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

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 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

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

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 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

Expression levels of miR-34c and miR-214 are changed when donor PBMCs are exposed to conditioned media from dying cells.

<p><b>A</b>: Changes in miR-34c, miR-214 and miR-155 expression in PMBCs (from one donor) exposed to conditioned media from HMGB1^+/+ and HMGB1^−/− MEF cells. PBMCs were pre- incubated with 50 µM glybenclamide (Glyb) for 30 minutes before being exposed to conditioned media (MEF CM) for 48 hrs. Heat shock (HS) was carried out at 42°C for 2 hrs. Data shown are average±s.d. from one independent experiment in triplicate, normalized to untreated control samples and RNU48 as endogenous control. <b>B</b>: Changes in miR-34c, miR-214 and miR-155 expression in PBMCs from another donor exposed to conditioned media. Data shown are average±s.d. from one independent experiment in triplicate, normalized to untreated control samples and RNU48 as endogenous control, where **indicates p<0.01, *indicates p<0.05, by paired Student’s t test. The two respective groups compared in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038899#pone-0038899-g005" target="_blank">Figure 5</a> are for each miR expression graph, the treatment at the beginning of the straight line with the treatment at the end of the line. The control lane is the first treatment in each graph, which is indicated by the white bar.</p

Differential expression of TNFα and hsa-miR-34c in human donor PBMCs following exposure to wild-type (wt) HCT116 or HMGB1 stable knock-down (kd) lysates.

<p><b>A</b>: HCT116 cells were stably transfected with a shHMGB1 vector in the presence of Puromycin (100 ug/ml). The clone with complete knockdown of HMGB1 (indicated by the arrow) was chosen to make necrotic lysates. <b>B</b>: TNFα ELISA showing differential release of TNFα in human donor PBMC cell cultures following exposure to HCT116 lysates (wild-type, wt or HMGB1 knockdown, kd) for 24 hrs. The amount of HCT116 lysates used was 104 cells/ml of human PBMC culture. Data shown are the average±s.d. of three independent experiments, each from a different donor, measured in triplicates, where **indicates p<0.01, by Student’s t test. <b>C</b>: Fold changes in expression (as log-2-transformed RQ values) of hsa-miR-34c and hsa-miR-214 in donor PBMCs exposed to the indicated HCT116 necrotic lysates for 48 hrs. Values were measured using TaqMan real-time RT-PCR. Data shown are the average±s.d. of three independent experiments, each from an individual donor, measured in triplicates, where **indicates p<0.01, and where ***indicates p<0.001, by paired Student’s t test.</p

Organ elimination half-life of exogenous mouse sRAGE administered by three different routes.

a<p>Not determined due to ambiguity, interruption, or lack of convergence of fit.</p