Endothelial periostin regulates vascular remodeling by promoting endothelial dysfunction in pulmonary arterial hypertension

Figures

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  Figure 1.. Upregulation of POSTN in the lung tissue of patients with PAH and PAH animal models.: a, POSTN mRNA level in the lung tissue of patients with PAH from public GEO dataset (GSE53408). b, Protein and c, mRNA expression of POSTN in the lung tissue of SuHx and MCT rat models. d, Representative images of POSTN/vWF co-localization in the lung tissue of SuHx and MCT rat models. Lung sections were stained for POSTN (red), vWF (green) and DAPI (blue). Scale bar = 50 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by the unpaired two-tailed Student’s t-test. Error bars, S.E.M.

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  Figure 2.. POSTN expression is increased in lysates and conditioned medium in PAECs stimulated with PAH-related stimuli.: a, Protein expression and b, Immunostaining images of FN and MMP2 in PAECs stimulated with of TGF-β2 (10 ng/mL) and IL-1β (1 ng/mL) for 5 days. Scale bar = 20 µm. c, Protein expression and d, Immunostaining images of FN in PAECs stimulated with IL-13 (10 ng/mL) for 4 days. Scale bar = 20 µm. ** P < 0.01, *** P < 0.001 determined by the unpaired two-tailed Student’s t-test. Error bars represent S.E.M.

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  Figure 3.. POSTN induces ECM deposition and endothelial abnormalities in PAECs.: a, Protein expression of FN and MMP2 in response to infection with the lentiviral POSTN vector for 4 days. b, Immunostaining images of FN after POSTN overexpression. Scale bar = 50 µm. c, Migration assay after overexpression of the POSTN lentivirus for 3 days. Scale bar = 200 µm. d, Representative images of F-actin after POSTN overexpression in PAECs. Cells were plated onto glass coverslips coated with or without rhPOSTN (10 µg/mL) and then stained with rhodamine-phalloidin. Scale bar = 50 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by the unpaired two-tailed Student’s t-test. Error bars represent S.E.M.

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  Figure 4.. POSTN knockdown ameliorates ECM deposition and endothelial function.: a, Protein expression of MMP2 after TGF-β2 (10 ng/mL) and IL-1β (1 ng/mL) co-treatment for 5 days with or without POSTN knockdown (2 nM). b, Migration assay after TGF-β2 (10 ng/mL) and IL-1β (1 ng/mL) co-treatment with or without POSTN knockdown (10 nM). Scale bar = 200 µm. c, Permeability assay after TGF-β2 (10 ng/mL) and IL-1β (1 ng/mL) co-treatment with or without POSTN knockdown (5 nM). d, Protein expression of phosphorylated p65 by co-treatment of TGF-β2 (10 ng/mL) and IL-1β (1 ng/mL) for 24 h with or without POSTN siRNA (5 nM) transfection. P < 0.05, ** P < 0.01, *** P < 0.001, as determined by unpaired two-tailed Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparison test. Error bars represent S.E.M.

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  Figure 5.. ILK interacts with POSTN and ILK inhibition reduces ECM deposition and attenuates endothelial function.: a, Interaction between ILK and POSTN in PAECs as determined by co-immunoprecipitation. b, Protein expression of ILK and FN after co-treatment of TGF-β2 (10 ng/ml) and IL-1β (1 ng/ml) for 3 days in conjugation with ILK siRNA (24 nM) transfection. c, Migration assay after co-treatment with TGF-β2 (10 ng/ml) and IL-1β (1 ng/ml) (upper) or IL-13 (10 ng/ml) (lower) with or without ILK knockdown (5 nM). Scale bar = 200 µm. d, Permeability assay after TGF-β2 (10 ng/ml) and IL-1β (1 ng/ml) co-treatment with or without ILK knockdown (5 nM). * P < 0.05, ** P < 0.01, *** P < 0.001 determined by unpaired two-tailed Student’s t test or one-way ANOVA with Bonferroni’s multiple comparison test. Error bars represent S.E.M.

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  Figure 6.. Schematic representation showing the proposed role of POSTN in PAECs. Upregulation of POSTN under PAH-related conditions induces ECM deposition and endothelial abnormalities. Proposed mechanism by which POSTN interacts with ILK and activates NF-κB signaling.