Sphingomyelinase Inhibitor Research Articles

Combination of Imipramine, a sphingomyelinase inhibitor, and β-caryophyllene improve their therapeutic effects on experimental autoimmune encephalomyelitis (EAE)

Multiple Sclerosis (MS) is one of the most common inflammatory diseases with the essential role of immune system in the demyelination, damage and inflammation of the central nervous system neurons (CNS). β-Caryophyllene (BCP), a natural and selective CB2 agonist, possesses several protective effects. In the present study, we evaluated the protective effects of low dose of BCP (5 mg/kg), sphingomyelinase (SMase) inhibitor imipramine (IMP, 10 mg/kg), and the combination of BCP (2.5 and 5 mg/kg) with IMP in the treatment of experimental autoimmune encephalomyelitis (EAE) mice as a known model of chronic MS. These effects were assessed on the levels of pro- or anti-inflammatory cytokines as well as the polarization of spleen lymphocytes and microglia, in EAE mice. Our results indicated that low dose of BCP, IMP and BCP combined with a SMase inhibitor IMP exert protective effects in treatment of EAE mice. We also found that they reduced the clinical and pathological defects in EAE mice through modulation of both local (microglia) and systemic (lymphocytes and blood) immunity from inflammatory (Th1/Th17/M1) towards anti-inflammatory (Th2/Treg/M2) phenotypes. Therefore, it can be suggested that a low dose of BCP alone or combined with IMP as a known SMase inhibitor deserve a therapeutic position for treatment of MS.

P2585Cardiac dysfunction after myocardial infarction: role of pro-inflammatory extracellular vesicles

Abstract Background Myocardial infarction (MI) is associated with significant loss of cardiomyocytes (CM), which are replaced by a fibrotic scar. Necrotic CM release damage-associated proteins that stimulate innate immune pathways and macrophages (MΦ) tissue infiltration, which drives to the progression of inflammation and myocardial remodeling process. Both, loss of CM and inflammatory response are determinants of the long term ventricle remodeling and heart failure. Circulating inflammatory extracellular vesicles (EV) play a crucial role in the acute and chronic phases of MI, in terms of inflammatory progression. In this study we examined whether reducing the generation of inflammatory EV within few hours from the ischemic event may ameliorate cardiac outcome at short and long time-point in LAD rat model. Methods Before coronary artery ligation, rats were injected IP with a chemical inhibitor of neutral sphingomyelinase (nSMase) which is essential for the biogenesis and release of EVs. The number and size profile of plasma-derived EV was assessed by NTA analysis at baseline and 24hrs after MI. Multiple EV cytokine levels were simultaneously determined using enzyme-linked immunosorbent assay (ELISA)-based protein array technology. Heart global function was assessed by echocardiography and hemodynamic analysis performed at 7, 14 and 28 days after MI. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langedorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory MΦ (MΦM1) were studied in vitro into primary rat neonatal CM. Results The induction of MI and the consequent inflammation, dramatically increase the number of circulating EV carrying inflammatory cytokines such as IL1α, ILβ and Rantes. Preventive inhibition nSMase significantly reduced the boost of inflammatory EV and cytokines in treated group as compared to control animals. The reduction of circulating EV post MI results in preserved LV ejection fraction at 7 and 28 days post-MI as compared to control group. Hemodynamic analysis confirmed functional recovery by displaying a higher velocity of LV relaxation and an improved contractility capacity in treated versus control group. The number of infiltrating CD68+ monocytes/macrophages in the infarct area was significantly reduced. Post-MI circulating EV induce cell death in adult CM when added to the perfusate of Langendorff, as assessed by the incresed level of cTnI into media. In vitro MΦM1-EV activated nuclear translocation of NF-kB. Specific inhibition of TLR4 receptor activity abrogated NF-kB translocation and reduced cell death. Indicating that the axis TRL4-NF-kB is essential in EV-mediated CM death. Conclusions Systemic inhibition of EV release during the acute phase of MI preserves heart function in an animal model of LAD. These findings suggest detrimental effects of exosomes in the acute phase of MI.

The Neuroprotective Effect of Amitriptyline on Radiation-Induced Impairment of Hippocampal Neurogenesis.

