Balloon-injured Rat Carotid Arteries Research Articles

Estrogen enhances IkBα synthesis and inhibits NF‐kB p65 binding to the promoters of inflammatory genes in rat aortic smooth muscle cells

BackgroundThe NFκB signaling pathway is critical for the expression of a variety of genes involved in the response to vascular injury. Estrogen (17β‐estradiol, E2) inhibits expression of these genes in an estrogen receptor‐dependent manner in balloon injured rat carotid arteries and in tumor necrosis factor (TNF)‐α treated rat aortic smooth muscle cells (RASMCs) in vitro. This study tested the hypothesis that E2 inhibits NFκB signaling and defined the machanisms.MethodsIκBα expression was determined using real time RT‐PCR and Western blot analysis. Interactions between NFκB p65 and promoters of inflammatory mediators were evaluated by chromatin immunoprecipitation (ChIP) assay.ResultsRASMCs treated with TNF‐α demonstrated rapid degradation of IκBα (10‐30 min), followed by a dramatic elevation in IκBα mRNA and protein synthesis (40~60 min). E2 enhanced TNF‐α induced IκBα mRNA and protein synthesis without affect IκBα degradation (Figure A~C). ChIP assay confirmed that TNF‐α increased levels of p65 significantly at all promoters analyzed (at 1hr) and these effects were reduced by E2 (Figure D).ConclusionsE2‐inhibited expression of inflammatory mediators in the setting of acute vascular injury may be mediated, in part, by accelerating IκBα re‐synthesis and inhibiting NFκB p65 binding to promoters of target genes.

Krüppel-like Factor 5 Shows Proliferation-specific Roles in Vascular Remodeling, Direct Stimulation of Cell Growth, and Inhibition of Apoptosis

Krüppel-like factor 5 (KLF5), originally isolated as a regulator of phenotypic modulation of vascular smooth muscle cells, induces pathological cell growth and is expressed in the neointima. Although induction of KLF5 up-regulates growth factors like platelet-derived growth factor-A chain, how KLF5 actually contributes to vascular remodeling, notably its direct effects on cell proliferation, had been poorly clarified. To investigate the effects of KLF5 on neointimal formation, we at first performed adenoviral overexpression of KLF5 to rats subjected to carotid balloon injury. Neointimal formation and proliferating cell nuclear antigen-positive rate were significantly increased at 14 days after injury in the KLF5-treated animals. At the cellular level, overexpression of KLF5 also resulted in markedly increased cell proliferation and cell cycle progression. As a molecular mechanism, we showed that KLF5 directly bound to the promoter and up-regulated gene expression of cyclin D1, as well as showing specific transactivation of cyclins and cyclin-dependent kinase inhibitors in cardiovascular cells. Conversely, knockdown of KLF5 by RNA interference specifically down-regulated cyclin D1 and impaired vascular smooth muscle cell proliferation. Furthermore, KLF5 attenuated cleavage of caspase-3 under conditions of apoptotic stimulation. Moreover, KLF5-administered animals exhibited a significant decrease in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling-positive cells in the medial layer, suggesting inhibition of apoptosis in the early phase after denudation. These findings collectively suggest that KLF5 plays a central role in cardiovascular pathologies through direct and specific stimulation of cell growth as well as inhibition of apoptosis.

RNA Interference Targeting STIM1 Suppresses Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat

Our objective was to study the expression and function of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum protein recently identified as the calcium sensor that regulated Ca(2+)-released activated channels in T cells. STIM1 was found to be upregulated in serum-induced proliferating human coronary artery smooth muscle cells (hCASMCs) as well as in the neointima of injured rat carotid arteries. Growth factors-induced proliferation was significantly lower in hCASMC transfected with STIM1 siRNA than in those transfected with scrambled siRNA (increase relative to 0.1% S: 116 +/- 12% and 184 +/- 16%, respectively, P < 0.01). To assess the role of STIM1 in preventing vascular smooth muscle cells (VSMCs) proliferation in vivo, we infected balloon-injured rat carotid arteries with an adenoviral vector expressing a short hairpin (sh) RNA against rat STIM1 mRNA (Ad-shSTIM1). Intima/media ratios reflecting the degree of restenosis were significantly lower in Ad-shSTIM1- infected arteries than in Ad-shLuciferase-infected arteries (0.34 +/- 0.02 vs. 0.92 +/- 0.11, P < 0.006). Finally, we demonstrated that silencing STIM1 prevents activation of the transcription factor NFAT (nuclear factor of activated T cell). In conclusion, STIM1 appears as a major regulator of in vitro and in vivo VSMC proliferation, representing a novel and original pharmacological target for prominent vascular proliferative diseases.

