHELIAL CELLS.
>> FOR MY STUDY WHAT WE HAVE
USED, IT'S NOT THE ENDOTHELIAL
CELL FROM THE HEART.
WE HAVE USED A COMMERCIALLY
AVAILABLE HUMAN DERMAL
ENDOTHELIAL CELLS.
SO ACCORDING TO THE SCREEN OF
CANDIDATES WHAT WE HAVE DONE IS
TO CONFIRM BY IMMUNOSTAINING
WHETHER THOSE MARKERS ORTHOS
CANDIDATES ARE HIGHLY EXPRESSED
IN CARDIAC AND LYMPHATIC
ENDOTHELIAL CELLS OR NOT.
I THINK THIS IS A VERY VALUABLE
QUESTION AS IN THE FUTURE, IT'S
VERY HELPFUL TO DO RN SEQ OR
SINGLE CELL RN SEQ FROM ORGAN
SPECIFIC LYMPHATIC AND TO
VALIDATE MAYBE THERE'S SOME
INFORMATION MISSING BY USING
THIS COMMERCIAL AVAILABLE -- OR
ORGAN SPECIFIC LECs.
THANK YOU FOR THE QUESTION.
>> THANK YOU.
JOSH, THERE'S A QUESTION FROM
YOU FROM MATTHEW BURCHILL.
IF YOU INDUCE LYMPHOANG GENESIS
IN CAPILLARIES IN WHICH YOU
DELETE VEGF RECEPTOR 3 AFTER
DEVELOPMENT --
>> AND THEN INDUCE WITH
ANGIOGENESIS DO I SEE GREATER
ZIPPERING THAN CONTROL, I DON'T
THINK THERE WILL BE A DIFFERENCE
BECAUSE EVEN IN CONTROL MICE --
I THINK THEY'RE ALL ZIPPERED UP
SO I DON'T THINK DELETION OF VEG
RECEPTOR 3 WILL HAVE A HUGE
DIFFERENCE IN THE GROWING
LYMPHATIC CAPILLARIES.
>> OKAY.
THANK YOU VERY MUCH.
AND THERE IS A QUESTION FROM --
THERE'S A COMMENT -- THERE'S
SUGGESTIONS TO OLIVER FROM
SATHISH.
HI, OLIVER.
JUST A SUGGESTION HERE.
I BELIEVE THAT THE PROX1 FLOX
LINE YOU HAVE USED IN YOUR PAPER
WILL EXPRESS GFP UPON
CRE-MEDIATED DELETION.
HAVE YOU DONE GFP EXPRESSION
ANALYSIS AFTER TEK-CRE OR
PAX3-CRE DELETION?
>> WE ACTUALLY DON'T HAVE THE
PROX1 ALLELE THAT EXPRESSES GFP
AFTER CRE RECOMBINATION, SO NO,
WE HAVEN'T DONE THAT.
BUT IN TERMS OF THE REST OF THE
QUESTION, SO WE ARE -- WE HAVE
BEEN BUILDING MODELS TO BE ABLE
TO MORE SPECIFICALLY LABEL JUST
THE ENDOTHELIAL CELLS DERIVED
FROM PRO AXIAL MESODERM, WHICH
WILL DEFINITELY MAKE IT MUCH
EASIER TO ACTUALLY ANALYZE WITH
IMAGING.
THANK YOU.
>> THANKS, OLI.
AND THERE'S A QUESTION FROM OUR
ORGANIZER, FROM ANDREA.
DO YOU WANT TO ASK YOUR
QUESTION?
>> THANKS.
GREAT JOB, EVERYONE.
SO MY QUESTION IS FOR ZORINA.
IT'S KIND OF A SELF-INTERESTED
QUESTION BECAUSE IT'S GETTING
MORE PEOPLE INVOLVED IN HUB MED.
SO IF YOU COULD PERHAPS SPEAK TO
IF THEY'RE INTERESTED IN JOINING
HUBMAP OR TO PARTICIPATING IN
HUBMAP, HOW THEY MIGHT GET
INVOLVED.
THANK YOU.
>> I THINK WE HAVE A QUESTION
FROM XIAOLEI.
>> I BELIEVE YOU HAVE IDENTIFIED
THE NOTCH SIGNALING HAS BEEN
AFFECTED IN YOUR SYSTEM.
I WONDER WHETHER YOU HAVE
CHECKED VEGF C IN YOUR SMOOT
MUSCLE CELLS?
>> THAT'S A GREAT QUESTION.
WE'VE IDENTIFIED NOTCH 3 I THINK
TWO WEEKS AGO.
WE ARE STILL ANALYZING ALL THE
MICE, BUT FOR VEGF C, WE
PERSONALLY HAVE NOT LOOKED BUT
WE HAVE -- AT THE STAGES WHEN
THE MUSCLE IS DIFFERENTIATED.
HOWEVER, WE HAVE LOOKED MUCH
EARLIER.
I KNOW YOU'RE PASSIONATE ABOUT
THE EARLY EMBRYONIC DEVELOPMENT
AND WE ALWAYS LOOK MUCH EARLY.
AND SO -- AND WE SEE THAT VEGF C
EXPRESSION BOTH -- ACTUALLY AT
THE LEVEL OF RNA IS
SIGNIFICANTLY REDUCED IN THE
PITX2 HYPOMORPHIN INTESTINES.
SO WE'RE EXCITED ABOUT NOT JUST
THE POSSIBILITY THAT IT
REGULATES CONTRACTILITY, SO WE
SHOWED IT REGULATES ROSS BUT
ALSO WE THINK THAT MAYBE IT
COULD BE REGULATING VEGF C
EXPRESSION SO REDUCTION IN THAT
COULD POTENTIALLY BE INVOLVED IN
WHY THESE MUSCLES DON'T LINE UP
PROPERLY WITH THE ADJACENT
LACTEALS.
>> THAT WOULD BE AWESOME.
THANK YOU.
>> THANK YOU.
AND THEN WE HAVE A QUESTION FROM
RACHEL.
GO AHEAD, RACHEL.
>> HI.
I ALSO HAD A QUESTION FOR YOU.
YOUR TALK WAS REALLY LOVELY.
I'M WONDERING IF YOU WERE ABLE
TO CAPTURE SUFFICIENT NUMBERS OF
LYMPHATIC ENDOTHELIAL CELLS IN
YOUR SINGLE CELL RNA SEQ TO THEN
LOOK AT OTHER RECEPTORS AND
LIGANDS THAT MAY BE ACTING IN
CONCERT AND ALSO HOW FAR
POSTNATALLY YOU TOOK THIS.
I KNOW THAT PARTICULARLY IN THE
INTESTINE, A LOT OF THE
DEVELOPMENT, PARTICULARLY THE
STEM CELL COMPARTMENT
DEVELOPMENT, HAVE BEEN
POSTNATALLY, SO I'M CURIOUS
ABOUT SIGNALING NETWORKS THAT
MAY BE ACTIVE DURING THOSE
TIMES.
>> RACHEL, THANKS SO MUCH.
SO THE DATA I PRESENTED WAS ON
THE MUSE KAU LAYERS BUT WE ARE
VERY, VERY INTERESTED.
ONE IS TO UNDERSTAND ET ROW JE
NATE IN THE INTESTINE, AS YOU
KNOW.
I DON'T KNOW WHETHER THIS WOULD
BE ENOUGH, BUT WE ACTUALLY
GOT -- THE ENDOTHELIAL CELLS
WERE ACTUALLY CLUSTERING QUITE
NICELY AND WE WERE ABLE TO
IDENTIFY ARTERIAL ENT THEED YELL
CELLS, ARTERIAL PROGENITOR
CELLS, VEINS, LYMPHATIC
ENDOTHELIAL CELLS, BUT OUR
CURRENT ANALYSIS STOP AT
POSTNATAL DAY 1.5, BUT WE JUST
ADDED POST DAY TALL DAY 9 AND
POSTNATAL DAY 21.
ONE OF THE REASONS WHY I'M
EXCITED ABOUT YOUR TALK IS
BECAUSE WE ALSO WANT TO ADD THE
SPATIAL TRANSCRIPTOMICS FOR SOME
OF THE EARLIER TIME POINTS,
BECAUSE WE THINK THAT THE
SPATIAL QUEUES ARE JUST GOING TO
BE ABSOLUTELY CRITICAL.
THE GENES COULD BE THE SAME BUT
THE SPATIALLY DIFFERENTIAL
MECHANISMS ARE GOING TO BE
LIKELY TO BE VERY DIFFERENT.
>> IT WOULD BE FUN TO TALK ABOUT
THAT SOME DAY.
>> ABSOLUTELY.
AND CAPILLARIES.
WE HAVE A LOT OF BLOT
CAPILLARIES WE'VE IDENTIFIED IN
THE INTESTINE.
WE HAVE A QUESTION FROM PATRICIA
GREENWEL.
>> I'M SORRY I JUST HAVE BEEN
UNMUTED SO ITIOUS SHOULD ACY
ANSWER THE QUESTION THAT WAS
MADE FOR ME EARLIER.
SO AS I WROTE, EVERYBODY IS
WELCOME TO PARTICIPATE IN
HUBMAP.
THERE IS A LOT OF DATA THAT IS
OPEN TO EVERYONE.
WE HAVE NOW SPECIFIC EFFORTS
THAT I'VE INDICATED TO LYMPHATIC
VAT CLA TOUR AND DR. SINGHALL IS
ONLINE.
YOU CAN ALSO BECOME AN ASSOCIATE
MEMBER AND ONE THING THAT I'M
JUST -- I WAS JUST TEXTING TO
SOME OF THE PEOPLE INVOLVED IN
HUBMAP IS THIS MEETING CAN HELP
US FURTHER DEFINE SPECIFIC
MARKERS FOR THE VASCULAR
ENDOTHELIAL CELLS WHICH HAVE
BEEN MY PASSION EVER SINCE GOD
KNOWS WHEN, I'M NOT GOING TO
DISCLOSE MY AGE.
BUT THAT'S VERY IMPORTANT
INFORMATION THAT WE SHOULD BE
ABLE TO CAPTURE ALSO IN HUBMAP
FOR THE BENEFIT OF EVERYONE.
SO HAPPY TO TALK ABOUT THAT
ANOTHER TIME, WHEN WE HAVE MORE
TIME.
THANKS.
>> SEE ZORINA, THANK YOU SO M.
PATRICIA.
>> THANK YOU.
THIS IS A QUESTION FOR RACHEL.
IT BEEN DEMONSTRATED THAT --
POSITIVE PELO CYTES ARE KEY
FACTORS IN -- THESE ARE ALSO IN
CLOSE PROXIMITY TO LYMPHATICS SO
MY QUESTION IS, DO YOU THINK
LYMPHATICS ALSO PLAY A ROLE IN
MAINTAINING TELOCYTES?
>> THAT'S A REALLY INTERESTING
QUESTION.
I THINK THE OTHER TWO PAPERS
THAT CAME OUT NEARLY AT THE SAME
TIME ADDRESSED THIS MORE
SPECIFICALLY.
IT SEEMS RSPONDIN BEING
EXPRESSED IS ACTING ON MULTIPLE
CELL TYPES IN THE NICHE.
I THINK THE OTHER NICHE FACTORS,
CANONICAL NICHE FACTORS BEING
PRODUCED BY LYMPHATICS IN THE
NICHE LIKELY HAVE PLURIPOTENT
ROLES AND I ANTICIPATE THEY'RE
ACTING NOT ONLY ON THE STEM
CELLS BUT ALSO IN OUR DATA IT
SUGGESTS THERE'S A ROLE FOR --
CELLS, OTHER -- EVEN IMMUNE
CELLS.
I'M EXCITED TO SORT THAT OUT
NEXT.
>> THANK YOU.
>> JOHN, DO WE HAVE TIME FOR ONE
LAST QUESTION TO MIKE DAVIS?
I WILL ASK.
SO DR. DAVIS, INTERESTING WORK.
YOU MENTIONED THE CONTRIBUTION
OF THE TWO TYPES OF PUMPS THAT
PROPEL LYMPH FLOW.
DO YOU KNOW IF THE DATA ARE THE
SAME ACROSS ALL LYMPHATIC
NETWORK IN THE BODY?
>> WELL, IT DEPENDS ON WHAT BODY
YOU'RE TALKING ABOUT.
IF IT'S THE MOUSE BODY, NO,
THEY'RE NOT THE SAME.
AS I MENTIONED, ALL THE
LYMPHATICS IN THE MOUSE VISCERAL
AND THORACIC CAVITIES DON'T
ACTIVELY PUMP AND THAT SEEMS TO
BE UNIQUE AMONG OTHER SPECIES.
SO IT'S NOT REPRESENTATIVE OF
HUMAN VESSELS CERTAINLY.
THERE ARE REGIONAL DIFFERENCES
IN THE STRENGTH OF PUMPING.
AND SO THE VESSELS IN THE LOWER
EXTREMITIES TEND TO BE STRONGER
PUMPS, IF STUDIES UNDER
IDENTICAL CONDITIONS.
WE HAD A 2018 PAPER THAT
DEMONSTRATED THAT AT LEAST.
>> OKAY, THIS IS GREAT.
SO I THINK WE'RE OUT OF TIME.
OOO, THERE IS ONE LAST QUESTION.
GO AHEAD.
FROM NADA.
>> FORGET IT.
I HAVE A PROBLEM HERE.
>> NADA, DID WE LOSE YOU?
HAD
>> LATER.
I'LL ASK MY QUESTION LATER.
I HAVE TO FIGURE OUT MY
COMPUTER.
>> OKAY, NADA, THAT'S NO
PROBLEM.
YOU CAN ALWAYS USE THE Q & A
LITTLE CHAT ROOM.
OKAY.
SO ONCE AGAIN, I WANT TO THANK
ALL THE SPEAKERS, ALL THE
PARTICIPANTS FOR JUST REALLY
LIVELY DISCUSSIONS.
I INVITE YOU TO TAKE A SHORT
BREAK.
I BELIEVE THAT WE WOULD LIKE TO
HAVE EVERYBODY COME BACK FOR
11:15 FOR OUR SESSION NUMBER
THREE.
>> SO WE ARE READY NOW TO START
SESSION NUMBER THREE, LYMPHATICS
IN INFLAMMATION AND DISEASES OF
THE GASTROINTESTINAL SYSTEM.
THE MODERATOR IS DR. YASUKO
IWAKIRI.
YALE SCHOOL OF MEDICINE.
>> THANK YOU, PATRICIA.
I'D LIKE TO THANK YOU FOR THE
OPPORTUNITY TO MODERATE THIS
EXCITING SESSION.
IT'S MY PLEASURE TO INTRODUCE
OUR FIRST SPEAKER, DR. RAFAEL
CZEPIELEWSKI.
WASHINGTON UNIVERSITY SCHOOL OF
MEDICINE IN ST. LOUIS.
HIS TALK ENTITLED ROLES OF
INTESTINAL LYMPHATIC DRAINAGE
AND REMODELING IN HEALTH AND
INFLAMMATORY BOWEL DISEASE.
RAFAEL, TAKE IT AWAY.
>> THANK YOU, EVERYONE.
I'M THRILLED TO BE INVITED TO
THIS OUTSTANDING SEMINAR.
AND TO BE TALKING TO YOU ABOUT
OUR RECENT DISCOVERIES ON THE
TOPIC OF LYMPHATIC DRAIN DRAE
REMODELING IN HEALTH AND
INFLAMMATORY BOWEL DISEASE.
THROUGHOUT MY TALK I'LL BE
SHOWING YOU THESE QR CODES,
LINKS FOR RELEVANT PUBLICATIONS
THAT WE HAVE AND SOMEONE ELSE
DID ON THE SUBJECT, AND YOU CAN
FOLLOW UP AND FIND IT PRETTY
FAST THROUGHOUT MY TALK.
SO AS OTHER PANELISTS MENTIONED,
THE INTESTINAL LYMPHATICS IS
SURVEYED BY -- ESPECIALLY IN THE
SMALL INTESTINE, THERE'S THIS
SPECIAL LYMPHATIC CAPILLARIES
CALLED LACTEALS PRESENT IN EACH
VILLI OF THE IF YOU GUT.
THIS SPECIAL CAPILLARIES,
LACTEALS, ARE IMPORTANT FOR
INTERSTITIAL FLUID DRAINAGE AND
TO FACILITATE IMMUNE CELL
MIGRATION BUT SPECIFIC TO THE
SMALL INTESTINE, THEY ARE KEY TO
MODULATE AND ALLOW DIETARY LIPID
UPTAKE AND TRANSPORT TO STEM
CIRCULATION AND KEY FOR THE
DEVELOPMENT OF ORAL TOLERANCE.
THESE FUNCTIONS ARE CONTROLLED
BY WHAT HAVE BEEN MENTIONED FROM
PREVIOUS PANELISTS WITH ITS
BUTTONS AND ZIPPER JUNCTIONS
BEING THE MODULATORS OF THE
ENTRANCE OF THE LYMPHATIC
SYSTEM.
SO WHERE IT STARTS IN EACH
VILLI, YOU HAVE AS YOU CAN SEE
HERE IN THIS VIDEO THE
SUBMUCOSAL LYMPHATICS IS FUELED
BY THE LYMPH DRAINING IN THIS
REGION OF THE GUT, AND THAT
DRAINS INTO LARGE LYMPHATIC
VESSELS THAT POSSESS VALVES THAT
CAN ENTER INTO DRAINING LYMPH
NODES, IN THIS CASE, THE
MESENTERY, AND THEY CAN GO
FORWARD TOWARDS THE THORACIC
DUCT AND SYSTEMIC CIRCLE
VAITION.
ESPECIALLY FOR INS TESSAL INTL
LYMPHATICS, THERE'S VAST ADIPOSE
TISSUE THAT LITERALLY CONNECTS
THE GUT TO THE REST OF THE BODY.
AND SEGMENTS OF THE INTESTINE.
TODAY I'M GOING TO BE TALKING
ABOUT WHAT HAPPENS DURING
INFLAMMATORY BOWEL DISEASE.
WHICH ARE CHRONIC INFLAMMATIONS
OF THE SMALL AND LARGE
INTESTINE.
I'M GOING TO BE FOCUSED ON
CROHN'S DISEASE BECAUSE CROHN'S
DISEASE ALSO POSSESSES ONE
PHENOTYPE WHICH IS AN ALTERATION
AND A PROLIFERATION OF THE
MESENTERIC FAT.
AND THE DISEASE IS MORE
PROMINENT TO THE -- HISTORICAL
FINDINGS -- LYMPHATIC
INVOLVEMENT DURING -- ONLY IN
THE REGIONS ASSOCIATED WITH THE
DISEASE, AND OTHERS AT THE SAME
TIME WOULD CHARACTERIZE THE
DISEASE SUGGESTING THERE MIGHT
BE AN OBSTRUCTION OF THE
MESENTERIC LYMPHATICS.
THINKING ABOUT THAT, WE DECIDED
TO STUDY MESENTERIC FACT FROM
CROHN'S DISEASE PATIENTS AND
CONTROL PATIENTS AND WHAT WE
FOUND IS OBSTRUCTION BY TERTIARY
LYMPHOID ORGANS, THEY'RE
POSITIONED AROUND OR WITHIN
THESE LARGE COLLECTING VESSELS
COLLECTING THE INTESTINE TO THE
DRAINING LYMPH NODES.
SO THAT LED US TO HYPOTHESIZE
THAT MAYBE THE FORMATION OF THIS
LINK OF ORGANS ASSOCIATED WITH
THIS ALTERATION THE MESENTERY
ONLY TO THE REGIONS OF CHRONIC
INFLAMMATION COULD BE ASSOCIATED
WITH OVERALL PATHOLOGY OF
CROHN'S DISEASE.
AND ALSO THAT WHEN YOU HAVE A
DISRUPTION IN THE COLLECTING
VESSEL -- THAT COULD LEAD INTO
OTHER PROBLEMS SUCH AS
ALTERATION OF TRAFFIC AND
MOLECULAR TRANSIT.
SO WE DECIDED TO USE A MOUSE
MODEL OF TERMINAL ILIUM
INFLAMMATION.
THIS IS A SURPLUS OF PRODUCTION
OF THE TNF CYTOKINE.
THIS MODEL WAS GENERATED BY
GEORGE KOLLIAS WHERE YOU HAVE
INFLAMMATION OF THE TERMINAL
ILIUM, ENCIRCLES THE TNF AND
DESCRIPTION OF ORGAN --
FORMATION IN THE GUT TOO, AND WE
OBTAINED THIS MODEL -- WHAT WE
DID IS WE TOOK THIS TNF IBD
MOUSE MODEL AND CROSSED WITH A
LYMPHATIC REPORTER.
UNDER THE CONTROL OF PROX1 GENE.
WHAT YOU SEE HERE IS THE IMAGING
OF THIS MESENTERY WHICH IS
CONNECT BEING THE TERMINAL ILIUM
TO THE MESENTERY, WE CAN SEE
THIS FINE RED LINE GOING THROUGH
THE MESENTERY.
AND THIS IS A -- MADE CONTROL.
HOWEVER YOU HAVE THIS ROUND
STRUCTURE THAT FORMS AND ALSO
HAS THE PRESENCE OF LYMPHATICS
AROUND IT.
UPON CLOSER LOOK, ORGANS ARE
HIGHLY ASSOCIATED ONLY TO
COLLECTING LYMPHATIC VESSELS AND
YOU CAN APPRECIATE -- ON ITS
FORMATION, WE SAW THE LYMPHATIC
VALVE REGION WAS UNDERGOING
LYMPH ANGIOGENESIS, CREATING
SPROUTS OUT OF THE COLLECTING
VESSEL WHICH IS QUITE UNIQUE FOR
THIS MODEL.
COULD ALTER FUNCTIONS OF THE
LYMPHATIC DRAINAGE.
THEN WE CROSSED THE TNF IBD
MODEL WITH PHOTO CONVERTIBLE
MOUSE.
THIS MOUSE HAS ALL ITS CELLS --
IN GREEN AND YOU CAN USE FLEE
CYTOMETRY TO CHECK FOR THE
LABELED CELLS.
SO WE TESTED DURING THE
MIGRATION OF CELLS GOING OUT OF
THE INTESTINE TO THE MESENTERY
LYMPH NODE AND ELSEWHERE COULD
BE COLLECTED BY THIS -- WE
LABELED THE TERMINAL -- WE DON'T
KNOW WHERE THE DISEASE IS --
WITH LIGHT AND THEN WE CHECK THE
DRAINAGE OF THESE MIGRATED CELLS
24 HOURS LATER.
WHAT WE CAN FIND IS THAT INSTEAD
OF GOING INTO THE SYSTEMIC
CIRCULATION, MOST OF THE CELLS
ARE NOW ARRESTED IN THE
MESENTERY.
AND THOSE ARE IMMUNE CELLS.
AND THIS IS TRUE FOR T-CELLS, B
CELLS, ESPECIALLY FOR DENDRITIC
CELLS.
YOU CAN FIND THESE RED LABELED
CELLS INSIDE THE COLLECTING
VESSELS COMING FROM THE REGION
OF THE INTESTINE.
NEXT WE COLLABORATED WITH
DR. MICHAEL DAVIS FROM
UNIVERSITY OF MISSOURI TO DO
COLLECTING VESSEL CANNULATION
AND CHECK THE LYMPHATIC DRAINAGE
FUNCTION FOR LYMPHATICS AND
OTHER MOLECULAR CARGO.
WHAT YOU CAN SEE IN THIS VIDEO,
THE CANNULATION WAS DONE CLOSE
TO THE GUT WALL AND THEN THE
DRAINAGE IS GOING TO THE LEFT
SIDE OF THE SCREEN WHERE THE
MESENTERIC LYMPH NODE IS.
THIS GOES SMOOTHLY WITHIN FIVE
MINUTE, THE TRASER WHICH IS OF
HIGH MOLECULAR WEIGHT CAN ARRIVE
TO THE MESENTERY -- HOWEVER THE
SAME IS NOT TRUE FOR A MOUSE
WITH CHRONIC INFLAMMATION AND IN
THE PRESENCE OF -- WHERE IT
STARTED GOING LEFT, SIDE, NOW
THE DRAINAGE IS ALSO GOING TO
THE RIGHT SIDE OF YOUR SCREEN,
GOING ALL THE WAY BACK TO A
DIFFERENT PART OF THE INTESTINE.
WE CAN ALSO APPRECIATE THERE IS
LEAKAGE AND BLOCKAGE.
SO WITH PHYSIOLOGICAL INPUT
PRESSURE, WE CANNOT SEE THE
DRAINAGE ALL THE WAY TO THE
MESENTERY.
WHAT YOU SEE IS PILLAGE AT
THE -- LEADING TO LYMPHATICS AND
ALSO YOU SEE THE DRAINAGE GOING
BACKWARDS ALL THE WAY TO THE
GUT, WHICH DEMONSTRATE THAT ALL
THE SEGMENT, IT HAS COMPLETELY
DEFECTIVE BOWELS.
SO NEXT WE DEVELOP ANOTHER
APPROACH USING AN INTRAVITAL
STEREO SCOPE SYSTEM TO USE AND
CHECK FOR INTESTINAL LYMPHATIC
DRAINAGE TOWARDS THE MESENTERY.
SO AFTER THE INJECTION, MICRO
INJECTION, WE CAN FOLLOW UP TO
AN HOUR THE DRAINAGE ALL THE WAY
TO THE MESENTERY.
IN THE LITTER MATE MOUSE, WE CAN
VISUALIZE THIS HAPPENS QUITE
FAST, WITHIN FIVE MINUTE WE
ALREADY SEE THE TRASER GOING ALL
THE WAY TO THE DRAIN LYMPH NODE.
HOWEVER, THE SAME IS NOT TRUE IN
THE IBD SETTING WHERE THEY'RE
PRESENT IN THE MESENTERY.
WHAT YOU SEE HERE IS THOSE ROUND
STRUCTURES NOW LIGHT UP IN
GREEN, WHICH PROVES THAT THE
ASSOCIATION OF THE LYMPHATICS
AND THE INTERSTITIAL ORGAN WITH
YOU BUT
YOU CAN ALSO SEE A LEAKAGE.
EVEN IF WE RECORD THIS VIDEO FOR
UP TO AN HOUR, WE DON'T SEE THE
DRAINAGE OF THE CARGO GOING ALL
THE WAY TO THE MESENTERIC LYMPH
NODE.
DEMONSTRATING THAT WE HAVE ORGAN
BLOCKAGE OF THE LYMPHATIC
SYSTEM.