The radioprotective effect of amitriptyline, an inhibitor of acid sphingomyelinase (ASMase), on radiation-induced impairment of hippocampal neurogenesis, loss of interneuron, and animal weight changes was investigated in BALB/c mice by immunostaining of biomarkers for cell division (Ki67), immature neurons (doublecortin or DCX), and interneurons (parvalbumin or PV) in the dentate gyrus (DG) of hippocampus. The results indicated that preirradiation (with 10 mg/kg, 2 times per day, for 7 consecutive days) or postirradiation (with 10 mg/kg, 2 times per day, for 14 consecutive days) treatment (pretreatment or posttreatment) with intraperitoneal injection of amitriptyline prevented the loss of newly generated neurons, proliferating cells, and interneurons in the subgranular zone of the DG. At the molecular level, pretreatment or posttreatment inhibited the expression of sphingomyelin phosphodiesterase 1 (SMPD1) gene which codes for ASMase. The pretreatment for 7 days also prevented radiation-induced weight loss from 2 to 3 weeks, but not within 1 week after irradiation. On the other hand, the posttreatment with amitriptyline for 14 days could improve animal weight gain from 4 to 6 weeks after irradiation. The present study suggests that amitriptyline may be a promising candidate radio-neuroprotective drug to improve radiation-induced impairment of hippocampal neurogenesis and relevant neurological and neuropsychological disorders.

P2587Cardiosomal microRNAs are essential in post-infarction myofibroblast phenoconversion

Abstract Background Circulating microRNAs (miRNAs) are actively transported, via inclusion in extracellular microvesicles/exosomes (herein named cardiosomes since they are deriving from cardiomyocytes), allowing protection from degradation and underlying cell-cell communications. After myocardial infarction (MI), quiescent cardiac fibroblasts can acquire an activated phenotype, and a recognized marker of such activation is the expression of α-smooth muscle actin (α-SMA); such cells are commonly known as myofibroblasts. Hypothesis We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Methods Using an established murine model of MI, obtained via permanent ligation of the left anterior descending coronary artery, we tested our hypothesis by measuring in primary isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, which we found to be upregulated in cardiomyocytes post-MI and to be involved in myofibroblast phenoconversion. Results Bioinformatic studies validated by biological assays identified three cardiomyocyte-specific miRNAs (namely miR-92a, miR-195, and miR-494) that are upregulated following cardiac ischemia and are also known to regulate α-SMA expression. We demonstrated that these miRNAs are upregulated in fibroblasts post-ischemia and are also contained in cardiosomes; to ensure that these miRNAs were confined inside cardiosomes, the samples were treated with RNase, showing that the level of miRNAs was not affected by RNase treatment, unless in presence of the detergent Triton X-100. The activation of myofibroblasts was confirmed by the increased expression of α-SMA. Using a luciferase assay, we also validated that miR-92a, miR-195, and mir-494 target the 3'UTR of α-SMA and regulate its transcription. Moreover, we observed that primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effects were detected following incubation with cardiosomes or medium from sham cardiomyocytes. Strikingly, myofibroblast phenoconversion was markedly attenuated when GW4869, an inhibitor of neutral sphingomyelinase 2 responsible for cardiosomes secretion, was added to cultured post-MI cardiomyocytes. Conclusion Taken together, our findings indicate that three cardiomyocyte-specific miRNAs (miR-92a, miR-195, and miR-494) are crucial in the activation of myofibroblasts.

Acid sphingomyelinase plays a critical role in LPS- and cytokine-induced tissue factor procoagulant activity

AbstractTissue factor (TF) is a cofactor for factor VIIa and the primary cellular initiator of coagulation. Typically, most TF on cell surfaces exists in a cryptic coagulant-inactive state but are transformed to a procoagulant form (decryption) following cell activation. Our recent studies in cell model systems showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells, and the hydrolysis of SM leads to decryption of TF. The present study was carried out to investigate the relevance of this novel mechanism in the regulation of TF procoagulant activity in pathophysiology. As observed in cell systems, administration of adenosine triphosphate (ATP) to mice enhanced lipopolysaccharide (LPS)-induced TF procoagulant activity in monocytes. Treatment of mice with pharmacological inhibitors of acid sphingomyelinase (ASMase), desipramine and imipramine, attenuated ATP-induced TF decryption. Interestingly, ASMase inhibitors also blocked LPS-induced TF procoagulant activity without affecting the LPS-induced de novo synthesis of TF protein. Additional studies showed that LPS induced translocation of ASMase to the outer leaflet of the plasma membrane and reduced SM levels in monocytes. Studies using human monocyte-derived macrophages and endothelial cells further confirmed the role of ASMase in LPS- and cytokine-induced TF procoagulant activity. Overall, our data indicate that LPS- or cytokine-induced TF procoagulant activity requires the decryption of newly synthesized TF protein by ASMase-mediated hydrolysis of SM. The observation that ASMase inhibitors attenuate TF-induced coagulation raises the possibility of their therapeutic use in treating thrombotic disorders associated with aberrant expression of TF.