Mrp4 Is A Transmembrane Export Pump Acting As An Endogenous Regulator Of Cyclic- Nucleotides Dependent Pathways

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers which regulate many biological processes. They can be eliminated by active efflux transporters, namely the multidrug resistance proteins MRP4 and MRP5. To delineate the role of MRP4/5, we studied arterial smooth muscle cells in which the role of cyclic nucleotide levels on proliferation has been well-established.Methods: Human SMCs were isolated from the coronary artery media from patients. Small interfering RNAs (siRNA) specific for MRP4 mRNA were designed and validated. Adenovirus encoding MRP4 short hairpin RNA (Ad-shMRP4) were used for in vivo studies.Results: MRP4 was over-expressed in serum-induced proliferating SMC as well as in atherosclerotic plaques in human coronary arteries and in neo-intima of injured rat carotid arteries. Inhibition of MRP4 by siRNA blocked VSMC proliferation in vitro. In balloon-injured rat carotid arteries, intima/media ratios were significantly lower in Ad-shMRP4-infected arteries than in Ad-shLuciferase-infected arteries (0.65 ± 0.1 vs 1.05 ± 0.2; p<0.03). In vitro, MRP4 inhibition significantly increased intracellular cAMP and cGMP levels. A PKA inhibitor (PKI) but not the PKG inhibitor (KT5823) completely reversed the anti-proliferative effect of MRP4 inhibition. The level of pCREB increased by 329 ± 18.8% (p=0.003) on MRP4 inhibition. Conclusion: We provide first evidences that MRP4 acts as an independent endogenous regulator of cyclic nucleotides intra-cellular levels in vascular smooth muscle cells

Retrovirally Mediated Overexpression of Glycosaminoglycan-Deficient Biglycan in Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation in Vitro and in Neointimae after Vascular Injury

Galactosamine-containing glycosaminoglycans (GAGs), such as the chondroitin sulfate chains of the proteoglycan versican, have been shown to inhibit elastogenesis. Another proteoglycan that may influence elastogenesis is biglycan, which possesses two GAG chains. To assess the importance of these chains on elastogenesis in blood vessels, rat aortic smooth muscle cells were transduced with a GAG-deficient biglycan cDNA-containing retroviral vector (LmBSN). Control cells were transduced with either biglycan or empty vector. Transduced cells were characterized in vitro and then seeded into balloon-injured rat carotid arteries to determine the effects on neointimal structure. Cultured cells overexpressing LmBSN showed marked up-regulation of tropoelastin and fibulin-5 mRNAs, increased amounts of desmosine and insoluble elastin, and increased deposition of elastic fibers as compared with empty vector- and biglycan-transduced cells. Conversely, collagen alpha(1) synthesis and the deposition of collagen fibers were both markedly decreased in LmBSN cultures. In vivo, neointimae formed from cells that overexpressed LmBSN and showed increased deposits of elastin that aggregated into parallel nascent fibers, generally arranged circumferentially. Neointimae that formed from cells with biglycan or empty vector contained fewer and less aggregated deposits of elastin. These findings suggest that the GAG chains of biglycan serve as inhibitors of elastin synthesis and assembly, and that biglycan can act as an important modulator of the composition of the extracellular matrix of blood vessels.

