Adult progenitor cells in vascular remodeling during atherosclerosis

The mobilization and recruitment of bone marrow-derived, circulating or tissue resident progenitor cells giving rise to smooth muscle-like cells have been implicated in neointima hyperplasia after arterial injury and in accelerated forms of arterial lesion formation, e.g., transplant arteriopathy or graft vasculopathy. By contrast, convincing evidence has emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to endothelial recovery, thus limiting neointima formation after arterial injury. In the chronic context of primary atherosclerosis, plaque progression and destabilization, a more complex picture has become apparent. In patients with coronary artery disease, the number and function of EPCs have been linked with an improved endothelial function or regeneration, but have been inversely correlated with cardiovascular risk. In animal models, however, the injection of bone marrow cells or EPCs, or the application of stem-cell mobilizing factors, have been associated with an exacerbation of atherosclerosis and unstable plaque phenotypes, whereas the contribution of bone marrow-derived smooth muscle progenitors to primary atherosclerosis appears to be rather confined. Here, we discuss crucial biochemical cues, namely chemokines, adhesion molecules, growth factors and pharmacological means that guide and control the context-specific mobilization, recruitment and fate of vascular progenitor cells in arterial remodeling during atherosclerosis.     

Trauma induced by nontraumatic coronary devices and its impact on vascular reactivity and morphology

This study evaluated the impact of low-pressure balloon devices on coronary morphology and function. An active coronary perfusion catheter (2.5-mm balloon diameter, inflation with 1 bar for 30 min) was placed in the left anterior descending coronary artery of 12 German landrace pigs under general anesthesia. After 3 mo, coronary segments with balloon contact were compared with control segments taken from the right coronary artery as to histology, vascular reactivity, and expression of endothelial nitric oxide synthase. Thirty-three balloon treated segments were analyzed. Twenty of these segments (61%) showed neointima formation. In these segments endothelium-independent relaxation induced by sodium nitroprusside was preserved. However, endothelium-dependent bradykinin-induced relaxation was significantly attenuated compared with both the control segments and the balloon-treated segments without neointima formation. In >60% of the ballooned arterial segments examined, low-pressure balloon devices induced neointima formation accompanied by reduced endothelium-dependent relaxation. Thus interventions with so-called nontraumatic coronary devices can induce relevant vascular injury, with potential adverse clinical consequences.     

Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT9‐mediated uric acid uptake

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti‐inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA‐induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP‐1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor‐kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose‐dependent manner. In contrast, phloretin significantly attenuated pro‐inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor‐kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA‐induced endothelial injury via a synergic mechanism including direct anti‐inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia‐related cardiovascular diseases.     

Macrophage‐derived exosomal miR‐4532 promotes endothelial cells injury by targeting SP1 and NF‐κB P65 signalling activation

Atherosclerosis is a complex pathological process involving macrophages, endothelial cells and vascular smooth muscle cells that can lead to ischemic heart disease; however, the mechanisms underlying cell‐to‐cell communication in atherosclerosis are poorly understood. In this study, we focused on the role of exosomal miRNAs in crosstalk between macrophages and endothelial cells and explored the rarely studied molecular mechanisms involved. Our in vitro result showed that macrophage‐derived exosomal miR‐4532 significantly disrupted human umbilical vein endothelial cells (HUVECs) function by targeting SP1 and downstream NF‐κB P65 activation. In turn, increased endothelin‐1 (ET‐1), intercellular cell adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) and decreased endothelial nitric oxide synthase (eNOS) expression in HUVECs increased attraction of macrophages, exacerbating foam cell formation and transfer of exosomal miR‐4532 to HUVECs. MiR‐4532 overexpression significantly promoted endothelial injury and pretreatment with an inhibitor of miR‐4532 or GW4869 (exosome inhibitor) could reverse this injury. In conclusion, our data reveal that exosomes have a critical role in crosstalk between HUVECs and macrophages. Further, exosomal miR‐4532 transferred from macrophages to HUVECs and targeting specificity protein 1 (SP1) may be a novel therapeutic target in patients with atherosclerosis.     

Influence of nebivolol and metoprolol on inflammatory mediators in human coronary endothelial or smooth muscle cells. Effects on neointima formation after balloon denudation in carotid arteries of rats treated with nebivolol.

