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.     

Role of c-Fyn in FGF-2-mediated tube-like structure formation by murine brain capillary endothelial cells.

Tube formation of endothelial cells is an important step of angiogenesis. However, little is known about the molecular mechanisms underlying growth factor-mediated tube formation by endothelial cells. FGF-2 stimulates tube formation by a murine brain capillary endothelial cell line, IBE cells, when cultured on collagen gels (differentiation-associated culture condition), whereas cells proliferate and migrate without forming tube on fibronectin-coated surface (proliferation/migration-associated condition). To elucidate FGF-2-mediated signal transduction pathways leading to tube formation by endothelial cells, we focused on the contribution of Src family kinases. Src family kinase inhibitor PP2 attenuated FGF-2-induced tube formation. Stable expression of kinase-inactive c-Src in IBE cells demonstrated no dominant negative effect on FGF-2-induced tube formation. In vitro kinase assay revealed that c-Fyn was activated by FGF-2 only in cells cultured on collagen gels. Three independent cell lines, expressing kinase-inactive c-Fyn, all exhibited attenuation of FGF-2-mediated tube formation. However, FGF-2-mediated proliferation or migration was not clearly perturbed in these cells. These results show the first time that c-Fyn plays a pivotal role in tube formation by endothelial cells.     

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

The p38 mitogen-activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor-dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2-induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2-induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2-induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2-induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2-driven angiogenesis.     

FGF-2 Inhibits Apoptosis in Human Teratocarcinoma Cells during Differentiation on Collagen Substratum

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24–48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop.In vivo,such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.     

Fibroblast growth factor-2 did not restore plasminogen system activity in endothelial cells on glycated collagen

People with diabetes experience morbidity and mortality from unregulated microvascular remodeling, which may be linked to hyperglycemia. Elevated glucose leads to extracellular matrix collagen glycation, which delays endothelial capillary-like tube formation in vitro. Glucose also increases endothelial cell fibroblast growth factor-2 (FGF-2) release and extracellular matrix storage, which should increase tube formation. In this study, we determined if FGF-2 could restore plasminogen system activity and angiogenic function in endothelial cells on glycated collagen. Human umbilical vein endothelial cells cultured on native or glycated collagen substrates were stimulated with FGF-2. Plasminogen system activity, cell migration, and capillary-like tube formation were measured, along with plasminogen system protein and mRNA levels. Glycated collagen decreased endothelial cell plasminogen system activity, cell migration, and tube length. FGF-2 did not restore plasminogen system activity or tube formation in cells on glycated collagen, despite decreasing plasminogen activator inhibitor-1 (PAI-1) protein level. We now show that PAI-1 binds to glycated collagen, which may localize PAI-1 to the extracellular matrix. These data suggest that FGF-2 may not restore angiogenic functions in endothelial cells on glycated collagen due to PAI-1 bound to glycated collagen.     

All-trans retinoic acid suppresses exocrine differentiation and branching morphogenesis in the embryonic pancreas

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.     

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.     

Beta1-integrin orients epithelial polarity via Rac1 and laminin

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.     

Role of the Src homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation.

To study the role of the Src homology 2 (SH2) domain-containing protein Shb in angiogenesis, wild-type Shb and SH2 domain-mutated Shb (R522K Shb) were overexpressed in murine immortalized brain endothelial cells. The wild-type Shb cells exhibited an increased rate of apoptosis on serum withdrawal. Both wild-type Shb and R522K Shb cells exhibited enhanced spreading concomitant with cytoskeletal rearrangements that occurred independently of fibroblast growth factor (FGF)-2 stimulation. However, these effects may partly be caused by altered regulation of Rac1 and Rap1 activation in the Shb cells. The Shb-induced cytoskeletal rearrangements were not dependent on phosphatidylinositol 3' kinase activity, but could be reversed by inhibition of Src family kinases. FGF-2 failed to further enhance migration of wild-type Shb and R522K Shb cells. The R522K Shb cells cultured in collagen gels exhibit diminished tubular morphogenesis when treated with FGF-2, implicating the need for a functional Shb molecule in this process. These data suggest that Shb plays a role in the proliferation and differentiation of endothelial cells and, hence, participates in angiogenesis.     

