1. INTRODUCTION
Pulmonary hypertension (PH) afflicts approximately 1% of the global population, but up to 10% of individuals older than 65 years(Hoeper et al., 2016). Pulmonary artery remodeling (PVR), characteristic of PH, inflicts increased pulmonary arterial pressure and ultimately results in right ventricle (RV) failure with breath shortness and syncope. General life-supporting measures, PH-specific drug therapies, balloon pulmonary angioplasty, pulmonary thromboendarterectomy and lung transplant are presently the main treatment strategies(Thenappan et al., 2018). Despite progresses, the present therapeutic avenues are far from satisfactory, and the prognosis for PH patients is dire. Specifically, the current measures are not curative, only to alleviate symptoms. Some of the available drugs, such as prostacyclin analogues and endothelin receptor antagonists, are plagued by off-target effects, severe side effects and high cost. Therefore, further dissection of the molecular mechanisms of PH is needed for translation into superior treatments.
Exosomes are nanoscale membranous vesicles with a diameter range of ~40 to 160 nm. They are of endosomal origin and released into the extracellular fluid compartment via exocytosis(Kalluri and LeBleu, 2020). A large number of cell types can secrete exosomes as a route for intercellular communication, such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), the two major resident cell types relevant in PH pathogenesis. Exosomes have been recognized as a signaling hub, since they can serve as transfer machinery for diverse cargos, such as DNA, RNA, lipids, metabolites, cytosolic or cell surface proteins. The access of cargos into adjacent or distant recipient cells profoundly impacts cellular functions. Owing to their functional diversity, exosomes have been extensively studied under various circumstances and have been documented to contribute to diseases as diverse as cardiovascular, mammalogical and central nervous system-related diseases and cancer(Kalluri and LeBleu, 2020). Studies have linked exosomes to PH initiation and development, and consider miRNAs and proteins as the cargos mediating exosomal function. Pulmonary artery endothelial cells (PAECs)-derived exosomal miR-181a-5p and miR-324-5p or pulmonary artery smooth muscle cells (PASMCs)-derived exosomal miR-143 has been shown to regulate PASMCs inflammation, apoptosis and proliferation(Deng et al., 2015; Sindi et al., 2020). Exosomal 15-lipoxygenase-2 (15-LO2), Wnt5a and translationally controlled tumor protein (TCTP), derived from PAECs, pulmonary microvascular endothelial cells (PMVECs) or blood outgrowth ECs (BOECs) respectively, have been demonstrated to play a pathogenic role in PH(Ferrer et al., 2018; Zhang et al., 2018a; Yuan et al., 2019). By using non-plasma-derived exosomes, these interesting studies suggest that exosomes contribute to PH by directly acting on pulmonary vascular cells or mediating pulmonary vascular cell interaction. Unfortunately, the conclusion was based on non-plasma exosomes and has not been validated in vivo . Plasma is a rich source of exosomes containing about 1010 exosomes/mL(Vicencio et al., 2015). Plasma exosomes from MCT-induced PH mice could induce PH in healthy mice(Aliotta et al., 2016); plasma-derived exosomal miR-211 was more abundant in hypoxic PH rats12. These studies, albeit informative, lacked mechanistic interrogation. PVR is the pathological basis of PH, and exosomes have been shown to affect this process. Mesenchymal stem cell-derived exosomes or exosomes release inhibitor (GW4869) inhibited PVR and thus mitigated PH(Lee et al., 2012; Zhang et al., 2020); plasma or lung homogenates-derived exosomes from MCT-induced PH mice instigated PVR(Aliotta et al., 2016); miR-211-overexpressed exosomes derived from PAECs also promoted PVR(Zhang et al., 2021a). Despite the demonstrated significance of exosomes in PVR process, the issues of exosome origin and pathogenic cargos have yet to be further addressed. Uncontrolled proliferation of PASMCs is the well-known pathological hallmark of PVR. PASMCs phenotypic switching emerges as an indispensable contributor to excessive PASMCs proliferation and plays a key role in the process of PVR(Zhang et al., 2018b). Studies have shown that ECs-, M2 macrophage-, or neural cells-derived extracellular vesicles (EVs) regulate VSMCs phenotypic switching(Hergenreider et al., 2012; Yan et al., 2020; Li et al., 2022). However, effects of exosomes on PASMCs phenotypic switching, an important pathological characteristic of PH, warrants clarification.
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a member scavenger receptor orchestrating versatile pathological processes in various cardiovascular diseases(Akhmedov et al., 2021); its expression is relatively low under basal conditions, but is inducible by various stimuli or microenvironment, with pro-inflammatory cytokines and shear stress eliciting rapid LOX-1 production. In chronic thromboembolic PH patients, expression of pulmonary arterial LOX-1 was markedly increased(Wynants et al., 2012). Consistent with the clinical data, we have demonstrated that pulmonary arterial LOX-1 expression is also increased in pulmonary arteries of hypoxia-induced PH rats and hypoxia-treated PASMCs(Zhang et al., 2018b). Therefore, LOX-1 holds promise as a valuable therapeutic target for PH, and deepened understanding of LOX-1 function is of significance. Our previous studies have shown that LOX-1 promotes PASMCs phenotypic switching under hypoxic conditions, and accelerates PASMCs proliferation and migration stimulated by hypoxia-activated platelets(Zhang et al., 2018b; Ge et al., 2021). Our data conclude that LOX-1 is required for the maintenance of PASMCs phenotype. Interestingly, LOX-1 expression in HEK-293 cells-derived exosomes was boosted by cholesterol depletion, but the mechanistic insights were lacking(Gioia et al., 2015). LOX-1 has been demonstrated to mediate the effects of EVs on recipient cells and be regulated by EVs(Spaans et al., 2018, 2020), but whether LOX-1 is transported by EVs is unclear.
Given the respective importance of plasma exosomes and LOX-1 in PH, the present study aimed to address two issues: whether plasma exosomes promotes PH by triggering PASMCs phenotypic switching, and whether LOX-1 is a novel pathogenic cargo of exosomes. Herein, LOX-1 has been shown to be transported by plasma exosomes, and exosomal LOX-1 has been demonstrated to confer PASMCs phenotypic switching, PVR, and PH via ERK1/2-KLF4 signaling axis.