Project Summary/Abstract Peripheral artery disease (PAD) is a public health crisis, affecting 20 million Americans and 200 million people worldwide, and continues to grow due to aging and the epidemic rise of diabetes and renal metabolic disorders. Vascular calcification (VC) is accelerated in these conditions, particularly in a distal pattern of medial artery calcification (MAC) in the lower leg and foot. In chronic limb-threatening ischemia (CLTI) — the most advanced form of PAD — we have shown that MAC poses a significant challenge to lower extremity revascularization and is a major risk factor for limb amputation. However, there remains no effective treatment for VC, and both the pathologic characteristics and mechanistic drivers of MAC in PAD are poorly understood. Emerging data suggest that Klotho — a regulator of insulin signaling and phosphate homeostasis — is lost in diabetes and renal disease, which may play an important role in osteogenic pathways promoting MAC. We hypothesize that Klotho is a central regulator in the pathogenesis of MAC, and its loss under conditions of hyperglycemia, hyperphosphatemia and low shear stress promotes VSMC osteogenic transdifferentiation and VC. This hypothesis will be rigorously investigated via two innovative and complementary central aims: Aim 1 takes a clinically-oriented approach to define molecular and cellular calcific changes occurring in the serum and vasculature of patients with CLTI. Pedal artery tissue and serum samples from patients with CLTI will be analyzed, focusing on the relationship between Klotho arterial tissue expression, serum Klotho levels, clinical pedal artery calcium burden (pMAC score), and VSMC phenotype. Aim 2 approaches the hypothesis from a basic science angle, using 3D human engineered vessels in culture to characterize de novo molecular alterations occurring in response to conditions of hyperglycemia, hyperphosphatemia and low shear stress. I will carry out this work with the close support of my mentors Dr. Michael Conte, an accomplished vascular surgeon-scientist and global leader in the field of PAD, as well as Dr. Matthew Kutys, who is an expert in state- of-the-art microfluidics and 3D vascular modeling. Both the Conte and Kutys labs are embedded within the rich UCSF research environment and provide access to the resources needed to execute my project. This investigation builds upon my career goal to become a vascular surgeon-scientist focused on the field of vascular calcification — a highly impactful area that remains a critical barrier to improving clinical outcomes in PAD. My prior research has laid important groundwork in this area, quantifying the problem of pedal artery calcification and establishing its impact on outcomes in CLTI. The proposed work provides an innovative opportunity to develop my unique investigator profile by delving translationally into the mechanisms governing MAC, leveraging the expertise of my mentors and my background in MAC clinical re...