The Effects of Exosomal Derived TSG-6 on Microglia Activation

Following a traumatic brain injury, microglia become overactive for long periods and display pathologic behavior. We have shown that concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (MSC-CCM) can suppress microglial activation. In this pilot study, we evaluated the efficacy of exosomes containing TNF-stimulated gene 6 (TSG6) derived from MSC-CCM on decreasing microglial activation in vitro via phagocytic activity and pro-inflammatory microglial gene expression

Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema

RATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development

Synthesis and Biological Evaluation of Novel Homoisoflavonoids for Retinal Neovascularization

Eye diseases characterized by excessive angiogenesis such as wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity are major causes of blindness. Cremastranone is an antiangiogenic, naturally occurring homoisoflavanone with efficacy in retinal and choroidal neovascularization models and antiproliferative selectivity for endothelial cells over other cell types. We undertook a cell-based structure–activity relationship study to develop more potent cremastranone analogues, with improved antiproliferative selectivity for retinal endothelial cells. Phenylalanyl-incorporated homoisoflavonoids showed improved activity and remarkable selectivity for retinal microvascular endothelial cells. A lead compound inhibited angiogenesis in vitro without inducing apoptosis and had efficacy in the oxygen-induced retinopathy model in vivo

Vascular and Cardiac Adult Stem Cell Therapy Center (VC-CAST)

poster abstractThe mission of the Vascular and Cardiac Adult Stem Cell Therapy Center (VC-CAST) is the discovery and clinical translation of therapies involving transplantation of adult stem cells into patients with debilitating diseases. VC-CAST fosters multidisciplinary research collaborations that address the biology of adult stem cells that are readily available, as well as the translation of their study from the laboratory into clinical trials. The use of such cells is highly feasible, and not ethically controversial, as they are derived from readily-available tissues such as fat and bone marrow. VC-CAST projects involve partners from multiple clinical and basic departments of the School of Medicine. VC-CAST projects are also collaborative externally, with most projects having one or more industrial or academic external partners. A key partnership has also been established at the Roudebush VA Medical Center in Indianapolis by creation of the Veterans Affairs Center for Regenerative Medicine (VACRM), which will provide a unique referral site focusing on research and implementation of first-in-human trials in the fields of poor circulation, stroke, arthritis, wound healing, diabetes, and emphysema. Given the focus on translation, the center is active in pursuit of intellectual property that is critical to building corporate engagement and thus the enablement of translation to clinical trials. Signature Center funding has allowed IUPUI investigators to try high-risk, high-reward ideas, which could not otherwise be funded readily, via either NIH or venture-capital methods. Most of these experiments have already led to discoveries of potentially critical significance to patients. The novelty of some of these discoveries has attracted new funding, as well as provided bases for potential licensing revenues and startup opportunities. This poster will highlight several such projects, representative of center activities in their multidisciplinary, translational, and potentially commercializable aspects. Several key projects are as follows: • Saving Legs from Amputation o Bone Marrow Stem Cells: Based on our completion of the Phase I/II clinical trial, “Stem cell Angiogenesis to promote limb salVagE (SAVE), we have initiated a randomized Phase III clinical trial testing one’s own bone marrow-derived stem cells to save legs from amputation, with Dr. Murphy as national PI. o Fat-derived (Adipose) Stem Cells– we are testing the hypothesis that these are more potent than Bone Marrow-derived stem cells with new funding from a corporate partner as well as the Department of Defense. o Endometrial Regenerative Cells– further extending above efforts, with new NIH funding to study this allogeneic (non-self, “off-the-shelf”) cell type. • Treatment of Heart Attack and prevention of Heart Failure. New data this year shows Adipose Stem Cells protect from heart damage when given systemically. • Treatment of Emphysema and other Lung Diseases. Adipose Stem Cells markedly protect from cigarette smoke-induced emphysema, a generally untreatable condition. • Prevention and Treatment of Diabetes– Adipose Stem Cells can ameliorate diabetes. This work has attracted new Veterans Affairs funding this past year. • Treatment of Parkinson’s Disease by rescue of dopaminergic neurons from death. New funding attracted in the past year by the Signature Center led to preclinical data that extended prior work in stroke models to models of Parkinson’s Disease. These data suggest that the conditioned medium from ASCs can be useful in this debilitating condition, and form the basis for a new NIH application. • Treatment of Diabetic Retinopathy by vascular stabilization using adipose stem cells. This is a new project in the past year, and has generated encouraging early data which is being used in seeking further (external) funding. • Human Placenta as a stem cell source: Isolation and Characterization of Endothelial and Mesenchymal Stem Cells from Term Placenta. • Human Saphenous Vein as a cell source: Isolation and Characterization of Endothelial Colony Forming Cells (ECFCs) from Human Saphenous Vein can form the basis for vascular network formation

