Rat Carotid Injury Model Research Articles

Abstract 103: Acly Inhibition As A Novel Therapeutic Approach For Vascular Remodeling In Coronary Artery Disease

Coronary artery (CoA) disease (CAD) is characterized by neointimal hyperplasia (NH), which is driven by a cancer-like pro-proliferative phenotype of smooth muscle cells (SMCs). A metabolic shift towards glycolysis and changes in the epigenetic landscape contribute to this abnormal phenotype. ATP Citrate Lyase (ACLY) is an enzyme that plays a role in promoting the Warburg effect, lipid synthesis, and chromatin remodeling in cancer. However, its role in CAD remains unknown. We hypothesized that ACLY is upregulated in CAD and supports the abnormal phenotype of CAD-CoASMCs. To investigate this hypothesis, we measured ACLY expression in human distal coronary arteries and isolated CoASMCs from CAD patients. We found increased expression and activation of ACLY in CAD-CoASMCs, with preferential localization in the nucleus. Inhibition of ACLY using BMS-303141 or siACLY decreased CAD-CoASMCs proliferation and survival. ACLY inhibition also reduced glycolysis markers and increased mitochondrial respiration. Additionally, ACLY inhibition lowered cholesterol levels and lipid droplet accumulation in CAD-CoASMCs. Further analysis showed that ACLY promotes nuclear acetyl-CoA production, leading to acetylation of specific histone proteins and GCN5-mediated transcriptional activation of genes involved in cell cycle progression. The transcription factor FOXM1 appeared to mediate this genetic signature. In vivo , pharmacological inhibition of ACLY using BMS-303141 attenuated NH in a rat model of carotid injury (CI). SMC-specific Acly K.O mice were protected against CI-induced NH. Consistently, tamoxifen-induced SMC-specific Acly deletion mitigated NH in mice subjected to CI. The therapeutic effects were associated with reduced histone acetylation. To assess the translational potential of our findings, we tested the effects of ACLY inhibition on ex vivo human coronary artery and saphenous vein explants stimulated with growth factors cocktail. We found that ACLY inhibition prevent NH development, supporting the potential of ACLY as a therapeutic target. In conclusion, our study reveals that ACLY plays a critical role in vascular remodeling in CAD and its pharmacological inhibition may represent a novel avenue as therapeutic treatment.

Perivascular application of sirolimus multilayer nanofibrous mat for prevention of vascular stenosis: Preparation, In vitro characterization, and In vivo efficacy evaluation

Graft stenosis, occurring after coronary artery bypass surgery (CABG), diminishes the efficacy and long-term patency of vascular interventions. Multilayer nanofibers (NFs) are developed to provide localized controlled release of sirolimus to prevent vascular stenosis, as well as limit drug exposure to other organs. NFs are composed of a mid-layer of drug and polycaprolactone (PCL), a bioadhesive layer of PCL and collagen, and a backing layer of PCL. NFs are characterized in vitro regarding morphology, mechanical strength, contact angle, water uptake, drug content, and drug release behavior. The average fiber diameter is around 300–500 nm. Sirolimus entrapment is confirmed by drug content, FTIR, and XRD. It is found that sirolimus is released from multilayer NFs in a sustained manner with about half of the payload released over one week. Sirolimus multilayer NFs are implanted in the rat carotid artery after inducing vascular injury. Histological, immunohistochemical (α-SMA, Ki-67, and CD31), and in vivo imaging analyses reveal that the administration of sirolimus multilayer NFs causes significant inhibition of intimal hyperplasia in the rat carotid injury model compared to the control groups and drug suspension. The obtained results reveal that the sirolimus multilayer nanosystem could be a promising carrier for local vascular drug delivery.

O-Linked β-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked β-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. These results suggest that O-GlcNAcylation of A20 plays a key role in the negative regulation of NF-κB signaling cascades in TNF-α-treated vascular smooth muscle cells in culture and in acutely injured arteries, thus protecting against inflammation-induced vascular injury.

