Proliferation Of Aortic Smooth Muscle Cells Research Articles

MicroRNA-99a-5p alleviates atherosclerosis via regulating Homeobox A1

AimsMicroRNAs have been demonstrated to be involved in the development of atherosclerosis. The present study aimed to evaluate the effect of miR-99a-5p and its target gene Homeobox A1 (HOXA1) in atherosclerosis. Main methodsThe biological functions of miR-99a-5p on human aortic smooth muscle cells (ASMCs) were assessed by MTT, wound healing and transwell assays. The target genes of microRNAs were predicted by TargetScan and miRDB. The binding of miR-99a-5p and HOXA1 was confirmed by luciferase reporter assay. In the in vivo study, high-fat diet-induced atherosclerosis model was established in Apolipoprotein E knockout mice. Hematoxylin-eosin (H&E), oil Red O and Masson trichrome staining were performed for determination of atherosclerotic lesion. The levels of miR-99a-5p and HOXA1 mRNA were detected by real-time PCR. HOXA1 and migration-associated protein levels were detected by western blot or immunohistochemistry analysis. Key findingsMiR-99a-5p inhibited HOXA1 expression by targeting 3′UTR of HOXA1 mRNA. Enforced HOXA1 significantly promoted the proliferation, migration, and invasion of ASMCs. Furthermore, miR-99a-5p overexpression inhibited the proliferation, migration, and invasion of ASMCs stimulated by HOXA1, whereas miR-99a-5p inhibition reversed the effects of HOXA1 knockdown on these behaviours of ASMCs. In vivo, the specific overexpression of miR-99a-5p significantly abated atherosclerotic lesions formatted, accompanied with a significant down-regulation of HOXA1 mRNA and protein expression levels. SignificanceWe demonstrate for first time that miR-99a-5p may serve as a potential inhibitor of the atherosclerosis, and miR-99a-5p plays its role partially through targeting HOXA1.

Synthesis of Clitocybin A, B and C and their Biological Evaluation for Antioxidant Activities

Clitocybin A, isolated from the culture broth of mushroom Clitocybe aurantiaca, showed various biological activities such as cell death inhibition effects, anti‐wrinkle effects, aortic smooth muscle cell proliferation inhibition effects. In an effort to further develop this promising compound toward a potent antioxidant, we have modified the structure of clitocybin A by changing the number of free phenolic hydrogen in isoindolinone moiety. In this paper, clitocybin A and its analogues, clitocybin B and C were synthesized and their antioxidant activities were evaluated. Clitocybin A possessing two phenolic hydrogens in isoindolinone moiety of clitocybin A analogues showed more potent radical scavenging activity than clitocybin B possessing one phenolic hydrogen. In inhibition of cellular DNA damage in H2O2treated cells, clitocybin C lacking free phenolic hydrogen in isoindolinone moiety showed more potent activity than clitocybin A possessing two phenolic hydrogens.Among the tested compounds, clitocybin A only showed dose‐dependently HNE inhibitory activity with an IC50 value of 78.8 uM. The presence of free phenolic hydrogens inisoindolinone moiety of clitocybin A analogues plays an important role in antioxidant activities.

STRIP2 silencing inhibits vascular smooth muscle cell proliferation and migration via P38-AKT-MMP-2 signaling pathway.

STRIP2 (FAM40B) was reported to regulate tumor cell migration. Our study aims to discuss the effect of STRIP2 in mouse aortic smooth muscle cell (MOVAS) proliferation and migration processes, which contributes greatly to atherosclerosis formation. In MOVAS cells, STRIP2 depletion suppressed cell proliferation and migration, which were related to a remarkable decrease in matrix metalloproteinases-2 (MMP-2)/MMP-9 expression. Additionally, P38mitogen-activated protein kinases and Protein kinase B (AKT) are inactivated while extracellular signal-regulated kinase (ERK1/2) and jun N-terminal kinase (JNK) are activated upon STRIP2 silencing. SB203580 (P38 inhibitor) further reduced AKT phosphorylation (p-AKT) while dehydrocorydaline chloride (Dc; P38 activator) reversed this effect. Furthermore, Dc significantly recovered MMP-2 expression in STRIP2-knockdown cells. As expected, overexpressing STRIP2 exhibited a contrary effect. Dc and AKT activator SC79 reversed the inhibition of cell proliferation and migration induced by STRIP2 silencing. Interestingly, STRIP2 depletion increased vascular endothelial growth factor level significantly. Taken together, STRIP2 contributed to cell proliferation and migration through P38-AKT-MMP-2 signaling in MOVAS cells, indicating the importance of STRIP2 in atherosclerosis.

