Release and Actions of Inflammatory Exosomes in Pulmonary Emphysema: Potential Therapeutic Target of Acupuncture.

BACKGROUND: Exosomes have been reported to mediate activation of the inflammatory response by secretion of inflammasome products such as IL-1ß or IL-18 and that changes in exosomes production or secretion may be a therapeutic target for treatment of a variety of different chronic diseases. The present study tested the hypothesis that exosome-mediated release of NLRP3 inflammasome products instigates the inflammatory response in the lung during emphysema, a type of chronic obstructive pulmonary disease (COPD) and that electroacupuncture (EA) may attenuate emphysema by inhibition of NLRP3 inflammasome activation and consequent inflammation. METHODS: The COPD mice model was developed by injecting porcine pancreatic elastase (PPE) via puncture tracheotomy and instillation. EA (4 Hz/20 Hz, 1 to 3 mA) was applied to the bilateral BL13 and ST36 for 30 min, once every other day for 2 weeks. Micro computed tomography (micro-CT) was performed to measure lung function. Histopathological changes in the lungs were displayed by HE staining. RESULTS: In a mouse model of porcine pancreatic elastase (PPE)-induced emphysema, the lung tissue was found to display several key features of emphysema, including alveolar septal thickening, enlarged alveoli, interstitial edema, and inflammatory cells infiltration. Lungs of mice receiving PPE exhibited substantially increased low attenuation area (LAA) in micro-CT images. The colocalization of NLRP3 vs ASC or caspase-1 detected by confocal microscopy was shown to increase in both bronchial and alveolar walls, indicating the increased formation of NLRP3 inflammasomes. IL-1ß, a prototype NLRP3 inflammasome activating product, was also found to have increased in the lung during emphysema, which was colocalized with CD63 (an exosome marker), an indicative of inflammatory exosome formation. By nanoparticle tracking analysis (NTA), IL-1ß-containing exosomes were shown to significantly increase in the bronchoalveolar lavage (BAL) from mice with emphysema. Therapeutically, IL-1ß production in the lung during emphysema was significantly reduced by EA at the acupoint Feishu (BL13) and Zusanli (ST36), accompanied by decreased colocalization of NLRP3 vs ASC or caspase-1. Increased exosome release into BAL during emphysema was shown to be significantly attenuated in EA-treated mice compared to their controls. However, EA of non-specific BL23 together with ST36 acupoint had no effects on NLRP3 inflammasome activation, exosome release and associated lung pathology during emphysema. CONCLUSION: NLRP3 inflammasome activation in concert with increased release of exosomes containing IL-1ß or other inflammasome products contributes to the development of lung inflammation and injury during PPE-induced emphysema and that EA of lung-specific acupoints attenuates inflammasome activation and exosome release, thereby reducing inflammatory response in the lung of mice with emphysema.

Regulatory role of mammalian target of rapamycin signaling in exosome secretion and osteogenic changes in smooth muscle cells lacking acid ceramidase gene.

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 (LEs)/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 (5 mg/kg/day), an mTOR inhibitor, significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin (OSP) and runt-related transcription factor 2 (RUNX2) and upregulation of smooth muscle 22α (SM22-α) in mice receiving high dose of Vitamin D (500 000 IU/kg/day). Asah1fl/fl /SMCre mice had markedly increased co-localization of mTORC1 with lysosome-associated membrane protein-1 (Lamp-1) (lysosome marker) and decreased co-localization of vacuolar protein sorting-associated protein 16 (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-MVB interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using coronary artery smooth muscle cells (CASMCs), Pi -stimulation significantly increased p-mTOR expression in Asah1fl/fl /SMCre CASMCs as compared to WT/WT cells associated with increased calcium deposition and mineralization. Torin-1 blocked Pi -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 Pi -stimulated CASMCs, associated with decreased exosome release. Functionally, Torin-1 significantly reduces 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.

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

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.

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

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 (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt 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 Asah1fl/fl/SMCre mice, high dose of Pi 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 Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ 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.

Medial calcification in the arterial wall of smooth muscle cell-specific Smpd1 transgenic mice: A ceramide-mediated vasculopathy.

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 Smpd1trg /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 (Smpd1trg /SMwt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1trg /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 Smpd1trg /SMcre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1trg /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.