The role of vascular stem cells in atherogenesis and post-angioplasty restenosis

It is well known that atherosclerosis prevails in elderly populations as ageing acts as a recognized risk factor for this disease. Although the pathogenic factors leading to atherosclerosis are highly heterogeneous, traditionally speaking, the causative risk factors include hyperlipidemia, hypertension, diabetes mellitus and smoking, which can damage to endothelial function, and subsequently promote lipid penetration and inflammatory cell infiltration. Damaged endothelial cells (ECs) may be replaced by neighboring cell division, while damaged smooth muscle cells (SMCs) may be replaced by medial SMCs emigrating into the intima during atherogenesis. However, this standpoint is challenged by recent findings that vascular progenitor/stem cells (VPCs) may contribute to atherogenesis and post-angioplasty restenosis. VPCs are a group of primitive cells that have the potential to produce mature, functional cells in the vascular wall. VPCs residing in bone marrow, vascular wall or circulating in the peripheral blood may be stimulated by a variety of pathogenic factors. These stem cells then participate in regeneration, repair and remodeling of the injured arterial wall. This new concept may bring about a great breakthrough in understanding the pathogenesis of atherosclerosis and develop novel therapeutic strategies for coronary heart disease. This article will mainly review the role of VPCs in atherogenesis, thus providing a novel understanding about the pathophysiology of atherosclerosis.     

Suppression of post-angioplasty restenosis with an Akt1 siRNA-embedded coronary stent in a rabbit model

Restenosis is the formation of blockages occurring at the site of angioplasty or stent placement. In order to avoid such blockages, the suppression of smooth muscle cells near the implanted stent is required. The Akt1 protein is known to be responsible for cellular proliferation, and specific inhibition of Akt1 gene expression results in the retardation of cell growth. To take advantage of these benefits, we developed a new delivery technique for Akt1 siRNA nanoparticles from a hyaluronic acid (HA)-coated stent surface. For this purpose, the disulfide cross-linked low molecular polyethyleneimine (PEI) (ssPEI) was used as a gene delivery carrier because disulfide bonds are stable in an oxidative extracellular environment but degrade rapidly in reductive intracellular environments. In this study, Akt1 siRNA showed efficient ionic interaction with the ssPEI carrier, which was confirmed by polyacrylamide gel electrophoresis. Akt1 siRNA/ssPEI nanoparticles (ASNs) were immobilized on the HA-coated stent surface and exhibited stable binding and localization, followed by time-dependent sustained release for intracellular uptake. Cellular viability on the nanoparticle-immobilized surface was assessed using A10 vascular smooth muscle cells, and the results revealed that immobilized ASNs exhibited negligible cytotoxicity against the adhering A10 cells. Transfection efficiency was quantified using a luciferase assay; the transgene expression of Akt1 suppression through the delivered Akt1 siRNA was measured using RT-PCR and western blot, demonstrating higher gene silencing efficiency when compared to other carriers. ASN coated on HA stents were deployed in the balloon-injured external iliac artery in rabbits in?vivo. It was shown that the Akt1 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis in the post-implantation phase.     

Coronary atherosclerosis and interventions: Pathological sequences and restenosis

