Regulatory Role of mTOR Signaling in Exosome Secretion and Osteogenic Changes in Smooth Muscle Cells Lacking Acid Ceramidase Gene

Abstract

Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N-terminal nucleophile hydrolase family. This enzyme is located in the lysosome, which mediates conversion of ceramide (CER) into sphingosine and free fatty acids at acidic pH. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation. The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LE)/lysosomes may control cargo selection, membrane biogenesis and exosome secretion, which may be fine controlled by lysosomal sphingolipids such as CER. This lysosomal-CER-mTOR signaling may be a crucial molecular mechanism responsible for development of arterial medial calcification (AMC). Torin-1 (5mg/kg/day), an mTOR inhibitor significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin and RUNX2 and upregulation of SM22-α in mice receiving high dose of Vitamin D (500,000 IU/kg/day). Asah1^fl/fl/SM^Cre mice had markedly increased co-localization of mTORC1 with Lamp-1(lysosome marker) and decreased co-localization of VPS16 (a multivesicular bodies (MVBs) marker) with Lamp-1, suggesting mTOR activation caused reduced MVBs interaction with lysosomes. Torin-1 significantly reduced the co-localization of mTOR vs Lamp-1, increased lysosome-MVBs interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using CASMCs, P_i-stimulation significantly increased p-mTOR expression in Asah1^fl/fl/SM^Cre CASMCs as compared to WT/WT cells, associated with increased calcium deposition and mineralization. Torin-1 blocked P_i-induced calcium deposition and mineralization. siRNA mTOR and Torin-1 significantly reduce co-localization of mTORC1 with Lamp-1, increased VPS16 vs Lamp-1 co-localization in P_i-stimulated CASMCs, associated with decreased exosome release. Functionally, Torin-1 significantly reduce arterial stiffening as shown by restoration from increased pulse wave velocity and decreased elastin breaks. These results suggest that lysosomal CER-mTOR signaling may play a critical role for the control of lysosome-MVB interaction, exosome secretion and arterial stiffening during AMC.

Introduction

Vascular calcification (VC) involved formation and accumulation of calcium phosphate salts within the vascular wall that has been associated with aging, chronic kidney disease (CKD), diabetes mellitus, and atherosclerosis¹. In response to various physiological stimuli or a mineral imbalance, vascular smooth muscle cells (VSMCs) secrete small, specialized membrane-bound bodies termed matrix vesicles (MVs) which act to nucleate calcium/phosphate (Ca/P_i) crystals in the form of hydroxyapatite. These MVs or exosomes (<200 nm) form the first nidus for mineralization². Literature reported that enhanced exosome excretion may trigger smooth muscle cell (SMC) phenotype changes^3–5, the apatite nucleation⁶, and calcifying nidus formation⁷, ultimately leading to extracellular matrix (ECM) mineralization^8–11. Recently, it has been reported that bioactive sphingolipids and ceramide (CER) are associated with cytoskeletal rearrangements in synthetic VSMCs lead to multivesicular bodies (MVBs) trafficking and elevated exosome secretion in VC^9,12,6.

Given that sphingolipids are involved in cardiovascular pathophysiology and biomineralization. Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N-terminal nucleophile hydrolase family. Deficiency of AC activity lead to Farber disease in humans¹³. Case reports have mentioned some manifestations of this disease including enlargement and calcification of axillary lymph nodes¹⁴. This enzyme is located in the lysosome, which mediates conversion of CER into sphingosine and free fatty acids at acidic pH¹⁵. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation¹⁶. In human femoral arterial SMCs, it was reported that CER enhanced the Ox-LDL-induced VSMC mineralization via p38 MAPK signaling pathway, both CER and Ox-LDL stimulated phosphorylation of p38 MAPK. However, inhibition of p38 MAPK blocked this mineralization process¹⁷. In Ox-LDL stimulated human VSMCs, a significantly reduced calcium deposition was observed with PDTC, a NF-κB inhibitor. However, this effect of PDTC on calcification was reversed by C2-ceramide treatment, indicating that NF-κB regulates VC in a ceramide-dependent manner¹⁸. Recently, a study identified serum- and glucocorticoid-inducible kinase 1 (SGK1) which is up-regulated in VSMCs during pro-calcifying conditions and act as downstream of ASM/CER mediated calcification effects. The study demonstrated that knockdown of SGK1 suppresses the pro-calcific effects of ASM/CER treatment in VSMCs. Hence, concluded that ASM/ceramide via promoting SGK1-dependent osteo-/chondrogenic transdifferentiation of VSMCs contributes to the development of VC during hyperphosphatemia¹⁹. Kapustin et al reported that neutral sphingomyelinase 2 (N-SMase2) inhibition reduces mineralization in response to osteogenic medium in human coronary artery SMC⁹. Recently, our group demonstrated that SMC specific lysosomal acid ceramidase (Ac) and acid sphingomyelinase (ASM) controlled CER metabolism play an important role in the development and progression of arterial medial calcification (AMC)^20,21. These studies demonstrated that either deletion of lysosomal Asah1 gene or over‐expression of Smpd1 gene specifically in SMCs cause reduction of lysosome-MVB interaction and consequent prolongation of MVBs fate resulting in enhanced SMCs-derived exosome/small extracellular vesicle release, SMC phenotypic transition initiating AMC. However, the finer molecular mechanisms how lysosomal-sphingolipid metabolism regulates exosome excretion from MVBs in SMCs during the development and progression AMC is still unknown.

