Deficiency of cold-inducible RNA-binding protein exacerbated monocrotaline-induced pulmonary artery hypertension through Caveolin1 and CAVIN1

Abstract

Cold-inducible RNA-binding protein (CIRP) was a crucial regulator in multiple diseases. However, its role in pulmonary artery hypertension (PAH) is still unknown. Here, we first established monocrotaline (MCT)-induced rat PAH model and discovered that CIRP was down-regulated predominantly in the endothelium of pulmonary artery after MCT injection. We then generated Cirp-knockout (Cirp-KO) rats, which manifested severer PAH with exacerbated endothelium damage in response to MCT. Subsequently, Caveolin1 (Cav1) and Cavin1 were identified as downstream targets of CIRP in MCT-induced PAH, and the decreased expression of these two genes aggravated the injury and apoptosis of pulmonary artery endothelium. Moreover, CIRP deficiency intensified monocrotaline pyrrole (MCTP)-induced rat pulmonary artery endothelial cells (rPAECs) injuries both in vivo and in vitro, which was counteracted by Cav1 or Cavin1 overexpression. In addition, CIRP regulated the proliferative effect of conditioned media from MCTP-treated rPAECs on rat pulmonary artery smooth muscle cells, which partially explained the exceedingly thickened pulmonary artery intimal media in Cirp-KO rats after MCT treatment. These results demonstrated that CIRP acts as a critical protective factor in MCT-induced rat PAH by directly regulating CAV1 and CAVIN1 expression, which may facilitate the development of new therapeutic targets for the intervention of PAH.

Keywords: CAVIN1; Caveolin1; cold-inducible RNA-binding protein; monocrotaline; pulmonary artery endothelial cells; pulmonary artery hypertension.

FIGURE 1

Reduced CIRP expression in the pulmonary artery endothelium of MCT‐induced PAH rats. A, qPCR analysis of Cirp expression in rat lungs at week four after saline‐ or MCT‐ injection. (n = 4 per group). B, Western blotting and quantitation of CIRP expression in rat lungs at week four after saline or MCT injection. β‐ACTIN served as loading control. (n = 3 per group). C, Immunofluorescence of CIRP in rat lungs at week four after saline or MCT injection. Black arrows point to CIRP positive signals in endothelium. Scale bar, 50 µm. D, Immunofluorescence of CIRP in rPAECs and rPASMCs. DAPI stains nuclei. Scale bar, 100 µm. E, Western blotting and quantitation of CIRP in rPAECs and rPASMCs following 48 h culture with or without MCTP. β‐ACTIN served as loading control. (n = 3 per group). DAPI, 4′,6‐diamidino‐2‐phenylindole; MCT, Monocrotaline; MCTP, Monocrotaline Pyrrole. The data are shown as mean ± SD from three independent experiments. **P <.01, *P <.05. ns, not significant

FIGURE 2

CIRP deficiency exaggerated MCT‐induced PAH in rats. A and B, Right ventricular systolic pressure (RVSP) was assessed by invasive haemodynamic measurements. C, Right ventricular hypertrophy (Fulton index) was evaluated by the right ventricle over the left ventricle + septum weight ratio (RV/(LV + S)). D, Doppler echocardiology was performed to measure the pulmonary artery acceleration time (PAT) and pulmonary artery ejection time (PET) of Cirp‐KO and WT rats at week two and four after MCT injection. E, The systole and diastole blood pressure of rats in quiet environment was measured noninvasively to calculate mean artery pressure (MAP). F, Transthoracic two‐dimensional echocardiography was applied on rats to evaluate left ventricular function. Top: representative echocardiographic images of WT and Cirp‐KO rats at week four after saline or MCT injection. Bottom: fractional shortening (FS) and the ejection fraction (EF) of left ventricle, left ventricular posterior wall at diastole (LVPWd) were measured on the M‐mode echocardiogram and calculated with standard formula. WT, wild type; KO, Cirp‐knockout; MCT, monocrotaline. The data are shown as mean ± SD from three independent experiments. ***P <.001, **P <.01, *P <.05