Abstract 192: The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis

Abstract Current research suggests that on tumor-related extracellular vesicles (EV) play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms including angiogenesis. Our previous study demonstrated that IL6 induced secretion of VEGF and contributing angiogenesis via activation of Stat3. To further clarify the biological activities of the lung cancer derived EVs, we analyzed the content of IL6 and VEGF in exosome using ELISA kit. We found that IL-6 and VEGF were more enriched in EVs than non-EVs in AS2 and CL1-5 cells. And the isolated EV from AS2 and CL1-5 cells increased vascular permeability in miles assay and promotes angiogenesis in tubular formation assay. We further investigated if the inhibition of exosome secretion affects the autocrine IL6/Stat3/VEGF pathway. GW4869 inhibits secretion of exosome by playing an inhibitor of neutral sphingomyelinase (nSMase). Treatment of either cell with GW4869 significantly decreased the levels of released exosomes in AS2 but not in CL1-5 cell. Consistent with the result, the treatment GW4869 dampened autocrine IL6/Stat3/VEGF pathway in AS2 but not in CL1-5 cell. Bevacizumab, the recombinant anti- VEGF monoclonal antibody, were approved for treating patients with advanced NSCLC, has been shown to be efficient in suppressing the accumulation of pleural fluid (malignant pleural effusion). To investigate if the exosomal VEGF has a different affinity for bevacizumab, we treated AS2, CL1-5 and MPE-derived cancer cells with bevacizumab and EV or non-EV part from cell-free medium. We found VEGF dimer form were more abundant in EV part and cannot be neutralized by bevacizumab. Conversely, VEGF mono-form was more abundant non-EV part and can be neutralized by bevacizumab. In animal model, AS2 was injected intraperitoneal and then malignant ascites developed as follow. We found the administration of bevacizumab after AS2 injection i.p. inhibited peritoneal seeding and ascites formation compared to control group. But the inhibition was reversed when we injected EV as well as bevacizumab in combination group. In breast cancer cell, microvesicles (MVs) activates VEGF receptors through a unique 90 kDa form of VEGF (VEGF90K) which has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor. And an Hsp90 inhibitor (17-AAG) was demonstrated to release VEGF90K from MVs without interaction with VEGF restoring the sensitivity of VEGF90K to Bevacizumab. However, our study found 17-AAG alone inhibited VEGF secretion in EV as well as the activity of upstream Stat3, and Akt pathway in lung cancer. Our preliminary data implied EV-VEGF and the existence of different isoform of VEGF in EV contribute to tumor angiogenesis and limit the effectiveness of bevacizumab therapy which is not dependent on Hsp90. Citation Format: Chien-Chung Lin, Yu-Ting Huang, Chun-Hua Hung, Mei-Ling Tsai, Wu-Chou Su. The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 192.

