Periostin‐related progression of different types of experimental pulmonary hypertension: A role for M2 macrophage and FGF ‐2 signalling

Figures

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  FIGURE 1. Pulmonary haemodynamics and muscularization of small pulmonary arteries in pulmonary arterial hypertension mice, induced with Sugen5416 and chronic hypoxia (SuHx) and monocrotaline pyrrole (MCT‐P). Effect of periostin gene knockout on RVSP, RV hypertrophy (RV/[LV + S]) in SuHx wild‐type (WT) and periostin−/− (PN−/−) mice (A), and in MCT‐P WT mice and MCT‐P periostin−/− mice (B); RVSP, right ventricular systolic pressure; RV/(LV + S), right ventricle/(left ventricle + septum) weight ratio. Data are presented as means ± SD (n = 6 or 7 per group). Statistical comparisons were conducted using one‐way ANOVA followed by Tukey's multiple comparisons test versus WT normoxia or WT SuHx and are represented above each plot. *p <0.05, ***p <0.001.****p <0.0001. (C) Representative photomicrographs of Elastica van Gieson staining and quantitative analysis of the pulmonary small vessels muscularization in WT normoxia, WT SuHx and periostin−/− SuHx groups; 50–100 pulmonary vessels (with an outer diameter of less than 50 μm) per section were analysed, blinded to the tissue source. Scale bar = 50 μm. Data are presented as means ± SD (n = 6 per group) and statistical comparisons were performed…

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  FIGURE 2. mRNA expression of macrophage markers and chemokines associated with macrophage migration to pulmonary arteries in SuHx wild‐type (WT) and periostin−/− mice. (A) mRNA levels of CCL2, CCL4 and CCL7 in whole lung tissue of WT and periostin−/− normoxia mice, SuHx WT and periostin−/− mice. RNA was extracted from lung tissue 3 or 7 days after SuHx treatment and applied to quantitative real‐time (RT) PCR. Data are presented as fold changes relative to the 18S ribosomal mRNA level; PN−/−, periostin−/−. Results are presented as means ± SD from five to 12 samples. Statistical comparisons were conducted using multiple groups ANOVA followed by Tukey's multiple comparisons test. *p <0.05, **p <0.01, ****p <0.0001. (B) The mRNA expression levels of macrophage markers in lung tissues from WT and periostin−/− SuHx mice. RNA was extracted from lung tissue 1 or 3 weeks after SuHx treatment and quantitative RT‐PCR was performed to assess the mRNA levels of inducible nitric oxide synthase (iNOS) (M1 macrophage) and Fizz1 (M2 macrophage) marker genes. Data are presented as fold changes relative to the 18S ribosomal mRNA levels. Results are presented as means ± SD from five to 11 samples. Statistical…

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  FIGURE 3. Localization of CD68‐ and CD206‐positive macrophages to pulmonary arteries (PAs) and CD68‐positive cells to right ventricle (RV) in SuHx wild‐type (WT) and periostin−/− mice. (A) Representative fluorescence immunostaining of peri‐PAs from WT normoxia (Nx), WT SuHx and periostin−/− SuHx mice 2 weeks post‐stimulation with Sugen5416 and hypoxic exposure. Lung sections were stained with anti‐CD31 Ab, anti‐CD68 Ab and anti‐CD206 Ab (M2 macrophage marker), and counterstained with DAPI. Representative immunofluorescence images are shown (white, CD31; green, CD68; red, CD206; blue, DAPI, yellow, co‐localization of CD68 with CD206); scale bar 50 μm. The percentage of CD68+ and CD206+ double‐positive cells was calculated as the ratio of yellow cells to blue (DAPI) cells. Results are presented as means ± SD from 10 to 12 vessels per group. Statistical comparisons were conducted using ANOVA followed by Tukey's multiple comparisons test. ***p <0.001; ††† p <0.001; ‡ p <0.05. (B) Representative sections of RV tissue from WT SuHx and periostin−/− SuHx mice analysed by CD68 immunostaining. Sections of RV tissue were stained with anti‐CD68 Ab. The number of CD68‐positive cells (pointed by black…

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  FIGURE 4. Regulation of migration by periostin in human pulmonary arterial smooth muscle cells (HPASMCs), human pulmonary microvascular endothelial cells (HPMVECs) and human and mice macrophages. (A) Dose‐dependent effects of periostin on chemotaxis in HPASMCs, HPMVECs, phorbol 12‐myristate 13‐acetate (PMA)‐treated U937 and RAW264.7 cells. (B) The number of migrated HPASMCs following treatment with various concentrations of periostin in the presence or absence of anti‐CD11b and anti‐CD51 antibodies. The concentration of recombinant periostin (r‐periostin) varied from 50 to 1000 ng/ml. The chemotaxis vertical axis shows the number of migrated cells per five high‐power fields (5 HPF) as the fold change compared to control (media alone). Results are presented as means ± SD from four to six samples. Statistical comparisons were conducted using multiple groups ANOVA followed by Dunnett's multiple comparisons test, Dunn's multiple comparisons test or Tukey's multiple comparisons test. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001; †† p <0.01

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  FIGURE 5. Expression of inflammatory cytokines and neutrophil‐associated chemokines in whole lung tissue of SuHx wild‐type (WT) and periostin−/− mice. mRNA levels of (A) IL‐6, TNF‐α, IL‐1β and (B) CXCL1, CXCL2 and (C) Fibroblast growth factor‐2 in whole lung tissue from SuHx WT and periostin−/− (PN−/−) mice. RNA was extracted from lung tissue 3 or 7 days or 3 weeks after SuHx treatment and quantitative real‐time PCR was performed. Data are presented as fold changes relative to the 18S ribosomal mRNA level; PN−/−, periostin−/−. Results are presented as means ± SD from five to 12 samples. Statistical comparisons of multiple groups were conducted using ANOVA followed by Tukey's multiple comparisons test. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001

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  FIGURE 6. Serum periostin levels in patients with pulmonary hypertension (PH). (A) Serum periostin levels according to the presence of interstitial pneumonia (IP) and systemic sclerosis (SSc). IP(+), presence of interstitial lung disease; IP(−), absence of interstitial lung disease; SSc(+), presence of SSc; SSc(−), absence of SSc. Results are presented as means ± SD. Statistical comparisons of multiple groups were performed using ANOVA followed by Tukey's multiple comparisons test. *p <0.05, **p <0.01, ***p <0.001 versus control. (B) Receiver operating characteristic curve for best‐fit value of serum periostin level to predict mortality. (C) Overall survival from the time of blood sampling in relation to periostin serum levels in 49 patients with PH. Patients with periostin levels measuring 149.4 ng/ml or higher had a poor prognosis