3.6 KLF4 mediated exosomes-induced PASMCs phenotypic switching,
proliferation and migration
The transcription factor Krüppel-like factor 4 (KLF4) is a known
powerful negative regulator of VSMCs marker genes(Salmon et al., 2012).
KLF4 is significantly upregulated in PH patient pulmonary arterioles and
serum, and is highly correlated with PASMCs proliferation(Sheikh et al.,
2015; Dou et al., 2021). Knockout of KLF4 in PASMCs prevented PH and RV
hypertrophy(Sheikh et al., 2015), and KLF4 knockdown ameliorated PH(Sun
et al., 2021). KLF4 expression was therefore examined to dissect its
role in exosomal LOX-1-induced PASMCs phenotypic switching. Expression
of KLF4 in PASMCs-WT was dramatically increased by
Exos-WTHypo. (Fig. 6A-B), but inhibited by
Exos-KOHypo. (Fig. 6C-D). Paradoxically, KLF4 was
downregulated in the pulmonary arteries of WT PH rats, which was
prevented by LOX-1 deletion (Fig. 6E and SFig. 4A). The disparity
between animal and cell results may originate from the fact that KLF4 is
also abundant and mediates dichotomous effects in other pulmonary
arterial cells such as ECs(Shatat et al., 2014; Sheikh et al., 2015).
Also, KLF4 in PASMCs was reported to be dynamically modulated in hypoxic
PH, with its expression peaked on day 7 and decreased on day 21 after
hypoxia(Sheikh et al., 2015). This expression profile may partially
account for the observed inconsistency. Furthermore, KLF4 knockdown in
PASMCs-WT with small interference RNA (siRNA) reversed
Exos-WTHypo.-induced PASMCs phenotypic switching,
proliferation and migration (Fig. 6F-H and SFig. 4B-D). These data
further substantiated the regulatory role of KLF4 in exosomal
LOX-1-induced PASMCs phenotypic switching. PDGF-BB, a potent mitogen,
induces KLF4 expression via ERK1/2 signaling and thus represses SM22α,
thereby driving phenotypic switching in cultured human aortic SMCs(Yu et
al., 2011; Wang et al., 2017). Herein, we speculated that ERK1/2 acted
as a kinase upstream of KLF4 to orchestrate exosomal LOX-1-induced
PASMCs phenotypic switching. Of note, ERK1/2 inhibitor SCH772984
inhibited KLF4 up-regulation in Exos-WTHypo.-treated
PASMCs-WT (Fig. 6I-J). Therefore, ERK1/2-KLF4 singling axis acted
mechanistically in exosomal LOX-1-induced PASMCs phenotypic switching.