hESC衍生间充质干细胞可显著促进体外血管生成

人胚胎干细胞(hESC)是具有体外无限增殖能力和多向分化潜能的一类亚全能干细胞,在特定的条件下可被诱导分化为机体的各种细胞包括间充质干细胞(MSC)。该研究以人脂肪间充质干细胞(ADSC)和脐带间充质干细胞(UC-MSC)为对照,对hESC衍生间充质干细胞(hESC-MSC)在体外血管生成中的作用进行了系统的研究,包括其条件培养上清对脐静脉血管内皮细胞(HUVEC)的功能和向血管内皮细胞分化潜能的影响。研究发现,hESC-MSC的条件培养上清可显著促进HUVEC的增殖和迁移,其促进HUVEC增殖的作用显著高于UC-MSC的条件培养上清;hESC-MSC经不同血管内皮细胞诱导方案诱导后均具有体外类血管网络生成的能力,其网络长度均显著高于ADSC和UC-MSC经单一血管内皮细胞诱导方案诱导后的长度。因此,hESC-MSC可显著促进体外血管生成,提示该细胞有望成为一种有效的治疗新型心血管疾病的细胞。     

内皮细胞增殖抑制因子研究进展

内皮细胞过度增殖引起的病理性血管生成是肿瘤、类风湿性关节炎等发病的关键环节。内皮细胞增殖由血管内皮细胞生长因子等促血管生成因子提供促增殖信号,而新近发现的多种内皮增殖抑制因子,如血管内皮抑素、血管抑素、血小板反应蛋白-1、微囊蛋白1、某些microRNAs和某些抑癌基因等,则通过抑制促增殖信号、调节细胞周期、诱导细胞凋亡等途径下调内皮细胞的增殖及血管生成。内皮增殖抑制因子可望成为病理性血管生成防治的新靶点。     

趋化因子及其受体对血管内皮细胞的作用

趋化因子是机体内一类重要的生物活性物质,参与多种生理病理活动的调控。趋化因子可通过对血管内皮细胞的趋化作用,引起血管内皮细胞增殖、迁移、毛细血管形成而促进血管生成;部分趋化因子可通过凋亡和抑制多种促血管生成因子的活性而发挥抑制血管生成的作用。现将趋化因子及其受体对血管内皮细胞的作用进行综述。     

趋化因子在血管生成中的作用

趋化因子可影响血管生成,或通过对内皮细胞的趋化作用,引起血管内皮细胞增殖,毛细血管形成而促进血管生成;或通过抑制多种促血管生成因子活性而发挥抑制血管生成的作用。趋化因子和其他多种血管生成相关性因子组成网络,共同参与血管生成的调控。此外,CXC簇趋化因子SDF－1α也参与胃肠血管的发育过程。     

几丁聚糖和透明质酸钠对血管内皮细胞增殖的影响

目的：比较几丁聚糖和透明质酸钠对血管内皮细胞增殖的影响。方法：用含不同浓度的几丁聚糖和透明质酸钠的培养液对血管内皮细胞（EV304）进行培养,以四唑盐比色法测细胞增殖,并用流式细胞仪测定细胞周期。结果：几丁聚糖在≥0.1mg/ml时促进血管内皮细胞的增殖,透明质酸钠对血管内皮细胞的增殖有抑制作用,几丁聚糖可使细胞周期中G1期比例下降,而透明质酸钠使细胞周期中G1期比例上升。结论：几丁聚糖促进血管内皮细胞的增殖,而透明质酸钠对血管内皮细胞的增殖有抑制作用。     

血管内皮生长因子受体信号转导通路与肿瘤血管生成

血管内皮生长因子是促进血管生成的重要调节因子.它能促进内皮细胞增殖、迁移,阻止内皮细胞凋亡、管腔网状结构退化,增加血管渗透性.所有这些作用都是通过血管内皮生长因子受体信号转导通路实现的.它们在肿瘤血管生成、肿瘤生长中起着重要的作用.以血管内皮生长因子受体信号转导通路为靶点是开发肿瘤血管生成抑制剂的理想策略.     

