Vascular smooth muscle cells (VSMC) when cultured in vitro undergo a process known as phenotypic modulation. This is basically a process of dedifferentiation of VSMC from specialized contractile cells to more fibroblastic synthetic or secretory cells. These two phenotypic extremes are readily distinguishable by morphologic, ultrastructural, and biochemical criteria. In particular, synthetic VSMC express low levels of contractile proteins and high levels of extracellular matrix proteins such as collagen and fibronectin. Phenotypic modulation occurs in vivo as well during the response of the blood vessel to injury. Restenosis following angioplasty and atherosclerosis are both characterized by the dedifferentiation of media smooth muscle cells and the development of thick extracellular matrices. The factors regulating the phenotypic characteristics of VSMC are largely unknown, although growth factors (e.g., platelet-derived growth factor) and other paracrine mediators (e.g., transforming growth factor-beta are known to play roles in promoting VSMC proliferation and dedifferentiation both in vitro and in vivo. Nitric oxide and the second messenger cyclic GMP have been proposed to inhibit VSMC proliferation, but their role in phenotypic regulation and modulation is completely unknown. One possible reason for the lack of information on the role of the NO-cGMP signaling system in VSMC phenotype is that cultured VSMC cease to express the major receptor protein for cGMP - namely the cGMP-dependent protein kinase (cGMP kinase) after only a few passages. Hence the cells are unresponsive to the effects of cGMP elevations. In this proposal, we will test the hypothesis that the down- regulation of cGMP kinase expression in cultured VSMC is a response of the cells to iNOS induction (due to culture conditions, cytokines, or other factors), and that this down-regulation leads to the development of the synthetic phenotype. In addition, we will directly test the hypothesis that cGMP kinase expression and activity is critical for the manifestation of the differentiated phenotype of VSMC. We will determine the levels of marker proteins specific for the """"""""contractile"""""""" phenotype using VSMC stably-transfected with cDNAs encoding cGMP kinase. In particular, we will examine whether cGMP kinase expression and activity regulate the production of extracellular matrix proteins in vitro, since these proteins have been implicated in the regulation of phenotypic modulation of VSMC both in vitro and in vivo. The availability of transfected cell model systems makes this study feasible for the first time. These results will shed new light on the mechanisms underlying VSMC phenotypic modulation and proliferation which can ultimately be applied to the study of VSMC in vivo in response to vascular injury.
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