Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism.

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%-80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

Vitreous composition modification after transpalpebral electrical stimulation of the eye: Biochemical analysis.

Electrical stimulation (ES) of the eye represents a therapeutic approach in various clinical applications ranging from retinal dystrophies, age-related macular degeneration, retinal artery occlusion and nonarteritic ischemic optic neuropathy. In clinical practice, ES of the eye is mainly performed with a transcorneal or transpalpebral approach. These procedures are non-invasive and well-tolerated by the patients, reporting only minimal and transient adverse events, while serious adverse effects were not observed. Despite the growing literature on animal models, only clinical parameters have been investigated in humans and few data are available about biochemical changes induced by ES of the eye. The purpose of this study is to investigate the possible mechanism that regulates the beneficial effects of ES on retinal cells function and survival in humans. 28 patients undergoing pars plana vitrectomy (PPV) for idiopathic epiretinal membrane (iERM) were randomly divided in two groups: 13 patients were treated with transpalpebral ES before surgery and 15 underwent surgery with no prior treatment. Vitreous samples were collected for biochemical analysis during PPV. ES treatment leads to a reduction in the vitreous expression of both proinflammatory cytokines, namely IL-6 and IL-8, and proinflammatory lipid mediators, such as lysophosphatidylcholine. Indeed, we observed a 70% decrease of lysophosphatidylcholine 18:0, which has been proven to exert the greatest proinflammatory activities among the lysophosphatidylcholine class. The content of triglycerides is also affected and significantly decreased following ES application. The vitreous composition of patients undergoing PPV for iERM displays significant changes following ES treatment. Proinflammatory cytokines and bioactive lipid mediators expression decreases, suggesting an overall anti-inflammatory potential of ES. The investigation of the mechanism by which this treatment alters the retinal neurons leading to good outcomes is essential for supporting ES therapeutic application in various types of retinal diseases.

Inhibition of Ceramide Synthesis Reduces α-Synuclein Proteinopathy in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) is a proteinopathy associated with the aggregation of α-synuclein and the formation of lipid-protein cellular inclusions, named Lewy bodies (LBs). LB formation results in impaired neurotransmitter release and uptake, which involve membrane traffic and require lipid synthesis and metabolism. Lipids, particularly ceramides, are accumulated in postmortem PD brains and altered in the plasma of PD patients. Autophagy is impaired in PD, reducing the ability of neurons to clear protein aggregates, thus worsening stress conditions and inducing neuronal death. The inhibition of ceramide synthesis by myriocin (Myr) in SH-SY5Y neuronal cells treated with preformed α-synuclein fibrils reduced intracellular aggregates, favoring their sequestration into lysosomes. This was associated with TFEB activation, increased expression of TFEB and LAMP2, and the cytosolic accumulation of LC3II, indicating that Myr promotes autophagy. Myr significantly reduces the fibril-related production of inflammatory mediators and lipid peroxidation and activates NRF2, which is downregulated in PD. Finally, Myr enhances the expression of genes that control neurotransmitter transport (SNARE complex, VMAT2, and DAT), whose progressive deficiency occurs in PD neurodegeneration. The present study suggests that counteracting the accumulation of inflammatory lipids could represent a possible therapeutic strategy for PD.

Inhibition of Sphingolipid Synthesis as a Phenotype-Modifying Therapy in Cystic Fibrosis.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells. METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned. RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin. CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.

Iron and Sphingolipids as Common Players of (Mal)Adaptation to Hypoxia in Pulmonary Diseases.

Hypoxia, or lack of oxygen, can occur in both physiological (high altitude) and pathological conditions (respiratory diseases). In this narrative review, we introduce high altitude pulmonary edema (HAPE), acute respiratory distress syndrome (ARDS), Chronic Obstructive Pulmonary Disease (COPD), and Cystic Fibrosis (CF) as examples of maladaptation to hypoxia, and highlight some of the potential mechanisms influencing the prognosis of the affected patients. Among the specific pathways modulated in response to hypoxia, iron metabolism has been widely explored in recent years. Recent evidence emphasizes hepcidin as highly involved in the compensatory response to hypoxia in healthy subjects. A less investigated field in the adaptation to hypoxia is the sphingolipid (SPL) metabolism, especially through Ceramide and sphingosine 1 phosphate. Both individually and in concert, iron and SPL are active players of the (mal)adaptation to physiological hypoxia, which can result in the pathological HAPE. Our aim is to identify some pathways and/or markers involved in the physiological adaptation to low atmospheric pressures (high altitudes) that could be involved in pathological adaptation to hypoxia as it occurs in pulmonary inflammatory diseases. Hepcidin, Cer, S1P, and their interplay in hypoxia are raising growing interest both as prognostic factors and therapeutical targets.

