Knockdown of eIF3a alleviates pulmonary arterial hypertension by inhibiting endothelial-to-mesenchymal transition via TGFβ1/SMAD pathway.

OBJECTIVE

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by vascular remodeling and involves Endothelial-to-Mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs). EndMT is a complex cell differentiation process, mainly showing the detachment of endothelial cell migration and reducing endothelial cell characteristics to varying degrees, acquiring mesenchymal cell characteristics. In addition, numerous studies have reported that eIF3a over expression plays an important role in the occurrence and development of fibrotic diseases, cancer, and degenerative lesions, however, the mechanisms of eIF3a affecting the dysfunction of pulmonary arterial endothelial cells remains largely unknown. Therefore, we aimed to demonstrate the underlying mechanisms of eIF3a-knockdown inhibiting EndMT by regulating TGFβ1/SMAD signal pathway in PAH.

METHODS

In this study, we screened the potential target genes associated with idiopathic pulmonary arterial hypertension (IPAH) by WGCNA to provide a reference for the diagnosis and treatment of PAH. By constructing WGCNA, which indicated that the blue module (module-trait associations between modules and clinical feature information were calculated to selected the optimum module) is most closely associated with IPAH, we further screened out 10 up-regulated candidate biomarker genes. Male SD rats were randomly assigned to four groups: Control, Monocrotaline (MCT), AAV1-shRNA-NC group and AAV1-shRNA-eIF3a group. The eIF3a-knockdown rat model was constructed by adeno-associated virus type-1 (AAV1) infection, PAH was evaluated according to hemodynamic alteration, right heart hypertrophy and histopathological changes in the lung tissue. Hematoxylin eosin (H&E) staining was used to assess the morphological changes of pulmonary arteries in rats of each treatment group. Co-localization of eIF3a with alpha-small muscle action (α-SMA) and co-localization of eIF3a with endothelial marker (CD31) were detected by double-label immunofluorescence. Immunohistochemistry (IHC) and Western blot (WB) experiments were performed to assess the expression of eIF3a, EndMT and TGFβ1/SMAD signal related proteins. In vitro, primary rat pulmonary artery endothelial cells (PAECs) were transfected with si-eIF3a to investigate the effects of eIF3a-knockdown on hypoxia-induced EndMT in PAECs and further elucidate its underlying molecular mechanisms.

RESULTS

By WGCNA analysis, we screened the up-regulated hub genes of TMF1, GOLGB1, ARMC8, PRPF40 A, EIF3 A, ROCK2, EIF5B, CCP110, and KRR1 associated with PAH, and in order to verify the potential role of eIF3a in the development of pulmonary arterial hypertension, MCT-induced PAH rat model was constructed successfully. The expression of eIF3a was increased in MCT-treated lungs. Knockdown of eIF3a significantly inhibited the pulmonary arterial hypertension and vascular remodeling in MCT-induced PAH rat model, ameliorated MCT-induced increases of right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) in rats. Double-labeled immunofluorescence showed eIF3a was mostly co-localized with CD31, this result indicated that the development of MCT-induced PAH was related to the regulation of PAECs function (most likely associated with the change of EndMT in endothelial cells). WB showed that the expressions of EndMT related proteins were significantly increased by regulating TGFβ1/SMAD signaling pathway in MCT-induced PAH rat lung tissues, however, knockdown of eIF3a markedly attenuated these changes. In addition, we observed the same results in rat PAECs with chronic hypoxia exposure. These results indicate that eIF3a-knockdown inhibited EndMT by regulating TGFβ1/SMAD signaling pathway in PAECs, thereby improving the development of MCT-induced PAH.

CONCLUSIONS

Knockdown of eIF3a inhibited EndMT in PAECs regulating TGFβ1/SMAD signaling pathway, significantly alleviated the changes of RVSP, RVH and vascular remodeling in MCT-induced PAH rats, eIF3a may be a promising and novel therapeutic target for the treatment of PAH.