养心通脉有效部位方对心肌缺血大鼠心脏血管内皮细胞血管生成的影响

目的:探讨养心通脉有效成分部位方(YTⅡ)促血管生成的作用靶点及其机理.方法:制备养心通脉有效部位方(YTⅡ)和各对照组(YT Ⅰ组、SBW组、bFGF组、KX组)含药血清,并进行心肌缺血模型大鼠心脏血管内皮细胞的培养;观察比较在血管生成的过程中YTⅡ及各对照组对心脏血管内皮细胞"增殖"、"迁移"和"管腔结构形成"不同环节的干预作用.结果:各组药物干预后,心肌缺血大鼠冠状血管内皮细胞的增殖率、迁移率、成管率3类指标均呈现出bFGF组>YTⅡ组>YTⅠ组>SBW组>NS组>KX组递减的趋势.YTⅡ和bFGF对血管生成的不同环节均有明显的促进作用,且显著优于其他各组.结论:养心通脉有效部位方(YTⅡ)益气活血,能刺激血管内皮细胞"增殖"、"迁移"和"管腔结构形成"不同环节,促进缺血肌的血管生成.     

沙利度胺通过诱导G1阻滞和凋亡抑制血管内皮细胞增殖

沙利度胺是一种抗血管生成药物,临床上用于治疗多种肿瘤,但其抗肿瘤血管生成机制尚不十分清楚. 本文采用MTT法观察沙利度胺对体外培养的血管内皮细胞增殖的影响. 结果发现,沙利度胺能够抑制血管内皮细胞的增殖,其IC50为16.47 μg/mL;然后采用Hoechst染色和流式细胞仪检测细胞凋亡和细胞周期,发现沙利度胺能够诱导内皮细胞凋亡,并干扰细胞的周期,出现G0/G1期阻滞. 最后,通过Western印迹方法分析沙利度胺对血管内皮细胞Bcl-2蛋白表达的影响,发现抗凋亡的Bcl-2蛋白表达水平随沙利度胺浓度增大而降低. 初步结果提示,沙利度胺可能通过阻遏抗凋亡分子Bcl-2表达,激活诱导G1期阻滞的信号通路而抑制内皮细胞新生,从而抑制肿瘤生长. 诱导内皮细胞凋亡及G1期阻滞的具体分子机制正在研究中.     

人脐静脉血管内皮细胞的分离、培养、鉴定及试验研究

血管内皮细胞体外培养,在血管再生、新血管生成机理、内皮细胞在心血管系统疾病中的作用、内皮细胞与造血的关系等方面的研究中有很高的应用价值。本研究借鉴了前人的工作,建立了胶原酶灌流消化获得人脐静脉血管内皮细胞并进行体外培养的方法,探讨了影响人脐静脉血管内皮细胞（HUVEC）分离培养的影响因素。同时应用建立的HUVEC体外培养进行了内皮抑素的抑制活性检测,证明了内皮抑素对于激活增殖的HUVEC的抑制作用,为今后的试验研究打下了基础。     

牡砺糖胺聚糖对血管内皮细胞损伤的保护作用研究

目的：探讨牡蛎糖胺聚糖（O—GAG）对血管内皮细胞损伤的保护作用,观察它对损伤的血管内皮细胞内一氧化氮（NO）、丙二醛（iDA）含量及乳酸脱氢酶（LDH）活性的影响。方法：采用人脐静脉内皮细胞株ECV304体外培养的方法,建立过氧化氢（H2O2）诱导的内皮细胞损伤模型,噻唑蓝（MTT）比色法观察牡蛎糖胺聚糖对血管内皮细胞增殖活性的影响,硝酸还原酶法、硫代巴比妥酸法和硝基苯肼法分别检测细胞内NO的含量、细胞培养液内MDA的含量和LDH的活性。结果：模型组较正常对照组细胞增殖活性明显降低（P〈0．01）。与模型组相比,经牡蛎糖胺聚糖预处理的各保护组（除10μg／ml）细胞增殖活性明显增加（P〈0．05,P〈0．01）,NO的含量增加,MDA的含量和LDH的活性降低（P〈0．01）。牡蛎糖胺聚糖（100、200μg／ml）对于正常的内皮细胞有促增殖作用（P〈0．05）。结论：牡蛎糖胺聚糖对氧化损伤的血管内皮细胞具有保护作用,其作用机制可能与增加细胞NO含量、减少MDA生成和LDH释放有关。牡蛎糖胺聚糖对正常血管内皮细胞在一定剂量范围内有促增殖作用．     

Lens-specific VEGF-A expression induces angioblast migration and proliferation and stimulates angiogenic remodeling

Vascular endothelial growth factor (VEGF) is a secreted mitogen which specifically stimulates proliferation of vascular endothelial cells in vitro and in vivo. Its expression pattern is consistent with it being an important regulator of vasculogenesis and angiogenesis, and targeted disruption of VEGF-A has demonstrated that it is essential for vascular development. To determine if VEGF-A was sufficient to alter vascularization in the eye we generated transgenic mice which express human VEGF-A(165) specifically in the lens. Expression of transgenic VEGF-A led to excessive proliferation and accumulation of disorganized angioblasts and endothelial cells around the lens. The results support the hypothesis that VEGF-A can initiate the process of vascularization by stimulating chemoattraction and proliferation of angioblasts and endothelial cells and that VEGF-A expression can stimulate angiogenic remodeling. However, VEGF-A alone was not sufficient to direct blood vessel organization or maturation.     

