Fig 1.
In vitro transfection efficiency and cytotoxicity of rPOA as a gene carrier.
(A) Optimization of pCMV-Luc transfection into H9c2 cardiomyocytes using three different weight ratios (1:1, 1:2, and 1:4) of rPOA and PEI25K (1:1) as a positive control. Transfection efficiencies were measured by luciferase assay. Data are expressed as the mean ± SD of three experiments. *P < 0.001 vs. PEI. (B) Green fluorescence protein (GFP) expression in rPOA-transfected H9c2 cardiomyocytes. pEGFP-C1 plasmids were mixed with indicated carrier and ratio. A 465–495 nm excitation filter was used to detect GFP expression. (C) GFP-expressing cells were analyzed by flow cytometry to determine the percentage of GFP-expressing cells. (D) Cytotoxicity of rPOA vs. PEI in H9c2 cells. Polymer/pDNA complexes were transfected into H9c2 cells, and cell viability was measured 24 h later by MTT assay. Data are expressed as the mean ± SD. *P < 0.005 vs. control and PEI transfections.
Fig 2.
In vivo transfection efficiency and cytotoxicity of rPOA as a gene carrier.
(A) Representative cardiac tissue sections with H&E staining and GFP fluorescence analysis 3 days after injection of pEGFP-C1, PEI/GFP, or rPOA/GFP into rat hearts (400× magnification). Scale bar, 100 μm. (B) Quantitative analysis of GFP expression across different groups. *P < 0.005, **P < 0.001 vs. control. (C) H&E staining of saline-, PEI-, or rPOA-injected rat myocardial tissue. (D) Quantitative analysis of nuclear stained cells as a measure of left ventricular cellular infiltration post injection with saline (n = 4), PEI (n = 5), or rPOA (n = 6). **P < 0.001 between groups.
Fig 3.
Therapeutic application of rPOA in rat MI model with VEGF expression.
(A) VEGF expression in the rat myocardium 1 week after the injection of PEI/pVEGF or rPOA/pVEGF complexes, as visualized by anti-VEGF antibody labeling (200×). Red arrows indicated brown staining of VEGF expression. (B) Quantitative analysis of VEGF immunoreactivity per mm2 tissue. Average values were obtained from five random high magnification fields in the infarct border zone from each animal. Data represent mean ± SD. *P < 0.05.
Fig 4.
Histological analysis of gene therapy-treated MI hearts.
(A) Representative picture of myocardial sections stained with 2,3,5-triphenyltetrazolium chloride (TTC). Pale yellow indicates infarct region. (B) Ratio of infarcted to non-infarcted left ventricular myocardium (%) from LAD-ligated rats injected with saline, PEI/pVEGF, or rPOA/pVEGF (n = 5/group). *P < 0.0005, **P < 0.00001 vs. saline. (C) Representative myocardium sections stained with Masson’s trichrome (lower panel, 200×). Scale bar, 200 μm. (D) Percent of myocardial collagen fibrosis expressed as the ratio of fibrotic area to left ventricle area in LAD-ligated rats injected with saline, PEI/pVEGF, or rPOA/pVEGF (n = 5/group). *P < 0.005, **P < 0.00001. (E) Myocardial sections labeled with an antibody against CD31 to detect neovascularization 1 week after injection (400×). Scale bar, 100 μm. (F) Quantitative analysis of CD31-positive cells in ischemic myocardia treated with saline, PEI/pVEGF, or rPOA/pVEGF (n = 5/group). *P < 0.05, **P < 0.005.