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.

Summary Cloned CD4 T cell lines that recognize the Ac1-16 peptide of myelin basic protein bound to I-A" were isolated and used to analyze the immunopathogeuesis of experimental autoimmune encephalomyelitis (EAE). T helper type 1 (Thl) clones induced disease, while Th2 clones did not. Using variants of a single cloned Thl line, the surface expression of o~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 a4 integrin and in their ability to bind to endothelial cells and recombinant vascular cell adhesion molecule 1 (VCAM-1). The cx4 integrin-high, disease-indudng cloned Thl T cells enter brain parenchyma in abundance, while ~x4 integrin-low, nonencephalitogeuic Thl cells do not. Moreover, antibodies to o~4 integrin, its ligand VCAM-1, and intercellular adhesion molecule 1 all influence the pathogenicity of this eucephalitogenic 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, o~4 integrin levels did not affect antigen responsiveness or production of the Thl cytokines interleukin 2, interferon 7, and lymphotoxin/tumor necrosis factor fl; and antibodies against o~4 integrin did not block antigen recognition in vitro. Thus, we conclude that surface expression of oe4 integrin is important in CD4 T cell entry into brain parenchyma. A general conclusion of these studies is that or4 integrins may be crucial in allowing activated effector T cells to leave blood and enter the brain and other tissues to clear infections.

No abstract

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.Capillary endothelial cells in vivo are surrounded by and rest on basement membranes composed of several extracellular matrix components (10,24,28,37). These cells are normally quiescent and are constrained by their investing basement membranes. Following injury or in response to other stimuli these cells somehow escape the constraints of the basement membrane, undergo several phenotypic changes, migrate through and proliferate in the interstitium, and ultimately form a new capillary network complete with investing basement membranes (2). Thus, throughout the reparative process capillary endothelial cells are in intimate contact with matrix components. Therefore it is not unreasonable to suggest that the matrix composition and possibly organization in the microenvironment surrounding such cells may play important roles in directing the dynamic responses of capillary endothelial cells following injury.

Angiogenesis, which is the formation of new blood vessels from those pre-existing, occurs during development, wound healing, and tumor growth, and in response to stimuli such as exercise and hypoxia (1-3). This process involves complex signaling events that cause the endothelial cells comprising capillaries to initiate proliferative and migratory phenotypes. The sprouting endothelial cells must break through their existing basement membrane and form contacts with and migrate along different extracellular matrix components, finally establishing a new, patent capillary (1, 2).In an effort to define the molecular mechanisms underlying these events, a number of in vitro cell systems have been established. These include growth of endothelial cells or blood vessel fragments in fibrin clots, on amnionic membranes, in collagen matrices, and on Matrigel ® matrices (4-6). These models are characterized by re-organization that requires significant endothelial cell migration and/or invasion, and remodeling of the surrounding matrix molecules. It has been shown that microvascular endothelial cells undergo morphological changes that can include organization into tubelike structures when grown within a type I collagen matrix (7,8). The morphological changes are accompanied by changes in growth factor receptor profiles and extracellular matrix protein production (9, 10).Matrix metalloproteinases (MMPs) 1 belong to a family of enzymes with diverse substrate specificity, ranging from multiple extracellular matrix components to growth factors, cytokines, and other proteinases (11,12). It was first recognized that matrix metalloproteinases play a role in angiogenesis, based on the observation that inhibition of MMP activity by endogenous tissue inhibitors of metalloproteinases or synthetic compounds could inhibit in vitro tube formation (13,14). Several studies have demonstrated that the gelatinases, MMP-2 and MMP-9, are involved in vascular cell migration and invasion assays (15, 16). However, the regulation of MMP expression and activation during angiogenic events is not well understood. The recent cloning of several membrane-type MMPs (MT-MMPs) (17-19) that each contain a putative transmembrane domain and appear to have substrate specificity for the pro-MMP-2 has led to considerable speculation concerning the role of the cell surface in regulating proteolytic activity, and the extent to which MT-MMPs may be involved in controlling proteolytic cascades involving .We hypothesized that the controlled activation of metalloproteinases would be necessary for the in vitro remodeling of endothelial cells cultured in a collagen matrix, and that this activation would require the transcriptional up-regulation of specific MMPs, including MT-MMPs. Utilizing primary cultures of rat microvascular endothelial cells, we demonstrated a coordinate increase in expression of MMP-2 and MT1-MMP (MMP-14) following culture within a type I collagen matrix, and that activation of MMP-2 correlated temporally with MT1-MMP induction. MMP inhibitors blocked t...

