Gary K Owens

Publications of Gary K Owens

• Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease.

  Authors: Matthew R Alexander, Gary K Owens

  Annual review of physiology. 03/2012; 74:13-40.

  The vascular smooth muscle cell (SMC) in adult animals is a highly specialized cell whose principal function is contraction. However, this cell displays remarkable plasticity and can undergo profoundThe vascular smooth muscle cell (SMC) in adult animals is a highly specialized cell whose principal function is contraction. However, this cell displays remarkable plasticity and can undergo profound changes in phenotype during repair of vascular injury, during remodeling in response to altered blood flow, or in various disease states. There has been extensive progress in recent years in our understanding of the complex mechanisms that control SMC differentiation and phenotypic plasticity, including the demonstration that epigenetic mechanisms play a critical role. In addition, recent evidence indicates that SMC phenotypic switching in adult animals involves the reactivation of embryonic stem cell pluripotency genes and that mesenchymal stem cells may be derived from SMC and/or pericytes. This review summarizes the current state of our knowledge in this field and identifies some of the key unresolved challenges and questions that we feel require further study.

• Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

  Authors: Matthew R Alexander, Meera Murgai, Christopher W Moehle, Gary K Owens

  Physiological genomics. 02/2012; 44(7):417-29.

  Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration,Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

• Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice.

  Authors: Matthew R Alexander, Christopher W Moehle, Jason L Johnson, Zhengyu Yang, Jae K Lee, Christopher L Jackson, Gary K Owens

  The Journal of clinical investigation. 12/2011; 122(1):70-9.

  Clinical complications of atherosclerosis arise primarily as a result of luminal obstruction due to atherosclerotic plaque growth, with inadequate outward vessel remodeling and plaque destabilizationClinical complications of atherosclerosis arise primarily as a result of luminal obstruction due to atherosclerotic plaque growth, with inadequate outward vessel remodeling and plaque destabilization leading to rupture. IL-1 is a proinflammatory cytokine that promotes atherogenesis in animal models, but its role in plaque destabilization and outward vessel remodeling is unclear. The studies presented herein show that advanced atherosclerotic plaques in mice lacking both IL-1 receptor type I and apolipoprotein E (Il1r1⁻/⁻Apoe⁻/⁻ mice) unexpectedly exhibited multiple features of plaque instability as compared with those of Il1r1⁺/⁺Apoe⁻/⁻ mice. These features included reduced plaque SMC content and coverage, reduced plaque collagen content, and increased intraplaque hemorrhage. In addition, the brachiocephalic arteries of Il1r1⁻/⁻Apoe⁻/⁻ mice exhibited no difference in plaque size, but reduced vessel area and lumen size relative to controls, demonstrating a reduction in outward vessel remodeling. Interestingly, expression of MMP3 was dramatically reduced within the plaque and vessel wall of Il1r1⁻/⁻Apoe⁻/⁻ mice, and Mmp3⁻/⁻Apoe⁻/⁻ mice showed defective outward vessel remodeling compared with controls. In addition, MMP3 was required for IL-1-induced SMC invasion of Matrigel in vitro. Taken together, these results show that IL-1 signaling plays a surprising dual protective role in advanced atherosclerosis by promoting outward vessel remodeling and enhancing features of plaque stability, at least in part through MMP3-dependent mechanisms.

• Bone marrow-derived MCP1 required for experimental aortic aneurysm formation and smooth muscle phenotypic modulation.

  Authors: Christopher W Moehle, Castigliano M Bhamidipati, Matthew R Alexander, Gaurav S Mehta, James N Irvine, Morgan Salmon, Gilbert R Upchurch, Irving L Kron, Gary K Owens, Gorav Ailawadi

  The Journal of thoracic and cardiovascular surgery. 12/2011; 142(6):1567-74.

