Long-term effect of PROLI/NO on cellular proliferation and phenotype after arterial injury.
ABSTRACT: Vascular interventions are associated with high failure rates from restenosis secondary to negative remodeling and neointimal hyperplasia. Periadventitial delivery of nitric oxide (NO) inhibits neointimal hyperplasia, preserving lumen patency. With the development of new localized delivery vehicles, NO-based therapies remain a promising therapeutic avenue for the prevention of restenosis. While the time course of events during neointimal development has been well established, a full characterization of the impact of NO donors on the cells that comprise the arterial wall has not been performed. Thus, the aim of our study was to perform a detailed assessment of proliferation, cellularity, inflammation, and phenotypic cellular modulation in injured arteries treated with the short-lived NO donor, PROLI/NO. PROLI/NO provided durable inhibition of neointimal hyperplasia for 6 months after arterial injury. PROLI/NO inhibited proliferation and cellularity in the media and intima at all of the time points studied. However, PROLI/NO caused an increase in adventitial proliferation at 2 weeks, resulting in increased cellularity at 2 and 8 weeks compared to injury alone. PROLI/NO promoted local protein S-nitrosation and increased local tyrosine nitration, without measurable systemic effects. PROLI/NO predominantly inhibited contractile smooth muscle cells in the intima and media, and had little to no effect on vascular smooth muscle cells or myofibroblasts in the adventitia. Finally, PROLI/NO caused a delayed and decreased leukocyte infiltration response after injury. Our results show that a short-lived NO donor exerts durable effects on proliferation, phenotype modulation, and inflammation that result in long-term inhibition of neointimal hyperplasia.
Project description:BACKGROUND:The microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice. METHODS:Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods. RESULTS:All GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson's trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-?-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response. CONCLUSIONS:GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.
Project description:Despite the use of the sirolimus (rapamycin) drug-eluting coronary stent, diabetics are at increased risk of developing in-stent restenosis for unclear reasons. Hyperleptinemia, which often coexists with diabetes and metabolic syndrome, is an independent risk factor for progression of coronary artery disease. It has not been determined whether elevated circulating leptin decreases the efficacy of the sirolimus drug-eluting stent in inhibiting neointimal hyperplasia, the process underlying restenosis after stenting. Here we show that leptin activates the mammalian target of rapamycin (mTOR) signaling pathway in primary murine vascular smooth muscle cells (VSMC) and stimulates VSMC proliferation in a PI3K-dependent fashion. Exogenous leptin, administered at levels comparable to those found in obese humans, promotes neointimal VSMC hyperplasia in a murine femoral artery wire injury model. Leptin significantly increases the dose of the mTOR inhibitor sirolimus that is required for effective inhibition of neointimal formation. Combination therapy with LY294002, a PI3K inhibitor, and sirolimus effectively inhibits leptin-enhanced neointimal hyperplasia. These data show that, in the setting of hyperleptinemia, higher doses of an mTOR inhibitor, or combination therapy with mTOR and PI3K inhibitors, inhibits neointimal hyperplasia after arterial injury. These studies may explain the higher rates of restenosis observed in diabetics treated with a sirolimus-eluting coronary stent and suggest a potential novel therapeutic approach for inhibiting in-stent restenosis in such patients.
Project description:BACKGROUND:Neointimal hyperplasia is a prominent pathological event during in-stent restenosis. Phenotype switching of vascular smooth muscle cells (VSMCs) from a differentiated/contractile to a dedifferentiated/synthetic phenotype, accompanied by migration and proliferation of VSMCs play an important role in neointimal hyperplasia. However, the molecular mechanisms underlying phenotype switching of VSMCs have yet to be fully understood. METHODS:The mouse carotid artery ligation model was established to evaluate Sema3A expression and its role during neointimal hyperplasia in vivo. Bioinformatics analysis, chromatin immunoprecipitation (ChIP) assays and promoter-luciferase reporter assays were used to examine regulatory mechanism of Sema3A expression. SiRNA transfection and lentivirus infection were performed to regulate Sema3A expression. EdU assays, Wound-healing scratch experiments and Transwell migration assays were used to assess VSMC proliferation and migration. FINDINGS:In this study, we found that semaphorin-3A (Sema3A) was significantly downregulated in VSMCs during neointimal hyperplasia after vascular injury in mice and in human atherosclerotic plaques. Meanwhile, Sema3A was transcriptionally downregulated by PDGF-BB via p53 in VSMCs. Furthermore, we found that overexpression of Sema3A inhibited VSMC proliferation and migration, as well as increasing differentiated gene expression. Mechanistically, Sema3A increased the NRP1-plexin-A1 complex and decreased the NRP1-PDGFR? complex, thus inhibiting phosphorylation of PDGFR?. Moreover, we found that overexpression of Sema3A suppressed neointimal hyperplasia after vascular injury in vivo. INTERPRETATION:These results suggest that local delivery of Sema3A may act as a novel therapeutic option to prevent in-stent restenosis.
