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.