Statin-induced vascular smooth muscle cell apoptosis: a possible role in the prevention of restenosis?

Growing evidence suggests that statins are more than simple lipid-lowering drugs. The so called pleiotropic effects of statins include multiple actions on cells of the vasculature. A large number of studies have confirmed that these compounds exert beneficial effects by mechanisms unrelated to cholesterol metabolism. For example, statins have been shown to inhibit the migration and proliferation of vascular smooth muscle cells (VSMC), and to induce apoptosis in this cell type. It is not yet clear if the induction of apoptosis in VSMC by statins is beneficial or detrimental. In the context of post-angioplasty restenosis, recurrent plaque growth after intervention, the inhibition of neointimal proliferation as well as a reduction of neointimal cell numbers by apoptosis is appealing. Multiple animal studies and clinical trials have therefore been undertaken to investigate effects of statin treatment on the development of restenosis, with very controversial results. Conversely, in advanced atherosclerotic lesions VSMC in the intima may stabilize the plaque and prevent plaque rupture by synthesizing collagen. VSMC in media adjacent to plaque areas or restenotic lesions should not be exposed to apoptosis promoting agents. In this context, recent evidence suggests that pravastatin protects such lesions by inhibiting inflammation and macrophage activation Our recent findings together with observations from other groups suggest that neointima cells are more sensitive to the induction of apoptosis than media VSMC. Importantly, statins were found to preferentially induce apoptosis in neointimal VSMC in our study. The purpose of the present review is to summarize statin effects on proliferation and apoptosis in VSMC in vitro and in vivo. Furthermore, the development of drug-coated stents may help to deliver high local doses of statins to enhance their effectiveness in the treatment of post-angioplasty restenosis.     

Cytokines and growth factors involved in apoptosis and proliferation of vascular smooth muscle cells

This review focuses on the role of cytokines and growth factors involved in the regulation of smooth muscle cells in an atherosclerotic plaque. As a plaque begins to develop, upon endothelial injury inflammatory cells within the lesion interact with the accumulating LDL, other inflammatory cells and smooth muscle cells and release cytokines and growth factors. The mediators released from the activated cells regulate the proliferation and/or survival of smooth muscle cells. This determines the stability and integrity of a plaque. New data emerging from various studies have provided novel insights into many of the cellular interactions and signaling mechanisms involving apoptosis of smooth muscle cells in the atherosclerotic plaques. A number of these studies, focusing on activation of inflammatory cells and the roles of chemokines, cytokines and growth factors, are addressed in this review.     

糖尿病大鼠冠状动脉平滑肌细胞体外培养方法的探讨

目的探讨糖尿病大鼠冠状动脉平滑肌细胞体外培养方法,建立糖尿病大鼠冠状动脉平滑肌细胞模型,为糖尿病性冠心病的研究奠定基础。方法建立糖尿病大鼠模型,用酶消化法体外培养冠状动脉血管平滑肌细胞。结果糖尿病大鼠造模成功后分离冠状动脉,用酶消化法培养糖尿病大鼠血管平滑肌细胞,24h更换培养液液,培养7~10d细胞重叠生长达多层,高低起伏呈“峰—谷”状。细胞α-actin免疫组织化学染色鉴定为平滑肌细胞。结论糖尿病大鼠血管平滑肌细胞增殖速度快,培养条件要求严格,在形态学上与正常大鼠平滑肌细胞相同。     

Osteopontin is required for angiotensin II-induced migration of vascular smooth muscle cells

Migration and proliferation of vascular smooth muscle cells (VSMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Angiotensin II (Ang-II) is typically associated with excessive proliferation and migration of VSMCs and vascular remodeling. High levels of osteopontin (OPN) mRNA and protein were reported in human atherosclerotic plaque from the aorta, carotid and coronary arteries. However whether OPN plays a role in VSMCs migration induced by Ang-II is unknown. Here we show that, in primary cultured rat VSMCs, Ang-II exhibits chemotactic effect on cultured VSMCs and induces OPN expression dose-dependently. With a lentiviral shRNA specifically targeting OPN and transwell migration assay, we find that blockade of OPN with shRNA inhibits Ang-II-induced MMP9 upregulation and VSMCs migration. Our results demonstrated that OPN is required for Ang-II to induce VSMCs migration and suggested OPN as a potential target in preventing atherosclerotic development.     

