Figures & data
Table 1 Demographic and clinical characteristics of participants with pulmonary hypertension and controls
Figure 1 The increased circulating levels of miR-190a-5p in patients with COPD-PH correlate with disease severity.
Notes: (A) Levels of miR-190a-5p in patients with PH were significantly higher than those in the healthy controls. The increase in miR-190a-5p levels was more significant in the COPD-PH groups than in the healthy controls. In contrast, there were no significant differences in the miR-190a-5p levels of the IPAH, PAH-CTD, CTEPH, and healthy controls groups. *P<0.05, in comparison with healthy controls. ***P<0.001, in comparison with healthy controls. (B) The relative expression of KLF15 mRNA was significantly lower in the global cohort of patients with PH and the COPD-PH subgroup than in the healthy controls. In contrast, there were no significant differences in the relative expression of KLF15 mRNA of the IPAH, PAH-CTD, CTEPH, and healthy controls groups. **P<0.01, in comparison with healthy controls. ****P<0.0001, in comparison with healthy controls. (C) Compared with COPD-PH patients with PO2 above 60 mm Hg, those with PO2 below 60 mm Hg had significantly higher levels of miR-190a-5p and significantly lower relative expression levels of KLF15 mRNA. *P<0.05 compared with COPD-PH patients with PO2 above 60 mm Hg. **P<0.01 compared COPD-PH patients with PO2 above 60 mm Hg. (D) Compared with non-severe COPD-PH patients, patients with severe COPD-PH had significantly higher levels of miR-190a-5p and significantly lower relative expression levels of KLF15 mRNA. *P<0.05 compared with non-severe COPD-PH patients. (E) Higher levels of miR-190a-5p were significantly correlated with lower relative expression levels of KLF15 mRNA in COPD-PH patients.
Abbreviations: COPD-PH, pulmonary hypertension secondary to chronic obstructive pulmonary disease; CTEPH, chronic thromboembolic pulmonary hypertension; IPAH, idiopathic pulmonary arterial hypertension; KLF15, Krüppel-like factor 15; PAH-CTD, connective tissue disorder-associated pulmonary arterial hypertension; PaO2, partial pressure of arterial oxygen; PH, pulmonary hypertension.
Abbreviations: COPD-PH, pulmonary hypertension secondary to chronic obstructive pulmonary disease; CTEPH, chronic thromboembolic pulmonary hypertension; IPAH, idiopathic pulmonary arterial hypertension; KLF15, Krüppel-like factor 15; PAH-CTD, connective tissue disorder-associated pulmonary arterial hypertension; PaO2, partial pressure of arterial oxygen; PH, pulmonary hypertension.
Table 2 Cox survival analysis of the COPD-PH cohort
Figure 2 Circulating miR-190a-5p levels have diagnostic and prognostic value in COPD-PH patients.
Notes: (A) ROC curve showing that the levels of miR-190a-5p have a significant value in the prediction of patients with PH. (B) ROC curve showing that the levels of miR-190a-5p in patients with COPD-PH have a better predictive value than that in patients with PH. (C) Kaplan–Meier survival curves indicate there is no relationship between the miR-190a-5p levels and all-cause death in patients with PH. The cutoff point (2.0) is derived from the ROC curve. (D) Kaplan–Meier survival curves indicate that patients with lower levels of miR-190a-5p have better event-free survival in the COPD-PH subgroup. The cutoff point (2.75) is derived from the ROC curve.
Abbreviations: COPD-PH, pulmonary hypertension secondary to chronic obstructive pulmonary disease; PH, pulmonary hypertension; ROC, receiver operator characteristic.
Abbreviations: COPD-PH, pulmonary hypertension secondary to chronic obstructive pulmonary disease; PH, pulmonary hypertension; ROC, receiver operator characteristic.
Figure 3 Effects of hypoxia on the levels of miR-190a-5p, KLF15, eNOS, and Arg2 in HPAECs and HPMECs.
Notes: (A) The expression levels of KLF15, eNOS, and Arg2 were determined in HPAECs and HPMECs exposed to normoxia and hypoxia by Western blot analysis. (B) Quantification of the relative expression levels of KLF15, eNOS, and Arg2 protein in HPAECs. The values obtained by densitometric measurements were normalized to the GADPH expression levels. ****P<0.0001 compared with the normoxic condition. (C) Quantification of the relative expression levels of KLF15, eNOS, and Arg2 protein in HPMECs. The values obtained by densitometric measurements were normalized to the GADPH expression levels. ****P<0.0001 compared with the normoxic condition. (D) The miR-190a-5p levels were determined in HPAECs, HPMECs, HPASMCs, and HPAFs exposed to normoxia and hypoxia by using RT-qPCR. ****P<0.0001 compared with the normoxic condition.
