EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.     

中药促缺血心肌血管新生机制研究进展

中药在缺血性心脏病的治疗方面具有重要作用,能够缓解症状并改善预后,促血管新生可能是其发挥作用的机制之一。已证实通过促进血管新生,可使心肌梗死面积缩小,减少细胞坏死和凋亡,改善心功能等,但其机制尚需进一步明确。目前中医药在促进梗死心肌血管新生研究方面已经取得了一定的成果,研究发现,大量中药单体、方剂及中成药具有促进血管新生作用。随着对中药促血管新生研究的不断深入,其机制的研究亦愈趋广泛,已深入到细胞及分子水平:涉及骨髓干细胞,促进血管生长的因子及其受体(血管内皮细胞生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子等),抑制血管生长的因子(血管抑素、内皮抑素)及Akt/NOS/NO等通路。本文就相关研究回顾中药对缺血心肌血管新生影响并对机制进行探讨。     

Delta样配体4与血管生成

Notch信号转导途径参与了许多重要的发育过程,如神经发育、血管形成等,最近的一系列研究表明Notch受体与邻近细胞配体--Delta样配体4（Dll-4）共同参与了新生血管的生长和分支,通过抑制血管内皮顶端细胞的形成,细胞密度、位置以及行为方式的调节,而有效促进血管内皮细胞的正常分化和血管网的及时形成.Dll4在血管生成过程中的作用为相关疾病发生机制的理解、临床诊断及治疗提供了重要的帮助.     

Wnt5a及其信号通路与血管新生的研究进展

Wnt5a是Wnt蛋白家族中的成员之一,在细胞成熟、胚胎发育等过程中发挥着重要作用。研究表明Wnt5a的表达调控及其信号通路与血管新生密切相关,并且在血管新生性相关疾病中发挥了重要作用。本文从Wnt5a与其相关信号转导通路对血管新生的影响以及分子机制等方面进行阐述和展望,旨在为以Wnt5a为靶点进行血管新生性疾病的防治提供理论依据。     

血管平滑肌细胞的分化与血管内壁的构建

血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的发育与血管壁的构建是目前相关领域中的重要学科前沿．国内外同行的工作多集中在血管发育初始阶段内皮细胞及其前体细胞在血管新生中的作用、调节因素及生物学机制．VSMCs参与血管壁早期构建,特别是VSMCs的募集与分化机制已经成为血管新生研究中的一个新领域． 本期发表的《 抑制Rac1蛋白活化阻碍胚胎发育早期血管新生 》(见696～701页)报道了韩雅玲教授及其合作者在这一领域取得的最新研究结果．Rac1是真核细胞内重要的一类信号传递分子,在细胞信号传递过程中发挥分子开关作用．他们采用胚胎干细胞(ESCs)为模型,建立稳定表达持续型Rac1和显性失活型Rac1编码序列的小鼠ESCs并制备胚胎小体,诱导分化后观察其对内皮细胞分化和迁移的影响,发现抑制Rac1可以干扰血管内皮细胞连接成血管网状结构,细胞骨架F-actin排列紊乱,细胞的迁移受到明显抑制,表明Rac1在胚胎早期血管发育过程中与内皮细胞的迁移有关[1]． 近年来,韩雅玲教授及其研究集体在VSMCs发育与血管构建、胚胎干细胞来源的拟胚体血管平滑肌发育与血管新生机制以及胚胎主动脉VSMCs起源等方面开展了研究,取得了一系列有价值的成果[2～11],可能为闭塞性和增生性血管病的发生及防治提供理论依据和候选基因．详见“相关链接”．     

肿瘤饥饿疗法的新靶标——内分泌腺衍生血管内皮生长因子

“肿瘤饥饿疗法”是通过抑制促肿瘤血管新生细胞因子的作用,阻断肿瘤血管形成,最终实现“饿死”肿瘤细胞的一种治疗方法．内分泌腺衍生血管内皮生长因子(EG-VEGF)是在2001年被发现的一个组织选择性促血管新生因子．近年来的研究表明,EG-VEGF还兼有促进造血干细胞分化、刺激胃肠道收缩及影响肠神经系统发育等多种生理功能．EG-VEGF的异常表达与多种肿瘤及血管新生依赖性疾病的发生发展密切相关,有望作为相应的治疗靶点开发诊断及治疗试剂．本文对有关研究进展及应用前景作一简要综述．     

内皮祖细胞在新血管形成中的作用及其影响因素

内皮祖细胞是血管内皮的前体细胞,具有迁移,增殖、分化、黏附和吞噬功能,在体内外均可以诱导分化为成熟的内皮细胞,参与血管损伤的修复,病理及生理性新生血管的形成等过程,从而在多种心血管疾病、器官移植、肿瘤发生发展中起到重要作用。本文就EPC的特性、功能,在血管新生中所起的作用及其影响因素做一综述,以期为更深入的研究EPC提供依据。     

HIF-2α诱导MSCs向内皮细胞分化的机制

缺氧诱导因子-2（hypoxia—inducible factor-2,HIF-2）是一种转录因子。其主要活性作用部位是HIF-2α亚基,与HIF-10L有高度同源性,但二者在基因调节中有不同的作用,HIF-2α能优先表达某些靶基因如EPO、IGF、VEGFR等,是近年来研究较热门的一种促血管新生因子。骨髓间充质干细胞（mesenchy—real stem cells,MSCs）向内皮细胞分化、促进血管新生及管腔形成在很大程度上取决于HIF-2α的调节作用,此调节过程主要依赖于VEGF／VEGFR信号途径。     

