骨髓基质干细胞种植体外修复内皮及对血管平滑肌细胞增生的影响

目的探讨骨髓基质干细胞(MSCs)种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔血管内皮、平滑肌和人MSCs,通过细胞共培养模拟血管内皮修复过程,用流式细胞仪分析MSCs分子表型特征,免疫荧光细胞化学法观察与内皮共培养的MSCsFlk1和vWF蛋白表达,根据下室内皮生长状态及是否接种MSCs将其分为对照组、单纯MSCs组、融合内皮组、对数内皮组和MSCs种植组。氚胸腺嘧啶脱氧核苷(3HTdR)掺入检测平滑肌细胞DNA合成,Westernblot检测平滑肌细胞中增殖细胞核抗原蛋白表达。结果分离的MSCs表达基质细胞标志CD105和CD166,不表达造血干祖细胞和内皮细胞标志CD34、Flk1、vWF;与内皮共培养5天时,vWF染色仍为阴性,但约25.71%MSCs开始表达Flk1;MSCs种植组平滑肌细胞3HTdR掺入虽高于融合内皮组,但与对数内皮组比较显著降低;MSCs种植组平滑肌细胞PCNA蛋白吸光度相对值虽高于融合内皮组,但与对数内皮组比较明显减少。结论MSCs种植能抑制平滑肌细胞增生,种植在成熟内皮中的MSCs具有微环境依赖向内皮分化的能力。     

Mesenchymal stem cells in ex vivo cord blood expansion

Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34(+) cell doses transplanted. Here, current strategies directed at improving TNC and CD34(+) cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo co-culture expansion system.     

紫杉醇联合骨髓基质干细胞种植体外修复内皮及对血管平滑肌增生的影响

目的探讨紫杉醇联合骨髓基质干细胞种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔主动脉内皮、平滑肌和人骨髓基质干细胞,通过细胞共培养将内皮/骨髓基质干细胞接种于下室、平滑肌细胞接种于上室模拟血管内皮修复过程,分别用3H TdR掺入和Westernblot检测紫杉醇(1,10,100nmol/L)干预20min后第10天平滑肌DNA合成和PCNA蛋白表达,用免疫荧光细胞化学法观察与紫杉醇干预内皮共培养的骨髓基质干细胞vWF和Flk1蛋白表达。结果骨髓基质干细胞种植组平滑肌细胞3H TdR掺入和PCNA蛋白光密度相对值均高于融合内皮组(n=6,P<0.05),低于对数内皮组(n=6,P<0.05)。共培养前骨髓基质干细胞不表达vWF和Flk1蛋白,与紫杉醇干预内皮共培养10天时vWF染色阴性,但部分骨髓基质干细胞开始表达Flk1蛋白。结论骨髓基质干细胞种植能部分抑制紫杉醇引起的平滑肌细胞延迟增生,与紫杉醇干预内皮共培养的骨髓基质干细胞有向内皮分化的能力。     

AngⅡ,NO对VEC,VSMC增殖及凋亡的影响

目的 了解血管活性物质AngⅡ、NO对VEC、VSMC增殖及凋亡的影响。方法 在培养的VEC、VSMC细胞中加入NO、AngⅡ等物质,观察细胞生长及凋亡情况。结果 NO诱导VSMC凋亡而AngⅡ抑制VSMC凋亡,NO拮抗AngⅡ对VEC凋亡的诱导作用。结论 血管活性物质NO、AngⅡ调节VEC及VSMC增殖与凋亡的能力与调节血管张力的相互抵消作用一致,这些血管活性物质的表达失去平衡可使调节血管张力、细胞增殖及细胞死亡的内环境稳定发生紊乱。     

In vitro and ex vivo regulation of vascular smooth muscle cell growth and phenotypic modulation by sulphated polysaccharides

Heparin and pentosan polysulphate (PPS), a semi-synthetic sulphated polysaccharide, affected the phenotypic modulation of primary or subcultured rabbit smooth muscle cells (SMC) and at the same time inhibited their proliferation in culture. PPS was 5 fold more potent than heparin. Ex vivo, after subcutaneous administration of PPS or heparin (8 mg/kg/day for 13 days), SMC isolated from treated rabbits were growth-inhibited. At the same time, they reversed promptly to the contractile state as evidenced by immunofluorescent detection of intracellular myosin or electron microscopy. This ex vivo inhibitory effect was related to the dose and the duration of treatment, and was lost if 6 hours elapsed between the final dose and removal of the aorta. Such an effect was also observed after oral treatment with PPS (200 mg/kg/day for 7 days).     

