Coadministration of Adenoviral Vascular Endothelial Growth Factor and Angiopoietin-1 Enhances Vascularization and Reduces Ventricular Remodeling in the Infarcted Myocardium of Type 1 Diabetic Rats
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Figures
- FIG. 1.
A: Representative micrographs showing the in vivo transfection efficiency of Ad.LacZ in the nondiabetic sham-operated groups. Robust infection of the myocardium as assessed by β-galactosidase staining in the viable cardiac muscle surrounding the sites of gene transfer can be seen in the Ad.LacZ-transfected myocardium. B: Expression of VEGF as assessed by immunohistochemical staining. C: Expression of Ang-1 as assessed by immunohistochemical staining. The decrease in the expression of VEGF and Ang-1 is evident in the diabetic DS and DLZMI groups compared with the respective nondiabetic CS and CLZMI groups. Increase in the expression of VEGF and Ang-1 can be seen in the groups that received the combination gene therapy (CVAMI and DVAMI) compared with their respective (CLZMI and DLZMI) Ad.LacZ-treated groups. (−) represents representative micrographs showing the sections in which primary antibody was not added to verify the specificity of the staining protocol. CS, nondiabetic control sham; DS, diabetic sham; CLZMI, nondiabetic control animals that received Ad.LacZ injections; DLZMI, diabetic animals that received Ad.LacZ injections; CVAMI, nondiabetic control animals that received combination gene therapy; and DVAMI, diabetic animals that received combination gene therapy. (A high-quality color digital representation of this figure is available in the online issue.)
- FIG. 2.
Effect of combination gene therapy on myocardial fibrosis 7 and 30 days after gene therapy. A: Representative images show myocardial infarct and fibrosis after 7 days of MI and combination gene therapy. B: Representative images show myocardial infarct and fibrosis after 30 days of MI and combination gene therapy. There is no evident fibrosis in the CS and DS groups. A thinner infarct and significant fibrosis is evident in the CLZMI and DLZMI groups. Combination gene therapy in the CVAMI and DVAMI groups resulted in a thicker infarct containing islands of viable cardiac tissue. CS, nondiabetic control sham; DS, diabetic sham; CLZMI, nondiabetic control animals that received Ad.LacZ injections; DLZMI, diabetic animals that received Ad.LacZ injections; CVAMI, nondiabetic control animals that received combination gene therapy; and DVAMI, diabetic animals that received combination gene therapy. (A high-quality color digital representation of this figure is available in the online issue.)
- FIG. 3.
Effect of combination gene therapy on capillary and arteriolar density 7 days after the intervention. Representative images (A) and graphical representation (B) of capillary density analysis among the different groups (counts/mm2). Representative images (C) and graphical representation (D) of arteriolar density analysis among the different groups (counts/mm2). There was a significant increase in the capillary and arteriolar density in the CVAMI and DVAMI groups compared with the CLZMI and DLZMI groups, respectively. CS (black solid bar), nondiabetic control sham; DS (gray solid bar), diabetic sham; CLZMI (white bar), nondiabetic control animals that received Ad.LacZ injections; DLZMI (diagonal brick bar), diabetic animals that received Ad.LacZ injections; CVAMI (wave bar), nondiabetic control animals that received combination gene therapy; and DVAMI (wide upward diagonal bar), diabetic animals that received combination gene therapy. Values are presented as mean ± SEM. *P ≤ 0.05 when DS is compared with CS, #P ≤ 0.05 compared with CLZMI, †P ≤ 0.05 when DVAMI is compared with DLZMI. (A high-quality color digital representation of this figure is available in the online issue.)
- FIG. 4.
