Neutral Sphingomyelinase Inhibitor Research Articles

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

Chronic nSMase2 Inhibition Suppresses Neuronal Exosome Spreading In Vivo & Induces Sex‐Specific Attenuation of AD Pathology in APPNL‐F/NL‐F Knock‐In Mice

AbstractBackgroundAltered ceramide metabolism is implicated in Alzheimer’s disease (AD) progression. Although prior work shows that inhibition of neutral sphingomyelinase (nSMase) 2, a key enzyme responsible for hydrolysis of sphingomyelin to ceramide, reduces exosome secretion, attenuates Aβ pathology, and improves cognitive function, these studies have been limited to in vitro application or short‐term treatments in fast‐progressing models.MethodWe employed the slow‐progressing APPNL‐F/NL‐F knock‐in (APP KI) AD mouse model and cell type‐specific exosome reporter CD63‐GFPf/f mice to investigate the impact of chronic long‐term nSmase2 inhibition on in vivo neuron‐derived exosome dynamics, region‐specific Aβ plaque load, and cognitive function. Male and female APPNL‐F/NL‐F‐CD63‐GFPf/+ (APP KI‐CD63) mice were treated daily with the nSmase2 inhibitor, GW4869 (GW; 1.25mg/kg BW; i.p.), or vehicle (VEH, DMSO in saline), from 8 months old (mo.) to 13mo. and from 10 mo. to 18mo. We also analyzed ceramide levels in both APP KI and WT mice. Brain‐derived exosomes of 14mo. APP KI females versus males and age‐ and sex‐matched WT mice were also isolated and quantified.ResultWe found significant elevations in multiple ceramide species from somatosensory cortex (SC) samples of 14mo. APP KI mice compared to age‐ and sex‐matched WT controls. We also found a sex‐specific increase in total exosomes in 14mo. APP KI females versus males that was absent in WT mice. Chronic inhibition of nSMase2 by GW4869 leads to significantly suppression of neuronal exosome spreading. Non‐linear regression analyses of size distribution curves showed consistent shifts to smaller plaque sizes in GW‐treated APP KI females, which were accompanied by decreased plaque numbers in each region examined. Conversely, plaque load was largely unaffected by GW treatment in APP KI males. T maze trials to examine spatial working memory revealed a female‐specific reduction in spontaneous alternation rate in APP KI vs. WT mice, which was significantly reversed with GW treatment.ConclusionOur results demonstrate that chronic nSMase2 inhibition suppresses neuronal exosome spreading in APPNL‐F/NL‐F knock‐in mice and preferentially attenuates Aβ pathology and cognitive dysfunction in females, pointing to a strong contribution of sex‐specific alterations in ceramide metabolism and exosome dynamics to the progression of AD‐like pathology in this model.

Exosomal miR-331-3p derived from chemoresistant osteosarcoma cells induces chemoresistance through autophagy

BackgroundOsteosarcoma is a common malignant bone tumor, and chemotherapy can effectively improve the prognosis. MicroRNA-331 (MiR-331) is associated with poor cancer outcomes. However, the role of miR-331 in osteosarcoma remains to be explored.MethodsDrug-resistant osteosarcoma cells were cultured, and their exosomes were purified. The secretion and uptake of exosomes by drug-resistant osteosarcoma and osteosarcoma cells were confirmed using a fluorescence tracking assay and Transwell experiments. The effects of drug-resistant exosomes on cell proliferation were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. siRNA-Drosha and neutral sphingomyelinase inhibitor GW4869 were used to determine the transfer of miRNAs. qRT-PCR and western blotting were used to detect the role of autophagy in the regulation of drug-resistant cell-derived exosomal miR-331-3p.ResultsExosomal miR-331-3p levels in drug-resistant cells were higher than in exosomes from osteosarcoma cells. The exosomes secreted by the drug-resistant osteosarcoma cells could be absorbed by osteosarcoma cells, leading to acquired drug resistance in previously non-resistance cells. Inhibition of miRNAs resulted in reduced transmission of drug resistance transmission by exosomes. Exosomes from drug-resistant osteosarcoma cells transfected with siRNA-Drosha or treated by GW4869 could not enhance the proliferation of MG63 and HOS cells. Finally, miR-331-3p in the exosomes secreted by drug-resistant osteosarcoma cells could induce autophagy of osteosarcoma cells, allowing them to acquire drug resistance. The inhibition of miR-331-3p decreased drug resistance of osteosarcoma cells.ConclusionExosomes secreted from chemoresistant osteosarcoma cells promote drug resistance through miR-331-3p and autophagy. Inhibition of miR-331-3p could be used to alleviate drug resistance in osteosarcoma.

Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

BackgroundCell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC).MethodsHere, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport.ResultsFollowing twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus.ConclusionOverall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs.8WXtUmcgzqdrdTPfGd7tbXVideo

Neutral sphingomyelinase blockade enhances hematopoietic stem cell fitness through an integrated stress response

AbstractHematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2–dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

Inhibition of neutral sphingomyelinase 2 impairs HIV-1 envelope formation and substantially delays or eliminates viral rebound

Although HIV-1 Gag is known to drive viral assembly and budding, the precise mechanisms by which the lipid composition of the plasma membrane is remodeled during assembly are incompletely understood. Here, we provide evidence that the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) interacts with HIV-1 Gag and through the hydrolysis of sphingomyelin creates ceramide that is necessary for proper formation of the viral envelope and viral maturation. Inhibition or depletion of nSMase2 resulted in the production of noninfectious HIV-1 virions with incomplete Gag lattices lacking condensed conical cores. Inhibition of nSMase2 in HIV-1-infected humanized mouse models with a potent and selective inhibitor of nSMase2 termed PDDC [phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl) pyrrolidin-3-yl)-carbamate] produced a linear reduction in levels of HIV-1 in plasma. If undetectable plasma levels of HIV-1 were achieved with PDDC treatment, viral rebound did not occur for up to 4 wk when PDDC was discontinued. In vivo and tissue culture results suggest that PDDC selectively kills cells with actively replicating HIV-1. Collectively, this work demonstrates that nSMase2 is a critical regulator of HIV-1 replication and suggests that nSMase2 could be an important therapeutic target with the potential to kill HIV-1-infected cells.

Chronic nSMase inhibition suppresses neuronal exosome spreading and sex-specifically attenuates amyloid pathology in APP knock-in Alzheimer's disease mice

Female biased pathology and cognitive decline in Alzheimer's disease (AD) have been consistently observed with unclear underlying mechanisms. Although brain sphingolipid ceramide is elevated in AD patients, whether and how ceramide may contribute to sex-specific differences in amyloid pathology is unknown. Here we investigated the sex-specific impact of chronic pharmacological inhibition of neutral sphingomyelinase (nSMase), a key enzyme responsible for ceramide metabolism, on in vivo neuron-derived exosome dynamics, Aβ plaque load, and cognitive function in the APPNL-F/NL-F knock-in (APP NL-F) AD mouse model. Our results found sex-specific increase of cortical C20:0 ceramide and brain exosome levels only in APP NL-F but not in age-matched WT mice. Although nSMase inhibition similarly blocks exosome spreading in male and female mice, significantly reduced amyloid pathology was mostly observed in cortex and hippocampus of female APP NL-F mice with only modest effect found on male APP NL-F mice. Consistently, T maze test to examine spatial working memory revealed a female-specific reduction in spontaneous alternation rate in APP NL-F mice, which was fully reversed with chronic nSMase inhibition. Together, our results suggest that disease induced changes in ceramide and exosome pathways contribute to the progression of female-specific amyloid pathology in APP NL-F AD models.

