Development Of Neointima Formation Research Articles

MicroRNA-92a -mediated endothelial to mesenchymal transition controls vein graft neointimal lesion formation

PurposeLong-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation. MethodsmiR-92a and EndMT-related proteins detected by qRT-PCR and Western blot in vitro and in vivo. Adeno-associated virus 6 (AAV6) delivery gene therapy was used to inhibit neointimal formation in vivo. The intimal hyperplasia of vein grafts was measured by HE staining, the expression of EndMT-related protein in vein grafts was measured by immunofluorescence. Immunohistochemistry and luciferase assay were used to detect potential targets of miR-92a. ResultsThe expression of miR-92a was found to be upregulated in neointimal hyperplasic lesions after vein grafting. Using cultured human umbilical vein endothelial cells (HUVECs), we show that TGF-β1 treatment of HUVECs significantly increased miR-92a expression and induced EndMT, characterized by suppression of endothelial-specific markers (CD31 and VE-cadherin) and an increase in mesenchymal-specific markers (a-SMA and vimentin), while inhibition of miR-92a expression blunted EndMT in cultured HUVECs. Furthermore, AAV6 mediated miR-92a suppression gene therapy effectively resulted in decreased EndMT and less neointimal formation in vein grafts in vivo. We further identified that integrin alpha 5 (ITGA5) is a potential target gene involved in the development of neointima formation in these vein grafts. ConclusionThis data suggests that neointimal formation does not solely rely on vascular smooth muscle cell phenotypic switching but is also related to EndMT, and miR-92a-mediated EndMT is an important mechanism underlying neointimal formation in vein grafts.

Oral Charcoal Adsorbents Attenuate Neointima Formation of Arteriovenous Fistulas.

Chronic kidney disease (CKD) accelerates the development of neointima formation at the anastomosis site of arteriovenous (AV) fistulas. Accumulation of certain uremic toxins has a deleterious effect on the cardiovascular system. The oral charcoal adsorbent, AST-120, reduces circulating and tissue uremic toxins, but its effect on neointima formation at an AV fistula is unknown. To understand the effect of CKD and AST-120 on neointima formation, we created AV fistulas (common carotid artery to the external jugular vein in an end-to-side anastomosis) in mice with and without CKD. AST-120 was administered in chow before and after AV fistula creation. Administration of AST-120 significantly decreased serum indoxyl sulfate levels in CKD mice. CKD mice had a larger neointima area than non-CKD mice, and administration of AST-120 in CKD mice attenuated neointima formation. Both smooth muscle cell and fibrin components were increased in CKD mice, and AST-120 decreased both. RNA expression of MMP-2, MMP-9, TNFα, and TGFβ was increased in neointima tissue of CKD mice, and AST-120 administration neutralized the expression. Our results provided in vivo evidence to support the role of uremic toxin-binding therapy on the prevention of neointima formation. Peri-operative AST-120 administration deserves further investigation as a potential therapy to improve AV fistula patency.

Dedicator of cytokinesis 2 silencing therapy inhibits neointima formation and improves blood flow in rat vein grafts

ObjectiveThe high rate of vein graft failure due to neointimal hyperplasia is a major challenge for cardiovascular surgery. Finding novel approaches to prevent neointimal hyperplasia is important. Thus, the purpose of this study was to investigate whether dedicator of cytokinesis 2 (DOCK2) plays a role in the development of neointima formation in the vein grafts. Methods and resultsWe found that DOCK2 levels were significantly elevated in the vein grafts following grafting surgery. In addition, overexpression of DOCK2 promoted venous smooth muscle cell (SMC) proliferation and migration. Conversely, knocking-down endogenous DOCK2 expression in venous SMCs inhibited SMC proliferation and migration. Consistent with this, knocking-down DOCK2 expression in the grafted veins significantly reduced neointimal formation compared with the controls 28 days after vein transplantation. Moreover, DOCK2 silencing treatment improved hemodynamics in the vein grafts. Mechanistically, knockdown of DOCK2 significantly alleviated the vein graft-induced down regulation of SMC contractile protein expression and impeded the vein graft-induction of both Cyclin D1 and PCNA expression. In particular, to ensure high efficiency when transferring the DOCK2 short hairpin RNA (shDOCK2) into the grafted veins, a 30% poloxamer F-127 gel incorporated with 0.25% trypsin was smeared around the vein grafts to increase the adenovirus contact time and penetration. ConclusionsDOCK2 silencing gene therapy effectively attenuates neointimal hyperplasia in vein grafts. Knock-down of DOCK2 would be a potential therapeutic approach for the treatment of vein graft failure.

