There is clear evidence that altered 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 during vascular injury or in disease states [see review by Alexander et al.1]. For example, a major focus of previous studies has been to identify molecular mechanisms that control the coordinate expression of genes such as smooth muscle α-actin (SM α-actin), SM22α, 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, hypoxia, inflammatory cytokines, etc.) in regulation of SMC differentiation in vivo during vascular development, as well as following vascular injury, or with 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 simultaneously target conditional knockout (or over-expression) of genes which we postulate regulate differentiation and phenotypic plasticity of SMCs and also perform rigorous SMC-pericyte (SMC-P) lineage tracing experiments to define mechanisms that control phenotypic transitions of these cells during injury-repair and in diseases such as atherosclerosis4. Remarkably, using these model systems, we have recently shown that SMC-pericytes de-differentiate, and undergo phenotypic transitions to cells resembling macrophages, myofibroblasts, mesenchymal stem cells, and other cell types yet to be determined during development of experimental atherosclerosis, as well as in various models of injury-repair including vascular injury and myocardial infarction/cardiac remodeling. Moreover, we have shown that the phenotypic transitions of SMC-pericytes in these models is regulated by activation of stem cell pluripotency genes, including Oct4 (manuscript in review), and Klf43, 5, factors also shown to be involved in reprogramming of somatic cells into induced pluripotential stem (iPS) cells.
Our lab has also pioneered studies of the role of epigenetic mechanisms in control of SMC differentiation and phenotypic switching1, 6, as well as lineage determination of multiple specialized cell types from embryonic stem cells (ESC)7. 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 into alternative cell types involves reversing a subset of these histone modifications and transcriptional silencing of SMC marker genes. However, these cells retain certain histone modifications that we hypothesize serve as a mechanism for "cell lineage memory" during reversible phenotypic switching. That is, a mechanism that allows a SMC to undergo transient transitions to alternative phenotypes necessary for vascular repair, but which biases the cell into re-differentiating into a SMC once the repair is complete. Of major significance, we have recently developed a powerful new assay that for the first time allows assessment of specific histone modifications within single cells within fixed histological tissue specimens4 (referred to as ISH-PLA), and using this system along with our SMC specific lineage tracing mice, have shown that de-differentiated (phenotypically modulated) SMC within advanced atherosclerotic lesions of ApoE-/- mice retain an epigenetic signature of SMC even when expressing no detectable expression of SMC marker genes such as Acta2 or Myh11.
Finally, a major long term emphasis of the lab is to translate results of our basic science studies into advancing clinical practice. Current projects in this area include testing how inhibition of IL-1β signaling may or pro-atherogenic oxidized phospholipids may promote increased stability of atherosclerotic plaques thus reducing the probability of a heart attack or stroke. In addition, we are investigating ways to therapeutically modulate phenotypic transitions of SMC-P as a means to treat a wide range of major human diseases including atherosclerosis, hypertension, aortic aneurysms, peripheral vascular disease, cancer, and microvascular complications of diabetes/obesity that in aggregate account for >75% of all deaths worldwide.
For the most current list of Dr. Owen's publications, please click here.
Washington University of St. Louis
Dr. Gary Owens has established a collaborative partnership with Dr. Gwen Randolph of Washington University where she will commit effort toward experiments related to his recent RO1 titled, “Role of IL1B in Regulating SMC and Macrophage Differentiation in Atherosclerosis”. The central focus of this grant is to determine the effects of genetic or pharmacological inhibition of IL1β and IL1R1 signaling on phenotypic transitions of SMC and macrophages, as well as on the overall size and stability of late stage atherosclerotic lesions. Whereas there is good evidence that disruption of IL1β signaling inhibits formation of fatty streaks and early stage lesions, the role of IL1 in late stage lesions is unclear. Aim 1a will use novel utilize SMC and myeloid specific lineage tracing IL1R1 knockout mouse lines generated by our lab to test the hypothesis that IL1R1-dependent transitions in phenotype of SMC and macrophages within advanced atherosclerotic lesions play a critical role in determining overall plaque and lumen size, as well as lesion composition including multiple indices of plaque stability.
In collaboration and partnership, Dr. Randolph will design and refine experimental plans in regards to mouse and human macrophage and monocyte analyses in atherosclerotic plaques, while sharing and refining protocols to help us troubleshoot approaches based on her expert knowledge of macrophages, monocytes, and how these cells are best studied.
(1) Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective.
Cherepanova OA, Gomez D, Shankman LS, Swiatlowska P, Williams J, Sarmento OF, Alencar GF, Hess DL, Bevard MH, Greene ES, Murgai M, Turner SD, Geng YJ, Bekiranov S, Connelly JJ, Tomilin A, Owens GK. Nat Med. 2016 Jun;22(6):657-65. doi: 10.1038/nm.4109. Epub 2016 May 16. PMCID: PMC4899256
Link to paper
(2) KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis.
Shankman LS, Gomez D, Cherepanova OA, Salmon M, Alencar GF, Haskins RM, Swiatlowska P, Newman AA, Greene ES, Straub AC, Isakson B, Randolph GJ, Owens GK. Nat Med. 2015 Jun;21(6):628-37. doi: 10.1038/nm.3866. Epub 2015 May 18. PMID: 25985364
(3) Alexander MR, Owens GK. Epigenetic Control of Smooth Muscle Cell Differentiation and Phenotypic Switching in Vascular Development and Disease. Annu Rev Physiol 2012 February 15;74:13-40.
(4) 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.
(5) 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.
(6) Gomez D, Shankman LS, Nguyen AT, Owens GK. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat Methods 2013 January 13;10:171-7.
(7) Salmon M, Gomez D, Greene E, Shankman L, Owens GK. Cooperative Binding of KLF4, pELK-1, and HDAC2 to a G/C Repressor Element in the SM22alpha Promoter Mediates Transcriptional Silencing During SMC Phenotypic Switching In Vivo. Circ Res 2012 August 31;111(6):685-96.
(8) 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.
(9) 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.