Human endothelial cells are chemotactic to endothelial cell growth factor and heparin

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.     

Inhibition of endothelial cell proliferation by gamma-interferon

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose- dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor- ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.     

Serial propagation of human endothelial cells in vitro

Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.     

The characterization of the receptor for endothelial cell growth factor by covalent ligand attachment

Cellular receptors for endothelial cell growth factor (ECGF) have been demonstrated on several cell types by binding of 125I-ECGF in a specific and saturable manner (Schreiber, A. B., Kennedy, J., Kowalski, J., Friesel, R., Mehlman, T., and Maciag, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6138-6142). Here we report the covalent cross-linking of 125I-ECGF to a polypeptide present on the surface of the plasma membrane of murine lung capillary endothelial cells by the homobifunctional reagent, disuccinimidyl suberate. Cross-linking of cell surface associated 125I-ECGF yields a major polypeptide with an apparent molecular weight of 150,000. Experiments demonstrated that the cross-linked polypeptide complex represents 125I-ECGF covalently bound specifically to a cell surface receptor because: covalent modification of the polypeptide was inhibited by excess, unlabeled ECGF; preincubation of cells with unlabeled ECGF at 37 degrees C significantly reduced cross-linking while incubation at 4 degrees C did not; other polypeptide growth factors do not compete with 125I-ECGF for cross-linking to the ECGF receptor; labeling of the polypeptide did not take place in the absence of DSS; and cells previously shown to have a paucity of ECGF receptors did not yield a cross-linked labeled receptor. These data suggest that the mitogenic events mediated by ECGF occur after occupancy of the specific cell surface polypeptide and suggest that these events are relevant to ECGF-induced signal transduction across the endothelial cell plasma membrane.     

Inhibitory effect of human recombinant interleukin-1 alpha and beta on growth of human vascular endothelial cells

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen which stimulates the growth of endothelial cells. The mitogenic effect of ECGF was inhibited by addition of recombinant interleukin-1 (rIL-1) alpha or beta in a concentration dependent manner. The morphological change was not observed distinctly. In the condition without ECGF, both types of rIL-1 enhanced [3H]-thymidine uptake slightly, but failed to increase cell numbers. These data suggest the possibility that the effect of rIL-1 on EC is modulated by the presence of ECGF.     

Interleukin-1β-Induced Wnt5a Enhances Human Corneal Endothelial Cell Migration through Regulation of Cdc42 and RhoA

Wnt5a can activate β-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1β (IL-1β) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.     

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.     

Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in focal adhesion turnover and integrin activation

We have asked whether the Nck and Crk adaptor proteins play important roles in the vascular endothelial growth factor (VEGF)-induced signaling pathways that lead to an enhancement in cell migration. The introduction into human umbilical vein endothelial cells of a dominant-negative inhibitor for either Nck or Crk blocked the recruitment of both endogenous proteins to the KDR VEGF receptor subtype indicating that both proteins are recruited to the same docking site. The Nck and Crk dominant-negatives led to the formation of abnormally large focal adhesion, blocked VEGF-induced integrin activation, and blocked VEGF-induced actin dynamics. The dominant-negatives had no effects on these properties in cells expressing constitutively active Rac1 or RhoA. Since a DN to either Nck or Crk blocks the cellular responses mediated by both proteins, we performed experiments directed at clarifying signaling pathways specifically mediated by each protein. Inhibition of the interaction between Nck with its downstream effector PAK led to abnormally large focal adhesions, but had no effect on integrin activation or cell adhesiveness. Evidence is presented that Crk complexes with C3G in control cells, and VEGF treatment leads to the recruitment of the complex to the cell surface. Inhibition of the C3G downstream effector Rap1 leads to enlarged focal adhesions and blocks VEGF-induced integrin activation. We conclude that Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in cell migration.     

The proteomic analysis of mouse choroid plexus secretome reveals a high protein secretion capacity of choroidal epithelial cells

Choroid plexuses (CP) are involved in multiple functions related to their unique architecture and localization at the interface between the blood and cerebrospinal fluid compartments. These include the release by choroidal epithelial cells (CEC) of biologically active molecules, such as polypeptides, which are distributed globally to the brain. Here, we have used a proteomic approach to get an unbiased overview of the proteins that are secreted by primary cultures enriched in epithelial cells from mice CP. We identified a total of 43 proteins secreted through the classical vesicular pathway in CEC -conditioned medium. They include transport proteins, collagen subunits and other cell matrix proteins, proteases, protease inhibitors and neurotrophic factors. Treating CEC cultures with lipopolysaccharide, increased the secretion of four protein species and induced the release of two additional proteins. Our study also reveals a higher protein secretion capacity of CECs compared with other CP cells or cultured astrocytes. In conclusion, this study provides for the first time the characterization of the major proteins that are secreted by CECs. These proteins may play a critical role in neuronal growth, differentiation and function as well as in brain pathologies.     

Heparin and endothelial cell growth factor modulate collagen and proteoglycan production in human smooth muscle cells

The effects of heparin and endothelial cell growth factor (ECGF) on extracellular matrix production were examined in human iliac smooth muscle cells. The cells were grown in (a) medium supplemented with heparin (100 micrograms/ml) and ECGF (75 micrograms/ml), (b) medium supplemented with ECGF (75 micrograms/ml) alone, or (c) unsupplemented medium. In the presence of heparin and ECGF, collagen production was inhibited 91-95% as compared to cultures incubated with ECGF alone or without both supplemental factors. In contrast, the production of proteoglycans was elevated 2.5 fold in the presence of heparin and ECGF. Enzymatic digestion of the proteoglycans indicated that both large and small molecular weight chondroitin sulfate proteoglycans were markedly elevated, while dermatan sulfate and heparan sulfate proteoglycans were increased to a lesser extent. The results suggest that the combination of heparin and ECGF elicits potent modulation of extracellular matrix production, with divergent effects on collagen and proteoglycan synthesis.     

