sphingosine-kinase and Reperfusion-Injury

Topics: Animals; Apoptosis; Ceramides; Humans; Lysophospholipids; Monoamine Oxidase; Myocardial Reperfusion Injury; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Sphingosine

The lysosphingolipid sphingosine 1-phosphate (S1P) is a component of HDL. Findings from a growing number of studies indicate that S1P is a mediator of many of the cardiovascular effects of HDL, including the ability to promote vasodilation, vasoconstriction, and angiogenesis, protect against ischemia/reperfusion injury, and inhibit/reverse atherosclerosis. These latter cardioprotective effects are being shown to involve the S1P-mediated suppression of inflammatory processes, including reduction of the endothelial expression of monocyte and lymphocyte adhesion molecules, decreased recruitment of polymorphonuclear cells to sites of infarction, and blocking of cardiomyocyte apoptosis after myocardial infarction. This review article summarizes the evidence that S1P as a component of HDL serves to regulate vascular cell and lymphocyte behaviors associated with cardiovascular (patho)physiology.

Topics: Animals; Atherosclerosis; ATP-Binding Cassette Transporters; Cardiovascular System; Cell Movement; Cystic Fibrosis Transmembrane Conductance Regulator; Endothelial Cells; Humans; Immunologic Factors; Lipoproteins, HDL; Lysophospholipids; Muscle, Smooth, Vascular; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction; Sphingosine; Vasoconstriction; Vasodilation

Microglial hyperactivation mediated by sphingosine kinase 1/sphingosine-1-phosphate (SphK1/S1P) signalling and the consequent inflammatory mediator production serve as the key drivers of cerebral ischaemia-reperfusion injury (CIRI). Although SphK1 reportedly controls autophagy and microglial activation, it remains uncertain as to whether SphK1 is similarly capable of regulating damage mediated by CIRI-activated microglia. In the current study, we adopted both in vitro oxygen-glucose deprivation reperfusion (OGDR) models and in vivo rat models of focal CIRI to ascertain this possibility. It was found that CIRI upregulated SphK1 and induced autophagy in microglia, while inhibiting these changes significantly impaired to prevented neuronal apoptosis. Results of mechanistic investigation revealed that SphK1 promoted autophagy via the tumour necrosis factor receptor associated factor 2 (TRAF2) pathway. Altogether, our findings unfolded to reveal a novel mechanism, whereby SphK1-induced autophagy in microglia contributed to the pathogenesis of CIRI, potentially highlighting novel avenues for future therapeutic intervention in ischaemic stroke patients.

Topics: Animals; Autophagy; Brain Ischemia; Microglia; Phosphotransferases (Alcohol Group Acceptor); Rats; Reperfusion; Reperfusion Injury; Stroke

Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury.. We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting.. DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR).. This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.

Topics: Animals; Coenzyme A; Ferroptosis; Flavonols; Glucose; Infarction, Middle Cerebral Artery; Iron; Ligases; Lipids; Mammals; Oxygen; Phosphatidylethanolamine Binding Protein; Phospholipid Hydroperoxide Glutathione Peroxidase; Phosphotransferases (Alcohol Group Acceptor); Rats; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; TOR Serine-Threonine Kinases; Water

Topics: Animals; Liver; Lysophospholipids; Microcirculation; Phosphotransferases (Alcohol Group Acceptor); Rats; Reperfusion Injury; Signal Transduction; Sphingosine

Sphingosine kinase 1 (Sphk1) and the signaling molecule sphingosine-1-phosphate (S1P) are known to be key regulators of a variety of important biological processes, such as neovascularization. Nitric oxide (NO) is also known to play a role in vasoactive properties, whether Sphk1/S1P signaling is able to alter angiogenesis in the context of cerebral ischemia-reperfusion injury (IRI), and whether such activity is linked with NO production, however, remains uncertain.. We used immunofluorescence to detect the expression of Sphk1 and NOS in cerebral epithelial cells (EC) after IR or oxygen-glucose deprivation (OGDR). Western blotting was used to detect the Sphk1 and NOS protein levels in brain tissues or HBMECs. Adenovirus transfection was used to inhibit Sphk1 and NOS. An NO kit was used to detect NO contents in brain tissues and epithelial cells. Tube formation assays were conducted to measure angiogenesis.. We determined that EC used in a model of cerebral IRI expressed Sphk1, and that inhibiting this expression led to decreased expression of two isoforms of NO synthase (eNOS and iNOS), as well as to decrease neovascularization density and NO production following injury. In HBMECs, knocking down Sphk1 markedly reduced NO production owing to reduced eNOS activity, and inhibiting eNOS directly similarly decreased NO production in a manner which could be reversed via exogenously treating cells with S1P. We further found that knocking down Sphk1 reduced HBMEC eNOS expression, in addition to decreasing the adhesion, migration, and tube formation abilities of these cells under OGDR conditions.. Based on these results, we therefore postulate that Sphk1/S1P signaling is able to mediate angiogenesis following cerebral IRI via the regulation of eNOS activity and NO production. As such, targeting these pathways may potentially represent a novel means of improving patient prognosis in those suffering from cerebral IRI.