1638-P: Oxidized Lipids Cause Ceramide Accumulation and Insulin Resistance in Skeletal Muscle

Accumulation of muscle lipids promotes insulin resistance and type 2 diabetes (T2D). Ceramides are important muscle lipids that decrease insulin sensitivity and it was previously shown that accumulation of ceramides in the sarcolemma negatively impacts insulin sensitivity in humans. The mechanisms explaining accumulation of lipids in specific compartments in skeletal muscle are unknown, but one possibility is through circulating oxidized lipids. POVPC, a major circulating oxidized phospholipid, was exposed to rat myotubes (1-50 μM) for 4h and the total cellular ceramide levels in myotubes measured by LC-MS/MS. Total ceramide content increased dose dependently at POVPC doses in the physiological range (5 μM, p <0.05). To determine the mechanism of POVPC induced ceramide accumulation, ceramide was measured in myotubes after treatment with POVPC (25 μM) in the presence of inhibitors of neutral and acid sphingomyelinase (GW4869 and ARC39), ceramide synthase (fumonisin B1) and serine palmitoyltransferase (myriocin). The individually administered inhibitors in the presence of POVPC each showed a decrease (41-55%) in ceramide accumulation. When all of inhibitors were administered together along with POVPC, an almost complete inhibition of ceramide accumulation (95%) was observed indicating that the sphingomyelinase, de novo, and salvage pathways of ceramide synthesis are activated by POVPC. The role of oxidized lipids on insulin sensitivity was examined after treatment of rat myotubes with 1-25 μM POVPC. Increasing doses of POVPC at doses as low as 1 μM significantly decreased insulin sensitivity in vitro (p <0.05). These results reveal circulating oxidized lipids promote ceramide accumulation and insulin resistance in muscle cells and may help explain variability in total and localized muscle ceramide levels between individuals. These data also suggest that therapies designed to lower circulating oxidized lipids could be beneficial for the treatment and prevention of T2D. Disclosure K.Zemski berry: None. B.C.Bergman: Research Support; Eli Lilly and Company. Funding National Institutes of Health (P30DK048520); University of Colorado (25N0350)

Photosensitive small extracellular vesicles regulate the immune microenvironment of triple negative breast cancer

Currently, the treatment of triple-negative breast cancer (TNBC) is limited by the special pathological characteristics of this disease. In recent years, photodynamic therapy (PDT) has created new hope for the treatment of TNBC. Moreover, PDT can induce immunogenic cell death (ICD) and improve tumor immunogenicity. However, even though PDT can improve the immunogenicity of TNBC, the inhibitory immune microenvironment of TNBC still weakens the antitumor immune response. Therefore, we used the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by TNBC cells to improve the tumor immune microenvironment and enhance antitumor immunity. In addition, bone mesenchymal stem cell (BMSC)-derived sEVs have good biological safety and a strong drug loading capacity, which can effectively improve the efficiency of drug delivery. In this study, we first obtained primary BMSCs and sEVs, and then the photosensitizers Ce6 and GW4869 were loaded into the sEVs by electroporation to produce immunomodulatory photosensitive nanovesicles (Ce6-GW4869/sEVs). When administered to TNBC cells or orthotopic TNBC models, these photosensitive sEVs could specifically target TNBC and improve the tumor immune microenvironment. Moreover, PDT combined with GW4869-based therapy showed a potent synergistic antitumor effect mediated by direct killing of TNBC and activation of antitumor immunity. Here, we designed photosensitive sEVs that could target TNBC and regulate the tumor immune microenvironment, providing a potential approach for improving the effectiveness of TNBC treatment. Statement of SignificanceWe designed an immunomodulatory photosensitive nanovesicle (Ce6-GW4869/sEVs) with the photosensitizer Ce6 to achieve photodynamic therapy and the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by triple-negative breast cancer (TNBC) cells to improve the tumor immune microenvironment and enhance antitumor immunity. In this study, the immunomodulatory photosensitive nanovesicle could target TNBC cells and regulate the tumor immune microenvironment, thus providing a potential approach for improving the treatment effect in TNBC. We found that the reduction in tumor sEVs secretion induced by GW4869 improved the tumor-suppressive immune microenvironment.Moreover, similar therapeutic strategies can also be applied in other kinds of tumors, especially immunosuppressive tumors, which is of great value for the clinical translation of tumor immunotherapy.

