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Angiogenesis is necessary for solid tumor growth and dissemination. In addition to angiogenesis, it has become increasingly clear that inflammation is a key component in cancer insurgence that can promote tumor angiogenesis. We noted that angiogenesis is a common and key target of most chemopreventive molecules, where they most likely suppress the angiogenic switch in premalignant tumors, a concept we termed angioprevention. We have shown that various molecules, such as flavonoids, antioxidants, and retinoids, act in the tumor microenvironment, inhibiting the recruitment and/or activation of endothelial cells and phagocytes of the innate immunity. N-acetyl-cysteine, and the green tea flavonoid epigallocatechin-3-gallate (EGCG) and the beer/ hops-derived chalcone Xanthohumol all prevent angiogenesis in the Matrigel sponge angiogenic assay in vivo and inhibit the growth of the highly angiogenic Kaposi's sarcoma tumor cells (KS-Imm) in nude mice. The synthetic retinoid 4-hydroxyfenretinide (4HPR) also shows anti-angiogenic effects. We analyzed the regulation of gene expression they exert in primary human umbilical endothelial cells (HUVEC) in culture with functional genomics. Expression profiles obtained through Affymetrix GeneChip arrays identified overlapping sets of genes regulated by anti-oxidants. In contrast, the ROS-producing 4HPR induced members of the TGFbeta-ligand superfamily, which, at least in part, explains its anti-angiogenic activity. NAC and the flavonoids all suppressed the IkB/NF-kappaB signaling pathway even in the presence of NF-kappaB stimulation by TNFalpha, and showed reduced expression of many NF-kappaB target genes. A selective apoptotic effect on transformed cells, but not on endothelial cells, of the anti-oxidants may be related to the reduced expression of the NF-kappaB-dependent survival factors Bcl2 and Birc5/surviving, which are selectively overexpressed in transformed cells by these factors. The repression of the NF-kappaB pathway suggests anti-inflammatory effects for the antioxidant compounds that may also represent an indirect role in angiogenesis inhibition. The green tea flavonoid EGCG does target inflammatory cells, mostly neutrophils, and inhibits inflammation-associated angiogenesis. The other angiopreventive molecules are turning out to be effective modulators of phagocyte recruitment and activation, further linking inflammation and vascularization to tumor onset and progression and providing a key target for cancer prevention.

The intricate interplay between the endothelium and immune cells has been well recognized in the context of immune responses. However, the fact that this inter-relation extends well beyond immune regulation is becoming increasingly recognized, with particular regards to the influence of the immune system on the essential endothelial process of angiogenesis, where the contribution of cytokines drives the angiogenic process. As angiogenesis is an important component of numerous pathological states, among these chronic inflammatory conditions and cancer, understanding the role of cytokines and chemokines in guiding new vessel formation provides key insight into novel therapeutic modalities. Here we review the actions of principal cytokines and chemokines on the angiogenic process and discuss how both can be considered potential pharmaceutical targets or pharmaceuticals themselves for modulation of angiogenesis in chronic inflammation associated with cancer, rheumatoid arthritis and other inflammatory diseases.

The importance of angiogenesis for the growth of tumors is widely recognized. Drugs that successfully target the endothelium, such as antivascular endothelial growth factor antibodies, are beginning to have an effect on the life expectancy of cancer patients. However, the endothelial cell is not the only possible target for antiangiogenic therapy or prevention of vascularization (angioprevention). It is evident from the literature that native immune cells recruited into tumors in turn stimulate the endothelium and are responsible for an indirect pathway of tumor vascularization. Inflammation-dependent angiogenesis seems to be a central force in tumor growth and expansion, a concept supported by the observation that the use of "classic" anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs, leads to angiogenesis inhibition. The mechanisms of inflammatory angiogenesis provide new approaches to target, cure, or even better, prevent tumor angiogenesis by treatment with synthetic or natural agents with anti-inflammatory properties. We propose chemoprevention of inflammatory angiogenesis as a way of checking the cancer before it progresses.

Purpose: Green tea consumption has been linked to a reduced occurrence of some tumor types. Current data indicate that the principal mediator of this chemopreventive effect is epigallocatechin-3-gallate (EGCG), the most abundant polyphenol found in dried tea leaves. Here, we examined the effects of this compound on the two key cell populations typically involved in tumor growth: tumor cells and endothelial cells., Experimental Design: The effects of green tea and EGCG were tested in a highly vascular Kaposi's sarcoma (KS) tumor model and on endothelial cells in a panel of in vivo and in vitro assays., Results: EGCG inhibited KS-IMM cell growth and endothelial cell growth, chemotaxis, and invasion over a range of doses; high concentrations also induced tumor cell apoptosis. EGCG inhibited the metalloprotease-mediated gelatinolytic activity produced by endothelial cell supernatants and the formation of new capillary-like structures in vitro. Green tea or purified EGCG when administered to mice in the drinking water inhibited angiogenesis in vivo in the Matrigel sponge model and restrained KS tumor growth. Histological analysis of the tumors were consistent with an anti-angiogenic activity of EGCG and green tea., Conclusions: These data suggest that the green tea gallate or its derivatives may find use in the prevention and treatment of vascular tumors in a chemoprevention or adjuvant setting.

