Migration of vascular smooth muscle cells (VSMCs) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. The lipoxygenase products of polyunsaturated fatty acids (PUFA) were shown to play a role in these diseases. Cyclic AMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. The aim of the present study was to test the role of CREB in an oxidized lipid molecule, 15(S)-HETE-induced VSMC migration and neointima formation.
Methods and Results
15(S)-HETE stimulated VSMC migration in CREB-dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1 or p38MAPK inhibited 15(S)-HETE-induced CREB phosphorylation and VSMC migration. 15(S)-HETE induced expression and secretion of interleukin-6 (IL-6), as analyzed by RT-PCR and ELISA, respectively. Neutralizing anti-IL-6 antibodies blocked 15(S)-HETE-induced VSMC migration. Dominant-negative mutant-mediated blockade of ERK1/2, JNK1, p38MAPK or CREB suppressed 15(S)-HETE-induced IL-6 expression in VSMCs. Serial 5′ deletions and site-directed mutagenesis of IL-6 promoter along with chromatin immunoprecipitation using anti-CREB antibodies showed that cAMP response element is essential for 15(S)-HETE-induced IL-6 expression. Dominant-negative CREB also suppressed balloon injury-induced IL-6 expression, SMC migration from media to intimal region and neointima formation. Adenovirus-mediated transduction of 15-lipoxygenase 2 (15-LOX2) caused increased production of 15-HETE in VSMCs and enhanced IL-6 expression, SMC migration from media to intimal region and neointima formation in response to arterial injury.
The above results suggest a role for 15-LOX2-15-HETE in the regulation of VSMC migration and neointima formation involving CREB-mediated IL-6 expression.
Periostin is dramatically upregulated in rat carotid arteries after balloon injury. The objective of the present study was to understand mechanisms underlying periostin upregulation in balloon injured rat carotid arteries and in cultured vascular smooth muscle cells (VSMCs).
Methods and Results
Periostin protein was strongly expressed at 3d (in the medial SMCs) and 7d (in the neointima) after injury. It was also abundantly expressed in the neointima in the late phase (at 14d and 28d) after injury. Periostin upregulation was mediated through PI3-kinase-dependent signaling pathway. In vivo, wortmannin, a PI-3-kinase inhibitor, inhibited balloon injury-induced Akt phosphorylation and periostin mRNA expression. In vitro, periostin mRNA expression in cultured VSMCs was stimulated by growth factors (TGF-β1, FGFs, PDGF-BB, and angiotensin II). This stimulatory effect was inhibited by the PI3-kinase inhibitor LY294002. Further, periostin protein was mostly located in the cytoplasma of VSMCs in culture and abundantly secreted into the culture medium (CM) after stimulation with FGF-2, which significantly promoted VSMC migration in vitro. Immunodepletion of periostin from the VSMC-CM or blockade of periostin function with an anti-periostin antibody significantly reduced VSMC migration.
Upregulation of periostin expression in rat carotid arteries following balloon injury and in cultured VSMCs after stimulation by growth factors is mediated through PI-3 kinase-dependent signaling pathway. Periostin protein secreted by VSMCs plays a significant role in regulating VSMC migration in vitro.
periostin; PI-3 kinase; smooth muscle; migration
3, 3′diindolylmethane (DIM), a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs) remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.
DIM dose-dependently inhibited the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4/6 as well as an increase in p27Kip1 levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptorβ (PDGF-Rβ) and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK)3β, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3). Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.
These results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-Rβ and the activities of downstream signaling pathways. The results suggest that DIM has the potential to be a candidate for the prevention of restenosis.
Dihydroaustrasulfone alcohol (DA), an active compound firstly isolated from marine corals, has been reported to reveal anti-cancer and anti-inflammation activities. These reported activities of DA raised a possible application in anti-restenosis. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) and the stimulation of platelet-derived growth factor (PDGF)-BB play major pathological processes involved in the development of restenosis. Experimental results showed that DA markedly reduced balloon injury-induced neointima formation in the rat carotid artery model and significantly inhibited PDGF-BB-stimulated proliferation and migration of VSMCs. Our data further demonstrated that translational and active levels of several critical signaling cascades involved in VSMC proliferation, such as extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, and signal transducer and activator of transcription (STAT), were obviously inhibited. In addition, DA also decreased the activation and expression levels of gelatinases (matrix metalloproteinase (MMP)-2 and MMP-9) involved in cell migration. In conclusion, our findings indicate that DA can reduce balloon injury-neointimal hyperplasia, the effect of which may be modulated through suppression of VSMC proliferation and migration. These results suggest that DA has potential application as an anti-restenotic agent for the prevention of restenosis.