SO WE CAN MEASURE THE LENGTH OF
TIME THAT IT TAKES TO GO FROM
INJECTION SITE TO THE MESENTERIC
LYMPH NODE AND WE CAN APPRECIATE
THAT THROUGH THE AGE OF THE
MOUSE, THIS PHENOTYPE OF
BLOCKAGE INCREASES, AND IF WE
TREAT THE MICE WITH ANTI-TNF,
STANDARD THERAPY FOR CROHN'S
DISEASE A AT AN AGE WHERE TLOs
ARE JUST FORMING WE CAN BLOCK -T
LED US TO ALSO THINK THAT MAYBE
TNF ITSELF MIGHT BE IMPACTING
ENDOTHELIAL CELLS.
SO WE DECIDED TO TEST TO PERFORM
AN IN VITRO STUDY USING HUMAN
ENDOTHELIAL CELLS TO CHECK
WHETHER TNF CYTOKINE CAN ALTER
HUMAN LYMPHATIC ENDOTHELIAL
CELLS.
AS MENTIONED BY PREVIOUS
PANELISTS, WE CULTURE THE CELLS
UNDER DIFFERENT CONDITIONS TO
TEST WHETHER THE LYMPHATIC
OSCILLATORY FLOW AND STATIC
CONDITIONS COULD BE ALTERED BY
THE PRESENCE OR ABSENCE OF T.V.
SO AS TNF.
A KEY FACTOR UPREGULATED UNDER
OSCILLATORY FLOW CONDITIONS, AND
THEY CONTROL THE ECK PRETION F
OTHER PROTEINS ESSENTIAL FOR
BOWEL MAINTENANCE.
SO WE USED THESE DIFFERENT
STATIC -- TO MIMIC THE
ENVIRONMENT OF A VALVE -- THE
ONES WE SEE IN MICE THAT ARE
BEING DEFECTIVE.
SO IN COLLABORATION WITH
DR. YING YANG AND DR. JOSH
SCALLAN, WE PERFORMED THE SAME
STUDIES WITH AND WITHOUT TNF.
WHAT WE FIND IS THE
OVEREXPRESSION OR UPREGULATION
OF GATA2 AND EXPRESSION OF PROX1
IS DOWNREGULATED IN FLOW
CONDITIONS WITH THE PRESENCE OF
TNF, AND ALTHOUGH FOX C IT2 DOET
GET ALTERED, ITS EXPRESSION IS
ALSO ALTERED BY BEING STATIC
WITH TNF, SHOWING THAT TNF
ITSELF CAN MODULATE THESE KEY
TRANSCRIPTION FACTORS THAT
CONTROL -- WE CAN SEE USING QPR
THAT KEY GENES ARE EXTREMELY
DOWNREGULATED IN THE PRESENCE OF
TNF.
DEMONSTRATING THE DEFECTS WE'RE
SEEING MIGHT BE DERIVED FROM A
DIRECT IMPACT OF TNF CYTOKINE
INTO LYMPHATIC ENDOTHELIAL
CELLS.
SO WITH THAT, I HOPE THAT YOU
COULD APPRECIATE THAT THE
LYMPHATIC SYSTEM IN THE
INTESTINE IS QUITE IMPORTANT FOR
LEUKOCYTE TRAFFICKING AND THE
DRAINAGE OF MOLECULAR SOLUTE,
AND DURING INFLAMMATION AND
ALTERATION NOT ONLY AT THE GUT
WALL BUT ALSO IN ITS DRAINING
MESENTERY, CAN INDUCE THE
FORMATION OF -- ANGIOGENESIS AT
THE COLLECTING VALVES THAT LEADS
TO -- AND -- DEFECTIVE CAN LEAD
TO RETROGRADE FLOW, LIPID REACH
WITH CARGO AND POTENTIALLY HAS
BACTERIA WHICH CAN ENHANCE
INFLAMMATION AT THE SITE AND THE
TLOs CAN BLOCK AND OBSTRUCT
THE NORMAL LYMPHATIC FLOW OF
CELLS IN CARGO.
AND WE COULD EXPLAIN THAT BY
VERIFYING TNF ITSELF CAN
DOWNREGULATE PROX1 AND OTHER KEY
TRANSCRIPTION FACTORS THAT CAN
MODULATE LYMPHATIC VALVE
MAINTENANCE.
IN TERMS OF OPPORTUNITIES OF OUR
WORK, I BELIEVE THAT THE
MECHANISM GOVERNING THIS LYMPH
ANGIOGENESIS AT THE VALVE SITE
AND COLLECTING VESSEL -- AM I
OUT?
>> YOU'RE OKAY.
>> AND COLLECTING VESSELS IS
VERY IMPORTANT TO STUDY FURTHER,
AND UNDERSTANDING THE MECHANISM
OF HOW TNF IS MODULATED IS DOWN
REGS LAEVE -- THAT WILL BE QUITE
ESSENTIAL TO STUDY THE EFFECTS
OF INFLAMMATION INTO THE
LYMPHATIC BEDS.
ALSO IT'S IMPORTANT TO HIGHLIGHT
THAT THERE MIGHT BE UNEXPLORED
ROLES OF LYMPHATICS IN
INTESTINAL PATHOLOGIES, AND THE
SMALL ADD LARNLGD INTESTINE,
IMPORTANT TO HIGHLIGHT THE
DIFFERENCE BETWEEN CROHN'S
DISEASE AND ULCERATIVE COLITIS,
AND IN TERMS OF GENERAL GAPS, IT
WILL BE INTERESTING TO EXPLORE
ARE THERE RELATIONSHIPS BETWEEN
INTESTINAL LYMPHATICS AND OTHER
LYMPHATIC -- AND OTHER CELLS
AROUND IT, THAT SURROUND IT.
LACTEALS AND OTHER LYMPHATICS,
AND I THINK IMPORTANT TO
HIGHLIGHT, HOW THEY CAN SENSE
AND RESPOND TO THESE TISSUE
ALTERATIONS, MICROBIOME, IMMUNE
CELL QUEUES AND HOW CHRONIC
INFLAMMATION WOULD IMPACT THAT
AND HOW AGING CAN BE IMPACTING
ALSO THESE RELATIONSHIPS.
WITH THAT, I'D LIKE TO THANK MY
WHOLE LABORATORY, MY MENTOR, GED
LIN RANDOLPH AND SEVERAL KEY
COLLABORATORS AT WASH U AND
DR. MICHAEL DAVIS, YING YANG AND
JOSH SCALLAN FOR HELPING WITH
THIS WORK.
OUR FUNDING SOURCES.
I WOULD LOVE TO ANSWER QUESTIONS
RELATED TO OUR WORK.
THANK YOU SO MUCH.
>> THANK YOU, RAFAEL, FOR THIS
FANTASTIC TALK.
I WANT TO REMIND THE VIDEO
VIEWERS THAT YOU CAN ALSO SEND
QUESTIONS VIA THE LIVE FEEDBACK
BUTTON.
IT'S MY GREAT PLEASURE TO
INTRODUCE OUR SECOND SPEAKER,
DR. DARIA ESTERHAZY FROM THE
UNIVERSITY OF CHICAGO.
HER TALK IS ENTITLED IMPACT OF
INTESTINAL HELMINTHS ON DUODENAL
LYMPHATIC FUNCTION.
>> THANK YOU A LOT.
MY SYSTEM IS NOT ALLOWING ME TO
DO THIS.
DO YOU HAVE A WAY TO SHARE IT?
>> THERE SHOULD BE A SHARE
BUTTON AT THE BOTTOM OF YOUR
SCREEN.
DO YOU HAVE YOUR SLIDES OPEN
ALREADY?
>> I DO.
IT SAYS MY SOFT MICROSOFT PS
UNKNOWN.
I'M REALLY SORRY.
>> DARIA, ARE YOU ABLE TO SHARE
YOUR SLIDES WITH US?
>> MY COMPUTER WAS SWITCHED FROM
MY INSTITUTION.
>> MAYBE WE CAN SWITCH TO THE
NEXT SPEAKER AND I CAN HELP YOU.
>> NO PROBLEM.
SO THE NEXT SPEAKER IS ACTUALLY
MYSELF.
TALKING ABOUT LYMPHATIC SYSTEM
IN CHRONIC LIVER DISEASE.
LET ME SHARE MY SCREEN.
CAN YOU SEE MY SCREEN?
>> THE LIVER IS THE KEY ORGAN OF
LYMPH PRODUCTION, WITH 25 TO 50%
OF LYMPH PASSING THROUGH THE
THORACIC DUCT ORIGINATING IN THE
LIVER.
IN CIRRHOTIC PATIENTS, THE LYMPH
PRODUCTION FROM THE LIVER
INCREASES TO SIX FOLD LEADING TO
THE SERIOUS COMPLICATION CALLED
ASCITES.
COMPARED TO OTHER ORGAN, THE
LYMPHATIC SYSTEM IN THE LIVER IS
UNDERSTUDIED.
THAT IS BECAUSE OF THE LIMITED
NUMBER OF LYMPHATIC ENDOTHELIAL
CELL MARKERS.
THE TYPICAL LYMPHATIC
ENDOTHELIAL CELL MARKERS SUCH AS
PROX1, LYVE-1, FIT4 -- WHICH IS
A MAJOR CELL TYPE, MAJOR
ENDOTHELIAL CELL TYPE IN THE
LIVER.
ANOTHER REASON WOULD BE THE
COMPLEX STRUCTURE OF THE
VASCULATURE IN THE LIVER.
HERE THERE ARE OTHER VESSELS IE
AREA AS WELL.
SO IN THIS DIAGRAM, I WOULD LIKE
TO SHOW YOU THE -- OF THE LIVER.
MOST OF THE LYMPHATIC VESSELS
ARE INDICATED IN THE PORTAL
TRACT LIVER WHICH CONSIST OF
PORTAL VEIN -- ARTERY, BILE
DUCT.
TRANSPORT TO THE DUODENUM FOR
ABSORPTION.
THE BLOOD COMING FROM THE PORTAL
VEIN AND HEPATIC ARTERIES ARE
MIXED WITHIN THE CIRCULATION.
THE HYDROSTATIC PRESSURE IN THE
CIRCULATION ALLOWS THE PLASMA
COMPONENT OF THE BLOOD FILTER
INTO THE SPACE OF -- WHICH IS
LOCATED BETWEEN THE HEPATOCYTE
AND THE SIGN SOY DAL AND
ENDOTHELIAL CELLS.
LIVER CIRRHOSIS SITUATION,
THERE'S INCREASING HYDROSTATIC
PRESSURE DUE TO THE MASSIVE
ACCUMULATION OF SCAR TISSUE
FORMATION OR FIBROSIS THAT
INCREASES THE PRODUCTION DCH.
NOT ONLY THE -- BUT ALSO
INFILTRATED IN CELLS --
PARTICULARLY IN THE PORTAL TRACT
AREA.
THEREFORE, THE LYMPHATIC SYSTEM
IN THE LIVER IS ALSO NOT ONLY
IMPORTANT FOR THE HOMEOSTASIS
BUT ALSO IMPORTANT FOR THE
DECREASING THE LOCAL
INFLAMMATION WITHIN THE LIVER.
APPROXIMATELY 80% OF THE LYMPH
PRODUCED WITHIN THE LIVER ARE
ACCOMMODATED BY THE PORTAL --
LYMPHATIC VESSEL IN -- AREA.
THIS DIAGRAM SHOWS THE THREE
DIMENSIONAL IMAGE OF LYMPHATIC
VESSELS WRAPPING AROUND THE
PORTAL VEIN AND HEPATIC
ARTERIES, AND BILE DUCT.
BECAUSE OF THE CLOSE VICINITY TO
THE BILE DUCT, LYMPHATIC VESSEL
ALSO SERVES AS THE SITE --
PARTICULARLY THE BILE DUCT.
THE THEY'RE TRANSFERRED OUTSIDF
THE LIVER AND THEN FILTER
THROUGH THE REGIONAL -- HEPATIC
REGIONAL LYMPH NODE THEN MOVE TO
THE SYSTEM -- AND CONNECT TO THE
THORACIC DUCTS AND RETURN TO THE
SYSTEMIC CIRCULATION EVENTUALLY.
THERE ARE MULTIPLE STUDIES THAT
HAVE SHOWN THAT INCREASED
LYMPHATIC VESSEL NUMBERS OR LIM-
ASCITES, HYPERTENSION, SERIOUS
COMPLICATION OF CIRRHOSIS,
CANCERS, LIVER FIBROSIS WHICH
I'M GOING TO TALK LATER ON, AND
ISCHEMIA FACTORS OF INJURY,
HEPATIC ENCEPHALOPATHY, WHICH IS
THE SERIOUS COMPLICATION OF
LIVER DISEASES.
NOT ONLY TO INCREASE
ANGIOGENESIS BUT ALSO SEVERAL
STUDIES HAVE REPORTED THE
DECREASED -- IDENTITY AND
REDUCED LYMPH TRANSPORT IN --
OBSERVED IN THE PATHWAY TO
DISEASE, SHOWN VERY NICELY FROM
THE UNIVERSITY OF COLORADO.
THERE ARE A LOT TO BE ANSWERED
IN THE LIVER FIELD, FOR EXAMPLE,
WHAT ARE THE MOLECULAR
MECHANISMS OF INCREASED LYMPH
ANGIOGENESIS, WHAT ARE THE
FUNCTIONAL ROLES OF HEPATIC
LYMPHATIC VESSELS IN DIFFERENT
ETIOLOGIES OF LIVER DISEASES,
WHETHER INCREASED LYMPH
ANGIOGENESIS IS BENEFICIAL OR
HAS INCREASED CONSEQUENCES.
I'D LIKE TO SHOW YOU A PROJECT
WHICH SHOWS THE EFFECT OF --
HEPATIC VESSELS IN LIVER
FIBROSIS.
THIS PROJECT WAS MOSTLY DONE BY
A VERY TALENTED AND HARD WORKING
POSTDOC, JAIN JEONG.
INFLAMMATION DRIVES LIVER
FIBROSIS, CHRONIC INFLAMMATION
FROM CHRONIC ALCOHOL
CONSUMPTION, HCV OR HBV, DRUGS
AND DIETS, CAUSING CIRRHOSIS,
CARCINOGENESIS AND PORTAL
HYPERTENSION.
BECAUSE THE LYMPHATIC SYSTEM IS
IMPORTANT FOR REDUCING THE LOCAL
INFLAMMATORY RESPONSES, WE
THOUGHT THAT PERHAPS LYMPHATIC
SYSTEM PLAYS A ROLE FOR REDUCING
LIVER FIBROSIS.
TO STUDY THIS, WE USED TWO TYPES
OF EXPERIMENTAL MODEL OF LIVER
FIBROSUS.
ONE IS BILE DUCT LIGATION MODELS
AND THE OTHER IS THE CARBON
TETRACHLORIDE INHALATION MODEL.
BECAUSE OF THE TIME LIMIT I'M
GOING TO SHOW YOU SOME OF THE
DATA RELATED TO THE BILE DUCT
LIGATION OR BDL.
SO IN THIS MODEL WE LIGATE THE
BILE DUCT WHICH CAUSES THE --
INDUCES MASSIVE INFLAMMATION AND
LEADING TO INJURY AND FIBROSIS.
FIRST WE DETERMINE THE EVOLUTION
OF -- BDL.
AS YOU CAN SEE HERE, THE SERUM
ALT WHICH IS USED AS THE
INDICATOR OF LIVER FIBROSIS WAS
INCREASED UP TO TWO WEEKS AND IT
STAYS FOUR WEEKS AFTER.
ACTUALLY BY TWO WEEKS BILE DUCT
LIGATION, THOSE ANIMALS BECOME
VERY, VERY SICK.
IN THE LIVER FIBROSIS, YOU CAN
SEE HERE THERE'S INCREASE IN THE
FIBROSIS BY FOUR WEEKS AFTER THE
BILE DUCT LIGATION IN A
TIME-DEPENDENT MODEL.
LYMPHATIC VESSELS, SO HERE LEFT
SIDE SHOWS THE LYMPHATIC VESSELS
AROUND THE -- AREA OF THE SHAM
ANIMALS AND THE RIGHT SIDE SHOWS
THE BILE DUCT LIGATED ANIMALS.
RED SHOWS THE HEPATIC VESSELS.
AS YOU CAN SEE HERE IN BILE DUCT
LIGATION MODEL, THERE'S
SIGNIFICANT INDUCTION OF
LYMPHATIC VESSELS AROUND THE
PORTAL TRACT AREA AND THEY ARE
MAXIMALLY DILATED AS WELL TOO.
ALTHOUGH YOU CAN SEE THERE'S SO
MANY LYMPHATIC VESSELS GROWING
AROUND THE PORTAL TRACT AREA,
BUT THEY ARE NOT REALLY
RECOGNIZED, WHAT YOU CAN NOTICE
IN THE LIVER FIELD, BECAUSE
UNLESS YOU STAY WITH THE LYMPH
ENDOTHELIUM MARKER YOU CANNOT
ORGANIZE BECAUSE THEY ARE A VERY
THIN LAYER OF THE VESSELS.
WE THEN LOOKED AT THE LYMPHATIC
VESSEL NUMBERS IN THE DIFFERENT
STAGE OF FIBROSIS.
ALONG WITH THE BILE DUCT
PROLIFERATION.
PERFORATION.
SO WE NO IT'S INCREASING BECAUSE
OF -- BILE DUCT SHOWN IN GREEN,
LYMPHATIC VESSEL SHOWN IN RED.
BILE DUCT INCREASES ITS
PROLIFERATION EVEN AT FOUR WEEKS
AFTER THE BILE DUCT LIGATION.
HOWEVER, THE LYMPH VESSEL ALSO
BEGIN MAXIMUM ABOUT TWO WEEKS
AFTER THE BILE DUCT LIGATION,
HOWEVER, SIGNIFICANTLY DROPPED
BY FOUR WEEKS AFTER THE BILE
DUCT LIGATION, SO WE THOUGHT
THAT PERHAPS THIS SIGNIFICANT
DROP IN THE LYMPH VESSEL NUMBERS
ALLOW THE FIBROSIS TO PROGRESS
FARTHER TO CIRRHOSIS.
ALTHOUGH UNTIL TWO WEEKS AFTER
THE BILE DUCT LIGATION, THE
VESSEL TRIES TO PROTECT THE
LIVER FROM FIBROSIS.
SO TO TEST THIS, WE LOOKED AT
THE EFFECT OF LYMPH
ANGIOGENESIS -- EXPANSION OF
LIVER FIBROSIS.
FOR THAT, WE GAVE THE VEGF
3 RECEPTOR AND LOOKED AT THE
EFFECT ON LIVER FIBROSIS.
AS WE EXPECTED -- IN FIBROSIS
THERE'S SIGNIFICANT INDUCTION --
AS A RESULT OF BLOCKING LYMPH
ANGIOGENESIS.
AND LIVER INJURY SIGNIFICANTLY
INCREASED BY THE BLOCKING OF THE
VEGF RECEPTOR 3, AND DRAINAGE
FUNCTION ALSO DECREASED
SIGNIFICANTLY BY GIVING THE VEGF
3 INHIBITOR.
WHAT ABOUT VEGF C -- WHAT ABOUT
THE EFFECT OF VEGF3 EXPRESSION
BY FIBROSIS?
WE LOOKED AT THE EFFECT ON LIVER
FIBROSIS.
AS YOU CAN SEE HERE, BY
EXPRESSION, YOU SIGNIFICANTLY
DECREASED LIVER FIBROSIS AND
ALSO DECREASED THE LIVER INJURY,
INDICATING THAT THE VEGF-C
OVEREXPRESSION AND LYMPH
ANGIOGENESIS DECREASES LIVER
FIBROSIS AND INJURY.
WE ALSO LOOK AT WHAT IS THE
FUNCTIONAL CHANGES OF THE
HEPATIC ENDOTHELIAL CELLS IN
DIFFERENT STAGE OF A BILIARY
FIBROSIS?
SO WE ISOLATED ENDOTHELIAL CELLS
FROM THIS CHANNEL AND THEN
PERFORMED RNA SEQUENCING
ANALYSIS.
TO HIGHLIGHT THE MOST IMPORTANT
FINDING FROM THIS PATHWAY
ANALYSIS, IS THAT IN FOUR WEEKS
AFTER THE BILE DUCT LIGATION,
THERE WAS A SIGNIFICANT
INDUCTION OF THE COAGULATION OR
THROMBOTIC PATHWAY AND WE
CONFIRMED THIS BY STAINING THE
FIBRINOGEN IN THE LIVER, AS YOU
CAN SEE THERE'S -- FIBRIN SIN.
THIS INCREASED THROMBOTIC
RESPONSE MAY ACTUALLY BLOCK THE
HEPATIC DRAINAGE BY -- FOR THE
LIGATED ANIMALS.
JUST TO CONCLUDE, LYMPH
ANGIOGENESIS -- I WOULD LIKE TO
CONCLUDE WITH THE KNOWLEDGE GAP,
NEEDS AND OPPORTUNITIES FOR THE
STUDY OF DISEASES AND ITS
COMPLICATIONS.
FIRST IDENTIFICATION OF
ENDOTHELIAL MARKERS NOT
EXPRESSING OTHER LIVER CELL
TYPES IS URGENTLY NEEDED.
MECHANISMS OF LYMPH ANGIOGENESIS
IN THE LIVER ARE NOT FULLY
UNDERSTOOD.
ROLE OF LYMPHATIC SYSTEM IN
DIFFERENT ETIOLOGIES OF LIVER
DISEASES REMAIN TO BE
UNDERSTOOD.
FINALLY, MODULATION OF HEPATIC
LYMPHATIC SYSTEM COULD BE THE
NOVEL THERAPEUTIC STRATEGIES FOR
THE TREAT M OF LIVER DISEASES
AND ITS COMPLICATION.
I WOULD LIKE TO THANK ALL THE
PEOPLE WHO CONTRIBUTED TO THE
STUDY, PARTICULARLY JAIN AND
YILIN, AND NIH AND NIDDK FOR
SUPPORTING THIS PROJECT.
THANK YOU VERY MUCH FOR YOUR
ATTENTION.
I WOULD LIKE TO MOVE TO THE NEXT
SPEAKER.
DR. NADA ABUMRAD.
FROM WASHINGTON UNIVERSITY
SCHOOL OF MEDICINE.
HER TALK IS ENTITLED MAINTENANCE
OF LYMPHATIC VESSELS PREVENTS
LYMPH LEAKAGE AND OBESITY.
NADA, PLEASE GO AHEAD.
SO WHAT I'M GOING TO BE TALKING
ABOUT TODAY IS HOPEFULLY
RELEVANT TO SEVERAL OF THE TALKS
THAT I'VE LISTENED TO.
IT'S BEEN A GREAT MEETING SO
FAR, AND I THANK YOU FOR
INCLUDING ME IN THIS MEETING.
AND I HOPE THAT WE'LL BE ABLE TO
GET AN INTERESTING DISCUSSION AT
THE END OF THE SESSION.
THAT'S WHAT I HAD IN MIND WITH
THESE SLIDES.
SO WE'VE COVERED THE OLYMPICS,
WE'VE HAD SEVERAL INTRODUCTIONS
SO I AM NOT GOING TO COVER THEM.
EXCEPT TO MENTION THAT WE HAVE
TO REMEMBER THAT THEIR MAIN
FUNCTION IS BASICALLY TO PASS
THE LIPIDS, THESE GO TO TISSUES
WHERE THEY ARE BROKEN DOWN TO
FATTY ACIDS THAT ARE TAKEN UP BY
THE TISSUES AND METABOLIZED AND
USED FOR MAINTAINING HEALTH OF
THE TISSUE AND ITS RENEWAL.
HOWEVER, THEY ALSO CAN BECOME
TOXIC IF THEY ARE IN GREAT
CONCENTRATION AND NOT PROPERLY
USED.
SO I WILL SKIP THIS, ACTUALLY
I'LL MENTION IT BRIEFLY.
IT'S THAT WHAT WE KNOW ABOUT HOW
ABSORPTION IS REGULATED BY THE
LYMPHATICS IS THAT IF YOU IMPAIR
THE LACTEALS BY DIFFERENT
DEVELOPMENTAL MANIPULATION GENES
THAT ARE -- MANIPULATING GENES
THAT ARE INVOLVED IN
DEVELOPMENT, YOU CAN BLOCK LIPID
UPTAKE, AND THIS IS
METABOLICALLY PROTECTED.
THE SAME IS IF YOU ZIPPER THE
LACTEAL, THE JUNCTION THAT JOSH
WAS TALKING ABOUT, THE BUTTONS,
IF YOU ZIPPER THEM, THIS IS
WORK -- HOWEVER IF YOU EMBED THE
TRANSPORT BY -- YOU IMPAIR THE
TRANSPORT BY CREATING
DYSFUNCTION IN THE FUNCTION OF
THESE -- ESPECIALLY IF YOU HAVE
THE JUNCTIONS BETWEEN THE CELLS
AND YOU HAVE LEAKAGE, THIS
CREATES A RISK OF OBESITY
BECAUSE IT CAN CONTRIBUTE TO
VISCERAL OBESITY AND RISK OF
TYPE 2 DIABETES.
SO THE PROTEIN I'M GOING TO TALK
ABOUT IS CD36, WHICH IS A FATTY
ACID TRANSPORTER, IT DOESN'T
ONLY TRANSFER FATTY ACID BUT IT
ALSO TRANSPORTS SOME
LIPOPROTEINS AND VARIETY OF
NON-LIPID LIGANDS.
THE IMPORTANT THING ABOUT THIS
PROTEIN IS THAT IT SIGNALS, IT
ASSOCIATES WITH MULTIPLE KINASES
BUT BECAUSE SITE ASSOCIATION --
RPS THROUGH ITS CYTOPLASMIC
DOMAIN, AND THE STRUCTURE L
STRUCTURE WAS IDENTIFIED AND WE
HAVE IDENTIFIED IT AS A FATTY
ACID TRANSPORTER, AND THEY FOUND
THE FATTY ACID IN THIS INTERNAL
TUNNEL WITHIN THE PROTEIN.
THE FAMILY OF PROTEIN, THE CD36
FAMILY OF PROTEIN WHICH ALL S
CHANNEL INSIDE THE PROTEIN FOR
TRANSPORTING THE LIPID TO THE
BILAYER.
ANOTHER THING THAT IS IMPORTANT
ABOUT THIS PROTEIN IS THAT IT
CONNECTS TO THE CYTOSKELETON, SO
IT CAN CREATE SIGNALING THAT AT
THE SAME TIME INFLUENCES
MORPHOLOGY OF THE CELL,
INFLUENCES MIGRATION OF CELLS,
AND SECRETION, ET CETERA.
