Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1-4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1-4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.     

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.     

Respiratory syncytial virus stimulation of vascular endothelial cell growth Factor/Vascular permeability factor

We hypothesized that respiratory syncytial virus (RSV)-induced pathologies could be mediated, in part, by vascular active cytokines elaborated during virus infection. To address this hypothesis, we determined whether RSV stimulated vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF) elaboration in vitro. Supernatants from unstimulated A549 cells and normal human bronchial epithelial cells contained modest levels of VEGF. In contrast, supernatants from RSV-infected cells contained elevated levels of VEGF/VPF. This stimulation was seen after as little as 2 h, was still prominent after 48 h, and, by immunoblot, was specific for the 165- and 121-amino acid isoforms of VEGF/VPF. It was not associated with significant cell cytotoxicity or alterations in VEGF messenger RNA. It did, however, require new protein biosynthesis. In accordance with these findings, the 165- and 121-amino acid isoforms of VEGF/VPF were also found in the nasal washings from patients with RSV infections. These studies demonstrate that RSV is a potent stimulator of VEGF/VPF elaboration and that, in vitro, this stimulation is mediated via a noncytotoxic translational and/or post-translational biosynthetic mechanism. VEGF/VPF may play an important role in the pathogenesis of RSV-induced disorders.     

Participation of glomerular endothelial cells in the capillary repair of glomerulonephritis.

In many glomerular diseases severe injury to the mesangium may occur, leading to matrix dissolution and damage to the glomerular capillaries. Although the destruction of glomerular architecture may lead to permanent injury, in some cases spontaneous recovery occurs. The mechanisms that mediate this recovery are unknown. In this study we provide evidence for glomerular capillary repair (angiogenesis) in the adult injured glomerulus. Injection of anti-Thy 1 antibody into rats results in severe mesangiolysis with capillary ballooning, microaneurysm formation, and loss of endothelial cells in addition to mesangial cells. Although mesangial proliferation is a major response to injury, proliferation of endothelial cells also can be documented from days 2 to 14 in association with repair of the capillaries. The endothelial cell proliferation peaks on days 2 and 7, when it is seven- to ninefold greater than normal. Many of the endothelial cells display morphological features of angiogenesis. The initial wave of endothelial cell proliferation can be reduced by 40% with neutralizing anti-basic fibroblast growth factor antibodies (P < 0.001). The later glomerular endothelial cell proliferation is associated with upregulated expression of vascular permeability factor/endothelial cell growth factor (VPF/VEGF) and an increase of flk, a VPF/VEGF receptor. Although PDGF is expressed in this model, anti-PDGF antibody treatment did not affect the endothelial cell proliferative response. In summary, glomerular endothelial cells have an active role in the glomerular response to injury. Glomeruli are capable of healing microaneurysms, and the mechanism involves basic fibroblast growth factor- and VPF/VEGF-mediated endothelial proliferative responses.     

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial-cell-specific mitogen; as such, its role in angiogenesis has been studied extensively. VEGF/VPF may also serve as a local, endogenous regulator of large-vessel endothelial cell integrity. Surprisingly, however, VEGF/VPF expression in normal and/or atherosclerotic vessels has not been previously characterized. Accordingly, we studied normal human arteries and veins as well as atherosclerotic and restenotic human coronary arteries for evidence of VEGF/VPF expression. VEGF/VPF was detected immunohistochemically in sections of normal human aorta, mammary artery, and saphenous vein. Moreover, VEGF/ VPF expression was identified in 32 (97%) of 33 pathological coronary arterial specimens; the extent of VEGF/VPF staining was graded as moderate to strong in 21 of the 32 (66%) positive specimens. VEGF/VPF double immunostaining and in situ hybridization demonstrated that smooth muscle cells constitute the principal cellular source of VEGF/VPF. VEGF/VPF immunostaining among primary atherosclerotic lesions localized predominantly to the extracellular matrix. In restenotic specimens, VEGF/VPF immunostaining was more prominently cellular, particularly among proliferating smooth muscle cells. Although VEGF/VPF expression was observed in areas of macrophage infiltration, double immunostaining failed to localize VEGF/VPF to macrophages in these foci; instead, double immunostaining clearly identified CD45RO-positive cells as responsible for VEGF/VPF expression in such areas. No correlation could be demonstrated between VEGF/VPF immunostaining and extent of vasa vasorum. These findings thus establish that postnatal VEGF/VPF expression is a feature of normal human arteries and veins and is often extensively expressed in arteries narrowed by atherosclerotic plaque. VEGF/VPF expression in the wall and/or plaque of medium to large vessels suggests a role for VEGF/VPF other than promoting angiogenesis. This role may involve maintenance and repair of luminal endothelium.     

Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery

Glomeruloid bodies are a defining histological feature of glioblastoma multiforme and some other tumors and vascular malformations. Little is known about their pathogenesis. We injected a nonreplicating adenoviral vector engineered to express vascular permeability factor/vascular endothelial growth factor-164 (VPF/VEGF(164)) into the ears of athymic mice. This vector infected local cells that strongly expressed VPF/VEGF(164) mRNA for 10 to 14 days, after which expression gradually declined. Locally expressed VPF/VEGF(164) induced an early increase in microvascular permeability, leading within 24 hours to edema and deposition of extravascular fibrin; in addition, many pre-existing microvessels enlarged to form thin-walled, pericyte-poor, "mother" vessels. Glomeruloid body precursors were first detected at 3 days as focal accumulations of rapidly proliferating cells in the endothelial lining of mother vessels, immediately adjacent to cells expressing VPF/VEGF(164). Initially, glomeruloid bodies were comprised of endothelial cells but subsequently pericytes and macrophages also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid bodies severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF(164) expression declined, glomeruloid bodies devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid bodies and indicate that VPF/VEGF(164) is sufficient for their induction and necessary for their maintenance.     

Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine that is overexpressed in many tumors, in healing wounds, and in rheumatoid arthritis. VPF/VEGF is thought to induce angiogenesis and accompanying connective tissue stroma in two ways: 1), by increasing microvascular permeability, thereby modifying the extracellular matrix and 2), as an endothelial cell mitogen. VPF/VEGF has been reported in animal corpora lutea and we investigated the possibility that it might be present in human ovaries and have a role in corpus luteum formation. We here report that VPF/VEGF mRNA and protein are expressed by human ovarian granulosa and theca cells late in follicle development and, subsequent to ovulation, by granulosa and theca lutein cells. Therefore, VPF/VEGF is ideally positioned to provoke the increased permeability of thecal blood vessels that occurs shortly before ovulation. VPF/VEGF likely also contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/corpus albicans formation. Finally, VPF/VEGF was overexpressed in the hyperthecotic ovarian stroma of Stein-Leventhal syndrome in which it may also have a pathophysiological role.     

Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors.

The overexpression in tumor cells of (proto)-oncogenic receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or ErbB2/neu (also known as HER-2) is generally thought to contribute to the development of solid tumors primarily through their effects on promoting uncontrolled cell proliferation. However, agents that antagonize the function of the protein products encoded by these (proto)-oncogenes are known to behave in vivo in a cytotoxic-like manner. This implies that such oncogenes may regulate critical cell survival functions, including angiogenesis. The latter could occur as a consequence of regulation of relevant growth factors by such oncogenes. We therefore sought to determine whether EGFR or ErbB2/neu may contribute to tumor angiogenesis by examining their effects on the expression of vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF), one of the most important of all known inducers of tumor angiogenesis. We found that in vitro treatment of EGFR-positive A431 human epidermoid carcinoma cells, which are known to be heavily dependent on VEGF/VPF in vivo as an angiogenesis growth factor, with the C225 anti-EGFR neutralizing antibody caused a dose-dependent inhibition of VEGF protein expression. Prominent suppression of VEGF/VPF expression in vivo, as well as a significant reduction in tumor blood vessel counts, were also observed in established A431 tumors shortly after injection of the antibody as few as four times into nude mice. Transformation of NIH 3T3 fibroblasts with mutant ErbB2/neu, another EGFR-like oncogenic tyrosine kinase, resulted in a significant induction of VEGF/VPF, and the magnitude of this effect was further elevated by hypoxia. Moreover, treatment of ErbB2/neu-positive SKBR-3 human breast cancer cells in vitro with a specific neutralizing anti-ErbB2/neu monoclonal antibody (4D5) resulted in a dose-dependent reduction of VEGF/VPF protein expression. Taken together, the results suggest that oncogenic properties of EGFR and ErbB2/neu may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors such as VEGF/VPF. These genetic changes may cooperate with epigenetic/environmental effects such as hypoxia to maximally stimulate VEGF/VPF expression. Therapeutic disruption of EGFR or ErbB2/neu protein function in vivo may therefore result in partial suppression of angiogenesis, a feature that could enhance the therapeutic index of such agents in vivo and endow them with anti-tumor effects, the magnitude of which may be out of proportion with their observed cytostatic effects in monolayer tissue culture.     

