Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma.

Cloned CD4 T cell lines that recognize the Ac1-16 peptide of myelin basic protein bound to I-Au were isolated and used to analyze the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). T helper type 1 (Th1) clones induced disease, while Th2 clones did not. Using variants of a single cloned Th1 line, the surface expression of alpha 4 integrins (very late antigen 4 [VLA-4]) was identified as a major pathogenic factor. Encephalitogenic clones and nonencephalitogenic variants differ by 10-fold in their level of surface expression of alpha 4 integrin and in their ability to bind to endothelial cells and recombinant vascular cell adhesion molecule 1 (VCAM-1). The alpha 4 integrin-high, disease-inducing cloned Th1 T cells enter brain parenchyma in abundance, while alpha 4 integrin-low, nonencephalitogenic Th1 cells do not. Moreover, antibodies to alpha 4 integrin, its ligand VCAM-1, and intercellular adhesion molecule 1 all influence the pathogenicity of this encephalitogenic clone in vivo. The importance of the expression of VLA-4 for encephalitogenicity is not unique to cloned T cell lines, as similar results were obtained using myelin basic protein-primed lymph node T cells. alpha 4 integrin levels did not affect antigen responsiveness or production of the Th1 cytokines interleukin 2, interferon gamma, and lymphotoxin/tumor necrosis factor beta; and antibodies against alpha 4 integrin did not block antigen recognition in vitro. Thus, we conclude that surface expression of alpha 4 integrin is important in CD4 T cell entry into brain parenchyma. A general conclusion of these studies is that alpha 4 integrins may be crucial in allowing activated effector T cells to leave blood and enter the brain and other tissues to clear infections.

Endothelial cells interact with the core protein of basement membrane perlecan through beta 1 and beta 3 integrins: an adhesion modulated by glycosaminoglycan.

Aortic endothelial cells adhere to the core protein of murine perlecan, a heparan sulfate proteoglycan present in endothelial basement membrane. We found that cell adhesion was partially inhibited by beta 1 integrin-specific mAb and almost completely blocked by a mixture of beta 1 and alpha v beta 3 antibodies. Furthermore, adhesion was partially inhibited by a synthetic peptide containing the perlecan domain III sequence LPASFRGDKVTSY (c-RGD) as well as by GRGDSP, but not by GRGESP. Both antibodies contributed to the inhibition of cell adhesion to immobilized c-RGD whereas only beta 1-specific antibody blocked residual cell adhesion to proteoglycan core in the presence of maximally inhibiting concentrations of soluble RGD peptide. A fraction of endothelial surface-labeled detergent lysate bound to a core affinity column and 147-, 116-, and 85-kD proteins were eluted with NaCl and EDTA. Polyclonal anti-beta 1 and anti-beta 3 integrin antibodies immunoprecipitated 116/147 and 85/147 kD surface-labeled complexes, respectively. Cell adhesion to perlecan was low compared to perlecan core, and cell adhesion to core, but not to immobilized c-RGD, was selectively inhibited by soluble heparin and heparan sulfates. This inhibition by heparin was also observed with laminin and fibronectin and, in the case of perlecan, was found to be independent of heparin binding to substrate. These data support the hypothesis that endothelial cells interact with the core protein of perlecan through beta 1 and beta 3 integrins, that this binding is partially RGD-independent, and that this interaction is selectively sensitive to a cell-mediated effect of heparin/heparan sulfates which may act as regulatory ligands.

The induction of 72-kD gelatinase in T cells upon adhesion to endothelial cells is VCAM-1 dependent.

