Inhibits Neointima Formation Research Articles

Ca2+ Entry Through Reverse Mode Na+/Ca2+ Exchanger Contributes to Store Operated Channel-Mediated Neointima Formation After Arterial Injury

BackgroundNa+/Ca2+ exchange (NCX) reversal-mediated Ca2+ entry is a critical pathway for stimulating proliferation in many cell lines. However, the role of reverse-mode NCX1 in neointima formation and atherosclerosis remains unclear. The aims of the present study were to investigate the functional role of NCX1 in the pathogenesis of atherosclerosis and vascular smooth muscle cell (VSMC) proliferation, and to determine the interaction between NCX1 and store depletion in VSMCs. MethodsA rat balloon injury model was established to examine the effect of the knockdown of NCX1 on neointima formation after injury. VSMCs were cultured to verify that NCX1 knockdown suppressed serum-induced VSMC proliferation. ResultsThe results showed that balloon injury induced neointima formation and upregulated NCX1 expression at 7 and 14 days after injury in rat carotid arteries (1.18- and 1.45-fold, respectively). A lentivirus vector expressing short hairpin (sh)RNA against rat NCX1 dramatically downregulated NCX1, proliferating cell nuclear antigen (PCNA) and Ki-67 expression, and suppressed neointima formation in vivo (62% at 7 days and 70% at 14 days). KB-R7943 (an inhibitor of reverse-mode NCX1) and NCX1 knockdown significantly inhibited serum-induced VSMC proliferation (65% at 72 hours and 41% at 72 hours, respectively), determined according to PCNA and Ki-67 expression and cell counting in vitro, and markedly suppressed store depletion-mediated Ca2+ entry and peripheral cytosolic Na+ transients in VSMCs. ConclusionsReverse-mode NCX1 is activated by store depletion and is required for proliferative VSMC proliferation and neointima formation after arterial injury.

Cholinergic anti-inflammatory pathway inhibits neointimal hyperplasia by suppressing inflammation and oxidative stress

Neointimal hyperplasia as a consequence of vascular injury is aggravated by inflammatory reaction and oxidative stress. The α7 nicotinic acetylcholine receptor (α7nAChR) is a orchestrator of cholinergic anti-inflammatory pathway (CAP), which refers to a physiological neuro-immune mechanism that restricts inflammation. Here, we investigated the potential role of CAP in neointimal hyperplasia using α7nAChR knockout (KO) mice. Male α7nAChR-KO mice and their wild-type control mice (WT) were subjected to wire injury in left common carotid artery. At 4 weeks post injury, the injured aortae were isolated for examination. The neointimal hyperplasia after wire injury was significantly aggravated in α7nAChR-KO mice compared with WT mice. The α7nAChR-KO mice had increased collagen contents and vascular smooth muscle cells (VSMCs) amount. Moreover, the inflammation was significantly enhanced in the neointima of α7nAChR-KO mice relative to WT mice, evidenced by the increased expression of tumor necrosis factor-α/interleukin-1β, and macrophage infiltration. Meanwhile, the chemokines chemokine (C-C motif) ligand 2 and chemokine (CXC motif) ligand 2 expression was also augmented in the neointima of α7nAChR-KO mice compared with WT mice. Additionally, the depletion of superoxide dismutase (SOD) and reduced glutathione (GSH), and the upregulation of 3-nitrotyrosine, malondialdehyde and myeloperoxidase were more pronounced in neointima of α7nAChR-KO mice compared with WT mice. Accordingly, the protein expression of NADPH oxidase 1 (Nox1), Nox2 and Nox4, was also higher in neointima of α7nAChR-KO mice compared with WT mice. Finally, pharmacologically activation of CAP with a selective α7nAChR agonist PNU-282987, significantly reduced neointima formation, arterial inflammation and oxidative stress after vascular injury in C57BL/6 mice. In conclusion, our results demonstrate that α7nAChR-mediated CAP is a neuro-physiological mechanism that inhibits neointima formation after vascular injury via suppressing arterial inflammation and oxidative stress. Further, these results imply that targeting α7nAChR may be a promising interventional strategy for in-stent stenosis.