Elevated Estrogen Induces Senescence in Male Endothelial Cells Via Neutral Sphingomyelinase-Mediated Ceramide Formation

Prevalence of cardiovascular disease is higher among trans-females undergoing estrogen therapy compared to cis-females and males. Our previous work has shown that chronic exposure to elevated estrogen induces microvascular endothelial dysfunction in arterioles collected from healthy biological males, however the mechanism contributing to dysfunction is unknown. Conflicting evidence has shown that estrogen can both promote or prevent cell senescence, a phenotypic change characterized by replicative arrest and endothelial dysfunction. Since estrogen is capable of activating neutral sphingomyelinase (NSmase), an enzyme that increases cellular levels of the pro-senescent sphingolipid ceramide, we hypothesized that elevated estrogen induces endothelial cell senescence in biological males through the activation of NSmase. To study this, primary male human umbilical vein endothelial cells (HUVECs) were treated with increasing doses of 17-beta estradiol (E2; 0.5nM to 100nM) for 24hrs with or without a neutral sphingomyelinase inhibitor (GW4869 5μM). Cells were also exposed to C2-ceramide (5μM) or inactive dihidro-C2-ceramide (5μM) and maintained for 48hrs in 1% serum EBM-2 media prior to beta-galactosidase staining (biomarker of senescence). Treatment groups were imaged using 20x bright-field microscopy. Total number of positive beta-galactosidase-stained cells were counted and normalized to total number of cells per image field (mean%±SEM reported; n=3 patients for all); counters were blinded to treatment group. One-Way ANOVA with alpha<0.05 was used to compare between groups. Male HUVECs exhibited no change in percent positive cells with 0.5nM E2 treatment (25±11.4% vs 21.5±2.4% vehicle control), however had significantly higher percentage of positive cells when exposed to higher doses of E2 (1nM, 45.±11.5%; 50nM, 48.1±6.1%; 100nM, 45.5±2.6%; p<0.05 all). Treatment with ceramide (40.44±7.8% vs 11.2±4.3% control, p<0.05) but not dihydroceramide (14.89±12.1%) increased percentage of positive cells. Inhibition of neutral sphingomyelinase alone did not influence percent positivity (12.4±2.7% vs 11.2±4.3% control), however in comparison to 100nM E2 treatment, those treated with GW4869 and 100nM E2 exhibited a lower percentage of positive cells (45.5±2.6% 100nM E2 vs 19.22±7.0% E2+GW4869, p<0.05). These results suggest that elevated estrogen may cause an increase in endothelial cell senescence among biological males through the upregulation of neutral sphingomyelinase-mediated ceramide formation. Our work adds mechanistic insight by which estrogen may promote endothelial dysfunction among trans-females undergoing estrogen therapy and proposes the inhibition of NSmase-mediated ceramide formation as a novel therapeutic avenue for reducing CVD risk associated with gender transition. This work is supported by National Institutes of Health (NHLBI) K08 HL141562 (JKF) and American Heart Association Predoctoral Fellowship 909315 (GSK). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanisms of pulmonary microvascular endothelial cells barrier dysfunction induced by LPS: The roles of ceramides and the Txnip/NLRP3 inflammasome.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are characterized by pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction and proinflammatory cytokine influx into lung tissue, resulting in pulmonary oedema. Ceramide overproduction is an important mediator of pulmonary hyperinflammation and pulmonary oedema in Acute lung injury (ALI). Ceramides induce NLRP3 inflammasome activation are essential for the hyperinflammatory response. However, the roles and specific mechanisms of ceramide-induced NLRP3 inflammasome activation, proinflammatory cytokine manufacturing and PMVECs barrier dysfunction in ALI are unclear. Herein, pretreatment with the acid sphingomyelinase (ASMase) inhibitor imipramine (but not a neutral sphingomyelinase (NSMase) inhibitor or de novo pathway inhibitor) significantly inhibited ceramide early production in rats with lipopolysaccharide (LPS)-induced ALI; Furthermore, the Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury were significantly decreased. Verapamil, a Txnip inhibitor, substantially inhibited Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury in rats with C8-ceramide-induced ALI. In vitro, short hairpin RNA-mediated Txnip silencing significantly inhibited C8-ceramide-induced Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages (AMs) and early secretion of the proinflammatory cytokines IL-1β (4-12h) as well as IL-6 and TNF-α at subsequent times (later than 12h). However, C8-ceramide significantly increased the early secretion (within 8h) of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in a co-culture model of NR8383 AMs and PMVECs, and Txnip silencing of NR8383 AMs inhibited the secretion of pro-inflammatory cytokines and reduced cytoskeletal rearrangements, intercellular connection breakage and hyperpermeability in PMVECs. Overall, our results suggest that in LPS-induced ALI, ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 AMs leads to early IL-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI.

