Vital Roles of Gremlin‐1 in Pulmonary Arterial Hypertension Induced by Systemic‐to‐Pulmonary Shunts

Figures

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  Figure 1. Pulmonary hypertensive status in rats with persistent systemic‐to‐pulmonary shunt or cervical shunt ligation. A through J, Hemodynamic indexes derived from right heart catheterization procedure and ratio of the weight of right ventricle/that of left ventricle plus septum (RV/LV+S). A, Right ventricular systolic pressure (RVSP). B, Right ventricular developed pressure (RVDP). C, Positive maximum RV dP/dt (+RV dp/dtmax). D, Negative maximum RV dP/dt (−RV dp/dtmax). E, Pulmonary arterial systolic pressure (PASP). F, Pulmonary artery diastolic pressure (PADP). G, Mean pulmonary arterial pressure (mPAP). H, Ratio of pulmonary arterial systolic pressure/systemic arterial systolic pressure (Pp/Ps). I, mPAP/mean systemic blood pressure (mSBP). J, RV/LV+S. K, Proportion of pulmonary vasculopathies of different grades presented in intra‐acinar pulmonary arteries <50 μm in external diameter. L, Representative morphological imagines of pulmonary arterioles (hematoxylin‐eosin stain, ×1000). L1 to L3, Intra‐acinar pulmonary arteries from sham operation group (SOG) with normal morphological features; L4 to L6, intra‐acinar pulmonary arteries from combined surgery group reserving for 8 weeks…

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  Figure 2. Expression change of BMP (bone morphogenetic protein) cascade and gremlin‐1 in rats with persistent systemic‐to‐pulmonary shunt or cervical shunt ligation. A, Representative Western blotting image of gremlin‐1, BMP cascade (BMP receptor 2 [BMPR2], P‐Smad1/5/8 [phosphorylated drosophila mothers against decapentaplegic protein 1/5/8], T‐Smad1/5/8 [total drosophila mothers against decapentaplegic protein 1/5/8], and inhibitor of DNA‐binding 1 [Id1]) in lungs from sham operation group (SOG) and combined surgery group (CSG). B through D, Quantitative analysis of BMP cascade by densitometric scanning: BMPR2 (B), P‐Smad1/5/8 (C), and Id1 (D). E, Protein level of gremlin‐1 in lungs from SOG and CSG. F, Plasma gremlin‐1 concentration presented a stepwise increase in a time‐dependent manner and decreased after cervical shunt closure. G through P, Regression scatterplot results demonstrate the correlation between plasma gremlin‐1 level and right ventricular systolic pressure (RVSP) (G), right ventricular developed pressure (RVDP) (H), positive maximum RV dP/dt (+RV dp/dtmax) (I), negative maximum RV dP/dt (−RV dp/dtmax) (J), pulmonary arterial systolic pressure (PASP) (K), pulmonary artery…

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  Figure 3. Mechanical stretch increased gremlin‐1 expression and suppressed BMP (bone morphogenetic protein) cascade in distal pulmonary arterial smooth muscle cells (dPASMCs) from healthy rats. A through C, Mechanical stretch (24 hours, 1 Hz) increased the mRNA level of gremlin‐1 (A) but decreased the mRNA level of BMP receptor 2 (BMPR2) (B) and inhibitor of DNA‐binding 1 (Id1) (C) in an amplitude‐dependent way and achieved the optimal effect at 15%. D through F, Mechanical stretch (15%, 1 Hz) increased the mRNA level of gremlin‐1 (D) but decreased the mRNA level of BMPR2 (E) and Id1 (F) in a time‐dependent manner and exerted an optimal effect at 24 hours. G, Representative Western blotting image showing that the protein level changes of gremlin‐1 and BMP cascade in dPASMCs under the condition of mechanical stretch (15%, 24 hours, and 1 Hz). H through K, Quantification analysis presented the protein level change of gremlin‐1 (H), BMPR2 (I), P‐Smad1/5/8 ([phosphorylated drosophila mothers against decapentaplegic protein 1/5/8) (J), and Id1 (K) in dPASMCs under the condition of mechanical stretch (15%, 24 hours, and 1 Hz). Data are mean±SD of 5 to 6 independent experiments. NS indicates not…

