scispace - formally typeset
Search or ask a question

Showing papers on "Angiogenesis published in 1990"


Journal ArticleDOI
Judah Folkman1
TL;DR: Method of treating a wound or burn which comprises directly dressing its surface with non-woven fabric comprising staple fibers of spun, regenerated collagen substantially free of telopeptides is disclosed.

4,637 citations


Journal ArticleDOI
06 Dec 1990-Nature
TL;DR: Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.
Abstract: Neovascularization is critical for the growth of tumours and is a dominant feature in a variety of angiogenic diseases such as diabetic retinopathy, haemangiomas, arthritis and psoriasis. Recognition of the potential therapeutic benefit of controlling unabated capillary growth has led to a search for safe and effective angiogenesis inhibitors. We report here the synthesis of a family of novel inhibitors that are analogues of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius. We first isolated this fungus from a contaminated culture of capillary endothelial cells. Purified fumagillin inhibited endothelial cell proliferation in vitro and tumour-induced angiogenesis in vivo; it also inhibited tumour growth in mice, but prolonged administration was limited because it caused severe weight loss. Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.

1,358 citations


Journal ArticleDOI
TL;DR: A secreted inhibitor of angiogenesis that is controlled by a tumor suppressor gene in hamster cells has been found to be similar to a fragment of the platelet and matrix protein thrombospondin, which demonstrates a function for the ubiquitous adhesive glycoprotein thromBosponin that is likely to be important in the normal physiological down-regulation of neovascularization.
Abstract: A secreted inhibitor of angiogenesis that is controlled by a tumor suppressor gene in hamster cells has been found to be similar to a fragment of the platelet and matrix protein thrombospondin. The two proteins were biochemically similar and immunologically crossreactive and could substitute for one another in two functional assays. Human thrombospondin inhibited neovascularization in vivo and endothelial cell migration in vitro, as does the hamster protein, gp140. gp140 sensitized smooth muscle cells to stimulation by epidermal growth factor, as does human thrombospondin. The thrombospondin gene has been localized on human chromosome 15. These results demonstrate a function for the ubiquitous adhesive glycoprotein thrombospondin that is likely to be important in the normal physiological down-regulation of neovascularization. In addition, they raise the possibility that thrombospondin may be one of a number of target molecules through which a tumor suppressor gene could act to restrain tumor growth.

1,100 citations


Journal ArticleDOI
19 Jul 1990-Nature
TL;DR: Evidence is presented that resident mouse peritoneal mast cells constitutively contain large amounts of TNF-α bioactivity, whereas cultured, immature mast cells vary in their T NF-α content, and that release of TTF-α by mast cells may contribute to host defence, the pathophysiology of allergic diseases and other processes dependent on TFB.
Abstract: Tumour necrosis factor-alpha (TNF-alpha)/cachectin is a multifunctional cytokine that has effects in inflammation, sepsis, lipid and protein metabolism, haematopoiesis, angiogenesis and host resistance to parasites and malignancy. TNF-alpha was first described in activated macrophages, but certain mouse or rat mast cell populations (reviewed in refs 4,5) and some in vitro-derived human cells with cytochemical features of mast cells-basophils may also contain products similar to TNF-alpha. Here we present evidence that resident mouse peritoneal mast cells constitutively contain large amounts of TNF-alpha bioactivity, whereas cultured, immature mast cells vary in their TNF-alpha content. IgE-dependent activation of cultured or peritoneal mast cells induces extracellular release of TNF-alpha and augments levels of TNF-alpha messenger RNA and bioactivity. These findings identify mouse mast cells as an important source of both preformed and immunologically inducible TNF-alpha, and suggest that release of TNF-alpha by mast cells may contribute to host defence, the pathophysiology of allergic diseases and other processes dependent on TNF-alpha.

960 citations


Journal ArticleDOI
05 Jan 1990-Science
TL;DR: Recombinant human platelet factor-4 (rhPF4), purified from Escherichia coli, inhibited blood vessel proliferation in the chicken chorioallantoic membrane in a dose-dependent manner and suggested that the angiostatic effect was due to specific inhibition of growth factor-stimulated endothelial cell proliferation.
Abstract: Recombinant human platelet factor-4 (rhPF4), purified from Escherichia coli, inhibited blood vessel proliferation in the chicken chorioallantoic membrane in a dose-dependent manner. Treatment of several cell types with rhPF4 in vitro suggested that the angiostatic effect was due to specific inhibition of growth factor-stimulated endothelial cell proliferation. The inhibitory activities were associated with the carboxyl-terminal, heparin-binding region of the molecule and could be abrogated by including heparin in the test samples, an indication that sulfated polysaccharides might modulate the angiostatic activity of platelet factor-4 in vivo. Understanding of the mechanisms of control of angiogenesis by endogenous proteins should facilitate the development of effective treatments for diseases of pathogenic neovascularization such as Kaposi's sarcoma, diabetic retinopathy, and malignant tumor growth.

