PIKfyve inhibition alters the osteogenic phenotype of vascular smooth muscle cells in a YAP-dependent manner

Background: Cardiovascular calcification is a contributor to cardiovascular disease. The phenotypic transition of vascular smooth muscle cells (SMCs) from a contractile to an osteogenic phenotype, characterized by the production of calcifying extracellular vesicles (EVs) that promote extracellular mineral deposition, is a critical process in vascular calcification. The endolysosomal system, a network of endomembrane organelles, plays a role in regulating essential cellular functions. FYVE-Type Zinc Finger Containing (PIKfyve), a lipid kinase and endosome maturation regulator, is a key enzyme in the endolysosomal system. We hypothesize that PIKfyve plays a crucial role in regulating the SMC phenotype.

Methods and Results: Both human calcified carotid plaques and calcifying SMCs expressed more PIKfyve compared to non-calcified controls. The PIKfyve substrate, phosphatidylinositol 3-phosphate (PI3P), was reduced (-40%) in the cellular membranes of calcifying SMCs and was restored by the small molecule PIKfyve inhibitor Apilimod. Assessing the SMC phenotype demonstrated a reduction of osteogenic hallmarks such as matrix mineralization (-77%, p<0.001), collagen secretion (-99%, p<0.001), tissue non-specific alkaline phosphatase (TNAP) protein expression (-86%, p=0.015) and activity (-92%, p<0.001) by Apilimod in calcifying SMCs. Moreover, Apilimod decreased the number of mineral-positive EVs, which had a lower aggregation potential (-80%, p<0.001). Transcriptomics was used to evaluate other phenotypic characteristics of calcifying SMCs after PIKfyve inhibition. Apilimod promoted adipogenic markers (PPARG: +218%, CD36: +2300%, FABP3: +2250%, all p<0.001) as well as fatty acid uptake (+86%, p=0.019; fluorescent C12 probe) and intracellular lipid droplets (Oil Red O), expression of the lipid droplet associated gene PLIN2, and increased the expression of macrophage-like SMC markers (CD68: +500%, p=0.028; LGALS3: +132%, p=0.027) and cytokines (IL1B: +471%, p=0.010; TNF: +442%, p=0.022), suggesting transition towards an alternative SMC phenotype. The underlying molecular mechanisms were investigated using in silico analyses and a kinase array, which pointed to the involvement of the hippo pathway and yes-associated protein 1 (YAP1). Western blot analysis supported the induction of YAP1-Ser127 phosphorylation and subsequent repression of YAP1 transcriptional targets (TEAD, Lamin B2, Axl) by Apilimod in SMCs. Pharmacological re-activation of YAP1 reduced adipogenic-like and macrophage-like features to control levels and partially restored EV nucleation but had no effect on matrix mineralization. In vivo, Apilimod increased PI3P levels and decreased vascular calcification in Ldlr-deficient mice fat a high-fat, high-cholesterol diet. Although Apilimod did not change plaque size in atherosclerotic plaques, it did alter lipid distribution. 

Conclusion: Our findings suggest that PIKfyve regulates the phenotypic characteristics of plaque SMCs. PIKfyve inhibition reduces the osteogenic transition of SMCs while promoting an adipogenic/pro-inflammatory phenotype mediated by YAP1. The endolysosomal system emerges as a crucial regulator of various SMC phenotypes.