Isolation of human umbilical vein endothelial cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.     

In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).     

Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells

Angiogenesis is the sprouting of new capillary blood vessels from pre-existing ones. The kinin family of vasoactive peptides, formed by the serine protease tissue kallikrein from its endogenous multifunctional protein substrate kininogen, is believed to regulate the angiogenic process. The aim of this study was to determine the expression of tissue kallikrein and kinin receptors in an in vitro model of angiogenesis. Microvascular endothelial cells from the bovine mature and regressing corpus luteum were used only if they reacted with known endothelial cell markers. At first the cultured endothelial cells began sprouting, and within four weeks formed three-dimensional, capillary-like structures. Immunolabelling for tissue prokallikrein and the mature enzyme was intense in the angiogenic endothelial cells derived from mature corpora lutea. Immunoreactivity was lower in non-angiogenic endothelial cells and least in angiogenic endothelial cultures of the regressing corpus luteum. Additionally, using specific antisense DIG-labelled probes, tissue kallikrein mRNA was demonstrated in cells of the angiogenic phenotype. Immunolabelled kinin B2 receptors, but not kinin B1 receptors, were visualised on angiogenic endothelial cells. Our results suggest an important regulatory role for kinins in the multiple steps of the angiogenic cascade that may occur in wound healing and cancer cell growth.     

Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro

Tightly controlled proteolytic degradation of the extracellular matrix by invading microvascular endothelial cells is believed to be a necessary component of the angiogenic process. We have previously demonstrated the induction of plasminogen activators (PAs) in bovine microvascular endothelial (BME) cells by three agents that induce angiogenesis in vitro: basic FGF (bFGF), PMA, and sodium orthovanadate. Surprisingly, we find that these agents also induce plasminogen activator inhibitor-1 (PAI-1) activity and mRNA in BME cells. We also find that transforming growth factor-beta 1 (TGF-beta 1), which in vitro modulates a number of endothelial cell functions relevant to angiogenesis, also increases both PAI-1 and urokinase-type PA (u-PA) mRNA. Thus, production of both proteases and protease inhibitors is increased by angiogenic agents and TGF-beta 1. However, the kinetics and amplitude of PAI-1 and u-PA mRNA induction by these agents are strikingly different. We have used the ratio of u-PA:PAI-1 mRNA levels as an indicator of proteolytic balance. This ratio is tilted towards enhanced proteolysis in response to bFGF, towards antiproteolysis in response to TGF-beta 1, and is similar to that in untreated cultures when the two agents are added simultaneously. Using an in vitro angiogenesis assay in three-dimensional fibrin gels, we find that TGF-beta 1 inhibits the bFGF-induced formation of tube-like structures, resulting in the formation of solid endothelial cell cords within the superficial parts of the gel. These results suggest that a net positive proteolytic balance is required for capillary lumen formation. A novel perspective is provided on the relationship between extracellular matrix invasion, lumen formation, and net proteolytic balance, thereby reflecting the interplay between angiogenesis-modulating cytokines such as bFGF and TGF-beta 1.     

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.     

Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells

We have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of three-dimensional collagen gels to invade the underlying matrix as capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary-like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel-like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.     

Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors

We have previously shown that the tumor promoter 4 beta-phorbol 12-myristate 13-acetate (PMA) induces capillary endothelial cells grown to confluency on the surface of three-dimensional collagen gels to invade the underlying matrix and to form capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell, 42:469-477, 1985). Since angiogenesis frequently occurs within a fibrin-rich extracellular matrix, we have examined the ability of PMA-treated endothelial cells to invade fibrin gels. Control endothelial cells grown on fibrin gels formed a confluent monolayer on the gel surface and did not invade the underlying matrix. Treatment of the cultures with PMA resulted in a progressive lysis of the substrate without invasion of the fibrin matrix. However, if the cells were treated with PMA either in the presence of fibrinolytic inhibitors (Trasylol, epsilon-aminocaproic acid) or in the absence of detectable plasminogen, dissolution of the substrate was prevented, and the endothelial cells invaded the fibrin gel, forming vessel-like tubular structures similar to those previously observed with collagen gels. These results demonstrate that the invasive and morphogenetic events induced by PMA do not necessarily require an interaction between endothelial cells and collagen fibrils but can also occur with other biologically relevant substrata. They also suggest (1) that invasion may occur via a plasmin-independent mechanism and (2) that in vivo, neutralization of excess proteolytic activity may play an important permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.     

Tumor-promoting phorbol esters induce angiogenesis in vitro

A crucial event during angiogenesis is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. To investigate the possible role of proteases in endothelial cell invasiveness in vitro, bovine microvascular endothelial cells (BMEC) grown on collagen gels were treated with phorbol myristate acetate (PMA), a tumor promoter that markedly increases their production of collagenase and plasminogen activator. Whereas control BMEC were confined to the surface of the gels, PMA-treated BMEC invaded the underlying collagen matrix, where they formed an extensive network of capillary-like tubular structures. This phenomenon, which mimics some of the events occurring during angiogenesis in vivo, required protein synthesis and intercellular contact, was accompanied by collagen degradation, and was prevented by the metalloprotease inhibitor 1,10-phenanthroline.     

In Vitro Angiogenic and Proteolytic Properties of Bovine Lymphatic Endothelial Cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-l, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

Caveolin-1 expression enhances endothelial capillary tubule formation.

