Acid Ceramidase Gene Research Articles

Acid ceramidase gene therapy ameliorates pulmonary arterial hypertension with right heart dysfunction

BackgroundUp-regulation of ceramides in pulmonary hypertension (PH), contributing to perturbations in sphingolipid homeostasis and the transition of cells to a senescence state. We assessed the safety, feasibility, and efficiency of acid ceramidase gene transfer in a rodent PH model.MethodsA model of PH was established by the combination of left pneumonectomy and injection of Sugen toxin. Magnetic resonance imaging and right heart catheterization confirmed development of PH. Animals were subjected to intratracheal administration of synthetic adeno-associated viral vector (Anc80L65) carrying the acid ceramidase (Anc80L65.AC), an empty capsid vector, or saline. Therapeutic efficacy was evaluated 8 weeks after gene delivery.ResultsHemodynamic assessment 4 weeks after PH model the development demonstrated an increase in the mean pulmonary artery pressure to 30.4 ± 2.13 mmHg versus 10.4 ± 1.65 mmHg in sham (p < 0.001), which was consistent with the definition of PH. We documented a significant increase in pulmonary vascular resistance in the saline-treated (6.79 ± 0.85 mm Hg) and empty capsid (6.94 ± 0.47 mm Hg) groups, but not in animals receiving Anc80L65.AC (4.44 ± 0.71 mm Hg, p < 0.001). Morphometric analysis demonstrated an increase in medial wall thickness in control groups in comparison to those treated with acid ceramidase. After acid ceramidase gene delivery, a significant decrease of pro-inflammatory factors, interleukins, and senescence markers was observed.ConclusionGene delivery of acid ceramidase provided tropism to pulmonary tissue and ameliorated vascular remodeling with right ventricular dysfunction in pulmonary hypertension.Graphical

E70 A new mutation of uncertain significance in Farber disease

Abstract Introduction Farber disease is a rare autosomal recessive lysosomal storage disorder. It is caused by a mutation in the ASAH1 gene, which results in a deficiency of acid ceramidase and subsequently an accumulation of ceramide in various tissues. The manifestations are diverse and the cardinal symptoms of the Farber disease triad include subcutaneous nodules, joint pain and hoarseness. Aims To recall the clinical features of this rare disease in order to avoid a late diagnosis and to inform about the identification of a novel mutation in a Moroccan patient known until now as a mutation of uncertain significance in the acid ceramidase gene. Method About a clinical case reported retrospectively with a prospective follow-up. Results We report the case of a fourteen-year-old boy born from healthy, consanguineous Moroccan parents after an uneventful pregnancy and delivery. He had normal development until the age of 9 months. The first symptoms appeared at the end of the first year of life with joint stiffness, hoarseness, subcutaneous nodules, blepharitis resulting in amblyopia, dental enamel defect, gingival hyperplasia, and severe failure to thrive, without any visceral manifestations. Routine laboratory investigations revealed normal hydroelectolytic, glycemic, hepatic, renal, and thyroid status. Standard radiography revealed diffuse bone demineralization, laryngoscopy showed laryngeal dyskinesia, echocardiography and abdominal ultrasound were normal. Biopsy of a nodule revealed hyalinosis deposition leading initially to the hypothesis of systemic hyalinosis. The diagnosis of Farber disease was suspected at the age of eight and definitively confirmed at the age of fourteen after a genetic study of ASAH1 revealing a novel compound homozygous mutation in exon 3 c.194T&amp;gt;G (p.Leu65Trp) whose significance was considered uncertain to date with an acid ceramidase enzyme assay that showed a markedly reduced leucocyte acid ceramidase activity, consistent with a homozygous status for acid ceramidase deficiency. Treatment with analgesics based on paracetamol and nonsteroidal anti-inflammatory drugs, with iron, calcium and vitamin D supplements was prescribed. Physiotherapy was recommended but was very difficult as the child is so painful. Discussion and Conclusion Farber disease is a rare condition with only about 200 cases identified worldwide. It is caused by mutations in the ASAH1 gene. The disease has a wide range of clinical presentations, and this case report highlights the challenge of diagnosis, with a long delay due to lack of awareness of the disease among clinicians. Furthermore, this case describes a novel homozygous mutation in the ASAH1 gene: c.194T&amp;gt;G (p.Leu65Trp) of the ASAH1 gene in a Moroccan patient, expanding the phenotypic spectrum of the disease. Genetic testing should be considered in patients with a clinical presentation consistent with the disease, supplemented by an acid ceramidase enzyme assay in case of doubt as was done in our patient. To date, there is no cure for Farber disease, and treatment is symptomatic and supportive. However, early diagnosis and appropriate management can help improve the quality of life for patients and their families. This case highlights the importance of further research in order to develop more effective treatments for this rare condition. Clinical trials are planned in the close future. Ethics The patient and his family members provided informed consent for publication of indirectly identifiable data.

