Background: Cardiovascular diseases are a leading cause of mortality globally, with cardiovascular calcification as a prominent predictor and contributor. Particularly microcalcifications might cause atherosclerotic plaque rupture, leading to myocardial infarction. Recent work indicated that extracellular vesicles (EVs) released from calcifying vascular smooth muscle cells (SMCs) are pivotal for microcalcification formation. EVs originate from the endolysosomal system and FYVE-Type Zinc Finger Containing Phosphoinositide Kinase (PIKfyve), a lipid kinase, plays a role in the endolysosomal maturation and emerged to be upregulated in calcifying SMCs and human tissue. We hypothesize that alterations in endomembrane homeostasis will modulate EV cargo and thereby SMC calcification.

Methods and Results: Phosphatidylinositol 3-phosphate (PI3P) - the substrate of PIKfyve - was decreased in cellular membranes of calcifying SMCs and recovered by Apilimod, a small molecule PIKfyve inhibitor. Apilimod prevented matrix mineralization and collagen network formation in calcifying SMCs accompanied by reduced pro-collagen 1A1 secretion (-98%, p<0.001). Apilimod inhibited tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA (-79%, p<0.01), protein (-96%, p<0.01) and activity (-92%, p<0.01) level. PIKfyve silencing RNA showed similar results. Structural modeling and zymography assays suggest no direct effect of Apilimod on enzymatic TNAP activity. Furthermore, Apilimod caused a dose-dependent increase of EV release assessed by nanoparticle tracking analysis (up to 5.2 fold, p<0.05). EVs size was not affected. Analysis of the EV cargo demonstrated that Apilimod reduces TNAP protein abundance and activity (-58%, p<0.01) - a characteristic cargo that may determine EV calcification potential. Apilimod-induced EVs exhibited lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Next, we performed transcriptome and kinome analyses to discover the underlying mechanism of PIKfye-mediated SMC calcification. These data revealed a link to adipocyte-like differentiation and SMC phenotype specifying pathways. Validation supported increased expression of adipogenic transcription factors (CEBPA, PPARG) and genes of the cholesterol and fatty acid metabolism pathways (FABP3, CD36) by Apilimod; while SMC-specific markers (ACTA2, SM22) and key SMC transcription factors (GATA6, MYOCD) were downregulated. Furthermore, Apilimod promoted nuclear translocation of PPARg, the main regulator of adipogenic differentiation, suggesting a phenotype switch from osteogenic-like to adipogenic-like SMCs. Finally, in Ldlr-deficient mice fed a high-fat high-cholesterol diet for 15 weeks, Apilimod application for 5 weeks increased PI3P levels but did not alter plaque size or collagen content in atherosclerotic plaques. However, we found differentially distributed plaque lipid depositions detected by Oil Red O staining. Apilimod treatment decreased triglyceride serum levels, while cholesterol levels did not altered.

Conclusion: Disrupting endolysosomal maturation with Apilimod promotes the release of EVs with reduced calcification potential and induces a phenotypic adaption towards adipocyte-like SMCs, causing reduced SMC calcification. Anti-arteriosclerotic effects of Apilimod in vivo remain to be further investigated.