A Polysaccharide Isolated from the Medicinal Herb Bletilla striata Induces Endothelial Cells Proliferation and Vascular Endothelial Growth Factor Expression in vitro

A polysaccharide from the traditional Chinese medicinal herb, Bletilla striata (Thunb.) Reichb. f., was isolated, purified and characterized. It induced the proliferation of human umbilical vascular endothelial cells and the expression of vascular endothelial growth factor up to 156% and 147% of control after 72 h, respectively. Revisions requested 8 November 2005; Revisions received 13 January 2006     

Mathematical modeling of capillary formation and development in tumor angiogenesis: penetration into the stroma

The purpose of this paper is to present a mathematical model for the tumor vascularization theory of tumor growth proposed by Judah Folkman in the early 1970s and subsequently established experimentally by him and his coworkers [Ausprunk, D. H. and J. Folkman (1977) Migration and proliferation of endothelial cells in performed and newly formed blood vessels during tumor angiogenesis, Microvasc Res., 14, 53–65; Brem, S., B. A. Preis, ScD. Langer, B. A. Brem and J. Folkman (1997) Inhibition of neovascularization by an extract derived from vitreous Am. J. Opthalmol., 84, 323–328; Folkman, J. (1976) The vascularization of tumors, Sci. Am., 234, 58–64; Gimbrone, M. A. Jr, R. S. Cotran, S. B. Leapman and J. Folkman (1974) Tumor growth and neovascularization: an experimental model using the rabbit cornea, J. Nat. Cancer Inst., 52, 413–419]. In the simplest version of this model, an avascular tumor secretes a tumor growth factor (TGF) which is transported across an extracellular matrix (ECM) to a neighboring vasculature where it stimulates endothelial cells to produce a protease that acts as a catalyst to degrade the fibronectin of the capillary wall and the ECM. The endothelial cells then move up the TGF gradient back to the tumor, proliferating and forming a new capillary network. In the model presented here, we include two mechanisms for the action of angiostatin. In the first mechanism, substantiated experimentally, the angiostatin acts as a protease inhibitor. A second mechanism for the production of protease inhibitor from angiostatin by endothelial cells is proposed to be of Michaelis-Menten type. Mathematically, this mechanism includes the former as a subcase. Our model is different from other attempts to model the process of tumor angiogenesis in that it focuses (1) on the biochemistry of the process at the level of the cell; (2) the movement of the cells is based on the theory of reinforced random walks; (3) standard transport equations for the diffusion of molecular species in porous media. One consequence of our numerical simulations is that we obtain very good computational agreement with the time of the onset of vascularization and the rate of capillary tip growth observed in rabbit cornea experiments [Ausprunk, D. H. and J. Folkman (1977) Migration and proliferation of endothelial cells in performed and newly formed blood vessels during tumor angiogenesis, Microvasc Res., 14, 73–65; Brem, S., B. A. Preis, ScD. Langer, B. A. Brem and J. Folkman (1997) Inhibition of neovascularization by an extract derived from vitreous Am. J. Opthalmol., 84, 323–328; Folkman, J. (1976) The vascularization of tumors, Sci. Am., 234, 58–64; Gimbrone, M. A. Jr, R. S. Cotran, S. B. Leapman and J. Folkman (1974) Tumor growth and neovascularization: An experimental model using the rabbit cornea, J. Nat. Cancer Inst., 52, 413–419]. Furthermore, our numerical experiments agree with the observation that the tip of a growing capillary accelerates as it approaches the tumor [Folkman, J. (1976) The vascularization of tumors, Sci. Am., 234, 58–64]. An erratum to this article is available at .     

Nicotine enhances neovascularization and promotes tumor growth

Solid tumors require vascularization for their growth. Bone marrow-derived endothelial progenitor cells participate in tumor angiogenesis. Here, we show that nicotine markedly accelerated growth of colon cancer cells inoculated subcutaneously in mice but had no effect on proliferation of carcinoma cells in vitro. We found that the tumor growth was associated with increased vascularization of the tumor and that bone marrow-derived cells contributed to the formation of the new blood vessels. Our findings show that nicotine promotes tumor growth, at least in part, by stimulating tumor-associated neovascularization.     

