Search

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

We used the aortic ring model of angiogenesis to investigate the role of β1 and β3 integrins in postangiogenic vascular survival in collagen and fibrin matrices. Confocal microscopy studies showed that both β1 and β3 integrins were expressed in endothelial cells and pericytes of sprouting neovessels. Antibody blocking experiments demonstrated that β1 integrins but not β3 integrins were required for angiogenic sprouting in collagen. Conversely, in fibrin, blockade of both integrins was needed to inhibit angiogenesis whereas treatment with either antibody alone was ineffective. Antibody-mediated blockade of β1 but not β3 integrins accelerated vascular regression in collagen. In contrast, both anti-β1 and -β3 integrin antibodies were required to promote neovessel breakdown in fibrin. These results demonstrate that angiogenic sprouting and postangiogenic neovessel survival in collagen are critically dependent on β1 integrins. They also indicate that these processes involve a redundant repertoire of β1 and β3 integrins when angiogenesis occurs in fibrin. Thus, pharmacologic targeting of integrin receptors aimed at blocking neovessel formation and survival must be tailored to the specific extracellular matrix environment in which angiogenesis takes place. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This chapter describes protocols developed in our laboratory to prepare and analyze angiogenic cultures of rat aorta. Rings of rat aorta cultured in collagen gels produce neovessel outgrowths which reproduce ex vivo key steps of the angiogenic process including endothelial migration, proliferation, proteolytic digestion of the extracellular matrix, capillary tube formation, pericyte recruitment, and vascular regression. The angiogenic response of aortic explants can be stimulated with growth factors or inhibited with anti-angiogenic molecules. Aortic ring cultures can also be used to study tumor angiogenesis. Protocols outlined in this chapter describe how this assay can be modified to investigate the angiogenic activity of cancer cells.

Protocols outlined in this chapter illustrate how to prepare and analyze angiogenic cultures of rat aorta. Aortic rings embedded in gels of extracellular matrix generate vascular outgrowths that can easily be monitored over time with inverted microscopy. The angiogenic response can be measured by counting vessels or with image analysis. Aortic ring cultures can be used to investigate molecular mechanisms of angiogenesis and test the efficacy of stimulators and inhibitors of the angiogenic process. As such this assay is an invaluable tool for both basic and applied angiogenesis research.

Objective: The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the cascade of gene activation that regulates aortic angiogenesis in response to injury., Methods and Results: Angiogenesis was studied by culturing rat or mouse aortic rings in collagen gels. Gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction, microarray analysis, immunocytochemistry, and ELISA. TNFα gene disruption and recombinant TNFα or blocking antibodies against vascular endothelial growth factor (VEGF) or TNF receptors were used to investigate TNFα-mediated angiogenic mechanisms. Resident aortic macrophages were depleted with liposomal clodronate. Angiogenesis was preceded by overexpression of TNFα and TNFα-inducible genes. Studies with isolated cells showed that macrophages were the main source of TNFα. Angiogenesis, VEGF production, and macrophage outgrowth were impaired by TNFα gene disruption and promoted by exogenous TNFα. Antibody-mediated inhibition of TNF receptor 1 significantly inhibited angiogenesis. The proangiogenic effect of TNFα was suppressed by blocking VEGF or by ablating aortic macrophages. Exogenous TNFα, however, maintained a limited proangiogenic capacity in the absence of macrophages and macrophage-mediated VEGF production., Conclusions: Overexpression of TNFα is required for optimal VEGF production and angiogenesis in response to injury. This TNFα/VEGF-mediated angiogenic pathway requires macrophages. The residual capacity of TNFα to stimulate angiogenesis in macrophage-depleted aortic cultures implies the existence of a VEGF-independent alternate pathway of TNFα-induced angiogenesis.

Angiogenesis during reactive and pathologic processes is characteristically associated with inflammation. Macrophages and dendritic cells present in the inflammatory infiltrate contribute to the angiogenic process by multiple mechanisms. Macrophages produce a broad array of angiogenic growth factors and cytokines, generate conduits for blood flow through proteolytic mechanisms, and promote the remodeling of arterioles into arteries. They can also inhibit angiogenesis and cause reabsorption of neovessels by inducing endothelial cell death. Dendritic cells can stimulate or inhibit angiogenesis depending on their activation status and subset specificity. Dendritic cells stimulate angiogenesis by secreting angiogenic factors and cytokines, promoting the proangiogenic activity of T lymphocytes, and trans-differentiating into endothelial cells. Inflammatory infiltrates associated with angiogenesis also contain Tie2+, VEGFR2+, and GR1+ myelomonocytic cells which actively regulate the angiogenic process through paracrine mechanisms. In this paper we review our current knowledge of this field and discuss how recent advances have provided the rationale for novel therapeutic approaches against cancer.

Angiogenesis is regulated by a complex cascade of cellular and molecular events. The entire process can be reproduced in vitro by culturing rat or mouse aortic explants in three-dimensional biomatrices under chemically defined conditions. Angiogenesis in this system is driven by endogenous growth factors released by the aorta and its outgrowth. Sprouting endothelial cells closely interact with pericytes, macrophages, and fibroblasts in an orderly sequence of morphogenetic events that recapitulates all stages of angiogenesis. This model can be used to study the basic mechanisms of the angiogenic process and to test the efficacy of proangiogenic or antiangiogenic compounds. Aortic cultures can be evaluated with a range of morphologic and molecular techniques for the study of gene expression. In this chapter we describe basic protocols currently used in our laboratory to prepare, quantify, and analyze this assay.

We used the aortic ring model of angiogenesis to investigate the role of beta(1) and beta(3) integrins in postangiogenic vascular survival in collagen and fibrin matrices. Confocal microscopy studies showed that both beta(1) and beta(3) integrins were expressed in endothelial cells and pericytes of sprouting neovessels. Antibody blocking experiments demonstrated that beta(1) integrins but not beta(3) integrins were required for angiogenic sprouting in collagen. Conversely, in fibrin, blockade of both integrins was needed to inhibit angiogenesis whereas treatment with either antibody alone was ineffective. Antibody-mediated blockade of beta(1) but not beta(3) integrins accelerated vascular regression in collagen. In contrast, both anti-beta(1) and -beta(3) integrin antibodies were required to promote neovessel breakdown in fibrin. These results demonstrate that angiogenic sprouting and postangiogenic neovessel survival in collagen are critically dependent on beta(1) integrins. They also indicate that these processes involve a redundant repertoire of beta(1) and beta(3) integrins when angiogenesis occurs in fibrin. Thus, pharmacologic targeting of integrin receptors aimed at blocking neovessel formation and survival must be tailored to the specific extracellular matrix environment in which angiogenesis takes place., (Copyright (c) 2007 S. Karger AG, Basel.)