The HIV-Tat protein can be released extracellularly where it is able to recruit leukocytes and induce angiogenesis. These activities are mediated by the direct interaction of Tat with VEGFR2 on endothelial cells and chemokine receptors on leukocytes. We have recently shown that angiostatin, an anti-angiogenic peptide fragment of plasminogen, is able to inhibit the recruitment of neutrophils induced by bacterial fMLP and alpha chemokines both in vitro and in vivo. In vivo this was associated with an inhibition of the angiogenic response by angiostatin. These observations suggested that angiostatin could be a suitable inhibitor of Tat-induced angiogenesis, as it acts on both endothelial and neutrophil at the same time. In vitro, chemotaxis assays demonstrated that angiostatin inhibited Tat-induced chemotaxis of neutrophils with an inverse bell shaped profile. In vivo the injection of matrigel plugs containing Tat or its chemokine-like peptide (CysL24-51) caused the infiltration of neutrophils and a strong angiogenic response. Angiostatin completely blocked this inflammatory response, inhibiting the recruitment of inflammatory and endothelial cells inside the implant. Taken together, these results indicate that angiostatin can act as an inhibitor of both endothelial and neutrophil recruitment. As these cell types are also the targets of extracellularly released Tat, angiostatin could be used to contrast Tat-associated vasculopathies.

Kaposi's sarcoma is a highly angiogenic, AIDS-associated neoplasm that is more frequent in male than in female patients. Cases of spontaneous regression during pregnancy have been reported and the pregnancy hormone human chorionic gonadotropin (hCG) has shown anti-Kaposi's sarcoma activity in several, but not all, clinical trials. Antiproliferative and proapoptotic activities specific for Kaposi's sarcoma (KS) cells have been shown. We report here further analyses of the anti-KS activity of the hormone and show that urinary hCG, the hCG beta-subunit, the hCG beta-core, and to a lesser extent a recombinant hCG, directly inhibit the activity of matrix metalloproteases of different origin. The hCG hormone also inhibited angiogenesis in vivo in the matrigel sponge assay as well as growth of KS cell xenografts in nude mice. The effect of the pure recombinant hormone dimer on xenograft growth was transient, indicating that the activity of intact hCG alone is not sufficient to overcome the growth potential of this tumor and suggesting that active hCG fragments or other anti-KS activities contribute to the activity of urinary hCG.

We developed an in vivo gene therapy approach to characterize and optimize the anti-angiogenic activity of class I interferons (IFNs), using packaging cell lines producing an amphotropic LXSN-based retrovirus expressing either IFN-alpha1 (alpha1Am12), IFN-beta (betaAm12) murine cDNAs, or the vector alone (neoAm12). Pretreatment of endothelial-like Eahy926 cells in vitro with conditioned media (CM) from alpha1Am12 or betaAm12 cells for 48 hours significantly inhibited their migration and invasion as compared to neoAm12-CM-treated cells. betaAm12-CM also inhibited the formation of capillary-like structures on Matrigel by EAhy926 cells. In vivo, inclusion of the betaAm12 cells strongly inhibited, and alpha1Am12 partially inhibited, the angiogenic response in the Matrigel sponge model in both immune-competent and athymic nude mice. Electron microscopy showed a reduction of host cell infiltration in alpha1Am12- and betaAm12-containing sponges and reduction of invading tubular clefts of host cells as compared to controls. Finally, inoculation of either alpha1Am12 or betaAm12 cells (10%) along with a highly angiogenic Kaposi's sarcoma cell line (90%) resulted in a powerful reduction of tumor growth in nude mice in vivo, as did infection with the interferon-alpha-producing retroviruses. These data suggest that a gene therapy approach using class I interferons can effectively inhibit tumor angiogenesis and growth of vascular tumors.

Tumor-induced angiogenesis is a key event for neoplastic progression. In vitro assays are important for identification of potential angiogenic agents and rapid screening for pharmacological inhibitors. The increased interest in this field of study has generated several in vitro assays that recapitulate the steps of endothelial cell activation and differentiation. In this short report we emphasize the utility of Matrigel, a reconstituted basement membrane, to define two different steps in the angiogenic process: invasion in response to growth factors and organization of microvessels into a network with branching morphology on a Matrigel substrate.

Kaposi's sarcoma (KS), a neoplasm often associated with iatrogenic and acquired immunosuppression, is characterized by prominent angiogenesis. Angiogenic factors released by both KS and host cells, as well as HHV-8 and HIV viral products, have been implicated in the pathogenesis of this lesion. Angiogenesis is the result of imbalance among angiogenesis promoters and inhibitors, which disrupts homeostasis. The aim of this study was to investigate the expression and mechanism of KS control of thrombospondin-1 (TSP), a physiological inhibitor of angiogenesis. Immunohistochemical analysis of four KS lesions showed only spotty reactivity for TSP in the stroma and in less than 10 per cent of lesional blood vessels. In addition, the typical KS spindle cells were not stained. In agreement with these findings, decreased levels of TSP were measured with an ELISA assay in the supernatants of cultured KS cells, compared with endothelial cells. In vitro, TSP inhibited the endothelial cell proliferation and motility induced by KS cell supernatants. TSP also prevented endothelial cell motility induced by Tat, a product of HIV-1 endowed with angiogenic potential and implicated in the pathogenesis of AIDS-KS. In vivo, TSP inhibited the angiogenic activity exerted by Tat in the Matrigel sponge model. These results suggest that TSP down-regulation might be permissive for the development of KS-associated angiogenesis., (Copyright 1999 John Wiley & Sons, Ltd.)

Kaposi's sarcoma (KS) is a highly angiogenic lesion which frequently presents as an aggressive form in HIV-infected male patients. We have previously shown that the HIV-1 Tat protein induces endothelial cell migration and invasion in vitro and a rapid angiogenic response in vivo, suggesting that it acts as a cofactor in epidemic KS. In this study we tested beta interferon (IFN beta) and retinoic acid (RA) for the inhibition of Tat-induced angiogenesis using in vivo and in vitro models. IFN beta, at a concentration above 2500 U/ml, was an effective inhibitor of Tat-stimulated growth, migration and morphogenesis of an endothelial cell line in vitro and of angiogenesis in vivo. A strong reduction of properties associated with neovascularisation was induced by 10,000 U/ml. In vivo, RA alone was on ineffective inhibitor of angiogenesis, and in vitro gave only a limited inhibition of endothelial cell growth. However, 13-cis RA used in combination with IFN beta impressively potentiated its effects. A combination of lower doses of IFN beta (2500 U/ml) and 13-cis RA induced a virtually complete inhibition of the Tat-related angiogenic phenotype both in vivo and in vitro. The potentiation of the anti-angiogenic activity of IFN beta by 13-cis RA suggests that this combination could be a useful approach for the therapy of epidemic KS.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

Abundant vasculature with increased permeability is a prominent histological feature of Kaposi's sarcoma (KS), a multifocal, cytokine-regulated tumor. Here we report on the role of vascular endothelial growth factor (VEGF) in AIDS-KS angiogenesis and vascular permeability. We demonstrate that different cytokines, which were previously shown to be active in KS development, modulate VEGF expression in KS spindle cells and cooperate with VEGF on the functional level. Northern blot analysis as well as studies on single cells using in situ hybridization revealed that VEGF expression in cultivated AIDS-KS spindle cells is up-regulated by platelet-derived growth factor-B and interleukin-1 beta. Western blot and enzyme-linked immunosorbent assay analysis of cell culture supernatants demonstrated that the VEGF protein is secreted by stimulated AIDS-KS spindle cells in sufficiently high amounts to activate proliferation of human dermal microvascular endothelial cells. Basic fibroblast growth factor did not increase VEGF expression but acted synergistically with VEGF in the induction of angiogenic KS-like lesions in a mouse model in vivo. Angiogenesis and cellularity of KS-like lesions were clearly increased when both factors were injected simultaneously into the flanks of mice, compared with separate injection of each factor. A comparable angiogenic reaction as obtained by simultaneous injection of basic fibroblast growth factor and VEGF was observed when cell culture supernatants of AIDS-KS spindle cells were used for these experiments. Finally, analysis of primary human AIDS-KS lesions revealed that high amounts of VEGF mRNA and protein were present in KS spindle cells in vivo. These data provide evidence that VEGF, in concert with platelet-derived growth factor-B, interleukin-1 beta, and basic fibroblast growth factor, is a key mediator of angiogenesis and vascular permeability in KS lesions in vivo.