Lipids and mental disorders: Evidence, uncertainties and perspectives

Brain is an organ with the highest lipid concentration in the body. Cellular membrane lipids can affect both the positioning and the functioning of membrane proteins, thus regulating several cell actions. Changes in the lipid composition of cell membrane can modulate the microenvironment and consequently the function of its proteins, e.g. neurotransmission. Some of the first studies on the subject have shown a negative correlation between serum cholesterol levels and depression, suicidality and behavioral disorders. Several studies -but not all- have found decreased concentrations of ω-3 polyunsaturated fatty acids (ω-3-PUFA) in plasma or erythrocyte membranes of patients with depression, bipolar disorder or after a suicide attempt. In some cases, positive results after their administration have been reported. The effect of ω-3-PUFA in affective disorders is attributed to their action on neurotransmission, neuroplasticity as well as to their anti-inflammatory and anti-oxidative properties. Besides, decreased levels of ω-3-PUFA have been found in erythrocytes or platelets of schizophrenic patients. Some studies have shown in schizophrenics an increased rate of membrane phospholipids breakdown and a decreased ratio of ω-3-PUFA incorporation in phospholipids, possibly because of increased activity of phospholipase A2, an enzyme with crucial role in signaling transduction. Deficient dietetic ingestion of ω-3-PUFA may increase the risk for development of schizophrenia, while a diet rich in ω-3-PUFA may have a preventive role for the disease or improve its course. Although there is no evidence for their action as a monotherapy, they may be useful as an add-on therapy to drug treatment. Some authors suggest that abnormal sphingolipid metabolism, leading to accumulation of ceramides, may be responsible for the development of mood and anxiety disorders, as well as for induction of inflammation or oxidative stress, mechanisms possibly responsible for the physical symptoms of depression. Some drugs seem to combine inhibition of sphingomyelinase (an enzyme catalyzing the production of ceramides) and antidepressant effect. Despite the multitude of related studies, many aspects of the subject remain obscure. Current research focuses on the validity of preventive (especially in the perinatal period) or therapeutic administration of ω-3-PUFA as well as to the pharmacological manipulation of enzymes involved in lipid metabolism (e.g. sphingomyelinase) for the treatment of psychiatric disorders.

Acid sphingomyelinase inhibition alleviates muscle damage in gastrocnemius after acute strenuous exercise.

[Purpose]Strenuous exercise often induces skeletal muscle damage, which results in impaired performance. Sphingolipid metabolism contributes to various cellular processes, including apoptosis, stress response, and inflammation. However, the relationship between exercise-induced muscle damage and ceramide (a key component of sphingolipid metabolism), is rarely studied. The present study aimed to explore the regulatory role of sphingolipid metabolism in exercise-induced muscle damage.[Methods]Mice were subjected to strenuous exercise by treadmill running with gradual increase in intensity. The blood and gastrocnemius muscles (white and red portion) were collected immediately after and 24 h post exercise. For 3 days, imipramine was intraperitoneally injected 1 h prior to treadmill running.[Results]Interleukin 6 (IL-6) and serum creatine kinase (CK) levels were enhanced immediately after and 24 h post exercise (relative to those of resting), respectively. Acidic sphingomyelinase (A-SMase) protein expression in gastrocnemius muscles was significantly augmented by exercise, unlike, serine palmitoyltransferase-1 (SPT-1) and neutral sphingomyelinase (N-SMase) expressions. Furthermore, imipramine (a selective A-SMase inhibitor) treatment reduced the exercise-induced CK and IL-6 elevations, along with a decrease in cleaved caspase-3 (Cas-3) of gastrocnemius muscles.[Conclusion]We found the crucial role of A-SMase in exercise-induced muscle damage.

Contribution of Ceramide Signaling to Activation of the mTORC1 Pathway and Calcification Nidus Formation in Coronary Arterial Smooth Muscle Cells

The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LE)/lysosomes may control cargo selection, membrane biogenesis, exosome secretion and lysosome trafficking, which is fine controlled by lysosomal sphingolipids such as ceramide. This lysosomal sphingolipid‐regulated mTOR signaling may be a crucial molecular mechanism responsible for development of the arterial medial calcification (AMC) given the crucial role of exosome secretion from vascular smooth muscle cells (SMCs). In the present study, we used coronary artery SMCs (CASMCs) from Asah1fl/fl/SMcre mice with SMC‐specific deletion of lysosomal acid ceramidase (mouse gene code: Asah1) to demonstrate the role of lysosomal ceramide in mTOR signaling and arterial calcification. First, we demonstrated that mTORC1, a 289 kD cytoplasm protein is enriched in CASMCs by Western blot analysis and treatment of CASMCs isolated from Asah1fl/fl/SMcre mice with high Pi increased more calcium deposition compared to WT/WT cells as shown by the presence of Alizarin Red‐stained nodules. This calcium deposition was significantly decreased by specific mTOR inhibitor (Torin‐1) and by acid sphingomyelinase (Asm) inhibitor (Imipramine). Correspondingly, Asah1 gene deletion induced phenotypic change in Pi‐treated CASMCs, as depicted by decreased SM22‐α (SMC marker) expression in Western blot analysis, which was reversed by Torin‐1 or Imipramine. Using confocal microscopy, we further found that Pi markedly increased colocalization of mTORC1 with Lamp‐1, a lysosome targeting of mTORC1, but decreased colocalization of VPS16 with Lamp‐1, an indicative of multivesicular bodies (MVBs) interaction with lysosomes in CASMCs from Asah1fl/fl/SMcre mice, suggesting that activation of mTOR signaling inhibits lysosome trafficking or fusion with MVBs if AC was deficient. However, mTOR and Asm inhibition produced opposite effects on lysosome trafficking and interaction with MVBs compared to CASMCs treated with AC deficiency, further suggesting the role of ceramide‐mTOR signaling in lysosome trafficking or interactions with MVBs. Using nanoparticle tracking analysis, Pi‐induced excretion of exosomes (100–150 nm in size) were found to be significantly increased in CASMCs of Asah1fl/fl/SMcre mice compared to CASMCs from their control littermates. These results suggest that lysosomal ceramide‐mTOR signaling critically controls MVB fate and exosome release and that AC deficiency may lead to increased mTOR activation, thereby increasing exosome release and leading to calcification nidus formation in CASMCs.Support or Funding Information(Supported by NIH grants HL057244, HL075316 and DK120491)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defining the roles of host lipids in flavivirus infection

The flavivirus family includes Zika virus (ZIKV), Dengue virus, West Nile virus, Japanese Encephalitis and Yellow Fever virus, each of which causes a major global health threat. During their long‐lasting co‐existence with their host, these viruses have developed sophisticated strategies to co‐opt thecellular machinery including, host lipid metabolism, to effectively establish infection. They enter the host cells through receptor‐mediated endocytosis. In endosomes, the acidic environment induces fusion between the virus and the host membrane resulting in viral genome release. The viral RNA is translated into a polyprotein, which is post‐translationally cleaved by cellular and viral proteases into three structural proteins including capsid, membrane, and envelope, and seven nonstructural proteins. During viral infection cell membranes are co‐opted for virtually every step of the viral life cycle: entry, replication, assembly and release. The role of host factors, especially that of lipids, in pathogenesis of flaviviruses is largely unknown, and there exists a gap in understanding the basic biology of these viruses. This is especially true for ZIKV, which until recently was one of the most understudied flaviviruses. Because lipids are an important interface in host‐virus interactions, studying these biomolecules may provide strategies to combat diseases caused by these pathogens. Zika virus (ZIKV) is a positive‐sense RNA virus that remodels host membrane to establish sites of replication and assembly. To determine how ZIKV modulates the host lipid repertoire in an unbiased manner, we performed comprehensive lipid profiling of human cells infected with ZIKV. We found that ZIKV replication triggers significant changes in the cellular sphingolipidomes especially that of ceramide and sphingomyelin highly enriched during infection. Disruption of sphingolipid biosynthesis with CRISPR/Cas9 gene‐editing or small molecule inhibitors effectively blocks ZIKV replication. Both sphingomyelin and ceramide redistribute to ZIKV replication sites, and the sphingomyelinase‐catalyzed degradation of sphingomyelin to ceramide is the critical pathway to establish infection effectively. Inhibition of sphingomyelinase attenuated ZIKV replication, while the accumulation of intracellular ceramide through knockout of sphingomyelin synthesis enhanced ZIKV infection. Thus, we identify a sphingolipid salvage pathway with a critical role in ZIKV replication and show that ceramide is a key mediator of ZIKV pathogenesis.Support or Funding InformationThis work was supported by the Collins Medical Trust and NIH grant R21AI133631. W.B.M. and J.B.W. were supported by the Medical Research Foundation of Oregon, the Oregon Clinical and Translational Research Institute, and the Sunlin and Priscilla Chou Foundation. D.K. was supported by the Summer Equity Internship Program of the OHSU Center for Diversity and Inclusion. E.B. was supported by NIH grants R01GM060170 and R01GM101983. E.A.D. and A.R.N. were supported by NIH grants R01DK105961, U19AI106754, and U54GM069338.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Involvement of exosomes in dopaminergic neurodegeneration by microglial activation in midbrain slice cultures