768 citations



Journal Article
TL;DR: It is reported that rings of rat aorta embedded in gels of fibrin or collagen and cultured in MCDB 131, an optimized growth medium for microvascular endothelial cells, generate branching microvessels in the absence of serum or other soluble protein supplements.

641 citations


Journal ArticleDOI
15 Jun 1990-Science
TL;DR: A protein derived from cartilage was purified that inhibits angiogenesis in vivo and capillary endothelial cell proliferation and migration in vitro in three separate bioassays, and may help elucidate the mechanisms by which neovascularization is controlled in both normal and pathological states.
Abstract: Certain tissues such as cartilage are resistant to vascular invasion, yet no single tissue-derived molecule that can inhibit angiogenesis has been reported. A protein derived from cartilage was purified that inhibits angiogenesis in vivo and capillary endothelial cell proliferation and migration in vitro in three separate bioassays. This protein is also an inhibitor of mammalian collagenase. These findings may help elucidate the mechanisms by which neovascularization is controlled in both normal and pathological states.

535 citations


Journal ArticleDOI
TL;DR: The results suggest that FN controls capillary endothelial cell proliferation based on its ability to support tension-dependent alterations of cell shape--i.e., both by binding to cell-surface integrins and by resisting mechanical loads that are applied to these receptors.
Abstract: An in vitro system has been developed to study the mechanism by which fibronectin (FN) regulates capillary endothelial cell growth in the presence of soluble angiogenic mitogens. Endothelial cells were cultured in chemically defined medium containing a constant, saturating amount of basic fibroblast growth factor. Formation of cell-FN contacts was then varied in a controlled fashion by three different techniques: (i) nonadhesive, bacteriological dishes were precoated with increasing densities of FN; (ii) soluble RGD peptides were used to progressively inhibit binding of cell-surface integrin receptors to adsorbed FN; and (iii) FN-coated surfaces were covered with increasingly thick layers of polyhydroxyethylmethacrylate (a nonadhesive polymer) to physically restrict cell access to FN binding sites. Endothelial cells became more extended and proliferated more rapidly as FN coating concentrations were raised from approximately 250 to approximately 10,000 FN molecules per micron 2. Computerized morphometric analysis confirmed that cell shape (projected cell areas) was determined by the density of FN contacts and that DNA synthetic levels were tightly coupled to the extent of cell spreading, regardless of the method used to perturb cell adhesion. In contrast, neither soluble FN nor cell-surface binding of FN-coated microbeads (diameter, 4.5 microns) had any effect on growth when cells were grown in suspension and cell spreading was prohibited. These results suggest that FN controls capillary endothelial cell proliferation based on its ability to support tension-dependent alterations of cell shape--i.e., both by binding to cell-surface integrins and by resisting mechanical loads that are applied to these receptors.

511 citations


Journal ArticleDOI
TL;DR: The data suggest that, in the chicken CAM, TGF beta 1 initiates a sequence of cellular responses that results in growth inhibition, cellular accumulation through migration, and microvascular angiogenesis.
Abstract: Application of TGF beta 1 (10-100 ng) to the chicken chorioallantoic membrane (CAM) for 72 h resulted in a dose-dependent, gross angiogenic response. The vascular effects induced by TGF beta 1 were qualitatively different than those induced by maximal doses of basic FGF (bFGF) (500 ng). While TGF beta 1 induced the formation of large blood vessels by 72 h, bFGF induced primarily small blood vessels. Histologic analysis revealed that TGF beta 1 stimulated pleiotropic cellular responses in the CAM. Increases in fibroblast and epithelial cell density in the area of TGF beta 1 delivery were observed as early as 4 h after TGF beta 1 treatment. By 8 h, these cell types also demonstrated altered morphology and marked inhibition of proliferation as evidenced by 3H-thymidine labeling. Thus, the TGF beta 1-stimulated accumulation of these cell types was the result of cellular chemotaxis from peripheral areas into the area of TGF beta 1 delivery. Microscopic angiogenesis in the form of capillary sprouts and increased endothelial cell density first became evident at 16 h. By 24 h, capillary cords appeared within the mesenchyme of the CAM, extending towards the point of TGF beta 1 delivery. 3H-thymidine labeling revealed that the growth of these capillary cords was due to endothelial cell proliferation. Finally, perivascular mononuclear inflammation did not become evident until 48 h of treatment, and its presence correlated spatially and temporally with the gross and histological remodelling of newly formed capillary cords into larger blood vessels. In summary, these data suggest that, in the chicken CAM, TGF beta 1 initiates a sequence of cellular responses that results in growth inhibition, cellular accumulation through migration, and microvascular angiogenesis.

