Date of Award

5-2006

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Lucy Liaw

Second Committee Member

Carla Mouta-Bellum

Third Committee Member

Jeong Yoon

Abstract

Vascular smooth muscle cell (VSMC) proliferation is an important feature of atherosclerotic plaque formation and restenosisl. Notchl, Notch2, Notch3 and Notch4 are up-regulated in adult intimal smooth muscle cells and regenerating endothelial cells following injury using the rat balloon catheter denudation model2. In this study, the objective was to determine the mechanisms by which Notch signaling regulates the proliferation of VSMC. We employed an in vitro VSMC model for Notch gain-offunction experiments. We used this model to assess alterations in VSMC proliferation using growth curve and DNA synthesis analysis following expression of the intracellular -do mains (m)o f Notchl (NlICD) and Notch4 (N4ICD) which act as constitutively active signaling receptors, or expression of the downstream effector HES-related transcript-2 ( H R T ~ )O~v.e r-expression of NlICD, N4ICD and HRT2 was demonstrated to positively regulate S-phase entry in a rat smooth muscle cell line (PAC-1). Expression of NlICD and N4ICD was analyzed by quantitative PCR and shown to up-regulate the VSMC specific Notch target genes HRTl and HRT2. The loss of Go/Glarrest was associated with a reduction in protein levels of the cyclin dependent kinase inhibitors p27 and p21. Over-expression of p27 resulted in a dose dependent rescue of the Notchlinduced loss of cell cycle regulation. Finally, direct repression of the p27 promoter by HRT2 was demonstrated using reporter assays, gel retardation assays and chromatin immunoprecipitation. Transcriptional repression is demonstrated to occur within the minimal p27 promoter region 435 - 774bp upstream of the start codon, and mutational analysis demonstrates that repression is dependent on a conserved class-C domain within the minimal promoter region. In order to assess the relevance of in vitro Notch induced proliferation to other smooth muscle hyperplasias, human smooth muscle neoplasms were examined in order to determine whether Notch receptorslligands were expressed. Using in situ hybridization, expression of Notch receptors, particularly Notch 1 and 2 as well as the Notch ligand Jagged 1 was observed in both gastrointestinal stromal tumors and leiomyosarcomas. Lastly, in order to identify novel targets of Notch signaling in smooth muscle cells microarray analysis of human aortic smooth muscle cells was performed following transduction with HRTl and HRT2 adenovirus. Microarray experiments verified the repression of p27 by HRTl and HRT2 in primary human cells. In addition, several novel targets of HRT transcription factors were identified including Jaggedl, Sprouty2 and Sprouty4, which will be useful for generating hypotheses designed to identify smooth muscle-specific Notch signaling pathways.

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