MicroRNA-663 Regulates Human Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Neointimal Formation

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor-induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

MicroRNA 21 is upregulated following coronary artery stenting and genetic deletion limits neointima formation

Purpose: Atherosclerosis in coronary arteries causes luminal narrowing, restriction of blood flow and the development of angina. Percutaneous Coronary Intervention (PCI) with stents is a common treatment. In-Stent Restenosis (ISR) or luminal renarrowing following PCI is due to the proliferation and migration of smooth muscle cells (VSMCs) and deposition of extracellular matrix (ECM). MicroRNAs (miRs) are small non-coding 19-25 nucleotide RNA species that post-transcriptionally regulate gene expression in health but have also been implicated in many disease settings. MiR-21 levels are dysregulated in atherosclerotic lesions, following vascular injury and have been implicated in VSMC proliferation and migration. We hypothesised that miR-21 promotes ISR. Methods: Male Welsh Landrace swine 18-22kg received bare-metal stents (BMS) and were euthanised at 0, 7 or 28 days (n=4/group). Total RNA was extracted from one half of the stented porcine coronary arteries. MiR-21 expression levels were measured by quantitative real-time polymerase chain reaction. In situ hybridisation was also conducted on stented vessels. For mouse studies, miR-21 knockout (KO) and wild type (WT) controls were used. The aorta of a donor mouse was stented and interposition grafted into a recipient mouse of identical genotype. Grafts were harvested at 0 or 28 days. Following electrochemical dissolution of the stent, tissues were paraffin embedded and morphometric analysis conducted on serial sections throughout the stented segment. Results: In the porcine model, miR-21 levels were upregulated at both 7 and 28 days by a factor of 5.3±1.1 and 3.8±1.3 respectively (p<0.001 for both). Insitu hybridisation demonstrated elevated expression of miR-21 throughout the vessel wall but at a high level in the developing neointima. No difference was observed between miR-21 KO (n=3) and WT (n=9) mice in total vessel, luminal, medial or neointimal area at day 0. Comparing miR-21 KO (n=6) v miR-21 WT (n=12) mice at 28 days significantly less neointimal area and neointimal:medial ratio was observed (0.12±0.02mm2 v 0.18±0.02mm2; 1.47±0.22 v 2.11±0.17, p<0.05 for both). As a consequence luminal area was significantly reduced in the miR-21 WT mice (0.22±0.02mm2 v 0.32±0.03mm2, p<0.01). No significant difference was observed in total vessel or medial area at 28 days. Conclusions: Mir-21 is upregulated in porcine coronary arteries following stenting with BMS. Genetic deletion of miR-21 in a murine model of stenting attenuates the development of neointimal formation and ISR. Targeting miR-21 to reduce ISR maybe of use in a clinical setting.

