Reactive oxygen species regulate osteopontin expression in a murine model of postischemic neovascularization

Abstract

Objective: Previous findings from our laboratory demonstrated that neovascularization was impaired in osteopontin (OPN) knockout animals. However, the mechanisms responsible for the regulation of OPN expression in the setting of ischemia remain undefined. Therefore, we sought to determine whether OPN is upregulated in response to ischemia and hypothesized that hydrogen peroxide (H(2)O(2)) is a critical component of the signaling mechanism by which OPN expression is upregulated in response to ischemia in vivo.

Methods and results: To determine whether ischemic injury upregulates OPN, we used a murine model of hindlimb ischemia. Femoral artery ligation in C57BL/6 mice significantly increased OPN expression and H(2)O(2) production. Infusion of C57BL/6 mice with polyethylene glycol-catalase (10 000 U/kg per day) or the use of transgenic mice with smooth muscle cell-specific catalase overexpression blunted ischemia-induced OPN, suggesting ischemia-induced OPN expression is H(2)O(2)-dependent. Decreased H(2)O(2)-mediated OPN blunted reperfusion and collateral formation in vivo. In contrast, the overexpression of OPN using lentivirus restored neovascularization.

Conclusions: Scavenging H(2)O(2) blocks ischemia-induced OPN expression, providing evidence that ischemia-induced OPN expression is H(2)O(2) dependent. Decreased OPN expression impaired neovascularization, whereas overexpression of OPN increased angiogenesis, supporting our hypothesis that OPN is a critical mediator of postischemic neovascularization and a potential novel therapeutic target for inducing new vessel growth.

Figure 1. Hind Limb Ischemia Increases OPN expression in the Proximal Region of the Ischemic Limb – Contributing Cell Types Include Macrophages, Endothelial Cells, and Smooth Muscle Cells

Femoral artery ligation was performed on C57Bl/6 mice and OPN expression was assessed in the adductor muscles of the non-ischemic (NIL) and ischemic limbs (IL) at the time points indicated. A. Full-length (FL) OPN protein expression in the IL was measured by Western blot (WB) at 0, 3, 5, 7, 14, and 21 days post-surgery. (*p<0.05 vs. day 0, 3, 14, & 21; #p<0.05 vs. day 0, 14, & 21; n=3-4). GAPDH demonstrates equal loading. B. OPN mRNA was assessed by qRT-PCR (*p<0.05, n=3). C. FL and cleaved OPN protein expression at 5d was measured by WB. (FL, *p<0.001; cleaved, #p<0.05; n=4). GAPDH demonstrates equal loading. D-F. OPN expression and distribution was assessed in the adductor muscles of the IL at 5 days in C57Bl/6 mice. In all panels, DAPI appears blue and stains the nuclei, OPN appears green, the designated cell marker appears as red, and co-localization is yellow and denoted with arrows. All panels include an image taken at 20x, to show anatomical structure, and 63x, to show co-localization detail. D. OPN (green) co-localizes with Mac3 (red), a marker for macrophages. E. OPN (green) co-localizes with lectin (red), a marker for endothelial cells. F. OPN (green) co-localizes with smooth muscle α-actin (red), a marker for SMCs. Bars are means ± S.E.M.

Figure 2. Hind Limb Ischemia Increases ROS in the Proximal Region of the Ischemic Hind Limb

ROS production was assessed in the adductor muscles of the non-ischemic (NIL) and ischemic limbs (IL) of C57Bl/6 mice at postoperative day 3, 5, and 7. A. O₂^•- production was measured using DHE-HPLC. (ns; n=5-6) B. The Amplex Red Assay was used to measure H₂O₂ levels. H₂O₂ measurements were normalized to tissue wet weight. H₂O₂ was increased in the IL compared to the NIL at 3, 5, and 7 days (*p<0.001), with a peak at 5d (#p<0.05 vs. 3d). n=3-6. Bars are means ± S.E.M.

