Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice

Abstract

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1 fl/fl /SM cre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1 fl/fl /SM cre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1 fl/fl /SM wt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1 fl/fl /SM cre mice, high dose of P i led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1 fl/fl /SM cre CASMCs as shown by GCaMP3 Ca 2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC. Arterial calcification involves the accumulation or deposition of apatite calcium salts within the vascular wall that has been associated with aging, atherosclerosis, diabetes mellitus and chronic kidney disease (CKD) 1. Anatomically, arterial calcification is classified into intimal and medial calcification 2. The intimal calcification often causes occlusion of the arteries, which is characterized by accumulation of lipids, inflammation, and fibrosis observed as irregular scattered deposits in the atherosclerotic plaques 3. The arterial medial calcification (AMC) increases arterial stiffness, which is detected as continuous linear hydroxyapatite deposits in the absence of inflammatory cells along the internal elastic lamina. It is frequently observed in the elderly people or in patients with diabetic mellitus and chronic renal failure 4,5. Literature cites that under normal physiological conditions, spontaneous accumulation of calcium and phosphate levels doesn't induce arterial calcification because minerals (Ca/P i) are tightly balanced in the vasculature 6,7. Increased intracellular phosphate levels in vascular smooth muscle cells (VSMCs) directly drive their osteogenic differentiation and mineralization, inducing expression of osteogenic markers like RUNX2 and osteopontin 8,9 , associated with secretion of matrix vesicles as primary nucleation sites for calcification 7,9. Several studies including ex vivo human samples and ...

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

“…Our findings in podocytes demonstrated that inhibition or activation of the TRPML1 channel modulates the interaction of lysosomes and MVBs, thereby regulating the exosome release from these cells [31]. Also, recently, we found that inhibition of TRPML1 channel activation in coronary arterial SMCs due to deficiency of acid ceramidase may lead to the reduction of lysosome-MVB interaction and consequent prolongation of MVB fate, increasing SMC-derived small extracellular vesicle (sEV) release initiating arterial medial calcification (AMC) [55]. Based on these findings, we hypothesized and found that Mcoln1 −/− mice exhibited abnormal lysosome positioning and increased sEV levels in the arterial medial SMCs, which may contribute to the arterial stiffness during the development of AMC.…”

“…Isolation of mouse CASMCs was described previously [34]. Briefly, CASMCs were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco Gaithersburg, MD, USA) in humidified 100% air and 5% CO2 mixture at 37 • C. Confluent cells (70%-80%) were treated with or without high phosphate (Pi) (3 mmol/L) [35] and then incubated with TRMPL1 channel agonist, MLSA-1 (10 µM, Sigma-Aldrich Chemicals, St. Louis, MO, USA) and TRMPL1 channel blocker, verapamil (10 µM, Cayman Chemical, Ann Arbor, MI, USA) incubated for 24 h [31,55] . The average body weight for the male mice aged 12-14 weeks ranged from~25-30 g. All mice were grossly normal; the homozygous (Mcoln1 −/− ) mice, however, were prone to developing hind-limb paralysis by the age of 28 weeks, and the average life expectancy was 8 months.…”

On a Magical Mystery Tour with 8-Bromo-Cyclic ADP-Ribose: From All-or-None Block to Nanojunctions and the Cell-Wide Web

“…In short, if TPC2 plays a role in acute HPV it is a subtle one. Alternatively, there may be redundancy of function within the system, perhaps through the expression of alternative lysosome-resident, calcium permeable channels such as TRPML1 [ 129 , 130 , 131 , 132 ] and TRPM2 [ 133 , 134 ], each of which has been proposed to couple to RyRs and impact smooth muscle function and vascular reactivity. In this respect it is notable that mTORC1-dependent modulation of autophagy impacts pulmonary vascular remodeling [ 127 , 128 ], given that inhibition of mTORC1 [ 135 ] may regulate lysosomal TPC2 [ 121 ] and TRPML1 [ 136 ].…”

“…Endolysosome targeted TRPML1 [ 129 , 130 , 131 , 132 ] and TRPM2 [ 133 , 134 ] by mTORC1 and/or ADP-ribose;…”

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“…Indeed, lysosome dysfunction or injury by various lysosomotropic agents such as bafilomycin A and chloroquine are reported to increase the exosome release. Recently, we demonstrated that inhibition of lysosomal AC causes the lysosome trafficking dysfunction leading to impaired fusion of lysosomes with MVBs Yuan et al, 2019b;Bhat et al, 2020). AC inhibition blocks the lysosomal TRPML1-Ca 2+ channel activity and disturbs lysosome-MVB interaction resulting in increased exosome release in podocytes and VSMC Bhat et al, 2020).…”