Indoxyl Sulfate-Induced Extracellular Vesicles Released from Endothelial Cells Stimulate Vascular Smooth Muscle Cell Proliferation by Inducing Transforming Growth Factor-Beta Production

Vascular access stenosis predominantly occurs as a result of neointimal hyperplasia (NH) formation at the anastomosis. Moreover, in the presence of NH, transforming growth factor-beta (TGF-β) promotes vascular smooth muscle cell (VSMC) proliferation. Extracellular vesicles (EVs) released by endothelial cells are closely associated with vascular dysfunction. Here, we investigated the effects of EVs on TGF-β signaling and VSMC proliferation. Specifically, EVs were collected from the culture medium of indoxyl sulfate (IS)-treated human umbilical vein endothelial cells and used (2 × 10⁶) to stimulate human aortic smooth muscle cells (SMCs) (1 × 10⁶). Western blotting was performed to assess the levels of Akt, ERK1/2, p38 MAPK, and Smad3. BrdU proliferation assays, quantitative PCR, and ELISA assays were performed to evaluate SMC proliferation and TGF-β production. The IS-induced EVs stimulated the proliferation of aortic SMCs in a concentration-dependent manner. The EVs both contained TGF-β and promoted TGF-β production by SMCs by phosphorylating Akt, ERK1/2, p38 MAPK, and Smad3, which was significantly inhibited by an anti-TGF-β antibody. SMC proliferation was suppressed by both an anti-TGF-β antibody and inhibitors of the downstream factors. These results suggest that EVs are involved in the pathogenesis of vascular access stenosis by modulating TGF-β signaling in VSMCs under uremic conditions.

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation.

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversioninducing cysteinerich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.

RECK suppresses interleukin‐17/TRAF3IP2‐mediated MMP‐13 activation and human aortic smooth muscle cell migration and proliferation

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular proliferative/occlusive disorders. The proinflammatory cytokine interleukin (IL)‐17 signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various transcription factors critical for inflammatory responses, including AP‐1 and NF‐κB. Here we investigated whether IL‐17A/TRAF3IP2 signaling promotes MMP‐13‐dependent human aortic smooth muscle cell (SMC) migration and proliferation, and whether RECK overexpression blunts its mitogenic and migratory potential. Indeed, IL‐17A treatment induced (i) AP‐1, NF‐κB, CREB and p38 MAPK activation, (ii) miR‐21 induction, (iii) miR‐27b and miR‐320 inhibition, (iv) MMP‐13 expression and activation, (v) RECK suppression, and (vi) SMC migration and proliferation, all in a TRAF3IP2‐dependent manner. RECK (REversion‐inducing‐Cysteine‐rich protein with Kazal motifs) is a membrane‐anchored MMP inhibitor, and forced expression of TRAF3IP2 suppressed its expression, but induced MMP‐13 expression and activation. Importantly, forced expression of RECK promoted IL‐17‐mediated MMP‐13/RECK physical association, reduced MMP13 activity and inhibited MMP‐13‐dependent SMC migration and proliferation. Further supporting its causative role in vascular proliferative diseases, addition of recombinant MMP‐13 stimulated SMC migration and proliferation, in part via ERK activation. These results indicate that increased MMP‐13 and decreased RECK contribute to IL‐17A/TRAF3IP2‐mediated SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.Support or Funding InformationVA‐I01‐BX002255IK6BX004016‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1–100 μM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

Warburg effect is involved in apelin-13-induced human aortic vascular smooth muscle cells proliferation.

Apelin is the endogenous ligand for the G protein-coupled receptor APJ. Both apelin and APJ receptor are distributed in vascular smooth muscle cells (VSMCs) and play important roles in the cardiovascular system. Our previous reports have indicated that apelin-13 promoted the proliferation of VSMCs, but its exact mechanism remains to be further explored. The results of the present study demonstrated that the Warburg effect plays a pivotal role in apelin-13-induced human aortic vascular smooth muscle cells (HA-VSMCs) proliferation. Apelin-13 promoted the expression of glucose transporter type 1 (GLUT1), pyruvate kinase 2 (PKM2), lactate dehydrogenase A (LDHA), monocarboxylate transporter 1 (MCT1), and monocarboxylate transporter 4 (MCT4) in a dose- and time-dependent manner. Moreover, apelin-13 increased the extracellular, intracellular lactate level, and decreased adenosine triphosphate level in HA-VSMCs. Furthermore, siRNA-PKM2 reversed extracellular and intracellular lactate generation and inhibited the proliferation of HA-VSMCs induced by apelin-13. Downregulation of LDHA also significantly prevented extracellular and intracellular lactate generation and inhibited the proliferation of HA-VSMCs induced by apelin-13. Taken together, our results demonstrated a novel mechanism for HA-VSMCs proliferation induced by apelin-13 via Warburg effect.

