Wire-mediated Vascular Injury Research Articles

Inhibition of activated factor X by rivaroxaban attenuates neointima formation after wire-mediated vascular injury

Accumulating evidence suggests that activated factor X (FXa), a key coagulation factor, plays an important role in the development of vascular inflammation through activation of many cell types. Here, we investigated whether pharmacological blockade of FXa attenuates neointima formation after wire-mediated vascular injury. Transluminal femoral artery injury was induced in C57BL/6 mice by inserting a straight wire. Rivaroxaban (5mg/kg/day), a direct FXa inhibitor, was administered from one week before surgery until killed. At four weeks after surgery, rivaroxaban significantly attenuated neointima formation in the injured arteries compared with control (P<0.01). Plasma lipid levels and blood pressure were similar between the rivaroxaban-treated group and non-treated group. Quantitative RT-PCR analyses demonstrated that rivaroxaban reduced the expression of inflammatory molecules (e.g., IL-1β and TNF-α) in injured arteries at seven days after surgery (P<0.05, respectively). In vitro experiments using mouse peritoneal macrophages demonstrated that FXa increased the expression of inflammatory molecules (e.g., IL-1β and TNF-α), which was blocked in the presence of rivaroxaban (P<0.05). Also, in vitro experiments using rat vascular smooth muscle cells (VSMC) demonstrated that FXa promoted both proliferation and migration of this cell type (P<0.05), which were blocked in the presence of rivaroxaban. Inhibition of FXa by rivaroxaban attenuates neointima formation after wire-mediated vascular injury through inhibition of inflammatory activation of macrophages and VSMC.

Abstract 13438: Rivaroxaban, a Direct Factor Xa Inhibitor, Attenuates Neointima Formation after Mechanical Vascular Injury

Bachground: Activated factor X (FXa) is a key member in the coagulation cascade responsible for thrombin generation, although accumulating evidence suggests it also has various biological functions in many cell types, contributing to the pathogenesis of neointima formation after vascular injury. In this study, we assessed the hypothesis that rivaroxaban, a direct FXa inhibitor, attenuates neointima formation after mechanical vascular injury by inhibiting both pro-inflammatory activation of macrophages and proliferative activation of vascular smooth muscle cells (VSMCs). Methods and Results: Rivaroxaban (5 mg/kg/day) was administered for 4 weeks to C57BL/6 mice after wire-mediated vascular injury. Plasma lipid levels and blood pressure were similar between rivaroxaban group and the control group. Rivaroxaban significantly reduced neointima area (30452 ± 2531 μm2 vs. 19582 ± 2531 μm2; P &lt; 0.05) and intima/media ratio (4.37 ± 0.50 vs. 2.60 ± 0.50; P &lt; 0.05) in the injured arteries at four weeks after injury, as determined by elastica van Gieson staining, compared with the control group. In vitro experiments using mouse peritoneal macrophages or murine macrophage cell line RAW264.7 demonstrated that FXa increased mRNA expression of inflammatory molecules (e.g., IL-1β and TNF-α) and vasoproliferative molecules (e.g., PDGF and BMP-2), which was blocked in the presence of rivaroxaban. In vitro experiments using rat VSMCs demonstrated that FXa stimulation promoted both mitogenesis and migration of this cell type, as determined by MTS assay and scratch-wound assay, respectively. Rivaroxaban attenuated these FXa-induced mitogenesis and migration. Conclusions: Rivaroxaban attenuates neointima formation in a mouse model of vascular injury. Our analyses suggest that FXa contributes to both pro-inflammatory activation of macrophages and proliferative activation of VSMCs, participating in the progression of neointima formation after vascular injury. FXa may serve as a potential therapeutic target for neointima formation after vascular injury.

HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9

ObjectiveEndogenous ligands such as high-mobility group box 1 (HMGB1) and nucleic acids are released by dying cells and bind to Toll-like receptors (TLRs). As TLR9 is involved in both microbial and sterile inflammation by detecting both bacterial and endogenous DNA, we investigated its role in inflammation and lesion formation in a mouse model of vascular injury. Methods and resultsC57BL/6 (WT) and TLR9 KO mice were subjected to wire-mediated vascular injury. Anti-HMGB1 antibody and purified HMGB1 protein were chronically delivered around the injured arteries by gelatin hydrogel, and neointima formation at 4 weeks after injury was evaluated. In addition, the same vascular injury was performed in bone-marrow chimeric mice (WT bone marrow into TLR KO mice; TLR9 KO bone marrow into WT mice). We also evaluated the production of inflammatory cytokines by mouse macrophages in response to HMGB1 and CpG-ODN. In wild-type mice after vascular injury, anti-HMGB1 antibody significantly reduced neointima formation and HMGB1 protein accelerated neointima hyperplasia. HMGB1 failed to accelerate lesion formation in TLR9 KO mice. The bone marrow transplantation study revealed that TLR9 in bone marrow-derived cells played a fundamental role in neointima formation. In vitro, HMGB1 and CpG-ODN synergistically induced the production of inflammatory cytokines by macrophages. ConclusionsHMGB1 serves as an endogenous mediator of inflammation and lesion formation via the TLR9 pathway in response to vascular injury. Blockade of HMGB1 and/or TLR9 may represent a novel approach to treating atherosclerosis.

Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

BackgroundHyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.Methods and ResultsHA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.ConclusionVSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.

Targeting N-acetylglucosamine-bearing polymer-coated liposomes to vascular smooth muscle cells

The targeted delivery of anti-inflammatory agents has great therapeutic potential for treating restenosis following percutaneous coronary intervention. To develop a drug delivery system targeted to injured blood vessels, we examined whether N-acetylglucosamine (GlcNAc)-bearing polymer-coated liposomes (GlcNAc-Ls) are specifically taken up by vascular smooth muscle cells (VSMCs). Flow cytometric analysis revealed that GlcNAc-Ls were taken up by VSMCs in vitro. Furthermore, GlcNAc-Ls were intravenously administered to mice that had undergone wire-mediated vascular injury. GlcNAc-Ls markedly accumulated at the intramural site of the injured vessel walls but not at the contralateral (uninjured) vessel walls. These results demonstrated that GlcNAc-Ls can be specifically taken up by VSMCs both in vitro and in vivo. We propose a novel strategy of using GlcNAc-Ls that has potential for application in drug delivery targeted to injured blood vessels.

Targeted Disruption of the Prostaglandin E 2 E-Prostanoid 2 Receptor Exacerbates Vascular Neointimal Formation in Mice

Restenosis after angioplasty remains a major clinical problem. Prostaglandin E(2) (PGE(2)) plays an important role in vascular homeostasis. The PGE(2) receptor E-prostanoid 2 (EP2) is involved in the proliferation and migration of various cell types. We aimed to determine the role of EP2 in the pathogenesis of neointimal formation after vascular injury. Wire-mediated vascular injury was induced in the femoral arteries of male wild-type (EP2+/+) and EP2 gene-deficient (EP2-/-) mice. In EP2+/+ mice, EP2 mRNA expression was increased in injured vessels for at least 4 weeks after vascular injury. Neointimal hyperplasia was markedly accelerated in EP2-/- mice, which was associated with increased proliferation and migration of vascular smooth muscle cells (VSMCs) and increased cyclin D1 expression in the neointima layer. Platelet-derived growth factor-BB (PDGF-BB) treatment resulted in more significant cell proliferation and migration in VSMCs of EP2-/- mice than in those of EP2+/+ mice. Activation and overexpression of EP2 attenuated PDGF-BB-elicited cell proliferation and migration, induced G(1)→S-phase arrest and reduced PDGF-BB-stimulated extracellular signal-regulated kinase phosphorylation in EP2+/+ VSMCs. These findings reveal a novel role of the EP2 receptor in neointimal hyperplasia after arterial injury. The EP2 receptor may represent a potential therapeutic target for restenosis after angioplasty.

Fluvastatin accelerates re-endothelialization impaired by local sirolimus treatment

Sirolimus-eluting stent reduces restenosis after percutaneous coronary intervention. However, accumulating evidence suggests that sirolimus potentially affects re-endothelialization, leading to late thrombosis. Statins have protective effects on endothelium. Recently, statins are reported to increase the number of circulating endothelial progenitor cells (EPCs) and accelerate re-endothelialization after vascular injury. Here, we tested the hypothesis that fluvastatin has beneficial effect on re-endothelialization after local sirolimus treatment. We performed wire-mediated vascular injury to both sides of femoral arteries of wild-type mice and bone marrow chimeric mice. Either sirolimus (100 μg) or DMSO was administered locally to the perivascular area of the injured arteries. All mice received either fluvastatin (5 mg/kg/day) or vehicle by gavage starting at one week before the surgery until sacrifice. At 4 weeks after the surgery, re-endothelialization of the sirolimus-treated artery was significantly less than that of DMSO-treated one in the vehicle-treated mice as determined by the percentage of CD31-positive area ( P < 0.05). Systemic administration of fluvastatin accelerated the re-endothelialization in the sirolimus-treated artery to the similar degree of that in the DMSO-treated artery ( P = NS). Contribution of bone marrow-derived cells to re-endothelialization was seldom observed in bone marrow chimeric mice regardless of fluvastatin administration. Fluvastatin significantly ameliorated proliferation (2.5-folds) and migration activities (2.3-folds) of mature endothelial cells impaired by sirolimus treatment ( P < 0.05, respectively). Fluvastatin increased endothelial nitric oxide synthase expression and decreased plasminogen activator inhibitor-1 expression in mature endothelial cell in the presence of sirolimus ( P < 0.05, respectively). Our findings suggest that fluvastatin has protective effects against impaired re-endothelialization after sirolimus treatment.