Bone tissue engineering using adipose‐derived stem cells and endothelial cells: Effects of the cell ratio

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.     

In vitro and in vivo effects of rat kidney vascular endothelial cells on osteogenesis of rat bone marrow mesenchymal stem cells growing on polylactide-glycoli acid (PLGA) scaffolds

It is well established that vascularization is critical for osteogenesis. However, adequate vascularization also remains one of the major challenges in tissue engineering of bone. This problem is further accentuated in regeneration of large volume of tissue. Although a complex process, vascularization involves reciprocal regulation and functional interaction between endothelial and osteoblast-like cells during osteogenesis. This prompted us to investigate the possibility of producing bone tissue both in vitro and ectopically in vivo using vascular endothelial cells because we hypothesized that the direct contact or interaction between vascular endothelial cells and bone marrow mesenchymal stem cells are of benefit to osteogenesis in vitro and in vivo. For that purpose we co-cultured rat bone marrow mesenchymal stem cells (MSC) and kidney vascular endothelial cells (VEC) with polylactide-glycolic acid scaffolds. In vitro experiments using alkaline phosphatase and osteocalcin assays demonstrated the proliferation and differentiation of MSC into osteoblast-like cells, especially the direct contact between VEC and MSC. In addition, histochemical analysis with CD31 and von-Willebrand factor staining showed that VEC retained their endothelial characteristics. In vivo implantation of MSC and VEC co-cultures into rat's muscle resulted in pre-vascular network-like structure established by the VEC in the PLGA. These structures developed into vascularized tissue, and increased the amount and size of the new bone compared to the control group (p < 0.05). These results suggest that the vascular endothelial cells could efficiently stimulate the in vitro proliferation and differentiation of osteoblast-like cells and promote osteogenesis in vivo by the direct contact or interaction with the MSC. This technique for optimal regeneration of bone should be further investigated.     

Immunolocalization of basic fibroblast growth factor during chicken cardiac development

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.     

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.     

VEGF-R2 and neuropilin-1 are involved in VEGF-A-induced differentiation of human bone marrow progenitor cells

Tumor growth and metastasis require the generation of new blood vessels, a process known as neo-angiogenesis. Recent studies have indicated that early tumor vascularization is characterized by the differentiation and mobilization of human bone marrow cells. Vascular endothelial growth factor-A (VEGF-A) is one of the growth factors, which enhances their differentiation into endothelial cells, but little is known about the implication of the VEGF-receptor tyrosine kinases and about the implication of the VEGF-R co-receptor, neuropilin-1, in this process. In this context, the identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells was investigated in order to define the pharmaceutical targets involved in tissue vascularization associated with this process. For this purpose, an in vitro model of differentiation of human bone marrow AC133+ (BM-AC133+) cells into vascular precursors was used. In this work, we have demonstrated for the first time that the effect of VEGF-A on BM-AC133+ cells relies on an early action of VEGF-A on the expression of its tyrosine kinase receptors followed by an activation of a VEGF-R2/neuropilin-1-dependent signaling pathway. This signaling promotes the differentiation of BM-AC133+ cells into endothelial precursor cells, followed by the proliferation of these differentiated cells. Altogether, these results strongly suggest that VEGF inhibitors, acting at the level of VEGF-R2 and/or neuropilin-1, by inhibiting differentiation and proliferation of these cells, could be potentially active compounds to prevent progenitor cells to be involved in tumor angiogenesis leading to tumor growth.     

Specific inhibition of endothelial cell proliferation by thrombospondin.

Angiogenesis is a multi-step event involving endothelial cell migration, attachment, and proliferation. A thrombospondin (TSP)-like protein has recently been described as a naturally-occurring inhibitor of angiogenesis. We now report that human platelet TSP inhibits the in vitro proliferation of endothelial cells from the rabbit corpus luteum, bovine adrenal cortex and pulmonary artery, and human umbilical vein. The antiproliferative effect of TSP was neutralized by monoclonal antibodies against TSP. The growth arrest seen with TSP was specific for endothelial cells since TSP actually stimulated the growth of vascular smooth muscle cells and human foreskin fibroblasts. These results imply that the angiogenesis-inhibiting effect of TSP is mediated through an inhibition of endothelial cell proliferation. Elucidation of the mechanism of action of TSP on endothelial cell proliferation may lead to potential therapeutic approaches for the control of neovascular diseases.