Emodin induces growth arrest and death of human vascular smooth muscle cells through reactive oxygen species and p53

Percutaneous coronary intervention is the main therapy for revascularization of occluded coronary arteries. However, a progressive artery restenosis caused by abnormal proliferation and migration of vascular smooth muscle cells (VSMC) hinders the effective treatment. In this study, we examined the effect of emodin, a natural anthraquinoid compound, on cultured VSMC. Lower doses of emodin suppressed cell proliferation and induced unscheduled DNA synthesis. Higher doses of emodin increased lumpy chromatin condensation and lysosomes in VSMC, suggesting the occurrence of apoptosis and autophagy. Emodin increased production of reactive oxygen species (ROS), which was abolished by an NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI could also decrease the number of apoptosis induced by emodin, suggesting the involvement of ROS in emodin-induced apoptosis. Emodin upregulated the protein levels of p53 in a dose-dependent manner. Laser confocal microscope showed most of emodin scattering in the cytoplasms and a little within the nuclei. These findings collectively indicated that emodin induces both growth arrest and death of human VSMCs in 2 independent manners, implying it as a promising therapy for preventing restenosis.     

Telomere/Telomerase system: a new target of statins pleiotropic effect?

Statins are well established drugs for primary and secondary prevention of coronary artery disease (CAD). Despite the well-known ability of statins to lower cholesterol, it is now clear that clinical benefits are also substantially higher than expected and several clinical trials, like JUPITER (Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin trial) have indicated that such clinical effects are independent of cholesterol reduction. These cholesterol-independent actions have been named "pleiotropic effects" and include: anti-oxidation and anti-inflammatory effects, modulation of immune activation, stabilization of atherosclerotic plaque, decreased platelet activation, inhibition of cardiac hypertrophy, reduction of cytokine-mediated vascular smooth muscle cell (VSMC) proliferation and improvement of endothelial function. Recently, additional pleiotropic effects of statins on "cellular senescence" have been seen in different cell types, including endothelial progenitor cells (EPC), endothelial cells (EC), VSMC and chondrocytes. At the molecular level, the effect of statins on cellular senescence could be mediated by their interaction with the telomere/telomerase system. Recent evidence suggests that the anti-aging effects of statins are linked to their ability to inhibit telomere shortening by reducing either directly and indirectly oxidative telomeric DNA damage, as well as by a telomere capping proteins dependent mechanism. In this review, we discuss the pleiotropic effects of statins, focusing on the telomere/telomerase system. We will also present our current findings regarding leukocyte telomere length in very old people with myocardial infarction on statin therapy.     

气血并治方有效组分对载脂蛋白E基因敲除小鼠动脉粥样硬化不稳定斑块的干预作用

目的探讨气血并治方水提取物有效组分(CWQB)配伍对载脂蛋E基因敲除(ApoE-)小鼠晚期动脉粥样硬化不稳定斑块的干预作用及其可能的作用机制。方法6周龄ApoE-小鼠,给予高脂饲料,随机分为模型组、辛伐他汀2.5mg·kg-1组、CWQB 72和360mg·kg-1组。从24周龄起灌胃给药,每日1次,连续12周。同时取同龄C57BL/6小鼠作为正常对照。36周龄时麻醉处死,检测血脂水平和主动脉病理变化,免疫组织化学及计算机图像处理系统分析主动脉斑块中巨噬细胞内CD68的表达和平滑肌细胞内α-肌动蛋白的表达。结果CWQB及辛伐他汀均能降低小鼠的血脂水平,降低其胆固醇含量,升高高密度脂蛋白水平。CWQB大剂量组和辛伐他汀组可见主动脉斑块纤维帽厚度增加,斑块面积和管腔面积的比值下降;斑块中平滑肌细胞内α-肌动蛋白表达增加,巨噬细胞内CD68表达减少。结论CWQB具有一定的消减和稳定主动脉斑块的作用,其机制可能与其降低血脂水平、减少斑块内巨噬细胞浸润和增加血管平滑肌细胞数量有关。     