Sildenafil attenuates hypoxic pulmonary remodelling by inhibiting bone marrow progenitor cells.

The recruitment of bone marrow (BM)-derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM-derived progenitor cells, we induced pulmonary remodelling in rats by two-week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM-derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH-induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH-induced increase in c-kit+ cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c-kit+ cells that colocalize with tyrosine kinase receptor 2 (VEGF-R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia-induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia-inducible factor (HIF)-1α. Furthermore, sildenafil depressed the number of CXCR4+ cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM-derived c-kit+ cells in the pulmonary tissue. The attenuation of the recruitment of BM-derived c-kit+ cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.

Inhibition of ceramide de novo synthesis by myriocin produces the double effect of reducing pathological inflammation and exerting antifungal activity against A. fumigatus airways infection.

BACKGROUND: Fungal infections develop in pulmonary chronic inflammatory diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF). The available antifungal drugs may fail to eradicate fungal pathogens, that can invade the lungs and vessels and spread by systemic circulation taking advantage of defective lung immunity. An increased rate of sphingolipid de novo synthesis, leading to ceramide accumulation, was demonstrated in CF and COPD inflamed lungs. The inhibitor of sphingolipid synthesis myriocin reduces inflammation and ameliorates the response against bacterial airway infection in CF mice. Myriocin also inhibits sphingolipid synthesis in fungi and exerts a powerful fungistatic effect. METHODS: We treated Aspergillus fumigatus infected airway epithelial cells with myriocin and we administered myriocin-loaded nanocarriers to A. fumigatus infected mice lung. RESULTS: We demonstrate here that de novo synthesized ceramide mediates the inflammatory response induced by A. fumigatus infection in airway epithelia. CF epithelial cells are chronically inflamed and defective in killing internalized conidia. Myriocin treatment reduced ceramide increase and inflammatory mediator release whereas it upregulated HO1 and NOD2, allowing the recovery of a functional killing of conidia in these cells. Myriocin-loaded nanocarriers, intratracheally administered to mice, significantly reduced both the inflammatory response induced by A. fumigatus pulmonary challenge and fungal lung invasion. CONCLUSIONS: We conclude that inhibition of sphingolipid synthesis can be envisaged as a dual anti-inflammatory and anti-fungal therapy in patients suffering from chronic lung inflammation with compromised immunity. GENERAL SIGNIFICANCE: Myriocin represents a powerful agent for inflammatory diseases and fungal infection.

Unexpected distribution of CA19.9 and other type 1 chain Lewis antigens in normal and cancer tissues of colon and pancreas: Importance of the detection method and role of glycosyltransferase regulation.

BACKGROUND: CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. METHODS: Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. RESULTS: CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, together with Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1kb away from the B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. CONCLUSIONS: CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. GENERAL SIGNIFICANCE: CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels.

Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q-trap mass spectrometry.

Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r2 = 0.9996). The intra- and between-day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra-tracheal delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n = 4) to 11.7 (median; 7.6-22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.

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.

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis.

BACKGROUND: Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects. METHODS: The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models. RESULTS: We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation. CONCLUSIONS: The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection. GENERAL SIGNIFICANCE: Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm.

BACKGROUND: The human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents. Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections. RESULTS: Antifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25-64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4-16) for both planktonic and sessile cells. Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms. An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles. CONCLUSIONS: The direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements.

We focused on transcription factors and epigenetic marks that regulate the B3GALT5 gene through its retroviral long terminal repeat (LTR) promoter. We compared the expression levels of the B3GALT5 LTR transcript, quantitated by competitive RT-PCR, with those of the candidate transcription factors HNF1α/ß and Cdx1/2, determined by Western blot analysis, in colon cancer biopsies, various cell lines, and cell models serving as controls. We found that HNF1α/ß were easily detected, irrespective of the amount of LTR transcript expressed by the source, whereas Cdx1/2 were undetectable, and no sample lacking HNF1α/ß expressed the LTR transcript. On transfection in proper host cells, both HNF1α and HNF1ß provided detectable LTR transcript, whereas shRNA-mediated silencing of HNF1ß impaired transcription. Treating cells with 5'-aza-2'-deoxycytidine (5AZA) strongly reduced expression, without affecting HNF1α/ß, despite the lack of CpG islands in the LTR and proximal sequences. By electrophoresis mobility shift and luciferase reporter assays, the LTR promoter binding and activity did not correlate with the amounts of LTR transcript expressed in the cells and depended on the levels of the transcription factors. We conclude that HNF1α/ß are necessary but insufficient to activate and regulate B3GALT5 LTR transcription, which depends on unknown regulatory elements that are active when methylated and located outside of and far from the LTR promoter.