Suppression of c-Cbl Tyrosine Phosphorylation Inhibits Neointimal Formation in Balloon-Injured Rat Arteries

c-Cbl, a ubiquitously expressed protooncogene, is tyrosine phosphorylated in response to a variety of stimuli, including growth factors such as platelet-derived growth factor (PDGF), and consequently activates signaling proteins such as phosphatidylinositol-3 kinase (PI3K) and Akt. In the present study, we examined the role of c-Cbl tyrosine phosphorylation in vascular injury. Western blotting showed that the tyrosine phosphorylation of c-Cbl was increased in balloon-injured rat carotid arteries and in cultured smooth muscle cells on stimulation by PDGF-BB, followed by the activations of Akt and the mammalian target of rapamycin. Adenovirus-mediated overexpression of a c-Cbl mutant that ablates the major tyrosine phosphorylation sites attenuated the Akt and the mammalian target of rapamycin activation and decreased the proliferation and migration of smooth muscle cells in response to PDGF-BB or fibroblast growth factor. These effects could be reversed by constitutively active PI3K or Akt, suggesting that c-Cbl phosphorylation promotes the PDGF-BB-induced proliferation and migration of smooth muscle cells through the PI3K/Akt pathways. In addition, overexpression of c-Cbl-m increased the ubiquitination of the PDGF and fibroblast growth factor receptors. Importantly, in balloon-injured rat carotid arteries, local delivery of c-Cbl-m reduced the phosphorylation of Akt and the mammalian target of rapamycin, inhibited the migration and proliferation of smooth muscle cells, and prevented neointimal hyperplasia. Our results demonstrate a novel role of c-Cbl in vascular remodeling after injury and suggest that modulation of c-Cbl tyrosine phosphorylation may be a therapeutic approach to treat vascular neointimal hyperplasia such as restenosis after angioplasty.

Activation Transcription Factor-4 Induced by Fibroblast Growth Factor-2 Regulates Vascular Endothelial Growth Factor-A Transcription in Vascular Smooth Muscle Cells and Mediates Intimal Thickening in Rat Arteries Following Balloon Injury

Activation transcription factor (ATF)-4 is a member of the ATF/CREB family of basic leucine zipper transcription factors that regulates cellular responses to a variety of stresses. The role of ATF-4 in smooth muscle cells of the vessel wall is completely unknown. Here, we show that ATF-4 expression is induced in smooth muscle cells in response to injury, both in vitro using a model of mechanical injury and in the media of balloon-injured rat carotid arteries. We demonstrate that ATF-4 is activated by fibroblast growth factor (FGF)-2, an injury-induced mitogen, through the phosphatidylinositol 3-kinase pathway. Injury also activates vascular endothelial growth factor (VEGF)-A, whose expression is stimulated by ATF-4 overexpression and exposure to FGF-2. FGF-2 induces ATF-4 binding to a recognition element located in the VEGF-A gene at +1767 bp and luciferase reporter gene expression dependent on this site. Moreover, ATF-4 knockdown with small interfering RNA or ATF-4 deficiency ameliorates FGF-2-inducible VEGF-A expression. Intraluminal delivery of ATF-4 small interfering RNA in rat carotid arteries blocks balloon injury-inducible ATF-4 and VEGF-A expression after 4 hours and intimal thickening after 14 days. These findings reveal, for the first time, the induction of ATF-4 by both vascular injury and FGF-2. ATF-4 serves as a conduit for the inducible expression of 1 growth factor by another during the process of intimal thickening.

A Systemic Combination Therapy with Granulocyte-Colony Stimulating Factor Plus Erythropoietin Aggravates the Healing Process of Balloon-Injured Rat Carotid Arteries

