Human Aortic Smooth Muscle Cells Migration Research Articles

RETRACTED: Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p

Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. Linc-ROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.

LncRNA TNK2-AS1 regulated ox-LDL-stimulated HASMC proliferation and migration via modulating VEGFA and FGF1 expression by sponging miR-150-5p.

Long non‐coding RNAs (lncRNAs) have been indicated for the regulatory roles in cardiovascular diseases. This study determined the expression of lncRNA TNK2 antisense RNA 1 (TNK2‐AS1) in oxidized low‐density lipoprotein (ox‐LDL)‐stimulated human aortic smooth muscle cells (HASMCs) and examined the mechanistic role of TNK2‐AS1 in the proliferation and migration of HASMCs. Our results demonstrated that ox‐LDL promoted HASMC proliferation and migration, and the enhanced proliferation and migration in ox‐LDL‐treated HASMCs were accompanied by the up‐regulation of TNK2‐AS1. In vitro functional studies showed that TNK2‐AS1 knockdown suppressed cell proliferation and migration of ox‐LDL‐stimulated HASMCs, while TNK2‐AS1 overexpression enhanced HASMC proliferation and migration. Additionally, TNK2‐AS1 inversely regulated miR‐150‐5p expression via acting as a competing endogenous RNA (ceRNA), and the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by miR‐150‐5p overexpression. Moreover, miR‐150‐5p could target the 3’ untranslated regions of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) to regulate FGF1 and VEGFA expression in HASMCs, and the inhibitory effects of miR‐150‐5p overexpression in ox‐LDL‐stimulated HASMCs were attenuated by enforced expression of VEGFA and FGF1. Enforced expression of VEGFA and FGF1 also partially restored the suppressed cell proliferation and migration induced by TNK2‐AS1 knockdown in ox‐LDL‐stimulated HASMCs, while the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by the knockdown of VEGFA and FGF1. Collectively, our findings showed that TNK2‐AS1 exerted its action in ox‐LDL‐stimulated HASMCs via regulating VEGFA and FGF1 expression by acting as a ceRNA for miR‐150‐5p.

Long non-coding RNA SNHG16 regulates human aortic smooth muscle cell proliferation and migration via sponging miR-205 and modulating Smad2.

The present study investigated the role of long non‐coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) in the human aortic smooth muscle cell (HASMC) proliferation and migration and explored the potential link between SNHG16 and atherosclerosis. Our results showed that platelet‐derived growth factor (PDGF)‐bb treatment promoted cell proliferation and migration with concurrent up‐regulation of SNHG16 in HASMCs. Small nucleolar RNA host gene 16 overexpression promoted HASMC proliferation and migration, while SNHG16 knockdown suppressed cell proliferation and migration in PDGF‐bb‐stimulated HASMCs. The bioinformatic analyses showed that SNHG16 possessed the complementary binding sequence with miR‐205, where the interaction was confirmed by luciferase reporter assay and RNA pull‐down assay in HASMCs, and SNHG16 inversely regulated miR‐205 expression. MiR‐205 overexpression attenuated the enhanced effects of PDGF‐bb treatment on HASMC proliferation and migration. Moreover, Smad2 was targeted and inversely regulated by miR‐205, while being positively regulated by SNHG16 in HASMCs. Smad2 knockdown attenuated PDGF‐bb‐mediated actions on HASMC proliferation and migration. Both miR‐205 overexpression and Smad2 knockdown partially reversed the effects of SNHG16 overexpression on HASMC proliferation and migration. Moreover, SNHG16 and Smad2 mRNA were up‐regulated, while miR‐205 was down‐regulated in the plasma from patients with atherosclerosis. Small nucleolar RNA host gene 16 expression was inversely correlated with miR‐205 expression and positively correlated with Smad2 expression in the plasma from atherosclerotic patients. In conclusion, our data showed the up‐regulation of SNHG16 in pathogenic‐stimulated HASMCs and clinical samples from atherosclerotic patients. Small nucleolar RNA host gene 16 regulated HASMC proliferation and migration possibly via regulating Smad2 expression by acting as a competing endogenous RNA for miR‐205.

Inhibitory effects of herbal decoction Ojeoksan on proliferation and migration in vascular smooth muscle cells.

