Corilagin inhibits the proliferation of vascular smooth muscle and excessive matrix deposition following vascular injury via the MAPK pathway.

The role of nitric oxide in the pathophysiology of intimal hyperplasia

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

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

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.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

Nesfatin-1 promotes VSMC migration and neointimal hyperplasia by upregulating matrix metalloproteinases and downregulating PPARγ

microRNA (miR) -22-3p has been confirmed to be engaged in the phenotype transformation and proliferation of vascular smooth muscle cells (VSMCs), which is intimately correlated with restenosis. The current research set out to explore the detailed mechanism and function of miR-22-3p in VSMC proliferation, phenotype transformation, and migration via the translocase of outer mitochondrial membrane (TOMM40). Peripheral blood samples were acquired from patients with in-stent restenosis (ISR) after percutaneous coronary intervention (PCI), with subsequent quantitative reverse transcription (qRT) -polymerase chain reaction (PCR) and Western blot analyses of miR-22-3p and TOMM40 expression. After miR-22-3p-inhibitor, oe-TOMM40, and sh-TOMM40 were transfected into VSMCs, Cell Counting Kit (CCK) -8 assay, scratch test, and Western blot analysis were implemented to measure the VSMC proliferation, migration, and matrix metallopeptidase 9 (MMP9), α-smooth muscle actin (SMA), smooth muscle-myosin heavy chain (SM-MHC), and osteopontin (OPN) expressions, respectively. In addition, human umbilical vein endothelial cell (HUVEC) proliferation was examined by CCK-8 assay. The binding relationship between miR-22-3p and TOMM40 was assessed by dual luciferase reporter and RNA immunoprecipitation assays. The peripheral blood of patients with ISR after PCI had low expression of miR-22-3p and high expression of TOMM40. The mechanistic analysis reported the negative targeting relationship between miR-22-3p and TOMM40. Down-regulating miR-22-3p or up-regulating TOMM40 elevated the proliferation, migration, and phenotype transformation of VSMCs. miR-22-3p inhibitor had no evident impact on HUVEC proliferation. In addition, rescue assays displayed that TOMM40 silencing annulled miR-22-3p inhibition-enhanced VSMC proliferation, migration, and phenotype transformation. Conclusively, miR-22-3p could repress VSMC proliferation, phenotypic transformation, and migration by targeting TOMM40, which might be a possible treatment candidate for restenosis after PCI in patients with cardiovascular disease.

Buyang huanwu decoction attenuates arterial in-stent restenosis via transforming growth factor beta receptor 1-mediated suppression of neointimal hyperplasia.

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

Prevention of stenosis after vascular reconstruction: Pharmacologic control of intimal hyperplasia — A review

BackgroundMicroRNA miR‐214 has been implicated in many biological cellular functions, but the impact of miR‐214 and its target genes on vascular smooth muscle cell (VSMC) proliferation, migration, and neointima smooth muscle cell hyperplasia is unknown.Methods and ResultsExpression of miR‐214 was closely regulated by different pathogenic stimuli in VSMCs through a transcriptional mechanism and decreased in response to vascular injury. Overexpression of miR‐214 in serum‐starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miR‐214 dramatically increased VSMC proliferation and migration. Gene and protein biochemical assays, including proteomic analyses, showed that NCK associated protein 1 (NCKAP1)—a major component of the WAVE complex that regulates lamellipodia formation and cell motility—was negatively regulated by miR‐214 in VSMCs. Luciferase assays showed that miR‐214 substantially repressed wild‐type but not the miR‐214 binding site mutated version of NCKAP1 3′ untranslated region luciferase activity in VSMCs. This result confirmed that NCKAP1 is the functional target of miR‐214 in VSMCs. NCKAP1 knockdown in VSMCs recapitulates the inhibitory effects of miR‐214 overexpression on actin polymerization, cell migration, and proliferation. Data from cotransfection experiments also revealed that inhibition of NCKAP1 is required for miR‐214–mediated lamellipodia formation, cell motility, and growth. Importantly, locally enforced expression of miR‐214 in the injured vessels significantly reduced NCKAP1 expression levels, inhibited VSMC proliferation, and prevented neointima smooth muscle cell hyperplasia after injury.ConclusionsWe uncovered an important role of miR‐214 and its target gene NCKAP1 in modulating VSMC functions and neointima hyperplasia. Our findings suggest that miR‐214 represents a potential therapeutic target for vascular diseases.

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.

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are key pathological factors in the initiation and progression of vascular disorders, including arteriosclerosis and restenosis following percutaneous coronary intervention (PCI). Hydroxysafflor yellow A (HSYA), the main component of the safflower yellow pigments, has widely been used for the treatment of cardiovascular diseases in traditional Chinese medicine. However, to the best of our knowledge, there are no studies investigating the pharmaceutical effect of HSYA on VSMCs or the underlying molecular mechanism. The present study aimed to investigate the effect of HSYA on platelet‑derived growth factor (PDGF)‑BB‑stimulated VSMC proliferation and migration. HSYA significantly inhibited PDGF‑BB‑stimulated VSMC proliferation and, in response to PDGF‑BB‑stimulation, VSMCs dedifferentiated into a proliferative phenotype. However, HSYA effectively reversed this phenotype switching. In addition, the production of nitrous oxide and cyclic guanosine monophosphate induced by PDGF‑BB was also suppressed by HSYA, and HSYA markedly inhibited PDGF‑BB‑stimulated VSMC migration. Investigation of the molecular mechanism revealed that HSYA inhibited PDGF‑BB‑induced activation of Akt signaling. In addition, HSYA also suppressed PDGF‑BB‑stimulated upregulation of cell cycle related proteins and heme oxygenase‑1. In conclusion, HSYA was able to inhibit PDGF‑BB‑stimulated VSMC proliferation and migration, partially via suppressing PDGF‑BB‑induced Akt signaling activation. Therefore, HSYA may be useful for the prevention and treatment of cardiovascular diseases, including atherosclerosis and restenosis following PCI.

It is well known that atherosclerosis and restenosis are common cardiovascular diseases and major health care problems. Vascular remodeling and migration and proliferation of vascular smooth muscle cells (VSMCs) are key features of these pathologies. There has been enormous progress in drug development and clinical management of these disorders, with angioplasty and drug eluting stents being standard procedures to treat vascular obstruction. However, despite their benefits, these current treatment modalities are not always efficacious. Furthermore, they can also be associated with postoperative complications and graft failures, some of which can be life threatening. Evaluation of newer mechanisms involved in VSMC proliferation aimed at uncovering additional therapeutic approaches to curb VSMC dysfunction in cardiovascular diseases is clearly warranted. See accompanying article on page 851 Accumulating evidence suggests that several common diseases, including cardiovascular disorders, diabetes, and the vascular complications of diabetes, are governed by a combination of genetic and environmental factors and that epigenetic mechanisms, such as DNA methylation and histone modifications in chromatin, form a key link between them.1–3 Epigenetics is the added layer of gene regulation that occurs in chromatin without changes in the actual underlying DNA sequence and plays a major role in dictating cell-specific gene expression patterns and transcriptional outcomes.4,5 Along with DNA methylation, key posttranslational modifications of histone N-terminal tails can alter chromatin structure to form an added layer of gene regulation and modulate gene transcription.5 Therefore, gene transcription depends on chromatin structure, which is very dynamic, depending on a multitude of histone posttranslational modifications that allow for the conversion of inaccessible, compact, or …