4. DISCUSSION
The present study aims to unravel the role of plasma-derived exosomes in
PH pathogenesis and decipher the underlying mechanisms whereby exosomes
regulate PVR. We found that: (a) plasma-derived exosomes promoted PASMCs
phenotypic switching and PVR, thus accelerating PH; (b) the cell-surface
protein LOX-1 was loaded into plasma exosomes, and exosomal LOX-1 levels
were markedly up-regulated in hypoxic PH rats; (c) exosomal LOX-1
powerfully induced PASMCs phenotypic switching, PVR and PH by activating
ERK1/2/KLF4 signaling axis. PVR, the well-documented pathological basis
of PH, manifests as excessive PASMCs
proliferation in pulmonary vascular lesions. Phenotypically modulated
PASMCs are believed to be a major source of over-proliferative cells in
remodeled pulmonary arteries. This study sheds light on exosomes, given
that exosomal cargos may participate in PH pathogenesis by driving
endothelial dysfunction, RV hypertrophy, PVR(Aliotta et al., 2016; Zhang
et al., 2018a; Yuan et al., 2019). Scarce information on exosomes is
available in the context of PH. In particular, whether exosomes modulate
PASMCs phenotypic switching remains a hitherto open question. This study
dealt with this issue by executing detailed in vivo and in
vitro experiments. Our data, for the first time, have generated the
proof that plasma-derived exosomes are driving force of PASMCs
phenotypic switching, thereby deteriorating PVR and PH. Of intrigue,
exosomes have been demonstrated to work by unloading LOX-1 cargo into
PASMCs and engaging ERK1/2-KLF4 signaling axis.
Exosomes, defined as EVs of 40-160 nm in diameter, were initially
regarded as a disposal mechanism for unwanted cellular components, but
evolving evidence illuminated their functions far beyond. Exosomes are
able to act as transfer machinery for proteins, lipids and nucleic
acids. Dysregulation of exosomes is intimately associated with a number
of cardiovascular diseases, including myocardial infarction,
cardiomyopathy, hypertension, diabetes, atherosclerosis(Kalluri and
LeBleu, 2020). In the case of MCT-induced PH, exosomes derived from
animal plasma or lung homogenates induced RV hypertrophy and PVR in
healthy mice; the effects of exosomes were attributable to microRNA
cargo but no specific microRNA was identified(Aliotta et al., 2016). In
hypoxic PH, hypoxia time-dependently promoted exosomes secretion in
either PAECs or the pulmonary artery intima of PH mice; exosomes release
inhibitor GW4869 prevented RV hypertrophy and PVR in hypoxic PH mice,
and restrained PAECs proliferation and migration(Zhang et al., 2018a).
These studies, albeit important, arguably did not explore PASMCs
phenotypic switching, a central pathology of PVR. Hypoxia is an
essential hallmark of a multitude of diseases such as
cardiovascular and metabolic
disorders(Abe et al., 2017; Mesarwi et al., 2019). It modulates exosomes
by influencing their secretion, composition and function(Zhang et al.,
2018a; Bister et al., 2020); indeed, enhanced secretion of
plasma-derived exosomes were observed in hypoxic PH rats. Hypoxia is a
well-recognized potent inducer of VSMCs phenotypic modulation(Zhang et
al., 2018b; Yan et al., 2021). Herein, we demonstrated that hypoxia
increased the secretion of plasma exosomes and thus drove PASMCs
phenotypic modulation, excessive proliferation and migration. These
data, from the perspective of exosomes, represent novel insights into
hypoxia-induced pathologies.
The loaded cargos determine the functions of exosomes. MiR-211 in
plasma-derived exosomes was increased in hypoxic PH rats, and
PAECs-derived miR-211-overexpressed exosomes promoted RV hypertrophy and
PVR in healthy rats(Zhang et al., 2021a). miR-181a-5p and miR-324-5p in
KLF2-overexpressed PAECs-derived exosomes inhibited PAECs apoptosis,
proliferation and inflammation, and suppressed PASMCs
proliferation(Sindi et al., 2020). PAECs-derived exosomal 15-LO2 was
increased under hypoxia, and affected the synthesis and secretion of
exosomes(Zhang et al., 2018a). BOECs-derived exosomal TCTP was
transferred to PASMCs, thus promoting proliferation and inhibiting
apoptosis(Ferrer et al., 2018). Exosomal Wnt5a was decreased in PH
PMVECs, and exacerbated PH by suppressing pericyte recruitment to
PMVECs(Yuan et al., 2019). While those exosomal cargos have been
explored, their relation to PASMCs is unclear and their mechanistic
validity is limited by a lack of in vivo functional validation.
The present study projected spotlight on exosomal LOX-1, based on: (a)
our previous studies have recognized LOX-1 as an important culprit in
PASMCs phenotypic switching; (b) LOX-1 could be loaded into HEK-293
cells-derived exosomes(Gioia et al., 2015; Zhang et al., 2018b). Our
data have answered the question of whether LOX-1 is a pathogenic cargo
of plasma-derived exosomes in PH and how exosomal LOX-1 modulates PASMCs
phenotypic switching, PVR and PH. We set out to characterize exosomal
LOX-1 by means of distinct and reliable methodologies. Our detailedin vitro experiments demonstrated that exosomal LOX-1
etiologically confers PASMCs phenotypic switching, PVR and PH.