Topics: Animals; Brain Ischemia; Cells, Cultured; Humans; Lysophospholipids; Male; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Reperfusion Injury; Sphingosine

Endoplasm reticulum stress and inflammation response have been found to be linked to cerebral ischemia-reperfusion (IR) injury. Sphingosine kinase 1 (SPHK1) has been reported to be a novel endoplasm reticulum regulator. The aim of our study is to figure out the role of SPHK1 in cerebral IR injury and verify whether it has an ability to regulate inflammation and endoplasm reticulum stress. Hydrogen peroxide was used to induce cerebral IR injury. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, western blots, and immunofluorescence were used to measure the alterations of cell viability, inflammation response, and endoplasm reticulum stress. The results demonstrated that after exposure to hydrogen peroxide, cell viability was reduced whereas SPHK1 expression was significantly elevated. Knockdown of SPHK1 attenuated hydrogen peroxide-mediated cell death and reversed cell viability. Our data also demonstrated that SPHK1 deletion reduced endoplasm reticulum stress and alleviated inflammation response in hydrogen peroxide-treated cells. In addition, we also found that SHPK1 modulated endoplasm reticulum stress and inflammation response to through the NF-κB signaling pathway. Inhibition of NF-κB signaling pathway has similar results when compared with the cells with SPHK1 deletion. Altogether, our results demonstrated that SPHK1 upregulation, induced by hydrogen peroxide, is responsible for cerebral IR injury through inducing endoplasm reticulum stress and inflammation response in a manner working through the NF-κB signaling pathway. This finding provides new insight into the molecular mechanism to explain the neuron death induced by cerebral IR injury.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; Inflammation; Mice; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction

Nonalcoholic fatty liver (NAFL) is emerging as a leading risk factor of hepatic ischemia/reperfusion (I/R) injury lacking of effective therapy. Lipid dyshomeostasis has been implicated in the hepatopathy of NAFL. Herein, we investigate the bioactive lipids that critically regulate I/R injury in NAFL.. Lipidomics were performed to identify dysregulated lipids in mouse and human NAFL with I/R injury. The alteration of corresponding lipid-metabolizing genes was examined. The effects of the dysregulated lipid metabolism on I/R injury in NAFL were evaluated in mice and primary hepatocytes.. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) were uncovered to be substantially activated by I/R in mouse NAFL. Sphingosine kinase 1 (Sphk1) was found to be essential for hepatic S1P generation in response to I/R in hepatocytes of NAFL mice. Sphk1 knockdown inhibited the hepatic S1P rise while accumulating ceramides in hepatocytes of NAFL mice, leading to aggressive hepatic I/R injury with upregulation of oxidative stress and increase of reactive oxygen species (ROS). In contrast, administration of exogenous S1P protected hepatocytes of NAFL mice from hepatic I/R injury. Clinical study revealed a significant activation of S1P generation by I/R in liver specimens of NAFL patients. In vitro studies on the L02 human hepatocytes consolidated that inhibiting the generation of S1P by knocking down SPHK1 exaggerated I/R-induced damage and oxidative stress in human hepatocytes of NAFL.. Generation of S1P by SPHK1 is important for protecting NAFL from I/R injury, which may serve as therapeutic targets for hepatic I/R injury in NAFL.

Topics: Animals; Hepatocytes; Humans; Ischemia; Lysophospholipids; Mice; Non-alcoholic Fatty Liver Disease; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Sphingosine

Hepatic ischemia/reperfusion (I/R) injury remains a significant problem in clinical practice. Sphingosine kinase 1 (SphK1) phosphorylates sphingosine to sphingosine-1-phosphate (S1P) which participates in multiple bioactive processes. However, little is known about the role of SphK1 in hepatic I/R injury. This study aimed to investigate the effect of SphK1 knockout on liver I/R injury and to explore underlying mechanisms.. SphK1 knockout and wild type mice were subjected to 70% partial hepatic I/R. Serum alanine aminotransferase was determined to indicate the degree of liver damage. Hematoxylin-eosin staining and TUNEL assay were used to assess histological changes and hepatocellular apoptosis, respectively. Immunohistochemistry was performed to detect the expression and translocation of phosphorylated p65 and signal transducer and activator of transcription 3 (STAT3). Western blotting was used to determine the expression of S1P receptor 1 (S1PR1), phosphorylated p65 and STAT3. Real-time PCR was used to demonstrate the changes of proinflammatory cytokines. Oxidative stress markers were also determined through biochemical assays.. SphK1 knockout significantly ameliorated I/R-induced liver damage, mitigated liver tissue necrosis and apoptosis compared with wild type control. I/R associated inflammation was alleviated in SphK1 knockout mice as demonstrated by attenuated expression of S1PR1 and reduced phosphorylation of nuclear factor kappa B p65 and STAT3. The proinflammatory cytokines interleukin-1β, interleukin-6 and tumor necrosis factor-α were also inhibited by SphK1 genetic deletion. The oxidative stress markers were lower in SphK1 knockout mice after I/R injury than wild type mice.. Knockout of SphK1 significantly alleviated damage after hepatic I/R injury, possibly through inhibiting inflammation and oxidative stress. SphK1 may be a novel and potent target in clinical practice in I/R-related liver injury.

Topics: Animals; Apoptosis; Cytokines; Disease Models, Animal; Gene Knockout Techniques; Hepatitis; Inflammation Mediators; Liver; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction; Sphingosine-1-Phosphate Receptors; STAT3 Transcription Factor; Transcription Factor RelA

New strategies are required to combat neuronal ischemia-reperfusion injuries. K6PC-5 is a novel sphingosine kinase 1 (SphK1) activator whose potential activity in neuronal cells has not yet been tested.. Cell survival and necrosis were assessed with a Cell Counting Kit-8 assay and lactate dehydrogenase release assay, respectively. Mitochondrial depolarization was tested by a JC-1 dye assay. Expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling components were examined by quantitative real-timePCR and western blotting.. K6PC-5 protected SH-SY5Y neuronal cells and primary murine hippocampal neurons from oxygen glucose deprivation/re-oxygenation (OGDR). K6PC-5 activated SphK1, and SphK1 knockdown by targeted short hairpin RNA (shRNA) almost completely abolished K6PC-5-induced neuronal cell protection. Further work showed that K6PC-5 inhibited OGDR-induced programmed necrosis in neuronal cells. Importantly, K6PC-5 activated Nrf2 signaling, which is downstream of SphK1. Silencing of Nrf2 by targeted shRNA almost completely nullified K6PC-5-mediated neuronal cell protection against OGDR.. K6PC-5 activates SphK1-Nrf2 signaling to protect neuronal cells from OGDR. K6PC-5 might be a promising neuroprotective strategy for ischemia-reperfusion injuries.

