Fig 1.
Time course and sources of MMP-2 expression in MMP-2+/+ livers.
MMP-2 mRNA expression (panel A) and MMP-2 activity (panel B) were readily detected in wild-type livers before and after hepatic IRI. MMP-2 positive staining was detected in the vasculature of naïve MMP-2+/+ livers (panel C); MMP-2 in red (a; Alexa Fluor 594), PECAM-1 in green (b; Alexa Fluor 488), nuclear stain in blue (c; Dapi), and staining overlay (d). In addition to the vasculature, MMP-2 positive staining was also detected in Ly6G neutrophils in MMP-2+/+ livers at 6h post-IRI (panel D); MMP-2 in red (a and b), Ly6G in green (c and d) and staining overlay (e and f); (arrows and arrow heads denote MMP-2 positive staining in the liver vasculature and in infiltrating leukocytes, respectively; n = 5/group; *p<0.05 relative to naïve livers).
Fig 2.
Survival, liver histology and serum transaminases in MMP-2-/- and MMP-2+/+ mice after liver IRI.
MMP-2 expression (panel A) was detected in MMP-2+/+ livers (lanes 1 and 2) and absent in MMP-2-/- livers (lanes 3 and 4) post-IRI. Survival (panel B) of MMP-2-/- deficient mice (dotted line) was significantly reduced at 7 days post-IRI, as compared with MMP-2+/+ mice (solid line). Representative H&E staining (panel C) of MMP-2+/+ (a, c, and e) and MMP-2-/- (b, d, and f) livers at 6h (a, and b), 24h (c, and d), and 48h (e, and f) post-I/R injury showed that disruption of lobular architecture was significantly exacerbated in MMP-2-/- livers post-reperfusion. The serum transaminase levels, sAST (panel D) and sALT (panel E), were markedly increased in MMP-2-/- mice, particularly at 6h and 24h after IRI; (n = 5–6 mice/group *p<0.05).
Fig 3.
Leukocyte infiltration in MMP-2-/- and MMP-2+/+ livers.
Representative immunostaining of Ly-6G+ neutrophils (panel A) and Mac-1+ leukocytes (panel B) in MMP-2+/+ (a, and b) and MMP-2-/- (c, and d) livers at 6h (a, and c) and 24h (b, and d) post-reperfusion; infiltrating leukocytes were detected in significantly higher numbers in livers of MMP-2-/- mice after IRI (panel C). MPO enzymatic activity (panel D) was also markedly upregulated in MMP-2-/- livers post-IRI (n = 5/group; *p<0.05).
Fig 4.
Pro-inflammatory cytokine expression in MMP-2-/- and MMP-2+/+ livers.
IFN-γ, TNF-α, and IL-6, were upregulated in MMP-2-/- deficient livers post-IRI, compared to respective controls (n = 5/group; *p<0.05).
Fig 5.
Serum transaminases, liver histology and leukocyte infiltration/ activation in anti-MMP-2 antibody treated mice.
AST (panel A) and ALT (panel B) levels were elevated in blood samples of anti-MMP-2 antibody treated mice collected 6h after liver IRI. Liver histological preservation (panel C) was further impaired in anti-MMP-2 antibody treated mice (b), compared to IgG treated controls (a). Moreover, MPO activity (panel D), Mac-1 leukocyte infiltration (panel E) and proinflammatory cytokine expression (panel F) were all increased in anti-MMP-2 antibody treated mice, compared to IgG treated controls (n = 4/group; *p<0.05).
Fig 6.
Effect of MMP-2 selective inhibition on MMP-9 activity and MMP-9-dependent leukocyte migration.
MMP-9 activity (panel A) was increased in MMP-2-/- livers (lanes 3, 4, 7, 8, 11, and 12), compared to respective MMP-2+/+ controls (lanes 1, 2, 5, 6, 9, and 10); naïve livers (lanes 1 to 4), and livers harvested at 6h (lanes 5 to 8) and 24h (lanes 9 to 12) post-IRI. Infiltrating MMP-9+ leukocytes (panel B) were detected in higher numbers in MMP-2-/- livers than in MMP-2+/+ livers. Representative immunostaining (panel C) shows that MMP-9+ leukocytes were virtually absent from MMP-2+/+ naïve livers (a), but readily detected in MMP-2-/- naïve livers (b); MMP-9+ leukocyte infiltration was also lower in MMP-2+/+ livers (c) when compared to MMP-2-/- livers (d) at 6h post-reperfusion. MMP-9 activity (panel D) was significantly increased in isolated MMP-2+/+ neutrophils treated with a selective MMP-2 inhibitor in doses of 100nM (lane 2) and 500nM (lane 3) and in MMP-2-/- neutrophils (lane 4), compared to respective MMP-2+/+ controls (lane1). Moreover, in vitro cell migration (panel E) was significantly enhanced in MMP-2+/+ neutrophils treated with a MMP-2 selective inhibitor and in MMP-2-/- neutrophils; on the other hand, the addition of a MMP-9 selective inhibitor to MMP-2-/- neutrophils significantly reduced their migration (n = 5/group; in vitro data is expressed as mean ± SD of four independent experiments; *p<0.05 relative to untreated MMP-2+/+ samples and **p<0.05 relative to untreated MMP-2-/- samples).
Fig 7.
Effect of MMP-2 activity inhibition on PARP-1 degradation.
The cleaved form of PARP-1 (panel A) was detected in the MMP-2+/+ livers (lanes 1 and 2) and virtually absent in MMP-2-/- livers (lanes 3, and 4) at 6h post-IRI; the cleaved PARP/total PARP ratio was profoundly depressed in the MMP-2-/- livers post-reperfusion. The cleaved PARP/total PARP ratios (panel B) were also markedly decreased in lysates of MMP-2-/- SECs and MMP-2 inhibitor-treated MMP-2+/+ SECs. The LDH levels (panel C) were increased in MMP-2-/- SECs and in MMP-2 inhibitor-treated MMP-2+/+ SECs, compared with controls (n = 4–5/group; in vitro data is expressed as mean ± SD of four independent experiments; *p<0.05 relative to MMP-2+/+ samples and **p<0.05 relative to MMP-2-/- samples).
Fig 8.
Effect of PJ34 (PARP inhibitor) administration in hepatic IRI.
Administration of PJ34 to wild-type mice and to MMP-2-/- mice at reperfusion reduced the serum AST levels (panel A) in these mice at 6h post-IRI. MMP-2-/- mice treated with PJ34 showed improved liver histological preservation (panel B) post-IRI; H&E staining of vehicle treated-MMP-2-/- (a) and PJ34 treated-MMP-2-/- (b) livers post-reperfusion. MMP-9 activity was significantly depressed in MMP-2 null mice treated with PJ34 at 6h after IRI (n = 4–5/group; *p<0.05).
Fig 9.
Anti-MMP-2 antibody therapy in MMP-9 null mice.
The serum AST levels (panel A) in MMP-9-/- mice treated with anti-MMP-2 antibodies at 6h of liver IRI were significantly reduced when compared to wild-type mice treated with anti-MMP-2 antibodies and increased when compared to MMP-9-/- mice treated with IgG. Histological preservation (panel B) was markedly improved in MMP-9 null livers treated with MMP-2 mAb at 6h after IRI (b), when compared to the extensively damaged anti-MMP-2 mAb treated WT livers (a), but inferior when compared to MMP-9-/- livers treated with control IgG (c) (n = 4/group; *p<0.05 relative to WT+MMP-2mAb; **p<0.05 relative to MMP-9-/-+IgG).