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
The Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia.
Methods and Results
Wire injury was performed in wild type (WT) and EBP50 knockout (KO) mice. Two weeks after injury, neointima formation was 80% lower in KO than in WT mice. Proliferation of KO VSMC was significantly lower than WT cells and overexpression of EBP50 increased VSMC proliferation. Akt activity and expression of S-phase kinase protein 2 (Skp2) decreased in KO cells resulting in the stabilization of the cyclin-dependent kinase inhibitor, p21cip1. Consequently, KO cells were arrested in G0/G1 phase. Consistent with these observations, p21cip1 was detected in injured femoral arteries of KO but not WT mice. No differences in apoptosis between WT and KO were observed.
EBP50 is critical for neointima formation and induces VSMC proliferation by decreasing Skp2 stability, thereby accelerating the degradation of the cell cycle inhibitor p21cip1.
proliferation; smooth muscle; EBP50; Skp2; p21cip1
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
FRNK, the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and PYK2 in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling.
Methods and Results
Adenoviral-mediated gene transfer of GFP-tagged, wildtype (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenoviral-mediated expression of specific shRNAs were used to “knock down” FAK vs. PYK2 in cultured VSMCs, but only FAK shRNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Y168F-, Y232F-, and Y168,232F-) GFP-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Y168 phosphorylation was required for FRNK to inhibit invasion. Preventing Y168 phosphorylation also increased FRNK-paxillin interaction, as determined by co-immunoprecipitation, total internal reflection fluorescence (TIRF)-microscopy, and fluorescence recovery after photobleaching (FRAP). Furthermore, wtFRNK competed with FAK for binding to p130Cas (a critically important regulator of cell migration), and prevented its phosphorylation. However, Y168F-FRNK was unable to bind p130Cas.
We propose a 3-stage mechanism for FRNK inhibition – FA targeting, Y168 phosphorylation, and competition with FAK for p130Cas binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.
focal adhesion kinase; PYK2; paxillin; vascular remodeling
The aim of this study was to explore the antitumor effect of Gan-Lu-Yin (GLY), a traditional Chinese herbal formula, on leukemia. Ethanolic extract of GLY was applied to evaluate its regulatory mechanisms in proliferation, migration, and differentiation of WEHI-3 leukemic cells as well as antitumor effect on BALB/c mice model. The results showed that GLY markedly reduced cell proliferation and migration with induced differentiation of WEHI-3 cells. The expression level of phosphorylated FAK, Akt, ERK1/2, and Rb was decreased p21 expression while level was increased in WEHI-3 treated with GLY. The results of cell cycle analysis revealed that GLY treatment could markedly induce G1 phase arrest and decrease cell population in S phase. Moreover, experimental results demonstrated that GLY decreased the protein expression and enzyme activity of MMP-2 and MMP-9. GLY treatment also reduced WEHI-3 leukemic infiltration in liver and spleen and tumor growth in animal model. Accordingly, GLY demonstrated an inhibitory effect on tumor growth with a regulatory mechanism partially through inhibiting FAK, Akt, and ERK expression in WEHI-3 cells. GLY may provide a promising antileukemic approach in the clinical application.
Arginase stimulates the proliferation of cultured vascular smooth muscle cells (VSMCs); however, the influence of arginase on VSMC growth in vivo is not known. This study investigated the impact of arginase on cell cycle progression and neointima formation following experimental arterial injury.
Methods and Results
Balloon injury of rat carotid arteries resulted in a sustained increase in arginase activity in the vessel wall and the induction of arginase I protein in both the media and neointima of injured vessels. Furthermore, local perivascular application of the potent and selective arginase inhibitors S-(2-boronoethyl)-l-cysteine (BEC) or NG-hydroxy-nor-L-arginine (L-OHNA) immediately after injury markedly attenuated medial and neointimal DNA synthesis and neointima formation. Substantial arginase I protein and arginase activity was also detected in rat cultured aortic VSMCs. Moreover, treatment of VSMCs with BEC or L-OHNA, or knockdown of arginase I protein, arrested cells in the G0/G1 phase of the cell cycle and induced the expression of the cyclin-dependent protein kinase inhibitor, p21.
This study demonstrates that arginase is essential for VSMCs to enter the cell cycle and that arginase I contributes to the remodeling response following arterial injury. Arginase I represents a potentially new therapeutic target for the treatment of vasculoproliferative disorders.