The primary cause of cardiac morbidity and mortality in developed countries is ischemic (coronary) heart disease. The incidence of this disease is virtually all due to atherosclerosis, and ischemic heart disease is also the most prevalent disease in the industrialized world, causing over 40% of all deaths in the United States and Western Europe. In Japan, the incidence of ischemic heart disease due to coronary atherosclerosis is gradually increasing as well. Compared with the classical nomenclature of atherosclerosis; that is, fatty streak, fibrous plaque and complicated lesions, the term Stary's classification has been universally accepted because it reflects the more recently acquired knowledge about the morphological and biochemical details of the processes in coronary atherosclerosis, which have been obtained by new strategies such as angioscopy, intravascular ultrasound and molecular biological methods. The term Stary's classification has been applied for the coronary atherosclerosis of patients with acute coronary syndrome at the National Cardiovascular Center, for the analysis of predisposing atherosclerosis of these patients. The recent findings regarding acute coronary syndrome resulting from a rupture of coronary atherosclerotic plaques indicate that this syndrome is probably the most important mechanism underlying the sudden onset. It has been found that the risk of plaque rupture may depend more on plaque composition than on plaque size. Plaques rich in soft extracellular lipids and macrophages are possibly more vulnerable to plaque rupture. Two of the goals of the present review are to clarify how plaque disruption occurs and to elucidate the relationship between plaque disruption and coronary risk factors in elderly Japanese patients with acute coronary syndrome. Coronary stents have been shown to be efficacious in the treatment of acute and threatened closure complicating percutaneous transluminal coronary angioplasty (PTCA) and have produced encouraging initial results in the prevention of restenosis. In the autopsy study of restenosis after PTCA, it was observed that dense caps of collagen fibers in the adventitia in the vicinity of the disrupted internal elastic laminae were present in all of the remodeling lesions. It is suggested that remodeling, which resulted in adventitial scarring, is one of the major causative factors of restenosis after PTCA. The long-term success of stenting, however, remains limited by the occurrence of late in-stent restenosis, with an incidence of 20-42% depending on the stent design and the patient population studied. Another aim of the present review is to describe the pathological mechanism of restenosis after PTCA and/or stent replacement and, consequently, the vascular remodeling that occurs around adventitial tissue after PTCA and intimal hyperplasia that is chronically irritated by a foreign body granulomatous reaction after stenting. Finally, the results of the investigation of the effect of a tissue factor pathway inhibitor on the prevention of interventional restenosis is described.     

Gene therapy of restenosis and atherosclerosis: hopes and facts

Stents are the main technique of coronary revascularization in France and western countries. However, a better understanding of the pathophysiology of in-stent restenosis and the well-recognized roles played by inflammation and cell proliferation led to the development of drug-eluting stents, which have nearly eliminated the risk of restenosis. In this context, the success of gene therapy will depend on our ability to simplify and optimize current protocols of arterial gene transfer. For the time being, arterial gene therapy remains a powerful tool for deciphering the complex pathophysiology of restenosis and will certainly have far-reaching implications in the fields of vascular biology and therapeutics.     

转导骨桥蛋白短发夹状RNA预防动脉粥样硬化模型兔血管成形后的再狭窄

背景：血管成形后再狭窄严重限制了经皮冠状动脉介入治疗的应用和远期疗效。平滑肌细胞的表型转变,增殖是血管成形后再狭窄的重要机制。 目的：探讨利用球囊在体转导骨桥蛋白短发夹状RNA,通过抑制实验性动脉粥样硬化模型兔损伤血管部位骨桥蛋白的表达,预防血管成形后再狭窄。 方法：构建动脉粥样硬化模型兔20只,随机等分成空质粒组和OPN-shRNA质粒组,分别利用球囊在腹主动脉导入OPN-shRNA质粒和空载体。 结果与结论：2组兔球囊扩张后血管平滑肌层出现特异性绿色荧光,且随转染后时间的延长荧光强度逐渐降低,与空质粒组相比,OPN-shRNA质粒组兔扩张动脉的管腔面积明显增加,而斑块负荷明显减小。提示在动脉粥样硬化兔模型局部血管利用球囊导管能成功地转导OPN-shRNA质粒,被扩张血管的再狭窄程度减轻,血栓负荷减轻。这对于预防模型兔血管成形后再狭窄的发生具有十分重要的意义。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

实验性再狭窄动物模型研究

目的　为弄清经皮冠状动脉腔内形成术后动脉再狭窄的发生机制 ,本实验研究建立再狭窄动物实验模型。方法　采用右髂动脉球囊内皮剥脱术加高胆固醇喂饲家兔。结果　经血管造影检查 ,血管成形术前右髂动脉平均狭窄程度在6 0 %以上 ,血管成形术后 5周右髂动脉平均再狭窄程度在 5 0 %以上 ;病理切片和组织生化显示 ,内膜明显增厚 ,可见大量泡沫细胞 ,脂质沉积显著增加 ;透射电镜和免疫组织化学鉴定显示 ,增生内膜主要是平滑肌细胞增殖。结论　采用本方法成功地建立了再狭窄动物实验模型。     

A tumor necrosis factor-induced model of human primary demyelinating diseases develops in immunodeficient mice

We have reported previously that in the central nervous system (CNS) local expression of tumor necrosis factor (TNF) transgenes can trigger the development of oligodendrocyte apoptosis, primary inflammatory demyelination and neurological dysfunction, accompanied by lymphocyte and macrophage infiltration into the CNS. To distinguish between the local effects of transgene-encoded TNF and the potential encephalitogenic effects of immune infiltrates upon CNS disease pathogenesis, we have backcrossed Tg6074 TNF-transgenic mice to mice deficient in CD4, beta2-microglobulin (beta2m), immunoglobulin mu chain (Igmu) or recombination activation gene-1 (Rag-1). TNF was capable of triggering undiminished primary demyelination in all of the immunodeficient mice, in the presence of activated cells of the macrophage/microglial lineage. We conclude that TNF is sufficient to induce primary inflammatory demyelination and neurological deficits even in the absence of adaptive immunity.     