Epigallocatechin-3-gallate regulates mitofusin 2 expression through the peroxisome proliferator-activated receptor-γ coactivator-1α and estrogen-related receptor-α pathway.

Our previous study showed that epigallocatechin-3-gallate (EGCG) inhibition of human aortic smooth muscle cell (HASMC) proliferation might be mediated via upregulation of mitofusin 2 (Mfn-2). Studies on the mechanism of Mfn-2 inhibition of cell proliferation have mainly focused on downstream signaling. However, it is still not clear how upstream signaling molecules regulate Mfn-2. The promoter region of the Mfn-2 gene contains cis-acting elements of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and estrogen-related receptor-α (ERR-α), suggesting a possible link between EGCG, Mfn-2, and PGC-1α/ERR-α. However, the effect of EGCG on PGC-1α/ERR-α remains unknown. In this study, we investigated the role of PGC-1α/ERR-α in the regulation of Mfn-2 induced by EGCG and assessed the underlying mechanisms. The effects of EGCG on cell proliferation of cultured HASMCs were observed by a cell counting kit-8 (CCK8) and 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. Mfn-2, PGC-1α, and ERR-α gene and protein levels were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis. PGC-1α gene-silencing (PGC-1α small interfering RNA [siRNA]) was achieved by RNA interference and Mfn-2 promoter and peroxisome proliferator response element (PPRE) functional activity was achieved by a luciferase transfection assay. The results showed that the ERR-α-specific antagonist XCT-790 and PGC-1α siRNA decreased the expression of Mfn-2, thus antagonizing the inhibition of HASMC proliferation induced by EGCG. EGCG enhanced Mfn-2 promoter (-352 to 459) activity, while XCT-790 and PGC-1α siRNA abrogated this effect. PGC-1α stimulating Mfn-2 expression was dependent on intact ERR-α binding in the Mfn-2 promoter. The transcriptional effect of PGC-1α on the Mfn-2 promoter required the integrity of the -432 to 459 region and supported that Mfn-2 was a key target gene of PGC-1α. These results imply that PGC-1α/ERR-α played important physiological roles in inhibiting the proliferation of HASMCs by modulating Mfn-2 gene expression. Hence, EGCG regulated Mfn-2 expression likely through the PGC-1α/ERR-α pathway.

SFRP5 serves a beneficial role in arterial aging by inhibiting the proliferation, migration and inflammation of smooth muscle cells

Secreted frizzled-related protein5(SFRP5) is one of the anti-inflammatory adipokines secreted from white adipose tissue. However, little is known about the effect of SFRP5 on the cardiovascular system. The aim of the present study was to determine the effect of SFRP5 on smooth muscle cell (SMC) proliferation, migration and inflammation. The plasma levels of SFRP5 were evaluated in a cohort‑based elderly population using ELISA, and the expression of SFRP5 in Sprague‑Dawley rat aortas was detected using immunohistochemistry. SMC proliferation and migration were evaluated invitro using 5‑ethynyl‑2'‑deoxyuridine cell proliferation and wound‑healing assays, respectively, while reactive oxygen species (ROS) production and cell signaling were assessed using a 2',7'‑dichlorodihydrofluorescein diacetate assay and immunoblotting, respectively. The results revealed that plasma levels of SFRP5 were positively correlated with age in the elderly Chinese cohort. Similarly, aorta SFRP5 expression was significantly higher in 15‑month‑old rats compared with 6‑month‑old rats. Invitro, SFRP5 significantly inhibited rat aortic SMC proliferation and migration that were induced by platelet‑derived growth factor (PDGF)‑BB, as well as inhibiting ROS generation. Compared with the effect of PDGF‑BB on SMCs, SFRP5 at 100 and 200ng/ml significantly decreased SMC proliferation by 31.5 and 34.8%, respectively (P<0.05). SFRP5 at 100 and 200ng/ml also inhibited the migration of SMCs by 24.9 and 28.4%, respectively, when compared with the effects of PDGF‑BB. SFRP5 attenuated the PDGF‑BB‑induced expression of β‑catenin and proliferating cell nuclear antigen, while p38 phosphorylation was significantly attenuated. Together, the present results suggested that SFRP5 may inhibit SMC proliferation, migration and inflammation by suppressing the Wnt/β‑catenin and p38/mitogen‑activated protein kinase signaling pathways.