Secretion of vascular endothelial growth factor/vascular permeability factor from human luteinized granulosa cells is human chorionic gonadotrophin dependent

Vascularization is a prominent event during corpus luteum formation, providing low density lipoproteins for steroid biosynthesis and enabling transport of secreted steroids. The process of vascularization is controlled by specific regulators. Vascular endothelial growth factor (VEGF), otherwise named vascular permeability factor (VPF), induces endothelial cell proliferation as well as angiogenesis in vivo and increases capillary permeability. Here we report the expression of VEGF/VPF mRNA by cultured human luteinized granulosa cells (GC) for at least 10 days. Without HCG VEGF/VPF expression declined after day 4 and by day 10 was reduced to approximately 30% of the value at day 4. However, after culture in the presence of 1 U/ml human chorionic gonadotrophin (HCG), expression of VEGF/VPF mRNA by GC was four times greater than control experiments by day 10, and increased 100% from day 4 to day 10. Simultaneously, HCG supplementation increased VEGF/VPF secretion by GC. Medium VEGF/VPF on day 3 was 13 pM without and 11 pM with HCG. Medium VEGF/VPF on day 10 was 6 pM without HCG and 29 pM with HCG. These results suggest that vascularization of the corpus luteum is induced by HCG-mediated effects of VEGF/VPF.      

The contribution of Harold F. Dvorak to the study of tumor angiogenesis and stroma generation mechanisms.

In 1983, Harold Dvorak and his colleagues were the first to show that tumor cells secreted vascular permeability factor (VPF) and that a blocking antibody to VPF could prevent the edema and fluid accumulation that is characteristic of human cancers. In 1986, Dvorak went on to demonstrate that VPF was secreted by a variety of human tumor cell lines and proposed that VPF was in part responsible for the abnormal vasculature seen in human tumors. As a result, he and other investigators demonstrated that VPF was capable of stimulating endothelial cell growth and angiogenesis. These fundamental discoveries led to additional research conducted by Napoleone Ferrara and his laboratory, confirming the cloning of VPF and renaming the protein vascular endothelial growth factor (VEGF). In 1986, Dvorak proposed that by secreting VPF, tumors induce angiogenesis by turning on the wound healing response. He noted that wounds, like tumors, secrete VPF, causing blood vessels to leak plasma fibrinogen, which stimulates blood vessel growth and provides a matrix on which they can spread. Unlike wounds, however, that turn off VPF production after healing, tumors did not turn off their VPF production and instead continued to make large amounts of VPF, allowing malignant cells to continue to induce new blood vessels and so to grow and spread. Thus, tumors behave like wounds that fail to heal. This work is again extremely significant for patients worldwide, as Dvorak's scientific research is leading his colleagues all over the world to examine how to treat a tumor through its blood supply.     

Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin.

We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and beta3 integrin subunits resulting in increased levels of the alphavbeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavbeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavbeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavbeta3 integrin, the alphavbeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.     

血管内皮生长因子在毛囊生物学中的研究现状

血管内皮生长因子(vascular endothelial growth factor,VEGF)又名血管通透性因子(vascular permeability factor,VPF),是重要的血管生成正性调节因子。作为毛乳头细胞的一种自分泌生长因子,其对毛囊的生长亦有重要作用。血管内皮生长因子不仅能促进毛囊的生长,还参与毛囊生长周期的调控。在内皮细胞中,血管内皮生长因子发挥作用主要是通过与其受体的结合,诱导受体二聚体化和自身磷酸化,从而激活胞内信号转导通路,但在毛囊细胞中是否如此,仍需进一步研究。     

Expression of vascular endothelial growth factor by human renal cancer cells enhances angiogenesis of primary tumors and production of ascites but not metastasis to the lungs in nude mice

We determined the role that vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), plays in the progression of human renal cell cancer in nude mice. Low metastatic and low VEGF/VPF-expressing human renal cancer cells SN12C were transfected with the VEGF165 cDNA or plasmid alone as control. VEGF165-transfected SN12C cells produced large amounts of biologically active VEGF in culture that did not affect cell doubling time or confluence. Subsequent to implantation into the renal subcapsule of nude mice, the VEGF165-transfected SN12C cells produced fast-growing (PCNA labeling), large tumors that expressed high levels of VEGF/VPF and were well vascularized (CD3-positive vessels). The tumors produced hyperpermeability of peritoneal blood vessels (Evans blue dye-leak assay), bloody ascites, and short survival time. Parental or control transfected SN12C cells produced less vascularized, slower growing tumors with no ascites. Regardless of in vivo expression level of VEGF, the incidence of spontaneous lung metastasis was low, suggesting that in itself, the expression of VEGF/VPF by renal cancer cells is not sufficient to produce metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.     

Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.     