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopamine neurons in the substantia nigra. Microglial activation is frequently observed in the brains of patients with PD and animal models. Interferon-γ (IFN-γ)/lipopolysaccharide (LPS) treatment triggers microglial activation and the reduction of dopamine neurons in midbrain slice cultures. We have previously reported that nitric oxide (NO) is mainly involved in this dopaminergic degeneration. However, this degeneration was not completely suppressed by the inhibition of NO synthesis, suggesting that factors other than NO also contribute to dopaminergic neurodegeneration. Exosomes are extracellular vesicles with diameters of 40–200 nm that contain various proteins and micro RNAs and are regarded as a novel factor that mediates cell-to-cell interactions. Previous studies have demonstrated that exosome release is enhanced by microglial stimulation and that microglia-derived exosomes increases neuronal apoptosis. In the present study, we investigated whether exosomes are involved in dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. IFN-γ/LPS treatment to the midbrain slice cultures activated microglia, increased exosomal release, and decreased dopamine neurons. GW4869, an inhibitor of a neutral sphingomyelinase 2, decreased exosomal release and significantly prevented dopaminergic neurodegeneration by IFN-γ/LPS without affecting NO production. In contrast, D609, an inhibitor of sphingomyelin synthase and NO synthase, did not affect dopaminergic neurodegeneration, although it strongly inhibited NO production. The protective effect mediated by inhibition of NO synthase would be counteracted by enhanced exosomal release caused by D609 treatment. In addition, dopaminergic neurodegeneration is triggered by the treatment of exosomes isolated from culture media of IFN-γ/LPS-treated slices. These results suggest that exosomes are involved in dopaminergic neurodegeneration by microglial activation.

Lysosomal regulation of extracellular vesicle excretion during d-ribose-induced NLRP3 inflammasome activation in podocytes

The NLRP3 inflammasome is activated in the cytoplasm of cells and its products such as IL-1β are exported through a non-classical ER-Golgi pathway. Several mechanistically distinct models including exocytosis of secretory lysosomes, microvesicles (MVs) and extracellular vehicles (EVs) have been proposed for their release. In this study, we hypothesized that the NLRP3 inflammasome product, IL-1β in response to exogenously administrated and endogenously produced d-ribose stimulation is released via extracellular vesicles including EVs via a sphingolipid-mediated molecular mechanisms controlling lysosome and multivesicular body (MVB) interaction. First, we demonstrated that both endogenous and exogenous d-ribose induced NLRP3 inflammasome activation to produce IL-1β, which was released via EVs in podocytes. Then, we found that colocalization of marker MVB marker VPS16 with IL-1β within podocytes increased upon d-ribose stimulation, which was accompanied by decreased colocalization of lysosome marker Lamp-1 and VPS16, suggesting decrease in MVB inclusion of IL-1β due to reduced lysosome and MVB interaction. All these changes were mimicked and accelerated by lysosome v-ATPase inhibitor, bafilomycin. Moreover, ceramide in podocytes was found elevated upon d-ribose stimulation, and prior treatments of podocyte with acid sphingomyelinase (Asm) inhibitor, amitriptyline, acid ceramidase (AC) inducer, genistein, or AC CRISPR/cas9 activation plasmids were found to decrease d-ribose-induced ceramide accumulation, EVs release and IL-1β secretion due to reduced interactions of lysosome with MVBs. These results suggest that inflammasome-derived products such as IL-1β during d-ribose stimulation are released via EVs, in which lysosomal sphingolipid-mediated regulation of lysosome function plays an important role.