dihydroaustrasulfone alcohol; anti-restenosis; neointimal hyperplasia; marine origin
The proliferation and migration of vascular smooth muscle cells (VSMCs) contributes importantly to the development of in-stent restenosis. Lithium has recently been shown to have beneficial effects on the cardiovascular system, but its actions in VSMCs and the direct molecular target responsible for its action remains unknown. On the other hand, PGC-1α is a transcriptional coactivator which negatively regulates the pathological activation of VSMCs. Therefore, the purpose of the present study is to determine if lithium chloride (LiCl) retards VSMC proliferation and migration and if PGC-1α mediates the effects of lithium on VSMCs. We found that pretreatment of LiCl increased PGC-1α protein expression and nuclear translocation in a dose-dependent manner. MTT and EdU incorporation assays indicated that LiCl inhibited serum-induced VSMC proliferation. Similarly, deceleration of VSMC migration was confirmed by wound healing and transwell assays. LiCl also suppressed ROS generation and cell cycle progression. At the molecular level, LiCl reduced the protein expression levels or phosphorylation of key regulators involved in the cell cycle re-entry, adhesion, inflammation and motility. In addition, in vivo administration of LiCl alleviated the pathophysiological changes in balloon injury-induced neointima hyperplasia. More importantly, knockdown of PGC-1α by siRNA significantly attenuated the beneficial effects of LiCl on VSMCs both in vitro and in vivo. Taken together, our results suggest that LiCl has great potentials in the prevention and treatment of cardiovascular diseases related to VSMC abnormal proliferation and migration. In addition, PGC-1α may serve as a promising drug target to regulate cardiovascular physiological homeostasis.
Dihydroaustrasulfone alcohol is the synthetic precursor of austrasulfone, which is a marine natural product, isolated from the Taiwanese soft coral Cladiella australis. Dihydroaustrasulfone alcohol has anti-inflammatory, neuroprotective, antitumor and anti-atherogenic properties. Although dihydroaustrasulfone alcohol has been shown to inhibit neointima formation, its effect on human vascular smooth muscle cells (VSMCs) has not been elucidated. We examined the effects and the mechanisms of action of dihydroaustrasulfone alcohol on proliferation, migration and phenotypic modulation of human aortic smooth muscle cells (HASMCs). Dihydroaustrasulfone alcohol significantly inhibited proliferation, DNA synthesis and migration of HASMCs, without inducing cell death. Dihydroaustrasulfone alcohol also inhibited platelet-derived growth factor (PDGF)-induced expression of cyclin-dependent kinases (CDK) 2, CDK4, cyclin D1 and cyclin E. In addition, dihydroaustrasulfone alcohol inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it had no effect on the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/(Akt). Moreover, treatment with PD98059, a highly selective ERK inhibitor, blocked PDGF-induced upregulation of cyclin D1 and cyclin E and downregulation of p27kip1. Furthermore, dihydroaustrasulfone alcohol also inhibits VSMC synthetic phenotype formation induced by PDGF. For in vivo studies, dihydroaustrasulfone alcohol decreased smooth muscle cell proliferation in a rat model of restenosis induced by balloon injury. Immunohistochemical staining showed that dihydroaustrasulfone alcohol noticeably decreased the expression of proliferating cell nuclear antigen (PCNA) and altered VSMC phenotype from a synthetic to contractile state. Our findings provide important insights into the mechanisms underlying the vasoprotective actions of dihydroaustrasulfone alcohol and suggest that it may be a useful therapeutic agent for the treatment of vascular occlusive disease.
dihydroaustrasulfone alcohol; vascular smooth muscle cell; proliferation; migration; phenotypic modulation; cell cycle; restenosis
Vascular smooth muscle cell (VSMC) migration and proliferation are the hallmarks of restenosis pathogenesis after angioplasty. Cyclooxygenase (COX)-derived prostaglandin (PG)E2 is implicated in the vascular remodeling response to injury. However, its precise molecular role remains unknown.
This study investigates the impact of COX-2-derived PGE2 on neointima formation after injury.
