Welcome to the Clinical Center Grand Rounds, a weekly series of educational lectures for physicians and health care professionals broadcast from the Clinical Center at the National Institutes of Health in Bethesda, MD. The NIH Clinical Center is the world's largest hospital totally dedicated to investigational research and leads the global effort in training today's investigators and discovering tomorrow's cures. Learn more by visiting us online at http://clinicalcenter.nih.gov OUR FIRST SPEAKER IS DR. BETH KOZEL, INVESTIGATOR AND LASKER CLINICAL SCHOLAR ARE THE LABORATORY OF VASCULAR AND MATRIX GENETICS IN NATIONAL HEART, LUNG AND BLOOD INSTITUTE, EARNED HER MEDICAL DEGREE WITH DOCTORATE IN CELL BIOLOGY AT THE WASHINGTON UNIVERSITY IN 2004, RESIDENCY IN PEDIATRICS ST. LOUIS CHILDREN'S HOSPITAL, FELLOWSHIP IN CLINICAL GENETICS AT WASHINGTON UNIVERSITY SCHOOL OF MEDICINE IN ST. LOUIS CHILDREN'S HOSPITAL. SHE WAS ASSISTANT PROFESSOR OF PEDIATRICS AND GENETICS AND GENOMIC MEDICINE AT WASHINGTON UNIVERSITY, BECAME A LASKER RESEARCH SCHOLAR AND CAME TO THE NIH IN 2015. AS A MATRIX BIOLOGIST, VASCULAR BIOLOGIST AND GENETICS SEEKS TO UNDERSTAND A RARE DISORDER SUCH AS WILLIAMS SYNDROME IN MULTI-GENE DELETION DISORDER. DR. KOZEL RECEIVED THE HEART TO HEART AWARD IN 2015, MEMBER OF SEVERAL SOCIETIES INCLUDING AMERICAN SOCIETY OF MATRIX BIOLOGY, SERVES ON THE COUNCIL AND BIOLOGY ORGANIZATION, WILL PRESENT ON VASCULAR REMODELING IN MICE AND MAN, KATP CHANNEL OPENERS AS RATIONAL THERAPEUTICS FOR TREATMENT OF WILLIAMS BEUREN SYNDROME. LET'S WELCOME DR. KOZEL. [APPLAUSE] >> THANK YOU ALL FOR COMING TOGETHER. THANK YOU FOR THAT LOVELY INTRODUCTION. AS YOU HEARD, I'VE BEEN HERE FOR ABOUT A YEAR. MY TRAINING IS A COMBINATION OF VASCULAR BIOLOGY, CLINICAL GENETICS AND MATRIX BIOLOGY, IN THAT COMBINATION I FOUND THIS VERY INTERESTING DISORDER THAT I STUDY WHICH IS WILLIAMS SYNDROME. ONE OF THE PICTURES THAT YOU CAN SEE HERE IS A LITTLE GIRL I TAKE CARE OF IN OUR CLINIC IN ST. LOUIS WHO HAS WILLIAMS SYNDROME. AND FIRST UP I JUST DON'T HAVE ANY DISCLOSURES TO GIVE. THE OBJECTIVES THAT WE HAD FOR TODAY'S LECTURE ARE BEING ABLE TO RECOGNIZE VASCULAR FEATURES AND MEDICATIONS INCLUDING BETA BLOCKERS, ANGIOTENSIN BLOCKERS AND CALCIUM CHANNEL BLOCKERS, IT'S A RARE CONDITION, MICRODELETION, OCCURS DUE TO LOSS OF 26 TO 28 CONTIGUOUS GENES ON CHROMOSOME 7. THE FEATURES THAT WE'RE GOING TO TALK MOST ABOUT TODAY ARE CARDIOVASCULAR ANOMALIES INCLUDING STENOSIS OR NARROWING OF THE GREAT VESSELS, HIGH BLOOD PRESSURE AND VASCULAR THICKNESS. THERE ARE CHARACTERISTIC NEUROCOGNITIVE FINDINGS INCLUDING LOWER IQ, HIGHLY SOCIAL BEHAVIOR, HIGH EMPATHY AND LACK OF SOCIAL FEAR. THEY ALSO HAVE OTHER MEDICAL PROBLEMS INCLUDING POOR WEIGHT GAIN IN INFANCY, AND OBESITY AND DIABETES LATER ON AS WELL AS ENDOCRINOLOGIC ABNORMALITIES. THE BLOOD VESSEL DISEASE OCCUR DUE TO HAPLOINSUFFICIENCY OF ELASTIN GENE, IMPORTANT IN TISSUES THAT STRETCH. SO IT'S SECRETED AS A MONOMER AND CREATES A WEB OF PROTEIN THAT STRETCHES AND RECOILS IN TISSUES. WHAT YOU CAN SEE HERE IS ELECTRON MICROGRAPH OF BLOOD VESSEL CUT IN CROSS-SECTION, AND THE BLACK PLACES HERE, THE TISSUE IS AUTOCLAVED, BLACK PLACES ARE WHERE CELLS SHOULD BE AND ALL OF THE WHITE IS THE ELASTIN. IT'S A VERY STRONG MATERIAL. IT ACTUALLY HAS A HALF-LIFE OF 74 YEARS. WHAT YOU MAKE IN INFANCY AND EARLY CHILDHOOD IS ALL OF THE ELASTIN THAT YOU GET FOR YOUR LIFE. IN THE PATIENTS THAT WE TAKE& CARE OF THEY ARE MISSING ONE COPY OF THE ELASTIN ALLELE, AND ELASTIN GENE, SO THEIR BODY TRIES TO MAKE UP FOR THE FACT THAT EACH CELL AND CELL LAYER ISN'T MAKING ENOUGH ELASTIN, BY MAKING MORE AND MORE LAYERS. SO WHAT YOU CAN SEE HERE IN THE PATIENTS WHO HAVE ELASTIN INSUFFICIENCY, BLOOD VESSEL WALLS ARE THICKER. WHAT EVERY PEDIATRIC RESIDENT AND PROBABLY MOST CARDIOLOGY RESIDENTS WILL TELL YOU IS PATHOKNEW PNEUMONIC FEATURE YOU CAN SEE HERE, SUPRA VALVULAR NARROWING. YOU CAN SEE NARROWING AND YOU CAN SEE HERE THESE INDIVIDUALS HAVE STINTS IN PULMONARY VESSELS, CAN YOU SEE NARROWING IN ANY LARGE ELASTIC TISSUE. AND IF YOU ASK MOST PEDIATRIC RESIDENTS, AND MOST OF THE PEOPLE WHO CARE FOR INDIVIDUALS WITH WILLIAMS SYNDROME THEY WOULD SAY FOR THE MOST PART THAT'S WHAT THE DISEASE IS IN INDIVIDUALS WITH ELASTIN INSUFFICIENCY. SOME WORK WE AND OTHERS WHO HAVE LOOKED AT INDIVIDUALS WITH THIS DISEASE FOUND VASCULOPATHY IS MORE WIDESPREAD. WE CAN SEE A LARGE NUMBER DO INDEED HAVE EITHER CURRENTLY OR HISTORY OF SUPER VALVULAR STENOSIS OR PULMONARY STENOSIS WITH A RANGE OF BLOOD VESSEL NARROWING IN OTHER LOCATIONS IN THE BODY. AROUND 30% OF THE FOLKS HAVE MIDDLE AORTIC SYNDROME, NARROWING OF VESSELS GOING TO THE GUT, GOING TO THE KIDNEY. IN ADDITION WE SEE SOME CORONARY ABNORMALITIES AND HYPERTENSION IN THESE FOLKS. THESE FINDINGS SUGGEST ELASTIN INSUFFICIENCY CAUSES ARTERIO PATHY AND STIFFNESS IN THE BLOOD VESSELS. WHEN