Abstract 212: MicroRNA-663 is a Novel Regulator of Human Vascular Smooth Muscle Cell Phenotypic Switch in vitro and Neointimal Formation in vivo

Background Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Recently, microRNAs (miRNAs) emerge as critical regulators for vascular smooth muscle cell (VSMC) function. Our initial study identified miR-663 as one of the most sharply downregulated miRNAs in human proliferative aortic smooth muscle cells. Hypothesis MiR-663 is implicated in human VSMC phenotypic switch and the development of neointima formation. Methods and Results By using quantitative real-time PCR (qRT-PCR), we found that microRNA-663 (miR-663) was significantly downregulated in cultured human aortic VSMCs upon platelet-derived growth factor (PDGF) treatment, whereas its expression was markedly increased during VSMC differentiation as induced by either retinoid acid or SMC differentiation medium, a condition which induces SMC differentiation and inhibits cell proliferation. Furthermore, we demonstrated that overexpression of miR-663 significantly increased the expression of VSMC differentiation marker genes, such as SM22α, SM α-action, calponin, and SM myosin heavy chain, suggesting that miR-663 is a novel modulator implicated in human VSMC phenotypic switch. Moreover, miR-663 potently inhibited PDGF induced VSMC proliferation and migration. Mechanistically, we identified JunB as a downstream target of miR-663 in human VSMCs. Indeed, overexpression of miR-663 markedly inhibited the expression of the transcription factor JunB as well as its downstream molecules including matrix metallopeptidase-9 (MMP-9) and myosin light chain-9 (Myl9), thus inhibiting VSMC proliferation and migration. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by approximately 50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. Conclusion These results indicate that miR-663 is a novel modulator implicated in human VSMC phenotypic switch through targeting JunB expression and suggest that specific modulation of miR-663 in human VSMCs may represent a novel and attractive approach for the treatment of vascular proliferative diseases.

Predictors of Ductus Arteriosus In-Stent Stenosis in the Hybrid Approach to Hypoplastic Left Heart Syndrome

The hybrid approach to palliation of hypoplastic left heart syndrome using pulmonary artery bands, a patent ductus arteriosus (PDA) stent, and atrial septostomy has been well described. One potential complication of hybrid stage 1 palliation is the development of neointimal formation and in-stent stenosis (ISS). This study aimed to identify predictors of ISS development. Patients who underwent hybrid stage 1 palliation between 2002 and 2010 were included in the study. The clinical information included oxygen saturation, weight, vital signs, and medications. Echocardiographic data included ventricular function, degree of tricuspid regurgitation, and velocity through the PDA stent and pulmonary artery bands. Hemodynamic data from interstage catheterizations were similarly noted. Patients who developed clinically significant ISS requiring either transcatheter intervention or early stage 2 repair were compared with those who did not. Of the 66 patients included in the study, 40 were boys (61%). The median age at hybrid palliation was 7days (range, 1-93days), and the median initial weight was 3.2kg (range, 1.4-5kg). In 13 patients (20%), ISS developed. The mean initial weight was significantly greater in the ISS group (3.5±0.5 vs. 3.0±0.6kg) (p = 0.03). The mean oxygen saturations did not differ significantly between the no-ISS group (82.2 %±5.7%) and the ISS group (81.4%±2.0%) (p=0.31). The mean PDA velocities were higher in the ISS group (2.7±0.4m/s) and increased at a faster rate than in the no-ISS group at (2.4±0.4m/s) (p=0.01). The degree of tricuspid regurgitation, ventricular function, and pulmonary artery band gradients shown by echocardiography were similar in the two groups. The development of ISS after hybrid stage 1 palliation can lead to interstage interventions or earlier comprehensive stage 2 repair. Patients with greater initial weight and a lower stent-to-weight ratio are more likely to develop ISS. The cause of ISS is complex, and additional investigation of its etiology currently is ongoing.