T cell extravasation from the bloodstream into the perivascular tissue during inflammation involves transmigration through the endothelial cell layer and basement membrane into the interstitial matrix. The specific mechanisms by which T cells transmigrate, however, are poorly understood. Matrix degradation by enzymes such as 72-kD gelatinase has been implicated as an important component in tissue invasion by various types of cells. In this study, we have demonstrated that 72-kD gelatinase is induced in T cells upon adhesion to endothelial cells. We also provide evidence that the induction of 72-kD gelatinase is mediated by binding to vascular cell adhesion molecule-1 (VCAM-1). The T cells used in this study were cloned murine Th1 cells antigenic to myelin basic protein. These cells express very late antigen-4 on their cell surface and have been shown to infiltrate the brain parenchyma and cause experimental autoimmune encephalomyelitis when infused into normal mice (Baron, J. L., J. A. Madri, N. H. Ruddle, G. Hashim, and C. A. Janeway. 1993. J. Exp. Med. 177:57-68). In the experiments presented here, T cells were cocultured with VCAM-1-positive and -negative endothelial cells grown in a monolayer in order to study the expression of 72-kD gelatinase upon T cell adhesion. Additional experiments were conducted in which T cells were cocultured with VCAM-1 positive cells grown on microporous membranes suspended in transwells to study 72-kD gelatinase following T cell transmigration. T cells were also incubated with recombinant VCAM-1 in order to study the role of VCAM-1 in inducing 72-kD gelatinase. The results demonstrated that T cells adhered to both VCAM-1-positive and -negative endothelial cells. T cells that adhered to the VCAM-1-positive endothelial cells exhibited an induction in 72-kD gelatinase protein, activity, and mRNA whereas 72-kD gelatinase was not induced in the T cells that adhered to the VCAM-1-negative endothelial cells. Incubating T cells with recombinant VCAM-1 coated onto tissue culture plastic showed that T cells adhered to the molecule and that adhesion to recombinant VCAM-1 was sufficient to induce 72-kD gelatinase. Further, T cells that had transmigrated through a VCAM-1-positive endothelial cell monolayer exhibited 72-kD gelatinase activity but not mRNA expression. In addition, 72-kD gelatinase was detected on the cell surface of the transmigrated T cells by FACS analysis. In other experiments, TIMP-2 was added to the transmigration studies and was shown to reduce T cell transmigration.(ABSTRACT TRUNCATED AT 400 WORDS)

Capillary endothelial cell cultures: phenotypic modulation by matrix components.

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.

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."

Distribution of a 69-kD laminin-binding protein in aortic and microvascular endothelial cells: modulation during cell attachment, spreading, and migration.

Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity column. SDS-PAGE analysis of the eluted proteins identified a 69-kD band as the major binding protein, along with minor components migrating at 125, 110, 92, 85, 75, 55, and 30 kD. Polyclonal antibodies directed against a peptide sequence of the 69-kD laminin-binding protein isolated from human tumor cells identified this protein in BAEC lysates. In frozen sections, these polyclonal antibodies and monoclonal antibodies raised against human tumor 69-kD stained the endothelium of bovine aorta and the medial smooth muscle cells, but not surrounding connective tissue or elastin fibers. When nonpermeabilized BAEC were stained in an in vitro migration assay, there appeared to be apical patches of 69 kD staining in stationary cells. However, when released from contact inhibition, 69 kD was localized to ruffling membranes on cells at the migrating front. Permeabilized BAEC stained for 69 kD diffusely, with a granular perinuclear distribution and in linear arrays throughout the cell. During migration a redistribution from diffuse to predominanately linear arrays that co-distributed with actin microfilaments was noted in double-label experiments. The 69-kD laminin-binding protein colocalized with actin filaments in permeabilized cultured microvascular endothelial cells in a continuous staining pattern at 6 h postplating which redistributed to punctate patches along the length of the filaments at confluence (96 h). In addition, 69 kD co-distribution with laminin could also be demonstrated in cultured subconfluent cells actively synthesizing matrix. Endothelial cells express a 69-kD laminin-binding protein that is membrane associated and appears to colocalize with actin microfilaments. The topological distribution of 69 kD and its cytoskeletal associations can be modulated by the cell during cell migration and growth suggesting that 69 kD may be a candidate for a membrane protein involved in signal transduction from extracellular matrix to cell via cytoskeletal connections.

Codistribution of collagen types IV and AB2 in basement membranes and mesangium of the kidney. an immunoferritin study of ultrathin frozen sections.

Affinity-purified rabbit antibodies specific for collagen types I, III, AB2 and for a partially characterized type IV collagen derived from a murine tumor were used to study the distribution of collagens in the normal mouse kidney. Immunofluorescence staining of conventional frozen sections demonstrated that types I and III were present in bundles around large vessels and in fibers surrounding glomeruli and tubules, whereas types IV and AB2 were distributed in a linear fashion along basement membranes of tubules, glomeruli, and Bowman's capsule and in the mesangial stalk. The distribution of types IV nd AB2 was examined at the ultrastructural level by staining of 600- to 800-A thick frozen sections with a three-stage procedure employing specific collagen antibodies, biotinyl sheep antirabbit IgG, and avidin-ferritin conjugates. Labeling by this procedure demonstrated codistribution of types AB2 and the putative type IV in all three basement membranes. In addition, mesangial matrix was shown to contain both of these collagen types. These results support recent biochemical evidence of collagen heterogeneity in basement membranes, and also support the concept of a structural relationship between mesangial matrix and glomerular basement membranes.

