Date of Award
Level of Access
Doctor of Philosophy (PhD)
Second Committee Member
Third Committee Member
Vascular smooth muscle cells (VSMC) populate the medial layer of blood vessels and are able to modulate their phenotype in response to environmental cues. Defining the molecular pathways that influence the phenotypic state VSMC is important for understanding cardiovascular development, vascular repair of injury and numerous vascular pathologies. Notch receptors are expressed throughout the lifecycle of VSMC and contribute to their differentiation and proliferation. Activation of Notch receptors by the ligand Jagged-1 (Jag-1) is believed to be a pro-differentiation signal for VSMC; however, the mechanisms downstream of Notch signaling influencing the phenotypic state of VSMC are not defined. I sought to identify novel gene targets of Jag-1/Notch signaling that promote VSMC differentiation, and to define potential non-overlapping functions of Notch receptors in regulating these genes.
I observed decreased proliferation and increased contraction upon activation of Notch signaling by Jag-1 in human aortic VSMC. Using microRNA (miR) arrays, I identified miR-143 and miR-145 (miR-143/145) as enriched by Notch signaling. Blocking the induction of miR-143/145 by Jag-1/Notch signaling using specific antagomiRs inhibited contractile protein expression but had no effect on proliferation. Chromatin immunoprecipitation and miR-143/145 reporter assays indicate miR-143/145 is a transcriptional target of canonical Notch signaling in VSMC.
The cell cycle inhibitor p27kip1 was up regulated by Jag-1/Notch signaling in VSMC. While Notch1, Notch2 and Notch3 can interact with Jag-1, only Notch2 activation increased p27kip1 and inhibited VSMC proliferation. Both Notch2 and Notch3 activate miR-143/145 however. Jag-1/Notch2 signaling suppressed Skp2, a negative regulator of p27kip1, resulting in increased p27kip1 stability and accumulation VSMC. In vivo, human arteries with VSMC hyperplasia and injured murine carotid arteries express significantly higher levels of Notch2 and p27kip1 than control arteries, suggesting a potential negative feedback function for Notch2.
Collectively, Jag-1 promotes VSMC exit from the cell cycle via activation of Notch2 and stabilization of p27kip1, while Notch2 and Notch3 activate miR-143/145 and increase contractile proteins. I propose that Notch2 and Notch3 signal in parallel in hyperplastic arteries to resolve the injury and maintain the contractile phenotype of VSMC. These findings provide novel insights into the molecular regulation of cardiovascular disease.
Boucher, Joshua Michael, "Regulation of Vascular Smooth Muscle Phenotype by Notch Signaling" (2013). Electronic Theses and Dissertations. 1944.