458 citations


Journal ArticleDOI
TL;DR: 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.
Abstract: 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.

Journal ArticleDOI
10 Aug 1990-Cell
TL;DR: It is suggested that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.

Journal ArticleDOI
TL;DR: The data discussed here suggest that modulation of expression or function of cell-cell adhesive molecules could be critical both to morphogenic changes and to mitogenesis by release of cells from cell- cell contact.
Abstract: This review tries to provide a general, and very speculative, view of growth control mechanisms that may be common to the development of blood vessels and to pathological processes including cell proliferation. From a developmental point of view, vascular growth is most likely to include local autocrine or paracrine mechanisms that permit the two cells of the vessel wall to grow, organize into the characteristic tubular and layered structures of the vessel wall, and eventually achieve a return to quiescence. The "real" mechanisms controlling growth in vivo are difficult to ascertain from studies in culture. For example, a large list of angiogenesis molecules must be able to generate endothelial replication, but in culture many of these molecules are inhibitory for each endothelial replication. Similarly, in culture, we have a long list of smooth muscle mitogens, but none of these have as of yet been proven to control smooth muscle growth in vivo. Endothelial growth control has been attributed to the presence of membrane molecules able to inhibit endothelial replication and to the actions of soluble growth factors and their receptors. Unfortunately for the former hypothesis we still lack specific molecules with the properties of contact inhibition of replication. The data discussed here, however, suggest that modulation of expression or function of cell-cell adhesive molecules could be critical both to morphogenic changes and to mitogenesis by release of cells from cell-cell contact. Moreover, our data and data from other laboratories suggest that angiogenic factors, including the HBGFs and TGF-beta, may function in angiogenesis by altering cell-cell and cell-cell substrate interactions rather than via a primary effect on cell replication. This view of angiogenesis is consistent with the absence of a mitogenic effect of some angiogenic factors. Although endothelial cell replication is obviously necessary to angiogenesis, the lack of mitogenic effect of some factors suggests a need for a more general explanation of the actions of angiogenic factors. Endothelial injury may be interrelated with smooth muscle growth. The simplest possibility is that a failure of the endothelial cell barrier function, due either to denudation or an increase in adhesivity for leukocytes, would permit access of platelets or leukocytes to the vessel wall. These extrinsic cells, in turn, would stimulate smooth muscle cell replication by release of growth factors. The second possibility is that the endothelial cell may itself release growth factors into the vessel wall.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: Thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary and bovine aortic endothelial cells and has the potential to modulate the angiogenic process.
Abstract: Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.

Journal ArticleDOI
TL;DR: It is shown that IL-6 mRNA is produced in vivo in two self-limiting physiologic angiogenic processes: the formation of the vascular system accompanying development of ovarian follicles and theformation of a capillary network in the maternal decidua following embryonic implantation.
Abstract: Interleukin 6 (IL-6) is a cytokine that acts on various cell types. Here we show that IL-6 mRNA is produced in vivo in two self-limiting physiologic angiogenic processes: (i) the formation of the vascular system accompanying development of ovarian follicles and (ii) the formation of a capillary network in the maternal decidua following embryonic implantation. In situ and RNA blot hybridization analyses detected transient expression of IL-6 mRNA in gonadotropin-primed hyperstimulated ovaries, with maximal mRNA levels coinciding with the period of formation of a capillary network around follicles. Expression of IL-6 mRNA was detected in the vasculature extending from the ovarian medulla to the forming capillary sheath in the thecal layer of individual growing follicles. No expression was detected in more-developed preovulatory follicles once angiogenesis had been completed. IL-6 mRNA was also detected in the uterus of pregnant mice 9.5 days postcoitum, and there was no appreciable IL-6 mRNA at later stages of embryonic development. Expression in the uterus was confined to cords of endothelial cells in the process of formation of an anastomosing network that traversed the maternal decidua towards the developing embryo. The expression of IL-6 mRNA in two independent physiological angiogenic processes and the transient nature of its expression in endothelial cells suggest a role for IL-6 in angiogenesis.

Journal ArticleDOI
TL;DR: It is suggested that C GRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.
Abstract: The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.

Journal ArticleDOI
TL;DR: The results demonstrate the expression of VEGF in the CL but not in mural granulosa cells, suggesting a temporal relation between V EGF expression and growth of capillary vessels, and suggest that VegF is involved in the process of CL angiogenesis.
Abstract: In the course of the development of the ovarian follicle and differentiation of granulosa cells into corpus luteum (CL), extensive changes in the microvasculature of these structures take place. This suggests the local release of angiogenic factors. In the present work we examined whether a newly described secreted vascular endothelial growth factor (VEGF) is expressed in normal rat ovary by in situ hybridization. Our results demonstrate the expression of VEGF in the CL but not in mural granulosa cells, suggesting a temporal relation between VEGF expression and growth of capillary vessels. The hybridization pattern in the CL was consistent with localization of VEGF message to luteal cells. Expression of VEGF was detected also in cumulus oophorus cells. These findings suggest that VEGF is involved in the process of CL angiogenesis.

Journal Article
TL;DR: The distribution of basic fibroblast growth factor was studied immunohistochemically in fresh frozen sections of normal human tissues and the presence of bFGF in the extracellular compartment of a diverse variety of organs may play a role in angiogenesis.

Journal ArticleDOI
TL;DR: A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of approximately 1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line, suggesting that this secretable growth factor could be readily available in the extracellular space under normal physiological conditions in vivo.
Abstract: A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of approximately 1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with blood-vessel growth and maintenance

Journal ArticleDOI
TL;DR: The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis.
Abstract: Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of3H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis.

Journal Article
TL;DR: Results suggest that, in conditions associated with vascular proliferation, pericytes acquire HMW-MAA and the number ofpericytes may be increased as compared with normal tissues.
Abstract: In the course of immunohistochemical characterization of murine monoclonal antibodies recognizing the human high molecular weight-melanoma associated antigen (HMW-MAA), a striking reactivity with blood vessels in the tumor stroma was noted. Immunocytochemical analysis by light and electronmicroscopy of a panel of tissues and cell lines showed that the staining of microvessels by anti-HMW-MAA monoclonal antibodies was restricted to pericytes. Correspondingly, anti-HMW-MAA monoclonal antibodies were found to react with cultured pericytes from human brain, but not with endothelial cells in serologic assays, and to immunoprecipitate from biosynthetically labeled pericytes an antigen with the characteristic structural profile of HMW-MAA. At the subcellular level, the expression of HMW-MAA in cultured pericytes was mainly restricted to microspikes that are localized in clusters on the cellular membrane. Staining by anti-HMW-MAA monoclonal antibodies of pericytes was not only found in the tumor stroma, but also in other lesions associated with angiogenesis, such as granulation tissue of wound healing and synovitis. In these lesions, microvascular staining for another marker of pericytes, ie, alpha-smooth muscle actin, also was observed. These results suggest that, in conditions associated with vascular proliferation, 1) pericytes acquire HMW-MAA and 2) the number of pericytes may be increased as compared with normal tissues.

Journal ArticleDOI
TL;DR: It is suggested that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected and may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.
Abstract: Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a soilid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM-resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM producedin vitro and in basement membranes of the cornea and blood vesselsin vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM-immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.