Intermedin reduces neointima formation by regulating vascular smooth muscle cell phenotype via cAMP/PKA pathway

Background and aimsVascular smooth muscle cell (VSMC) dedifferentiation contributes to neointima formation, which results in various vascular disorders. Intermedin (IMD), a cardiovascular paracrine/autocrine polypeptide, is involved in maintaining circulatory homeostasis. However, whether IMD protects against neointima formation remains largely unknown. The purpose of this study is to investigate the role of IMD in neointima formation and the possible mechanism. MethodsIMD1-53 (100ng/kg/h) or saline water was used on rat carotid-artery balloon-injury model. The mouse left common carotid-artery ligation-injury model was established using IMD-transgenic and C57BL/6J mice. Immunohistochemistry and immunofluorescence staining was used to detect the protein expression in rat carotid arteries. Radioimmunoassay was used to determine the serum IMD level. The hematoxylin andeosin staining was used for carotid arteries morphological testing. In vitro, for rat primary cultured VSMC phenotype transition, proliferation and migration assays, platelet-derived growth factor-BB (PDGF-BB) reagent and IMD1-53 peptide were added to the culture media at the final concentration of 20 ng/mL and 10-7mol/L respectively. Quantification of VSMC proliferation involved MTT and BrdU assay and migration was detected by wound-healing assay. Western blot and realtime PCR were used to detect the protein and mRNA levels of tissues or cells. ResultsWith the rat carotid-artery balloon-injury model, IMD was significantly downregulated in injured arteries and plasma. Exogenous IMD1-53 greatly inhibited neointima formation and prevented VSMC from switching to a synthetic phenotype. With the left common carotid-artery ligation-injury model, IMD-transgenic mice showed less neointima formation than C57BL/6J mice. PDGF-BB reduced IMD mRNA expression in rat primary cultured VSMCs but increased that of its receptors, calcitonin receptor-like receptor or receptor activity-modifying proteins. Furthermore, PDGF-BB promoted VSMC proliferation and migration and transformed VSMCs to the synthetic phenotype, which was reversed with IMD1-53 treatment. Mechanistically, IMD1-53 maintained the contractile VSMC phenotype via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. ConclusionsIMD attenuated neointima formation both in the rat model of carotid-artery balloon injury and mouse model of common carotid-artery ligation injury. IMD protection may be mediated by maintaining a VSMC contractile phenotype via the cAMP/PKA pathway.

Small molecule-mediated induction of miR-9 suppressed vascular smooth muscle cell proliferation and neointima formation after balloon injury.

Pathologic proliferation and migration of vascular smooth muscle cells (VSMCs) exacerbate cardiovascular disease. MicroRNAs (miRNAs), as endogenous inhibitors of protein synthesis, are expected to modulate pathologic proliferation of VSMCs. Here we report that both platelet-derived growth factor receptor (PDGFR) targeting miR-9 and a small molecule that increases miR-9 can inhibit the serum-induced proliferation of VSMCs. First, based on miRNA-target prediction databases and empirical data, we have selected miR-9 as a potent anti-proliferative miRNA in VSMCs. Further examination indicated that miR-9 directly targets PDGFR disrupting downstream signaling cascades, and this resulted in inhibition of VSMC proliferation and migration. Exogenous delivery of miR-9 inhibited VSMC proliferation in vitro, and a small molecule that increased miR-9 expression also inhibited neointima formation following balloon injury in vivo. We provide evidence of miRNA-mediated modulation of VSMC proliferation and further demonstrate that small molecule-mediated regulation of miRNA targeting a key regulator of VSMC proliferation is a viable therapeutic strategy for treating vascular disease involving pathologic VSMC proliferation such as restenosis.

Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser163 and Ser177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.