Contribution of Hepatic Steatosis–Intensified Extracellular Vesicle Release to Aggravated Inflammatory Endothelial Injury in Liver-Specific Asah1 Gene Knockout Mice

To study the mechanism by which nonalcoholic fatty liver disease (NAFLD) contributes to vascular endothelial Nod-like receptor pyrin domain 3 (NLRP3) inflammasome activation and neointima hyperplasia, NAFLD was established in high-fat diet (HFD)-treated Asah1fl/fl/Albcre (liver-specific deletion of the acid ceramidase gene Asah1) mice. Compared with Asah1 flox [Asah1fl/fl/wild type (WT)] and wild-type (WT/WT) mice, Asah1fl/fl/Albcre mice exhibited significantly enhanced ceramide levels and lipid deposition on HFD in the liver. Moreover, Asah1fl/fl/Albcre mice showed enhanced expression of extracellular vesicle (EV) markers, CD63 and annexin II, but attenuated lysosome-multivesicular body fusion. All these changes were accompanied by significantly increased EV counts in the plasma. In a mouse model of neointima hyperplasia, liver-specific deletion of the Asah1 gene enhanced HFD-induced neointima proliferation, which was associated with increased endothelial NLRP3 inflammasome formation and activation and more severe endothelial damage. The EVs isolated from plasma of Asah1fl/fl/Albcre mice on HFD were found to markedly enhance NLRP3 inflammasome formation and activation in primary cultures of WT/WT endothelial cells compared with those isolated from WT/WT mice or normal diet-treated Asah1fl/fl/Albcre mice. These results suggest that the acid ceramidase/ceramide signaling pathway controls EV release from the liver, and its deficiency aggravates NAFLD and intensifies hepatic EV release into circulation, which promotes endothelial NLRP3 inflammasome activation and consequent neointima hyperplasia in the mouse carotid arteries.

Enhanced Vascular Endothelial NLRP3 Inflammasome Activation by Plasma Exosomes from Liver‐specific Acid Ceramidase Gene Knockout Mice on the High Fat Diet

Circulatory exosomes have been reported to induce vascular endothelial injury and neointima proliferation in mice with liver‐specific deletion of acid ceramidase gene (Asah1fl/fl/Albcre, Asah1 is mouse code of this gene). However, the precise mechanism mediating this exosome‐induced endothelial injury remains unknown. The present study tested a hypothesis that plasma exosomes from Asah1fl/fl/Albcre mice on the high fat diet (HFD) activate endothelial NLRP3 inflammasome activation leading to endothelial dysfunction. To test this hypothesis, wild type (WT/WT) mice and Asah1fl/fl/Albcre mice were fed with normal diet (ND) or HFD for 2 months. Using immunofluorescence confocal microscopy, we confirmed that ceramide levels significantly increased in the liver of Asah1fl/fl/Albcre mice compared to its WT/WT littermates and that HFD further enhanced lysosome ceramide levels in the liver as shown by the colocalization of ceramide with lysosome marker, lamp‐1. Then, isolated exosomes from the plasma of these mice were used to treat the primary cultured WT/WT endothelial cells from the carotid artery. It was found that exosomes isolated from HFD‐treated mice (HFD‐Exo) even from WT/WT mice significantly stimulated NLRP3 inflammasome formation and activation as shown by the significant increases in the colocalization of NLRP3 with Caspase‐1 or ASC, caspase‐1 activity and interleukin 1 beta (IL‐1β) level in the endothelial cells compared to exosomes from ND treated mice (ND‐Exo). This inflammasome formation and activation were remarkably enhanced if endothelial cells were treated by HFD‐Exo isolated from Asah1fl/fl/Albcre mice. To confirm whether lysosomal ceramide contributes to HFD‐Exo‐induced endothelial NLRP3 inflammasome activation, we further treated endothelial cells with C(2)‐ceramide, which was shown similar effects comparable to that induced by HFD‐Exo. Using dibutyryl‐cAMP, a blocker of ceramide‐induced action significantly attenuated C(2)‐ceramide‐ or HDF‐Exo‐induced enhancement of endothelial NLRP3 inflammmasome activation. Together, these results suggest that liver‐specific deletion of lysosomal Asah1 gene promotes release of HFD‐Exos that may contain high ceramide, activating endothelial NLRP3 inflammasome and thereby resulting in endothelial dysfunction during HFD‐induced liver steatosis.

