Project Summary/Abstract The current proposal represents a combined clinical patient-oriented and experimental murine investigation which seeks to examine mechanisms of vascular dysfunction in obesity. Obesity has developed into one of our most critical health care problems as 69% of the US population is currently overweight or obese. Adipose tissue dysfunction, inflammation, and insulin resistance are essential hallmarks linking obesity to the pathogenesis of cardiovascular disease. Our preliminary data demonstrate marked up-regulation of a unique pro-inflammatory Wnt signaling pathway that may play a major role in mechanisms of vascular dysfunction in obesity. In this proposal, we will examine the role of Wnt signaling in the regulation of microvascular endothelial function in intact blood vessels and isolated endothelial cells acquired from living subjects. We will utilize a multidisciplinary approach and complementary expertise between clinical and basic scientists to characterize the pathophysiological role of dysfunctional Wnt5a signaling. In aim 1, we will characterize depot-specific mechanisms of vascular endothelial dysfunction in human adipose tissue arterioles using videomicroscopy of small vessels isolated from subcutaneous and visceral fat compartments during elective surgical procedures in 150 obese and 50 age- and gender-matched lean subjects. We will characterize vascular phenotypes in relation to Wnt signaling and test the hypothesis that over-activation of Wnt5a-mediated signaling is a dominant regulatory feature that leads to vascular dysfunction. In aims 2 and 3, specific pharmacological and biological inhibitors of the Wnt5a and TGFβ pathways will be employed using arterioles and endothelial cells from aim 1 to test the hypothesis that antagonism of Wnt5a reverses vascular dysfunction, in part through its ability to modulate EndoMT in adipose tissue, and seek to identify novel regulators and therapeutic targets in obesity. To corroborate these findings in genetic models, we will explore EndoMT and the vascular and metabolic phenotypes of mice that are engineered to conditionally ablate or overexpress Wnt5a in myeloid cells. In aim 4, studies of endothelial phenotyping will be repeated 6-months after life-saving bariatric weight loss surgical intervention in the same 150 obese subjects from aim 1 to examine the effects of marked weight reduction on arteriolar responses and relevant Wnt molecular pathways identified in aims 2 and 3. The overall project combines studies of cellular signaling and whole vessel physiology using primary tissues from severely obese individuals where clinically very little vascular data currently exist. Our proposal may identify the Wnt5a-Sfrp5 axis as a novel modulator of vascular biology and potentially lead to the identification of new targets and approaches to combat obesity-induced cardiovascular disease.