The proliferation of vascular smooth muscle cells plays a crucial role in pathogenesis of cardiovascular disease. The principal objective of this study was to determine the effects of Ojeoksan (OJS) on human aortic smooth muscle cell (HASMC) proliferation induced by tumor necrosis factor α (TNF-aα). Thymidine incorporation after TNF-α treatment was increased and this effect was inhibited significantly by OJS treatment. HASMC proliferation and migration by kinetic live cell imaging were also reduced by treatment with OJS. TNF-α induced the expression of cyclins/cyclin-dependent kinases (CDKs) and reduced the expression of p21waf1/cip1/p27kip1. However, OJS also attenuated the expression of TNF-α-induced cell-cycle regulatory proteins. The results of Western blot analysis demonstrated that the TNF-α treated HASMC secreted gelatinases, probably including MMP-2/-9, which may be involved in the invasion and migration of HASMC. Additionally, OJS suppressed the mRNA expression levels of matrix metalloproteinase-2/-9 (MMP-2/-9) in a dose-dependent manner. OJS inhibited the production of TNF-α-induced hydrogen peroxide (H2O2) and the formation of DCF-sensitive intracellular reactive oxygen species (ROS). Further, OJS suppressed the nuclear translocation and phosphorylation of inhibitor of kappa B-α (IκB-α) of nuclear factor κB (NF-κB) under TNF-α conditions. Our results demonstrate that OJS exerts inhibitory effects on TNF-α-induced HASMC proliferation and migration, suggesting the involvement of the inhibition of both MMP-2 and MMP-9 expressions, and the downregulation of ROS/NF-κB signaling. Thus, herbal decoction OJS may be a possible therapeutic approach to the inhibition of cardiovascular disease including atherosclerosis.

Knockdown of TRIM28 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.

Experimental study of blood pressure and its impact on spontaneous hypertension in rats with Xin Mai Jia

AimThe aim of this study was to evaluate the antihypertensive effect of Xin Mai Jia (XMJ) and to explore the mechanism of its hypotensive effect. MethodsA total of 50 spontaneously hypertensive rats (SHR) were randomised into five groups. A total of 30 Wistar-Kyoto rats were randomised into three groups, comprising the control group. All of the rats were administered medicine through a gastrogavage once a day for 8 weeks. The tail-cuff method was applied to their monitor blood pressure. After 8 weeks of treatment, serum NO, SOD activity, MDA level, ET, ALD, AngII, RE, and CGRP in the serum were detected in all of the rats. Pathological changes in the aorta were observed via haematoxylin-eosin (HE) and immunohistochemical staining. Vasodilation function was assessed by measuring acetylcholine-induced vessel relaxation in the rats’ organ chambers. The function of the mesenteric arteries was measured using DMT wire myography. Human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) injury models were induced by hydrogen peroxide (H2O2). HASMCs and HUVECs were injured by H2O2 and then exposed to various drugs. HASMC and HUVEC migration was evaluated using the cell scratch test. The expression of the AT1 receptors (AT1R) in the HASMCs was detected via immunofluorescence (IFC) assay. ResultsAfter 8 weeks of treatment, XMJ reduced the systolic blood pressure of the SHR. XMJ significantly reduced the serum RE, AngII, ALD, and ET-1 levels and increased the content of CGRP and NO in the SHR, upregulated the SOD content, and downregulated MDA level of the SHR. XMJ improved pathological damage of the aorta to varying degrees, decreased the expression of AT1R in the SHR aortic vessels, and improved the mesenteric microvascular relaxation of the SHR. Cell experiments confirmed that XMJ inhibited the migration of the HUVECs and HASMCs induced by H2O2 and the expression of AT1R in the HASMCs. ConclusionXMJ had satisfactory hypotensive action on the SHR in this study. Its mechanism may be associated with inhibiting RAAS activity and improving RAAS function, inhibiting hypertensive-induced vascular diastolic dysfunction, and improving vascular endothelial function.

