Vascular smooth muscle cell (SMC) differentiation is a very important process during vasculogenesis and angiogenesis, and it is recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular diseases including atherosclerosis, hypertension, and restenosis. The overall goal of the proposed studies is to identify the mechanisms by which environmental signals regulate SMC differentiation marker gene expression. Serum response factor (SRF) is the only transcription factor that has been shown to regulate all of the SMC differentiation marker genes. Since RhoA regulates SRF, and because RhoA activity is affected by many environmental cues (matrix, contractile agonists, growth factors, mechanical stretch) we hypothesize that Rho signaling is important for regulating SMC-specific transcription, and ultimately, SMC phenotype.
Our specific aims are as follows:
Aim 1 - to identify the Rho-dependent signaling pathways that contribute to the regulation of SMC-specific gene expression. We will measure Rho activation in SMC, and we will use dominant negative approaches to determine the extent to which Rho and Rho effectors contribute to the regulation of SMC-specific promoter/reporter constructs.
Aim 2 - to identify the mechanisms by which Rho affects SRF-dependent transcription in SMC. We will study the effects of Rho on the regulation of SRF phosphorylation and DNA binding, and on SRF's interaction with other transcription factors.
Aim 3 - to determine the effects of RhoA signaling on SMC differentiation in vivo. Because SMC phenotype is regulated by many diverse environmental cues that cannot be accurately reproduced in culture models, it is important to study SMC differentiation in vivo. We will express dominant negative and constitutively active forms of Rho specifically in SMC in mice using the SM myosin heavy chain promoter. Measuring SMC differentiation marker gene expression, examining vessel and organ morphology, and determining proliferation indices will assess effects on SMC differentiation. Completion of these aims should lead to a better understanding of the signaling and transcription mechanisms that regulate SMC differentiation and perhaps to novel targets for cardiovascular therapeutic intervention.
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