Degradation and resynthesis of the extracellular matrix (ECM) are essential during tissue remodeling. Expansion of the vascular intima in atherosclerosis and restenosis following injury is dependent upon smooth muscle cell (SMC) proliferation and migration. The migration of SMC from media to intima critically depends on degradation of ECM protein by matrix metalloproteinases (MMPs). MMP inhibitors and eNOS gene transfer have been shown to inhibit SMC migration in vitro and neointima formation in vivo. Nitric oxide (NO) and cyclic-GMP have been implicated in the inhibition of VSMC migration. But, there are few studies addressing the role of NO signaling pathways on the expression of MMPs. Here we reported the involvement of cyclic-GMP-dependent protein kinase (PKG) (an important mediator of NO and cGMP signaling pathway in VSMC) on MMP-2 expression in rat aortic SMC. The goal of the present study was to gain insight into the possible involvement of PKG on MMP-2 in rat aortic SMC. MMP-2 protein and mRNA level and activity were downregulated in PKG-expressing cells as compared to PKG-deficient cells. In addition, the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased in PKG-expressing cells as compared to PKG-deficient cells. PKG-specific membrane permeable peptide inhibitor (DT-2) reverses the process. Interestingly, little or no changes of MMP-9 were observed throughout the study. Taken together our data suggest the possible role of PKG in the suppression of MMP-2.
Matrix metalloproteinases-2; PKG; Transfection; Vascular smooth muscle cells; DT-2
Background and Objectives
Vascular smooth muscle cell (VSMC) proliferation is responsible for the restenosis of previously inserted coronary stents. Angiotensin II (Ang II) is known to regulate VSMC proliferation. LKB1, a serine/threonine kinase, interacts with the p53 pathway and acts as a tumor suppressor.
Materials and Methods
We assessed the association of Ang II and the expression of LKB1 in primary cultured murine VSMCs and neointima of the Sprague Dawley rat carotid artery injury model. We created carotid balloon injuries and harvested the injured carotid arteries 14 days after the procedure.
Ang II increased LKB1 expression in a time-dependent manner and peaked at an Ang II concentration of 10-7 mole/L in VSMCs. In the animal experiment, neointima was markedly increased after balloon injury compared to the control group. Immunohistochemical studies showed that LKB1 expression increased according to neointima thickness. Ang II augmented LKB1 expression after the injury. Western blot analysis of LKB1 with carotid artery lysate revealed the same pattern as LKB1 immunohistochemistry. Increased LKB1 expression started at 5 days after the balloon injury, and peaked at 14 days after the injury. Although LKB1 expression was increased after the injury, LKB1 kinase activity was not increased. Ang II or balloon-injury increased the expression of LKB1 although the LKB1 activity was reduced.
Ang II increased LKB1 expression in VSMCs and neointima. These findings were not kinase dependant.
Angiotensin II; LKB1 protein, rat; Coronary restenosis
Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have only been performed in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete media, male VSMC proliferated at greater rates than female VSMC. In hormone-deplete media, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male versus female VSMC. These findings correlated with greater G0/G1 cell cycle arrest and changes in cell cycle protein expression in male versus female VSMC following exposure to NO. Next, the rat carotid artery injury model was performed to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males versus females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in both sexes in castrated animals. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males than females and in hormonally-intact compared to castrated rats, indicating that the effect of NO in the vasculature may be sex- and hormone-dependent.
neointimal hyperplasia; vascular smooth muscle proliferation; nitric oxide; hormones; cell cycle
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.
Angiotensin converting enzyme (ACE) activity contributes to the vascular response to injury because ACE inhibition limits neointima formation in rat carotid arteries after balloon injury. To investigate the mechanisms by which ACE may contribute to vascular smooth muscle cell (VSMC) proliferation, we studied expression of ACE in vivo after injury and in vitro after growth factor stimulation. ACE activity 14 d after injury was increased 3.6-fold in the injured vessel. ACE expression, measured by immunohistochemistry, became apparent at 7 d in the neointima and at 14 d was primarily in the most luminal neointimal cells. To characterize hormones that induce ACE in vivo, cultured VSMC were exposed to steroids and growth factors. Among steroids, only glucocorticoids stimulated ACE expression with an 8.0 +/- 2.1-fold increase in activity and a 6.5-fold increase in mRNA (30 nM dexamethasone for 72 h). Among growth factors tested, only fibroblast growth factor (FGF) stimulated ACE expression (4.2 +/- 0.7-fold increase in activity and 1.6-fold increase in mRNA in response to 10 ng/ml FGF for 24 h). Dexamethasone and FGF were synergistic at the indicated concentrations inducing 50.6 +/- 12.4-fold and 32.5-fold increases in activity and mRNA expression, respectively. In addition, when porcine iliac arteries were transfected with recombinant FGF-1 (in the absence of injury), ACE expression increased in neointimal VSMC, to the same extent as injured, nontransfected arteries. The data suggest a temporal sequence for the response to injury in which FGF induces ACE, ACE generates angiotensin II, and angiotensin II stimulates VSMC growth in concert with FGF.
