Ceramide Expression Research Articles

Barrier Abnormalities in Type 1 Diabetes Mellitus: The Roles of Inflammation and Ceramide Metabolism

Xerosis is a common sign of both type 1 and type 2 diabetes mellitus (DM), and patients with DM and mouse models for DM show a compromised epidermal permeability barrier. Barrier defects then allow the entry of foreign substances into the skin, triggering inflammation, infection, and worsening skin symptoms. Characterizing how barrier abnormalities develop in DM could suggest treatments for xerosis and other skin disease traits. Because the proper ratio, as well as proper bulk amounts, of heterogeneous ceramide species are keys to forming a competent barrier, we investigated how ceramide metabolism is affected in type 1 DM using a mouse model (induced by streptozotocin). Chronic inflammation, evident in the skin of mice with DM, leads to (i) decreased de novo ceramide production through serine racemase activation-mediated attenuation of serine palmitoyl transferase activity by D-serine; (ii) changes in ceramide synthase activities and expression that modify the ratio of ceramide molecular species; and (iii) increased ceramide-1-phosphate, a proinflammatory lipid mediator, that stimulates inflammatory cytokine expression (TNFα and IFN-γ). Together, chronic inflammation affects ceramide metabolism, which attenuates epidermal permeability barrier formation, and ceramide-1-phosphate could amplify this inflammation. Alleviation of chronic inflammation is a credible approach for normalizing barrier function and ameliorating diverse skin abnormalities in DM.

Blocking SphK/S1P/S1PR1 axis signaling pathway alleviates remifentanil-induced hyperalgesia in rats

Recent research shows a correlation between altered sphingolipid metabolism and nociceptive processing. Activation of the sphingosine-1-phosphate receptor 1 subtype (S1PR1) by its ligand, sphingosine-1-phosphate (S1P), causes neuropathic pain. However, its role in remifentanil-induced hyperalgesia (RIH) has not been investigated. The purpose of this research was to establish if the SphK/S1P/S1PR1 axis mediated remifentanil-induced hyperalgesia and identify its potential targets. This study examined the protein expression of ceramide, sphingosine kinases (SphK), S1P, and S1PR1 in the spinal cord of rats treated with remifentanil (1.0 μg/kg/min for 60 min). Prior to receiving remifentanil, rats were injected with SK-1 (a SphK inhibitor); LT1002 (a S1P monoclonal antibody); CYM‐5442, FTY720, and TASP0277308（the S1PR1 antagonists); CYM-5478 (a S1PR2 agonist); CAY10444 (a S1PR3 antagonist); Ac-YVAD-CMK (a caspase-1 antagonist); MCC950 (the NOD-like receptor protein 3 (NLRP3) inflammasome antagonist); and N-tert-Butyl-α-phenylnitrone (PBN, a reactive oxygen species (ROS) scavenger). Mechanical and thermal hyperalgesia were evaluated at baseline (24 h prior to remifentanil infusion) and 2, 6, 12, and 24 h following remifentanil administration. The expression of the NLRP3-related protein (NLRP3, caspase-1), pro-inflammatory cytokines (interleukin-1β(IL-1β), IL-18), and ROS was found in the spinal dorsal horns. In the meantime, immunofluorescence was used to ascertain if S1PR1 co-localizes with astrocytes. Remifentanil infusion induced considerable hyperalgesia in addition to increased ceramide, SphK, S1P, and S1PR1, NLRP3-related protein (NLRP3, Caspase-1, IL-1β, IL-18) and ROS expression, and S1PR1 localized astrocytes. By blocking the SphK/S1P/S1PR1 axis, remifentanil-induced hyperalgesia was reduced, as was the expression of NLRP3, caspase-1, pro-inflammatory cytokines (IL-1β, IL-18) and ROS in the spinal cord. In addition, we observed that suppressing NLRP3 or ROS signal attenuated the mechanical and thermal hyperalgesia induced by remifentanil. Our findings indicate that the SphK/SIP/S1PR1 axis regulates the expression of NLRP3, Caspase-1, IL-1β, IL-18 and ROS in the spinal dorsal horn to mediate remifentanil-induced hyperalgesia. These findings may contribute to pain and SphK/S1P/S1PR1 axis research positively, and inform the future study of this commonly used analgesic.

Abnormal fatty acid metabolism and ceramide expression may discriminate myocardial infarction from strangulation death: A pilot study

Determining myocardial infarction (MI) and mechanical asphyxia (MA) was one of the most challenging tasks in forensic practice. The present study aimed to investigate the potential of fatty acid (FAs) metabolism, and lipid alterations in determining MI and MA. MA and MI mouse models were constructed, and metabolic profiles were obtained by LC-MS-based untargeted metabolomics. The metabolic alterations were explored using the PCA, OPLS-DA, the Wilcoxon test, and fold change analysis. The contents of lipid droplets (LDs) were detected by the transmission scanning electron microscope and Oil red O staining. The immunohistochemical assay was performed to detect CD36 and dysferlin. The ceramide was assessed by LC-MS. PCA showed considerable differences in the metabolite profiles, and the well-fitting OPLS-DA model was developed to screen differential metabolites. Thereinto, 9 metabolites in the MA were reduced, while metabolites were up- and down-regulated in MI. The increased CD36 suggested that MI and MA could enhance the intake of FAs and disturb energy metabolism. The increased LDs, decreased dysferlin, and increased ceramide (C18:0, C22:0, and C24:0) were observed in MI groups, confirming the lipid deposition. The present study indicated significant differences in myocardial FAs metabolism and lipid alterations between MI and MA, suggesting that FAs metabolism and related proteins, certain ceramide may harbor the potential as biomarkers for discrimination of MI and MA.

370-OR: Proinflammatory Stress Activates Neutral Sphingomyelinase 2 Based Generation of Ceramide-Enriched ß-Cell Exosomes

Data suggest that β cell exosomes, extracellular vesicles released by exocytosis of multivesicular endosomes (MVEs) , may act as paracrine effectors in islet health. However, mechanisms linking β cell stress to changes in exosomes and whether activation of these pathways can impact diabetes remain less explored. In other cells, the enzyme neutral spingomyelinase 2 (nSMase2) induces ceramide dependent MVE vesicle formation and release of ceramide-enriched exosomes. We hypothesized that β cell inflammatory stress engages ceramide-dependent exosomal formation pathways, and that ceramide-enriched exosomes could then impact surrounding β cells. To test this we treated INS-1 β cells with 24 hrs of 5 ng/mL of IL1β. nSMase 2 and ceramide expression were quantified with immunoblot and/or flow cytometry and exosomes were isolated using a tetraspanin bead-based pulldown. IL1β increased β cell nSMase2 expression and ceramide expression in concert. Direct nSMase2 activation with 24 hrs of 5-ng/mL Caffeic acid phenethyl ester (CAPE) also increased β cell ceramide staining. IL1β and CAPE treatments also yielded increased ceramide expression on β cell exosomes. Human islets exhibited similar increases in cellular nSMase2 and ceramide staining after 24-hr cytokine mix treatment (5ng/mL IL1β, 10ng/mL TNFα, and 100ng/mL IFNγ) . To test the potential for transfer of β cell exosomes to surrounding cells, we generated INS-1 cells with GFP tagged CD9 (exosome transmembrane protein) , which release GFP labeled exosomes. Flow cytometry staining in wild-type INS-1 cells after 24-hr transwell coculture with CD9-GFP cells showed that cocultured wild-type cells exhibited increased green fluorescence, suggesting transfer of CD9-GFP exosomes. Our findings suggest that nSMase2 activity may regulate β cell exosome subpopulations under conditions of inflammatory stress. Future work will interrogate impacts on exosome cargo and physiologic impacts of transfer on recipient cells. Disclosure J.Xu: None. E.K.Sims: Other Relationship; Medscape, Patent. Funding 5 R01DK121929

Ceramide expression in relation to breast cancer molecular subtypes in Saudi women

Background/IntroductionDespite advances in the diagnosis and management of breast cancer (BC), it is still associated with high mortality rates. New biomarkers are being developed for the diagnosis, treatment, and prediction of responses of BC. Ceramide (CER), a bioactive sphingolipid, has emerged recently as a useful diagnostic tool in several types of tumors. In this study, we evaluated CER expression in invasive BC and assessed its relation to the molecular subtypes of BC. Materials and methodsThe clinical data and histopathological slides of 50 patients with invasive ductal carcinoma were retrieved and reviewed. The cases were then stained with a mouse monoclonal anti-ceramide antibody. Pearson correlation was used to assess the correlation between CER percentage and intensity and other clinical and pathological variables. ResultsCER expression showed a direct relationship with estrogen and progesterone receptors Allred scores. However, it showed an inverse relation with tumor grade, HER2/neu status and Ki-67 index. ConclusionsCER expression is likely to be associated with luminal BC molecular subtypes. However, more research is needed to confirm these results and to explore its relation to the different clinical outcomes, including response to treatment and prognosis.

Ceramide accumulation accelerates nucleus pulposus cells degradation by p38MAPK activation.

Ceramide is a lipid molecule that regulates life activities such as cell differentiation, proliferation, apoptosis, and aging. However, whether ceramide plays a role in the intervertebral disc degeneration (IDD) is not clear. The aim of this study is to explore the effect of ceramide during the nucleus pulposus (NP) cells degeneration. We used human NP cells and passaged them until the fourth generation to analyze the content of ceramide. Cell-permeable C6-ceramide was used to upregulate ceramide expression, and myriocin was used to inhibit the accumulation of ceramide. To understand the relation between p38MAPK and ceramide, SB203580 was used to inhibit the activation of p38MAPK. We tested the viability of NP cells by the detection of collagen II and p16 expression, the proliferation, and the apoptosis of NP cells. Ceramide content was increased in NP cells from passage 1 (P1) to P4. The upregulation of ceramide accelerated the P1 NP cell degeneration by the reduction of collagen II production and proliferative cells population, increased p16 expression, and apoptotic cells population. However, the suppression of ceramide delayed the degeneration of P4 NP cells in the previous aspects. The accumulation of ceramide activated the phosphorylation of p38MAPK, and the inhibition of p38MAPK activation also alleviated the C6-ceramide-induced NP cell degeneration. Ceramide accumulates during NP cell degradation, and the upregulated ceramide contributes to the NP cells degeneration by p38MAPK activation.

A tissue-specific screen of ceramide expression in aged mice identifies ceramide synthase-1 and ceramide synthase-5 as potential regulators of fiber size and strength in skeletal muscle.

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi‐organ investigation of age‐associated changes of ceramide species, a unique and distinctive change pattern of C16:0 and C18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C16:0 and C18:0 acyl chains, respectively, was down‐regulated in skeletal muscle of aged mice. Similarly, an age‐dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1 Δ/Δ and CerS5 Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1‐ and CerS5‐deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age‐related muscle wasting disorders in humans.

C2-ceramide influences alveolar epithelial barrier function by downregulating Zo-1, occludin and claudin-4 expression

Noncardiogenic lung edema is a key factor affecting the prognosis of acute lung injury (ALI). Previous studies have been focused on regulatory roles of ceramide on lung vascular endothelial barrier functions and had already identified ceramide as mediator involved in the formation of lung edema. However, the effects of ceramide on lung epithelial barrier were still unknown. This study aimed to investigate the effects of ceramide on the barrier function of alveolar epithelial cells. Primary mouse alveolar type II epithelial cells (AECII) were grown on Transwell polyester membranes to construct monolayer, and stimulated with different concentrations of ceramide. Transepithelial resistance (TER) was measured to assess the epithelial cell permeability. Western blotting and real-time quantitative polymerase chain reaction were used to detect the mRNA and protein levels of tight junction, respectively. After incubation with different concentrations of c2-ceramide, TER of AECII monolayer decreased significantly in a dose-dependent manner. Moreover, expressions of ZO-1, occludin and claudin-4 were significantly reduced by c2-ceramide in the study. This study demonstrated that ceramide could increase alveolar epithelial cell monolayer permeability by downregulation of tight junction proteins. Therefore, modulation of ceramide expression may serve as a new therapeutic approach to treat acute lung injury.

Early PQQ supplementation has persistent long-term protective effects on developmental programming of hepatic lipotoxicity and inflammation in obese mice.

Nonalcoholic fatty liver disease (NAFLD) is widespread in adults and children. Early exposure to maternal obesity or Western-style diet (WD) increases steatosis and oxidative stress in fetal liver and is associated with lifetime disease risk in the offspring. Pyrroloquinoline quinone (PQQ) is a natural antioxidant found in soil, enriched in human breast milk, and essential for development in mammals. We investigated whether a supplemental dose of PQQ, provided prenatally in a mouse model of diet-induced obesity during pregnancy, could protect obese offspring from progression of NAFLD. PQQ treatment given pre- and postnatally in WD-fed offspring had no effect on weight gain but increased metabolic flexibility while reducing body fat and liver lipids, compared with untreated obese offspring. Indices of NAFLD, including hepatic ceramide levels, oxidative stress, and expression of proinflammatory genes (Nos2, Nlrp3, Il6, and Ptgs2), were decreased in WD PQQ-fed mice, concomitant with increased expression of fatty acid oxidation genes and decreased Pparg expression. Notably, these changes persisted even after PQQ withdrawal at weaning. Our results suggest that supplementation with PQQ, particularly during pregnancy and lactation, protects offspring from WD-induced developmental programming of hepatic lipotoxicity and may help slow the advancing epidemic of NAFLD in the next generation.-Jonscher, K. R., Stewart, M. S., Alfonso-Garcia, A., DeFelice, B. C., Wang, X. X., Luo, Y., Levi, M., Heerwagen, M. J. R., Janssen, R. C., de la Houssaye, B. A., Wiitala, E., Florey, G., Jonscher, R. L., Potma, E. O., Fiehn, O. Friedman, J. E. Early PQQ supplementation has persistent long-term protective effects on developmental programming of hepatic lipotoxicity and inflammation in obese mice.

Treatment with diet or insulin induces a different placental Ceramide expression during gestational diabetes mellitus (GDM)

Gestational diabetes mellitus (GDM) is associated with a number of factors that prevent the placenta from transporting necessary nutrients and oxygen important for fetal development. Ceramides are a family of lipids that consist of sphingosine covalently linked to a fatty acid that is known to be associated with the development of diabetes and the induction of cell apoptosis. Our objective was to determine levels of ceramide and apoptosis during gestational diabetes treated with insulin or diet. Placental biopsies and paraffin embedded placental tissues slides for GDM‐I (gestational diabetes mellitus treated with insulin), GDM‐D (gestational diabetes mellitus treated with diet), and term controls were obtained. Immunohistochemistry was preform for ceramide localization in the placenta. Immunoblot was done for serine palmitoyltransferase (SPT; enzyme involve in ceramide production), active caspase 3 and XIAP protein (inhibitor of caspase activation). There were no significant differences in maternal age, gestational weeks and fetal weight between control and GDM pregnancies. Immunohistochemistry showed an increased in ceramide in the villi trophoblast of the placenta during GDM‐I but not in the GDM‐D tissues. Similarly, an upregulation for nuclear SPT expression, the rate‐limiting enzyme in de novo ceramide biosynthesis, was present only in the GDM‐I placenta (3.4‐fold; p<0.05) as compared to controls. In contrast, no significant differences were observed for cytosolic SPT expression between control and GDM tissues. There was a similar upregulation for active caspase 3 (1.2‐fold; p<0.05) in the placenta of both GDM‐I and GDM‐D when compared to control placental tissue. Interestingly no significant differences were observed for cytosolic XIAP between samples, while there was a significant decrease for nuclear XIAP only (1.7‐fold; p<0.05) in the GDM‐I placenta as compared to controls. Our findings confirm the presence of ceramide in the human placenta of control and GDM patients. Furthermore our results demonstrated that ceramide is increased in the placental trophoblast during insulin treatment and that this increased correlate with the increased caspase activation and decreased XIAP observed during this treatment. Our results also suggest that the increased activation of placental caspase 3 during GDM‐D is occurring in a ceramide independent manner. Treatment variation between diet and insulin tissues suggest that an equally effective but different mechanism is exerted in the placenta leading to the alleviating of the symptoms/consequences present during GDM in the placenta.

De novo ceramide synthesis is involved in acute inflammation during labor.

Gestation is regulated by an inflammatory process that allows implantation and parturition. The comprehension of such inflammatory switches is important for the identification of therapeutic targets in pregnancy defects. Sphingolipids are a class of structural membrane components with important signaling functions. Among sphingolipids, ceramide is a well-known mediator of stress signals and pro-inflammatory responses. In this paper, we evaluated the association between ceramide increase and the inflammatory process of labor, comparing placentas from vaginal deliveries, including both spontaneous and induced labor, versus elective cesarean. We demonstrated that: (i) the inflammatory marker IL-6 is upregulated in labored placentas; (ii) IL-6 content inversely correlates with labor duration; (iii) ceramide content and expression of serine palmitoyl transferase (SPT, rate limiting enzyme for de novo ceramide synthesis) are increased in labored placentas; (iv) the expression of SPT directly correlates with inflammation and inversely with labor duration. These observations suggest that ceramide metabolism and signaling may be implicated in controlling important inflammatory mechanisms driving gestation: we hypothesize that ceramide can be a therapeutic target in inflammatory complications of parturition.

Cyclooxygenase-2 and Inflammation Mediators Have a Crucial Role in Reflux-Related Esophageal Histological Changes and Barrett’s Esophagus

Gastroesophageal reflux (GER) causes injury of the esophageal squamous epithelium, a condition called reflux esophagitis. The sequence reflux-esophagitis-intestinal metaplasia-dysplasia-invasive cancer is widely accepted as the main adenocarcinogenetic pathway in the esophagus; however, the mechanisms of this progression need to be better defined. We evaluated COX-2 expression and activity in biopsies from patients affected with GER, and these parameters have been correlated with the stage of the disease, ceramide expression, apoptotic process, and angiogenesis. The effects of celecoxib on bile acid- and EGF-induced mucosal proliferation, apoptosis and angiogenesis have been also investigated. Four groups of patients were distinguished: non esophagitis, non erosive esophagitis, erosive esophagitis, and Barrett's esophagus. COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis were evaluated in esophageal biopsies. COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis progressively increase from non esophagitis patients to patients with non erosive and erosive esophagitis, to those with BE. Incubation of the cells with DCA/EGF increases PGE2 production, proliferative activity and VEGF production, effects prevented by celecoxib pretreatment. Ceramide expression increased from non esophagitis patients to patients with non erosive and erosive esophagitis, and decreased in BE; caspase-3 activity progressively decreased from non esophagitis to BE patients, suggesting an impairment of the apoptotic process with disease progression. These results stand for a close relationship between progression of initial steps of gastroesophageal reflux disease (GERD) and COX-2, proliferative activity and EGF/VEGF expression and could have implications in GERD treatment in order to prevent its neoplastic evolution.

Ceramide mediates smoking‐induced insulin resistance

Environmental toxins are increasingly implicated in adverse health outcomes. Considering the lung's apposition with the environment, we sought to test the hypothesis that ceramide functions as an intermediate between smoking and systemic insulin resistance. To determine the effect of cigarette smoke on lung ceramide metabolism, common lung cell lines including A‐549 and Beas2B were exposed to cigarette smoke extract (CSE). CSE treatment elicited a robust increase in ceramide and elevated expression of several enzymes involved with de novo ceramide synthesis. Moreover, myoblasts incubated with conditioned medium from CSE‐treated lung cells became completely insensitive to insulin stimulation, suggesting the presence of an insulin antagonist in the medium of CSE‐treated lung cells. Lastly, we measured systemic lipids in mice following nasal instillation of CSE and found an increase in muscle and hepatic ceramides; however, such findings were not observed in lung‐RAGE KO mice. This work identifies ceramide as a possible intermediate between the lung and peripheral tissues. Future work will determine the role of lung‐secreted ceramides on systemic effects in vivo, such as cardiovascular function and insulin signaling. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI; PRR) and a BYU Mentoring Environment Grant (BTB).

Inhibitory Effect of Polyphenol-Rich Fraction from the Bark of Acacia mearnsii on Itching Associated with Allergic Dermatitis.

We examined the inhibitory effect of polyphenol-rich aqueous extract from the bark of Acacia mearnsii (PrA) on itching associated with atopic dermatitis (AD). HR-1 mice were fed a normal diet, special diet (AD group), or special diet containing 3% PrA (PrA group) for 6 weeks. In the AD group, itching frequency and transepidermal water loss increased compared to the control group. In the PrA group, an improvement in atopic dermatitis symptoms was observed. Ceramide expression in the skin decreased in the AD group compared to the control group, but no decrease was observed in the PrA group. mRNA expression of ceramidase decreased in the PrA group compared to the AD group. The results of this study have revealed that PrA inhibits itching in atopic dermatitis by preventing the skin from drying. It is considered that the mechanism by which PrA prevents the skin from drying involves the inhibition of increased ceramidase expression associated with atopic dermatitis.

Weight loss amelioration of non‐alcoholic steatohepatitis linked to shifts in hepatic ceramide expression and serum ceramide levels

Non-alcoholic steatohepatitis (NASH) is associated with increased hepatic insulin resistance. Ceramides and other toxic sphingolipids promote inflammation, lipotoxicity and insulin resistance; however, the role of ceramides in the pathogenesis of NASH has not been determined. This study characterizes expression of ceramide-related genes in human livers with NASH and examines the effects of weight loss on NASH and pro-ceramide gene expression in liver. Liver biopsies were obtained to assess the histopathological status of non-alcoholic fatty liver disease/NASH prior to and following completion of a 1-year course of implementing either lifestyle changes or a standard enrichment protocol designed to encourage weight loss. Liver biopsy samples were used to measure pro-ceramide gene expression by quantitative reverse transcriptase polymerase chain reaction analysis (qRT-PCR), and serum was used to measure ceramide immunoreactivity. At baseline, serine palmitoyltransferase (SPTLC)2 (P = 0.02) and ceramide synthase (CER)1 (P = 0.001) mRNA transcripts were less abundantly expressed in livers with NASH relative to normal controls. After weight loss (average 9.3%), SPTLC1 (P = 0.005) and uridine diphosphate glucose ceramide glucosyltransferase (UGCG) (P = 0.001) expression significantly declined while CER1 increased (P = 0.001) among subjects randomized to the lifestyle change subgroup. Reductions in calorie and fat consumption were significantly correlated with changes in ceramide-related gene expression. Finally, both net and relative reductions in serum ceramide levels were significantly greater in the lifestyles compared with the standard enrichment (control) protocol group (both P < 0.005). NASH is associated with increased insulin resistance and altered ceramide gene expression in liver. Weight loss-mediated reversal of NASH is associated with reduced pro-ceramide gene expression in liver.

Temporal expression profiles of ceramide and ceramide-related genes in wild-type and mPer1/mPer2 double knockout mice

Most living organisms exhibit circadian rhythms in physiology and behavior. These oscillations are generated by an endogenous circadian clock and control many biological processes. Ceramide has attracted attention as a signal mediator in diverse cell processes including cell death and differentiation. The relationships between ceramide expression levels and the circadian clock have not previously been investigated. To determine if there are circadian variations in the content of ceramide, we measured ceramide concentrations in the livers of wild-type (WT) and mPer1/mPer2 double knockout (DKO) mice. The ceramide concentration in WT mice was dramatically increased at Zeitgeber Time 9 (ZT9; 9h after lights-on time) and ZT21 but no rhythmicity in ceramide expression was seen in DKO mice. Because ceramide can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase), or by ceramide synthase (CerS)-mediated synthesis, we assayed the expression patterns of ceramide-related genes using real-time PCR. CerS2 expression levels showed a biphasic pattern of expression in WT mice but no rhythmicity in DKO mice. While the neutral SMase (nSMase) and acidic SMase (aSMase) mRNA in WT mice were expressed in a circadian manner, the correlation between the expression levels of these SMases with times of day was weak in DKO mice. Collectively, our findings suggest that both SMases and CerS2 mRNA expression are regulated by the presence of mPer1/mPer2 circadian clock genes in vivo, and imply that ceramide may play a vital role in circadian rhythms and physiology.

Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice

alpha1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of alpha1,6-fucosyltransferase (Fut8) knockout (Fut8(-/-)) mice. Phenotypically, Fut8(-/-) mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8(-/-) cells also show marked dysregulation of the TGF-beta1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8(-/-) mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8(-/-) mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8(-/-) mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8(-/-) mice.

Targeted Induction of Lung Endothelial Cell Apoptosis Causes Emphysema-like Changes in the Mouse

Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

Anti-Inflammatory and Anti-Apoptotic Effects of Fumonisin B1, an Inhibitor of Ceramide Synthase, in a Rodent Model of Splanchnic Ischemia and Reperfusion Injury

Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study sought to determine whether pharmacological inhibition of ceramide biosynthesis in the intestine attenuates pathophysiological sequelae of shock induced by splanchnic artery occlusion and reperfusion. Ischemia and reperfusion injury was induced in anesthetized rats by clamping both the superior mesenteric artery and the celiac artery for 45 min followed by reperfusion. Within 6 min after reperfusion, animals developed significant systemic hypotension with 100% of the animals dying during the 4-h period of reperfusion. In parallel experiments, animals were necropsied after 60 min of reperfusion, and the ileum was harvested for histological examination and assessment of biochemical changes. Administration of fumonisin B1 (FB1), a competitive and reversible inhibitor of ceramide synthase (3 mg/kg, 15 min before reperfusion), significantly reduced i) the increased ceramide expression as detected by immunohistochemistry; ii) peroxynitrite-mediated protein nitration; iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils following a decrease in intercellular adhesion molecule-1 expression; iv) production of the proinflammatory cytokine tumor necrosis factor-alpha; and v) apoptosis in the ileum. Overall, tissue-protective effects were clearly observed upon histological examination of the ileum. These beneficial events were ultimately linked to decreases in both the development of hypotension and overall mortality. These results implicate ceramide as a key signaling molecule in splanchnic arterial ischemia and reperfusion-induced shock. The broader implications of our results provide a pharmacological rationale for the development of inhibitors of ceramide biosynthesis as novel therapeutics for ischemia and reperfusion-induced shock of several etiologies.

Significance of Ceramide and DNA Ploidy in Laryngeal Carcinogenesis

Objective: To investigate the expression of ceramide produced by sphingomyelin and DNA ploidy in normal laryngeal mucosa, laryngeal precancerous lesions and laryngeal carcinoma. Methods: Eighty consecutive patients with leukoplakia larynx were identified from the archived pathology files of the Eye, Ear, Nose and Throat Hospital of Fudan University from 1990 to 2001. Among them, 23 patients developed laryngeal carcinoma. Flow cytometry and immunohistochemistry were performed to test DNA content and ceramide expression on normal tissue, precancerous lesions and laryngeal carcinoma. Results: Among 80 patients with laryngeal carcinoma, 23 cases were all aneuploids, and 57 diploids. Expression of ceramide decreased gradually from normal tissue via precancerous lesions to laryngeal carcinoma. Cell staining per high-power field was 400 ± 30, 180 ± 20, and 10 ± 10, respectively (p < 0.05, t test). The expression of ceramide in the DNA diploid cell was higher than that in the aneuploid cell (p < 0.05, t test). Conclusions: Ceramide, the second messenger in apoptosis, plays a significant role in the progression from precancerous lesion to carcinoma of the larynx. Reduction in ceramide may be the key factor in laryngeal carcinogenesis.