Natural grape extracts regulate colon cancer cells malignancy.

Natural dietary components are evolutionary-selected molecules able to control inflammation and cancerous transformation and progression. Because many studies assessed the beneficial properties of key molecules extracted from grapes, we aimed at investigating the properties of Liofenol™, a natural red wine lyophilized extract, devoid of alcohol and composed by a miscellaneous of components (polyphenols, flavonoids, anthocyanins). We proved that the colon cancer cell line HCT116 responded to Liofenol™ treatment by reducing their proliferation, in association with an increase of p53 and p21 cell cycle gate keepers. Liofenol™ increased dihydroceramides, sphingolipid mediators involved in cell cycle arrest and reduced proliferation rate. We observed a strong induction of antioxidant response, with the activation of the transcriptional factor Nrf2, involved in redox homeostasis and differentiation, without altering tumor sensitivity to chemotherapy. Liofenol™ induced an important morphology change in HCT116 cells, migration inhibition, undifferentiated stem/stem-like cells markers downregulation, and E-cadherin downregulation, interested in epithelia to mesenchymal malignant transition. We conclude that lyophilized grape extract, at dose comparable to putative dietary doses, can activate molecular pathways, involving Nrf2 signaling and the modulation of structural and signaling sphingolipid mediators that cooperate in promoting differentiation and reducing proliferation of digestive tract cancer cells.

Role of Sphingolipids in the Pathobiology of Lung Inflammation.

Sphingolipid bioactivities in the respiratory airways and the roles of the proteins that handle them have been extensively investigated. Gas or inhaled particles or microorganisms come into contact with mucus components, epithelial cells, blood barrier, and immune surveillance within the airways. Lung structure and functionality rely on a complex interplay of polar and hydrophobic structures forming the surfactant layer and governing external-internal exchanges, such as glycerol-phospholipids sphingolipids and proteins. Sphingolipids act as important signaling mediators involved in the control of cell survival and stress response, as well as secreted molecules endowed with inflammation-regulatory activities. Most successful respiratory infection and injuries evolve in the alveolar compartment, the critical lung functional unit involved in gas exchange. Sphingolipid altered metabolism in this compartment is closely related to inflammatory reaction and ceramide increase, in particular, favors the switch to pathological hyperinflammation. This short review explores a few mechanisms underlying sphingolipid involvement in the healthy lung (surfactant production and endothelial barrier maintenance) and in a selection of lung pathologies in which the impact of sphingolipid synthesis and metabolism is most apparent, such as acute lung injury, or chronic pathologies such as cystic fibrosis and chronic obstructive pulmonary disease.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats.

BACKGROUND: Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage. STUDY DESIGN AND METHODS: The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage. We exposed rats to exchange transfusion (ET; 50% blood volume and isovolemic replacement with Hextend [negative colloid control], MP4OX [PEGylated HBOC with high oxygen affinity], and ααHb [αα-cross-linked HBOC with low oxygen affinity; n=4-6/group]). Sham rats acted as control. Animals were euthanized at 2, 6, and 12 hours after ET; brain tissue was harvested and processed for analysis. RESULTS: In MP4OX animals, the number of neurons that overexpressed the hypoxia-inducible factor (HIF)-1α was higher than in ααHb, particularly at the early time points. In addition, MP4OX was associated with greater phosphorylation of protein kinase B (Akt), a well-known cytoprotective factor. Indeed, the degree of apoptosis, measured as terminal deoxynucleotidyl transferase-positive neurons and caspase-3 cleavage, ranked in order of MP4OX < Hextend < ααHb. CONCLUSION: Even though both HBOCs showed increased levels of HIF-1α compared to shams or Hextend-treated animals, differences in signaling events resulted in very different outcomes for the two HBOCs. ααHb-treated brain tissue showed significant neuronal damage, measured as apoptosis. This was in stark contrast to the protection seen with MP4OX, apparently due to recruitment of Akt and neuronal specific HIF-1α pathways.