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&lt;0.05) and migration (~1.8‐fold, P&lt;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&lt;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&lt;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.

The CCL5/CCR5 Axis Promotes Vascular Smooth Muscle Cell Proliferation and Atherogenic Phenotype Switching

Background/Aims: Hyperlipidemia induces dysfunction in the smooth muscle cells (SMCs) of the blood vessels, and the vascular remodeling that ensues is a key proatherogenic factor contributing to cardiovascular events. Chemokines and chemokine receptors play crucial roles in vascular remodeling. Here, we examined whether the hyperlipidemia-derived chemokine CCL5 and its receptor CCR5 influence vascular SMC proliferation, phenotypic switching, and explored the underlying mechanisms. Methods: Thoracoabdominal aorta were isolated from wild-type, CCL5 and CCR5 double-knockout mice (CCL5^–/–CCR5^–/–) fed a high-fat diet (HFD) for 12 weeks. Expression of the contractile, synthetic, and proliferation markers were assayed using immunohistochemical and western blotting. The effects of CCL5 and palmitic acid on cultured SMC proliferation and phenotypic modulation were evaluated using flow cytometry, bromodeoxyuridine (BrdU), and western blotting. Results: Wild-type mice fed an HFD showed markedly increased total cholesterol, triglyceride, and CCL5 serum levels, as well as significantly increased CCL5 and CCR5 expression in the thoracoabdominal aorta vs. normal-diet-fed controls. HFD-fed CCL5^-/-CCR5^-/- mice showed significantly decreased expression of the synthetic phenotype marker osteopontin and the proliferation marker proliferating cell nuclear antigen, and increased expression of the contractile phenotype marker smooth muscle α-actin in the thoracoabdominal aorta vs. wild-type HFD-fed mice. Human aorta-derived SMCs stimulated with palmitic acid showed significantly increased expression of CCL5, CCR5, and synthetic phenotype markers, as well as increased proliferation. CCL5-treated SMCs showed increased cell cycle regulatory protein expression, paralleling increased synthetic and decreased contractile phenotype marker expression. Inhibition of CCR5 activity by the specific antagonist maraviroc or its expression using small interfering RNA significantly inhibited human aortic SMC proliferation and synthetic phenotype formation. Therefore, CCL5 induces SMC proliferation and phenotypic switching from a contractile to synthetic phenotype via CCR5. CCL5-mediated SMC stimulation activated ERK1/2, Akt/p70S6K, p38 MAPK, and NF-κB signaling. NF-κB inhibition significantly reduced CCR5 expression along with CCR5-induced SMC proliferation and synthetic phenotype formation. Conclusions: Hyperlipidemia-induced CCL5/CCR5 axis activation serves as a pivotal mediator of vascular remodeling, indicating that CCL5 and CCR5 are key chemokine-related factors in atherogenesis. SMC proliferation and synthetic phenotype transformation attenuation by CCR5 pharmacological inhibition may offer a new approach to treatment or prevention of atherosclerotic diseases associated with hyperlipidemia.

Reduction of In-Stent Restenosis by Cholesteryl Ester Transfer Protein Inhibition.

