miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

Abstract

Pulmonary artery hypertension (PAH) pathology involves extracellular matrix (ECM) remodeling in cardiac tissues, thus promoting cardiac fibrosis progression. miR-29a-3p reportedly inhibits lung progression and liver fibrosis by regulating ECM protein expression; however, its role in PAH-induced fibrosis remains unclear. In this study, we aimed to investigate the role of miR-29a-3p in cardiac fibrosis progression in PAH and its influence on ECM protein thrombospondin-2 (THBS2) expression. The diagnostic and prognostic values of miR-29a-3p and THBS2 in PAH were evaluated. The expressions and effects of miR-29a-3p and THBS2 were assessed in cell culture, monocrotaline-induced PAH mouse model, and patients with PAH. The levels of circulating miR-29a-3p and THBS2 in patients and mice with PAH decreased and increased, respectively. miR-29a-3p directly targets THBS2 and regulates THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis. The circulating levels of miR-29a-3p and THBS2 were correlated with PAH diagnostic parameters, suggesting their independent prognostic value. miR-29a-3p targeted THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis, indicating miR-29a-3p acts as a messenger with promising therapeutic effects.

Keywords: THBS2; cardiomyocytes; fibrosis; miR-29a-3p; pulmonary arterial hypertension.

Figure 1

Monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) in mice. C57BL/6 mice were treated with or without MCT (right side, 60 mg/kg; IP, once) for 30 days. (A) Hematoxylin and eosin (H&E) staining revealed a thicker pulmonary artery wall (blue arrows) following MCT-induced PAH. A, alveolar; B, bronchial; black arrows, vein. (B) The 3D lung image and vascular tree image were obtained by contrast-enhanced micro-computed tomography (MicroCT). (C,D) The mean pulmonary artery (mPA) and aortic artery (AO) image and PA/AO ratio; (E,F) left ventricle (LV) and right ventricle (RV) image and RV/LV ratio; (G,H) PA area was measured using MicroCT and Image J software. Data are representative of three independent experiments (n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05).

Figure 2

Myocardial fibrosis in MCT-induced PAH animal model. (A,B) Interstitial and perivascular collagen deposition in the transverse cardiac section was examined by Masson’s trichrome staining. Collagen fibers are indicated in blue. (C,D) Interstitial and perivascular collagen fiber accumulation in cardiac tissue was determined and quantified by transmission electron microscopy (TEM). Collagen fibers are indicated with blue arrows. (E,F) Cardiac collagen deposition and arrangement from the endocardium to epicardium were identified and quantified by second-harmonic generation (SHG) microscopy. Data are representative of three independent experiments (n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01).

Figure 3

Cardiomyocyte-derived exosomes were detected in vitro and in vivo. (A,B) Multivesicular body (MVB) and exosome presence in myocardial tissue and human cardiomyocytes (HCM) were examined and quantified using TEM and Image J software. MVB, red arrow; exosome, blue arrow. (C) HCMs were treated with or without MCT for 24 h, and then the secreted exosomes were isolated from the culture medium. The exosome size was measured by nanoparticle tracking analysis. (D) The expression of exosomal proteins CD81 and HSP90 was examined by western blotting analysis. Data are representative of three independent experiments (n = 3).

Figure 4

Proteomics profiling of HCM-derived exosomes. HCMs were treated with or without MCT for 24 h, and then the exosomal proteins were isolated and analyzed by high-resolution liquid chromatography-mass spectrometry (LC-MS/MS). (A) The intersection of the differentially expressed proteins between the control and MCT-treated samples was subjected to Venn diagram analysis. (B) The fold changes in gene expression between the experimental groups are illustrated with a heatmap. Genes are listed on the y-axis. (C–E) Functional annotation of differentially expressed genes in MCT-treated HCMs were analyzed using gene ontology (GO) barplot, dotplot, and emapplot analyses. (F–H) Extracellular structure organization and regeneration-associated genes identified using these analyses of differentially expressed genes in MCT-treated HCMs. Data are representative of three independent experiments (n = 3).