The level of caveolin-1 expression closely correlates with the oncogenic transformation of NIH 3T3 cells, the proliferation of human cancer cells, and the differentiation of adipocytes and muscle cells. However, the role of caveolin-1 in endothelial cell proliferation and differentiation remains unknown. Here, we have shown that angiogenic growth factors that stimulate endothelial cell proliferation lead to dramatic reductions in caveolin-1 expression. In addition, using an in vitro Matrigel assay system, we studied the potential role of caveolin-1 in capillary-like tubule formation (i.e. endothelial cell differentiation) using human microvascular endothelial cells (HMEC-1). We showed that the level of endogenous caveolin-1 expression increased in a time-dependent manner when endothelial cells underwent differentiation and that the maximum level of caveolin-1 expression occurred just prior to the formation of capillary-like tubules. Interestingly, overexpression of caveolin-1, via an adenoviral gene delivery system, clearly accelerated endothelial cell differentiation/tubule formation and led to a dramatic approximately 3-fold increase in the number of capillary-like tubular structures. Conversely, down-regulation of caveolin-1 expression, via an antisense adenoviral approach, reduced the number of capillary-like tubules formed by >10-fold. Consistent with the unique function of caveolin-1 in interacting with key signaling molecules, delivery of the caveolin-1 scaffolding domain into the cytoplasm of living endothelial cells was also sufficient to enhance capillary-like tubule formation. Taken together, these results clearly demonstrate that caveolin-1 and the caveolin-1 scaffolding domain play an important positive role in the regulation of endothelial cell differentiation, a prerequisite step in the process of angiogenesis.     

Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix

Transforming growth factor beta (TGF-beta) is angiogenic in vivo. In vitro, endothelial cell proliferation is inhibited by TGF-beta. We have correlated this inhibitory effect with an increase in cellular fibronectin synthesis and deposition in a two-dimensional culture system using specific matrix coatings. The inhibitory effect was mimicked by addition of soluble fibronectin to cultures. In contrast, TGF-beta was found to elicit the formation of tube-like structures (mimicking angiogenesis) when microvascular endothelial cells were grown in three-dimensional collagen gels. In this culture system TGF-beta elicited rapid extensive formation of complex, branching, tube-like structures, while cell proliferation was not inhibited. These data confirm and support the hypothesis that TGF-beta is angiogenic and may exert some of its effects through modulation of matrix synthesis and are consistent with the hypothesis that the organization of the extracellular environment influences cellular responses to this "panregulin."     

Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells

The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in “in vitro” tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.     

Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro

The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized-reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary-like tubes (CLT) formed by endothelial cells was reduced by up to 10-fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP-2 and MMP-9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25-70% of the capillary-like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF-blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast-conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary-like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three-dimensional cell organization.     

Optimized fibrin gel bead assay for the study of angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo. Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel. Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads. After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.     

Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.     

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.     

TNF-related activation-induced cytokine (TRANCE) induces angiogenesis through the activation of Src and phospholipase C (PLC) in human endothelial cells.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.     

Plasmalemmal vacuolar H+-ATPase is decreased in microvascular endothelial cells from a diabetic model

Angiogenesis requires invasion of extracellular matrix (ECM) proteins by endothelial cells and occurs in hypoxic and acidic environments that are not conducive for cell growth and survival. We hypothesize that angiogenic cells must exhibit a unique system to regulate their cytosolic pH in order to cope with these harsh conditions. The plasmalemmal vacuolar type H(+)-ATPase (pmV-ATPase) is used by cells exhibiting an invasive phenotype. Because angiogenesis is impaired in diabetes, we hypothesized that pmV-ATPase is decreased in microvascular endothelial cells from diabetic rats. The in vitro angiogenesis assays demonstrated that endothelial cells were unable to form capillary-like structures in diabetes. The proton fluxes were slower in cells from diabetic than normal model, regardless of the presence or absence of Na(+) and HCO(3) (-) and were suppressed by V-H(+)-ATPase inhibitors. Immunocytochemical data revealed that pmV-ATPases were inconspicuous at the plasma membrane of cells from diabetic whereas in normal cells were prominent. The pmV-ATPase activity was lower in cells from diabetic than normal models. Inhibition of V-H(+)-ATPase suppresses invasion/migration of normal cells, but have minor effects in cells from diabetic models. These novel observations suggest that the angiogenic abnormalities in diabetes involve a decrease in pmV-ATPase in microvascular endothelial cells.     

A novel approach for studying angiogenesis: A human skin equivalent with a capillary-like network

Angiogenesis results from an ordered set of events that can be modulated in vivo by a variety of angiogenesis-enhancing or inhibiting agents. We review in vitro angiogenesis models and the agents that enhance or inhibit angiogenesis. We also discuss a new in vitro angiogenesis model created within a skin equivalent. Briefly, endothelial cells were combined with the cutaneous cells of a standard skin equivalent and cultured in a chitosan cross-linked collagen-glycosaminoglycan scaffold of this endothelialized skin. This model enables the formation of capillary-like structures in a coculture environment containing newly synthesized extracellular matrix by fibroblasts and keratinocytes. Several morphological characteristics associated with the microvasculature in vivo were observed in the endothelialized skin equivalent such as histotypic organization of tubular structures, basement membrane deposition, and intercellular junction formation. This revised version was published online in July 2006 with corrections to the Cover Date.