>> GOOD AFTERNOON, IT'S MY PLEASURE TO INTRODUCE TODAY'S NIH DIRECTOR LECTURER MANFRED BOEHM. HE GOT HIS H M.D. FROM UNIVERSITY OF HIDLEBERG IN GERMANY AND DID HIS RESIDENCY IN MEDICINE IN BERLIN BEFORE BECOMING A RESEARCH FELLOW AT THE MAX STOVER CENTER FOR MOLECULAR MEDICINE ALSO IN BERLIN AND LATER AT UNIVERSITY OF MICHIGAN. SEVERAL YEARS AGO HE WAS RECRUITED TO BECOME AN INVESTIGATOR AT NHLBI AND RECENTLY TENURED AS A SENIOR INVESTIGATOR. HISlz RESEARCH INTERESTS HAS FOCUSED ON INJURY AND REMODELING OF BLOOD VESSELS AND HE STUDIED A VARIETY OF DIFFERENT SYSTEMS FOR BLOOD VESSEL INJURY AND EMBRYO LOGICAL DEVELOPMENT AND ALSO HAS BEEN INTERESTED IN HEREDITARY GENETIC DISORDERS, CONGENITAL DISORDERS WHICH BLOOD VESSELS ARE ALTERED. RECENTLY BECAME ENGAGED IN THE STUDY OF INTERESTING GROUP OF PATIENTS WHO CAME HERE FROM THE UNDIAGNOSED DISEASES PROGRAM. WE HAVE A NEW DISEASE CALLED ACDC. I DON'T KNOW IF YOU'LL TALK ABOUT THAT. OKAY. GREAT. SO WITHOUT FURTHER ADIEU, I'M DELIGHTED TO INTRODUCE DR. BOEHM WHO WILL SPEAK TO US ABOUT GENOTYPE TO MECHANISM TO THERAPY, MOLECULAR INSIGHT IN RARE GENETIC VASCULAR DISEASES. >> THANK YOU, DR. GOTTESMAN FOR THE KIND INTRODUCTION AND GREAT HONOR FOR ME TO PRESENT HERE. GREAT TO SEE SO MANY COLLEAGUES AND MORE IMPORTANTLY SO MANY FRIENDS HERE IN THE AUDIENCE. SO TODAY I WILL TALK ABOUT TWO RARE DISEASES. ONE WHERE WE WENT FROM A PHENOTYPE TO A GENOTYPE DISEASE MECHANISM THAT LEADS TO IDENTIFICATION OF NEW DISEASE CALLED ACDC. ARTERIAL CALCIFICATION DUE TO DEFICIENCY IN CD 73. THIS IS A COLLABORATION WITH UNDIAGNOSED DISEASE PROGRAM HEADED BY BILL GOFF. THE SECOND PROTOTYPE -- THE FIRST ONE IS PUBLISHED. THE SECOND ONE IS UNPUBLISHED WORK, A COLLABORATION WITH STEVE HOLLAND FROM NI NIAID AND THE ROLE OF STATS IN VAST REMODELING. SO MY LAB, THE MAIN FOCUS IS REMODELING. WE WORK A LOT IN MOUSE MODELS AND WE ARE EXPLORING CERTAIN TISSUE SIGNALING PATHWAYS LIKE TNF ALPHA, TGF BETA, NK KAPPA B, CHECK STAT PATHWAYS AN THINK ACCELERATE CELL PHENOTYPE PROLIFERATION, CELL FATE CHANGES DEATH OF CELLS, AND WE ARE IN RECENT YEARS VERY INTERESTED IN TRANSLATING THESE FINDINGS IN TO HUMAN CONTEXT BY STUDYING GENETIC DISEASES WHO HAVE A VASCULAR IMPLICATIONS. APPROXIMATE I WILL AS INDICATED TALK ABOUT TWO DISEASES IN THIS CONTEXT. SO THE FIRST PART IS ON ARTERIAL CALCIFICATION DUE TO DEFICIENCY IN CD 73 OR ACDC. SO THIS IS THE STORY OF TWO SISTERS FROM KENTUCKY WHO HAVE BEEN BATTING A HER -- BATTLING A MYSTERIOUS DISEASE MOST OF THEIR LIFE. SO IT STARTED IN THEIR 20s WHEN -- OR TEENSND THEY HAD PAIN IN THEIR LOWER LEGS WHILE WALKING. SO IT STARTED WHEN THEY WERE WALKING MAYBE 8 OR 10 BLOCKS FOR QUITE SOME TIME, THEN THIS PAIN SLOWLY STARTS TO APPEAR. SO AS THE TWO SISTERS GREW OLDER THE PAIN GOT WORSE AND WORSE THAT AT THE END THEY COULDN'T WALK A BLOCK AND THEY ALSO BECAME PAIN IN THEIR HANDS. THE TWO SISTERS CAME FROM A FAMILY WITH FIVE SIBLINGS AND ALL THE YOUNGER SIBLINGS MAKE FUN OF THEM AND SAID YOU'RE GETTING OLD PRETTY FAST, YOU CAN'T EVEN WALK A BLOCK. HOWEVER, SOONER OR LATER ALL OF THESE THREE OTHER SIBLINGS DEVELOPED THE SAME SYMPTOMS. THE SYMPTOMS ARE CONSISTENT WITH SOMETHING THAT'S CALLED (INDISCERNIBLE) INTERMITTENT. THE ANATOMICAL CAUSE Z WAS OBVIOUS TO THE PHYSICIAN WHO STUDIED -- WHO TREATED -- SAW THIS FAMILY. THIS SHOWS AN X-RAY OF ONE OF THE SISTERS AND YOU SEE THIS MASSIVE CALCIFICATION IN THE KNEE AREA. THIS CALCIFICATION IS A CALCIFIED ARTERY AND WHAT YOU -- SO YOU SEE HOW NORMAL KNEES LOOK LIKE, ON THE OTHER SIDE YOU USUALLY DON'T -- YOU ARE UNABLE TO SEE A NORMAL ARTERY. SO IT WAS PRETTY CLEAR TO THE PHYSICIAN THAT THIS CAUSES