Drugs Targeting Epigenetic Histone Acetylation in Vascular Smooth Muscle Cells for Restenosis and Atherosclerosis

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

The phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a pivotal role in hypertension-induced vascular changes including vascular remodeling. The precise mechanisms underlying VSMC phenotypic modulation remain elusive. Here we test the role of peroxisome proliferator-activated receptor (PPAR)-gamma in the VSMC phenotypic modulation during hypertension. Both spontaneously hypertensive rat (SHR) aortas and SHR-derived VSMCs exhibited reduced PPAR-gamma expression and excessive VSMC phenotypic modulation identified by reduced contractile proteins, alpha-smooth muscle actin (alpha-SMA) and smooth muscle 22alpha (SM22alpha), and enhanced proliferation and migration. PPAR-gamma overexpression rescued the expression of alpha-SMA and SM22alpha, and inhibited the proliferation and migration in SHR-derived VSMCs. In contrast, PPAR-gamma silencing exerted the opposite effect. Activating PPAR-gamma using rosiglitazone in vivo up-regulated aortic alpha-SMA and SM22alpha expression and attenuated aortic remodeling in SHRs. Increased activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling was observed in SHR-derived VSMCs. PI3K inhibitor LY294002 rescued the impaired expression of contractile proteins, and inhibited proliferation and migration in VSMCs from SHRs, whereas constitutively active PI3K mutant had the opposite effect. Overexpression or silencing of PPAR-gamma inhibited or excited PI3K/Akt activity, respectively. LY294002 counteracted the PPAR-gamma silencing induced proliferation and migration in SHR-derived VSMCs, whereas active PI3K mutant had the opposite effect. In contrast, reduced proliferation and migration by PPAR-gamma overexpression were reversed by the active PI3K mutant, and further inhibited by LY294002. We conclude that PPAR-gamma inhibits VSMC phenotypic modulation through inhibiting PI3K/Akt signaling. Impaired PPAR-gamma expression is responsible for VSMC phenotypic modulation during hypertension. These findings highlight an attractive therapeutic target for hypertension-related vascular disorders.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The receptor for advanced glycation end products (RAGE) and its ligands have been implicated in the activation of oxidant stress and inflammatory pathways in vascular smooth muscle cells (VSMCs) leading to the initiation and augmentation of atherosclerosis. Here we report that non-receptor Src tyrosine kinase and the membrane protein caveolin-1 (Cav-1) play a key role in the activation of RAGE by S100B in VSMCs. S100B increased the activation of Src kinase and tyrosine phosphorylation of caveolin-1 in VSMCs. A RAGE-specific antibody blocked both these effects. An inhibitor of Src kinase, PP2, significantly blocked S100B-induced activation of Src kinase, mitogen-activated protein kinases, transcription factors NF-kappaB and STAT3, superoxide production, tyrosine phosphorylation of Cav-1, VSMC migration, and expression of the pro-inflammatory genes monocyte chemotactic protein-1 and interleukin-6. Cholesterol depletion also inhibited S100B-induced effects indicating the requirement for intact caveolae in RAGE-specific signaling. Nucleofection of either a Src dominant negative mutant, or a Cav-1 mutant lacking the scaffolding domain, or Cav-1 short hairpin RNA significantly reduced S100B-induced inflammatory gene expression in VSMCs. Furthermore, VSMCs derived from insulin-resistant and diabetic db/db mice displayed increased RAGE expression, Src activation, and migration compared with those from control db/+ mice. The RAGE antibody blocked enhanced migration in db/db cells. These studies demonstrate for the first time that, in VSMCs, Src kinase and Cav-1 play important roles in RAGE-mediated inflammatory gene expression and migration, key events associated with diabetic vascular complications.

