High glucose exposure drives intestinal barrier dysfunction by altering its morphological, structural and functional properties.

High dietary glucose consumption and hyperglycemia can result in chronic complications. Several studies suggest that high glucose (HG) induces dysfunction of the intestinal barrier. However, the precise changes remain unclear. In our study, we used in vitro models composed of Caco-2 and/or HT29-MTX cells in both monoculture and co-culture to assess the effects of long-term HG exposure on the morphological, structural, and functional properties of the intestinal barrier. Cells were grown in medium containing normal physiologic glucose (NG, 5.5 mM) or a clinically relevant HG (25 mM) concentration until 21 days. Results demonstrated that HG induced morphological changes, with the layers appearing denser and less organized than under physiological conditions, which is in accordance with the increased migration capacity of Caco-2 cells and proliferation properties of HT29-MTX cells. Although we mostly observed a small decrease in mRNA and protein expressions of three junction proteins (ZO-1, OCLN and E-cad) in both Caco-2 and HT29-MTX cells cultured in HG medium, confocal microscopy showed that HG induced a remarkable reduction in their immunofluorescence intensity, triggering disruption of their associated structural network. In addition, we highlighted that HG affected different functionalities (permeability, mucus production and alkaline phosphatase activity) of monolayers with Caco-2 and HT29-MTX cells. Interestingly, these alterations were stronger in co-culture than in monoculture, suggesting a cross-relationship between enterocytes and goblet cells. Controlling hyperglycemia remains a major therapeutical method for reducing damage to the intestinal barrier and improving therapies.

Sphingolipid distribution at mitochondria-associated membranes (MAMs) upon induction of apoptosis.

The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis.

Secretion of Acid Sphingomyelinase and Ceramide by Endothelial Cells Contributes to Radiation-Induced Intestinal Toxicity.

Ceramide-induced endothelial cell apoptosis boosts intestinal stem cell radiosensitivity. However, the molecular connection between these two cellular compartments has not been clearly elucidated. Here we report that ceramide and its related enzyme acid sphingomyelinase (ASM) are secreted by irradiated endothelial cells and act as bystander factors to enhance the radiotoxicity of intestinal epithelium. Ceramide and the two isoforms of ASM were acutely secreted in the blood serum of wild-type mice after 15 Gy radiation dose, inducing a gastrointestinal syndrome. Interestingly, serum ceramide was not enhanced in irradiated ASMKO mice, which are unable to develop intestinal failure injury. Because ASM/ceramide were secreted by primary endothelial cells, their contribution was studied in intestinal epithelium dysfunction using coculture of primary endothelial cells and intestinal T84 cells. Adding exogenous ASM or ceramide enhanced epithelial cell growth arrest and death. Conversely, blocking their secretion by endothelial cells using genetic, pharmacologic, or immunologic approaches abolished intestinal T84 cell radiosensitivity. Use of enteroid models revealed ASM and ceramide-mediated deleterious mode-of-action: when ceramide reduced the number of intestinal crypt-forming enteroids without affecting their structure, ASM induced a significant decrease of enteroid growth without affecting their number. Identification of specific and different roles for ceramide and ASM secreted by irradiated endothelial cells opens new perspectives in the understanding of intestinal epithelial dysfunction after radiation and defines a new class of potential therapeutic radiomitigators. SIGNIFICANCE: This study identifies secreted ASM and ceramide as paracrine factors enhancing intestinal epithelial dysfunction, revealing a previously unknown class of mediators of radiosensitivity.

Plasma membrane reorganization links acid sphingomyelinase/ceramide to p38 MAPK pathways in endothelial cells apoptosis.

The p38 MAPK signaling pathway is essential in the cellular response to stress stimuli, in particular in the endothelial cells that are major target of external stress. The importance of the bioactive sphingolipid ceramide generated by acid sphingomyelinase is also firmly established in stress-induced endothelial apoptotic cell death. Despite a suggested link between the p38 MAPK and ceramide pathways, the exact molecular events of this connection remain elusive. In the present study, by using two different activators of p38 MAPK, namely anisomycin and ionizing radiation, we depicted how ceramide generated by acid sphingomyelinase was involved in p38 MAPK-dependent apoptosis of endothelial cells. We first proved that both anisomycin and ionizing radiation conducted to apoptosis through activation of p38 MAPK in human microvascular endothelial cells HMEC-1. We then found that both treatments induced activation of acid sphingomyelinase and the generation of ceramide. This step was required for p38 MAPK activation and apoptosis. We finally showed that irradiation, as well as treatment with exogenous C16-ceramide or bacterial sphingomyelinase, induced in endothelial cells a deep reorganization of the plasma membrane with formation of large lipid platforms at the cell surface, leading to p38 MAPK activation and apoptosis in endothelial cells. Altogether, our results proved that the plasma membrane reorganization leading to ceramide production is essential for stress-induced activation of p38 MAPK and apoptosis in endothelial cells and established the link between the acid sphingomyelinase/ceramide and p38 MAPK pathways.

