Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth.

Two heparin-binding proteolytic fragments of fibronectin--an amino-terminal 29-kd segment and a carboxyl-terminal 40-kd segment-are apparently specific, potent inhibitors of the growth of cultured bovine aortic endothelial cells and inhibit growth in a reversible, dose-dependent manner. In contrast, native fibronectin at higher dosages neither inhibits nor interferes with the effects of the 29-kd fragment. The data, therefore, suggest that fibronectin fragments may participate in the regulation of vascular growth.     

Role of material-driven fibronectin fibrillogenesis in cell differentiation

Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials.     

Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.     

Adhesion and growth of cultured human endothelial cells on perfluorosulphonate: role of vitronectin and fibronectin in cell attachment.

The suitability of neutralized perfluorosulphonic acid (Nafion) as a surface for the attachment and growth of human cells was investigated in tissue culture. Nafion was equivalent to tissue culture polystyrene (TCP), and markedly better than polytetrafluoroethylene (Teflon), for the attachment and growth of human umbilical artery endothelial (HUAE) cells. The attachment and growth of HUAE cells on Fn-coated Nafion was equivalent to that on Fn-coated TCP. The contribution to the attachment and spreading of HUAE cells that is due to adsorption of serum fibronectin (Fn) or vitronectin (Vn) on to the Nafion or TCP was directly tested by selective removal of Fn or Vn from the serum before addition to the culture medium. HUAE cells seeded on to Nafion or TCP in medium depleted of Vn failed to attach and spread on to these surfaces, as measured after 4 or 24 h of culture. HUAE cells seeded in medium depleted of Fn, but containing Vn, attached and spread on to Nafion, albeit to a decreased extent as compared to that in intact serum when measured after 4 h of culture, and there was no effect of depletion of Fn when measured after 24 h of culture. HUAE cells seeded on to TCP in medium depleted of Fn became attached and spread during 4 h of culture. Our results show that Nafion is a suitable polymeric surface for the attachment and growth of human cells, including endothelial cells. For HUAE cells, adsorption on to the surface of an adhesive glycoprotein, such as Vn or Fn, is an essential step for attachment and spreading of the cells onto the Nafion surface.     

Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.     

妊高征患者子宫静脉血浆对血管内皮细胞膜上纤维结合蛋白的影响

本研究于体外培养的人脐静脉血管内皮中加入妊高征病人子宫静脉血浆，培养一定时间后，免疫组织化学法检测细胞膜上纤维结合蛋白(Fn)。结果发现经妊高征病人子宫静脉血浆处理过的血管内皮表面Fn增加。表明妊高征胎盘能够合成某种因子刺激血管内皮合成Fn；内皮细胞膜上的Fn增多可能是妊高征病人凝血异常的原因。     

Role of subcellular shear-stress distributions in endothelial cell mechanotransduction

The endothelium of blood vessels presents a wavy surface to the flowing blood. The subcellular distribution of shear stress depends on the shape and orientation of the cells and on their spatial arrangement within the monolayer. By studying details of the distribution of stress at this scale and the morphological responses that serve to modify the distribution, we can gain insight into the physical mechanisms by which the cell senses its fluid mechanical environment. The rapidly growing body of evidence indicates that endothelial cells discriminate between subtle variations in the exact loading conditions including differences in temporal and spatial gradients of shear stress, steady and pulsatile laminar flow, and laminar and turbulent flows. While in a few studies the effects of these individual flow characteristics have been carefully isolated, it is difficult to assess the relative importance of any one parameter. To interpret the relationships between isolated flow characteristics or the integrated effects of combined loading conditions and the biochemical signaling events that mediate the cell response, a full stress analysis of the cell is needed. The microscopic distribution of shear stress acting upon the cell surface provides the boundary condition for such an analysis. Experimental and analytical tools are being developed to assess the stress distribution throughout the cellular structures that might be involved in mechanotransduction. © 2002 Biomedical Engineering Society. PAC2002: 8716Xa, 8719Uv, 8719Xx     

Effects of endothelial removal and regeneration on smooth muscle glycosaminoglycan synthesis and growth in rat carotid artery in organ culture

