Drug-eluting stents have revolutionized revascularization of coronary artery lesions by largely preventing restenosis. Importantly, emerging long term safety data suggests that rapamycin-eluting stents pose an elevated risk of late thrombosis, likely due to incomplete healing of the endothelium. An ideal stent drug would, therefore, selectively inhibit proliferation and promote differentiation of vascular smooth muscle cells (VSMC), without inhibiting re-endothelialization. In our studies of the molecular mechanisms underlying VSMC phenotypic modulation, a process necessary for angiogenesis, atherosclerosis, and restenosis, we have discovered that the mTOR inhibitor rapamycin promotes VSMC differentiation by inducing a new program of gene expression, including contractile proteins. We have found that rapamycin inhibition of the mTOR effector S6K1, and the resulting activation of Akt2 is necessary for this effect. Surprisingly, Akt1 inhibited VSMC differentiation. We have also made the exciting discovery that rapamycin activates a VSMC transcription factor, GATA-6, and that this factor is necessary for rapamycin-induced differentiation. This transcription factor is specific to VSMC and not found in endothelial cells. We find that rapamycin also induces expression of the master regulatory transcriptional coactivator myocardin that promotes VSMC differentiation. The mTOR pathway is well known to regulate protein synthesis. Notably, we identify regulation of VSMC-specific transcription as a novel function for this pathway. We hypothesize that rapamycin regulation of transcription factors via Akt2 is a critical mechanism by which this drug inhibits proliferation and promotes differentiation in VSMC.
We aim to understand the mechanisms by which rapamycin regulates prodifferentiation transcription factors in VSMC. (1) In addressing this hypothesis, we aim to determine how rapamycin activation of Akt2 regulates GATA-6. We hypothesize that rapamycin induces phosphorylation of GATA-6 that leads to its nuclear translocation and activation. We will use siRNA, VSMC from Akt1 or Akt2 knockout mice, and GATA-6 phosphorylation site mutants to determine which kinase phosphorylates GATA-6, and how phosphorylation influences GATA-6 activity. We hypothesize that a phospho-mimetic mutant GATA-6 may be constitutively active, and therefore a potential VSMC-specific prodifferentiation therapeutic. (2) We aim to use siRNA methods to determine the role of myocardin in rapamycin-induced expression. We will determine whether and how rapamycin promotes myocardin expression and/or activity using DNA binding, chromatin immunoprecipitation, and promoter reporter methods. (3) We will determine whether Akt2 deletion exacerbates, and Akt1 deletion diminishes intimal hyperplasia, and whether the therapeutic response to rapamycin requires Akt2, using an injury model in wild type, Akt1 or Akt2 knockout mice. We propose that understanding these downstream targets of rapamycin and the molecular mechanisms by which they are regulated will provide key targets for development of improved stent therapeutics.
Cardiovascular disease is a major cause of morbidity and mortality in the western world. While stents coated with the drug rapamycin have greatly reduced the risk of restenosis (re-blockage of the vessel) after coronary artery angioplasty, recent findings have revealed that they confer a small but significant risk of heart attack or death. This project aims to understand the molecular mechanisms underlying the beneficial anti-restenotic response of vascular smooth muscle cells to rapamycin, as this knowledge may allow us to tailor future therapeutics to inhibit these cells specifically, avoiding detrimental side effects on other cell types that can cause the dangerous complications.
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