A clinically applicable adjuvant for an atherosclerosis vaccine in mice

Vaccination with MHC‐II‐restricted peptides from Apolipoprotein B (ApoB) with complete and incomplete Freund's adjuvant (CFA/IFA) is known to protect mice from atherosclerosis. This vaccination induces antigen‐specific IgG1 and IgG2c antibody responses and a robust CD4 T cell response in lymph nodes. However, CFA/IFA cannot be used in humans. To find a clinically applicable adjuvant, we tested the effect of vaccinating Apoe‐deficient mice with ApoB peptide P6 (TGAYSNASSTESASY). In a broad screening experiment, Addavax, a squalene‐based oil‐in‐water adjuvant similar to MF59, was the only adjuvant that showed similar efficacy as CFA/IFA. This was confirmed in a confirmation experiment for both the aortic arch and whole aorta analyzed by en face analysis after atherosclerotic lesion staining. Mechanistically, restimulated peritoneal cells from mice immunized with P6 in Addavax released significant amounts of IL‐10. Unlike P6 in CFA/IFA, vaccination with P6 in Addavax did not induce any detectable IgG1 or IgG2c antibodies to P6. These data suggest that squalene‐based adjuvants such as MF59 are good candidate adjuvants for developing a clinically effective atherosclerosis vaccine.     

Proprotein convertases furin and PC5: targeting atherosclerosis and restenosis at multiple levels

Several growth factors, chemokines, adhesion molecules, and proteolytic enzymes important for cell–cell/cell–matrix interactions in atherosclerosis and restenosis are initially synthesized as inactive precursor proteins. Activation of proproteins to biologically active molecules is regulated by limited endoproteolytic cleavage at dibasic amino acid residues. This type of activation typically requires the presence of suitable proprotein convertases (PCs). The PC-isozymes furin and PC5 are expressed in human atherosclerotic lesions and have been found to be up-regulated, following vascular injury in animal models in vivo. In vitro, these PCs can regulate vascular smooth muscle cell and macrophage functions and signaling events, through activation of pro--integrins and/or pro-membrane-type matrix metalloproteinases. Integrins link the cytoskeleton with the extracellular matrix and mediate bidirectional signaling and mechanotransduction, whereas matrix metalloproteinases are the major matrix-degrading enzymes. Both activities are required for cell recruitment to the intima. Furthermore, cleavage of extracellular matrix molecules by matrix metalloproteinases potentially contributes to weakening of the fibrous cap, promoting plaque rupture. Based on these recent in vitro and in vivo data, furin and PC5 are potential contributors to the initiation, progression, and complications of atherosclerosis and restenosis. Targeting these PCs may provide future anti-atherosclerotic therapies.     

Effect of protamine on blood lipoproteins in A model of atherosclerosis

Laboratory of Biochemistry, Research Institute of Neurology and Psychiatry, Khar'kov. (Presented by Academician of the Academy of Medical Sciences of the USSR A. A Korzh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 7, pp. 44–46, July, 1991.     

The primary prevention of atherosclerosis in general practice

The prevention of atherosclerosis, especially ischaemic heart disease, in general practice is important. The evidence for and against the various risk factors is reviewed, and the rationale for screening and health education is examined. I conclude that health education and screening for risk factors are likely to be more successful in decreasing morbidity and mortality than treating established disease.

There are arguments for and against screening and health education and about the effectiveness of various schemes. Much of the routine work of health education and screening can be carried out by suitably trained health visitors, practice nurses, or community nurses.