TCT-293 Neointimal Tissue Characteristics Based on Optical Coherence Tomography Analysis Following Paclitaxel Coated Balloon Treatment in a Porcine Iliofemoral Vascular Territory

Background: The optical coherence tomography (OCT) characteristics of in-stent neointimal tissue have been associated with histological markers of healing. We aimed to investigate the differences in neointimal characteristics based on OCT analysis of Paclitaxel coated balloon (PCB) treatment versus POBA in the Iliofemoral territory of the familial hypercholesterolemic swine model (FHS). Methods: 14 Iliofemoral arterial segments of 7 FHS were balloon injured followed by a BMS placement. At 14 days, the injured sites were treated with either a PCB or POBA (control). OCT images, acquired at 28 days post inflation, were classified at every 2mm according to previously published classification of neointimal types (homogeneous, heterogeneous and layered). Quantitative morphometric evaluation of vessel, stent, microvessel and peristrut low intensity areas (PLI) presence was also performed. A multivariable logistic regression model was performed dividing the neointima into homogeneous and non-homogeneous. Results: AA total of 150 OCT cross sections were included in the final analysis. PCB resulted in a reduction of percent area stenosis (%AS) by 50% (PCB: 34 18% versus POBA 68 21%, p 0.05). The homogeneous pattern was more frequent in PCB (25%) than in the POBA group (3%, p 0.001). Conversely, the layered pattern was more prevalent in the POBA group (50.7% versus 4%, p 0.001). The homogeneous pattern correlated with less degree of neointimal formation [%AS: 16.68 5.3%, p 0.001) than the non-homogeneous group (heterogeneous: 49.01 24.9% and layered: 67.29 15.6%). The presence of micro vessels (89% versus 58%, p 0.001) and PLI (39% versus 24%, p 0.1) were more frequently found in the POBA group than in the PCB group. The only independent predictor of non-homogeneous patterns was the %AS [OR (95% CI)] [3.98(1.21-13.09)]. Conclusions: In vivo OCT analysis suggests that the biological effect of Paclitaxel delivered via DCB frequently results in the homogeneous development of neointimal formation and lower frequency of PLI and micro-vessels compared to a POBA control. These in vivo findings support the biological efficacy observed in clinical trials using this technology.

Pathological Importance of the Endothelin-1/ETBReceptor System on Vascular Diseases

Activation of the endothelin (ET)-1/ET receptor system is involved in the development of vascular diseases such as atherosclerosis, vascular hypertrophy, and restenosis. Some issues still remain unresolved including whether ET receptor antagonists are expected to become the new therapeutic tools for the treatment of vascular diseases. One of the unresolved critical points is the functional role of ET receptor subtypes on each vascular disease, in particular the pathophysiological roles of the ETB receptor. We recently demonstrated that selective inhibition of the ETB receptor system showed harmful effects in the development of neointimal formation after vascular injury. However, there was no apparent difference in the therapeutic effects between a nonselective ETA/ETB receptor antagonist and selective ETA receptor antagonist. These findings indicate that antagonism of the ETA receptor system is essential for suppressing vascular remodeling, irrespective of the presence of ETB-receptor-mediated actions, although the selective ETB receptor antagonist worsens vascular remodeling. In addition, we found that ET receptor systems contribute to sex differences in the severity of vascular disease, thereby suggesting that the efficacy of ET receptor antagonists for vascular diseases may differ between sexes. In this paper, we outline the roles of the ET-1/ETB receptor system on vascular diseases and its sex differences.

Neointimal hyperplasia in a porcine model of vein graft disease: comparison between organ culture and coronary artery bypass grafting

BACKGROUND: Neointima formation is a crucial problem in the early phase after coronary artery bypass grafting which may result in vein graft stenosis and occlusion 10 years after surgery. METHODS: Six pigs underwent aortocoronary bypass surgery by grafting the left internal mammary vein to the left anterior descending coronary artery (LAD). The remnants of internal mammary veins were cultured for 14 days under standardized conditions. Pigs were sacrificed two weeks after surgery. Histological and immunohistological investigations were performed with special regard to morphometric analysis, differences of the vein wall structure and connective tissue changes. RESULTS: Histological examination revealed similar amounts of collagen, elastic and reticulin fibers in intima and media of native and cultured vein segments with an accumulation in the neointima of vessel specimens after bypass surgery (p<0.01). Moreover, veins at day 14 after bypass surgery showed a significant increase of the intima (220.74 µm vs. 1.91 µm in native and 2.14 µm in cultured veins; p<0.01) and a thickening of the media (334.07 µm vs. 52.44 µm in native and 76.29 µm in cultured veins; p<0.01). CONCLUSIONS: Contrary to cultured vessel specimens, venous conduits are characterized by the development of neointima formation, thickening of the media and a higher percentage of elastic, collagen, reticulin fibers and vascular smooth muscle cells in the intima.