The primary graft-related complication during the first clinical trial evaluating the use of tissue-engineered vascular grafts (TEVGs) was stenosis. We investigated the role of macrophages in the formation of TEVG stenosis in a murine model. We analyzed the natural history of TEVG macrophage infiltration at critical time points and evaluated the role of cell seeding on neovessel formation. To assess the function of infiltrating macrophages, we implanted TEVGs into mice that had been macrophage depleted using clodronate liposomes. To confirm this, we used a CD11b-diphtheria toxin-receptor (DTR) transgenic mouse model. Monocytes infiltrated the scaffold within the first few days and initially transformed into M1 macrophages. As the scaffold degraded, the macrophage infiltrate disappeared. Cell seeding decreased the incidence of stenosis (32% seeded, 64% unseeded, P=0.024) and the degree of macrophage infiltration at 2 wk. Unseeded TEVGs demonstrated conversion from M1 to M2 phenotype, whereas seeded grafts did not. Clodronate and DTR inhibited macrophage infiltration and decreased stenosis but blocked formation of vascular neotissue, evidenced by the absence of endothelial and smooth muscle cells and collagen. These findings suggest that macrophage infiltration is critical for neovessel formation and provides a strategy for predicting, detecting, and inhibiting stenosis in TEVGs.

Abstract. Transforming growth factor beta (TGF-13) is angiogenic in vivo. In vitro, endothelial cell proliferation is inhibited by TGF-13. 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-13 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-[3 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-13 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" GIOGENESIS (the formation of new blood vessels from endothelial cells) is a complex process involving endothelial cell activation (including synthesis and secretion of proteases), migration, and proliferation, as well as matrix synthesis, differentiation, multicellular organization, stabilization, and capillary endothelial cell regression of local endothelial cell populations during healing and repair (Furcht, 1986;. Previously, we have demonstrated the importance of extracellular matrix (ECM) t composition and organization (laminin and type IV collagen) in the regulation of endothelial cell migration, proliferation, and multicellular organization during angiogenesis (Madri and Williams, 1983;Pratt et al., 1984Pratt et al., , 1985Form et al., 1986). Although ECM appears to be an important modulator of angiogenesis, it is but one element in a complex control mechanism which also makes use of various soluble factors including platelet-derived growth factor and transforming growth factor beta (TGF-[3). Recently, TGF-[3 (a 25-kD homodimer that has been shown to control cell growth and differentiation) has been noted to elicit a striking angiogenic response in mice as well as to dramatically affect synthesis in vivo and in vitro . Ignotz and Massague (1986) have demonstrated a functional involvement of fibronectin in mediating some cellular responses to TGF-13 in fibroblasts and have suggested a model for TGF-13 action based on the modulation of ECM in the target cell. Since TGF-13 is present in platelets and inflammatory cells it is reasonable to postulate that this protein may function as 1. Abbreviations used in this paper: ECM, extracellular matrix; Fn, fibronectin; Ln, laminin; TGF-[3, transforming growth factor beta; IV, type IV collagen; V, type V collagen. a paracrine mediator of the repair process, affecting local endothelial cell behavior by modulating matrix synthesis and degradation in the local cell populations in the injured area Saksela et al., 1987). In this report we show that in two-dimensi...

Activated components of the complement system are potent mediators of inflammation that may play an important role in numerous disease states. For example, they have been implicated in the pathogenesis of inflammatory joint diseases including rheumatoid arthritis (RA). To target complement activation in immune-mediated joint inflammation, we have utilized monoclonal antibodies (mAbs) that inhibit the complement cascade at C5, blocking the generation of the major chemotactic and proinflammatory factors CSa and C5b-9. In this study, we demonstrate the efficacy of a mAb specific for murine C5 in the treatment of collagen-induced arthritis, an animal model for RA. We show that systemic administration of the anti-C5 mAb effectively inhibits terminal complement activation in vivo and prevents the onset of arthritis in immunized animals. Most important, anti-C5 mAb treatment is also highly effective in ameliorating established disease. These results demonstrate a critical role for activated terminal complement components not only in the induction but also in the progression of collagen-induced arthritis and suggest that C5 may be an attractive therapeutic target in RA.