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) (50kDa) 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 (5mg/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 (500000IU/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.

Subendothelial Accumulation of Exosomes and Coronary Microvascular Dysfunction in Mice Lacking Acid Ceramidase

Coronary microvascular dysfunction (CMD) has been demonstrated to be one of the major factors contributing to ischemic heart disease. However, the mechanisms initiating CMD remain poorly understood. The present study tested the hypothesis that lysosomal acid ceramidase finely controls subendothelial accumulation of exosomes leading CMD and subsequent myocardial ischemia. Using an endothelial-specific acid ceramidase (AC) gene (mouse gene code: Asah1) knockout (Asah1fl/fl/ECcre) mouse model fed with the Western diet (WD), we found that Asah1fl/fl/ECcre mice on the WD have more thickening of mid-layer in their small coronary arteries as shown significantly increased media/lumen ratio compared to the WD-fed wild type littermates (WT/WT). By nanoparticle tracking analysis (NTA) using NanoSight NS300 nanoparticle analyzer, we also observed that exosomes (~80-130 nm) in the blood stream were markedly increased in WD-fed Asah1fl/fl/ECcre mice compared to WT/WT mice. There were also more exosomes detected in the subendothelial space of coronary arterial wall, as shown by remarkable increase in colocalization of exosome marker CD63 or Annexin II with proteoglycans, bioglycan or decorin (subendothelial matrix markers) in Asah1fl/fl/ECcre mice. By electron microscopy (EM), we further confirmed that Asah1fl/fl/ECcre mice on the WD also significantly augmented the accumulation of exosomes under the endothelium small coronary arteries. Functionally, electrocardiogram (ECG) recording showed that in response to ergonovine, a coronary artery constrictor, the elevation of S-T segment was more remarkable in WD-fed Asah1fl/fl/ECcre than WT/WT mice. Using HypoxyProbe-1, the myocardial ischemic response to ergonovine was also detected more in the heart of WD-fed Asah1fl/fl/ECcre than WT/WT mice. Together, these results indicate that subendothelial exosome accumulation due to defect of lysosomal AC gene expression and activity may be a crucial pathogenic factor for CMD.

Reversal of Endothelial Extracellular Vesicle-Induced Smooth Muscle Phenotype Transition by Hypercholesterolemia Stimulation: Role of NLRP3 Inflammasome Activation.

Recent studies reported that vascular endothelial cells (ECs) secrete NLR family pyrin domain-containing 3 (NLRP3) inflammasome products such as interleukin-1β (IL-1β) via extracellular vesicles (EVs) under various pathological conditions. EVs represent one of the critical mechanisms mediating the cell-to-cell communication between ECs and vascular smooth muscle cells (VSMCs). However, whether or not the inflammasome-dependent EVs directly participate in the regulation of VSMC function remains unknown. In the present study, we found that in cultured carotid ECs, atherogenic stimulation by oxysterol 7-ketocholesterol (7-Ket) induced NLRP3 inflammasome formation and activation, reduced lysosome-multivesicular bodies (MVBs) fusion, and increased secretion of EVs that contain inflammasome product IL-1β. These EC-derived IL-1β-containing EVs promoted synthetic phenotype transition of co-cultured VSMCs, whereas EVs from unstimulated ECs have the opposite effects. Moreover, acid ceramidase (AC) deficiency or lysosome inhibition further exaggerated the 7-Ket-induced release of IL-1β-containing EVs in ECs. Using a Western diet (WD)-induced hypercholesterolemia mouse model, we found that endothelial-specific AC gene knockout mice (Asah1fl/fl/ECCre) exhibited augmented WD-induced EV secretion with IL-1β and more significantly decreased the interaction of MVBs with lysosomes in the carotid arterial wall compared to their wild-type littermates (WT/WT). The endothelial AC deficiency in Asah1fl/fl/ECCre mice also resulted in enhanced VSMC phenotype transition and accelerated neointima formation. Together, these results suggest that NLRP3 inflammasome-dependent IL-1β production during hypercholesterolemia promotes VSMC phenotype transition to synthetic status via EV machinery, which is controlled by lysosomal AC activity. Our findings provide novel mechanistic insights into understanding the pathogenic role of endothelial NLRP3 inflammasome in vascular injury through EV-mediated EC-to-VSMC regulation.

