PRDX2 in Myocyte Hypertrophy and Survival is Mediated by TLR4 in Acute Infarcted Myocardium

Figures

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  Figure 1. Hypoxia-induced oxidative stress inhibits H9c2 viability and promotes H9c2 apoptosis. H9c2 were exposed to hypoxia conditions for 0, 3, 6, 12 or 24 h before cell activity, ROS production and apoptosis-related protein were measured. (A and B) Mitochondrial ROS production in H9c2 cells at different times of hypoxic treatment. The data are presented as means ± SD of the mean from three separate cell experiments. ***p < 0.001 vs control. Magnification 200×. (C) The effects of hypoxia treatment on H9c2 cell viability. The data are presented as means ± SD of the mean from three separate cell experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. (D–H) The expression of apoptosis-related proteins was measured by western blot. GAPDH was used as a loading control. The results show that hypoxia treatment promotes the expression of caspase-3 (E) and Bax (F) in H9c2 cells but inhibits Bcl-2 expression (G). The results also show that hypoxia promotes the expression of PRDX2 which may be related to anti-oxidative stress. The data are presented as means ± SD of the mean from three separate cell experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

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  Figure 2. The expression of PRDX2 inhibit hypoxia-mediated oxidative stress injury to H9c2. (A) H9c2 cells were transfected with PRDX2 siRNA (siPRDX2) and control siRNA (siSCR) at a concentration of 80 pmols. Cells were harvested 36 h after the transfection, and the silencing effect on PRDX2 mRNA levels was determined by RT-PCR. The silencing effect of PRDX2 siRNA at the protein level was determined 48 h post-transfection using western blotting. The data are presented as means ± SD of the mean from five separate cell experiments. ***p < 0.001 vs control. (B) H9c2 cells were transfected with a PRDX2 overexpression vector and control (SCR). Cells were harvested 48 h after the transfection, and the PRDX2 mRNA levels were determined by RT-PCR. The overexpression effect of PRDX2 at the protein level was determined 48-h post-transfection using western blotting. GAPDH was used as a loading control. The data are presented as means ± SD of the mean from five separate cell experiments. ***p < 0.001 vs control. (C and D) Mitochondrial ROS production in H9c2 cells after culture in normal hypoxic conditions for 6 h with/without PRDX2 overexpression or down-regulation. The data are presented as means ±…

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  Figure 3. PRDX2 expression promote TLR4 mediated VEGF and inflammatory factor expression in hypoxia condition. (A) H9c2 cells were transfected with TLR4 siRNA (siTLR4) and control siRNA (siSCR) at a concentration of 80 pmols. Cells were harvested 36 h after the transfection, and the silencing effect on TLR4 mRNA levels was determined by RT-PCR. The silencing effect of TLR4 siRNA at the protein level was determined 48 h post-transfection using western blotting. GAPDH was used as a loading control. The data are presented as means ± SD of the mean from five separate cell experiments. ***p < 0.001 vs control. (B) Total protein was collected from H9c2 cells which were exposed in hypoxic condition for 6 h then cultured in normal condition for 24 h. The control group was exposed to normal conditions. VEGF protein levels were determined by performing western blot. The data are presented as means ± SD of the mean from three separate cell experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. (C–F) Supernatants from H9c2 cells were harvested and analyzed by enzyme-linked immunosorbent assay (ELISA) for TNF-α, IL-6, IL-1β and VEGF after exposure to hypoxic conditions for 6 h and then cultured…

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  Figure 4. PRDX2 overexpression in H9c2 cells promote HUVEC migration, vasculogenic mimicry formation and myocardial hypertrophy related protein expression. H9c2 cells were cultured in serum-free medium and exposed to hypoxic conditions for 6 h then in normal conditions for another 24 h, then the medium was harvested for transwell and vasculogenic mimicry formation of HUVECs. (A) Migration of HUVECs was assessed using transwell chambers. The invasive capabilities of HUVECs are significantly increased with chemotaxis of PRDX2 overexpression in H9c2 cells. The data are presented as means ± SD of the mean from ten separate cell experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. (B) Images of tube formation. Tube length is presented as percent of total tube length per field versus untreated control cells. The data are presented as means ± SD of the mean from ten separate cell experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. (C) VEGFR2 phosphorylation in the surface of HUVECs was detected by western blots. The phospho-specific bands were quantified and normalized by the intensities of the corresponding VEGFR2 bands. The data are presented as means ± SD of the mean from…

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  Figure 5. The overexpression of PRDX2 inhibits ROS levels and myocardial injury after AMI but promotes inflammatory responses. After 2 weeks of coronary artery ligation, serum from mice was harvested for ELISA analysis and after 1 month, myocardial tissue was used for immunohistochemical analysis. (A–C) ELISA for inflammatory cytokines. The data are presented as means ± SD from three separate experiments from five to six mice in each group. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. (D) Representative images of infarct size as stained by TTC. (E) PRDX2 decreased infarct size compared with other groups. The data are presented as means ± SD from five to six separate mice in each group. **p < 0.01, ***p < 0.001 vs control. (F) Immunofluorescence for ROS generation by DCF-DA. (G) The results show that the expression of PRDX2 inhibits generation of oxidative stress and the inhibition of PRDX2 on ROS was enhanced with TLR4 knock out. The data are presented as means ± SD from ten separate fields. ***p < 0.001 vs control. (H and I) TUNEL analysis of apoptosis in myocardial infarction. Statistical analysis of apoptosis. AB, aortic banding. The data are presented as means ± SD from three…

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  Figure 6. The overexpression of PRDX2 promote angiogenesis and myocardial hypertrophy. After 2 weeks of coronary artery ligation, serum from mice was harvested for RT-PCR analysis and myocardial tissue were harvested 4 weeks after artery ligation for immunohistochemical analysis. (A) Effects of PRDX2 on cell surface area of cardiomyocytes. (B) Cell surface area was calculated by measuring 10 random cardiomyocytes. (C–E) RT-PCR for ANP (C), PRDX2 (D) and VEGF (E) expression. The data are presented as means ± SD from three separate experiments from five to six mice in each group. *p < 0.05, ***p < 0.001 vs normal WT group. (F) The formation of microvascular cells in infarction region were stained with CD31. AB, aortic banding. The data are presented as means ± SD from 10 fields in each group. *p < 0.05, ***p < 0.001 vs normal WT group.