Regulatory mechanisms in vascular injury and repair

Abstract

Proliferation of SMC after vascular injury accounts for clinical conditions in transplant vasculopathy, in-stent restenosis and vein bypass graft failure. Vascular injury upregulates the expression of many transcription factors, two important ones are Yin yang-1 (YY-1) and early growth response-1 (Egr-1). The aims of this thesis were (1) to examine a possible mechanism by which a transcriptional repressor YY-1 inhibits SMC proliferation and (2) the development of a phospho-specifc antibody to a transcription activator, Egr-1 found to be a positive regulator of SMC proliferation. The results show that YY-1 inhibits p21WAF1/Cip1 transcription that perturbs the formation of p21WAF1/Cip1/cdk4/cyclin D1 complex thus blocking the downstream pRBSer249/Thr252 phosphorylation and expression of PCNA and TK-1. This inhibition was observed only in SMCs and not in ECs. Moreover, inhibition of endogenous YY-1 was performed to show the gain- and loss-of function of this transcription factor. YY-1 binds with Sp1 and prevents its occupancy of a Sp1 binding element in the p21WAF1/Cip1 promoter without YY-1 itself binding to the promoter. YY- 1 suppression of p21WAF1/Cip1 also involves p53 ubiquitination and proteasomal degradation. Further studies showed that overexpression of the first two-zinc finger region of YY-1 can inhibit smooth muscle cell proliferation and not ECs. This cell-type specific effect of YY-1 could be a potential tool in controlling SMC proliferation in drug eluting stent. The second part of this dissertation is the generation of phosphospecific antibody to Egr-1. Egr-1 controls a variety of genes implicated to SMC proliferation. Phosphorylation of Egr-1 can induce or repress the expression of its target gene depending on what type of kinase is involved. Preliminary data show that a phospho-specific antibody to Egr-1, pS26, can detect the Egr-1 phosphorylated protein from cell extract. Specificity of pS26 was determined also using slot blots of synthetic peptides and recombinant proteins, peptide blocking and phosphatase treatment. Further validation is needed to 6 fully confirm the specificity of this new phospho-specific antibody to Egr-1. A phospho-specific antibody to Egr-1 will serve as a tool to dissect mechanism by which this immediate early gene product exerts it control on fibroproliferative vasculopathies. The results generated by this thesis have added a new layer on the understanding of mechanism on inhibition of SMC proliferation and a generation of potential tool to dissect the mechanism of phosphorylation of a transcription factor implicated to SMC proliferative vasculopathy.