AGE-DEPENDENT ALTERATIONS IN VASCULAR SMOOTH MUSCLE CELL RESPONSIVENESS TO PLATELET-DERIVED GROWTH FACTOR IN GENETIC HYPERTENSION

1. This study examined and compared the growth characteristics of vascular smooth muscle cells (VSMC) isolated from 4 and 12 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats following stimulation with platelet-derived growth factor-BB (PDGF-BB). 2. Vascular smooth muscle cells from 4 week old SHR proliferated at a slower rate than VSMC isolated from 12 week old SHR (1.08±0.06/day vs 1.89±0.13/day respectively, P < 0.05). In contrast, there was no difference in the proliferation of VSMC from 4 and 12 week WKY rats (0.62±0.06/day vs 0.82±0.13/day, respectively, P>0.05). 3. The cell density at which VSMC from 4 and 12 week old SHR become refractory to the mitogenic effects of PDGF-BB was similar and approximately two-fold greater than VSMC from age-matched WKY rats. 4. This study concludes that there is an age-dependent, differential and specific upregulation of growth rate mechanisms in VSMC from SHR which enhance proliferation in response to PDGF-BB.     

他汀类药物与血管平滑肌细胞凋亡

血管平滑肌细胞（VSMC）的异常增殖和迁移在粥样斑块形成和冠状动脉介入治疗（PCI）后再狭窄中起非常重要的作用。他汀类药物不仅是一种有效的调脂药，同时还有多种调脂以外的作用。如他汀类药物可以抑制VSMC的增殖和迁移，并诱导其凋亡。临近病变部位的中层VSMC的凋亡可能造成斑块纤维帽容易破裂，进而引起斑块不稳定和临床事件。但研究证实他汀类药物有稳定斑块的作用，且新生内膜VSMC对他汀类药物诱导的凋亡作用比普通的血管中层VSMC更敏感。因此，他汀类药物的促新生内膜VSMC凋亡作用可能对预防PCI术后再狭窄起有益作用。具体作用机制以及临床上如何合理发挥此类作用尚待进一步研究。     

Extract from the leaf of nucifera reduced the development of atherosclerosis via inhibition of vascular smooth muscle cell proliferation and migration

Nelumbo nucifera GAERTN, a perennial aquatic plant, has been used as a medicinal herb in China and India. We have previously reported that consumption of nucifera leaf extract (NLE) reduced the development of atherosclerosis in cholesterol-fed rabbits; however, the molecular mechanisms involved were unclear. Atherosclerotic plaque is generated partly by proliferation and migration of vascular smooth muscle cells (VSMC). Herein, we demonstrated that VSMC treated with NLE-triggered apoptosis and affected the JNK and p38 Mitogen-Activated Protein Kinase (MAPK) pathways. Pre-treating VSMC with inhibitors of JNK, p38, and p53 reduced NLE-induced apoptosis. Non-cytotoxic doses of NLE also abolished secretion of MMP-2/9 and inhibited cell migration via restraining the FAK/PI 3-kinase/small G protein pathway. Histopathological examination showed that 1.0% of NLE reduced neointima formation conspicuously and inhibited smooth muscle cell proliferation and MMP-2 secretion in the blood vessel of rabbits fed with a high cholesterol diet (HCD). We also verified that the extract’s total phenolic acids and the total flavonoids were approximately about 70%. In conclusion, our results shed light on the molecular mechanisms whereby the polyphenol-rich water extract of nucifera leaves could inhibit both proliferation and migration of VSMC, and it might serve as a potential anti-atherogenic agent.     

Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

Reoccurrence of symptoms occurs in 30–50% of coronary artery disease patients receiving vein grafts or bare-metal stents due to intimal thickening (restenosis). Restenosis is caused by vascular smooth muscle cell (VSMC) migration and proliferation. New therapeutic approaches that reduce VSMC migration and proliferation while promoting endothelial cell (EC) coverage are required. We assessed the effect of a soluble form of N-cadherin (SNC-Fc, a fusion of the extracellular portion of N-Cadherin to a mutated Fc fragment of IgG), a cell–cell junction molecule, on human saphenous VSMC proliferation and migration in vitro. We also assessed its effect on intimal thickening in a validated human ex vivo organ culture model. We observed that SNC-Fc significantly inhibited VSMC proliferation and to a lesser extent migration. The anti-proliferative effect of SNC-Fc was mediated by the interaction of SNC-Fc with the FGFR, rather than through inhibition of β-catenin signalling. SNC-Fc also significantly reduced intimal thickening by ~ 85% in the ex vivo organ culture model. SNC-Fc treatment inhibited proliferation of the intimal cells but did not affect migration. SNC-Fc reduced EC apoptosis, without detrimental effects on EC proliferation and migration in vitro. Importantly SNC-Fc increased EC coverage in the ex vivo model of intimal thickening. In conclusion, we suggest that SNC-Fc may have potential as an anti-proliferative therapeutic agent for reducing restenosis which has no detrimental effects on endothelial cells.     

Foam cell formation by particulate matter (PM) exposure: a review

Increasing evidence suggests that exposure of particulate matter (PM) from traffic vehicles, e.g., diesel exhaust particles (DEP), was associated with adverse vascular effects, e.g., acceleration of atherosclerotic plaque progression. By analogy, engineered nanoparticles (NPs) could also induce similar effects. The formation of lipid laden foam cells, derived predominately from macrophages and vascular smooth muscle cells (VSMC), is closely associated with the development of atherosclerosis and adverse vascular effects. We reviewed current studies about particle exposure-induced lipid laden foam cell formation. In vivo studies using animal models have shown that exposure of air pollution by PM promoted lipid accumulation in alveolar macrophages or foam cells in plaques, which was likely associated with pulmonary inflammation or systemic oxidative stress, but not blood lipid profile. In support of these findings, in vitro studies showed that direct exposure of cultured macrophages to DEP or NP exposure, with or without further exposure to external lipids, promoted intracellular lipid accumulation. The mechanisms remained unknown. Although a number studies found increased reactive oxygen species (ROS) or an adaptive response to oxidative stress, the exact role of oxidative stress in mediating particle-induced foam cell formation requires future research. There is currently lack of reports concerning VSMC as a source for foam cells induced by particle exposure. In the future, it is necessary to explore the role of foam cell formation in particle exposure-induced atherosclerosis development. In addition, the formation of VSMC derived foam cells by particle exposure may also need extensive studies.     

Allograft-induced proliferation of vascular smooth muscle cells: potential targets for treating transplant vasculopathy

Despite advances in immunosuppressive drugs, coronary artery transplant vasculopathy (CATV) is the major cause of graft failure that limits long-term survival of cardiac transplantation. The pathogenesis of CATV involves a chronic immune response of the recipient to the donor vasculature in which activated recipient immune cells damage the endothelium and produce cytokines, resulting in vascular smooth muscle cell (VSMC) activation. Activated VSMC migrate from the media into the lumen, proliferate, and elaborate cytokines and matrix proteins, resulting in loss of lumen diameter and vascular contractility. Because of its extensive nature, interventions which are successful in patients with conventional coronary artery disease are often not applicable to the majority of patients with CATV. Although intended for immune suppression, many immunosuppressive agents owe at least part of their efficacy to their anti proliferative effects on VSMC, including rapamycin, mycophenolic acid, cyclosporin, calcium channel blockers, and HMG CoA reductase inhibitors. Because activation of VSMC is responsible for most of the obliterative arterial intimal thickening present in solid organ allografts, the induction of expression of a selected set of genes may reflect the status of acceptance of the vasculature by the recipient, and the activation, migration, and proliferation of VSMC represent potential points for therapeutic intervention. The risk of infection and malignancy associated with immunosuppressive therapy further promote the need to identify a molecular target which directly modulates the VSMC response to injury. This review will summarize the anti proliferative effects that immunosuppressive drugs have on VSMC proliferation. We will also describe efforts to define the genes which regulate the vascular response to allograft injury, and describe how some of these proteins may represent targets to reduce VSMC proliferation and attenuate CATV.     