Topics: Animals; Apoptosis; Cell Line; Cell Survival; Cells, Cultured; Enzyme Activators; Glucose; Humans; Mice; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Oxygen; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction

This study aimed to evaluate the effects of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury, as well as the underlying mechanisms.. Exosomes derived from hiPSC-MSCs were isolated and characterized both biochemically and biophysically. hiPSC-MSCs-Exo were injected systemically into a murine ischemia/reperfusion injury model via the inferior vena cava, and then the therapeutic effects were evaluated. The serum levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Primary hepatocytes and human hepatocyte cell line HL7702 were used to test whether exosomes could induce hepatocytes proliferation in vitro. In addition, the expression levels of proliferation markers (proliferation cell nuclear antigen, PCNA; Phosphohistone-H3, PHH3) were measured by immunohistochemistry and Western blot. Moreover, SK inhibitor (SKI-II) and S1P1 receptor antagonist (VPC23019) were used to investigate the role of sphingosine kinase and sphingosine-1-phosphate-dependent pathway in the effects of hiPSC-MSCs-Exo on hepatocytes.. hiPSCs were efficiently induced into hiPSC-MSCs that had typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 100 to 200 nm and expressed exosome markers (Alix, CD63 and CD81). After hiPSC-MSCs-Exo administration, hepatocyte necrosis and sinusoidal congestion were markedly suppressed in the ischemia/reperfusion injury model, with lower histopathological scores. The levels of hepatocyte injury markers AST and ALT were significantly lower in the treatment group compared to control, and the expression levels of proliferation markers (PCNA and PHH3) were greatly induced after hiPSC-MSCs-Exo administration. Moreover, hiPSC-MSCs-Exo also induced primary hepatocytes and HL7702 cells proliferation in vitro in a dose-dependent manner. We found that hiPSC-MSCs-Exo could directly fuse with target hepatocytes or HL7702 cells and increase the activity of sphingosine kinase and synthesis of sphingosine-1-phosphate (S1P). Furthermore, the inhibition of SK1 or S1P1 receptor completely abolished the protective and proliferative effects of hiPSC-MSCs-Exo on hepatocytes, both in vitro and in vivo.. Our results demonstrated that hiPSC-MSCs-Exo could alleviate hepatic I/R injury via activating sphingosine kinase and sphingosine-1-phosphate pathway in hepatocytes and promote cell proliferation. These findings represent a novel mechanism that potentially contributes to liver regeneration and have important implications for new therapeutic approaches to acute liver disease.

Topics: Animals; Cell Line; Cell Proliferation; Cells, Cultured; Exosomes; Hepatocytes; Humans; Induced Pluripotent Stem Cells; Liver; Lysophospholipids; Male; Mesenchymal Stem Cells; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction; Sphingosine

Stroke is associated with high morbidity and mortality, and much remains unknown about the injury-related mechanisms that occur following reperfusion. This study aimed to explore the roles of Toll-like receptor 2 (TLR2) and sphingosine kinase 1 (Sphk1) in microglial cells in inflammatory responses induced by cerebral ischemia/reperfusion (I/R). For this purpose, C57BL/6 mice were randomly divided into 4 groups as follows: the sham-operated group, the I/R group, the I/R group treated with TLR2 antibody, and the I/R group treated with N,N-dimethylsphingosine. Focal cerebral I/R was induced by middle cerebral artery occlusion. Double-labeling immunofluorescence was used to observe the protein expression of TLR2 and Sphk1 in the ischemic brain tissue. Quantitative polymerase chain reaction was performed to determine the mRNA levels of TLR2 and Sphkl in ischemic brain tissue. Enzyme-linked immunosorbent assay was carried out to detect the protein contents of interleukin (IL)-1β, tumor necrosis factor-α (TNF‑α), IL-17 and IL-23 in ischemic brain tissue. The results revealed that I/R upregulated TLR2 and Sphk1 expression in microglial cells, and the inhibition of either TLR2 or Sphk1 inhibited the expression of the pro-inflammatory cytokines, IL-1β, TNF-α, IL-17 and IL-23. Notably, the inhibition of TLR2 activity also decreased Sphk1 expression. These results thus indicate that the activation of microglial cells, via a TLR2→Sphk1→pro-inflammatory cytokine (IL-1β, TNF-α, IL-17 and IL-23) pathway, may participate in I/R injury.

Topics: Animals; Brain; Brain Ischemia; Cytokines; Inflammation; Male; Mice; Mice, Inbred C57BL; Microglia; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Toll-Like Receptor 2

