Compensatory roles of Protein Related to DAN and Cerberus (PRDC) decrease in pulmonary arterial hypertension

Figures

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  Figure 1. PRDC level in human hypertensive lungs with IPAH. (A) mRNA level of PRDC expression in human lungs (n=6). (B) Representative image of western blot analyses in human lungs. (C) Densiometric analysis of PRDC normalized to GAPDH in human lungs (n=6). (D) Immunofluorescence for PRDC (green), smooth muscle α-actin (α-SMA, red) and nuclei (blue) in normal pulmonary arteries of control lungs (D1-D4) and remodeled pulmonary arteries of lungs with IPAH (D5-D8) (Scale bar, 25 µm). White arrows indicate colocalization of PRDC and α-SMA. (E) Immunofluorescence for PRDC (green), von Willebrand factor (vWF; red) and nuclei (blue) in normal pulmonary arteries of control lungs (E1-E4) and remodeled pulmonary arteries of lungs with IPAH (E5-E8) (Scale bar, 25 µm). White arrows indicate the colocalization of PRDC and vWF. ***P < 0.001.

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  Figure 2. PRDC and BMP signal in rat hypertensive lungs. (A-C) PRDC expression in rat lungs: mRNA level of PRDC (A), representative immunoblotting image of PRDC (B) and pooled data of densitometric analysis of PRDC (C) (n=3-5). (D) Correlation between lung PRDC band density and RVSP (D1), PASP (D2), mPAP (D3) and RVHI (D4). (E) Immunofluorescence for PRDC (green), smooth muscle α-actin (α-SMA, red) and nuclei (blue) in normal pulmonary arteries and remodeled pulmonary arteries of rat lungs (Scale bar, 25 µm). White arrows indicate colocalization of PRDC and α-SMA. (F) Immunofluorescence for PRDC (green), von Willebrand factor (vWF; red) and nuclei (blue) in normal pulmonary arteries and remodeled pulmonary arteries of rat lungs (Scale bar, 25 µm). White arrows indicate the colocalization of PRDC and vWF. (G-I) Expression pattern of BMP cascade in lungs with monocrotaline induced PAH: mRNA level of BMPR2 and Id-1 (G); Representative immunoblotting image of BMP cascade (H) and pooled data of densitometric analysis (I) (n=3-5). **P < 0.01, ***P < 0.001, NS p > 0.05.

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  Figure 3. PRDC reversed the anti-proliferation effects of BMP2/4 on dPASMCs. (A-B) BMP2/4 suppressed dPASMCs proliferation (n=12). (C) Direct roles of PRDC in dPASMCs proliferation (n=3). (D-E) PRDC antagonized the inhibitory effects of BMP2/4 on dPASMCs proliferation (n=12). (F-G) Representative immunofluorescence staining (F) and quantitative analysis (G) for Ki-67 in dPASMCs exposed to BMP2 or BMP2+PRDC (Scale bar, 50 µm) (n=4). (H-I) Representative immunofluorescence staining (H) and quantitative analysis (I) for Ki-67 in dPASMCs exposed to BMP4 or BMP4+PRDC (Scale bar, 50 µm) (n=4). *P < 0.05, **P < 0.01, ***P < 0.001, NS p > 0.05.

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  Figure 4. Expression changes of proliferating cell nuclear antigen and cell cycle cytokines. (A) Representative image of western blot analyses in dPASMCs exposed to BMP2 or BMP2+PRDC. (B) Densiometric analysis of PCNA, CyclinD1, p21 and p27 normalized to GAPDH in dPASMCs exposed to BMP2 or BMP2+PRDC (n=3-4). (C) Representative image of western blot analyses in dPASMCs exposed to BMP4 or BMP4+PRDC. (D) Densiometric analysis of PCNA, Cyclin D1, p21 and p27 in dPASMCs exposed to BMP4 or BMP4+PRDC (n=3). *P < 0.05, **P < 0.01, ***P <0.001, NS p > 0.05.

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  Figure 5. PRDC partially abolished anti-migration induced by BMP2/4 in dPASMCs. (A) Representative images of dPASMCs migrated and attached to the membrane bottom surface of transwell chamber exposed to BMP2 or BMP2+PRDC, Scale bar, 50 µm. (B) Number of migrated dPASMCs exposed to BMP2 or BMP2+PRDC, which was averaged from six randomly selected fields (n=6). (C) Representative images of wound-healing assay in dPASMCs exposed to BMP2 or BMP2+PRDC. (D) Quantitative analysis of migration area of dPASMCs exposed to BMP2 or BMP2+PRDC (n=6). (E) Representative images of dPASMCs migrated and attached to the membrane bottom surface of transwell chamber exposed to BMP4 or BMP4+PRDC, Scale bar, 50 µm. (F) Number of migrated dPASMCs exposed to BMP4 or BMP4+PRDC, which was averaged from six randomly selected fields (n=6). (G) Representative images of wound-healing assay in dPASMCs exposed to BMP4 or BMP4+PRDC. (H) Quantitative analysis of migration area of dPASMCs exposed to BMP4 or BMP4+PRDC (n=6). *P < 0.05, **P < 0.01, ***P < 0.001, NS p >0.05.