THE DISEASE. HOWEVER, WHAT WAS UNCLEAR IS WHAT IS THE REAL MOLECULE OR MECHANISM BEHIND THIS. SO THESE TWO SISTERS CAME TO THE NIH UNDIAGNOSED DISEASE PROGRAM. THIS SHOWS SOME VASCULAR EVALUATION THAT WE DID HERE. YOU SEE HERE THIS IS A CT KALES L YUM SCAN, YOU SEE MASSIVE CALCIFICATION HERE IN THE ILIAC ARTERY AND THE POPLITEAL ARTERY. THIS ONE IS IN CT -- WHERE YOU SHOW WHERE IT SHOWS BLOOD FLOW. YOU CAN SEE HERE THE BLOOD FLOW STOPS HERE AND ALL THE LOWER LEG IS PERFUSED BY CHOLERA STATS FORMED OVER THE YEARS. WHAT WAS SURPRISING TO US THAT THIS APPEARS TO BE LOCALIZED ONLY IN THE LOWER EXTREMITIES. AND WE COULD NOT FIND ANY CALCIFIED ARTERY FROM THE WASTE UP. SPECIFICALLY THE CORONARIES WHERE WE SEE A LOT OF CALCIFICATION DUE TO ATHEROSCLEROSIS WAS FREE OF CALCIUMFUL SO THIS SLIDE SUMMARIZES THE CLINICAL PRESENTATION OF THESE PATIENTS. CORONARY ARTERIES AND ALTER ARE COMPLETELY NORMAL. HOWEVER THEY HAVE HEAVY CALCIFIED BLOOD VESSELS FROM THE WASTE UP, THE IT YAK ARTERIES FEMORAL ARTERIES, TICK ALREADY ARTERIES AND THEY DID NOT -- TICK ALREADY ARTERIES AND THEY DIDN'T HAVE -- TICK ALREADY ARTERIES. THEY HAD COMPLETELY NORMAL KIDNEY FUNCTION AS SEEN HERE. HOWEVER, THEY HAD VERY ABNORMAL PATHOLOGICAL ABIs, THIS IS MEASURING THE BLOOD PRESSURE IN THE UPPER EXTREMITIES AND COMPAREED TO THE LOWER EXTREMITIES. SOME OF THEM HAD THAT LOW PRESSURE, THIS ONE THAT'S ALREADY CRITICAL AND THESE PATIENTS ARE TO ACTUALLY LOSE THEIR LIMBS. 90% OF ATHEROSCLEROTIC BLOCKS ARE KALES L FEWED. THIS IS A STRONG MARKER FOR FUTURE CARDIO VASCULAR EVENTS AS INDICATED IN DIABETES, SPECIFICALLY TYPE 2 AND THE PARTICULAR PATTERN IN DIABETES PATIENTS IS CALLEDED (INAUDIBLE) CIRRHOSIS. ALSO PATIENTS WITH END STAGE RENAL DISEASE DEVELOP VASCULAR CALCIFICATION. THERE IT'S A STRONG MARKER FOR DISEASE PROGRESSION. SO WHAT CAUSES PATHOLOGICAL VASCULAR CALCIFICATION? THE KEY IS CONCENTRATION OVERCALLS L YUM AND PHOSPHATE EXTRA CELLULAR. THIS IS RELATIVELY HIGH IN NORMAL BODY FLUID AND VERY CLOSE TO THE CONCENTRATION THAT PRECIPITATES AND AUTOMATICALLY FORMS CALCIUM CRYSTALS. SO IT'S EASY TO UNDERSTAND THERE ARE PROBABLY A LOT OF ANTI-KALES L FEWCATION FACTORS PRESENT TO PREVENT CALCIFICATION TO HAPPEN. THERE ARE ALSO EFFECTORS THAT REGULATE THE CALCIUM AND PHOSPHATE HOMEOSTASIS AND CELL DEATH PLAYS AN IMPORTANT ROLE RELEASING CALCIUM FROM APOPTOTIC BODY WHICH IS THOUGHT TO BE THE MAIN CAUSE OF CALCIFICATION IN ATHEROSCLEROSIS. THERE ARE ALSO OTHER PROGRAM LIKE OSTEOGENIC DIFFERENTIATION THAT MEANS CELL DIFFERENTIATES TO WHAT IS AN OSTEOBLAST AND START TO MAKE BONE. FOR THIS TO HAPPEN IT CHANGES TO MICROENVIRONMENT AND TO ENABLE PRECIPITATION AND CALCIUM CRYSTAL FORMATION. ALSO MATRIX DEGRADATION IS BELIEVED TO PLAY AN IMPORTANT PART IN THIS PROCESS. THERE ARE TWO MAIN FORMS OF VASCULAR CALCIFICATION. ONE IS (INDISCERNIBLE) CALCIFICATION MOSTLY SEEN IN ATHEROSCLEROSIS. SO IF YOU SEE HERE THE VESSEL WALL, THIS IS WHERE THE CALCIFICATION TAKES PLACE. THIS IS PROBABLY 90, 95% OF ALL CALCIFICATION USUALLY A CLINICIAN COMES ACROSS. WHICH IS VERY DIFFERENT FROM ANOTHER FORM OF CALCIFICATION, WHICH IS A MEDIA CALCIFICATION, WHERE THE CALCIUM CRYSTALS ARE FORMING WITHIN THE WALL, NOT IN THE NEO(INDISCERNIBLE) OVER THERE, THIS CALCIFICATION IS OFTEN INDICATED HERE ASSOCIATED WITH THE ELASTIC FIBERS. AND BREAK DOWN OF THESE ELASTIC FIBERS INDUCES THIS PROCESS. CAN INDUCE THIS PROCESS. THIS SHOWS A NORMAL ARTERY. SO YOU SEE THE MEDIA OVER HERE AND THEN ENDOTHELIAL LAYER HERE, PERIVASCULAR LAYER HERE, WHICH IS VERY DIFFERENT FROM AN ATHEROSCLEROTIC ARTERY OR ARTERY FROM ACDC PATIENT. SO THESE ATHEROSCLEROTIC ARTERY, THE