Renal ischemia-reperfusion is a main cause of acute kidney injury (AKI), which is associated with high mortality. Here we show that extracellular vesicles (EVs) secreted from hiPSC-MSCs play a critical role in protection against renal I/R injury. hiPSC-MSCs-EVs can fuse with renal cells and deliver SP1 into target cells, subsequently active SK1 expression and increase S1P formation. Chromatin immunoprecipitation (ChIP) analyses and luciferase assay were used to confirm SP1 binds directly to the SK1 promoter region and promote promoter activity. Moreover, SP1 inhibition (MIT) or SK1 inhibition (SKI-II) completely abolished the renal protective effect of hiPSC-MSCs-EVs in rat I/R injury mode. However, pre-treatment of necroptosis inhibitor Nec-1 showed no difference with the administration of hiPSC-MSCs-EVs only. We then generated an SP1 knockout hiPSC-MSC cell line by CRISPR/Cas9 system and found that SP1 knockout failed to show the protective effect of hiPSC-MSCs-EVs unless restoring the level of SP1 by Ad-SP1 in vitro and in vivo. In conclusion, this study describes an anti-necroptosis effect of hiPSC-MSCs-EVs against renal I/R injury via delivering SP1 into target renal cells and intracellular activating the expression of SK1 and the generation of S1P. These findings suggest a novel mechanism for renal protection against I/R injury, and indicate a potential therapeutic approach for a variety of renal diseases and renal transplantation.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Differentiation; Cell Line, Transformed; Epithelial Cells; Extracellular Vesicles; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells; Kidney; Lysophospholipids; Male; Mesenchymal Stem Cells; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sp1 Transcription Factor; Sphingosine

Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid mediator involved in modulating inflammatory process. We investigated the effects of FTY720, a structural analogue of S1P after phosphorylation, on lung injury induced by hindlimb ischemia reperfusion (IR) in rats.. Fifty Sprague-Dawley rats were divided into groups SM, IR, F3, F5, and F10. Group SM received sham operation, and bilateral hindlimb IR was established in group IR. The rats in groups F3, F5, and F10 were pretreated with 3, 5, and 10 mg/kg/d FTY720 for 7 days before IR. S1P lyase (S1PL), sphingosine kinase (SphK) 1, and SphK2 mRNA expressions, wet/dry weight (W/D), and polymorphonuclear/alveolus (P/A) in lung tissues were detected, and the lung injury score was evaluated.. W/D, P/A, and mRNA expressions of S1PL, SphK1, and SphK2 were higher in group IR than in group SM, while these were decreased in both groups F5 and F10 as compared to IR (. FTY720 pretreatment could attenuate lung injury induced by hindlimb IR by modulating S1P metabolism and decreasing pulmonary neutrophil infiltration.

Topics: Animals; Blood Gas Analysis; Fingolimod Hydrochloride; Hindlimb; Immunosuppressive Agents; Lung Injury; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger

Previous studies have demonstrated that hypoxic preconditioning (HPC) alleviates hypoxia/reoxygenation (H/R) injury. However, the impact and mechanism involved were not fully understood. This study aimed to evaluate the effect of HPC on H/R injury in cardiomyocytes and investigate the molecular mechanisms involved. In our study, primary neonatal rat cardiomyocytes were isolated and characterized by immunofluorescence staining. We established H/R models in vitro to mimic ischemia/reperfusion (I/R) injury in vivo. Primary cardiomyocytes were exposed to HPC and then subjected to H/R. SphK2 expression was determined by quantitative real-time PCR and Western blotting. Cell apoptosis was measured by Hoechst staining. H9c2 cells were transfected with SphK2 siRNA or pcDNA3.1-SphK2 plasmid. The transfection efficiency was evaluated 48h post-transfection. After H/R, cell apoptosis rate was determined by Annexin V-FITC/PI and caspase-3/-9 activity was measured. The activation of FAK/AKT pathway was evaluated by Western blotting. Our results showed that HPC significantly increased SphK2 expression in primary cardiomyocytes under normal or H/R condition and protected against H/R-induced cell apoptosis, whereas SphK2 inhibitor K145 abolished the cardioprotective effect of HPC. HPC markedly reduced the cell apoptosis rate of H9c2, decreased the activities of caspase-3 and -9 and increased p-FAK and p-AKT levels, which were reversed by SphK2 knockdown. Additionally, SphK2 overexpression exerted a similar effect with HPC on cell apoptosis and FAK/AKT. Inhibition of H9c2 cell apoptosis induced by HPC and SphK2 overexpression was abolished by PI3K/AKT inhibitor LY294002. These results indicate that HPC may protect cardiomyocytes against H/R injury via SphK2 and the downstream FAK/AKT signaling pathway. Our findings provided important evidences for the protective role of HPC in ameliorating myocardial H/R injury.

Topics: Animals; Animals, Newborn; Apoptosis; Caspase 3; Chromones; Focal Adhesion Kinase 1; Hypoxia; Ischemic Preconditioning; Morpholines; Myocytes, Cardiac; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

Microglial activation is one of the causative factors of neuroinflammation in cerebral ischemia/reperfusion (IR). Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in the regulation of proinflammatory cytokines in activated microglia. Recent research demonstrated that S1P increased IL-17A-secretion and then worsened CNS (central nervous system) inflammation. Thus, in the present study, we sought to use microglial cells as the object of study to discuss the molecular mechanisms in Sphk1/S1P-regulated IL-17A-secretion in IR.. We used immunofluorescence and confocal microscopy to detect whether Sphk1 is expressed in microglia after cerebral IR or oxygen-glucose deprivation (OGDR). Western blot analysis was used to estimate the total Sphk1 protein level at different time points after OGDR. To detect cytokine secretion in microglial supernatants in response to OGDR, we measured the concentration of IL-17A in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). To evaluate whether microglia subjected to OGDR exhibited neuronal injury, we used a commercially available terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) kit to detect apoptotic neurons.. Sphk1 was expressed in microglia in response to cerebral IR or OGDR at appointed time. Pre-injection with PF-543, an inhibitor of Sphk1, before IR clearly reduced the expression of Sphk1 in microglia relative to brain IR alone. The number of TUNEL-positive neurons was also decreased in the PF-543-pretreated animals before IR compared to the animals with IR alone. When S1P was administered in OGDR microglia, IL-17A expression and neuronal apoptosis were increased compared to OGDR alone and the administration of S1P alone. ELISA further confirmed the above results. Moreover, the inhibition of Sphk1 by siRNA reduced IL-17A production and relieved neuronal apoptosis in OGDR microglia.. These results indicated that Sphk1/S1P regulates the expression of IL-17A in activated microglia, inducing neuronal apoptosis in cerebral ischemia/reperfusion. The microglial Sphk1/S1P pathway may thus be a potential therapeutic target to control neuroinflammation in brain IR.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cells, Cultured; Glucose; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Interleukin-17; Lysophospholipids; Male; Methanol; Microglia; Neurons; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Small Interfering; Sphingosine; Sulfones

Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.

