Project description:Lung transplantation remains the only viable therapy for patients with end-stage lung disease; however, full utilization of this treatment strategy is severely compromised by the lack of donor lung availability. For example, the vast majority of donor lungs available for transplantation are obtained from brain death (BD) individuals. Unfortunately, the autonomic storm which accompanies BD often results in neurogenic pulmonary edema (NPE), thereby either producing irreversible lung injury or leading to primary graft dysfunction following lung transplantation. We previously demonstrated that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor and major barrier-enhancing agent, as well as S1P analogues serve to reduce vascular permeability and ischemia/reperfusion (I/R) lung injury in rodents via ligation of the S1P1 receptor, S1PR1. As primary lung graft dysfunction is induced by lung vascular endothelial cell barrier dysfunction, we hypothesized that SEW-2871, a S1PR1 agonist, may attenuate NPE when administered to the donor shortly after BD. Significant lung injury was observed 4h after BD in a rat BD model with ~60% increases in BAL total protein, BAL cell counts, and lung tissue W/D weight ratios. In contrast, rats receiving SEW-2871 (0.1 mg/kg) 15 minutes after the induction of BD and assessed 4h later exhibited significant lung protection (~50% reduction, p=0.01) reflected by reduced BAL total protein, BAL cytokines concentrations, BAL albumin, BAL total cell count and lung tissue wet/dry (W/D) weights ratio. Microarray analysis at 4hrs revealed a global impact of both BD and SEW on lung gene expression with differential expression of a subclass of genes enriched in immune/inflammation response pathways across the 4 experimental groups. Overall, SEW served to attenuate the BD-mediated ie gene expression upregulation. Two potentially useful biomarkers, Tnf and Ccrl2, exhibited gene dysregulation by microarray analysis, which was validated by qPCR. We conclude that SEW-2871 significantly attenuates BD-induced lung injury and may serve as a potential candidate to improve human lung donor availability and transplantation outcomes. Animals were divided into four groups: sham, BD (A Fogarty catheter 4 Fr. was inserted and secured into the extradural space and inflated to induce BD), SEW (injection of SEW-2871), and BD/SEW. Three replicates each.

Project description:Lung transplantation remains the only viable therapy for patients with end-stage lung disease; however, full utilization of this treatment strategy is severely compromised by the lack of donor lung availability. For example, the vast majority of donor lungs available for transplantation are obtained from brain death (BD) individuals. Unfortunately, the autonomic storm which accompanies BD often results in neurogenic pulmonary edema (NPE), thereby either producing irreversible lung injury or leading to primary graft dysfunction following lung transplantation. We previously demonstrated that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor and major barrier-enhancing agent, as well as S1P analogues serve to reduce vascular permeability and ischemia/reperfusion (I/R) lung injury in rodents via ligation of the S1P1 receptor, S1PR1. As primary lung graft dysfunction is induced by lung vascular endothelial cell barrier dysfunction, we hypothesized that SEW-2871, a S1PR1 agonist, may attenuate NPE when administered to the donor shortly after BD. Significant lung injury was observed 4h after BD in a rat BD model with ~60% increases in BAL total protein, BAL cell counts, and lung tissue W/D weight ratios. In contrast, rats receiving SEW-2871 (0.1 mg/kg) 15 minutes after the induction of BD and assessed 4h later exhibited significant lung protection (~50% reduction, p=0.01) reflected by reduced BAL total protein, BAL cytokines concentrations, BAL albumin, BAL total cell count and lung tissue wet/dry (W/D) weights ratio. Microarray analysis at 4hrs revealed a global impact of both BD and SEW on lung gene expression with differential expression of a subclass of genes enriched in immune/inflammation response pathways across the 4 experimental groups. Overall, SEW served to attenuate the BD-mediated ie gene expression upregulation. Two potentially useful biomarkers, Tnf and Ccrl2, exhibited gene dysregulation by microarray analysis, which was validated by qPCR. We conclude that SEW-2871 significantly attenuates BD-induced lung injury and may serve as a potential candidate to improve human lung donor availability and transplantation outcomes.

Project description:Background: Renal lipid dysmetabolism contributes to glomerular disease progression, including Alport Syndrome. We recently identified alterations in the apolipoprotein M/sphingosine-1-phosphate/sphingosine-1-phosphate receptor 4 signaling axis in glomeruli from patients with glomerular disease. Methods: We utilized Col4a3 knockout mice and immortalized podocytes derived from these mice as a mouse model of Alport Syndrome. Mice and podocytes were treated with recombinant apolipoprotein M or the sphingosine-1-phosphate receptor 4 antagonist, CYM50358. Results: Col4a3-/- glomeruli and podocytes exhibited reduced apolipoprotein M and increased sphingosine-1-phosphate receptor 4 expression and increased sphignsoine-1-phosphate levels, mirroring findings in patients with glomerular disease. Treatment with apolipoprotein M or CYM50358 reduced albuminuria, BUN, and plasma creatinine, and ameliorated glomerulosclerosis, tubulointerstitial fibrosis, podocyte loss and foot process effacement. Both treatments reduced triglyceride and cholesterol accumulation in glomeruli and podocytes. RNA-seq analysis of Col4a3-/- revealed that sphingosine-1-phosphate receptor 4 antagonism upregulated lysosomal and autophagy-related genes. Western blot analysis confirmed increased LC3-II/LC3-I ratios and decreased p62, indicating enhanced autophagic flux. Treated podocytes showed increased lysosome numbers and co-localization with lipid droplets. In contrast, apolipoprotein M had no effect on autophagy but promoted cholesterol efflux. Conclusions: The apolipoprotein M/sphingosine-1-phosphate axis is dysregulated in Col4a3-/- podocytes. Targeting this pathway through apolipoprotein M supplementation or sphingosine-1-phosphate receptor 4 antagonism improves renal function and reduces lipid accumulation by enhancing either cholesterol efflux or autophagy, respectively. These findings suggest that restoring lipid homeostasis via targeting the APOM/S1P/S1PR4 axis may be a promising therapeutic strategy for Alport Syndrome and other glomerular diseases.

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