MEDIA IS OVER HERE. AND IT'S DELAMBNATED FROM THE LAMINA ELASTIC ANGINA OVER HERE. SO IT TRIES TO PROCESS THE OCCLUSION OF THE VESSEL FORMATION AND MOST CALCIFICATION HAPPEN TO BE IN THIS AREA. HOWEVER, IN SOME CIRCUMSTANCE CAN GO OUT INTO THE MEDIA. ON THE OTHER HAND HERE YOU YOU HAVE A SAMPLE OF ACDC PATIENTS BEFORE WE DIAGNOSE WITH ARCCDC UNDERWENT BYPASS OPERATION SO WE WERE LUCKY OBTAIN THE SAMPLE. YOU CAN SEE THE LAMINA ELASTICA IS HERE AND CALCIFYCATION PROCESS TAKES PLACE IN MEDIA AND THIS PROCESS COMPLETELY DESTROYS THE MEDIA, LEADING TO A PATHOLOGY THAT WE SEE. THESE TWO DISEASES ARE VERY DIFFERENT. SO ACDC IS NOT AN EARLY FORM ATHEROSCLEROSIS, IT'S A VERY DIFFERENT DISEASE. SO THIS FAMILY CAME TO UNDIAGNOSED DISEASE PROGRAM, THAT USES GENETIC TOOLS TO EVALUATE AMONG OTHERS TO EVALUATE THEIR PATIENTS. THIS FAMILY AS INDICATED THERE ARE FIVE SIBLINGS AFFECTED. FIVE SIBLINGS AFFECTED AND THE THIRD GENERATION FAMILY SO IT'S THE IDEA -- IDEAL CASE WE USE GENETICS TO EVALUATE OR FIND OUT WHAT CAUSES THIS CALCIFICATION. SO THE GROUP BY BILL GAL DID A HAPLOTYPE METHOD TO LOCK WHICH PART OF THE CHROMOSOME IS INHERITED FRo9 BOTH PATIENTS IN SIBLINGS AND IS THE SAME ALL FIVE SIBLINGS AND THE BLOOD THAT SHOWS THAT IS SHOWN HERE AND YOU SEE THERE IS ONLY ONE REGION OF HOMOZYGOSITY, THESE ARE THE TWO PART, THESE ARE FIVE SIBLINGS AND THIS PATIENT SHARE -- THESE FIVE PATIENTS SHARED WITH EACH OTHER. SINCE ALL FIVE PATIENTS HAVE THE SAME CLINICAL PHENOTYPE WE CAN ASSUME THAT THE GENE THAT CAUSES IT LIES IN THESE REGION. THIS REGION HAD 90 GENES SO ONE OF THESE 90 GENES CAUSES ACDC. THAT IS WHERE WE COME INTO PLAY, WORK FROM (INDISCERNIBLE) WHO GOT SKIN BUY I DON'T KNOWSIS FROM ALL FIVE PATIENTS AND FROM THEIR PARENTS. THE FIRST THING WE DID IS SKIN FIBROBLASTS SO WE HAVE CELLULAR CORRELATE TO LOOK ARE THERE ANY CHANGES IN TRANSCRIPTIONAL PROFILE IN THESE PATIENTS THAT HELP US TO NARROW DOWN WHICH GENE ARE AFFECTED. SO WE RUN A MICROARRAY, AND WE LOOK FOR GENES, THERE WAS ONLY ONE GENE DIFFERENT WHICH WAS CD 73 OR FIVE ECTO NUCLEARTIDASE. THIS WAS VERY SURPRISING THAT WE DID ONE EXPERIMENT AND LOOKED MAYBE HIT IT ALREADY AND CINDY WENT ON AND CONFIRMED THESE DIFFERENCE IN EXPRESSION BY RUNNING A PCR BY USING TWO CONTROL SAMPLES, TWO SAMPLES FROM AFFECTED MEMBERS. EXPRESSION OF THIS GENE IS DRAMATICALLY DECREASED. WE THEN RUN A WESTERN AND YOU CAN SO E IN THREE CONTROLS CD 73 OVER HERE, BUT NOT IN AFFECTED PATIENTS. WITH THIS INFORMATION WE WENT BACK TO BILL AND SAID HEY, BILL, SEQUENCE CD 73. HE SAID WHY SEQUENCE P CD 73? WE THINK THIS IS THE THE GENE THAT ACTUALLY CAUSES ACDC AND HE WAS VERY SKEPTICAL BUT HE SEQUENCED IT AND CALLED BECOME AND SAID YOU WON'T BELIEVE IT BUT CD 73 HAS THERE IS A VARIANT ALL FIVE SIBLINGS SHARE VERY RARE NOT SEEP BEFORE. SOUP LATER WE IDENTIFY TO OTHER FAMILIES MEMBERS OF THEM HAD THE SAME PHENOTYPE WITH AGAIN MUTATION IN CD 73. SO CD 73 IS CAUSING GENETIC VARIANCE, CD 73 IS CAUSING ARCCDC. WHAT IS CD 73 DOING? CD 73 IS INVOLVED IN EXTRA CELLULAR PARAMETER METABOLISM AND CONVERTS ANP TO ADEAN SCENE AND P PHOSPHATE. WHAT IS INTERESTING IS IT'S DIRECTLY DOWNSTREAM OF ANOTHER ENZYME CALLED ENPB-1. ENPB-1 ME TAB RISES ATP TO ARCNP AND PIROPHOSPHATE PROVIDES THE SUBSTRATE FOR CD 73. AND THERE ARE GENETIC VARIANTS KNOWN IN ENPB-1 GENERALIZED TO TIER CALCIFICATION IN INFANCY. SO THESE ARE KIDS WHO ARE WHO DEVELOP MASSIVE VASCULAR CALCIFICATION SHORTLY AFTER LIFE AND THIS COMPLICATION IS SO SEVERE MANY KIDS ACTUALLY DIE OF MYOCARDIAL INFARCTION BEFORE THREE MONTHS OLE. -- OLD. SO PUTTING THESE FINDINGS FROM WHAT WE KNEW SO FAR FROM FUNCTION OF CD 