Organized type I collagen influences endothelial patterns during “spontaneous angiogenesis in vitro”: Planar cultures as models of vascular development

Summary Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries—a phenomenon referred to as “spontaneous angiogenesis in vitro”. We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to indentify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.     

Endothelial progenitor cells: A source for therapeutic vasculogenesis?

Angiogenesis has been defined as sprouting of blood vessels from pre-existing vascular structures. Risau and co-workers defined the term vasculogenesis while studying the formation of new blood vessels in embryoid bodies. This process is characterized by the recruitment of endothelial progenitor cells (EPC) to sites of new vessel formation with subsequent differentiation of EPC into mature endothelial cells, extensively proliferating in situ. Data from recent years provided evidence that EPC also exist in the adult and contribute to new vessel formation, a process called post-natal vasculogenesis. The existence of EPC has been convincingly shown in both, animals and humans. They represent a perfect cellular progenitor cell population for the ex vivo generation of EC, which in turn serve as cellular source for therapeutic vasculogenesis or tumor targeting. This review provides an overview on this hot topic of cellular-based therapeutic concepts and the therapeutic potential of ex vivo generated EPC.     

Clock controls angiogenesis

Circadian rhythms control multiple physiological and pathological processes, including embryonic development in mammals and development of various human diseases. We have recently, in a developing zebrafish embryonic model, discovered that the circadian oscillation controls developmental angiogenesis. Disruption of crucial circadian regulatory genes, including Bmal1 and Period2, results in marked impairment or enhancement of vascular development in zebrafish. At the molecular level, we show that the circadian regulator Bmal1 directly targets the promoter region of the vegf gene in zebrafish, leading to an elevated expression of VEGF. These findings can reasonably be extended to developmental angiogenesis in mammals and even pathological angiogenesis in humans. Thus, our findings, for the first time, shed new light on mechanisms that underlie circadian clock-regulated angiogenesis.     

血管内皮细胞微粒研究进展

内皮细胞微粒（endothelial microparticle,EMPs）是内皮细胞活化或凋亡时,从其表面释放的小囊泡,其作为反映内皮细胞功能的新标记物,在炎症反应、心血管疾病和糖尿病等多种疾病中都有所增加。本文就EMP可能的形成机制、组成成分和主要作用作一概述。     

Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions

Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.     

Mesenchymal stem cells are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction

Summary Both cell therapy and angiogenic growth factor gene therapy have been applied to animal studies and clinical trials. Little is known about the direct comparison between cell therapy and angiogenic growth factor gene therapy. The goal of this study was to compare the effects of human bone marrow-derived mesenchymal stem cells (hMSCs) transplantation and injection of angiogenic growth factor genes in a model of acute myocardial infarction in mice. The hMSCs were obtained from adult human bone marrow and expanded in vitro. The purity and characteristics of hMSCs were identified by flow cytometry and immunophenotyping. Immediately after ligation of the left anterior descending coronary artery in male severe combined immunodeficient (SCID) mice, culture-expanded hMSCs or angiogenic growth factor genes were injected intramuscularly at the left anterior free wall. The engrafted hMSCs were positive for cardiac marker, desmin. Infarct size was significantly smaller in the hMSCs-treated group than in the angiopoietin-1 (Ang-1) or vascular endothelial growth factor (VEGF)-treated group at day 28 after infarction. hMSCs transplantation was better in decreasing left ventricular end-diastolic dimension and increasing fractional shortening than Ang1 or VEGF gene therapy. Capillary density was markedly increased after hMSCs transplantation than Ang1 and VEGF gene therapy. In conclusion, intramyocardial transplantation of hMSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium. hMSCs are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction. Transplantation of MSCs may become the future therapy for acute myocardial infarction for myocardial regeneration.     

心脏血管的形成

心脏的血 管 形成 是 血管 发生 (vasculogenesis)、血 管 生成 (angiogenesis)及 动 脉生 成 (arteriogenesis)三种 机制 共同 作 用的 结 果 .血管 发 生是 指在 胚 胎期 ,来 源 于中 胚 层的 干细 胞增 殖 和分 化 ,形 成 内皮 细胞 ,进而 与其 他细 胞形 成 原始 的 心血 管系 统 .血 管生 成 出现 在血 管 发生 之后 ,是指 通过 内 皮细 胞的 增 殖由 原始 血 管丛 或已 存在 的血 管 形成 无 完好 血管 的 膜中 的毛 细 血管 .而 动 脉生 成是 指 具有 完好 的 动脉 中膜 的 小动 脉 的生 成,也包 括原 有的 侧 支循 环 的改 建及 成 熟 .总结 了 出生 前后 心 脏脉 管系 统 形成 的细 胞 及分 子机 理 ,并 从生 物 学及 临床 治疗 上就 一 些内 皮 前体 细胞 及 其它 脉管 起 源相 关问 题 进行 简单 的 介绍 .     