Segments of rat carotid artery were maintained in serum-free and serum-supplemented media with endothelium both present and substantially removed by air drying. At intervals of 3, 7, and 14 days the synthesis of glycosaminoglycan across the vessel walls was determined by autoradiographic detection of incorporated [3H]glucosamine. In control carotids the typical pattern of incorporation was 40% of label in the intima, consisting of endothelium and subendothelial matrix, 23, 13, and 15% in the three medial layers (M1, M2, M3, respectively), and 9% in the adventitia. During the first week in culture the proportion, and often the amount, of label in M1 increased significantly. Following air drying labeling decreased markedly in M1 but often increased in M2 and M3. By 14 days residual endothelial cells had regenerated, and the pattern of incorporation in the medial layers beneath this new endothelium was the same as for the controls with a high level of labeling in M1. In areas free of endothelium incorporation in M1 remained at a low level. Digestion with chondroitinase ABC and Streptomyces hyaluronidase showed that the changes in M1-labeling levels were due to changes in the amounts of both hyaluronic acid and sulfated glycosaminoglycan, whereas pulse and continuous labeling studies showed that the different labeling levels for the various layers and conditions were due to different rates of synthesis and not degradation. Carotids were also labeled with [3H]thymidine. Control and regenerating endothelia were active in serum-free and serum-supplemented media and had similar mitotic indices. Indices for smooth muscle cells in M1, however, were generally very low and were not affected by the presence or absence of endothelium. We conclude that endothelial removal results in decreased glycosaminoglycan synthesis in the inner media, that mitotically active endothelium correlates with increased glycosaminoglycan synthesis in the inner media, and that these changes occur independently of smooth muscle cell growth.     

Nanopatterning of fibronectin and the influence of integrin clustering on endothelial cell spreading and proliferation

Investigating stages of maturation of cellular adhesions to the extracellular matrix from the initial binding events to the formation of small focal complexes has been challenging because of the difficulty in fabricating the necessary nanopatterned substrates with controlled biochemical functionality. We present the fabrication and characterization of surfaces presenting fibronectin nanopatterns of controlled size and pitch that provide well-defined cellular adhesion sites against a nonadhesive polyethylene glycol background. The nanopatterned surfaces allow us to control the number of fibronectin proteins within each adhesion site from 9 to 250, thereby limiting the number of integrins involved in each cell-substrate adhesion. We demonstrate the presence of fibronectin on the nanoislands, while no protein was observed on the passivated background. We show that the cell adheres to the nanopatterns with adhesions that are much smaller and more evenly distributed than on a glass control. The nanopattern influences cellular proliferation only at longer times, but influences spreading at both early and later times, indicating adhesion size and adhesion density play a role in controlling cell adhesion and signaling. However, the overall density of fibronectin on all patterns is far lower than on homogeneously coated control surfaces, showing that the local density of adhesion ligands, not the average density, is the important parameter for cell proliferation and spreading.     

血管内皮生长因子在毛囊生物学中的研究现状

血管内皮生长因子(vascular endothelial growth factor,VEGF)又名血管通透性因子(vascular permeability factor,VPF),是重要的血管生成正性调节因子。作为毛乳头细胞的一种自分泌生长因子,其对毛囊的生长亦有重要作用。血管内皮生长因子不仅能促进毛囊的生长,还参与毛囊生长周期的调控。在内皮细胞中,血管内皮生长因子发挥作用主要是通过与其受体的结合,诱导受体二聚体化和自身磷酸化,从而激活胞内信号转导通路,但在毛囊细胞中是否如此,仍需进一步研究。     

Alternative splicing of endothelial cell fibronectin mRNA in the IIICS region. Functional significance.

Transforming growth factor-beta 1 (TGF-beta 1) is thought to play a role in modulating vascular cell function in vivo. In vitro, it decreases endothelial cell proliferation and migration. We postulated that these biologic activities could be mediated through TGF-beta 1 modulation of specific gene expression. Therefore we differentially screened a human umbilical vein endothelial cell cDNA library with cDNAs prepared from both untreated and TGF-beta 1-treated bovine aortic endothelial cells. Using this technique, we isolated many TGF-beta 1-induced cDNA clones. Sequence analysis of these cDNAs showed that many of them corresponded to alternatively spliced fibronectin mRNAs. These fibronectin clones all contained the extradomain I (ED I) but three different forms of the type III connecting segment (IIICS). These different fibronectin cDNAs were expressed in bacteria and the recombinant proteins used to study the effects of IIICS alternative splicing on cell attachment, spreading, and migration in bovine aortic endothelial and smooth muscle cells and B16F10 melanoma cells. The results of these experiments show that attachment and spreading of bovine aortic endothelial and smooth muscle cells depend primarily on the presence of the Arg-Gly-Asp-Ser (RGDS) sequence in the recombinant fibronectin proteins. However attachment and spreading of bovine aortic endothelial cells are modulated by alternative splicing in the IIICS region. Specifically splicing of the IIICS region decreases spreading and increases migration rates of the endothelial cells. On the contrary, using a cell line (B16F10 melanoma cells) that is known not to require the RGDS sequence for adhesion confirmed previous findings that B16F10 melanoma cells do not require the presence of the RGDS sequence for attachment and spreading. Indeed B16F10 cells were able to attach and spread on two recombinant proteins that did not contain the RGDS sequence. However attachment and spreading of B16F10 were dramatically inhibited when a 75-base pair DNA fragment was removed from the 5' end of the IIICS region. These results suggest that various regions of the fibronectin molecule may be able to interact with different cell populations to promote cell attachment and spreading, and that alternative splicing may modulate this process.     

