Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene.

Owais M Bhat,Pin-Lan Li,Sarah Camus,Fadi N Salloum,Xinxu Yuan

doi:10.3390/ijms21051713

Abstract

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1−/− mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1−/− mice as compared to their wild-type littermates. Besides, Mcoln1−/− mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1−/− mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1−/− mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Highlights

Vascular calcification (VC) was considered to be a passive and degenerative process, whereas it is considered to be an actively regulated process associated with the pathological deposition of calcium minerals in the vascular system [1,2]
We investigated whether mucolipin-1 mediates lysosome positioning leading to small extracellular vesicle (sEV) release in arterial smooth muscle cells (SMCs), contributing to their phenotype switch and arterial stiffness during the development of arterial medial calcification (AMC)
We observed markedly increased immunostaining of osteogenic markers such as “bone” transcription factors (e.g., RUNX2) and matrix proteins in the arterial medial SMCs of vitamin D (Vit D)-treated Mucolipin 1 (Mcoln1)−/− mice as compared to their wild-type littermates. These results showed that osteogenic shifts in arterial medial SMCs with Mcoln1 gene deletion aid in the development of AMC

Summary

Introduction

Vascular calcification (VC) was considered to be a passive and degenerative process, whereas it is considered to be an actively regulated process associated with the pathological deposition of calcium minerals in the vascular system [1,2]. One of the possible mechanisms of VC is phenotypic change of VSMCs from a contractile to a synthetic and osteochondrogenic phenotype followed by augmented expression of osteochondrogenic markers, for example, osteocalcin, osteopontin, RUNX2, and alkaline phosphatase and reduced expression of VSMC contractile markers such as smooth muscle α-actin and smooth muscle 22α (SM22-α). Another proposed mechanism is the release of matrix vesicles from viable VSMCs to initiate the VC [6]. During VC progression, the phenotypic transition process involves cytoskeleton remodeling, such as intracellular modifications, which enhanced exosome secretion [11,12]

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Results

Conclusion