Slit/Robo信号在血管新生中的功能研究

分泌型糖蛋白Slit及其受体Roundabout(Robo)最初是作为一类重要的发育中神经元轴突导向分子而被发现的。目前为止对Slit/Robo信号对神经系统发育过程中轴突吸引或排斥的导向功能研究比较多,而对在发育中生长方式与其非常相似的血管发生过程中研究比较少。现有研究提示两者在发育过程中可能存在共同的信号调控机制,是Slit/Robo信号通路在血管新生中充当着重要的角色。该文就Slit/Robo信号对血管内皮细胞迁移的调节、对血管新生的作用及其可能介导的信号通路进行综述,以期进一步推动Slit/Robo信号通路在血管发生中的研究。     

外源性电场对组织修复过程中细胞行为的影响

组织修复涉及一系列复杂的生理学、免疫学及细胞生物学过程。研究表明,受损组织周围存在着一定强度的内源性电场,类似生理强度的外源性电场能指导细胞定向迁移、控制细胞极化、调节细胞增殖和分化等一系列生物学行为。该文针对外源性电场在伤口愈合、骨组织愈合以及血管新生过程中的细胞生物学作用及其对修复过程中组织水平的影响进行综述,以期为外源性电场在今后临床中的应用提供参考。     

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.     

EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best‐understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine‐tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF‐R2 activation by VEGF nor its internalization, but it modulates VEGF‐R2 downstream signaling through phospho‐ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF‐induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.     

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.Eph/ephrin work in the laboratory of the authors is supported by a grant from the Deutsche Forschungsgemeinschaft (Au83/3–2 within the SPP1069 "Angiogenesis")     

EphB4 signaling is capable of mediating ephrinB2-induced inhibition of cell migration

Signaling between the ligand ephrinB2 and the respective receptors of the EphB class is known to play a vital role during vascular morphogenesis and angiogenesis. The relative contribution of each EphB receptor type present on endothelial cells to these processes remains to be determined. It has been shown that ephrinB2-EphB receptor signal transduction leads to a repulsive migratory behavior of endothelial cells. It remained unclear whether this anti-migratory effect can be mediated by EphB4 signaling alone or whether other EphB receptors are necessary. It also remained unclear whether the kinase activity of EphB4 is pivotal to its action. To answer these questions, we developed a cellular migration system solely dependent on ephrinB2-EphB4 signaling. Using this system, we could show that EphB4 activation leads to the inhibition of cell migration. Furthermore we identified PP2, a known inhibitor of kinases of the Src family, and PD 153035, a known inhibitor of EGF receptor kinase, as inhibitors of EphB4 kinase activity. Using PP2, the inhibition of cell migration by ephrinB2 could be relieved, demonstrating that the kinase function of EphB4 is of prominent importance in this process. These results show that EphB4 activation is not only accompanying ephrinB2 induced repulsive behavior of cells, but is capable of directly mediating this effect.     

Cardiovascular ephrinB2 function is essential for embryonic angiogenesis

EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. We have generated mice in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. We find that such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

Dll4-selective Notch signaling induces ephrinB2 gene expression in endothelial cells

The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation. Here, we show that Notch stimulation instructively induces arterial characteristics in endothelial cells (EC). Forced expression of Notch intracellular domain (NICD, activated form of Notch) induced mRNA expression for a subset of arterial-specific markers such as ephrinB2, connexin40, and HERP1 only in EC but not other cell lines. In co-culture experiments using EC and either Dll4- or Jagged1-expressing cells, we found that Dll4 stimulation but not Jagged1 markedly induced ephrinB2 expression. An inducible expression of HERP1 and HERP2 by NICD has no measurable effects on expression of ephrinB2 and venous marker EphB4 although either HERP1 or HERP2 overexpression exerts potent inhibitory effects on EphB4 expression without ephrinB2 induction. We also found no functional interaction between Notch and TGF-beta-ALK1 signalings in an induction of ephrinB2 expression. These results suggest that Dll4-stimulated Notch signaling induces a part of arterial characteristics only in EC via HERP-independent mechanism. Our data provide new insight into the molecular mechanism of ligand-selective Notch activation during differentiation of arterial EC.     

EphB4 promotes site-specific metastatic tumor cell dissemination by interacting with endothelial cell-expressed ephrinB2

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4-expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. AACR.     

Flow regulates arterial-venous differentiation in the chick embryo yolk sac

Formation of the yolk sac vascular system and its connection to the embryonic circulation is crucial for embryo survival in both mammals and birds. Most mice with mutations in genes involved in vascular development die because of a failure to establish this circulatory loop. Surprisingly, formation of yolk sac arteries and veins has not been well described in the recent literature. Using time-lapse video-microscopy, we have studied arterial-venous differentiation in the yolk sac of chick embryos. Immediately after the onset of perfusion, the yolk sac exhibits a posterior arterial and an anterior venous pole, which are connected to each other by cis-cis endothelial interactions. To form the paired and interlaced arterial-venous pattern characteristic of mature yolk sac vessels, small caliber vessels of the arterial domain are selectively disconnected from the growing arterial tree and subsequently reconnected to the venous system, implying that endothelial plasticity is needed to fashion normal growth of veins. Arterial-venous differentiation and patterning are controlled by hemodynamic forces, as shown by flow manipulation and in situ hybridization with arterial markers ephrinB2 and neuropilin 1, which show that expression of both mRNAs is not genetically determined but plastic and regulated by flow. In vivo application of ephrinB2 or EphB4 in the developing yolk sac failed to produce any morphological effects. By contrast, ephrinB2 and EphB4 application in the allantois of older embryos resulted in the rapid formation of arterial-venous shunts. In conclusion, we show that flow shapes the global patterning of the arterial tree and regulates the activation of the arterial markers ephrinB2 and neuropilin 1.