Importantly, the conclusion was substantially fortified by the in
vivo rescue experiments that Olr1-/- rats were
supplied with either Exos-WTHypo. or
Exos-KOHypo.. In addition, endogenously expressed
LOX-1 contributed minimally to all the processes, as revealed by
applying Exos-WTHypo. to PASMCs-KO. These data
faithfully concluded that exosomal LOX-1 underlined exosomes-mediated
deleterious effects on PH. However, whether other exosomal contents than
LOX-1 also exerts effects and where the exosomal LOX-1 originates awaits
further investigation. Novel methodologies may help to (a) trace the
precise origin of exosomal LOX-1, (b) exclude the involvement of
non-exosomal LOX-1, such as unknown particles, plasma proteins and/or
protein aggregates contaminated in our exosome preparations. In PH,
PAECs dysfunction compromises intercellular tight junction and hence
renders pulmonary endothelium permeable to plasma exosomes. In this
scenario, the medial PASMCs are able to communicate directly with and
hence be influenced by exosomes. Whether PAECs per se are another
direct target of exosomes, however, remains an interesting yet
unaddressed issue.
Aberrant ERK1/2 activation has been shown to be a pathogenic mechanism
of PH(Kwapiszewska et al., 2012). ERK1/2 signaling is critical for VSMCs
proliferation and tightly associated with LOX-1: ERK1/2 activation was
induced by LOX-1 but inhibited by dominant-negative LOX-1(Tanigawa et
al., 2006a); ERK1/2 was stimulated by ox-LDL via LOX-1 signaling
pathway(Tanigawa et al., 2006b). We have demonstrated that endogenous
LOX-1 drives PASMCs phenotypic switching by activating ERK1/2(Zhang et
al., 2018b). The gleaned data herein suggest that exosomal LOX-1, like
endogenous LOX-1, also engages ERK1/2 signaling for downstream
detrimental effects.
As has been well recognized, KLF4 is a pivotal transcriptional regulator
of VSMCs marker genes. It acts principally by binding to the G/C
repressor element in VSMC marker gene (such as Acta2 ,Tagln , and Myh11 ) promoters, and inhibiting SRF binding to
CARG elements(Wamhoff et al., 2004; Salmon et al., 2012). KLF4 was
significantly increased in hypoxia-exposed primary human PASMCs and
pulmonary arteries of hypoxic PH mice(Sheikh et al., 2015); KLF4
contributed to cigarette smoke-induced PVR and PH(Sun et al., 2018);
KLF4 was up-regulated by PDGF-BB, oxidized phospholipids, miR-29 or
HIF-1α and promoted VSMCs phenotypic switching or migration(Pidkovka et
al., 2007; Cushing et al., 2015; Shan et al., 2020); KLF4 knockdown
attenuated VSMCs dedifferentiation(Yoshida et al., 2008). We showed that
hypoxia downregulated KLF4 expression and promoted PASMCs
proliferation(Zhu et al., 2017). Given those data, we reasoned that KLF4
may mediate plasma exosomes-induced PASMCs phenotypic switching. While
our data has unveiled the relevance of KLF4 in exosomal LOX-1-induced
phenotypic switching, in vivo data did not reconcile with somein vitro cell results. As stated in the results, the dynamic
expression pattern of PASMCs KLF4 (peaked on day 7 and decreased on days
14-21) may bring about discrepancy(Sheikh et al., 2015). Additionally,
cell type-determined (PAECs and PASMCs) effects of KLF4 may be another
confounding factor; whereas PASMCs deletion of KLF4 prevented PH and RV
hypertrophy in hypoxia-induced PH, endothelial deficiency did exacerbate
the disease(Shatat et al., 2014); KLF4 was thought to be protective yet
detrimental in endothelial cells and VSMCs respectively. The opposing
effects of KLF4 in PAECs and PASMCs may thus underline the
animal-to-cell inconsistency. Molecularly, context-specific recruitment
of interacting partner proteins, and differential transactional control
may have caused the irreconciliation. As reported, KLF4 expression was
induced by all-trans retinoic acid (ATRA) or PDGF-BB, but these two
stimuli exhibited an opposite effect on
SM22α expression and VSMCs
proliferation; ATRA activated, but PDGF-BB repressed the
SM22α promoter, through KLF4
binding to or dissociating from its cis-DNA elements(Yu et al., 2011).
ERK1/2 lies between PDGF-BB (a potent SM22α suppressor and proliferation
inducer) and KLF4 deacetylation(Yu et al., 2011), and ERK/KLF4 signaling
downstream of PDGF maintained VSMCs in the contractile status(Wang et
al., 2017). ERK/KLF4-mediated VSMCs phenotypic modulation could
ameliorate neurovascular injury(Zhang et al., 2021b). Our results
suggest that ERK1/2/KLF4 signaling cascade mediates exosomal
LOX-1-induced PASMCs phenotypic switching. Future studies would
investigate other signaling pathways such as PKC/Stat3/Pim-1(Ge et al.,
2021), SRF, myocardin, RhoA, PDGF-BB, TGF-β, Notch, extracellular matrix
and microRNA(Mack, 2011; Xie et al., 2011), given their function in
phenotypic modulation.
In summary, our data have established that plasma-derived exosomes are
an important pathogenic driver of hypoxic PH, that LOX-1 cargo co-opts
exosomes to gain access into PASMCs and confers PVR and PH, and that
exosomal LOX-1 acts by activating
ERK1/2/KLF4 signaling axis. Hence, this study offers novel insights into
the pathogenesis of hypoxic PH and points to exosomal LOX-1 as an
interesting target amenable to therapeutic intervention.