Delta4, an endothelial specific Notch ligand expressed at sites of physiological and tumor angiogenesis

Delta-Notch signalling regulates cell-fate choices in a variety of tissues during development. We report the expression of Delta4 (D14) in arterial endothelium during mouse embryogenesis and in the endothelium of tumor blood vessels. The expression of D14 in the mouse begins at 8 dpc in the dorsal aortae, umbilical artery and the heart. Subsequent expression is restricted to smaller vessels and capillaries and is reduced in most adult tissues. However, it is high in the vasculature of xenograft human tumors in the mouse, in endogenous human tumors and is regulated by hypoxia. These data implicate D14 and the Notch signalling pathway in angiogenesis and suggest possible new targets for antiangiogenic tumor therapy.     

Primitive endothelial cell lines from the porcine embryonic yolk sac

Endothelial cell lines have been established from cells that were isolated from porcine yolk sacs from day 18 and day 22 embryos and propagated in vitro under various growth conditions. After expansion in vitro, the general properties of the cells proved similar for the different media used. The endothelial cells expressed cell surface receptors for acetylated low-density lipoprotein and also expressed cell surface-associated angiotensin-converting enzyme. The cells showed a characteristically high level of binding for Bandeiraea simplicifolia lectin I and Dolichos biflorus agglutinin but did not bind significant amounts of Ulex europaeus lectin I. The cells expressed low but serologically detectable levels of Class I major histocompatibility complex (MHC) antigens but failed to bind antibodies directed against Class II MHC antigens. Alpha5beta1 integrins were weakly expressed, whereas vascular cell adhesion molecule-1 (CD106) and alphavbeta3 integrins were not detected. Three-dimensional tube formation was readily observed in cultures grown on Matrigel and occurred even in uncoated plastic dishes in the absence of Matrigel. In contrast to most of the adult porcine endothelial cells, yolk sac-derived endothelial cells did not possess serologically detectable receptors for porcine growth hormone (GH), an observation consistent with the finding that GH did not increase the proliferative rate of these cells. Electron microscopic examination demonstrated the presence of Weibel-Palade bodies, tight endothelial cell junctions, and typical rough endoplasmic reticulum. Exposure of the cells to either concanavalin-A-stimulated porcine splenocyte culture supernatants or to human tumor necrosis factor alpha did not cause upregulation of VCAM-1 or Class II MHC antigens. Addition of porcine interferon-gamma led to an increase in the level of expression of Class I MHC. Yolk sac endothelial cells from day 22 embryos showed a low but detectable level of expression of Class II MHC antigens, whereas the endothelial cells from day 18 embryos showed no expression of Class II antigens after interferon-gamma stimulation. The cells maintained competence to develop vascular structures in vitro and could do so after coinjection with murine tumor cells into adult, immunocompromised mice.     

Smad7 Misexpression during Embryonic Angiogenesis Causes Vascular Dilation and Malformations Independently of Vascular Smooth Muscle Cell Function

Numerous in vitro and in vivo studies implicate transforming growth factor-beta (TGFbeta) superfamily signaling in vascular development and maintenance. Mice and humans with mutations in TGFbeta superfamily signaling pathway genes exhibit a range of vascular defects that include dilated, fragile and hemorrhagic vessels, defective angiogenic remodeling, severe vascular malformations including arterio-venous malformations, and disrupted vascular smooth muscle cell recruitment and maintenance. Despite a wealth of data, the functions of TGFbeta superfamily signals during angiogenesis are poorly defined, since early embryonic lethality and difficulty distinguishing between primary and secondary defects frequently confound phenotypic interpretation. To perturb TGFbeta superfamily signaling during angiogenesis, we have misexpressed Smad7, an intracellular antagonist of TGFbeta superfamily signaling, in the developing chick limb and head. We find that the great vessels are strikingly dilated and frequently develop intra and intervascular shunts. Neither noggin nor dominant negative BMP receptor misexpression causes similar vascular phenotypes. However, simultaneous misexpression of constitutively active BMP receptors with Smad7 suppresses the Smad7-induced phenotype, suggesting that a BMP-like intracellular pathway is the target of Smad7 action. Despite the gross morphological defects, further analyses find no evidence of hemorrhage and vessel structure is normal. Furthermore, enlarged vessels and vascular malformations form in either the presence or absence of vascular smooth muscle, and vascular smooth muscle cell recruitment is unperturbed. Our data define the TGFbeta superfamily pathway as an integral regulator of vessel caliber that is also essential for appropriate vessel connectivity. They demonstrate that dilation need not result in vessel rupture or hemorrhage, and dissociate vessel maintenance from the presence of a vascular smooth muscle cell coat. Furthermore they uncouple vascular smooth muscle cell recruitment and differentiation from TGFbeta superfamily signaling.     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.