Angioplasty and stent implantation, the most common treatment for atherosclerotic lesions, have a significant failure rate because of restenosis. This study asks whether increasing plasma high-density lipoprotein (HDL) levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, prevents stent-induced neointimal hyperplasia. New Zealand White rabbits received normal chow or chow supplemented with 0.14% (wt/wt) des-fluoro-anacetrapib for 6 weeks. Iliac artery endothelial denudation and bare metal steel stent deployment were performed after 2 weeks of des-fluoro-anacetrapib treatment. The animals were euthanized 4 weeks poststent deployment. Relative to control, dietary supplementation with des-fluoro-anacetrapib reduced plasma cholesteryl ester transfer protein activity and increased plasma apolipoprotein A-I and HDL cholesterol levels by 53±6.3% and 120±19%, respectively. Non-HDL cholesterol levels were unaffected. Des-fluoro-anacetrapib treatment reduced the intimal area of the stented arteries by 43±5.6% (P<0.001), the media area was unchanged, and the arterial lumen area increased by 12±2.4% (P<0.05). Des-fluoro-anacetrapib treatment inhibited vascular smooth muscle cell proliferation by 41±4.5% (P<0.001). Incubation of isolated HDLs from des-fluoro-anacetrapib-treated animals with human aortic smooth muscle cells at apolipoprotein A-I concentrations comparable to their plasma levels inhibited cell proliferation and migration. These effects were dependent on scavenger receptor-B1, the adaptor protein PDZ domain-containing protein 1, and phosphatidylinositol-3-kinase/Akt activation. HDLs from des-fluoro-anacetrapib-treated animals also inhibited proinflammatory cytokine-induced human aortic smooth muscle cell proliferation and stent-induced vascular inflammation. Inhibiting cholesteryl ester transfer protein activity in New Zealand White rabbits with iliac artery balloon injury and stent deployment increases HDL levels, inhibits vascular smooth muscle cell proliferation, and reduces neointimal hyperplasia in an scavenger receptor-B1, PDZ domain-containing protein 1- and phosphatidylinositol-3-kinase/Akt-dependent manner.

Uncoupling Protein 2 Inhibits Myointimal Hyperplasia in Preclinical Animal Models of Vascular Injury.

BackgroundIntracoronary stent restenosis, characterized by excessive smooth muscle cell (SMC) proliferation and myointimal hyperplasia, remains a clinical challenge. Mitochondrial membrane potential has been linked to the proliferative rate of SMCs. This study aimed to screen a critical gene regulating mitochondrial potential and to confirm its effects on myointimal formation in preclinical animal models.Methods and ResultsWe performed transcriptome screening for genes differentially expressed in ligated versus unligated mouse carotid arteries. We observed that uncoupling protein 2 gene (Ucp2) mRNA, encoding UCP2, was transiently upregulated during the first 3 days after ligation and then significantly downregulated from day 7 through day 21, during which time neointima formed remarkably. The UCP2 protein level also declined after day 7 of ligation. In ligated carotid arteries, Ucp2 −/− mice, compared with wild‐type littermates, exhibited accelerated myointimal formation, which was associated with increased superoxide production and can be attenuated by treatment with antioxidant 4‐hydroxy‐2,2,6,6‐tetramethyl‐piperidinoxyl (TEMPOL). Knockdown of UCP2 enhanced human aortic SMC migration and proliferation that can also be attenuated by TEMPOL, whereas UCP2 overexpression inhibited SMC migration and proliferation, along with decreased activity of nuclear factor‐κB. Moreover, nuclear factor‐κB inhibitor attenuated UCP2 knockdown‐enhanced SMC proliferation. Adenovirus‐mediated overexpression of UCP2 inhibited myointimal formation in balloon‐injured carotid arteries of rats and rabbits and in‐stent stenosis of porcine coronary arteries. Moreover, UCP2 overexpression also suppressed neointimal hyperplasia in cultured human saphenous vein ex vivo.ConclusionsUCP2 inhibits myointimal hyperplasia after vascular injury, probably through suppressing nuclear factor‐κB–dependent SMC proliferation and migration, rendering UCP2 a potential therapeutic target against restenosis.

BP.06.01] THE EFFECTS OF FEBUXOSTAT ON ANGIOTENSIN II-INDUCED VASCULAR REMODELING

Objective: A xanthine oxidase (XO) inhibitor, febuxostat (FEB) is often used in patients with hyperuricemia. Several reports have suggested that uric acid contributes to endothelial dysfunction and kidney injury. Because XO produces hydrogen peroxide with uric acid in the nucleic acid metabolism, XO inhibitors can also have the anti-oxidative properties. From these reasons, XO inhibitors have the possibilities to suppress cardiovascular diseases. Actually, the effects of FEB on cardiovascular events have been examined in several clinical trials in Japan and several basic researches have suggested that FEB suppressed cardiac fibrosis and renal inflammation, however, those mechanisms remain unclear. In this study, we investigated the effects of FEB on angiotensin II (Ang II)-induced vascular remodeling. Design and method: To induce vascular remodeling, Ang II (2 mg/kg/day, s.c.) was infused by osmotic minipump into the mice. FEB was suspended in 1% carboxymethyl cellulose and administrated daily for 14 days. The mice were divided into four groups: control, FEB alone, Ang II alone, and Ang II+FEB. Medial thickening and vascular fibrosis were assessed by Elastica van Gieson stain. Blood pressure was measured by tail-cuff method. The macrophage infiltration and XO localization in the aortic tissue were assessed by immunofluorescence. In our in vitro studies, protein phosphorylation and cell proliferation were measured by western blotting and MTT assay, respectively. Results: Serum XO activity was lower in the Ang II+FEB group than in the Ang II alone group. FEB administration suppressed Ang II-induced blood pressure elevation and aortic fibrosis, however it did not affect medial thickening of the aortae. In our in vitro studies, FEB did not change the aortic smooth muscle cell proliferation and ERK1/2 phosphorylation induced by Ang II stimulation. We found that XO was expressed strongly in the macrophages, and Ang II-induced macrophage infiltration in the aortae tended to be suppressed by FEB. Conclusions: Our results suggested that FEB affects the mechanisms of fibrosis in vascular cells more than those of smooth muscle cell proliferation induced by Ang II. We supposed that FEB suppresses macrophage-derived XO activity in the mouse model of Ang II-induced vascular remodeling.