Role of Peptide YY in blood vessel function and atherosclerosis in a rabbit model

Cardiovascular disease remains a burden for Westernized countries. Peptide YY (PYY) raises blood pressure, yet its role has not yet been determined in diseased arteries. This study aimed at identifying PYY and eNOS in diseased blood vessels and to determine which blood vessels respond to PYY. New Zealand White rabbits were fed an atherogenic diet (n = 6, 0.5% cholesterol + 1% methionine + 5% peanut oil) and control animals fed a normal diet (n = 6) for 4 weeks. Immunohistochemistry was used to determine the localization of PYY and eNOS in the aorta. The aorta, carotid, renal, iliac, inferior mesenteric, and renal interlobular arteries were removed, mounted in organ baths, and subjected to doses of PYY (10^?9–10^?7 mol/L) and then acetylcholine (10^?6 mol/L). Immunohistochemistry of the aorta shows PYY staining in plaque macrophages, smooth muscle cells and endothelium, and these cells co‐expressed eNOS. PYY caused a minor vasoconstrictive response in all blood vessels studied but was blunted in arteries from control animals. Acetylcholine caused relaxation of PYY constricted blood vessels. This data clearly shows that PYY is present in atherosclerotic plaque and is a minor constrictor of the vasculature tree. Further studies aimed at understanding the role of PYY in cardiovascular disease are warranted.     

苦参碱对纤维蛋白纤维蛋白原降解产物诱导血管细胞损伤、增殖及腹腔巨噬细胞释放IL-1的影响

目的：研究苦参碱(Mat)对纤维蛋白纤维蛋白原降解产物(FFDP)作用的影响。 方法：大鼠主动脉内皮细胞损伤以乳酸脱氢酶释放测定;大鼠主动脉平滑肌细胞增殖采用结晶紫染色法测定;白细胞介素-1活性采用小鼠胸腺细胞增殖法测定。结果：FFDP能促进大鼠主动脉内皮细胞释放乳酸脱氢酶,诱导大鼠主动脉平滑肌细胞增殖,并促使小鼠腹腔巨噬细胞分泌IL-1增加。结论：Mat可抑制FFDP的作用。对动脉粥样硬化的防治可能有一定意义。     

Contribution of Gut Microbiota-Derived Uremic Toxins to the Cardiovascular System Mineralization

Chronic kidney disease (CKD) affects more than 10% of the world population and leads to excess morbidity and mortality (with cardiovascular disease as a leading cause of death). Vascular calcification (VC) is a phenomenon of disseminated deposition of mineral content within the media layer of arteries preceded by phenotypic changes in vascular smooth muscle cells (VSMC) and/or accumulation of mineral content within the atherosclerotic lesions. Medial VC results in vascular stiffness and significantly contributes to increased cardio-vascular (CV) morbidity, whereas VC of plaques may rather increase their stability. Mineral and bone disorders of CKD (CKD-MBD) contribute to VC, which is further aggravated by accumulation of uremic toxins. Both CKD-MBD and uremic toxin accumulation affect not only patients with advanced CKD (glomerular filtration rate (GFR) less than 15 mL/min/1.72 m², end-stage kidney disease) but also those on earlier stages of a disease. The key uremic toxins that contribute to VC, i.e., p-cresyl sulphate (PCS), indoxyl sulphate (IS) and trimethylamine-N-oxide (TMAO) originate from bacterial metabolism of gut microbiota. All mentioned toxins promote VC by several mechanisms, including: Transdifferentiation and apoptosis of VSMC, dysfunction of endothelial cells, oxidative stress, interaction with local renin–angiotensin–aldosterone system or miRNA profile modification. Several attractive methods of gut microbiota manipulations have been proposed in order to modify their metabolism and to limit vascular damage (and VC) triggered by uremic toxins. Unfortunately, to date no such method was demonstrated to be effective at the level of “hard” patient-oriented or even clinically relevant surrogate endpoints.     