Cisplatin-induced oxidative stress stimulates renal Fas ligand shedding

Acute kidney injury (AKI), a significant complication of cisplatin chemotherapy is associated with reactive oxygen species (ROS)-dependent renal cell death, but the cellular targets of ROS in cisplatin nephrotoxicity are not fully resolved. Here, we investigated cisplatin-induced oxidative renal damage and tested the hypothesis that ROS-dependent shedding of death activator Fas ligand (FasL) occurs in cisplatin nephropathy. We show that intraperitoneal injection of sulfobutyl ether-β-cyclodextrin (Captisol™)-solubilized cisplatin elevated the level of lipid peroxidation product malondialdehyde in mouse kidneys and urinary concentration of oxidative DNA damage biomarker 8-hydroxy-2′-deoxyguanosine. Cisplatin increased mouse kidney-to-body weight ratio and the plasma or urinary levels of predictive biomarkers of AKI, including creatinine, blood urea nitrogen, microalbumin, neutrophil gelatinase-associated lipocalin, and cystatin C. Histological analysis and dUTP nick end labeling of kidney sections indicated tubular injury and renal apoptosis, respectively in cisplatin-treated mice. Whereas the plasma concentration of soluble FasL (sFasL) was unaltered, urinary sFasL was increased ∼4-fold in cisplatin-treated mice. Real-time quantitative live-cell imaging and lactate dehydrogenase assay showed that cisplatin stimulated caspase 3/7 activation and cytotoxicity in a human proximal tubule epithelial cell line which were attenuated by inhibitors of the FasL/Fas system and poly [ADP-ribose] polymerase-1. Moreover, TEMPOL, an intracellular free radical scavenger mitigated cisplatin-induced renal oxidative stress and injury, AKI biomarker and urinary sFasL elevation, and proximal tubule cell death. Our findings indicate that cisplatin-induced oxidative stress triggers the shedding of membrane-bound FasL to sFasL in the kidney. We demonstrate that cisplatin elicits nephrotoxicity by promoting FasL/Fas-dependent oxidative renal tubular cell death

A Comprehensive Profiling of Cellular Sphingolipids in Mammalian Endothelial and Microglial Cells Cultured in Normal and High-Glucose Conditions

Sphingolipids (SPLs) play a diverse role in maintaining cellular homeostasis. Dysregulated SPL metabolism is associated with pathological changes in stressed and diseased cells. This study investigates differences in SPL metabolism between cultured human primary retinal endothelial (HREC) and murine microglial cells (BV2) in normal conditions (normal glucose, NG, 5 mM) and under high-glucose (HG, 25 mM)-induced stress by sphingolipidomics, immunohistochemistry, biochemical, and molecular assays. Measurable differences were observed in SPL profiles between HREC and BV2 cells. High-glucose treatment caused a >2.5-fold increase in the levels of Lactosyl-ceramide (LacCer) in HREC, but in BV2 cells, it induced Hexosyl-Ceramides (HexCer) by threefold and a significant increase in Sphingosine-1-phosphate (S1P) compared to NG. Altered SPL profiles coincided with changes in transcript levels of inflammatory and vascular permeability mediators in HREC and inflammatory mediators in BV2 cells. Differences in SPL profiles and differential responses to HG stress between endothelial and microglial cells suggest that SPL metabolism and signaling differ in mammalian cell types and, therefore, their pathological association with those cell types