Topics: Animals; Cell Proliferation; Ceramidases; Exosomes; Hepatocytes; Lysophospholipids; Male; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Reperfusion Injury; Sphingomyelin Phosphodiesterase; Sphingosine

We investigated the hypothesis that postconditioning by FTY720 (FTY) in isolated perfused mouse hearts is independent of the sphingosine 1-phosphate (S1P) pathway.. Ex vivo hearts were exposed to postconditioning (POST) by either ischemia or FTY720. Protection against ischemia/reperfusion (IR) injury was measured by recovery of left ventricular developed pressure (LVDP) and infarct size.. FTY effectively postconditioned (POST) ex vivo hearts against ischemia/reperfusion (IR) injury as measured by recovery of LVDP and a low infarct size. FTY protection, unlike S1P but like sphingosine (Sph), was insensitive to inhibition of S1P G-Protein Coupled Receptors (GPCRs) or inhibition of PI3 kinase. Protection by FTY and Sph was however blocked by inhibitors of PKA and PKG. Thus, FTY follows the same cardioprotective pathway as Sph. This was further supported by studies of FTY POST in knockout (KO) mice lacking the SphK2 form of Sph kinase that is needed for phosphorylation of FTY to an S1P analog. In the absence of SphK2, FTY (and Sph) POST was still cardioprotective. This differed from the effect of SphK2 KO on protection by ischemic POST (IPOST). IPOST was not effective in KO hearts. To see if the GPCR signaling pathway to protection is normal in KO hearts, we looked at POST by GPCR agonists S1P and adenosine. Both provided effective protection even in KO hearts suggesting that the problem with IPOST in KO hearts is a low level of S1P available for release during IPOST. Thus, pharmacologic POST with FTY or Sph, like adenosine and S1P, is unaffected in the KO.. FTY720 administered in vivo might behave in a dual manner showing both S1P-like effects and sphingosine-like effects. It appears that the latter may have been overlooked and may be the more important in aging hearts.

Topics: Adenosine; Algorithms; Animals; Fingolimod Hydrochloride; Immunosuppressive Agents; In Vitro Techniques; Ischemic Postconditioning; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Perfusion; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Reperfusion Injury; Sphingosine

The mitochondrial permeability transition (MPT) and inflammation play important roles in liver injury caused by ischemia-reperfusion (IR). This study investigated the roles of sphingosine kinase-2 (SK2) in mitochondrial dysfunction and inflammation after hepatic IR.. Mice were gavaged with vehicle or ABC294640 (50 mg/kg), a selective inhibitor of SK2, 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion.. Following IR, hepatic SK2 mRNA and sphingosine-1-phosphate (S1P) levels increased ~25- and 3-fold, respectively. SK2 inhibition blunted S1P production and liver injury by 54-91%, and increased mouse survival from 28% to 100%. At 2 h after reperfusion, mitochondrial depolarization was observed in 74% of viable hepatocytes, and mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. SK2 inhibition decreased mitochondrial depolarization and prevented MPT onset. Inducible nitric oxide synthase, phosphorylated NFκB-p65, TNFα mRNA, and neutrophil infiltration, all increased markedly after hepatic IR, and these increases were blunted by SK2 inhibition. In cultured hepatocytes, anoxia/re-oxygenation resulted in increases of SK2 mRNA, S1P levels, and cell death. SK2 siRNA and ABC294640 each substantially decreased S1P production and cell death in cultured hepatocytes.. SK2 plays an important role in mitochondrial dysfunction, inflammation responses, hepatocyte death, and survival after hepatic IR and represents a new target for the treatment of IR injury.

Topics: Adamantane; Animals; Cell Death; Enzyme Inhibitors; Gene Knockdown Techniques; Hepatocytes; In Vitro Techniques; Inflammation; Liver; Lysophospholipids; Male; Mice; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Reperfusion Injury; RNA, Messenger; RNA, Small Interfering; Sphingosine

Regulatory T cells (Tregs) are key components of the peripheral tolerance system and have become an immunotherapeutic agent for treating inflammatory processes. This therapeutic option, however, is hampered by problems arising from isolating and expanding desirable Tregs. Here we used an alternative approach with a pharmacologic agent to stimulate Tregs to achieve immunosuppressive effects. Pretreatment of mice with the naturally occurring sphingosine N,N-dimethylsphingosine (DMS) was found to increase both tissue-infiltrating T effectors (Teffs, CD4(+)Foxp3(-)) and Tregs (CD4(+)Foxp3(+)) in the early phase of bilateral renal ischemia/reperfusion injury. DMS itself had no effects on renal function or histopathology, but rapidly and transiently increased both Teffs and Tregs and increased the expression of chemokines CXCL9, CCL5, and CXCL10 in non-ischemic kidneys (sham operation). This renoprotection was abolished by administration of the Treg suppressing agents, anti-CTLA-4 or anti-CD25 monoclonal antibodies, suggesting that Tregs play a key role in DMS-induced renoprotection. Thus, Tregs recruited to the kidney by DMS ameliorate acute kidney injury and provide a new approach to control inflammatory diseases.