     

移植静脉的重塑与再狭窄

血管再狭窄是影响PTCA和静脉移植远期疗效的重要因素 ,既往认为血管再狭窄是由于血管平滑肌迁移至内膜 ,增殖和分泌细胞外基质造成的。最近实验研究表明血管非适应性重塑是造成血管狭窄的主要原因 ,包括早期血管壁细胞如内皮细胞、平滑肌细胞、成纤维细胞表型改变 ,通过增殖与凋亡参与损伤修复及合成分泌细胞外基质与胶原机化与重排共同参与血管重塑。血管再狭窄不是单纯孤立的内膜、中膜及外膜变化的结果 ,而是血管壁构成在修复损伤过程中重塑的结果 ,而适应性重塑血管代偿性扩张不形成再狭窄     

A minipig model of high-fat/high-sucrose diet-induced diabetes and atherosclerosis

Summary Type 2 diabetes is a major risk factor of the development of atherosclerosis in humans. However, studies examining mechanisms underlying diabetes-accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. Pigs have a cardiovascular system that is very similar to that of humans and is useful as a model for human physiology and pathophysiology. In this study, we established a new miniature pig model for studying dyslipidaemia and atherosclerosis in diabetes. Chinese Guizhou minipigs were fed a normal control diet or a high-fat/high-sucrose diet (HFSD) for 6 months. Plasma total cholesterol (TC), high-density lipoprotein cholesterol, triglyceride (TG), insulin and glucose were quantified at monthly intervals. The induction of insulin resistance and dysfunction of the pancreatic beta-cell were assessed by oral glucose tolerance test and insulin sensitivity test. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. During the feeding period, mild high plasma TC and TG were induced. At the end of 6 months, in HFSD-fed animals, the adipocytes were hypertrophic, fat deposit in the liver was observed, loss of pancreatic beta-cells was observed, and the aortic fatty streak lesions were clearly present in the animals' aortas. Our study established that miniature pigs that were fed a HFSD without adding dietary cholesterol developed insulin resistance, mild diabetes and atherosclerotic lesions. HFSD-fed miniature pigs may be good animal models for research on the treatment of diabetic dyslipidaemia complicated with atherosclerosis.     

动脉粥样硬化并发血栓形成家兔模型的研究

目的： 建立AS并发血栓形成的动物模型，为开展急性冠脉综合症（ACS）发病机制的研究奠定一定的实验基础。 方法： 实验组20只雄性新西兰兔，高胆固醇喂养18周，建立AS性兔模型；另取5只雄性新西兰兔用普通颗粒饲料喂养作为对组照。18周末采用血管紧张素Ⅱ 30 μg/kg静脉注射诱导，24 h后重复静脉注射1次。观察两组血脂、动脉壁斑块、血栓形态和AS血栓形成模型成功率。 结果： 实验组第9、18周血清TC、LDL-C水平明显高于对照组；实验组AS模型成功率为100%，而并发血栓的形成率为60％，可见血栓形成处血管内膜被掀起及内膜的连续性中断，伴有相应节段中膜的断裂、坏死和组织脱落，血栓与斑块相邻，而对照组未见血栓形成。 结论： 血管紧张素Ⅱ可导致血流动力学异常和血管壁结构的破坏，能成功诱导AS并发血栓形成模型，为ACS发生、发展和干预提供一个方便可行的研究方法。     

PACAP deficiency aggravates atherosclerosis in ApoE deficient mice

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays an important role in cytoprotection, inflammation and cardiovascular regulation. Thus, we studied the involvement of PACAP in atherogenesis. Differentiated human THP-1 macrophages (MΦ) were stimulated with oxidized low-density lipoproteins (oxLDL) and the influence of PACAP38 treatment on lipid content and TNF release was determined. To test the effect of PACAP deficiency (PACAP^?/?) on the development of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP^?/? mice were crossbred with ApoE^?/? to generate PACAP^?/?/ApoE^?/? mice. Blood cholesterol and triglyceride levels were quantified. Lumen stenosis in the brachiocephalic trunk, cellularity and amounts of pro-inflammatory as well as autophagy-, apoptosis- and necroptosis-relevant proteins were analysed in atherosclerotic plaques by quantitative immunohistochemistry. In vitro, PACAP38 inhibited oxLDL-induced intracellular lipid storage as well as TNF release in MФ. In vivo, after SC, but not under CED, PACAP^?/?/ApoE^?/? mice showed an increased lumen stenosis compared to ApoE^?/? mice. In atherosclerotic plaques of PACAP^?/?/ApoE^?/? mice, the immunoreactive areas of TNF⁺, IL-1β⁺, autophagic, apoptotic and necroptotic cells were increased. In contrast, the overall cell density was decreased compared to ApoE^?/? under SC, while no differences were seen under CED. Similar plasma cholesterol levels were observed in PACAP^?/?/ApoE^?/? and ApoE^?/? mice under the respective feeding regime. Thus, PACAP^?/-/ApoE^?/? mice represent a novel mouse model of accelerated atherosclerosis where CED is not required. Our data indicate that PACAP acts as an endogenous atheroprotective neuropeptide. Thus, stable PACAP agonists may have potential as anti-atherosclerotic therapeutics. The specific PACAP receptor(s) mediating atheroprotection remain(s) to be identified.     