Recent studies showed that the mammalian target of rapamycin complex (mTORC) signaling on late endosomes (LE)/lysosomes may control cargo selection, membrane biogenesis and lysosome trafficking in lysosomal degradation pathways²², which is fine controlled by sphingolipids, ceramide and sphingosine-1-phosphate (S1P)²³. This sphingolipid-regulated mTORC signaling has been reported to be involved in VC²⁴, which may be a crucial molecular mechanism responsible for the development of calcification of atheromatous plaques and even in arterial medial layer due to lysosome dysfunction that we observed recently^25–27. In fro/fro (N-SMase2^−/−) fibroblasts, study anticipated that N-SMase2 and CER are the important mediators in the regulation of hyaluronan synthesis, via microdomains and the Akt/mTOR pathway²⁸. An in vitro study demonstrated that ceramide-1-phosphate (C1P) causes phosphorylation of mTORC1 thereby regulating macrophage proliferation. mTORC1 signaling on late endosomes (LE)/lysosomes seems to control vesicular formation and trafficking in lysosomal degradation pathways²². In VSMCs and mice model, miR-30b was shown to markedly inhibit activation of the mTOR signaling pathway which attenuated the VC during hyperphosphatemia in CKD²⁹. Another study in rat cultured VSMCs demonstrated that high P_i -induced expression of Akt /mTOR pathway are involved in VSMC calcification through the activation of Cbfα1³⁰. Exosomes isolated from high glucose-induced human umbilical vein endothelial cell (HUVEC) promoted VSMCs calcification involving Notch3 protein mediated mTOR signaling pathway³¹. In this regard, rapamycin was reported to decrease VC by inhibiting mTOR pathway under in vitro and in vivo²⁴. Recent studies have indicated that inhibition of lysosomal and endosomal function also promoted osteogenic transformation of VSMCs leading to VC³². Based on these observations, we hypothesized that lysosomal sphingolipids may contribute to mTOR activation on lysosomes and blunt their fusion to MVBs involved in exosome excretion from SMCs during AMC.

Material and Methods

Animal Studies

SMC-specific Asah1 gene knockout (Asah1^fl/fl/SM^Cre) mice (N-Acylsphingosine Amidohydrolase 1 (Asah1) were employed for this study, characterized in our previous study²⁰. Male C57BL/6 J wild-type and Asah1^fl/fl/SM^Cre mice of 12–14 weeks were used. All protocols and experiments were carried out in compliance with appropriate guidelines and regulations by Institutional Animal Care and Use Committee of Virginia Commonwealth University. Animals were thereafter categorized into 8 groups, for each mouse strain (Asah1^fl/fl/SM^Cre and WT/WT) subcutaneously injected active vitamin D (Vit D) (500,000 IU/Kg/bw/day) or matched vehicle (5% v/v ethanol) for 3–4 days to develop AMC^33,34. Following 16–17 days of post injection period, animals were sedated with 2% isoflurane through a nose cone. Started 3 days prior to Vit D treatment, intraperitoneally injection of Torin-1 (a pharmacological inhibitor of mTORC1) (5 mg/kg BW)^35,36 were continued for two weeks (n = 5–6 per group). The Vit D solution was prepared as described previously^20,33. Blood samples were collected, and plasma was isolated, stored at −80 °C. Mice were sacrificed, heart and aorta were retrieved, with a portion of each preserved in 10% buffered formalin for histopathological observations and immunostaining. The remaining portion of heart and aorta were frozen in liquid nitrogen and stored at −80 °C to perform dual fluorescence staining and confocal microscopy.

Calcium staining, mineralization and immunohistochemistry

Alizarin Red S staining was performed³³ for detection of AMC. Different washes of alcohol and xylene followed by distilled water was used for deparaffinization of samples. The arterial sections were stained by 1% Alizarin Red S solution for 5 min followed by incubation with acetone for 20 sec and another 20 sec with acetone-xylene. The reddish color indicated positively stained area. Mineralization was observed by Von Kossa staining as previously described³⁷ and following manufacturer’s protocol (ab150687, Abcam, USA). To perform the histological examinations, paraffin sections were made using fixed mouse heart and aortas, where tissues were dehydrated and embedded in paraffin and cut into 5 μm sections. Immunohistochemical examinations were performed as stated earlier³⁸ or according to the manufacturer’s protocol for CHEMICON IHC Select HRP/DAB Kit (EMD Millipore, MA)³³. Briefly, after antigen recovery with citrate buffer, 3% H₂O₂ was used to quench endogenous peroxidase activity. The tissue was blocked by using 2.5% horse serum for 1 hour at RT followed by incubation with primary antibodies against SM22-α (1:1500), RUNX2 (1:300), OSP (1:100), CD63 (1:50) and AnX2 (1:50) overnight at 4 °C and subsequently with biotinylated secondary antibodies and a streptavidin peroxidase complex (PK-7800, Vector Laboratories, Burlingame, CA, USA). The tissue slides were treated in a sequential manner in conformity with manufacturer’s protocol. Finally, the tissues were counterstained with hematoxylin, dehydrated and mounted using DPX. The area percentage of the positive staining was measured using Image Pro Plus 6.0 software^33,39.

Confocal Microscopy.

Co-localization of the MVB protein marker VPS16 against mTORC1 and lysosomal protein marker Lamp-1, indicating mTOR activation and MVB-lysosome interaction in SMCs was evaluated by indirect immunofluorescent staining. Primary antibodies VPS16 (1:300), mTORC1 (1:100) and Lamp-1 (1:200) were used to incubate acetone fixed frozen aortic tissue slides overnight at 4 °C. The aortic tissue slides were incubated with Alexa-488 or Alexa-555-labeled secondary antibodies for 1 hour at RT to perform the double immunofluorescent staining. Finally, slides were mounted by DAPI-mounting solution, and then analyzed by confocal laser scanning microscope (Fluoview FV1000, Olympus, Japan). As mentioned previously^39,40 the Image Pro Plus 6.0 software was used for analysis of images (Media Cybernetics, Bethesda, MD), which measured and represented co-localization as the Pearson Correlation Coefficient (PCC)³³.

Primary culture of mouse CASMCs and Alizarin Red S staining

Isolated mouse CASMCs as described previously⁴¹ were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% FBS (Gibco) and 1% penicillin‐streptomycin (Gibco) in humidified 100% air and 5% CO₂ mixture at 37°C. Confluent cells were treated with phosphate (P_i) (3 mmol/L)²⁰, and osteogenic medium was changed every two days for 2 weeks to develop in vitro calcification model⁴². Briefly, CASMCs cells were incubated with 1 mL of 1% Alizarin Red S stain for 5 minutes, washed with diH₂O and visualized using a phase microscope. CASMCs were treated with Torin-1 (a pharmacological inhibitor of mTORC1) (100 nm)⁴³ and incubated for 24 hours. Besides, CASMCs were also treated with mouse specific Silencer® Select siRNA mTOR (Assay ID s80903, Thermoscientific, USA) and Silencer® Select Negative siRNA. Manufactures protocol was followed during siRNA transfection process [sc-29528 (siRNA Transfection Medium: sc-36868, siRNA Transfection Reagent: sc-29528)].