Teniposide regulates the phenotype switching of vascular smooth muscle cells in a miR-21-dependent manner

The switch of vascular smooth muscle cells (SMCs) from the contractile phenotype to proliferative one can make contributions to atherosclerosis and neointima formation. MiR-21 can prevent the rupture of advanced lesion plaques. We previously reported the protection of DNA topoisomerase II (Topo II) inhibitors against atherosclerosis and vascular calcification. However, it remains unknown if Topo II inhibitors can change SMC phenotypes. Herein, we show that teniposide protected SMC phenotype switching during atherosclerosis by enhancing expression of smooth muscle α-actin (SMA) while reducing osteopontin (OPN) expression in aortic lesion plaques. In vitro, teniposide induced expression of smooth muscle protein 22-α and calponin 1, but inhibited expression of OPN and epiregulin in human aortic SMCs (HASMCs). Moreover, teniposide attenuated platelet derived growth factor-BB-induced HASMC proliferation and migration. Mechanistically, the effect of teniposide on SMC phenotypes was completed, at least in part, by activating miR-21 expression. In addition, teniposide ameliorated ligation-induced carotid artery remodeling in C57BL/6J mice by regulating SMA and OPN expression. Taken together, our study demonstrates that teniposide regulates SMC phenotype switching by upregulating expression of contractile genes in a miR-21-dependent manner, and this function is an important anti-atherogenic mechanism of teniposide.

Abstract 317: Nlrc5 Positively Regulates Intimal Formation via Interaction With PPARã in Vascular Smooth Muscle Cells

Background: Innate immunity and vascular smooth muscle cells play crucial role in the development of vascular remodeling and intimal hyperplasia. NLR family CARD domain containing 5 (Nlrc5), known as a pattern recognition receptor, has been noted for its implication in immune disease in recent studies. However, whether Nlrc5 regulates intimal hyperplasia is still unclear. Aim: We aimed to determine the role of Nlrc5 in intimal hyperplasia and its underlying molecular mechanism. Methods and Results: We showed that an elevated expression of Nlrc5 in neointima induced by carotid ligation compared normal carotid artery. In human aortic smooth muscle cells (HASMCs), PDGF induced Nlrc5 expression and nuclear translocation. By utilizing Nlrc5 knockout mice, we found Nlrc5 deficiency prominently aggravated neointimal formation compared to wild type (WT) mice. On the contrary, gain of function of Nlrc5 based on local adenovirus injection attenuated intimal hyperplasia. The neointimal area was comparable in WT mice receiving either WT or Nlrc5-/- bone marrow transplantation, demonstrating that hematopoietic cell-derived Nlrc5 did not affect vascular hyperplasia. In vitro experiments unveiled that knockdown of Nlrc5 resulted in excessive proliferation, migration and dedifferentiation of HASMCs. Nevertheless, depletion of Nlrc5 in HASMCs did not influence proinflammatory cytokine excretion and immune activation reflected by major histocompatibility complex-I expression. Mechanistic study revealed that Nlrc5 bound to nuclear transcription factor PPARγ and positively regulated PPARγ luciferase activity, ensuing contributing to KLF4 ubiquitination and degradation. Furthermore, we generated five deletion constructs of Nlrc5 and identified that the functional domain containing DD and NACHT constructs was responsible for the interaction with PPARγ. Conclusions: Our study reveals that Nlrc5 alleviates intimal formation through interaction with PPARγ and inhibition of smooth muscle cell dedifferentiation, suggesting that Nlrc5 constitutes a novel molecular target for vascular remodeling.

Minocycline Inhibits PDGF‐BB‐induced Human Aortic Smooth Muscle Cell Proliferation and Migration via Induction of RECK