Exosomes derived from SDF1‐overexpressing mesenchymal stem cells inhibit ischemic myocardial cell apoptosis and promote cardiac endothelial microvascular regeneration in mice with myocardial infarction

Exosomes extracted from mesenchymal stem cells (MSCs) was reported to reduce myocardial ischemia/reperfusion damage. Besides, stromal-derived factor 1 (SDF1a) functions as cardiac repair after myocardial infarction (MI). Therefore, the present study aims to identify whether exosomes (Exo) released from SDF1-overexpressing MSCs display a beneficial effect on ischemic myocardial infarction. Initially, a gain-of-function study was performed to investigate the function of SDF1 in ischemic myocardial cells and cardiac endothelial cells. Coculture experiments were performed to measure potential exosomic transfer of SDF1 from MSCs to ischemic myocardial cells and cardiac endothelial cells. During the coculture experiments, exosome secretion was disrupted by neutral sphingomyelinase inhibitor GW4869 and upregulated exosomal SDF1 using SDF1 plasmid. Effects of Exo-SDF1 on cardiac function in MI mice were investigated in vivo. MSCs suppressed myocardial cell apoptosis and promoted microvascular regeneration of endothelial cells through secretion of exosomes. The addition of GW4869 led to increased apoptotic capacity of myocardial cells, decreased microvascular formation ability of endothelial cells, enhanced autophagy ability, and elevated Beclin-1 level as well as ratio of LC3II/LC3I. Overexpression of SDF1 and Exo-SDF1 inhibited apoptosis and autophagy of myocardial cells, but promoted tube formation of endothelial cells. The interference of PI3K signaling pathway promoted apoptosis and autophagy of myocardial cells, but inhibited tube formation of endothelial cells. SDF1 activated the PI3K signaling pathway. Exo-SDF1 protected cardiac function of MI mice and inhibited myocardial tissue damage. This study provided evidence that SDF1 overexpression in MSCs-derived exosomes inhibited autophagy of ischemic myocardial cells and promoted microvascular production of endothelial cells.

Gemcitabine Induces Microvesicle Particle Release in a Platelet-Activating Factor-Receptor-Dependent Manner via Modulation of the MAPK Pathway in Pancreatic Cancer Cells.

Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.

Neutral Sphingomyelinase Inhibition Alleviates LPS-Induced Microglia Activation and Neuroinflammation after Experimental Traumatic Brain Injury.

Neuroinflammation is one of the key secondary injury mechanisms triggered by traumatic brain injury (TBI). Microglial activation, a hallmark of brain neuroinflammation, plays a critical role in regulating immune responses after TBI and contributes to progressive neurodegeneration and neurologic deficits following brain trauma. Here we evaluated the role of neutral sphingomyelinase (nSMase) in microglial activation by examining the effects of the nSMase inhibitors altenusin and GW4869 in vitro (using BV2 microglia cells and primary microglia), as well as in a controlled cortical injury (CCI) model in adult male C57BL/6 mice. Pretreatment of altenusin or GW4869 prior to lipopolysaccharide (LPS) stimulation for 4 or 24 hours, significantly downregulated gene expression of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, iNOS, and CCL2 in microglia and reduced the release of nitric oxide and TNF-α These nSMase inhibitors also attenuated the release of microparticles and phosphorylation of p38 MAPK and ERK1/2. In addition, altenusin pretreatment also reduced the gene expression of multiple inflammatory markers associated with microglial activation after experimental TBI, including TNF-α, IL-1β, IL-6, iNOS, CCL2, CD68, NOX2, and p22phox Overall, our data demonstrate that nSMase inhibitors attenuate multiple inflammatory pathways associated with microglial activation in vitro and after experimental TBI. Thus, nSMase inhibitors may represent promising therapeutics agents targeting neuroinflammation.

