Vascular smooth muscle cells (VSMC) serve as key constituents of the developing vasculature, ensuring stability of the nascent vessels, and are essential for the proper performance of the mature cardiovascular system. Pathological alterations in SMC biology, manifesting as perturbations in the differentiation state of SMC, play a pivotal role in the development and progression of various vascular diseases. Thus an understanding of the mechanisms and factors that control SMC differentiation underlies the potential for diagnostic and therapeutic advances. As such, this thesis aimed to implicate the c-Myb transcription factor in the process of SMC differentiation. For this purpose, in vitro assays were developed using the embryoid body (EB) model system, suitable for clarifying the molecular pathways and extrinsic factors that promote or obstruct SMC lineage specification and maturation to the contractile phenotype.
Contractile activity was observed in the developing EBs, and further characterized as smooth muscle and cardiac contractions. Temporal induction of SM-specific markers preceded and paralleled contractile SMC appearance and their mRNA levels predicted the relative ability of distinct mouse ES cell (mESC) lines to generate EBs with contracting SMC. Using fluorescent SM-specific promoter-reporter gene strategy it was shown that spontaneous SM-contractions coincide with SM-myosin-heavy-chain (SM-MHC) promoter activity. Moreover, this technology was employed to generate SMC (SM-MHC+) in a scalable stirred-suspension culture system.
This thesis addressed the effects of c-myb ablation on SMC differentiation both in vitro and in vivo. c-myb-/- mESC were unable to produce EBs with spontaneously contracting SMC, but gave rise to contracting cardiomyocytes unimpaired. Temporal patterns of myogenic and SM-specific gene expression levels during the course of differentiation revealed that, unlike wild-type EBs, c-myb-/- EBs lacked robust induction of these critical markers. Accordingly, c-myb-/- EBs exhibited significantly reduced SMC numbers. Additionally, in chimaeric EB model substantial contribution by c-myb-/- mESC was detrimental to the development of SM-contractions. To corroborate the in vitro data, chimaeric embryos and adult mice were used to demonstrate significantly reduced c-myb-/- cell contribution to vascular and visceral SMC lineages in vivo.
Collectively, this thesis assigns a novel role for the c-Myb transcription factor and advances knowledge of in vitro SMC differentiation.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/26282 |
| Date | 18 February 2011 |
| Creators | Kolodziejska-Baginska, Karolina Maria |
| Contributors | Husain, Mansoor |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis, Video |
Page generated in 0.0015 seconds