Figure 3. OPN Expression in the Proximal Region of the Ischemic Limb is H₂O₂-dependent

H₂O₂ production and OPN expression were measured in the adductor muscles of the non-ischemic (NIL) and ischemic limbs (IL) at postoperative day 5 in C57Bl/6 mice infused with Saline or 10,000 U/kg/day PEG-Catalase (PEG-Cat). A. H₂O₂ was measured by Amplex Red Assay. H₂O₂ was increased in the IL of both treatment groups (*p<0.001 vs. Saline NIL; #p<0.05 vs. PEG-Catalase NIL); however, PEG-catalase blunted ischemia-induced H₂O₂ compared to control (†p<0.001). n=5. B. OPN mRNA levels were measured by qRT-PCR. OPN was increased in the IL of saline controls (*p<0.001 vs. Saline NIL), but not animals infused with PEG-catalase. PEG-catalase treatment blocked increased OPN mRNA in the IL (†p<0.01). n=5. C. Full-length (FL) and cleaved OPN protein expression in the IL of saline infused animals were increased at 5d by WB analysis (FL, *p<0.001; cleaved, *p<0.001; n=8). PEG-catalase (PEG-Cat) treatment blunted this increase (FL, #p<0.0001; cleaved, #p<0.0001; n=8). GAPDH demonstrates equal loading. D. PEG-catalase decreased OPN protein expression in the IL compared to control at 5d, as detected by immunofluorescence. Blue=DAPI-nuclei, Red=OPN, Green=autofluorescence. Bars are means ± S.E.M.

Figure 4. OPN Expression in the Proximal Region of the Ischemic Limb is Decreased by SMC-specific Catalase Overexpression

H₂O₂ production and OPN expression were measured at postoperative day 5 days in Tg^SMC-Cat or wild-type (WT) littermates. A. H₂O₂ production was increased in the IL of WT and Tg^SMC-Cat animals, as measured by Amplex Red (*p<0.001); however, Tg^SMC-Cat animals exhibit decreased H₂O₂ production in the IL compared to WT (†p<0.0001). H₂O₂ measurements were normalized to tissue wet weight. n=5. B. OPN mRNA levels were increased in the WT IL (*p<0.001 vs. NIL); however, OPN mRNA levels in the Tg^SMC-Cat IL was not increased compared to the NIL. Tg^SMC-Cat animals had decreased OPN mRNA in the IL compared to WT (†p<0.05). n=6. C. Full-length (FL) and cleaved OPN protein expression in the WT IL was increased at 5d by WB analysis (FL, *p<0.001; cleaved, *p<0.001; n=5). This increase was blunted in Tg^SMC-Cat animals (FL, #p<0.01; cleaved, #p<0.01; n=5). GAPDH demonstrates equal loading. D. OPN expression in the Tg^SMC-Cat IL is decreased compared to WT IL at 5d by immunofluorescence. Blue=DAPI - nuclei, Red=OPN, Green=autofluorescence. Bars are means ± S.E.M.

Figure 5. H₂O₂-dependent OPN Mediates Collateral Formation

LASER Doppler Perfusion Imaging (LDPI) was used to evaluate perfusion and Micro-CT to quantify collateral formation in the IL. Perfusion and collateral formation was assessed in the proximal regions of the IL and normalized to the NIL. All measurements were performed at 5d post-surgery. Ischemic legs were compared between treatment groups. A. Representative LDPI tracings from WT and Tg^SMC-Cat animals at day 5. B. Quantitative analysis of WT and Tg^SMC-Cat LDPI tracings (*p<0.05, n=5). C. Representative Micro-CT angiographs from WT and Tg^SMC-Cat mice at postoperative day 5. To determine the relative contribution of OPN to collateral formation compared to other H₂O₂-dependent pathways, we performed an OPN add-back experiment. Tg^SMC-Cat+LV-GFP treatment group had less reperfusion than WT+LV-GFP mice. Add-back of OPN to Tg^SMC-Cat mice using LV-OPN (Tg^SMC-Cat+LV-OPN) restored collateral formation and perfusion to the level of WT animals. D. Representative LDPI tracings from WT+LV-GFP control, Tg^SMC-Cat+LV-GFP, and Tg^SMC-Cat+LV-OPN treated animals. E. Quantitative analysis of WT+LV-GFP control, Tg^SMCCat+LV-GFP, and Tg^SMC-Cat+LV-OPN LDPI tracings. (*p<0.001 vs. Tg^SMC-Cat+LV-GFP, n=6). Bars are means ± S.E.M.

Figure 6. H₂O₂ Increases OPN Expression in Mouse Aortic Smooth Muscle Cells

Murine SMCs were used as an in vitro system. SMCs were quiesced for 48 hours prior to no treatment ( − ) or stimulation ( + ) with 100 μM H₂O₂ for 4 hours. Cells were harvested for mRNA and protein and DMEM collected to assess secreted OPN by ELISA. A. H₂O₂ stimulation increased OPN mRNA expression (*p<0.01), B. cellular OPN protein expression (*p<0.0001), measured by WB, and C. secreted OPN protein expression, measured by ELISA (*p<0.001). Bars are means ± S.E.M., n=4.