PROJECT SUMMARY Varicose veins (VV), which affects approximately 23% of adults in the USA are non-lethal however, it negatively impacts the quality of life in many patients due to its debilitating symptoms. With limited methods for prevention and early treatments for VV, there is an unmet need to understand the mechanisms of VVs. VV pathogenesis is multifactorial and is a combination of epigenetic and genetic factors. In addition, hemodynamics and inflammation play an important role in venous pathology. Vein wall dilation occurs concurrently or as a consequence of the venous hypertension and inflammation, further exacerbating the venous reflux. These changes results in the overexpression of matrix metalloproteinases (MMPs), which causes degradation of the extracellular matrix proteins, thereby further affecting the structural integrity of the vein wall. This effect is due to changes in the endothelium (ECs) and smooth muscle cells (SMCs) is the alterations in cross talk which plays an important role in venous constriction. Alterations in venous tone also contribute to the development of VV. Our group identified 30 independent genetic variants associated with VV in a previous genome-wide association study (GWAS). In complex disease traits, the disease-associated loci may be in non-coding regions even if they may be responsible for gene expression regulation. It is difficult to know in which cell types or physiological contexts this regulation occurs. Thus, candidate genes require validation to establish causality. One way of doing this is with single-cell genomic assays. In our study, we propose to elucidate the molecular mechanisms behind the development of varicose veins by leveraging the recent advances in single-cell sequencing technologies. By using single cell RNA-seq, we propose to examine the transcriptomic changes at the single cell level in varicose veins. Moreover, we propose to decipher the key regulators effecting the different components of the varicose vein and their effects on ECSMC crosstalk (Aim 1). Due to limited access to ECs and SMCs from patients (given that the isolation and long-term culture of vascular cells from patient’s blood is an extremely difficult task), the mechanisms underlying vascular dysfunction in varicose veins remain largely unknown. As part of Aim 2, we will utilize the induced pluripotent stem cell technology to recapitulate the disease phenotype of varicose veins in vitro.