SO IT DOESN'T ONLY TRANSFER
LIPIDS, IN PARTICULAR, FATTY
ACID, IT HAS A BUNCH OF
ACTIVITIES THAT MAKE IT
COMPLICATED, BUT INTERESTING.
SO THE THING THAT MOTIVATES ME
IN STICKING WITH THIS PROTEIN IS
THAT IT'S HIGHLY POLYMORPHIC.
THE DEFICIENCY OCCURS IN 3% OF
ASIANS OR AFRICAN AMERICANS, AND
HERE IS SHOWING YOU A PERSON WHO
IS DEFICIENT IN CD36 AND YOU
FIND THAT FATTY ACID UPTAKE,
WHICH IS HERE, IT'S ASSESSED BY
PET SCANNING, YOU FIND THAT IT
IS ABSENT IN THE HEART OF THIS
PERSON THAT IS VERY -- WE HAVE
SHOWN THIS IN MICE AND THEN IT
WAS -- THIS IS WORK OF A GROUP
IN JAPAN AND ANOTHER GROUP AT
THE MAYO CLINIC BASICALLY SHOWED
THAT IT DOES FUNCTION IN UPTAKE
AND ESPECIALLY IN THE HEART
MUSCLE AND ADIPOSE TISSUE BUT
ALSO IN IMMUNE CELLS, AND IT'S
RELEVANT TO -- I WILL SHOW YOU
THE LYMPHATICS.
SO WE MADE THE OBSERVATION A
WHILE BACK THAT WHEN WE HAD
CD36-DEFICIENT MICE IN
COLLABORATION WITH PATRICK TSO,
HE HAS DEVELOPED THE
METHODOLOGY, WHO CANNULATE THE
MISS ENTERIC LYMPHATIC DUCTS AND
THE DISTRICT IS TO CANNULATE IT
OPPOSITE TO THE FLOW OF THE
LYMPH SO YOU CAN COLLECT THE
LYMPH AS YOU ARE -- SO WHAT WE
LEARNED IS THE CD36 DEFICIENT
MICE DO NOT PASS THE LIPID WELL
INTO THE LYMPH, BECAUSE THE
LYMPH HE WAS COLLECTING WAS LOW
IN TRIGLYCERIDE AND LOW IN
CHOLESTEROL.
INTERESTINGLY, IF WE FOLLOW THE
ABSORPTION OF LIPID IN THE
BLOOD, YOU SEE THAT THE
CD36-DEFICIENT MOUSE HAS THE
TRIGLYCERIDES APPEARING VERY
QUICKLY IN THE BLOOD, SO I KNOW
WE MENTIONED -- ONE OF THE
SPEAKERS MENTIONED EARLIER IS
THAT THERE ARE TWO WAYS FOR THE
LIPIDS TO GO INTO THE
CIRCULATION FROM THE GUT.
THEY EITHER GO VIA LIMB FA AT
THIS TIME LYMPHATICSR
THEY CAN, UNDER CERTAIN
CIRCUMSTANCES, GO DIRECTLY TO
THE PORTAL VEIN, SO WE SEE IN
THE CD36 KO MOUSE, THE LIPIDS
ARE IMPAIRED AND INABILITY TO
ENTER THE LYMPH AND END UP
QUICKLY IN THE CIRCULATION.
NOW THESE LIPIDS ARE A LITTLE
DIFFERENT BECAUSE THERE IS A
DEFECT IN MAKING THEM, THEY ARE
SMALLER, AND WHAT WE SEE IS THAT
CLEARANCE IS ALSO A LITTLE BIT
DELAYED.
BUT THAT'S THE LYMPH -- THAT'S
THE LYMPH PHENOTYPE OF CD36
DEFICIENT MOUSE.
SO THAT OBSERVATION WE FORGOT
ABOUT IT FOR A WHILE UNTIL WE
GOT INTERESTED AGAIN IN THE
ENDOTHELIUM.
THERE WERE A LUNCH OF PAPERS ,
I'M TALKING HERE ABOUT THE
VASCULAR ENDOTHELIUM, THE BLOOD
VESSELS, IS IMPORTANT FOR
REGULATING UPTAKE OF LIPIDS BY
TISSUES IE. SHOWING YOU HERE
THERE ARE TWO WAYS THAT LIPID
CAN GET INTO THE BLOOD BY
SECRETION OF FATTY ACID -- BY
FAT CELLS OR BY THE LYMPHATICS
EMPTYING INTO THE CIRCULATION.
THEN IN THE CASE OF THE
LYMPHATICS, THEY HAVE TO BE
PROCESSED BY LIPOPROTEIN -- TO
UNDERLYING TISSUES AND POSSIBLY
OTHER RECEPTORS.
SR-B1, IN COLLABORATION WITH
NYU, ACTUALLY THIS IS HIS WORK,
THE SRB1, WE JUST ARE
COLLABORATORS.
SO HE SHOWED THAT SRB-1 ACTUALLY
TAKES -- MICRONS.
SORRY?
>> TWO MINUTES REMAINING.
>> SO I SHOULD GO FAST.
WE KNOW A LOT ABOUT ENDOTHELIAL
CELLS, THAT THEY HAVE VERY --
ARE VERY HETEROGENEOUS AND CD36
MARKS THE MICRO VASCULATURE THAT
IS INVOLVED IN THE LIPIDS.
WHEN WE DELETED THE ENDOTHELIAL
CELL CD36, WHAT WE FOUND IS THAT
THERE WAS A DECREASE IN UPTAKE
OF FATTY ACID BY THE TISSUE, AND
THERE WAS AN INCREASE IN GLUCOSE
UPTAKE AT A COMPENSATORY
RESPONSE TO THE LACK OF FATTY
ACID UPTAKE.
SO THEN WE WANTED TO SEE WHAT
HAPPENS TO THE LACTEALS, AND
WHAT WE FOUND, AND THAT'S THE
WORK OF VINCENZ EA. CIFARELLI
WHO HAS HER OWN LAB AT ST. LOUIS
UNIVERSITY, WHAT WE FOUND IS
THAT THEY'RE HIGHLY FRAGMENTED.
IT WAS VERY INTERESTING TO NOTE
THAT ITS HIGHEST EXPRESSION WAS
IN THE COLLECTING VESSELS, YOU
SEE HERE FLUORESCENCE IN THE
COLLECTING VESSELS, ALTHOUGH IT
WAS ALSO EXPRESSED IN THE
LACTEALS.
WHAT THE PHENOTYPE OF THESE MICE
WAS, THEY HAD SHORTER LACTEALS
AND THEY HAD MORE OPENINGS.
SO THEN WE CREATED, IN
COLLABORATION WITH GWEN
RANDOLPH, WE CREATED A MOUSE
WITH CD36 DELETION IN THE
LACTEALS, AND WE FOUND THAT
THESE MICE ACCUMULATE VISCERAL
FAT, WITHOUT HIGH FAT DIET, THIS
IS JUST FED CHOW, THEY BECOME
RESISTANT AND HAVE INFLAMMATION
AND MACROPHAGE INFILTRATION.
SO THEY ARE -- WHAT WE FIGURED
OUT WAS THAT THEY ARE LEAKY,
THERE IS A LEAKAGE OF LYMPH BY
THE COLLECTING DUCT AND AB
SORGS,
ABSORPTION, IT IS DELAYED, . WT
WAS INTERESTING IS WE FOUND
METABOLISM IN THESE CELLS IS
DIFFERENT.
THEY HAVE A DECREASE IN CELL
RESPIRATION AND THEY DON'T
RESPOND TO VEGF BY INCREASING
THEIR CELLULAR RESPIRATION.
THEY ALSO HAVE INCREASED
GLYCOLYSIS, AGAIN, THEY DON'T
RESPOND TO VEGF.
THERE ARE DIFFERENCES ON VEGF
AND MIGRATION OF THE CELLS.
THESE CELLS DO NOT TIGHTEN UP
JUNCTIONS IN RESPONSE TO VEGF
WHEN THEY DON'T HAVE CD36 WHEN
IT'S DEPLETED.
SO IN SUMMARY, CD36 IS EXPRESSED
IN THE LACTEAL, BUT IT
PARTICULARLY HIGH IN THE
COLLECTING DUCTS.
ITS DELETION -- I WILL SHOW YOU,
INHIBITS VEGF R2 AND AKT
PHOSPHORYLATION, IT ENTERS
LYMPHATIC CELL INTEGRITY AND
RESULTS IN LEAKAGE.
SO THIS IS BASICALLY SHOWING YOU
THAT -- SO I'M GOING TO CLOSE ON
JUST MAKING A QUICK COMPARISON
BETWEEN THE LYMPHATIC
ENDOTHELIAL CELL AND BLOOD
ENDOTHELIAL CELLS.
WE SEE SOME DIMINUTION OF VEGF
R2 PHOSPHORYLATION, BUT WE SEE
REALLY ALMOST AN ABOLISHING OF
THE SIGNALING PHOSPHOAKT AND
THAT'S THE SAME WE SEE IN BLOOD
ENDOTHELIAL CELLS, SO WE ARE
FOCUSING ON THIS.
WE THINK THE CONNECTION TO FOS
COAKT AND ALSO FOX 01 IS ONE OF
THE FACTORS REGULATED BY CD36 IS
GOING TO HELP US UNDERSTAND HOW
CD36 REGULATE MAINTENANCE OF THE
JUNCTIONS IN BOTH, BECAUSE IT
ALSO CAUSES A LEAKAGE IN THE
BLOOD VESSELS.
I WILL SKIP THAT.
I WANT TO TELL YOU WHY WE ARE
FOCUSING ON THIS.
WE HAVE SHOWN ARE REGULATES AMPK
ACTIVITY, ALSO REGULATES FOX
01 DELIVERY OF FATTY ACIDS, SO
WE ARE GOING TO FIGURE OUT HOW
IT HELPS MAINTAIN HOMEOSTASIS OF
THE LYMPHATIC CELLS.
SO WHAT ARE THE KNOWLEDGE GAPS
TO MY PERSPECTIVE?
IT'S THE FACT WE DON'T KNOW A
LOT ABOUT HETEROGENEITY OF
LYMPHATIC ENDOTHELIAL CELLS IN
TERMS OF GENE EXPRESSION,
SIGNATURE, FUNCTION.
IN TERMS OF HOW THIS CHANGES
FROM, LET'S SAY, THE LACTEALS TO
THE COLLECTING TO THE
MESENTERIC, ET CETERA.
WE DON'T KNOW VERY MUCH ABOUT
THE MECHANISM THAT MAINTAINS
VESSEL HOMEOSTASIS AND OPTIMIZES
FUNCTION, AND WE ALSO DON'T KNOW
MUCH ABOUT THE METABOLIC FACTOR
THAT ALTERS THE LEC PHENOTYPE.
AND THE QUESTION I WAS GOING TO
ASK BEFORE IS THAT IF SOME OF
THE SPEAKERS ARE TALKING ABOUT
THE STEM CELLS AND REGULATION OF
DEVELOPMENTAL ASPECTS, HAVE
LOOKED AT FATTY ACID OXIDATION
AND THE METABOLIC ASPECTS OF THE
RELATIONSHIP.
SO I THINK I HAVE MENTIONED THE
PEOPLE WHO DID THE WORK I HAVE
SHOWN YOU.
THIS IS MY LAB MANAGER WHO IS A
WONDERFUL PARTNER IN THIS WORK.
BUT I WANT TO MENTION VINCENZA
IS PURSUING THE LYMPHATIC
SYSTEM, SHE IS DEVELOPING HER
LAB SO HOPEFULLY WE HEAR A LOT
FROM HER IN THE FUTURE.
SHE IS NOW AT ST. LOUIS
UNIVERSITY.
THANK YOU.
>> THANK YOU, NADA, FOR THIS
FASCINATING WORK.
SO WE'RE GOING TO MOVE TO THE
NEXT SPEAKER, DR. DARIA
ESTERHAZY.
FROM THE UNIVERSITY OF CHICAGO.
HER TALK IS ENTITLED IMPACT OF
INTESTINAL HELMINTHS ON DUO DEAL
LYMPHATIC FUNCTION.
GO AHEAD.
>> YES, NOW I SHOULD BE ABLE TO.
I REALLY APOLOGIZE.
THIS WAS A SYSTEM SETTING,
BELIEVE IT OR NOT.
OKAY.
SO MANY THANKS FOR THE
OPPORTUNITY.
SO I'LL TELL YOU ABOUT HOW WE
STUDIED THE INTESTINAL
LYMPHATICS OR HOW OW LAB OUD
UP DOING SO.
I'LL GIVE YOU A BRIEF OVERVIEW
AS TO WHAT OUR WORK IS ABOUT IN
GENERAL.
WE LOOK AT THE LYMPHATICS FROM A
LYMPH NODE PERSPECTIVE, AND IT
STARTED OFF WITH THIS
UNDERSTANDING THAT THE LYMPH
NODES ALONG THE GASTROINTESTINAL
TRACT ARE ANATOMICALLY BUT ALSO
FUNCTIONALLY DISTINCT BECAUSE
YOU'RE EXPOSED TO DIFFERENT
THINGS ALONG THE INTESTINE AND
OBVIOUSLY EACH INTESTINAL
SEGMENT ALSO HAS DIFFERENT
PURPOSES.
AND WE OBSERVED THAT OVERALL,
THE UPPER SMALL INTESTINAL LYMPH
MODES MOUNT BY DEFAULT AN
IMMUNOIMPRESSIVE IMMUNE RESPONSE
AND FOR THE DOWN, THE FUE
MORE INFLAMMATORY.
THAT BALL ABC SEEMS TO BE
HEALTHY BECAUSE ON THE FLIP SIDE
IF THAT BALANCE IS PERTURBED,
THAT CAN LEAD TO, AGAIN,
SEGMENT-SPECIFIC DISEASE IN THE
UPPER SMALL INTESTINE AND MORE
PROMINENT INFLAMMATORY BOWEL
DISEASES IN THE DISTAL INTESTINE
AND ALSO CANCER.
THE TWO PROJECT AREAS IN THE LAB
ARE ON THE ONE HAND
UNDERSTANDING THE CROSSTALK
BETWEEN THE GUT, PANCREAS AND
LIVER AND SO THAT IS AN
INTERESTING DISCUSSION, WE ALSO
LOOK AT LYMPHATICS.
THAT'S NOT THE AREA I WILL TALK
ABOUT TODAY.
I WILL TALK ABOUT THIS OTHER
AREA WE'VE ONLY RECENTLY STARTED
TO TALK ABOUT, DRIVING FORCES
AND CELLULAR SUBSTRATES OF
TISSUE-SPECIFIC LYMPH NODES.
WHAT I WILL FOCUS ON TODAY IS
THIS UPPER REGION, BECAUSE IT'S
INTERESTING IN THAT IT'S
IMMUNOSUPPRESSIVE OR TOLERANCE
PROMOTING AND HOW THAT THING CAN
BE PERTURBED BY LUMINAL EVENTS
SUCH AS HELMINTH INFECTIONS.
THE HELMINTH THAT STARTED US OFF
IS THIS ORIGINALLY RAT HELMINTH
CALLED STRONGYLOIDES
VENEZUELENSIS.
WE CAN LOOK AT IT FROM THE
IMMUNE CELL SIDE WITHOUT GOING
INTO ALL THE DETAILS OF THE
IMMUNE SYSTEM, UPON INFECTION,
REALLY ONLY IN THIS -- EACH OF
THESE LYMPH -- THEY ARE MARKED
BY BEING CD11B POSITIVE, AND OF
COURSE THEY ARE MIGRATORY BY
BEING POSITIVE OVER HERE.
AGAIN THAT'S A PHENOTYPE WE ONLY
SEE IN THE NICHE THAT DRAINS THE
INFLAMED GAP SEGMENT BEING THE
DUODENUM.
WE HAVE AN INCREASE OF TH2 CELLS
AND ONLY A LITTLE -- THE ONE --
LOCALIZATION, AND THIS ALSO
AFFECTS WHAT WE CALL HOW YOU
REACT TO YOUR FOOD, WHERE ALL OF
A SUDDEN IF YOU HAVE A WORM IN
YOUR DUODENUM, YOU ARE LESS ABLE
TO MOUNT THESE IMMUNE RESPONSES
AND THIS ACTUALLY LEADS TO FOOD
ALLERGY IN THE MOUSE MODEL.
NOW THE INTERESTING THING ABOUT
THE WORM IS OF COURSE EVERY
INFECTION LEADS TO SOME KIND OF
LYMPH NODE SWELLING BUT HONESTLY
WE DON'T EVER SEE IT AS EXTREME
AS UPON HELMINTH INFECTION.
THIS IS A LYMPH NODE JUST WITH
AN IPHONE LOOKS LIKE, JUST BY
VISUALIZING, WE CAN TELL THE
INFECTION -- IF YOU STAIN FOR
THIS, THIS REALLY GOES IN HAND
AND THAT'S WHERE IT'S DIFFERENT
FROM, SAY, TRANSIENT INFECTIONS
WITH VIRUSES, WITH ANGIOGENESIS
AND LYMPH ANG GENESIS. ANGIOG.
BASICALLY ASKED THIS VERY
FUNDAMENTAL QUESTION OF WHERE
DOES THIS ALL START, IN OTHER
WORDS, WHAT ARE THE GUT TISSUE
EVENTS THAT GOVERN THIS
ADAPTATION TO HELMINTH INFECTION
AND I'LL SHOW YOU IN A MINUTE
WHY WE ACTUALLY STARTED ASKING
MORE SPECIFICALLY, ARE THE
TISSUE LYMPHATICS REALLY
AFFECTED BY THE HELMINTH
INFECTION AND IF SO, WHAT ARE
THE IMMEDIATE DOWNSTREAM
CONSEQUENCES?
AND I'LL NOT TALK ABOUT THE
UPSTREAM EVENTS THAT WE'RE
OBVIOUSLY ALSO STUDYING, AND
THEN FINALLY WHAT IT TAKES TO
RESOLVE THIS.
AND THEN WE ONLY OBSERVE THIS
WITH THIS ONE SO WE REALLY
WANTED TO KNOW IS THIS SOMETHING
THAT ALL HELMINTHS DO OR JUST --
AND HOW DOES THAT COMPARE
POTENTIALLY TO OTHER INSULTS IN
THE GUT INDUCED BY PATHOGENS,
AND IS IT SPECIFIC TO THIS
DUODENAL NICHE OR DOES IT GO
BEYOND THAT.
SO WHY STUDY THE LYMPHATICS?
THIS ACTUALLY LINKS QUITE NICELY
TO THE PREVIOUS TALK AND ITS
THEME, AND THAT IT DOES ALSO
AFFECT LIPID ABSORPTION.
SO ON THE ONE HAND WE COULD
TEASE OUT THAT PHENOTYPE BY
PUTTING MICE ON A HIGH FAT DIET
WHERE NORMALLY THEY GAIN WEIGHT,
BUT IF YOU INFECT WITH A WORM,
EIGHT DAYS POST INFECTION
INSTEAD OF CONTINUING TO GAIN
WEIGHT, THE MICE IF ANYTHING
LOSE WEIGHT.
INDICATING THEY HAVE SOME AT
LEAST MINIMAL METABOLIC DEFECT
AND WE DON'T THINK IT'S BECAUSE
THEY BURN MORE, BECAUSE ACTUALLY
WHEN YOU LOOK MORE CLOSELY, BY
GAVAGE AND MEASURING
TRIGLYCERIDES IN THE SERUM, THE
OIL WILL NOT HAVE REACHED MUCH
BEYOND THE DUODENUM IN A MOUSE,
THIS IS 8 DAYS POST INFECTION,
YOU HAVE A DECREASE IN UPTAKE
INTO THE SERUM.
THIS PERSISTS AT LEAST FIVE DAYS
AFTER THE WORM CLEARANCE, SO
AGAIN, SUGGESTING THAT THERE
MUST HAVE BEEN MASSIVE
RESTRUCTURING AND THEN IF YOU
LOOK OVER FOUR WEEKS OUT, IF
ANYTHING IT'S INCREASED BUT IT'S
CERTAINLY NOT DECREASED ANY
MORE, SO EVENTUALLY THAT ISSUE
RESOLVES.
THIS WAS ALL WORK I SHOULD SAY
ABOUT A PH.D. STUDENT WHO IS --
HAS BEEN WORKING VERY HARD ON
THIS FOR THREE YEARS.
SO WE KNOW THAT THE UPPER
INTESTINAL LYMPHATICS ARE
HEAVILY IMPLICATED IN LIPID
ABSORPTION.
AND BECAUSE WE HAVEN'T HAD THE
PICTURE UP YET, I WOULD JUST
LIKE TO MENTION THAT THE
LACTEALS ALONG THE INTESTINE
ACTUALLY DECREASE.
OF COURSE THE VILLUS LENGTH
DECREASES BUT ALSO THE DUODENAL
LYMPHATICS, AND WE'RE NOT THE
FIRST ONE TO OBSERVE THAT,
SOMETIMES EVEN HAVE TWO LACTEALS
IN ORDER TO ACCOMMODATE FOR THIS
INCREASED NEED FOR LIPID
TRANSPORT.
WE ALSO KNOW THAT MOST LIPIDS
ARE TAKEN UP IN THE DUODENUM
FOLLOWED BY THE JU KNEW NIM, W
THAT YOU NEED TO BASICALLY
PACKAGE THIS INTO MICRONS
BECAUSE IF YOU HAD INHIBITOR TO
THE GAVAGE CALLED PL81, YOU'LL
ACCUMULATE THE FATS.
YOU SEE MOST LIPIDS IN THE
DUODENAL LYMPH NODES BUT IF YOU
PREVENT INFLAMMATION, THAT'S NO
LONGER TRUE.
AND SO ONE THING THAT WAS
ALREADY MENTIONED BUT MAYBE NOT
IN THAT DETAIL, IT FURNS OUT TUT
OBVIOUSLY ON THE LYMPHATIC SIDE
OF THINGS, NOT ONLY THE VASTNESS
OF THE VASCULATURE, HOW MANY
LIPIDS YOU CAN TAKE UP SO YOU
HAVE THESE MORE PER PERMISSIVE
BUTTON-LIKE JUNCTIONS, THIS IS
HOW WE QUANTIFY THIS, BY
BASICALLY ITS IMAGE
QUANTIFICATION IS A LOT OF WORK
BUT THAT'S WHAT THE FIELD SEEMS
TO DO.
SO WHEN YOU QUANTIFY ALONG THE
INTESTINE, THIS SORT OF
CORRELATES ALONG WITH WHAT WE
THINK IS THE INVOLVEMENT OF THE
LIPID ABSORPTION, AS YOU GO
ALONG THE SMALL INTESTINE, THE
LACTEALS BECOME MORE
ZIPPER-LIKE.
NOW IF WE LOOK AT THAT FROM A
DISTENDED LYMPHATICS POINT OF
VIEW, THE LACTEALS ARE MUCH MORE
DISTENDED AND INTERESTINGLY THEY
BECOME MUCH MORE ZIPPER-LIKE SO
THAT WOULD CORRELATE WITH THIS
DECREASED FAT AND PHENOTYPE.
WE WANTED TO KNOW IF ONE
HELMINTH OR SEVERAL HELMINTHS DO
THIS.
IN SHORT -- DO THE SAME THING
WHERE YOU BECOME MORE
ZIPPER-LIKE UPON THE HELMINTH
INFECTION AND THE LACTEALS
BECOME MORE DISTENDED.
I SHOULD SAY WE DID NOT SEE THIS
UPON BACTERIA OR VIRUSES
SUGGESTING THIS IS A HELMINTH
INFECTION WHICH WE'RE NOW TRYING
TO WORK OUT.
THE FINAL THING THAT I WANTED TO
TOUCH ON IS HOW WE LOOKED AT NOT
ONLY FAT TRANSPORT BUT ALSO
IMMUNE CELL TRAFFICKING BUT WE
MADE THE INTESTINE GREAN GREN
INDUCIBLE ROSA26 MOUSE AND THE
IDEA IS THAT BECAUSE YOU HAVE
INTESTINAL CELL TURNOVER, SOME
DENDRITIC CELLS WILL BECOME
GREEND MIGRATE TO
GREEN AND MIGRATE TO THE LYMPH
NODES.
THREE DAYS AFTER INFECTION WITH
WORM, ANOTHER FIVE DAYS LATER,
WE CAN SEE THIS ACTUALLY ALSO
CORRELATES WITH A MASSIVE
MIGRATION DEFECT.
SUGGESTING THAT THE ZIPPER-LIKE
FUNCTION OF THE -- OR CLOSING OF
THE DUODENAL LYMPHATICS ALSO
AFFECT HOW IMMUNE CELLS TRAFFIC.
AND THAT'S ALL OF COURSE ONLY
THE TIP OF THE ICE ICEBERG, SI
WOULD JUST LIKE TO FINISH WITH
THE GAPS AND NEEDS AND BIGGER
PICTURE.
WE HAVE NO IDEA WHAT THIS LOOKS
LIKE IN HUMANS BUT IT'S
INTRIGUING TO THINK THAT
HELMINTHS, IN MANY PARTS OF THE
WORLD, ARE STILL A DAILY BREAD
AND BUTTER AND CAN COINCIDE WITH
PHENOTYPES OF MALABSORPTION AND
VITAMIN E DEFICIENCY IN PARTS OF
THE WORLD.
I WOULD SAY IN THE BROADER
SENSE, SOME THINGS HAVE BEEN
TOUCHED ON BUT THIS REALLY
RELATES TO THIS SITE-SPECIFIC --
WE LACK THIS INFORMATION OF
SITE-SPECIFIC TRANS CRYPTOMIX IN
GUT LYMPHATICS, PARTICULARLY
HARD TO STUDY JUST BECAUSE OUR
RNA INTEGRITY IS SO MUCH MORE
COMPROMISED.
IDENTIFYING RELEVANT MOLECULES
AND CELLS WITH WHICH THEY
COMMUNICATE AND SORT OF REGULATE
THEIR DYNAMICS.
I DO THINK WE STILL LACK
FUNDAMENTAL UNDERSTANDING OF
WHAT THE GUT LYMPH COMPOSITION
IS ALONG THE INTESTINE.
SPATIAL TRANSCRIPTOMICS, SOME OF
THOSE ISSUES IN PART BECAUSE
THEY DON'T RELY ON LIVE TISSUE.
WITH THAT, I WOULD LIKE TO THANK
THE PEOPLE IN MY TEAM, MOST
NOTABLY JORDAN LANE, AND I'M
HAPPY TO TAKE QUESTIONS LATER
ON.
THANK YOU.
>> THANK YOU, DARE DARIA.
REALLY INTERESTING WORK.