Minocycline is an FDA‐approved tetracycline antibiotic that exerts anti‐inflammatory and anti‐apoptotic effects independent of its bactericidal activity. For example, it attenuates neointima formation after arterial injury and atherosclerotic plaque development in an animal model of diet‐induced atherosclerosis via inhibition of smooth muscle cell (SMC) proliferation and migration; however, the molecular mechanisms underlying this inhibition are not completely known. Here we investigated whether minocycline inhibits PDGF‐BB‐induced primary human aortic SMC (HASMC) proliferation and migration in vitro. Our results show that PDGF‐BB stimulates proliferation (~2.92‐fold, P<0.05) and migration (~1.8‐fold, P<0.05) of HASMC via a redox‐sensitive mechanism involving NOX2 (inhibited by gp91dstat) and NOX4 (inhibited by GKT137831). Reversion‐Inducing‐Cysteine‐Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored matrix metalloproteinase inhibitor. For the first time, our results show that PDGF‐BB downregulates RECK expression in a time‐dependent manner (by ~80% at 6 h, n=3, P<0.01), but induces miR‐221 and miR‐222 expression (~1.8‐fold and ~2.23‐fold, respectively). The reporter assays revealed that both miRs bind the 3′UTR of RECK mRNA, inhibiting its expression by ~50% (P<0.01). Supporting these observations, transduction of miR‐221 or miR‐222 mimics suppress RECK expression, but their antagomirs reverse PDGF‐BB‐induced inhibition of HASMC proliferation and migration. Further investigations revealed that PDGF‐BB activates the redox‐sensitive nuclear transcription factors NF‐kB and AP‐1 in HASMC, and shRNA directed against the AP‐1 subunit c‐Jun and the p65 subunit of NF‐kB each inhibit PDGF‐BB‐induced miR‐221 or miR‐222 expression, suggesting that PDGF‐BB suppresses RECK expression in HASMC in part via AP‐1‐ and NF‐kB‐dependent miR‐221 and miR‐222 induction. Notably, minocycline increases RECK expression dose‐dependently within the therapeutic dose of 1–100 μM, and inhibits PDGF‐BB‐induced HASMC proliferation and migration. Intriguingly, silencing RECK reverses the inhibitory effects of minocycline on PDGF‐BB‐induced migration and proliferation. Together, these results demonstrate that PDGF‐BB downregulates RECK expression in HASMC via AP‐1‐ and NF‐kB‐dependent miR‐221 and miR‐222 induction, and minocycline reverses PDGF‐BB‐induced ROS generation, miR‐221 and miR‐222 induction, and RECK suppression. These findings suggest that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.Support or Funding InformationNIH R01‐HL‐070241 (PD)VA Merit Award I01‐BX002255 (BC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Critical role of Rap1 in triggering the effects of microparticles from metabolic syndrome patients on vascular smooth muscle cell functions

The metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular disease and atherosclerosis including hyperglycemia, dyslipidemia, hypertension and obesity. We have previously shown that circulating levels of microparticles (MPs), small vesicles released from plasma membrane, from MetS patients induce endothelial dysfunction. Here, we analyze whether MPs from MetS patients may participate to the alteration of smooth muscle cells (SMC) function described during the atherosclerosis development. Circulating MPs of non-MetS subjects and MetS patients have been isolated from plasma and characterized by proteomic analysis. Then, the involvement of Rap1 in the effects of MPs on human aortic SMC (HASMC) proliferation and migration was analyzed. Differential proteomic analysis of MPs from both types of individuals identifies Rap1, a small GTPase, as two-fold overexpressed in MPs from MetS compared with non-MetS subjects. In addition, Rap1 is in active state, that is, GTP-associated, in both type of MPs. When HASMC are incubated with MPs for 24 h, both types of MPs significantly promote proliferation and migration. Even more, MetS MPs are able to increase the expression of the pro-inflammatory molecules MCP-1 and IL-6. Neutralization of Rap-1 by specific antibody or pharmacological inhibition of Rap-1 with GGTI-298 either partially or completely prevents the effects of MPs from MetS patients but not those from non-MetS MPs. These effects includes HASMC proliferation, migration, inflammation and increase of p38 and ERK5 phosphorylation. These data suggest that overexpression of Rap1 in MetS MPs might participate in the enhanced SMC proliferation, migration and activation of MAPK/ERK pathway leading to atherosclerosis.

P115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine–induced vascular smooth muscle cell migration and proliferation

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-Gi/o-Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling.

Hypoxia-inducible factor 1a induces phenotype switch of human aortic vascular smooth muscle cell through PI3K/AKT/AEG-1 signaling.

To date, hypoxia-inducible factor 1a (HIF-1a) and astrocyte elevated gene-1 (AEG-1) have been involved in the proliferation, migration and morphological changes of vascular smooth muscle cells. However, the potential relationship of HIF-1a-AEG-1 pathway in human aortic smooth muscle cell (HASMC) has not been reported. In the present study, in-vitro assays were utilized to explore the potential impact of HIF-1a-AEG-1 signaling on HASMC phenotype. Here, we found that HIF-1a expression was up-regulated in the media of thoracic aortic dissection tissues as compared with normal aortic tissues, and was associated with increased apoptotic SMCs and decreased AEG-1 expression. Mechanically, hypoxia promoted the expression of HIF-1a by PI3K-AKT pathway in HASMCs; HIF-1a further suppressed the expressions of AEG-1, a-SMA and SM22a, and promoted osteopontin (OPN) expression. Functionally, HIF-1a inhibited the proliferation and migration of HASMCs. However, si-HIF-1a or Akt inhibitor abrogated HIF-1a-mediated related expressions and biological effects above. In conclusion, HIF-1a induces HASMC phenotype switch, and closely related to PI3K/AKT and AEG-1 signaling, which may provide new avenues for the prevention and treatment of aortic dissection diseases.