Percutaneous vascular interventions lead inevitably to a destruction of the endothelial lining at the site of injury. There are conflicting data on the therapeutic benefit of hematopoietic growth factors aiming at mobilisation of circulating endothelial cells to accelerate the reendothelialisation process. Aim of our study was to evaluate the impact of a maximised 7 day-combination therapy with G-CSF plus EPO on the healing process following balloon injury of the rat carotid artery. Osmotic pumps to systemically deliver G-CSF and EPO at saturating doses during the first seven days post injury were implanted into the peritoneal cavity of splenectomised male Sprague-Dawley rats. Cytokine treatment resulted in significantly elevated numbers of white blood cells, hematocrit levels, circulating hematopoietic stem cells, and-temporarily-circulating endothelial precursors. Functional reendothelialisation as assessed by Evan's blue staining on day 14 post injury was not affected by the cytokine treatment. The neointimal response was analysed on day 7 and 28 post injury, and was significantly higher at the day 7 timepoint (Cytokines: I/M-ratio 1.10+/-0.09 vs. Saline: 0.36+/-0.06). Cytokine treated rats also displayed higher rates of thrombotic occlusion (Cytokines: 25-33% vs. Saline: 0%). Serum levels of PAI-1, TGFbeta1, and PDGF-BB were not elevated in the cytokine treated rats. The proliferative rates both in the injured vessel wall and the surrounding adventitia as assessed by BrdU incorporation were significantly higher in the cytokine treated animals. The number of CD45(pos) hematopoietic cells was significantly higher in the adventitia of the cytokine treated rats. Vasa vasorum were not found to be significantly different. The increased neointimal response was not due to expression of G-CSF- or EPO-receptors on VSMCs within the vessel wall or myofibroblasts in the adventitia. The systemic administration of G-CSF plus EPO during the first week after balloon-injury impairs the vascular healing process by increasing the neointimal response and the risk for thrombotic occlusion.

RNA Interference for Discoidin Domain Receptor 2 Attenuates Neointimal Formation in Balloon Injured Rat Carotid Artery

Discoidin domain receptor 2 (DDR2) plays potential roles in the regulation of collagen turnover mediated by smooth muscle cells (SMCs) in atherosclerosis. Little is known about the function of DDR2 in vascular system. We investigated whether inhibition of DDR2 by small interfering RNA (siRNA) can reduce neointimal formation after arterial injury. SMCs from thoracic aorta of adult Wistar rats were cultured. The carotid artery from adult Wistar rats was injured by balloon catheter. DDR2 significantly increased migration and proliferation of SMCs. DDR2 siRNA inhibited 86% of DDR2 protein expression in cultured SMCs. DDR2 protein and mRNA expression significantly increased at 14 days after carotid injury. DDR2 siRNA significantly reduced DDR2 protein and mRNA expression induced by balloon injury. The immunohistochemical stain demonstrated that DDR2 siRNA decreased MMP2 protein labeling induced by balloon injury, a pattern similar to that of DDR2 protein labeling. Neointimal area was significantly increased after carotid injury and was significantly reduced by DDR2 siRNA. DDR2 increases migration and proliferation of SMCs, and expression of DDR2 in carotid artery significantly increases after injury. DDR2 siRNA attenuates neointimal formation after carotid injury. DDR2 may play a pivotal role in the pathogenesis of neointimal thickening after mechanical injury.

Increased proteinO-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury

Inflammation plays a major role in vascular disease. We have shown that leukocyte infiltration and inflammatory mediator expression contribute to vascular remodeling after endoluminal injury. This study tested whether increasing protein O-linked-N-acetylglucosamine (O-GlcNAc) levels with glucosamine (GlcN) and O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) inhibits acute inflammatory and neointimal responses to endoluminal arterial injury. Ovariectomized rats were treated with a single injection of GlcN (0.3 mg/g ip), PUGNAc (7 nmol/g ip) or vehicle (V) 2 h before balloon injury of the right carotid artery. O-GlcNAc-modified protein levels decreased markedly in injured arteries of V-treated rats at 30 min, 2 h, and 24 h after injury but returned to control (contralateral uninjured) levels after 14 days. Both GlcN and PUGNAc increased O-GlcNAc-modified protein levels in injured arteries compared with V controls at 30 min postinjury; the GlcN-mediated increase persisted at 24 h but was not evident at 14 days. Proinflammatory mediator expression increased markedly after injury and was reduced significantly (30-50%) by GlcN and PUGNAc. GlcN and PUGNAc also inhibited infiltration of neutrophils and monocytes in injured arteries. Chronic (14 days) treatment with GlcN reduced neointima formation in injured arteries by 50% compared with V controls. Acute GlcN and PUGNAc treatment increases O-GlcNAc-modified protein levels and inhibits acute inflammatory responses in balloon-injured rat carotid arteries; 14 day GlcN treatment inhibits neointima formation in these vessels. Augmenting O-GlcNAc modification of proteins in the vasculature may represent a novel anti-inflammatory and vasoprotective mechanism.

CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery

We previously showed that cellular repressor of E1A-stimulated genes (CREG) is up-regulated during serum starvation-induced vascular smooth muscle cell (SMC) differentiation. The aim of this study was to determine the role of CREG in maintaining the quiescent, differentiated phenotype of SMCs both in culture and in balloon-injured rat carotid artery. In cultured SMCs recombinant virus-mediated CREG expression enhanced cellular differentiation, inhibited proliferation, and reduced synthesis of extracellular matrix component fibronectin. In contrast, CREG knockdown via retroviral transfer of short hairpin RNAs abrogated serum starvation-induced SMC differentiation and growth arrest. Both immunostaining and Western analysis demonstrated marked down-regulation of CREG in the vascular media after balloon injury to the rat carotid artery. Retrovirus-mediated CREG transfer to the injured artery inhibited SMC dedifferentiation and proliferation, and reduced neointimal hyperplasia. These results suggest that CREG participates in the maintenance of quiescent mature SMC phenotype in the arterial media by promoting SMC differentiation and growth arrest and that CREG gene transfer has therapeutic potential for vascular diseases associated with neointimal hyperplasia.

Effect of Udenafil on Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia in Rat Carotid Artery Injury Model

Background and Objectives: Neointimal hyperplasia, which was caused by smooth muscle cell proliferation, was noted to occur after performing percutaneous coronary intervention. Phosphodiesterase type 5 (PDE5) inhibitor has been shown to inhibit smooth muscle cell proliferation. Udenafil is one of the PDE5 inhibitors, and it is also expected to inhibit smooth muscle cell proliferation and reduce neointimal hyperplasia. We investigated the effect of udenafil on the smooth muscle cell proliferation and neointimal hyperplasia that occurs after balloon injury in the carotid arteries of rats. Materials and Methods: Smooth muscle cells were treated with 1 mM, 100 μM, 10 μM, 1 μM and 100 nM concentrations of udenafil. The viability of the smooth muscle cells was evaluated by MTT assay. The carotid arteries of rats were injured with a balloon catheter. Udenafil (100 μM, 10 μM and 1 μM) was applied on the carotid artery adventitia after balloon injury. At 21 days after treatment, the carotid arteries were harvested and stained with H & E. The neointima and media area were measured with a computerized image analysis program. Results: In the in vitro experiment, treatment with 1 mM udenafil reduced smooth muscle cell viability by 68.8±4.42% compared to the control group. In the balloon injured rat carotid artery, treatment with 100 μM udenafil reduced the neointima area by 71.8% compared to the control group. Conclusion: Udenafil administration effectively inhibited smooth muscle cell proliferation and it reduced neointimal hyperplasia in the balloon-injured rat carotid artery. (Korean Circ J 2008;38:320-324)

Regulation of cell–matrix contacts and β-catenin signaling in VSMC by integrin-linked kinase: implications for intimal thickening

Vascular smooth muscle cell (VSMC) proliferation and migration is responsible for intimal thickening that occurs in restenosis and atherosclerosis. Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in signaling pathways involved in cell proliferation and migration. We studied the involvement of ILK in intimal thickening. ILK expression was significantly increased in two models of intimal thickening: balloon-injured rat carotid arteries and human saphenous vein organ cultures. Over-expression of a dominant negative ILK (DN-ILK) significantly reduced intimal thickening by approximately 50% in human saphenous vein organ cultures, demonstrating an important role in intimal thickening. ILK protein and activity was reduced on laminin and up-regulated on fibronectin, indicating ILK protein expression is modulated by extracellular matrix composition. Inhibition of ILK by siRNA knockdown and DN-ILK significantly decreased VSMC proliferation and migration while wild type ILK significantly increased proliferation and migration on laminin, confirming an essential role of ILK in both processes. Localization of paxillin and vinculin and protein levels of FAK and phospho-FAK indicated that inhibition of ILK reduced focal adhesion formation. Additionally, inhibition of ILK significantly attenuated the presence of the cell-cell complex proteins N-cadherin and beta-catenin, and beta-catenin signaling. We therefore suggest ILK modulates VSMC proliferation and migration at least in part by acting as a molecular scaffold in focal adhesions as well as modulating the stability of cell-cell contact proteins and beta-catenin signaling. In summary, ILK plays an important role in intimal thickening by modulating VSMC proliferation and migration via regulation of cell-matrix and cell-cell contacts and beta-catenin signaling.