An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.     

Human and bovine vascular endothelial cells. Comparative effect on cell growth and longevity of an eye-derived growth factor (EDGF) and of extracellular matrix

Human and bovine vascular endothelial cells from the umbilical vein and the aorta, respectively, were cultured in the presence of EDGF (a growth factor prepared from bovine retina) on plastic or on extracellular matrix (ECM). Both EDGF and ECM are required to allow the maximal proliferation of human cells and their organization in a typical monolayer. Conversely, bovine aortic endothelial cells grow perfectly in the absence of both factors in 6% fetal calf serum. However, a requirement for EDGF can also be demonstrated in low serum conditions, or in cells at high passage number. ECM had no growth promoting activity by itself. Thrombin acts similarly to EDGF on bovine serum-starved cells. EDGF prolongs the in vitro lifespan of both types of cells. Cells at all stages still synthesize factor VIII antigen as revealed by immunofluorescence. Thus EDGF, like other growth factors from brain, FGF or ECGF, may have an important role in angiogenesis, a critical problem in pathological retinas.     

Modulation of smooth muscle cell behaviour by platelet-derived factors and the extracellular matrix

We have studied the combined effects of platelet-derived soluble factors and three types of macromolecular substrata on the proliferation and migration of smooth muscle cells in vitro. Bovine aortic smooth muscle cells were plated onto three-dimensional gels of type I collagen or onto cell-free extracellular matrices deposited on such gels by either bovine aortic endothelial cells or smooth muscle cells. The cells were cultured in the presence of whole-blood serum (WBS) or platelet-poor plasma (PPP). Smooth muscle cell proliferation on type I collagen gels was dependent on the presence of platelet-derived factors, i.e. the cells proliferated in the presence of WBS but not in PPP. In contrast, cell proliferation on the extracellular matrices occurred at the same rate in PPP and WBS. Smooth muscle cells plated onto collagen gels rapidly migrated down into the gel matrix; the percentage of cells migrating was inversely proportional to cell density. The presence of extracellular matrices did not alter the rate of cell migration into the underlying gel matrix. Irrespective of the substratum used, smooth muscle cell migration was independent of platelet-derived or plasma factors and occurred in the absence of proliferation. These results indicate that possible chemotactic, chemokinetic, and/or mitogenic factors produced by the vascular cells and deposited within the extracellular matrix may play an important role in modulating smooth muscle cell behaviour in the vascular wall.     

Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine

Corneal endothelial dysfunctions occurring in patients with Fuchs'' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na⁺/K⁺-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.     

Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.     

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.     

角膜内皮细胞的检测、受损因素及治疗新进展

摘要：角膜内皮细胞(corneal endothelial cells,CECs)通过其屏障作用及离子泵功能维持角膜透明,但年龄、疾病及创伤等因素会使其功能有所降低,导致角膜失代偿、角膜水肿、大泡性角膜病变,甚至视力的丧失。因此,CECs可谓是角膜的"生命"。近年来,CECs的检测方法逐渐新增,并且不同检测指标具有不同意义;导致CECs受损的因素也有所增加,同时CECs损伤后的治疗也逐渐成为近年来的研究热点。早期发现CECs受损、明确病因并进行干预或治疗会减少CECs的损伤,对指导临床工作有重要的意义。本文主要对CECs检测方法及指标、损伤因素及其治疗的最新进展进行了综述。     

Thrombospondins function as regulators of angiogenesis

Thrombospondins (TSPs) -1 and -2 were among the first protein inhibitors of angiogenesis to be identified, a property that was subsequently attributed to the interactions of sequences in their type I repeats with endothelial cell-surface receptors. The interactions of TSPs-1 and -2 with cell-surface receptors, proteases, growth factors, and other bioactive molecules, coupled with the absence of direct structural functions that can be attributed to these matrix proteins, qualify them for inclusion in the category of ‘matricellular proteins’. The phenotypes of TSP-1, TSP-2, and double TSP-1/2-null mice confirm the roles that these proteins play in the regulation of angiogenesis, and provide clues to some of the other important functions of these multi-domain proteins. One of these functions is the ability of TSP-1 to activate the latent TGFβ1 complex, a property that is not shared by TSP-2. A major pathway by which TSP1 or TSP2 inhibits angiogenesis involves an interaction with CD 36 on endothelial cells, which leads to apoptosis of both the liganded and adjacent cells. However a homeostatic mechanism, which inhibits endothelial cell proliferation, and may be physiologically preferable under some circumstances, has also been elucidated, and involves interaction with the very low density lipoprotein receptor (VLDLR). The interaction of TSP1with its receptor, CD47, further inhibits angiogenesis by antagonizing nitric oxide signaling in endothelial and vascular smooth muscle cells. Paradoxically, there is also evidence that TSP-1 can function to promote angiogenesis. This apparent contradiction can be explained by the presence of sequences in different domains of the protein that interact with different receptors on endothelial cells. The anti-angiogenic function of TSPs has spurred interest in their use as anti-tumor agents. Currently, peptide mimetics, based on sequences in the type I repeats of TSPs that have been shown to have anti-angiogenic properties, are undergoing clinical testing.