Notch signaling regulates the differentiation of bone marrow-derived cells into smooth muscle-like cells during arterial lesion formation

Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle α-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with γ-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC.

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.

Deficiency of tumour necrosis factor- and interferon- in bone marrow cells synergistically inhibits neointimal formation following vascular injury

Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since it is now known that vascular injury involves an inflammatory response, we examined the role of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the neointimal formation after injury. Control (BALB/c), TNF-alpha-deficient (Tnf(-/-)), IFN-gamma-deficient (Ifng(-/-)), or double-deficient (Tnf(-/-)Ifng(-/-)) mice were subjected to wire-mediated vascular injury of the right femoral artery. Neointimal formation after injury was significantly reduced after the injury in the Tnf(-/-)Ifng(-/-) mice, compared to that in the control, Tnf(-/-), and Ifng(-/-) mice. Immunohistochemical analysis showed that TNF-alpha and IFN-gamma were expressed in neointimal lesions in the control mice, but not in mice with deficiency of the corresponding cytokine. No significant difference in re-endothelialization was observed among these groups. The number of proliferating cell nuclear antigen in the neointimal lesions was significantly decreased in the Tnf(-/-)Ifng(-/-) mice. Bone marrow transplantation experiments revealed that deficiency of TNF-alpha and IFN-gamma specifically in bone marrow cells significantly inhibited neointimal formation after vascular injury. The absence of TNF-alpha and IFN-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury, and thus, may provide new insights into the mechanisms underlying restenosis after PCI.

Critical Role of Bone Marrow Apoptosis-Associated Speck-Like Protein, an Inflammasome Adaptor Molecule, in Neointimal Formation After Vascular Injury in Mice

Inflammatory cytokines such as interleukin (IL)-1 beta and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1-dependent IL-1 beta and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC(-/-)) mice. Wire-mediated vascular injury was produced in the femoral artery of ASC(-/-) and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC(-/-) mice after injury. IL-1 beta and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC(-/-) mice. To investigate the contribution of bone marrow-derived cells, we developed bone marrow-transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC(-/-) bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC(-/-) vascular smooth muscle cells was not impaired. These findings suggest that bone marrow-derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.

Abstract 1175: Critical Role of Bone Marrow ASC (Apoptosis-associated Speck-like Protein Containing a CARD) in Neointimal Formation After Vascular Injury in Mice

Background: ASC is an adaptor protein that forms inflammasome complex and regulates caspase-1-dependent interleukin (IL)-1β and IL-18 generation; however, the role of ASC in the pathophysiology of cardiovascular diseases remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC −/− ) mice. Methods and Results: Wire-mediated vascular injury was produced in the femoral artery of ASC −/− and wild-type (WT: C57BL/6) mice. Immunohistochemical analysis revealed that ASC was markedly expressed in the infiltrated macrophages in the neointima. Histological analysis revealed that neointimal formation was significantly reduced in ASC −/− mice as compared to that in the WT mice (I/M ratio, p = 0.007). IL-1β and IL-18 were clearly expressed in the neointimal lesion of the WT mice but showed decreased expression in the lesion of ASC −/− mice. However, TUNEL staining showed no significant difference in apoptotic cells of the media between these mice. Further, no significant differences were observed in early reendothelialization (CD31) and the number of macrophages (F4/80) and smooth muscle cells (SMCs: αSMA) per unit of the neointima. Flow cytometry analysis also showed no difference in the number of peripheral CD34 + /Flk-1 + cells (EPCs) after injury. To determine the contribution of bone marrow cells, we developed 3 types of bone marrow transplantation (BMT Wild+Wild , BMT Wild+ASC−/− , and BMT ASC−/− → Wild ) mice. The neointimal formation in BMT ASC−/−+Wild mice mice was remarkably reduced as compared to that in BMT Wild+Wild (I/M ratio, p = 0.0003) and BMT Wild+ASC−/− mice (p = 0.015), indicating that bone marrow ASC is critical for neointimal formation. In vitro experiments showed that the lipopolysaccharide-induced production of inflammatory cytokines such as IL-1β, IL-6, MCP-1, and IFN-γ, but not TNF-α, in ASC −/− bone marrow cells was significantly decreased. Further, serum-stimulated proliferation activity and MAP kinase (ERK1/2 and p38) activation of ASC −/− SMCs were not impaired. Conclusions: These findings suggest that bone marrow-derived ASC is critical for the neointimal formation after vascular injury and identify ASC as a novel therapeutic target for cardiovascular diseases.