Syndecan-4 Deficiency Limits Neointimal Formation After Vascular Injury by Regulating Vascular Smooth Muscle Cell Proliferation and Vascular Progenitor Cell Mobilization

Syndecan-4 (Syn4) is a heparan sulfate proteoglycan and works as a coreceptor for various growth factors. We examined whether Syn4 could be involved in the development of neointimal formation in vivo. Wild-type (WT) and Syn4-deficient (Syn4-/-) mice were subjected to wire-induced femoral artery injury. Syn4 mRNA was upregulated after vascular injury in WT mice. Neointimal formation was attenuated in Syn4-/- mice, concomitantly with the reduction of Ki67-positive vascular smooth muscle cells (VSMCs). Basic-fibroblast growth factor- or platelet-derived growth factor-BB-induced proliferation, extracellular signal-regulated kinase activation, and expression of cyclin D1 and Bcl-2 were impaired in VSMCs from Syn4-/- mice. To examine the role of Syn4 in bone marrow (BM)-derived vascular progenitor cells (VPCs) and vascular walls, we generated chimeric mice by replacing the BM cells of WT and Syn4-/- mice with those of WT or Syn4-/- mice. Syn4 expressed by both vascular walls and VPCs contributed to the neointimal formation after vascular injury. Although the numbers of VPCs were compatible between WT and Syn4-/- mice, mobilization of VPCs from BM after vascular injury was defective in Syn4-/- mice. Syn4 deficiency limits neointimal formation after vascular injury by regulating VSMC proliferation and VPC mobilization. Therefore, Syn4 may be a novel therapeutic target for preventing arterial restenosis after angioplasty.

Abstract 5551: Double Deficiency of Tumor Necrosis Factor-α and Interferon-γ in Bone Marrow-Derived Cells Prevents Neointimal Formation in a Murine Model of Vascular Injury

Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Recent evidence indicates that inflammatory responses induced by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), are involved in the progression of neointimal formation. However, the role of TNF-α and IFN-γ in the restenosis after PCI has not been fully understood. The purpose of this study is to examine the impact of TNF-α and IFN-γ in bone marrow-derived cells in the development of neointimal formation after vascular injury in mice. Wild-type (WT), TNF-α-deficient (TNF-α −/− ), IFN-γ-deficient (IFN-γ −/− ), and TNF-α/IFN-γ double-deficient (DKO) mice were subjected to wire-mediated vascular injury of the right femoral artery. Immunohistochemical analysis showed the expression of TNF-α and IFN-γ was detected in the neointimal lesion of WT mice, but these cytokines were not detected in the lesion of the corresponding deficient mice. Neointimal formation was significantly reduced after the injury in the DKO mice, compared to that in the WT, TNF-α −/− , and IFN-γ −/− mice (I/M ratio, WT: 2.28±0.17, TNF-α −/− : 2.13±0.20, IFN-γ −/− : 2.37±0.16, DKO: 1.32±0.10, p&lt;0.05, each n=14–17). No significant difference in reendothelialization (CD31 staining) was observed among these groups. Further, vascular smooth muscle cell (α-SMA) and macrophage (F4/80) contents in the neointimal area also did not differ among the groups. The number of proliferating cell nuclear antigen (PCNA) and Ki-67 positive cells in the neointimal lesion was significantly decreased in DKO mice. To determine the contribution of bone marrow cells, we developed 3 types of bone marrow chimeric (BMT Wild→Wild , BMT DKO→Wild , and BMT Wild→DKO ) mice. The neointimal formation in BMT DKO→Wild mice was significantly reduced as compared to that in BMT Wild→Wild (I/M ratio, p&lt;0.05, each n=7) and BMT Wild→DKO mice (p&lt;0.05). These results suggest that the lack of TNF-α and IFN-γ in bone marrow-derived cells synergistically prevents neointimal formation after vascular injury and provide new insights into the mechanisms underlying the restenosis after PCI.