SO I'D LIKE TO INTRODUCE OUR
LAST SPEAKER, DR. JORGE
CASTORENA-GONZALEZ FROM TULANE
UNIVERSITY SCHOOL OF MEDICINE.
HIS TALK ENTITLED MECHANISMS OF
IMPAIRED LYMPH TRANSPORT IN
OBESITY HAD BEEN -INDUCED L.
>> THANK YOU FOR A NICE
INTRODUCTION.
I WOULD LIKE TO BEGIN MY
PRESENTATION BY THANKING THE
ORGANIZERS FOR ALLOWING US TO
PRESENT TODAY.
I ALSO WANT TO START MY
PRESENTATION BY ACKNOWLEDGING
THOSE -- THE MEMBERS OF MY LAB.
THE WORK CH VICTORIA AND KELLI
HAS BEEN ESSENTIAL FOR THE WORK
OF MY LAB SINCE WE STARTED TWO
YEARS AGO.
I ALSO WANT TO THANK MY
COLLABORATORS AND MY FORMER
MENTORS, ESPECIALLY DR. MICHAEL
DAVIS, AND OF COURSE I WANT TO
THANK THE NIH FOR THE FUNDING
THAT MAKES THIS RESEARCH
POSSIBLE.
SO I DON'T HAVE TO CONVINCE YOU
THAT OBESITY IS A MAJOR HEALTH
PROBLEM WORLDWIDE.
IN THE LAST REPORT BY THE WORLD
HEALTH ORGANIZATION, ABOUT
650 MILLION ADULTS ARE OBESE.
AND THERE IS A VERY CLEAR
CONNECTION BETWEEN OBESITY AND
LYMPHATICS DYSFUNCTION.
THIS IS EVEN MORE CLEAR IN
MORBIDLY OBESE PATIENTS, THOSE
PRESENT WITH A BMI OF OVER
40 KILOGRAMS PER METER SQUARED,
AT HIGHER RISK OF DEVELOPING
LOWER EXTREMITY LYMPHEDEMA.
SO THE CONNECTION BETWEEN
OBESITY AND LYMPHATIC
DYSFUNCTION IS REALLY A
DYSFUNCTION AND
DISEASE-PROMOTING FEEDBACK LOOP.
IN THIS RECENT STUDY BY
DR. KATARU AND THE GROUP BY
DR. BABAK MEHRARA.
SO IN MANY PLACES OF THE BODY,
WE KNOW THAT LYMPH HAS TO BE
TRANSPORTED AGAINST AN ADVERSE
PRESSURE DRIVEN AND THIS IS DUE
TO GRAVITATIONAL FORCES.
AT THE CLINIC, THE PHYSICIANS
ARE ABLE TO ASSESS DRAINAGE OR
TRANSPORT OF LYMPH BY ASSESSING
DRAINAGE INTO THE SPECIFIC LYMPH
NODES.
SO IN TERMS OF OBESITY INDUCED
LYMPHEDEMA, WHICH IS THE CENTRAL
TOPIC OF THIS TALK, IN PARALYMPH
DRPT OR DRAINAGE CAN RESULT FROM
NORMAL OR IMPAIRED UPPER TAKE OF
INTERSTITIAL FLUID AFTER
CAPILLARIES, VFL DEFICIENCIES,
INCREASED PERMEABILITY,
OBSTRUCTION OR DELAYED FLOW
AMONG OTHERS.
LYMPHEDEMA IS USUALLY ASSOCIATED
WITH A FEW OTHER CONDITIONS LIKE
THE ONES LISTED HERE.
SO IN MY LAB, ONE OF THE
TECHNIQUES WE USE IS THE STUDY
OF MICRO DISSECTED LYMPHATIC
VESSELS FROM DIFFERENT SPECIES.
IN OUR MOST RECENT PUBLICATION,
WE INCLUDED FULL VIDEO IN THE
SUPPLEMENTAL MATERIALS
EXPLAINING THIS PROCESS.
ONCE WE HAVE OUR DISSECTED
LYMPHATIC SEGMENT, WE TRANSFER
IT ON TO AN OBSERVATION CHAMBER,
AND BY USING TWO GLASS MICRO
PIPETTES, WE CAN STUDY THESE
LYMPHATIC SEGMENTS.
IN MY LAB, WE USE A MICRO
FLUIDICS FLOW CONTROLLER THROUGH
HAVING INDEPENDENT CONTROL OF
INPUT AND OUTPUT FLOW.
SO WE CAN STUDY ASPECTS OF
FUNCTION OF COLLECTING LYMPHATIC
DEVELOPS ELSE FROM
VESSELS FROM DIFFERENT SPECIES.
INCLUDING HUMANS, WE CAN CONTROL
FOR DIFFERENT AND SPECIFIC AN
ANATOMICAL REGIONS, WE CAN
CONTROL TEMPERATURE, FLOW AND
PRESSURE KENTUCKYS AND EVALUATE
THE EEFNGHTS OF EFFECTS OL
AGENTS.
WITH THESE TECH TECHNIQUES WN
QUANTITATIVELY ASSESS THE
CONTRACTILE FUNCTION OF THESE
VESSELS, THEIR AMPLITUDE,
FREQUENCY, EJECTION FRACTION AND
SOME OTHER FACTORS.
A FEW YEARS AGO WE HAD A
COLLABORATION WITH A SURGEON.
AT THE TIME WE WERE -- PATIENTS
WHO HAD A BMI OF OVER 40 MG PER
METERS SQUARED.
HOW THE LYMPHATIC CONTRACTILITY
OF THESE HUMAN VESSELS COMPARED
TO THAT THAT WE NORMALLY OBSERVE
IN LEAN WILD TYPE MICE.
AS YOU CAN SEE, INITIALLY, WE
DID NOT SEE ANY SIGNIFICANT
DIFFERENCES WHEN WE COMPARE
HUMAN AND MOUSE DATA AS EVIDENT
BY THESE TWO PLOTS WHERE WE'RE
PRESENTING CONTRACTION FREQUENCY
AND EJECTION FRACTION AS A
FUNCTION OF INTRALUMINAL
PRESSURE.
HOWEVER, A RECENT STUDY
SHOWED -- BY DR. RAFAEL
CZEPIELEWSKI WHO PRESENTED
EARLIER TODAY IN COLLABORATION
WITH DR. MICHAEL DAVIS, THEY
RECENTLY MEASURED THE IN VIVO
PRESSURES IN THE MOUSE AND THEY
FOUND THAT THE PRESSURES THEY
WERE ABLE TO MEASURE WERE ALL
BELOW REALLY 5 CENTIMETERS OF
WATER.
SO THAT WILL PUT THE MOUSE
LYMPHATIC VESSELS IN THIS
REGION.
WHEN WE'RE TALKING ABOUT HUMAN
VESSELS, A COUPLE THINGS WE HAVE
TO CONSIDER IS FIRST ALTHOUGH WE
DIDN'T OBSERVE ANY CONTRACTILE
DEFECTS, WE HAVE TO CONSIDER
THAT WE TESTED THESE LYMPHATIC
VESSELS OUTSIDE THEIR NATIVE
ENVIRONMENT, WHICH IS LIKELY A
VERY PRO INFLAMMATORY
ENVIRONMENT, AND SECOND, IF
OBESITY RESULTS IN THE OVERLOAD
OF LYMPHATIC NETWORKS WITH FLUID
BACKING UP, LEADING TO A
SIGNIFICANT INCREASE OF
TRANSMURAL PRESSURE, THAT WOULD
PUT THE HUMAN LYMPHATIC ME SALS
IN OBESE PATIENTS IN THIS AREA
OF THE BLOOD, WHERE THE EJECTION
FRACK SHUPS ARE SO LOW, THAT
LIKELY HAVE A VERY, VERY
COMPROMISED PUMPING CAPABILITY.
SO WE KNOW IN ADDITION TO
CONTRACTIONS, LYMPHATIC VALVES
ARE CRITICAL FOR EFFICIENT LYMPH
TRANSPORTS.
SO THESE ARE THREE DIFFERENT
CONSTRUCTIONS THAT WE HAVE
GENERATED IN MY LAB FROM
COLLECTING LYMPHATIC VALVES.
AS YOU CAN SEE, THIS IS A
LYMPHATIC VALVE IN AN OPEN
POSITION, HOWEVER, WE KNOW THESE
VALVES CLOSE IN THE PRESENCE OF
DECREASED PRESH REMEMBER
GRADIENT, FORMING A VERY, VERY
TIGHT SEAL WHICH PREVENTS
BACKFLOW.
BUT LYMPHATIC VALVES ARE NOT
ONLY CRITICAL AT MINIMIZING RM
BLOOD FLOW.
THESE ARE ALSO CRITICAL FOR --
BECAUSE THEY ALLOW FOR THE
GENERATION OF PROPULSIVE
PRESSURE DURING THE CONTRACTION
CYCLE.
THIS IS DISPLAYED IN THIS VIDEO,
THIS LYMPHATIC VESSEL OF THE
MOUSE.
SO AS YOU WERE ABLE TO SEE IN
DR. MICHAEL DAVIS' PRESENTATION,
THE GOLD STANDARD TECHNIQUE FOR
ASSESSING LYMPHATIC VALVE
FUNCTIONING EX-VO VO -- -Y
COMPLEX, COMPLICATED SYSTEMS TO
USE AND VERY FEW ARE STILL IN
GOOD WORKING CONDITION AROUND
THE WORLD.
SO A FEW YEARS ARC WE DEVELOPED
A SIMPLIFIED METHOD FOR
QUANTITATIVE ASSESSMENT FOR
LYMPHATIC VALVE FUNCTION.
THIS ALLOWS US TO DETERMINE THE
AMOUNT OF PRESSURE ACROSS A
VALVE.
WE HAVE A SINGLE VALVE SYSTEM
AND INITIALLY WE START WITH THE
SAME PRESSURE IN THE INPUT AND
THE OUTPUT AND WE START
INCREASING THE OUTPUT PRESSURE,
CHALLENGING THIS VALVE.
SO THIS IS DISPLAYED IN THIS
VIDEO OVER HERE.
SO AS I WOULD -- AS OUTPUT E
BEGINS TO INCREASE, THE LYMPH
VALVE INCREASES, AND PRESSURE
CONTINUES TO INCREASE, THIS
SECTION OF THE VESSEL TENDS TO
AFFECT THE REST OF THE LYMPHATIC
SYSTEM.
SOME OF THE WORK WE DID IN MY
LAB WAS THAT WE SUBJECTED MICE
FOR 14 WEEKS TO EITHER A CONTROL
OR A WESTERN DIET, AND TO INDUCE
OBESITY.
SO THESE MICE BECAME OBESE.
THEY ALSO DISPLAYED HIGH BLOOD
GLUCOSE AND HIGH CHOLESTEROL
LEVELS.
OH, THE COMPETENCE OF LYMPHATIC
VALVES FROM TWO DIFFERENT
ANATOMICAL REGIONS.
WE FIRST LOOK AT POPLITEAL AREA
AND TO OUR SURPRISE, WE DID NOT
SEE ANY LYMPHATIC VALVE
DEFICIENCIES AS EVIDENT BY THE
SIMILAR AND VERY LOW PRESSURE --
THAT WE OBSERVE HERE.
BACK LEAKS WE OBSERVED HERE.
THESE ANIMALS HAD SIGNIFICANT
PRESSURE BACK-LEAK.
SO THESE ARE WHAT WE REFER TO AS
LEAKY VALVES.
SO FURTHERMORE, IF WE GO BACK TO
THE VIDEO I JUST SHOWED YOU
BEFORE, THIS TIME THE VIDEO IS
GOING TO STOP WHEN THE VALVE
ENGAGES AND GOES INTO A CLOSED
POSITION.
SO IN THIS SPECIFIC CASE, WE CAN
SEE THAT FOR THIS PARTICULAR
VESSEL SEGMENT, IT REQUIRES
ABOUT 0.5 CENTIMETERS OF ADVERSE
PRESSURE GRADIENT TO DRIVE THIS
LYMPHATIC VALVE INTO THE CLOSED
POSITION.
THIS IS ACTUALLY A VERY -- FOR
HEALTHY CONTROL VALVES, BUT AS
YOU CAN SEE, WHEN WE COMPARED
CONTROL VERSUS WESTERN DIET, WE
FOUND THOSE LEAKY MESENTERIC
VALVES, THEY ALSO REQUIRE
SIGNIFICANTLY HIGHER LEVELS OF
ADVERSE PRESSURE TRYING TO DRIVE
THEM INTO THE CLOSED POSITION.
IF WE GO BACK TO THE STUDY I WAS
REFERRING TO EARLIER IT WAS
REPORTED PRESSURES IN THE MOUSE
ARE BELOW 5 CENTIMETERS OF
WATER, THAT WOULD MEAN IN OUR
DATA, ABOUT ONE-THIRD OF OUR
VALVES FROM OBESE LIFE ARE NEVER
LIKELY ABLE TO -- AS THEY ARE
REQUIRING MORE THAN
5 CENTIMETERS OF WATER TO CLOSE.
SO FOR THESE VALVES WE ACTUALLY
WERE NOT ABLE TO IDENTIFY ANY
STRUCTURAL OBVIOUS ABNORMALITIES
THAT COULD BE ASSOCIATED WITH
THIS LEAKINESS AND INCREASED
LEVELS OF ADVERSE PRESSURE
REQUIRED TO CLOSE THESE VALVES,
SO WHAT WE'RE HIGH POT HYPS
IS A QUESTION FROM EARLIER
TODAY, WOULD WE BELIEVE THE
WORK -- THE WORKING HIGH
POTASSIUM THESE IS LYMPHATIC
VALVE STIFFENING MAY BE AN
IMPORTANT FACTOR IN OBESITY AND
OBESITY INDUCED LYMPHEDEMA.
ONE OF THE THINGS WE WERE ABLE
TO FIND IN THESE ANIMALS WAS
WHEN COLLECTING THESE LYMPH
VALVE FROM THE -- WAS
SIGNIFICANTLY INCREASED.
PAI-1 IS A MAJOR INHIBITOR OF
TISSUE AND UROKINASE UROKI-
METABOLIC SYNDROME AND OTHERS.
SO WE SUBJECTED PAI-1 SUFFICIENT
MICE TO THE SAME DIET REGIMEN
AND WHAT WE WERE ABLE TO FIND IS
THAT BY ONE KNOCKOUT MICE FED A
WESTERN BUY ET WERE SUFFICIENTLY
PROTECTED AGAINST OBESITY
COMPARED TO -- PRODUCES NORMAL
BLOOD GLUCOSE AND CHOLESTEROL
LEVELS.
MORE IMPORTANTLY, WE BELIEVE THE
OVERALL IMPROVEMENT IN METABOLIC
FUNCTION AND THIS ALLOWED THESE
MICE TO PREVENT THE LYMPHATIC
FUNCTION THAT WAS OAR OBSERVEN
KNOCKOUT MICE.
YOU CAN SEE THEY ARE LOW AS THE
CONTROL DIET AND ALL THE VALVES
ARE REQUIRING THE SAME ADVERSE
PRESSURE DURING THE CLOSING.
SO SOME OF THE CURRENT AND
FUTURE WORK WE'RE DOING IN MY
LAB IS, WE'RE TRYING TO
DETERMINE THEN OR ANSWER THE
QUESTION WHAT IS CHANGING IN THE
LYMPHATIC ENDOTHELIUM OF
COLLECTING LYMPH AND
UNDERSTANDING THE MECHANISMS
INVOLVED IN VALVE DYSFUNCTION IN
OBESITY.
WE ARE UTILIZING A TECHNIQUE
THAT WAS INITIALLY DEVELOPED BY
DR. STEVEN SEGAL AT UNIVERSITY
OF MISSOURI.
THIS WAS LATER ADOPTED BY YOURS
FOR LYMPHATICS, BASICALLY --
RECENTLY WE PUBLISHED THIS PAPER
WHERE WE'RE ABLE TO FURTHER
OPTIMIZE THIS TECHNIQUE TO APPLY
IT FOR DIFFERENT SPECIES
INCLUDING HUMANS AND SO NOW
WE'RE TRYING TO CHARACTERIZE
CHANGES IN THE INTACT
ENDOTHELIUM OF LYMPHATIC VESSELS
FROM DIFFERENT SPECIES AND
DIFFERENT ANATOMICAL REGIONS,
EITHER IN THE INTACT STATE OR BY
FURTHER CULTURING THESE
LYMPHATIC ENDOTHELIAL TUBES.
SO THIS IS BASICALLY WITH A
PURPOSE OF UNDERSTANDING AND
CHARACTERIZING CHANGES IN THE
LYMPHATIC ENDOTHELIUM OF
COLLECTING VESSELS FROM LEAN
CONTROLS AND DIET-INDUCED OBESE
WILD TYPE AND PAI-1-DEFICIENT
MICE.
WE'RE TRYING TO DO THESE AND
TRYING TO LOOK AT THINGS THAT
ARE KNOWN TO INTERACT WITH
PAI-1.
SO WITH THIS TECHNIQUE, WE'RE
ABLE TO UTILIZE THESE TYPE OF
TECHNIQUES AND WE START
BASICALLY WITH A MORE --
POPULATION SO WE DON'T HAVE TO
DEAL WITH TRYING TO CLEAN OUT OR
SELL A SAMPLE BASICALLY.
SO ONE OF THE OTHER THINGS THAT
WE'RE DOING IN MY LAB IS, AS
HIGHLIGHTED IN PREVIOUS
PRESENTATIONS, WE KNOW THAT A
MAJOR FUNCTION OF THE LYMPHATIC
SYSTEM IS THAT OF LIPID
ABSORPTION.
SO WE'RE ALSO INTERESTED IN
TRYING TO DETERMINE WHETHER
PAI-1 PLAYS A ROLE IN REGULATING
LACTEAL INTEGRITY AND FUNCTION
IN DIET-INDUCED OBESITY.
THESE ARE JUST SOME RECENT
PICTURES WE WERE EABL ABLE TT
IN MY LAB.
I WANT TO THANK HER FOR SHARING
HER EXPERTISE.
SO TO CLOSE MY PRESENTATION,
SOME OF THE GAPS AND
OPPORTUNITIES, SOME OF THEM THAT
ARE SPECIFIC TO THE CURRENT
FOCUS OF THE RESEARCH PERFORMED
IN MY LAB IS TO DETERMINE THE
LYMPHATIC-SPECIFIC ROLE OF PAI-C
AND METABOLIC DYSFUNCTION IN
DIET-INDUCED OBESITY.
ASSESS THE POTENTIAL OF
PHARMACOLOGICAL PAI-1 INHIBITORS
AS A NOVEL THERAPEUTIC STRATEGY
IN OBESITY AND OBESITY-INDUCED
LYMPHATIC DYSFUNCTION.
OTHER FACTORS ARE LONG TERM AND
CLINICALLY RELEVANT FOR
SECONDARY LYMPHEDEMA PATIENTS,
DETERMINE THE EXTENT OF
LYMPHATIC VALVE DYSFUNCTION AND
STIFFENING OF COLLECTING
LYMPHATICS IN OBESE AND
NON-OBESE PATIENTS WITH
SECONDARY LYMPHEDEMA.
HOPEFULLY AT SOME POINT WE CAN
IDENTIFY THE UNDERLYING
MECHANISMS AND DEVELOP
PHARMACOLOGICAL TREATMENTS, BUT
EVEN THE SIZE OF THIS ENDEAVOR,
WE BELIEVE THAT THESE WOULD
REQUIRE THE DEVELOPMENT OF NOVEL
TECHNICAL AND APPROACHES AND O
ANALYTICAL METHODOLOGIES, AS
WELL AS A MULTIDISCIPLINARY AND
MULTI-INSTITUTIONAL
COLLABORATIVE EFFORTS.
THANK YOU FOR YOUR ATTENTION.
>> THANK YOU VERY MUCH, JORGE,
FOR THIS REALLY GREAT TALK.
SO NOW WE MOVE ON TO THE Q & A
SESSION.
WE HAVE RECEIVED A LOT OF
QUESTIONS SO I WILL TRY TO
DISTRIBUTE TO EACH INDIVIDUAL AS
MUCH AS POSSIBLE.
SO THE FIRST QUESTION FOR
RAFAEL.
BUT FOR THOSE WHO RESEARCH
LYMPHATIC FUNCTION AND IBD, ARE
THERE ANY CONCERNS FOR TAKING
THE OMENTUM AS A LYMPH NODE
TRANSFER AND CAUSING IATROGENIC
ABDOMINAL LYMPH FLOW ISSUES?
>> I'M NOT SURE I UNDERSTOOD THE
COMPLETE CONNECTION.
SO THE OMENTUM ITSELF, IT'S NOT
COMPLETELY DRAINING THE SAME
NETWORK THAT MESENTERY OR OTHER
PERITONEAL CALFITY CAVITY TO
THE OMENTUM WILL END UP DRAINING
INTO THE ROOT OF THE NETWORK,
BUT USUALLY AT LEAST WE DON'T
SEE IT'S COMPLETELY WIRED THE
SAME WAY.
BUT I DID DIDN'T UNDERSTAND TE
LYMPH NODE TRANSFER PART OF THE
QUESTION, SO IN TERMS OF IBD, I
DON'T THINK THAT THE FIELD HAS
THINGS THAT -- REMOVING THE
OMENTUM OR ITS LYMPHATICS WOULD
DIRECTLY IMPACT THE GUT.
>> OKAY.
THANK YOU.
THE NEXT QUESTION ALSO FOR
RAFAEL.
THIS IS FROM ANDREA.
WOULD YOU LIKE TO UNMUTE AND ASK
YOUR QUESTION?
AN ANDREA, IF YOU ARE STILL THE?
>> BEAUTIFUL TALK, ELEGANT WORK,
LOVE YOUR USE OF MULTIPLE
MODALITIES.
QUESTION S WITH THE LYMPH
LEAKAGE, ARE YOU ABLE TO PARTNER
THAT WITH A REPORTER ANIMAL THAT
MAY ALLOW YOU TO TRACK
INFILTRATION OF DIFFERENT
MYELOID CELLS OR, YOU KNOW, I
MEAN, THE GUESS THE QUESTION
TECHNICALLY, HOW MANY CHANNELS,
HOW MANY THINGS CAN YOU LOOK AT
IN YOUR TWO-PHOTON IMAGING?
JUST REALLY LOVED IT.
>> YES, SO I HAVE MAJORLY
FOCUSED MY 15-MINUTE TALK INTO
MORE THE LYMPHATIC, WHICH WAS
THE OVERARCHING QUESTION THAT WE
WERE DISCUSSING, BUT I SAW SOME
OTHER QUESTIONS RELATED TO WHAT
DO IMMUNE CELLS LOOK LIKE IN
THIS SCENARIO, AND I'VE HAD
IMMUNOLOGIST TRAINING SO I'VE
CHECKED SEVERAL OF THESE IMMUNE
CELLS THAT ARE PRESENT IN THE
TELOS AND THE LYMPHATICS.
SO SO FAR, WE HAVE BEEN DOING --
IT'S STILL RESTRICTED TO A
COUPLE CALLERS.
WE ARE TRYING TO EXPAND THIS A
LITTLE BIT MORE TO SEE IF THE
IMMUNE CELLS CAN COME OUT INTO
THE PERITONEAL CALFITY AND G
THESE PHOTO TAGGING TECHNIQUES
TO TRY TO TRACE THAT, BUT AS FAR
AS WE CAN SEE NOW, THERE'S ALSO
ACCUMULATION AND BLOCKAGE, IS
THAT WE CAN SEE SO FAR, BUT IF
YOU ALLOW THE MOUSE MODEL TO
PROGRESS IN TERMS OF DISEASE AND
AGE, THEN YOU END UP DEVELOPING
ASCITES, SO IN THAT PAPER, I
REPORT A PERCENTAGE OF THE MICE
WHEN THEY ARE REALLY OLD, THEY
DEVELOP ASCITES, WHICH IS AN
ACCUMULATION OF LYMPH IN THE
PERITONEAL CALFITY.
BUT CAVITY.
BUT WE ARE TRAINED SO WE CAN ADD
MORE MARKERS, WE CAN DO
HOPEFULLY MORE.
>> THANK YOU.
WE HAVE A QUESTION FROM A
PANELIST, TIM.
PLEASE UNMUTE AND ASK QUESTION.
>> HI, RAPHA.
YOU MENTIONED DID YOU START
LOOKING AT THE CELL TYPES IN
THESE TLOs.
ARE THEY NAIVE, ACTIVATED,
RESPONDING TO SOMETHING IN
PARTICULAR?
>> YES.
SO THE ANSWER HAS TWOFOLD.
ONE IS IF WE THINK ABOUT THE
LEUKOCYTE CIRCULATION WITHIN
LYMPHATICS, MOST OF THE CELLS
THAT ARE CIRCLE CIRCULATINGB
AND T-CELLS THAT KEEP
CIRCULATING THROUGH SEVERAL
TISSUES AND THEY JUST KEEP
GOING, ESPECIALLY AT THE GUT,
THEY JUST KEEP COMING AND GOING.
SO THERE IS A BUNCH OF NAIVE
T-CELLS.
THEY ARE AT THE END OF
ACCUMULATING DUE TO THIS EFFECT
BUT WE ALSO HAVE -- WHEN WE LOOK
FOR THE ACTIVATED CELLS, WE HAVE
MORE ACTIVATED T-CELLS
ESPECIALLY.
AND THE OTHER WORK STUDYING THE
B CELLS, ALTERATION IN THE B
CELL AND ANTIBODIES.
SO WE SEE A MORE ACTIVATED --
SINCE THIS MOUSE MODEL IS HIGHLY
INFLAMMATORY, BUT IF IF YOU JT
COUNT BY THE NUMBER, BECAUSE YOU
HAVE THIS OBSTRUCTION, YOU END
UP ACCUMULATING THE CELLS THEY
MOVE THE MOST TO, SO WE END UP
HAVING A LOT OF NAIVE CELLS.
AND IT'S IMPORTANT TO MENTION
THAT THESE TELOS ARE ALSO,
BECAUSE THEY'RE -- WITH A
DIFFERENT WEIRD STRUCTURE, THEY
ALLOW CELLS GOING FROM THE BLOOD
CIRCULATION GOING INTO IT.
IN A SMALLER PACE.
>> AND IS THERE OUTFLOW TRACT,
LIKE S1P DEPENDENT AND ALL THOSE
THINGS?