YOU HAVE LESS ELASTIN YOU GET LESS STRETCH. THIS IS A TEST ON 100 INDIVIDUALS WITH WILLIAMS SYNDROMES LOOKING AT VELOCITY, THE MARKER FOR VASCULAR THICKNESS. DOTTED LINE IS MEAN FOR NORMAL UNAFFECTED INDIVIDUALS. YOU CAN SEE PULSE WAVE VELOCITY OR VASCULAR THICKNESS GETS HIGHER AS WE GET OLDER. THE BLUE SQUARES ARE MALE PATIENTS IN THE COHORT, YELLOW ARE FEMALES. INCREASED VASCULAR STIFFNESS OCCURS FROM THE YOUNGEST AGE THAT WE CAN GET THEM TO SIT STILL FOR THE TEST, SO THAT STIFFNESS IS A DEVELOPMENTAL PHENOTYPE THAT EXISTS FROM THE VERY BEGINNING. SOME OF THE QUESTIONS THAT WE WOULD LIKE TO ANSWER IN OUR PATIENTS WITH ELASTIN INSUFFICIENCY ARE HEART TO DO. WE HAVE A GOOD ANIMAL MODEL FOR THIS DISEASE. THIS IS SHOWING YOU THE ELASTIN MUTANT IN MICE. LIKE THE PEOPLE, THERE ARE INCREASED NUMBERS OF SMOOTH MUSCLE LAYERS IN THE BLOOD VESSELS. YOU CAN ALSO SEE ELASTIN MUTANT CROSSED INTO VARIOUS STRAINS OF GENETIC BACKGROUNDS OF MICE WHERE WE SEE HYPERTENSION IN THEM AND CAN MEASURE PULSE WAVE VELOCITY USING AN ULTRASOUND ECHO APPROACH AND WE SEE LIKE THE HUMAN PATIENTS, THE ELASTIN HETEROZYGOUS MICE HAVE INCREASED PULSE WAVE VELOCITY AND INCREASED VASCULAR STIFFNESS. VASCULAR STIFFNESS IS IMPORTANT IN GENERAL TO THINK ABOUT BECAUSE OF ITS IMPACT ON CARDIOVASCULAR OUTCOMES SUCH AS STROKE, HEART ATTACK, SUDDEN DEATH AND IN SOME CASES DEMENTIA. MANY FEATURES ARE FEATURES WE SEE A PREPONDERANCE IN IN OUR WILLIAMS POPULATION, A 100-FOLD INCREASE OF SUDDEN DEATH RELATIVE TO AGE MATCHED PEERS. WE WANTED TO THINK BEYOND THE FOCAL STENOSIS TO THE VASCULOPATHY. WHAT I TAKE AWAY, LIKE I TOLD YOU BEFORE, THEY ARE STIFFER BUT NOT EVERYBODY IS. SO YOU SEE SOME INDIVIDUALS THAT ARE HERE SORT OF TWO TO SIX STANDARD DEVIATIONS ABOVE THE MEAN IN VASCULAR THICKNESS, A COMPONENT ARE RELATIVELY NORMAL, A GROUP OF INDIVIDUALS WHO HAVE LOW PULSE WAVE VELOCITY. SO WE WONDERED WHAT WAS IT THAT SORT OF MODIFIED THAT EFFECT, WHAT ARE PEOPLE DOING, WHAT ARE DIFFERENCES THAT CONTRIBUTE TO THAT FINDING. ONE OF THE THINGS WE FOUND IN THAT HUMAN STUDY IS IF YOU LOOKED AT THE PATIENT, THE PULSE WAVE VELOCITY, PATIENTS ON BLOOD PRESSURE MEDICATIONS, AS OPPOSED TO THOSE THAT WEREN'T, THAT THOSE WHO WERE ON BLOOD PRESSURE MEDICATIONS HAD A DIFFERENT TRAJECTORY FOR PULSE WAVE VELOCITY THAN PATIENTS WHO WEREN'T WHICH SUGGESTED TREATMENT OF BLOOD PRESSURE MIGHT BE SOMETHING WE COULD USE TO IMPACT VASCULAR STIFFNESS IN OUR PATIENT POPULATION. THIS PARTICULAR STUDY HOWEVER WASN'T POWERED TO BE ABLE TO DETERMINE DIFFERENCES BETWEEN CLASSES. AND SO THAT'S SOMETHING THAT WE WANTED TO LOOK AT DIRECTLY, WE USED OUR ANIMAL MODEL TO ASSESS THIS. SO THIS IS A STUDY PERFORMED BY CARMEN HALABI AND MY GROUP WHEN WE WERE IN ST. LOUIS IN WHICH WE LOOKED AT TREATING OUR MICE WITH BLOOD PRESSURE MEDICATIONS. SO WE STARTED TREATING THEM AT WEENING, AND TREATED THEM UNTIL THREE MONTHS OF AGE WITH LOSARTIN, OR A BETA BLOCKER OR CALCIUM CHANNEL BLOCKER. WE SEE A LOWERING OF BLOOD PRESSURE, STATISTICALLY SIGNIFICANT WITH LOSARTIN AND NICOPARDINE WE NEEDED MORE ANIMALS OR HIGHER DOSE. THEY ARE GETTING THE MEDICATION BECAUSE WE SEE A HEART RATE DROP IN THEM. IN GENERAL WE SEE TRENDS OR SIGNIFICANT DECREASE IN BLOOD PRESSURE WITH THESE MEDICATIONS AS WELL AS DECREASE IN PULSE PRESSURE. WHEN WE LOOK AT THE VESSELS THEMSELVES THOUGH WHAT WE DON'T SEE IS CHANGE IN THE BIOMECHANICAL PROPERTIES OF THE VESSEL, SO THIS TEST, IN THIS TEST WE TAKE THE BLOOD VESSELS OUT OF THE MOUSE, CANNULATE THEM ON TWO CANNULA AND PUFF UP WITH FLUID TO DIFFERENT AMOUNTS OF PRESSURE AND LOOK AT THE SIZE OF THAT BLOOD VESSEL. SO IN THE WILDTYPE ANIMALS YOU SEE HERE YOU CAN SEE THERE'S AN ELASTIC PART OF THE CURVE HERE, AND THEN EVENTUALLY THE VESSEL STRETCHES AS FAR AS IT'S GOING TO STRETCH AND LEVELS OUT AND BECOMES STIFF AT HIGHER PRESSURES. THE ELASTIN HETEROZYGOUS ANIMALS HOWEVER ARE NARROWER AT EVERY POINT ALONG THIS CURVE. AND YOU CAN SEE THAT THE PORTION OF THE CURVE IN WHICH THEY STRETCH IS TO SOME DEGREE SHORTER BECAUSE THEY HAVE LESS ELASTIN AND THEY GET TO THIS SORT OF STIFF PHASE WHERE COLLAGEN DRIVES THE BIOMECHANICS OF THE VESSEL AND MUCH EARLIER PRESSURES. THE MEDICATIONS THAT WE GIVE DO LOWER BLOOD PRESSURE BUT THEY DON'T SEEM TO DO MUCH IN TERMS OF CHANGING THE SLOPE OF THIS CURVE SO THEY ARE NOT CHANGING THE BIOMECHANICAL PROPERTIES OF THE VESSEL PER SE. HOWEVER, WHEN WE GIVE MICE THESE MEDICATIONS, SO IN THIS CASE LOSARTIN WE CAN SEE A DECREASE IN PULSE WAVE VELOCITY, DECREASE IN STIFFNESS EVEN THOUGH WE'RE NOT CHANGING THE VESSEL ITSELF. THE WAY WE THINK THAT'S HAPPENING IS BASICALLY CHANGING THE BLOOD PRESSURE SO IF YOU TAKE A VESSEL OR ANIMAL WHO IS HYPERTENSIVE OPERATING UP HERE IN THE HIGHER STIFFER PART OF ITS PRESSURE DIAMETER RELATIONSHIP, AND YOU TREAT IT WITH A BLOOD PRESSURE MEDICATION, YOU'RE MOVING IT DOWN TO BASICALLY A MORE COMPLIANT PART OF ITS PRESSURE