A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist\u27s toolbox. Keywords: biorepository; cancer, ceramide; lipidomics; lung adenocarcinoma; lung squamous cell carcinoma; mass spectrometry; non-small cell lung cancer. Copyright © 2020 Rohrbach et al

Fenretinide Causes Emphysema, Which Is Prevented by Sphingosine 1-Phoshate

Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs

Sphingosine-1-phosphate signalling drives an angiogenic transcriptional programme in diffuse large B cell lymphoma

Although the over-expression of angiogenic factors is reported in diffuse large B-cell lymphoma (DLBCL), the poor response to anti-VEGF drugs observed in clinical trials suggests that angiogenesis in these tumours might be driven by VEGF-independent pathways. We show that sphingosine kinase-1 (SPHK1), which generates the potent bioactive sphingolipid sphingosine-1-phosphate (S1P), is over-expressed in DLBCL. A meta-analysis of over 2000 cases revealed that genes correlated with SPHK1 mRNA expression in DLBCL were significantly enriched for tumour angiogenesis meta-signature genes; an effect evident in both major cell of origin (COO) and stromal subtypes. Moreover, we found that S1P induces angiogenic signalling and a gene expression programme that is present within the tumour vasculature of SPHK1-expressing DLBCL. Importantly, S1PR1 functional antagonists, including Siponimod, and the S1P neutralising antibody, Sphingomab, inhibited S1P signalling in DLBCL cells in vitro. Furthermore, Siponimod, also reduced angiogenesis and tumour growth in an S1P-producing mouse model of angiogenic DLBCL. Our data define a potential role for S1P signalling in driving an angiogenic gene expression programme in the tumour vasculature of DLBCL and suggest novel opportunities to target S1P-mediated angiogenesis in patients with DLBCL

A Rapid and Adaptable Lipidomics Method for Quantitative UPLC-mass Spectrometric Analysis of Phosphatidylethanolamine and Phosphatidylcholine in Vitro, and in Cells

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are highly prevalent phospholipids in mammalian membranes. There are currently no methods for detection of minute levels of these phospholipids or simultaneously with products of the utilization of these phospholipid substrates by phospholipase A2 (PLA2) enzymes. To examine the substrate utilization of PE and PC by PLA2, we developed a method to accurately detect and measure specific forms of PE and PC as low as 50 femtomoles. Validation of this method consisted of an enzymatic assay to monitor docosahexaenoic acid and arachidonic acid release from the hydrolysis of PE and PC by group IV phospholipase A2 (cPLA2α) coupled to the generation of Lyso-PE (LPE) and Lyso-PC (LPC). In addition, the PE and PC profiles of RAW 264.7 macrophages were monitored with zymosan/lipopolysaccharide-treatment. Finally, genetic validation for the specificity of the method consisted of the downregulation of two biosynthetic enzymes responsible for the production of PE and PC, choline kinase A (CHKA) and ethanolamine kinase 1 (ETNK1). This new UPLC ESI-MS/MS method provides accurate and highly sensitive detection of PE and PC species containing AA and DHA allowing for the specific examination of the substrate utilization of these phospholipids by PLA2in vitro and in cells

Immunohistochemical analysis of HIF-1α.

<p>The number of the HIF-1α positive cells is counted in vehicle control, S1P, fenretinide, and fenretinide with S1P treated rat lungs. Then they were referenced to the total length of the alveolar perimeters (A). Representative images of immunohistochemical staining of HIF-1α are shown (B). Bars = 50 µm, Original Magnification x100 Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P, TLAP =  total length of the alveolar perimeters.</p

Effect of exogenous S1P on phospho-sphingosine kinase 1 (pSphk1) and the balance of dihydroceramide and dihydro-S1P (n = 4, each for group).

<p>Densitometric data and representative protein bands are shown. Phospho-Sphk1 was adjusted to Sphk1 (A). The balance of dihydroceramide/dihydro-S1P was investigated by mass spectrometric analysis (B). Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P.</p

The schematic represents the concept of how fenretinide and exogenously administered sphingosine 1 phosphate (S1P) affect the adult lung structure maintenance.

<p>Administration of fenretinide changes the ceramide/S1P ratio by increasing the generation of ceramide, which in turn decreases HIF-1α and VEGF expression in the lung. Reduction of HIF-1alpha and VEGF -both central to the adult lung structure maintenance program- causes emphysema (A). Exogenously administered S1P increases the amount of intracellular S1P via sphingosine kinase 1 (Sphk1) activation (B), thus protecting against lung cell apoptosis.</p

Morphological analysis of the lungs from fenretinide challenged rats treated with or without sphingosine 1-phosphate (S1P).

<p>When compared to control lungs (A), the low power magnification shows air-space enlargement in the chronic fenretinide treated rat lungs (B). Examples of data based on concurrent S1P administration and S1P alone treatment are shown in (C) and (D), respectively. Quantitative analysis is shown in (E). Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P Bars = 250 µm, Original Magnification x40.</p

A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell-dependent mouse model of allergic asthma

Background: Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cells and IgE-dependent murine model of allergic asthma has not yet been examined. Objective: We used an isoenzyme-specific SphK1 inhibitor,SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. Methods: Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. Results: SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor-kB (NF-kB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13,IFN-g, and TNF-a and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-kB in the lungs.Fil: Price, Megan M.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Oskeritzian, Carole A.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Falanga, Yves T.. Virginia Commonwealth University. Department of Microbiology and Immunology; Estados UnidosFil: Harikumar, Kuzhuvelil B.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Allegood, Jeremy C.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Alvarez, Sergio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Conrad, Daniel. Virginia Commonwealth University. Department of Biology; Estados UnidosFil: Ryan, John J.. Virginia Commonwealth University. Department of Microbiology and Immunology; Estados UnidosFil: Milstien, Sheldon. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Spiegel, Sarah. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados Unido