Mitofusin 2 Triggers Vascular Smooth Muscle Cell Apoptosis via Mitochondrial Death Pathway

Previous studies have shown that mitofusin 2 (Mfn-2) (or hyperplasia suppressor gene [HSG]) inhibits vascular smooth muscle cell (VSMC) proliferation. Here, we demonstrate that Mfn-2 is a primary determinant of VSMC apoptosis. First, oxidative stress with H2O2, inhibition of protein kinase C with staurosporine, activation of protein kinase A with forskolin, and serum deprivation concurrently elevate Mfn-2 expression and induce VSMC apoptosis. Second, overexpression of Mfn-2 also triggers apoptosis of VSMCs in culture and in balloon-injured rat carotid arteries, thus contributing to Mfn-2-mediated prevention of neointima formation after angioplasty. Third, Mfn-2 silencing protects VSMCs against H2O2 or Mfn-2 overexpression-induced apoptosis, indicating that upregulation of Mfn-2 is necessary and sufficient for oxidative stress-mediated VSMC apoptosis. The Mfn-2 proapoptotic effect is independent of its role in mitochondrial fusion but mainly mediated by inhibition of Akt signaling and the resultant activation of the mitochondrial apoptotic pathway, as manifested by decreased Akt phosphorylation, increased mitochondrial Bax/Bcl-2 ratio, cytochrome c release, and activation of caspases-9 and caspase-3. Furthermore, Mfn-2-induced apoptosis was blocked by overexpression of an active phosphoinositide 3-kinase mutant or Bcl-xL or inhibition of caspase-9 but not caspases-8. Thus, in addition to its antiproliferative effects, Mfn-2 constitutes a primary determinant of VSMC apoptosis.

CTRP3/cartducin promotes proliferation and migration of endothelial cells

CTRP3/cartducin, a novel secretory protein, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. CTRP3/cartducin gene is transiently up-regulated in a balloon-injured rat carotid artery tissue. In this study, we report a new function of CTRP3/cartducin as a regulator of angiogenic processes. CTRP3/cartducin promoted proliferation and migration of mouse endothelial MSS31 cells in a dose-dependent manner. Further, stimulation of MSS31 by CTRP3/cartducin led to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). MAPK/ERK kinase 1/2 (MEK1/2) inhibitor, U0126, and p38 MAPK inhibitor, SB203580, blocked the CTRP3/cartducin-induced cell proliferation, and migration was blocked by U0126, but not the SB203580. Taken together, these results suggest that CTRP3/cartducin may be involved as a novel angiogenic factor in the formation of neointima following angioplasty.

MicroRNA Expression Signature and Antisense-Mediated Depletion Reveal an Essential Role of MicroRNA in Vascular Neointimal Lesion Formation

MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.

Butylated hydroxyanisole stimulates heme oxygenase-1 gene expression and inhibits neointima formation in rat arteries.

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation. The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries. Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in an HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation. These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease.

Inhibition of Neointima Formation by Anti-Vascular Endothelial Growth Factor and Receptor-1 Peptides in a Balloon-Injured Rat Carotid Artery