DIAMETER RELATIONSHIP. YOU'RE NOT CHANGING THE VESSEL ITSELF BUT YOU'RE CHANGING WHERE IT FUNCTIONALLY WORKS. AND SO WE THOUGHT, WELL, THEN ARE THESE BLOOD PRESSURE MEDICATIONS A GOOD WAY TO ACTUALLY TREAT BOTH HYPERTENSION AND VASCULAR STIFFNESS IN OUR PATIENTS? COULD WE SORT OF KILL TWO BIRDS WITH ONE STONE? IN WE WANTED TO TREAT AN ELAST INSUFFICIENT INDIVIDUAL WE WOULD HAVE TO THEIR BLOOD PRESSURE TO LOWER NUMBERS THAN SOMEBODY WITH NORMALLY COMPLIANT VESSELS BECAUSE YOU HAVE TO GET PAST THEIR PRESSURES TO THE AREA OF THE CURVE WHERE ELASTIN HET IS COMPLIANT, AND AT THE SAME TIME PUSHING THAT BLOOD VESSEL DOWN TO SMALLER AND SMALLER DIAMETERS SO YOU'RE ACTUALLY MAKING THE BLOOD VESSEL SMALLER AS YOU'RE TREATING IT. AND THAT HAS IMPACT IN TERMS OF HOW THAT VESSEL LOOKS. SO IN THIS CASE WHAT YOU CAN SEE IS LUMEN DIAMETER AS DETECTED BY ULTRASOUND TESTING OF THE HEART, LOOKING AT AORTIC DIAMETER, ELASTIN HET VESSEL IS SMALLER BUT BLOOD PRESSURE TREATED ELASTIN HAS A NARROWER DIAMETER. SO THERE'S SOME SUGGESTION THAT SAYS POTENTIALLY THAT HYPERTENSION THAT IS PRESENT IN THE ELASTIN HET MAY STINT THEM OVER, TREATING THEM WITH BLOOD PRESSURE MEDICATIONS COULD POTENTIALLY MAKE THEM WORSE. AND THE REASON THAT OCCURS IS THAT BLOOD FLOW IS BASED ON TWO THINGS. ONE, THE PRESSURE THAT YOU PUSH BLOOD THROUGH WITH, BUT EVEN MORE SO BY THE DIAMETER OR IN THIS CASE THE RADIUS OF THE BLOOD VESSEL. SO FLOW IN THIS CASE IS IMPACTED TO THE FOURTH POWER BASED ON RADIUS, SO VERY SMALL CHANGES IN LUMEN DIAMETER CAN HAVE FAIRLY LARGE IMPACT ON BLOOD FLOW TO END ORGANS. WHEN WE PUT OUR NUMBERS INTO THE EQUATION WE PREDICT THAT YOU WILL GET REDUCED BLOOD FLOW AROUND 40%, BASED ON THE PRESSURE AND RADIUS RELATIONSHIPS IN OUR ANIMALS. AND THAT'S IMPORTANT BECAUSE IN OUR PATIENTS IN ADDITION TO HAVING FOCAL STENOSES WHAT WE SEE ARE LONG CONDUIT DIFFERENCES IN BLOOD VESSELS. AND SO THIS IS THE CT SCAN FROM ONE OF OUR PATIENTS, AND YOU CAN SEE THEIR AORTA HERE LOOKS RELATIVELY NORMAL. LET'S SEE IF I CAN MAKE THIS MOVE. BUT IF YOU LOOK AT THE BACK HERE YOU CAN SEE AS YOU GET THROUGH THE ARCH IT BECOMES LARGELY DIMINISHED AND YOU END UP WITH A VERY SMALL BLOOD VESSEL, ACTUALLY GOING DOWN INTO THE GUT. AND THIS INDIVIDUAL HAD YEARS OF FAIRLY SIGNIFICANT BELLY PAIN UNTIL HE DEVELOPED SOME COLLATERALS IN HIS GUT. AND SO THE BLOOD FLOW CHANGES THAT WE SEE HAVE THE POTENTIAL TO IMPACT DISEASE IN THESE FOLKS AND THAT THEY ARE STARTING WITH THESE LOWER SIZE LUMENS ALLOWING LOWER BLOOD DELIVERY AND THAT POTENTIALLY HAS AN EFFECT MORE IN SITUATIONS WHERE YOU'RE ASKING AN ORGAN BED, GUT, EXTREMITIES TO DO MORE WORK THAN BASELINE. WE TESTED THIS IN OUR ANIMAL MODEL, WE LOOKED AT THE BLOOD FLOW USING CAROTID PROBE AND FOUND SIMILAR TO WHAT WE CALCULATED WE SAW 30% REDUCTION IN BLOOD FLOW GOING TO THE BRAIN OR AT LEAST THROUGH THE CAROTID OF THESE MICE. WHEN WE LOOKED AT THE BRAINS OF THESE MICE WE ACTUALLY SAW SOME DIFFERENCES IN THAT WE SAW EVIDENCE OF GFAP AND SMALL AMOUNTS OF MICROHEMORRHAGE. WE DID BEHAVIORAL TESTING AND SAW DIFFERENCES IN THE ELASTIN HET VERSUS WILD TYPE. THIS IS ELASTIN MUTANT ONLY AS OPPOSED TO WILLIAMS SYNDROME, SEEING SOME PHENOTYPES IN THE MICE DUE TO VASCULAR DISEASE AS OPPOSED TO ENTIRE WILLIAMS SYNDROME. LOOKING AMOUNT BLOOD FLOW WITH THE INSTITUTE OF RADIOLOGY AT WASH U WE SAW DECREASED TISSUE PERFUSION IN WILD TYPE VERSUS ELASTIN HET MICE. IN THE WHOLE BRAIN QUANTIFICATIONS AND IN THE CORTEX, WITH TRENDS GOING DOWNWARD IN THE HIPPOCAMPUS AND THALAMUS. BUT THAT SUGGESTED LIKE WE WERE TALKING ABOUT BEFORE THAT POTENTIALLY WE HAVE SOME REDUCED BLOOD FLOW AROUND THAT INDIVIDUALS WITH ELASTIN INSUFFICIENCY MAY HAVE POOR ABILITY TO RESPOND TO CHANGES IN BLOOD FLOW. WE SAW SIMILAR FINDINGS IN THE HEART IN THAT WE HAVE REDUCED VASCULATURE, UNFORTUNATELY WE WERE NOT ABLE TO MAKE THESE ONES SPIN EARLIER SO I CAN'T SHOW YOU THAT. BUT WE DO SEE DECREASE -- EVIDENCE OF DECREASED BLOOD FLOW AND SMALLER VESSELS IN THE CARDIAC TISSUE WHICH MAY SUGGEST SOME DIFFERENCES IN HOW THE HEART RESPONDS TO STRESS SITUATIONS AS WELL. SO IN THINKING ABOUT TREATING THIS DISEASE, WE WERE AT THE SAME TIME TAKING CARE OF A GROUP OF PATIENTS WHO HAD ANOTHER GENETIC SYNDROME CALLED CANTU SYNDROME OCCURS DUE TO ACTIVATING CHANNELS IN THE KATP CHANNELS, THEY HAVE WIDE OPEN LARGE CALIBER BLOOD VESSELS, LOW BLOOD PRESSURE AND VASCULAR STIFFNESS AND DATA FROM A FRENCH GROUP SHOWED ANIMALS GIVEN MANOXIDIL SHOWED INCREASED DEPOSITION OF ELASTIN IN EXTRACELLULAR MATRIX AND THOUGHT THAT COMBINATION OF THINGS WOULD POTENTIALLY BE USEFUL FOR TREATING VASCULAR DISEASE IN ELASTIN INSUFFICIENCY. SO UNLIKE THE CURVES I SHOWED FOR THE OTHER BLOOD PRESSURE MEDICATIONS WHEN WE TREAT OUR ELASTIN HET MICE FROM WEANING TO THREE MONTHS OF AGE WITH MINODIXIL, THE TREATED MICE HAVE A DIAMETER CLOSE TO WILD TYPE THROUGH THE WORKING BLOOD PRESSURE OF THE