BACKGROUND: Cardiovascular calcification is a significant predictor and contributor to cardiovascular diseases. Vascular smooth muscle cell (SMC)–derived extracellular vesicles (EVs) play a crucial role in microcalcification formation. EVs can originate from the endosomal system, and PIKFYVE (1-phosphatidylinositol 3-phosphate 5-kinase), a lipid kinase, plays a key role in endomembrane maturation. We hypothesize that PIKFYVE inhibition will modulate EV cargo and, thereby, arterial calcification. METHODS: Human coronary artery SMCs were cultured in osteogenic media to induce calcification. PIKFYVE inhibition was achieved using the pharmacological inhibitor apilimod and siRNA. We characterized the SMC phenotype and EVs through proteomics, transcriptomics, and kinomics. Ldlr -deficient mice fed a high-fat, high-cholesterol diet received apilimod for 5 weeks. RESULTS: Calcified human arteries and SMCs exhibited increased PIKFYVE protein expression compared with controls. In calcifying SMCs, phosphatidylinositol 3-phosphate levels were reduced but restored by apilimod. Apilimod prevented matrix mineralization and collagen deposition in calcifying SMCs, accompanied by reduced procollagen 1A1 secretion. Apilimod inhibited TNAP (tissue-nonspecific alkaline phosphatase) at mRNA, protein, and activity levels. EVs released from apilimod-treated calcifying SMCs exhibited lower mineral cargo, reduced aggregation potential, and diminished TNAP cargo. Phenotypic omics analyses revealed that apilimod induced a shift toward an adipocyte-like SMC phenotype, marked by upregulation of adipogenic TFs (transcription factors), fatty acid metabolism genes, and increased fatty acid uptake. Reactivation of YAP (Yes-associated protein)-TEAD (transcriptional enhancer factor) signaling partially reversed these phenotypic changes. In vivo, apilimod reduced vascular calcification and plaque TNAP activity but increased plaque lipid accumulation. CONCLUSIONS: Pharmacological inhibition of PIKFYVE disrupts YAP-TEAD signaling, thereby reducing arterial calcification by limiting the calcification potential of EVs and suppressing osteogenic SMC programming, but also induces an adipocyte-like SMC phenotype with lipid accumulation. These findings emphasize the need to consider SMC phenotypic plasticity and potential adverse effects when developing therapeutic strategies for arterial calcification.
Hense et al. (Wed,) studied this question.