Project Grant F32AR085954

MECHANISMS REGULATING TENDON FIBROSIS AND ADHESION FORMATION - PROJECT SUMMARY INJURIES TO FLEXOR TENDONS IN THE HAND CAN RESULT IN SIGNIFICANT LOSS OF FUNCTION, IMPACTING DAILY ACTIVITIES AND THE OVERALL QUALITY OF LIFE. OVER 100,000 FLEXOR TENDON INJURIES OCCUR EACH YEAR, REPORTED MAINLY IN MALES BELOW AGE 30, ALTHOUGH PRELIMINARY DATA FROM HOUSTON METHODIST REPORTS 72% OF INJURIES OCCURRING AFTER AGE 50. UP TO 40% OF FLEXOR TENDON INJURIES FORM ADHESIONS VIA FIBROSIS TO THE SURROUNDING TISSUE THAT HINDER TENDON GLIDING AND IMPEDE FUNCTION. PROLONGED AND EXCESSIVE INFLAMMATION PROMOTES FIBROSIS, WHICH IS EXACERBATED BY AGING. CURRENTLY, NO TREATMENTS EXIST TO PREVENT ADHESION. PHYSICAL THERAPY REDUCES ADHESION BUT CAN RUPTURE THE REPAIR. AN ADDITIONAL SURGERY, TENOLYSIS, IS REQUIRED TO REMOVE THE ADHESION AND IMPROVE FUNCTION. THEREFORE, THERE IS A CRITICAL NEED TO DEVELOP THERAPIES TO PREVENT ADHESION FORMATION WHILE PROMOTING FLEXOR TENDON HEALING. TWO KEY PATHWAYS INVOLVED IN ADHESION FORMATION INCLUDE PRO-INFLAMMATORY NUCLEAR FACTOR KAPPA-LIGHT- CHAIN-ENHANCER OF ACTIVATED B CELLS (NF-KB) AND PRO-FIBROTIC TRANSFORMING GROWTH FACTOR BETA (TGF-B) SIGNALING. THIS PROPOSAL WILL INVESTIGATE CYCLIN-DEPENDENT KINASE 9 (CDK9) - A MASTER REGULATOR OF TRANSCRIPTION THAT IS REQUIRED FOR BOTH NF-KB AND TGF-B SIGNALING. CDK9 INHIBITION REDUCES INFLAMMATION WHILE PROTECTING MECHANICAL PROPERTIES IN MUSCULOSKELETAL INJURIES. HOWEVER, THE ROLE OF CDK9 IS COMPLETELY UNEXPLORED IN TENDON HEALING. I WILL TEST THE HYPOTHESIS THAT EXCESSIVE CDK9 ACTIVITY DRIVES ADHESION FORMATION VIA INFLAMMATORY NF-KB SIGNALING AND PROMOTING MYOFIBROBLAST DIFFERENTIATION VIA THE TGF-B PATHWAY, WHICH IS EXACERBATED IN AGED MICE. IN AIM 1, I WILL DETERMINE THE ROLE OF CDK9 IN TENOCYTE-TO-MYOFIBROBLAST DIFFERENTIATION USING UNDER- AND OVER-EXPRESSION EXPERIMENTS IN VITRO AND INVESTIGATE INTERACTIONS WITH TGF-B MEDIATORS. IN AIM 2, I WILL DEMONSTRATE GENETIC EVIDENCE OF CDK9-MEDIATED TENDON ADHESION RNA INHIBITION TO KNOCKDOWN CDK9 EXPRESSION AND DETERMINE THE ROLE OF CDK9 IN INFLAMMATION AND MYOFIBROBLAST DIFFERENTIATION IN VIVO. IN AIM 3, I WILL COMPARE CDK9 ACTIVITY IN YOUNG AND AGED MICE DURING TENDON HEALING USING SINGLE-CELL RNA-SEQUENCING, SPATIAL TRANSCRIPTOMICS, AND SPATIAL PROTEOMICS TO MAP ROLE OF CDK9 IN THE INFLAMMATORY RESPONSE AND MATRIX REMODELING AND IDENTIFY THE KEY CELL POPULATIONS AND PATHWAYS REGULATING ADHESION TO THE SURROUNDING TISSUE. THE COMPLETION OF THIS PROPOSED WORK WILL PROVIDE INSIGHT INTO THE MECHANISMS NECESSARY TO CREATE EFFECTIVE ANTI-FIBROTIC THERAPIES THAT COULD PREVENT ADHESION FORMATION TO THE SURROUNDING TISSUE AFTER INJURY. FURTHER, THESE RESULTS WILL INFORM FUTURE STUDIES TARGETING CDK9 SIGNALING IN MUSCULOSKELETAL AND INFLAMMATORY DISEASES. AN OUTSTANDING MENTORING TEAM WILL PROVIDE ALL NECESSARY EXPERTISE TO TRAIN ME. COMPLETING THE PROPOSED STUDIES WILL PROVIDE ME WITH THE OPPORTUNITIES AND TRAINING TO SUPPORT MY CAREER GOALS AND DEVELOP THE SKILLS I NEED TO ACHIEVE MY LONG-TERM GOAL OF BECOMING AN INDEPENDENT INVESTIGATOR IN THE AREAS OF MECHANOBIOLOGY, TISSUE ENGINEERING, AND FIBROSIS.