ANIMAL. BLOOD PRESSURE IS DOWN, PULSE PRESSURE IS DOWN, THE SAME IS TRUE NO MATTER WHAT BLOOD VESSEL WE LOOK AT, ASCENDING AORTA, CAROTID, INCREASING THE CONDUIT THROUGH WHICH BLOOD IS TRAVELING IN THESE ANIMALS. WHEN WE QUANTIFIED ELASTIN THERE IS AN INCREASE IN DESMODINE, WITHOUT A SUBSTANTIAL CHANGE IN COLLAGEN. AND WHEN WE DID IN VIVO STUDIES IN THESE WE SHOWED UNLIKE LOSARTIN, THE MINODIXIL HETS ARE NORMALIZATION IN PERFUSION TO BRAIN WHICH SUGGESTS TAKEN TOGETHER, THAT THIS MEDICATION MAY BE USEFUL FOR TREATING BLOOD PRESSURE, VASCULAR STIFFNESS AND PERFUSION IN THIS POPULATION WHICH MAKES IT A VERY EXCITING MEDICATION TO THINK ABOUT IN USE FOR THESE PATIENTS. IN SUMMARY I THINK I TOLD YOU TODAY THAT ELASTIN INSUFFICIENCY PREDISPOSES TO A WHOLE SET OF VASCULAR FEATURES WITH STENOSIS BEING ONE OF THEM BUT WE NEED TO THINK ABOUT ELASTIN INSUFFICIENCY BOTH IN WILLIAMS SYNDROME PATIENT AND POTENTIALLY AGING AS ELASTIN TURNS OVER AT A WIDESPREAD VASCULAR DISEASE THAT AFFECTS MULTIPLE ENDPOINTS FROM BLOOD PRESSURE TO VASCULAR STIFFNESS TO BLOOD FLOW TO ORGANS. WE KNOW THAT WE CAN IMPACT FUNCTIONAL STIFFNESS BY TREATMENTS WITH DIFFERENT MEDICATIONS, THERE'S PHARMACOLOGIC THINGS WE TALKED ABOUT. BLOOD PRESSURE MEDICATIONS, ALSO SOME GENETIC CHANGES THAT WE DIDN'T TALK ABOUT TODAY THAT CAN ALSO IMPROVE THESE FINDINGS. BUT WE NEED TO BE CAREFUL IN THE TREATMENT OF THESE INDIVIDUALS WITH BLOOD PRESSURE MEDICATIONS THAT WE'RE NOT BASICALLY PUSHING THEM BY LOWERING THEIR BLOOD PRESSURES TO STOPPING THAT SORT OF INNATE NEED TO STINT OPEN NARROWER BLOOD VESSELS, THAT WE'RE NOT PUSHING THEM TOWARDS ACTUALLY HAVING EVEN SMALLER BLOOD VESSELS AND REDUCED FLOW. OTHER TREATMENTS LIKE THE KATP CHANNEL OPENERS THAT WE TALKED ABOUT MAY BE A USEFUL ADJUNCT OR USEFUL THERAPY FOR FOLKS GOING FORWARD, BECAUSE OF THEIR ABILITY TO AFFECT BLOOD FLOW PRESSURE AND STIFFNESS. THEY DO COME WITH SIDE EFFECTS WE HAVE TO THINK ABOUT IN THIS POPULATION. THEY DO HAVE SIDE EFFECTS OF HIRSITISM BUT IT'S EXCITING TO MODIFY. THE FUTURE WORK WILL BE ASSESSING PHENOTYPES WE'RE SEEING IN THE MICE, AND LOOKING AT THEM IN PATIENTS, SO WE HAVE A STUDY OPEN RIGHT NOW LOOKING AT THE SAME TYPES OF ASL STUDIES IN HUMAN PATIENTS THAT WE DID IN THE MICE AS WELL AS ALL OF THE OTHER PHENOTYPING MECHANISMS TO SEE IF WE'RE SEEING THE SAME THINGS NOTICE HUMAN PATIENTS THAT WE ARE IN THE MICE AND CAN USE THOSE TO HELP GUIDE US AS WE TRY TO MOVE FORWARD INTO A CLINICAL TRIAL WITH THIS MEDICATION. SO THIS CONCLUDES MY PRESENTATION. I WANT TO THANK ALL THE PEOPLE THAT DID THE WORK. A LOT OF THE WORK WAS DONE AT WASH U, AND HERE, SO THE IMAGING WORK WAS DONE LIKE I SAID AT THE CENTER IN ST. LOUIS, RUSS KNUDSEN DID THE BULK OF STUDIES AND BLOOD FLOW WORK. I WILL CONCLUDE AND TAKE ANY QUESTIONS. >> THANK YOU, DR. KOZEL. CAN WE HAVE THE LIGHTS UP AND IF YOU HAVE A QUESTION PLEASE GO TO THE MICROPHONE. >> WERE DIABETIC WILLIAMS PATIENTS -- (INAUDIBLE). >> IT'S A GOOD QUESTION. I DON'T KNOW. I'M TRYING TO THINK. A LOT OF WILLIAMS PATIENTS END UP BECOMING DIABETIC, THE BULK DON'T END UP ON MEDICATION SO I DON'T THINK IF THERE'S GOOD DATA ONE WAY OR THE OTHER BASED ON PATIENT STUDIES. >> YOU MENTIONED SOME PHENOTYPES THE PATIENTS HAVE SORT OF -- (INAUDIBLE). >> THAT'S A GREAT QUESTION. KAREN MIGHT WANT TO SPEAK TO THAT. THERE'S BEEN A LOT OF WORK LOOKING INTO WHAT GENES MIGHT BE INVOLVED IN THAT, AND THE ANSWER IS PROBABLY THAT THERE ARE MULTIPLE THAT ARE INVOLVED IN SOME OF THE VISUAL/SPATIAL DEFICITS AS WELL AS HYPERSOCIALITY PARTS. DOES THE VASCULAR DISEASE IMPACT ANY OF THAT IS OUR INTEREST. CERTAINLY WE DON'T THINK VASCULAR DISEASES CAUSES ALL THE SOCIAL FEATURES OF WILLIAMS SYNDROME, THAT'S ABSOLUTELY NOT TRUE. BUT THEY MAY CONTRIBUTE TO SOME FEATURES OF THE CONDITION INCLUDING ANXIETY AND SOME OTHER ISSUES THAT WE'RE SEEING THAT& CAN OVERLAP WITH THAT AND IT'S ALSO A POSSIBILITY YOU COULD HAVE SYNERGY OF THE VASCULAR DISEASE WITH OTHER GENETIC FINDINGS THAT YOU SEE AS PART OF THE LARGER DELETION. >> THANK YOU AGAIN. [APPLAUSE] >> IF HAVE YOU OTHER QUESTIONS YOU CAN STICK AROUND TILL THE END OF THE SECOND LECTURE. SO WITH THAT I'LL INTRODUCE DR. MANFRED BOEHM WHO EARNED MEDICAL DEGREE AT THE UNIVERSITY OF HEIDELBERG IN GERMANY, RESIDENCY IN INTERNAL MEDICINE AT THE& FRANZ VOLAR CLINIC IN BERLIN, RESEARCH FELLOWSHIPS AT THE MAX CENTER FOR MOLECULAR MEDICINE IN BERLIN 1997 AND FOLLOWED BY A FELLOWSHIP AT THE UNIVERSITY OF MICHIGAN IN 1999. HE JOINED THE NATIONAL HEART, LUNG AND BLOOD INSTITUTE AS RESEARCH FELLOW IN THE CARDIOLOGY BRANCH 1999, AND BECAME INVESTIGATOR WITH THE NHLBI CENTER FOR MOLECULAR MEDICINE IN 2003. DR. BOEHM'S LABORATORY ESTABLISHED PATIENT CENTERED VASCULAR PROGRAM TO CONDUCT RESEARCH ON QUESTIONS FOCUSED ON VASCULAR INJURY, REMODELING AND REPAIR, AND TRANSLATE RESEARCH FINDINGS INTO NEW THERAPEUTIC STRATEGIES. DR. BOEHM RECEIVED A FOUNDATION