3, TOGETHER WE PULLED UP THE FOLLOWING WORKING HYPOTHESIS. WE THINK THAT WE COME UP WITH WITH ATP IS CONVERTED TO ANP AND PIROPHOSPHATES THROUGH EMPB-1, AMP IS CONVERTED TO ADEN SCENE PHOSPHATE BY CD 73. PIROPHOSPHATE IS STRONG INHIBITOR OF CALCIFICATION. PHOSPHOTASE ON THE OTHER HAND IS A COENZYME INDUCING CALCIFICATION. AND IT'S KNOWN THAT PHOSPHOAT A TIME CREATES BY ROW PHOSPHATE AND THAT'S HOW IT LEADS TO CALCIFICATION. IN GAKI WE HAVE NO PIROPHOSPHATE PRODUCED SO WE HAVE HIGH LEVELS OF TISSUE CALCIFICATION. IN ACDC WE HAVE NO CD 73, THERE'S NO ADEAN SCENE, WE THINK ADEN SCENE INHIBITS PHOSPHOTASE. IF YOU DONE HAVE THIS INHIBITION PHOSPHOTASE GOES UP AND CREATES BY ROW PHOSPHATE, LEADS TO TISSUE KALES L FEWCATION. TO TEST THAT A FIRST -- CALCIFICATION. WE LOOKED FOR ALKALINE PHOSPHOTASE ACTIVITY IN THE SKIN FIBROBLAST THAT WE HAD. AND YOU CAN SEE HERE THESE ARE CONTROL CELLS, WE INDUCE KALES L FEWCATION USING A YOU CAN SEE HERE THERE IS NO BLUE STAINING INDICATEDDED FOR TISSUE NON-SPECIFIC ARGININE FOESTASE. IN ACDC PATIENTS YOU SEE BASELINE THERE'S SOME WHICH DRAMATICALLY INCREASE AFTER WE INDUCE CELLS WITH CALCIFICATION MEDIA. THIS SLIDE OVER HERE SHOWS THE QUANTIFICATION OF THAT IN BLACK ARDC, THE STRIPES OVER THERE CONTROL, YOU SEE THERE'S A SLIGHT INCREASE OF ARGININE PHOSPHOTASE IN ACDC COMPARED TO NORMAL CONDITION. WHICH IS THEN INCREASED DRAMATICALLY INCREASED WHEN YOU PUT CELLS IN KALES L FEWING CONDITIONS. THE NEXT SET OF EMPERIMENTS WE HOOKED -- LOOKED AT THESE CELLS IF THEY CALCIFY. WE DID CALCIFICATION ASSAY WHERE WE SUBCHECKED THE CELLS, CONTROL CELLS AND ACDC CELLS TO THESE CONDITIONS AND YOU CAN SEE HERE IN RED WHICH IS INDICATIVE OF CALCIUM CRYSTAL FORMATION THAT'S HEAVY CALCIUM CALCIFY KISS OF THESE CELLS UNDER OSTEOGENIC STEMLATION. WHEN WE INDUCE THE SAME CALCIFICATION AT THE PRODUCT OF DISMISSING IN CD 73 DEFICIENT PATIENT ADEN SCENE, IT CAN INHIBIT CALCIFICATION INDICATING THE LACK OF ADENOSENE CAUSES CALCIUM CRYSTAL FORMATION TO HAPPEN. THIS LEADS TO THE FOLLOWING MECHANISM, IN THE EXTRA CELLULAR SPACE, ATP IS CREATED TO METABOLIZE TO AMP AND PIE RUE PHOSPHATE BY EMTP-1. IT'S THEN FURTHER METABOLIZED TO ADENOSINE AND PHOSPHATE BY CD 73. THEN ADENOSINE THROUGH ADENOSINE RECEPTOR, INHIBITS ARGININE PHOSPHOTASE. AND THIS INHIBITION OF ALKALINE PHOSPHOTASE ALLOWS PIROPHOSPHATE TO ACCUMULATE AND INHIBIT CALCIFICATION. SO THE PATIENTS THAT THAT WE HAD ACCESS TO WITH ACDC DEVELOP VERY SLOWLY OVER THE YEARS THINK SYMPTOMS. BUT THE DISEASE IS PROGRESSIVE AND MORE AND MORE OF THESE PATIENTS HAVE PROBLEMS WITH THEIR LIMP AND SOME HAD TO GO IN SURGERY. SO WE NEED TO LOOK FOR SOMETHING TO HELP THESE PATIENTS. SO AS I INDICATED ARGININE PHOSPHOTASE AND PIROPHOSPHATE IS A KEY IN THIS DISEASE. WE THOUGHT TO TEST, ANALOG OF PIROPHOSPHATE IF THIS DECREASE CALCIFICATION IN VITRO AND MAYBE AN IMPORTANT TRACK TO TREAT THESE PATIENTS. SO WE TOOK OUR SKIN FIBROBLASTS AND INDUCE CALCIFICATION SHOWN OVER HERE AND AFTER THE CALCIFICATION WAS ESTABLISHED WE GAVE THEM PHOSPHOTASE WHICH NOT ONLY BLOCKS IT, IT EVEN REVERSE CALCIFICATION. SO THIS WAS ENOUGH RATIONALE FOR US TO PUT THESE PATIENTS ACTUALLY ON THE CLINICAL PROTOCOL. SO WE HAVE NOW CLINICAL PROTOCOL, IT'S AN OPEN LABEL NON-RANDOMIZED PILOT STUDY WHERE WE LOOK FOR TO EVALUATE EFFICACY OFTYTRYNATE TO TREAT ARC CCD PATIENTS. THE PRIMARY OBJECTIVE IS ATTENUATION OR EVEN DECREASE IN THE LOWER EXTREMITY CALCIFICATION BY CT CALCIUM SCORE. WE ALSO LOOK FOR CHANGES IN BLOOD FLOW AND IMPROVEMENT FUNCTIONAL IMPROVEMENTS THROUGH RETINOL TEST. WE ACTUALLY HAVE ALREADY ENROLLED 50% OF ALL KNOWN ACDC PATIENTS WHICH IS FOUR PATIENTS. SO