Journal Article
TL;DR: CDPT failed to inhibit tumor growth and the vascularization of the VX2 carcinoma in the thigh muscle or the metastases to the lung, findings that may reflect regional differences in the responsiveness of the endothelium, the distribution of copper, or the activity of cuproenzymes.
Abstract: Microvascular proliferation, a hallmark of malignant brain tumors, represents an attractive target of anticancer research, especially because of the quiescent nonproliferative endothelium of the normal brain. Cerebral neoplasms sequester copper, a trace metal that modulates angiogenesis. Using a rabbit brain tumor model, normocupremic animals developed large vascularized VX2 carcinomas. By contrast, small, circumscribed, relatively avascular tumors were found in the brains of rabbits copper-depleted by diet and penicillamine treatment (CDPT). The CDPT rabbits showed a significant decrease in serum copper, copper staining of tumor cell nuclei, microvascular density, the tumor volume, endothelial cell turnover, and an increase in the vascular permeability (breakdown of the blood-brain barrier), as well as peritumoral brain edema. In non-tumor-bearing animals, CDPT did not alter the vascular permeability or the brain water content. CDPT also inhibited the intracerebral growth of the 9L gliosarcoma in F-344 rats, with a similar increase of the peritumoral vascular permeability and the brain water content. CDPT failed to inhibit tumor growth and the vascularization of the VX2 carcinoma in the thigh muscle or the metastases to the lung, findings that may reflect regional differences in the responsiveness of the endothelium, the distribution of copper, or the activity of cuproenzymes. Metabolic and pharmacologic withdrawal of copper suppresses intracerebral tumor angiogenesis; angiosuppression is a novel biologic response modifier for the in situ control of tumor growth in the brain.

Journal ArticleDOI
TL;DR: It is suggested that a precisely regulated proteolytic balance is necessary for normal capillary morphogenesis, and that when unchecked by protease inhibitors, excessive proteolysis is incompatible with normal capilla morphogenesis.

Journal Article
TL;DR: Findings suggest that CD34 is an endothelial marker for EAM present during angiogenesis, and is preferentially found on a small subset of cells with the morphologic appearance of migrating cells.

Journal ArticleDOI
Werner Risau1
TL;DR: Several factors that stimulate the proliferation of endothelial cells in vitro have been shown to induce angiogenesis in vivo, and the differential expression, release and activation of these factors might regulate ang iogenesis under various physiological and pathological conditions.

Journal ArticleDOI
TL;DR: It is demonstrated that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo, and autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells.
Abstract: Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, we demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth and suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells.

Journal ArticleDOI
TL;DR: The hypothesis that TGF‐β1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube‐like structures with apparent tight junctions and abluminal basal lamina deposition in three‐dimensional cultures is confirmed.
Abstract: Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with anti-ZO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-beta 1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures.

Journal ArticleDOI
TL;DR: In this paper, the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF was described.
Abstract: Acidic and basic fibroblast growth factors (aFGF and bFGF) are angiogenic polypeptide mitogens for cells of mesodermal and neuroectodermal origin. In this report we describe the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF. Further evidence includes (a) prevention of mitogenicity by protamine and by anti-bFGF, (b) displacement of 125I-bFGF from cell membranes, and (c) stimulation of capillary endothelial cell migration. Specific immunohistochemistry localized bFGF to endothelial cells and, surprisingly, to cardiac myocytes, with almost no immunoreactivity in smooth muscle cells. These peptides may function in cardiac embryogenesis, hypertrophy, atherogenesis, angiogenesis, and wound healing, and may also have endocrine, neurotropic, or vasomotor functions.

Journal ArticleDOI
TL;DR: The observed antitumor effects of rHuPF 4 might be due to the inhibition of angiogenesis, which could have implications for the development of novel therapeutic approaches to angiogenic diseases.
Abstract: Although it is well established that angiogenesis is essential to tumor development, no human protein with high specificity and efficacy for prevention of angiogenesis has been characterized. In a previous study, we demonstrated that recombinant platelet factor 4 (rPF 4) inhibited angiogenesis in the chicken chorioallantoic membrane. In the present study, we have extended that finding to the use of recombinant human platelet factor 4 (rHuPF 4) to inhibit solid tumor growth in the mouse. rHuPF 4 effectively suppressed the growth of the B16-F10 murine melanoma in syngeneic C57BL/6J hosts and prevented the growth of primary tumors of both B16-F10 murine melanoma and HCT 116 human colon carcinoma in semisyngeneic CByB6F1/J female athymic nude mice. These two transformed cell lines were completely insensitive to rHuPF 4 in vitro at levels (50 micrograms/mL) that extensively inhibit normal endothelial cell proliferation. The migration of human endothelial cells was also inhibited at these concentrations of rHuPF 4, suggesting a second mechanism by which rHuPF 4 may modulate capillary development. The observed antitumor effects of rHuPF 4 might be due to the inhibition of angiogenesis. This finding could have implications for the development of novel therapeutic approaches to angiogenic diseases. Alternative, and possibly concurrent, mechanisms of the rHuPF 4 antitumor effect include lymphokine-activated killer cell activation and the induction of other cytokines.