Abbreviations: Arg2, Arginase 2; eNOS, endothelial nitric oxide synthase; HPAECs, human pulmonary arterial endothelial cells; HPAFs, human pulmonary artery fibroblasts; HPASMCs, human pulmonary artery smooth muscle cells; HPMECs, human pulmonary microvascular endothelial cells; KLF15, Krüppel-like factor 15; RT-qPCR, real-time quantitative PCR.
Abbreviations: Arg2, Arginase 2; eNOS, endothelial nitric oxide synthase; HPAECs, human pulmonary arterial endothelial cells; HPAFs, human pulmonary artery fibroblasts; HPASMCs, human pulmonary artery smooth muscle cells; HPMECs, human pulmonary microvascular endothelial cells; KLF15, Krüppel-like factor 15; RT-qPCR, real-time quantitative PCR.
Figure 4 miR-190a-5p correlated with the expression levels of Arg2 and eNOS by targeting KLF15.
Notes: (A) miRNA luciferase reporter assays in HPAECs co-transfected with WT or Mut KLF15 reporters and miR-190a-5p are shown. The medium panel indicates the WT forms of putative miR-190a-5p target sequences in the 3′-UTR of KLF15. The bottom panel indicates putative miR-190a-5p-binding sites with the 3′-UTR of KLF15. The shaded portion indicates mutations introduced into the 3′-UTR of KLF15. (B) Dual luciferase reporter assay in HPAECs. The luciferase activity of each group was calculated by normalizing the firefly luciferase activity to renilla luciferase activity. (C) The expression levels of KLF15, eNOS, and Arg2 were determined in HPAECs transfected with negative controls, miR-190a-5p inhibitor, miR-190a-5p mimic, and KLF15 siRNA by using Western blot. (D) Quantification of the relative expression levels of KLF-15, eNOS, and Arg2 protein in each group. The values obtained by densitometric measurements were normalized to GADPH expression levels. ****P<0.0001 compared with control.
Abbreviations: Arg2, Arginase 2; eNOS, endothelial nitric oxide synthase; HPAECs, human pulmonary arterial endothelial cells; KLF15, Krüppel-like factor 15; Mut, mutant; WT, wild type.
Abbreviations: Arg2, Arginase 2; eNOS, endothelial nitric oxide synthase; HPAECs, human pulmonary arterial endothelial cells; KLF15, Krüppel-like factor 15; Mut, mutant; WT, wild type.
Figure 5 In vivo effect of antagomir-190a-5p treatment on hemodynamic parameters, right heart hypertrophy, and the expression of KLF15, eNOS, and Arg2 in the hypoxia-induced PAH mouse model.
Notes: (A) Representative tracings of RV pressures in the normoxia group. (B) Representative tracings of RV pressures in the hypoxia-antagomir-NC group. (C) Representative tracings of RV pressures in the hypoxia-antagomir-190a-5p group. (D) Quantitative analyses of RVSP in each group. ****P<0.0001 compared with the normoxia group; ##P<0.01 compared with the hypoxia-antagomir-NC group. (E) Quantitative analyses of RV/(LV+S) in each group. ***P<0.001 compared with the normoxia group; #P<0.05 compared with the hypoxia-antagomir-NC group. (F) The miR-190a-5p levels were determined in the lung tissue of mice by using RT-qPCR. ****P<0.0001 compared with the normoxia group; ####P<0.0001 compared with the hypoxia-antagomir-NC group. (G) The expression levels of KLF15, eNOS, and Arg2 were determined in the lung tissue of mice by using Western blot. (H) Quantification of the relative expression levels of KLF-15, eNOS, and Arg2 protein in each group. The values obtained by densitometric measurements were normalized to the GADPH expression levels. ****P<0.0001 compared with the normoxia group; ####P<0.0001 compared with the hypoxia-antagomir-NC group.
Abbreviations: Arg2, Arginase 2; eNOS, endothelial nitric oxide synthase; KLF15, Krüppel-like factor 15; NC, negative control; PAH, pulmonary arterial hypertension; RVSP, right ventricular systolic pressure; RV/(LV+S), the ratio of the free wall of the right ventricle weight to the weight of the left ventricle and septum.
Abbreviations: Arg2, Arginase 2; eNOS, endothelial nitric oxide synthase; KLF15, Krüppel-like factor 15; NC, negative control; PAH, pulmonary arterial hypertension; RVSP, right ventricular systolic pressure; RV/(LV+S), the ratio of the free wall of the right ventricle weight to the weight of the left ventricle and septum.