>> THAT IS THE PART THAT I STILL
HAVEN'T FOLLOWED UP ON, AND WE
REALLY WANT TO UNDERSTAND THE
PART LIKE HOW -- BECAUSE AT SOME
LEVEL CELLS EXIT OTHERWISE YOU
WILL EXPLODE, SO WE ARE TRYING
TO UNDERSTAND HOW MUCH IS THE
EXIT, AND IF THERE ARE ANY OTHER
REROUTING OF THE LYMPHATIC
DRAINAGE THAT IS HAPPENING IN
THE GUT, IF IT'S MORE MALLABLE,
SO THERE IS A SIZE WE ARE
EXPLORING.
>> SELEN, WOULD YOU LIKE TO
UNMUTE AND ANSWER THE QUESTION?
>> THANKS FOR ANSWERING MY
ORIGINAL QUESTION AND I WOULD
LIKE TO FOLLOW UP ON THAT.
I WAS WONDERING IF YOU CHECKED
OR ARE YOU PLANNING TO CHECK THE
LVR RECEPTIVE FAMILIES
BASICALLY, EXPRESSION ON THE
LYMPHATIC VESSEL WALL, BECAUSE
BASE KRI MY QUESTION IS IN TWO
PARTS.
THE PAI-1 AND UATPA, THEY ARE
REGULATED BY THE ALVEOLI
RECEPTORS SO PROBABLY YOU HAVE
SOME EFFECT, SO THAT YOU CAN
SEE, I KNOW THAT YOU GUYS ARE
USING APOE BUT YOU MIGHT WANT TO
CONSIDER USING LDR RECEPTOR
KNOCKOUT BACKGROUNDS.
>> THAT'S A GREAT QUESTION, AND
GREAT FOLLOW-UP.
SO YES, ACTUALLY ONE OF THE LAST
SLIDES OF MY PRESENTATION, I
WENT NO IT REALLY INTO II
HAD A DIAGRAM OF THE DIFFERENT
PROTEINS KNOWN TO INTERACT WITH
PAI-1 AND 1 IS WHAT YOU'RE
SAYING IS THE LDR.
SO WE'RE DEFINITELY LOOKING INTO
ALL THESE PROTEINS KNOWN TO
REGULATE FIBROSIS AND PERHAPS
THOSE COULD BE THE ONES
RESPONSIBLE FOR STIFFENING OF
WHAT WE BELIEVE IS STIFF EPING
OF THE LYMPHATIC VALVES.
WHEN WE'RE DOING THESE
SPAIRNLTS, WE
EXPERIMENTS LIE LIKE THOSE PAE
PRESSURE CURVES JUST TO TRY TO
SEE IF THERE WAS EVIDENCE OF
STIFFENING THE HOLDING FATTY
WALL, WE COULDN'T REALLY
DETECT -- THERE WAS A SMALL
TREND FOR VESSELS TO BE STIFFER
BUT IT WAS NOT STATISTICALLY
SIGNIFICANT, BUT WE BELIEVE THAT
JUST -- THE DATA SHOWING THAT
THESE LYMPHATIC VALVES FROM THE
OBESE MICE REQUIRES SO MUCH
ADVERSE PRESSURE TO CLOSE THEM,
WE BELIEVE THAT THAT'S -- WE
COULDN'T FIND ANY OTHER
STRUCTURAL ABNORMALITIES.
>> THANK YOU.
>> NATASZA.
>> I HAVE A QUESTION FOR NADA, A
COMMENT IT WAS A BEAUTIFUL TALK
AND A VERY IMPORTANT ONE AS
WELL.
DO YOUR MUTANTS DEVELOP FATTY
LIVER DISEASE AND DO YOU KNOW
HOW THE LIPIDS ENTER THE PORTAL
CIRCULATION?
HAVE YOU THOUGHT ABOUT VLDL OR
PERHAPS HDL AS A CARRIER?
>> THEY DEVELOP FATTY LIVER AFER
THEY EAT -- YOU KNOW, AFTER A
MEAL, BUT NOT -- WELL, WE
HAVEN'T REALLY LOOKED AT IT
EXTENSIVELY, BUT WE KNOW THAT
THEY HAVE MORE FAT -- THE LIVER
POST PRAN
POSTPRANDIALLY, AFTER A MEAL.
BUT --
>> DO YOU HAVE ANY IDEA HOW THEY
ENTER THE PORTAL VEIN?
DO THEY ENTER --
>> WELL, WE WE KNOW THAT THE
ENDOTHELIUM IS LEAKY TOO.
THAT'S REALLY WHY.
AND THAT'S WHAT I WAS TRYING TO
GET TO AT THE END.
IT'S HARD TO BE CLEAR IN A
15-MINUTE TALK, BUT THE
ENDOTHELIUM IS ALSO LEAKY.
SO THIS IS PROBABLY HOW THEY GET
THERE.
>> THANK YOU.
>> THE PERICELLULAR PATHWAY IS
PROBABLY NOT AS -- AS IT SHOULD
BE.
THIS IS WHAT WE ARE TESTING
RIGHT NOW.
ONE THING THAT I WOULD LIKE TO
SAY, WE KNOW LIPIDS GO INTO --
WE ACTUALLY ARE NOW WORKING ON
LIPIDS BEING DELIVERED THROUGH
EXOSOMES, AND I WAS WONDERING,
IS THERE ANYBODY WORKING ON THE
LYMPHATIC SYSTEM RELEASING
EXOSOMES WHICH COULD REGULATE
OTHER CELLS IN -- BECAUSE THE
BLOOD ENDOTHELIUM RELEASES A LOT
OF EXOSOMES, INCLUDING LIPID
CONTAINING EXOSOMES.
MAYBE SOMEBODY CAN COMMENT WHEN
WE HAVE TIME LATER.
>> YEAH, THAT'S INTRIGUING.
SO HAVE YOU LOOKED AT YOUR
SYSTEM USING TRANSMISSION
ELECTROMICROSCOPY?
>> YES.
WITH RESPECT, YOU MEAN, TO
STRUCTURE OF --
>> YEAH, WITH RESPECT TO
STRUCTURE TO LOOK AT THESE
VESICLES.
INTO
>> YES, WE HAVE.
YEAH.
WE CAN DISCUSS THAT LATER ON.
WE DID CON FOE KOL AND ALSO
TRANSMISSION ELECTROMICROSCOPY.
>> THANKS VERY MUCH.
>> OKAY.
SO THE NEXT QUESTION FOR DARIA.
GIVEN THE INTESTINAL PARASITIC
WORM INFECTION ORDERS THE GUT
MICROBIOME, CAN YOU SEPARATE THE
INFERENCE OF THE WORM FROM THE
GUT MICROBIOME ON LYMPHATIC
ALTERATIONS?
>> OKAY.
IT'S SOMETHING I ANSWERED IN THE
CHAT BUT BECAUSE FOR OTHERS AS
WELL, I WILL BRIEFLY SAY T IT'S
TOUGH TO DO THAT, AND IT'S A
GREAT QUESTION BECAUSE IF YOU
HAVE GERM-FREE MICE OR
ANTIBIOTIC-TREATED MICE, THAT ON
ITS OWN CHANGES THE LYMPHATICS
MASSIVELY, SO YOU'RE GOING TO
LOOK AT A VERY DIFFERENT
SITUATION.
THE OTHER IS THAT IN ORDER TO
GROW THE WORM, WE NEED MICROBES.
THERE ARE PEOPLE OUT THERE WHO
HAVE THOSE SPECIFIC MICROBES
THAT WON'T EXIST IN THE GUT, SO
ONE COULD TRY THAT WAY.
BUT WHAT I WILL SAY IS THAT THE
DUODENUM, WHILE I'M THE LAST ONE
TO SAY THE DUODENUM PLY CROW
BIOME ISN'T IMPORTANT BUT IT'S
NOT VERY HIGHLY POPULATED BY
MICROBES.
WHAT I WOULD SUSPECT, SOMETHING
WE'RE PURSUING NOW WITH AN IDEA,
WHY YOU MIGHT WANT TO CLOSE UP
THE LYMPHATICS IS TO ACTUALLY
PROTECT AGAINST LEAKAGE OF
BACTERIA, WHETHER YOU CHANGE THE
COMPOSITION OR NOT, BECAUSE YOU
OBVIOUSLY DAMAGE THE EPITHELIUM
WITH A WORM VERY HEAVILY, AND
THAT MIGHT BE ONE OF THE
PROTECTIVE MECHANISMS.
BUT IT SOMETHING THAT IS VERY
VALID AND A GOOD QUESTION AND
THE
YET TO BE DETERMINED FULLY.
>> 2 MINUTES REMAINING.
GLL HOW
>> HOW MUCH, JOHN?
>> 2 MINUTES.
>> OKAY.
ZORINA?
>> HI.
I THOUGHT I USED TO TWO MINUTES
TO SEE IF I CAN FIND OUT WHETHER
WE COULD LEARN MORE ABOUT THE
ROLE OF THE MICROBIOME IN
INTERTESSAL ABSORPTION,
SPECIFICALLY LYMPHATICS SEEMS TO
BE VERY IMPORTANT, YET I DIDN'T
HEAR MUCH AND IS THAT BECAUSE OF
CHALLENGES IN EXPERIMENTATION,
SO IF ANYBODY WOULD LIKE TO
COMMENT.
THANK YOU.
>> ANYBODY WOULD LIKE TO COMMENT
ABOUT THIS?
>> SO THE ONE THING I COMMENT ON
IS THAT I THINK IT IS
CHALLENGING, BECAUSE AS YOU
CHANGE THE MICROBIOME, THE
LYMPHATICS ARE JUST PART OF
WHAT'S BEING CHANGED.
IF THE EPITHELIUM STRUCTURE
CHANGES MASSIVELY, THE WHOLE GUT
STRUCTURE CHANGES MASSIVELY.
SMALL INTESTINE -- THE DUODENUM
HAS TWEIST AS BIG TWICE .
SO TO ASSOCIATE BETWEEN THE
EFFECT OF THE MICROBIOTA ON THE
EPITHELIUM VERSUS THE LYMPHATICS
OR INDIRECTLY ON HOW WE ABSORB
FOOD, AND HOW THE GUT ADAPTS TO
THE FACT THAT YOU FOR EXAMPLE
CAN'T FERMENT FIBERS ANYMORE,
THAT'S THE CHALLENGING PART
THERE.
ONE WOULD PROBABLY HAVE TO --
AND AGAIN, IT'S NOT PERFECT --
DO EITHER LYMPHATIC SPECIFIC
KNOCKOUTS OF BACTERIAL SENSORS
LIKE -- VERSUS THE EPITHELIUM,
AGAIN -- ALREADY CHANGES SO
MUCH.
BUT I WOULD SUSPECT THAT THE
MICROBIOTA PLAYS A MORE DIRECT
ROLE ON THE LYMPHATICS LOWER
DOWN IN THE INTESTINE.
ON THE UPPER INTESTINE BECAUSE
IT'S SO IMPORTANT FOR FOOD
ABSORPTION YOU ALWAYS HAVE THAT
SORT OF INTERMEDIATE INTERPLAY
AS WELL.
>> THANK YOU, DARIA.
SO TAKING ADVANTAGE AS
MODERATOR, I'D LIKE TO FINISH
WITH THE LAST QUESTION ABOUT THE
LIVER LYMPHATICS.
WHETHER YOU THINK THE MECHANISM
OF LYMPHATIC REGRESSION BETWEEN
TWO WEEKS AND FOUR WEEKS OF
THE -- DOES THAT OCCUR IN VEGF C
MICE?
I WANT TO MENTION THAT -- IS
SIGNIFICANTLY INCREASED, THAT
ACTUALLY SUR PRISS THE VESSEL
GROWTH.
THAT IS ONE OF THE MECHANISMS
THAT WE HAVE SEEN A REGRESSION
OR DECREASE IN LYMPHATIC VESSELS
AS AND .
AND THE OTHER QUESTION ISING
FROM PATRICIA, THE LYMPHATIC
VESSEL IN THE LIVER REGENERATE
IN TRANSPLANTED LIVER?
THIS IS A VERY GOOD QUESTION,
ACTUALLY WE ARE STUDYING THIS.
WE TRANS TRANSPLANTED THE LIM
MICE AND DOES IT REGROW OR
CONNECT TO THE HOST, SO
HOPEFULLY WE CAN SHOW THAT SOME
TIME IN THE FUTURE.
NOW I'D LIKE TO CONCLUDE WITH
SESSION THREE WILL.
I'D .
I'D LIKE TO THANK ALL THE
FANTASTIC SPEAKERS AND ALSO THE
ACTIVE DISCUSSION BY THE
AUDIENCE.
THANK YOU VERY MUCH.
IT IS MY PLEASURE TO INTRODUCE
DR. TIMOTHY LE CRAS, ASSOCIATE
DEAN GRADUATE EDUCATION
UNIVERSITY OF CINCINNATI COLLEGE
OF MEDICINE, ALSO PROFESSOR IN
THE DEPARTMENT OF PEDIATRICS.
HE'S EXPERTS IN VAS QUEUE RASH
MALFORMATIONS AND DEVELOP.
HIS LAB MADE IMPORTANT
CONTRIBUTIONS TO DIAGNOSE AND
TREATMENT OF LYMPHATIC DISEASE
INCLUDING DEVELOPMENT OF
BIOMARKERS THAT ARE AVAILABLE TO
CLINICAL BLOOD TESTS.
I WANT TO THANK DR. LE CRAS FOR
JOINING US AND MODERATING THIS
EXCITING SESSION.
DR. LE CRAS.
>> THANK YOU VERY MUCH.
CAN YOU HEAR ME OKAY?
>> YES.
>> GREAT.
I'LL SHARE MY SLIDES.
CAN YOU SEE THE SLIDE OKAY?
GREAT.
GOOD AFTERNOON, EVERYBODY.
THANK YOU FOR YOUR ATTENTION.
MY NAME IS TIM LE CLAS AND I'M
GOING TO BE MODERATING SESSION 4
THIS AFTERNOON.
SO E WE'LL START WITH MY TALK
THEN THAT WILL BE FOLLOWED BY
PRESENTATION DR. MICHAEL
DELLINGER, THEN DR. EDWARD
SCHWARZ AND THEN DR. SUSAN
QUAGGIN.
I'LL GET STARTED.
TODAY I'M GOING TO TALK TO YOU
ABOUT THE PATHOGENESIS AND
TREATMENT OF COMPOSITE FORM
LYMPHANGIOMATOSIS.
BEFORE I FOCUS ON THAT
PARTICULAR DISEASE I WILL GIVE A
QUICK INTRODUCTION TO VASCULAR
ANOMALIES AND THAT AFFECT
CHILDREN AND CHUNG ADULTS.
VASCULAR ANOMALIES ARE IN TWO
MAIN GROUPS. VASCULAR TUMORS
BENIGN LIKE HEMANGIOMAS LOCALLY
AGGRESSIVE LICK HE MANAGE MESS
SEW THILL OWE MA AND
ANGIOSARCOMA.
THE MALFORMATIONS CAN COMPRISE
OF CAPILLARY MALFORMATIONS,
VENUS ARTERIO VENUS AND THEN
LYMPHATIC MALFORMATIONS.
SO SOME EXAMPLES INCLUDE STOT
DISEASE AND DR. DELLINGER WILL
TALK ABOUT THAT DISEASE GORHAM
IN HIS PRESENTATION H.
GENERALIZED LYMPHATIC ANOMALY, I
WILL MENTION TODAY AND I WILL
FOCUS ON KAPOSIFORM OR KLA.
THEY CAN BE COMBINED
MALFORMATIONS THAT ENCOMPASS A
NUMBER OF COMPONENTS OF THE
VASCULAR SYSTEM SO LIKE CAPI
VENUS LYMPHATIC MALFORMATIONS.
SO KLA WAS ORIGINALLY DESCRIBED
BY CRETER ET AL IN 2014 SO
RELATIVELY RECENTLY IDENTIFIED.
IT AFFECTS CHILDREN AND YOUNG
ADULTS.
NO MALE FEMALE BIAS.
THE FA THOLINGS CHARACTERIZED BY
DIFFUSE MULTI-FOCAL LESIONS WITH
FOCAL AREAS OF LYMPHATIC
ENDTHELIAL CELLS AND MALFORMED
LYMPHATIC CHANNELS.
YOU CAN SEE IN THE TOP RIGHT
PANEL WHICH SHOWS PATHOLOGY FROM
A PATIENT WITH KLA.
PATIENTS SUFFER FROM HIGH
MORBIDITY AND MORTALITY,
PARTICULARLY CAUSED BY
HEMORRHAGIC EFFUSIONS, THAT CAN
INVOLVE THE LUNGS AND
PERICARDIUM AND CONSUMPTIVE
COAGULOPATHY.
ON THE RIGHTEOUS SEE L I HAVE
BEEN CONTRIBUTIONMPH ANGIOGRAM
THAT SHOWS THE NORMAL LYMPHATIC
VESSELS IN THIS PATIENT WITH
KLA.
IT IS A VERY CHALLENGING DISEASE
TO DIAGNOSE AND TREAT.
AND PATIENTS OFTEN RECEIVE
INCORRECT DIAGNOSIS AND THE
CORRECT DIAGNOSIS MAYBE DELAYED
FOR SEVERAL YEARS.
SO WE BECAME INTERESTED IN KLA
WHEN WE WERE INVOLVED IN A
CLINICAL TRIAL, A PHASE 2
CLINICAL TRIAL FOR THE TREATMENT
OF COMPLICATED VASCULAR
ANOMALIES RAP P MYOSIN, THIS IS
AN MTOR INHIBITOR LED BY DR.
DENISE ADAMS PUBLISHED IN 2016.
IN THIS STUDY CHILDREN AND YOUNG
ADULTS WERE TREATED WITH
COMPLICATED ANOMALIES WITH ORAL
SIROLIMUS THERAPY.
THE STUDY WAS TO DETERMINE
EFFICACY AND SAFETY OF SIROLIMUS
BUT A SECONDARY AIM OUR LAB WAS
INVOLVED IN WAS TO IDENTIFY
BLOOD BIOMARKERS.
SO BLOOD BIOMARKERS MIGHT AVOID
THE NEED FOR BIOPSY IN THESE
PATIENTS, POTENTIALLY PROGNOSIS
TREATMENT RESPONSE AND ALSO
IDENTIFY FACTORS AND PATHWAYS
INVOLVED IN THE PATHOGENESIS.
SO WE MEASURED KNOWN ANGIOGENIC
FACTORS IN THE PATIENT SERUM
USING ELISA.
WE FOUND VERY INTERESTING
DISCOVERY.
SO WE FOUND THAT IN TWO
PARTICULAR PATIENT GROUPS,
ANGIOPOIETIN 2 LEVELS,
ANGIOPOIETINS ARE ANGIOGENIC
FACTOR HIGHLY ELEVATED IN THE
BLOOD OF PATIENTS WITH KLA,
KAPOSIFORM HYMANGI ENDOTHELIOMA.
THE LABS WERE HIGHLY HE WILL
INVESTIGATED IN KLA PATIENTS
COMPARED TO PATIENT WITH
GENERALIZED LYMPHATIC ANOMALY
AND CONTROLS.
THERE'S CONTROVERSY WHETHER KLA
IS A MORE SEVERE FORM OF GLA BUT
THE LEVELS OF ANGIOPOIETIN 2
WERE HIGHER IN KLA PATIENTS AND
THE IDENTIFICATION OF MUTATIONS
IN THESE DISEASES NOW SUGGEST
THAT THEY ARE NOT DIFFERENT
SEVERITY FORMS.
SO WITH THE FINDING OF HIGHLY
ELEVATED ANGIOPOIETIN 2 LEVELS
THIS RAISED THE QUESTION OF WHAT
IS DRIVING THE ELEVATED
ANGIOPOIETIN 2 LEVELS IN THESE
KLA PATIENTS.
SO ABOUT TWO YEARS LATER AFTER
WE PUBLISHED THE ELEVATED
ANGIOPOIETIN 2 LEVELS, THIS
PAPER WAS PUBLISHED BY BARCLAY
ET AL AND THEY IDENTIFIED A
SOMATIC NRAS ACTIVATING MUTATION
IN THE KLA PATIENT LESIONS.
SO BY WHOLE EXOME SEQUENCING
THEY IDENTIFIED THE NRAS Q 61R
MUTATION IN 10 OUT OF 11 K,LA
PATIENTS.
THE ALLELIC FREQUENCY OF NRAS
MUTATION RANGED FROM ONE TO 28%.
SO NRAS IS A PROTO ONCOGENE IN
THE RAS GENE FAMILY THAT CODES
SMALL GTPASE THAT REGULATES CELL
PROLIFERATION.
MANY OF YOU MAY KNOW THIS NRAS Q
61R MUTATION IS FOUND IN GREATER
THAN 20% MELANOMAS, THYROID
CANCER AND OTHER TUMORS.
IT INCREASES MAP KINASE AND
KINASE SIGNALING PATHWAYS IN
UNCHECKED CELL PROLIFERATION.
SO AT THIS POINT WE KNEW THAT
KLA WAS CHARACTERIZED BY
SPINDLED EPITHELIAL CELLS THAN
NORMAL LYMPHATICS.
THE PAPER BY BARCLAY ET AL
IDENTIFY ID THIS MUTATION IN THE
PATIENT LESIONS.
AND WE FOUND THAT ANGIOPOIETIN 2
LEVELS WERE ELEVATED IN THE
BLOOD OF THE PATIENTS.
SO WE WERE INTERESTED IN
DETERMINING THE ROLE OF THIS
NRAS Q 61R MUTATION IN
PATHOGENESIS KLA.
TO ADDRESS THE QUESTIONS WE
DEVELOPED DOXY SIGH KLINE NRAS Q
61R AND WILD TYPE LENTO VIRAL
VECTORS.
YOU CAN SEE THE CONSTRUCTINGS
THE NRAS Q 61R VECTOR AT TOP AND
IN THE BOTTOM RIGHT HAND CORNER
THE NRS WILD TYPE EXPRESSING
VECTOR.
THESE ARE DOCK SIGH CYCLINE
INDUCIBLE VECTORS AND IT EBB
CODES RESISTANCE GENE.
SO WE PACKAGED THESE LENTIVIRAL
VECTORS INTO LEAN SHY VIRAL
PARTICLES AND USED THEM TO
TRANCES IF HE CAN HUMAN
ENDOTHELIAL PROGENITOR CELLS.
THESE ARE COMMONLY USED
ENDOTHELIAL CELL TYPE TO STUDY
ANGIOGENESIS.
SO WE ADDED PIRMICIN AND
SELECTED THE CELLS TAKEN UP NRAS
MUTANT OR WILD TYPE CONSTRUCTS.
TO INDUCE EXPRESSION OF MUTANT
OR WILD TYPE PROTEIN WE ADDED
DOXY CYCLINE TO THE MEDIA.
SO I'LL SHOW YOU DATA ON THE
CHANGES IN CELL SHAPE AND
SIGNALING PATHWAYS IN THESE
CELLS, MIGRATION OF
PROLIFERATION OF THE CELLS
ANGIOGENESIS MODELING IN V 20 IN
VIVO AND FINALLY SHOW YOU
EXPRESSION OF ANGIOPOIETIN 2.
SO TOP PANEL HERE, YOU CAN SEE
THAT WHEN WE ADD DOXY CYCLINE TO
THE MEDIA OF THE NRAS Q 61R
CELLS AND INDUCE NRAS BUT THE
CELLS BECOME SPINDLED AND CHANGE
SHAPE.
WHEREAS CELLS EXPRESS IN NRAS
WILD TYPE GENE DON'T.
WHEN WE QUANTITATED THAT BELOW
HERE, SO YOU CAN SEE THAT THE
NRAS EXPRESSING WILD TYPE CELLS
IN GREEN SO AND THEN THE NRAS
MUTANT CELLS IN RED.
SO YOU CAN SEE THAT THEY CHANGE
THEIR CIRCULARITY WHEN WE
MEASURE THE CIRCULARITY INDESK.
SO AT 24 AND 48 HOURS THE CELLS
EXPRESSING THE NRAS Q 61R MUTANT
PROTEIN BECOME SPINDLE AND
CHANGE CIRCULARITY.
WE ALSO LOOKED AT SIGNALING
PATHWAYS IN THE MUTANT CELLS.
YOU CAN SEE OUTLINED IN THE RED
BOX HERE THAT WE PARTICULARLY
SAW INCREASES IN PHOSPHOERG
WHICH IS PART OF THE MAP KINASE
PATHWAY, IN THE NRAS Q 61R
EXPRESSING CELLSN'T ON THE RIGHT
YOU CAN SEE A GRAPH WE CORRECTED
THE PHOSPHOERG TO TOTAL NRAS,
TWO DAYS AND 17 DAYS ON DOXY
CYCLINE WE SUSTAINED LEVELS IN
THE MUTANTS CELLS.
SO WE WENT TO LOOK AT MIGRATION
OF THE CELLS, SO YOU CAN SEE
HERE WE PLATE CELLS AND THEN
ALLOW THEM TO CONFLUENT, REMOVE
A CHAMBER TO EXPOSE A GAP ON THE
PLATE AND THEN CELLS MIGRATED
INTO THAT SPACE AND WE BLOCK
PROLIFERATION.
SO YOU CAN SEE THAT IN RED HERE
THAT THE NRAS Q 161R EXPRESSING
MUTANT CELLS MIGRATED FASTER
RATE AND CLOSED THAT GAP QUICKER
THAN NRAS WILD TYPE CELLS.
SIMILARLY WITH CELL
PROLIFERATION WE SAW THAT THE
NRAS K Q 61R MUTANT CELLS
PROLIFERATED HIGHER RATES THAN
THE NRAS WILD TYPE CELLS.
SO WE THEN WENT TO STUDY THE
NRAS WILD TYPE VERSUS MUTANT
CELLS, AND IN VITRO ANGIOGENESIS
ASSAY.
THIS WORK WAS DONE BY DR. SAM
AND LISA BUSCALO'S LAB.
YOU TAKE THE ENDOTHELIAL CELLS
AND COAT ON TO BEADS THEN THEY
SUSPENDED IN FIBRIN GEL WITH
FIBROBLASTS AT THE TOP.
WE ADDED DOXY CYCLINE TO THE
MEDIA AND IMAGED THE CELLS SEVEN
DAYS LATER.
SO HERE YOU CAN SEE CONFOCAL
IMAGING BUT YOU SEE THE CELLS
EXPRESSING THE WILD TYPE NRAS
FORM TUBES BETWEEN BEADS IN
GREEN.
THE NRAS Q 361R EXPRESSING CELLS
FORM LARGE DISORGANIZED
STRUCTURES.
WHEN YOU OUTLINE THESE STRUCTURE
AND THEN QUANTITATE IT, YOU CAN
SEE THAT THE VASCULAR AREAS
SIGNIFICANTLY INCREASE IN THE
CELLS EXPRESSING THE MUTANT NR
NRAS.