Prostacyclin synthase gene transfer inhibits neointimal formation by suppressing PPARδ expression

Objective Prostacyclin (PGI 2) is a potent ligand of peroxisome proliferator-activated receptor δ (PPARδ) that regulates cell growth and differentiation. The aim of this study was to elucidate how endogenous PGI 2 overexpression affects the expressions of PPARδ and mitogen-activated protein kinases (MAPKs) in the development of neointimal formation in experimental angioplasty with adenovirus-mediated PGI 2 synthase (Ad-PGIS) gene transfer. Methods and results In human aortic smooth muscle cells, protein blotting analysis showed that PGI 2 overproduction decreased the levels of phosphorylated p38 MAPK (P-p38 MAPK) (2.0-fold versus 0.83-fold relative to control). Immunohistochemical analysis in balloon-injured arteries revealed diffuse expression of PPARδ in the neointima of control vessels, with no expression in uninjured vessels. The level of PPARδ expression was lower in Ad-PGIS-treated arteries than in control vessels, with the PPARδ localized in the neointima adjacent to endothelium. Staining of P-p38 MAPK showed a similar pattern to PPARδ among the three groups. Morphometric analysis at day 14 revealed that Ad-PGIS reduced the intima-to-media ratio by up to 59%. Conclusions Ad-PGIS gene transfer reduced PPARδ expression and inhibited neointimal formation after balloon injury in accordance with the reduction in the phosphorylation of p38 MAPK.

The effect of interleukin-10 knock-out and overexpression on neointima formation in hypercholesterolemic APOE*3-Leiden mice

Objective Inflammatory factors are thought to play a regulatory role in restenosis. Interleukin-10 (IL10) is an important anti-inflammatory cytokine with anti-atherogenic potentials. The aim of this study was to assess the effects of IL10 modulation on cuff-induced neointima formation in hypercholesterolemic APOE*3-Leiden mice. Methods The involvement of IL10 in neointima formation was studied in a hypercholesterolemic mouse model of cuff-induced stenosis of the femoral artery by IL10 knocking-out or overexpression procedures. IL10 +/− mice were crossbred with APOE*3-Leiden mice to generate hypercholesterolemic APOE*3-LeidenIL10 −/− mice. To achieve IL10 overexpression in APOE*3-Leiden mice, a single intramuscular injection of a murine IL10 overexpression plasmid was performed followed by electroporation. Results Knocking-out IL10, in hypercholesterolemic APOE*3-Leiden mice, resulted in a significant 1.9-fold increase of neointima surface as compared to APOE*3-LeidenIL10 +/+ littermates ( p = 0.02). Conversely, a marked 45% inhibition on cuff-induced neointima formation was obtained after IL10 overexpression ( p = 0.02). Electrodelivery of IL10 vector leads to detectable IL10 serum levels, with a sustained expression over the experimental period of 3 weeks. IL10 overexpression reduced plasma cholesterol levels in APOE*3-Leiden mice, whereas IL10 deficiency in these mice did not lead to altered cholesterol levels as compared to the IL10 +/+ group. Finally, IL10 overexpression stimulated endogenous IL10 mRNA expression in the spleen and reduced the transcriptional responses of several pro-inflammatory cytokines. Conclusion Here, we clearly demonstrate the role of IL10 in the development of neointima formation in hypercholesterolemic mice and the potential therapeutic effect of non-viral electrodelivery of IL10 cDNA to inhibit post-angioplasty restenosis.

