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. Author manuscript; available in PMC: 2020 Oct 22.
Published in final edited form as: Circulation. 2019 Aug 29;140(17):1409–1425. doi: 10.1161/CIRCULATIONAHA.119.040629

Figure 2. Transient AdAlox5 infection generates a remodeled neointima which expresses endogenous non-viral 5-LO.

Figure 2.

Representative immunofluorescent confocal images and quantification of lung sections collected weekly following viral instillation. (A and B) Distal pulmonary arterioles (PAs) were stained with early apoptosis marker, cleaved caspase 3 (green) and endothelial marker, von Willebrand factor (vWF, red). Boxed area with split channels highlights intimal cell injury 1 week after viral injection. n=6; scale bar, 50μm. (C and D) Representative confocal staining and quantification of Ki67 (green) expressing proliferative vWF+ (red) cells. Boxed area with split channels indicates neointimal proliferation at postoperative week 3. n=6; scale bar, 50μm. (E and F) Representative images and quantification of α-SMA (green) and vWF (red) staining. Colocalization of α-SMA and vWF in the obliterative lesion is highlighted. n=6; scale bar, 50μm. (G and H) Representative images and quantification of 5-LO (green) and vWF (red) costaining. Boxed area with split channels demonstrates endogenous 5-LO expression in the transformed neointima. Note the nuclear envelope localization of 5-LO. n=6; scale bar, 50μm. (I and J) Representative images and quantification of GFP-tagged virus (GFP, green) and epithelium marker, E-Cadherin (red) staining. Note GFP-tagged adenoviral particles are located in the airway epithelium 1 week after infection and gradually receded. n=6; scale bar, 50μm. (K) Proposed model: GFP-labeled AdAlox5 infects airway epithelium and alveolar macrophages after intratracheal instillation and secretes LTB4. Exogenous LTB4 diffuses to PAs and causes intimal cell apoptosis. Maladaptive intimal cell proliferation forms the neointima, which expresses endogenous 5-LO with de novo synthesis of LTB4 independent of viral activities. Extensive vascular remodeling in advanced disease features an obliterative and proliferative intima with signature of endothelium to mesenchymal transition (EndMT). As adenoviral 5-LO disappears from the lung, it is replaced by endogenous 5-LO which is sustained. DAPI (blue) identifies nuclei. Data are presented as means and SEM; Kruskal-Wallis test; ns, not significant, *p< 0.05, **p < 0.01.