Objective To investigate the effect of Astragaloside IV (AS-IV) on tumor necrosis factor-α (TNF-α)-induced expressions of matrix metalloproteinases (MMPs) in a rat vascular smooth muscle cells (VSMCs) proliferation model and its mechanism. Methods VSMCs were prepared from the thoracoabdominal aorta of rats by using issue-sticking method. Morphology of cells was observed by inverted microscope, and identified by immunohistochemical methods with antibody against SM-α-actin. The model of VSMCs proliferation and migration was established by TNF-α inducer in vitro, and randomly divided into the following groups: the control group, the TNF-α group, the TNF-α+ AS-IV (0.5 μg/ml) group, the TNF-α+ AS-IV (5 μg/ml) group, the TNF-α+ AS-IV (25 μg/ml) group, and the TNF-α+ AS-IV (50 μg/ml) group. The effect of AS-IV on TNF-α-induced VSMCs proliferation activity was detected by the caerulein and cholecystokinin octapeptide (CCK-8) method. The quantitative real-time polymerase chain reaction (real-time PCR) and Western blotting were used to examine the effects of AS-IV on the VSMCs-secreted mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2), respectively. Results The proliferative activity, migratory distance and invasive capacity of VSMCs were obviously increased in TNF-α stimulation group versus in control group (all P<0.01), which suggested that TNF-α can promote VSMCs proliferation and migration, and that the rat model of VSMCs proliferation in vitro was successfully established. The results of CCK-8 tests showed that VSMCs proliferation was obvious and the optical density (OD) value was elevated (P<0.01) after a preset time incubation with TNF-α. VSMCs proliferation was inhibited in each AS-IV treatment group, and the OD value was decreased as compared with the TNF-α group. And the inhibitive effect was increased along with the increments of AS-IV concentration and the acting time, which indicated that AS-IV can inhibit TNF-α-induced VSMCs proliferation in a time- and dose-dependent manner. The results of real-time PCR and Western blotting assays indicated that TNF-α changed the ratio of MMPs to the tissue inhibitors of metalloproteinases (TIMPs) by down-regulating active MMP-2 expression without influencing proMMP-2 and TIMP-2 expressions, and thus promoted the degradation of ECM. AS-IV (0.5-50 μg/ml) inhibited VSMCs proliferation and migration by down-regulating the TNF-α-induced MMP-2 overexpression in a dose-dependent manner, up-regulating the mRNA and protein expressions of TIMP-2, and modulating the ratio of MMPs to TIMPs, thereby inhibited the degradation of ECM. Conclusions AS-IV inhibits VSMCs proliferation and migration in a time- and dose-dependent manner. AS-IV inhibits VSMCs proliferation and migration by down-regulating TNF-α-induced MMP-2 overexpression, up-regulating TIMP-2 expressions, and normalizing the ratio of MMPs to TIMPs. Therefore, AS-IV inhibits the degradation of ECM, which may play a role in the prevention and treatment of in-stents restenosis after PCI. Key words: Astragalus; Muscle, smooth, vascular; Tumor necrosis factor-alpha; Matrix

To elucidate the physiological role of protein kinase C (PKC) delta, a ubiquitously expressed isoform in vascular smooth muscle cells (VSMC), PKC delta was stably overexpressed in A7r5 cells, rat clonal VSMC. The [3H]thymidine incorporation in A7r5 overexpressed with PKC delta (DVs) was suppressed to 37.1 +/- 16.3% (mean +/- S.D.) of the level in control or A7r5 transfected with vector alone (EVs). The reduction of [3H]thymidine incorporation was strongly correlated with overexpressed PKC levels. Moreover, transient transfection of a dominant negative mutant of PKC delta restored the reduced proliferation in DVs. Flow cytometry analysis demonstrated that DVs were arrested in the G0/G1 phase of the cell cycle. Expression of cyclins D1 and E and retinoblastoma protein phosphorylation were reduced, while the protein levels of p27 were elevated in DVs as compared with EVs. There were no significant differences in the expression of c-fos, c-jun, c-myc, cyclin D2, D3, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and p21 among the clones. We conclude that PKC delta inhibits the proliferation of VSMC by arresting cells in G1 via mainly inhibiting the expression of cyclin D1 and cyclin E.

