USP9X-enriched MSC-sEV inhibits LSEC angiogenesis in MASH mice by downregulating the IκBα/NF-κB/Ang-2 pathway

Pathological angiogenesis of liver sinusoidal endothelial cells (LSEC) plays a crucial role in the progression of metabolic dysfunction-associated steatohepatitis (MASH)-induced liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) have shown promising therapeutic potential against MASH. This study aimed to investigate the impact of MSC-sEV on LSEC angiogenesis and elucidate the underlying molecular mechanisms. The effects of MSC-sEV on LSEC angiogenesis were evaluated in Tumor Necrosis Factor- alpha (TNF-α)-treated LSECs in vitro and in Methionine and Choline Deficient Diet (MCD)-induced MASH mice in vivo. Herein, we found that MSC-sEV effectively suppressed LSEC angiogenesis by targeting the angiogenesis marker Angiogenin 2 (Ang-2) in both TNF-α-treated LSECs and MASH mice. Gene manipulation experiments revealed that the primary mechanism by which MSC-sEV inhibited LSEC angiogenesis was through the modulation of nuclear factor kappa B inhibitor alpha (IκBα) / nuclear factor kappa B (NF-κB) / Ang-2 pathway. Additionally, mass spectrometry and co-immunoprecipitation (Co-IP) data suggested that MSC-sEV delivered the ubiquitin specific peptidase 9 X-linked (USP9X) protein to LSECs, leading to enhanced IκBα deubiquitination and NF-κB in activation, ultimately resulting in the inhibition of Ang-2-mediated LSEC angiogenesis. Knockdown of USP9X attenuated the regulatory effects of MSC-sEV on Ang-2 expression, LSEC angiogenesis, and the progression of MASH. In conclusion, our findings indicate that USP9X delivered via MSC-sEV can suppress LSEC angiogenesis and alleviate MASH-induced liver fibrosis through the IκBα/NF-κB/Ang-2 signaling pathway. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) transport USP9X to liver sinusoidal endothelial cells (LSECs), which further leads to IκBα deubiquitination and NF-κB activation, ultimately resulting in the inhibition of Ang-2-mediated LSEC angiogenesis and MASH progression. • MSC-sEV suppresses LSEC angiogenesis and alleviates MASH in mice. • MSC-sEV-transferred USP9X is responsible for reducing LSEC angiogenesis. • USP9X inhibits LSEC angiogenesis via IκBα/NF-κB/Ang-2 pathway.