Restenosis Inhibition and Re-differentiation of TGF\u03b2/Smad3-activated Smooth Muscle Cells by Resveratrol

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies — a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

PET Study of Sphingosine-1-Phosphate Receptor 1 Expression in Response to Vascular Inflammation in a Rat Model of Carotid Injury.

Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [11C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [11C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction of the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [11C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [11C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [11C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.

Epothilones Suppress Neointimal Thickening in the Rat Carotid Balloon-Injury Model by Inducing Vascular Smooth Muscle Cell Apoptosis through p53-Dependent Signaling Pathway.

Microtubule stabilizing agents (MTSA) are known to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and effectively reduce neointimal hyperplasia and restenosis. Epothilones (EPOs), non-taxane MTSA, have been found to be effective in the inhibition of VSMC proliferation and neointimal formation by cell cycle arrest. However, effect of EPOs on apoptosis in hyper-proliferated VSMCs as a possible way to reduce neointimal formation and its action mechanism related to VSMC viability has not been suited yet. Thus, the purposes of the present study was to investigate whether EPOs are able to inhibit neointimal formation by inducing apoptosis within the region of neointimal hyperplasia in balloon-injured rat carotid artery, as well as underlying action mechanism. Treatment of EPO-B and EPO-D significantly induced apoptotic cell death and mitotic catastrophe in hyper-proliferated VSMCs, resulting in cell growth inhibition. Further, EPOs significantly suppressed VSMC proliferation and induced apoptosis by activation of p53-dependent apoptotic signaling pathway, Bax/cytochrome c/caspase-3. We further demonstrated that the local treatment of carotid arteries with EPOs potently inhibited neointimal lesion formation by induction of apoptosis in rat carotid injury model. Our findings demonstrate a potent anti-neointimal hyperplasia property of EPOs by inducing p53-depedent apoptosis in hyper-proliferated VSMCs.

Effect of imatinib mesylate and rapamycin on the preformed intimal hyperplasia in rat carotid injury model.

PurposeIntimal hyperplasia (IH) is the main cause of restenosis or occlusion after vascular procedures. Imatinib mesylate and rapamycin are known to prevent IH. The purpose of this study was to evaluate the effect of these drugs on the regression of preformed IH in rat carotid injury model.MethodsIH was established in rat carotid arteries using a balloon catheter. The drug effects were assessed in vitro on proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMC) in the neointima. And in vivo studies were carried out in 4 groups: imatinib, rapamycin, combined, and no medication. After 2-week oral medication, morphometric analysis evaluated the number and density of neointimal cells, intima-to-media (I/M) ratio and cross-sectional area. Cell proliferation, apoptosis, and collagen changes were also investigated by immunohistochemical staining (IHCS).ResultsImatinib and rapamycin significantly inhibited VSMC proliferation and migration, and promoted apoptosis in vitro. In morphometric analysis, the number and density of neointimal cells decreased significantly in all medication groups compared with control group (P < 0.01). However, there was no significant difference in neointimal cross-sectional area and I/M ratio among groups. In IHCS, imatinib and rapamycin inhibited neointimal cell proliferation significantly. However, there was no significant change in cell apoptosis and collagen composition.ConclusionCombined treatment of with imatinib and rapamycin induced reduction of cell mass in preformed intimal hyperplasia, but failed to induce regression of intimal mass in this short-term medication study. Further studies will be needed with additional strategies of inducing lysis of the extracellular matrix.

Beclin 1 knockdown retards re-endothelialization and exacerbates neointimal formation via a crosstalk between autophagy and apoptosis