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  Figure 6. Expression changes of MMPs/TIMPs in dPASMCs. (A) Western blotting images of MMP2, MMP9, TIMP1 and TIMP2 in dPASMCs exposed to BMP2 or BMP2+PRDC. (B) Densiometric quantification of MMP2, MMP9, TIMP1and TIMP2 in dPASMCs exposed to BMP2 or BMP2+PRDC (n=3). (C) Western blotting images of MMP2, MMP9, TIMP1 and TIMP2 in dPASMCs exposed to BMP4 or BMP4+PRDC. (D) Densiometric quantification of MMP2, MMP9, TIMP1 and TIMP2 in dPASMCs exposed to BMP4 or BMP4+PRDC (n=3-4). *P < 0.05, **P < 0.01, ***P < 0.001, NS p > 0.05.

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  Figure 7. PRDC antagonized the pro-apoptotic effects of BMP2/4 on dPASMCs. (A-B) Representative images of the TUNEL assay and quantitative analysis of TUNEL positive in dPASMCs exposed to BMP2 or BMP2+PRDC, Scale bar, 50 µm (n=4). (C) Early and late apoptosis of dPASMCs exposed to BMP2 or BMP2+PRDC measured by flow cytometry with Annexin V-FITC/propidium iodide (PI) staining. (D) Percentage of apoptosis rate in dPASMCs exposed to BMP2 or BMP2+PRDC (n=4). (E-F) Representative images of the TUNEL assay and quantitative analysis of TUNEL positive in dPASMCs exposed to BMP4 or BMP4+PRDC, Scale bar, 50 µm (n=4). (G) Early and late apoptosis of dPASMCs exposed to BMP4 or BMP4+PRDC measured by flow cytometry with Annexin V-FITC/propidium iodide (PI) staining. (H) Percentage of apoptosis rate in dPASMCs exposed to BMP4 or BMP4+PRDC (n=4). *P < 0.05, **P < 0.01, ***P < 0.001, NS p >0.05.

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  Figure 8. PRDC restored the expression of apoptosis-associated proteins in dPASMCs induced by BMP2/4. (A) Caspase3/7 activity in dPASMCs exposed to BMP2 or BMP2+PRDC (n=6). (B-C) Representative western-blotting images and densitometry results of caspase3, caspase9, Bax, Bcl-2 and the ratio of Bax to Bcl-2 in dPASMCs exposed to BMP2 or BMP2+PRDC (n=3). (D) Caspase3/7 activity in dPASMCs exposed to BMP4 or BMP4+PRDC (n=6). (E-F) Representative western-blotting images and densitometry results of caspase3, caspase9, Bax, Bcl-2 and the ratio of Bax to Bcl-2 in dPASMCs exposed to BMP4 or BMP4+PRDC (n=3). *P < 0.05, **P < 0.01, ***P < 0.001, NS p > 0.05.

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  Figure 9. PRDC administration deteriorated rat pulmonary hemodynamics and pulmonary vasculopathies. (A-E) Effect of PRDC administration on PASP (A), PADP (B), mPAP (C), Pp/Ps (D), mPAP/mSBP (E). (F) Representative morphological imagines from MCT-Saline and MCT-PRDC (hematoxylin-eosin staining, F1-F3, F5-F7, ×1000; Weigert and Van Gieson staining, F4, F8, ×1000); (G) Percent medial thickness of PAs < 75 µm IED; (H) Distribution of non-, partial- and fully-muscularization of PAs < 75 µm IED; (I) Proportion of pulmonary vasculopathies stages in PAs < 75 µm IED; (J) Percent of PAs < 75 µm IED with or without neointimal formation in rats with stage II or III pulmonary vasculopathies; (K) Percent of PAs <75 µm IED with < 50% or > 50% luminal occlusion in rats with stage II or III pulmonary vasculopathies. MPA, muscularized pulmonary artery; PMPA, partial muscularized pulmonary artery; NMPA, non-muscularized pulmonary artery. *P < 0.05, **P < 0.01, ***P < 0.001, NS p > 0.05.

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  Figure 10. PRDC administration aggravated the right ventricular hypertrophy and function induced by monocrotaline injection in rats. (A) H&E staining of rat heart transversal section; (B) Quantitative analysis of right ventricular hypertrophy index (RVHI); (C) WGA staining of rat heart (bar=50 µm); (D) Quantitative analysis of right ventricular cardiac myocytes by WGA staining; (E) Effect of PRDC administration on RVSP; (F-G) Quantitative analysis of right ventricular contraction function (+dp/dtmax, F) and relaxation function (-dp/dtmax, G). *P < 0.05, **P < 0.01, ***P < 0.001, NS p > 0.05.

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  Figure 11. Schematic diagram of findings of this study. (A) Lung PRDC physiologically antagonizes lung BMP cascade to maintain the homeostasis of pulmonary vascularture; (B) Monocrotaline-induced suppression of lung BMP cascade resulted into the phenotype transformation of dPASMCs, pulmonary vascular remodeling and PAH; (C) PRDC administration further exacerbated monocrotaline-induced PAH.