Co‑expression of tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 improves myocardial ischemia‑reperfusion injury by promoting angiogenesis and inhibiting oxidative stress

Figures

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  Figure 1.. Ad-hTK1/hTIMP1 significantly ameliorates myocardial injury and improves cardiac function in myocardial I/R model rats. (A) H&E, Masson and Sirius red staining were performed on cardiac tissue sections of rats in the sham, I/R and I/R + Ad-hTK1/hTIMP1 groups. Blue in Masson-stained sections and red in Sirius red-stained sections represent collagen deposition. Scale bar, 100 µm. (B) Myocardial infarction size was measured via triphenyl-tetrazolium-chloride staining. Scale bar, 5 mm. Echocardiographic measurement of the two cardiac function parameters (C) LVFS and (D) LVEF. *P<0.05 and ***P<0.001 vs. Sham; #P<0.05 and ##P<0.01 vs. I/R. Ad, adenovirus; hTK1, human tissue kallikrein 1; hTIMP1, human tissue inhibitors of matrix metalloproteinase 1; I/R, ischemia/reperfusion; LVFS, left ventricular fractional shortening; LVEF, left ventricular ejection fraction.

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  Figure 2.. Ad-hTK1/hTIMP1 promotes microvessel formation, and reduces MMP2 and MMP9 expression levels in myocardial I/R model rats. Microvessels in the myocardium of rats in the sham, I/R and I/R + Ad-hTK1/hTIMP1 groups were (A) detected via immunofluorescence staining using an anti-CD31 primary antibody and (B) quantified. Scale bar, 100 µm. (C) Immunohistochemical staining of vWF-positive cells in the myocardium of rats in the sham, I/R and I/R + Ad-hTK1/hTIMP1 groups. Scale bar, 100 µm. (D) MMP2 and MMP9 mRNA expression levels in rat hearts. MMP2 and MMP9 protein expression levels in rat hearts were (E) determined via western blotting and (F) semi-quantified. *P<0.05 and **P<0.01 vs. Sham; #P<0.05 vs. I/R. Ad, adenovirus; hTK1, human tissue kallikrein 1; hTIMP1, human tissue inhibitors of matrix metalloproteinase 1; MMP, matrix metallopeptidase; I/R, ischemia/reperfusion; vWF, von Willebrand Factor.

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  Figure 3.. Ad-hTK1/hTIMP1 significantly reduces oxidative stress in myocardial I/R model rats. Measurement of serum levels of oxidative stress biomarkers, including (A) GSH, (B) SOD, (C) CAT, (D) GST and (E) MDA, in rats in the sham, I/R and I/R + Ad-hTK1/hTIMP1 groups. *P<0.05 and **P<0.01 vs. Sham; #P<0.05 vs. I/R. Ad, adenovirus; hTK1, human tissue kallikrein 1; hTIMP1, human tissue inhibitors of matrix metalloproteinase 1; I/R, ischemia/reperfusion; GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; GST, glutathione-S-transferase; MDA, malondialdehyde.

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  Figure 4.. Ad-hTK1/hTIMP1 significantly enhances H/R-treated CMVEC proliferation, migration and tube formation. The cell model with H/R treatment was established in CMVECs. (A) CMVEC proliferation was detected by performing the MTT assay. (B) CMVEC migration was assessed by conducting wound healing assays. The area between the two red lines indicates the wound area. Scale bar, 200 µm. CMVEC migration was also (C) measured by performing a Transwell assay and (D) quantified. Scale bar, 100 µm. CMVEC tube formation was (E) assessed by conducting a tube formation assay and (F) quantified. Scale bar, 100 µm. *P<0.05 and **P<0.01 vs. Control; #P<0.05 vs. H/R. Ad, adenovirus; hTK1, human tissue kallikrein 1; hTIMP1, human tissue inhibitors of matrix metalloproteinase 1; H/R, hypoxia/reoxygenation; CMVEC, cardiac microvascular endothelial cells; I/R, ischemia/reperfusion.

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  Figure 5.. Ad-hTK1/hTIMP1 significantly decreases intracellular ROS production and γ-H2A.X expression levels in H/R-treated CMVECs. (A) Measurement of intracellular ROS (green fluorescence) in CMVECs. Nuclei were labelled using DAPI (blue fluorescence). Scale bar, 100 µm. (B) The ratio of green fluorescence/nuclei staining was quantified. γ-H2A.X and H2A.X protein expression levels in CMVECs were (C) determined via western blotting and (D) semi-quantified. **P<0.01 vs. Control; #P<0.05 vs. H/R. Ad, adenovirus; hTK1, human tissue kallikrein 1; hTIMP1, human tissue inhibitors of matrix metalloproteinase 1; ROS, reactive oxygen species; H/R, hypoxia/reoxygenation; CMVEC, cardiac microvascular endothelial cells; H2A.X, H2A.X variant histone; TBP, TATA binding protein.