  This study tested the hypothesis that monocyte chemotactic protein 1 (MCP1) is required for abdominal aortic aneurysm (AAA) and smooth muscle phenotypic modulation in a mouse elastase perfusionThis study tested the hypothesis that monocyte chemotactic protein 1 (MCP1) is required for abdominal aortic aneurysm (AAA) and smooth muscle phenotypic modulation in a mouse elastase perfusion model. Infrarenal aortas of C57BL/6 (wild type [WT]) and MCP1 knockout (KO) mice were analyzed at 14 days after perfusion. Key cellular sources of MCP1 were identified using bone marrow transplantation. Cultured aortic smooth muscle cells (SMCs) were treated with MCP1 to assess its potential to directly regulate SMC contractile protein expression and matrix metalloproteinases (MMPs). Elastase perfused WT aortas had a mean dilation of 102% (n = 9) versus 53.7% for MCP1KO aortas (n = 9, P < .0001) and 56.3% for WT saline-perfused controls (n = 8). Cells positive for MMP9 and Mac-2 were nearly absent in the KO aortas. Complimentarily, the media of the KO vessels had abundant differentiated smooth muscle and intact elastic fibers and markedly less MMP2. Experiments in cultured SMCs showed MCP1 can directly repress smooth muscle markers and induce MMP2 and MMP9. Bone marrow transplantation studies showed that KO of MCP1 in bone marrow-derived cells protects from AAA formation. Moreover, KO in the bone was significantly more protective than global KO, suggesting an unexpected benefit to selectively depleting MCP1 in bone marrow-derived cells. These results have shown that MCP1 derived from bone marrow cells is required for experimental AAA formation and that retention of nonbone marrow MCP1 limits AAA compared with global depletion. This protein contributes to macrophage infiltration into the AAA and can act directly on SMCs to reduce contractile proteins and induce MMPs.

• WD repeat-containing protein 5, a ubiquitously expressed histone methyltransferase adaptor protein, regulates smooth muscle cell-selective gene activation through interaction with pituitary homeobox 2.

  Authors: Qiong Gan, Pierre Thiébaud, Nadine Thézé, Li Jin, Guofeng Xu, Patrick Grant, Gary K Owens

  The Journal of biological chemistry. 06/2011; 286(24):21853-64.

  WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), whichWD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.

• Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes.

  Authors: Mark H Hoofnagle, Ronald L Neppl, Erica L Berzin, G C Teg Pipes, Eric N Olson, Brian W Wamhoff, Avril V Somlyo, Gary K Owens

  American journal of physiology. Heart and circulatory physiology. 02/2011; 300(5):H1707-21.

  Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardinMyocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.

• Smooth and cardiac muscle-selective knock-out of Kruppel-like factor 4 causes postnatal death and growth retardation.

  Authors: Tadashi Yoshida, Qiong Gan, Aaron S Franke, Ruoya Ho, Jifeng Zhang, Y Eugene Chen, Matsuhiko Hayashi, Mark W Majesky, Avril V Somlyo, Gary K Owens

  The Journal of biological chemistry. 05/2010; 285(27):21175-84.

  Krüppel-like factor 4 (Klf4) is a transcription factor involved in differentiation and proliferation in multiple tissues. We demonstrated previously that tamoxifen-induced deletion of the Klf4 geneKrüppel-like factor 4 (Klf4) is a transcription factor involved in differentiation and proliferation in multiple tissues. We demonstrated previously that tamoxifen-induced deletion of the Klf4 gene in mice accelerated neointimal formation but delayed down-regulation of smooth muscle cell differentiation markers in carotid arteries following injury. To further determine the role of Klf4 in the cardiovascular system, we herein derived mice deficient for the Klf4 gene in smooth and cardiac muscle using the SM22alpha promoter (SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice). SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice were born at the expected Mendelian ratio, but they gradually died after birth. Although approximately 40% of SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice survived beyond postnatal day 28, they exhibited marked growth retardation. In wild-type mice, Klf4 was expressed in the heart from late embryonic development through adulthood, whereas it was not expressed in smooth muscle. No changes were observed in morphology or expression of smooth muscle cell differentiation markers in vessels of SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice. Of interest, cardiac output was significantly decreased in SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice, as determined by magnetic resonance imaging. Moreover, a lack of Klf4 in the heart resulted in the reduction in expression of multiple cardiac genes, including Gata4. In vivo chromatin immunoprecipitation assays on the heart revealed that Klf4 bound to the promoter region of the Gata4 gene. Results provide novel evidence that Klf4 plays a key role in late fetal and/or postnatal cardiac development.

• The actin associated protein palladin is important for the early smooth muscle cell differentiation.