Virtual screening reveals aprepitant to be a potent inhibitor of neutral sphingomyelinase 2: implications in blockade of exosome release in cancer therapy.

Exosomes are membrane-derived nano-vesicles upregulated in pathological conditions like cancer. Therefore, inhibiting their release is a potential strategy for the development of more efficient combination therapies. Neutral sphingomyelinase 2 (nSMase2) is a key component in exosome release; however, a clinically safe yet efficient nSMase2 inhibitor remains to be used discovered. Accordingly, we made an effort to identify potential nSMase2 inhibitor(s) among the approved drugs. Virtual screening was performed and aprepitant was selected for further investigation. To evaluate the reliability of the complex, molecular dynamics were performed. Finally, using the CCK-8 assay in HCT116 cells, the highest non-toxic concentrations of aprepitant were identified and the nSMase2 activity assay was performed to measure the inhibitory activity of aprepitant, in vitro. To validate the screening results, molecular docking was performed, and the retrieved scores were in line with the screening results. The root-mean-square deviation (RMSD) plot of aprepitant-nSMase2 showed proper convergence. Following treatment with different concentrations of aprepitant in both cell-free and cell-dependent assays, nSMase2 activity was remarkably decreased. Aprepitant, at a concentration as low as 15µM, was able to inhibit nSmase2 activity in HCT116 cells without any significant effects on their viability. Aprepitant is therefore suggested to be a potentially safe exosome release inhibitor.

Ceramide-dependent trafficking of Epstein-Barr virus LMP1 to small extracellular vesicles

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with a multitude of cancers. The primary EBV oncogene latent membrane protein 1 (LMP1) is secreted from infected cancer cells in small extracellular vesicles (EVs). Additionally, the tetraspanin protein CD63 forms a complex with LMP1 and CD63 can be trafficked to EVs through a ceramide-dependent manner. Therefore, we hypothesize that ceramide is required for efficient packaging of LMP1 into small EVs. Following treatment with the neutral sphingomyelinase inhibitor GW4869, LMP1 cellular localization was disrupted and immunoblotting of EV lysates revealed a significant reduction in extracellular LMP1. NTA of EVs from the LCLs treated with GW4869 demonstrated a significant decrease in particle secretion. Additionally, ceramide inhibition resulted in enhanced LMP1-mediated NFkB activation in EV producing cells. Taken together, these data reveal a critical role for the lipid ceramide in LMP1 exosomal trafficking and the oncogenic signaling properties of the viral protein.

Engineered cardiac tissue as a novel in vitro human myocardium model for studying cardiac stretch.