Methods and Results
Vascular remodeling was induced by wire-injury in femoral arteries of mice. Both neointima formation and the restenosis ratio were diminished in COX-2 KO mice as compared to controls, whereas these parameters were enhanced in COX-1>COX-2 mice where COX-1 is governed by COX-2 regulatory elements. PG profile analysis revealed that the reduced PGE2 by COX-2 deficiency, but not PGI2, could be rescued by COX-1 replacement, indicating COX-2-derived PGE2 enhanced neointima formation. Through multiple approaches, the EP3 receptor was identified to mediate the VSMC migration response to various stimuli. Disruption of EP3 impaired VSMC polarity for directional migration by depressing small GTPase activity and retarded vascular neointimal hyperplasia while overexpression of EP3α and EP3β aggravated neointima formation. Inhibition or deletion of EP3α/β, a Gαs protein-coupled receptor, activated thecAMP/PKA pathway and depressed activation of RhoA in VSMCs. PGE2 could stimulate PI3K/Akt/GSK3β signaling in VSMCs through Gβγ subunits upon EP3α/β activation. Abolition of EP3 suppressed PI3K signaling and reduced GTPase activity in VSMCs, and altered cell polarity and directional migration.
COX-2-derived PGE2 facilitated the neointimal hyperplasia response to injury through EP3α/β-mediated cAMP/PKA and PI3K pathways, indicating EP3 inhibition maybe a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.
neointima formation; PGE2; EP3; VSMC migration; polarity
Superoxide anion is elevated during neointima development and is essential for neointimal vascular smooth muscle cell (VSMC) proliferation. However, little is known about the role of manganese superoxide dismutase (MnSOD, SOD2) in the neointima formation following vascular injury. SOD2 in the mitochondria plays an important role in cellular defense against oxidative damage. Because of its subcellular localization, SOD2 is considered the first line of defense against oxidative stress and plays a central role in metabolizing superoxide. Because mitochondria are the most important sources of superoxide anion, we speculated that SOD2 may have therapeutic benefits in preventing vascular remodeling. In this study, we used a rat carotid artery balloon-injury model and an adenoviral gene delivery approach to test the hypothesis that SOD2 suppresses vascular lesion formation. SOD2 was activated along with the progression of neointima formation in balloon-injured rat carotid arteries. Depletion of SOD2 by RNA interference markedly promoted the lesion formation, whereas SOD2 overexpression suppressed the injury-induced neointima formation via attenuation of migration and proliferation of VSMCs. SOD2 exerts its inhibitory effect on VSMC migration induced by angiotensin II by scavenging superoxide anion and suppressing the phosphorylation of Akt. Our data indicate that SOD2 is a negative modulator of vascular lesion formation after injury. Therefore, SOD2 augmentation may be a promising therapeutic strategy for the prevention of lesion formation in proliferative vascular diseases such as restenosis.
Manganese superoxide dismutase; Oxidative stress; Neointima; Migration; Proliferation; Vascular smooth muscle cells; Signal transduction; Free radicals
Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyperplasia in vivo. Troglitazone (1 microM), a member of the thiazolidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bromodeoxyuridine incorporation (6.5+/-3.9 vs. 17.6+/-4.3% cells labeled, P < 0.05) and c-fos induction. This effect was associated with an inhibition (by 73+/-4%, P < 0.01) by troglitazone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70+/-6%, P < 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglitazone-treated rats had 62% (P < 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC proliferation and migration and, thus, may be a useful agent to prevent restenosis and possibly atherosclerosis.
Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.
Methods and Results
Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.
This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
This manuscript describes our latest work investigating the role of the Skp2, an F-box protein component of the SCFskp2 ubiquitin-ligase, in promoting VSMC proliferation, and neointima formation in response to vascular injury in vivo. Our previous work has identified a major role for Skp2 as a key target for numerous positive and negative growth regulatory signals in vitro. These signals converge to regulate the expression of Skp2, which then controls cell-cycle progression by promoting degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Until now, there has been no data in the literature on the role played by Skp2 in the regulation of VSMC proliferation and neointima formation in vivo. Our current manuscript describes, we believe for the first time, the important role played by Skp2 in these processes, using both mouse and rat arterial injury models. This is important because proliferation of VSMCs underlies the development of postangioplasty or post-stenting restenosis, venous graft failure, and transplant arteriosclerosis. Our work demonstrates for the first time that Skp2 is a major regulator of VSMC proliferation and neointimal thickening in vivo in response to vascular injury and highlights Skp2 as a potential target for future strategies designed to combat vasculoproliferative diseases.
Background and Purpose
Neointima is considered a critical event in the development of vascular occlusive disease. Nectandrin B from nutmeg functions as a potent AMP-activated protein kinase (AMPK) activators. The present study addressed whether nectandrin B inhibits intimal hyperplasia in guide wire-injured arteries and examined its molecular mechanism.
Neointima was induced by guide wire injury in mouse femoral arteries. Cell proliferation and mechanism studies were performed in rat vascular smooth muscle cells (VSMC) culture model.