ObjectiveEndothelial regeneration is an essential process for the prevention of excessive neointimal formation following endothelial denudation. Beclin 1, a mammalian autophagy gene, is a link between autophagy and apoptosis. We hypothesized that the interference of Beclin 1 can influence re-endothelialization and ultimately affect neointimal formation by regulating autophagy and apoptosis. MethodsA rat carotid injury model of endothelial denudation was used, and small interfering RNA of Beclin 1 was perivascularly administered. Neointima was evaluated by morphological analysis. von Willebrand factor, Beclin 1, LC3, autophagic substrate p62 and caspase-3 levels were detected by immunofluorescence or Western blotting. Terminal deoxynucleotidyl transferase-mediated digoxigenin–dUTP–biotin nick-end labeling assay was performed to evaluate apoptosis. ResultsCarotid injury induced an upregulation of Beclin 1 protein which was down regulated by more than 50% with small RNA interference. Beclin 1 knockdown significantly retarded re-endothelialization 7 days after injury and subsequently augmented neointima by more than 2 folds at 14 and 21 days. Autophagy and apoptosis were detected to reveal the regulatory effect of Beclin 1. The injury-activated autophagy, shown by the increased levels of punctate LC3 and LC3II as well as decreased p62 expression, was significantly inhibited by Beclin 1 knockdown. Meanwhile, the apoptotic endothelial cell number was increased and caspase-3 was up-regulated, though the expression of truncated BID was not significantly influenced. ConclusionBeclin 1 knockdown exacerbated neointimal formation after rat carotid injury, associated with retarded re-endothelialization due to enhanced apoptosis, while simultaneously prohibiting autophagic activation. The data suggested an essential role of Beclin 1 as a regulator between autophagy and apoptosis in the setting of neointimal formation.

High-Throughput Screening Identifies Idarubicin as a Preferential Inhibitor of Smooth Muscle versus Endothelial Cell Proliferation

Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection). We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization.

Sirolimus-loaded stealth colloidal systems attenuate neointimal hyperplasia after balloon injury: A comparison of phospholipid micelles and liposomes

Restenosis after angioplasty remains a serious complication in clinical cardiology. This study aims to investigate the stealth colloidal systems for local intra-arterial drug delivery. Micelles from polyethylene glycol conjugated with phosphatidylethanolamine and PEGylated liposomes loaded with sirolimus were prepared and characterized with regard to their loading efficiency, particle size distribution, zeta potential, morphology, nuclear magnetic resonance spectroscopy, drug release profile and stability. The antirestenotic effects of the sirolimus-loaded micelles (14 nm) and liposomes (90 nm) were evaluated and compared in the rat carotid injury model following local intravascular delivery. In comparison to control groups, treatment of balloon injured rats with drug loaded micelles and nanoliposomes significantly reduced vascular stenosis by 42% and 19%, respectively (P<0.05). In addition, the luminal area was significantly enlarged by 39% and 60% following treatment with sirolimus-loaded liposomes and micelles, respectively (P<0.05). Immunohistochemistry revealed that sirolimus-loaded nanocarriers suppressed cell proliferation (Ki67-positive cells) as compared to control groups without affecting the density of smooth muscle actin staining. These results suggest that both colloidal nanocarriers could serve as effective intramural drug delivery systems for the treatment of restenosis; however, phospholipid based micelles provided better antirestenotic effects than PEGylated liposomes.

DNA Enzyme ED5 Depletes Egr-1 and Inhibits Neointimal Hyperplasia in Rats

Objectives: Depletion of early growth response factor-1 (Egr-1) by a DNA enzyme, ED5, inhibits neointimal hyperplasia (NH) following vascular injury by an unknown mechanism. The aim of this study was to characterize the effects of ED5 in a rat carotid injury model in order to elucidate the mechanism by which ED5 inhibits NH. Methods: ED5 was transfected into the arterial wall of Wistar rats using FuGENE6 transfection reagent following artery balloon injury. Hematoxylin and eosin staining, immunohistochemistry, real-time reverse transcription polymerase chain reaction and Western blotting analysis were used to characterize the response to ED5. Results: NH decreased significantly in the ED5- plus FuGENE6-treated rats (p < 0.05) compared with the control groups, and this was accompanied by a reduced inflammatory response. Egr-1 mRNA and protein levels were significantly decreased in the ED5-treated group, as expected. The decrease in Egr-1 was accompanied by decreases in the mRNA and protein levels of PDGF-BB, Cyclin D1, CDK4, MCP-1, and ICAM-1 (p < 0.05). Conclusions: Transfection of the Egr-1-specific synthetic DNA enzyme ED5 significantly reduced NH after injury in rats, at least in part, as a result of decreased expression of downstream proliferative genes such as PDGF-BB, Cyclin D1, CDK4, and the inflammatory factors MCP-1 and ICAM-1.

