内皮细胞粘附分子与血管壁通透性

内皮细胞是血管壁的主要通透屏障,对血管壁通透性有多种调节作用。细胞粘附分参与细胞间连接形成以及细胞－基底膜间的粘附,与内皮层连续完整性以及通透性密切相关。参与内皮细胞中间连接的粘附分子有三大家庭,即整合素家庭、钙依赖性粘附素家族和免疫球蛋白家族,对这些粘附分子家族的研究有助于阐明内皮细胞损伤修复、新生血管形成、血管通透性调节以及循环血细胞游出的机制。     

体外研究血管内皮细胞单层通透性的新方法

血管壁通透性增高足创伤、烧伤、感染、休克和炎症等病理过程的重要变化和特征,其机理尚不十分清楚。内皮细胞是血管壁的主要通透屏障,研究内皮单层的通透性特征及其在病理情况下的变化机制对人们了解血管壁的通透性增高机制和寻找临床防治方法有着极为重要的意义。我们建立了用体外培养的内皮细胞单层研究通透性的装置,可以较为准确地测定内皮单层对液体的滤过系数Kf,便于体外研究各种体液因子和炎症介质对血管通透性的影响及其机制,也可用于研究白细胞-内皮细胞的相互作用。     

低氧条件下大鼠血管内皮细胞的形态观察

原代培养大鼠血管内皮细胞,将培养箱内通入5%CO2-95%N2混合气体（氧分压为18.3 mmHg）并培养大鼠血管内皮细胞12、24h,使用MTT法、LDH活力测定及细胞骨架染色对低氧细胞模型鉴定,研究大鼠血管内皮细胞低氧模型的建立条件及其形态学特点。在低氧12h条件下,血管内皮细胞存活率降低、LDH释放增加,但细胞骨架保持完整;在低氧24h条件下,血管内皮细胞存活率降低、LDH释放增加,细胞骨架破碎。结果表明在低氧（氧分压为18.3 mmHg）24h条件下,可以建立大鼠血管内皮细胞低氧模型。     

血管内皮细胞生长因子及临床应用策略

血管内皮细胞生长因子（VEGF）是一种特异作用于血管内皮细胞的多功能细胞因子,它能引起血管通透性增加,引起细胞外基质成分改变,诱导血管形成,在炎症,创伤愈合,心脏缺血,动脉粥亲硬化,糖尿病性视网膜病变及肿瘤形成等与血管生成和病变有关的诸多病理过程中起重要作用。     

肌肽对低氧所致大鼠血管内皮细胞损伤的保护作用

目的：研究肌肽对低氧所致大鼠血管内皮细胞损伤的影响。方法：建立低氧条件下大鼠血管内皮细胞损伤模型,用MTT法观察肌肽对低氧损伤的血管内皮细胞活性的影响,测定细胞培养基中LDH活力,并对细胞骨架进行考马斯亮蓝R-250染色观测其细胞结构。结果：浓度为10mmol/L～20mmol/L肌肽孵育血管内皮细胞6h后,可以抑制缺氧12h和24h引起的血管内皮细胞活性下降,同时减少LDH的释放,保持细胞骨架完整。结论：肌肽对低氧所致的血管内皮细胞损伤具有保护作用。     

血管内皮细胞生长因子研究进展

从不同侧面阐述了血管内皮细胞生长因子（VEGF）在新生血管形成中的作用.VEGF诱导新生血管形成,具有血管渗透性,是新生血管形成的主要调控者之一.VEGF mRNA不同剪接,形成5种VEGF变异体(isoform)即VEGF121-206.VEGF诱导新生血管的调控过程、拮抗VEGF成为大家竞相研究的领域.     

内皮细胞抑制素的研究进展

内皮细胞抑制素是一种新发现的特异的血管内皮细胞增殖抑制剂,它可以有效地抑制内皮细胞生长,对肿瘤血管形成具有强烈的抑制作用,在抗实体瘤方面有显著的效果。本文就它的发现、结构、生物学活性、作用机理及研究进展进行综述。     

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。     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Protein kinase C affects reformation of endothelial junctions in xenopus XTH-2 cells.