HDL signaling and protection against coronary artery atherosclerosis in mice

Atherosclerosis is a leading underlying factor in cardiovascular disease and stroke, important causes of morbidity and mortality across the globe. Abundant epidemiological studies demonstrate that high levels of high density lipoprotein (HDL) are associated with reduced risk of atherosclerosis and preclinical, animal model studies demonstrate that this association is causative. Understanding the molecular mechanisms underlying the protective effects of HDL will allow more strategic approaches to development of HDL based therapeutics. Recent evidence suggests that an important aspect of the ability of HDL to protect against atherosclerosis is its ability to trigger signaling responses in a variety of target cells including endothelial cells and macrophages in the vessel wall. These signaling responses require the HDL receptor, scavenger receptor class B type 1 (SR-B1), an adaptor protein (PDZK1) that binds to the cytosolic C terminus of SR-B1, Akt1 activation and (at least in endothelial cells) activation of endothelial NO synthase (eNOS). Mouse models of atherosclerosis, exemplified by apolipoprotein E or low density lipoprotein receptor gene inactivated mice (apoE or LDLR KO) develop atherosclerosis in their aortas but appear generally resistant to coronary artery atherosclerosis. On the other hand, inactivation of each of the components of HDL signaling (above) in either apoE or LDLR KO mice renders them susceptible to extensive coronary artery atherosclerosis suggesting that HDL signaling may play an important role in protection against coronary artery disease.     

应用基因敲除鼠研究动脉粥样硬化

ApoE和LDL-R基因敲除鼠可自发形成动脉粥样硬化斑块,是动脉粥样硬化研究的常用模型。在此基础上应用基因打靶技术建立的白介素-1、C反应蛋白、清道夫受体、IgGFc、补体C1q及CD44等模型对研究动脉粥样硬化中炎症和免疫因子的作用机制起了重要作用。     

Anti-inflammatory effect of abciximab-coated stent in a porcine coronary restenosis model

The aim of this study was to examine the anti-inflammatory effect of abciximab-coated stent in a porcine coronary overstretch restenosis model. Ten abciximab-coated stents, ten sirolimus-eluting stents (SES), and ten paclitaxel-eluting stents (PES) were deployed with oversizing (stent/artery ratio 1.3:1) in porcine coronary arteries, and histopathologic analysis was done at 28 days after stenting. There were no significant differences in the neointima area normalized to injury score and inflammation score among the three stent groups (1.58 +/- 0.43 mm(2), 1.57 +/-0.39 mm(2) in abciximab-coated stent group vs. 1.69 +/- 0.57 mm(2), 1.72 +/- 0.49 mm(2) in the SES group vs. 1.92 +/- 0.86 mm(2), 1.79 +/- 0.87 mm(2) in the PES group, respectively). In the neointima, most inflammatory cells were lymphohistiocytes. Significant positive correlations were found between the extent of inflammatory reaction and the neointima area (r=0.567, p<0.001) and percent area stenosis (r=0.587, p<0.001). Significant correlations were found between the injury score and neointimal area (r=0.645, p<0.001), between the injury score and the inflammation score (r=0.837, p<0.001), and between the inflammation score and neointimal area (r=0.536, p=0.001). There was no significant difference in the inflammatory cell counts normalized to injury score among the three stent groups (75.5 +/- 23.1/microL in abciximabcoated stent group vs. 78.8 +/- 33.2/microL in the SES group vs. 130.3 +/- 46.9/microL in the PES group). Abciximab-coated stent showed comparable inhibition of inflammatory cell infiltration and neointimal hyperplasia with other drug-eluting stents in a porcine coronary restenosis model.