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.