Cyclic biaxial strain of pulmonary artery endothelial cells causes an increase in cell layer-associated fibronectin

Bovine pulmonary artery endothelial (PAE) cells were cultured on an artificial compliant substrate (Mitrathane) and were strained biaxially at a frequency of 1/s for 2, 4, 6, 7, or 24 h. Total protein synthesis, determined by estimating the incorporation of radiolabeled precursors into nondialyzable protein, was increased in cultures that had been biaxially strained for 6, 7, or 24 h, with differences more apparent in the cell layer fraction than in the medium fraction. Medium and cell layer-associated fibronectin were quantitated by enzyme-linked immunosorbent assay and by densitometric analysis of the autoradiograms of electrophoresed protein. Fibronectin levels in the medium of biaxially strained cells were initially depressed in comparison to nonstrained controls but, with time, began to approach control values. Cell layer-associated fibronectin of biaxially strained cultures was significantly elevated at 24 h, whereas DNA synthesis was not altered. Immunohistochemical localization of fibronectin and factor VIII-von Willebrand antigen revealed a more intense staining pattern in strained cultures. Distribution of stress fibers containing fibrous actin was visualized by staining with rhodamine-phalloidin and was altered in strained cultures. These observations indicate that cells respond to cyclic biaxial strain by selectively enhancing structural components associated with cell adhesion.     

Role of vascular endothelial growth factor during breast cancer

We review the results of experimental and clinical observations on neoangiogenesis in patients with breast cancer. Vascular endothelial growth factor is an important positive regulator of this process. Experiments showed the possibility of using various direct and indirect antiangiogenic means in the therapy of breast cancer, but clinical efficiency of these methods was not proved. Expression of vascular endothelial growth factor can serve as a prognostic criterion in breast cancer. Antiangiogenic preparations should not be used as monotherapy, but as the treatment complementary to standard therapy.     

血管内皮生长因子在子宫内膜异位症发病中的作用

目的探讨血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)在子宫内膜异位症(endometriosis,EM)发病中的作用。方法应用免疫组织化学方法并结合图像分析技术。结果正常子宫内膜和EM在位内膜腺上皮细胞的VEGF随月经周期呈现规律性变化,分泌期腺上皮VEGF蛋白表达量显著高于增殖期(P<0.05)。在增殖期,EM在位子宫内膜腺上皮VEGF的表达与正常子宫内膜相比无明显差别,但在分泌期,EM在位子宫内膜腺上皮细胞中VEGF的表达强度明显高于正常子宫内膜(P<0.01)。EM在位内膜腺上皮的VEGF含量显著高于同组卵巢子宫内膜异位囊肿的异位腺上皮(P<0.01)。结论表明VEGF的表达异常与EM的发病有关。     

Role of fibronectin-stimulated tumor cell migration in glioma invasion in vivo: clinical significance of fibronectin and fibronectin receptor expressed in human glioma tissues

In order to clarify the role of fibronectin in glioma invasion in vivo, we analyzed the relationship between fibronectin-stimulated cell migration and adhesion in 14 primary glioma cells and the expression of fibronectin and the fibronectin receptor in the corresponding tumor tissues. The tumors comprised nine glioblastomas (GB) and five anaplastic gliomas (AG) consisting of two astrocytomas, two oligoastrocytomas and one ependymoma. All glioma cells tested in the primary cell culture were found to migrate to fibronectin in a dose-dependent manner. The extent of cell migration to fibronectin was not significantly different for the GB and AG groups. On the other hand, cell adhesion to fibronectin in the AG was much stronger than that in the GB group. Immunohistochemistry demonstrated that fibronectin positively stained in the extra-cellular matrix (ECM) in eight cases and that the fibronectin receptor was positive in tumor cell membranes in 10 cases. In addition, cellular fibronectin isoforms containing ED-A and ED-B sequences were found to be immunolocalized in the tumor cells and the ECM of GB. These isoforms were also specifically expressed in tumor vessels within tumor tissues, but not in those within normal brain tissues. Cell migration tended to be expressed more strongly by glioma cells derived from tumor tissues in which fibronectin was posi-tively immunolocalized in the ECM than from tissues with negative fibronectin in the ECM. Four glioma cells derived from GB whose tumor cells did not positively stain for fibronectin receptors migrated much less extensively to fibronectin than other glioma cells whose tissues showed positive staining for the fibronectin receptor. Of these four GB, two had loss of heterozygosity in the locus of fibronectin receptor b1 gene. These results suggest that fibronectin deposited in the extracellular matrix of tumors, which can be derived from both plasma and the tumor cell itself, strongly promotes the migration of glioma cells, and that expression of the fibronectin receptor may play a critical role in the biological behavior of the tumor cells, particularly in fibronectin-stimulated cell migration in vivo.© Kluwer Academic Publishers 1998     