Novel Pathological Role of hnRNPA1 (Heterogeneous Nuclear Ribonucleoprotein A1) in Vascular Smooth Muscle Cell Function and Neointima Hyperplasia.

hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) plays a variety of roles in gene expression. However, little is known about the functional involvement of hnRNPA1 in vascular smooth muscle cell (VSMC) function and neointima hyperplasia. In this study, we have attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC function, injury-induced vessel remodeling, and human atherosclerotic lesions, as well as discern the molecular mechanisms involved. APPROACH AND RESULTS: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation, and migration were regulated by hnRNPA1. Our data show that hnRNPA1 exerts its effects on VSMC functions through modulation of IQGAP1 (IQ motif containing GTPase activating protein 1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through 2 mechanisms: upregulating microRNA-124 (miR-124) and binding to AU-rich element of IQGAP1 gene. Further evidence suggests that hnRNPA1 upregulates miR-124 by modulating miR-124 biogenesis and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. Our data show that hnRNPA1 is a critical regulator of VSMC function and behavior in the context of neointima hyperplasia, and the hnRNPA1/miR-124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.

Interferon Regulatory Factor 4 Inhibits Neointima Formation by Engaging Krüppel-Like Factor 4 Signaling.

The mechanisms underlying neointima formation remain unclear. Interferon regulatory factors (IRFs), which are key innate immune regulators, play important roles in cardiometabolic diseases. However, the function of IRF4 in arterial restenosis is unknown. IRF4 expression was first detected in human and mouse restenotic arteries. Then, the effects of IRF4 on neointima formation were evaluated with universal IRF4-deficient mouse and rat carotid artery injury models. We performed immunostaining to identify IRF4-expressing cells in the lesions. Smooth muscle cell (SMC)-specific IRF4-knockout (KO) and -transgenic (TG) mice were generated to evaluate the effects of SMC-IRF4 on neointima formation. We used microarray, bioinformatics analysis, and chromatin immunoprecipitation assay to identify the downstream signals of IRF4 and to verify the targets in vitro. We compared SMC-IRF4-KO/Krüppel-like factor 4 (KLF4)-TG mice with SMC-IRF4-KO mice and SMC-specific IRF4-TG/KLF4-KO mice with SMC-specific IRF4-TG mice to investigate whether the effect of IRF4 on neointima formation is KLF4-dependent. The effect of IRF4 on SMC phenotype switching was also evaluated. IRF4 expression in both the human and mouse restenotic arteries is eventually downregulated. Universal IRF4 ablation potentiates neointima formation in both mice and rats. Immunostaining indicated that IRF4 was expressed primarily in SMCs in restenotic arteries. After injury, SMC-IRF4-KO mice developed a thicker neointima than control mice. This change was accompanied by increased SMC proliferation and migration. However, SMC-specific IRF4-TG mice exhibited the opposite phenotype, demonstrating that IRF4 exerts protective effects against neointima formation. The mechanistic study indicated that IRF4 promotes KLF4 expression by directly binding to its promoter. Genetic overexpression of KLF4 in SMCs largely reversed the neointima-promoting effect of IRF4 ablation, whereas ablation of KLF4 abolished the protective function of IRF4, indicating that the protective effects of IRF4 against neointima formation are KLF4-dependent. In addition, IRF4 promoted SMC dedifferentiation. IRF4 protects arteries against neointima formation by promoting the expression of KLF4 by directly binding to its promoter. Our findings suggest that this previously undiscovered IRF4-KLF4 axis plays a key role in vasculoproliferative pathology and may be a promising therapeutic target for the treatment of arterial restenosis.

The role of monocyte subpopulations in vascular injury following partial and transient depletion.

The innate immunity system plays a critical role in vascular repair and restenosis development. Liposomes encapsulating bisphosphonates (LipBPs), but not free BPs, suppress neointima formation following vascular injury mediated in part by monocytes. The objective of this study was to elucidate the role of monocyte subpopulations on vascular healing following LipBP treatment. The potency- and dose-dependent treatment effect of clodronate (CLOD) and alendronate (ALN) liposomes on restenosis inhibition, total monocyte depletion, and monocytes subpopulation was studied. Rats subjected to carotid injury were treated by a single IV injection of LipBPs at the time of injury. Low- and high-dose LipALN treatment (3 and 10mg/kg, respectively) resulted in a dose-dependent effect on restenosis development after 30days. Both doses of LipALN resulted in a dose-dependent inhibition of restenosis, but only high dose of LipALN depleted monocytes (-60.1±4.4%, 48h post injury). Although LipCLOD treatment (at an equivalent potency to 3mg/kg alendronate) significantly reduced monocyte levels (72.1±6%), no restenosis inhibition was observed. The major finding of this study is the correlation found between monocyte subclasses and restenosis inhibition. Non-classical monocyte (NCM) levels were found higher in LipALN-treated rats, but lower in LipCLOD-treated rats, 24h after injury and treatment. We suggest that the inhibition of circulating monocyte subpopulations is the predominant mechanism by which LipBPs prevent restenosis. The effect of LipBP treatment on the monocyte subpopulation correlates with the dose and potency of LipBPs.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