FOR ADVANCED EDUCATION SCIENCE AWARD FOR RESEARCH EXCELLENCE FROM NIH IN THE PAST. HE SERVED ON EDITORIAL BOARDS OF A NUMBER OF JOURNALS INCLUDING TRENDS IN CARDIOVASCULAR MEDICINE AND JOURNAL OF MOLECULAR MEDICINE. HE HOLDS TWO PATENTS, PARTICIPATED IN COOPERATIVE RESEARCH AND DEVELOPMENT AGREEMENTS WITH BIOTECHNOLOGY COMPANIES AND SEVERAL ACTIVE CLINICAL PROTOCOLS STUDYING GENETIC VARIANTS ASSOCIATED WITH VASCULAR DISEASE. HE WILL PRESENT ON VASCULAR REMODELING IN MICE AND MEN, NEW INSIGHTS FROM THE RARE INHERITED VASCULAR CALCIFICATION DISEASE DUE TO DEFICIENCY IN CD73 ALSO KNOWN AS ACDC. NOW LET'S WELCOME DR. BOEHM. >> THANK YOU. [APPLAUSE] THANK YOU FOR INVITING ME. DISCLOSURE, I HAVE NO FINANCIAL RELATIONSHIP WITH ANY COMMERCIAL INTERESTS. AND OBJECTIVE TODAY IS TO TALK AND TO UNDERSTAND THE ROLE OF EXTRACELLULAR PURINE METABOLISM IN VASCULAR MODELING, SPECIFICALLY IN VASCULAR CALCIFICATION. SO, AS FRED ALREADY MENTIONED, MY GROUP WORKS ON VASCULAR DISEASE ON MENDELIAN VASCULAR DISEASES, VERY RARE FORM OF VASCULAR DISEASES THAT LIE A LITTLE BIT OUTSIDE THE USUAL SPECTRUM, AND WE USE FUNCTIONAL GENOMICS TO BETTER UNDERSTAND THIS DISEASE. OUR PROGRAM IS PATIENT-CENTERED TRANSLATIONAL PROGRAM, WHERE THE FOCUS IS TO IDENTIFY DISEASE MECHANISM, AND THEN MOVE FROM THERE TO COME UP WITH NEW TREATMENT STRATEGIES. SO THIS SHOWS YOU THE PIPELINE THAT WE DEVELOPED FOR OUR PATIENTS THAT ARE COMING HERE INTO THE CLINICAL CENTER. SO MOST IMPORTANTLY, WHEN A PATIENT COMES HERE WE PERFORM CLINICAL EVALUATION, SO I HAVE A CLINICAL CREW, AS WELL AS A BASIC LAB CREW. AND THEN THESE CLINICAL -- DOING THIS CLINICAL EVALUATION, WE COLLECT SAMPLES FOR WHOLE EXOME SEQUENCING, WHOLE GENOME SEQUENCING TO IDENTIFY DISEASE-CAUSING VARIANTS, MANY PATIENTS IF NOT ALL THE DISEASE IS UNKNOWN SO WE'RE LOOKING FOR WHAT IS CAUSING THIS DISEASE. AT THE SAME TIME, WE DEVELOP IN VITRO MODELS TO BETTER MECHANISMS, AND THESE DISEASE- MODELS HAVE VERY CLOSELY TAILORED TO THE CLINICAL PRESENTATION OF THE PATIENTS. THE IN VITRO DISEASE MODELS THAT WE UTILIZE ARE PRIMARY CELLS, WE USE BLOOD CELLS, INFLAMMATORY CELLS, ALSO FIBROBLASTS FROM THE SKIN. AND THEN WE ALSO GENERATED iPS CELLS, PATIENT-SPECIFIC, WHERE WE GENERATE A VARIETY OF DIFFERENT CELL TYPES THAT WE USE TO BETTER UNDERSTAND THE DISEASE. WE RUN THEM THEN THROUGH HIGH THROUGHPUT FUNCTIONAL EVALUATION, AND THEN THIS GIVES US INFORMATION ON WHAT KIND OF POTENTIAL PATHWAY MIGHT BE AFFECTED IN THESE PATIENTS. AND THIS INFORMATION HELPS US TO BETTER RANK AND UNDERSTAND THE GENETIC VARIANTS THAT WE FIND IN THESE PATIENTS. AFTER WE HAVE IDENTIFIED THE GENETIC VARIANTS, WE USE PATIENT-SPECIFIC -- MORE SPECIFIC IN VITRO DISEASE MODELS, SOMETIMES IN VIVO DISEASE MODELS TO COME UP WITH THERAPY TARGETS THAT THEN GOES BACK TO THE PATIENT. SO I WORK HERE AT THE NIH. I HAVE THE PRIVILEGE TO BE NOT AS FOCUSED AS IF I WOULD BE ON THE OUTSIDE, SO I WORK -- OR THE LAB WORKS ON A VARIETY OF MANY DIFFERENT DISEASES, ONE OF THEM IS ACDC, A DISEASE OF CALCIFICATION AND WE'LL TALK ABOUT THAT MORE TODAY BUT ALSO DATA THAT IS TOO OUT INFLAMMATORY DISEASES THAT CAUSES STROKE IN KIDS OR INFLAMMATORY DISEASE THAT CAUSES MALIGNANT FORM OF VASCULITIS, WE WORK WITH (INDISCERNIBLE) AND THE ROLE OF THE MODELING OF DOMINANT SYNDROME PATIENTS, PATIENTS FROM STEVE HOLLAND. SO TODAY I TALK ABOUT GENETICS OF ECTOPIC CALCIFICATION, WHERE WE WORKED FROM THE GENOTYPE TO THE DISEASE MECHANISM TO THE THERAPY. WE BASICALLY RUN THROUGH THIS PIPELINE THAT I SHOWED YOU BEFORE, RIGHT FROM THE START UNTIL THE END. VASCULAR DISEASE, EVERYBODY HAS THIS PREVALENCE AND ORGAN SYSTEM BUT I WOULD SAY VASCULAR IS PARTICULARLY IMPORTANT. VASCULAR DISEASES ARE VERY COMMON AND VASCULAR-RELATED DEATH IS MORE THAN ONE-QUARTER OF ALL DEATH IN DEVELOPED COUNTRIES, INCLUDING THE UNITED STATES. ONE FORM OF VASCULAR DISEASE, VASCULAR REMODELING, IS VASCULAR CALCIFICATION. VASCULAR CALCIFICATION IS A PREDICTOR OF EVENTS. PATIENTS COME WITH CORONARY ARTERY DISEASE ONE EVALUATION WE DO IS WE LOOK FOR CALCIFICATION OF THE BLOOD VESSEL IN THE HEART, AND DEVELOP A SCORING SYSTEM THAT GIVES US VERY IMPORTANT INFORMATION ON THE PROGRESSION OF THE DISEASE, DIFFERENT TREATMENT STRATEGIES, AND SO ON. ANOTHER FORM OF VASCULAR CALCIFICATION IS MEDIAL ARTERIAL CALCIFICATION. YOU FIND THAT PREDOMINANTLY IN DIABETES TYPE 2 DISEASES, IT'S OFTEN CALLED ALSO MONKEBERG SCLEROSIS, STRONG MARKER FOR FUTURE CARDIOVASCULAR EVENTS. ALSO END STAGE RENAL DISEASE PATIENTS ARE DEVELOPING VERY MALIGNANT FORM OF VASCULAR CALCIFICATION. SO VASCULAR CALCIFICATION IS NOT ONLY A BY-PRODUCT OF VASCULA DISEASE; IT IS ONE OF THE MAIN COMPONENTS OF IT, AND IN MANY CASES IS STRIVING. THERE ARE TWO TYPES OF VASCULAR CALCIFICATION, NOT BASED ON CHEMICAL COMPOSITION BUT MOSTLY ON ITS LOCATION. WHAT YOU SEE HERE IS BLOOD VESSEL, SCHEMATIC FORM OF A BLOOD VESSEL, AND THIS ONE