Western blot analysis

Briefly, equal amount of protein was resolved on 4–20% precast polyacrylamide gels (Bio-Rad, USA) and transferred to nitrocellulose using Trans-Blot turbo transfer system (Bio-Rad, USA). The membrane was blocked and incubated with primary antibodies rabbit polyclonal to p-mTOR (Ser2448) (1:1000 Cell Signaling, USA) and rabbit anti-vinculin (1:1000, Thermoscientific, USA) at 4 °C overnight followed by incubation with donkey anti-rabbit-HRP IgG (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour at room temperature. The chemiluminescence technique using LI-COR Odyssey Fc was then used for identification of bands and the band intensity of target proteins were normalized to vinculin and determined by Image J software version 1.44p (NIH, Bethesda, MD, USA)⁴⁴.

Nanoparticle Tracking Analysis (NTA)

The exosome size and concentration in the cell culture media was evaluated by Nanoparticle Tracking Analysis (NTA) using the light scattering mode of the NanoSight LM10 (Nano Sight Ltd., Enigma Business Park, Amesbury, United Kingdom). The detached cells or debris were removed from cell culture supernatant by centrifugation at 300 g at 4 oC for 10 min. Further filtered through 0.22 μm filters to remove apoptotic bodies and microvesicles, and finally ultracentrifuged at 100,000 × g for 90 min at 4°C (Beckman 70.1 T1 ultracentrifuge rotator)⁴⁵. In order to carry out the NTA, the exosome pellet was diluted in PBS. Each diluted sample was captured in 5 frames (30 s each) with background level 10, camera level 12, and shutter speed 30 respectively. The captured video was evaluated with NTA software (Version 3.2 Build 16). The PRISM software (GraphPad, San Diego, CA, USA) was used to plot an average particle size distribution graph⁹.

Non-Invasive, in Vivo Measurement of Aortic Pulse Wave Velocity (PWV)

Arterial stiffness was measured by Pulse Wave Velocity (PWV) using a high-resolution Doppler ultrasound instrument (Vevo2100, Visual-Sonics, Toronto, ON, Canada)⁴⁶. Briefly, anesthetization of animals was managed by 2% isoflurane laid on a temperature-controlled platform where the body temperature of mice was maintained at 37 °C to eliminate the movement artifacts. Furthermore, parameters like electrocardiogram (ECG), heart rate (HR), and respiratory rate were observed. The isoflurane concentration was adjusted to 1.5% to maintain the heart rate (HR) of about 450 bpm. Flow wave Doppler measurement of aortic outflow was performed by a 6 mm, 30 MHz pulsed Doppler probe with a pulse repetition frequency of 40 kHz. Aortic outflow profiles were taken longitudinally at proximal and distal locations to the heart. Similar heart rate was observed in both proximal and distal point measurements. PWV was measured at both the proximal and distal locations from the peak of the ECG R wave to the foot of the flow waves for 5 replicates in each mouse. PWV (mm/ms) is based on TT(Dt) and the distance between the measurement sites (Dd), where TT (Transit time) is the difference between the time of arrival at proximal and distal points⁴⁶.

Elastin Staining by Verhoeff’s Van Gieson stain

The elastic fibers in aortic tissue sections were stained using Verhoeff’s Van Gieson staining kit following manufacturer’s instructions (Abcam, Cambridge, MA, USA).

Statistical analysis.

All of the values are expressed as mean ± SEM. Significant differences among multiple groups were examined by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. P < 0.05 was considered statistically significant.

Results

mTOR signaling inhibition reduces lysosomal-CER related arterial stiffness in Asah1^fl/fl/SM^Cre mice.

Arterial stiffness measured by non-invasive ultrasonography and assessed by in vivo aortic pulse-wave velocity (PWV), was significantly increased due to SMC specific Asah1 gene deletion in Asah1^fl/fl/SM^Cre mice under control conditions compared with wild type littermates (WT/WT) as shown (Fig. 1A). The summarized data are depicted in the bar graph (Fig.1C). Besides, Vit D treatment significantly increased arterial stiffness in Asah1^fl/fl/SM^Cre mice compared with littermates (WT/WT). Moreover, Torin-1 (a pharmacological inhibitor of mTORC1) treatment significantly decreased PWV in all the above-mentioned genotypes of mice during AMC under Vit D‐treated conditions. The arterial stiffness change was more noticeable in Asah1^fl/fl/SM^Cre mice with or without progression of AMC. These results indicate that lysosomal-CER may contribute to arterial stiffness mediated via mTOR pathway during development and progression of AMC.

Figure 1.

Effect of SMC‐specific Asah1 deletion on arterial stiffness in Vit D‐treated SMC specific Asah1 KO mice. (A) Representative Doppler images showed Doppler velocity signals. (B) Representative images of aortic sections showed damage to elastic fibers. (C, D) Summarized bar graph showing the effect of Torin-1 on the PWV and elastin breaks. n=5, SMC: smooth muscle cell; Vehl: vehicle; Vit D: vitamin D; Tor: Torin-1, ‘n’ is mouse number. *P < 0.05 vs WT/WT Vehl; #P < 0.05 vs WT/WT Vit D group; $P< 0.05 vs. Asah1^fl/fl/SM^Cre Vit D group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown as mean ± SEM of values.

Further, we observed the effect of SMC specific Asah1 gene deletion on the structural changes of aortic media such as elastin degradation during arterial stiffness in Asah1^fl/fl/SM^Cre mice. Elastin staining by Verhoff’s Van Geison stain was determined on histological aortic sections. Due to SMC specific Asah1 gene deletion, integrity of the elastic laminae in the aortic media seems to be compromised, and treatment with Vit D enhanced thinning and increased breaks of elastin as shown in Fig. 1B, distortion of junctions in between inner and outermost layers, and solid plates formation or sheaths disorganization. Fig. 1D shows the summarized data in the bar graph. These histological changes were easily found in the calcified aortas of Asah1^fl/fl/SM^Cre mice as compared to WT/WT, and Torin-1 treatment efficiently averted these histopathological changes in the aortic media during AMC, as shown by significant decrease in PWV and elastic breaks, suggesting that mTOR signaling may be crucial pathway in lysosomal-CER related arterial stiffness.

mTOR signaling inhibition suppresses lysosomal-CER related AMC in Asah1^fl/fl/SM^Cre mice.