Platelet, Not Endothelial, P-Selectin Is Required for Neointimal Formation After Vascular Injury

P-selectin blockade significantly inhibits inflammation and neointimal formation after arterial injury; however, the independent roles of platelet and endothelial P-selectins in this process are unknown. In atherosclerosis, both platelet and endothelial cell P-selectins are important. This study was designed to determine whether P-selectin expression on platelet, endothelial, or both surfaces is critical to the inflammatory response and neointimal formation after arterial injury. Using wild-type (WT) and P-selectin-knockout (Psel(-/-)) mice, we performed bone marrow transplantation to generate chimeric mice that expressed either platelet P-selectin (Plt-Psel) or endothelial P-selectin (EC-Psel). Double injury of the carotid artery was performed in these mice as well as in WT and Psel(-/-) mice. Animals were euthanized 4 or 21 days after arterial injury. Morphometric data showed that there was more neointimal formation in the WT mouse group when compared with the Psel(-/-) mouse group (0.015+/-0.004 vs 0.004+/-0.004 mm2, P<0.001). Further comparison showed significantly less neointimal area in EC-Psel mice (0.006+/-0.004 mm2) compared with Plt-Psel mice (0.011+/-0.005 mm2, P=0.026) and WT mice (0.015+/-0.004 mm2, P=0.001). No significant differences were observed between WT and Plt-Psel mice or between Psel(-/-) and EC-Psel mice. Decreased neointimal formation was accompanied by a reduced inflammatory response, as evidenced by immunostaining of RANTES and MCP-1 4 days after injury. Platelet P-selectin expression, but not endothelial P-selectin, plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting leukocyte-platelet interactions could be effective in inhibiting restenosis.

W09-P-004 EFfect of pravastatin on sympathetic innervation in hyperlipidemic rabbits

Inflammatory factors are thought to play a regulatory role in restenosis. Interleukin-10 (IL10) is an important anti-inflammatory cytokine with anti-atherogenic potentials. The aim of this study was to assess the effects of IL10 modulation on cuff-induced neointima formation in hypercholesterolemic APOE*3-Leiden mice.The involvement of IL10 in neointima formation was studied in a hypercholesterolemic mouse model of cuff-induced stenosis of the femoral artery by IL10 knocking-out or overexpression procedures. IL10+/− mice were crossbred with APOE*3-Leiden mice to generate hypercholesterolemic APOE*3-LeidenIL10−/− mice. To achieve IL10 overexpression in APOE*3-Leiden mice, a single intramuscular injection of a murine IL10 overexpression plasmid was performed followed by electroporation.Knocking-out IL10, in hypercholesterolemic APOE*3-Leiden mice, resulted in a significant 1.9-fold increase of neointima surface as compared to APOE*3-LeidenIL10+/+ littermates (p = 0.02). Conversely, a marked 45% inhibition on cuff-induced neointima formation was obtained after IL10 overexpression (p = 0.02). Electrodelivery of IL10 vector leads to detectable IL10 serum levels, with a sustained expression over the experimental period of 3 weeks. IL10 overexpression reduced plasma cholesterol levels in APOE*3-Leiden mice, whereas IL10 deficiency in these mice did not lead to altered cholesterol levels as compared to the IL10+/+ group. Finally, IL10 overexpression stimulated endogenous IL10 mRNA expression in the spleen and reduced the transcriptional responses of several pro-inflammatory cytokines.Here, we clearly demonstrate the role of IL10 in the development of neointima formation in hypercholesterolemic mice and the potential therapeutic effect of non-viral electrodelivery of IL10 cDNA to inhibit post-angioplasty restenosis.

Essential role of vascular endothelial growth factor and Flt-1 signals in neointimal formation after periadventitial injury.