WE THEN WENT AND LOOKED IN MORE DETAIL HOW DOES ADENOSINEI/w ACTUALLY MODULATE ENDOCELLULAR CELL TO LEAD TO CALCIFICATION? THIS IS ON GOING WORK, UNPUBLISHED WORK. AND ONE THING THAT'S IMPORTANT IS TO SEE WHAT KIND OF CALCIFICATION DO WE ACTUALLY HAVE. IT'S KNOWN AS MEDIA CALCIFICATION BUT DO WE HAVE THEIR STONE, JUST FORMATION OF A ROCKY SUBSTRATE IN THE BLOOD VESSEL, IS THERE ACTUALLY BONE FORMATION. THE BONE FORMATION THERE ARE SPECIFIC PROGRAM THAT ARE KNOWN TO BE INDUCED IN CELLS THAT INDUCE THAT. WE GOT ANOTHER TISSUE SAMPLE FROM A VERY RECENT PATIENT, SO THIS IS A CHRONIC CALCIFIED BLOOD VESSEL WHICH IS HARD AS STONE AND CALCIFICATION IS SEEN HERE IN BROWN. IF YOU LOOK AT H AND SOON IN WHAT YOU SEE HERE IS VERY SOLID MASS OF CALCIFICATION IN THESE SAMPLES. SO FAR IT'S INDICATIVE THAT IT'S NOT BONE FORMATION SO THAT THERE IS ALMOST LIKE A STONE FORMING SUBSTANCE FORMED IN THESE BLOOD VESSELS. THIS WAS IMPORTANT INFORMATION FOR US BECAUSE CINDY WAS TRYING A LONG TIME TO LOOK FOR HOW IS AMP AFFECTED IN THESE PATIENTS BECAUSE CYCLIC AMP WAS ASSOCIATE WITH THE THE BONE FORMATION PROGRAM. WE COULDN'T MAKE ANY SENSE OUT OF THAT. AT SOME POINT SHE CAME TO ME AND SAID I'M GETTING TIRED OF THE CYCLIC AMP, SO SHE BOUGHT 15, 20 DIFFERENT INHIBITORS OF ALL DIFFERENCE PATHWAY AND THROUGH IT ON THE CELL AND LOOK WHICH OF THAT ACTUALLY AFFECTS ARGININE PHOSPHOTASE. SO THIS IS THE COMPLEX SIGNALING OF ADENOSINE. THIS IS FOUR DIFFERENT RECEPTOR RECEPTORS, TWO ACTIVATE CYCLIC AMP TWO INHIBIT IT. ADENOSINE SIGNALS THREW AKT, KNOCKS AND PLC PATHWAY TO CHANGE TARGETS THAT MAY REGULATE VASCULAR CALCIFICATION. SO PUTTING A LOT OF EXPERIMENTS INTO ONE SLIDE SHE CRIMINAL OUT NOT CYCLIC AMP CONSISTENT WITH WHAT WE SEE IN HISTOLOGY. AND ONLY PATHWAY THAT ARE LEFT ARE PI-3 KINASE PATHWAY MTOR AND P MEK ERK PATHWAY. SHE THEN WHEN ON AN THIS SHOWS ONE OF THESE EMPERIMENTS SO ONLY POSITIVE RESULT ARE SHOWN. YOU SEE INCREASE ON STIMULATION OF ARGININE PHOSPHOTASE AND YOU SEE HERE THAT THESE THREE INHIBITORS MEK AND PI-3 PATHWAY AND RAPAMYCIN NICELY DECREASE ARGININE PHOSPHOTASE. THIS SHOWS STAINING FOR THAT AND SHOWS DEFECTIVE RAP MYSIS CONTROL CELLS TO ACDC YOU SEE ARGININE PHOSPHOTASE POSITIVE CELLS AND RAPAMYCIN BLOCKS THAT. MOREOVER IF WE INDUCE CALCIFICATION YOU SEE HERE CALCIFICATION FORMATION IN THE CONTROL WHICH IS COMPLETELY BLOCKED BY ALL THREE INHIBITORS. WE ARE INTERESTED IN MOVING ON WITH RAPAMYCIN FOR POTENTIAL CLINICAL TRIAL TO TREAT PATIENTS WHO HAVE SOME KIND OF IMMUNE OR AUTOIMMUNE DISEASE WITH INCREASE INFLAMMATION, IT'S ALSO H OFTEN ASSOCIATED WITH CALCIFICATION, WE PUT IN A BENCH TO BEDSIDE TO TREAT, TO THE CHEST TO PUT ON THE WORK TO DEVELOP A TREATMENT PROTOCOL FOR JUVENILE MYSITIS TOGETHER WITH NEAHS. SO THIS SHOWS YOU AND IN VITRO MODEL WE DEVELOPED TO EXPLORE CALCIFICATION. HOWEVER, TO TEST DRUGS LIKE RAPAMYCIN IT WOULD BE GOOD TO HAVE AN ANIMAL MODEL. TO DO THAT WE ACTUALLY DEVELOP IPS CELLS FROM THESE ACDC PATIENTS. AND ONE CRITERIA, WE WILL GET BACK TO THAT LATER THESE IPS CELLS IS THEY HAVE TO FORM TERATOMAS, THESE TERATOMAS FORM ALL CONFERENCE KINDS OF LAYER, GERM LAYERS AND WE ARE WONDERING MAYBE ACDC TERATOMA IN THESE MICE CALCIFY. THIS IS ACTUALLY A MICROCT EVALUATION OF ONE OF THESE TERATOMAS, YOU CAN SEE ROLL IPS, THIS HUGE MAST, THAT'S SOME LITTLE MICROCALCIFICATION WHICH IS OFTEN SEEN IN TERATOMAS. BUT IN ACDC YOU SEE THESE VERY PROMINENT VERY MASSIVE CALCIFICATION IN THE TISSUE. WHAT WE CAN DO ISKo NOW TAKE OUR MOUSE WITH THESE ACDC CELLS, TREAT THEM WITH RAPAMYCIN AND SEE IF YOU CAN MODULATE CALCIFICATION. SO I'M SWITCHING TABERS AND TALK ABOUT SECOND DISEASE