Neutrophil MMP-9 Proenzyme, Unencumbered by TIMP-1, Undergoes Efficient Activation in Vivo and Catalytically Induces Angiogenesis via a Basic Fibroblast Growth Factor (FGF-2)/FGFR-2 Pathway

Ghislain Opdenakker,Philippe E Van Den Steen,Bernhard Schweighofer,Veronica C Ardi,Elena I Deryugina,James P Quigley

doi:10.1074/jbc.m109.033472

Ghislain Opdenakker, Philippe E Van Den Steen + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.033472

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Abstract

The structural and catalytic requirements for neutrophil MMP-9 proenzyme (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 tissue inhibitor of metalloproteinase (TIMP)-free status of the 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 the onplants containing TIMP-free neutrophil proMMP-9 indicated that in vivo activation of the zymogen occurred during the first 24 h after 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 basic fibroblast growth factor (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 involvement of a FGF-2/FGFR-2 pathway in neutrophil proMMP-9-induced angiogenesis. The enhanced angiogenesis catalyzed by neutrophil MMP-9 appears to evoke also a localized, low threshold level vascular endothelial growth factor (VEGF)/VEGFR-2 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 quantitative mouse angiogenesis model employing subcutaneous collagen implants, thus implicating the novel TIMP-free MMP-9/FGF-2/FGFR-2 pathway in proMMP-9-induced angiogenesis in a mammalian setting.

Highlights

At the site of primary tumor formation, the in vivo source of angiogenic matrix metalloproteinase-9 (MMP-9) has not been precisely ascribed to a particular cell type
The gelatinolytic activity of ⌬OGMMP-9 in zymographs is reduced, its native enzymatic activity against soluble substrates has been shown similar to that of wild type (WT)-MMP-9 [29]. When both OG and hemopexin domains of MMP-9 were absent (⌬OG⌬Hem), the angiogenic capability of MMP-9 was reduced to the low levels manifested by the ⌬Hem-MMP-9 variant (Fig. 1B). These results demonstrate that the catalytic activity of MMP-9 is required for its angiogenic ability, indicating that activation of proMMP-9 must occur in vivo
These findings demonstrate that the presence of the hemopexin domain is necessary for full angiogenesis-inducing capacity of the MMP-9 enzyme, whereas the MMP-9-distinct OG domain appears unnecessary

Summary

Introduction

At the site of primary tumor formation, the in vivo source of angiogenic MMP-9 has not been precisely ascribed to a particular cell type. Timed applications of TIMP-1 into collagen onplants containing TIMP-free neutrophil proMMP-9 completely abrogated induction of angiogenesis over control levels when applied at 1, 4, or 12 h after grafting of onplants, whereas the addition of TIMP-1 at 24 h (Fig. 3A) or later (data not shown) had little or no effect on MMP-9-induced angiogenesis.

Results

Conclusion