WE THEN DID IN VIVO MODEL
XENOGRAPH MODEL?
MICE.
WE SUSPEND IN MATRIGEL AND
INJECT ON THE SKIN OF NUDE MICE.
THEY WERE FED DOXY CYCLINE DIET
AND REMOVE THE MATRIGEL PLUGS
EIGHT DAYS.
YOU CAN SEE THE PLUGS REMOVED
AFTER 8 DAYS AND THE CELLS, NRAS
Q 61R CELLS DEVELOP LARGE PLUGS
THAT ARE BLOODY.
WHEN WE SECTION THROUGH THE
PLUGS HERE YOU CAN SEE IN THE
BOTTOM RIGHT HAND CORNER HERE
THAT NRAS Q 61R MUTANT CELLS ON
DOXY CYCLINE HAVE LARGE CHANNELS
THAT ARE BLOODY AND WE
QUANTITATED THAT ON THE RIGHT
AND YOU CAN SEE THE INCREASE IN
VASCULAR AREA.
>> TWO MINUTES REMAIN.
>> THANK YOU.
SO FINALLY WE LOOKED AT
EXPRESSION OF ANGIOPOIETIN 2 AND
FOUND THE EXPRESSION OF
ANGIOPOIETIN 2 BY WESTERN BLOT
ANALYSIS WAS SIGNIFICANTLY
INCREASE IN NRAS MUTANT CELLS
COMPARED TO WILD TYPE.
WHEN WE MEASURE ANGIOPOIETIN 2
IN CELL CULTURE YOU CAN SEE
MUTANT CELLS ARE SECRETING MUCH
HIGHER LEVELS OF ANGIOPOIETIN 2
INTO THE CELL CULTURE NEEDSIA.
NOW THIS SHOWS A NUMBER OF
MUTATIONS THAT WE IDENTIFIED
VARIOUS VASCULAR ANOMALIES AND
SIGNALING PATHWAYS DOWNSTREAM.
SO DOWNSTREAM NRAS WHICH IS
IDENTIFIED IN KLA IS BOTH MAP
KINASE SIGNALING AND PI 3 KINASE
SIGNALING PATHWAYS.
SO I WILL SHOW BRIEFLY SOME DATA
USING INHIBITORS UO 1D 6 AND
TRINATNIP AND RAPAMYCIN MTOR.
SO IN THIS WESTERN BLOT HERE YOU
SEE UO 126 BLOCKED PHOSPHOERG
LEVELS IN MUTANT CELLS AND
RAPAMYCIN BLOCKS DOWNSTREAM OF
PI 3 KINASE PATHWAY.
SO WE LOOK AT MORPHOLOGY OF THE
CELLS YOU CAN SEE THAT THE MAP
KINASE INHIBITOR UO 126 BLOCKS
THE SPINDLING OF THE NRAS MUTANT
CELLS WHICH RAPAMYCIN DOESN'T.
YOU CAN SEE THAT QUANTITATIVE
BELOW HERE WHERE UO 126 MAP
KINASE INHIBITOR BOXES THE
CHANGE IN CELL SHAPE.
WHEREAS RAPAMYCIN DOES NOT.
THEN WE LOOK AT EFFECT OF UO 126
IN TRIMATNIB INHIBITOR USED TO
TREAT PATIENTS.
YOU CAN SEE THE BOTH MAP KINASE
INHIBITORS BLOCK THE INCREASES
IN ANGIOPOIETIN 2 IN THE MUTANT
CELLS.
SO IN SUMMARY WHAT I HAVE SHOWN
YOU IS THAT THE NRAS Q 61R
MUTATION INTRODUCED TO HUMAN
ENDOTHELIAL CELLS INDUCES MAP
KINASE SIGNALING INDUCES A
SPINDLING OF THE ENDOTHELIAL
CELLS, AND INCREASES EXPRESSION
OF ANGIOPOIETIN 2.
SO FUTURE WE ARE INTERESTED IN
DETERMINING THE ROLE OF
ANGIOPOIETIN 2 AND
PATHOPHYSIOLOGY OF KLA.
SO IN SUMMARY I SHOWN YOU THESE
NRAS K 61R MODELS ARE USED TO
UNDERSTAND THE PATHOGENESIS OF
KLA AND TWO, POTENTIALLY SCREEN
NEW DRUGS AND TREATMENTS FOR
THIS DEVASTATING DISEASE.
THIS WORK WAS RECENTLY PUBLISHED
IN THE JOURNAL ANGIOGENESIS.
SO LASTLY, I WOULD LIKE TO
MENTION WE DEVELOPED A CLINICAL
ANGIOPOIETIN 2 TEST AT SINCENATY
CHILDRENS AND THIS CAN BE
ORDERED BY PHYSICIANS AND THEY
CAN SEND PATIENT SERUM TO OUR
HEMOSTASIS LABORATORY.
USUALLY RESULTS ARE REPORTED IN
TWO WEEKS.
SO THANKS TO ALL MY
COLLABORATORS, DR. DENISE ADAMS,
CHILDREN'S HOSPITAL
PHILADELPHIA, DR. LISA BUSCALO,
A LOT OF WORK DONE IN
COMBINATION WITH HER LAB,
DEVELOPED THE LENTIVIRAL VECTORS
TRISHA, SONDRA, GRIFFITH
MCDANIEL WHO JOINED THIS LAB,
DR. ADRIENNE AAMMILL AND THE
STEEL FAMILIES FOR SUPPORTING
THE WORK OVER SEVERAL YEARS.
FUNDING WAS FINAL THE LMI AND
LGDA ORIGINALLY AND MOST
RECENTLY FROM NATIONAL HEART
LUNG AND BLOOD INSTITUTE.
THANK YOU SO MUCH FOR YOUR
ATTENTION.
IN TERMS OF GABS NEEDS AND
OPPORTUNITIES, WE STILL NEED TO
IDENTIFY OTHER GENES AND
PATHWAYS AND FACTORS THAT
CONTRIBUTE TO THE PATHOGENESIS
OF THESE VASCULAR ANOMALIES.
WE HAVE MADE A GOOD START BUT
THERE'S CERTAINLY MORE GENES AND
PATHWAYS OUT THERE.
THAT COULD ALSO BE TARGETED FOR
TREATMENT.
WE DON'T KNOW HOW MUTANT
ENDOTHELIAL CELLS AFFECT NORMAL
ENDOTHELIAL CELLS.
WE NEED TO BE ABLE TO IDENTIFY
AND TARGET MUTANT ENDOTHELIAL
CELLS TO REDUCE SIDE EFFECTS OF
DRUGS ON NORMAL ENDOTHELIAL
CELLS.
WE ALSO NEED MORE NON-INVASIVE
DIAGNOSTIC TESTS FOR THESE
DISEASES.
THANKS SO MUCH.
I WILL TURN IT OVER TO DR.
MICHAEL DELLINGER.
YOU READY?
>> I AM.
THANK YOU, TIM.
I WOULD LIKE TO THANK THE
ORGANIZERS FOR GIVING ME THIS
OPPORTUNITY TO PRESENT MY LAB'S
WORK ON COMPLEX LYMPHATIC
ANOMALIES.
COMPLEX LYMPHATIC ANOMALIES ARE
RARE, SPORADICALLY OCCURRING
DISEASES THAT ARE CHARACTERIZED
BY PRESENCE OF MULTI-FOCAL
LYMPHATIC MALFORMATIONS.
COMPLEX LYMPHATIC ANOMALIES
INCLUDE GENERALIZED LYMPHATIC
ANOMALY, GORHAM SCOUT DISEASE,
KAPOSIFORM LYMPHANGIOMATOSIS AND
LYMPHATIC ANOMALY.
THESE DISEASES ARE DIAGNOSED IN
CHILDREN AND YOUNG ADULTS AND
ARE DIFFICULT TO TREAT.
THESE DISTINCT YET SIMILAR
DISEASE SHARE SEVERAL
OVERLAPPING FEATURES AND SOME OF
THE COMMON CHARACTERISTICS OF
KLA ARE LYMPHATIC MALL
FORMATIONS IN THE LUNG AND SOFT
TISSUE, INK INVASION OF BONE AND
COLLAR THORAX.
IN THE ANOMALY MY LAB IS
INTERESTED IN IS GORHAM STOUT
DISEASE.
IT IS CHARACTERIZED BY LYMPHATIC
INVASION OF BONE AND MASSIVE
BONE LOSS AND PATIENTS CAN HAVE
LYMPHATIC MALL FORMATIONS IN
SOFT TISSUES AND ORGANS.
SINCE FORHAM AND STOUT PUBLISHED
THEIR PAPER IN 1955, MORE THAN
300 CASES HAVE BEEN PUBLISHED.
INs THIS CASE REPORTS
INCREASED OUR UNDERSTANDING OF
THE CLINICAL FEATURES OF THE
DISEASE.
GORHAM STOUT DISEASE COURSE OF
THE DISEASE IS UNPREDICTSABLE IN
SOME PATIENTS THE OSTEOLYTIC
PROCESS OCCURS SLOWLY OVER
PERIOD OF YEARS, WHEREAS OTHERS
OCCURS RAPIDLY OVER MONTHS.
ONE INTERESTING FEATURE OF
DISEASE IS IT IS REPORTED TO
SPONTANEOUSLY ARREST AFTER A
PERIOD OF INACTIVITY.
NO EVIDENCE OF BONE FORMATION
AFTER THE DISEASE ARRESTS ONCE
THE BONE DISAPPEARS IT DOESN'T
REGENERATE.
THIS DISEASE CAN AFFECT ONE BONE
BUT USUALLY AFFECTS MULTIPLE
BONES.
WHEN MULTIPLE BONES ARE AFFECTED
THEY ARE TYPICALLY BONES NEXT TO
ONE ANOTHER.
WE DON'T TYPICALLY OBSERVE SKIP
LESIONS IN PATIENTS.
THE DISEASE CAN AFFECT ANY BONE
IN THE BODY BUT TYPICALLY
AFFECTS MOST FREQUENTLY AFFECTS
THE RIBS AND VARIETY BRAY.
AND UNFORTUNATELY THORACIC
INVOLVEMENT IS ASSOCIATED WITH
POOR PROGNOSIS BECAUSE PATIENTS
DEVELOP COLLAR THORAX WHICH
CAUSES RESPIRATORY DISTRESS,
FAILURE AND DEATH.
AT THE TIME WE STARTED OUR WORK
ON GORHAM STOUT DISEASE
MOLECULAR MECHANISMS DRIVING
LYMPHOANGIOGENESIS DRIVING THE
DISEASE ARE NOT KNOWN.
A LOT WAS KNOWN ABOUT MOLECULAR
MECHANISMS CONTROLLING THE
DEVELOPMENT OF LYMPHATIC VESSELS
AND OVER 20 YEARS VEGF C EMERGED
AS PRINCIPLE DRIVER OF LIMB FANG
OWE GENESIS.
COMPARISON OF VESSELS WHICH
CONVERSELY OVEREXPRESSION OF
VEGF C STIMULATES FORMATION OF
LYMPHATIC VESSELS.
BASED ON THOSE OBSERVATIONS
WHICH HYPOTHESIZE OVEREXPRESSION
OF VEGF C IN BONE CELLS WHICH
STIMULATE LYMPHATIC VESSEL
INVASION OF BONE AND BONE LOSS.
TO TEST THIS HYPOTHESIS WE BRED
MICE TOGETHER TO OVEREXPRESS
VEGF C IN OSTEOBLASTS,
OSTEOCYTES AND CHONDROCYTES.
WE FOUND THAT OVEREXPRESSION OF
VEGF C BY BONE CELL STIMULATED
LYMPHATIC INVASION OF BENEFIT
AND OSTEOCLAST MEDIATED BONE
RESORPTION.
WE ALSO FOUND INHIBITION OF VEGF
RECEPTOR 3 TO PREVENT INVASION
OF BONE AND BONE LOSS.
WE ALSO FOUND THAT INHIBITING
EXPRESSION OF VEGF C REVERSE
DISEASE IN MICE WITH ESTABLISHED
DISEASE.
WE THEN WENT TO FURTHER
CHARACTERIZE THE FORMATION OF
BONE ASSOCIATED LYMPHATICS AND
FOUND THESE LYMPHATICS DEVELOP
IN STEP WISE FASHION WHERE
REGIONAL LYMPHATICS GROW, BREECH
THE PERIOUTSIDE YUM, BECOME
ASSOCIATED WITH OSTEOCLASTS,
WHICH APPEAR TO CREATE A PATH
THROUGH THE BONE OR LYMPHATICS
UNTIL THEY REACH THE MARROW
CALFTY.
-- CAVITY.
WHILE WE WERE DOING THIS WORK WE
FORM AD COLLABORATION WITH MIKA
AND HIS GROUP SAW PATIENT
RECEIVED DIAGNOSIS OF GORHAM
STOUT DISEASE AFTER PRESENTING
WITH OSTEOLYTIC LESIONS IN
MALFORMATIONS.
BONES WERE AFFECTED IN THIS
PATIENT AND SHE WAS TREATED WITH
SEVERAL MEDICATIONS ALL WITH
MINOR OR NO RESPONSE.
AND UNFORTUNATELY THIS PATIENT
PASSED WHEN SHE WAS 37 YEARS
OLD.
TO TEST THE HYPOTHESIS THAT
GORHAM STOUT DISEASE IS CAUSED
BY GENETIC MY TALKS HEARSAY
GROUP SEQUENCED AFFECTED AND
UNAFFECTED TISSUE FROM PATIENT
AND IDENTIFIED SOMATIC
ACTIVATING MUTATION IN KRAS.
THE VARIANT IDENTIFIED IS G 12B
VARIANT, A WELL KNOWN ONCOGENIC
MUTATION IN KRAS.
KRAS IS A SMALL G PROTEIN THAT
FUNCTIONS AS MOLECULAR SWITCH,
WHEN BOUND TO GDP IT IS
INACTIVE, WHEN BOUND TO GTP IT
UNDERGOES A CONFIRMATIONAL
SWITCH WHICH ALLOWS INTERACTING
WITH DOWNSTREAM EFFECTOR
PROTEINS AND PROMOTES SIGNAL
TRANSDUCTION.
TWO RECOGNIZED BEST UNDERSTOOD
PATHWAYS DOWNSTREAM OF KRAS ARE
PI 3 KINASE AND MAP KINASE
PATHWAYS.
MUTATION THAT THE PATIENT HAD
IMPAIRS GTP HYDROLYSIS RESULTING
IN CONSTITUTIVE RAS SIGNALING.
TO DETERMINE THE EFFECT
HYPERACTIVITY RAS SIGNALING HAS
ON FORMATION OF LYMPHATIC
VESSELS, WE USE A CRE LOCK
SYSTEM TO EXPRESS MUTANT FORM OF
KRAS IN LYMPHATIC ENDOTHELIAL
CELLS.
THIS EXPERT WE FED NEWBORN MICE
TAMOXIFEN TO INDUCE EXPRESSION
OF KRAS LIMB LYMPHATIC
ENDOTHELIAL CELLS AND COLLECTED
BONE FROM MICE AT 7 WEEKS.
WE STAINED AND PUT INTO MARK
LYMPHATICS AND FOUND THAT BONES
FROM CONTROL ANIMALS DIDN'T
CONTAIN LYMPHATIC VESSELS,
WHEREAS BONES FROM OUR MUTANT
ANIMALS DID CONTAIN LYMPHATIC
VESSELS.
SO THESE RESULTS SUGGEST THAT
HYPERACTIVE RAS SIGNALING IN
LYMPHATIC ENDOTHELIAL CELLS
STIMULATE FORMATION OF LYMPHATIC
VESSELS IN BONE.
WHEN PERFORMING THIS EXPERIMENT,
WE FOUND TWO INTERESTING
OBSERVATIONS, FIRST WE FOUND
MUTANT ANIMALS DEVELOPED LARGE
CYSTS AND THESE CYSTS WERE
CONSISTENT WITH MACRO CYSTIC
LYMPHATIC MALFORMATIONS AND
THESE TYPICALLY OCCURRED IN THE
HIND REGIONS OF THE ANIMALS BUT
WE SAW THEM ALSO ON THE FACE.
WE ALSO FOUND THAT OUR MUTANT
ANIMALS HAD SHORTER LIFE SPAN
THAN CONTROL ANIMALS.
THIS IS BECAUSE OUR MUTANT
ANIMALS DEVELOP THORAX WHICH
CONTINUE RESPONSIBLE FOR THEIR
DEATH.
THAT'S ASSOCIATESSED WITH
LYMPHATIC VALVE DEFECTS SO THAT
LED US TO LOOK AT LYMPHATIC
VALVES IN MUTANT ANIMALS.
WE TREATED NEWBORN ANIMALS WITH
TAMOXIFEN AND COLLECTED EAR SKIN
FROM MICE AT 21 DAYS AND STAINED
FOR GFP TO MARK FOR LYMPHATIC
VESSELS.
WE FOUND THAT THE LYMPHATIC
NETWORK IN OUR MUTANT ANIMALS
WAS LESS COMPLEX THAN THAT IN
CONTROL ANIMALS SO THAT MUTANT
ANIMALS HAD SIGNIFICANTLY FEWER
LYMPHATIC BRANCH POINTS THAN
CONTROL ANIMALS AND LYMPHATIC
VESSEL DIAMETERS WERE GREATER IN
MUTANT ANIMALS AND CONTROL
ANIMALS.
WHEN WE LOOK AT LYMPHATIC
VALVES, WE FOUND MUTANT ANIMALS
HAD FEWER LYMPHATIC VALVES THAN
CONTROL ANIMALS SO THESE RESULT
SUGGEST HYPERACTIVE KRAS
SIGNALING IN LYMPHATIC
ENDOTHELIAL CELLS CAN AFFECT
FORMATION OF CELLS.
DO PATIENTS WITH GORHAM STOUT
HAVE THOSE EFFECTS?
THEY ARE LIMITED NUMBERS OF
STUDIES THAT CHARACTERIZED
LYMPHATIC FUNCTION IN PATIENT
WITH GORHAM STOUT DISEASE,
CONTRAST AGENTS ARE REPORTED TO
REFLUX IN THE BONES WITH
PATIENTS WITH GORHAM STOUT
DISEASE AND RETRO GRADE
LYMPHOWAS OBSERVED IN PATIENTS
WITH THE DISEASE.
THESE RESULTSES SUGGEST THAT
PATIENTS WITH THE DISEASE CAN
HAVE LYMPHATIC VALVE DEFECTS.
HYPE ACTIVE RAS SIGNALING IN
LYMPHATIC ENDOTHELIAL CELLS
AFFECT FORMATION OF LYMPHATIC
VALVES.
WE ASK WHETHER IT AFFECTS
MAINTENANCE OF LYMPHATIC VALVES.
WE EXAMINED THE VALVES IN
MESENTARY IN CONTROL OF THESE
ANIMALS BECAUSE THESE VESSELS
ARE REPORTED TO DEVELOP
LYMPHATIC VALVES DURING
EMBRYONIC DEVELOPMENT SO WE FED
NEWBORN MICE TAMOXIFEN TO INDUCE
EXPRESSION OF K, AREAS IN
LYMPHATIC ENDOTHELIAL CELLS AND
ISOLATED MESENTARY FROM 12 DAY
OLD MICE AND FOUND MUTANT
ANIMALS HAD FEWER LYMPHATIC
VALVES THAN CONTROL ANIMALS.
SO THESE RESULTS SUGGEST THAT
HYPERACTIVE RAS SIGNALING AFFECT
THE MAINTENANCE OF LYMPHATIC
VALVES.
WE THEN ASSESSED THE ABILITY OF
LYMPHATIC IN CONTROL MUTANT
ANIMALS TO PROMOTOR FORWARD
LYMPH FLOW.
WE INJECTED MESENTARYIC DYE IN
THORACIC DUCT.
YOU CAN SEE THE NICE APPEARING
NORA SICK DUCT.
WHEREAS MUTANT THE EVIDENCE DYE
WEPT UP AND REFLUXED TO THE
INTERCOSTAL LYMPHATICS.
SO THESE RESULT SUGGEST THAT
HYPERACTIVE RAS SIGNALING IN
CELLS CAN CAUSE RETRO GRADE
LYMPH FLOW.
NEXT WE ASSESSED TRAMATNIB ON
MICE.
IT IS AN FDA APPROVED MEK
INHIBITOR USED TO TREAT DISEASE
CAUSED BY HYPE ACTIVE RAS MAP
KINASE SIGNALING.
IN THIS EXPERIMENT, WE FOUND
THAT TRAMATNIB CAN PREVENT
LYMPHATIC VALVE LOSS IN OUR
MUTANT ANIMALS.
SO TO SUMMARIZE, IDENTIFIED
ACTIVATING MUTATION IN KRAS IN A
PATIENT WITH GORHAM STOUT
DISEASE, IN CONCURRENT WITH OUR
STUDY ODE GROUP IDENTIFIED
SOMATIC ACTIVATING MUTATION IN
KRAS IN A DIFFERENT PATIENT.
WE WENT ON TO SHOW THAT HYPE
ACTIVE KRAS SIGNALING IN
LYMPHATIC ENDOTHELIAL CELLS
LEADS TO FORMATION OF TOPIC
LYMPHATICS IN BONE AND HYPE
ACTIVE RAS SIGNALING AFFECTING
DEVELOPMENT AND MAINTENANCE OF
LYMPHATIC VALVES.
AND LASTLY,RAMATNIB CAN PREVENT
LOSS IN MUTANT ANIMALS.
WE PERFORMED THE FIRST MOUSE
MODELS.
THE FIRST GENERATION MODEL WAS
VEGF C TRANSGENIC MODEL USING
VEGFC TO DRIVE ACTIVATION OF
PATHWAYS MISREGULATED IN
PATIENTS WITH COMPLEX LYMPHATIC
ANOMALIES.
IN OUR SECOND GENERATION MODEL,
WE USED GENETIC TOOLS TO DRIVE
EXPRESSION OF MUTANT FORM OF
KRAS WHICH WE IDENTIFIED IN A
PATIENT TO PROMOTE DISEASE IN
MICE.
SO THESE ANIMAL MODELS ARE
HELPING US DEVELOP BETTER
UNDERSTANDING OF THE
PATHOPHYSIOLOGY OF LYMPHATIC
ANOMALIES AND OUR HOPE IS THAT
OUR WORK LEAD TO REPURPOSING OF
EPI APPROVED DRUGS AND ENABLE A
PRECISION MEDICINE APPROACH TO
THE TREATMENT OF COMPLEX
LYMPHATIC ANOMALIES.
WITH RESPECT TO GAPS
OPPORTUNITIES AND NEEDS, A BIG
QUESTION IN THE FIELD IS HOW IS
THE MICROENVIRONMENT ALTERED IN
LESIONS AND DOES IT CONTRIBUTE
TO DISEASE PROGRESSION?
SO BETTER UNDERSTANDING OF THE
CELLULAR MOLECULAR CHANGES IN
LESIONS COULD YIELD KNEW
TREATMENT TO USE IN COMBINATION
WITH TARGETED THERAPIES, ANOTHER
QUESTION IS HOW CLA POSITIVE
GENETIC MUTATIONS AFFECT
ENDTHELIAL CELL POPULATIONS AND
HOW DO MUTANT LECs AFFECT
NORMAL LECs.
ONE AREA WE ARE INTERESTED IN
EXPLORING IS AFFECT MUTANT
MUTATIONS HAVE WHETHER MUTATION
OCCURS IN A LIMB ANGIOBLAST OR
PART OF EXISTING VESSEL AND
LASTLY MORE VASCULAR ANOMALY
CENTERS ARE NEEDED FOR ADULT
PATIENTS. PATIENTS WITH A
COMPLEX LYMPHATIC ANOMALY ARE
SEEN AT VAS QUEUE ALREADY
ANOMALY CENTER AT CHILDREN'S
HOSPITALS, SO YOUNG ADULTS NEED
A PLACE TO GO TO CONTINUE THEIR
CARE AFTER THEY AGE OUT
CHILDREN'S HOSPITAL AND NEWLY
DIAGNOSED ADULT PATIENTS NEED
SPECIAL CENTERS WITHIN
EXPERIENCE MULTI-DISCIPLINARY
TEAM FOR THEIR CARE.
WITH THAT THANKS TO PEOPLE IN MY
LAB THAT WORKED ON THESE
PROJECTS.
MY COLLABORATORS AND FUNDING
RESOURCES.
THANK YOU FOR YOUR ATTENTION.
HAPPY TO ANSWER YOUR QUESTIONS.
>> THANKS VERY MUCH, MIKE.
WE WILL TAKE THE QUESTIONS AT
THE END SO WE'LL MOVE TO DR.
EDWARD SCHWARZ.
TAKE IT AWAY, DR. SCHWARZ.
>> HANKS SO MUCH TIM AND FOR THE
ORGANIZERS FOR GIVING ME THE
OPPORTUNITY TO TELL YOU ABOUT
ARTHRITIS.
THESE ARE MY DISCLOSURES.
SO RHEUMATOID ARTHRITIS WE TEACH
IS THE MOST PREVALENT AUTOIMMUNE
DISEASE AFFECTING UP TO 1%
POPULATION IN GENERAL AND AS WE
AGE PATIENTS OVER 65, 53 TO 5%.
THE -- 3 TO 5%.
THE ETIOLOGY REMAINS UNKNOWN BUT
THE DISEASE LARGELY MANIFESTSIST
BY INFLAMMATION IN AFFECTED
JOINTS, DRIVEN BY
PROINFLAMMATORY CYTOKINES INTO A
VICIOUS CYCLE AND RESULTS IN
THIS LARGE TISSUE WHICH WE CALL
PANES TISSUE WHICH NOW EXPERTS
AT OSTEOCLAST AT THE LEADING
EDGE THAT ERODE THE BONE AND
CARTILAGE.
NOW, I AM MY METEOROLOGIST BY
TRAINING AND NOBODY WANTED
RHEUMATOID ARTHRITIS TO BE AN
AUTOIMMUNE DISEASE MORE THAN ME.
THE PROBLEM IS THAT IT IS NOT.
SO MY LAB IS FOLLOWING AND
TESTING MULTIPLE HYPOTHESES TO
PROVE THAT THEORY.
WE KNOW THIS BECAUSE THE
AUTOIMMUNE HYPOTHESIS DOESN'T
FULFILL POSTULATES.
THERE ARE PATIENTS WITHOUT
AUTOANTIBODIES WHICH IS WHERE
AUTO-IMMUNITY TERM COMES FROM,
SERO NEGATIVE.