Mitoxantrone suppresses vascular smooth muscle cell (VSMC) proliferation and balloon injury-induced neointima formation: An in vitro and in vivo study.

Neointima formation, which occurs after vascular injury due to vascular disease or interventions such as angioplasty and stent placement, is a complex process that involves multiple molecular and cellular mechanisms. The inhibition of neointima formation is vital to prevent restenosis of blood vessels. In the present study, we investigated whether the systemic administration of mitoxantrone can inhibit neointima formation, and evaluated the underlying mechanisms under in vitro and in vivo experimental conditions. In vitro, rat and human vascular smooth muscle cells (RVSMCs and HVSMCs) were stimulated with platelet-derived growth factor-BB (PDGF-BB) and treated with mitoxantrone or DMSO as a control. In vivo, 54 male Sprague-Dawley rats were subjected to carotid artery balloon injury and then intravenously administered with mitoxantrone. Cell proliferation was determined using the CCK-8 assay. Cell cycle analysis was performed using flow cytometry, and protein expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We used monoclonal mouse anti-bromodeoxyuridine (BrdU) antibody for the detection of BrdU and anti-Topoisomerase II antibody for staining Type II topoisomerase (Topo II), one week after the ballon injury. In both RVSMCs and HVSMCs, mitoxantrone treatment induced Topo II degradation, as well as suppressed DNA replication, cell cycle progression, and VSMC proliferation. A reduction in intimal hyperplasia, intimal-to-medial area ratio, and Topo II level was observed in mitoxantrone-treated rats, as compared to the control (saline) group. Overall, our results indicate that systemic administration of mitoxantrone can reduce neointimal hyperplasia and, thus, represents a suitable option for restenosis treatment.

214 A histone deacetylase 7-derived 7-amino acid peptide acts as a phosphorylation carrier

Histone deacetylase 7 (HDAC7) belongs to the class II HDAC family and plays a pivotal role in the maintenance of endothelium integrity. There are 8 splicing variants in mouse HDAC7...

Atorvastatin treatment modulates p16 promoter methylation to regulate p16 expression.

Intimal hyperplasia, the key event of arterial restenosis, is a result of cell proliferation and cell migration. Atorvastatin exerts an inhibitory effect on cell proliferation and migration, but the mechanism remains largely unknown. p16, as a well-known tumor suppressor, was also reported to suppress cell growth and migration, but with an unclear mechanism. In this study, we demonstrated that atorvastatin represses cell proliferation and migration in vascular smooth muscle cells (VSMCs) and that this process is mediated by p16. Furthermore, we found that DNA methylation in the p16 promoter was reduced and p16 expression was restored in VSMCs treated with 5-aza-2'-deoxycytidine or atorvastatin. However, the effect was absent when DNA methyltransferase 1 (DNMT1) was knocked down with RNA interference. These observations demonstrated that atorvastatin regulates p16 expression via DNMT1-induced DNA methylation in the p16 promoter. In addition, we found that the mitogen-activated protein kinase (MAPK) pathway was involved in the regulation of p16 by DNMT1, and MAPK inhibitors partially released the effects of atorvastatin on p16 and DNMT1. Finally, we illustrated that atorvastatin inhibits neointima formation and modulates p16 expression in balloon catheter-injured rat carotid artery. Taken together, we demonstrated that atorvastatin inhibits neointima formation through inducing p16 expression by affecting DNA methylation in the p16 promoter region.

