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.