THERE ARE LOTS OF PEOPLE THAT
HAVE AUTOANTIBODIES, MOST DON'T
HAVE RHEUMATOID ARTHRITIS AND ON
AVERAGE PATIENTS DEVELOP
AUTOANTIBODIES AT LEAST TEN
YEARS BEFORE THEY DEVELOP ANY
SIGNS AND SYMPTOMS.
AUTOANTIBODIES FOUND IN PATIENTS
DON'T DIRECTLY INDUCE JOINT
INFLAMMATION AND DAMAGE
DIRECTLY.
THE AUTOANTIBODIES WE USE IN
ANIMAL MODELS RHEUMATOID
ARTHRITIS DO NOT EXIST IN
PEOPLE.
MOST SIGNIFICANTLY CLINICALLY
AND FOR THIS TALK, THE
AUTOANTIBODIES THAT EXISTED IN
RHEUMATOID ARTHRITIS, THE
TITLERS DON'T INCREASE, THERE'S
NO CHANGE IN AUTOIMMUNE PROFILE
DURING A FLAIR, AFFECTIVE
THERAPY THAT TREATS RHEUMATOID
ARTHRITIS DOES NOT DECREASE
AUTOANTIBODY TITERS.
THERE IS A FUNDAMENTAL
DISCONNECT BETWEEN THAT.
STARTING AS POST DOC 30 YEARS
AGO, STILL TODAY I USE TNF
TRANSGENIC MOUSE MODEL,
PRECURSOR TO THE ONE YOU ALREADY
HEARD THIS MORNING.
IT DOESN'T HAVE INFLAMMATORY
BOWEL DISEASE BUT CHRONIC LOW
LEVEL TNF EXPRESSION WHICH LEADS
IN TO THE JOINT DESTRUCTION YOU
CAN SEE HERE IN THE KNEE JOINT
OF ME IN THIS CUSS, TIBIAL AND
FEMUR SIDES.
SO THIS UNABATING MODEL WAS MOST
INTERESTING TO ME BECAUSE IT
LOOKS MOST LIKE RHEUMATOID
ARTHRITIS.
AND OF COURSE THE GREAT
VALIDATION OF IT CAME FROM W THE
ANTI-TNF THERAPY.
AROUND 2006 WE WERE STILL
WORKING ON THIS MODEL AND MOST
TNF SIGNALING HAVE BEEN
ELUCIDATED BY VARIOUS FIELDS.
SO THERE IS ONE QUESTION
REMAINING FOR US TO ANSWER IN
TERMS OF UNDERSTANDING THE ROLE
TNF AND OSTEOCLAST GENESIS AND
JOINT DESTRUCTION THIS THIS
MODEL.
THAT WAS WHAT WAS THE FACTORS
THAT RELEASING THESE OSTEOCLAST
PRECURSORS FROM BONE MARROW BY
TNF.
I THINK STILL TODAY WHEN STUCK
YOU DO A MICROARRAY OR SPATIAL
TRANSCRIPTOMICS SO THIS WORK WAS
DONE BY LEEANN'S LAB WHERE SHE
DID MICROARRAY ANALYSIS OF THESE
BONE MARROW DERIVED OSTEOCLAST
PRECURSORS AND BLOOD CELLS FROM
THE WILD TYPE AND TNF MICE THE
FACT SHE FOUND THE GENE FOUND
MOST LIELY INDUCED IN OSTEOCLAST
PRECURSORS WAS VEGF C WHICH ALL
OF YOU KNOW IS LYMPHATIC GROWTH
FACTOR.
THAT ESSENTIALLY CHANGED THE
DIRECTION OF OUR LABORATORY EVER
SINCE.
HER LAB WENT TO SPECIFICALLY
SHOW THAT VEGF C IS HIGHLY
EXPRESSED IN OSTEOCLAST
PRECURSORS AND OSTEOCLAST TNF
TRANSGENIC MICE AND PATIENTS
WITH RHEUMATOID ARTHRITIS WITH
FOCAL EROSIONS AN THESE ARE THE
OSTEOCLASTS THAT THE -- IN THE
JOINT ERODING THE BONE AWAY.
THE NEXT APPROACH THAT WE
DECIDED TO DO IS IF YOU STUDY
FLAIR YOU HAVE TO HAVE OUTCOME
MEASURES WHAT HAPPENS BEFORE THE
FLAIR DURING THE FLAIR AND AFTER
THE FLAIR.
SO I WAS BREADS TO HAVE STEVEN
PRU AS Ph.D. SUNT IN MY LAB,
YOU KNOW HIM BETTER THAN ME AND
HE'S MADE SEVERAL MAJOR
CONTRIBUTIONS TO THIS FIELD, THE
FIRST IS DEVELOPING LONGITUDINAL
CONTRAST ENHANCED MRI FOR THE
MOUSE.
THIS IS ACTUALLY UTILIZING THE
CLINICAL PRE-TEST THE MAGNET
DEVELOP SURFACE COILS THE MOUSE
CAN GO INTO CLINICAL INSTRUMENT.
AT THAT TIME WE ARE LOOKING TO
REALLY 3-D RENDERING OF SYNOVIAL
TISSUE IN THESE MICE WITH ANKLE
ARTHRITIS, YOU CAN SEE HE
SUCCEEDED IN THAT BY GENERATING
THIS CONTRAST ENHANCEMENT WHICH
ENABLES THE 3-D REMEMBER
DEERING.
-- RENDERING.
I TOLD HIM HE HAD A TUMOR IN HIS
MOUSE, I HAD NO IDEA WHAT THIS
WAS, PROBABLY SURE MOST KNOW
THIS THE POPLY TEAL LYMPH NODE.
SO -- THIS DRAINS THE ANKLE SO
HE WENT TO DEVELOP ADDITIONAL
OUTCOME MEASURES INCLUDING 3-D
CT AND NEAR INFRARED ICG IMAGING
WHICH I'M SURE WE WILL HEAR MORE
ABOUT DURING THIS MEETING WHICH
THIS SIMPLE FLUORESCENT DYE IS
INJECTED IN THE FOOT PAD OF THE
MOUSE AND TRAVELS THROUGH THE --
TO DOMINANT LYMPHATIC VESSELS TO
THE POPLITEAL LYMPH NODE AND
PERFORMED REAL TIME TO QUANTIFY
VARIOUS METRICS OF IT INCLUDING
THE CONTRACTIONS WHICH ARE
OBVIOUSLY INCREASED IN THE
SETTING OF INFLAMMATION AND
OTHER ASPECTS I WILL TALK ABOUT
IN A LITTLE BIT.
HAVING DEVELOPED THESE THREE
LONGITUDINAL OUTCOME MEASURES,
STEVE WAS ABLE TO IDENTIFY SOME
VERY INTERESTING BEHAVIORS IN
THIS MOUSE MODEL OF CHRONIC
INFLAMMATORY EROSIVE ARTHRITIS.
WHAT HE FOUND IS THAT IN THE
BEGINNING OF THE DISEASE, BEFORE
THERE IS ANY INFLAMMATION IN THE
KNEE JOINT HERE POPLITEAL LYMPH
NODE GETS LARGE AND CONTRAST
ENHANCING.
AND THEN ALL OF A SUDDEN
SPONTANEOUSLY HE OBSERVE IT IS
COLLAPSE OF THAT LYMPH NODE SUCH
THAT IT IS NO LONGER TAKING UP
CONTRAST AND SIMULTANEOUS WITH
THAT WE COULD SEE THE FLAIR AS
EVIDENCED BY IMAGING OF THE MRI
OF THE SYNOVIAL.
THIS IS AN EXAMPLE OF THE
LONGITUDINAL DATA IN WHICH YOU
CAN SEE EXPANSION OF THE LYMPH
NODE BOTH IN SIZE AND CONTRAST
ENHANCEMENT AND THEN SUDDEN
DECREASE IN THAT WHICH WE CALL
THE COLLAPSE.
HERE YOU CAN SEE THERE IS NO
KNEE ARTHRITIS DURING THIS
INFLAMMATORY PERIOD UNTIL THE
COLLAPSE OCCURS.
YOU CAN SEE OF COURSE THE
INDIVIDUAL IMAGES OF THIS, SO
DURING THE EXPANDING PHASE YOU
HAVE DRAINAGE FROM FOOT PAD.
NOT MUCH ARTHRITIS.
YOU HAVE A LARGE LYMPH NODE WITH
LARGE SINUS SPACES, AND VERY FEW
OSTEOCLASTS AND DURING THE
COLLAPSE THERE IS NO MORE
DRAINAGE THE LYMPH NODE IS
SMALL, THIS IS THAT TISSUE I WAS
TELLING YOU ABOUT AND THESE ARE
ALL THE OSTEOCLASTS DEVOURING
THE BONE.
THE LAST INTERESTING POINT ABOUT
IMAGING AND TNF MOUSE MODEL I
PREFERENTIALLY USE AMONG ALL
ELSE, BOTH THINGS CAN BE
HAPPENING IN THE SAME ANIMAL AT
THE SAME TIME.
SO THIS IS A MOUSE WHICH IN LEFT
LEG EXPANDING WITH NO EVIDENCE
OF ARTHRITIS AND EROSION AND AT
THE SAME TIME RIGHT LEG HAS
COLLAPSED POPLITEAL LYMPH NODE
WITH A KNEE FLAIR IN TERMS OF
SYNOVITIS AND FOCAL EROSIONS.
HAVING DONE THIS NOW WENT TO
CHARACTERIZE WHAT IS GOING ON IN
THESE LYMPH NODES, I WILL
SUMMARIZE A LOT OF IN SITU AND
IN VITRO AND EXVIVO DATA TO SAY
THE MAJOR FINDING OF THIS IS
EXPANSION OF B CELLS.
SO IN THE EXPANDING LYMPH NODE
THESE ARE THE FOLLICLES WHERE
NON-ACTIVATED B CELLS SHOULD BE
LIVING.
THESE ARE VERY LARGE SINUS
SPACES, THE ONLY CELLS WE
PRIMARILY FIND IN THE SINUSES
ARE ACTIVATED MACROPHAGES COMING
FROM INFLAMED JOINTS.
THEN COLLAPSE PHASE IT APPEARS B
CELLS MIGRATED OUT OF FOLLICLES
AND ARE NOW CLOGGING THE
SINUSES.
IN COLLABORATION WITH ANDREA WHO
IS A BONA FIDE CELL
IMMUNOLOGIST, HE SCREENED THESE
B CELLS BY HUNDREDS OF SURFACE
MARKERS AND IDENTIFIED THIS
PHENOTYPE WHICH HE TERMED B CELL
AND INFLAMED LYMPH NODES OR BN
CELLS, THAT'S WHAT IMMUNOLOGISTS
DO.
BUT AGAIN, AUTOIMMUNE HYPOTHESIS
FAILS IN THAT ANY EXPERIMENT WE
CAN DO IN BULK WE FOUND NO
SIGNIFICANT DIFFERENCES IN THE
NUMBER OF THESE B CELLS, THERE
WERE NO ACTIVATION MARKERS, B
CELLS ARE POLYCLONAL.
THEY DON'T HAVE MARKERS FOR
PROLIFERATION OR CHANGES IN
CHEMOKINES OR CHEMOKINE
RECEPTORS.
SO THE SOLUTION WAS THEY WERE
PLAYING THAT PASSIVE ROLE IN
COLLAPSE AND ARTHRITIC FLAIR.
THE NEXT ON THE SCENE WAS A
Ph.D. STUDENT IN MY LAB WHO
DEVELOPED THIS IMMUNOFLUORESCENT
INTRAVITAL MICROSCOPY OF
POPLITEAL LYMPHATIC VESSELS
INJECTING FLUORESCENTLY LABELED
ANTI-CD 11B ANTIBODY INTO THE
FOOT PAD, YOU CAN SEE FROM THE
REAL TIME VIDEO, THE MACROPHAGES
ARE FLYING THROUGH THIS
POPLITEAL VESSEL BEING EXPANDED
BUT NO MOVEMENT AT ALL IN THE
COLLAPSED.
SO BECAUSE WE KNEW THERE WERE B
CELLS IN THE LYMPH NODE THAT WE
PROPOSED THAT WERE CLOGGING AND
BECAUSE ANTI-CD 20 IS AN FDA
APPROVED DRUG FOR PATIENTS WHEN
THEY FAIL ANTI-TNF THERAPY, IT
IS CALLED RETUXIMAB, IF YOU
TREAT THESE TNF MICE WITH
PLACEBO OBVIOUSLY THEY WILL
FLAIR.
BUT IF YOU TREAT THEM WITH
ANTI-TNF -- ANTI-CD 20 THEY WILL
NOT FLAIR.
MORE INTERESTINGLY HE SHOWED IF
YOU WAIT FOR MICE TO FLAIR,
MEANING THEY HAVE A COLLAPSE OF
THE LYMPH NODE, THEY HAVE THE
LARGE SYNOVITIS AND ICG ISN'T
DRAINING, TWO WEEKS AFTER B CELL
DEPLETION THERAPY THE FIRST SIGN
IS INCREASE IN GADOLINIUM WHICH
IS OCCUPYING THE SINUS SPACES
THAT ARE CLEARED OF B CELLS.
THE SYNOVITIS IN THE KNEE MELTS
AWAY AND YOU RECOVER THE
LYMPHATIC FLOW FROM THE FOOT
PAD.
SHE ALSO WENT ON TO SHOW EVEN
THOUGH THIS IS ANTI-CD 20
DOESN'T ACTUALLY TOUCH THESE
MACROPHAGES, THIS IS A B CELL
SPECIFIC MARKER.
THESE MACROPHAGES APPEAR STUCK
OR ELIMINATED FOLLOWING ANTI-CD
20 THERAPY.
BUT SHE DID GO ON TO SHOW THAT
EVEN THOUGH YOU RESTORE SOME
FLOW YOU DO NOT RESTORE
CONTRACTIONS.
SO THIS WAS PRESUMED TO BE
PASSIVE FLOW IN ORDER TO
UNDERSTAND THAT BETTER, ECHO AND
KAREN BENTLEY PERFORMED
TRANSMISSION ELECTRON MICROSCOPY
TO CHARACTERIZE AND CROSS
SECTIONAL ANALYSIS THE NATURAL
HISTORY OF WHAT HAPPENS.
THESE ARE ACTIVATED MACROPHAGES
THAT ARE INITIALLY ADHERING.
WE CAN SEE DEGENERATION OF THE
ENDOTHELIAL CELLS IN SMOOTH
MUSCLE AND ULTIMATELY APOPTOSIS
OF THE CELLS.
THEN JUST LIKE EVERYBODY ELSE WE
JUMPED ON A PLANE AND WENT TO
COLUMBIA TO MIKE DAVIS' LAB AND
WORK WITH JOSH TO CAN LATE
VESSELS FROM WILD TYPE TNF
TRANSGENIC MICE WITH EXPANDING
LYMPH NODES AND THE COLLAPSE AND
YOU CAN SEE THAT THIS IS AN
INTRINSIC PHENOTYPE TO THE
VESSELS THAT SEEM TO BE DAMAGED
FROM THE CHRONIC INFLAMMATION.
SO MOST RECENTLY MARK AND UA
PAGE IN THE LAB HAVE DONE AN
EXPERIMENT THE SEE IF THIS IS
REVERSIBLE WITH THE ANTS TNF
THERAPY.
THEY TREATED MICE WITH DISEASE
WE ALREADY KNEW WE HAD LOST
LYMPHATIC SMOOTH MUSCLE ACTIN ON
THESE ANTI-TNF THERAPY DOESN'T
RESTORE THAT.
WE LOOKED CAREFULLY TO SEE IF WE
CAN SEE ANY REPAIR REGENERATION
BY BRD LABELING, WE DIDN'T SEE
EVIDENCE OF REPAIR REGENERATION.
OF THESE VESSELS.
THOUGH WE CAN SEE REPAIR AND
REGENERATION OF THE BONE.
LASTLY I WOULD LIKE TO POINT OUT
WITH PUBLISH PILOT STUDIES
EVERYTHING IN THE TNF MICE WE
ARE ABLE TO RECAPITULATE IN
HUMANS THIS IS A PATIENT UPON
INITIAL DIAGNOSIS OF RHEUMATOID
ARTHRITIS, YOU CAN SO E THE
INFLAMED SYNOVIUM BONE MARROW
EDEMA WHERE EROSIONS ARE AND THE
LARGE LYMPH NODES AND THIS IS AN
RA PATIENT PRE-TOTAL KNEE
REPLACEMENT AFTER 32 YEARS
DISEASE.
THE SYNOVIUM AND IF YOU WEREN'T
LOOKING CAREFULLY YOU WOULDN'T
FIND THESE COLLAPSED LYMPH
NODES.
RESECTION OF THOSE LYMPH NODES
CONFIRM THEY DO HAVE THE SAME BN
CELLS.
LASTLY WITH THE HELP OF CHRIS
RICHMOND, RICHARD BELL AND RON
WOOD WE COMPLETED A PILOT STUDY
OF ICG IMAGING IN HEALTHY
CONTROLS AND RHEUMATOID
ARTHRITIS PATIENTS AND WERE ABLE
TO CONFIRM THE LYMPHATIC DEFECTS
IN THE HANDS OF PATIENTS WITH
FLARE.
IN SUMMARY, WE CURRENTLY HAVE A
MODEL OF IMPORTANCE OF
LYMPHATICS IN PARTICULAR
LYMPHATIC FLOW.
IN INDUCTION OF FLARE.
WE BELIEVE RHEUMATOID ARTHRITIS
IS A COMPLICATED DYNDROME, IT IS
POSSIBLE NO TWO PATIENT VERSUS
THE SAME DISEASE BUT IT MAN
FASTS IN CHRONIC INFLAMMATION IN
THE JOINTS AND THE LYMPHATICS
ANDLY FANG OWE GENESIS IS A
PROTECTIVE MECHANISM TO REPAIR
THE JOINT WHICH WORKS FOR MANY
YEARS.
DUE TO THE CHRONIC INFLAMMATION
AND BREAK DOWN OF VESSELS, THE
LYMPHATIC FLOW IS LOST, WE HAVE
STAGNATION OF THESE ACTIVATED
MACROPHAGES THAT PRODUCE AMONG
OTHER THINGS CXCL 13 WHICH
RECRUIT B CELLS IN THIS MODEL
INCLUDES UNPUBLISHED SPATIAL
TRANSCRIPTOMIC DATA WHICH WE
IDENTIFIED IMPORTANT P CELL AND
PLASMA CELL DIFFERENTIATION AS
BEING INVOLVED IN THIS.
SO THIS WORK IS PURSUING.
WITH THAT I WOULD LIKE TO THANK
A LONG LIST OF COLLEAGUES AND
COLLABORATORS, I WOULD LIKE TO
POINT OUT I'M LARGELY FUNDED BY
THE NATIONAL INSTITUTE OF
ARTHRITIS AND MUSCULOSKELETAL
SKIN DISEASE.
THAT IN TERMS OF THE THREE
QUESTIONS I WAS ASKED TO ADVANCE
THE FIELD, BE VERY DIFFICULT TO
MOVE FORWARD WITHOUT FIRST AND
FOREMOST TRANSGENIC MODELS WITH
CONDITIONAL INDUCIBLE CRE IN
LYMPHATIC MUSCLE CELLS,
LYMPHATIC MUSCLE CELL LINES IN
MICE, AND ICG IMAGING, THE
CLINICAL IMAGING WE NEED SOME
MEANS TO DEVELOP IT INTO MORE
QUANTITATIVE SYSTEM.
WITH THAT, I'M HAPPY TO ANSWER
ANY QUESTIONS.
>> THANKS VERY MUCH, DR.
SCHWARZ.
WE WILL TAKE THE QUESTIONS AT
THE END SO WE'LL MOVE TO DR.
QUAGGIN.
>> THANKS, TIM.
JUST GOING TO FIND MY CAN YOU
SEE MY SLIDES?
>> WE CAN.
>> TERRIFIC.
THANK YOU, VERY MUCH, I WOULD
LIKE TO THANK THE ORGANIZERS FOR
INCLUDING ME IN THIS WORKSHOP
THAT'S BEEN ABSOLUTELY FANTASTIC
TODAY AND I WAS ASKED TO TALK
ABOUT LYMPHATICS IN THE KIDNEY.
WHAT I THOUGHT I WOULD DO IS TO
GIVE A RELATIVELY BROAD HIGH
LEVEL OVERVIEW AND FLAVOR WHERE
WE ARE WITH PARTICULAR FOCUS HOW
WE MIGHT BE ABLE TO MODULATE
LYMPHATICS TO TREAT KIDNEY
DISEASE.
IN MY OWN GROUP VASCULAR
BIOLOGIST AS WELL AS NEFFOLOGIST
AND WE ARE VERY INTERESTED IN
THE VASCULATURE IN THE KIDNEY.
DURING DEVELOPMENT AND IN
DISEASE.
WHAT I WANT YOU TO LEAVE HERE
AFTER THE NEXT 13 MINUTES OR SO
IS REALLY UNDERSTANDING THAT
THERE IS INCREDIBLE
HETEROGENEITY OF ENDOTHELIAL
CELLS WITHIN THE KIDNEY AND
DEVELOPMENT IN DISEASE
LYMPHATICS AND BLOOD.
THAT MODULATING KIDNEY
LYMPHATICS CAN AFFECT OUTCOME
FROM MULTIPLE DIFFERENT KIDNEY
DISEASES AND LYMPHATICS ARE
PRETTY EXCITING CANDIDATE
THERAPEUTIC TARGETS.
SO TO INTRODUCE YOU TO KIDNEY WE
HEARD ABOUT SOME OTHER ORGANS
TODAY.
THE KIDNEY IS PRETTY AMAZING
ORGAN, THE SIZE OF YOUR FIST, IT
RECEIVES ABOUT 25% CARDIAC
OUTPUT SO IT IS INCREDIBLY
VASCULAR ORGAN.
WHEN WE THINK ABOUT THE NORMAL
FUNCTION, THIS IS JUST A
SCHEMATIC OF A SPECIALIZED
MICROVASCULAR BEDS, THE
GLOMERULAR.
YOU HAVE ON THE ORDER OF A
MILLION OF THESE IN YOUR TUBE
KIDNEY, AND PRODUCE ON THE ORDER
OF 18 180 LITERS OF FILTRATE EAH
DAY.
YOU DON'T EXCRETE AT YOUR END SO
IT REQUIRES A COMPLEX NETWORK OF
SPECIALIZED MICROVASCULAR BEDS
TO ABSORB THE FLUID, SOLUTES AND
OTHER SUBSTANCES.
THE CORTEX OUTER PART OF THE
KIDNEY YOU HAVE PERITUBULAR
CAPILLARIES AND IN THE MEDULLA
OR INNER PART OF THE KIDNEY,
IMPORTANTLY FOR THIS GROUP
THERE'S NO CLASSIC LIMB
LYMPHATICS, BUT YOU DO HAVE
DESCENDING AND ASCENDING
VASORECK TIN AND THIS IS WHERE
YOU FINE TUNE SOLUTES, EXCRETION
AS WELL AS RESORB A LOT OF WATER
AND WATER BALANCE.
KIDNEY HAS A LOT OF ADDITIONAL
ROLES, REGULATING BLOOD PRESSURE
THROUGH THE RAS SYSTEM AND
PRODUCING HORMONES TO REGULATE
BLOOD CELL DEVELOPMENT AS WELL
AS (INAUDIBLE).
SO IF WE OVERLAY LYMPHATICS NOW,
WHICH WAS SHOWN IN DARK BLUE IN
THIS SCHEMATIC YOU CAN NOTICE
LOTS SPARSER IN THE BLOOD
VASCULATURE, THEY ARE ABSENT
FROM THE MEDULLA.
PRESENT IN THE CORTEX.
THEN TRACK ALONG ARTERIALS AND
WE THROUGH THE HYLUM.
THE PAST DECADES WE CAN
RECOGNIZE THE DIFFERENT VASCULAR
BEDS THROUGH HISTOLOGIC METHODS
BUT NOT REALLY UNTIL OMICS CAME
ON THE SCENE THAT WE COULD
REALLY START TO UNDERSTAND THE
MOLECULAR FINGER PRINTS OF THESE
DIFFERENT VASCULAR BEDS.
THIS IS JUST AN IMAGE FROM ONE
OF SHAHIM'S PAPER WHERE HE'S
LATED ENDOTHELIAL CELLS FROM
DEVELOPING ADULT KIDNEY ABLE
DEMONSTRATE MULTIPLE CLUSTERS
AND INDIVIDUAL MOLECULAR
FINGERPRINTS FOR DIFFERENT
ENDOTHELIAL SUBPOPULATIONS.
IMPORTANTLY, MOST OF THE DATA
SETS OUT THERE INCLUDING FROM
HUMAN DATA SETS THAT CLUMP
ENDOTHELIAL CELLS TOGETHER BUT
NOTICE IN THIS DATA SET, WE ARE
MISSING LYMPHATIC ENDOTHELIAL,
BUT BECAUSE THEY ARE SPARSE THEY
ARE HARD TO FIND IN SINGLE CELL
DATA SETS SO THIS IS A SLIDES
THAT WAS PROVIDED BY A NEW
INVESTIGATOR AT NORTHWESTERN AND
HE DEVELOPED A NUMBER OF NEW
TRANSGENIC MOUSE MODELS WHERE HE
IS ABLE TO BOOST DEVELOPMENT OF
LYMPHATICS IN THE BOTTOM HERE.
WITHOUT MORPHOLOGY OF KIDNEY.
YOU CAN DO IT IN CELL AND TIME
SPECIFIC WAYS.
BECAUSE HE'S BEEN ABLE TO BOOST
THIS POPULATION WITHIN KIDNEY,
WHEN HE PERFORMS SINGLE CELL
ANALYSIS HE IS ABLE THE IDENTIFY
DIFFERENT CLUSTERS OF BLOOD
ENDOTHELIAL CELLS BUT VARIOUS
POPULATION OF KIDNEY SPECIFIC
LYMPHATIC ENDOTHELIAL CELLS AND
HE INCREASED PROPORTION.
NOT ONLY GIVING HIM THE ABILITY
TO LOOK AT MOLECULAR
FINGERPRINTS IN HEALTH AND
DISEASE IN THE KIDNEY BUT ALSO
ALLOWS HIM TO USE THIS DATA SET
TO INTERROGATE OTHER DATA SETS
AND PULL OUT SMALL POPULATIONS
OF BLOCKS IN PUBLISHED HUMAN AND
OTHER CLINICAL MODELS.
I WANT TO TALK ABOUT DEVELOPMENT
OF THE LYMPHATICS IN THE KIDNEY,
AND WE HAVE HEARD A LOT OFFER
BEAUTIFUL TALKS EARLIER TODAY.
