Abnormalities of vascular smooth muscle cell (SMC) growth and migration play an important role in hypertension, atherosclerosis and restenosis after coronary intervention. SM-20 is a member of novel multigene family that encodes a 239 amino acid intracellular protein induced by platelet-derived growth factor in SMC culture. SM20 is developmentally regulated in the heart, arterial wall, and skeletal muscle and is induced during differentiation of skeletal myoblasts. In the arterial wall, SM-20 is expressed only in SMC and is upregulated in the intima of atherosclerotic plaques and injured arteries. Preliminary studies using antisense approaches suggest that SM-20 underexpression results in increased SMC size and decreased saturation density; in skeletal myoblast culture, it is associated with decreased fusion. We have identified a mutant strain of Drosophila with P-element insertion in the fly SM-20 homologue that has delayed and reduced frequency of hatching, abnormal larbval movement, delayed larval development, decreased larval viability, and pupal lethality. We hypothesize that SM-20 encodes a structural protein that is important in the maintenance of cell structure and is critical for normal growth and development of sarcomeric and smooth muscle. SM-20 may also serve as an important smooth muscle-specific target within th arterial wall.
Aim 1 will continue work in Drosphila to establish that the lethal mutation is due to SM-20 and to determine the precise cause of lethality.
Aim 2 will generate an SM-20 knockout mouse. We hypothesize that the absence of SM-20 will result in developmental abnormalities in the heart, muscular arteries, and skeletal muscle. In addition, we hypothesize that the absence of SM-20 will result in an altered vascular response to arterial injury and atherogenic stimuli and an altered cardiac response to elevated pressure.
Aim 3 will employ the Tet-Off system to overexpress SM-20 sense and antisense RNA in SMC and skeletal myoblasts and to express it in fibroblasts, which do not normally synthesize SM- 20.
Aim 4 will continue work begun in cell culture to characterize positive and negative regulatory regions in the SM-20 promoter and will us transgenic mice to identify and characterize tissue-specific and developmental-specific elements in vivo.
Aim 5 will examine the expression of SM-20 isoforms and will develop isoform-specific antibodies.
These aims represent a comprehensive approach to establish the function of a novel gene family expressed in the cardiovasculature and to provide new insights into SMC gene regulation in vivo.
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