Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming

Atherosclerosis is an inflammatory disease that preferentially forms at hemodynamically compromised regions of altered shear stress patterns. Endothelial cells (EC) and smooth muscle cells (SMC) undergo phenotypic modulation during atherosclerosis. An in vitro coculture model was developed to determine the role of hemodynamic regulation of EC and SMC phenotypes in coculture. Human ECs and SMCs were plated on a synthetic elastic lamina and human-derived atheroprone, and atheroprotective shear stresses were imposed on ECs. Atheroprone flow decreased genes associated with differentiated ECs (endothelial nitric oxide synthase, Tie2, and Kruppel-like factor 2) and SMCs (smooth muscle α-actin and myocardin) and induced a proinflammatory phenotype in ECs and SMCs (VCAM-1, IL-8, and monocyte chemoattractant protein-1). Atheroprone flow-induced changes in SMC differentiation markers were regulated at the chromatin level, as indicated by decreased serum response factor (SRF) binding to the smooth muscle α-actin-CC(a/T)6GG (CArG) promoter region and decreased histone H4 acetylation. Conversely, SRF and histone H4 acetylation were enriched at the c- fos promoter in SMCs. In the presence of atheroprotective shear stresses, ECs aligned with the direction of flow and SMCs aligned more perpendicular to flow, similar to in vivo vessel organization. These results provide a novel mechanism whereby modulation of the EC phenotype by hemodynamic shear stresses, atheroprone or atheroprotective, play a critical role in mechanical-transcriptional coupling and regulation of the SMC phenotype.

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CHIP Represses Myocardin-Induced Smooth Muscle Cell Differentiation via Ubiquitin-Mediated Proteasomal Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.01737-08 ◽

2009 ◽

Vol 29 (9) ◽

pp. 2398-2408 ◽

Cited By ~ 51

Author(s):

Ping Xie ◽

Yongna Fan ◽

Hua Zhang ◽

Yuan Zhang ◽

Mingpeng She ◽

...

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Molecular Mechanisms ◽

Serum Response Factor ◽

Ex Vivo ◽

Critical Role ◽

Physiological Control ◽

Interacting Protein

ABSTRACT Myocardin, a coactivator of serum response factor (SRF), plays a critical role in the differentiation of vascular smooth muscle cells (SMCs). However, the molecular mechanisms regulating myocardin stability and activity are not well defined. Here we show that the E3 ligase C terminus of Hsc70-interacting protein (CHIP) represses myocardin-dependent SMC gene expression and transcriptional activity. CHIP interacts with and promotes myocardin ubiquitin-mediated degradation by the proteasome in vivo and in vitro. Furthermore, myocardin ubiquitination by CHIP requires its phosphorylation. Importantly, CHIP overexpression reduces the level of myocardin-dependent SMC contractile gene expression and diminishes arterial contractility ex vivo. These findings for the first time, to our knowledge, demonstrate that CHIP-promoted proteolysis of myocardin plays a key role in the physiological control of SMC phenotype and vessel tone, which may have an important implication for pathophysiological conditions such as atherosclerosis, hypertension, and Alzheimer's disease.

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YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation

Scientific Reports ◽

10.1038/s41598-020-78544-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jian-Pu Zheng ◽

Xiangqin He ◽

Fang Liu ◽

Shuping Yin ◽

Shichao Wu ◽

...

Keyword(s):

Smooth Muscle ◽

Gene Transcription ◽

Molecular Mechanisms ◽

Serum Response Factor ◽

Specific Gene ◽

Phenotypic Modulation ◽

Differentiation Markers ◽

Carg Box

AbstractYin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

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Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00654.2013 ◽

2014 ◽

Vol 307 (3) ◽

pp. H337-H345 ◽

Cited By ~ 6

Author(s):

Lara Gotha ◽

Sang Yup Lim ◽

Azriel B. Osherov ◽

Rafael Wolff ◽

Beiping Qiang ◽

...

Keyword(s):

Smooth Muscle ◽

Critical Role ◽

Arterial Injury ◽

Side Chains ◽

Wild Type ◽

Injury Response ◽

Migratory Response

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2Δ3/Δ3 (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type ( P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB ( P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

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Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH

10.2337/figshare.15108567.v1 ◽

2021 ◽

Author(s):

Rory P. Cunningham ◽

Mary P. Moore ◽

Ryan J. Daskek ◽

Grace M. Meers ◽

Takamune Takahashi ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Critical Role ◽

Acid Oxidation ◽

Mitochondrial Fatty Acid Oxidation ◽

Nonalcoholic Fatty Liver ◽

Mitochondrial Fatty Acid ◽

Endothelial Nitric Oxide

Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H<sub>2</sub>O<sub>2</sub> emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated western diet induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.