WHICH IS STROKE SYNDROME, THE STATS IN VAST REMODELING. THIS STEMS FROM EARLY WORK IN MY LAB WHERE WE EXPLORED MOUSE VASCULAR REMODELING AND WE WERE SPECIFICALLY INTERESTED IN WHAT REGULATES THE INFLAMMATORY RESPONSE WITHIN THE BLOOD VESSEL AFTER INJURY. THAT WAS THEN TELLING THE IMMUNE SYSTEM HOW MUCH INFLAMMATION IS NECESSARY TO HAVE A BALANCED REMODELING PROCESS. AND WITH SERIES OF STUDIES WE IDENTIFIED THAT UPON TNF ALPHA ACTIVATION AND STIMULATION OF SMOOTH MUSCLE CELL, MEDIA SMOOTH MUSCLE CELL, THAT IN THESE CELLS THERE IS AN ACTIVATION SIMULTANEOUS ACTIVATION OF NF KAPPA B AND STAT 3 AND IT FORMS A COMPLEX AND BINDS TO PROMOTERS OF VARIOUS CYTOKINES AMONG STF-1 AND THEN REGULATES EARLY MACROPHAGE INFILTRATION AND T-CELL INFILTRATION. SO WE WERE INTERESTED IN HOW DO WE TRANSLATE THIS INTO PATIENTS? SO WE CONTACTEDDED STEVE HOLLANDS WHO ACTUALLY IDENTIFIED AND WORKED ON AUTOSOMEAL DOMINANT HYPER E SYNDROME OR CHOKE SYNDROME. IT BECAME THE NAME BECAUSE IN THE BIBLE THERE WAS SOME PHRASE THAT SATAN PUNISHED POOR CHOKES WITH SOIL BOILS FROM THE SOIL TO THE FOOT UNTO HIS GROUND. AND IF YOU LOOK AT PATIENTS YOU CAN EASILY UNDERSTAND WHY DID THE PERSON WHO GAVE THIS DISEASE THE NAME, BECAUSE THESE PATIENTS DEVELOP BACTERIAL INFECTION, STAPHYLOCOCCUS AUREUS SKIN INFECTION. YOU SEE THE RASH AND NEW BONE AND MASSIVE ABSCESS IN THAT PATIENT POPULATION. MANY OF THESE PATIENTS DEVELOP PNEUMONIA AND ACTUALLY PNEUMONIA CAN BE TREATED QUITE EFFICIENTLY WITH WITH ANTIBIOTICS. HOWEVER, THE PROBLEM, I TALK TO STEVE AND TO OTHER PHYSICIANS WHO TAKE CARE OF THESE, THE PROBLEM IS THAT THESE LUNGS DO NOT HEAL VERY PROPERLY. AND FURTHERMORE THESE PATIENTS ALSO HAVE SOME KIND OF VASCULAR DEFECTS TO HAVE SMOOTH MUSCLE FORMATION, CORONARY ARTERIES IN THE TIRTUOSITY AND WOUND HEELING PROBLEMS. SO IT WAS A SURGICAL INTERVENTION AND THESE WOUNDS JUST DIDN'T HEAL. SO ABOUT STAT 3 MUTATION THAT CAUSES STROKE WASjF IN STAT 3 AND THIS WAS IDENTIFIED BY STEVE HOLLAND. THESE PATIENTS HAVE VASCULAR ABNORMALITY, VASCULAR TIRTUOSITY AND A LOT OF NON-IMMUNE SYMPTOMS WHICH YOU THINK SOMEBODY WITH A VARIANT IN STAT 3 HAVE. THEY HAVE DELAYED WOUND HEALING, DERMAL RASH AT BIRTH AND THEY HAVE MULTIPLE CONNECTIVE TISSUE AND SKELETAL ABNORMALITIES. SIMILAR TO YOU ARE APPROACH IN ACDC PATIENTS. WE GOT SKIN SAMPLE BIOPSIES FROM THESE PATIENTS AND WE GENERATED SO FAR MORE THAN 12 FIBROBLAST LINES FROM THESE PATIENTS THE HOT SPOT FOR THE MUTATION IN STAT 3 EITHER IN THE DNA BINDING DOMAIN OR IN THE SH-2 DOMAIN AND WE HAVE PROBABLY 50% OF BOTH OF THEM. HOWEVER, THERE IS FUNCTIONALLY FOR ALL THE STUDY WHAT I SHOWED YOU NO DIFFERENCE BETWEEN THESE TWO MUTATIONS AND ALSO PATIENTS ARE INDISTINGUISHABLE IF THEY HAVE MESSAGE 2 DOMAIN OR DNA BINDING MUTATION. VERY SIMILAR TO THE ACDC STUDY THE FIST THING WE DID, WE LOOKED FOR WHAT IS DIFFERENT? HOW ARE THESE CELLS REGULATING THEIR TRANSCRIPTOME DIFFERENTLY. WE KNOW FROM SMOOTH MUSCLE CELL TNF GOOD START POINT TO LOOK AT THAT. SO WE TREATED CELLS WITH TNF ALPHA AND WE LOOK FOR DIFFERENCE AND WHAT POPPED UP WAS THAT BY DOING PATHWAY ANALYZERS ANGIOGENIC PROGRAM AND MEMBERS OF THAT KEY GENES THAT SHOWS THE HIGHEST CHANGES WHERE SEVERAL MMPs AND KDR AND THAT SOMEHOW THIS PROGRAM IS ON THE HIGHER CONTROL OF HIF 1 ALPHA. WE CHOOSE THESE 5, FOUR GENES TO LOOK AT THE SIGNATURE TO EXPLORE IN MORE DETAIL WHAT IS GOING ON IN THESE CELLS. THIS SUMMARIZES A WHOLE SET OF EXPERIMENT WHERE WE USE THESE FOUR GENES AND LOOK FOR CHANGES IN HOW SIGNALING REGULATES IT SO HERE YOU SEE THE FIRST THREE ARE WILD TYPE. YOU SEE NICE UP REGULATION OF TNF ALPHA SIGNALING STIMULATION HOWEVER THIS IS