The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.     

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.     

人参汤提取物对血管内皮细胞保护作用的研究

探讨人参汤提取物对血管内皮细胞的保护作用.采用体内、外血管内皮细胞损伤模型,测定体外血管内皮细胞破裂MTT染色后的OD值、体内血管内皮细胞损伤时血中CEC数及血清中NO的浓度.体外实验人参汤各剂量组的OD值比模型对照组降低(P<0.05)、体内实验人参汤各剂量组CEC数量、NO浓度比模型对照组都显著降低(P<0.01).实验结果表明,人参汤提取物对血管内皮细胞的损伤具有保护作用.     

利用γ-丁内酯衍生物研究空泡化与细胞分化

从化学遗传学的角度,利用γ-丁内酯衍生物研究了成纤维细胞生长因子（fibroblast growth factor, FGF）诱导鸡胚盘细胞向血管内皮细胞分化过程中,空泡化对血管内皮细胞分化的影响。中性红染色及吖啶橙染色结果表明,在血管内皮细胞分化过程中出现的膜泡为酸性膜泡,γ-丁内酯衍生物可能通过抑制这些酸性膜泡,抑制了鸡胚盘细胞向血管内皮细胞的分化。该研究结果进一步证实了空泡化在细胞分化中的作用,并为研究γ-丁内酯衍生物的靶蛋白在鸡胚盘细胞向内皮细胞分化中的作用打下了基础。     

Phagosome acidification blocked by intracellular Toxoplasma gondii

Toxoplasma gondii belongs to a group of highly virulent intracellular parasites that reside in host cell vacuoles which resist typical phagosome-lysosome fusion. Live Toxoplasma replicate prodigiously within modified phagocytic vacuoles formed during invagination of the host plasma membrane. In contrast, heat-killed Toxoplasma or specific antibody (heat-inactivated)-coated live Toxoplasma-containing vacuoles readily undergo lysosome fusion and digestion in normal macrophages. Of newly recognized significance to Toxoplasma survival is the microbicidal effect of phagosome acidification, which reportedly can occur independently of fusion with other acidic vesicles. We report here that modified live Toxoplasma-containing vacuoles fail to acidify in normal macrophages, as indicated by the sensitive pH probe fluorescein. In contrast, when live Toxoplasma are coated with specific antibody (heat-inactivated), they trigger phagosome acidification when entering normal macrophages. A similar acidification is observed when normal phagocytes ingest dead Toxoplasma. Extracellular Toxoplasma are highly susceptible to acidic pH conditions, indicating that the acidification block in the modified vacuoles may be important for intracellular survival.     

Establishment of the model of vascular endothelial cell membrane chromatography and its preliminary application

A model of vascular endothelial cell membrane chromatography was established by using an ECV304 cell membrane stationary phase (ECV304 CMSP) prepared by immobilizing the ECV304 cell membrane onto the surface of silica carrier. The surface and chromatographic characteristics of ECV304 CMSP were studied. The active component from Caulophyllum robustum was screened by using the model of vascular endothelial cell membrane chromatography. The interaction between the active component and membrane receptor was determined by using a replace experiments. The effect of the active component was tested by using tube formation of ECV304 cell. The results indicated that the model of ECV304 cell membrane chromatograph (ECV304 CMC) can stimulate the interaction between drug and receptor in vitro and the retention characteristics of taspine as active component was similar to that of model molecule in the model of ECV304 CMC. And therefore, taspine acted on VEGFR2 and inhibited the tube formation of ECV304 cell induced by VEGF. This model can be used to screen definite active component as a screening model.     

Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Recent studies have suggested that bone marrow cells possess a broad differentiation potential, being able to form new liver cells, cardiomyocytes and neurons. Several groups have attributed this apparent plasticity to 'transdifferentiation'. Others, however, have suggested that cell fusion could explain these results. Using a simple method based on Cre/lox recombination to detect cell fusion events, we demonstrate that bone-marrow-derived cells (BMDCs) fuse spontaneously with neural progenitors in vitro. Furthermore, bone marrow transplantation demonstrates that BMDCs fuse in vivo with hepatocytes in liver, Purkinje neurons in the brain and cardiac muscle in the heart, resulting in the formation of multinucleated cells. No evidence of transdifferentiation without fusion was observed in these tissues. These observations provide the first in vivo evidence for cell fusion of BMDCs with neurons and cardiomyocytes, raising the possibility that cell fusion may contribute to the development or maintenance of these key cell types.     

Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.     

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.     

Haemodynamic shear stress activates a K+ current in vascular endothelial cells

The endothelial lining of blood vessels is subjected to a wide range of haemodynamically-generated shear-stress forces throughout the vascular system. In vivo and in vitro, endothelial cells change their morphology and biochemistry in response to shear stress in a force- and time-dependent way, or when a critical threshold is exceeded. The initial stimulus-response coupling mechanisms have not been identified, however. Recently, Lansman et al. described stretch-activated ion channels in endothelial cells and suggested that they could be involved in the response to mechanical forces generated by blood flow. The channels were relatively nonselective and were opened by membrane stretching induced by suction. Here we report whole-cell patch-clamp recordings of single arterial endothelial cells exposed to controlled levels of laminar shear stress in capillary flow tubes. A K+ selective, shear-stress-activated ionic current (designated Ik.s) was identified which is unlike previously described stretch-activated currents. Ik.s varies in magnitude and duration as a function of shear stress (half-maximal effect at 0.70 dyn cm-2), desensitizes slowly and recovers rapidly and fully on cessation of flow. Ik.s activity represents the earliest and fastest stimulus-response coupling of haemodynamic forces to endothelial cells yet found. We suggest that localized flow-activated hyperpolarization of endothelium involving Ik.s may participate in the regulation of vascular tone.     

WAVE2 is required for directed cell migration and cardiovascular development

WAVE2, a protein related to Wiskott-Aldrich syndrome protein, is crucial for Rac-induced membrane ruffling, which is important in cell motility. Cell movement is essential for morphogenesis, but it is unclear how cell movement is regulated or related to morphogenesis. Here we show the physiological functions of WAVE2 by disruption of the WAVE2 gene in mice. WAVE2 was expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos showed haemorrhages and died at about embryonic day 10. Deficiency in WAVE2 had no significant effect on vasculogenesis, but it decreased sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity formed in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries was severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo.     

微血管形成在腰椎间盘退变过程中的表现特征及影响因素

目的观察微血管形成在腰椎间盘退变过程中的病理表现特征,并分析其相关影响因素.方法选取48例行单节段腰椎后路经椎间孔减压椎间植骨融合内固定术患者,术中切取患处组织,冷冻切片后HE染色,观察病理改变,Weidner微血管计数法统计微血管密度.根据是否有微血管分为有微血管组、无微血管组,比较微血管形成的可能影响因素,对差异具有统计学意义的因素进一步行Logistics回归分析.结果 25例患者椎间盘内存在明显微血管形成,组织切片中存在典型的条形管腔结构,血管内皮细胞核有明显蓝染,微血管形成率为52.08%,MVD值平均为11.9±2.5.有微血管组椎间盘钙化率明显高于无微血管组,差异具有显著统计学意义(P0.01);有微血管组JOA评分明显低于无微血管组,腰椎疼痛VAS评分明显高于无微血管组,差异具有统计学意义(P0.05);椎间盘退变程度、椎间盘疝出程度两组间比较差异无统计学意义(P0.05).多因素回归分析显示:椎间盘钙化、腰椎疼痛VAS评分与退变椎间盘内微血管形成有明显相关性(OR值分别8.992,7.359,P值均0.05).结论椎间盘退变伴随着微血管形成,椎间盘钙化、腰椎疼痛VAS评分与退变腰椎间盘微血管形成有关.     

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.     

Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.     

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.     

Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases

Previous studies suggest that neuronal activity may guide the development of synaptic connections in the central nervous system through mechanisms involving glutamate receptors and GTPase-dependent modulation of the actin cytoskeleton. Here we demonstrate by in vivo time-lapse imaging of optic tectal cells in Xenopus laevis tadpoles that enhanced visual activity driven by a light stimulus promotes dendritic arbor growth. The stimulus-induced dendritic arbor growth requires glutamate-receptor-mediated synaptic transmission, decreased RhoA activity and increased Rac and Cdc42 activity. The results delineate a role for Rho GTPases in the structural plasticity driven by visual stimulation in vivo.     

The emergence of geometric order in proliferating metazoan epithelia

The predominantly hexagonal cell pattern of simple epithelia was noted in the earliest microscopic analyses of animal tissues, a topology commonly thought to reflect cell sorting into optimally packed honeycomb arrays. Here we use a discrete Markov model validated by time-lapse microscopy and clonal analysis to demonstrate that the distribution of polygonal cell types in epithelia is not a result of cell packing, but rather a direct mathematical consequence of cell proliferation. On the basis of in vivo analysis of mitotic cell junction dynamics in Drosophila imaginal discs, we mathematically predict the convergence of epithelial topology to a fixed equilibrium distribution of cellular polygons. This distribution is empirically confirmed in tissue samples from vertebrate, arthropod and cnidarian organisms, suggesting that a similar proliferation-dependent cell pattern underlies pattern formation and morphogenesis throughout the metazoa.     

小鼠胚肝基质细胞在体外培养中的动态演化

胚肝基质细胞是研究胚肝造血调控的重要工具，本实验对小鼠胚肝基质细胞在体外培养过程中的生长、细胞类型组成及其组成比例的演变过程进行了观察．实验显示小鼠原代培养初期基质细胞是由平滑肌样上皮细胞、巨噬细胞以及成纤维样细胞、树突状细胞、内皮细胞构成的，经过多次传代后，巨噬细胞等类型的细胞逐渐丢失，平滑肌样上皮细胞和成纤维样细胞成为主体，表明体外培养的小鼠胚肝基质细胞在4代以内都能较真实的反映体内造血微环境的构成，适用于胚肝造血研究．此为进一步研究胚肝基质细胞在胚胎期造血过程中的作用奠定了工作基础．     

Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth

Angiogenesis, the formation of new capillaries, which is observed in embryonic and injured tissue and is particularly prominent in the vicinity of solid tumours, involves the migration and proliferation of capillary endothelial cells. It is probably triggered by agents, such as basic fibroblast growth factor (bFGF), thought to be released from tissues adjacent to proliferating capillaries. As well as being a potent inducer of cell division in capillary endothelial cells in vitro, bFGF can act as an angiogenic agent in vivo. It is present in a wide variety of richly vascularized tissues including brain, pituitary, retina, adrenal gland, kidney, corpus luteum, placenta and various tumours. So far, however, the normal bFGF-producing cell species in these tissues have not been identified. We report here that capillary endothelial cells express the bFGF gene, that they produce and release bFGF and that bFGF derived from them can stimulate the proliferation of capillary endothelial cells. We conclude that bFGF can act as a self-stimulating growth factor for capillary endothelial cells, and that it is possible that the formation of new capillaries is induced by capillary endothelial cells themselves.     

Angiogenesis selectively requires the p110alpha isoform of PI3K to control endothelial cell migration

Phosphoinositide 3-kinases (PI3Ks) signal downstream of multiple cell-surface receptor types. Class IA PI3K isoforms couple to tyrosine kinases and consist of a p110 catalytic subunit (p110alpha, p110beta or p110delta), constitutively bound to one of five distinct p85 regulatory subunits. PI3Ks have been implicated in angiogenesis, but little is known about potential selectivity among the PI3K isoforms and their mechanism of action in endothelial cells during angiogenesis in vivo. Here we show that only p110alpha activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110alpha led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110alpha exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110alpha activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110beta in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1alpha, whereas p110delta is expressed at low level and contributes only minimally to PI3K activity in endothelial cells. These results provide the first in vivo evidence for p110-isoform selectivity in endothelial PI3K signalling during angiogenesis.