Objective: To test the influence of homocysteine on the production and activation of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) and on cell migration of cultured rat vascular smooth muscle cells (VSMCs). Also, to explore whether rosuvastatin can alter the abnormal secretion and activation of MMP-2 and TIMP-2 and migration of VSMCs induced by homocysteine. Methods: Rat VSMCs were incubated with different concentrations of homocysteine (50–5 000 μmol/L). Western blotting and gelatin zymography were used to investigate the expressions and activities of MMP-2 and TIMP-2 in VSMCs in culture medium when induced with homocysteine for 24, 48, and 72 h. Transwell chambers were employed to test the migratory ability of VSMCs when incubated with homocysteine for 48 h. Different concentrations of rosuvastatin (10−9–10−5 mol/L) were added when VSMCs were induced with 1 000 μmol/L homocysteine. The expressions and activities of MMP-2 and TIMP-2 were examined after incubating for 24, 48, and 72 h, and the migration of VSMCs was also examined after incubating for 48 h. Results: Homocysteine (50–1 000 μmol/L) increased the production and activation of MMP-2 and expression of TIMP-2 in a dose-dependent manner. However, when incubated with 5 000 μmol/L homocysteine, the expression of MMP-2 was up-regulated, but its activity was down-regulated. Increased homocysteine-induced production and activation of MMP-2 were reduced by rosuvastatin in a dose-dependent manner whereas secretion of TIMP-2 was not significantly altered by rosuvastatin. Homocysteine (50–5 000 μmol/L) stimulated the migration of VSMCs in a dose-dependent manner, but this effect was eliminated by rosuvastatin. Conclusions: Homocysteine (50–1 000 μmol/L) significantly increased the production and activation of MMP-2, the expression of TIMP-2, and the migration of VSMCs in a dose-dependent manner. Additional extracellular rosuvastatin can decrease the excessive expression and activation of MMP-2 and abnormal migration of VSMCs induced by homocysteine.
Matrix metalloproteinase-2 (MMP-2); Vascular smooth muscle cells (VSMCs); Migration; Rosuvastatin; Homocysteine
Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration.
Results and Methods
The effects of increased HO-1 expression as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches including detection of intracellular superoxide, NADPH oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased PDGF-stimulated, redox-sensitive signaling pathways.
Herein we demonstrate that increased HO-1 expression and CO decreases PDGF-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury.
heme oxygenase-1; carbon monoxide; NADPH oxidase; vascular smooth muscle; Nox1
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.
Atherosclerosis is characterized by hyperplastic neointima and an inflammatory response with cytokines such as TNFα. TNFα is a pleiotropic cytokine that mediates inflammatory, proliferative, cytostatic and cytotoxic effects in a variety of cell types, including endothelial cells and vascular smooth muscle cells (VSMCs). Interestingly, TNFα has been shown to play two very opposing roles in these cell types; it inhibits proliferation and induces apoptosis in endothelial cells, while it enhances the proliferation and migration of VSMCs. Here we show that TNFα is capable of stimulating proliferation of rat VSMCs as well as human VSMCs in a Raf-1/MAPK-dependent manner. TNFα could increase the expression of E2F-regulated proliferative cdc6, Thymidylate synthase (TS) and cdc25A genes in Aortic smooth muscle cells (AoSMC), as seen by real time PCR assays. There is an activation of the stress-induced kinase, JNK1, in VSMCs upon TNFα stimulation. TNFα was capable of inducing binding of the Raf-1 kinase to Rb, and treatment with the Rb-Raf-1 inhibitor, RRD-251, could prevent TNFα-induced S-phase entry in AoSMCs. In addition, inhibition of Raf-1 or Src kinases using pharmacologic inhibitors could also prevent S-phase entry, while inhibition of JNK was not as effective. These results suggest that inhibiting the Rb-Raf-1 interaction is a potential avenue to prevent VSMC proliferation associated with atherosclerosis.
cell cycle; RRD-251; Rb-Raf interaction; MAP kinase cascade; E2F1 transcription factor
Adenosine monophosphate-activated protein kinase (AMPK), a metabolic and redox sensor, is reported to suppress cell proliferation of non-malignant and tumor cells. Whether AMPKα alters vascular neointima formation induced by vascular injury is unknown.
The aim of this study was to determine the roles of AMPKα in the development of vascular neointima hyperplasia and to elucidate the underlying mechanisms.
