Schema of IFNα treatment protocols.

<p>(A) Schema of prevention and therapeutic protocols for IFNα treatment in SU5416/hypoxia-induced PH in rats. (B) Schema of prevention and therapeutic protocols for IFNα treatment in hypoxia-induced PH in mice.</p

IFNα inhibits pulmonary vascular cell proliferation.

<p>(A–D) Representative 40x images of lung sections from 3 week SUH rat, 3 week SUH rat + IFNα, 5 weeks SUH rat, and 5 week SUH rats+ IFNα stained for PCNA (brown) as an indicator of proliferating cells. WB analysis for PCNA and p21 in whole lung lysates from (E) 3-week SUH rats or (F) 5-week SUH rats with or without IFNα (n = 4 animals per group). (G) Control or IPAH HPASMC were serum starved 24 h and then stimulated with PDGF (10 ng/ml) with or without increasing IFNα for 24 hours. (H) Control or IPAH HPAEC were serum starved overnight and then stimulated with VEGF (50 ng/ml) with or without increasing IFNα for 24 hours. Proliferation was assessed by measuring [H3]-thymidine incorporation. Analysis of variance *<i>P</i><0.05.</p

Human IFNα attenuates PH in mice in a IFNAR-dependent fashion.

<p>Effect of IFNα on (A) RVSP and (B) RVH in normoxic and hypoxic WT or IFNAR1 −/− mice (n = 6 mice per group). (C) Relative expression of IFNα (normalized to GAPDH) in total lung from C57BL/6J mice exposed to 0, 7, or 21 days of CH as determined by qRT-PCR. (D) Serum concentration of IFNα in C57BL/6J mice exposed to 0, 7, or 21 days CH as determined by ELISA. n = 8 animals per group. Analysis of variance *P<0.05. (E) Serum concentration of IFNα in control vs. IPAH human serum as determined by ELISA.</p

IFNα prevents and reverses pulmonary vascular remodeling in SUH rats.

<p>(A) Representative photomicrographs of small pulmonary arterioles (≤50 μm) from an SUH rat with vascular occlusion (V.O.) of 0%, <50%, and >50%. (B) Percent of small pulmonary arterioles (≤50 μm) with V.O. 0%, <50%, or >50% in SUH treatment groups (50 arterioles per animal, n = 4 animals per group). (C) Representative photomicrographs of pulmonary arterioles (≤100 μm) from SUH treatment groups demonstrating differences in wall thickness. (D) % Wall thickness in pulmonary arterioles (≤100 μm) from SUH treatment groups (20 arterioles per animal, n = 4 animals per group).</p

IFNα prevents and reverses experimental PH.

<p>(A) Effect of IFNα on RVSP and (B, C) RVH in SUH rats treated with IFNα or vehicle (n = 6 rats per group). (D–F) Representative Images of hearts from normoxic, 5 week SUH, and 5 week SUH rats treated with IFNα. Effect of IFNα on (G) RVSP and (H–I) RVH in hypoxic mice treated with IFNα or vehicle (n = 8 mice per group). Analysis of variance *<i>P</i><0.05.</p

Human IFNα stimulates STAT1 phosphorylation in mice and rats.

<p>WB analysis of STAT1, phospho-STAT1 in whole lung homogenates from: (A) normoxic rats, 5 week SUH rats, and 5 week SUH rats treated with IFNα (n = 4 rats per group); or (B) normoxic mice, 6 week hypoxic mice, and 6 week hypoxic mice treated with IFNα. Densitometric ratio of phospho-STAT1 to STAT1 and phospho-STAT3 to STAT3 in lung tissue of different treatment groups in (C) SUH rats and (D) hypoxic mice.</p

IFNα reduces the number of TUNEL positive cells in the pulmonary arterioles of SUH rats and inhibits HPAEC apoptosis.

<p>Representative photomicrographs of pulmonary arterioles stained for TUNEL (red) and nuclei (blue) in (A) normoxic control rats; (B, C) 3 week SUH rats; (D, E) 3 week SUH rats treated with IFNα; (F, G) 5 week SUH rats; and (H, I) 5 week SUH rats treated with IFNα. Photomicrographs are representative of 4–6 animals per group. (J) WB analysis for total AKT and phospho-AKT in whole lung lysates from 3-week SUH rats or 5-week SUH rats with or without IFNα (n = 4 animals per group). Control or IPAH HPASMC were grown in complete media and apoptosis was induced by (K) serum starvation or (L) cycloheximide plus hydrogen peroxide with or without IFNα. Control or IPAH HPAEC were grown in complete media and apoptosis was induced by (M) serum starvation or (N) cycloheximide plus hydrogen peroxide. Percent apoptotic cells was assessed by the ratio of TUNEL positive nuclei to total nuclei.</p

C3d deposition in chronic hypoxia-induced PH in mice.

<p>(A–D) Lungs from normoxic and hypoxic C57Bl/6J mice were stained with α-C3d and α-SMA antibody and counterstained with DAPI to detect nuclei(n = 4). (E) Quantification of C3d staining in WT vs. C3−/− mice in normoxia and hypoxia. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05. (F) Representative Western blot for C3d in normoxic vs. hypoxic C57Bl/6J mice. (G) Quantification of Western blots for C3d in normoxic vs. hypoxic mice. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05. (H) C5a was immunoprecipitated from lung or plasma of normoxic and hypoxic C57Bl/6J mice and analyzed by Western blot (rC5a = recombinant mouse C5a).</p

Markers of Inflammation and endothelial dysfunction in WT vs. C3 −/− mice.

<p>(A) IL-6 mRNA was measured by quantitative rtPCR in RNA prepared from normoxic or hypoxic WT and C3−/− lungs. IL-6 levels were normalized to the house keeping gene β2-microglobulin. (B) ICAM-1 was quantified by ELISA in lung homogenates from normoxic or hypoxic WT or C3−/− mice. (C) ET-1 was quantified by ELISA in plasma from normoxic or hypoxic WT or C3−/− mice. Bars represent mean ± SD (n = 4) for A–C. *<i>P</i><0.05.</p

Loss of C3 prevents platelet activation caused by CH.

<p>(A) Bleeding time of WT and C3−/− mice exposed to normoxia or CH (n = 5–7). (B) Scatter plot showing platelet population in platelet rich plasma. All experiments were similarly gated to the area encircled. (C) Flow cytometry histogram demonstrating that the gated cell population is positive for the platelet marker CD41. (D–E) Representative flow cytometry histograms of platelets from (D) hypoxic WT or (E) hypoxic C3−/− mice stained with P-selectin antibody or isotype control. (F) Percent P-selectin positive platelets in PRP isolated from normoxic and hypoxic WT or C3−/− mice (n = 6). Bars represent mean ± SD. *<i>P</i><0.05; n.s. = not significant.</p