  Authors: Li Jin, Qiong Gan, Bartosz J Zieba, Silvia M Goicoechea, Gary K Owens, Carol A Otey, Avril V Somlyo

  PloS one. 01/2010; 5(9):e12823.

  Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet itsPalladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs. The goal of the study was to determine the molecular mechanisms underlying palladin's ability to regulate the expression of SMC marker genes. Results showed that palladin expression was rapidly induced in an A404 cell line upon retinoic acid (RA) induced differentiation. Suppression of palladin expression with siRNAs inhibited the expression of RA induced SMC differentiation genes, SM α-actin (SMA) and SM22, whereas over-expression of palladin induced SMC gene expression. Chromatin immunoprecipitation assays provided evidence that palladin bound to SMC genes, whereas co-immunoprecipitation assays also showed binding of palladin to myocardin related transcription factors (MRTFs). Endogenous palladin was imaged in the nucleus, increased with leptomycin treatment and the carboxyl-termini of palladin co-localized with MRTFs in the nucleus. Results support a model wherein palladin contributes to SMC differentiation through regulation of CArG-SRF-MRTF dependent transcription of SMC marker genes and as previously published, also through actin dynamics. Finally, in E11.5 palladin null mouse embryos, the expression of SMA and SM22 mRNA and protein is decreased in the vessel wall. Taken together, our findings suggest that palladin plays a key role in the differentiation of SMCs in the developing vasculature.

• Smooth muscle phenotypic modulation is an early event in aortic aneurysms.

  Authors: Gorav Ailawadi, Christopher W Moehle, Hong Pei, Sandra P Walton, Zequan Yang, Irving L Kron, Christine L Lau, Gary K Owens

  The Journal of thoracic and cardiovascular surgery. 12/2009; 138(6):1392-9.

  OBJECTIVES: Vascular smooth muscle cells can undergo profound changes in phenotype, defined by coordinated repression of smooth muscle cell marker genes and production of matrix metalloproteinases inOBJECTIVES: Vascular smooth muscle cells can undergo profound changes in phenotype, defined by coordinated repression of smooth muscle cell marker genes and production of matrix metalloproteinases in response to injury. However, little is known of the role of smooth muscle cells in aortic aneurysms. We hypothesized that smooth muscle cells undergo phenotypic modulation early in the development of aortic aneurysms. METHODS: Abdominal aortas from C57B6 mice (n = 79) were perfused with elastase or saline (control) and harvested at 1, 3, 7, or 14 days. Aortas were analyzed by means of quantitative polymerase chain reaction and immunohistochemistry for smooth muscle cell marker genes, including SM22A, smooth muscle alpha-actin, and matrix metalloproteinases 2 and 9. In complimentary experiments human aneurysms (n = 10) and control aorta (n = 10) were harvested at the time of surgical intervention and analyzed. RESULTS: By 14 days, aortic diameter was larger after elastase perfusion compared with control diameter (100% +/- 9.6% vs 59.5% +/- 18.9%, P = .0002). At 7 days, elastase-perfused mice had a 78% and 85% reduction in SM22 alpha and smooth muscle alpha-actin expression, respectively, compared with that seen in control animals well before aneurysms were present, and these values remained repressed at 14 days. Immunohistochemistry confirmed less SM22 alpha and smooth muscle alpha-actin in experimental aneurysms at 14 days in concert with increased matrix metalloproteinase 2 and 9 expression at 7 and 14 days. Similarly, human aneurysms had less SM22 alpha and smooth muscle alpha-actin and increased matrix metalloproteinase 2 and 9 staining, compared with control values, as determined by means of quantitative polymerase chain reaction. CONCLUSIONS: Aneurysms demonstrate smooth muscle cell phenotypic modulation characterized by downregulation of smooth muscle cell marker genes and upregulation of matrix metalloproteinases. These events in experimental models occur before aneurysm formation. Targeting smooth muscle cells to a reparative phenotype might provide a novel therapy in the treatment of aortic aneurysms.

• Bone Marrow-Derived Cell-Specific Chemokine (C-C motif) Receptor-2 Expression is Required for Arteriolar Remodeling.