Background and Objectives: Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen. This study was undertaken to test the hypothesis that the neointima hyperplasia induced by a balloon injury is inhibited by blocking VEGF and VEGF receptor-1 (VEGFR-1) with anti-VEGF peptides. Materials and Methods: Anti-VEGF RRKRRR peptide (dRK6) and anti-VEGFR-1 peptide (anti-flt-1) were synthesized at Pohang University of Science and Technology, Korea. Male Sprague-Dawley rats, weighing 300-350 g, were subcutaneously injected 0.5 mg/kg of dRK6 or 0.5 mg/kg of anti-flt-1, dissolved in phosphate buffer solution, 2 days before induction of a carotid balloon-injury, and then daily in the same manner post carotid balloon injury for 2 weeks. Results: Neointima formation was suppressed in both the dRK6 and anti-flt-1 groups compared to that in the untreated controls at 2 weeks post carotid balloon-injury (neointimal area; control group 0.44±0.09 mm 2 , dRK6 group 0.25±0.05 mm 2 , anti-flt-1 group 0.19±0.05 mm 2 , p<0.01). Anti-flt-1 peptide and dRK6 reduced the numbers of proliferative bromodeoxyuridine-labeled cells in the neointima (control group 16.4±10.6%, dRK6 group 3.7±2.1%, anti-flt-1 group 5.9±3.4%, p<0.05). In addition, an inflammatory response, as determined by monocyte chemoattractant protein-1 and interleukin-6 upregulation, which was evident in the controls, was inhibited by both dRK6 and anti-flt-1. Conclusion: This study suggests anti-vascular endothelial growth factor peptides can reduce the inflammation and neointima formation in balloon injured rat carotid arteries. (Korean Circulation J 2007;37:475-482)

Hepatocyte Growth Factor Fusion Protein Having Collagen-Binding Activity (CBD-HGF) Accelerates Re-endothelialization and Intimal Hyperplasia in Balloon-injured Rat Carotid Artery

Hepatocyte growth factor (HGF) is known to stimulate endothelial cell proliferation. However, re-endothelialization is not enhanced when the native protein is administered to the injured artery, probably due to the short half-life of HGF at the site of injury. Therefore, the effects of an HGF fusion protein having collagen-binding activity (CBD-HGF) on re-endothelialization and neointimal formation was studied in the balloon-injured rat carotid artery. The left common carotid artery of male Sprague-Dawley rats was injured with an inflated balloon catheter, and then treated with CBD-HGF 10 microg/mL), HGF (10 micro g/mL) or saline (control) for 15 min. After 14 days, the rats were injected with Evans blue and sacrificed. The re-endothelialized area was significantly greater in the CBD-HGF- treated rats than in the control or HGF -treated rats. Neointimal formation was significantly more pronounced in the CBD-HGF treated rats than in other rat groups. Both HGF and CBD-HGF stimulated proliferation of vascular smooth muscle cells as well as endothelial cells in vitro. Consistent with this, cultured smooth muscle cells were shown to express the HGF receptor (c-Met). CBD-HGF accelerates re-endothelialization and neointimal formation in vivo. CBD fusion protein is a useful vehicle to deliver vascular growth factors to injured arteries.

The lipid peroxidation product 4‐hydroxy‐trans‐2‐nonenal (HNE) promotes unique ER stress responses

Aldehydes such as HNE are major end products of lipid peroxidation. Protein-HNE adducts accumulate in tissues under oxidative stress; however, the mechanisms by which lipid peroxidation-derived aldehydes incite tissue injury remain unclear. We tested the hypothesis that electrophilic aldehydes trigger the unfolded protein response. Exposure of rat aortic smooth muscle cells to HNE (25–200 μM) led to the appearance of multiple anti-protein-HNE immunopositive proteins. Two-dimensional Western blots, immunoprecipitation, and MALDI-TOF MS analyses demonstrated the modification of several ER proteins including Grp78, Grp58, PDI, and reticulocalbin. Exposure to HNE led to activation of the PERK pathway. HNE activated p38 and JNK and increased the expression of HO-1. Pretreatment with Tiron prevented HNE-induced PERK phosphorylation, and inhibition of JNK abrogated HO-1 induction. Balloon-injured rat carotid arteries showed increased neointimal immunoreactivity with anti-protein-HNE antibodies and anti-phospho-PERK antibodies. These results suggest that HNE causes ER stress and activates the alarm and adaptive phases of the unfolded protein response via redox-sensitive mechanisms. These effects of HNE could contribute to the proinflammatory effects of oxidized lipids and to the induction of the unfolded protein response in atherosclerotic lesions and restenotic vessels.