Nectandrin B increased AMPK activity in VSMC. Nectandrin B inhibited the cell proliferation induced by PDGF and DNA synthesis. Moreover, treatment of nectandrin B suppressed neointima formation in femoral artery after guide wire injury. We have recently shown that Pin1 plays a critical role in VSMC proliferation and neointima formation. Nectandrin B potently blocked PDGF-induced Pin1 and cyclin D1 expression and nectandrin B‘s anti-proliferation effect was diminished in Pin1 overexpressed VSMC. PDGF-induced phosphorylation of ERK and Akt was marginally affected by nectandrin B. However, nectandrin B increased the levels of p53 and its downstream target p21 and, also reversibly decreased the expression of E2F1 and phosphorylated Rb in PDGF-treated VSMC. AMPK inhibition by dominant mutant form of adenovirus rescued nectandrin B-mediated down-regulation of Pin1 and E2F1.
Conclusions and Implications
Nectandrin B inhibited VSMC proliferation and neointima formation via inhibition of E2F1-dependent Pin1 gene transcription, which is mediated through the activation of an AMPK/p53-triggered pathway.
AMPK; nectandrin B; neointima; Pin1
To understand the effects of lithospermic acid (LA), a potent antioxidant from the water-soluble extract of Salvia miltiorrhiza, on the migration and proliferation of rat thoracic aorta vascular smooth muscle cells (VSMCs).
VSMC migration, proliferation, DNA synthesis and cell cycle progression were investigated by transwell migration analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) incorporation assay, and flow cytometric detection, respectively. Intracellular reactive oxygen species (ROS) generation was detected using 2′,7′-dichlorofluorescin diacetate (DCFH-DA). The expression of cyclin D1 protein and matrix metalloproteinase-9 (MMP-9) protein, as well as the phosphorylation state of ERK1/2, were determined using Western blots. The activity of MMP-9 and the expression of MMP-9 mRNA were assessed by gelatin zymography analysis and RT-PCR, respectively.
LA (25−100 μmol/L) inhibited both lipopolysaccharide (LPS)- and fetal bovine serum (FBS)-induced ROS generation and ERK1/2 phosphorylation. By down-regulating the expression of cyclin D1 and arresting cell cycle progression at the G1 phase, LA inhibited both VSMC proliferation and DNA synthesis as induced by 5% FBS. Furthermore, LA attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression and its enzymatic activity.
LA is able to inhibit FBS-induced VSMC proliferation and LPS-induced VSMC migration, which suggests that LA may have therapeutic effects in the prevention of atherosclerosis, restenosis and neointimal hyperplasia.
lithospermic acid; ERK1/2; matrix metalloproteinase-9; vascular smooth muscle cells; proliferation; migration
BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. In this study, we tested the hypothesis that localized arterial infection at the time of balloon angioplasty with an adenovirus (ADV-tk) encoding the herpes simplex virus thymidine kinase gene (HSV-tk), followed by systemic ganciclovir administration, can inhibit VSMC proliferation and neointima formation in a well-characterized model of arterial injury and restenosis. MATERIALS AND METHODS: The left carotid arteries of 31 male Sprague-Dawley rats were subjected to balloon angioplasty and immediately infected with 2 x 10(9) pfu of either ADV-tk or a control adenovirus that does not encode a recombinant protein (ADV-delta E1). Twenty-four hours after injury, animals from each experimental group were randomized to receive a course of systemic ganciclovir (ADV-tk/+GC, ADV delta E1/+GC) or saline (ADV-tk/-GC, ADV-delta E1/-GC). VSMC DNA synthesis was measured by 5'-bromodeoxuridine (BrdU) incorporation 2-4 days after balloon injury. The extent of restenosis, expressed as the neointima to media (I/M) area ratio was determined by digital planimetry 20 days after balloon injury in each of the four treatment groups. Immunohistochemistry using a mAb to von Willebrand factor (vWF) was used to determine the effects of ADV-tk infection and ganciclovir treatment on re-endothelialization of the carotid arteries 20 days following balloon angioplasty. RESULTS: Forty-one percent of the medial VSMCs in the ADV-tk/-GC arteries were labeled with BrdU 4 days after balloon injury. In contrast, ADV-tk infected animals that were treated with systemic ganciclovir (ADV-tk/+GC) displayed a 40% reduction in BrdU-staining medial VSMCs (p < 0.03). I/M area ratios of the three control groups were 1.17 +/- 0.18 (ADV-tk/-GC, n = 5), 1.15 +/- 0.10 (ADV-delta E1/+GC, n = 6), and 0.91 +/- 0.08 (ADV-delta E1/-GC, n = 6). These differences were not statistically significant (p > 0.05). In contrast, the ADV-tk/+GC animals (n = 6) displayed an I/M area ratio of 0.49 +/- 0.13 which was significantly lower than that seen in each of the three control groups (p < 0.02). None of the treated animals showed evidence of significant organ toxicity at autopsy. A regenerated endothelium was observed in the ADV-tk/+GC animals 20 days after balloon injury. CONCLUSIONS: Localized arterial infection with ADV-tk at the time of balloon angioplasty followed by systemic ganciclovir therapy reduces VSMC proliferation and neointimal expansion in the rat carotid artery injury model. Moreover, combined treatment with ADV-tk and systemic ganciclovir does not result in systemic toxicity and appears to selectively eliminate proliferating VSMCs, while preserving the capacity of the injured arterial segments to re-endothelialize within 3 weeks of injury. Taken together, these results support the feasibility of using this gene therapy approach for the treatment of human vascular proliferative disorders.
