Abstract
High phosphate levels promote vascular smooth muscle cell (VSMC) calcification, a major contributor to cardiovascular mortality in chronic kidney disease. MicroRNAs, particularly miR-21-5p, are key epigenetic regulators of cell phenotype. This study investigated whether inhibiting miR-21-5p attenuates hyperphosphatemia-induced VSMC calcification and identified the underlying molecular pathway. Primary human aortic VSMCs were cultured under high inorganic phosphate (2.5 mM) conditions to model calcification. Real-time quantitative PCR demonstrated a significant time-dependent upregulation of miR-21-5p in calcified VSMCs. Transfection with a miR-21-5p inhibitor significantly decreased intracellular calcium accumulation, Alizarin Red S staining, and alkaline phosphatase activity. Concurrently, miR-21-5p inhibition suppressed osteogenic marker expression (RUNX2 and osteopontin) while preserving smooth muscle lineage markers (α-smooth muscle actin and SM22α). Bioinformatic analysis combined with dual-luciferase reporter assays identified SMAD7 as a direct target of miR-21-5p. Restoring SMAD7 expression through miR-21-5p inhibition attenuated TGF-β1-induced Smad2/3 phosphorylation and downstream extracellular matrix mineralization. Furthermore, siRNA-mediated knockdown of SMAD7 blunted the anti-calcific effects of miR-21-5p inhibition. Our findings demonstrate that miR-21-5p inhibition mitigates high phosphate-induced VSMC osteogenic transdifferentiation by targeting SMAD7 and modulating TGF-β signaling, suggesting a novel therapeutic pathway for hyperphosphatemic vascular calcification.