Inhibition of Smooth Muscle β-Catenin Hinders Neointima Formation After Vascular Injury.

Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although β-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of β-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC β-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of β-catenin inhibitors on vascular SMC growth. We used an inducible, conditional genetic deletion of β-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC β-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC β-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking β-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, β-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. SMC β-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological β-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting β-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

Adipose differentiation‑related protein knockdown inhibits vascular smooth muscle cell proliferation and migration and attenuates neointima formation.

Vascular smooth muscle cells (VSMCs) have an important role in atherosclerosis development. Evidence has demonstrated that adipose differentiation-related protein (ADRP) is associated with foam cell formation and atherosclerosis progression. However, to the best of our knowledge, no previous studies have investigated the role of ADRP knockdown in platelet-derived growth factor (PDGF)-stimulated proliferation and migration of VSMCs in vitro. Furthermore, the effect of ADRP knockdown on neointima formation in vivo remains unclear. In the present study, primary human aortic VSMCs were incubated with PDGF following ADRP small interfering (si)RNA transfection. Cell viability, migration and cell cycle distribution were analyzed by MTT, wound healing and Transwell assays and flow cytometry, respectively. Extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, Akt, p-Akt, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 protein levels were determined by western blotting. Apolipoprotein E−/− mice fed an atherogenic diet were injected with siADRP or control siRNA twice a week. After 3 weeks of therapy, aortas were excised and ADRP mRNA and protein expression was determined. Neointima formation was assessed by hematoxylin and eosin staining. The results of the current study demonstrated that ADRP knockdown significantly inhibited PDGF-induced increases in VSMC viability, caused G1 phase cell cycle arrest and decreased PCNA expression. Knockdown of ADRP inhibited PDGF-induced migration of VSMCs by reducing MMP protein expression and activity. In addition, the present study also demonstrated that ADRP knockdown inhibited ERK and Akt signaling pathways in response to PDGF. Furthermore, siADRP administration suppressed neointima formation in the mouse model. The results of the present study indicate that ADRP may be a potential target for the treatment of atherosclerosis.

A novel lipoprotein (a) lowering drug, D-47, decreases neointima thickening after vascular injury.

Although Lp(a) have been thought to be a cardiovascular risk factor, it is unclear whether lowering Lp(a) levels reduces the risk of cardiovascular diseases. No pharmacological agents which selectively reduce serum Lp(a) levels, and Lp(a) is present in primate but absent in common laboratory animals such as mice and pigs. In the present study we used transgenic mice of human Lp(a) and tested effect a novel Lp(a) lowering drug D-47 on neointima formation after vascular injury. D-47 successfully decreased plasma levels of Lp(a) and possibly inhibited neointima formation in Lp(a) transgenic mice. The results indicate that we can modulate plasma Lp(a) levels by pharmacologic agents and inhibit atherogenic properties of Lp(a) by reducing plasma levels of Lp(a). J. Med. Invest. 64: 64-67, February, 2017.

Suppression of neointima formation by targeting β-catenin/TCF pathway.

Coronary artery disease is treated by vein grafting and stent implantation. Late vein graft failure and restenosis of stented arteries reduce the success rates of these approaches and are caused by neointima formation. We have previously shown that Wnt proteins are up-regulated during intimal thickening, and have speculated that these lead to activation of downstream genes with β-catenin/T-cell factor (TCF)-responsive promoters. In the present study, we aimed to provide evidence that β-catenin/TCF signalling promotes neointima formation and assess whether targeting this pathway has potential for reducing neointima formation. We utilized a gene therapy approach selectively targeting cells in which the β-catenin/TCF pathway is activated by using a recombinant adenovirus Ad-TOPTK, which carries a herpes simplex virus thymidine kinase (HSV-TK) gene under the control of a β-catenin/TCF-response promoter. Cells with activated β-catenin will therefore be selectively killed. Ad-TOPTK and ganciclovir (GCV) treatment significantly suppressed the growth of the neointima in a murine model of left carotid artery ligation. In summary, we demonstrated that Wnt/β-catenin/TCF signalling promotes neointima formation, by showing that the selective death of cells with activated β-catenin suppressed neointima formation. This highlights the therapeutic potential for reducing late vein graft failure and in-stent restenosis by targeting β-catenin/TCF signalling.

