Matrix metalloproteinase-9 is required for adequate angiogenic revascularization of ischemic tissues: potential role in capillary branching

Abstract

Angiogenesis, an essential component of a variety of physiological and pathological processes, offers attractive opportunities for therapeutic regulation. We hypothesized that matrix metalloproteinase-9 genetic deficiency (MMP-9-/-) will impair angiogenesis triggered by tissue ischemia, induced experimentally by femoral artery ligation in mice. To investigate the role of MMP-9, we performed a series of biochemical and histological analyses, including zymography, simultaneous detection of perfused capillaries, MMP-9 promoter activity, MMP-9 protein, and macrophages in MMP-9-/- and wild-type (WT) mice. We found that ischemia resulted in doubling of capillary density in WT and no change in the MMP-9-/- ischemic tissues, which translated into increased (39%) perfusion capacity only in the WT at 14 days after ligation. We also confirmed that capillaries in the MMP-9-/- presented significantly (P<0.05) less points of capillary intersections, interpreted by us as decreased branching. The combined conclusions from simultaneous localizations of MMP-9 expression, capillaries, and macrophages suggested that macrophage MMP-9 participates in capillary branching. Transplantation of WT bone marrow into the MMP-9-/-, restored capillary branching, further supporting the contribution of bone marrow-derived macrophages in supplying the necessary MMP-9. Our study indicates that angiogenesis triggered by tissue ischemia requires MMP-9, which may be involved in capillary branching, a potential novel role for this MMP that could be exploited to control angiogenesis.

Figure 1.

MMP-9 expression and activity are induced at early time points in hindlimb muscle tissues exposed to ischemia. MMP-9 protein expression was assayed in the nonischemic (day 0) or ischemic (1, 3, 7, and 14 days) WT adductor muscle tissue lysates using SDS-PAGE gelatin zymography (top) and Western blotting (middle). Both indicate early induction of MMP-9 (day 1), with levels peaking at 3 days and returning to baseline by 7 days after ischemia (latent “Pro” MMP-9 and activated MMP-9). Quantification of total gelatinolytic activity associated with MMP-9 or MMP-2 (both pro- and activated bands) in WT and MMP-9^−/− tissue lysates, normalized using standards (“MMP Stds”) (n=4 animal for each time point), illustrates the significant early induction of MMP-9 in response to ischemia in the WT, and similar induction of MMP-2 in the WT and MMP-9^−/−. *P<0.05 vs 0 day time point. For a detailed separate analysis of MMP-2 and MMP-9, see online Figure 1.

Figure 2.

Capillary histochemical affinity staining indicates lack of an ischemia-induced angiogenic response in MMP-9^−/− mice. Examination of tissue cross-sections stained with endothelial specific Griffonia simplicifolia lectin (green) indicates that capillary density increases in WT, but not in MMP-9^−/− tissues in response to ischemia (scale bar=20 μm). All nuclei counterstained with Hoechst (blue). Insets illustrate in addition BrdU-positive (red) immunodetection of proliferating cells (pink, due to localization to nuclei counterstained in blue) with capillary staining (green), as well as a negative control with no primary antibody (“No 1⁰”). Proliferating cells were found only in ischemic WT tissues supporting the presence of a robust angiogenic response. Quantification of capillary density displays a significant increase in capillary number within WT but not within MMP-9^−/− ischemic tissues (n=4 for each point, *P<0.05 vs MMP-9^−/−), supporting MMP-9 role in ischemia-induced angiogenic response.

Figure 3.