In vivo up-regulation of the unfolded protein response after hypoxia.

BACKGROUND: Low oxygen (O2) availability, a condition called hypoxia, has different and profound consequences in tissues and organs. Besides the hypoxia-inducible response, mammalian cells induce a coordinated cytoprotective pathway called Unfolded Protein Response (UPR). We studied the molecular basis of UPR and apoptosis in animal models exposed to different hypoxic stresses and assessed the ability of liver and myocardium to respond to low oxygen by activating different arms of the UPR according to the severity of the insults in a tissue specific manner. METHODS: We assessed the levels of several UPR markers in hypoxic animals by Real Time PCR and Western blotting. RESULTS: While the hepatocytes activate the apoptotic pathway mediated, in part, by CHOP and p-JNK, we could not detect an UPR-dependent apoptosis in myocytes. Moreover, severe hypoxia results in ATF4 translation, and induction of CHOP and GADD34 transcripts in liver, by contrast in the myocardium, the ATF4-CHOP-GADD34 signaling pathway is not detectably activated. GENERAL SIGNIFICANCE: Comparison of several UPR markers in liver and myocardium enabled to underscore the ability of hepatocytes and myocites to selectively activate and fine tune the UPR signaling pathway during hypoxia in vivo.

Dysregulated lipid metabolism in lymphangioleiomyomatosis pathogenesis as a paradigm of chronic lung diseases.

A chronic inflammatory condition characterizes various lung diseases. Interestingly, a great contribution to inflammation is made by altered lipids metabolism, that can be caused by the deregulation of the mammalian target of rapamycin complex-1 (mTORC1) activity. There is evidence that one of mTOR downstream effectors, the sterol regulatory element-binding protein (SREBP), regulates the transcription of enzymes involved in the de novo fatty acid synthesis. Given its central role in cell metabolism, mTOR is involved in several biological processes. Among those, mTOR is a driver of senescence, a process that might contribute to the establishment of chronic lung disease because the characteristic irreversible inhibition of cell proliferation, associated to the acquisition of a pro-inflammatory senescence-associated secretory phenotype (SASP) supports the loss of lung parenchyma. The deregulation of mTORC1 is a hallmark of lymphangioleiomyomatosis (LAM), a rare pulmonary disease predominantly affecting women which causes cystic remodeling of the lung and progressive loss of lung function. LAM cells have senescent features and secrete SASP components, such as growth factors and pro-inflammatory molecules, like cancer cells. Using LAM as a paradigm of chronic and metastatic lung disease, here we review the published data that point out the role of dysregulated lipid metabolism in LAM pathogenesis. We will discuss lipids' role in the development and progression of the disease, to hypothesize novel LAM biomarkers and to propose the pharmacological regulation of lipids metabolism as an innovative approach for the treatment of the disease.

Ceramide present in cholangiocarcinoma-derived extracellular vesicle induces a pro-inflammatory state in monocytes.

Cholangiocarcinoma (CCA) is a rare cancer characterized by a global increasing incidence. Extracellular vesicles (EV) contribute to many of the hallmarks of cancer through transfer of their cargo molecules. The sphingolipid (SPL) profile of intrahepatic CCA (iCCA)-derived EVs was characterized by liquid chromatography-tandem mass spectrometry analysis. The effect of iCCA-derived EVs as mediators of inflammation was assessed on monocytes by flow cytometry. iCCA-derived EVs showed downregulation of all SPL species. Of note, poorly-differentiated iCCA-derived EVs showed a higher ceramide and dihydroceramide content compared with moderately-differentiated iCCA-derived EVs. Of note, higher dihydroceramide content was associated with vascular invasion. Cancer-derived EVs induced the release of pro-inflammatory cytokines in monocytes. Inhibition of synthesis of ceramide with Myriocin, a specific inhibitor of the serine palmitoyl transferase, reduced the pro-inflammatory activity of iCCA-derived EVs, demonstrating a role for ceramide as mediator of inflammation in iCCA. In conclusion, iCCA-derived EVs may promote iCCA progression by exporting the excess of pro-apoptotic and pro-inflammatory ceramides.

An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells.

The main concerns in targeted "sphingolipidomics" are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences.