Abstract
Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)‐derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1 trg/SMcre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1 trg/SMwt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1 trg/SMcre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1 trg /SMcre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1 trg/SMcre mice, increased Pi concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome‐MVB interaction. However, amitriptyline prevented these changes in Pi‐treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.
Highlights
Vascular calcification is the build‐up or accumulation of apatite cal‐ cium salts in the media and/or intima of arteries that has been as‐ sociated with ageing, chronic kidney disease, diabetes mellitus and atherosclerosis.[1]
The present study showed that phenotypic switch in SMCs during arterial medial calcification (AMC) was characterized by decreased expression of the vascular smooth muscle cells (VSMCs) lineage marker, smooth muscle 22α (SM22‐α) (Figure 3A) and up‐regulation of both OSP (Figure 3C) and RUNX2 (Figure 3E) while as amitriptyline treatment prevented this phenotype change
We found that CD63 (Figure 6C), Annexin‐II (AnX2) (Figure 6E) and alkaline phosphatase (ALP) (Figure 6G) staining as small extracellular vesicle (sEV) markers were significantly increased in the coronary arterial wall of vi‐ tamin D (Vit D)‐treated Smpd1trg/SMcre mice than their littermates (Smpd1trg/SMwt and WT/WT) which were de‐ creased by amitriptyline as shown in the bar graphs (Figure 6D,F,H)
Summary
Vascular calcification is the build‐up or accumulation of apatite cal‐ cium salts in the media and/or intima of arteries that has been as‐ sociated with ageing, chronic kidney disease, diabetes mellitus and atherosclerosis.[1] It has been reported that the arterial calcification pathology mimics the bone formation process and that the earliest phase involves the osteogenic differentiation of vascular SMCs.[1,2,3,4] Various human[5] and animal[5,6] studies have reported that osteogenic conversion of SMCs in the medial region appears prior to mineraliza‐ tion in arterial medial calcification (AMC), suggesting a critical role for smooth muscle cell (SMCs) phenotypic transition in this vascular pathologic change.[7] It is known that both intimal and medial vascular calcification may be mediated by a common mechanism, namely the large increases in extracellular vesicles (EVs) in the vascular intersti‐ tial space, in particular, the small extracellular vesicles (sEVs) (with size of 40‐100 or to 140 nm) These sEVs are mainly produced and secreted from arterial SMCs.[8] role of sphingolipids (SLs) such as ceramide in particular lysosomal ceramide in SMCs and asso‐ ciated pathogenic role in AMC is still poorly understood. Administration of amitriptyline, a pharmacological inhibitor of ASM, to these animals ameliorates AMC
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