  Authors: Meghan M Nickerson, Ji Song, Joshua K Meisner, Sameer Bajikar, Caitlin W Burke, Casey W Shuptrine, Gary K Owens, Thomas C Skalak, Richard J Price

  Arteriosclerosis, thrombosis, and vascular biology. 10/2009;

  OBJECTIVE: Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatoryOBJECTIVE: Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle. METHODS AND RESULTS: Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2(-/-)-WT, WT-CCR2(-/-), and EGFP(+)-WT. One day after implantation, tissue MCP-1 levels rose from "undetectable" to 463pg/mg, and the number of EGFP(+) cells increased more than 4-fold, indicating marked inflammation. A 66% (28 microm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2(-/-)-WT mice but largely rescued in WT-CCR2(-/-) mice. EGFP(+) BMCs were numerous throughout the tissue, but we found no evidence that EGFP(+) BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. CONCLUSIONS: BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle.

• Sp1-Dependent Activation of KLF4 is Required for PDGF-BB-Induced Phenotypic Modulation of Smooth Muscle.

  Authors: Rebecca Ann Deaton, Qiong Gan, Gary K Owens

  American journal of physiology. Heart and circulatory physiology. 02/2009;

  There is clear evidence that phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs toThere is clear evidence that phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by loss of SMC marker gene expression. The transcription factor KLF4 is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing expression of the SRF co-factor, myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate expression of KLF4 in phenotypically modulated SMCs. Results demonstrated that the transcription factor Sp1 regulated expression of KLF4 in SMC. The KLF4 promoter contains 3 consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, over-expression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with siRNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, results suggest a novel role for Sp1 in increasing expression of KLF4 in phenotypically modulated SMCs. Key words: smooth muscle, Krpple-like factor 4, Sp1, PDGF-BB.

• Derivation of contractile smooth muscle cells from embryonic stem cells.

  Authors: Sanjay Sinha, Mark H Hoofnagle, Gary K Owens

  Methods in molecular biology (Clifton, N.J.). 02/2009; 482:345-67.

  Smooth muscle cells (SMCs) play a key role in vascular physiology and pathology. An appreciation of normal SMCs developmental mechanisms will likely lead to a better understanding of diseaseSmooth muscle cells (SMCs) play a key role in vascular physiology and pathology. An appreciation of normal SMCs developmental mechanisms will likely lead to a better understanding of disease processes and potentially to novel treatment strategies. We present a method for generating relatively pure populations of SMCs from embryonic stem cells (ESC) which display appropriate excitation and contractile responses to vasoactive agonists. We also present protocols for assessment of SMCs purity and identity by immunofluorescence, quantitative RT-PCR, and FACS. This ESC-based system has tremendous potential for studying developmental regulation of SMC lineage, as well as for possible SMC tissue engineering.

• Oxidized Phospholipids Induce Type VIII Collagen Expression and Vascular Smooth Muscle Cell Migration.

  Authors: Olga A Cherepanova, Nataliya A Pidkovka, Olga F Sarmento, Tadashi Yoshida, Qiong Gan, Eser Adiguzel, Michelle P Bendeck, Judith Berliner, Norbert Leitinger, Gary K Owens

  Circulation research. 02/2009;

  Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a critical role in the development of atherosclerosis. However, the factors present within lesions that mediate VSMCPhenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a critical role in the development of atherosclerosis. However, the factors present within lesions that mediate VSMC phenotypic switching are unclear. Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are active components of minimally modified low density lipoprotein and have been previously shown to induce multiple proatherogenic events in endothelial cells and macrophages, but their effects on VSMCs have been largely unexplored until recently. We previously showed that OxPLs induced phenotypic switching of VSMCs, including suppression of SMC differentiation marker genes. The goal of the present studies was to test the hypothesis that OxPLs alter extracellular matrix production and VSMC migration. Results showed that POVPC activated expression of several extracellular matrix proteins in VSMC. POVPC increased expression of type VIII collagen alpha1 chain (Col8a1) mRNA in cultured VSMCs and in vivo in rat carotid arteries by 9-fold and 4-fold, respectively. POVPC-induced activation of Col8a1 gene expression was reduced by small interfering RNA-mediated suppression of Krüppel-like factor 4 (Klf4) and Sp1, and was abolished in Klf4-knockout VSMCs. POVPC increased Klf4 binding to the Col8a1 gene promoter both in vivo in rat carotid arteries and in cultured VSMCs based on chromatin immunoprecipitation assays. Moreover, POVPC-induced VSMC migration was markedly reduced in Klf4- or type VIII collagen-knockout VSMCs. Given evidence that OxPLs are present within atherosclerotic lesions, it is interesting to suggest that OxPL-induced changes in VSMC phenotype may contribute to the pathogenesis of atherosclerosis at least in part through changes in extracellular matrix composition.