Firstly, we observed p-mTOR expression in the aorta of WT/WT and Asah1^fl/fl/SM^Cre mice, and found that p-mTOR expression was significantly increased in Vit D treated Asah1^fl/fl/SM^Cre mice compared with littermates (WT/WT) as shown in Fig. 2A, 2B. Besides, we explored the role of mTOR signaling inhibition in lysosomal-CER related AMC. Consistent with our previous findings, (Fig. 2C, 2D) showed calcium/mineral deposition in the arterial media, as assessed by Alizarin Red S and Von Kossa staining was significantly increased in Vit D treated Asah1^fl/fl/SM^Cre mice compared with littermates (WT/WT), whereas Torin-1 treatment significantly ameliorated the calcium/mineral deposition in these mice. Importantly, decrease in calcium/mineral deposition was observed in all the genotypes of mice, however much more prominent changes were observed in mice with SMC specific Asah1 gene deletion. The summarized data are shown in the bar graph (Fig. 2E, 2F).This AMC was accompanied with phenotypic switch of arterial medial SMCs, because OSP, RUNX2 immunostaining was markedly increased and SM22-α was decreased in the aortic medial wall of Asah1^fl/fl/SM^Cre mice compared with wild type (Fig. 3A, 3B, 3C). However, Torin-1 treatment reversed these changes in the phenotype of aortic medial SMCs as shown by significantly decreased immunostaining of OSP, RUNX2 associated with increased SM22-α. The bar shows summarized data in (Fig. 3D, 3E, 3F). These results indicate that SMC specific Asah1 gene deletion which perturbates the sphingolipid rheostat in arterial medial SMC’s lead to phenotype change in SMCs mediated via mTOR activation during the development of AMC.

Figure 2.

Effect of mTOR-inhibition on AMC in the Asah1^fl/fl/SM^Cre mice. (A, B) Western blot analysis of enhanced expression of p-mTOR (Ser2448) in Vit D-treated Asah1^fl/fl/SM^Cre mice. n=4. Representative photomicrographs showed (C) calcium deposition by Alizarin Red S staining (red color) and (D) mineral deposition by Von Kossa staining (black color). (E, F) Summarized bar graph showed Torin-1 significantly decreased calcium deposition and mineralization in aortic medial wall. n=5, SMC: smooth muscle cell; Vehl: vehicle; Vit D: vitamin D; Tor: Torin-1. ‘n’ is mouse number. *P < 0.05 vs WT/WT Vehl; #P < 0.05 vs WT/WT Vit D group; $P< 0.05 vs. Asah1^fl/fl/SM^Cre Vit D group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown as mean ± SEM of values.

Figure 3.

mTOR-inhibition prevented phenotype switch in the Asah1^fl/fl/SM^Cre mice. Representative images from the aortic sections depicts changes in the expression of (A) SM22‐α (brown stain) (B) OSP and (C) RUNX2 in the aortic media of Asah1^fl/fl/SM^Cre mice. Summarized bar graph showed Torin-1 significantly increased (D) SM22‐α and decreased (E) OSP (F) RUNX2 in aortic medial wall of mice receiving Vit D injections. n=5, SMC: smooth muscle cell; Vehl: vehicle; Vit D: vitamin D; Tor: Torin-1; OSP, osteopontin; RUNX2, runt‐related transcription factor 2, ‘n’ is mouse number. *P < 0.05 vs WT/WT Vehl; #P < 0.05 vs WT/WT Vit D group; $P< 0.05 vs. Asah1^fl/fl/SM^Cre Vit D group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown as mean ± SEM of values.

mTOR activation reduced lysosome-MVBs interactions in lysosomal-CER related AMC in Asah1^fl/fl/SM^Cre mice.

Next, we explored whether SMC specific Asah1 gene deletion in the aortic medial SMCs lead to mTOR activation indicated by co-localization of mTOR with lysosome as shown in Fig. 4A. Here, we observed significantly increased co-localization of mTORC1 (green) with Lamp‐1 (lysosome marker, red) in the aortic medial SMCs in Asah1^fl/fl/SM^Cre mice (larger yellow spots) compared with littermates (WT/WT) (fewer yellow spots) under control conditions which was enhanced by Vit D treatment as summarized in the bar diagram (Fig. 4C). Furthermore, we found that mTOR activation in aortic medial SMCs is associated with markedly decreased co-localization of VPS16 (a multivesicular bodies (MVBs) marker, green) with Lamp‐1 (lysosome marker, red) in Asah1^fl/fl/SM^Cre mice (fewer yellow spots)compared with wild type littermates (larger yellow spots) with or without Vit D treatment, indicating reduced MVBs interaction with lysosomes (Fig. 4B). However, Torin-1 treatment markedly decreased co-localization of mTORC1 (green) with Lamp‐1 (lysosome marker, red) associated with significantly increased co-localization of VPS16 (MVBs marker, green) with Lamp‐1 (lysosome marker, red) in aortic media with or without Vit D treatment in all these mice as depicted in the bar graph (Fig. 4D), suggesting that mTOR signaling pathway may be implicated in the lysosome‐MVBs interaction in lysosomal-CER related AMC.

Figure 4.

mTOR activation and lysosome-MVB interaction in the Asah1^fl/fl/SM^Cre mice. Representative confocal images showed (A) co-localization of mTORC1 (green) and Lamp-1(red) in aortic medial wall (B) co-localization of VPS16 (green) and Lamp-1(red) and, (C) Bar graph shows Torin-1 significantly decreased the co-localization of mTORC1/Lamp-1 which was enhanced due SMC Asah1 gene deletion in Asah1^fl/fl/SM^Cre mice. (D) Bar graph shows Torin-1 significantly increased the co-localization of VPS16/Lamp-1 which was reduced due SMC Asah1 gene deletion in Asah1^fl/fl/SM^Cre mice. n=5–6, SMC: smooth muscle cell; Vehl: vehicle; Vit D: vitamin D; Tor: Torin-1, PCC: Pearson correlation coefficient, ‘n’ is mouse number. *P < 0.05 vs WT/WT Vehl; #P < 0.05 vs WT/WT Vit D group; $P< 0.05 vs. Asah1^fl/fl/SM^Cre Vit D group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown as mean ± SEM of values.

mTOR signaling inhibition decreased exosome markers in lysosomal-CER related AMC in Asah1^fl/fl/SM^Cre mice.