OBJECTIVE AND BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis. Proliferating vascular smooth muscle cells (SMC) and endothelial cells (EC) enhancing the inflammatory response, both contribute to the progression of atherosclerosis. Anti-proliferative, anti-inflammatory and anti-oxidative therapy seems to be a promising therapeutic strategy. The aim of this study was to assess the anti-proliferative and anti-inflammatory effect of the beta-blocker nebivolol in comparison to metoprolol in vitro and to find out whether nebivolol inhibits neointima formation in vivo. METHODS AND RESULTS: Real-time-RT-PCR revealed a decrease in VCAM-1, ICAM-1, PDGF-B, E-selectin and P-selectin mRNA expression in human coronary artery EC and SMC incubated with nebivolol for 72 hours while metoprolol did not have this effect. Nebivolol reduced MCP-1 and PDGF-BB protein in the culture supernatant of SMC and EC, respectively. Sprague-Dawley rats were treated with nebivolol for 0 or 35 days before and 28 days after carotid balloon injury. Immunohistological analyses showed that pre-treatment with nebivolol was associated with a decreased number of SMC layers and macrophages and an increased lumen area at the site of the arterial injury. The intima area was reduced by 43% after pre-treatment. CONCLUSION: We found that nebivolol reduced the expression of proinflammatory genes in endothelial cells and vascular smooth muscle cells in vitro whereas metoprolol did not. In vivo, nebivolol inhibited neointima formation by reducing SMC proliferation and macrophage accumulation.     

Endothelial cell barrier regulation by sphingosine 1-phosphate

Disruption of vascular barrier integrity markedly increases permeability to fluid and solute and is the central pathophysiologic mechanism of many inflammatory disease processes, including sepsis and acute lung injury (ALI). Dynamic control of the endothelial barrier involves complex signaling to the endothelial cytoskeleton and to adhesion complexes between neighboring cells and between cells and the underlying matrix. Sphingosine 1-phosphate (S1P), a biologically active lipid generated by hydrolysis of membrane lipids in activated platelets, organizes actin into a strong cortical ring and strengthens both intercellular and cell-matrix adherence. The mechanisms by which S1P increases endothelial barrier integrity remain the focus of intense basic research. The downstream structural changes induced by S1P interact to decrease vascular permeability to fluid and solute, which translates into a reduction lung edema formation in animal models of ALI, thus suggesting a potentially life-saving therapeutic role for vascular barrier modulation in critically ill patients.     

Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia.     

Serotonin-induced endothelial cell proliferation is blocked by omega-3 fatty acids.

Serotonin (5HT) released from aggregating platelets at sites of vascular injury is a known mitogen for vascular endothelial cells. Recent studies have indicated that regenerating endothelial cells at sites of vessel wall injury may play a role in the development of restenosis by synthesizing and releasing growth factors for vascular smooth muscle cells, proliferation of which may result in the development of neointima. Diets rich in fish oils (omega-3 fatty acids) are associated with reduced risk of cardiovascular disease including atherosclerosis and restenosis. This study examined the effect of the omega-3 and other fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on 5HT induced endothelial cell proliferation. Among the fatty acids examined only EPA and DHA could reverse the mitogenic effect of 5HT on vascular endothelial cells, whereas oleic acid or palmitic acid did not have any effect. When added together, EPA and DHA potentiate each other in reversing the mitogenic effect of 5HT. EPA and DHA also inhibited the 5HT-induced increase in the 5HT2 receptor mRNA, without a change in the receptor density or affinity. This data suggests that one of the mechanisms by which omega-3 fatty acids may attenuate the development of atherosclerosis or restenosis is to inhibit the mitogen induced growth of vascular endothelial cells, which attenuates the release of growth factors for vascular smooth muscle cells.     