• PDGF-DD, a Novel Mediator of Smooth Muscle Cell Phenotypic Modulation, is Upregulated in Endothelial Cells Exposed to Atherosclerotic-Prone Flow Patterns.

  Authors: James Alexander Thomas, Rebecca Anne Deaton, Nicole E Hastings, Yueting Shang, Christopher W Moehle, Ulf J Eriksson, Stavros Topouzis, Brian R Wamhoff, Brett R Blackman, Gary K Owens

  American journal of physiology. Heart and circulatory physiology. 12/2008;

  Platelet-Derived Growth Factor-BB (PDGF-BB) is a well-known smooth muscle cell (SMC) phenotypic modulator that signals by binding to platelet-derived growth factor-alphaalpha (PDGF-alphaalpha),Platelet-Derived Growth Factor-BB (PDGF-BB) is a well-known smooth muscle cell (SMC) phenotypic modulator that signals by binding to platelet-derived growth factor-alphaalpha (PDGF-alphaalpha), -alphabeta and -betabeta membrane receptors. Platelet-Derived Growth Factor-DD (PDGF-DD) is a recently-identified PDGF family member but its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited the expression of multiple SMC genes including SM alpha-actin and SMMHC and upregulated the expression of the potent SMC differentiation repressor gene Kruppel-Like Factor-4 (KLF-4) at both the mRNA and protein levels. Based on the results of promoter reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. The SMC phenotypic modulatory activity of PDGF-DD was attenuated by pharmacological inhibitors of ERK phosphorylation and by an siRNA to KLF-4, highlighting the role of these two pathways in this process. PDGF-DD failed to repress SM alpha-actin and SMMHC in mouse SMCs lacking a functional PDGF-beta receptor. Importantly, PDGF-DD was increased within neointimal lesions within the aortic arch region of ApoE(-/-) mice. Furthermore, human endothelial cells (ECs) exposed to an atherosclerosis-prone flow pattern, as occurs in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in expression of PDGF-DD. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow. Key words: smooth muscle, atherosclerosis, shear stress, PDGF.

• The actin associated protein palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies.

  Authors: Li Jin, Tadashi Yoshida, Ruoya Ho, Gary K Owens, Avril V Somlyo

  The Journal of biological chemistry. 11/2008;

  Palladin is a widely expressed actin associated protein localized at stress fibers, focal adhesions, and other actin based structures, playing a significant role in cell adhesion and cell motility.Palladin is a widely expressed actin associated protein localized at stress fibers, focal adhesions, and other actin based structures, playing a significant role in cell adhesion and cell motility. Knock out of palladin in mice is embryonic lethal, demonstrating the importance of palladin in development yet its role in the vasculature is not known. In the present study, smooth muscle cell (SMC) genes, such as myosin, actin, caldesmon, calponin and LPP are downregulated in embryoid bodies (EBs) derived from embryonic stem cells (ESCs) lacking palladin. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of a SM alpha-actin (SMA) promoter. Negative selection, was then used to purify SMCs from EBs. Purified SMCs expressing multiple SMC markers were designated APSCs (SMA-puromycin-selected cells). Palladin null APSCs expresses significantly less myosin, actin, calponin, and h-caldesmon. The filamentous (F) to globular (G) actin ratio, known to regulate myocardin family transcription factors, was also decreased. Palladin null APSCs showed increased cell adhesion and decreased cell motility. Importantly, palladin null APSCs within collagen gels generated less maximum contractile force when stimulated with endothelin-1, sphingosine 1-phosphate (S1P) and thrombin. Myosin light chains (MLC20) were phosphorylated by lysophosphatidic acid to the same extent in palladin null and wild type APSCs but myosin content/total protein was reduced by >50%, consistent with the observed decreases in contractility. All together, these results suggest that palladin is essential for expression of the full complement of contractile proteins necessary for optimal force development of SMCs derived from EBs.