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Regulation of SM22α expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00306.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. H1444-H1452 ◽

Cited By ~ 19

Author(s):

Nihal Kaplan-Albuquerque ◽

Chrystelle Garat ◽

Vicki Van Putten ◽

Raphael A. Nemenoff

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Arginine Vasopressin ◽

Promoter Activity ◽

Serum Response Factor ◽

Phenotypic Modulation ◽

Carg Box ◽

Pdgf Signaling

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM α-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22α appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22α expression by AVP and PDGF in cultured VSMC. Levels of SM22α mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22α promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22α promoter. Expression of constitutively active Ras produced similar suppressions on SM22α promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.

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Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH

10.2337/figshare.15108567 ◽

2021 ◽

Author(s):

Rory P. Cunningham ◽

Mary P. Moore ◽

Ryan J. Daskek ◽

Grace M. Meers ◽

Takamune Takahashi ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Critical Role ◽

Acid Oxidation ◽

Mitochondrial Fatty Acid Oxidation ◽

Nonalcoholic Fatty Liver ◽

Mitochondrial Fatty Acid ◽

Endothelial Nitric Oxide

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Three Constituents of Moringa oleifera Seeds Regulate Expression of Th17-Relevant Cytokines and Ameliorate TPA-Induced Psoriasis-Like Skin Lesions in Mice

Molecules ◽

10.3390/molecules23123256 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3256 ◽

Cited By ~ 3

Author(s):

Nuan Ma ◽

Qin Tang ◽

Wan-Ting Wu ◽

Xin-An Huang ◽

Qin Xu ◽

...

Keyword(s):

Moringa Oleifera ◽

Skin Diseases ◽

Critical Role ◽

Folk Medicine ◽

Skin Lesions ◽

Keratinocyte Differentiation ◽

Therapeutic Application ◽

Differentiation Markers

As a folk medicine, Moringa oleifera L. is used effectively to treat inflammatory conditions and skin diseases. However, its mechanism of action is not well understood, limiting its medical use. We isolated and identified three compounds, namely niazirin, marumoside A and sitosterol-3-O-β-d-glucoside, from the seeds of Moringa oleifera, and studied their effects on the expression of Th17-relevant cytokines (IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19) using lipopolysaccharide-stimulated THP-1 cells. Additionally, as Th17 plays a critical role in the pathogenesis of psoriasis, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced psoriasis-like skin lesion mouse model to study their potential therapeutic application in vivo. The compounds suppressed the expression of IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19 in vitro, and in vivo they ameliorated psoriasis-like skin lesions, decreased IL-17A mRNA expression, and increased the expression of keratinocyte differentiation markers. To our knowledge, this is the first report regarding the mechanism and therapeutic application of Moringa oleifera seeds to treat psoriasis-like lesions in vivo.

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A role for platelet-derived growth factor β-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00180.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. L1162-L1170 ◽

Cited By ~ 46

Author(s):

Robert P. Jankov ◽

Crystal Kantores ◽

Rosetta Belcastro ◽

Soojin Yi ◽

Ross A. Ridsdale ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Smooth Muscle ◽

Rat Model ◽

Vascular Remodeling ◽

Critical Role ◽

Receptor Expression ◽

Newborn Rat ◽

Arterial Smooth Muscle

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-α and -β receptors (PDGF-αR and -βR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-βR, but not PDGF-αR, on arterial smooth muscle. PDGF-BB, a major PDGF-βR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-βR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-βR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-βR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.

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A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6260 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6260-6273 ◽

Cited By ~ 176

Author(s):

V M Rivera ◽

C K Miranti ◽

R P Misra ◽

D D Ginty ◽

R H Chen ◽

...

Keyword(s):

Growth Factor ◽

Serum Response Factor ◽

Critical Role ◽

Response Factor ◽

Serum Response Element ◽

Response Element ◽

Ribosomal S6 Kinase ◽

Serum Response

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

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Apoptotic myocytes generate monocyte chemoattractant protein-1 and mediate macrophage recruitment

Journal of Applied Physiology ◽

10.1152/japplphysiol.00254.2007 ◽

2008 ◽

Vol 104 (3) ◽

pp. 601-609 ◽

Cited By ~ 13

Author(s):

Miyuki Kobara ◽

Nahoko Sunagawa ◽

Masaki Abe ◽

Nana Tanaka ◽

Hiroe Toba ◽

...

Keyword(s):

Critical Role ◽

Neonatal Rat ◽

Terminal Deoxynucleotidyl Transferase ◽

Nuclear Staining ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Protein Levels ◽

Monocyte Chemoattractant Protein

The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-1 (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2–1 μmol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 μmol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-1 mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-1. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a trans-well system. The culture medium of STS-treated myocytes recruited monocytes in a MCP-1-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 min of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 ± 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody showed that 69.8 ± 3.9% of TUNEL-positive myocytes expressed MCP-1 protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-1, which have a critical role in the active recruitment of macrophages.

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