Exosomal hsa-miR-299-3p from endothelium mediates high phosphorus-induced vascular calcification in mice model of CKD via phosphorylated JAK2/STAT5 pathway

Introduction Vascular calcification (VC) is a prevalent and life-threatening complication of chronic kidney disease (CKD), yet the mechanisms by which hyperphosphatemia drives VC remain incompletely understood. This study investigates the role of endothelial cells (ECs)-derived exosomal microRNAs in mediating osteogenic differentiation of vascular smooth muscle cells (VSMCs) under high phosphate (HP) conditions. Methods A CKD-VC mouse model was established using a HP and high-adenine diet. Exosomes (Exos) were isolated from ECs cultured under normal or HP conditions. The effects of Exos on calcification of VSMCs were evaluated using in vitro co-culture systems and in vivo administration. miRNA sequencing, dual-luciferase reporter assays, and loss/gain of function experiments were performed to identify key exosomal miRNAs and their downstream targets. Western blotting, qRT-PCR, and histological analyses were used to assess molecular and pathological changes. Results HP-stimulated ECs released Exos (HP-Exos) that were internalized by VSMCs and significantly promoted VC in both in vitro and in vivo models. miRNA sequencing identified miR-299-3p as significantly upregulated in HP-Exos. Functional studies demonstrated that exosomal miR-299-3p directly targeted membrane-associated RING-CH3 (MARCH3), leading to activation of the p-JAK2/STAT5 signaling pathway. This cascade subsequently upregulated osteogenic markers and downregulated contractile marker, thereby promoting osteogenic differentiation of VSMCs. Knockdown of miR-299-3p in vivo attenuated VC in CKD mice. Discussion These findings reveal a previously unrecognized mechanism by which HP drives CKD-VC through ECs-derived exosomal miR-299-3p. The miR-299-3p/MARCH3/p-JAK2/STAT5 signaling axis represents a critical regulatory pathway in VC pathogenesis and offers a potential therapeutic target for this life-threatening complication of CKD.