Figure 1.
Intratracheal instillation of Ad viral vectors causes lung injury and lung inflammation.
C57bl/6 mice were intratracheally instilled with Ad viral vectors at the doses of 1×107/ifu, 1×108 ifu, and 1.625×109 ifu per mouse. PBS and bleomycin were administered as negative and positive controls. 14 and 21 days after administration of PBS, bleomycin and Ad viral vectors, mouse bronchoalveolar lavage fluids (BALF) were collected and used to determine the protein concentration (A and B). Aliquots of 180 µ BALF were applied to cytospin and Wright staining to determine total cell counts and differential cell counts under microscope (200X magnification) (C, D, E, F). The cell counts were expressed as numbers of cells/high power field (hpf). n≥5; * and #, p<0.05; ** and ##, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. #, ##, and n.s. compare the difference between the high dose AD vector group and the bleomycin group.
Figure 2.
Intratracheal instillation of Ad viral vectors causes pulmonary fibrosis.
21 days after administration of PBS, bleomycin and Ad viral vectors (1×107/ifu, 1×108 ifu, and 1.625×109 ifu per mouse), mouse lungs were harvested, fixed, embedded and sectioned for H&E and Trichrome staining (A). The harvested lungs of these mice were used for collagen analysis by the Sircol assay (B). The content of collagen was normalized to the total amount of protein. We plotted the collagen contents against the natural logarithm of the dose of Ad virus and calculated the equation of the line of the best-fit as indicated in the insert (C). We scan the slides of the Masson’s Trichrome staining on the Aperio ScanScope CS at 20x magnification at standard resolution and measured the percent of tissue area that was classified as fibrosis with Aperio ImageScope v11 software. We presented the score of fibrosis as the percentage of fibrotic tissue area over the total lung tissue area (D). Mouse lungs were homogenized and are used to extract total RNA for the measurement of mRNA levels of mouse Col1a1 (E), Col1a2 (F), PCNA (G), Acta2 (H) by real time qRT-PCR. RPL19 gene was used as internal control for qRT-PCR. n≥5; *, p<0.05; **, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. n.s. compares the difference between the high dose AD vector group and the bleomycin group.
Figure 3.
Intratracheal instillation of Ad viral vectors induces TGF-β production in mouse lungs.
14 and 21 days after administration of PBS, bleomycin and Ad viral vectors, mouse BALFs were collected and used to determine the concentration of active TGF-β (A and B). (C–E) 21 days after administration with PBS, bleomycin, and Ad vectors, mice lungs were used to extract total RNA for the measurement of mRNA levels of mouse TGF-β1, TGF-β2, and TGF-β3 by real time qRT-PCR. RPL19 gene was used as internal control for qRT-PCR. n≥5; * and #, p<0.05; ** and ##, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. #, ##, and n.s. compare the difference between the high dose AD vector group and the bleomycin group.
Figure 4.
Intratracheal instillation of Ad viruses increases expression levels of profibrotic cytokines.
(A–F) 21 days after administration with PBS, bleomycin, and Ad vectors, mice lungs were harvested and were used to extract total RNA for the measurement of mRNA levels of mouse TNFα (A), IL-1β (B), IL-1α (C), IL-13 (D), IL-10 (E), and IL-16 (F) by real time qRT-PCR. RPL19 gene was used as internal control for qRT-PCR. (G–H) 21 days after administration of PBS, bleomycin and high dose Ad viral vectors (1.625 ifu/mous), mouse BALFs were collected and used to determine the concentration of TNFα (G) and IL-1β (H). n≥5; *, p<0.05; ** and ##, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. ## and n.s. compare the difference between the high dose AD vector group and the bleomycin group.
Figure 5.
Intratracheal instillation of Ad viruses causes dysregulation of ECM proteins and MMPs.
21 days after administration with PBS, bleomycin, and Ad vectors, mice lungs were harvested and were used to extract total RNA for the measurement of mRNA levels of mouse Integrin α1 (A), Integrin α5 (B), Integrin αv (C), MMP-2 (D), and MMP-9 (E) by real time qRT-PCR. RPL19 gene was used as internal control for qRT-PCR. n≥5; * and #, p<0.05; **, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. # and n.s. compare the difference between the high dose AD vector group and the bleomycin group.
Figure 6.
Intratracheal instillation of Ad viruses causes elevated expression of Wnt signaling genes associated with fibrosis.
21 days after administration with PBS, bleomycin, and Ad vectors, mice lungs were harvested and were used to extract total RNA for the measurement of mRNA levels of mouse Lrp6 (A), Wnt2 (B), Wnt2b (C), and Wnt5b (D) by real time qRT-PCR. RPL19 gene was used as internal control for qRT-PCR. n≥5; * and #, p<0.05; **, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. # and n.s. compare the difference between the high dose AD vector group and the bleomycin group.
Figure 7.
Intratracheal instillation of Ad viruses induces cytokine levels in BALF and protein expression of ECM and Wnt proteins.
21 days after administration of PBS, bleomycin and high dose Ad viral vectors (1.625 ifu/mous), mice lungs were homogenized for the measurement of protein levels of Integrin α5, Wnt5b, and MMP-9 by Western blot analysis. The amount of tubulin was used as control for equal loading. The representative blots are shown in (A) and the quantification of relative expression of MMP-9, Integrin α5 and Wnt5b are shown in B, C, and D, respectively. n≥5; *, p<0.05; **, p<0.01; n.s, no significance. * and ** compare the difference between PBS and other groups. n.s. compares the difference between the high dose AD vector group and the bleomycin group.
Figure 8.
Intratracheal instillation of Ad viruses induces DNA damages.
21 days after administration of PBS, bleomycin and high dose Ad viral vectors (1.625 ifu/mous), mouse lungs were harvested, fixed, embedded and sectioned for TUNEL staining (green color) and DAPI counterstaining (blue color). Fluorescence microscopy images were taken with 10X objective lens.
Figure 9.
Ad viral infection-mediated fibrosis is not limited to the infection sites.
C57bl/6 mice were intratracheally instilled with PBS and Ad-GFP viral vectors at the doses of 1.625×109 ifu per mouse. 5 (5D) and 14 (14D) days after administration of PBS and Ad-GFP viral vectors, mouse lungs were harvested, fixed, embedded and sectioned for co-immunofluorescence staining for GFP (red color) and α-SMA (green color) and DAPI (blue) counterstaining. Fluorescence microscopy images were taken with 10X objective lens (A) and 20X objective lens (B). White arrows indicate the position of α-SMA-positive cells.