血管去内皮损伤后平滑肌细胞凋亡的实验观察

目的 了解血管去内皮损伤后新内膜形成及平滑肌细胞(SMCs)凋亡的时相变化。方法用氮气干燥剥脱大鼠颈动脉内皮复制血管损伤模型,HE染色光镜形态计量内膜/中膜比(I/M),末端脱氧核苷酸转移酶(TdT)介导的荧光素d-uTP缺口末端标记(TRNEL)方法观察不同时间新内膜形成(I/M)及VSMC凋亡。结果 血管去内皮后四天,在SMCs增生形成的新内膜中有TUNEL法证实的细胞凋亡,I/M加比,凋亡指数(TUNELI)分别为0.12±0.06,2.4±1.98。在七天,内膜明显增厚(I/M为0.6±0.15,与四天比,P<0.05),TUNELI达到最大值(为9.3±3.8,与四天比,P<0.05)。到第14天,内膜明显增厚约为中膜的1.25倍(I/M比1.25±0.14,与七天比,P<0.05),TUNELI逐渐减小(为8.75±4.01;与七天比,P>0.05)。损伤后21天,内膜开始变薄(I/M比为0.98±0.41,与14天比,P<0.05),凋亡水平进一步下降(TUNELI为6.58±3.97,但差异无显著性,P=NS)。结论 血管壁对损伤刺激诱发增生反应的同时激活凋亡机制。凋亡调节血管壁细胞数和内膜增厚演变。细胞凋亡的平衡失调可能是动脉粥样硬化(AS)、再狭窄(RS)等血管疾病发生的机制之一。     

Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells

Ex vivo expansion of hematopoietic stem cells was suggested as the best way of overcoming problems caused by limited hematopoietic cell number for cord blood transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) as a cell feeder layer for support of UCB-derived committed hematopoietic progenitor cells (HPCs) in the absence or presence of recombinant cytokines. The UCB-derived MSCs used in the study differentiated into osteoblast, chondrocytes, and adipocytes under proper conditions. Frequencies in colony forming unit-granulocyte, macrophage, colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte, burst forming unit-erythrocyte, and colony forming unit-erythrocyte increased to 3.46-, 9.85-, 3.64-, and 2.03-folds, respectively, only in culture supplemented by UCB-derived MSCs as a cell feeder layer without recombinant cytokines (culture condition C). Identified expansion kinetics in all kinds of committed HPCs showed plateaus at 7 culture days, suggesting some consumable components were required for the expansion. Physiological importance and different roles for different committed HPCs of UCB-derived MSCs as a cell feeder layer were revealed by a distinguished expansion capacity for colony forming unit-megakaryocyte. The preferred maintenance of CD33⁻CD34⁺ in culture condition C was also identified. The presence of cobblestone-like areas as hematopoietic microenvironment and various cell feeder layer-originated hematopoietic cytokines including interleukin-1β and granulocyte, macrophage-colony stimulating factor were suggested as underlying mechanisms for the identified expansion capacity. The present numeric and biological information about intrinsic expansion capacity for UCB-derived committed HPCs will increase further biological and clinical applications of UCB-derived MSCs. An erratum to this article can be found at     

Human embryonic stem cell-derived vascular smooth muscle cells in therapeutic neovascularisation

Ischemic diseases remain one of the major causes of morbidity and mortality throughout the world. In recent clinical trials on cell-based therapies, the use of adult stem and progenitor cells only elicited marginal benefits. Therapeutic neovascularisation is the Holy Grail for ischemic tissue recovery. There is compelling evidence from animal transplantation studies that the inclusion of mural cells in addition to endothelial cells (ECs) can enhance the formation of functional blood vessels. Vascular smooth muscle cells (SMCs) and pericytes are essential for the stabilisation of nascent immature endothelial tubes. Despite the intense interest in the utility of human embryonic stem cells (ESCs) for vascular regenerative medicine, ESC-derived vascular SMCs have received much less attention than ECs. This review begins with developmental insights into a range of smooth muscle progenitors from studies on embryos and ESC differentiation systems. We then summarise the methods of derivation of smooth muscle progenitors and cells from human ESCs. The primary emphasis is on the inherent heterogeneity of smooth muscle progenitors and cells and the limitations of current in vitro characterisation. Essential transplantation issues such as the type and source of therapeutic cells, mode of cell delivery, measures to enhance cell viability, putative mechanisms of benefit and long-term tracking of cell fate are also discussed. Finally, we highlight the challenges of clinical compatibility and scaling up for medical use in order to eventually realise the goal of human ESC-based vascular regenerative medicine.     