IS THE STRUCTURE OF THE BLOOD VESSEL THAT IS CALLED INTIMA, AND IN SOME CASES OF DISEASE THE CALCIFICATION IS LOCALIZED IN THE INTIMA, THIS IS CLASSIC CALCIFICATION THAT YOU SEE IN VERY COMMON DISEASE LIKE DYSLIPIDEMIA, HYPERCHOLESTEROLEMIA, OFTEN ASSOCIATED WITH ATHEROSCLEROSIS. WE HAVE MEDIAL CALCIFICATION DIFFERENT IN LOCALIZATION OF CALCIFICATION WHERE THE CALCIFICATION OCCUR IN THE MEDIA AND IT'S IN MANY CASES INITIATED AT THE ELASTIC STRUCTURES, IN THE BLOOD VESSEL. THIS FORM OF CALCIFICATION IS MUCH, MUCH LESS FREQUENT THAN THE INTIMA CALCIFICATION. YOU FIND IT IN DIABETES, RENAL FAILURE, OSTEOPOROSIS AND HYPERTENSION. IF YOU DO AN X-RAY OF THE PATIENT YOU CANNOT NECESSARILY DISTINGUISH IF THE CALCIFICATION IS PRESENT IN THE MEDIA OR IF THIS IS NEOINTIMMA CALCIFICATION. CALCIFICATION IS COMPLEX AND NOT ONLY A BY-PRODUCT OF DISEASE, IT'S AN ACTIVE PROCESS THAT IS HAPPENING IN THE TISSUE AND IN THE BLOOD VESSEL. IT'S HIGHLY REGULATED, AND THERE ARE MANY DIFFERENT REGULATORS THAT DEFINE CALCIFICATION TO OCCUR AND TO PROGRESS. ONE IMPORTANT FACTORS ARE FAILED ANTI-CALCIFIC PROCESSES THAT BLOCK CALCIFICATION TO BE INITIATED AND ACTUALLY THIS BALANCE IS VERY, VERY NOT STABLE, SO IF THERE WOULD NOT BE THESE FAILED ANTI-CALCIFIC ANY SMALL ALTERATIONS IN TISSUE CAN TIP THE CALCIFICATION, INITIATE CALCIFICATION PROCESS. ANOTHER IMPORTANT REGULATOR IS MATRIX DEGRADATION, OFTEN EVEN INITIATING CALCIFICATION. SO CALCIFICATION REQUIRES MATRIX TO BE BROKEN DOWN, AND THEN THESE BROKEN DOWN MATRIX COMPONENTS OFTEN USE INITIATION POINTS FOR CALCIFICATION. CELLS ALSO ACTIVATE OSTEOGENIC DIFFERENTIATION PROGRAMS SIMILAR TO BONE FORMATION TO INITIATE CALCIFICATION, AND ALSO INFLAMMATION AND APOPTOSIS, IT'S A VERY IMPORTANT REGULATOR OF AN INITIATOR OF CALCIFICATION, ALSO WE DO NOT KNOW WHAT EXACTLY ARE THE UNDERLYING MECHANISMS. ALSO, ALTERATIONS IN PHOSPATE OR CALCIUM HOMEOSTASIS, SPECIFICALLY IN END STAGE RENAL DISEASE, ARE VERY IMPORTANT REGULATORS OF CALCIFICATION. SO I'M FOCUSING NOW ON EXTRACELLULAR PURINE METABOLISM, BASICALLY DEGRADATION OF METABOLIZATION OF ATP IN THE EXTRACELLULAR SPACE THAT WHEN YOU LOOK FOR EXAMPLE FOR TISSUE INJURY YOU HAVE A LARGE AMOUNT OF ATP RELEASED INTO THE EXTRACELLULAR SPACE, THEN SIGNAL THROUGH CELLULAR -- SEVERAL RECEPTORS, IMMEDIATE RESPONSE INFLAMMATION RESPONDS THAT GOES WITHIN MINUTES IN THE SURROUNDING TISSUE. THEN ATPs METABOLIZE TO FORM ANOTHER PRODUCT WHICH IS ADENOSINE. ADENOSINE PLAYS A ROLE IN THE RESOLUTION OF INFLAMMATION AND THEN MORE CHRONIC FIBROSI AND ANGIOGENESIS. ADENOSINE SIGNALS THROUGH RECEPTORS WITH DIFFERENT DOWNSTREAM PATHWAYS WHICH MAKES IT DIFFICULT TO MODEL AND TO UNDERSTAND WHAT IS HAPPENING IN THE CELL. ADENOSINE FUNCTION WITHIN HOURS AND LEADS TO CHANGES IN THE TISSUE THAT THEN GOES ON FOR DAYS OR EVEN WEEKS AND MONTHS. SO PURINE METABOLISM ABILITIES AS PERIMETER METABOLISM WELL ESTABLISHED FOR INFLAMMATION, HOWEVER IF YOU LOOK FOR WHAT IS THE ROLE IN CALCIFICATION YOU FIND THAT MOST MONOGENIC FORMS WITH VASCULAR CALCIFICATION HAVE SOMEHOW ALTERATION EXTRACELLULAR PURE EVEN METABOLISM. THE RELEASE OF ATP FROM EXTRACELLULAR TO INTRACELLULAR COMPONENT IS AMONG OTHERS REGULATED BY ABCC6 TRANSPORTERS, ATP IS METABOLIZED BY ENPP1 INTO PYROPHOSPHATE, AND INTO AMP. AMP THEN IS FURTHER METABOLIZED WITH ANOTHER ENZYME CALLED CD73, INTO ADENOSINE THAT THEN IS FURTHER METABOLIZED INTO ADENOSINE BY HYDROGENASE, AND OTHER ENZYMES ARE IMPORTANT, ALKALINE PHOSPHATASE, METABOLIZED PYROPHOSPHATE RELEASED THROUGH METABOLIZATION, RELATED TO PHOSPATE TRANSPORTERS. AND ONE RARE DISEASE CAUSED BY VASCULAR WHERE THE PATIENTS DEVELOP STRONG VASCULAR CALCIFICATION IS GACI WHERE THE LEG OF ENZYME LEADS TO ININABILITY TO GENERATE PYROPHOSPHATE THAT LEADS TO CALCIFICATION IN NEWBORNS WITH MYOCARDIAL INFARCTION AND OTHER THINGS. THE OTHER ONE THAT IS VASCULAR CALCIFICATION MORE LOCALIZED IN THE BRAIN IS FAHR'S DISEASE LINKED TO PHOSPATE TRANSPORTERS. THE DISEASE WE'RE WORKING ON IS CAUSED BY MUTATION IN FUNCTIONAL LOSS OF CD73, THE DISEASE IS CALLED ACDC, ARTERIALAL CALCIFICATION DUE TO DEFICIENCY OF CD73. AND AS I SAID, CD73 METABOLIZES AMP TO ADENOSINE AND PHOSPATE. THIS SHOWS OUR FIRST TWO PATIENTS, LOUISE AND PAULA, , THIS WAS A COLLABORATION WITH BILLY GALL. CINDY WAS LEADING THAT PROJECT. SO THESE TWO WOMEN WERE THE INITIAL PATIENTS WITH ACDC, THEY CAME HERE, THEY HAD NO IDEA WHAT IS THE UNDERLYING CAUSING DISEASE. AND THEY WERE STRUCK FOR A VERY LONG TIME. SO THEY STARTED WHEN THEY WERE MAYBE 20 YEARS OLD, THAT THEY HAD PROBLEMS BY WALKING. THE MORE THEY WALKED, THEY CERTAINLY DEVELOPED PAIN IN THEIR LEGS CALLED CLAUDEICATION, SWELLING IN SMALL JOINTS IN HANDS AS WELL AS IN THEIR FEET. AND WHEN THEY GO TO A DOCTOR, THEY IDENTIFY MASSIVE VASCULAR CALCIFICATION WITH LOWER ANKLE-BRACHIAL INDEX, THE CAUSE OF THE DISEASE WAS UNKNOWN. I WOULD LIKE TO SHOW YOU HERE