内皮祖细胞在支架术后再内皮化中应用的研究进展

经皮冠状动脉介入治疗的应用改善了冠心病患者的临床症状及预后,但现在困扰人们的问题是作为其术后并发症之一的支架内再狭窄发病率仍然很高。大量的研究证实,内膜增生在支架内再狭窄的形成中起主导作用,所以提高受损内膜再内皮化的速度是防止支架内再狭窄的一个重要措施。新近的研究表明,内皮祖细胞能参与损伤后血管内皮修复,促进受损血管内膜的再内皮化,因此,在防止支架内再狭窄中将得到进一步的研究与应用。因此,本文就内皮祖细胞在支架术后再内皮化中应用的研究进展做一综述。     

The cyclic adenosine monophosphate elevating medicine,forskolin, reduces neointimal formation and atherogenesis in mice

Neointimal formation and atherogenesis are major vascular complications following percutaneous coronary intervention, and there is lack of pharmacological therapy. This study was aimed to examine the effect of forskolin (FSK), a cyclic adenosine monophosphate (cAMP)‐elevating agent, on vascular response to angioplasty wire injury and on atherogenesis in mice. Forskolin treatment reduced neointima formation at 7 and 28 days after wire injury. Early morphometrics of the injured vessels revealed that FSK treatment enhanced endothelial repair and reduced inflammatory cell infiltration. In vitro treatment of primary aortic cells with FSK, at 3‐100 μmol/L, increased endothelial cell proliferation, whereas FSK, at 30‐100 μmol/L, inhibited smooth muscle cell proliferation. FSK inhibited lipopolysaccharide‐induced leucocyte‐endothelial interaction in vitro and in vivo. In a mouse model of atherosclerosis driven by dyslipidaemia and hypertension, FSK administration increased endothelial repair and reduced atherosclerotic plaque formation, without affecting blood pressure, plasma lipids or aortic aneurysms formation. In summary, FSK, at doses relevant to human therapeutic use, protects against neointimal hyperplasia and atherogenesis, and this is attributable to its activities on pro‐endothelial repair and anti‐inflammation. This study raises a potential of clinical use of FSK as an adjunct therapy to prevent restenosis and atherosclerosis after percutaneous coronary intervention.     

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.     

血管外膜在动脉粥样硬化中的作用

动脉粥样硬化被认为是受损的内皮细胞释放粘附因子,吸引单核细胞粘附浸润到内膜下吞噬脂质,同时平滑肌细胞进行增殖迁移并形成新生内膜的过程,但目前越来越多的证据提示血管外膜作为反应的先导者从外向内参与了这一过程。在诸多血管疾病模型中,均能检测到外膜的早期激活。成纤维细胞作为血管外膜的主要细胞成分,在血管损伤早期会进行增殖迁移至中膜和内膜,还可以通过释放活性氧、各种细胞因子、基质金属蛋白酶等来影响炎症反应,导致内膜增生,最终促进了血管重塑及一些心血管疾病的发生。因此,越来越多的研究关注外膜成纤维细胞对于动脉粥样硬化、糖尿病、腹主动脉瘤等疾病中的作用及其机制,本文对该领域新近研究进展做一综述。     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

Regulated expression of endothelial cell-derived lipase

A lipoprotein lipase-like gene was recently cloned from endothelial cells. In vitro functional experiments have suggested that this endothelial-derived lipase (EDL) has phospholipase activity, and preliminary in vivo studies have suggested a role in the regulation of high-density lipoprotein metabolism. To investigate local control of lipase activity and lipid metabolism in the blood vessel wall, we have examined the regulation of EDL expression in cultured human umbilical vein and coronary artery endothelial cells. EDL mRNA levels were upregulated in both cell types by inflammatory cytokines implicated in vascular disease etiology, including TNF-alpha and IL-1beta. In addition, both fluid shear stress and cyclic stretch were found to increase the EDL mRNA levels in these cultured cells. This highly regulated expression of EDL in vascular endothelial cells suggests that this recently identified lipase is intricately involved in modulating vessel wall lipid metabolism and may play a role in vascular diseases such as atherosclerosis.     

Akap200 suppresses the effects of Dv-cbl expression in the Drosophila eye

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.     

Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

Patients with diabetes have an increased risk of vascular complications. Suv39h1, a histone methyltransferase, plays a protective role against myocardial injury in diabetes. Herein, we intend to explore whether Suv39h1 could affect neointimal formation after vascular injury in diabetic rats and reveal the underlying mechanism. In this study, we generated adenovirus expressing Suv39h1 as well as lentivirus expressing Suv39h1‐targeting shRNA and evaluated the significance of Suv39h1 in vascular smooth muscle cells (VSMCs) under diabetic conditions. In vitro, we examined proliferative and migratory behaviours as well as the underlying signalling mechanisms in VSMCs in response to high glucose treatment. In vivo, we induced diabetes in SD rats with streptozocin and established the common carotid artery balloon injury model. Suv39h1 was found to be both necessary and sufficient to promote VSMC proliferation and migration under high glucose conditions. We observed corresponding changes in intracellular signalling molecules including complement C3 and phosphor‐ERK1/2. However, either up‐regulating or down‐regulating Suv39h1, phosphor‐p38 level was not significantly affected. Consistently, Suv39h1 overexpression led to accelerated neointima formation, while knocking down Suv39h1 reduced it following carotid artery injury in diabetic rats. Using microarray analyses, we showed that altering the Suv39h1 level in vivo dramatically altered the expression of myriad genes mediating different biological processes and molecular function. This study reveals the novel role of Suv39h1 in VSMCs of diabetes and suggests its potential role as a therapeutic target in diabetic vascular injury.     

Novel model of inflammatory neointima formation reveals a potential role of myeloperoxidase in neointimal hyperplasia

Atherosclerosis, which is characterized by neointima formation, is an inflammatory disease. However, there is no inflammatory product-elicited neointimal model to support the causal role of inflammation in atherogenesis. We reported previously that leukocyte-derived MPO induces vascular injury responses such as endothelial dysfunction. We now test the role of MPO in inflammatory neointima formation. We infused temporarily isolated rat common carotid arteries with MPO (200 nM) and incubated for 1 h. We found that although MPO itself did not induce any neointima formation 2 wk after treatment, in the presence of its substrate, hydrogen peroxide, MPO was able to elicit neointimal hyperplasia. We further confirmed that MPO-induced neointimal hyperplasia is mediated by its product, hypochlorous acid (HOCl). HOCl elicited apoptosis both in intima and media followed by vascular proliferative response and resulted in neointima formation with a heterogeneous cell population. Both histological and functional features of HOCl-treated vessels are similar to those in atherosclerotic lesions. To our knowledge, this is the first direct in vivo demonstration of neointimal formation induced by a product of the inflammatory cascade. The results suggest that MPO may be a mediator for pathological neointima growth. This novel neointimal model could be useful for studying inflammation and atherosclerosis.     

Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin

Adrenomedullin (AM) and corticotrophin (ACTH) are both vasoactive peptides produced by a variety of cell types, including endothelial cells. Although AM and ACTH are considered to be important in the control of blood pressure and the response to stress, respectively, their role in inflammation and the immune response has not been clarified. This study shows, with the use of a cell-based ELISA, that AM and ACTH induce cell surface expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVEC). Furthermore, this effect appears to be mediated in part via elevation of cAMP, given that both peptides elevate cAMP, the cell-permeable cAMP analog dibutyryl cAMP is able to mimic induction of all three cell adhesion molecules and the effect of AM and ACTH is inhibited by the adenylyl cyclase inhibitor SQ-22536. These findings demonstrate a role for AM and ACTH in the regulation of the immune and inflammatory response. E-selectin; intercellular adhesion molecule-1; vascular cell adhesion molecule-1; adrenomedullin; adrenocorticotropic hormone; human umbilical vein endothelial cells     

Towards the treatment of saphenous vein bypass graft failure--a perspective of the Bristol Heart Institute

Coronary artery bypass graft surgery (CABG) is widely used for the treatment of atheromatous stenosis of coronary arteries. However, as many as 50% of grafts fail within 10 years after CABG due to neointima (NI) formation, a process involving the proliferation and migration of vascular smooth muscle cells (VSMCs). Superimposed on neointima formation is accelerated atherogenesis which ultimately results in late vein graft failure. To date no therapeutic intervention has proved successful in treating late vein graft failure and as such is a matter of some urgency. However, in recent years, several diverse approaches aimed at preventing neointimal formation have been devised which have yielded promising results. These include the use of external stents, gene therapy as well as conventional pharmacological interventions. The objective of this article, therefore, is to review these recent approaches and their potential clinical applications in the treatment of vein graft disease.