3.3 LOX-1-intact exosomes exacerbated hypoxia-induced PH inOlr-/- rats
A set of in vivo experiments conducted inOlr1-/- rats, as designed in Fig. 3A, aimed to
reinforce the in vitro conclusion that exosomal LOX-1 contributed
to hypoxic PH. Olr1 deletion has been demonstrated to improve the
hemodynamic indexes, RVSP and PVR in hypoxia-treated rats(Ge et al.,
2021). Of note, supply of Olr1-/- rats with
Exos-WTHypo. significantly increased RVSP, PAWT, RVWT
(Fig. 3C-D, F-G and SFig. 2E), RV/LV+IS and RV/ tibial length (SFig.
2A-B), and considerably decreased PAT/PET (Fig. 3B, E) and TAPSE (SFig.
2C, F), but exerted no effects on body weight gain (SFig. 2D). In
contrast, Exos-KOHypo. administered toOlr1-/- rats largely normalized the PH
indicators (Fig. 3B-G and SFig. 2A-F). PVR, a pathological hallmark of
PH resulting from excessive PASMCs proliferation, was absent in the
pulmonary arteries of hypoxic Olr1-/- rats, as
revealed by HE staining (Fig. 3H); Exos-WTHypo.aggravated the PVR in hypoxic Olr1-/- rats, in
contrast to Exos-KOHypo. (Fig. 3H and SFig. 2G-H).
Thus, supply of Olr1-/- rats with
Exos-WTHypo. phenocopied PH, as evidenced by increased
RVSP, PVR and RV hypertrophy. Compared with hypoxic WT rats, hypoxicOlr1-/- rats exhibited increased SM22α
expression and decreased PCNA levels in the media of pulmonary arteries,
suggesting that Olr1-/- deletion prevented
PASMCs phenotypic switching (Fig. 3I). Exos-WTHypo.,
rather than Exos-KOHypo., administered toOlr1-/- rats aggravated phenotypic switching
(Fig. 3I). These results were strengthened by immunoblotting of PASMCs
phenotypic makers (SM22α and PCNA) (Fig. 3J-K). Collectively, thesein vivo data suggested that exosomal LOX-1 aggravated PVR by
promoting PASMCs phenotypic switching, thus exacerbating PH.
3.4PASMCs-expressed
LOX-1 contributed negligibly to exosomes-induced PASMCs phenotypic
switching
Whereas exosomal LOX-1 transferred to PASMCs was able to trigger
phenotypic switching, LOX-1 expressed in PASMCs may also play a role in
this process. To be more precise, PASMCs derived from WT (PASMCs-WT) orOlr1-/-rats (PASMCs-KO) were treated with Exos-WTHypo..
Interestingly, evaluation of α-SMA, SM22α and PCNA with
immunofluorescence and Western blotting showed that the effects of
Exos-WTHypo. on PASMCs-WT and PASMCs-KO were
indiscernible (Fig. 4A-C). Also, Exos-WTHypo. exerted
indistinguishable effects on cell proliferation and migration between
PASMCs-WT and PASMCs-KO, as revealed by EdU staining, flow cytometry and
scratch wound healing assay (Fig. 4D-I). Hence, the results suggested
that the endogenously expressed LOX-1 in PASMCs, as opposed to exosomal
LOX-1, played a limited role in exosomes-induced PASMCs phenotypic
switching.
3.5 ERK1/2signaling mediated
exosomes-induced PASMCs
phenotypic switching
Having
established a pivotal role of exosomal LOX-1 in PH pathogenesis,
exploration of the underlying mechanisms ensued. Extracellular
signal-regulated kinases 1/2 (ERK1/2) regulates a plethora of cellular
processes, such as cell proliferation, migration, survival,
transcription(Roskoski, 2012); all these processes somehow play roles in
PVR and PH. We have demonstrated that PAMSCs-residing LOX-1 promotes
ERK1/2 phosphorylation (p-ERK1/2), thereby enhancing PASMCs phenotypic
switching(Zhang et al., 2018b). Therefore, we tested whether exosomal
LOX-1 may also implicate ERK1/2 signaling. As revealed,
Exos-WTHypo. markedly elevated ERK1/2 phosphorylation
(Fig. 5A-B), whereas p-ERK1/2 was lowered in
Exos-KOHypo.-treated PASMCs-WT (Fig. 5C-D). Pulmonary
arterial p-ERK1/2 levels were increased in WT PH rats, which was
abrogated in Olr1-/- rats (Fig.
5E-F and SFig. 3A). Administration
of Exos-WTHypo. to Olr1-/- rats upregulated pulmonary arterial p-ERK1/2, an effect abolished by
Exos-KOHypo. (Fig. 5E-F). Hence, in vitro andin vivo evidence convergently demonstrated that ERK1/2 signaling
mediated the pathological effects of exosomal LOX-1. A potent and
selective ERK1/2 inhibitor SCH772984 was used to examine the causal link
between ERK1/2 activation and PASMCs phenotypic switching(Morris et al.,
2013). CCK8 assay and Western blotting showed that SCH772984 optimally
blocked ERK1/2 at a concentration of 100nM, but had no significant
effect on PASMCs viability under normoxic condition (SFig. 3B-D).
SCH772984 inhibited Exos-WTHypo.-induced p-ERK1/2
upregulation (Fig. 5G-H), and alleviated Exos-WTHypo.-
induced PAMSCs phenotypic switching as indicated by α-SMA, SM22α and
PCNA (Fig. 5I-J). Thus, these results indicated that exosomal LOX-1
aggravated PASMCs phenotypic switching by engaging ERK1/2 signaling.