Exosomes Play a Key Role in Multiple Myeloma Bone Disease and Tumor Development

Multiple Myeloma (MM) is an often incurable plasma cell proliferative disorder, representing about 10% of all hematologic malignancies. Osteolysis is one of the hallmarks of this malignancy, meaning that almost every patient will manifest with an osteolytic lesion during their disease course, resulting in increased morbidity and pain with ultimately a severe impact on the quality of life.The development and progression of MM is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles (EVs) takes place. This crosstalk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease.Here we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. This syngeneic murine model highly mimics human MM disease and presents with typical MM characteristics such as osteolysis, angiogenesis and a serum M-spike.We first investigated the effects of 5TGM1 small EVs or exosomes, sized 50-120nm in diameter, on osteoclasts and osteoblasts in vitro. 5TGM1 exosomes were able to enhance the resorptive active of osteoclasts. In contrast, these exosomes induced apoptosis in pre-osteoblasts while also blocking their differentiation to mature osteoblasts. RT-PCR showed a downregulation of Runx2, Osterix and Collagen 1A1 expression, while DKK-1 expression was upregulated. Mechanistically, we confirmed the presence of DKK-1 on the 5TGM1 exosomes, which led to a downregulation of the Wnt pathway in osteoblasts.In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. We injected C57BL/KalwRij intravenously during three weeks with 5TGM1 exosomes, and analyzed the femurs by micro-computed tomography. These mice had a significantly lower trabecular bone volume, as the result of a lower trabecular number combined with a higher trabecular separation.Next, we inhibited exosome secretion in 5TGM1-inoculated mice to prevent bone loss and increase sensitivity to bortezomib. For blocking exosome secretion we used the neutral sphingomyelinase inhibitor GW4869. This increased cortical bone volume and decreased bone resorption markers (C-terminal telopeptide of collagen type I) without significantly affecting tumor load. This indicates that inhibiting exosome secretion in the MM microenvironment has a protective effect on myeloma bone disease, not associated with effects on tumor load. Furthermore, blocking exosome secretion by GW4869 also sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response in vivo when GW4869 and bortezomib were combined.Altogether, our results indicate a key role for exosomes in the myeloma BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy. DisclosuresNo relevant conflicts of interest to declare.

Tricyclic antidepressant amitriptyline inhibits autophagic flux and prevents tube formation in vascular endothelial cells.

Amitriptyline is a tricyclic antidepressant and an inhibitor of lysosomal acid sphingomyelinase (ASM). Amitriptyline is well known for its cardiovascular side effects and toxicity in psychiatric patients. However, the mechanisms underlying the cardiovascular side effects of amitriptyline remain largely undefined. This study aimed to determine the effects of amitriptyline on angiogenic capability of vascular endothelial cells in physiological settings and identify its mechanism of action. The exvivo aortic ring angiogenesis and invitro-cultured endothelial cell tube formation assay were used to assess the effects of amitriptyline on endothelial angiogenic capability. It was demonstrated that amitriptyline impaired the angiogenesis of aortic rings, which was similar to that found in aortic rings with haploinsufficiency of the ASM gene. In cultured mouse microvascular endothelial cells (MVECs), amitriptyline impaired the proliferation and tube formation under basal condition, which were accompanied by attenuated angiogenic signalling pathways such as endothelial nitric oxide synthase, Akt and Erk1/2 pathways. Mechanistically, amitriptyline inhibited autophagic flux without affecting autophagosome biogenesis at basal condition. ASM gene silencing or autophagy inhibition mimics the inhibitory effects of amitriptyline on endothelial cell proliferation and tube formation. Collectively, our data suggest that amitriptyline inhibits endothelial cell proliferation and angiogenesis via blockade of ASM-autophagic flux axis. It is implicated that the cardiovascular side effects of amitriptyline may be associated with its inhibitory action on physiological angiogenesis.

Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts

Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or ‘exosomes’, not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy.

Adipose tissue browning in cancer-associated cachexia can be attenuated by inhibition of exosome generation

Cancer-associated cachexia (CAC) is a disorder characterized by unintended weight loss due to skeletal muscle wasting and adipose tissue loss. Although muscle atrophy in this condition has been well studied, the mechanisms underlying adipose tissue loss, which include browning, have not been investigated in detail. In this respect, though recent studies have shown that exosomes from cancer cells can promote lipolysis, the link between exosomes from cancer cells and CAC has not been clearly established. In this study, we investigate if exosomes from Lewis lung carcinoma (LLC) cells can induce lipolysis in vitro (in 3T3-L1 adipocytes) and in vivo (in LLC tumor-bearing mice). We find that exosomes from LLC cells do induce lipolysis in 3T3-L1 adipocytes and that the white adipose tissues of mice with LLC tumors show clear signs of lipolysis. We also find that this lipolysis can be inhibited using the neutral sphingomyelinase inhibitor GW4869. Our results indicate that GW4869 not only inhibits exosome generation and release from LLC cells, but can also inhibit lipolysis induced by LLC-derived exosomes in 3T3-L1 adipocytes. Furthermore, we also demonstrate that LLC tumor-bearing mice treated with GW4869 did not develop CAC. In summary, our results suggest that inhibiting exosome generation and release can inhibit lipolysis and adipose tissue browning, and may be useful as a novel strategy for treating CAC.