In vivo transduction of ETV2 improves cardiac function and induces vascular regeneration following myocardial infarction

Figures

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  Fig. 1. In vivo transduction of ETV2 into MI hearts. Representative confocal immunofluorescent images of sectioned heart tissues harvested 3 days after injection of control or ETV2-Ires-VENUS lentivirus into MI hearts. Anti-ACTN2, VIMENTIN, α-SMA, or CD31 in red and DAPI for nuclei in blue. Scale bars: 50 µm for merged and 20 µm for enlargement (far right panels)

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  Fig. 2. Delivery of lentiviral ETV2 into MI hearts enhances the expression of pro-angiogenic genes but decreases the expression of genes involved in fibrosis and inflammation. Heart tissues harvested 7 days after injection of lentiviral particles of ETV2-Ires-VENUS or control into MI hearts were subjected to qRT-PCR analyses. a ETV2 gene, b angiogenesis, c fibrosis, and d inflammation. The y-axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *p < 0.05 and **p < 0.01 compared to control group, n = 3

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  Fig. 3. Overexpression of ETV2 significantly improves cardiac function and reduces cardiac fibrosis in a mouse model of myocardial infarction. Mice undergoing MI were injected with lentiviral particles of ETV2-Ires-VENUS or control followed by echocardiography analysis. a, b Ejection fraction (EF) and fractional shortening (FS) were significantly higher in the ETV2-injected group compared to those in control virus-injected group, n = 7–10 for each group. c, d Representative images showing collagen deposition (blue) in the left ventricle of mouse hearts by Masson’s Trichrome staining. Bar graphs summarizing the percentage of fibrosis in the left ventricle of mouse hearts. For each heart, three randomly selected sections from the apex to ligated area were measured, n = 6 for each group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control

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  Fig. 4. ETV2 overexpression increases capillary density in MI hearts. MI hearts of mice receiving lentiviral particles of ETV2-Ires-VENUS or VENUS were perfused with rhodamine conjugated IB4 to visualize vessels. They were subjected to immunohistochemical staining with anti-GFP antibody, IB4 (red), ETV2 (green), and DAPI for nuclei (blue). a Representative images of capillaries 8 weeks after MI (upper panels) and summarized quantification (lower panels), scale bars: 100 µm. For quantification, the number of capillaries in five randomly selected fields in each heart was counted, n = 5–7. b Representative images of capillaries (upper panels) and summarized quantification (lower panels) in the border zone and infarct zone in individual test groups, scale bars: 100 µm. n = 5. c A significant number of vessels were double-positive for both IB4 (red) and ETV2 (green) in ETV2-injected heart tissues. Representative confocal images of double-positive capillaries (upper panels) and summarized quantification (lower panels) in heart tissue harvested 8 weeks after ETV2 injection, scale bars: 50 µm. *p < 0.05, **p < 0.01 compared to control group

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  Fig. 5. Gene expression analysis in cardiac fibroblasts infected with ETV2. Rat neonatal cardiac fibroblasts infected with lentiviral ETV2 were subjected to gene expression analysis. The y-axis represents fold change in mRNA expression compared to that in control virus-infected cells. p-values were calculated compared to the control group, n = 3

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  Fig. 6. ETV2 promotes migration and proliferation of endothelial cells. a, b Scratch assay. Culture supernatant harvested from rat neonatal cardiac fibroblasts infected with lentiviral ETV2 or control was incubated with mouse endothelial cells upon ‘scratch’ (see Methods). a Representative images under an inverted microscope and b quantification of scratch wound coverage. c, d Increased proliferation of endothelial cells in response to ETV2. HUVECs transfected with control virus or ETV2 lentivirus were incubated with BrdU for 3 h and subjected to FACS-BrdU analysis. c Representative flow analysis and d quantification

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  Fig. 7. Myocardial injection of AAV9-ETV2 improves cardiac function in a rat model of myocardial infarction. AAV9-ETV2 and control virus were injected into MI hearts of rats followed by image and functional analysis. a Optical images of sacrificed hearts 8 weeks after injection. White circle shows the area of fibrotic scar formation. b Representative images of echocardiography. c Quantitative analysis of EF and FS, ***p < 0.001. d Analysis of 18F-FDG cardiac uptake PET images with left ventricular polar maps (upper panels). Color on the map represents summed value of 18F-FDG uptake in the left ventricle. Cardiac 18F-FDG PET image was reoriented to the short-axis (SA), the horizontal long-axis (HLA), and the vertical long-axis (VLA). Quantification of 18F-FDG PET image in 20-segment polar map (bottom panels). Scores (%) of 18F-FDG uptake in the infarct area showed significant reduction in AAV-ETV2 group compared to that in the control group (n = 6, p < 0.05)