Molecular Regulation of Differentiation of Vascular Smooth Muscle Cells in Development and in Disease

Dr. Gary K. Owens, Department of Molecular Physiology and Biological Physics and the Robert M. Berne Cardiovascular Research Center

There is clear evidence that abnormal control of the differentiated state of vascular smooth muscle cells (SMC), or SMC phenotypic switching, plays a critical role in development of a number of major human diseases including atherosclerosis, hypertension, asthma, and cancer. However, the mechanisms and factors that regulate SMC phenotypic switching in these diseases are poorly understood. A major long-term goal of our laboratory has been to elucidate cellular and molecular mechanisms that control the growth and differentiation of SMC during normal vascular development, and to determine how these control processes are altered in disease states [see review by Owens et al. ¹]. Current studies are focused on identifying molecular mechanisms that control the coordinate expression of genes such as smooth muscle α–actin (SM a-actin), SM22a, and smooth muscle myosin heavy chains (SM MHC) that are required for the differentiated function of the SMC. Studies involve use of a wide repertoire of molecular-genetic techniques and include identification of cis elements and trans regulatory factors that regulate cell-type specific expression of SMC differentiation marker genes both in cultured cell systems and in vivo in transgenic mice. In addition, we use a variety of gene knockout, mouse chimeric, and gene over-expression approaches to investigate the role of specific transcription factors and local environmental cues (e.g. growth factors, mechanical factors, cell-cell and cell-matrix interactions, etc.) in regulation of SMC differentiation in vivo during vascular development, or in association with vascular injury or cardiovascular disease ², ³. A particularly exciting recent development is that we have employed SMC specific promoters originally cloned and characterized in our laboratory to create mice in which we can target knockout (or over-expression) of genes of interest specifically to SMCs. Such systems permit development of unique and powerful genetic mouse model systems with which to directly explore mechanisms that contribute to vascular development in vivo, as well as to investigate the etiology of a variety of major cardiovascular diseases including atherosclerosis.

An area of major focus in the lab is studies of the role of epigenetic mechanisms in control of SMC differentiation and phenotypic switching ⁴, as well as lineage determination of multiple specialized cell types from embryonic stem cells (ESC) ⁵. Of major interest, we have shown that lineage determination of SMC, as well as other specialized cells from ESC, involves acquisition of locus- and cell-type selective histone modifications that influence chromatin structure and permissiveness of genes for transcriptional activation. Moreover, we have demonstrated that phenotypic switching of SMC involves reversal of a subset of these histone modifications and transcriptional silencing of SMC marker genes, but cells retain certain histone modifications that we hypothesize serve as a mechanism for “cell lineage memory” during reversible phenotypic switching. Of major interest, we have recently shown that SMC phenotypic switching involves activation of pluripotency gene networks including KLF43, Sox2, and Oct4, genes also shown to be involved in reprogramming somatic cells into induced progenitor stem cells or IPS cells.

Another project in the lab is focused on identification of mechanisms that lead to abnormal maturation of tumor blood vessels (i.e. defective SMC/pericyte investment and differentiation), a process linked with high rates of tumor cell shedding and metastasis. Of major interest, we have identified a novel human g-retrovirus that selectively infects individuals with inactivating polymorphisms in the cancer susceptibility gene RNAseL, and that this infection increases multiple properties of tumor cells associated with increased tumor growth and metastasis, including secretion of soluble factors that inhibit SMC/pericyte differentiation.

Finally, we have a series of projects focused on investigating mechanisms that regulate the stability of advanced atherosclerotic plaques, a process of critical importance in determining if a plaque is likely to rupture and set off a heart attack. Of major interest, we have recently obtained unexpected results showing that knockout of interleukin-1 receptor signaling in an ApoE knockout atherosclerotic mice resulted in reduced outward remodeling and de-stabilization of lesions. These results are opposite to the dogma that IL-1 signaling is pro-atherosclerotic and suggest that ongoing clinical studies investigating possible use of IL-1 antagonists to treat atherosclerosis may actually be detrimental rather than beneficial to patients. We are in the process of developing small molecule inhibitors of these de-stabilization pathways, with the goal of identifying exciting new therapies to prevent or reduce the chance atherosclerotic plaque rupture and a heart attack, which account for nearly 50% of all deaths in this country.

Reference List

(1) Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004 July;84(3):767-801.

(2) Wamhoff BR, Hoofnagle MH, Burns A, Sinha S, McDonald OG, Owens GK. A G/C Element Mediates Repression of the SM22a Promoter Within Phenotypically Modulated Smooth Muscle Cells in Experimental Atherosclerosis. Circ Res 2004 November 12;95(10):981-8.

(3) Yoshida T, Kaestner KH, Owens GK. Conditional Deletion of Kruppel-Like Factor 4 Delays Downregulation of Smooth Muscle Cell Differentiation Markers but Accelerates Neointimal Formation Following Vascular Injury. Circ Res 2008 June 20;102(12):1548-57.

(4) McDonald OG, Wamhoff BR, Hoofnagle MH, Owens GK. Control of SRF binding to CARG-box chromatin regulates smooth muscle gene expression in vivo. J Clin Invest 2006;116:36-48.

(5) Gan Q, Yoshida T, McDonald OG, Owens GK. Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells. Stem Cells 2007 January;25(1):2-9.