Bindarit is an original compound with peculiar anti-inflammatory activity due to a selective inhibition of a subfamily of inflammatory chemokines, including the monocyte chemotactic proteins MCP-1/CCL2, MCP-3/CCL7, and MCP-2/CCL8. In this study, we investigated the effect of bindarit on neointima formation using two animal models of arterial injury: rat carotid artery balloon angioplasty and wire-induced carotid injury in apolipoprotein E-deficient (apoE−/−) mice.
Methods and results
Treatment of rats with bindarit (200 mg/kg/day) significantly reduced balloon injury-induced neointima formation by 39% at day 14 without affecting re-endothelialization and reduced the number of medial and neointimal proliferating cells at day 7 by 54 and 30%, respectively. These effects were associated with a significant reduction of MCP-1 levels both in sera and in injured carotid arteries of rats treated with bindarit. In addition, in vitro data showed that bindarit (10–300 µM) reduced rat vascular smooth muscle cell (VSMC) proliferation, migration, and invasion, processes contributing to the injury-induced neointima formation in vivo. Similar results were observed in hypercholesterolaemic apoE−/− mice in which bindarit administration resulted in a 42% reduction of the number of proliferating cells at day 7 after carotid injury and in a 47% inhibition of neointima formation at day 28. Analysis of the cellular composition in neointimal lesions of apoE−/− mice treated with bindarit showed that the relative content of macrophages and the number of VSMCs were reduced by 66 and 30%, respectively, compared with the control group.
This study demonstrates that bindarit is effective in reducing neointima formation in both non-hyperlipidaemic and hyperlipidaemic animal models of vascular injury by a direct effect on VSMC proliferation and migration and by reducing neointimal macrophage content. All of these data were associated with the inhibition of MCP-1 production.
Bindarit; Neointima hyperplasia; Monocyte chemoattractant protein-1; Macrophages; Vascular smooth muscle cells
We tested the hypothesis whether selective blunting of platelet-derived growth factor (PDGF)–dependent vascular smooth muscle cell (VSMC) proliferation and migration is sufficient to prevent neointima formation after vascular injury.
To prevent neointima formation and stent thrombosis after coronary interventions, it is essential to inhibit VSMC proliferation and migration without harming endothelial cell function. The role of PDGF—a potent mitogen and chemoattractant for VSMC that does not affect endothelial cells—for neointima formation remains controversial.
To decipher the signaling pathways that control PDGF beta receptor (βPDGFR)–driven VSMC proliferation and migration, we characterized 2 panels of chimeric CSF1R/βPDGFR mutants in which the binding sites for βPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase [PI3K], GTPase activating protein of ras, SHP-2, phospholipase Cγ 1 [PLCγ]) were individually mutated. Based on in vitro results, the importance of PDGF-initiated signals for neointima formation was investigated in genetically modified mice.
Our results indicate that the chemotactic response to PDGF requires the activation of Src, PI3K, and PLCγ, whereas PDGF-dependent cell cycle progression is exclusively mediated by PI3K and PLCγ. These 2 signaling molecules contribute to signal relay of the βPDGFR by differentially regulating cyclin D1 and p27kip1. Blunting of βPDGFR-induced PI3K and PLCγ signaling by a combination mutant (F3) completely abolished the mitogenic and chemotactic response to PDGF. Disruption of PDGF-dependent PI3K and PLCγ signaling in mice expressing the F3 receptor led to a profound reduction of neointima formation after balloon injury.
Signaling by the activated βPDGFR, particularly through PI3K and PLCγ, is crucial for neointima formation after vascular injury. Disruption of these specific signaling pathways is sufficient to attenuate pathogenic processes such as vascular remodeling in vivo.
PI-3 kinase; PLCγ; platelet-derived growth factor; proliferation; restenosis
A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM.
Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did.