THIS IS AN IMAGE FROM ONE OF DR.
DANIEL JAFFRY AND DAVID LONGS
WONDERFUL PAPERS AND THEY HAVE
DONE MOST OF THE WORK LOOKING AT
THE DEVELOPMENT OF LYMPHATICS IN
THE KIDNEY USING IMAGING AND
OTHER TECHNIQUES.
WHAT THEY WERE ABLE TO SHOW IS
LYMPHATIC DEVELOPMENT STARTS
AFTER THE BLOOD ENDOTHELIAL
DEVELOPMENT, FOUR DAYS INTO META
OR ADULT KIDNEY DEVELOPMENT IN
THE MOUSE EMBRYO.
IT BEGINS FROM CLUSTERS OF THE
LYMPHATIC CELLS WITHIN THE
KIDNEY, AROUND THE HYLUM AND
EXPANDS UP OVER THE FOLLOWING
FOUR DAYS.
I WANTED TO PROVIDE DATA FROM
OUR OWN GROUP, WE HEARD IN TIM'S
TALK ABOUT ANGIOPOIETIN 2, WE
WORK ON VASCULAR TYROSINE KINASE
SIGNALING PATHWAYS AND CLEARLY
IMPORTANT BOTH FOR BLOOD AND
LYMPHATIC VASCULAR DEVELOPMENT
AND OVER THE YEARS WE DEVELOPED
A NUMBER OF DIFFERENT MOUSE
MODELS SO WE CAN MANIPULATE THE
LIGANDS AND RECEPTORS IN TIME
CELL SPECIFIC WAYS.
ONE BIG SURPRISES WE HAD WHEN
REMOVED MOST BOTH MAJOR
ANGIOPOIETIN LIGANDS, THEY
BREED, THEY APPEAR RELATIVELY
OKAY EXCEPT THEY DO HAVE
INTERESTING PHENOTYPES.
THESE ARE BULGING PHENOTYPES AS
WE CALL IN THE EYES OF THE MOUSE
AND ALSO IN THE KIDNEY.
I'M EMBARRASSED TO SAY THOUGH
I'M NEFFOLOGIST, WE FIGURED OUT
THE I PHENOTYPE FIRST DUE TO
BULGING OF THE ANTERIOR CHAMBER
BECAUSE MICE ARE LACKING
CIRCULAR VESSEL SHOWN IN GREEN
HERE, THIS VESSEL IS LYMPHATIC
LIKE, IT IS DESCRIBED AS A
HYBRID VESSEL THAT DERIVE THE
VENUS VEINS AROUND THE EYE.
THERE ARE SPROUTS TIP CELLS
AGGREGATE AND WHEN CANLYZES IT
STARTS TO TAKE ON LYMPHATIC FATE
SO IT STARTS TO EXPRESS PROX 1
VEGF RECEPTOR 3 BUT CONTINUES TO
EXPRESS BLOOD ENDOTHELIAL
MARKERS AND NEVER EXPRESSES LIVE
ONES SO DOESN'T GO ALL THE WAY,
A TRUE HYBRID VESSEL.
CLEARLY IN OUR MICE THEY ARE
COMPLETELY MISSING, THIS BIG
JUICY SCHLEMM'S CANAL AND THE
MICE HAVE A SEVERE GLAUCOMA FORM
WHICH TURNS OUT TO BE RELATIVELY
SIGNIFICANT CAUSE OF CONGENITAL
GLAUCOMA IN CHILDREN.
AFTER WE FIGURED OUT THE EYE
PHENOTYPE, WE WERE ABLE TO GO
BACK AND LOOK AT THESE KIDNEYS,
AND AS YOU CAN SEE THERE ARE
CYSTS IN KIDNEYS BUT NOT TUBULAR
CYSTS, INSTEAD THEY ARE
INTERSTITIAL SPACES THAT ARE
FILLED WITH FLUID.
THE REASON THEY DEVELOP THESE
BULGING KIDNEYS IS BECAUSE THEY
ARE MISSING THE ASCENDING
VASORECTA.
IN THE MEDULLA OF THE KIDNEY NO
CLASSIC LYMPHATICS THERE'S
ASCENDING VASORECTA THAT LACK
COVERAGE BUT ARE FIN STRAIT AND
HAVE FLOW, MOLECULARLY IDENTICAL
TO SCHLEMM'S CANAL SO THEY TAKE
ON MARKERS, PHLOX 1 VEGFR 3 BUT
NEVER EXPRESS POTOPLATIN.
SO SIMILAR TO SCHLEMM'S CANAL,
ANOTHER SWEAR MEDIATE WITH
ENDOTHELIAL CHARACTERISTICS.
IN THE FINAL FEW MINUTES I
WANTED TO TURN TO THE ROLE OF
LYMPHATICS IN KIDNEY DISEASE.
THERE'S BEEN A LOT OF WORK ON
THE ENDOTHELIUM AND KIDNEY
DISEASE MOSTLY WITH DROP OUT
BLOOD ENDOTHELIUM, LOTS OF
PHARMA INTERESTED IN IF BOOSTING
BLOOD OR VASCULAR CAPILLARY
DEVELOPMENT TO PROTECT KIDNEY.
CONVERSELY, UNLIKE THE DROP OUT
OF CAPILLARIES THERE IS
EXPANSION OF LYMPHATICS IN MANY
DIFFERENT KIDNEY DISEASES, AS WE
HAVE HEARD, IN OTHER ORGAN
DISEASES WHERE THERE'S
INCLINATION.
SIMILAR TO THE LIVER AS FIBROSIS
GETS WORSE, INFLAMMATION GETS
WORSE THERE ARE MORE LYMPHATICS
SO YOU MIGHT THINK THAT
EXPANSION OF LYMPHATICS IS A BAD
THING IN KIDNEY DISEASE.
HOWEVER THERE'S BEEN NUMBER OF
RELATIVELY RECENT PAPERS THAT
ARE CHALLENGING THIS.
THIS IS ONE PAPER FROM DAVID
LONG'S GROUP SHOWING POLYCYSTIC
KIDNEY DISEASE WHICH IS THE
COMMONEST CAUSE OF GENETIC
KIDNEY DISEASE.
THERE IS PRETTY ROBUST EXPANSION
OF LYMPHATICS AROUND THE CYST.
WHEN THEY DELIVER VEGF C AND
ABLE TO BOOST ANGIOGENESIS
AROUND THE CYST, THEY REDUCE THE
INJURY IN THIS MODELS.
TRANSPLANT WHERE KIDNEY IS TAKEN
FROM ANOTHER INDIVIDUAL, LOTS OF
INFLAMMATION, AGAIN, BOOSTING
LYMPHATICS HAS PRODUCED IMPROVED
OUTCOMES.
ACUTE KIDNEY INJURY, VERY, VERY
COMMON CAUSE OF HOSPITAL
MEDIATED KIDNEY FAILURE
LYMPHATICS START TO BECOME
APPARENT AROUND SEVEN DAYS AND
AGAIN PRE-CLINICAL MODELS IF YOU
BOOST LYMPHATICS YOU CAN PROTECT
THE KIDNEYS, MOST IMPORTANTLY WE
LIKE TO PREVENT PROGRESSION OF
ACUTE KIDNEY INJURY ON CHRONIC
KIDNEY DISEASE WE SEE LYMPHATIC
GROWTH AND EMERGING EVIDENCE
THAT IS BENEFICIAL.
I DON'T HAVE TIME TO GO INTO ALL
THE PAPERS PRESENTED.
SUFFICE TO SAY IN THIS CHART
FROM MIKE DAWN'S REVIEW THERE'S
BENEFICIAL STUDIES AS WELL AS
SOME DETRIMENTAL CONSEQUENCES SO
REALLY HIGHLIGHTING THAT WE NEED
MORE INVESTIGATION BEFORE WE CAN
HARNESS.
AND LIKE THE OTHER ORGANS YOU
HAVE HEARD, WE KNOW LYMPHATICS
ARE NOT SIMPLY PASSIVE CONDUITS
OF EXCESS FLUID OR DAMAGE CELLS,
BUT ALSO CELLULAR CROSS TALK AND
WE BELIEVE PRODUCING SECRETED
FACTORS THAT LIKELY ARE GOING TO
BE PROTECTIVE.
IN THE FINAL 30 SECONDS I WANTED
TO LEAVE YOU WITH A PROVOCATIVE
THOUGHT.
WE HEARD FROM (INAUDIBLE) AND
RACHEL EARLIER TODAY, THIS
EMERGING MODEL LYMPHATIC
ENDOTHELIAL CELLS PRODUCE
SUBSTANCES THAT PROTECT PROMOTE
DEVELOPMENT GROWTH OF ORGANS AND
E PAIR AND (INAUDIBLE) PAPER.
SHE DEVELOPED IN THIS RISK
FACTOR FROM LYMPHATICS, THEY HAD
SMALLER HEARTS THAN EMBRYO SIZE.
UNPUBLISHED WORK FROM DR.
MCDONNA WHEN HE REMOVES
LYMPHATICS FROM DEVELOPING
KIDNEYINGS THE KIDNEYS ARE
SMALLER COMPARED TO TOTAL BODY.
SO IN CLOSING I HOPE THAT I HAVE
CONVINCED YOU THERE'S A HUGE LET
REGENEITY OF ENDOTHELIAL CELLS.
A LOT OF EVIDENCE THAT EMERGING
EVIDENCE THAT PROMOTING WITH
ANGIOGENERAL AT THIS MIGHT BE
BENEFICIAL BUT WE REALLY NEED TO
UNDERSTAND BENEFITS VERSUS
ADVERSE EFFECTS IF WE ARE GOING
TO HARNESS THIS FOR PATIENTS.
MY FINAL SLIDE FROM DR. MIKE
DONAN WHO SHARED HIS SLIDES AND
PRELIMINARY DATA, OTHER AMAZING
INDIVIDUALS IN GROUP WHO HAVE
DONE A LOT OF WORK IN LYMPHATICS
OVER THE YEARS.
WITH THAT I WILL CLOSE AND THANK
THE FUNDING SOURCES.
THANKS VERY MUCH.
>> THANK YOU, DR. QUAGGIN,
FANTASTIC.
WE ARE NOW GOING TO MOVE ON TO
ANSWER QUESTIONS.
I JUST LIKE TO REMIND ANYBODY
THAT IS USING THE NIH VIDEOCAST
THAT THEY CAN SEND QUESTIONS
USING THE LIVE FEEDBACK
FUNCTION.
PLEASE SEND ADDITIONAL QUESTIONS
THAT WAY.
BUT WE WILL GO AHEAD AND GET
START WITH QUESTIONS WE RECEIVE
MISDEMEANOR THE Q&A AND THE
RECEIVEDIN THE Q&A IN THE CHAT.
I WILL START WITH THE ONES I
RECEIVED.
SO HAD A GREAT QUESTION FROM
YONG ASKING WHAT IS THE SENTINEL
ORIGIN OF KLA, BLOOD OR
LYMPHATIC ENDOTHELIAL CELLS
CONTRIBUTING TO THE DISEASE?
ANSWER IS WE DON'T KNOW.
IT IS DIFFICULT TO GET TISSUE
FROM PATIENTS TAKING TISSUE IS
RISKY.
SO WE -- I THINK WE NEED MORE
MODELS TO BEGIN TO ADDRESS THAT
QUESTION.
MY LAB IS WORKING ON THAT NOW,
WE DON'T KNOW IT COULD BE BLOOD
ENDOTHELIAL CELLS
DIFFERENTIATING TO LYMPHATIC
ENDOTHELIAL CELLS OR COULD BE
LYMPHATIC ENDOTHELIAL CELLS NRS
THAT CONTRIBUTE TO DISEASE.
GREAT QUESTION.
SECOND I HAD WAS FROM
(INDISCERNIBLE), HE ASKED
WHETHER WHAT KIND OF DRUGS COULD
BE USED FOR KLA PATIENTS WITH
THE NRAS 61QR MUTATION BASED ON
OUR WORK.
SO AGAIN GREAT QUESTION.
I KNOW THAT SOME PATIENTS ARE
BEING TREATED WITH TRANATNIB
FROM OUR STUDIES IS A MEK
INHIBITOR, I KNOW SIDE EFFECTS
CAN BE AN ISSUE.
SEROLAMU SARKS IS USED FOR A
NUMBER OF YEARS IN PATIENT AND
SHOWN SIGNIFICANT IMPROVEMENTS.
DR. ADAMS PUBLICATION IN JOURNAL
PEDIATRICS SHOWED THAT.
SO IT IS CERTAINLY EVOLVING
FIELD IN TERMS OF DRUG
TREATMENTS FOR PATIENTS.
UNDERSTANDING MUTATIONS, SHOULD
BE VERY HELPFUL IN FUTURE AND
USING PRE-CLINICAL MODELS THAT
LIKE THE ONES WE HAVE BEEN
DEVELOPING.
MOVE TO QUESTIONS FOR DR.
DELLINGER NEXT.
I SEE A QUESTION FROM DR.
QUAGGIN THAT SHE ASKED ME.
>> I CAN ASK YOU, I'M REALLY
INTERESTED IN SIGNALING SO
REALLY INTERESTED IN THE ANG 2
WORK AND WONDER IF YOU LOOKED AT
PHLOX L 1 EXPRESSION IN THE
ACTUAL LESIONS.
THE REASON BEING PHLOX L 1 IS A
POTENT TRANSCRIPTIONAL REGULATOR
AND CLEARLY INVOLVED IN SOME
VASCULAR MALFORMATIONS AS WELL.
>> THAT IS A GREAT QUESTION,
SUE.
SO THE SHORT ANSWER IS WE HAVE
NOT.
BUT IT IS ON OUR LIST OF THINGS
TO DO.
THANKS TO NHLBI FUNDING WE ARE
ABLE TO DO THAT NOW.
GREAT QUESTION.
IT IS KNOWN TRANSCRIPTIONAL
REGULATOR OF ANGIOPOIETIN 2.
IN TERMS OF ROLE OF ANGIOPOIETIN
2, THAT IS AGAIN ANOTHER
QUESTION WE ARE ANSWERING.
GETTING NEUTRALIZING ANTIBODIES,
TO BLOCK ANG 2 IN OUR MODELS.
THAT IS AN EXCITING IF IT COULD
BE CONTRIBUTING TO THE DISEASE
PROCESS, THE PATHOPHYSIOLOGY
SINCE NEUTRALIZING ANTI-BOYS
HAVE BEEN DEVELOPED IN NUMBER OF
TUMORS AND SOME CLINICAL TRIALS
DONE WITH NEUTRALIZING ANN
ANGIOANTIBODIES.
SO SHORT ANSWER, WE DON'T KNOW
YET, WE HOPE WE CAN FIGURE IF
THIS COULD BE A POTENTIAL
TREATMENT.
GREAT QUESTION.
>> WHILE YOU ARE LOOKING FOR
QUESTIONS I CAN ASK MICHAEL.
CLEARLY LIGAND IS A DOMINANT
FACTOR FOR OSTEOCLAST AND THE
SUCCESS OF LIGAND THERAPY IN
OSTEOPOROSIS TUMOR OSTEOLYSIS
AND OTHER OSTEOLYTIC DISEASES.
HAS BEEN EXTRAORDINARY.
HAVE PATIENTS BEEN TREATED WITH
DENASMIB?
>> I AM AWARE OF ONE CASE REPORT
WHERE PATIENT HAS BEEN TREATED
WITH THAT AND THE OSTEOLYTIC
PROCESS WAS REPORTED TO NOT
PROGRESS WHILE PATIENT WAS ON
THE DRUG.
THANK YOU FOR YOUR QUESTION.
>> MIKE, I SEE TALK IN THE CHAT
FROM DR. (INDISCERNIBLE) HE
ASKED IF YOU WAITED UNTIL MUTANT
MICE BECOME ADULTS BEFORE
TAMOXIFEN AND INDUCE THE MUTANT
KRAS.
>> WE HAVE.
WE ARE REALLY EXCITED BY THIS
RESULT.
THE LATEST WE HAVE LOOKED IS SIX
WEEKS AFTER GIVING MICE
TAMOXIFEN ADULT MICE TAMOXIFEN
AND LYMPHATICS LOOK COMPLETELY
NORMAL.
THERE APPEARS TO BE WINDOW OF
SENSITIVITY TO THE RAS MUTATION.
AND HYPOTHESIS IS IN ORDER
MUTATION TAB HAVE GREATEST
EFFECT NEEDS TO BE ACTIVELY
GROWING LYMPHATIC NETWORK.
THAT IS A HYPOTHESIS WE TEST NOW
LITTLE EFFECT IN ADULT MICE.
>> MIKE, I SEE ANOTHER QUESTION
FROM NADA.
QUESTION DO YOU SEE CHANGES IN B
CAD HEREIN AT THE LEC FUNCTION
OR PI 3K PATHWAY?
>> WE HAVEN'T STAINED FOR E CAD
HEREIN IN ANIMAL MODEL.
HOWEVER, IN TISSUE CULTURE, E
CAD HEREIN DOESN'T APPEAR TO BE
LOCALIZED TO THE CELL CELL
JUNCTIONS AND IT IS SOMETHING WE
ARE CURRENTLY EXPLORING IN
COLLABORATION WITH JOSH LOOKING
AT THE EFFECT OF RAS MUTATION ON
E CAD HEREIN AND HOW THAT COULD
IMPACT DEVELOPMENT AND
MAINTENANCE.
>> DO YOU SEE CHANGES IN THE PI
3K PATHWAY?
BECAUSE --
>> WE ALSO -- WE DO SEE IN
TISSUE CULTURE WE HAVE DONE
WESTERN BLOT FOR PI 3 KINASE
PATHWAY AND INCREASED ACTIVATION
OF THAT PATHWAY IN OUR KRAS
MUTANT CELLS.
ONE THING WE ARE DOING IS
TREATING CELLS WITH EITHER PI 3
KINASE INHIBITORS OR MAP KINASE
INHIBITORS TO WEED OUT WHICH
PATHWAYS IS CONTROLLING THE
DIFFERENT PHENOTYPES WE OBSERVE.
>> I SEE YOU HAD A QUESTION FOR
DR. SCHWARZ.
WOULD YOU LIKE TO INMUTE
YOURSELF AND ASK IT?
>> THANK YOU SO MUCH.
FANTASTIC TALK DR. SCHWARZ.
I WONDER IF YOU CAN SEE
CORRELATION IN TIME WHICH COMES
FOR EFFECT ON TNF ON THE B
CELLS.
CHICKEN OR EGG KIND OF SITUATION
AN WHETHER YOU CAN FIND ANY
DIFFERENT -- CELLS THAT YOU CAN
SEE SYNOVIAM OR AWAY FROM THE
POPLITEAL LYMPH NODE?
>> SIMPLE ANSWER, THERE ARE
ECTOPIC B CELL FOLLICLES IN THE
TNF MICE AND IN PATIENTS WITH
RHEUMATOID ARTHRITIS.
SO THAT IS CERTAINLY EXISTS.
IN TERMS OF WHICH COMES FIRST
THERE'S HARD THE FINDS EFFECTS
IN OUR MODEL AND AN DRYIA HAS
ADOPTED TNF 1 AND 2 KNOCK OUT B
CELLS INTO TNF MICE IN THE
DISEASE AND THE DISEASE IS
UNCHANGED.
MOST RECENTLY I WAS ELUDED TO
SOME UNPUBLISHED PATIENT
TRANSCRIPTOMIC DATA.
REPEAT AGAIN WE CANNOT FIND
ANYTHING AT THE BULK LEVEL.
AT THE SPATIAL I CAN ONLY TELL
YOU THE MARK POSITIVE SPOTS
WHICH HAVE THESE ENDOTHELIAL
CELLS, ARE SHOWING SOME VERY
INTERESTING SIGNS.
I WOULD SAY THE STROMA OF TNF
EFFECTS ON STROMA HAPPEN FIRST,
THESE CELLS ARE LARGELY
UNCHANGED, I'M TOLD THEY
TRANSLOCATE FROM THE FOLLICLES
IN TO THE POSITIVE SINUSES
SPACES WHERE THEY INTERACT WITH
T-CELLS AND THEN THAT IS THE
FIRST TIME WE SEE CLASS
SWITCHING IN B CELL ACTIVATION.
GREAT I SEE ANOTHER QUESTION,
DR. SCHWARZ FROM DR. MARARRA.
WOULD YOU LIKE TO INMUTE AND ASK
YOUR QUESTION?
>> THANK YOU SO MUCH.
I WAS WONDERING IF YOU LOOKED AT
KRAS EXPRESSION IN THE
ENDOTHELIAL CELLS POPLITEAL
LYMPHODISSECTION.
MOSTLY CONCERNED WHAT HAPPENS
WITH LYMPHEDEMA BECAUSE WE HAVE
A LOT OF SAME PHENOTYPES IN THAT
SETTING.
>> WE HAVEN'T LOOKED AT KRAS
ACTIVATION IN LYMPHATIC VESSELS
POST INJURY.
OR LYMPHEDEMA MODELS.
HOWEVER I WILL SAY COMPLEX
LYMPHATIC ANOMALY OF PATIENTS
HAVE LYMPHEDEMA.
THOUGH THE PATIENTS THAT -- THE
PATIENT THAT I DESCRIBED TODAY
DID NOT HAVE LYMPHEDEMA I
IMAGINE AS WE CONTINUE TO
SEQUENCE COMPLEX LYMPHATIC
ANOMALY SOME WHICH DO HAVE
LYMPHEDEMA MA WILL HAVE RAS
PATHWAY ACTIVATING PATIENTS.
>> THANKS.
>> SO NOW I SEE SOME QUESTIONS
FOR DR. QUAGGIN.
I SEE A QUESTION HOW YOU
DISTINGUISH BETWEEN LIVE BORN
POSITIVE LECs AND LIVE BORN
POSITIVE MACROPHAGES?
ARE THEY ABUNDANT POPULATION
CERTAIN TISSUES THESE ARE
MARKERS IN MULTIPLEX IMAGING
ASSAYS AND MORPHOLOGY.
>> GREAT QUESTION.
MULTIPLE MARKERS AND THAT IN
PART THAT IS WHY SO GREAT TO
HAVE THESE DATA SETS WHERE WE
CAN TAKE IT BACK TO TISSUE.
AND DISTINGUISH.
>> MAYBE I HAVE ONE QUESTION FOR
ALL THE PANELISTS.
AND MAYBE ANYBODY WHO STILL ON
FROM THIS MORNING.
I AM INTERESTED TO KNOW WHAT
PEOPLE'S THOUGHTS ARE ABOUT
ORGAN SPECIFIC LYMPHATICS AND
MICHAEL, I AM LOOKING AT YOU
ALSO ABOUT IN THE BONE WHEN YOU
ARE GROWING THESE, HOW DIFFERENT
ARE THOSE LACKS IN THAT SETTING
THAN -- AND IS THERE AN ISSUE
WHEN WE START TO THINK ABOUT
THESE EXPERIMENTAL MODELS WHERE
WE ARE BOOSTING LYMPHATIC
GROWTH, ARE THEY THE SAME AS
NATIVE LYMPHATIC OR ARE WE
ADDING A LEVEL OF ARTIFACT?
ANYONE CAN ANSWER.
I SEE MY ZOOM SCREEN.
>> SURE, I CAN START OFF.
THAT IS ONE THING WE ARE
INTERESTED IN, THE DIFFERENCES
BETWEEN LYMPHATIC VESSELS IN
BONE VERSUS OTHER TISSUES.
WE ACTUALLY SEE AMAZING
DIFFERENCES IN OUR VEGF C
TRANSGENIC MODEL, WHEREAS IF WE
INDUCE VEGF C EXPRESSION IN BONE
IT SIMULATES THE GROWTH OF
LYMPHATICS OUTSIDE OF BONE BUT
ALSO STIMULATES FOR MISSION OF
LYMPHATICS IN BONE.
THEN WE WITHDRAW VEGF C THE
LYMPHATICS IN BONE DISAPPEAR BUT
THE LYMPHATIC NEXT TO BONE STAY
ABNORMAL AND PERSIST SO THAT
SUGGESTS THERE ARE TISSUE
SPECIFIC DIFFERENCES AND IT CAN
BE DUE TO THE MICROENVIRONMENT
AND THAT CAN BE A COMBINATION OF
INHIBITORY FACTORS PRESENT IN
TISSUE, OR FACTORS THAT
STABILIZE THE LYMPHATIC NETWORK.
IN THE SETTING OF DISEASES WE
WORK ON, WE THINK THAT THIS
SOMATIC GENETIC MUTATION IS THAT
GAS PEDAL THAT CAN OVERCOME ANY
INHIBITOR FACTORS, AND PROMOTE
OR MAINTAIN LYMPHATIC VESSEL IN
ENVIRONMENT WHICH TYPICALLY
CANNOT BE IN.
>> DR. SCHWARZ DO YOU WANT TO
COMMENT?
>> I WOULD SAY IT IS A
REMARKABLE FINDING BECAUSE BONE
DOESN'T HAVE LYMPHATICS.
SO THE BONE MARROW IS LYMPHATICS
AND USING SINUSOID.
THIS WOULD BE SPECIFIC, YEAH FOR
THIS DISEASE.
>> GREAT.
THANKS, THANK YOU EVERYBODY FOR
YOUR QUESTIONS, IT'S BEEN A
FANTASTIC SESSION.
WE APPRECIATE THE ORGANIZERS WHO
PUT ALL OF THIS TOGETHER.
AND THANKS FOR YOUR ATTENTION.
>> JUST I WOULD LIKE ON BEHALF
OF NIH TO THANK YOU ALL FOR
TAKING THE TIME TO ATTEND AND
PARTICIPATE IN DAY ONE OF THIS
WORKSHOP.
FOR ALL THE SPEAKERS, THANK YOU
FOR YOUR WONDERFUL INSIGHTFUL
PRESENTATIONS.
TO ALL ATTENDANTS FOR Q4
QUESTIONS AND CONTRIBUTIONS.
WE HOPE YOU WILL JOIN US
TOMORROW FOR DAY TWO OF THIS
WORKSHOP.
WE HAVE AN EXCITING PROGRAM IN
THE WORKS, INCLUDES SOME OF
OTHERS KEYNOTE AND SPECIAL TALKS
BY DR. SALLY AND SARA AND THREE
EXCITING SESSIONS INCLUDING ONE
LYMPHEDEMA, ONE COMMUNITY
PERSPECTIVE AND ONE ON NEW
TECHNOLOGIES IN LYMPHATIC
IMAGING AND MAPPING.
LOOKING FORWARD TO SEEING Y'ALL
AGAIN TOMORROW AND THANK YOU
AGAIN.
GOOD EVENING.