Topics: Acute Kidney Injury; Animals; Antibodies, Monoclonal; Chemokine CCL5; Chemokine CXCL10; Chemokine CXCL9; Chemotaxis, Leukocyte; CTLA-4 Antigen; Cytoprotection; Disease Models, Animal; Enzyme Inhibitors; Forkhead Transcription Factors; Immunologic Factors; Interleukin-2 Receptor alpha Subunit; Ischemia; Kidney; Male; Mice; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Sphingosine; T-Lymphocytes, Regulatory; Time Factors; Tumor Necrosis Factor-alpha

Renal ischemia-reperfusion injury is a major cause of acute kidney injury. We previously found that renal A(1) adenosine receptor (A(1)AR) activation attenuated multiple cell death pathways including necrosis, apoptosis, and inflammation. Here, we tested whether induction of cytoprotective sphingosine kinase (SK)-1 and sphingosine-1-phosphate (S1P) synthesis might be the mechanism of protection. A selective A(1)AR agonist (CCPA) increased the synthesis of S1P and selectively induced SK1 in mouse kidney and HK-2 cells. This agonist failed to protect SK1-knockout but protected SK2-knockout mice against renal ischemia-reperfusion injury indicating a critical role of SK1 in A(1)AR-mediated renal protection. Inhibition of SK prevented A(1)AR-mediated defense against necrosis and apoptosis in HK-2 cells. A selective S1P(1)R antagonist (W146) and global in vivo gene knockdown of S1P(1)Rs with small interfering RNA completely abolished the renal protection provided by CCPA. Mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(f)(/)(f) PEPCK(Cre/-)) were not protected against renal ischemia-reperfusion injury by CCPA. Mechanistically, CCPA increased nuclear translocation of hypoxia-inducible factor-1α in HK-2 cells and selective hypoxia-inducible factor-1α inhibition blocked A(1)AR-mediated induction of SK1. Thus, proximal tubule SK1 has a critical role in A(1)AR-mediated protection against renal ischemia-reperfusion injury.

Topics: Acute Kidney Injury; Adenosine; Adenosine A1 Receptor Agonists; Animals; Kidney; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Statistical; Phosphotransferases (Alcohol Group Acceptor); Receptor, Adenosine A1; Receptors, Lysosphingolipid; Reperfusion Injury

Activation of A(1) adenosine receptors (ARs) protects against renal ischemia-reperfusion (I/R) injury by reducing necrosis, apoptosis, and inflammation. However, extrarenal side effects (bradycardia, hypotension, and sedation) may limit A(1)AR agonist therapy for ischemic acute kidney injury. Here, we hypothesized that an allosteric enhancer for A(1)AR (PD-81723) protects against renal I/R injury without the undesirable side effects of systemic A(1)AR activation by potentiating the cytoprotective effects of renal adenosine generated locally by ischemia. Pretreatment with PD-81723 produced dose-dependent protection against renal I/R injury in A(1)AR wild-type mice but not in A(1)AR-deficient mice. Significant reductions in renal tubular necrosis, neutrophil infiltration, and inflammation as well as tubular apoptosis were observed in A(1)AR wild-type mice treated with PD-81723. Furthermore, PD-81723 decreased apoptotic cell death in human proximal tubule (HK-2) cells in culture, which was attenuated by a specific A(1)AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 induced sphingosine kinase (SK)1 mRNA and protein expression in HK-2 cells and in the mouse kidney. Supporting a critical role of SK1 in A(1)AR allosteric enhancer-mediated renal protection against renal I/R injury, PD-81723 failed to protect SK1-deficient mice against renal I/R injury. Finally, proximal tubule sphingosine-1-phosphate type 1 receptors (S1P(1)Rs) are critical for PD-81723-induced renal protection, as mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(flox/flox) PEPCK(Cre/-) mice) were not protected against renal I/R injury with PD-81723 treatment. Taken together, our experiments demonstrate potent renal protection with PD-81723 against I/R injury by reducing necrosis, inflammation, and apoptosis through the induction of renal tubular SK1 and activation of proximal tubule S1P(1)Rs. Our findings imply that selectively enhancing A(1)AR activation by locally produced renal adenosine may be a clinically useful therapeutic option to attenuate ischemic acute kidney injury without systemic side effects.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cells, Cultured; Humans; Kidney; Kidney Tubules, Proximal; Male; Mice; Phosphotransferases (Alcohol Group Acceptor); Receptor, Adenosine A1; Receptors, Lysosphingolipid; Reperfusion Injury; Thiophenes; Xanthines

Inflammation mediates/promotes graft injury after liver transplantation (LT). This study investigated the roles of sphingosine kinase-2 (SK2) in inflammation after LT. Liver grafts were stored in UW solution with and without ABC294640 (100 µM), a selective inhibitor of SK2, before implantation. Hepatic sphingosine-1-phosphate (S1P) levels increased ∼4-fold after LT, which was blunted by 40% by ABC294640. Hepatic toll-like receptor-4 (TLR4) expression and nuclear factor-κB (NF-κB) p65 subunit phosphorylation elevated substantially after transplantation. The pro-inflammatory cytokines/chemokines tumor necrosis factor-α, interleukin-1β and C-X-C motif chemokine 10 mRNAs increased 5.9-fold, 6.1-fold and 16-fold, respectively following transplantation, while intrahepatic adhesion molecule-1 increased 5.7-fold and monocytes/macrophage and neutrophil infiltration and expansion of residential macrophage population increased 7.8-13.4 fold, indicating enhanced inflammation. CD4+ T cell infiltration and interferon-γ production also increased. ABC294640 blunted TLR4 expression by 60%, NF-κB activation by 84%, proinflammatory cytokine/chemokine production by 45-72%, adhesion molecule expression by 54% and infiltration of monocytes/macrophages and neutrophils by 62-67%. ABC294640 also largely blocked CD4+ T cell infiltration and interferon-γ production. Focal necrosis and apoptosis occurred after transplantation with serum alanine aminotransferase (ALT) reaching ∼6000 U/L and serum total bilirubin elevating to ∼1.5 mg/dL. Inhibition of SK2 by ABC294640 blunted necrosis by 57%, apoptosis by 74%, ALT release by ∼68%, and hyperbilirubinemia by 74%. Most importantly, ABC294640 also increased survival from ∼25% to ∼85%. In conclusion, SK2 plays an important role in hepatic inflammation responses and graft injury after cold storage/transplantation and represents a new therapeutic target for liver graft failure.