These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.
The molecular correlate of the calcium release-activated calcium current (ICRAC), the channel protein Orai1, is upregulated in proliferative vascular smooth muscle cells (VSMC). However, the role of Orai1 in vascular disease remains largely unknown.
The goal of this study was to determine the role of Orai1 in neointima formation after balloon-injury of rat carotid arteries and its potential upregulation in a mouse model of VSMC remodeling.
Methods and Results
Lentiviral particles encoding short-hairpin RNA (shRNA) targeting either Orai1 (shOrai1) or STIM1 (shSTIM1) caused knockdown of their respective target mRNA and proteins and abrogated store-operated calcium entry and ICRAC in VSMC; control shRNA was targeted to luciferase (shLuciferase). Balloon-injury of rat carotid arteries upregulated protein expression of Orai1, STIM1 and calcium-calmodulin kinase IIdelta2 (CamKIIδ2); increased proliferation assessed by Ki67 and PCNA and decreased protein expression of myosin heavy chain in medial and neointimal VSMC. Incubation of the injured vessel with shOrai1 prevented Orai1, STIM1 and CamKIIδ2 upregulation in the media and neointima; inhibited cell proliferation and markedly reduced neointima formation 14 days post injury; similar results were obtained with shSTIM1. VSMC Orai1 and STIM1 knockdown inhibited nuclear factor for activated T-cells (NFAT) nuclear translocation and activity. Furthermore, Orai1 and STIM1 were upregulated in mice carotid arteries subjected to ligation.
Orai1 is upregulated in VSMC during vascular injury and is required for NFAT activity, VSMC proliferation and neointima formation following balloon-injury of rat carotids. Orai1 provides a novel target for control of VSMC remodeling during vascular injury or disease.
Calcium channels; CRAC channels; vascular smooth muscle proliferation; neointima formation
We have previously demonstrated that TGF-β in the presence of elevated levels of its primary signaling protein, Smad3, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. Moreover, we have shown that the mechanism in part, is through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluate the role of the ERK mitogen-activated protein kinase (ERK MAPK) in TGF-β-induced VSMC proliferation.
Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with ansiRNA. For in vivo experiments, Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK and Proliferating Cell Nuclear Antigen (PCNA).
In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time and concentration-dependent manner. Overexpression of the signaling protein, Smad3 enhanced TGF-β-induced activation of ERK MAPK whereas inhibition of Smad3 with ansiRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by PCNA.
Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that through a Smad3/ERK MAPK signaling pathway, TGF-β enhances VSMC proliferation. These findings explain at least in part, the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.
intimal hyperplasia; transforming growth factor-beta (TGF-β); Smad3; ERK MAPK; vascular smooth muscle cell
Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the development of vascular diseases. In the present study, we tested the efficacy and the mechanisms of action of gastrodin, a bioactive component of the Chinese herb Gastrodia elata Bl, in relation to platelet-derived growth factor-BB (PDGF-BB)-dependent cell proliferation and neointima formation after acute vascular injury. Cell experiments were performed with VSMCs isolated from rat aortas. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Eight-week-old C57BL/6 mice were used for the animal experiments. Gastrodin (150 mg/kg/day) was administered in the animal chow for 14 days, and the mice were subjected to wire injury of the left carotid artery. Our data demonstrated that gastrodin attenuated the VSMC proliferation induced by PDGF-BB, as assessed by WST assay and BrdU incorporation. Gastrodin influenced the S-phase entry of VSMCs and stabilised p27Kip1 expression. In addition, pre-incubation with sinomenine prior to PDGF-BB stimulation led to increased smooth muscle-specific gene expression, thereby inhibiting VSMC dedifferentiation. Gastrodin treatment also reduced the intimal area and the number of PCNA-positive cells. Furthermore, PDGF-BB-induced phosphorylation of ERK1/2, p38 MAPK, Akt and GSK3β was suppressed by gastrodin. Our results suggest that gastrodin can inhibit VSMC proliferation and attenuate neointimal hyperplasia in response to vascular injury. Furthermore, the ERK1/2, p38 MAPK and Akt/GSK3β signalling pathways were found to be involved in the effects of gastrodin.
gastrodin; vascular smooth muscle cell; neointima formation; platelet-derived growth factor
In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon.
Methods & Results
In endothelium-denudated mouse carotid arteries, oral morelloflavone—an active ingredient of the Thai medicinal plant Garcinia dulcis—significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis.
These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest.