Transplantation of EPCs overexpressing PDGFR-β promotes vascular repair in the early phase after vascular injury.

BackgroundEndothelial progenitor cells (EPCs) play important roles in the regeneration of the vascular endothelial cells (ECs). Platelet-derived growth factor receptor (PDGFR)-β is known to contribute to proliferation, migration, and angiogenesis of EPCs, this study aims to investigate effects of transplantation of EPCs overexpressing PDGFR-β on vascular regeneration.MethodsWe transplanted genetically modified EPCs overexpressing PDGFR-β into a mouse model with carotid artery injury. After 3 days of EPCs transplantation, the enhanced green fluorescent protein (EGFP)-expressing cells were found at the injury site and the lining of the lumen by laser scanning confocal microscope (LSCM). At 4, 7, and 14 days of the carotid artery injury, reendothelialization was evaluated by Evans Blue staining. Neointima formation was evaluated at day 14 with hematoxylin and eosin (HE) staining by calculating the neointimal area, medial area, and neointimal/media (NI/M) ratio. Intimal cell apoptosis was evaluated using TUNEL assay. Then we tested whether PDGF-BB-induced VSMC migration and PDGF-BB’s function in reducing VSMC apoptosis can be attenuated by EPCs overexpressing PDGFR-β in a transwell co-culture system.ResultsOur results showed that EPCs overexpressing PDGFR-β accelerates reendothelialization and mitigates neointimal formation at 14 days after injury. Moreover, we found that there is great possibility that EPCs overexpressing PDGFR-β enhanc VSMC apoptosis and suppress VSMC migration by competitive consumption of PDGF-BB in the early phase after carotid artery injury in mice.ConclusionsWe report the first in vivo and in vitro evidence that transplantation of genetically modified EPC can have a combined effect of both amplifying the reendothelialization capacity of EPCs and inhibiting neointima formation so as to facilitate better inhibition of adverse remodeling after vascular injury.

Optimal coating method for a dual-layer stent with sirolimus and alpha-lipoic acid in a porcine coronary restenosis model

The aim of this study was to evaluate the optimal method for coating a double-layer polymer coronary stent with sirolimus and alpha-lipoic acid (ALA) in a porcine coronary overstretch restenosis model. Pigs were randomized into three groups, in which the coronary arteries (10 in each group) were stented with a dual-layer stent with an inner layer of ALA and an outer layer of sirolimus (IAOS, n=10), a dual-layer stent with an inner layer of sirolimus and an outer layer of ALA (ISOA, n=10), or a commercial drug-eluting stent (biolimus-eluting stent, BES, n=10). Histopathological analysis was performed 28 days after stenting. There were no significant differences among the three groups in injury and inflammation scores. There were significant differences among the three groups in neointimal area (1.1±0.16 mm2 for IAOS vs. 1.3±0.41 mm2 for ISOA vs. 1.9±0.50 mm2 for BES, p<0.0001), and percentage of stenosis area (21.4±3.08% for IAOS vs. 29.9±7.72% for ISOA vs. 38.2±9.08% for BES, p<0.0001). Regarding the percentage of stenosis area, microcomputed tomography revealed significant differences between the three groups (23.8±3.51% for IAOS vs. 28.9±4.65% for ISOA vs. 36.4±8.07% for BES, p<0.05). Compared with commercial stent placement, both IAOS and ISOA resulted in significant inhibition of neointimal formation in a porcine coronary restenosis model. In addition, IAOS was more effective than ISOA in preventing anti-neointimal hyperplasia after stenting.