Methods and Results
Vascular smooth muscle cells (VSMCs) proliferation and neointimal hyperplasia were evaluated in cultured VSMCs and wire-injured mouse carotid arteries from wild-type (WT, C57BL/6J), AMPKα2−/−, and AMPKα1−/− VSMCs. Mouse VSMCs derived from aortas of AMPKα2−/− mice exhibited increased proliferation compared to either WT or AMPKα1−/− VSMCs. Further, deletion of AMPKα2, but not AMPKα1, reduced the level of p27Kip1, acyclin-dependent kinase inhibitor, and increased the level of S-phase kinase-associated protein 2 (Skp2), a known E3 ubiquitin ligase for p27Kip1, via activation of p52 nuclear factor kappa B (NF-κB)-2. Moreover, either pharmacological (i.e., via compound C) or genetical (i.e., via AMPKα2-specific siRNA) inhibition of AMPK decreased p27Kip1 levels, but increased the abundance of Skp2 in human VSMCs. Furthermore, gene silencing of Skp2 reversed the levels of p27Kip1 and VSMCs proliferation. Finally, neointima formation after mechanical arterial injury was increased in AMPKα2−/−, but not AMPKα1−/−, mice.
These findings indicate that deletion of AMPKα2 via p52-Skp2-mediated ubiquintination and degradation of p27Kip1 accentuates neointimal hyperplasia in response to wire injury.
Neointima formation; AMPK; VSMC; NF-κB; Skp2
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.
Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.
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.
Homeobox transcription factors specify body plan by regulating differentiation, proliferation, and migration at a cellular level. The homeobox transcription factor Gax is expressed in quiescent vascular smooth muscle cells (VSMCs), and its expression is downregulated by vascular injury or other conditions that lead to VSMC proliferation. Previous investigations demonstrate that Gax may regulate VSMC proliferation by upregulating the cyclin-dependent kinase (cdk) inhibitor p21. Here we examined whether Gax influences VSMC migration, a key feature in the development of stenotic lesions after balloon injury. Transduction of a Gax cDNA inhibited the migratory response of VSMCs toward PDGF-BB, basic fibroblast growth factor, or hepatocyte growth factor/scatter factor. Gax expression also inhibited migration of NIH·3T3 fibroblasts and embryonic fibroblasts lacking p53. Gax was unable to inhibit the migration of fibroblasts lacking p21, but this effect could be restored in these cells by providing exogenous p21 or by overexpressing another cdk inhibitor, p16. Flow cytometric analysis implicated a Gax-mediated downregulation of αvβ3 and αvβ5 integrin expression in VSMCs as a potential cause for reduced cell motility. Gax specifically downregulated β3 and β5 in VSMCs in culture and after acute vascular injury in vivo. Repression of integrin expression was also found in NIH 3T3 cells and p53 knockout fibroblasts, but not in p21-knockout fibroblasts, unless these cells express exogenous p21 or p16. These data suggest that cycle progression, integrin expression, and cell migration can be regulated in VSMCs by the homeobox gene product Gax.
J. Clin. Invest. 104:1469–1480 (1999).
Advanced age is a major risk factor for atherosclerosis, but how aging per se influences pathogenesis is not clear. Insulin-like growth factor-1 receptor (IGF-1R) promotes aortic vascular smooth muscle cell (VSMC) growth, migration, and extracellular matrix formation, but how IGF-1R signaling changes with age in VSMC is not known. We previously found age-related differences in the activation of Akt/FOXO3a and ERK1/2 pathways in VSMC, but the up-stream signaling remains unclear. Using explanted VSMC from Fischer 344/Brown Norway F1 hybrid rats shown to display age-related vascular pathology similar to humans, we compared IGF-1R expression in early passages of VSMC and found a constitutive activation of IGF-1R in VSMC from old compared to young rats, including IGF-1R expression and its tyrosine kinase activity. The link between IGF-1R activation and the Akt/FOXO3a and ERK pathways was confirmed through the induction of IGF-1R with IGF-1 in young cells and attenuation of IGF-1R with an inhibitor in old cells. The effects of three kinase inhibitors: AG1024, LY294002, and TCN, were compared in VSMC from old rats to differentiate IGF-1R from other upstream signaling that could also regulate the Akt/FOXO and ERK pathways. Genes for p27kip-1, catalase and MnSOD, which play important roles in the control of cell cycle arrest and stress resistance, were found to be FOXO3a-targets based on FOXO3a-siRNA treatment. Furthermore, IGF-1R signaling modulated these genes through activation of the Akt/FOXO3a pathway. Therefore, activation of IGF-1R signaling influences VSMC function in old rats and may contribute to the increased risk for atherosclerosis.
