AAV-Mediated Expression of AP-1-Neutralizing RNA Decoy Oligonucleotides Attenuates Transplant Vasculopathy in Mouse Aortic Allografts

Figure 1. hpAP-1 dODN Specificity and Generation in Aortic Grafts following Transduction (A) Transcription factor ELISA proving in situ the specificity of the designed sequence in binding to the transcription factor. (B) Schematic presentation of hairpin (hp) AP-1 dON generation following transduction. The H1 promoter drives the single-stranded RNA hpAP-1 dODN expression, which spontaneously base pairs to form hairpin (red, loop sequence). CMV promoter drives EGFP expression as positive control for transduction. (C) Representative images of fluorescence in situ hybridization experiments showing the presence of hpAP-1 dONs in transduced aortic tissue 30 days after initial surgery. A molecular beacon with red fluorescence (Cy5) was used to detect specifically dONs; nuclei were stained with DAPI (blue). Elastin autofluorescence was recorded on the green channel. Scale bar represents 25 μm (n = 9). (D) AAV9SLR transduction efficacy. Nuclei were marked using DAPI in order to count the number of cells/field of view. Four representative fields per section and two sections per graft were examined (n = 9).

Figure 2. Prevention of Neointima Formation following AAV9SLR-Mediated Delivery of hpAP-1 cons dONs (A) Representative images of H&E stainings of transplanted aortic grafts that received the mentioned treatments, 30 days after surgery. (B) Box-and-whisker plot of neointima/media ratios, as a measure of lumen obstruction. Horizontal bars in the boxes indicate median values, boxes indicate interquartile range, and whiskers indicate range of non-outlier values. Scale bar represents 100 μm (***p < 0.01, ****p < 0.0001; n = 14 for control, n = 7 for AAV hpAP-1 mut, n = 9 for AAV hpAP-1 cons).

Figure 3. Decreased Cell Proliferation following AAV9SLR-Mediated Delivery of hpAP-1 cons dONs (A) Representative images showing immunohistochemical staining against cyclin D2 (red) as a marker of proliferating cells. DAPI (blue) was used to mark cell nuclei, and elastin autofluorescence was recorded on the green channel. (B) Box-and-whisker plot of the level of proliferating cells, measured as percentage area of cyclin D2-positive cells in the neointima. Horizontal bars in the boxes indicate median values, boxes indicate interquartile range, and whiskers indicate range of non-outlier values (*p < 0.05, **p < 0.01; n = 14 for control, n = 7 for AAV hpAP-1 mut, n = 9 for AAV hpAP-1 cons).

Figure 4. Decreased VCAM-1 Protein Level following AAV9SLR-Mediated Delivery of hpAP-1 cons dONs (A) Representative images showing VCAM-1 (green) immunohistochemistry of grafts receiving the specified vectors. CD31 immunohistochemistry (red) was used to mark specifically endothelial cells, and DAPI (blue) labeled cell nuclei. (B) Box-and-whisker plot of mean fluorescence intensity detected in the green channel. Horizontal bars in the boxes indicate median values, boxes indicate interquartile range, and whiskers indicate range of non-outlier values (*p < 0.05 versus control; n = 14 for control, n = 7 for AAV hpAP-1 mut, n = 9 for AAV hpAP-1 cons). Scale bar represents 25 μm.

Figure 5. Decreased Immune Cell Infiltration following AAV9SLR-Mediated Delivery of hpAP-1 cons dONs (A and B) Representative images (A) and box-and-whisker plot (B) showing immunohistochemical analyses of CD4-positive cells in the aortic tissues of the mentioned treatment groups (*p < 0.05 and **p < 0.01 versus control). (C and D) Representative images (C) and box-and-whisker plot (D) showing immunohistochemical analysis of F4/80-positive monocytes/macrophages in the aortic tissues of the mentioned treatment groups. DAPI (blue) marks cell nuclei, and elastin autofluorescence is shown in green. Scale bar represents 25 μm. Horizontal bars in the boxes indicate median values, boxes indicate interquartile range, and whiskers indicate range of non-outlier values (*p < 0.05; n = 14 for control, n = 7 for AAV hpAP-1 mut, n = 9 for AAV hpAP-1 cons).

Figure 6. Decreased Pro-inflammatory Markers in Aortic Grafts Transduced with AAV9SLR-Expressing hpAP-1 cons dONs. (A–C) Box-and-whisker plots of quantitative real-time PCR analysis of MCP-1 (A), IL-6 (B), and IFN-γ (C) in the depicted treatment groups. (D–G) Representative images showing IL-6 (D) and MCP-1 (F) protein levels after transduction and corresponding box-and-whisker plots of the relative fluorescence intensity (E and G). Nuclei were stained with DAPI (blue), and elastin autofluorescence was recorded in the green channel. Scale bar represents 25 μm. Horizontal bars in the boxes indicate median values, boxes indicate interquartile range, and whiskers indicate range of non-outlier values (*p < 0.05; *p < 0.01, n = 14 for control, n = 7 for AAV hpAP-1 mut, n = 9 for AAV hpAP-1 cons).

Figure 7. Effect of AAV9SLR-Mediated of hpAP-1 cons dON Delivery on MMP Expression. (A) Representative MMP2 immunohistochemistry (red) images and (B) box-and-whisker plot showing the lack of effect of hpAP-1 consensus treatment. Elastin autofluorescence is pictured on the green channel, and DAPI (blue) marks cell nuclei. (C and D) Illustrative images (C) and box-and-whisker plot (D) of MMP9 immunostaining (red) in aortic cryosections. Single MMP9-positive cells, marked with arrows, were found mostly in the adventitia and media and infrequently in the neointima (*p < 0.05; n = 7 for AAV hpAP-1 mut, n = 9 for AAV hpAP-1 cons). Horizontal bars in the boxes indicate median values, boxes indicate interquartile range, and whiskers indicate range of non-outlier values. (E) Representative pictures demonstrating lack of colocalization of MMP9 signal (green) in the neointima of control grafts with the macrophage marker F4/80 (red, left image) and positive colocalization with ICAM-1-positive cells (red, center image). DAPI (blue) was used to label nuclei. Note that a small subset of MMP9-expressing cells as ICAM-1-positive immune cell population, while most of the MMP9 immunopositive cells were of…