Inhibition of intimal thickening after vascular injury with a cocktail of vascular endothelial growth factor and cyclic Arg-Gly-Asp peptide

BackgroundPercutaneous coronary intervention is widely used for the treatment of coronary artery disease; however, significant challenges such as restenosis remain. Key to solving these problems is to inhibit smooth muscle cell activation while enhancing re-endothelialization. Early growth response-1 (Egr-1) is a transcription factor that regulates vascular smooth muscle cell (SMC) proliferation and migration through its control of an array of downstream genes. MethodsA “cocktail” of vascular endothelial growth factor (VEGF)-A, VEGF-D and cyclic RGD was tested for its ability to inhibit neointima formation and accelerate re-endothelialization following balloon injury to carotid arteries of rats. ResultsIn vitro, the cocktail stimulated endothelial cell growth yet inhibited smooth muscle cell growth. In vivo, cocktail-treated injured arteries exhibited reduced intimal thickening by >50% (P<0.05). It increased both re-endothelialization and endothelial nitric oxide synthase (NOS) expression. Cocktail reduced Egr-1 expression, an effect blocked by the NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) that also prevented cocktail inhibition of neointima inhibition. ConclusionsThis combination may potentially be useful for the treatment of restenosis with concomitant stimulation of revascularization.

Co-overexpression of human tissue kallikrein 1 and human metalloproteinase 1 tissue inhibitor inhibits neointima formation in the rat artery after balloon angioplasty

To investigate the effects of adenovirus-mediated human tissue kallikrein 1(hTK-1) and/or human tissue metalloproteinase inhibitor 1 (hTIMP-1) gene delivery on the neointima formation in balloon-injured rat carotids and related mechanism. Forty-six male Sprague-Dawley rats were randomly assigned into 6 groups with the random number table: (1) sham-operated group(n=6), (2) angioplasty group (n=8), (3) vector virus group (n=8), (4) hTK-1 group (n=8), (5) hTIMP-1 group (n=8), (6) hTK-1-hTIMP-1 group (n=8). Except sham rats, all rats underwent carotid artery balloon injury and local delivery of saline or different recombined adenoviruses respectively. Rats were sacrificed 14 days later. Intima/media area ratio was assessed on hematoxylin-eosin stained tissue section. Immunofluorescence images stained for hTK-1, hTIMP-1 were obtained and analyzed by the confocal microscope for co-localization examination of hTK-1 and hTIMP-1. The protein expression levels of hTK-1, hTIMP-1, matrix metalloproteinases(MMP)-2 and MMP-9 were determined by Western blot. Immune histochemical staining for PCNA was also performed. (1)Intima area, intima/media area ratio, PCNA, MMP-2 and MMP-9 levels were all significantly increased in rats underwent angioplasty (did or did not receive vector virus) compared with sham-operated rats (all P<0.01) while above parameters were similar between rats underwent angioplasty or vector virus delivery (all P>0.05). (2) The intima area of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were (0.160±0.010), (0.110±0.015), (0.121±0.016) or (0.081±0.008) mm(2) respectively, intima area was similar between rats received hTK-1 or hTIMP-1 (P>0.05), differences were found between other groups (all P<0.01). The intima/media area ratio of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were 2.035±0.117, 1.443±0.097, 1.522±0.078 or 0.972±0.072 respectively, no difference was found between rats received hTK-1 or hTIMP-1 in intima/media area ratio (all P>0.05), differences were found between other groups (all P<0.01). The MMP-2 and MMP-9 expression of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were 0.817±0.036, 0.606±0.044, 0.571±0.061 or 0.455±0.030 and 0.745±0.057, 0.613±0.038, 0.582±0.050 or 0.473±0.038 respectively, no difference was found between rats received hTK-1 or hTIMP-1 in MMP-2 or MMP-9 expression (all P>0.05), differences were found between other groups (all P<0.01). The PCNA expression of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were 0.065±0.007, 0.052±0.004, 0.055±0.007 or 0.031±0.004 respectively, no difference was found between rats received hTK-1or hTIMP-1 in PCNA expression (all P>0.05), differences were found between other groups (all P<0.01). hTK-1 and hTIMP-1 co-overexpression may synergistically inhibit neointimal hyperplasia, attenuate vascular remodeling and reduce restenosis possibly via down regulating the expressions of PCNA, MMP-2 and MMP-9 in balloon-injured rat carotids.