We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.
restenosis; morelloflavone; migration; Garcinia dulcis; vascular smooth muscle cells
In vascular smooth muscle cells (VSMC) Axl is a key receptor tyrosine kinase since it is up-regulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed glutathiolation; GSSG) is an important post-translational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species (ROS) increase Axl-dependent VSMC function we assayed for glutathiolated proteins that associated with Axl in a redox-dependent manner. We identified glutathiolated non-muscle myosin heavy chain (MHC)-IIB as a novel Axl interacting protein. This interaction was specific in that other myosins did not interact with Axl. The endogenous ligand for Axl, Gas6, increased production of ROS in VSMC and also increased association of Axl with MHC-IIB. Antioxidants ebselen and N-acetylcysteine decreased association of Axl with MHC-IIB in response to both Gas6 and ROS. Blocking the Axl-MHC-IIB interaction with the specific myosin II inhibitor blebbistatin decreased phosphorylation of Axl and activation of ERK1/2 and Akt. Association of MHC-IIB with Axl was increased in balloon injured rat carotid vessels. Finally, expression of MHC-IIB was upregulated in the neointima of the carotid artery following balloon injury similar to upregulation of Axl protein expression as shown in our previous studies. These results demonstrate a novel interaction between Axl and MHC-IIB in response to ROS. This interaction provides a direct link between Axl and molecular motors crucial for directed cell migration, which may mediate increased migration in vascular dysfunction.
vascular smooth muscle; receptor protein tyrosine kinase; myosin heavy chains; reactive oxygen species; vascular disease
The small molecule indirubin-3′-monoxime (I3MO) has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and neointima formation in vivo. The influence of I3MO on VSMC migration and vascular inflammation, two additional key players during the onset of atherosclerosis and restenosis, should be investigated.
Methods and results
We examined the influence of I3MO on VSMC migration, with focus on monocyte-derived leukotrienes (LTs) and platelet-derived growth factors (PDGFs) as elicitors. Exogenous LTB4 and cysteinyl leukotrienes as well as LT-enriched conditioned medium of activated primary human monocytes induced VSMC migration, which was inhibited by I3MO. I3MO also blunted migration of VSMC stimulated with the PDGF, the strongest motogen tested in this study. Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC. Notably, I3MO not only interfered with the migratory response in VSMC, but also suppressed the production of pro-migratory LT in monocytes. Conditioned media from monocytes that were activated in the presence of I3MO failed to induce VSMC migration. In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range.
Our study reveals a novel dual inhibitory mode of I3MO on LT-mediated VSMC migration: (i) I3MO interferes with pro-migratory signalling in VSMC and (ii) I3MO suppresses LT biosynthesis in monocytes by direct inhibition of 5-LO. These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.
Indirubin-3′-monoxime; Vascular smooth muscle cells; Migration; Leukotrienes; 5-Lipoxygenase
Interferon regulatory factor 7 (IRF7), a member of the interferon regulatory factor family, plays important roles in innate immunity and immune cell differentiation. However, the role of IRF7 in neointima formation is currently unknown.
Methods and Results
Significant decreases in IRF7 expression were observed in vascular smooth muscle cells (VSMCs) following carotid artery injury in vivo and platelet‐derived growth factor‐BB (PDGF‐BB) stimulation in vitro. Compared with non‐transgenic (NTG) controls, SMC‐specific IRF7 transgenic (IRF7‐TG) mice displayed reduced neointima formation and VSMC proliferation in response to carotid injury, whereas a global knockout of IRF7 (IRF7‐KO) resulted in the opposite effect. Notably, a novel IRF7‐KO rat strain was successfully generated and used to further confirm the effects of IRF7 deletion on the acceleration of intimal hyperplasia based on a balloon injury‐induced vascular lesion model. Mechanistically, IRF7's inhibition of carotid thickening and the expression of VSMC proliferation markers was dependent on the interaction of IRF7 with activating transcription factor 3 (ATF3) and its downstream target, proliferating cell nuclear antigen (PCNA). The evidence that IRF7/ATF3‐double‐TG (DTG) and IRF7/ATF3‐double‐KO (DKO) mice abolished the regulatory effects exhibited by the IRF7‐TG and IRF7‐KO mice, respectively, validated the underlying molecular events of IRF7‐ATF3 interaction.
These findings demonstrated that IRF7 modulated VSMC proliferation and neointima formation by interacting with ATF3, thereby inhibiting the ATF3‐mediated induction of PCNA transcription. The results of this study indicate that IRF7 is a novel modulator of neointima formation and VSMC proliferation and may represent a promising target for vascular disease therapy.