COMPLETELY BLOCKED. SO WE DID A WHOLE -- THIS SUMMARIZES A WHOLE SERIES OF EXPERIMENT WITH GAIN AND LOSS OF GENETIC LIGAN AND LOSS OF FUNCTION EXPERIMENT CHROMOSOME, PHARMACOLOGICAL UP HI BIG WHERE WE FOUND TNF ALPHA ACTIVATES NF KAPPA AT THE SAME TIME STAT 3 GETS ACTIVATED BY IL-6. WE DOPE KNOW HOW -- THIS GOES WITHIN MINUTES. THIS ACTIVATION IS REQUIRED FOR THESE TRANSCRIPTIONAL PROGRAM TO BE ACTIVATED THEN STAT 3 IS PHOSPHORYLATED. BINDS TO NF KAPPA AND THIS BINDING IS REQUIRED TO BIND TO PROMOTERS OF THESE FOUR DIFFERENT GENES TO ACTIVATE THIS PROGRAM. IN ENDOTHELIAL CELLS, IL-6 WILL NOT ACTIVATE THESE PROGRAMS. THE ACTIVATION OF THIS PROGRAM IS SYNERGISTIC ACTIVATION OF NF KAPPA AND STAT 3 SIGNALING VERY SIMILAR WHAT WE FOUND IN THE -- IN OUR SMOOTH MUSCLE CELL IN THE MOUSE MODEL. SO WE HAD A VARIANT IN STAT 3 THAT PREVENTS THE BINDING OF STAT 3 TO NF CAP P AN BINDING TO THE CHROMATIN AND BLOCKS TRANSCRIPTIONAL ACTIVATION OF THESE FOUR GENES. WHAT IS THE ROLE IN THIS OVERALL PROCESS OF HICF-1 ALPHA? I WILL SHOW YOU THE DATA WHAT WE HAVE ON THAT SO FAR. WE PUT ATTENTION TO THAT BECAUSE THERE WAS A RESEN PAPER TO SHOWEDDED THAT HIF-1 ALPHA IS KEY REGULAR HAYTOR BETWEEN T REGULATORY CELLS AND TH-17 CELLS. AND IT REGULATES TH-17 CELLS TOGETHER WITH STAT 3. INTERESTINGLY PATIENTS HAVE A LACK OF TH 17 CELLS, INDICATING A TIF, IMMUNE CELLS PLAY A ROLE IN HOPES WHICH INDICATE A SIMILAR MEK ANYMORE SOMEHOW IN SKIN FIBROBLAST. SO WE FIRST LOOK IF HIF 1 ALPHA IS STABILIZED DIFFERENTLY BETWEEN NORMAL AND HYPOXIA CHANGING STIMULATION. SO WE FIRST HAD THE CELLS ON THE NOMOPIA THEN SWITCH TO HYPOXIA. YOU SEE STABILIZATION OF HIF IN THE CONTROL CELLS. HOWEVER THERE WAS EXACTLY THE SAME STABILIZATION IN CHOKES PATIENTS. HIF-1 ALPHA IS NOT HYPOXIA REGULATED, IT'S ALSO REGULATED BY CYTOKINE. IF IF WE FIRST STAFF THEM, BASICALLY PUT EVERYTHING TO THE SAME, THESE CELLS AND THEN STIMULATE WITH TNF ALPHA, TGF BETA, IL-6 YOU SEE STRONG INDUCTION OF HIF-1 ALPHA ABOUT SEPTEMBER THIS THE CHOKES PATIENT. MEANING IT'S CRITICAL FOR REGULATING CYTOKINE MEDIATED STABILIZATION OF HIF-1 ALPHA. WE THEN FURTHER LOCKED IF HIF-1 ALPHA IS REQUIRED FOR THESE PHENOTYPE TRANSCRIPTION LEVEL SIGNATURE HA WE IDENTIFIED AND USE OVER AN OVER AGAIN. SO IF WE KNOCK DOWN HIF-1 ALPHA AN OVEREXPRESS STAT 3, IN FIBROBLASTS YOU SEE TRANSCRIPTIONAL ACTIVATION OF THESE PROGRAM IS COMPLETELY DIMINISHED, COMPARED TO NORMAL FIBROBLASTS SO WE USE FIBROBLASTS BECAUSE WE CAN SELECT THEMTOR THAT. ON THE OTHER HAND WHEN WE KNOCK DOWN STAT 3 AN OVEREXPRESS HIF-1 ALPHA, THE TRANSCRIPTIONAL PROFILE IS COMPLETELY ABOLISHED IN CELLS INDICATING YOU NEED BOTH. HIF-1 ALPHA AND STAT 3 TO ACTIVATE THAT PROGRAM. AT THAT POINT WE THOUGHT WE NEED TO LEARN A LITTLE BIT MORE ABOUT HOW IS HIF-1 ALPHA REGULATED. WITH PATIENT WHOSE HAVE A GENETIC VARY I CAN'T WANT IN THE PATHWAY OF HIF-1 PATHWAY REGULATION, PATIENTS THAT HAVE MUTATIONS IN VHL TO INCREASE HIF-1 ALPHA AND LEADS TO TUMORS, THE OPPOSITE WHAT WE SEE IN HOPES AND ALSO A VERY INTERESTING POPULATION OF PATIENTS WHO HAD MUTATION IN FUMARATE HYDROTASE. AND THESE PATIENTS ALSO HAVE MASSIVE INCREASE IN HIF-1 ALPHA. THIS IS DUE TO ACCUMULATION OF FUMARATE THAT BLOCKS PH.D., KEY ENZYME TO REGULATE HIF FUNCTION AND THAT LEADS TO RENAL CELL CARCINOMA. THE PATIENTS THEMSELVES ARE INTERESTING TO US BUT THE FACT THAT WE HAVE A SUBSTANCE THAT CAN MODULATE HIF FUNCTION IS VERY USEFUL FOR OUR HOPES PATIENT, MORE IMPORTANTLY FUMARATE IS CLINICALLY USED TO TREAT PSORIASIS PATIENTS IN EUROPE AND IT WAS ALSO NOW USED FOR PATIENTS WITH MULTIPLE SCLEROSIS. WHICH IS CALLED BG-# ST. CXFC SO WE FIRST LOOKED WHAT