Abstract Background Pulmonary arterial hypertension (PAH) is a severe condition that leads to elevated blood pressure in the pulmonary circulation combined with an occlusive pulmonary arteriopathy. Dysfunction of endothelial cells (ECs) and aberrant proliferation of smooth muscle cells (SMCs), along with the accumulation of perivascular immune cells, contribute to arterial remodeling. We have shown that deficiency of the endosomal GTPase RAB7 in pulmonary artery ECs (PAECs), as found in PAH patients, leads to endothelial dysfunction, senescence, and spontaneous pulmonary hypertension (PH) in mice. We hypothesized that endothelial RAB7 exerts its effects on the growth of other mural cells by sequestering RNA-binding proteins that regulate the endothelial secretome. Methods We used in vitro experiments using pulmonary artery ECs (PAECs), co-culture with pulmonary artery SMCs (PASMCs) and adventitia fibroblasts (PAAFs) using well inserts, siRNA-mediated gene silencing, plasmid transfection, proximity-based biotinylation, proteomics, co-immunoprecipitation, transfer of conditioned media, DNA synthesis analysis, and a Ki-67 proliferation assay. In addition, in vivo experiments utilized the mouse chronic hypoxia/SU5416 (Hx/Su) model and intraperitoneal injections of Clofoctol. Results Knockdown of RAB7 in pulmonary artery ECs results in increased DNA synthesis of co-cultured PASMCs and PAAFs. Additionally, the gene expression profile of PASMCs shifted from a contractile to a synthetic phenotype, and the mRNA expression of fibroblast activation genes increased in PAAFs. Proteomic analysis of the secretome from RAB7-deficient PAECs revealed 205 differentially expressed proteins, with 196 proteins being upregulated or unique. Pathways that were affected included protein translation, mitosis, and interferon signaling. Proximity-based biotinylation, combined with proteomics, yielded a total of 69 upregulated or unique proteins. Among these, additional experiments identified cold shock domain containing E1 (CSDE1), an RNA-binding protein regulating target mRNA stability and translation, as a functionally associated partner of RAB7. We integrated data from secretomics of RAB7 siRNA-treated PAECs, mRNA analysis of RAB7 siRNA-treated PAECs, and the addition of CSDE1 siRNA to identify CSDE1 targets that likely have enhanced mRNA stability under RAB7 deficiency. Many of these targets were increased in the lung tissue of monocrotaline and chronic Hx/Su rats. Clofoctol docks to CSDE1, and Clofoctol treatment reduced CSDE1 targets in vitro and right ventricular systolic pressure in Hx/Su mice in vivo. Conclusions Our data suggest RAB7 as a sentinel CSDE1 gatekeeper driving the endothelial crosstalk with other mural cell types in the pulmonary artery via regulation of the endothelial secretome. This abstract is funded by: NIH, AHA
Farkas et al. (Fri,) studied this question.