Phenotypic modulation of aortic smooth muscle cells (SMC) is critical to the process of vascular wound repair and to the development of atherosclerotic plaques. In the aorta, SMC express several different phenotypes ranging from those characteristic of quiescent-contractile cells embedded in the aortic media to those characterized by migrating- proliferating-secreting cells present in the intima. The proliferating- secreting class of cells is thought to be a major contributor to the process of wound healing in the neointima and to the development of atherosclerotic plaques. In vitro, cultures of vascular smooth muscle cells also exhibit morphologically and phenotypically different phenotypes including those represented by density-inhibited quiescent cells, nodular cells embedded in a three dimensional extracellular matrix, cells in confluent monolayer culture in which the cells are actively engaged in protein synthesis, and sub-confluent cells. In order to gain understanding of factors that may regulate the transition between different SMC phenotypes we used cultured aortic SMC and, during the past funding period identified cloned and sequenced two genes (clusterin and gp38k) whose mRNA and protein expression is significantly increased in nodular cell cultures. Further, we have prepared an expression vector for clusterin and expressed the protein in mammalian cells. The expression of clusterin and gp38k, and nodule density, is increased in SMC cultures in the presence of reconstituted extracellular matrix suggesting a role for matrix components in their regulation. Neither of those genes was previously known to be expressed in SMC. In this application we propose to investigate the regulation of clusterin and gp38k mRNA and protein expression and function and determine if expression is correlated with the expression of genes reported to be specific indicators of differentiated smooth muscle. Further we propose to investigate the role,of clusterin and gp38k in the processes of multilayered growth and nodule formation via a combination of knockout and addition experiments, establish whether either protein functions to modify cell adhesion and cell migration, and to identify the domains of the proteins that are responsible for those activities. In addition to their functional roles, clusterin and gp38k may serve as phenotypic markers for modulation and an understanding of their functions in SMC in vitro may provide insight in to the regulation of that process in vivo. Therefore we propose to evaluate the distribution of each mRNA and antigen in normal and diseased vascular wall tissue. In this proposal we will investigate the regulation, synthesis, and expression of clusterin and gp38k, determine if either or both can regulate the process of nodule formation and establish if either one is uniquely associated with any of the SMC phenotypes that have been identified in vitro or in vivo.