Vascular endothelial growth factor (VEGF) is upregulated after arterial injury. Its role in the pathogenesis of neointimal formation after periadventitial injury, however, has not been addressed. Expression of VEGF and its receptors but not that of placental growth factor markedly increased with the development of neointimal formation in hypercholesterolemic mice after cuff-induced periarterial injury. Transfection with the murine soluble Flt-1 (sFlt-1) gene to block VEGF in vivo in mice inhibited early inflammation and later neointimal formation. The sFlt-1 gene transfer did not affect plasma lipid levels but attenuated increased expression of VEGF, Flt-1, Flk-1, monocyte chemoattractant protein-1, and other inflammation-promoting factors. Mice with Flt-1 kinase deficiency also displayed reduced neointimal formation. Inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury. VEGF might promote neointimal formation by acting as a proinflammatory cytokine.

Effect of a synthetic matrix metalloproteinase inhibitor (ONO-4817) on neointima formation in hypercholesterolemic hamsters.

Degradation of extracellular matrix (ECM) proteins plays an important role in the development of vascular remodeling. We investigated the alteration of matrix metalloproteinases (MMPs) on the development of neointima formation and the effect of a newly synthesized MMP inhibitor using hypercholesterolemic hamsters. Endothelial injury was achieved by a catheter in the hamster carotid artery. Two weeks after the injury, neointima was detected in all hamsters. Oral administration (twice a day) of ONO-4817 was started 2 hours before injury and continued for the next 2 weeks. The neointimal area, with appearance of maze-like structures, was markedly reduced by 52.4 +/- 8.4% by treatment with ONO-4817 at a dose of 20 mg/kg per day. The treatment by ONO-4817 (20 mg/kg per day) significantly reduced the indexes of histone H1 on day 1, 5, and 10 and the BrdU index of intimal smooth muscle cells on day 5 and 10, but not on day 1. Whereas DNA synthesis was not reduced by ONO-4817, in vitro SMC migration on the other hand was reduced dose dependently. According to the results of western-blotting analysis, the expressions of MMPs were increased 1 week after injury. Especially, MMP-12 was not detected in hamsters without cholesterol diet, but it was much increased after injury in hypercholesterolemic hamsters. Additionally, active form of MMP-12 increased in the injured artery of hypercholesterolemic hamsters. In conclusion, inhibition of MMPs results in the suppression of neointima following vascular injury via both prevention of SMC migration and SMC proliferation of late phase in hypercholesterolemic hamsters. MMP-12 plays an important role on vascular stenosis in hypercholesterolemia and ONO-4817 could be a useful compound for the therapy for this field.

Increased elastin-degrading activity and neointimal formation in porcine aortic organ culture. Reduction of both features with a serine proteinase inhibitor.

We investigated the association between tissue elastolytic activity and the development of neointimal formation using a previously described porcine aortic organ culture. Neointimal formation is associated with the presence of intact endothelium (nondenuded cultures) but is markedly reduced if endothelial cells are removed (denuded cultures). In nondenuded organ cultures, elastolytic activity assessed by using [3H]elastin increased sixfold at day 3 after initiation of the culture (P < .01), a time earlier than the previously published increase in intimal smooth muscle cells (ISMCs). Elastolytic activity did not increase from day 3 to day 7 despite doubling of ISMCs but did double by day 14 (P < .01) and remained elevated to day 28, correlating with increases in ISMCs. In denuded organ cultures, elastolytic activity was much lower than in nondenuded organ cultures at day 3 (P < .05) but increased fivefold in the presence of nondenuded organ culture conditioned medium (P < .01). Addition of alpha 1-proteinase inhibitor for 14 days caused a 60% decrease in elastolytic activity in nondenuded organ cultures and a 27% reduction in ISMCs compared with untreated controls (P < .05 for both). The elastolytic activity, resolved as lytic bands on an elastin substrate gel, reflected candidate enzymes, one at 76 kD and perhaps a doublet at 43 and 50 kD. Our study suggests that endothelial cells release a soluble agent that enhances elastin-degrading activity in the aorta and may at least partially account for the initiation of neointimal formation.