• PIAS1 Mediates TGF{beta}-Induced SM {alpha}-Actin Gene Expression Through Inhibition of KLF4 Function-Expression by Protein Sumoylation.

  Authors: Keiko Kawai-Kowase, Takayuki Ohshima, Hiroki Matsui, Toru Tanaka, Takehisa Shimizu, Tatsuya Iso, Masashi Arai, Gary K Owens, Masahiko Kurabayashi

  Arteriosclerosis, thrombosis, and vascular biology. 11/2008;

  OBJECTIVE: TGFbeta and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We haveOBJECTIVE: TGFbeta and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) alpha-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFbeta activates SM alpha-actin through PIAS1. METHODS AND RESULTS: An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFbeta-induced expression of SM alpha-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM alpha-actin promoter activity in a TGFbeta control element (TCE)-dependent manner. Because the TCE within the SM alpha-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM alpha-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. CONCLUSIONS: These results provide evidence that PIAS1 promotes TGFbeta-induced activation of SM alpha-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.

• Kruppel-like factor 4, Elk-1, and histone deacetylases cooperatively suppress smooth muscle cell differentiation markers in response to oxidized phospholipids.

  Authors: Tadashi Yoshida, Qiong Gan, Gary K Owens

  American journal of physiology. Cell physiology. 10/2008;

  Phenotypic switching of vascular smooth muscle cells (SMCs), such as increased proliferation, enhanced migration, and downregulation of SMC differentiation marker genes, is known to play a key rolePhenotypic switching of vascular smooth muscle cells (SMCs), such as increased proliferation, enhanced migration, and downregulation of SMC differentiation marker genes, is known to play a key role in the development of atherosclerosis. However, the factors and mechanisms controlling this process are not fully understood. We recently showed that oxidized phospholipids including 1-palmytoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), which accumulate in atherosclerotic lesions, are potent repressors of expression of SMC differentiation marker genes in cultured SMCs as well as in rat carotid arteries in vivo. Here, we examined the molecular mechanisms whereby POVPC induces suppression of SMC differentiation marker genes in cultured SMCs. Results showed that POVPC induced phosphorylation of Erk1/2 and Elk-1. The MEK inhibitors, U0126 and PD98059, attenuated POVPC-induced suppression of smooth muscle (SM) alpha-actin and SM-myosin heavy chain. POVPC also induced expression of Kruppel-like factor 4 (Klf4). Chromatin immunoprecipitation assays revealed that POVPC caused simultaneous binding of Elk-1 and Klf4 to the promoter region of the SM alpha-actin gene. Moreover, co-immunoprecipitation assays showed a physical interaction between Elk-1 and Klf4. Results in Klf4-null SMCs showed that blockade of both Klf4 induction and Elk-1 phosphorylation completely abolished POVPC-induced suppression of SMC differentiation marker genes. POVPC-induced suppression of SMC differentiation marker genes was also accompanied by hypoacetylation of histone H4 at the SM alpha-actin promoter, which was mediated by the recruitment of histone deacetylases (HDACs), HDAC2 and HDAC5. Co-immunoprecipitation assays showed that Klf4 interacted with HDAC5. Results provide evidence that Klf4, Elk-1, and HDACs coordinately mediate POVPC-induced suppression of SMC differentiation marker genes. Key words: gene transcription, Erk1/2, histone acetylation.

• Conditional deletion of Krüppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury.

  Authors: Tadashi Yoshida, Klaus H Kaestner, Gary K Owens

  Circulation research. 07/2008; 102(12):1548-57.