Objective: To characterize the T53 cell line and its clones derived from an adenocarcinoma of BK virus (BKV)/tat transgenic mice and to establish the role of native Tat in tumorigenicity, induction of metastases and angiogenesis., Design and Methods: Tat was quantified by flow cytometry and chloramphenicol acetyltransferase (CAT) assays. Tumorigenicity and metastatic ability of cell lines were assayed in nude mice. Production of proteases was evaluated by a plasmin chromogenic assay and gelatinase zymography. The angiogenic effect was studied in vivo with conditioned medium from tumour cell lines., Results: Tat protein was detected in tumour cell lines in amounts from 600-7000 molecules/cell. Conditioned medium from tumour cell lines was able to transactivate an LTR-CAT in HL3T1 cells, indicating release of extracellular Tat. Tumour cell lines, inoculated into nude mice induced angiogenic tumours with remarkable recruitment of host endothelial cells. Metastases were detected in lymph nodes, lungs, kidneys, and heart. Cell lines produced relevant amounts of proteases. Conditioned medium implanted in mice with matrigel induced an angiogenic response, enhanced by addition of heparin. Preincubation with an anti-Tat antibody abolished the angiogenic effect., Conclusions: Tat from cells from BKV/tat transgenic mice promotes tumorigenesis and formation of metastases and induces angiogenic activity. Angiogenesis occurs at physiological concentrations of Tat lower than 20 ng/ml. The effects of Tat on induction of metastases and angiogenesis appear to be mediated by activation of proteases.

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS.

Extracellular human immunodeficiency virus type 1 (HIV-1) Tat protein promotes growth of spindle cells derived from AIDS-associated Kaposi sarcoma (AIDS-KS), an angioproliferative disease very frequent in HIV-1-infected individuals. Normal vascular cells, progenitors of AIDS-KS cells, proliferate in response to Tat after exposure to inflammatory cytokines, whose levels are augmented in HIV-1-infected individuals and in KS lesions. Here we show that Tat also promotes AIDS-KS and normal vascular cells to migrate and to degrade the basement membrane and stimulates endothelial cell morphogenesis on a matrix substrate. These effects are obtained at picomolar concentrations of exogenous Tat and are promoted by the treatment of the cells with the same inflammatory cytokines stimulating expression of the receptors for Tat, the integrins alpha 5 beta 1 and alpha v beta 3. Thus, under specific circumstances, Tat has angiogenic properties. As Tat and its receptors are present in AIDS-KS lesions, these data may explain some of the mechanisms by which Tat can induce angiogenesis and cooperate in the development of AIDS-KS.

Secretion of angiogenesis inhibitors and stimulators is modulated during in vitro differentiation of embryonic chick growth plate chondrocytes. Supernatants from dedifferentiated cells undergoing maturation to hypertrophic chondrocytes in suspension progressively inhibited vascular cell random migration and invasion of basement membrane matrix by endothelial cells. Maximal inhibition was exhibited by conditioned medium from hypertrophic chondrocytes. The same medium also repressed vascular cell migration induced by highly angiogenic Kaposi's sarcoma cell supernatants and prevented formation of an anastomosed network of tube-like structures by endothelial cells plated on matrigel. On the contrary, when the suspension culture of hypertrophic chondrocytes was supplemented with ascorbic acid, a condition leading to the formation of a mineralized tissue similar to calcified cartilage, a dramatic switch to production of angiogenic activity was observed. Medium conditioned by osteoblast-like cells derived from hypertrophic chondrocytes also induced vascular cell migration and invasion of basement membrane matrix. The presence of angiogenic activity in the conditioned medium was assessed also by an in vivo assay in mice using reconstituted basement membrane associated with heparin. Therefore, interactions of chondrocytes with their extracellular matrix are an absolute requirement for the expression of angiogenic activities by hypertrophic chondrocytes at late developmental stages.

In the initial phases of angiogenesis, endothelial cells must degrade and cross the vessel basement membrane, as do tumor cells during invasion and metastasis formation. Various metalloproteinases have been implicated in tumor cell invasion, in particular MMP-2 (72 kDa collagenase IV, gelatinase A), which has been demonstrated to be associated with tumor metastasis formation. Supernatants from AIDS-Kaposi sarcoma (KS) cells induce normal endothelial cells to invade through a reconstituted basement membrane (Matrigel) in vitro, which correlates with the angiogenic potential of KS cells in vivo. Here we demonstrate that two specific inhibitors of MMP-2, TIMP-2 and a peptide from the MMP-2 propeptide region (peptide 74), inhibit endothelial cell invasion induced by AIDS-KS cell supernatants. Smooth muscle cells were much less sensitive to these inhibitors. These data suggest that MMP-2 activation is a key event in endothelial cell invasion, the initial phase of tumor-associated neoangiogenesis. Inhibition of this enzyme could be an effective treatment for KS and tumor-associated angiogenesis.