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney.

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.

Multiple mechanisms of dissociated epidermal cell spreading.

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.

Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events.

Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.

Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor.

The modulation of enterocyte sheet migration was studied using Caco-2 cells, a well-differentiated human colonic cell line. Although Caco-2 cells attached and spread equivalently over collagen types I, III, IV, and V and laminin, migration over laminin was significantly slower than migration over the collagen types. Fibronectin was a poor substrate for attachment, spreading, and migration. Epidermal growth factor (EGF) stimulated migration over laminin but did not alter Caco-2 migration over collagen or fibronectin. This effect was independent of cell proliferation, which was stimulated equivalently on both laminin and collagen I. Expression and organization of cell surface receptors for matrix (integrins) were studied using antibodies specific for beta and alpha integrin subunits. Integrin surface expression was assessed by immunoprecipitation of surface 125iodinated control and EGF-treated cells. Beta 1 surface pools did not change substantially in any condition studied. Alpha 1 subunit pools were decreased after EGF treatment on collagen I but alpha 1 pools increased after EGF treatment on laminin. Surface pools of alpha 2 subunits were increased following EGF treatment whether cells were cultured on laminin or collagen I. However, traditional immunofluorescent and laser confocal imaging demonstrated substantial differences in the character of alpha 2 subunit organization between collagen and laminin in the migrating cell front. Furthermore, a functional antibody to the alpha 2 subunit inhibited EGF stimulation of migration over laminin without substantial effects on basal migration over laminin or collagen I. Thus, EGF appears to exert a matrix-specific effect on enterocyte migration by modulation of integrin expression and organization.

Modulation of platelet-derived growth factor receptor expression in microvascular endothelial cells during in vitro angiogenesis.

Microvascular endothelial cells in vivo exhibit a plastic phenotype, forming a nonproliferative, differentiated capillary network, while retaining their ability to respond to injury by proliferation, migration and neovascularization. The presence of PDGF receptors and PDGF responsiveness in microvascular endothelial cells and the significance of PDGF isoforms in the control of endothelial cell growth and differentiation remain controversial. Since culture of microvascular endothelial cells in a three-dimensional (3D) system induced cell differentiation and angiogenesis and inhibited proliferation, the present study investigates the role of different extracellular matrix environments in inducing different microvascular endothelial cell phenotypes on microvascular endothelial cell PDGF receptor expression and PDGF responsiveness. In conventional two-dimensional (2D) culture, microvascular endothelial cells expressed both PDGF receptor alpha and beta chains. Suramin treatment demonstrated continuous downregulation of the alpha receptor surface expression. PDGF BB and, to a lesser extent, PDGF AB were mitogenic in 2D-culture, PDGF AA failed to induce any proliferative response despite inducing receptor autophosphorylation. During in vitro angiogenesis induced by 3D-culture, both PDGF receptors were rapidly downregulated. Assessment of cell proliferation showed quiescent cells and PDGF unresponsiveness. We conclude that the induction of a differentiated phenotype during in vitro angiogenesis (tube formation) driven in part by the spatial organization of the surrounding matrix is associated with a downregulation of PDGF receptors. Identification of the molecular cell-matrix interactions involved in this receptor regulation may allow for targeted manipulation of cell growth in vivo and lead to novel therapeutic applications for PDGF.

Autocrine angiotensin system regulation of bovine aortic endothelial cell migration and plasminogen activator involves modulation of proto-oncogene pp60c-src expression.

Rapid endothelial cell migration and inhibition of thrombosis are critical for the resolution of denudation injuries to the vessel wall. Inhibition of the endothelial cell autocrine angiotensin system, with either the angiotensin-converting enzyme inhibitor lisinopril or the angiotensin II receptor antagonist sar1, ile8-angiotensin II, leads to increased endothelial cell migration and urokinase-like plasminogen activator (u-PA) activity (Bell, L., and J. A. Madri. 1990. Am. J. Pathol. 137:7-12). Inhibition of the autocrine angiotensin system with the converting-enzyme inhibitor or the receptor antagonist also leads to increased expression of the proto-oncogene c-src: pp60c-src mRNA increased 7-11-fold, c-src protein 3-fold, and c-src kinase activity 2-3-fold. Endothelial cell expression of c-src was constitutively elevated after stable infection with a retroviral vector containing the c-src coding sequence. Constitutively increased c-src kinase activity reconstituted the increases in migration and u-PA observed with angiotensin system interruption. Antisera to bovine u-PA blocked the increase in migration associated with increased c-src expression. These data suggest that increases in endothelial cell migration and plasminogen activator after angiotensin system inhibition are at least partially pp60c-src mediated. Elevated c-src expression with angiotensin system inhibition may act to enhance intimal wound closure and to reduce luminal thrombogenicity in vivo.

Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.

Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis and angiogenesis. To investigate the role of nitric oxide (NO) in VEGF-induced proliferation and in vitro angiogenesis, human umbilical vein endothelial cells (HUVEC) were used. VEGF stimulated the growth of HUVEC in an NO-dependent manner. In addition, VEGF promoted the NO-dependent formation of network-like structures in HUVEC cultured in three dimensional (3D) collagen gels. Exposure of cells to VEGF led to a concentration-dependent increase in cGMP levels, an indicator of NO production, that was inhibited by nitro-L-arginine methyl ester. VEGF-stimulated NO production required activation of tyrosine kinases and increases in intracellular calcium, since tyrosine kinase inhibitors and calcium chelators attenuated VEGF-induced NO release. Moreover, two chemically distinct phosphoinositide 3 kinase (PI-3K) inhibitors attenuated NO release after VEGF stimulation. In addition, HUVEC incubated with VEGF for 24 h showed an increase in the amount of endothelial NO synthase (eNOS) protein and the release of NO. In summary, both short- and long-term exposure of human EC to VEGF stimulates the release of biologically active NO. While long-term exposure increases eNOS protein levels, short-term stimulation with VEGF promotes NO release through mechanisms involving tyrosine and PI-3K kinases, suggesting that NO mediates aspects of VEGF signaling required for EC proliferation and organization in vitro.

Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis.

Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1.

Acidic fibroblast growth factor-Pseudomonas exotoxin chimeric protein elicits antiangiogenic effects on endothelial cells.

It has recently been shown that chimeric toxins composed of acidic fibroblast growth factor fused to mutant forms of Pseudomonas exotoxin (aFGF-PE) are cytotoxic to a variety of tumor cell lines with FGF receptors. Although aFGF-PE might be considered as a possible chemotherapeutic toxin, limited knowledge is available concerning its effect on endothelia. This study investigates whether one of the aFGF-PE fusion proteins, aFGF-PE664GluKDEL, can function as an anti-angiogenic agent. Protein synthesis studies using rat epididymal fat pad microvascular endothelial cells (RFCs) indicated that after 24 h in culture, aFGF-PE had a significant inhibitory effect on protein synthesis at concentrations greater than 100 ng/ml. In cultures incubated with 1000 ng/ml aFGF-PE, RFC protein synthesis was inhibited as much as 83%. RFCs were also cultured in a 3-dimensional type I collagen gel and incubated with either transforming growth factor beta 1, aFGF-PE, or a combination of both. Transforming growth factor beta 1 elicits in vitro angiogenesis in these 3-dimensional cultures which consist of rapid formation of complex tubular structures. Transforming growth factor beta 1-treated RFCs incubated with aFGF-PE were unable to produce this angiogenic response, nor were they able to migrate out of the 3-dimensional culture to form a monolayer as shown by controls. Cell viability analyses showed that aFGF-PE produced a dose-dependent toxic effect which ranged from 10 to 90% cell death. Competition assays in which the chimeric toxin was preincubated with antibodies to aFGF resulted in an 89% reversal of the inhibitory effects of aFGF-PE on endothelial cells. Acidic FGF-PE with a mutation in the ADP ribosylation domain of PE was inactive in both 2-dimensional and 3-dimensional cultures. These data show that aFGF-PE has specific in vitro cytotoxic, antiangiogenic, and antimigratory effects on microvascular endothelia.

Glutamine modulates phenotype and stimulates proliferation in human colon cancer cell lines.