Project description:We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8?×?106 cells, n?=?10) or vehicle (controls, n?=?10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3?×?106 cells, tail vein; n?=?4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67?+ proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.
Project description:BACKGROUND AND PURPOSE:The increased proliferation and migration of vascular smooth muscle cells (VSMCs) after arterial injury contributes greatly to the pathogenesis of neointimal hyperplasia. As a major component of epigenetics, histone methylation plays an important role in several cardiovascular diseases. However, its role in restenosis is still unclear. EXPERIMENTAL APPROACH:Human aortic VSMCs were challenged with PDGF-BB, and total histones were extracted and analysed by HPLC/MS. For the in vivo study, rats were subjected to wire-guided common carotid injury. KEY RESULTS:PDGF-BB markedly increased the H3K27me3 level, as demonstrated by use of HPLC/MS and confirmed by western blot analysis. Enhancer of zeste homologue 2 (EZH2), the histone H3K27 methyltransferase component of polycomb repressive complex 2, was also up-regulated by PDGF-BB in VSMCs, and in the neointimal hyperplasia induced by wire injury of the rat carotid artery. Furthermore, inhibiting H3K27me3 by treatment with 3-?M UNC1999, an EZH2/1 inhibitor, significantly suppressed PDGF-BB-induced VSMC proliferation compared with the PDGF-BB-treated group. Consistently, neointimal formation was significantly attenuated by oral or perivascular administration of UNC1999 compared with the sham group. Mechanistically, the increase in H3K27me3 inhibited the transcription of the cyclin-dependent kinase inhibitor p16INK4A and thus promoted VSMC proliferation. CONCLUSIONS AND IMPLICATIONS:Vascular injury elevated the expression of EZH2 and the downstream target H3K27me3, which suppressed p16INK4A expression in VSMCs and promoted VSMC proliferation and neointimal hyperplasia. EZH2 inhibition might be a potential therapeutic target for restenosis.
Project description:As a potent and selective allosteric inhibitor of MEK, TAK-733 has been shown to exert anti-cancer effects for a wide range of cancers both in vitro and in vivo. However, its effects on inhibiting growth have never been investigated in the cardiovascular system, where regulation of abnormal vascular smooth muscle cell growth in neointimal hyperplasia is an important area of focus. Angiotensin II was used to mimic inflammatory neointimal hyperplasia in an in vitro environment, and balloon catheter-induced injury with an infusion of angiotensin II was used to generate an in vivo rat restenosis model under inflammatory conditions. TAK-733 exerted anti-proliferative and anti-migratory effects on human vascular smooth muscle cells. These multiple effects of TAK-733 were evaluated using various assays, such as cell cycle analysis and wound healing. Interestingly, TAK-733 did not induce apoptosis in smooth muscle cells but only reduced the proliferation rate; additionally, it did not affect EC viability. TAK-733 also exhibited anti-inflammatory activity, as observed by attenuated monocyte adhesion to smooth muscle cells via inhibition of ICAM1 and VCAM1 overexpression. The in vivo study demonstrated that neointimal hyperplasia after balloon injury and angiotensin II stimulation was suppressed by TAK-733, and downregulation of the inflammatory signal and enhanced re-endothelialization were observed. TAK-733 may have therapeutic potential for treating neointimal hyperplasia by attenuating smooth muscle cell proliferation, migration, and inflammation. Thus, TAK-733 could be a promising drug candidate for treating patients with restenosis.
Project description:BACKGROUND:Restenosis is a serious problem in patients who have undergone percutaneous transluminal angioplasty. Endothelial injury resulting from surgery can lead to endothelial dysfunction and neointimal formation by inducing aberrant proliferation and migration of vascular smooth muscle cells. Exosomes secreted by mesenchymal stem cells have been a hot topic in cardioprotective research. However, to date, exosomes derived from mesenchymal stem cells (MSC-Exo) have rarely been reported in association with restenosis after artery injury. The aim of this study was to investigate whether MSC-Exo inhibit neointimal hyperplasia in a rat model of carotid artery balloon-induced injury and, if so, to explore the underlying mechanisms. METHODS:Characterization of MSC-Exo immunophenotypes was performed by electron microscopy, nanoparticle tracking analysis and western blot assays. To investigate whether MSC-Exo inhibited neointimal hyperplasia, rats were intravenously injected with normal saline or MSC-Exo after carotid artery balloon-induced injury. Haematoxylin-eosin staining was performed to examine the intimal and media areas. Evans blue dye staining was performed to examine re-endothelialization. Moreover, immunohistochemistry and immunofluorescence were performed to examine the expression of CD31, vWF and ?-SMA. To further investigate the involvement of MSC-Exo-induced re-endothelialization, the underlying mechanisms were studied by cell counting kit-8, cell scratch, immunofluorescence and western blot assays. RESULTS:Our data showed that MSC-Exo were ingested by endothelial cells and that systemic injection of MSC-Exo suppressed neointimal hyperplasia after artery injury. The Evans blue staining results showed that MSC-Exo could accelerate re-endothelialization compared to the saline group. The immunofluorescence and immunohistochemistry results showed that MSC-Exo upregulated the expression of CD31 and vWF but downregulated the expression of ?-SMA. Furthermore, MSC-Exo mechanistically facilitated proliferation and migration by activating the Erk1/2 signalling pathway. The western blot results showed that MSC-Exo upregulated the expression of PCNA, Cyclin D1, Vimentin, MMP2 and MMP9 compared to that in the control group. Interestingly, an Erk1/2 inhibitor reversed the expression of the above proteins. CONCLUSION:Our data suggest that MSC-Exo can inhibit neointimal hyperplasia after carotid artery injury by accelerating re-endothelialization, which is accompanied by activation of the Erk1/2 signalling pathway. Importantly, our study provides a novel cell-free approach for the treatment of restenosis diseases after intervention.
