Regulation of the vascular smooth muscle cell phenotype

Abstract

Smooth muscle cells (SMC) are present in many internal organs such as the blood vessels and the gastrointestinal channel. Their main functions are to provide stability to the tissue and to provide contractile capability. SMC are not terminally differentiated but can switch between several phenotypes, which is also known as phenotypic modulation. Unfavourable SMC phenotypes have been proposed to contribute to pathological processes such as atherosclerosis and asthma. The focus of this thesis has been to further elucidate the mechanisms that regulate gene expression in vascular SMC (VSMC).We developed a vascular differentiation system based on mouse embryonic stem cells and confirmed differentiation of VSMC as shown by expression of several marker genes. Moreover, VSMC markers and SMC markers were independently regulated in this system, which correlated with absence of CArG boxes in VSMC gene promoters. We could finally show that PDGF-B and TGFâ1, two molecules that have been implicated in VSMC development, were dispensable for VSMC differentiation.We have previously identified lipoma preferred partner (LPP) as a SMC-selective gene, and investigated its transcriptional regulation. We identified three evolutionary conserved CArG boxes and showed that two of these bind SRF in vivo. We further identified an alternative promoter that is regulated by one of the CArG boxes as indicated in reporter assays. We finally show that the alternative promoter directs expression specifically to SMC in vivo.Finally, the role of zinc finger protein 148 (ZFP148) as a regulator of extra-cellular matrix (ECM) gene expression was examined. Potential binding sites for ZFP148 were overrepresented in the promoter regions of 51 genes involved in production and regulation of ECM. We confirmed binding of ZFP148 to 10 ECM genes in vitro. siRNA-knockdown of zfp148 did however not affect the regulation of ECM genes in primary VSMC as indicated by quantitative PCR. Two genes, cspg4 and col5a1, were upregulated in the siRNA treated cells, which indicates that they might be regulated by ZFP148.