Endothelial cells can reversibly be forced to suppress the formation of endothelial junctions (EJ) by cultivation in a low calcium medium. The authors localized vinculin and cadherin as marker proteins of EJ and actin as a cytoskeletal component by fluorescence microscopy, and used this cell model to study the reformation of endothelial junctions under conditions of activation and inhibition of protein kinase C (PKC). Inhibition of PKC by H-7 leads to an acceleration of EJ reformation, while constitutive activation by TPA inhibits the reformation process.     

The permeability of the cell-to-cell membrane channel and its regulation in mammalian cell junctions

Summary Mammalian cell-to-cell channels show polar permselective properties discriminating against negatively charged 14 ?-wide molecules and are more restrictive than the channels of insect cell junctions. The channel permeability is modulated by conditions affecting the concentration of intracellular ionic Ca: elevation of the external Ca load (B cells), treatment of cell cultures with Ca-transporting ionophore (in the presence of external Ca, but not in its absence), treatment with a combination of cyanide and iodoacetate, or with high levels of carbon dioxide, all cause depression of channel permeability. Treatment of cell cultures with cyclic AMP or its more permeable derivative, dibutyryl cyclic AMP, produces increase in permeability. A similar channel up regulation is observed upon elevation of the endogenous level of cyclic AMP by serum deprivation or lowering of cell density. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center. This work was supported by grant number 5 R01 CA14464, awarded by the National Cancer Institute, DHEW.     

Simultaneous topography and recognition imaging on endothelial cells

Determining the landscape of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometres) is an important task in many fields of biological science. During the past five years, dynamic recognition imaging (e.g. simultaneous topography and recognition (TREC) imaging) has proven to be a powerful technique in biophysical research. This technique becomes an indispensable tool for high-resolution receptor mapping as it has been successfully demonstrated on different biomolecular model systems. In these studies, the topographical imaging of receptor molecules is combined with molecular recognition by their cognate ligands bound to the atomic force microscope (AFM) tip via a flexible and distensible tether. In this review, we describe the principles of TREC imaging and provide a flavour of its recent application on endothelial cells.     

Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.     

A coronary artery disease-associated gene product, JCAD/KIAA1462, is a novel component of endothelial cell-cell junctions

Cell–cell junctions play crucial roles in the organization and function of epithelial and endothelial cellular sheets. Here, we have identified the protein product for KIAA1462 gene, whose single nucleotide polymorphisms (SNPs) have recently reported to be associated with coronary artery disease, as a novel component of cell–cell junctions. We propose the name of KIAA1462 protein junctional protein associated with coronary artery disease (JCAD). JCAD is a ∼145 kDa protein without any known domains but contains a proline-rich region. Immunolocalization studies revealed that JCAD is specifically localized at cell–cell junctions in endothelial cells but not in epithelial cells. The accumulation of JCAD at cell–cell junctions in cultured endothelial cells was impaired by RNAi-mediated suppression of VE-cadherin expression. In cell adhesion-deficient mouse L fibroblasts, JCAD was recruited to cell–cell contacts when cadherin-mediated cell–cell adhesion was induced. These results indicate that JCAD is a component of VE-cadherin-based cell–cell junctions in endothelial cells. This study also suggests the implication of endothelial cell–cell adhesion in coronary artery disease.     

The role of the cytoskeleton in the formation of gap junctions by Connexin 30

Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.     

Regulation of podosome formation in aortic endothelial cells vessels by physiological extracellular cues

Invadosomes are specialised actin-based dynamic microdomains of the plasma membrane. Their occurrence has been associated with cell adhesion, matrix degrading and mechanosensory functions that make them crucial regulators of cell migration and invasion. Monocytic, cancer cell and Src-transformed cell invadosomes have been extensively described. Less well defined are the structures which form in other cell types, i.e., non-haematopoietic and non-transformed cells, exposed to specific stimuli. We herein describe the specificities of podosomes induced in aortic endothelial cells stimulated with TGFβ in vitro and in conditions that more closely resemble the in vivo situation. These podosomes display the typical architecture of monocytic podosomes. They organise into large rosette-shape superstructures where they exhibit collective dynamic behavior consisting in cycles of formation and regression. At the ultrastructural level, microfilament arrangements in individual podosomes were revealed. Oxygen levels and hemodynamic forces, which are key players in endothelial cell biology, both influence the process. In 3D environment, podosomes appear as globular structures along cellular extensions. A better characterization of endothelial podosomes has far-reaching implications in the understanding and, possibly, in the treatment of some vascular diseases.     

RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.