Effect of combination gene therapy on the expression of VEGF, Flk-1, Ang-1, Tie-2, and survivin 4 days after the intervention. A: Representative Western blots of VEGF, Flk-1, Ang-1, Tie-2, and survivin comparing CS and DS groups. B: Bar graphs show the quantitative difference in expression of VEGF, Flk-1, Ang-1, Tie-2, and survivin between the CS and DS groups. C: Representative Western blots for VEGF, Flk-1, Ang-1, Tie-2, and survivin comparing the CLZMI, DLZMI, CVAMI, and DVAMI groups, 4 days after the therapy. D: Bar graphs show the quantitative difference in expression of VEGF, Flk-1, Ang-1, Tie-2, and survivin among the CLZMI, DLZMI, CVAMI, and DVAMI groups. There was a significant increase in the expression of these proteins in the CVAMI and DVAMI groups compared with the CLZMI and DLZMI groups, respectively. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. CS (black solid bars), nondiabetic control sham; DS (gray solid bars), diabetic sham; CLZMI (white bar), nondiabetic control animals that received Ad.LacZ injections; DLZMI (diagonal brick bar), diabetic animals that received Ad.LacZ injections; CVAMI (wave bar), nondiabetic control animals that received combination gene therapy; and DVAMI (wide upward diagonal bar), diabetic animals that received combination gene therapy. Values given as mean ± SEM. *P ≤ 0.05 when DS is compared with CS, #P ≤ 0.05 compared with CLZMI, †P ≤ 0.05 when DVAMI is compared with DLZMI.
- FIG. 5.
Effect of combination gene therapy on phosphorylation of MK2 (Western blot) and DNA binding activity of NFκB (electrophoretic mobility shift assay [EMSA]). A: Representative Western blots for p-MK2 comparing CS and DS groups, 2 and 4 days after the surgery. B: Graphical representation of p-MK2 in the CS and DS groups, 2 and 4 days after the therapy. C: Representative Western blots for p-MK2 comparing CLZMI, DLZMI, CVAMI, and DVAMI groups, 2 and 4 days after the therapy. D: Graphical representation of p-MK2 in the CLZMI, DLZMI, CVAMI, and DVAMI groups, 2 and 4 days after the therapy. The gene therapy significantly increased the levels of p-MK2, 2 and 4 days after the therapy. E: EMSA analysis reveals increased DNA binding activity of NFκB in the CVAMI and DVAMI groups compared with the CLZMI and DLZMI groups, respectively. CS (black solid bars), nondiabetic control sham; DS (gray solid bars), diabetic sham; CLZMI (white bar), nondiabetic control animals that received Ad.LacZ injections; DLZMI (diagonal brick bar), diabetic animals that received Ad.LacZ injections; CVAMI (wave bar), nondiabetic control animals that received combination gene therapy; and DVAMI (wide upward diagonal bar), diabetic animals that received combination gene therapy. Values given as mean ± SEM. *P ≤ 0.05 when DS is compared with CS, #P ≤ 0.05 compared with CLZMI, †P ≤ 0.05 when DVAMI is compared with DLZMI.
- FIG. 6.
Effect of combination gene therapy on left ventricular myocardial functions (echocardiography). A: Representative echocardiograph pictures of parasternal short axis images after 30 days of surgery and therapy. Bar graphs represent LVIDs in systole (in mm) (B), % ejection fraction (C), % fractional shortening (D), and heart rate in beats/min (E). There was a significant increase in the myocardial functions in the CVAMI and DVAMI groups compared with the CLZMI and DLZMI groups, respectively. CS (black solid bars), nondiabetic control sham; DS (gray solid bars), diabetic sham; CLZMI (white bar), nondiabetic control animals that received Ad.LacZ injections; DLZMI (diagonal brick bar), diabetic animals that received Ad.LacZ injections; CVAMI (wave bar), nondiabetic control animals that received combination gene therapy; and DVAMI (wide upward diagonal bar), diabetic animals that received combination gene therapy. Values given as mean ± SEM. *P ≤ 0.05 when DS is compared with CS, #P ≤ 0.05 compared with CLZMI, †P ≤ 0.05 when DVAMI is compared with DLZMI.
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