Abstract 124: Integrated Omics Approaches to Elucidate Targets of Cyclic AMP-Epac-Rap1A Signaling in Microvascular Smooth Muscle Cells: Identification of Role in Production and Deposition of Extracellular Matrix Fibrillar Collagen

Abstract

Objective: Intracellular signaling by cyclic AMP (cAMP) has an essential role in vascular smooth muscle (VSM) physiology, including relaxation of large blood vessels such as aorta and pulmonary artery. Studies support a mechanism of signaling through e xchange p rotein a ctivated by c AMP (epac) and Ras-related small GTPase Rap1B down-regulation of RhoA activity. The role of cAMP in small blood vessels however was not examined, and remained unknown. This study elucidated the targets of cAMP signaling in peripheral blood vessels, specifically VSM explanted from healthy human (h) cutaneous arterioles and mouse (m) tail artery (microVSM). Results: Global changes in protein expression were assessed by Differential-in-Gel Electrophoresis in quiescent (h)-microVSM treated with the adenylate cyclase activator and cAMP elevating agent forskolin (10 μM, 30 min or 18 hr). Detectable changes were observed in proteins associated with the cytoskeleton, stress-response, protein-synthesis, -folding, -membrane transport, and extracellular matrix. Notably, actin modulating proteins caldesmon, ezrin-moesin, zyxin, gelsolin, and α-adducin, as well as cytoskeletal proteins, tubulin and vimentin were differentially expressed ( P< 0.05; 3-5 replicates). These results agreed with our recent demonstration of cAMP activation of epac-Rap1A, and RhoA-ROCK-F-actin signaling in (h)- and (m)-microVSM to increase expression and cell surface transport of functional α 2C -adrenoceptors (α 2C -ARs) that mediate vasoconstriction. Transcriptome analysis of Rap1A-null (knockout) (m)-microVSM transduced with constitutively active Rap1A showed connections to signaling linked to vessel production and deposition of extracellular matrix fibrillar collagen, compared with control cells ( P< 0.05, 4 replicates). Conclusions: Rap1 subtypes have a discrete role in the vasculature. This study links cAMP-Rap1A signaling to collagen and α 2C -AR expression in arteriole VSM. It suggests over-activation of Rap1A-coupled signaling in the peripheral circulation during chronic inflammation could lead to increased α 2C -AR mediated vessel reactivity, progressive perivascular fibrosis, and dysfunction as seen in human pathologies such as Raynaud’s phenomenon and scleroderma.