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

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.Gelatinase B, or matrix metalloproteinase-9 (MMP-9), 3 has been functionally and genetically linked to a number of critical biological functions, including wound healing, inflammation, tumor progression, vascular tissue remodeling, and angiogenesis (1-6). In particular, vascular tissue remodeling and angiogenesis, related histologically and mechanistically, have received a good deal of attention as MMP-9 has been reported to be a major trigger of the angiogenic switch (7). Phenotypic rescue of vasculogenic and angiogenic defects manifested in MMP-9 null mice has suggested a functional link between MMP-9 and the formation, structure, and remodeling of new blood vessels (7-15). The direct contribution of MMP-9 to angiogenesis and vascular performance is thought to involve catalytic activity of the enzyme resulting either in the cleavage of ECM components such as native and denatured collagens (16 -18) and processing of various cytokines and chemokines such as CXCL5, CXCL6, and CXCL8 (interleukin-8) (19,20) or release of angiogenic growth factors such as VEGF (7,12,15,(21)(22...

Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice

“…2,19,21,35 A recent study demonstrated that tissueinfiltrating neutrophil pro-MMP-9 induces angiogenesis catalytically via an FGF-2/FGFR2 pathway. 23 Consistent with that study, we have shown previously that pharmacologic PAI-1 inhibition results in the accumulation of FGF-2-and VEGF-A-expressing Gr-1 ϩ neutrophils within ischemic muscle tissues through an effect on endogenous tPA and MMP-9, and in an increase of plasma VEGF-A via up-regulation of endogenous tPA. 19 Neutrophils can secrete tissue inhibitors of metalloproteinase (TIMP)-free MMP-9 that can act in concert with, for example, macrophages to liberate proangiogenic growth factors such as VEGF and FGF-2 that are sequestered to the extracellular matrix.…”

“…FGF-2 signaling has been associated with neutrophil-mediated angiogenesis 23 and PAI-1 activity. 22 Immunohistochemical analysis of ischemic muscle tissues demonstrated that the number of F4/80 ϩ cells coexpressing FGF-2 or VEGF-A was not significantly different from vehicle-and PAI-1 inhibitor-treated tissues ( Figure 6B).…”

The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

“…After the Jak-2-STAT3 pathway is activated, the expression of downstream target genes may be up-regulated or down-regulated. These genes are related to tumor angiogenesis including Vegf, Hif-1a, bFgf, Mmp-2, Mmp-7, Mmp-9, and so on (Xie et al 2004, Jung et al 2007, Ardi et al 2009, Behera et al 2010, Ferrara 2010. It can be inferred that the activation of Jak-2-STAT3 can promote tumor angiogenesis.…”

Matrix metalloproteinases in head and neck cancer: current perspectives

“…117 Furthermore, thrombin-mediated invasion of U2 osteosarcoma cells involved a PEX9 and β 1 -integrin association, 118 while MMP9 catalytic and PEX domains have been reported to induce FGF2-mediated angiogenesis in neutrophils. 119 With respect to other MMPs, Suenaga et al 120 reported that binding of CD44 to the MMP14 PEX domain is critical for shedding of human fibrosarcoma and breast carcinoma cells. In addition, Eisenach et al described an MMP14-VEGFR2-Src complex formation that controls VEGFR2 cell-surface localization through hemopexin-dependent activity in breast cancer cells.…”