Nitrite activates AMP kinase to stimulate mitochondrial biogenesis independent of soluble guanylate cyclase

Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance, and vascular remodeling. Although the exact molecular mechanisms underlying nitrite’s actions are unknown, the current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from that of NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and deacetylation of PGC1α, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, 2 weeks of continuous oral nitrite treatment postinjury prevented the hyperproliferative response of smooth muscle cells. This protection was accompanied by a nitrite-dependent upregulation of PGC1α and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling compared to NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.

SERCA2a gene transfer prevents intimal proliferation in an organ culture of human internal mammary artery

Coronary restenosis, a major complication of percutaneous balloon angioplasty, results from neointimal proliferation of vascular smooth muscle cells (VSMCs). The sarco/endoplasmic reticulum calcium ATPase isoform 2a (SERCA2a), specific to contractile VSMCs, has been reported previously to be involved in the control of the Ca2+-signaling pathways governing proliferation and migration. Moreover, SERCA2a gene transfer was reported to inhibit in vitro VSMC proliferation and to prevent neointimal thickening in a rat carotid injury model.The aim of this study was to evaluate the potential therapeutic interest of SERCA2a gene transfer for prevention of in-stent restenosis using a human ex vivo model of left internal mammary artery (hIMA) intimal thickening.Left hIMAs, obtained at the time of aorto-coronary bypass surgeries, were subjected to balloon dilatation followed by infection for 30 min with adenoviruses encoding either human SERCA2 and GFP or control gene (beta-galactosidase) and GFP. Proliferation of subendothelial VSMCs and neointimal thickening were observed in balloon-injured hIMA maintained 14 days in organ culture under constant pressure and perfusion. SERCA2a gene transfer prevented vascular remodeling and significantly (p<0.01, n=5) reduced neointimal thickening in injured arteries (intima/media ratio was 0.07 ± 0.01 vs 0.40 ± 0.03 in βGal-infected arteries).These findings could have potential implications for treatment of pathological in stent-restenosis.

Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly 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 evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (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 small interfering RNA (siRNA). 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. In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA 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 proliferating cell nuclear antigen. 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 TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells.

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.

Modulation of Phenotypic Differentiation Accounts for the Durable Inhibition of Neointimal Hyperpasia By Nitric Oxide

Introduction: Neointimal hyperplasia is a major cause of restenosis following vascular interventions, often leading to procedure failure. Periadventitial delivery of the nitric oxide (NO) donor PROLI/NO provides durable inhibition of neointimal hyperplasia for up to 6 months despite its very short half-life. the aim of this study is to characterize the effect of NO on the phenotypic differentiation of vascular cells following injury. We hypothesize that modulation of cell phenotype within the arterial wall accounts for the durable efficacy of NO at inhibiting neointimal hyperplasia. Methods: The rat carotid injury model was performed in 10-week-old male Sprague Dawley rats. Groups included control, injury, or injury +PROLI/NO (10 mg). Arteries were harvested at 1, 2, 7, 14, and 56 days for immunohistochemistry. Proliferation was assessed by BrdU incorporation. Desmin was used to detect contractile vascular smooth muscle cells (VSMC), non-muscle myosin heavy chain (NM-MHC) was used to detect myofibroblasts (MF), and α -smooth muscle actin (α -SMA) was used to detect MF and VSMC (both synthetic and contractile phenotypes). Results: Overall, injury induced a significant increase in cell proliferation at 3 days in all layers. in the intima and in the media, NO inhibited proliferation for up to 8 weeks. Whereas in the adventitia, NO induced a surprising 60% increase in proliferation at 2 weeks (p<0.05). Assessment of the phenotypic markers revealed interesting patterns. Injury caused a significant increase in all three phenotypic markers (p<0.05). However, while contractile VSMC continued to increase by 8 weeks, MF returned to basal levels. Assessment of the individual layers of the arterial wall revealed distinct effects of NO on cell phenotype. in the intima, NO inhibited VSMC and MF to a similar extent up to 1 week (desmin 80%, α-SMA 80%, and NM-MHC 80% inhibition, p<0.05). However, by 8 weeks, NO inhibited MF to a greater extent than VSMC (NM-MHC 70% vs. desmin 50% inhibition, p<0.05). in the media, NO predominately inhibited the contractile VSMC phenotype (80% at 8 weeks; p<0.05), with little effect on MF (p=0.556). in the adventitia, NO appeared to have no effect on either VSMC or AF. Conclusions: The short-acting NO donor PROLI/NO inhibits neointimal hyperplasia by modulating proliferation and phenotypic differentiation in the arterial wall. While NO inhibited both VSMC and MF in the intima, NO predominately inhibited contractile VSMC in the media, and had no effect on VSMC or MF in the adventitia. Thus, the increase in proliferation observed in the adventitia is due to regulation of cell types other than VSMC and MF, such as endothelial cells of the vasa vasorum or resident or circulating progenitor cells. Further studies are needed to elucidate the effect of NO on the adventitia and its role in inhibiting neointimal hyperplasia.