Glutamine supplementation has been advocated for patients requiring parenteral nutritional support. However, the direct effect of glutamine on neoplastic cells is poorly understood. We therefore investigated the effects of glutamine on the proliferation, differentiation, and cell-matrix interactions of two human colon carcinoma cell lines (Caco-2 and SW620) adapted to glutamine-free media. Doubling times were calculated by logarithmic transformation of serial cell counts. Alkaline phosphatase, cathepsin C (dipeptidyl peptidase), lactase, and isomaltase expression (markers of differentiation) were assayed by digestion of synthetic substrates. Adhesion to matrix proteins was assessed by colorimetric quantitation of toluidine blue staining of adherent cells. Surface expression of Caco-2 receptors for matrix proteins (integrins) was studied by biotinylation and immunoprecipitation with specific antibodies. Glutamine (1-10 mM) dose-dependently stimulated Caco-2 proliferation on all matrices studied with maximal effect at 7 mM. For instance, Caco-2 doubling time on collagen IV decreased by 57 +/- 0.2% (SE) (P < 0.001). Glutamine inhibited the expression of all four digestive enzymes with maximal inhibition ranging from 10 to 40% (P < 0.05 for all). Adhesion to matrix proteins was markedly diminished (51 +/- 1%, P < 0.01) by glutamine (5 mM) treatment, correlating with decreased alpha 2 and beta 1 integrin subunit surface expression. Glutamine had similar effects on SW620 cells, stimulating proliferation, inhibiting digestive enzyme expression, and diminishing both adhesion and integrin surface expression. Glutamine supplementation modulates the phenotype of at least two human colon carcinoma cell lines, increasing proliferation, decreasing differentiation, and decreasing adhesion to matrix proteins in association with decreased integrin expression. Although the mechanisms of these effects await elucidation, such characteristics would appear to predict more aggressive tumor behavior and raise the possibility that nutritional supplementation with glutamine may be deleterious in patients with cancer.

New paradigms of signaling in the vasculature: ephrins and metalloproteases.

As our understanding of the control of vasculogenesis and angiogenesis continues to grow, we will be confronted with an increasing number of interacting and intersecting receptor-mediated signaling pathways. If we are to be successful in developing new and novel effective therapeutic reagents that can function as stimulators or inhibitors of these critically important processes, we will have to develop a sophisticated, full understanding of the complex interactions associated with ephrin-based and metalloprotease-based signaling pathways.

Extracellular matrix-driven matrix metalloproteinase production in endothelial cells: implications for angiogenesis.

The process of new blood vessel growth, angiogenesis, involves orchestrated alterations in endothelial cell interactions with adjacent cells and with components of the underlying basement membrane matrix. The activity of matrix metalloproteinases (MMPs), proteases that can cleave basement membrane and interstitial matrix molecules, has been shown to be necessary for angiogenesis as it occurs in several different in vivo and in vitro models. This review discusses the potential roles of two particular MMPs, MMP-2 and MT1-MMP, in angiogenesis, with emphasis on current understanding of how endothelial cell-extracellular matrix interactions may regulate the production of these MMPs via matrix-induced signaling leading to transcriptional activation and subsequent formation of active multiprotease complexes on the cell surface.

Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes.

Grafts of allogeneic dermis plus autologous epidermal cell cultures were used to replace extensively burned skin. Cryopreserved split-thickness cadaveric skin was grafted onto debrided burn wound, and autologous keratinocytes were cultured from uninjured donor sites. Several weeks later, allograft epidermis was abraded and replaced with the keratinocyte cultures. The final grafts were thus composites of autologous cultured epidermis and allogeneic dermis. In a case with 28 months follow-up, reconstitution of the dermal-epidermal (BMZ.1) and microvascular (BMZ.2) basement membrane zones was studied immunohistochemically and ultrastructurally. Immediately before grafting, thawed cryopreserved skin reacted with antibodies against laminin and type IV collagen in normal patterns. Twenty-nine days after grafting, BMZ.1 reacted weakly with both antibodies, and anticollagen type IV reactivity was absent from BMZ.2. Antilaminin reactivity of BMZ.2, however, was moderately intense, consistent with recent neovascularization. On day 29, the allograft epidermis was replaced with autologous keratinocyte cultures. Twenty-five days later (54 d after allografting), staining of both BMZs was intense with both antibodies. Ultrastructurally, at day 76 (47 d after culture placement) BMZ.1 revealed only small hemidesmosomes, few incipient anchoring fibrils, and a discontinuous lamina densa. BMZ.2, however, was fully reconstituted. By 124 d, both BMZs appeared normal. Observations in the dermis at 76 d included the presence of lymphocytes, organellar debris, and hyperactive collagen fibrillogenesis, all indicative of dermal remodelling. The microvasculature was well differentiated, but no elastic fibers or nerves were found. In the epidermis, melanocytes and evidence of melanosome transfer were seen at 5, 47, and 95 d after grafting of keratinocyte cultures. We conclude that the composite procedure reconstitutes skin with excellent textural and histologic qualities.