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. 2023 Dec 5;12(23):e031435.
doi: 10.1161/JAHA.123.031435. Epub 2023 Nov 28.

Transpulmonary Expression of Exosomal microRNAs in Idiopathic and Congenital Heart Disease-Related Pulmonary Arterial Hypertension

Wei-Ting Chang  1   2 , Wei-Chieh Lee  2   3 , Yu-Wen Lin  2 , Jhih-Yuan Shih  2   3 , Chon-Seng Hong  2   4 , Zhih-Cherng Chen  2   3 , Chun-Yuan Chu  5 , Chih-Hsin Hsu  6
Affiliations

Transpulmonary Expression of Exosomal microRNAs in Idiopathic and Congenital Heart Disease-Related Pulmonary Arterial Hypertension

Wei-Ting Chang et al. J Am Heart Assoc. .

Abstract

Background: Pulmonary artery hypertension (PAH) is a fatal disease characterized by a complex pathogenesis. Exosomes containing microRNAs (miRs) have emerged as a novel biomarker. Transpulmonary exosomal miRs offer valuable insights into pulmonary circulation microenvironments. Hereby, we aimed to explore the potentials of transpulmonary exosomal miRs as differentiating factors between idiopathic PAH and congenital heart disease (CHD)-related PAH.

Methods and results: During right heart catheterization, we collected exosomes at pulmonary arteries in 25 patients diagnosed with idiopathic PAH and 20 patients with CHD-related PAH. Next-generation sequencing identified several candidate exosomal miRs. Using quantitative polymerase chain reaction, we validated the expressions of these miRs and revealed significantly elevated expressions of miR-21, miR-139-5p, miR-155-5p, let-7f-5p, miR-328-3p, miR-330-3p, and miR-103a-3p in patients with CHD-related PAH, in contrast to patients with idiopathic PAH. Among these miRs, miR-21 exhibited the highest expression in patients with CHD-related PAH. These findings were further corroborated in an external cohort comprising 10 patients with idiopathic PAH and 8 patients with CHD-related PAH. Using an in vitro flow model simulating the shear stress experienced by pulmonary endothelial cells, we observed a significant upregulation of miR-21. Suppressing miR-21 rescued the shear stress-induced downregulation of the RAS/phosphatidylinositol 3-kinase/protein kinase B pathway, leading to a mitigation of apoptosis.

Conclusions: Our study identified a pronounced expression of transpulmonary exosomal miR-21, particularly in patients with CHD-related PAH, through next-generation sequencing analysis. Further investigation is warranted to elucidate the regulatory mechanisms involving miR-21 in the pathophysiology of PAH.

Keywords: congenital heart disease; exosome microRNAs; pulmonary artery hypertension; transpulmonary expression.

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Figures

Figure 1
Figure 1. Identification of exosomal miRs in patients with PAH.
A, Study design and flowchart of the right heart catheter–derived pulmonary circulating exosomal miRs in patients with PAH. B, Heat map clustering analysis of miR expression profiles between control patients, patients with CHD–related PAH, and IPAH. Genes are shown in the right column. The red bar indicates the miR expression level. C, The expression levels of target miRs across 3 groups (control, CHD‐related PAH, and IPAH); comparison was validated using quantitative polymerase chain reaction. CHD indicates congenital heart disease; IPAH, patients with idiopathic PAH; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRs, microRNAs; and PAH, pulmonary artery hypertension. ***P=0.005; ****P=0.001.
Figure 2
Figure 2. Validating the miR expression in endothelial cells under flow‐mediated shear stress stimuli.
A, The study design of flow‐mediated shear stress culture system. B, The expression levels of target miRs in HPMECs under flow‐mediated shear stress. HPMECs indicates human pulmonary microvascular endothelial cells; and miR, microRNA. *P<0.05, **P<0.01.
Figure 3
Figure 3. Suppression of miR‐21 mitigates cell apoptosis in HPMECs under flow‐mediated shear stress stimuli.
A, The study design of flow‐mediated shear stress culture system. The HPMECs were pretreated with or without miR‐21 inhibitor before flow‐mediated shear stress for 3 or 6 hours at 6 dyn/cm2. B, The miR‐21 expression in miR‐21 inhibitor under flow‐mediated shear stress was measured using qPCR. C, TUNEL assay conducted on HPMECs treated with miR‐21 inhibitor under flow‐mediated shear stress. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; HPMECs, human pulmonary microvascular endothelial cells; miR, microRNA; qPCR, quantitative polymerase chain reaction; and TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling. **P<0.01, ***P<0.005.
Figure 4
Figure 4. Suppression of miR‐21 attenuates apoptosis‐associated proteins in HPMECs under flow‐mediated shear stress stimuli.
A, Representative Western blot analyses and quantification of RAS, P‐JAK, P‐PI3K, and P‐AKT. B, Apoptosis‐associated protein expression in HPMECs pretreated with miR‐21 inhibitor under flow‐mediated shear stress. BAX indicates Bcl‐2‐associated X protein; BCL‐2, B‐cell lymphoma 2; HPMECs, human pulmonary microvascular endothelial cells; miR, microRNA; P‐JAK, phosphorylated Janus kinase; P‐AKT, phosphorylated protein kinase B; and P‐PI3K, phosphorylated phosphatidylinositol 3‐kinase. *P<0.05, **P<0.01.
Figure 5
Figure 5. The attenuation of apoptosis through suppression of miR‐21 was partially reversed by inhibiting RAS and PI3K in HPMECs under flow‐mediated shear stress stimuli.
A, TUNEL assay conducted on HPMECs treated by miR‐21 inhibitor concomitantly with and without RAS or PI3K inhibitors under flow‐mediated shear stress. B, Representative Western blot analyses and quantification of RAS, P‐JAK, P‐PI3K, and P‐AKT. C, Apoptosis‐associated protein expression in HPMECs pretreated with miR‐21 inhibitor under flow‐mediated shear stress. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; HPMECs, human pulmonary microvascular endothelial cells; miR, microRNA; P‐JAK, phosphorylated Janus kinase; P‐AKT, phosphorylated protein kinase B; P‐PI3K, phosphorylated phosphatidylinositol 3‐kinase; and TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.
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