Heart disease is the largest contributor to death worldwide and research is limited by a lack of adequate human tissue in vitro models. To address this, we developed a protocol for generating 3D, atrial-like engineered cardiac tissue (ECT) comprised of cardiomyocytes (CM) and cardiac fibroblasts, derived from human-induced pluripotent stem cells (hiPSC). Control and atrial-like hiPSC-CM were produced using GiWi differentiation supplemented without and with 0.75 µM retinoic acid, respectively. ECTs were generated using hiPSC-cardiac fibroblasts and Day 30 hiPSC-CM combined in a fibrin matrix and molded via a FlexCell Tissue TrainTM system. ECTs were cultured for 30 days and evaluated for action potentials (AP) as well as mRNA and protein expression profiles. A subset of control ECTs were cyclically and incrementally stretched for 7 days up to 18% elongation. Stretch-induced changes were evaluated via transmission electron microscopy (TEM) and cell culture medium (LC-MS/MS). Atrial-like ECTs recapitulated various atrial phenotypes. They exhibit an atrial-like electrophysiology with a shorter AP duration and increased repolarization fraction (p<0.05). Atrial-like ECT show atrial-specific mRNA/protein expression, such as lower ventricular-like MYL2 and higher atrial-like MYL7 levels (p<0.05). They also demonstrate distinct contraction mechanics with faster kinetics (p<0.05) and decreased normalized contraction force, compared to control ECT. Cyclic stretch reduced cardiomyocyte membrane convolution index (estimating membrane tension) and caveolae membrane structure density (p<0.05). The latter was rescued via neutral sphingomyelinase inhibitor GW4869 (20 µM, p<0.05). LC-MS/MS of stretch-conditioned culture medium revealed decreases in relative sphingomyelins and phosphatidylcholines (p<0.05), ostensibly indicating membrane damage which was reversed by GW4869. Overall, our findings demonstrate retinoic acid-induced atrial-like phenotypes in hiPSC-CM are sustained in ECT form. We also introduce a novel ECT stretch protocol that recapitulates changes observed in animal models of heart pressure overload.

Inhibition of Neutral Sphingomyelinase 2 by Novel Small Molecule Inhibitors Results in Decreased Release of Extracellular Vesicles by Vascular Smooth Muscle Cells and Attenuated Calcification.

Vascular calcification (VC) is an important contributor and prognostic factor in the pathogenesis of cardiovascular diseases. VC is an active process mediated by the release of extracellular vesicles by vascular smooth muscle cells (VSMCs), and the enzyme neutral sphingomyelinase 2 (nSMase2 or SMPD3) plays a key role. Upon activation, the enzyme catalyzes the hydrolysis of sphingomyelin, thereby generating ceramide and phosphocholine. This conversion mediates the release of exosomes, a type of extracellular vesicles (EVs), which ultimately forms the nidus for VC. nSMase2 therefore represents a drug target, the inhibition of which is thought to prevent or halt VC progression. In search of novel druglike small molecule inhibitors of nSMase2, we have used virtual ligand screening to identify potential ligands. From an in-silico collection of 48,6844 small druglike molecules, we selected 996 compounds after application of an in-house multi-step procedure combining different filtering and docking procedures. Selected compounds were functionally tested in vitro; from this, we identified 52 individual hit molecules that inhibited nSMase2 activity by more than 20% at a concentration of 150 µM. Further analysis showed that five compounds presented with IC50s lower than 2 µM. Of these, compounds ID 5728450 and ID 4011505 decreased human primary VSMC EV release and calcification in vitro. The hit molecules identified here represent new classes of nSMase2 inhibitors that may be developed into lead molecules for the therapeutic or prophylactic treatment of VC.

