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.