ABSTRACT Regulation of vascular smooth muscle cells (VSMC) phenotype and function plays important roles in the pathogenesis of atherosclerosis. Vascular calcification is a characteristic feature of atherosclerosis that predicts adverse cardiovascular outcome of atherosclerotic patients. Over the last two decades, increasing studies have demonstrated that vascular calcification is a regulated process; and osteogenic differentiation and calcification of VSMC contributes significantly to the development of vascular calcification. Calcium signaling is critical in regulating VSMC function. However, the role of the key intracellular calcium signaling mediator, phospholipase Cγ (PLCγ), in regulating VSMC calcification is entirely unknown. Our preliminary studies demonstrated that deletion of PLCγ2 markedly increased calcification in VSMC and in atherosclerotic lesions of ApoE-/- mice. Using VSMC from PLCγ2 deletion mice (PLCγ2-/-), we determined a direct effect of PLCγ2 deficiency on promoting VSMC calcification, which was independent of the PLCγ1 isoform or the known PLCγ- mediated signaling pathways, supporting a unique and novel function of PLCγ2 in regulating VSMC calcification. PLCγ2 deletion in VSMC altered cytoskeleton structure and increased secretion of matrix vesicles (MVs), membrane-bound nanoparticles that harbor calcium and matrix proteins. MV secretion is a key cellular event in osteogenesis that initiates extracellular matrix calcification during bone formation; and has recently been shown to play an important role in vascular calcification. However, the molecular regulations of MV secretion in VSMC are poorly understood. We found that restoring PLCγ2 normalized MV secretion and inhibited calcification of the PLCγ2-/- VSMC. Furthermore, PLCγ2 interacted with membrane-associated filamentous proteins, septin 4/5, which have been shown to dynamically interact with membrane phospholipids and exocytosis machinery proteins that regulate cytoskeleton arrangement and synaptic vesicle secretion. The roles of septins in VSMC MV secretion are unknown, our findings of PLCγ2/septin4/5 interactions and increased septin 4/5 in MVs from PLCγ2-/- VSMC support a new role of septins in regulating VSMC MV secretion and calcification. Therefore, we hypothesize that PLCγ2 deficiency induces VSMC calcification via altered septin/actin-cytoskeleton structure that leads to increased MV secretion. With our new mouse models, Aim 1 will determine the function of SMC-specific PLCγ2 in regulating vascular function in vivo; and Aim 2 will elucidate the molecular mechanisms underlying PLCγ2-regulated VSMC calcification. These studies will elucidate an integrative role of SMC-derived PLCγ2 in regulating cytoskeleton structure and MV secretion that lead to VSMC calcification. PLCγ2 mutations in humans have recently been identified to cause immunological diseases but the underlying mechanisms are not fully understood. Therefore, delineating the novel function and mechanism...