Plasma-derived exosomes were enriched in LOX-1 and promoted PASMCs phenotypic switching, proliferation and migration
To explore the contribution of plasma-derived exosomes to PH pathogenesis, a well-established rat model of PH was developed. Rats were exposed to hypoxia (10% O2) for 3 consecutive weeks. Hemodynamics, RVSP, PVR and RV hypertrophy converged to indicate the occurrence of PH (Fig. S1A-M). Exosomes were isolated and purified from rat plasma by ultracentrifugation and visualized by TEM. The plasma-derived exosomes were cup-shaped membranous vesicles with a diameter of about 100 nm (Fig.1A), as previously reported(Kalluri and LeBleu, 2020). Further characterization with NTA revealed: the size of plasma-derived exosomes was 123.9 ± 47.4 nm in normoxic rat (Exos-WTNormo.) and 130.4 ± 44.2 nm in hypoxic rat (Exos-WTHypo.); the concentration is 1.7×109 Particles/ml in Exos-WTNormo. and 2.8×109Particles/ml in Exos-WTHypo. (Fig. 1B). The identity of exosomes was verified as evidenced by robust CD9 (exosome marker) expression in the isolated exosomes, and CD9 was significantly enriched in Exos-WTHypo. (Fig. 1C). Hence, our results suggested that hypoxia promoted the release of plasma-derived exosomes.
To visualize the interplay between exosomes and PASMCs, exosomes were traced with PKH67 labeling and co-incubated with WT PASMCs (PASMCs-WT). Confocal microscopy revealed that exosomes were inside PASMCs-WT, indicating cytosol as the site of exosomes action (Fig. 1D). Phenotypic switching of PASMCs is defined by a downregulation of contractile markers such as α-SMA and SM22α, and an upregulation of proliferative markers such as PCNA. We found that Exos-WTHypo.down-regulated the expression of α-SMA and SM22α, accompanied by an increase in PCNA expression in PASMCs-WT (Fig. 1E-F). These results suggested that Exos-WTHypo. was a powerful trigger of phenotypic switching. Furthermore, Exos-WTHypo.hastened PASMCs-WT proliferation as revealed by EdU staining and flow cytometry (Fig. 1G and 1H). Exos-WTHypo. also promoted PASMCs-WT migration (Fig. 1I).
Exosomes are known to transfer cargos, such as microRNAs. Here, we focused on LOX-1, a ubiquitous plasma membrane protein expressed in PASMCs. We have demonstrated that PASMCs-expressed LOX-1 promoted PASMCs phenotypic switching(Zhang et al., 2018b). Given the cargo-loading capacity of exosomes, we were intrigued by the possibility of exosomes mediating intercellular LOX-1 transfer. LOX-1 was upregulated in pulmonary arteries of hypoxic rats as revealed by immunohistochemistry (SFig. 1N), but the origin of LOX-1 was uncertain. We reasoned that LOX-1 might be loaded into exosomes and unloaded into PASMCs, promoting PASMCs phenotypic switching. Indeed, LOX-1 was detectable in exosomes with immunoelectron microscopy. LOX-1-specific gold particles appeared to be predominantly clustered on exosomes surface, indicating that LOX-1 resides in exosomes membrane. Intriguingly, LOX-1 gold particles were enriched in Exos-WTHypo., as compared with Exos-WTNormo. (Fig. 1J). Western blotting reinforced that LOX-1 was a cargo of plasma-derived exosomes, and that hypoxia markedly heightened exosomal LOX-1 abundance (Fig. 1K). Moreover, Exos-WTHypo., as compared with Exos-WTNormo., upregulated LOX-1 protein in PASMCs-WT (Fig. 1L-M). The results suggested that plasma-derived Exos-WTHypo.acted as a transfer machinery for LOX-1, hence eliciting PASMCs-WT phenotypic switching, proliferation and migration.