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.     

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.     

Regulation of in vitro capillary tube formation by anti-integrin antibodies

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti- integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.     

Suramin inhibits C6 glioma-induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.     

Growth of brain microvessel endothelial cells on collagen gels: Applications to the study of blood-brain barrier physiology and CNS inflammation

Summary Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.     

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.     

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.     

Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.     

Contraction of fibrillar type I collagen by endothelial cells: A study in vitro

The formation of microvascular sprouts during angiogenesis requires that endothelial cells move through an extracellular matrix. Endothelial cells that migrate in vitro generate forces of traction that compress (i.e., contract) and reorganize vicinial extracellular matrix, a process that might be important for angiogenic invasion and morphogenesis in vivo. To study potential relationships between traction and angiogenesis, we have measured the contraction of fibrillar type I collagen gels by endothelial cells in vitro. We found that the capacity of bovine aortic endothelial (BAE) cells to remodel type I collagen was similar to that of human dermal fibroblasts—a cell type that generates high levels of traction. Contraction of collagen by BAE cells was stimulated by fetal bovine serum, human plasma-derived serum, bovine serum albumin, and the angiogenic factors phorbol myristate acetate and basic fibroblast growth factor (bFGF). In contrast, fibronectin and immunoglobulin from bovine serum, several nonserum proteins, and polyvinyl pyrrolidone (a nonproteinaceous substitute for albumin in artificial plasma) were not stimulatory. Contraction of collagen by BAE cells was diminished by an inhibitor of metalloproteinases (1, 10-phenanthroline) at concentrations that were not obviously cytotoxic. Zymography of proteins secreted by BAE cells that had contracted collagen gels revealed matrix metalloproteinase 2. Subconfluent BAE cells that were migratory and proliferating were more effective contractors of collagen than were quiescent, confluent cells of the same strain. Moreover, bovine capillary endothelial cells contracted collagen gels to a greater degree than was seen with BAE cells. Collectively, our observations indicate that traction-driven reorganization of fibrillar type I collagen by endothelial cells is sensitive to different mediators, some of which, e.g., bFGF, are known regulators of angiogenesis in vivo. © 1996 Wiley-Liss, Inc.     

Critical conditions for pattern formation and in vitro tubulogenesis driven by cellular traction fields

In vitro angiogenesis assays have shown that tubulogenesis of endothelial cells within biogels, like collagen or fibrin gels, only appears for a critical range of experimental parameter values. These experiments have enabled us to develop and validate a theoretical model in which mechanical interactions of endothelial cells with extracellular matrix influence both active cell migration--haptotaxis--and cellular traction forces. Depending on the number of cells, cell motility and biogel rheological properties, various 2D endothelial patterns can be generated, from non-connected stripe patterns to fully connected networks, which mimic the spatial organization of capillary structures. The model quantitatively and qualitatively reproduces the range of critical values of cell densities and fibrin concentrations for which these cell networks are experimentally observed. We illustrate how cell motility is associated to the self-enhancement of the local traction fields exerted within the biogel in order to produce a pre-patterning of this matrix and subsequent formation of tubular structures, above critical thresholds corresponding to bifurcation points of the mathematical model. The dynamics of this morphogenetic process is discussed in the light of videomicroscopy time lapse sequences of endothelial cells (EAhy926 line) in fibrin gels. Our modeling approach also explains how the progressive appearance and morphology of the cellular networks are modified by gradients of extracellular matrix thickness.     

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).     

Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: Association with vascular morphogenesis

Summary The growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.     

Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton

Inhibition of angiogenesis could be a treatment strategy for diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. PP2 is a pharmacological inhibitor of Src family kinases and was found to inhibit FGF-2 induced angiogenesis in vivo. Experiments in vitro showed that PP2 inhibited invasive growth and sprouting of both endothelial and vascular smooth muscle cells into a fibrin matrix. PP2 inhibited the formation of lamellopodia and expression of kinase inactive c-Src reduced phosphorylation of cortactin and paxillin, suggesting a model in which Src kinases are involved in organization of the actin cytoskeleton. Consequently, endothelial cells expressing kinase inactive c-Src failed to spread and form cord-like structures on a collagen matrix. These data suggest that pharmacological inactivation of Src family kinases inhibits FGF-2 stimulated angiogenesis by interference with organization of the actin cytoskeleton in both endothelial and vascular smooth muscle cells, which affects cell migration.     

Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix

Regulation of cell migration/invasion is important for embryonic development, immune function, and angiogenesis. However, migratory cells must also coordinately activate survival mechanisms to invade the extracellular matrix and colonize foreign sites in the body. Although invasive cells activate protective programs to survive under diverse and sometimes hostile conditions, the molecular signals that regulate these processes are poorly understood. Evidence is provided that signals that induce cell invasion also promote cell survival by suppressing apoptosis of migratory cells. Extracellular-regulated kinase (ERK) activation and molecular coupling of the adaptor proteins p130 Crk-associated substrate (CAS) and c-CrkII (Crk) represent two distinct pathways that induce cell invasion and protect cells from apoptosis in a three-dimensional collagen matrix. CAS/Crk-mediated cell invasion and survival requires activation of the small GTPase Rac, whereas ERK-induced cell invasion, but not survival requires myosin light chain kinase activation and myosin light chain phosphorylation. Uncoupling CAS from Crk or inhibition of ERK activity prevents migration and induces apoptosis of invasive cells. These findings provide molecular evidence that during invasion of the extracellular matrix, cells coordinately regulate migration and survival mechanisms through ERK activation and CAS/Crk coupling.     

Thrombospondin-1 Modulates Angiogenesisin Vitroby Up-Regulation of Matrix Metalloproteinase-9 in Endothelial Cells

Evidence suggests that thrombospondin-1 (TSP-1), a 450-kDa glycoprotein in platelets and extracellular matrix, is involved in angiogenesis. However, the mechanisms by which TSP-1 regulates angiogenesis are unknown, and the exact role of TSP-1 in angiogenesis has been controversial: both stimulatory and inhibitory effects of TSP-1 have been reported. In this study, we evaluated the effect of TSP-1 on the capacity of bovine aortic endothelial (BAE) cells to both invade and form microvessel-like tubes in collagen gels. BAE cell tube formation was enhanced by exogenous TSP-1 at relatively low concentrations (1–10 μg/ml) but inhibited at higher concentrations of TSP-1 (>15 μg/ml). In addition, we correlated this biphasic effect on tube formation with the capacity of TSP-1 to stimulate the activity of a matrix metalloproteinase-9 (MMP-9) in BAE cell collagen gel cultures. The TSP-1-mediated stimulation of MMP-9 activity was specific and dose- and time-dependent. Furthermore, TSP-1-stimulated BAE cell invasion and tube formation were reversed by antibodies against both TSP-1 and MMP-9, suggesting that TSP-1 modulates endothelial cell invasion and morphogenesisin vitroby a mechanism involving the regulation of MMP-9 activity. These findings support the conclusion that TSP-1 is a multifunctional modulator of angiogenesis and are consistent with the dynamic presence of TSP-1 in remodeling tissues in which matrix degradation is required.     

Collagen processing in ras-transfected mouse mammary epithelial cells

A mouse mammary epithelial cell line (NMuMG), after transfection with the c-rasH oncogene, forms invasive tumors in nude mice. NMuMG and NMuMG/p-rasH cells produce similar amounts of collagen (mostly type IV) when grown on plastic. NMuMG cells respond to growth on collagen gels by increasing the rate of collagen synthesis and deposition by 100%, unlike NMuMG/p-rasH cells which synthesize similar amounts of collagen whether grown on plastic or collagen gels. These results suggest that ras transformation partially inhibits the interaction between epithelial cells and the surrounding stroma that is necessary for basement membrane deposition in vivo and consequently may facilitate the invasion of the stroma by transfected cells.     

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.     

Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices

Endothelial cell invasion is a key step in angiogenic blood vessel formation. Sphingosine-1-phosphate (S1P) has been previously reported to play a role in endothelial cell proliferation, survival, migration, and angiogenesis. Here, we examine the ability of S1P to regulate human endothelial cell invasion into three-dimensional collagen or fibrin matrices. We show that S1P potently stimulated human endothelial cell invasion, lumen formation, and branching morphogenesis in collagen, and fibrin matrices, (5- and 15-fold increases in invasion were observed, respectively). The S1P-induced invasion response was pertussis-toxin sensitive and completely dependent on integrins. Addition of integrin blocking reagents revealed that the alpha2beta1 integrin regulated invasion in collagen matrices, while a combination of alphavbeta3 and alpha5beta1 integrins regulated invasion in fibrin. Additionally, the S1P-induced invasion response was dependent on matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinase-3 (TIMP-3) was the only physiologic inhibitor of metalloproteinases that completely inhibited the potent stimulation of invasion induced by S1P. In contrast, TIMP-1 had no blocking effect on invasion or morphogenesis, while TIMP-2 and TIMP-4 partially reduced invasion but completely blocked lumen formation events. Collectively, these data reveal a marked ability of S1P to induce metalloproteinase- and integrin-dependent human endothelial cell invasion and morphogenesis in both collagen and fibrin three-dimensional matrices, the two most physiologically relevant matrices for angiogenesis.