A Novel All-Trans Retinoic Acid Perivascular Wrap Reduces Neointimal Hyperplasia Following Arterial Balloon Injury

Introduction: All-trans retinoic acid (ATRA) is a vitamin A derivative that has many beneficial effects on the vasculature, including inhibition of vascular smooth muscle cell (VSMC) proliferation and stimulation of nitric oxide (NO) release. Thus, through a multidisciplinary collaboration, we developed a novel ATRA perivascular wrap utilizing a biodegradable citric-acid based polymer, poly (diol-citrate) (POC). The goal of our current study was to characterize the properties of ATRA wraps in vitro and evaluate the efficacy of our ATRA wrap to inhibit neointimal hyperplasia in vivo. We hypothesized that treatment with ATRA perivascular wraps would decrease the formation of neointimal hyperplasia with a concomitant decrease in proliferation. Methods: ATRA wraps were fabricated using POC wraps loaded with ATRA dissolved in DMSO to a final concentration of 20 mg/ml. ATRA release was measured over time and was confirmed using HPLC. VSMC proliferation was assessed by measuring DNA content. The rat carotid injury model was performed in 10 week old male Sprague Dawley rats. Treatment groups included: control, injury, and injury +ATRA wrap (1cm x 1cm). n=6-7/group. Arteries were harvested at 14 days. Morphometric analysis was performed on H&E stained cross-sections utilizing Image J software. Immunohistochemistry was performed to assess for BrdU incorporation and macrophage infiltration (ED1 staining). Results: In vitro, ATRA release from the wraps occurred for 14 days, with the greatest release in the first 7 days. ATRA wraps inhibited VSMC proliferation by approximately 75% at 7 days (p<0.05) compared to controls. In vivo, ATRA wraps resulted in significant inhibition of neointimal hyperplasia, with a 56.3% reduction in intimal area (p<0.001) and a 57% reduction in the intima/media area ratio (p<0.001) as compared to injury alone. We observed no significant changes in the lumen or medial area. With respect to proliferation, ATRA wraps resulted in a decreasing trend in proliferation for both the intimal and medial layers, with a slight increase in the adventitia. However, none of these changes reached statistical significance. With respect to inflammation, treatment with ATRA wraps resulted in increased macrophage infiltration compared to injury alone (1.8-fold, p<0.001). Conclusions: Neointimal hyperplasia was significantly inhibited by the citric acid-based biodegradable ATRA-eluting perivascular wrap without significant effect on vessel wall remodeling. This therapy represents a novel method to deliver ATRA to the vasculature to inhibit neointimal hyperplasia. the increased inflammatory response, while not anticipated, is mostly likely due to mass effect of the wrap in the small neck of the rat. Further study of this therapy is warranted in a large animal model.