Control of Instant Membrane Resealing by Lysosomal Acid Ceramidase in Endothelial Cells

Previous studies have shown that lysosome exocytosis and ceramide platform formation in the outer leaflet of the lipid bilayer are essential for plasma membrane repair. As a lysosomal enzyme catalyzing ceramide hydrolysis, the role of acid ceramidase (AC) in the control of cell membrane repair remains unknown. In the present study, we tested a hypothesis that lysosomal AC‐mediating signaling plays a crucial role in instant membrane resealing of endothelial cells (ECs), which help maintain the integrity of EC membrane and related functional activity. By Western blot analysis, it was found that carmofur, an AC inhibitor, decreased the AC expression in murine ECs. On the contrary, Genistein, an AC activator, elevated the level of AC in these cells. Using wavelength‐switching fluorescent microscopy with propidium iodide (PI), a fluorescent membrane‐impermeant dye as membrane resealing marker and fura‐2 as intracellular Ca2+ indicator, we monitored instant membrane resealing following saponin‐induced membrane disruption simultaneously with Ca2+ influx recording in murine ECs. The speed of PI fluorescence increase (df/dt) within ECs and time to a peak of PI fluorescence elevation were used to reflect the instant membrane resealing. Under Ca2+‐free condition, we found that membrane resealing in ECs was enhanced by carmofur as shown by df/dt reduction, but inhibited by genistein (increase in df/dt). In Hanks’ buffer containing Ca2+, however, carmofur and genistein failed to alter membrane resealing of murine ECs when Ca2+ influx was increased. In isolated coronary ECs from WT/WT and Asah1fl/fl/Endocre mice, we demonstrated that AC gene knockout specifically in ECs remarkably enhanced membrane resealing following saponin‐induced membrane interruption, which also mainly occurred under Ca2+‐free condition. These results suggest that lysosomal AC expression and activity are critical for the regulation of instant membrane resealing in ECs after acute injury, which represents a Ca2+‐independent membrane repairing mechanism.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.

Downregulation of Lysosomal Acid Ceramidase Mediates HMGB1-Induced Migration and Proliferation of Mouse Coronary Arterial Myocytes.

High-mobility group box 1 protein (HMGB1) has been reported to trigger lysosome destabilization causing a wide of inflammatory diseases. The present study tested whether a lysosomal enzyme, acid ceramidase (AC), plays a critical role in HMGB1-induced alteration in ceramide metabolism and whether such HMGB1-AC interaction is associated with abnormal migration and proliferation of vascular smooth muscle cells (SMCs). We first observed that the expression of AC in the medial layer of mouse coronary arterial wall and colocalization of AC with a lysosome marker Lamp-1. In primary cultured coronary arterial myocytes (CAMs), AC expression and colocalization with Lamp-1 were significantly up-regulated by AC inducer, genistein, but down-regulated by AC inhibitor, N-oleoylethanolamine (NOE). HMGB1 dose-dependently decreased the colocalization of AC with Lamp-1 and reduced mRNA and protein expressions of AC in CAMs, but reversed by genistein. Consistently, HMGB1 significantly induced increases in the levels of long-chain ceramides in CAMs, which were not further enhanced by NOE but blocked by genistein. More importantly, HMGB1 promoted migration and proliferation of CAMs, which were not further increased by NOE but reduced by genistein. Lastly, CAMs isolated from smooth muscle-specific AC knockout mice (AC gene Asah1) exhibited increased ceramide levels and enhanced the migration and proliferation, which resembles the effects of HMGB1 on wild-type CAMs. Together, these results suggest that HMGB1 promotes SMC migration and proliferation via inhibition of AC expression and ceramide accumulation.

Endothelial acid ceramidase in exosome-mediated release of NLRP3 inflammasome products during hyperglycemia: Evidence from endothelium-specific deletion of Asah1 gene

Exosomes have been demonstrated to be one of the mechanisms mediating the release of intracellular signaling molecules to conduct cell-to-cell communication. However, it remains unknown whether and how exosomes mediate the release of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome products such as interleukin-1 beta (IL-1β) from endothelial cells. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of multivesicular bodies (MVBs) to control the exosome-mediated release of NLRP3 inflammasome products during hyperglycemia. Using a streptozotocin (STZ)-induced diabetes mouse model, we found that endothelium-specific AC gene knockout mice (Asah1fl/fl/ECcre) significantly enhanced the formation and activation of NLRP3 inflammasomes in coronary arterial ECs (CECs). These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein compared to wild-type (WT/WT) littermates. We also observed the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was augmented in STZ-treated Asah1fl/fl/ECcre mice compared to WT/WT mice, which was accompanied by an increased IL-1β release of exosomes. In the primary cultures of CECs, we demonstrated that AC deficiency markedly enhanced the formation and activation of NLRP3 inflammasomes, but significantly down-regulated tight junction proteins when these cells were exposed to high levels of glucose. The CECs from Asah1fl/fl/ECcre mice had decreased MVB-lysosome interaction and increased IL-1β–containing exosome release in response to high glucose stimulation. Together, these results suggest that AC importantly controls exosome-mediated release of NLRP3 inflammasome products in CECs, which is enhanced by AC deficiency leading to aggravated arterial inflammatory response during hyperglycemia.

Podocyte‐Specific Rescuing and Deletion of Acid Ceramidase Gene in Mouse Glomeruli

We have recently developed a podocyte‐restricted acid ceramidase (AC) gene knockout mouse model (Asah1fl/fl/Podocre, Asah1 is a code of mouse AC gene), which was shown to produce podocyte dysfunction and consequent minimal change disease (MCD). The present study attempted to develop somatic gene intervention methods to perform podocyte‐specific gene deletion, rescuing and silencing of this AC gene in Asah1fl/fl/Podocre mice and their control littermates including Asah1fl/fl/WT mice, which could exclude the potential development or compensatory influence given that Asah1 gene was embryonically deleted in podocytes in those animal strains. We first constructed 2 different vectors which can be transfected to change AC gene expression including a podocyte‐specific AC gene (podo‐Asah1) expression vector and a podocyte‐specific Cre gene (podo‐Cre) expression vector. The pEZX vector containing podocin promoter was used to generate Asah1 and Cre full length gene expression plasmids, which were transfected into Asah1fl/fl/Podocre mice to rescue Asah1 gene and into Asah1fl/fl/WT mice to delete floxed Asah1 gene, respectively. Using isolated podocytes from 2 different mouse strains, we confirmed by immunofluorescence staining, real‐time PCR and Western blot analysis that these vectors could be successfully transfected into podocytes and produced corresponding effects to alter Asah1 gene expression, for example, rescuing of this gene in Asah1fl/fl/Podocre podocytes and deletion of this gene in Asah1fl/fl/WT podocytes. In animal experiments, we used microbubble ultrasound transfection techniques to introduce different plasmids into the kidneys. It was found that Asah1 gene was rescued in podocytes of Asah1fl/fl/Podocre mice by podo‐Asah1 plasmids and deleted in Asah1fl/fl/WT mice by podo‐Cre plasmids. Functionally, we demonstrated that increased Asah1 expression in podocytes decreased desmin levels, but enhanced podocin expression, indicating restoration of podocyte function. It also enhanced the interaction of multivesicular bodies (MVBs) and lysosomes as shown by increase in Vps16 and Lamp‐1 colocalization, which resulted in decreased exosome release from podocytes. Taken together, our results suggest that somatic transfection of podocin promoter‐driven Asah1 and Cre gene expression vectors produced podocyte‐restricted gene changes in different gene engineered animal models for AC function studies.Support or Funding Informationsupported by NIH grant DK54927, DK120491 and HL057244This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelium‐Specific Acid Ceramidase Gene Deletion Enhances Exosome Secretion and Release of NLRP3 Inflammasome Products during Hyperglycemia

Exosomes have been demonstrated to be one of the mechanisms mediating the release of a variety of intracellular signaling molecules to mediate cell‐to‐cell communication. However, it remains unknown whether exosomes mediate the release of Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome products such as IL‐1β from endothelial cells and how exosome‐mediated‐inflammatory signaling is regulated. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of the multivesicular body (MVB) to control the exosome‐mediated release of NLRP3 inflammasome products during hyperglycemia. Using streptozotocin (STZ)‐induced mouse model of diabetes mellitus (DM), we found that endothelium‐specific AC gene knock‐out mice (Asah1fl/fl/ECcre) had significantly enhanced formation and activation of NLRP3 inflammasomes including IL‐1β production in the coronary arterial endothelium. These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein (ZO‐1 and ZO‐2) compared to their wild‐type (WT/WT) littermates. Interestingly, we observed that the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was also augmented in STZ‐treated Asah1fl/fl/ECcre mice compared to WT/WT mice, which was accompanied by an increased IL‐1β release of exosomes into the blood. In the primary cultures of coronary endothelial cells (CECs) from WT/WT and Asah1fl/fl/ECcre mice, we demonstrated that the AC deficiency markedly enhanced the formation and activation of the NLRP3 inflammasomes, but significantly down‐regulated tight junction proteins when CECs were exposed to high levels of glucose. The CECs from Asah1fl/fl/ECcre mice also had decreased MVBs and increased exosome release in response to high glucose stimulation and these exosomes contained increased IL‐1β. These results from our in vivo and in vitro experiments suggest that AC importantly controls exosome secretion and release of NLRP3 inflammasome products in CECs and its deficiency enhances this exosome‐mediated release of NLRP3 inflammasome products to activate coronary arterial inflammatory response during hyperglycemia.Support or Funding InformationHL057244, HL075316, and DK120491This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced Exosome Release and Inhibited TRPML1 Channel Activity in Podocytes from Mice with Podocyte‐Restricted Deletion of Asah1 Gene

The ceramide hydrolysis by lysosomal acid ceramidase (AC) has been reported to regulate lysosome function, which determines the degradation of multivesicular bodies. In this regard, we found that urinary exosome excretion was remarkably increased in podocyte‐specific AC gene knockout (Asah1fl/fl/Podocre) mice. Given the importance of transient receptor potential mucolipin 1 (TRPML1) channel‐mediated Ca2+ release to lysosome function and its regulation by sphingolipids, the present study tested a hypothesis that podocyte‐specific Asah1 gene deletion inhibits TRPML1 channel activity, leading to lysosome dysfunction and exosome release from podocytes. It was found that exosomes released from Asah1fl/fl/Podocre podocytes were remarkably higher than WT/WT podocytes as detected by nanoparticle tracking analysis. Super‐resolution microscopy showed less fusion of multivesicular bodies and lysosomes in Asah1fl/fl/Podocre podocytes compared with WT/WT podocytes. Using GCaMP3 Ca2+ imaging, it was found that ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased Ca2+ release from lysosomes by 7.35 folds in WT/WT podocytes. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in WT/WT podocytes. In Asah1fl/fl/Podocre podocytes, however, ML‐SA1 was found to have no effects on TRPML1 channel‐mediated Ca2+ release, but sphingosine as an AC product of ceramide hydrolysis remarkably increased TRPML1 channel‐mediated Ca2+ release in both WT/WT podocytes (5.42 folds) and Asah1fl/fl/Podocre podocytes (4.52 folds). Using whole‐lysosome patch clamp recording, we detected ML‐SA1‐induced Ca2+ currents in a dose‐dependent manner through the membrane of lysosomes isolated from WT/WT podocytes. Compared to WT/WT podocytes, intact lysosomes isolated from Asah1fl/fl/Podocre podocytes had evidently blunted opening of TRPML1 channels in response to its agonist, ML‐SA1. AC associated sphingolipids, sphingomyelin, ceramide and sphingosine had different effects on TRPML1 channel activity in WT/WT podocytes, with inhibition by sphingomyelin, no effect from ceramide, but enhancement by sphingosine (2.25 folds). In Asah1fl/fl/Podocre podocytes, sphingosine remained to increase ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release (3.96 folds). These results suggest that the AC deficiency decreases TRPML1 channel activity due to reduction of sphingosine production, which may contribute to lysosome dysfunction and enhanced exosome release.Support or Funding InformationThis study was supported by NIH grants DK54927, DK120491, and HL057244.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Allogeneic hematopoietic cell transplantation in Farber disease.

Farber disease (FD) is a rare, lysosomal storage disorder caused by deficient acid ceramidase activity. FD has long been considered a fatal disorder with death in the first three decades of life resulting either from respiratory insufficiency as a consequence of airway involvement or from progressive neurodegeneration because of nervous system involvement. Peripheral symptoms associated with FD, including inflammatory joint disease, have been described to improve relatively rapidly after hematopoietic cell transplantation (HCT). To evaluate the disease-specific status and limitations in the long-term follow-up after HCT, investigate genotype/phenotype correlations and the benefit of allogeneic HCT in FD patients with nervous system involvement. Transplant- and disease-related information of ten FD patients was obtained by using a questionnaire, physicians' letters and additional telephone surveys. ASAH1 gene mutations were identified to search for genotype/phenotype correlations. After mainly busulfan-based preparative regimens, all patients engrafted with one late graft loss. The inflammatory symptoms resolved completely in all patients. Abnormal neurologic findings were present pre-transplant in 4/10 patients, post-transplant in 6/10 patients. Mutational analyses revealed new mutations in the ASAH1 gene and a broad diversity of phenotypes without a genotype/phenotype correlation. With a median follow-up of 10.4 years, overall survival was 80% with two transplant-related deaths. Allogeneic HCT leads to complete and persistent resolution of the inflammatory aspects in FD patients. It appears to have no beneficial effect on progression of nervous system involvement. New mutations in the acid ceramidase gene were identified. A genotype/phenotype correlation could not be established.

Zebrafish acid ceramidase: Expression in Pichia pastoris GS115and biochemical characterization

Acid ceramidase (N-acylsphingosine deacylase EC 3.5.1.23; AC) catalyzes the hydrolysis of ceramide into sphingosine (SPH) and free fatty acid. Zebrafish acid ceramidase (AC) has 60% homology with the human AC). Mutations in the human AC gene asah1 are known to cause Farber disease and spinal muscular atrophy with progressive myoclonic epilepsy. Zebrafish AC was overexpressed in Pichia pastoris by inserting asah1b gene into the genome. The majority of the overexpressed enzyme was secreted into the culture medium and purified to apparent homogeneity by stepwise chromatography. The recombinant protein was glycosylated precursor, that further undergoes limited autoproteolytic processing into two subunits (α and β) which are visible in SDS-PAGE. The zebrafish AC is heterodimer associated with an inter-subunit disulfide bond. SDS-PAGE estimated the mass of native enzyme to be approximately 50 kDa & size exclusion chromatography estimated the mass of the active enzyme as approximately 100 kDa, suggesting the formation of a dimer of heterodimers. The protein was secreted as a mixture of processed and unprocessed forms in the culture media. A preliminary characterization of purified zebrafish AC was done by an enzyme assay. The zebrafish AC expressed in Pichia pastoris would be used for further structural and functional analysis.

Genetic Variation in Acid Ceramidase Predicts Non-completion of an Exercise Intervention.

Genetic variation is associated with a number of lifestyle behaviours; it may be associated with adherence and individual responses to exercise training. We tested single nucleotide polymorphisms (SNPs) in the acid ceramidase gene (ASAH1) for association with subject adherence and physiologic benefit with exercise training in two well-characterised randomised, controlled 8-month exercise interventions: STRRIDE I (n = 239) and STRRIDE II (n = 246). Three ASAH1 non-coding SNPs in a linkage disequilibrium block were associated with non-completion: rs2898458(G/T), rs7508(A/G), and rs3810(A/G) were associated with non-completion in both additive (OR = 1.8, 1.8, 2.0; P < 0.05 all) and dominant (OR = 2.5, 2.6, 3.5; P < 0.05 all) models; with less skeletal muscle ASAH expression (p < 0.01) in a subset (N = 60); and poorer training response in cardiorespiratory fitness (peak VO2 change rs3810 r2 = 0.29, P = 0.04; rs2898458 r2 = 0.29, P = 0.08; rs7508 r2 = 0.28, p = 0.09); and similar in direction and magnitude in both independent exploratory and replication studies. Adherence to exercise may be partly biologically and genetically moderated through metabolic regulatory pathways participating in skeletal muscle adaptation to exercise training.

Deficiency of Lysosomal Ceramide Hydrolysis Contributes to Enhanced Exosome Release and Calcification in Coronary Artery Myocytes

Ceramide has been shown to contribute to vascular calcification under different pathological conditions. However, the precise mechanism mediating this process remains unknown. The present study investigated the role of lysosomal ceramide metabolism via acid ceramidase (AC) in the regulation of exosome excretion in coronary artery myocytes (CAMs) and the possible implication in the development of calcification. In cultured CAMs from smooth muscle (SM)‐specific Asah1 (mouse code of AC gene) knock‐out mice (Asah1fl/fl/SMcre), we observed that deletion of Asah1 gene increased lysosomal ceramide level compared to their control littermates, as detected by immunostaining of ceramide co‐localized with lysosome marker lamp‐1. Treatment of CAMs from wild type mice produced similar effects to reduce lysosomal ceramide given its effect to inhibit AC activity. Alizarin Red staining showed that more remarkable calcium deposition or calcification by 1.9 folds occurred in cultured CAMs from Asah1fl/fl/SMcre mice compared to cells from their control littermates, if these cells were treated with 3 mM NaH2PO3 and atherogenic stimulator, 7‐ket. Correspondingly, CAMs from Asah1fl/fl/SMcre mice were found to have significant phenotype transition as shown by Western blot analysis, increased expression of synthetic and osteogenic markers (more vimentin and osteopontin) and decreased contractile phenotypic marker (less SM22α), and this synthetic or osteogenic phenotype was enhanced by 7‐ket treatment. Using super‐resolution imaging with Structured Illumination Microscopy (SR‐SIM), we found that 7‐ket markedly reduced colocalization of Rab‐7a (multivesicular body (MVB) marker) with lamp‐1 in 7‐ket‐treated WT CAMs by 3 folds or CAMs from Asah1fl/fl/SMcre mice by 2.5 folds, suggesting inhibition of MVB‐lysosome encountering or fusion during inhibition of AC expression and activity. Using nanoparticle tracking analysis, 7‐ket‐induced excretion of exosomes (80–140 nm in size) was found to be significantly increased in CAMs of Asah1fl/fl/SMcre mice by 2.1 folds compared to CAMs from their control littermates. These results suggest that AC‐mediated ceramide hydrolysis in lysosomes critically controls MVB fate and exosome release and that insufficient AC expression and activity may lead to enhancement of exosome release, contributing to calcium deposition or calcification in CAMs or arterial media.Support or Funding Information(supported by NIH grants HL057244, HL075316 and HL122937).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

C26-Ceramide as highly sensitive biomarker for the diagnosis of Farber Disease

Farber disease (FD) is a rare autosomal recessive disease caused by mutations in the acid ceramidase gene (ASAH1). Low ceramidase activity results in the accumulation of fatty substances, mainly ceramides. Hallmark symptoms at clinical level are periarticular nodules, lipogranulomas, swollen and painful joints and a hoarse voice. FD phenotypes are heterogeneous varying from mild to very severe cases, with the patients not surviving past their first year of life. The diagnostic aspects of FD are poorly developed due to the rarity of the disease. In the present study, the screening for ceramides and related molecules was performed in Farber affected patients (n = 10), carriers (n = 11) and control individuals (n = 192). This study has the highest number of enrolled Farber patients and carriers reported to present. Liquid chromatography multiple reaction mass spectrometry (LC/MRM-MS) studies revealed that the ceramide C26:0 and especially its isoform 1 is a highly sensitive and specific biomarker for FD (p < 0.0001). The new biomarker can be determined directly in the dried blood spot extracts with low sample consumption. This allows for easy sample preparation, high reproducibility and use in high throughput screenings.

Dual Tumor-Targeting Nanocarrier System for siRNA Delivery Based on pRNA and Modified Chitosan.

Highly specific and efficient delivery of siRNA is still unsatisfactory. Herein, a dual tumor-targeting siRNA delivery system combining pRNA dimers with chitosan nanoparticles (CNPPs) was designed to improve the specificity and efficiency of siRNA delivery. In this dual delivery system, folate-conjugated and PEGylated chitosan nanoparticles encapsulating pRNA dimers were used as the first class of delivery system and would selectively deliver intact pRNA dimers near or into target cells. pRNA dimers simultaneously carrying siRNA and targeting aptamer, the second class of delivery system, would specifically deliver siRNA into the target cells via aptamer-mediated endocytosis or proper particle size. To certify the delivering efficiency of this dual system, CNPPs, pRNA dimers alone, chitosan nanoparticles containing siRNA with folate conjugation and PEGylation (CNPS), and chitosan nanoparticles containing pRNA dimers alone (CN) were first prepared. Then, we observed that treatment with CNPPs resulted in increased cellular uptake, higher cell apoptosis, stronger cell cytotoxicity, and more efficacious gene silencing compared to the other three formulations. Higher accumulation of siRNA in the tumor site, stronger tumor inhibition, and longer circulating time were also observed with CNPPs compared to other formulations. In conclusion, this dual nanocarrier system showed high targeting and favorable therapeutic efficacy both in vitro and in vivo. Thereby, a new approach is provided in this study for specific and efficient delivery of siRNA, which lays a foundation for the development of pRNA hexamers, which can simultaneously carry six different substances.

Effects of ceranib-2 on cell survival and TNF-alpha in colon cancer cell line.

The aim of this study was to investigate the effects of a novel anti-cancer drug, ceranib-2, which targets the acid ceramidase, in human colon cancer cell line. The cell lines were treated with 50 µM of ceranib-2. Relative mRNA expression of TNF-alpha, TNF-R1 and ASAH were assessed by quantitative RT-PCR. Ceranib-2 reduced cell viability in a dose-dependent manner and the apoptotic values of cells following treatment with the dose of 50 µM were reduced significantly both at 24 h and 48 h compared to the control cells (p < 0.001). TNF-alpha receptor 1 (TNF-R1) mRNA levels were reduced significantly in the cell lines treated with both 25 µM and 50 µM of ceranib-2 for 24 h compared to the control cells (p < 0.05), whereas the difference between the treatment and the control cell lines diminished at 48 h. The human acid ceramidase gene (ASAH) mRNA levels were significantly higher in the cell lines treated with 50 µM of ceranib-2 for 48 h than in the other cell lines (p < 0.001). The study shows that ceranib-2 increased apoptosis by inducing ASAH expression and reduced TNF-R1 expression in human colon cancer cell lines in a dose and time-dependent manner (Fig. 3, Ref. 17).