CO-releasing molecules CORM2 attenuates angiotensin II-induced human aortic smooth muscle cell migration through inhibition of ROS/IL-6 generation and matrix metalloproteinases-9 expression

Ang II has been involved in the pathogenesis of cardiovascular diseases, and matrix metalloproteinase-9 (MMP-9) induced migration of human aortic smooth muscle cells (HASMCs) is the most common and basic pathological feature. Carbon monoxide (CO), a byproduct of heme breakdown by heme oxygenase, exerts anti-inflammatory effects in various tissues and organ systems. In the present study, we aimed to investigate the effects and underlying mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on Ang II-induced MMP-9 expression and cell migration of HASMCs. Ang II significantly up-regulated MMP-9 expression and cell migration of HASMCs, which was inhibited by transfection with siRNA of p47phox, Nox2, Nox4, p65, angiotensin II type 1 receptor (AT1R) and pretreatment with the inhibitors of NADPH oxidase, ROS, and NF-κB. In addition, Ang II also induced NADPH oxidase/ROS generation and p47phox translocation from the cytosol to the membrane. Moreover, Ang II-induced oxidative stress and MMP-9-dependent cell migration were inhibited by pretreatment with CORM-2. Finally, we observed that Ang II induced IL-6 release in HASMCs via AT1R, but not AT2R, which could further caused MMP-9 secretion and cell migration. Pretreatment with CORM-2 reduced Ang II-induced IL-6 release. In conclusion, CORM-2 inhibits Ang II-induced HASMCs migration through inactivation of suppression of NADPH oxidase/ROS generation, NF-κB inactivation and IL-6/MMP-9 expression. Thus, application of CO, especially CORM-2, is a potential countermeasure to reverse the pathological changes of various cardiovascular diseases. Further effects aimed at identifying novel antioxidant and anti-inflammatory substances protective for heart and blood vessels that targeting CO and establishment of well-designed in vivo models properly evaluating the efficacy of these agents are needed.

Cytosolic Phospholipase A2 Alpha is Required for Human Smooth Muscle Cell Proliferation and Not Migration to Platelet Derived Growth Factor BB

Platelet derived growth factor BB (PDGF BB) has an important influence on smooth muscle cell migration and proliferation in restenosis and atherosclerosis. Our understanding of different signal transduction pathways involved in the response of smooth muscle cells to PDGF BB is potentially significant for understanding and manipulating these processes. Prior studies have demonstrated a crucial activation of cytosolic phospholipase A2 (cPLA2) in smooth muscle cells to PDGF BB with the production of arachidonic acid and prostaglandin E2. In this study, we first investigated the role of cPLA2α on human aortic smooth muscle cell (HASMC) migration using modified Boyden chamber assay and under agarose migration studies. AACOCF3 (cPLA2 and iPLA2 inhibitor), 1,2,4-trisubstituted pyrrolidine derivative (cPLA2 inhibitor), and Bromoenol lactone (iPLA2 inhibitor) had no effect on HASMC chemotaxis to PDGF-BB in a modified Boyden chamber. These results were confirmed with specific inhibition of cPLA2α using small interfering RNA (siRNA) showing that HASMC migration was not inhibited in modified Boyden chamber or under agarose migration studies. Using the same siRNA to cPLA2 alpha had very significant inhibition to PDGF-BB dependent HASMC proliferation. These data demonstrate there is a distinct role especially for cPLA2α on human aortic HASMC proliferation and not migration to PDGF-BB.

Viscolin Inhibits In Vitro Smooth Muscle Cell Proliferation and Migration and Neointimal Hyperplasia In Vivo.

Viscolin, an extract of Viscum coloratum, has anti-inflammatory and anti-proliferative properties against harmful stimuli. The aim of the study was to examine the anti-proliferative effects of viscolin on platelet derived growth factor-BB (PDGF)-treated human aortic smooth muscle cells (HASMCs) and identify the underlying mechanism responsible for these effects. Viscolin reduced the PDGF-BB-induced HASMC proliferation and migration in vitro; it also arrested HASMCs in the G0/G1 phase by decreasing the protein expression of Cyclin D1, CDK2, Cyclin E, CDK4, and p21Cip1 as detected by Western blot analysis. These effects may be mediated by reduced PDGF-induced phosphorylation of ERK1/2, JNK, and P38, but not AKT as well as inhibition of PDGF-mediated nuclear factor (NF)-κB p65 and activator protein 1 (AP-1)/c-fos activation. Furthermore, viscolin pre-treatment significantly reduced neointimal hyperplasia of an endothelial-denuded femoral artery in vivo. Taken together, viscolin attenuated PDGF–BB-induced HASMC proliferation in vitro and reduced neointimal hyperplasia in vivo. Thus, viscolin may represent a therapeutic candidate for the prevention and treatment of vascular proliferative diseases.