Blockade of Ceramide-Dependent Extracellular Vesicle Secretion Improves Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell (HSC) transplantation serves as a curative therapy for numerous benign and malignant hematopoietic diseases, and as a platform for HSC gene therapy. A main constraint is the limited quantity of HSCs collected from patients or donors and the inherent risk of graft failure, or oligocolonal reconstitution, especially after ex vivo manipulation. Current efforts to mitigate these limitations have predominantly focused on the discovery of extrinsic factors to maintain or expand hematopoietic stem and progenitor cells (HSPCs), whereas the role of the HSPC secretome in modulating niche function and improving occupancy have not been extensively studied. Recently, classes of nanometer-sized extracellular vesicles (EVs) have been shown to regulate tissue function by providing potent proangiogenic signals and remodeling cellular microenvironments. EV secretion additionally regulates homeostasis and stemness properties of HSPCs; however, in-depth studies into HSPC EV biogenesis and the impact on homeostatic and regenerative potency have been hindered by numerous technical challenges. Here, we utilize an optimized ex vivo expansion protocol for human and murine HSPCs to examine the impact of different EV biogenesis pathways on vesicle secretion. Our studies rapidly focused on ceramide-dependent vesicle secretion; blockade of ceramide biosynthesis with a widely used neutral sphingomyelinase inhibitor, GW4869 significantly reduced HSPC EV secretion. Treatment of HSPCs with GW4869 further inhibited mTOR signaling, altered autophagic flux, and decreased cell cycle entry. Increased ratios of lymphoid (MPP4):myeloid (MPP2/3) primed progenitors accumulated in culture. Upon competitive transplantation into myeloablated recipients, GW4869-exposed murine HSPCs demonstrated superior long-term engraftment potential (>8 steady-state fold change, p < 0.001), independent of bone marrow or splenic homing. Ceramide inhibition did not significantly alter long-term lineage output from transplanted HSPCs. Confirmatory genetic knockout (KO) of neutral sphingomyelinase (nSMase2) in murine and human HSPCs using Cas9 ribonucleoprotein complex delivery similarly demonstrated enhanced colony forming potential. To understand the impact of ceramide inhibition on vesicle cargo, comparative mass spectrometry was performed on HSPC nSMase2 KO EVs (EVnSmase2-KO). Nearly 900 proteins were depleted (fold change <2) in EVnSmase2-KO; these underrepresented proteins were noted to be involved in myeloid differentiation and activation, immune response, and exocytosis, suggesting vesicle modulation of these processes. These results suggest that ceramide inhibition not only leads to a reduction in EV release, but also impacts selective trafficking of EV cargo, ultimately altering HSPC function. While other EV biogenesis pathways have been demonstrated to promote HSC-self renewal, these findings implicate ceramide-dependent EV secretion as a novel negative regulator of HSPC repopulation. Transient disruption of ceramide-dependent EV secretion results in a more quiescent and less metabolically active HSPC pool, and additionally prevents bias toward a myeloid-primed progenitor pool seen with ex vivo expansion. These effects culminate in dramatically improved repopulation potential of HSPCs. Furthermore, unlike other small molecular inhibitors proposed to enhance engraftment of HSPCs, including rapamycin, no significant hinderance of ex vivo expansion was seen with GW4869 treatment. We therefore propose broadened exploration of ceramide inhibition for ex vivo maintenance of HSPCs prior to transplantation.

Allosteric Inhibition of Neutral Sphingomyelinase 2 (nSMase2) by DPTIP: From Antiflaviviral Activity to Deciphering Its Binding Site through In Silico Studies and Experimental Validation.

Flavivirus comprises globally emerging and re-emerging pathogens such as Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), among others. Although some vaccines are available, there is an unmet medical need as no effective antiviral treatment has been approved for flaviviral infections. The development of host-directed antivirals (HDAs) targeting host factors that are essential for viral replication cycle offers the opportunity for the development of broad-spectrum antivirals. In the case of flaviviruses, recent studies have revealed that neutral sphingomyelinase 2, (nSMase2), involved in lipid metabolism, plays a key role in WNV and ZIKV infection. As a proof of concept, we have determined the antiviral activity of the non-competitive nSMase2 inhibitor DPTIP against WNV and ZIKV virus. DPTIP showed potent antiviral activity with EC50 values of 0.26 µM and 1.56 µM for WNV and ZIKV, respectively. In order to unravel the allosteric binding site of DPTIP in nSMase2 and the details of the interaction, computational studies have been carried out. These studies have revealed that DPTIP could block the DK switch in nSMase2. Moreover, the analysis of the residues contributing to the binding identified His463 as a crucial residue. Interestingly, the inhibitory activity of DPTIP on the H463A mutant protein supported our hypothesis. Thus, an allosteric cavity in nSMase2 has been identified that can be exploited for the development of new inhibitors with anti-flaviviral activity.