Cardiovascular diseases, such as ischemic heart disease and stroke, represent the number one cause of death worldwide.1 In most cases, atherosclerosis and hypertension constitute the underlying causes for cardiovascular disease because it leads to arterial occlusion and impaired cardiac function, respectively. Therapeutic compensation of atherosclerotic artery occlusion by bypass grafting, angioplasty, or stenting bears the risk of intimal hyperplasia and subsequent restenosis.2 In general, a prerequisite for any pathological and physiological vascular remodeling process is the phenotypic switch of vascular smooth muscle cells (VSMCs).3 Even in adult blood vessels, VSMCs retain a remarkable plasticity that is essential for any changes in the vessel wall architecture. Contractile VSMCs representing the majority of VSMCs in healthy vessels guarantee maintenance of vascular tone and thereby the systemic blood pressure and blood flow by controlling the blood vessel diameter. As outlined in the Figure, a plethora of humoral factors, such as platelet-derived growth factor-BB and biomechanical stimuli, especially chronic changes in blood flow and wall stress, is capable to trigger the phenotypic switch of VSMCs from a quiescent, contractile differentiated state to a synthetic, proliferative, and dedifferentiated state.3,4 Figure. The effect of microRNA (miR)-633 on vascular homeostasis and remodeling. Major stimuli, transcription factors, and miRs that are involved in the differentiation and phenotypic switch of vascular smooth muscle cells (VSMCs) are depicted. miR-143/miR-145 promotes the contractile state by inhibiting suppressors of myocardin/serum response factor (SRF) activity (eg, ETS domain-containing protein-1 [Elk-1] and Kruppel-like factor [KLF] 4/5). Interestingly, myocardin activity seems to be regulated by an intrinsic loop because it stabilizes the expression of miR-145, whereas miR-143 seems to inhibit myocardin expression. miR-633 is a novel player with a dual effect on the VSMC phenotype not only by attenuating proliferation, migration, and proteolytic activity, but also by limiting their …

The role of nitric oxide in the pathophysiology of intimal hyperplasia

BackgroundOleic acid (OA) stimulates vascular smooth muscle cell (VSMC) proliferation and migration. The precise mechanism is still unclear. We sought to investigate the effects of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α) on OA-induced VSMC proliferation and migration.Principal FindingsOleate and palmitate, the most abundant monounsaturated fatty acid and saturated fatty acid in plasma, respectively, differently affect the mRNA and protein levels of PGC-1α in VSMCs. OA treatment resulted in a reduction of PGC-1α expression, which may be responsible for the increase in VSMC proliferation and migration caused by this fatty acid. In fact, overexpression of PGC-1α prevented OA-induced VSMC proliferation and migration while suppression of PGC-1α by siRNA enhanced the effects of OA. In contrast, palmitic acid (PA) treatment led to opposite effects. This saturated fatty acid induced PGC-1α expression and prevented OA-induced VSMC proliferation and migration. Mechanistic study demonstrated that the effects of PGC-1α on VSMC proliferation and migration result from its capacity to prevent ERK phosphorylation.ConclusionsOA and PA regulate PGC-1α expression in VSMCs differentially. OA stimulates VSMC proliferation and migration via suppression of PGC-1α expression while PA reverses the effects of OA by inducing PGC-1α expression. Upregulation of PGC-1α in VSMCs provides a potential novel strategy in preventing atherosclerosis.

Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor-induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. This is the first report detailing cross-interaction between toll-like receptor 2-mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.

Carvedilol Inhibits Platelet-Derived Growth Factor-Induced Extracellular Matrix Synthesis by Inhibiting Cellular Reactive Oxygen Species and Mitogen-Activated Protein Kinase Activation