2-Methoxycinnamaldehyde inhibits the TNF-α-induced proliferation and migration of human aortic smooth muscle cells.

The abnormal proliferation and migration of vascular smooth muscle cells(VSMCs) is a crucial event in the development of atherosclerosis, and tumor necrosis factor-α(TNF-α) is actively involved in this process by enhancing the proliferation and migration of VSMCs. 2-Methoxycinnamaldehyde(MCA) is a natural compound of Cinnamomum cassia. Although 2-hydroxycinnamaldehyde(HCA), another compound from Cinnamomum cassia, has been widely studied with regard to its antitumor activity, MCA has not attracted researchers' interest due to its mild toxic effects on cancer cells and its mechanisms of action remain unknown. In this study, we examined the effects of MCA on the TNF-α-induced proliferation and migration of human aortic smooth muscle cells(HASMCs). As shown by our results, MCA inhibited TNF-α-induced cell proliferation by reducing the levels of cyclinD1, cyclinD3, CDK4 and CDK6, and increasing the levels of the cyclin-dependent kinase inhibitors, p21 and p27, without resulting in cellular cytotoxicity. Furthermore, MCA decreased the level of secreted matrix metalloproteinase(MMP)-9 by inhibiting MMP-9 transcription. Unexpectedly, MCA did not affect the TNF-α-induced levels of mitogen-activated protein kinases(MAPKs). However, by showing that MCA potently inhibited the degradation of IκBα and the subsequent nuclear translocation of nuclear factor-κB(NF-κB), we demonstrated that MCA exerts its effects through the NF-κB signaling pathway. MCA also effectively inhibited platelet-derived growth factor(PDGF)-induced HASMC migration. Taken together, these observations suggest that MCA has the potential for use as an anti-atherosclerotic agent.

Synergistic effect of atorvastatin and cyanidin-3-glucoside against angiotensin II-mediated vascular smooth muscle cell proliferation and migration through MAPK and PI3K/Akt pathways.

This study aimed to investigate the mechanism of cyanidin-3-glucoside (C3G) in synergy with atorvastatin, even when it is used in low concentrations. Human aortic smooth muscle cells (HASMCs) were used to verify the synergistic mechanism of atorvastatin and C3G against angiotensin II-induced proliferation and migration. BrdU incorporation assay was used to evaluate cell proliferation. Wound healing and Boyden chamber assays were used to investigate cell migration. The cell cycle was examined using flow cytometry. The results revealed that atorvastatin and C3G exhibit a synergistic effect in ameliorating HASMC proliferation and migration by enhancing cell cycle arrest. In addition, these effects also decreased mitogen-activated protein kinase (MAPK) activity by attenuating the expression of phospho-p38, phospho-extracellular signaling-regulated kinase 1/2, and phospho-c-Jun N-terminal kinase. Furthermore, the combination of atorvastatin and C3G modulated the PI3K/Akt pathway and upregulated p21Cip1, which was associated with decreases in cyclin D1 and phospho-retinoblastoma expressions. The synergistic effect of atorvastatin and C3G induced anti-proliferation and anti-migration through MAPK and PI3K/Akt pathways mediated by AT1R. These results suggest that the synergistic effect of atorvastatin and C3G may be an alternative therapy for atherosclerosis patients.