THE MUSCULOSKELETAL PHENOTYPE WHICH IS QUITE PARTICULAR FOR THESE PATIENT POPULATIONS. SO YOU SEE THAT THESE PATIENTS DEVELOP LOSS OF JOINT SPACE. THEY DEVELOP JOINT CAPSULE CALCIFICATION. ATOPIC BONE FORMATION, AND ATHEROJOINT CHANGES. OVER PICTURE OF THESE CHANGES IN THE HAND OR IN THE FEET DOES NOT FIT WITH ANY KNOWN JOINT DISEASE LIKE RHEUMATOIDITIS. ALSO WHEN YOU LOOK AT THESE PATIENTS AND ASK HOW DOES THE DISEASE ACTUALLY START, YOU SEE THAT MOST OF THESE PATIENTS PRESENT INITIALLY WITH THESE JOINT CHANGES. AND ONLY ONE OF THEM PRESENTED INITIALLY WITH THE INTERMITTENT CLAUDICOT, PROBLEM OF BLOOD FLOW TO THE LOWER EXTREMITIES. THIS SHOWS YOU A NORMAL SPRAY OF A PATIENT, WHICH IS QUITE DIFFERENT WHEN YOU LOOK AT THE PATIENTS, ONE OF OUR ACDC PATIENTS, WHERE YOU SEE THIS VERY PROFOUND CALCIFICATION OF BLOOD VESSELS, COMPLETELY DESTROYS THE BLOOD VESSEL, DESTROYS THE BLOOD VESSEL. THIS SHOWS YOU CT CALCIUM SCAN AS WELL AS CT ANGIO, YOU SEE THE WHOLE DIMENSION OF THESE CALCIFICATIONS. IT STARTS OVER HERE GOING -- IN THE ILIAC, FEMORAL, POPLITEAL ARTERY, MOST CASES CALCIFICATION IS SYMMETRICALLY, LEADING TO PROFOUND VASCULAR IMPAIRMENT, SO MANY OF THESE MAIN BLOOD VESSELS WITH COMPLETELY OCCLUDED, COMPLETELY BECOMING STONES, AND THE PATIENTS DEVELOP COLLATERALS TO RESTORE VIABILITY TO THEIR LOWER LIMBS. SO THIS SHOWS YOU A NORMAL BLOOD VESSEL WITH THE MEDIA, AND IF YOU LOOK WHERE ARE THE CHANGES IN ACDC COMPARED TO OTHER CALCIFICATION DISEASES LIKE ATHEROSCLEROSIS THERE'S A REMARKABLE DIFFERENCE. THIS SHOWS ATHEROSCLEROTIC, THIS IS NEOINTIMA FORM THAT LARGELY OCCLUDES THE BLOOD VESSEL, CALCIFICATION IS FOUND IN THE NEOINTIMA. YOU SEE NO NEW INTIMA FORMATION, DISTINCT FROM ATHEROSCLEROSIS, MEDIA IS DESTROYED, LARGE CHUNKS OF CALCIUM GETTING INTO THE LUMINA OF THE PATIENT, CALCIUM IS LOCALIZED AT ELASTIC FIBER, WE DO NOT KNOW BREAKS FIRST AND CALCIFY, OR CALCIFY FIRST AND THEN BREAKS, BUT THIS IS WHERE CALCIFICATION FIRST OCCURS. PATIENTS CAME IN AND THIS IS A FAMILY OF FIVE, CONSANGUINEOUS FAMILY, ALL FIVE SIBLINGS AFFECTED, SAME CLINICAL FEATURES, FOR GENETICISTS LIKE BILLY GALL IT WAS A SLAM DUNK, WE IDENTIFIED LOSS OF CD73, NO CD73 CONVERTS AMP TO ADENOSINE AND WE THINK THAT IF THERE'S NO CD73, WE THINK THAT IT AFFECTS ALKALINE PHOSPHATASE, TISSUE SPECIFIC THAT LEADS TO CALCIFICATION. SO TO STUDY THAT WE MOVED ON AND DEVELOPED PATIENT-SPECIFIC CELL SYSTEMS. WE USED INDUCED PLEURIPOTENT STEM CELL TECHNOLOGY TO DEVELOP THE DISEASE MECHANISM IN A DISH. AS YOU KNOW, INDUCED PLEURIPOTENT STEM CELLS ARE SIMILAR TO EMBRYONIC STEM CELLS GENERATESSED FROM BLASTOCYTES, DIFFERENT TO THEM THEY ARE COMING FROM THE PATIENTS AND CAN BE GENERATED BY A VARIETY OF DIFFERENT CELL TYPES. THE OTHER ADVANTAGE OF INDUCED PLEURIPOTENT STEM CELLS, THEY ARE AMENABLE TO GENETIC CORRECTION, YOU CAN EASILY GENERATE -- NOT EASY BUT YOU CAN GENERATE ISOGENIC CONTROLS AND WORK WITH LOW PATIENT NUMBERS, BECAUSE YOU CAN USE EACH PATIENT AS ITS OWN CONTROL BY CORRECTING GENETIC DEFECT OR INDUCE GENETIC DEFECT IN A CONTROL PATIENT. SO WE USE MOSTLY ON MESODERM, MEANING BLOOD CELLS AND HEMATOPOIETIC CELLS, AND OUR PROGRAM IS TAILORED TOWARD DISEASE MODELING, THERAPEUTIC PRODUCTS AND DRUG SCREENING. SO THIS SHOWS OUR DIFFERENT PROTOCOL USING iPS CELLS AND DIFFERENTIATE iPS CELLS FOLLOWING DEVELOPMENTAL INTO CD31/CD34 CELL AND EASILY DIFFERENTIATE INTO MSC CELLS OR HEMATOPOIETIC-LIKE CELLS. SO FOR OUR ACDC PATIENTS WE WERE PARTICULARLY INTERESTED IN GENERATING MESENCHYMAL STROMAL CELLS BECAUSE THESE ARE CELLS THAT HAVE BEEN REPORTED TO HAVE OSTEOGENIC CALCIFICATION PROPERTIES. SO WE GENERATED THESE CELLS AND THEN WE INDUCED OSTEOGENIC STIMULATION AND UNDER OSTEOGENIC STIMULATION NORMAL MSC CALCIFIED AT AROUND DAY 21. WHEN YOU LOOK HERE, YOU HAVE HERE AT DAY 12, USUALLY NOT CALCIFY, NO CALCIFICATION INDICATED BY RED COLOR, iPS DERIVED MSC FROM CONTROL PATIENTS DO NOT CALCIFY. HOWEVER ACDC PATIENT CELLS CALCIFY AT DAY 12. YOU SEE TIME CALLS OF THE INCREASE OF CALLS CALCIFICATION SO WE DEVELOPED IN VITRO MODEL THAT MIMICS SOME CHARACTERISTICS OF WHAT WE SEE IN THE PATIENTS. SO WE THEN USE THAT TO TEST SOME HYPOTHESIS UNDERLYING IN THIS DISEASE PROGRESS, SPECIFICALLY THE ROLE OF TNAP, METABOLIZING PYROPHOSPHATE TO PHOSPATE, AND IF YOU LOSE THIS BREAK CALCIFICATION OCCURS. SO WE LOOKED IN OUR ACDC CELLS, COMPARED TO CONTROL CELLS, TO SEE IF THEY HAVE INCREASED TNAP ACTIVITY AND CLEARLY WE SEE MASSIVE INCREASE IN ACDC PATIENTS THAT LEAD TO DECREASE IN PYROPHOSPHATE LEVELS. SO INDICATING THAT ACDC THERE'S AN UPREGULATION OF TNAP THAT CAUSES PYROPHOSPHATE TO DECREASE. HOW IS THIS LINKED TO PRODUCTION OF ADENOSINE WHICH IS METABOLIZED BY CD73. ON THIS SIDE YOU SEE THE ADENOSINE LEVEL IN CONTROL PATIENTS, NORMAL ADENOSINE LEVELS AND DECREASE IN ACDC PATIENTS, MAKING LESS ADENOSINE. WE NEEDED TO CHALLENGE THE SYSTEM AND PROVIDE EXCESSIVE