American Society for Biochemistry and Molecular Biology
Journal of Biological Chemistry vol:284 pages:25854-25866
The structural and catalytic requirements for neutrophil proMMP-9 to induce angiogenesis were investigated using a quantitative angiogenesis model based on grafting of collagen onplants onto the chorioallantoic membrane of chick embryos. Both physiological activation of neutrophil proMMP-9 and proteolytic activity of the generated MMP-9 enzyme were critically dependent on the TIMP-free status of proMMP-9 zymogen. The presence of an intact active site and hemopexin domain were required for full angiogenesis-inducing activity of the MMP-9 enzyme. Timed additions of TIMP-1 to angiogenic onplants containing TIMP-free neutrophil proMMP-9 indicated that in vivo activation of the zymogen occurred during the first 24 h following onplant grafting. Within the onplant tissue, MMP-9 activation was accompanied by proteolytic modifications of fibrillar collagen and an influx of host proteins, the rate of which depended on the TIMP-free status of the zymogen. By quantifying the levels of host angiogenic factors, we demonstrated that FGF-2 was a major cytokine becoming bioavailable in the onplant tissue undergoing a neutrophil proMMP-9-mediated angiogenic switch. Inhibition of angiogenesis with specific function-blocking antibodies further indicated an initial involvement of a FGF-2/FGFR2 pathway in neutrophil proMMP-9-induced angiogenesis. The enhanced angiogenesis catalyzed by neutrophil MMP-9 appears to evoke also a localized, low threshold level, VEGF/VEGFR2 pathway, likely functioning in the formation and/or stabilization of blood vessels. That neutrophil proMMP-9, unencumbered by TIMP-1, directly mediates FGF-2-dependent angiogenesis was also demonstrated in our newly-established, quantitative mouse angiogenesis model employing subcutaneous collagen implants, thus implicating the novel TIMP-free MMP-9/FGF-2/FGFR2 pathway in neutrophil proMMP-9-induced angiogenesis in a mammalian setting.