Topics: Adamantane; Animals; Cell Adhesion Molecules; Chemokines; Enzyme Inhibitors; Inflammation; Leukocytes; Liver; Liver Transplantation; Lysophospholipids; Male; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Pyridines; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Reperfusion Injury; Sphingosine; Toll-Like Receptor 4; Up-Regulation

Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury and often leads to multiorgan dysfunction and systemic inflammation. Volatile anesthetics have potent antiinflammatory effects. We aimed to determine whether the representative volatile anesthetic isoflurane protects against acute kidney injury-induced liver and intestinal injury and to determine the mechanisms involved in this protection.. Mice were anesthetized with pentobarbital and subjected to 30 min of left renal ischemia after right nephrectomy, followed by exposure to 4 h of equianesthetic doses of pentobarbital or isoflurane. Five hours after renal IRI, plasma creatinine and alanine aminotransferase concentrations were measured. Liver and intestine tissues were analyzed for proinflammatory messenger RNA (mRNA) concentrations, histologic features, sphingosine kinase-1 (SK1) immunoblotting, SK1 activity, and sphingosine-1-phosphate concentrations.. Renal IRI with pentobarbital led to severe renal, hepatic, and intestinal injury with focused periportal hepatocyte vacuolization; small-intestinal apoptosis; and proinflammatory mRNA up-regulation. Isoflurane protected against renal IRI and reduced hepatic and intestinal injury via induction of small-intestinal crypt SK1 mRNA, protein and enzyme activity, and increased sphingosine-1-phosphate. We confirmed the importance of SK1 because mice treated with a selective SK inhibitor or mice deficient in the SK1 enzyme were not protected against hepatic and intestinal dysfunction with isoflurane.. Isoflurane protects against multiorgan injury after renal IRI via induction of the SK1/sphingosine-1-phosphate pathway. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated hepatic and intestinal protection and may lead to new therapeutic applications of volatile anesthetics during the perioperative period.

Topics: Acute Kidney Injury; Anesthetics, Inhalation; Animals; Enzyme Activation; Intestinal Diseases; Intestine, Small; Isoflurane; Kidney; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Up-Regulation

The roles of sphingosine kinases SK1 and SK2 in ischemia-reperfusion injury have not been fully elucidated since studies have found beneficial effects of SK1 while others showed no role in this injury. To help resolve this, we used SK1 or SK2 knockout mice and confirmed that renal ischemia-reperfusion injury induced SK1, but not SK2, in the kidneys. Furthermore, knockout or pharmacological inhibition of SK1 increased injury after renal ischemia-reperfusion injury. In contrast, lack of SK2 conferred renal protection following injury. In addition, we used lentiviral gene delivery to selectively express enhanced green fluorescent protein (EGFP) or human SK1 coexpressed with EGFP (EGFP-huSK1) in the kidney. Mice with kidney-specific overexpression of EGFP-huSK1 had significantly improved renal function with lower plasma creatinine, renal necrosis, apoptosis, and inflammation. Moreover, EGFP-huSK1 overexpression in cultured human proximal tubule (HK-2) cells protected against peroxide-induced necrosis. Selective overexpression of EGFP-huSK1 led to increased HSP27 mRNA and protein expression in vivo and in vitro. Functional protection as well as induction of HSP27 with EGFP-huSK1 overexpression in vivo was blocked with sphingosine-1-phosphate-1 receptor(1) (S1P(1)) antagonism. Thus, our findings suggest that SK1 is renoprotective by S1P(1) activation and perhaps HSP27 induction. Kidney-specific expression of SK1 through lentiviral delivery may be a viable therapeutic option to attenuate renal ischemia-reperfusion injury.

Topics: Animals; Apoptosis; Biomarkers; Cell Line; Creatinine; Disease Models, Animal; Gene Transfer Techniques; Genetic Vectors; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Inflammation Mediators; Kidney; Lentivirus; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Chaperones; Necrosis; Neutrophil Infiltration; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; Reperfusion Injury; Sphingosine-1-Phosphate Receptors; Time Factors

Sphingosine-1-phosphate (S1P), produced by sphingosine kinase 1 (SphK1) or kinase 2 (SphK2), mediates biological effects through intracellular and/or extracellular mechanisms. Here we determined a role for these kinases in kidney injury of wild-type mice following ischemia-reperfusion. SphK1 but not SphK2 mRNA expression and activity increased in the kidney following injury relative to sham-operated animals. Although SphK1(-/-) mice had no alteration in renal function following injury, mice with a disrupted SphK2 gene (SphK2(tr/tr)) had histological damage and impaired function. The immune-modulating pro-drug, FTY720, an S1P agonist failed to provide protection in SphK2(tr/tr) mice. Injured kidneys of these mice showed increased neutrophil infiltration and neutrophil chemokine expression along with a 3- to 5-fold increase in expression of the G-protein-coupled receptor S1P(3) compared to heterozygous SphK2(+/tr) mice. Kidney function and reduced vascular permeability were preserved in S1P(3)(-/-) compared to S1P(3)(+/-) mice after ischemia-reperfusion injury, suggesting increased S1P(3) mRNA may play a role in the injury of SphK2(tr/tr) mice. Our study suggests that constitutive expression of SphK2 may contribute to reduced ischemia-reperfusion injury of the kidney, and its absence may enhance injury due to increased neutrophil infiltration and S1P(3) activation. We also confirm that SphK2 is necessary to mediate the protective effects of FTY720.