Hemin inhibits hypertensive rat vascular smooth muscle cell proliferation through regulation of cyclin D and p21

We tested the hypothesis that HO-1 (heme oxygenase-1) activity varied between vascular smooth muscle cells (VSMC) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. HO-1 levels were measured under baseline and hemin-stimulated conditions and cell proliferation was monitored. Basal HO-1 levels in untreated cells were lower in SHR compared to WKY rats. Treatment with hemin increased HO-1 mRNA and protein levels in the cells obtained from WKY rats compared to that of SHR rats. However, hemin-treatment showed a greater inhibitory effect on VSMC proliferation in SHR rats than in WKY rats. Tin protoporphyrin IX (SnPPIX) showed a greater reversal of the anti-proliferative effect of hemin on cells from SHR rats than WKY. Similarly, VSMC proliferation from SHR was significantly inhibited in VSMC transfected with the HO-1 gene. These inhibitory effects were associated with cell cycle arrest in the G1 phase. The level of cyclin D, and cyclin dependent kinase inhibitor p21 was higher in SHR cells progressing through the G1 phase. Treatment of the cells with hemin down-regulated the expression of cyclin D and up-regulated that of p21. These results indicate that hemin, an HO-1 inducer, may play a more critical role in VSMC proliferation in SHR than WKY.     

藳本内酯和丁烯酜内酯对bFGF诱导的大鼠平滑肌细胞异常增殖的抑制作用

目的研究藳本内酯和丁烯酜内酯与大鼠主动脉平滑肌细胞的亲合活性，及其对主动脉平滑肌细胞增殖的抑制作用。方法采用大鼠主动脉细胞膜色谱模型观察藳本内酯和丁烯酜内酯的保留特性；培养并分离纯化大鼠主动脉平滑肌细胞，采用bFGF诱导平滑肌细胞增殖，以MTT比色法检测藳本内酯和丁烯酜内酯对平滑肌细胞增殖的抑制作用。结果藳本内酯和丁烯酜内酯与大鼠主动脉平滑肌细胞膜有亲和性，其保留行为与钙离子受体拮抗剂维拉帕米相似；藳本内酯和丁烯酜内酯不会引起正常大鼠主动脉平滑肌细胞增殖，但能明显抑制bFGF诱导的大鼠主动脉平滑肌细胞的增殖。其有效浓度分别为5.5和11.1 μmol·L^-1(P<0.05)。结论藳本内酯和丁烯酜内酯与大鼠主动脉平滑肌细胞具有亲和力，能抑制血管平滑肌细胞的异常增殖。     

Gene transfer of vascular endothelial growth factor and endothelial nitric oxide synthase--implications for gene therapy in cardiovascular diseases

Gene therapy is based on the delivery of exogenous genetic material in order to influence the endogenous genetic components involved in disease development. This new therapeutic approach has been suggested to have great potential in the treatment of insufficient angiogenesis or in prevention of restenosis after balloon angioplasty of atherosclerotic arteries. As both vascular endothelial growth factor (VEGF) and nitric oxide (NO) exert beneficial effects on vascular physiology, we studied the generation of both compounds after in vitro transfection of relevant genes. The plasmid vectors, containing VEGF cDNA were constructed and lipotransfected into vascular smooth muscle cells (VSMC). Transfected cells generated up to a few nanograms of VEGF, which induced proliferation of endothelial cells. VSMC transfected with another plasmid, containing endothelial constitutive NO synthase (ecNOS) cDNA generated micromolar quantities of nitrite. Moreover, such NO-producing cells synthesized significantly more VEGF than VSMC transfected with control plasmids. Thus, the study demonstrated that transfer of VEGF and/or NOS genes to VSMC led to the production of measurable amounts of both VEGF protein and NO. Additionally, we evidenced that NO can enhance the endogenous generation of VEGF. A new protective mechanism of NO has been thus revealed.