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.