PROJECT SUMMARY/ABSTRACT Thirteen percent of adults over 75 suffer from aortic valve stenosis (AVS), a progressive disease that leads to aberrant collagen deposition, leaflet stiffening, and eventual valve calcification at late stages. As it stands, surgical or transcatheter valve replacement is the only treatment option for severe AVS and is limited in durability and can require indefinite anticoagulation therapy. AVS is also sexually dimorphic with men having a two-fold higher risk for developing direct calcification, whereas women with equal disease severity tend to have more valvular fibrosis prior to calcification. The molecular mechanisms underlying sexual dimorphism in AVS progression remain poorly understood, but growing evidence suggests AVS is an inflammation-dependent disease. Recent studies have focused on the role that infiltrating immune cells such as macrophages play in AVS, with a growing appreciation that men and women undergo different immune responses and show differences in compositions of inflammatory cytokines. Combined, these observations suggest not only the need for alternative treatments for AVS (e.g., pharmaceutical therapies), but also the opportunity to develop personalized treatments based on sex and inflammatory state. Based on the extant evidence, we propose to investigate the role of inflammation in sex-specific valve disease progression. We hypothesize that AVS sexual dimorphism is perpetuated by: 1) sex differences in valve cell secreted factors promoting differential polarization of macrophages, 2) differences in infiltration behavior of male and female macrophages, and 3) direct macrophage-valve cell contact eliciting distinct phenotypic differentiation dependent upon sex, leading to the formation of a pro-fibrotic or pro-calcific niche. We will test these hypotheses with the following specific aims (1) Delineate how secreted factors from valve cells effect macrophage phenotype and vice versa in a sex-dependent manner, (2) Elucidate sex-specific differences in AVS disease initiation within an in vitro 3D co-culture model. The applicant, Dr. Alex Khang, will work with sponsor, Dr. Kristi S. Anseth, collaborating investigators with expertise in immunology (Dr. Laurel Hind) and sex-specific differences (Dr. Leslie Leinwand), and microscopy consultant Dr. Joseph Dragavon to carry out the experimental studies. All studies will be performed at the University of Colorado Boulder. All members of the research team and the required equipment for the proposed research are housed in the same building (Jennie Smoly Caruthers Biotechnology Building, JSCBB) which will facilitate constant interactions as well as frequent opportunities for mentorship, while eliminating logistical issues. The JSCBB also houses numerous faculty that are domain-experts and word-leaders in their respective field who Dr. Khang will interact with frequently at local seminars and events. During the funding period, Dr. Khang will gain expertise in en...