IS DMF DO IN OUR SKIN FIBROBLASTS? AND DMFF IS STRONGEST INDUCER OF HICF-1 ALPHA WE CAME ACROSS, MUCH STRONGER THAN HYPOXIA, STRONGER THAN CHLORIDE AND STABILIZES HIF 1 ALPHA INDEPENDENT OF STAT 3 FUNCTION. HOWEVER, WHEN WE USE DMF TO SEE IF THE IF WE CAN RESCUE THE CHOPE PHENOTYPE THE TRANSCRIPTIONAL SIGNATURE IN OUR CHOPE FIBROBLAST WE FOUND FOLLOWING. THE RED COLUMNS IS CONTROL PLUS TNF. YOU SEE NICE INDUCTION. THAT WHICH IS COMPLETELY ABOLISHED IN THE CHOPE PATIENT OVER HERE. IF YOU JUST USE DMF YOU DONE SEE ACTIVATION OF THESE PROGRAM CONTROL CELLS AS WELL AS IN CHOPE CELLS. HOWEVER, IF YOU COMBINE TNF AND DMF TOGETHER, IF YOU STABILIZE HIF UNDER THE STIMULATION OF TNF YOU THEN AGAIN SEE TNF DRIVEN STABILIZATION OF THE TRANSCRIPTION PROGRAM HOWEVER YOU ALSO SEE THE SAME TRANSCRIPTIONAL FACTORS COMING UP IN CHOPE PATIENTS. MEANING THAT STABILIZATION OF HIF IS CRITICAL FOR CHOPE AND THIS DRUG MAYBE POTENTIAL TREATMENT FOR CHOPE SO OVERALL THIS IS THE SAME SLIDE I SHOWED BEFORE, NOW WE PUT HIF INTO THE IT BECAUSE P IT'S SEN TRILLION TO REGULATE -- CENTRAL TO REGULATE THE PROGRAM. WE DONE KNOW YET IF STAT 3 TRANSCRIPTIONAL REGULATES HIF-1 ALPHA OR THROUGH PROTEIN STABILITY. BUT WE POSTULATE THAT THESE PROGRAM IS AN IMPORTANT IN ANGIOGENESIS. HOWEVER, WE HAVE SKIN FIBROBLASTS. HOW CAN WE MAKE FROM SKIN FIBROBLASTS THE NEXT STEP TO SKIN FIBROBLASTS ARE IMPORTANT IN ANGIOGENESIS? WE NEED SEVERAL CELL TYPES FOR THAT. WE USE IPS CELL TECHNOLOGY, WE GENERATED IPS FROM CHOPE PATIENTS AND YOU CAN USE THESE IPS CELLS FOR DISEASE MODELING, WHAT WE ARE DOING FOR DRUG DISCOVERY, AND YOU CAN FOR SOME PATIENT POPULATION, NOT CHOPE BUT MAYBE CELL REPLACEMENT THERAPY. SO WE DEVELOPED A DIFFERENTIATION PROGRAM WHERE WE TAKE IPS CELLS AND THEN THANK YOU CERTAIN MESODERM IN THE INTERMEDIATE WE CAN GENERATE ENDOTHELIAL CELLS, SMOOTH MUSCLE CELLS FIE PRO BLASTS AND HE MAT POIETIC STEM CELLS. WE FOUND INTERESTING FINDINGS IN THE -- HEMATOPOIETIC STEM CELLS. IN THE ENDOTHELIAL CELLS IN THE ROLE OF STAT 3 ON THAT. BUT WHAT I WANT TO SHOW YOU IS A DIFFERENT ASSAY CALLED TERATOMA ASSAY WHICH I EXPLAINED EARLIER BEFORE. SO WE LOOKED IF THESE IPS CELLS FROM DIFFERENT PATIENTS FORM THE SAME TERATOMAS. SO WE USE CONTROL IPS CELL CHOPE IPD CELLS AND IN STAT 3 USING DNA SIN NUCLEASE INTO THE AAVS 1 LOCUST. ANY6v DIFFERENCE BETWEEN CONTROL HERE AN RESCUE HERE INDICATE STAT 3 DEPENDENT. TO OUR SURPRISE TERATOMA HERE THEY FORM ALL THE SAME THREE LINEAGE TO SIZE -- THE SIZE AN APPEARANCE WAS DIFFERENCE SO YOU SEE MASSIVE TERATOMA IN THE CONTROL ORANGE, A LOT OF BLOOD VESSEL. YOU SEE A VERY PALE SMALL TERATOMA FROM CHOPE. TAKING THE SAME IPS CELLS WHEN OVEREXPRESS STAT 3 YOU GET MASSIVE TERATOMAS WITH EVEN FIVE WEEKS. HOWEVER, STAT 3 IS IMPORTANT REGULARLYTOR OF CELL GROWTH IN TUMOR. HOW CAN WE SAY IT HAS SOMETHING TO DO WITH ANGIOGENESIS. ONE FIRST THING WE DID, WE LOOK -- WHAT IS THE CAPILLARYIZATION OF THESE TERATOMA? THIS IS NORMAL STAINING FOR CD 31. FEW CD 31 POSITIVE CELLS SO DECREASED ENDOTHELIAL CELL IN THIS TERATOMA. THIS MIGHT BE TOO BECAUSE THE TUMOR GROWS SLOWER AND NEEDS LESS BLOOD VESSEL. SO WE DID ANOTHER RESCUE EXPERIMENT WHERE WE SAID IF STAT 3 REGULATES ANGIOGENESIS AND WE HAVE IN THE CHOPE IPS CELLS L DEFECT IN STAT 3, IF WE GIVE THESE TERATOMAS ENDOTHELIAL CELLS THEY SHOULD GROW NORMALLY, IT'S TRUE. SO IF YOU -- SO THIS IS TUMOR FORMATION RATE, THESE CHOPE IPS CELLS HAVE A VERY LOW EFFICIENCY OF FORMING TUMORS SO IF YOU PUT BACK IN STAT 3 IT GOES UP TO ALMOST 100%. WE CAN RESCUE THESE PHENOTYPE COMPLETELY WITH (INAUDIBLE) THAT REACHES NORMAL CONTROL VALUES. TUMOR WEIGHT IS RESCUE, WE'RE