MMP-9 deficiency decreases restoration of microvascular perfusion capacity in response to ischemia. Time course of perfusion capacity imaged on whole-mount tissue specimens using fluorescence microangiography (scale bars=200 μm). Nonischemic muscle contains numerous perfused capillaries with no apparent differences between WT and MMP-9^−/− mice. The number of perfused capillaries drops dramatically after femoral artery ligation, illustrated at day 3 in both WT and in MMP-9^−/− tissues, in spite of existing anatomical structures (illustrated by specific staining in Figure 2). Perfusion capacity is significantly better restored in WT tissues compared with the MMP-9^−/− tissues (day 7), and actually becomes higher than the baseline at day 14, whereas it remains deficient in MMP-9^−/− tissue, supporting the role of MMP-9 for proper revascularization. In addition, note the increased number of points of capillary intersections, indicative of branching, and increased tortuosity in WT compared with MMP-9^−/− ischemic tissue (day 14) or compared with nonischemic tissues. Vascular perfusion capacity of adductor muscle (left graph), quantified as fluorescence extracted from tissue homogenates (n=4 animals per point), dropped dramatically after femoral artery ligation in both WT and MMP-9^−/− tissue. By 14 days, WT vascular perfusion capacity surpassed initial capacity, indicating robust revascularization and angiogenesis, whereas in the MMP-9^−/− ischemic tissues, perfusion capacity remained lower than initial capacity (*P<0.05 vs WT 14 day), illustrating blunted revascularization. Quantification of capillary branching density (from confocal microscope images of tissues, n=4 animals for each point) indicated a significant increase in branching density in the WT after 14 days compared with normal nonischemic tissues (*P<0.05 vs nonischemic). On the other hand, MMP-9^−/− ischemic tissues had a decreased density of branches at 14 days (*P<0.05 vs WT), suggesting that lack of capillary branching contributed to decreased perfusion capacity.

Figure 4.

Detection of capillaries, MMP-9 promoter activity and protein, as well as macrophages suggests a role for macrophage MMP-9 in the early angiogenic response to ischemia. Left, Visualization by fluorescence microangiography of perfused capillaries (green) and MMP-9 promoter activity (red) in the GelB/LacZ Tg mice in nonischemic and ischemic tissue (day 3), indicates a perivascular localization of cells actively transcribing MMP-9 (arrows) in ischemic tissues. Inset, Typical finding of MMP-9 promoter activity at branching points. Middle, Positive detection for macrophages (red) in ischemic WT tissues was frequently observed at points of capillary (green) intersections. On the other hand, no macrophages were detected in MMP-9^−/− ischemic tissues. Inset, Negative control for staining with no primary antibody (No 1⁰). All nuclei are counterstained with Hoechst (blue). Right, Double immunohistochemistry illustrates colocalization (arrows) of (top) macrophages (green) and MMP-9 promoter activity (red, GelB/LacZ Tg mouse), as well as (bottom) macrophages (green) and MMP-9 protein (red, WT mouse). Insets demonstrate the specificity of immunostaining, ie, consecutive sections processes without primary antibody (No 1⁰) and the lack of MMP-9–positive signal in ischemic tissues harvested from the MMP-9^−/− mice. All scale bars=20 μm. Taken together, the results of these analyses suggest a link between MMP-9, macrophages, and angiogenic branching in ischemic tissues.

Figure 5.

Transplantation with WT bone marrow rescues ischemia-induced angiogenic capillary branching in the MMP-9^−/− mice. Nonischemic tissues illustrate similar levels of capillary perfusion capacity in the WT or MMP-9^−/−. Left, Fluorescence microangiography. Middle, Increased perfusion capacity was detected in WT ischemic tissues, whereas MMP-9^−/− ischemic tissues showed an impaired perfusion (day 14), consistent with inadequate angiogenesis. Transplantation (Tx) of WT bone marrow to MMP-9^−/− mice (right) rescued perfusion capacity, as suggested by the high capillary tortuosity and branching in the MMP-9^−/− ischemic tissue (top right). All scale bars=100 μm. Bottom right, MMP-9 (red)–positive macrophages (green) were detected (arrows) at early time points in the MMP-9^−/− ischemic tissues of mice transplanted with WT marrow by double immunohistochemistry (scale bar=20 μm). Inset, Demonstration that macrophages (green) are bone marrow–derived because they are positive for the donor rare allele CD45.1 (red). All nuclei are counterstained with Hoechst (blue). Graph, Quantification of capillary branching by fluorescence microangiography confirms increased branching in WT (solid bar) ischemic tissue (P<0.05 vs nonischemic) and decreased branching in MMP-9^−/− (white bar) ischemic tissue (P<0.05 vs nonischemic) and demonstrates that transplantation (Tx) of WT bone marrow into MMP-9^−/− mice (striped) increases capillary branching in MMP-9^−/− ischemic tissues vs MMP-9^−/− nonischemic tissues (*P<0.05), as well as ischemic tissues of nontransplanted MMP-9^−/− (*P<0.05), bringing it to levels comparable to WT ischemic tissues (P=NS).