Vascular smooth muscle cell (VSMC) migration and proliferation are involved in the intimal thickening responsible for late vein graft failure. In addition to growth and chemotactic factors, VSMCs require expression of matrix-degrading enzymes, e.g., metalloproteinases (MMP), to relieve the antiproliferative and antimigratory constraints of the extra-cellular matrix. Thapsigargin irreversibly inhibits Ca(2+)-ATPase, eliciting an increase in intracellular Ca2- and depletion of the intracellular calcium pools that are thought to be involved in the control of VSMC migration, VSMC proliferation, and MMP activity. We therefore studied the effect of thapsigargin on VSMC migration, VSMC proliferation, and MMP expression in human saphenous vein organ cultures. Vein segments were cultured for 14 days, and VSMC proliferation and migration were determined by autoradiography. Cell death was assessed using in situ end-labeling and lactate dehydrogenase release. Using Western blotting, we examined MMP-2 and MMP-9 and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression. Exposure to thapsigargin at 10 nmol/L for 60 minutes before culture significantly inhibited neointimal thickening (60%, P < .05), intimal and medial VSMC proliferation (32%, P < .05 and 37%, P < .05, respectively), and VSMC migration (36%, P < .05). Thapsigargin at 10 nmol/L did not significantly increase cell death or MMP-2, MMP-9, TIMP-1, and TIMP-2 expression. These results suggest that blockade of Ca(2+)-ATPase by thapsigargin inhibits VSMC migration and proliferation involved in neointimal formation without affecting MMP-2 and MMP-9 expression. Because short-term exposure to thapsigargin was sufficient to inhibit neointima formation, this drug may prove useful in the treatment of intimal thickening after arterial bypass graft surgery.

Vascular smooth muscle cell (VSMC) differentiation and proliferation are two important physiological processes during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contributes to the development of several major cardiovascular diseases including atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. Since the VSMC phenotype in these pathological conditions resembles that of developing VSMC during embryonic development, understanding of the molecular mechanisms that control VSMC differentiation will provide fundamental insights into the pathological processes of these cardiovascular diseases. Although VSMC differentiation is usually accompanied by an irreversible cell cycle exit, VSMC proliferation and differentiation occur concurrently during embryonic development. The molecular mechanisms simultaneously regulating these two processes, however, remain largely unknown. Our recent study demonstrates that cell division cycle 7, a key regulator of cell cycle, promotes both VSMC differentiation and proliferation through different mechanisms during the initial phase of VSMC differentiation. Conversely, Krüppel-like factor 4 appears to be a repressor for both VSMC differentiation and proliferation. This review attempts to highlight the novel role of cell division cycle 7 in TGF-β-induced VSMC differentiation and proliferation. The role of Krüppel-like factor 4 in suppressing these two processes will also be discussed.

Vascular smooth muscle cell (VSMC) migration and proliferation substantially contribute to neointimal hyperplasia related to in-stent restenosis. N6-methyladenosine (m6A) modification catalyzed by the METTL3 (methyltransferase-like 3)-containing methyltransferase complex is the most abundant RNA epigenetic modification in eukaryotes, but the role of m6A RNA methylation in VSMC migration and proliferation and neointima formation remains highly controversial. Primary human and rat VSMCs were utilized for in vitro experiments. VSMC-specific METTL3 knockout mice (Mettl3flox/floxMyh11-CreERT2) were generated to explore the role of METTL3 in carotid artery wire injury in vivo. Methylated RNA immunoprecipitation sequencing was performed to screen for genes targeted for METTL3-catalyzed m6A RNA methylation. Methylation site mapping, methylated RNA immunoprecipitation-quantitative polymerase chain reaction, chromatin immunoprecipitation-quantitative polymerase chain reaction, and reporter gene assays were used to explore how METTL3 modulates target gene expression. METTL3 expression was consistently upregulated in the neointima of mice subjected to carotid wire injury and in those of patients who underwent carotid endarterectomy. VSMC-specific METTL3 deficiency significantly attenuated neointima formation in mouse carotid arteries after wire injury. Accordingly, METTL3 ablation markedly repressed VSMC proliferation both in vitro and in vivo. Mechanistically, METTL3 directly catalyzed the m6A methylation of SGK1 (serum/glucocorticoid-regulated kinase 1) mRNA and subsequently facilitated its transcription, a process that was dependent on the established association between the SGK1 transcript and SGK1 promoter DNA via recruitment of the m6A reader YTHDC1 (YT521-B homology domain-containing protein 1). Conversely, SGK1 overexpression abolished the METTL3 deficiency-mediated suppression of VSMC proliferation and postinjury neointima formation. METTL3-catalyzed m6A RNA methylation promoted VSMC proliferation and exacerbated postinjury neointima formation by facilitating YTHDC1-dependent SGK1 gene transcription. Targeting the METTL3-YTHDC1-SGK1 axis to modulate VSMC proliferation may be a potential strategy for in-stent restenosis therapy.

Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system. To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide. Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs. These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.