An endothelial cell growth factor from the mouse neuroblastoma cell line NB41

A growth factor that stimulates the proliferation of endothelial cells from human umbilical vein but is not mitogenic for fibroblastic cells is present in medium conditioned by the mouse neuroblastoma cell line NB41. In a partially purified preparation, factor activity coeluted from a reverse-phase high-pressure liquid chromatography (HPLC) column with a reduced protein of about 24 kd. Activity recovered following electrophoresis of HPLC fractions corresponded to protein of 43-51 kd in the absence of reducing agent and to protein of 23-29 kd after reduction. Antiserum raised against a peptide corresponding to the putative N-terminal amino acid sequence of the 24-kd protein reacted with the 24-kd protein and with a protein of about 47 kd in the nonreduced preparation. After N-glycanase treatment, the immunoreactive 24-kd protein had a mobility corresponding to 19 kd. We infer that the native NB41 factor is a glycosylated dimer whose biochemical and biological properties distinguish if from other endothelial cell growth factors.     

Quantification of tumour-induced angiogenesis by image analysis

Quantitative techniques for in vivo and in vitro angiogenesis were developed using an image analyser. In the in vivo study, a Millipore chamber filled with mouse sarcoma 180 (S180) cells was transplanted subcutaneously to the dorsal side of a mouse, and the area of neovascularization induced by the tumour cells was quantified by image analysis. Images of vascular networks with poor contrast had their contrast improved by Laplacean transformation. The area of vascular network was 16.9 mm² in the control group without tumour cells and 44.2 mm² in the group with tumour cells, demonstrating a significant increase in neovascularized area by tumour cells. In the in vitro study, migration of vascular endothelial cells was induced with conditioned media of S180 cells. Image analysis was used to count automatically the nuclei of migrated endothelial cells, which were stained violet with Giemsa's solution. This automated measurement by image analyser is expected to save labour and time. Checkerboard analysis revealed that the endothelial cell migration induced by S180-conditioned medium was due to chemotaxis. The quantitation method using an automated image analyser is valuable in evaluating the induction of neovascularization by tumours and the effect of pharmacological agents on tumour angiogenesis in vivo and in vitro     

Vascular endothelial growth factor and basic fibroblast growth factor present in Kaposi's sarcoma (KS) are induced by inflammatory cytokines and synergize to promote vascular permeability and KS lesion development.

All forms of Kaposi''s sarcoma (KS) are characterized by spindle cell proliferation, angiogenesis, inflammatory cell infiltration, and edema. We have previously reported that spindle cells of primary KS lesions and KS-derived spindle cell cultures express high levels of basic fibroblast growth factor (bFGF), which is promoted by the inflammatory cytokines identified in these lesions. These cytokines, namely, tumor necrosis factor, interleukin-1, and interferon-gamma, induce production and release of bFGF, which stimulates angiogenesis and spindle cell growth in an autocrine fashion. Here we show that both AIDS-KS and classical KS lesions co-express vascular endothelial growth factor (VEGF) and bFGF. VEGF production by KS cells is promoted synergistically by inflammatory cytokines present in conditioned media from activated T cells and in KS lesions. KS cells show synthesis of VEGF isoforms that are mitogenic to endothelial cells but not to KS spindle cells, suggesting a prevailing paracrine effect of this cytokine. This may be due to the level of expression of the flt-1-VEGF receptor that is down-regulated in KS cells as compared with endothelial cells. KS-derived bFGF and VEGF synergize in inducing endothelial cell growth as shown by studies using both neutralizing antibodies and antisense oligodeoxynucleotides directed against these cytokines. In addition, VEGF and bFGF synergize to induce angiogenic KS-like lesions in nude mice and vascular permeability and edema in guinea pigs. These results indicate that inflammatory cytokines present in KS lesions stimulate the production of bFGF and VEGF, which, in turn, cooperate to induce angiogenesis, edema, and KS lesion formation.     

VEGF/Flk-1 interaction, a requirement for malignant ascites recurrence.

Vascular endothelial growth factor (VEGF) plays an important role in the production of ascitic fluid associated with malignant tumor growth. In an experimental model for malignant ascites formation, mice were inoculated intraperitoneally with syngeneic mouse sarcoma tumor cells. Ascites development was not prevented by administering tumor necrosis factor (TNF) simultaneously with the tumor cell inoculation. When the malignant ascites was first drained and renewal of ascites was monitored, however, a TNF dose-dependent inhibition of ascitic fluid accumulation was observed. Northern blot analyses indicated transient downregulation by TNF on the expression of VEGF mRNA in tumor cells. Monoclonal antibody, (mAb) DC101 generated against the mouse VEGF receptor Flk-1 prevented the recurrence of malignant ascites in mice similar to TNF inhibition. In addition, exogenous soluble human Flt-1 used as an inhibitor of endogenous VEGF binding also inhibited ascites recurrence. These data demonstrate that the observed inhibitory effect of TNF on reestablishment of malignant ascites can be achieved equally by inhibition of the interaction of VEGF with its receptor Flk-1.