Literature cites that exosomes or microvesicles plays a crucial role in the tissue calcification process. Next, we sought to determine whether exosomes during calcification are ameliorated via the mTOR signaling pathway, using Torin-1 as mTOR inhibitor. In consistent to our previous results we found that SMC specific Asah1 gene deletion markedly increased the accumulation of exosome markers such as CD63 and annexin 2 (AnX2) in the coronary arterial wall of Asah1^fl/fl/SM^Cre mice compared to their littermates (WT/WT) as shown in (Fig.5A, 5B). However, mTOR inhibition by Torin-1 significantly reduced accumulation of CD63 and AnX2 in the coronary arterial wall of these mice as depicted in the bar graph (Fig.5C, 5D). These results indicate that exosome increase in lysosomal-CER related AMC is mediated via the mTOR signaling pathway.

Figure 5.

Effect of mTOR inhibition on exosome markers in the Asah1^fl/fl/SM^Cre mice. Representative immunohistochemical images depicts that exosome makers (A) CD63 (brown stain) and (B) AnX2 staining in the coronary arterial wall. (C, D) The summarized bar graph showing area positive staining percentage demonstrates that Torin-1 significantly decreased expression of CD63 and AnX2 in the coronary arterial wall of mice receiving Vit D injection. n=5, SMC: smooth muscle cell; Vehl: vehicle; Vit D: vitamin D; Tor: Torin-1, ‘n’ is mouse number. *P < 0.05 vs WT/WT Vehl; #P < 0.05 vs WT/WT Vit D group; $P< 0.05 vs. Asah1^fl/fl/SM^Cre Vit D group by one-way one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown as mean ± SEM of values.

mTOR activation mediates P_i-induced lysosomal-CER related calcification in CASMCs in vitro.

To test whether lysosomal-CER related calcification is mediated via mTOR signaling in P_i treated CASMCs in vitro. Firstly, we demonstrated that p-mTOR (Ser2448), a 289 kD cytoplasm protein is enriched in CASMCs by western blot analysis, and treatment of CASMCs isolated from Asah1^fl/fl/SM^Cre mice with high P_i significantly increased p-mTOR (Ser2448) expression as compared to WT/WT CASMCs (Fig 6A,6B).

Figure 6.

mTOR activation and calcification in the P_i-treated Asah1^fl/fl/SM^Cre CASMCs. (A, B) Western blot analysis of enhanced expression of p-mTOR (Ser2448) in P_i-treated Asah1^fl/fl/SM^Cre CASMCs. (C) Representative images showed calcium deposition by Alizarin Red S staining (red color) and (D) mineral deposition by alkaline phosphatase (blue color) staining. (E, F) Summarized bar graph showed Torin-1 significantly decreased P_i-induced calcium deposition and mineralization in Asah1^fl/fl/SM^Cre CASMCs as compared to WT/WT cells. n=5, P_i: Phosphate; Tor: Torin-1, ‘n’ is experimental repeats. *P< 0.05 vs. WT/WT Vehl; #P< 0.05 vs. WT/WT P_i; $ P< 0.05 vs. Asah1^fl/fl/SM^Cre P_i group by one-way ANOVA followed by Holm-Sidak’s test. Data are shown mean ± SEM of values.

Moreover, we observed that Torin-1 treatment significantly reduced P_i-induced calcium and mineral deposition, as determined by alizarin red and alkaline phosphatase (ALP, blue color) both in Asah1^fl/fl/SM^Cre and WT/WT CASMCs as shown in Fig. 6C and 6D. The summarized data in the bar graph depicts that changes were more prominent with Ac gene deletion in P_i-treated Asah1^fl/fl/SM^Cre CASMCs as compared to WT/WT cells (Fig. 6E, 6F). These data confirm that lysosomal-CER-mTOR signaling via contributes to the development of VSMC calcification.

Regulation of lysosome-MVBs interaction by lysosomal-CER-mTOR signaling pathway in P_i-induced CASMCs in vitro.

Using confocal microscopy, we observed marked increase in the co-localization of mTORC1 (green) with Lamp‐1 (lysosome marker, red), a lysosome targeting of mTORC1, in the CASMCs isolated from Asah1^fl/fl/SM^Cre mice (larger yellow spots) compared with WT/WT (fewer yellows dots) under vehicle conditions and was enhanced during P_i -stimulation in the Asah1^fl/fl/SM^Cre CASMCs as shown in Fig. 7 and Fig. 8. However, siRNA against mTOR and Torin-1 significantly decreased co-localization of mTORC1 (green) with Lamp‐1 (lysosome marker, red) both in Asah1^fl/fl/SM^Cre and WT/WT CASMCs as shown in Fig. 7A, 8A. The summarized data is shown in the bar graph (Fig. 7C, 8C). Reduced lysosome-MVB interaction as indicated by markedly decreased co-localization of VPS16 (MVB marker, green) with Lamp‐1 (lysosome marker, red), was observed in Asah1^fl/fl/SM^Cre CASMCs (fewer yellows dots) as compared to WT/WT cells (larger yellow spots) with or without P_i treatment (Fig. 7B and 8B), which was reversed by siRNA mTOR and Torin-1 treatment as shown in the bar graph (Fig. 7D and 8D). These results suggest that lysosomal-CER-mTOR signaling pathway may contribute to lysosome-MVBs interaction in CASMCs during calcification.

Figure 7.

Pharmacological inhibition of mTOR signaling abrogates lysosome-MVBs interaction in P_i-treated Asah1^fl/fl/SM^Cre CASMCs. Representative confocal images showed (A) co-localization of mTORC1 (green) and Lamp-1(red), indicative of mTOR activation. (B) co-localization of VPS16 (green) and Lamp-1(red), indicative of lysosome-MVBs interaction (C, D) Bar graph shows significant increase in the co-localization of mTORC1/Lamp-1 associated with decreased co-localization of VPS16/Lamp-1 in P_i-treated Asah1^fl/fl/SM^Cre CASMCs as compared WT/WT cells (fewer yellows dots) whereas Torin-1 reversed this trend in these cells. n=5, P_i: Phosphate; Tor: Torin-1, ‘n’ is experimental repeats. *P< 0.05 vs. WT/WT Vehl; #P< 0.05 vs. WT/WT P_i; $P< 0.05 vs. Asah1^fl/fl/SM^Cre P_i group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown mean ± SEM of values.