Plasma ceramide, a real-time predictive marker of pulmonary and hepatic metastases response to stereotactic body radiation therapy combined with irinotecan.

BACKGROUND AND PURPOSES: Early biomarkers of tumour response are needed to discriminate between responders and non-responders to radiotherapy. We evaluated the ability of ceramide, a bioactive sphingolipid, to predict tumour sensitivity in patients treated by hypofractionated stereotactic body radiation therapy (SBRT) combined with irinotecan chemotherapy. MATERIALS AND METHODS: Plasma levels of total ceramide and of its subspecies were measured before and during treatment in 35 patients with liver and lung oligometastases of colorectal cancer included in a phase II trial. Cer levels were quantified by LC-ESI-MS/MS and compared to tumour volume response evaluated one year later by CT-scan. RESULTS: Pretreatment plasma ceramide levels were not indicative of tumour response. Nevertheless, the levels of total ceramide and of its 4 main subspecies were significantly higher at days 3 and 10 of treatment in objective responders than in non-responders. According to Kaplan-Meier curves, almost complete tumour control was achieved at 1year in patients with increased total ceramide levels whereas 50% of patients with decreased levels experienced an increase in tumour volume. CONCLUSIONS: Total plasma ceramide is a promising biomarker of tumour response to SBRT combined with irinotecan that should enable to segregate patients with high risk of tumour escape.

Inhibition of tumor angiogenesis by globotriaosylceramide immunotargeting.

Current antiangiogenic immunotherapeutic strategies mainly focus on the blockade of circulating cytokines or receptors that are overexpressed by endothelial cells. We proposed globotriaosylceramide (Gb3) as a viable alternative target for antiangiogenic therapies. In this setting, we developed an anti-Gb3 antibody and validated its therapeutic efficacy in metastatic tumor models.

Anti-Gb3 monoclonal antibody inhibits angiogenesis and tumor development.

Inhibiting the growth of tumor vasculature represents one of the relevant strategies against tumor progression. Between all the different pro-angiogenic molecular targets, plasma membrane glycosphingolipids have been under-investigated. In this present study, we explore the anti-angiogenic therapeutic advantage of a tumor immunotherapy targeting the globotriaosylceramide Gb3. In this purpose, a monoclonal antibody against Gb3, named 3E2 was developed and characterized. We first demonstrate that Gb3 is over-expressed in proliferative endothelial cells relative to quiescent cells. Then, we demonstrate that 3E2 inhibits endothelial cell proliferation in vitro by slowing endothelial cell proliferation and by increasing mitosis duration. Antibody 3E2 is further effective in inhibiting ex vivo angiogenesis in aorta ring assays. Moreover, 3E2 treatment inhibits NXS2 neuroblastoma development and liver metastases spreading in A/J mice. Immunohistology examination of the NXS2 metastases shows that only endothelial cells, but not cancer cells express Gb3. Finally, 3E2 treatment diminishes tumor vessels density, proving a specific therapeutic action of our monoclonal antibody to tumor vasculature. Our study demonstrates that Gb3 is a viable alternative target for immunotherapy and angiogenesis inhibition.

Enhancement of neutral lipid productivity in the microalga Isochrysis affinis Galbana (T-Iso) by a mutation-selection procedure.

Microalgae offer a high potential for energetic lipid storage as well as high growth rates. They are therefore considered promising candidates for biofuel production, with the selection of high lipid-producing strains a major objective in projects on the development of this technology. We developed a mutation-selection method aimed at increasing microalgae neutral lipid productivity. A two step method, based on UVc irradiation followed by flow cytometry selection, was applied to a set of strains that had an initial high lipid content and improvement was assessed by means of Nile-red fluorescence measurements. The method was first tested on Isochrysis affinis galbana (T-Iso). Following a first round of mutation-selection, the total fatty acid content had not increased significantly, being 262 ± 21 mgTFA (gC)-1 for the wild type (WT) and 269 ± 49 mgTFA (gC)-1 for the selected population (S1M1). Conversely, fatty acid distribution among the lipid classes was affected by the process, resulting in a 20% increase for the fatty acids in the neutral lipids and a 40% decrease in the phospholipids. After a second mutation-selection step (S2M2), the total fatty acid content reached 409 ± 64 mgTFA (gC)-1 with a fatty acid distribution similar to the S1M1 population. Growth rate remained unaffected by the process, resulting in a 80% increase for neutral lipid productivity.