Fibroblast growth factor receptor-1 signaling in pancreatic islet beta-cells is modulated by the extracellular matrix

Maintenance of pancreatic beta-cell mass depends on extracellular stimuli that promote survival and proliferation. In the islet, these stimuli come from the beta-cell microenvironment and include extracellular matrix deposited by associated vascular endothelial cells. Fibroblast growth factor receptor-1 (FGFR1) has recently been implicated as a signaling pathway that is important for normal beta-cell function. We would like to understand how extracellular matrix and FGFR1 signaling interact to promote beta-cell survival and proliferation. To examine beta-cell-specific receptor responses, we created lentiviral vectors with rat insulin promoter-driven expression of Venus fluorescent protein-tagged full-length (R1betav) and kinase-deficient (KDR1betav) FGFR1. Significant FGF-1-dependent activation of ERK1/2 was observed in betaTC3 cells, dispersed beta-cells, and beta-cells in intact islets. This response was enhanced by R1betav expression and reduced by KDR1betav expression. Plating-dispersed beta-cells on collagen type IV resulted in enhanced expression of endogenous FGFR1 that was associated with sustained activation of ERK1/2. Conversely, plating cells on laminin reduced expression of FGFR1, and this reduction was associated with transient activation of ERK1/2. Addition of neutralizing antibodies to inhibit beta-cell attachment to laminin via alpha(6)-integrin increased high-affinity FGF-1-binding at the plasma membrane and resulted in sustained ERK1/2 activity similar to cells plated on collagen type IV. These data show that the FGF-stimulated beta-cell response is negatively affected by alpha(6)-integrin binding to laminin and suggest regulation associated with vascular endothelial cell remodeling.     

Collagen gel overlay induces apoptosis of polarized cells in cultures: disoriented cell death

In this study, we attempted to investigate the response ofpolarized cells to inappropriate interaction with the extracellular matrix. Cell lines of epithelial [Madin-Darby canine kidney(MDCK) and LLC-PK₁],endothelial [bovine aortic endothelial cells (BAEC)], andmesenchymal (ESK-4 and NIH/3T3) origins were employed. With collagengel overlay, MDCK cells underwent membrane remodeling and graduallydeveloped lumen formation within 24 h. Apoptosis could also be observedfollowing cell remodeling. The ratio of apoptosis was enhanced from12.1 ± 2.4% within 24 h to 58.4 ± 9.8% atday 3, and finally the monolayer wasdisintegrated. Collagen gel overlay-induced apoptosis was not a resultof physical stress, since agarose gel overlay did not induce anymorphological alterations. All epithelial and endothelial cellsexamined developed apoptosis in response to collagen overlay. Incontrast, collagen overlay did not affect growth of fibroblasts at all,although their growth under agarose gel was slightly hindered due tophysical stress. Collagen overlay-induced apoptosis seems to be aunique phenomenon for polarized cells and thus is defined as"disoriented cell death." Furthermore,anti-₂-integrin antibody couldabolish collagen overlay-induced morphological changes and apoptosis inMDCK cells, indicating that signals through₂-integrin on the apicalmembrane are required for disoriented cell death. Finally, Bcl-2overexpression prolonged survival of MDCK cells in response to collagenoverlay, but these cells eventually developed apoptosis due todownregulation of Bcl-2 protein. These findings indicate thatinappropriate cell-matrix interaction results in apoptosis, which mayaccount for cell death mechanisms during developmental processes orunder pathological conditions.

     

Regulation of endothelial cell differentiation and transformation by H-Ras

Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.     

The glycoprotein-processing inhibitors bromoconduritol and N-methyl-1-deoxynojirimycin alter the adhesion phenotype of skeletal myoblasts

Treatment of chick myoblasts with the glucosidase inhibitors bromoconduritol (BCD) or N-methyl-1-deoxynojirimycin (MDJN), but not the mannosidase I inhibitor 1-deoxymannojirimycin (ManDJN), decreased their rate of adhesion to fibronectin and laminin and increased their rate of adhesion to collagen types I and IV. The adhesion of chick myoblasts to fibronectin, collagen type IV, and laminin was predominantly mediated by beta 1-type integrin(s) as judged by inhibition of adhesion with the beta 1-specific monoclonal antibody JG22. Collagen binding in inhibitor-treated cells remained JG22-sensitive suggesting the inhibitors promote increased activity of a beta 1-type collagen-selective integrin. The effects of BCD, MDJN, and ManDJN on myoblast beta 1-integrin detectable at the myoblast cell surface with JG22 antibody correlated well with their effects on adhesion to fibronectin and laminin, and paralleled the previously reported effects of these agents on myogenesis. Interaction of integrin with the extracellular matrix appears to be required for myoblast terminal differentiation. We found that Mn2+ ions increased the adhesion of myoblasts to extracellular matrix proteins and antagonized the effect of BCD and MDJN on myoblast differentiation, supporting a role for cell-matrix interactions in myogenesis. Inhibition of myogenesis by BCD or MDJN was not reversed by growth under low serum conditions, suggesting these agents do not act by maintaining myoblast in a proliferative state.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.