FUNDC1 mediated mitophagy alleviates rat aortic smooth muscle cell proliferation, migration induced by angiotensin II

Aim: VSMC hyperplasia caused by abnormal proliferation and migration is a major contributor to the development of atherosclerosis, and oxidative stress is a major mechanism of VSMC hyperplasia. Mitophagy is a critical cellular process that selectively targets damaged mitochondria for autophagosomal degradation in response to stress preventing oxidative damage. However, the role of mitophagy in VSMC in response to oxidative stress is unknown. The present study is designed to determine the role of FUNDC1 meditated mitophgy on VSMCs proliferation, migration as well as their underlying mechanisms.

Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the cardiovascular risk factor obesity may have relevant therapeutic implications.

MicroRNA-124 controls human vascular smooth muscle cell phenotypic switch via Sp1.

Phenotypic switch of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of atherosclerosis and aortic dissection. However, the mechanisms of phenotypic modulation are still unclear. MicroRNAs have emerged as important regulators of VSMC function. We recently found that microRNA-124 (miR-124) was downregulated in proliferative vascular diseases that were characterized by a VSMC phenotypic switch. Therefore, we speculated that the aberrant expression of miR-124 might play a critical role in human aortic VSMC phenotypic switch. Using quantitative RT-PCR, we found that miR-124 was dramatically downregulated in the aortic media of clinical specimens of the dissected aorta and correlated with molecular markers of the contractile VSMC phenotype. Overexpression of miR-124 by mimicking transfection significantly attenuated platelet-derived growth factor-BB-induced human aortic VSMC proliferation and phenotypic switch. Furthermore, we identified specificity protein 1 (Sp1) as the downstream target of miR-124. A luciferase reporter assay was used to confirm direct miR-124 targeting of the 3'-untranslated region of the Sp1 gene and repression of Sp1 expression in human aortic VSMCs. Furthermore, constitutively active Sp1 in miR-124-overexpressing VSMCs reversed the antiproliferative effects of miR-124. These results demonstrated a novel mechanism of miR-124 modulation of VSMC phenotypic switch by targeting Sp1 expression.NEW & NOTEWORTHY Previous studies have demonstrated that miR-124 is involved in the proliferation of a variety of cell types. However, miRNAs are expressed in a tissue-specific manner. We first identified miR-124 as a critical regulator in human aortic vascular smooth muscle cell differentiation, proliferation, and phenotype switch by targeting the 3'-untranslated region of specificity protein 1.

MiR-503 inhibits platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration through targeting the insulin receptor.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) is a common feature of disease progression in atherosclerosis. Here, we investigated the potential role of miR-503 in platelet-derived growth factor (PDGF)-induced proliferation and migration of human aortic smooth muscle cells and the underlying mechanisms of action. miR-503 expression was significantly downregulated in a dose- and time-dependent manner following PDGF treatment. Introduction of miR-503 mimics into cultured SMCs significantly attenuated cell proliferation and migration induced by PDGF. Bioinformatics analyses revealed that the insulin receptor (INSR) is a target candidate of miR-503. miR-503 suppressed luciferase activity driven by a vector containing the 3'-untranslated region of INSR in a sequence-specific manner. Downregulation of INSR appeared critical for miR-503-mediated inhibitory effects on PDGF-induced cell proliferation and migration in human aortic SMCs. Based on the collective data, we suggest a novel role of miR-503 as a regulator of VSMC proliferation and migration through modulating INSR.