粘着斑激酶在血管内皮细胞迁移中的作用

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase (PTK) that can localize indirectly to sites of clustering integrin family of heterodimeric receptors. As an important structure and signaling molecule in the adhesive complexes, which are large and stable referred as ‘focal adhesions‘ or relatively small and transient within filopodia and lamellipodia named ‘focal complexes‘, FAK is closely related with cell death, proliferation and migration. In this review, we discuss the function of FAK in the regulation of endothelial cell migration based on current data.     

Immunohistochemically localization of vascular endothelial growth factor, vascular endothelial growth factor receptor-2, collagen I and fibronectin in the epithelia-mesenchymal junction of the human tooth germ

During tooth development, tooth shape is mediated by the ECM through epithelial-mesenchymal interactions mediated by the ECM at the epithelia-mesenchymal junction. Blood vessel endothelium growth is mainly regulated by vascular endothelial growth factor (VEGF) and the relationship between tooth shape formation and VEGF are unknown. We examined immunohistochemical localization of VEGF and its receptor VEGF receptor-2 (VEGFR-2), collagen I and fibronectin, (both representative protein of ECM) at the epithelia-mesenchymal junction of human deciduous teeth from the cap stage to late bell stages in a human fetus at 16, 20, 24, 28 and 32 weeks of gestation. Immunoreactivity at the basement membrane for VEGF was detected from the cap stage to the bell stage. Immunoreactivity to fibronectin was weak in the cap stage and increased in the bell stage; collagen I was negative in the cap stage and slightly expressed in the bell stage in the basement membrane. We suggest that VEGF and ECM affect cooperatively in tooth shape formation at the basement membrane.     

Deposition of endothelial fibronectin on polymeric surfaces

Cellular fibronectin is deposited on tissue culture polystyrene during the adhesion and spreading of cultured human endothelial cells (HEC). Following the seeding of HEC upon this polymer, larger amounts of fibronectin are deposited as both cell density and incubation time increase. Our results indicate that the ability to deposit cellular fibronectin onto a polymeric surface is a condition for the spreading and proliferation of HEC.     

Matrix fibronectin disruption in association with altered endothelial cell adhesion induced by activated polymorphonuclear leukocytes

Sequestration of activated polymorphonuclear leukocytes (PMN) within the lung microcirculation may contribute to pulmonary vascular injury following trauma, sepsis, or disseminated intravascular coagulation. In this study cultured rat endothelial cells were utilized to evaluate the effect of PMN activation on endothelial cell attachment. The concept that disruption of the extracellular fibronectin matrix is associated with altered endothelial cell adhesion was also tested. Rat endothelial cells were grown in culture and identified by morphological techniques as well as immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with 51Cr in order to establish a cell injury assay based on release of free 51Cr or cell-associated 51Cr. PMN activation was verified microscopically and by chemiluminescence activity following phorbol myristate acetate (PMA) or opsonized zymosan exposure. Following incubation with PMA, the leukocytes aggregated, chemiluminesced vigorously, and caused endothelial cell injury and detachment as determined by release of 51Cr-labeled endothelial cells. PMNs exposed to serum-treated zymosan exhibited a more modest chemiluminescence burst which was consistent with their decreased activity to injure the endothelial monolayer. With PMA activation the degree of endothelial detachment from the monolayer increased as a function of time with a plateau observed by 3 hr. Microscopic immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin after incubation with PMA-activated neutrophils in association with endothelial cell disadhesion. Thus, exposure of activated rat PMN to rat endothelial cells in culture induces endothelial damage and an associated disruption of the fibronectin matrix which may contribute to endothelial cell detachment.