Exosomes: Potential executors of IL‐35 gene‐modified adipose‐derived mesenchymal stem cells in inhibiting acute rejection after heart transplantation

Heart transplantation has become the only 'cure' for end-stage heart diseases, but acute allograft rejection is the major obstacle to the survival of patients. Our previous studies showed that IL-35 gene-modified adipose-derived mesenchymal stem cells (IL-35-ASCs) can effectively inhibit graft rejection and prolong the survival of transplanted hearts in mice. This study further explored the mechanism of IL-35-ASCs, especially focusing on the important role of IL-35-ASC-derived exosomes (IL-35-ASCexos) in inhibiting acute rejection. IL-35-ASCs were constructed in vitro and pretreated with IL-35 neutralizing antibody and GW4869 (an inhibitor of neutral sphingomyelinase that impairs exosome biogenesis/release). Then, pretreated IL-35-ASCs and CD4+ T cells were cocultured in Transwell plates, and changes in regulatory T cells (Tregs) and cytokines were detected. Then, IL-35-ASCexos were extracted, identified and analysed, and their immunoregulatory effects on CD4+ T cells were studied through coculture experiments. Finally, IL-35-ASCexos were applied to a mouse heart transplantation model to investigate the therapeutic effects on acute rejection of the allograft. The coculture experiment showed that the IL-35-neutralizing antibody could not completely block the immunosuppressive function of IL-35-ASCs, while GW4869 could effectively reduce their immunoregulatory characteristics. Similar to IL-35-ASCs, IL-35-ASCexos also have powerful immunosuppressive properties, effectively upregulating the Treg ratio in vivo and in vitro and prolonging graft survival. As the main effectors of IL-35-ASCs, these findings highlight the therapeutic potential of IL-35-ASCexos in inhibiting acute cardiac rejection of the allograft. Although the specific mechanism remains unclear and needs to be further explored, IL-35-ASCexos therapy is expected to become a new method to inhibit acute graft rejection.

The Pathogenic Sphingolipid Psychosine is Secreted in Extracellular Vesicles in the Brain of a Mouse Model of Krabbe Disease.

Psychosine exerts most of its toxic effects by altering membrane dynamics with increased shedding of extracellular vesicles (EVs). In this study, we discovered that a fraction of psychosine produced in the brain of the Twitcher mouse, a model for Krabbe disease, is associated with secreted EVs. We evaluated the effects of attenuating EV secretion in the Twitcher brain by depleting ceramide production with an inhibitor of neutral sphingomyelinase 2, GW4869. Twitcher mice treated with GW4869 had decreased overall EV levels, reduced EV-associated psychosine and unexpectedly, correlated with increased disease severity. Notably, characterization of well-established, neuroanatomic hallmarks of disease pathology, such as demyelination and inflammatory gliosis, remained essentially unaltered in the brains of GW4869-treated Twitcher mice compared to vehicle-treated Twitcher controls. Further analysis of Twitcher brain pathophysiology is required to understand the mechanism behind early-onset disease severity in GW4869-treated mice. The results herein demonstrate that some pathogenic lipids like psychosine may be secreted using EV pathways. Our results highlight the relevance of this secretory mechanism as a possible contributor to spreading pathogenic lipids in neurological lipidoses.

Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)‐(1‐(3‐(3,4‐dimethoxyphenyl)‐2,6‐dimethylimidazo[1,2‐b]pyridazin‐8‐yl)pyrrolidin‐3‐yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1β or PDDC. IL-1β injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.