ATF3; IRF7; neointima formation; proliferation
The present studies were designed to measure the affinity of UP 269-6, a newly developed angiotensin AT1 receptor antagonist, for vascular AT1 receptors from normotensive and hypertensive rats and to investigate in vitro, its effects on angiotensin II (AII)-induced hyperplasia and hypertrophy of vascular smooth muscle cells (VSMC). In addition the in vivo effects of UP 269-6 on neointimal proliferation in a carotid artery balloon injury in normotensive rats were also investigated.UP 269-6 selectively inhibited [125I]-Sar-Ile8-AII binding to vascular AT1 receptors present on VSMC derived from normotensive Wistar rat and from SHR (Ki=16.6±3.6 nM and 7.5±2.0 nM, respectively). In comparison, losartan and its metabolite, EXP 3174, inhibited [125I]-Sar-Ile8-AII binding to vascular AT1 receptors derived from both cell models with Ki values slightly lower (losartan) and higher (EXP 3174), respectively, than that of UP 269-6.AII (1 μM) induced a weak and variable hyperplastic response (4 to 32% increase in cell number) in Wistar rat VSMC after 96 h.AII (1 μM) induced a time-dependent increase in cell number in VSMC from SHR. UP 269-6 inhibited concentration-dependently this effect with an IC50 value of 159±58 nM. Losartan was clearly less potent and EXP 3174 showed nearly the same inhibitory potency, compared to UP 269-6. UP 269-6 (1 μM) inhibited nearly completely the action of AII.AII (500 nM) caused maximal stimulation of protein synthesis in Wistar rat VSMC (117±36%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 28±6 nM, 3504±892 nM and 21±3 nM, respectively.AII (50 nM) induced maximal stimulation of protein synthesis in SHR VSMC (237±67%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 16±3 nM, 282±122 nM and 3.3±1.0 nM, respectively.UP 269-6 (75 mg kg−1 day−1) administered orally in the diet for 20 days induced a 38% reduction in neointimal area and a 36% reduction in neointima/media ratio associated with the intimal thickening induced by carotid artery balloon injury.In conclusion, UP 269-6 was shown to be a potent antiproliferative agent both in vitro on AII-induced hyperplasia and hypertrophy of VSMC derived from normotensive and hypertensive rats, and in vivo upon intimal thickening induced by carotid artery balloon injury in the rat.
Angiotenisin II receptor; vascular smooth muscle cells (VSMC); [125I]-Sar1-Ile8 binding; normotensive rat VSMC model; hypertensive rat VSMC model; hyperplasia; hypertrophy; neointimal proliferation; UP 269-6; losartan; EXP 3174
Proliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in neointimal formation which leads to restenosis of vein graft in venous bypass. STAT-3 is a transcription factor associated with cell proliferation. We hypothesized that silencing of STAT-3 by siRNA will inhibit proliferation of VSMCs and attenuate intimal thickening.
Rat VSMCs were isolated and cultured in vitro by applying tissue piece inoculation methods. VSMCs were transfected with STAT 3 siRNA using lipofectamine 2000. In vitro proliferation of VSMC was quantified by the MTT assay, while in vivo assessment was performed in a venous transplantation model. In vivo delivery of STAT-3 siRNA plasmid or scramble plasmid was performed by admixing with liposomes 2000 and transfected into the vein graft by bioprotein gel applied onto the adventitia. Rat jugular vein-carotid artery bypass was performed. On day 3 and7 after grafting, the vein grafts were extracted, and analyzed morphologically by haematoxylin eosin (H&E), and assessed by immunohistochemistry for expression of Ki-67 and proliferating cell nuclear antigen (PCNA). Western-blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression in vivo and in vitro. Cell apoptosis in vein grafts was detected by TUNEL assay.
MTT assay shows that the proliferation of VSMCs in the STAT-3 siRNA treated group was inhibited. On day 7 after operation, a reduced number of Ki-67 and PCNA positive cells were observed in the neointima of the vein graft in the STAT-3 siRNA treated group as compared to the scramble control. The PCNA index in the control group (31.3 ± 4.7) was higher than that in the STAT-3 siRNA treated group (23.3 ± 2.8) (P < 0.05) on 7d. The neointima in the experimental group(0.45 ± 0.04 μm) was thinner than that in the control group(0.86 ± 0.05 μm) (P < 0.05).Compared with the control group, the protein and mRNA levels in the experimental group in vivo and in vitro decreased significantly. Down regulation of STAT-3 with siRNA resulted in a reduced expression of Bcl-2 and cyclin D1. However, apoptotic cells were not obviously found in all grafts on day 3 and 7 post surgery.
The STAT-3 siRNA can inhibit the proliferation of VSMCs in vivo and in vitro and attenuate neointimal formation.
STAT-3, siRNA; vascular smooth muscle cells (VSMCs); intimal thickening