Contrasting effects of stanniocalcin-related polypeptides on macrophage foam cell formation and vascular smooth muscle cell migration

Stanniocalcin (STC) is a calcium- and phosphate-regulating hormone secreted by the corpuscles of Stannius, an endocrine gland of bony fish. Its human homologues, STC1 and STC2 showing 34% amino acid identity each other, are expressed in a variety of human tissues. To clarify their roles in atherosclerosis, we investigated the effects of their full-length proteins, STC1(18–247) and STC2(25–302), and STC2-derived fragment peptides, STC2(80–100) and STC2(85–99), on inflammatory responses in human umbilical vein endothelial cells (HUVECs), human macrophage foam cell formation, the migration and proliferation of human aortic smooth muscle cells (HASMCs) and the extracellular matrix expression. All these polypeptides suppressed lipopolysaccharide-induced expressions of interleukin-6, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in HUVECs. Oxidized low-density lipoprotein-induced foam cell formation was significantly decreased by STC1(18–247) and increased by STC2(80–100) and STC2(85–99), but not STC2(25–302), in human macrophages. Expression of acyl-CoA:cholesterol acyltransferase-1 (ACAT1) was significantly suppressed by STC1(18–247) but stimulated by STC2(80–100) and STC2(85–99). Expression of ATP-binding cassette transporter A1 was significantly stimulated by STC1(18–247). Neither STC1(18–247) nor STC2-derived peptides significantly affected CD36 expression in human macrophages or HASMC proliferation. STC2(80–100) and STC2(85–99) significantly increased HASMC migration, whereas STC1(18–247) significantly suppressed the angiotensin II-induced HASMC migration. Expressions of collagen-1, fibronectin, matrix metalloproteinase-2, and elastin were mostly unchanged with the exception of fibronectin up-regulation by STC2(80–100). Our results demonstrated the contrasting effects of STC1 and STC2-derived peptides on human macrophage foam cell formation associated with ACAT1 expression and on HASMC migration. Thus, STC-related polypeptides could serve as a novel therapeutic target for atherosclerosis.

Knockdown of SCARA5 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppression of the PDGF signaling pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration are critical in the progression of atherosclerosis and can be induced by platelet-derived growth factor (PDGF). Several studies have demonstrated that scavenger receptor class A, member 5 (SCARA5) is important in cancer cell migration and invasion. However, the role of SCARA5 in VSMCs remains to be elucidated in the development of atherosclerosis. Therefore, the role of SCARA5 was investigated in PDGF‑BB‑stimulated VSMC proliferation and migration. In the present study, it was shown that SCARA5 expression was enhanced by PDGF‑BB in human aortic smooth muscle cells (HASMCs). Knockdown of SCARA5 by small interfering (si)RNA significantly inhibited PDGF‑BB‑induced HASMC proliferation and migration. Furthermore, siRNA‑SCARA5 significantly inhibited the phosphorylation of PDGF receptor (PDGFR) β, AKT and extracellular signal‑regulated kinase1/2 in PDGF‑BB‑stimulated HASMCs. In conclusion, this study demonstrated that knockdown of SCARA5 inhibits PDGF‑BB‑induced HASMC proliferation and migration through suppression of the PDGF signaling pathway. Thus, SCARA5 may be a novel therapeutic target for preventing or treating vascular diseases involving VSMC proliferation and migration.

Chungtaejeon, a Korean fermented tea, prevents the risk of atherosclerosis in rats fed a high-fat atherogenic diet

ObjectiveHypercholesterolemia is one of the well-established risk factors for cardiovascular mortality and morbidity in coronary heart disease. The aim of this study was to investigate the anti-atherogenic effect of Chungtaejeon (CTJ, a Korean fermented tea) aqueous extract on proliferation and migration of human aortic smooth muscle cells (HASMCs) in vivo and in vitro. MethodsThe authors used high-fat atherogenic diet (HFAD) to induce hyperlipidemia in Wistar rats in in vivo animal experiments and used HASMCs for in vitro cell experiments. For the in vitro cell experiment, the proliferation of asms was evaluated using the MTT assay. Similarly, the expression of matrix metalloproteinases (MMPs) in HASMCs was measured using gelatin zymography. Antimigratory activity of CTJ was revealed using the wound-healing model and Boyden's chamber assay. In the in vivo experiment, CTJ was administered in three different doses for 20 d from the initiation of the HFAD. After 20 d, the serum lipid profile and total lipid contents in liver were measured. ResultsTreatment with CTJ for 24 h dose-dependently inhibited the proliferation and migration of HASMCs and expression of MMP-2 in HASMCs. The oral administration of CTJ at concentrations of 200 and 400 mg/kg decreased the levels of low-density lipoprotein cholesterol, total serum cholesterol and hepatic cholesterol of HFAD-fed rats. ConclusionCTJ possessed strong antiproliferative, antimigratory, as well as lipid-lowering activities. Thus, CTJ can be considered as a therapeutic option in the treatment of high-fat diet-induced atherosclerosis.