Topics: Animals; Fingolimod Hydrochloride; Gene Expression Regulation, Enzymologic; Kidney; Kidney Diseases; Lysophospholipids; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Reperfusion Injury; RNA, Messenger; Sphingosine

The importance of bioactive lipid signaling under physiological and pathophysiological conditions is progressively becoming recognized. The disparate distribution of sphingosine kinase (SphK) isoform activity in normal and ischemic brain, particularly the large excess of SphK2 in cerebral microvascular endothelial cells, suggests potentially unique cell- and region-specific signaling by its product sphingosine-1-phosphate. The present study sought to test the isoform-specific role of SphK as a trigger of hypoxic preconditioning (HPC)-induced ischemic tolerance.. Temporal changes in microvascular SphK activity and expression were measured after HPC. The SphK inhibitor dimethylsphingosine or sphingosine analog FTY720 was administered to adult male Swiss-Webster ND4 mice before HPC. Two days later, mice underwent a 60-minute transient middle cerebral artery occlusion and at 24 hours of reperfusion, infarct volume, neurological deficit, and hemispheric edema were measured.. HPC rapidly increased microvascular SphK2 protein expression (1.7+/-0.2-fold) and activity (2.5+/-0.6-fold), peaking at 2 hours, whereas SphK1 was unchanged. SphK inhibition during HPC abrogated reductions in infarct volume, neurological deficit, and ipsilateral edema in HPC-treated mice. FTY720 given 48 hours before stroke also promoted ischemic tolerance; when combined with HPC, even greater (and dimethylsphingosine-reversible) protection was noted.. These findings indicate hypoxia-sensitive increases in SphK2 activity may serve as a proximal trigger that ultimately leads to sphingosine-1-phosphate-mediated alterations in gene expression that promote the ischemia-tolerant phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the neurovascular unit in stroke.

Topics: Animals; Arterioles; Brain Edema; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Fingolimod Hydrochloride; Hypoxia-Ischemia, Brain; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Ischemic Preconditioning; Lysophospholipids; Male; Mice; Microcirculation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Reperfusion Injury; RNA, Messenger; Sphingosine

Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST.. Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+/-dP/dtmax), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +/-dP/dtmax were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 +/- 2% in the control group to 29 +/- 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 +/- 5%) was not different from the KO control group (53 +/- 5% of risk area, n = 4, P = NS).. A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1-KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.

Topics: Animals; Extracellular Signal-Regulated MAP Kinases; Ischemic Preconditioning, Myocardial; Isoenzymes; Male; Mice; Mice, Knockout; Models, Animal; Mutation; Myocardial Infarction; Myocardium; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Reperfusion Injury

Sphingosine kinase (SphK) is a key enzyme in the synthesis of sphingosine 1-phosphate (S1P), a bioactive sphingolipid. SphK is involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK1 in myocardial ischemia/reperfusion (IR) injury and IPC.. Wild-type and SphK1 null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), +/-dP/dtmax, and LV end-diastolic pressure (EDP) between SphK1 mutant and wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S1P levels were decreased by 41%. SphK1 null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and +/-dP/dtmax decreased, LVEDP increased, and infarct size increased (n=6, P<0.05). Apoptosis was markedly enhanced in SphK1 mutant IR mouse hearts. IPC was cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK1 null hearts. There was no change in infarct size in the IPC+IR group compared to the IR group in the null hearts (50.1+/-5.0% vs 45.0+/-3.8%, n=6, P=NS). IPC remained ineffective in the null hearts even when the index ischemia time was shortened by 10 min.. Deletion of the SphK1 gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These data provide the first genetic evidence that the SphK1-S1P pathway is a critical mediator of IPC and cell survival.

Topics: Animals; Apoptosis; Blotting, Western; Creatine Kinase; Disease Susceptibility; In Situ Nick-End Labeling; Ischemic Preconditioning, Myocardial; Lysophospholipids; Mice; Mice, Knockout; Mutation; Myocardium; Perfusion; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Sphingosine

The inhalational anesthetic isoflurane has been shown to protect against renal ischemia-reperfusion (IR) injury. Previous studies demonstrated that isoflurane modulates sphingolipid metabolism in renal proximal tubule cells. We sought to determine whether isoflurane stimulates sphingosine kinase (SK) activity and synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells to mediate renal protection via the S1P signaling pathway. Isoflurane anesthesia reduced the degree of renal failure and necrosis in a murine model of renal IR injury. This protection with isoflurane was reversed by SK inhibitors (DMS and SKI-II) as well as an S1P(1) receptor antagonist (VPC23019). In addition, mice deficient in SK1 enzyme were not protected from IR injury with isoflurane. SK activity as well as SK1 mRNA expression increased in both cultured human proximal tubule cells (HK-2) and mouse kidneys after exposure to isoflurane. Finally, isoflurane increased the generation of S1P in HK-2 cells. Taken together, our findings indicate that isoflurane activates SK in renal tubule cells and initiates S1P-->S1P(1) receptor signaling to mediate the renal protective effects. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.

Topics: Anesthetics, Inhalation; Animals; Cell Line; Creatinine; Enzyme Inhibitors; Humans; Isoflurane; Kidney Diseases; Kidney Tubules; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine