Angiogenesis: new targets for the development of anticancer chemotherapies

Angiogenesis is the process by which new blood vessels are formed from preexisting microvasculature. To ensure an adequate blood supply, tumor cells release angiogenic factors that are capable of promoting nearby blood vessels to extend vascular branches to the tumor. In addition, larger tumors have been shown to release angiogeneic inhibitory factors that prevent blood vessels from sending branches to smaller, more distant tumors that compete for oxygen and nutrients. Angiogenesis is a complex multistep biochemical process, and offers several potential molecular targets for non-cytotoxic anticancer therapies. Strategies for exploiting tumor angiogenesis for novel cancer drug discovery include: (i) inhibition of proteolytic enzymes that breakdown the extracellular matrix surrounding existing capillaries; (ii) inhibition of endothelial cell migration; (iii) inhibition of endothelial cell proliferation; (iv) enhancement of tumor endothelial cell apoptosis. There is also a host of miscellaneous agents that inhibit angiogenesis for which the specific mechanisms are not clear. Several methods have been developed for measuring antiangiogenic activity both in vitro and in vivo. Although there has been intensive research efforts focused at the phenomena of angiogenesis, as well as the search for antiangiogenic agents for more than two decades, many questions remain unanswered with regard to the overall biochemical mechanisms of the angiogenesis process and the potential therapeutic utility of angiogenic inhibitors. Nevertheless potent angiogenic inhibitors capable of blocking tumor growth have been discovered, and appear to have potential for development into novel anticancer therapeutics. However there are still hurdles to be overcome before these inhibitors become mainstream therapies.     

Current status and future prospects for anti-angiogenic therapies in cancer

Background: Angiogenesis is essential for tumour growth and development and since the pioneering work of Judah Folkman several anti-angiogenic strategies have now been successfully employed. Objective: This article aims to present a detailed review of current knowledge of the main pathways involved in angiogenesis, the strategies employed for inhibition and the current status of angiogenesis inhibitors in therapeutic use. Methods: A systematic review of the literature was undertaken including angiogenesis in cancer and angiogenesis inhibitors in pre-clinical and clinical trials. Conclusion: While angiogenic inhibitors are now in clinical use, their limited benefits mean we must urgently develop strategies to improve the efficacy of this approach.     

IBC’s 6th Annual Conference on Angiogenesis: Novel Therapeutic Developments

Angiogenesis is a process that is dependent upon co-ordinate production of angiogenesis stimulatory and inhibitory (angiostatic) molecules. Any imbalance in this regulatory circuit may lead to the development of a number of angiogenesis-mediated diseases. Angiogenesis is a multi-step process including activation, adhesion, migration, proliferation and transmigration of endothelial cells across cell matrices to or from new capillaries and from existing vessels. Angiogenesis is a process involved in the formation of new vessels by sprouting from pre-existing vessels. In contrast, vessel rudiments are sorted by a process termed vasculogenesis. Endothelial heterogeneity and organ specificity might contribute to differences in the response to different anti-angiogenic mechanisms (cultured EC versus microvascular EC isolated from different tissues). Under normal physiological conditions in mature organisms, endothelial cell turnover or angiogenesis is extremely slow (from months to years). However, angiogenesis can be activated for a limited time in certain situations such as wound healing and ovulation. In certain pathological states, such as human metastasis (oncology) and ocular neovascularisation, disorders including diabetic retinopathy and age-related macular degeneration (ophthalmology), there is excessive and sustained angiogenesis. Hence, understanding the mechanisms involved in the regulation of angiogenesis could have a major impact in the prevention and treatment of pathological angiogenic processes. Additionally, endothelial cells play a major role in the modelling of blood vessels. The interplay of growth factors, cell adhesion molecules, matrix proteases and specific signal transduction pathways either in the maintenance of the quiescent state or in the reactivation of endothelial cells is critical in physiological and pathological angiogenic processes.     

The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis, invasion and development of anti-angiogenic agents

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, progression and metastasis. The development of agents that target tumor vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. The chorioallantoic membrane (CAM) assay is well established and widely used as a model to examine angiogenesis, and anti-angiogenesis. This review 1) summarizes the currently used angiogenesis assays and the importance of CAM model among them; 2) summarizes the current knowledge about the development and structure of the CAM's capillary bed; 3) reports findings regarding the role played by molecular signaling pathways in angiogenesis process; 4) discusses the use, advantages and limitations of the CAM as a model for studying tumor angiogenesis and invasiveness, as well as development of angiogenic and/or anti-angiogenic agents; 5) discusses the importance of standardization of the major methodologies for all aspects of the use of the CAM in angiogenesis-related studies; 6) and finally, summarizes major findings regarding the agents developed by the use of CAM model in the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.     

Endothelin-1 and angiogenesis in cancer

Tumours require oxygenation, nutrition and a route for dissemination. This necessitates the development of new vessels or angiogenesis. High levels of new vessel development are indicators of poor prognosis in cancer; they also provide new avenues of anti-tumour therapy. Angiogenesis in cancer produces structurally different vessels from angiogenesis in wound healing and inflammation. This article reviews the differences between vessels in tumour angiogenesis and normal angiogenesis. The main focus of the article is the role of the vasoactive peptide endothelin-1 (ET-1) in tumour angiogenesis. The role of ET-1 in tumour development is reviewed, before the direct and indirect effects of ET-1 in angiogenesis are examined. ET-1 has a direct angiogenic effect on endothelial and peri-vascular cells. It also has an indirect action through the increased release of the potent pro-angiogenic substance vascular endothelial growth factor (VEGF), via hypoxia inducible factor-1. ET-1 also indirectly stimulates angiogenesis by stimulating fibroblasts and cancer cells to produce pro-angiogenic proteases. ET-1 is a novel stimulator of tumour angiogenesis and warrants further examination as an anti-angiogenic treatment target.     

Angiogenesis inhibitors in cancer therapy

The formation of new blood vessels is essential for tumor growth and progression. Angiogenesis inhibitors have been demonstrated to block tumor growth and/or metastasis. Although initial clinical data have been disappointing, new strategies to increase the efficacy of these drugs have ensured their ongoing clinical development. This review highlights the broad spectrum of angiogenesis inhibitors under investigation in both preclinical and clinical studies. From 'natural' inhibitors to thalidomide analogs, the number of compounds in development is extensive. Elucidating the mechanisms of action will enable their use in conjunction with existing/other novel therapies, thereby maximizing the potential efficacy of angiogenesis inhibitors to fulfill their promise in patients with cancer.     

Role of inflammatory mediators in angiogenesis

The angiogenic process involves several cell types and mediators, which interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Angiogenesis occurs in several physiological and pathological conditions, such as embryo development and wound healing, diabetic retinopathy and tumours. Inflammatory cells, namely monocytes/macrophages, T lymphocytes and neutrophils, fully participate in the angiogenic process by secreting cytokines that may affect endothelial cell (EC) functions, including EC proliferation, migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. With regards to inflammatory cells and endothelium cross-talk, such balance is conceptually very similar to that of pro-inflammatory and anti-inflammatory mediators that modulate an appropriate inflammatory response. In this review we will mainly discuss the relevance of both physiological and pathological inflammatory processes in angiogenesis, with particular regards to microenvironmental contribution. We will also describe some of the most relevant pro-inflammatory cytokines in the modulation of the angiogenic process. Furthermore, we will concentrate on what has been recently reported about the mechanism by which some of these cytokines are induced during inflammation to promote a suitable microenvironment for angiogenesis and tumour progression. Pro-angiogenic cytokines, such as IL-1 and TNF, and anti-angiogenic cytokines such as IFN-gamma and IL-12, will be briefly described. We will try to provide a rationale for the use of both cytokines and cytokine blockades as novel potential pharmaceutical targets to modulate angiogenesis in chronic inflammation as well as in cancer.     

Angiogenesis: regulators and clinical applications

Angiogenesis is a fundamental process in reproduction and wound healing. Under these conditions, neovascularization is tightly regulated. Unregulated angiogenesis may lead to several angiogenic diseases and is thought to be indispensable for solid tumor growth and metastasis. The construction of a vascular network requires different sequential steps including the release of proteases from "activated" endothelial cells with subsequent degradation of the basement membrane surrounding the existing vessel, migration of endothelial cells into the interstitial space, endothelial cell proliferation, and differentiation into mature blood vessels. These processes are mediated by a wide range of angiogenic inducers, including growth factors, chemokines, angiogenic enzymes, endothelial specific receptors, and adhesion molecules. Finally, when sufficient neovascularization has occurred, angiogenic factors are down-regulated or the local concentration of inhibitors increases. As a result, the endothelial cells become quiescent, and the vessels remain or regress if no longer needed. Thus, angiogenesis requires many interactions that must be tightly regulated in a spatial and temporal manner. Each of these processes presents possible targets for therapeutic intervention. Synthetic inhibitors of cell invasion (marimastat, Neovastat, AG-3340), adhesion (Vitaxin), or proliferation (TNP-470, thalidomide, Combretastatin A-4), or compounds that interfere with angiogenic growth factors (interferon-alpha, suramin, and analogues) or their receptors (SU6668, SU5416), as well as endogenous inhibitors of angiogenesis (endostatin, interleukin-12) are being evaluated in clinical trials against a variety of solid tumors. As basic knowledge about the control of angiogenesis and its role in tumor growth and metastasis increases, it may be possible in the future to develop specific anti-angiogenic agents that offer a potential therapy for cancer and angiogenic diseases.     

Endogenous angiogenesis inhibitors as therapeutic agents: historical perspective and future direction

Angiogenesis, the formation of new blood vessels from preexisting microvasculature, is a highly regulated process. Angiogenesis is controlled by both positive and negative factors thus providing several targets for drug discovery. The inhibition of angiogenesis represents a new approach to cancer therapy and several agents and approaches are in different stages of clinical development. These inhibitors were recently shown to constitute a new modality for cancer treatment. In this article, we will review angiogenesis inhibitors-related patent literature for the years 2000-2005. This review will cover specifically the discovery and development disclosures of endogenous inhibitors. The scope of this review is to give the reader a well-structured patent literature review of these agents targeting different steps of the angiogenic process. Finally, we have summarized the key attributes of the emerging endogenous angiogenesis inhibitors that make them potent antitumor agents.     

Cancer chemotherapeutics - angiogenesis agents: October 1999 - March 2000

Angiogenesis is often defined as the formation of capillary blood vessels from existing blood vessels. In the area of cancer research, approaches that seek to block angiogenesis are under intense investigation. In the past six months, a number of applications have appeared referring to small molecule inhibitors of matrix metalloproteinases (MMPs), the VEGF receptor tyrosine kinase, the integrin receptor α_vβ₃ and other defined targets. This review summarises the key features of these applications as well as other disclosures involving natural products claimed as angiogenesis inhibitors. The review also summarises angiogenesis-related disclosures involving endogenous inhibitors, novel targets and interesting technologies that have appeared in applications published between October 1999 and March 2000. If the patent activity is any indication, there is significant interest and a number of opportunities for treating cancer through inhibiting angiogenesis.     

Heparin derivatives as angiogenesis inhibitors

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot growth as a mass above few mm(3) unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.     

Imaging in the age of molecular medicine: monitoring of anti-angiogenic treatments

Angiogenesis is a complex multistep process and a crucial pre-requisite for tumor growth, invasion and metastasis. A profound knowledge of the mechanisms including the elucidation of markers for angiogenic vessels is essential for the generation of new anti-angiogenic chemotherapeutic agents and the improvement of specific imaging techniques. During the last decades, numerous angiogenesis inhibitors have been developed and some of them have shown promising results in preclinical and clinical trials. However, the response to anti-angiogenic treatment is often delayed and shows high inter-individual variations. In order to improve anti-angiogenic therapy, new specific surrogate markers are necessary that allow the characterization of different angiogenic steps, especially at the early stage. In this respect, non-invasive imaging is a potent tool for characterizing the tumor vascularization and for sensitive and longitudinal treatment monitoring. In particular, new molecular imaging techniques might ultimately improve the characterization of the angiogenic tumor phenotype and stage. This review summarizes the current status of different imaging modalities e.g. MRI, CT, US, nuclear and optical imaging with respect to the imaging of tumor angiogenesis and of anti-angiogenic treatments. It also includes new approaches in molecular imaging, which give deep insight into the tumor stage and the response of tumor vessels to anti-angiogenic therapy. Thus, this may lead to a more personalized cancer therapy in future.     

Angiogenesis - Research Frontiers. A Basic Science Conference of the New York Academy of Medicine

Angiogenesis, the development of new blood vessels, is essential for both tumour growth and metastasis. Recent advances in our understanding of the molecular mechanisms underlying the angiogenic process and its regulation have led to the discovery of a variety of targets for therapeutic intervention. The potential application of these angiogenic inhibitors is currently under intense preclinical and clinical investigation. Compelling evidence suggests that vascular endothelial growth factors (VEGFs) and their receptors play critical roles in tumour-associated angiogenesis. Tumour homing factors will drive the growth of new vessels, neoangiogenesis, to satisfy the demands of the growing tumour. By attacking the angiogenic process the tumour will be starved for oxygen and nutrients, thus impairing its growth. This has been demonstrated in a variety of animal tumour models in which disabling the function of VEGF or its receptor was shown to inhibit both tumour growth and metastasis. The New York Academy of Medicine organised a day-long meeting to discuss emerging ideas, currently available in vitro and animal models and evaluation of these therapies during their preclinical development and in clinical trials.     

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody

BACKGROUND AND PURPOSE

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect.

EXPERIMENTAL APPROACH

Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice.

KEY RESULTS

Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody.

CONCLUSION AND IMPLICATIONS

These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL.     

Angiogenesis-research frontiers. A basic science conference of the New York Academy of Medicine

Angiogenesis, the development of new blood vessels, is essential for both tumour growth and metastasis. Recent advances in our understanding of the molecular mechanisms underlying the angiogenic process and its regulation have led to the discovery of a variety of targets for therapeutic intervention. The potential application of these angiogenic inhibitors is currently under intense preclinical and clinical investigation. Compelling evidence suggests that vascular endothelial growth factors (VEGFs) and their receptors play critical roles in tumour-associated angiogenesis. Tumour homing factors will drive the growth of new vessels, neoangiogenesis, to satisfy the demands of the growing tumour. By attacking the angiogenic process the tumour will he starved for oxygen and nutrients, thus impairing its growth. This has been demonstrated in a variety of animal tumour models in which disabling the function of VEGF or its receptor was shown to inhibit both tumour growth and metastasis. The New York Academy of Medicine organised a day-long meeting to discuss emerging ideas, currently available in vitro and animal models and evaluation of these therapies during their preclinical development and in clinical trials.     

Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent

Angiogenesis, or the sprouting of new blood vessels, is a central process in the growth of solid tumors. For many cancers, the extent of vascularization of a tumor is a negative prognostic indicator signifying aggressive disease and increased potential for metastasis. Recent efforts to understand the molecular basis of tumor-associated angiogenesis have identified several potential therapeutic targets, including the receptor tyrosine kinases for the angiogenic factor vascular endothelial growth factor (VEGF). Here we review the approach taken at SUGEN, Inc. to discover and develop small molecule inhibitors of receptor tyrosine kinases as anti-angiogenic agents. We focus on SU5416, a selective inhibitor of VEGF receptors that is currently in clinical development for the treatment of advanced malignancies. Its biochemical, biological and pharmacological properties are reviewed and clinical implications discussed.     

A novel model of image acquisition and processing for holistic quantification of angiogenesis disrupted by application of mainstream and sidestream cigarette smoke solutions

Angiogenesis is a vital process in the growth of new blood vessels from pre-existing vasculature. Among several approaches being used for studies related to angiogenesis, chicken chorioallantoic membrane assay (CAM) is an excellent model system. However, its utility has been limited due to difficulty in quantifying putative angiogenic and anti-angiogenic response to an experimental compound in an objective and quantifiable manner. Herein, we report a novel approach of image acquisition and processing for better evaluation of neovascularization. The effects of mainstream cigarette smoke solutions (MSCSS) and sidestream cigarette smoke solutions (SSCSS) from different commercially available cigarettes on angiogenesis were quantified, using CAM assay. Different gross and nanometer scale topographies of CAMs were quantified, which are vital for 3D image scrutiny and can precisely enumerate angiogenesis. Pattern formation of blood vessels, diameter, area and 3D surface roughness of CAMs were substantially disrupted by application of cigarette-smoke extracts. An important point revealed in our study that SSCSS appeared to be significantly more toxic than MSCSS with respect to their effects on angiogenesis. This new imaging technique combined with other modalities, will provide a robust platform to optimize trial design and more patent studies in angiogenesis.     

Anti-Angiogenic Activity of Selected Receptor Tyrosine Kinase Inhibitors, PD166285 and PD173074: Implications for Combination Treatment with Photodynamic Therapy

Angiogenesis, the formation of new blood vessels from an existing vasculature, is requisite for tumor growth. It entails intercellular coordination of endothelial and tumor cells through angiogenic growth factor signaling. Interruption of these events has implications in the suppression of tumor growth. PD166285, a broad-spectrum receptor tyrosine kinase (RTK) inhibitor, and PD173074, a selective FGFR₁TK inhibitor, were evaluated for their anti-angiogenic activity and anti-tumor efficacy in combination with photodynamic therapy (PDT). To evaluate the anti-angiogenic and anti-tumor activities of these compounds, RTK assays, in vitro tumor cell growth and microcapillary formation assays, in vivo murine angiogenesis and anti-tumor efficacy studies utilizing RTK inhibitors in combination with photodynamic therapy were performed. PD166285 inhibited PDGFR--, EGFR-, and FGFR₁TKs and c-src TK by 50% (IC₅₀) at concentrations between 7–85nM. PD173074 displayed selective inhibitory activity towards FGFR₁TK at 26nM. PD173074 demonstrated (>100 fold) selective growth inhibitory action towards human umbilical vein endothelial cells compared with a panel of tumor cell lines. Both PD166285 and PD173074 (at 10nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo anti-angiogenesis studies in mice revealed that oral administration (p.o.) of either PD166285 (1–25 mg/kg) or PD173074 (25–100 mg/kg) generated dose dependent inhibition of angiogenesis. Against a murine mammary 16c tumor, significantly prolonged tumor regressions were achieved with daily p.o. doses of PD166285 (5–10 mg/kg) or PD173074 (30–60 mg/kg) following PDT compared with PDT alone (p<0.001). Many long-term survivors were also noted in combination treatment groups. PD166285 and PD173074 displayed potent anti-angiogenic and anti-tumor activity and prolonged the duration of anti-tumor response to PDT. Interference in membrane signal transduction by inhibitors of specific RTKs (e.g. FGFR₁TK) should result in new chemotherapeutic agents having the ability to limit tumor angiogenesis and regrowth following cytoreductive treatments such as PDT.     

Type I collagen and collagen mimetics as angiogenesis promoting superpolymers

Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligation and possibly clustering of endothelial cell (EC) surface alpha 1 beta 1/alpha 2 beta 1 integrin receptors by the GFPGER(502-507) sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating "angiogenic superpolymers", including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.     

Novel peptide-specific quantitative structure-activity relationship (QSAR) analysis applied to collagen IV peptides with antiangiogenic activity

Angiogenesis is the growth of new blood vessels from existing vasculature. Excessive vascularization is associated with a number of diseases including cancer. Antiangiogenic therapies have the potential to stunt cancer progression. Peptides derived from type IV collagen are potent inhibitors of angiogenesis. We wanted to gain a better understanding of collagen IV structure-activity relationships using a ligand-based approach. We developed novel peptide-specific QSAR models to study the activity of the peptides in endothelial cell proliferation, migration, and adhesion inhibition assays. We found that the models produced quantitatively accurate predictions of activity and provided insight into collagen IV derived peptide structure-activity relationships.