The protective effects of MSC‐EXO against pulmonary hypertension through regulating Wnt5a/BMP signalling pathway

Figures

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  Figure 1. Effect of MSC‐EXO on MCT‐induced pulmonary artery pressure and vascular remodelling. A, Comparative analysis of heart rate (HR), cardiac output (OC) and right ventricular systolic pressure (RVSP). B, Representative haematoxylin and eosin staining images in each group. C, Comparative analysis of the right ventricular (RV) to left ventricle (LV) plus the septum (LV+S) ratios [RV/(LV+S)] and the per cent of wall thickness and wall area. D, Double immunofluorescence staining analysis of a‐SMA(red) and p‐H3 (green) in lung tissue. E, Masson's staining, F, Vessel analysis by immunohistochemistry using anti‐CD31 antibodies in lung. G, Comparative analysis of optical density (OD) value to detection the fibrosis and the positive staining of CD3 in lung tissue. *P < 0.05 vs control group; # P < 0.05 vs MCT group. Red bar = 100 µm

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  Figure 2. The protein expression analysis by Western blot in lung tissue. A, a‐SMA, CD31 and V‐E cadherin; B, Bcl2, Bax and cleaved caspase‐3; C, Wnt5a, Wnt11, β‐catenin and cyclin D1; D, BMPR2, Smad1/5/8 and Smad2/3. *P < 0.05 vs control group; # P < 0.05 vs MCT group

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  Figure 3. Effect of MSC‐EXO on hypoxia‐PAEC in vitro. A, the protein expression analysis of Wnt5a, Wnt11, β‐catenin and cyclin D1 by Western blot. B, Detection of Wnt5a mRNA expression in PAEC and PASMC by PCR. C, Detection of Wnt5a protein expression in PAEC and PASMC by Western blot. **P < 0.01 vs Normal group

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  Figure 4. Effect of MSC‐EXO on hypoxia‐induced cell viability and proliferation. A, Cell counting kit (CCK)‐8 assay in PAEC. B, Proliferation analysis by BrdU cell proliferation assay in PAEC. C, Proliferation analysis by BrdU cell proliferation assay in PASMC in 24 h, 48 h and 72 h. D, Detection of proliferating cell nuclear antigen (PCNA) protein expression in PAEC by Western blot. E, Immunofluorescence staining with Ki67 antigen in PAEC. F, Comparative analysis of PCNA and ki67 protein expression. *P < 0.05 vs Normal group; # P < 0.05 vs hypoxia group; △ P < 0.05 vs MSC‐EXO group. Red bar = 100 µm

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  Figure 5. Effect of MSC‐EXO on PAEC invasion, migration and protein expression. A, Wound healing assay and Transwell assay in PAEC. B, Comparative analysis of invasion and migration ability. C, Protein expression and comparative of BMPR2, P/T‐Smad1/5/8 and P/T‐Smad2/3. D, Protein expression of TGF‐β1, BMP4 and BMP9. *P < 0.05 vs Normal group; # P < 0.05 vs hypoxia group; △ P < 0.05 vs MSC‐EXO group. Red bar = 100 µm

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  Figure 6. Effect of MSC‐EXO on hypoxia‐induced EndMT in PAECs. A, Immunofluorescence analysis of a‐SMA and V‐E cadherin. B, Protein expression and comparative analysis of CD31, a‐SMA and V‐E cadherin in PAEC. *P < 0.05 vs Normal group; # P < 0.05 vs hypoxia group; △ P < 0.05 vs MSC‐EXO group. Red bar = 100 µm