Aging; IGF-1R; Akt/FOXO3a; ERK/Fos; VSMC; Glucose
Alternative transcription and splicing of the allograft inflammatory factor-1 (AIF-1) gene results in the expression of two different proteins: AIF-1 and interferon responsive transcript-1 (IRT-1). Here, we explore the impact of AIF-1 and IRT-1 on vascular smooth muscle cell (VSMC) activation and neointima formation, the mechanisms underlying their alternative splicing, and associations of AIF-1 and IRT-1 mRNA with parameters defining human atherosclerotic plaque phenotype.
Methods and results
Translation of AIF-1 and IRT-1 results in different products with contrasting cellular distribution and functions. Overexpression of AIF-1 stimulates migration and proliferation of human VSMCs, whereas IRT-1 exerts opposite effects. Adenoviral infection of angioplasty-injured rat carotid arteries with AdAIF-1 exacerbates intima hyperplasia, whereas infection with AdIRT-1 reduces neointima. Expression of these variants is modulated by changes in nuclear factor of activated T-cells (NFAT) activity. Pharmacological inhibition of NFAT or targeting of NFATc3 with small interfering RNA (siRNA) lowers the AIF-1/IRT-1 ratio and favours an anti-proliferative outcome. NFAT acts as a repressor on the IRT-1 transcriptional start site, which is also sensitive to interferon-γ stimulation. Expression of AIF-1 mRNA in human carotid plaques associates with less extracellular matrix and a more pro-inflammatory plaque and plasma profile, features that may predispose to plaque rupture. In contrast, expression of IRT-1 mRNA associates with a less aggressive phenotype and less VSMCs at the most stenotic region of the plaque.
Inhibition of NFAT signalling, by shifting the AIF-1/IRT-1 ratio, may be an attractive target to regulate the VSMC response to injury and manipulate plaque stability in atherosclerosis.
AIF-1; NFAT; Restenosis; Atherosclerosis; Vascular smooth muscle
Intimal hyperplasia is defined as the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) with deposition of extracellular matrix. However, the cell cycle regulatory mechanisms of injury-induced VSMC proliferation are largely unknown. To examine the expression kinetics of cell cycle regulatory factors which is known to be worked positively or negatively, we used rat balloon injury model. Marked induction of proliferating cell nuclear antigen (PCNA), G1/S cyclin-dependent kinase (cdk2), and its regulatory subunit (cyclin E) occurred between 1 and 3 days after balloon arterial injury, and this was sustained for up to 7 days and then declined. However, the induction of the negative regulators, p21 and p27, occurred between 3 and 5 days of injury, peaked after 7 and 14 days and was then sustained. VSMC proliferation after balloon catheter injury of the rat iliac artery is associated with coordinated expression of positive (cdk2, cyclin E and PCNA) and negative (p21, p27) regulators. Cell cycle regulators such as cdk2, cyclin E, p21, p27 may be suitable targets for the control of intimal hyperplasia.
Endothelium, Vascular; Myocytes, Smooth Muscle; Tunica Intima; Cell Cycle Proteins; Rat
The aim of this study was to investigate the mechanisms by which nicotine increases vascular smooth muscle cell (VSMC) proliferation and post-injury neointimal formation.
Methods and results
Vascular injury was inflicted in the right iliac artery of nicotine-treated and control rats. Nicotine increased post-injury VSMC proliferation (Ki67+ cells) and neointimal formation (neointima/media ratio, 0.42 ± 0.23 vs. 0.14 ± 0.07, P= 0.02). To determine the mechanisms by which nicotine exacerbates VSMC proliferation, cultured cells were exposed to nicotine, and signalling pathways leading to cell proliferation were studied. Nicotine activated extracellular signal-regulated kinase (ERK) 1/2 in a dose- and time-dependent manner. The blockade of this signalling axis abolished nicotine-mediated proliferation. Functional nicotinic acetylcholine receptors and Ca2+ influx were necessary for ERK1/2 activation and nicotine-induced mitogenesis in VSMCs. Downstream to ERK1/2, nicotine induced the phosphorylation of Ets-like gene 1 in a timely co-ordinated manner with the up-regulation of the atherogenic transcription factor, early growth response 1 (Egr-1). The treatment of balloon-injured arteries with a lentivirus vector carrying a short hairpin RNA against Egr-1 abolished the deleterious effect of nicotine on vascular remodelling.
Nicotine acts through its receptors in VSMC to activate the ERK–Egr-1 signaling cascade that induces cell proliferation and exacerbates post-injury neointimal development.
Vascular smooth muscle cell; Extracellular signal-regulated kinase; Neointima; Egr-1; Nicotine
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