Figure 8.

siRNA mTOR inhibition abrogates lysosome-MVBs interaction in P_i-treated Asah1^fl/fl/SM^Cre CASMCs. Representative confocal images showed (A) co-localization of mTORC1 (green) and Lamp-1(red), indicative of mTOR activation. (B) co-localization of VPS16 (green) and Lamp-1(red), indicative of lysosome-MVBs interaction (C, D) Bar graph shows significant increase in the co-localization of mTORC1/Lamp-1 associated with decreased co-localization of VPS16/Lamp-1 in P_i-treated Asah1^fl/fl/SM^Cre CASMCs as compared WT/WT cells (fewer yellows dots) whereas siRNA mTOR reversed this trend in these cells. n=3–4, P_i: Phosphate; PCC: Pearson correlation coefficient, ‘n’ is experimental repeats. * P < 0.05 vs. WT/WT Vehl; #P < 0.05 vs. WT/WT P_i group; $P < 0.05 vs. Asah1^fl/fl/SM^Cre P_i group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown mean ± SEM of values.

Role of lysosomal-CER- mTOR signaling in the exosome secretion in P_i-induced CASMCs in vitro.

Using a nanoparticle tracking analysis system, we found that with or without P_{i -}stimulation, excretion of exosomes (<200 nm) were found to be significantly increased in CASMCs of Asah1^fl/fl/SM^Cre CASMCs compared to WT/WT, which was enhanced by P_i stimulation, as depicted by representative 3-D histograms in Fig. 9A and 9B. However, siRNA mTOR and Torin-1 significantly decreased the excretion of exosomes with or without P_{i -} stimulation in Asah1^fl/fl/SM^cre CASMCs. The summarized data in the bar graph depicted vesicle counts of <200 nm which shows a significant decrease upon prior treatment of siRNA mTOR and Torin-1 in P_i -stimulated Asah1^fl/fl/SM^Cre and WT/WT CASMCs (Fig. 9C and 9D). These results suggest that lysosomal CER -mTOR signaling regulates exosome release from CASMCs which may trigger calcification nidus formation.

Figure 9.

siRNA and pharmacological mTOR inhibition reduces exosome secretion in P_i-treated Asah1^fl/fl/SM^Cre CASMCs. (A, B) Representative 3-D histograms of secretion of exosomes (<200 nm) from CASMCs. (C, D) Bar graph show increased exosome release from Asah1^fl/fl/SM^cre CASMCs with or without P_i treatment as compared to WT/WT cells which was significantly decreased by siRNA mTOR and Torin-1. n=5–6, P_i: Phosphate; Tor: Torin-1, ‘n’ is experimental repeats. *P< 0.05 vs. WT/WT Vehl; #P< 0.05 vs. WT/WT P_i; $P< 0.05 vs. Asah1^fl/fl/SM^Cre P_i group by one-way ANOVA with Holm-Sidak’s test post-hoc analysis. Data are shown mean ± SEM of values.

Discussion

The present study first time revealed that lysosomal-CER‐mTOR signaling is critical for the control of lysosome fusion to MVBs, exosome secretion, osteogenic shift and arterial stiffening in AMC. Asah1 gene deletion that encodes for a lysosomal Ac to increase ceramide demonstrated that lysosomal-CER‐mTOR signaling may be one of the mechanistic pathways contributing to the development and progression of AMC. Non‐invasive imaging technique showed that PWV, which is correlated to arterial stiffness is significantly increased upon SMC specific Asah1 gene deletion in Asah1^fl/fl/SM^Cre mice as compared to their littermates which was further enhanced during hypercalcemia induced by high doses of Vit D. Histopathological analysis revealed that damage to elastin fibers in the aortic media of Asah1^fl/fl/SM^Cre mice, associated with distorted junctions and formation of solid plates or sheaths, resulting in damaged arterial architecture. However, Torin-1, a pharmacological inhibitor of mTOR efficiently averted these histopathological changes in the aortic media during AMC, as shown by significant decrease in PWV and elastic breaks. Moreover, pretreatment of these mice with Torin‐1 significantly decreased the Vit D-induced AMC and expression of osteogenic markers like osteopontin and RUNX2 associated with increased levels of SM22-α. Furthermore, we observed a significant increase in the co-localization of mTORC1 with Lamp‐1(lysosome marker) in Asah1^fl/fl/SM^Cre mice suggesting mTOR activation which was accompanied with decreased co-localization of VPS16 (MVBs marker) with Lamp‐1 indicating reduced MVBs interaction with lysosomes in these mice. However, mTOR inhibition by Torin‐1 showed marked decrease in the co-localization of mTOR vs Lamp‐1 associated with increased VPS16 vs Lamp‐1 interaction. Torin‐1 was also found to reduce accumulation of CD63 and annexin 2 (AnX2) (exosome markers) in the coronary arterial wall of Asah1^fl/fl/SM^Cre mice compared to wild type littermates. In CASMCs under in vitro conditions, we validated our in vivo findings. Firstly, we observed that P_i-stimulation significantly increased expression of p-mTOR in WT CASMCs which was further enhanced due to Ac gene deletion in P_i-treated Asah1^fl/fl/SM^Cre CASMC, indicating that CER contributes to activation of mTOR. Torin-1, an mTOR inhibitor markedly decreased the P_i-induced calcium and mineral deposition as shown by the presence of decreased Alizarin Red-stained nodules and ALP staining both in Asah1^fl/fl/SM^Cre cells and WT/WT cells. In addition, we found that siRNA mTOR and Torin-1 significantly decreased co-localization of mTORC1 with Lamp-1, but increased co-localization of VPS16 with Lamp-1 in these cells, signifying that activation of mTOR signaling inhibits lysosome fusion with MVBs. We further found that Ac gene deletion led to increased P_i-induced excretion of exosomes (<200), which was blocked by siRNA mTOR and Torin‐1 suggesting the role of lysosomal-CER-mTOR signaling in lysosome interactions with MVBs and exosome secretion. This lysosomal-CER-mTOR signaling in lysosome-MVB interactions, exosome secretion and arterial stiffening may represent a novel molecular mechanism involved in the development of AMC.

