Abstract 305: Role of Endothelin-1 Signaling in Osteogenic Differentiation and Matrix Calcification of Vascular Smooth Muscle Cells

Abstract

Vascular calcification (VC), the “hardening” of blood vessels and valves, is prevalent in patients with type II diabetes mellitus (T2DM). Although often asymptomatic at early stages, patients with VC have increased risk for cardiovascular morbidity and mortality. It is presently unknown whether diabetes is causative and what regulatory molecules and pathways are critical for initiation and progression of this complication. In a diabetic VC mouse model recently examined in our laboratory, vascular smooth muscle cells (SMCs) were found to be the major contributing cell source, with increased susceptibility to osteochondrogenic differentiation and calcification under hyperglycemia. Endothelin-1 (ET1) was high in diabetic mouse sera and cartilaginous areas of diabetic blood vessels, correlating with an increase in its type A receptor (ETA), osteochondrogenic transcription factor Runx2, and receptor for advanced glycation end-products (RAGE). Importantly, blocking the biological activity of ETA with its antagonists effectively inhibited matrix calcification of SMCs in culture associated with high glucose at levels of hyperglycemia. Addition of the ETA antagonists, atrasentan and BQ-123, to SMC cultures resulted in an inhibition of SMC calcification up to 71%, whereas ETB antagonism with BQ-788 resulted in only a slight reduction (~20%). Additionally, SMCs treated with atrasentan expressed higher mRNA levels of myocardin, a master transcription factor co-activator of SMCs, and lower expression levels of Runx2, suggesting a preservation of SMC phenotype in their original fate decision through ETA antagonism. Further exploration of ET1-ETA signaling through addition of ET1 to SMCs cultures showed a dose-dependent increase in SMC calcification, occurring only in high glucose cultures. Finally, blocking RAGE signaling with a neutralizing antibody reduced not only high glucose-accelerated SMC calcification but also the calcification enhanced by exogenous ET1. Taken together, our results showed a correlation of ET1-ETA with diabetic vascular osteochondrogenesis and its critical role in high glucose-associated SMC calcification. A possible interplay between the ET1-ETA and RAGE pathways is currently under investigation.