mTORC1-ATF4-MCP-1 Pathway as a Driver of Chronic Thromboembolic Pulmonary Hypertension in Rats through the Induction of Endothelial Dysfunction and Pulmonary Artery Thromboembolism

Abstract

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating condition caused by a putative mechanism associated with the activation of the mammalian target of rapamycin (mTOR). The current study aims to unravel the signaling pathway leading to CTEPH on the basis of mTOR activation. Methods: In vivo CTEPH models were established from rats exposed to repeated autologous thromboembolization. The rats were subjected to right ventricular pressure (RVP) and mean pulmonary artery pressure (mPAP) measurement as well as histopathological examination. To establish CTEPH cellular models for in vitro experiments, pulmonary artery endothelial cells were isolated from CTEPH rats and identified via immunofluorescence/flow cytometry. AZD8055, an mammalian target of rapamycin complex 1 (mTORC1) inhibitor, was used to treat both in vivo and in vitro models. Activating transcription factor 4 (ATF4) was overexpressed in in vitro models. Viability and apoptosis were detected using cell counting kit-8 assay and flow cytometry. Expression of mTOR, ATF4 and monocyte chemoattractant protein-1 (MCP-1) in pulmonary artery tissues/cells was measured by means of immunohistochemistry, Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The interaction between ATF4 and MCP-1 was predicted with bioinformatics approach and validated using Chromatin immunoprecipitation (ChIP) assay. Results: CTEPH rats exhibited elevated mPAP, activated mTORC1-ATF4-MCP-1 pathway, increased area/total area of pulmonary artery, and enhanced apoptosis and thromboembolism in the pulmonary artery tissues, which were all reversed by AZD8055 treatment. In in vitro models, ATF4 overexpression decreased cell viability, enhanced apoptosis and upregulated MCP-1 level, while AZD8055 exerted an opposite effect, normalizing these changes and suppressing the mTORC1-ATF4-MCP-1 pathway. ATF4 could bind to MCP-1 promoter region. Conclusion: The mTORC1-ATF4-MCP-1 pathway induces endothelial dysfunction and pulmonary artery thromboembolism to promote the development of CTEPH in rats.