Figure 5

miR-29a-3p regulates THBS2 expression in vitro and in vivo. (A,B) MCT-induced expression of miR-29a-3p and exosomal THBS2 mRNA, determined by real-time PCR. (C) THBS2 expression in MCT-treated HCM-secreted exosomes determined by western blotting. HSP70 was used as the internal control for exosomal proteins. (D) Alignment of the miR-29a-3p with the THBS2 3′-untranslated region, showing the putative binding sites. (E) Real-time PCR analysis of miR-29a-3p expression in HCMs after transfection with an miR-29a-3p mimic or inhibitor. Data are representative of three independent experiments (n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05). (F,G) After transfection, THBS2 expression was analyzed by real-time PCR and immunocytochemical staining. Data are representative of three independent experiments (n = 3), and values are expressed as median and interquartile range. Kruskal–Wallis test was used to compare two independent groups (* p < 0.05). (H,I) MCT-induced expression of miR-29a-3p and THBS2 mRNA, determined by real-time PCR. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01). (J) In situ hybridization (ISH) of miR-29a-3p expression (pink color) in myocardial tissues of MCT-induced PAH mice. U6, miRNA internal control. (K,L) THBS2 expression in myocardial tissues and circulating serum of MCT-induced PAH mice were examined by immunohistochemistry and western blotting, respectively. Transferrin was used as the internal control. Data are representative of three independent experiments (n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05 and ** p < 0.01).

Figure 6

Cardiomyocyte-derived exosomes regulate cardiac fibroblast to myofibroblast transformation. (A) Schematic diagram for studying the effects of human cardiomyocyte (HCM)-derived exosome uptake by human cardiac fibroblasts (HCF) using early endosome antigen 1(EEA1) staining. (B) Immunocytochemistry and confocal microscopy identified the time-dependent intake and regulation of HCM-derived exosomes by EEA1 in HCFs. Nuclear DNA, exosomes, and EEA1 were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue), Dil dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine, red), and GFP fluorescence dye (green), respectively. Data are representative of three independent experiments (n = 3). (C) HCFs were treated with or without HCM-derived exosomes, HCM+MCT-derived exosomes, or rTHBS2 (THBS2 recombinant protein, 1 μg/mL) for 48 h to assess cardiomyocyte-derived exosomal THBS2-mediated fibroblast to myofibroblast transformation. Expression of the myofibroblast marker proteins, α-SMA, β-catenin, fibronectin, and vimentin was examined by immunocytochemistry and confocal microscopy with Imaris software for confocal image 3D reconstruction. (D,E) Myofibroblast marker protein expression in myocardial tissues of MCT-induced PAH mice was examined by western blotting and immunohistochemistry. Data are representative of three independent experiments (n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05).

Figure 7

MiR-29a-3p/THBS2 axis regulates the progression of pulmonary artery hypertension (PAH). (A) Exosomal miR-29a-3p level in the serum of healthy controls (NR) and patients with PAH was determined by real-time PCR. (B) THBS2 expression in the serum was determined by ELISA. Data are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01). (C) Correlation coefficient between the miR-29a-3p and THBS2 expression levels. R, Pearson’s correlation coefficient. (D–F) The correlation of miR-29a-3p with the mean pulmonary artery pressure (mPAP), tricuspid annular plane systolic excursion (TAPSE), and pulmonary vascular resistance (PVR) in patients with PAH. (G–I) The correlation of the serum THBS2 level with mPAP, TAPSE, and PVR in patients with PAH. (J) Suggested mechanism underlying PAH-induced cardiac fibrosis mediated by exosomal miR-29a-3p and THBS2. Serum miR-29a-3p and THBS2 (secreted by cardiomyocytes) might increase cardiomyocyte hypertrophy and promote cardiac fibroblast to myofibroblast transformation, enhance collagen production/deposition, and augment cardiac fibrosis.