FIGURE 3

Cav1 and Cavin1 served as downstream targets of CIRP in regulating PAH. A, Proteomics was performed in lungs from WT and Cirp‐KO rats at week four after saline or MCT injection. (n = 3 per group). The volcano plot illustrated 131 proteins with significant difference between the two groups. B, Potential regulatory molecules were obtained by intersecting published CIRP targeted genes and protein spectrum conducted in this project. Blue circle represents 4376 CIRP target genes identified by PAR‐Clip and RNA sequencing; yellow circle represents 131 differentially expressed proteins in mass spectrometry data. C, Representative immunoblots and densitometric analysis of CAV1 and CAVIN1 in lungs of WT and Cirp‐KO rats at week four after saline or MCT injection. β‐ACTIN served as loading control. (n = 3 per group). D and E, The binding of CIRP to Cav1 (D) and Cavin1 (E) mRNAs in rPAECs with or without MCTP stimulation was determined by RIP. (n = 3 per group). F and G, Representative immunohistochemical images of lungs in WT and Cirp‐KO rats stained for CAV1 (F) and CAVIN1 (G) at week four after saline or MCT injection. Scale bar, 50 µm. WT, wild type; KO, Cirp‐knockout; MCT, monocrotaline. The data are shown as mean ± SD from three independent experiments. ***P <.001, **P <.01, *P <.05, ns, not significant

FIGURE 4

Cirp knockdown exacerbated MCTP‐induced rPAECs apoptosis and permeability that was counteracted by Cav1 or Cavin1 overexpression. A, TUNEL assay of lungs isolated from WT and Cirp‐KO rats at week four after saline or MCT injection. The apoptotic index was measured by counting puncta of TUNEL signal in randomly selected CD31‐positive cells (n ≥ 3 per group). Scale bar, 46 µm. B, Images of EB dye staining in lungs isolated from WT and Cirp‐KO rats at different time points after MCT injection respectively, and quantitative assessment of EB dye content in the lungs. C, Representative immunoblots and densitometric analysis of cleaved CASPASE‐3, CAV1 and CAVIN1 in Cirp knockdown and Cav1 or Cavin1 overexpressing rPAECs following MCTP treatment. β‐ACTIN served as loading control. (n = 3 per group). D, TUNEL assay of Cirp knockdown and Cav1 or Cavin1 overexpressing rPAECs following MCTP treatment. DAPI stains nuclei. Scale bars, 50.4 µm. E, The apoptotic index was measured by counting puncta of TUNEL positive signal. F and G, Permeability and cytotoxicity of Cirp knockdown and Cav1 or Cavin1 overexpressing rPAECs following MCTP treatment were assessed by quantifying absorbance of FITC‐dextran (F), LDH release (G). WT, wild type; KO, Cirp‐knockout; MCT, monocrotaline; MCTP, monocrotaline pyrrole; TUNEL, Terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labelling; LDH, lactate dehydrogenase; CD31, Platelet endothelial cell adhesion molecule‐1, PECAM‐1/CD31; DAPI, 4′,6‐diamidino‐2‐phenylindole; The data are shown as mean ± SD from three independent experiments. **** P <.0001, ***P <.001, ** P <.01, * P <.05. ns, not significant

FIGURE 5

Effects of conditioned media from MCTP‐treated rPAECs on rPASMCs proliferation. A, EdU staining of rPASMCs cultured in conditioned media from rPAECs. Top: conditioned media from Cirp knockdown and Cav1 or Cavin1 overexpressing rPAECs following MCTP treatment. Bottom: conditioned media from Cirp overexpressing and Cav1 or Cavin1 knockdown. DAPI stains nuclei. Scale bar, 63.9 µm. B and C, The proliferate index was measured by counting puncta of EdU signal. (n ≥ 3 per group). D, Immunofluorescence of SMA and CD31 in the lungs of WT and KO rats at week four after saline or MCT injection. DAPI stains nuclei. Scale bar, 100 µm. E, The thickness of intimal media was determined by intimal media area over pulmonary artery area of WT and Cirp‐KO rats at week four after saline or MCT injection. (n ≥ 3 per group). WT, wild type; KO, Cirp‐knockout; MCT, monocrotaline; EdU, 5‐ethynyl‐2’‐deoxyuridine; SMA, smooth muscle actin; CD31, platelet endothelial cell adhesion molecule‐1, PECAM‐1/CD31; DAPI, 4′,6‐diamidino‐2‐phenylindole. The data are shown as mean ± SD from three independent experiments. *** P <.001, ** P <.01, * P <.05