Project description:Coronary restenosis, a major complication of percutaneous balloon angioplasty, results from neointimal proliferation of vascular smooth muscle cells (VSMCs). The sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a), specific to contractile VSMCs, has been reported previously to be involved in the control of the Ca(2+)-signaling pathways governing proliferation and migration. Moreover, SERCA2a gene transfer was reported to inhibit in vitro VSMC proliferation and to prevent neointimal thickening in a rat carotid injury model. The aim of this study was to evaluate the potential therapeutic interest of SERCA2a gene transfer for prevention of in-stent restenosis using a ex vivo model of human left internal mammary artery (hIMA) intimal thickening. Left hIMAs, obtained at the time of aorto-coronary bypass surgeries, were subjected to balloon dilatation followed by infection for 30 min with adenoviruses encoding either human SERCA2 and green fluorescence protein (GFP) or control gene (?-galactosidase, ?-gal) and GFP. Proliferation of subendothelial VSMCs and neointimal thickening were observed in balloon-injured hIMA maintained 14 days in organ culture under constant pressure and perfusion. SERCA2a gene transfer prevented vascular remodeling and significantly (P<0.01, n=5) reduced neointimal thickening in injured arteries (intima/media ratio was 0.07±0.01 vs 0.40±0.03 in ?-gal-infected arteries). These findings could have potential implications for treatment of pathological in-stent restenosis.
Project description:Microtubule stabilizing agents (MTSA) are known to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and effectively reduce neointimal hyperplasia and restenosis. Epothilones (EPOs), non-taxane MTSA, have been found to be effective in the inhibition of VSMC proliferation and neointimal formation by cell cycle arrest. However, effect of EPOs on apoptosis in hyper-proliferated VSMCs as a possible way to reduce neointimal formation and its action mechanism related to VSMC viability has not been suited yet. Thus, the purposes of the present study was to investigate whether EPOs are able to inhibit neointimal formation by inducing apoptosis within the region of neointimal hyperplasia in balloon-injured rat carotid artery, as well as underlying action mechanism. Treatment of EPO-B and EPO-D significantly induced apoptotic cell death and mitotic catastrophe in hyper-proliferated VSMCs, resulting in cell growth inhibition. Further, EPOs significantly suppressed VSMC proliferation and induced apoptosis by activation of p53-dependent apoptotic signaling pathway, Bax/cytochrome c/caspase-3. We further demonstrated that the local treatment of carotid arteries with EPOs potently inhibited neointimal lesion formation by induction of apoptosis in rat carotid injury model. Our findings demonstrate a potent anti-neointimal hyperplasia property of EPOs by inducing p53-depedent apoptosis in hyper-proliferated VSMCs.
Project description:Restenosis caused by neointimal hyperplasia significantly decreases long-term efficacy of percutaneous transluminal angioplasty (PTA), stenting, and by-pass surgery for managing coronary and peripheral arterial diseases. A major cause of pathological neointima formation is abnormal vascular smooth muscle cell (VSMC) proliferation and migration. Notoginsenoside R1 (NGR1) is a novel saponin that is derived from Panax notoginseng and has reported cardioprotective, neuroprotective and anti-inflammatory effects. However, its role in modulating VSMC neointima formation remains unexplored. Herein, we report that NGR1 inhibits serum-induced VSMC proliferation and migration by regulating VSMC actin cytoskeleton dynamics. Using a mouse femoral artery endothelium denudation model, we further demonstrate that systemic administration of NGR1 had a potent therapeutic effect in mice, significantly reducing neointimal hyperplasia following acute vessel injury. Mechanistically, we show that NGR1's mode of action is through inhibiting the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Taken together, this study identified NGR1 as a potential therapeutic agent for combating restenosis after PTA in cardiovascular diseases.