Angiogenesis, the formation of new blood vessels, plays a critical role in the progression of cancer and other pathologic processes, but it also ameliorates coronary artery disease and other ischemic conditions caused by inadequate neovascularization. The outcome of angiogenesis depends on the capacity of neovessels to regress or survive. There is however a gap in our understanding of the molecular mechanisms regulating the fate of postangiogenic neovessels. We have reproduced the process of vascular regression by culturing rat or mouse aortic rings in collagen gels under chemically defined culture conditions. In this ex vivo culture system aortic rings generate microvessels which regress over time due to matrix metalloproteinase (MMP)-mediated proteolysis of the extracellular matrix (ECM). We have identified MMP-14 as one of the enzymes responsible for vascular regression, and shown that involution of neovessels can be accelerated with anti-21 and anti-23 integrin antibodies. We have successfully duplicated the vascular regression process in vivo by implanting aortic rings in the subcutaneous space of syngeneic rats or mice. We recently found that vascular regression and lysis of the ECM in the ex vivo aortic ring model are associated with perivascular accumulation of macrophages and overexpression of MMP-3, MMP- 12 and MMP-14. Macrophages isolated from regressing cultures inhibited the sprouting of freshly cut aortic rings. Molecular analysis of regressing aortic cultures demonstrated upregulated expression of type I interferons (INF-1 and IFN-2) and IFN response genes. Recombinant IFNs blocked the angiogenic response of the aortic explants and caused involution of neovessels. Since macrophages are required for angiogenic sprouting, we hypothesize that these cells change over time from an angiogenic to an angiostatic phenotype. We postulate that angiostatic macrophages are the main source of angiolytic MMPs. Finally we propose that type I IFNs promote the vascular regression process including the transformation of macrophages into an angiostatic and angiolytic phenotype.
Specific aims of this proposal are designed to investigate the following hypotheses: (1) Macrophages switch from an angiogenic to an angiostatic phenotype during vascular regression. (2) MMP-mediated vascular regression (angiolysis) following angiogenesis requires macrophages. (3) Interferons promote vascular regression following angiogenesis. Proposed experiments will be performed with in vitro, ex vivo and in vivo assays of angiogenesis. For in vitro studies we will use a capillary tube formation assay with isolated endothelial cells. The rat and mouse aortic models of angiogenesis will be used for ex vivo studies of angiogenesis and vascular regression. For studies in the live animal, we will use our newly developed in vivo assay in which subcutaneously implanted aortic rings stimulate formation of a richly vascularized granulation tissue which involutes over time. Our research plan includes genetically modified mice with disrupted MMP-3, MMP-12, MMP-14, or type I IFN receptor genes. Proposed experiments employ quantitative real-time RT-PCR, immunocytochemistry, confocal microscopy, Western blotting, TUNEL, ELISA, and pharmacological methods of ex vivo and in vivo macrophage ablation. Functional studies will be performed with anti-INF-1 and -IFN-2 blocking antibodies. Experiments proposed in this grant will further our understanding of the cellular and molecular mechanisms responsible for vascular regression following angiogenesis. This knowledge may contribute to the development of novel therapeutic approaches for the treatment of cancer and other angiogenesis-related disorders.
This grant is designed to identify key mechanisms of vascular regression following blood vessel growth (angiogenesis). Understanding how the fate of angiogenic neovessels is regulated at a molecular level has important clinical implications. Cancer patients will benefit from targeted disruption of the tumor vasculature with therapies aimed at inducing vascular regression. The same consideration applies to patients with wet age related macular degeneration or diabetic retinopathy. On the other hand, inducing the formation of regression-resistant blood vessels will benefit patients with coronary artery disease or other ischemic conditions. Studies proposed in this grant will contribute to the VA care mission since many diseases afflicting the veteran patient population are associated with abnormal persistence or loss of angiogenic neovessels.