AMOUNTS OF ANP TO GENERATE MORE ADENOSINE AND THEN WE LOOKED IN ADDITION OF THIS OTHER ENZYME WOULD AFFECT ADENOSINE PRODUCTION IN THAT CASE. SO YOU SEE HERE THAT NO TREATMENT WITH AMP, LOW LEVELS OF ADENOSINE. IF YOU GIVE AMP INCREASES TO PROBABLY A HUNDRED-FOLD OR TWO HUNDRED-FOLD, INHIBITOR OF TNAP DID NOT AFFECT BASE LEVEL OR AMP DEPENDENT LEVEL IN CONTROL PATIENTS. WE FOUND A VERY DIFFERENT SITUATION IN ACDC CELLS, IN ACDC CELLS UNDER NORMAL LEVEL ALREADY SIGNIFICANTLY DECREASED PRODUCTION OF ADENOSINE. SO IN NORMAL CELLS, CD73 IS THE MAIN PRODUCER OF ADENOSINE. ACDC CELLS THERE SEEMS TO BE ANOTHER ENZYME THAT IS ABLE TO GENERATE ADENOSINE, AND WHEN WE BLOCK AN INHIBITOR OF TISSUE NON-SPECIFIC ALKALINE PHOSPHATASE IT DRAMATICALLY DECREASES LEVELS OF ADENOSINE. IN SUMMARY, WHAT WE FIND SURPRISINGLY ON THE DISEASE MECHANISM THAT CD73 CONVERTS AMP TO ADENOSINE, AND LOW LEVELS OF TNAP TO SOME DEGREE MOVE PYROPHOSPHATE TO PHOSPATE. IF YOU DON'T HAVE CD73, TNAP IS UPREGULATED, PRODUCES SMALL AMOUNTS OF ADENOSINE, COMPENSATORY MECHANISM. HOWEVER, BECAUSE ITS PREFERRED SUBSTRATE IS PYROPHOSPHATE IT GOES DOWN AND YOU GET CALCIFICATION, THE CELL TRIES TO COMPENSATION FOR LOST OF CD73, UPREGULATES TNAP, THE MAIN FUNCTION OF TNAP IS IMPORTANT INHIBITOR OF CALCIFICATION. SO WE MOVED ON AND WANTED TO TEST THESE FINDINGS IN MICE. HOWEVER, NONE OF THE KNOWN MOUSE MODELS HAVE CALCIFICATION PHENOTYPE. SO WE CAME UP WITH I THINK A VERY COOL IDEA, AND WE GENERATED TERATOMA, INJECTING INTO MICE TO SEE IF THEY CAN DIFFERENTIATE IN ALL DIFFERENT GERM LAYERS. THE TERATOMA HAVE SOME VERY SMALL MICRO CALCIFICATION, HOWEVER IF WE INJECTED iPS CELLS FROM ACDC PATIENTS THEY SHOWED THESE REALLY LARGE AMOUNTS OF CALCIFICATION IN THE TERATOMA AND WHEN WE OVEREXPRESS CELLS, GAIN OF FUNCTION EXPERIMENT, IT RESCUED THIS PHENOTYPE MEANING CD73 IS DRIVING OR PREVENTING CALCIFICATION PROCESS. TERATOMAS REPRESENT SIMILARITIES TO WHAT WE SEE IN THE BLOOD VESSELS SO THIS IS STAINING FOR CALCIFICATION, ALIZARIN RED, VON KOSSA, ALKALINE PHOSPHATASE. YOU SEE HERE TNAP STAINING THAT'S JUST OUTLINING THE CALCIFICATION. THE BEAUTY OF THIS SYSTEM IS THAT THEN WE CAN TREAT THE MICE TO AFFECT PATIENT CELLS WE TRANSPLANTED INTO THESE MICE, WE USED A TRACK CALLED ETIDRONATE, FIRST GENERAL GENERATION BISPHOSPHONATE, INFANTILE FORM OF CALCIFICATION, AND ETIDRONATE DECREASED CALCIFICATION COMPARED TO CONTROLS. BASED ON THAT WE DEVELOPED A CLINICAL PROTOCOL WHERE WE USED BISPHOSPHONATE ON PATIENTS, SECONDARY OBJECTIVE REGRESSION INCREASE IN LOWER EXTREMITY BLOOD FLOW AND FUNCTIONAL IMPROVEMENT AND DECREASING HAND AND FOOT PAIN. WE'RE TREATING -- WE'VE ENROLLED 100 PER CENT OF PATIENTS REPORTED IN THE UNITED STATES, WHICH ARE 7 SO IT'S A VERY RARE DISEASE BUT WE BROUGHT THEM ALL TO THE NIH. THIS IS PRELIMINARY DATA FROM HOW THIS POTENTIALLY COULD IMPACT CALCIFICATION, REMEMBER THIS IS A MASSIVE CALCIFICATION IN THESE PATIENS HERE. HERE AGAIN IT'S CT CALCIUM SCAN. AND WE DID SOMETHING VERY SIMILAR WHAT YOU WOULD DO IN THE HEART, WE QUANTIFIED AMOUNT OF CALCIUM. YOU SEE HERE THAT BEFORE TREATMENT THE PATIENT HAS STEADY INCREASE IN CALCIFICATION, WHEN WE INITIATED TREATMENT IT PLATEAUS, IN SOME CASES EVEN DECREASED WHICH IS QUITE REMARKABLE BECAUSE THIS KIND OF CALCIFICATION HAVE NOT BEEN REPORTED TO REGRESS IN ANY CIRCUMSTANCES. SO WITH THAT I WOULD LIKE TO END AND THANK PARTICULARLY ACDC PATIENTS AND FAMILIES, MY CREW AND COLLABORATOR AND NIAMS, JOINS CALCIFICATION AND COLLABORATOR BILL GAHL AND I'M HAPPY TO ANSWER YOUR QUESTIONS. THANK YOU. [APPLAUSE] >> WE HAVE TIME FOR ONE QUICK QUESTION. PLEASE GO TO A MICROPHONE. IF YOU HAVE OTHER QUESTIONS BOTH SPEAKERS WILL BE HERE AFTER THE HOUR ENDS. THANK YOU. >> ONE BRIEF COMMENT. IN TERMS OF THE DIFFERENTIATION OF CALCIFICATION, I HAVE SEEN AT AUTOPSY CALCIFICATION OF HEART VALVES, WHERE THEY DIFFERENTIATE INTO BONE MARROW. I'M NOT THE ONLY ONE THAT HAS SEEN THIS BUT THAT ATTESTS TO THE PLURIPOTENCY OF DYSTROPHIC OR VALVULAR MITRAL VALVE CALCIFICATION, YOU MENTIONED THAT, IT'S AN EXTREME THING, BUT IT'S TRUE. IT LOOKS LIKE BONE MARROW BUT IT'S CALCIFICATION IF THE TRICUSPID VALVE. >> THERE ARE AS YOU INDICATED, THERE ARE -- A TISSUE CAN RESPOND IN DIFFERENT WAYS TO CALCIFICATION, ECTOPIC IS JUST FORMATION OF BONE OR MINERALS IN THE TISSUE. BUT THEN YOU CAN ALSO GET ECTOPIC OSSIFICATION, MEANING THEY REALLY FORM BONES AND TISSUE, IN TISSUE OF INFLAMMATION, IN SOME CASES IN ATHEROSCLEROSIS AND VASCULAR TISSUE. WHEN WE LOOK AT SAMPLES WE SEE INDEED WHAT YOU DESCRIBE, BONE FORMATION, BONE FORMATION THAT MIMICS BONE MARROW, SO WE SEE INFLAMMATORY CELLS ARISING AND ALSO IT'S A VERY COMPLEX PROCESS THAT IS HAPPENING IN THE BLOOD VESSELS. >> THANK YOU. >> AGAIN, WE THANK BOTH SPEAKERS. IF HAVE YOU ADDITIONAL SPEAKERS, COME DOWN, DR. KOZEL AND DR. BOEHM WILL HANDLE YOUR QUESTIONS. THANK YOU VERY MUCH. [APPLAUSE]