Growth factor–induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen

Abstract

The contribution of specific type I collagen remodeling in angiogenesis was studied in vivo using a quantitative chick embryo assay that measures new blood vessel growth into well-defined fibrillar collagen implants. In response to a combination of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), a strong angiogenic response was observed, coincident with invasion into the collagen implants of activated fibroblasts, monocytes, heterophils, and endothelial cells. The angiogenic effect was highly dependent on matrix metalloproteinase (MMP) activity, because new vessel growth was inhibited by both a synthetic MMP inhibitor, BB3103, and a natural MMP inhibitor, TIMP-1. Multiple MMPs were detected in the angiogenic tissue including MMP-2, MMP-13, MMP-16, and a recently cloned MMP-9-like gelatinase. Using this assay system, wild-type collagen was compared to a unique collagenase-resistant collagen (r/r), with regard to the ability of the respective collagen implants to support cell invasion and angiogenesis. It was found that collagenase-resistant collagen constitutes a defective substratum for angiogenesis. In implants made with r/r collagen there was a substantial reduction in the number of endothelial cells and newly formed vessels. The presence of the r/r collagen, however, did not reduce the entry into the implants of other cell types, that is, activated fibroblasts and leukocytes. These results indicate that fibrillar collagen cleavage at collagenase-specific sites is a ratelimiting event in growth factor-stimulated angiogenesis in vivo. IntroductionAngiogenesis is the process by which the preexisting vascular tree gives rise to new blood vessels. This process is tightly regulated during development and occurs only under highly specialized circumstances in the normal adult animal. Unregulated angiogenesis may be a pivotal element of disease etiology, such as during tumor growth and atherosclerosis. 1,2 All forms of angiogenesis, however, are thought to share certain basic features, including migration and mitogenesis of endothelial cells, lumen formation, connection of new vascular segments with the preexisting circulation, and extensive remodeling of the extracellular matrix by proteases. The detailed mechanisms by which proteolytic enzymes, such as the matrix metalloproteinases (MMPs), mediate some of these events in vivo remain unclear. 3,4 The MMPs constitute a large family of zinc-dependent endopeptidases that have been strongly implicated in both normal angiogenesis and tumor vascularization. 2,4 These enzymes are characteristically regulated at multiple levels, including gene expression, spatial localization, zymogen activation, and inhibition by the tissue inhibitors of metalloproteinases (TIMPs). 5 In vitro remodeling of extracellular matrices by cultured endothelial cells has consistently been shown to rely on MMPs. 4 However, it is unclear which aspects of cell culture models for angiogenesis may be directly extrapolated to the animal. In vivo studies also have foun...

Human neutrophils uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis

“…Therefore, no underlying mechanism has been proposed to explain why neutrophil MMP-9 might function as a critical proangiogenic protease. Previously, we used a quantitative angiogenesis model in the chicken embryo (28) and demonstrated that avian inflammatory cells and their MMPs, including monocyte MMP-13 and heterophil MMP-9, were critical for the induction of both physiologic and tumor angiogenesis (30,31). By taking advantage of this system, which allows for direct access to the angiogenic site and testing of intact viable cells and their delivered products, we directly analyzed for the involvement of functioning human neutrophils and the catalytic capabilities of their MMP-9 in angiogenesis.…”

“…We have previously demonstrated the functional role of distinct inflammatory cells and their MMPs during physiologic and tumorinduced angiogenesis by using a quantitative in vivo angiogenesis model (28), modified from the original assay described by Folkman and colleagues (29). In the model, angiogenesis is induced in 3D collagen rafts grafted on the chorioallantoic membrane (CAM) of the developing chicken embryo.…”

Metalloproteinases and their inhibitors in tumor angiogenesis

“…56 In addition to this, TIMP-1, -2 and -3 have all been shown to inhibit EC migration and invasion in type I collagen gel assays. 52,57 The repeated observations of a correlation between MMP inhibition and reduced tumor angiogenesis provide strong evidence for the involvement of the MMPs in this process. However, they do not provide specific information about which particular MMPs may be involved as these inhibitors are broad-range, often with not all of their targets clearly defined.…”

Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous melanoma

“…Col(I) turnover is important for correct alignment of sprouting endothelial cells. 18,28 Whereas its digestion allows elongation of vascular tubes, the level of Col(I) synthesis can limit neovascularization, because it contributes to the development of the matrix on which the vascular tube and vascular smooth muscle cells extend. [36][37][38] Halofuginone can exert an inhibitory effect on these various aspects of angiogenesis, as has been demonstrated in different experimental settings.…”

“…Because halofuginone has been reported to influence angiogenesis 18 and because Col(I) remodeling is important for blood vessel sprouting and formation of endothelial tubular structures, [26][27][28] we assessed development of the angiogenic vasculature in halofuginone-treated and control pig skin. Immunohistochemical analysis of CD31-positive endothelial cells in normal skin from control and halofuginone-treated piglets did not reveal differences in vascular density (75 6 5 and 75 6 13 vessels/mm 2 , respectively).…”