Earlier we found that increased lysosomal CER due to Ac gene deletion in SMCs markedly increased calcification in the arterial media of mice as well as isolated CASMCs from these mice. Here in the present study we postulate that mTOR signaling may be a mechanistic pathway through which lysosomal-sphingolipid pathway contribute to the AMC. However, the proper molecular mechanisms involved in AMC are still unknown. An important, change seen during the development and progression of AMC is stiffening of the large elastic arteries. Studies have shown increase in large elastic artery stiffness contributes to cardio vascular disease (CVD) risk and is associated with pathophysiological conditions like hypertension, left ventricular hypertrophy, stroke, sub-endocardial ischemia and cardiac fibrosis⁴⁷. Literature cites that arterial stiffness increase during AMC which is hallmark of aging process^48,49. Sphingolipid such as ceramide regulate actin cytoskeleton dynamics in VSMCs which is crucial for mechanosensing in these cells during aging process^50,51. In the current study, we determined whether lysosomal-CER-mTOR signaling contributes to arterial stiffness. We found that Ac gene deletion in arterial SMCs increased PWV, measure of arterial stiffness⁵² which was further increased due to Vit D treatment, and blocking mTOR signaling using Torin‐1, a pharmacological inhibitor of mTORC1, significantly decreased PWV in these mice model of AMC. In the mice and rabbits fed a western diet blockade of glycosphingolipid synthesis can lower arterial stiffness during the development of atherosclerosis⁵³. Recently, our laboratory demonstrated that SMC specific overexpression of Smpd1 gene that encodes for acid sphingomyelinase to increase CER in arterial media of Smpd1^trg/SM^Cre mice observed significantly increased arterial stiffness in these mice indicating lysosomal-CER contributes to arterial stiffness^20,21. In human ELN in a bacterial artificial chromosome (hBAC) hBAC-mNULL mice, mTOR inhibition by Rapalog reduces the aberrant mechanosignaling and aortic stiffening⁵⁴. In another study, increased mTOR activation was observed in the large arteries of the old mice which was associated with arterial stiffness in these arteries⁵⁵. Altogether, all these findings support our data which shows that sphingolipid-mTOR pathway contribute to arterial stiffness.

Literature reports that AMC is an actively regulated process controlled by osteogenic differentiation of SMCs and matrix mineralization^56,57. Recent reports from our laboratory described that lysosomal-sphingolipids may be important mediators of VC^20,21. But the exact molecular mechanism how lysosomal sphingolipids contribute to AMC is still poorly understood. A study revealed that the Lysosome Associated Protein Transmembrane 4B (LAPTM4B) interacts with CER, and is also involved in the 4F2 cell-surface antigen heavy chain (4F2hc)-mediated regulation of mTORC signaling⁵⁸. The findings of this study showed decreased phosphorylation of the mTORC1 substrate S6K in LAPTM4B knockout cells indicating that LAPTM4B promotes mTORC activation. Further, the study also reported that Asah1 gene deletion which can increase CER levels increased levels of phospho-S6K indicating mTORC activation, however silencing of Smpd1 gene which will block CER formation from sphingomyelin decreased the levels of phospho-S6K, suggesting that lysosomal-CER is associated with mTOR activation⁵⁸. In this context, we observed Asah1 gene deletion in the arterial SMCs led to mTOR activation and increased AMC both in vitro and in vivo while as blocking mTOR signaling by Torin-1 reduced AMC under these conditions. Under in vitro and in vivo conditions, inhibition of the mTOR activation signaling pathway by miR-30b was shown to markedly attenuated the VC in hyperphosphatemia of chronic kidney disease (CKD)²⁹. In rat cultured VSMCs high P_i induced expression of Akt /mTOR pathway result in VSMC calcification mediated via Cbfα1 actiavtion³⁰.VSMCS treated with exosomes isolated from high glucose-induced human umbilical vein endothelial cell promoted calcification involving Notch3 protein mediated through mTOR signaling pathway³¹. In Human aortic vascular smooth muscle cells (HAVSMCs), bone marrow mesenchymal stem cell (BMSC) derived exosomes inhibited high phosphorus-induced calcification via modifying miRNA profiles. The study further revealed that inhibition of calcification by BMSC derived exosomes involves mTOR, MAPK, and Wnt signaling pathway using functional miRNA-gene regulatory network⁵⁹. Also, rapamycin, a mTOR inhibitor was reported to decrease VC by inhibiting mTOR pathway both in vitro and in vivo²⁴. In patients with end-stage renal disease, it was found that inflammation induced-osteogenic differentiation in vascular tissues contributed to VC and also enhanced expression of bone formation biomarkers, which was regulated by the activation of the mTORC1 pathway. These findings support our hypothesis and suggest that lysosomal-CER pathway may contribute to mTOR activation which may be involved in calcification process during the development of AMC.

Arterial SMCs undergo phenotypic changes which simulates osteogenesis^5,60. During the AMC, medial VSMCs displayed osteogenic phenotype which mainly contributes to VC^61–63. In human VSMCs, C2-ceramide treatment result in Ox-LDL-induced VC through TLR4/NF-κB/CER signaling¹⁸. In human femoral arterial SMCs, increased N-SMase activity and CER levels increased Ox‐LDL‐induced matrix mineralization in these cultured SMCs¹⁷. Recently, our group reported that SMC specific lysosomal Asah1 or Smpd1 gene expression had remarkable effects on SMC marker (SM22-α) and phenotypic switch markers such OSP and RUNX2 expression suggesting that lysosomal sphingolipid‐CER pathway contribute to the phenotype change in arterial medial SMCs undergoing AMC^20,21. PDMP, a ceramide analogue, via inhibition of mTORC1 activity reduced osteoblastic proliferation and pre-osteoblastic cell differentiation by translocation of mTORC1 from late endosome/ Lysosome to the endoplasmic reticulum⁶⁴. In the present study, we found that pretreatment of SMC specific Asah1 KO mice with Torin-1, a pharmacological mTOR inhibitor, markedly decreased the expression of osteogenic markers like osteopontin and RUNX2 associated with increased levels of contractile marker SM22-α in Vit D-induced mice model. During aging process, VSMCs phenotypic transformation is closely linked with increased arterial stiffening and blood pressure⁶⁵. The mTOR signaling pathway plays a crucial role in VSMC proliferation, hypertension and atherosclerosis^66,67 and hence, has been identified as a therapeutic target^67,68. Furthermore, several studies have shown that the mTOR signalling pathway is involved in VSMC dedifferentiation^69–71. However, the regulation of the mTOR/p70S6K signalling pathway and its effects on the phenotypic switching of VSMCs in arterial medial calcification is still poorly understood. In VSMCs, homocysteine-induced proliferation, migration, and phenotypic transformation was reduced by overexpression of miR-145 mediated via decreased PI3K, Akt, and mTOR expression⁷². Recently, a study showed that rapamycin treatment markedly declined the kaempferol-induced osteogenetic activity associated with decreased RUNX2 and osterix expression involving mTOR, suggesting contribution of mTOR-RUNX2/osterix signaling in osteogenic effect under in vitro and in vivo⁷³. In another study, sirolimus (also known as rapamycin) diminished advanced glycation end product (AGE)-induced phenotypic transition of VSMCs indicated by increased expression OSP, ALP and decreased expression of α-SMA via inhibition of the ILK/mTOR/p70S6K signaling pathway⁷⁴. VSMC calcification has been observed to be a manifestation of cellular senescence, and replicative senescence of these cells enhances age-associated medial artery calcification^75,76. Several studies reported that replicative senescence of VSMCs was significantly inhibited via inhibition of mTOR-signaling pathway^77,78. In in CaCl2‐induced aortic aneurysm rat model, pretreatment with rapamycin decreased the OSP expression in aortic SMCs indicating phenotypic switch of aortic SMCs to contractile one via mTOR activation suggesting a crucial role of mTOR-signaling pathway in phenotypic transition of SMCs⁷⁹. Taken together, these data support our hypothesis that the mTOR-signaling is associated with the osteogenic effect of lysosomal-sphingolipids in SMC specific Asah1 gene KO mice.