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  Figure 4. Gremlin‐1 antagonized the inhibitory effect of BMP2 (bone morphogenetic protein 2) on distal pulmonary arterial smooth muscle cell (dPASMC) proliferation. A, Quantitative analysis of dPASMC distribution in cell phase at indicated time point. B, mRNA expression of gremlin‐1 in dPASMCs after recovery in CSMCM for 0, 12, and 24 hours. C, Representative Western blotting image (lower panel) and densitometric analysis (upper panel) showing the protein level change of gremlin‐1 in dPASMCs after recovery in CSMCM for 0, 12, and 24 hours. D, The concentration of gremlin‐1 secreted into culture medium, as detected by ELISA. E, BMP2 dose‐dependently suppressed dPASMC proliferation and procured the maximal inhibitory effect at 20 ng/mL. F, Gremlin‐1 dose‐dependently antagonized the proliferation suppressive effect of BMP2 (20 ng/mL) on dPASMCs and reached the maximally antagonistic effect at 100 ng/mL. G, Anti–gremlin‐1 (5 μg/mL) reversed the maximally antagonistic effect of gremlin‐1 (100 ng/mL) for BMP2 (20 ng/mL). H through J, Representative Western blotting image and densitometric analysis showing that BMP2 at 20 ng/mL induced significant Smad1/5/8 (drosophila mothers against…

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  Figure 5. Gremlin‐1 blocks the proapoptotic effects of BMP2 (bone morphogenetic protein 2) on distal pulmonary arterial smooth muscle cells (dPASMCs). A, Representative flow cytometry images showing the effects of BMP2, BMP2+gremlin‐1, and BMP2+gremlin‐1+anti–gremlin‐1 on the apoptosis of dPASMCs. B, Quantification of apoptotic cells. C through F, Effect of BMP2, BMP2+gremlin‐1, and BMP2+gremlin‐1+anti–gremlin‐1 on Bax mRNA level (C), B‐cell lymphoma‐2 (Bcl‐2) mRNA level (D), ratio of Bcl‐2/Bax (E), and Bcl‐2 protein level (F) in dPASMCs. Data are mean±SD of 4 to 5 independent experiments. NS indicates not significant. *P<0.05, **P<0.01, and NS P>0.05 vs control group.

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  Figure 6. Gremlin‐1 expression increased in patients with congenital heart disease with Eisenmenger syndrome (CHD‐ES). A, mRNA level of gremlin‐1 in patients with CHD‐ES (n=5) and control subjects (n=5). B, Representative Western blotting image of gremlin‐1 protein level (lower panel) and densitometric analysis (upper histogram) in patients with CHD‐ES (n=5) and control subjects (n=5). C, Representative images of immunohistological staining of gremlin‐1 in control lungs (C1 and C2, ×40) and lungs with CHD‐ES (C3 and C4, ×100) (bar=50 μm). D, Plasma level of gremlin‐1 demonstrated a stepwise increase with the extent of CHD‐associated pulmonary arterial hypertension (CHD‐PAH). E through L, Plasma level of gremlin‐1 positively correlated with pulmonary arterial systolic pressure (PASP) (E), mean pulmonary arterial pressure (mPAP) (F), pulmonary vascular resistance (G), total pulmonary vascular resistance (TPVR) (H), and ratio of pulmonary arterial systolic pressure/systemic arterial systolic pressure (Pp/Ps) (I), and negatively correlated with pulmonary/systemic shunt volume ratio (Qp/Qs) (J), cardiac output (CO) (K), and cardiac index (CI) (L). sePAH indicates severe PAH. **P<0.01.