M-CSF Accelerates Neointimal Formation in the Early Phase After Vascular Injury in Mice

Since the macrophage colony-stimulating factor (M-CSF) has been shown to stimulate differentiation and proliferation of monocyte/macrophage lineage and to be involved in the process of neointimal formation after vascular injury, we tested the effects of M-CSF on the recruitment of bone marrow-derived progenitor cells in neointimal formation after vascular injury in mice. Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. Recombinant human M-CSF [500 microg/(kg x day)] or saline (control) was administered for 10 consecutive days, starting 4 days before the injury. Treatment with M-CSF accelerated neointimal formation in the early phase after injury, and this neointimal lesion mainly consisted of bone marrow-derived cells. M-CSF treatment had no effect on the mobilization of endothelial progenitor cells (EPCs: CD34+/Flk-1+) and reendothelialization after injury. The stromal cell-derived factor-1 (SDF-1) was markedly expressed in the neointima and media after injury, whereas CXCR4+ cells were observed in the neointima. Further, a novel CXCR4 antagonist, AMD3100, significantly attenuated the M-CSF-induced neointimal formation. These findings suggest that M-CSF accelerated neointimal formation after vascular injury via the SDF-1-CXCR4 system, and the inhibition of this system has therapeutic potential for the treatment of cardiovascular diseases.

Cell Source in Stent Induced Restenosis

Traditionally, restenotic neointimal hypercellularity has been attributed to migration and proliferation of vessel wall smooth muscle cells (SMC). This scheme has been challenged by findings of circulating blood SMC progenitors that contribute to the formation of vascular lesions. In stent restenosis (ISR) limits the long-term success of percutaneous treatments and is caused by excessive accumulation of SMC. The pathobiological events underlying ISR are poorly understood. Recently, human tissue obtained by atherectomy, retrieved from ISR and de-novo primary lesions, were compared by immunohistochemistry analysis. Interestingly, the presence of bone marrow derived endothelial progenitor cells and dendritic cells, as well as neural crest derived cells were found in all ISR atherectomy specimens. Furthermore, specific staining was significantly increased in ISR tissue compared to primary plaque. This suggests the recruitment of primarily extravascular cells within ISR neointimal formation. Sirolimus-eluting stents (SES) have lately emerged as an effective strategy to prevent ISR. Despite increasing clinical use, little is known about the mechanism by which locally delivered Sirolimus prevents ISR. Recently, Sirolimus was shown to potently inhibit human vascular progenitor cell outgrowth in-vitro. In-vivo, Sirolimus elicited an inhibitory effect on both SMC and endothelial progenitor cell incorporation at sites of wire-mediated vascular injury of bone marrow chimeric mice. This potent inhibitory effect of sirolimus on circulating SMC progenitor cells may mediate in part the clinical efficacy of SES.

Potent Inhibitory Effect of Sirolimus on Circulating Vascular Progenitor Cells

Neointimal hyperplasia is the major cause of in-stent restenosis (ISR). The sirolimus-eluting stent (SES) has emerged as a promising therapy to prevent ISR; however, the exact mechanism by which locally delivered sirolimus, an immunosuppressive agent, prevents ISR remains unknown. Recent evidence suggests that circulating progenitor cells may contribute to neointimal formation. Mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers. Smooth muscle (SM)-like cells outgrew from the culture of MNCs (1x10(6)) in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas endothelial cell-like cells were obtained in the presence of vascular endothelial growth factor. Sirolimus potently inhibited SM-like cell outgrowth. The number of SM-like cells was significantly reduced at a concentration as low as 0.1 ng/mL (15.9+/-5.8% of control, P<0.001). Sirolimus also exerted an inhibitory effect on endothelial cell-like cells that originated from MNCs. Wire-mediated vascular injury was induced in femoral arteries of bone marrow chimeric mice. Either vehicle or sirolimus was administered locally to the perivascular area of the injured arteries. Sirolimus significantly reduced neointima hyperplasia at 4 weeks (intima/media ratio 2.0+/-0.3 versus 1.0+/-0.2, P<0.05) with a decreased number of bone marrow-derived SM-like cells and hematopoietic cells in the lesion. Reendothelialization was retarded in the arteries treated with sirolimus. The potent inhibitory effects of sirolimus on circulating smooth muscle progenitor cells may mediate the clinical efficacy of SES, at least in part. Sirolimus potentially may affect reendothelialization after stent implantation.