  Phenotypic switching of smooth muscle cells (SMCs) plays a key role in vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (Klf4) suppressed SMC differentiation markersPhenotypic switching of smooth muscle cells (SMCs) plays a key role in vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (Klf4) suppressed SMC differentiation markers in cultured SMCs. Here, we derive mice deficient for Klf4 by conditional gene ablation and analyze their vascular phenotype following carotid injury. Klf4 expression was rapidly induced in SMCs of control mice after vascular injury but not in Klf4-deficient mice. Injury-induced repression of SMC differentiation markers was transiently delayed in Klf4-deficient mice. Klf4 mutant mice exhibited enhanced neointimal formation in response to vascular injury caused by increased cellular proliferation in the media but not an altered apoptotic rate. Consistent with these findings, cultured SMCs overexpressing Klf4 showed reduced cellular proliferation, in part, through the induction of the cell cycle inhibitor, p21(WAF1/Cip1) via increased binding of Klf4 and p53 to the p21(WAF1/Cip1) promoter/enhancer. In vivo chromatin immunoprecipitation assays also showed increased Klf4 binding to the promoter/enhancer regions of the p21(WAF1/Cip1) gene and SMC differentiation marker genes following vascular injury. Taken together, we have demonstrated that Klf4 plays a critical role in regulating expression of SMC differentiation markers and proliferation of SMCs in vivo in response to vascular injury.

• Sphingosine-1-Phosphate Receptor Subtypes Differentially Regulate Smooth Muscle Cell Phenotype.

  Authors: Brian R Wamhoff, Kevin R Lynch, Timothy L Macdonald, Gary K Owens

  Arteriosclerosis, thrombosis, and vascular biology. 07/2008;

  OBJECTIVE: The role of S1P receptors in acute vascular injury and smooth muscle cell (SMC) phenotypic modulation is not completely resolved. METHODS AND RESULTS: S1P receptor antagonists were used toOBJECTIVE: The role of S1P receptors in acute vascular injury and smooth muscle cell (SMC) phenotypic modulation is not completely resolved. METHODS AND RESULTS: S1P receptor antagonists were used to test the hypothesis that specific S1P receptor subtypes differentially regulate SMC phenotypic modulation. In response to acute balloon injury of the rat carotid artery, S1P1/S1P3 receptor mRNA levels were transiently increased at 48 hours whereas S1P2 receptor expression was decreased. S1P2 expression was reinduced and increased at 7 to 10 days postinjury. Daily intraperitoneal injection of the S1P1/S1P3 antagonist VPC44116 decreased neointimal hyperplasia by approximately 50%. In vitro, pharmacological inhibition of S1P1/S1P3 receptors with VPC25239 attenuated S1P-induced proliferation of rat aortic SMCs. Conversely, inhibition of S1P2 with JTE013 potentiated S1P-induced proliferation. Inhibition of S1P1/S1P3 resulted in S1P-induced activation of the SMC differentiation marker genes SMalpha-actin and SMMHC, whereas inhibition of S1P2 attenuated this response. S1P2-dependent activation of SMalpha-actin and SMMHC was shown to be mediated by L-type voltage-gated Ca(2+) channels and subsequent RhoA/Rho kinase-dependent SRF enrichment of CArG box promoter regions. CONCLUSIONS: Results provide evidence that S1P1/S1P3 receptors promote, whereas S1P2 receptors antagonize, SMC proliferation and phenotypic modulation in vitro in response to S1P, or in vivo after vascular injury.

• Pitx2 is functionally important in the early stages of vascular smooth muscle cell differentiation.

  Authors: Yueting Shang, Tadashi Yoshida, Brad A Amendt, James F Martin, Gary K Owens

  The Journal of cell biology. 06/2008; 181(3):461-73.

  Mechanisms that control vascular smooth muscle cell (SMC) differentiation are poorly understood. We identify Pitx2 as a previously unknown homeodomain transcription factor that is rapidly induced inMechanisms that control vascular smooth muscle cell (SMC) differentiation are poorly understood. We identify Pitx2 as a previously unknown homeodomain transcription factor that is rapidly induced in an in vitro model of SMC differentiation from multipotent stem cells. Pitx2 induces expression of multiple SMC differentiation marker genes by binding to a TAATC(C/T) cis-element, by interacting with serum response factor, and by increasing histone acetylation levels within the promoters of SMC differentiation marker genes. Suppression of Pitx2 reduces expression of SMC differentiation marker genes in the early stages of SMC differentiation in vitro, whereas Prx1, another homeodomain protein, regulates SMC differentiation marker genes in fully differentiated SMCs. Pitx2, but not Prx1, knockout mouse embryos exhibit impaired induction of SMC differentiation markers in the dorsal aorta and branchial arch arteries. Our results demonstrate that Pitx2 functions to regulate the early stages of SMC differentiation.