Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo

Abstract

Matrix metalloproteinases (MMPs), a specialized group of enzymes capable of proteolytically degrading extracellular matrix proteins, have been postulated to play an important role in angiogenesis. It has been suggested that MMPs can regulate neovascularization using mechanisms other than simple remodeling of the capillary basement membrane. To determine the interplay between vascular endothelial growth factor (VEGF) and MMPs, we investigated the induction of angiogenesis by recombinant active MMPs and VEGF in vivo. Using a rat corneal micropocket in vivo angiogenesis assay, we observed that the active form of MMP-9 could induce neovascularization in vivo when compared with the pro- form of the enzyme as a control. This angiogenic response could be inhibited by neutralizing VEGF antibody, which suggests that MMPs acts upstream of VEGF. Additional in vitro studies using extracellular matrix loaded with radiolabeled VEGF determined that active MMPs can enzymatically release sequestered VEGF. Interestingly, in vivo angiogenesis induced by VEGF could be inhibited by MMP inhibitors, indicating that MMPs also act downstream of VEGF. In addition, inflammation plays an important role in the induction of angiogenesis mediated by both VEGF and MMPs. Our results suggest that MMPs act both upstream and downstream of VEGF and imply that potential combination therapies of VEGF and MMP inhibitors may be a useful therapeutic approach in diseases of pathological neovascularization.

Figure 1

Active MMP-2 and MMP-9 induces angiogenesis in a rat corneal micropocket assay. Representative photographs of rat corneas (n ≥ 5 for each treatment) at 7 days after implantation of pellets containing buffer (control, A), pro-MMP-9 (100 ng, B), active MMP-9 (100 ng, C), pro-MMP-2 (100 ng, D), and active MMP-2 (E). Mean vessel extension (F) and area (mm²G) were calculated as described in Materials and Methods. **P = 0.003, *P = 0.008.

Figure 2

Neutralization of MMP-9 induced angiogenesis by monoclonal anti-VEGF antibody. Representative photographs of rat corneas (n ≥ 5 for each treatment) at 7 days following implantation of pellets containing active MMP-9 (100 ng, A), active MMP-9 (100 ng) with MMP inhibitor (2 μg, B), active MMP-9 (100 ng) with nonspecific control IgG (1.5 μg, C), and active MMP-9 (100 ng) with anti-VEGF neutralizing antibody (1.5 g, D). E: Mean vessel extension and area (mm²) were calculated as described in Materials and Methods. *P = 0.005, **P = 0.001, ***P < 0.0001.

Figure 3

Increased VEGF expression in corneas implanted with pellets containing active MMP-9 and MMP-2. VEGF levels in corneas implanted with pellets containing active MMPs were analyzed by enzyme-linked immunosorbent assay (A) and expressed as picograms of VEGF per microgram of total protein. Immunohistochemical localization of VEGF protein in corneas 24 hours following implantation with control buffer (C), VEGF (D), active MMP-2 (E), and active MMP-9 (F). Representative image of cornea implanted with control buffer and stained with a nonspecific control IgG (B) to determine specificity of staining.

Figure 4

Active MMPs can release VEGF sequestered in the extracellular matrix. ECM preparations containing sequestered [¹²⁵I]VEGF were treated with active MMP-2 and active MMP-9 for 18 hours. Supernatant and ECM fractions were analyzed for [¹²⁵I]VEGF counts per minute.

Figure 5

Active MMPs can induce endothelial cell migration. The ability of active MMP-2 and MMP-9 to induce endothelial cell chemotaxis was examined using a modified Boyden chamber assay. Migrating cell number is expressed as fold increase in number of cells migrating toward control buffer ± SEM of quadruplicate samples. All experiments were done in triplicate.

Figure 6

Neutralization of VEGF induced angiogenesis by MMP inhibitor. Representative photographs of rat corneas (n ≥ 4 for each treatment) at 7 days following implantation of pellets containing VEGF (50 ng, A), VEGF (50 ng) with nonspecific control IgG (1.5 μg, B), VEGF (50 ng) with anti-VEGF neutralizing antibody (1.5 μg, C), and VEGF (50 ng) with MMP inhibitor (2 μg, D). Mean vessel extension (E) and area (mm², F) were calculated as described in Materials and Methods. *P = 0.0004, **P < 0.0001.

Figure 7

Increased MMP-2 and MMP-9 expression in corneas implanted with pellets containing VEGF. MMP-2 and MMP-9 levels in three corneas implanted with pellets containing VEGF or control buffer were analyzed by zymography (A). In situ zymography analysis of corneas 24 hours following implantation with control pellets (B, D) and VEGF pellets (C, E). Asterisks indicate the location of pellets.

Figure 8

VEGF- and active MMP-induced corneal neovascularization is mediated by inflammatory cells. Representative photographs of mouse corneas (n = 4 for each pellet) at 7 days following implantation of pellets in wild-type (WT) mice containing buffer (A), VEGF (100 ng, C), active MMP-9 (100 ng, E), active MMP-2 (100 ng, G), and in SCID mice containing buffer (B), VEGF (100 ng, D), active MMP-9 (100 ng, F), and active MMP-2 (100 ng, H). Immunohistochemical staining of CD11b in corneas of wild-type mice 24 hours following implantation with buffer (I), VEGF (50 ng, J), active MMP-9 (100 ng, K), and active MMP-2 (100 ng, L). M: Mean vessel extension was calculated as described in Materials and Methods. **P = 0.06, *P < 0.005.