睾酮抑制大鼠血管平滑肌细胞泡沫化及机制研究

目的研究睾酮对氧化型低密度脂蛋白(ox-LDL)诱导大鼠血管平滑肌细胞(VSMC)转化为泡沫细胞的抑制作用,并探讨其可能机制。方法培养大鼠VSMC,分为对照组、ox-LDL组、低(1nmol/L)、中(10nmol/L)、高(100nmol/L)浓度睾酮组。另外,设置睾酮组(100nmol/L)、氟他胺组(1μmol/L)、睾酮与氟他胺合用组,并与ox-LDL共培养建立泡沫细胞模型。利用油红O染色及酶荧光法检测不同浓度睾酮及氟他胺预处理对VSMC内脂质含量的影响,免疫印迹法检测各组细胞内线粒体融合素2(Mfn2)、清道夫受体CD36和ATP结合盒转运蛋白A1(ABCA1)的表达变化。结果与ox-LDL组比较,不同浓度睾酮组VSMC脂质蓄积减少,总胆固醇、游离胆固醇和胆固醇酯的含量均降低(P<0.05),Mfn2和ABCA1表达增加(P<0.05),CD36表达降低(P<0.05)。与睾酮组比较,睾酮与氟他胺合用组VSMC脂质蓄积增加,总胆固醇、游离胆固醇和胆固醇酯的含量均增加(P<0.05),Mfn2和ABCA1表达降低(P<0.05),CD36表达增加(P<0.05)。结论睾酮以雄激素受体依赖的方式,抑制ox-LDL诱导的VSMC泡沫化过程,其作用与睾酮上调Mfn2表达进而调节CD36和ABCA1表达有关。     

Introduction of vascular smooth muscle cells expressing recombinant genes in vivo

Vascular smooth muscle cells contribute to the formation of atherosclerotic plaques by proliferating in response to vascular injury and releasing growth-promoting factors. Because their autocrine and paracrine effects are not fully understood, expression of such growth factor genes in specific cell types in vivo would help to determine their mechanism of action. We describe a method to transfer vascular smooth muscle cells expressing recombinant gene products to localized segments of the arterial wall. Vascular smooth muscle cells from the inbred Yucatan minipig were infected in vitro with an amphotropic, replication-defective retrovirus transducing the gene for Escherichia coli beta-galactosidase. Vascular smooth muscle cells expressing this recombinant gene were implanted, using a catheter, into denuded iliofemoral artery segments of pigs in vivo. These arteries subsequently demonstrated beta-galactosidase activity in cells of the intima and media. This method, which provides for the introduction of genetically modified smooth muscle cells, can be used to define the biological effects of recombinant genes in the vessel wall and potentially to provide alternative treatments of vascular diseases.     

Apoptosis of vascular smooth muscle cells induced by cholesterol and its oxides in vitro and in vivo

The ability of cholesterol and its oxides to induce apoptosis in vascular smooth muscle cells in tissue culture and in a rabbit model of atherosclerosis was evaluated. Apoptosis was detected using DNA laddering and in situ end-labelling of fragmented DNA. Cholesterol oxides, but not cholesterol, were found to inhibit proliferation and induce apoptosis of vascular smooth muscle cells in tissue culture. 7-ketocholesterol was found to be the most potent inhibitor of proliferation, while 25-hydroxycholesterol was found to be the most potent inducer of apoptosis. These data suggest that the inhibition of proliferation and the induction of apoptosis by cholesterol oxides within vascular smooth muscle cells use different pathways, suggesting a differential role for these cholesterol oxides within the arterial wall. Cholesterol feeding after balloon injury in a rabbit model of atherosclerosis is known to result in the accumulation of cholesterol oxides. However, we found that cholesterol feeding had no effect on the level of apoptosis in the rabbit aortic wall after balloon injury, suggesting that the major factor determining apoptosis in our model was the balloon injury.     