Imbalanced mineral metabolism in SMCs enhanced secretion of calcifying exosomes via sphingolipid/CER pathway causing arterial calcification^9,80. These SMC‐derived vesicles were having increased levels of exosomes markers such as CD63, AnX2 and ALP⁸⁰. Several studies demonstrated that both intimal and medial calcifications is accompanied with increased exosomes (size of 40–100 or to 140 nm in size) in the vascular interstitial space^2,9,12. Evidences showed that augmented exosome secretion may elicit SMC phenotype changes^3,5 contributing to extracellular matrix (ECM) mineralization^9,11. It is already reported that sphingolipid pathway plays a curial role in the EV production in different cells both in vitro and in vivo^81,82. In our previous study, we demonstrated that deletion of lysosomal Asah1 gene specifically in SMCs caused reduction of lysosome-MVB interaction and enhanced SMCs-derived exosome release, SMC phenotypic transition during AMC²⁰. PDMP, a ceramide analogue, allows normal lysosomal functioning or activation, and their fusion with MVBs via inhibition of mTORC1 activity⁶⁴. In the present study, we investigated whether inhibition of mTOR-signaling pathway block lysosomal-CER mediated exosome secretion which act as a triggering mechanism in the AMC. Firstly, we observed enrichment of activated p-mTOR in P_i-stimulated CASMCs which was increased due to Ac gene deletion in these cells.

We also observed markedly increased co-localization of mTORC1 with lysosome marker (Lamp-1) in Asah1 gene KO CASMCs as compared WT/WT under vehicle conditions indicating mTOR activation which was associated with decreased co-localization of MVB marker (VPS16) with lysosome marker (Lamp-1) suggesting less lysosomal fusion with MVBs. However, mTOR inhibition at genetic and pharmacological level significantly decreased co-localization of mTORC1 with lysosome marker (Lamp-1), but increased co-localization of MVB marker (VPS16) with lysosome marker (Lamp-1) both in WT/WT and Asah1^fl/fl/SM^Cre CASMCs signifying that inhibition of mTOR signaling promotes lysosome fusion with MVBs. Furthermore, Nanoparticle tracking analysis demonstrated that mTOR inhibition in these CASMCs under osteogenic conditions decreased the excretion of exosome (<200 nm in size), suggesting the role of lysosomal-CER-mTOR signaling in lysosome interactions with MVBs/ exosome secretion. mTOR inhibition was also found to reduce the accumulation of CD63 and AnX2 (exosome markers) in the coronary arterial wall of Vit D-treated mice. High P_i-induced calcification in HAVSMCs was alleviated with treatment of BMSC-derived exosomes (CD63 and CD81 as exosome markers) through modifying miRNA profiles involving mTOR, MAPK, and Wnt signaling pathway⁵⁹. Cardiac progenitor cell-derived exosomes (CD63 and CD81 as exosome markers) promoted H9C2 cell growth via activation of Akt/mTOR pathway in a time-dependent manner suggesting that PI3K/AKT/mTOR pathway may have important regulatory function in cardioprotection⁸³. In cervical intraepithelial neoplasia patients, a non-significant difference of PI3k/Akt/mTOR gene expression was observed in cervical cancer tissue and the exosomes extracted from vaginal secretion but were significantly increased corresponding adjacent control tissues suggesting that PI3k/Akt/mTOR signaling pathway mediated by exosomes may provide candidate diagnostic biomarkers or potential therapeutic targets⁸⁴. Fibroblast-derived exosomes mediate mobilization of autocrine Wnt10b-mTOR pathway to promote axonal regeneration, a crucial step towards healing the injured central nervous system⁸⁵. Together, all these studies conclude that lysosomal-CER in association with mTOR activation enhance exosome release during calcification process as shown in Fig. 10. In this context, our findings indicate that lysosomal Ac associated CER-mTOR signaling may regulate lysosome fusion with MVBs, exosome secretion and arterial stiffening which may represent a novel molecular mechanism involved in the development of AMC.

Figure 10.

Schematic model indicates that lysosomal Ac associated CER-mTOR signaling may regulate lysosome fusion with MVBs and exosome secretion which may represent a novel molecular mechanism involved in the development of AMC. Ac: Acid ceramidase; SMCs: Smooth muscle cells; AMC: Arterial medial calcification.

Nonstandard Abbreviations:

AC

Acid Ceramidase

AMC

Arterial Medial Calcification

AnX2

Annexin-II

ASM

Acid Sphingomyelinase

CASMCs

Coronary Artery Smooth Muscle Cells

CER

Ceramide

ILV

Intraluminal Vesicles

Lamp-1

Lysosome Associated Membrane Protein-1

mTORC1

Mammalian Target of Rapamycin Complex 1

MVBs

Multivesicular Bodies

OSP

Osteopontin

RUNX2

Runt-related transcription factor 2

SM22-α

Smooth Muscle 22α

SMC

Smooth Muscle Cells

VC

Vascular Calcification

Vit D

Vitamin D

VPS16

Vacuolar Protein Sorting-associated Protein 16

VSMCs

Vascular Smooth Muscle Cells