In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid–polymeric nanoparticles

Following recent successes with percutaneous coronary intervention (PCI) for treating coronary artery disease (CAD), many challenges remain. In particular, mechanical injury from the procedure results in extensive endothelial denudation, exposing the underlying collagen IV-rich basal lamina, which promotes both intravascular thrombosis and smooth muscle proliferation. Previously, we reported the engineering of collagen IV-targeting nanoparticles (NPs) and demonstrated their preferential localization to sites of arterial injury. Here, we develop a systemically administered, targeted NP system to deliver an antiproliferative agent to injured vasculature. Approximately 60-nm lipid-polymeric NPs were surface functionalized with collagen IV-targeting peptides and loaded with paclitaxel. In safety studies, the targeted NPs showed no signs of toxicity and a ≥3.5-fold improved maximum tolerated dose versus paclitaxel. In efficacy studies using a rat carotid injury model, paclitaxel (0.3 mg/kg or 1 mg/kg) was i.v. administered postprocedure on days 0 and 5. The targeted NP group resulted in lower neointima-to-media (N/M) scores at 2 wk versus control groups of saline, paclitaxel, or nontargeted NPs. Compared with sham-injury groups, an ∼50% reduction in arterial stenosis was observed with targeted NP treatment. The combination of improved tolerability, sustained release, and vascular targeting could potentially provide a safe and efficacious option in the management of CAD.

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [ 3H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

Early Modulation Of The Injury Response By The Short-acting NO Donor PROLI/NO Leads To Durable Inhibition Of Neointimal Hyperplasia

Introduction: We have previously demonstrated that periadventitial delivery of the NO donor PROLI/NO provides durable inhibition of neointimal hyperplasia for up to 8 weeks in spite of its very short half-life of 2 seconds under physiologic conditions in vitro. The goal of this current study was to examine the effect of PROLI/NO on the arterial wall in the first 7 days following injury. Our hypothesis was that early modulation of cell populations in the arterial wall accounts for the durable efficacy of NO at inhibiting neointimal hyperplasia. Methods: The rat carotid injury model was performed in 10wk old male Sprague Dawley rats. Treatment groups included control, injury, or injury +PROLI/NO (10mg). Arteries were harvested at 2, 24, 72 hours and 7 days for immunohistochemistry as well as protein assessment via Western blot analysis. Total cell counts were performed on H&E stained sections. Proliferation was assessed with BrdU incorporation. Phenotypic differentiation was assessed by desmin and αSMC-actin expression. Results: Compared to uninjured arteries, overall cellularity was increased by 60% 7 days after injury (p<0.001). Increased cellularity was due to an 80% increase in cell number in the intima and adventitia, respectively (p<0.05). However, injury decreased cell number in the media at all time points by up to 85% (p<0.001). Treatment with NO prevented the increased cellularity produced with injury primarily by preventing neointima formation and repopulation of the media. The effects of NO were seen as early as 2 hours. At 7 days NO-treated arteries had cellularity similar to the uninjured artery and 69% fewer cells compared to injury alone (p≤0.009), with an 87% reduction in the intima and media respectively (p<0.05). NO treated arteries also had fewer cells in the adventitia compared to injury but to a lesser extent with a 54% reduction in cell number at 7 days. NO also inhibited proliferation up to 78%, 95% and 80% in the intima, media and adventitia, respectively (p<0.04). Compared to injury alone, NO-treated arteries inhibited the ubiquitous VSMC marker αSMC-actin at all time points with a 10-fold reduction seen at 7 days compared to injury. In contrast, there was a dramatic decrease in the VSMC specific marker desmin at 2 and 24 hours with NO compared to injury and a subsequent 2-fold increase in expression at the later time points. Conclusions: The short-acting NO-donor PROLI/NO inhibits neointimal hyperplasia via modulation of proliferation and phenotypic differentiation in the arterial wall as early as 2 hours following injury. These data suggest that the early effects of NO on the arterial injury response are ultimately responsible for the durability of this short-acting donor.