Feline immunodeficiency virus and retrovirus-mediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular smooth muscle cells

We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.     

Proliferative effect of lipoprotein lipase on human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.     

Serum starvation and growth factor receptor expression in vascular smooth muscle cells

BACKGROUND: Smooth muscle cell (SMC) proliferation in atherosclerosis is regulated through the interaction of growth factors like platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1) and their receptors (R). We hypothesized that serum starvation of SMCs may affect PDGFbeta-R and IGF-1-R expression and, consequently, the effect of their cognate ligands on SMC survival/proliferation. METHODS AND RESULTS: Serum starvation significantly increases PDGFbeta-R but not IGF-1-R mRNA and protein expression in SMCs. PDGF-BB stimulates cell survival but not proliferation in serum-starved SMCs of the synthetic phenotype, whereas SMCs of the contractile phenotype respond to PDGF-BB by a significant increase in proliferation. Immunohistochemical analysis of coronary atherosclerotic lesions reveals PDGFbeta-R expression in SMCs in the lamina fibromuscularis, but not in the media and in healthy parts of the arterial wall. No such differential expression was observed for IGF-1-R. CONCLUSIONS: Differential regulation of PDGFbeta-R and IGF-1-R expression by serum starvation might represent a mechanism for the control of SMC survival/proliferation in atherogenesis and restenosis. The distribution of PDGFbeta-Rs and IGF-1-Rs in atherosclerotic lesions may indicate an effect of serum starvation on SMCs in the arterial wall.     

Two platelet-derived growth factor receptors in vascular smooth muscle cells.

Distinct genes encode alpha and beta PDGF receptors which differ in their abilities to be triggered by three dimeric forms of the PDGF molecules. By use of a strategy involving introduction of expression vectors for alpha and beta PDGF receptor cDNA into the cells originally lacking these receptors, we demonstrated that each receptor was able to couple independently with mitogenic signal transduction pathways inherently present in these cells. Moreover, both receptors were capable of inducing a readily detectable chemotactic response. The vascular smooth muscle cells which express both types of PDGF receptors are mitogenic and chemotactic for PDGFs. Moreover, the alpha receptor is the preferred receptor for platelet PDGF-AB as well as the PDGF-AA isoform which is ubiquitously produced in many cells forming atherosclerotic plaques including macrophages, endothelial cells and even arterial smooth muscle cells. Our results indicated that the availability of specific PDGF isoforms and the relative expression of each receptor gene product appear to be major determinants of the PDGF response. An understanding of the mechanisms by which the expression of PDGF and their receptors on vascular smooth muscle cells are regulated will give greater insights as to how these gene products are involved in atherosclerosis.     

Smoothelin in vascular smooth muscle cells

Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that smoothelins are part of the contractile apparatus.     

白细胞表面分化抗原40配体在糖尿病患者下肢血管内皮细胞及平滑肌细胞中的表达

目的观察白细胞表面分化抗原40配体（CD40L）在糖尿病患者下肢血管内皮细胞及平滑肌细胞中的表达,探讨CD40L与糖尿病下肢血管病变发生发展的关系。方法选择2007年11月-2008年4月间因外伤或罹患糖尿病足、需要行截肢手术而在我院就诊的单纯糖尿病患者10例、糖尿病下肢血管病变者12例、非糖尿病单纯下肢血管病变者10例以及对照组8例。测定其血糖、血脂、糖化血红蛋白等,用免疫组织化学方法检测下肢动脉血管内皮细胞及平滑肌细胞中CD40L的表达,用计算机图像分析技术半定量检测CD40L的表达。结果糖尿病下肢血管病变组、单纯糖尿病组、单纯下肢血管病变组的CD40L表达均较对照组增强（P〈0．05）;糖尿病下肢血管病变组的CD40L表达较单纯糖尿病组和单纯下肢血管病变组增强（P〈0．05）;CD40L的表达与空腹血糖值呈正相关（r=0．542,P〈0．001）。结论CD40L是参与糖尿病血管病变的重要炎症因子,高血糖可促发CD40L过度表达。