Inhibition of organic anion transport in endothelial cells by hydrogen peroxide

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29758/1/0000096.pd

Metabolic inhibition potentiates oxidant injury

Toxic oxygen species have been implicated as important mediators of injury after reperfusion of an ischemic organ. The aim of this study was to determine if prior metabolic inhibition, such as that which occurs during ischemia, potentiates oxidant injury in vitro. Bovine pulmonary artery endothelial cells were metabolically inhibited for various periods of time with or without the mitochondrial inhibitor oligomycin (650 nM). The cells were rescued from metabolic inhibition by a wash step and subsequent addition of 5.5 mM glucose. At the same time that metabolic inhibition was relieved the cells were subjected to doses of H2O2 ranging from 0 to 100 [mu]M. ATP levels were monitored over a 2-hr time course after rescue from metabolic inhibition by the luciferin--luciferase assay. Cell viability at 2 hr after relief of metabolic inhibition was assessed by trypan blue exclusion. Intracellular pH during metabolic inhibition was determined with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein tetraacetomethoxymethyl ester. H2O2 consumption, a measure of H2O2 scavenging capability, was determined by a fluorescent assay. The viability and ATP levels of cells not subjected to metabolic inhibition were unaffected by these low concentrations of H2O2. Cells metabolically inhibited with glucose depletion and oligomycin were exquisitely sensitive to H2O2. Cells that were only deprived of glucose demonstrated no potentiation of injury, while cells subjected to mitochondrial inhibition with oligomycin alone also showed significant potentiation of oxidant injury. H2O2 consumption was not affected by metabolic inhibition. Conditions associated with mitochondrial inhibition consistently resulted in a decrease in intracellular pH. These experiments suggest that a synergism exists between metabolic inhibition and subsequent oxidant exposure. This synergism is dependent on inhibition of mitochondrial function but is independent of ATP levels. Intracellular acidosis correlated well with conditions which potentiated oxidant injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29401/1/0000474.pd

Arterial levels of oxidized glutathione (GSSG) reflect oxidant stress in vivo

Neutrophil-related, oxidant-mediated injury to the pulmonary microvasculature appears to follow endotoxemia, cutaneous thermal injury, and ischemia--reperfusion injury to the liver or intestine. Glutathione is an important endogenous intracellular oxygen radical scavenger. Plasma concentrations of oxidized glutathione (GSSG) reflect oxidant injury resulting from an overdose of certain oxidatively metabolized drugs. The purpose of this investigation was to evaluate plasma GSSG as an indicator of oxidant stress resulting from activation of the endogenous inflammatory response. An established model of neutrophil- and oxidant-related acute lung injury following intestinal ischemia and reperfusion in rats was used. Intestinal ischemia was induced by clip occlusion of the superior mesenteric artery (SMA) for 120 min. Reperfusion resulted from SMA clip removal. Following reperfusion for 0, 15, or 120 min, plasma GSSG levels in portal vein, inferior vena cava (IVC), and aorta were obtained. Plasma GSSG was undetectable in sham animals and those with intestinal ischemia alone. Following reperfusion, all plasma samples had significant elevations in GSSG. Aortic plasma GSSG after 15 min of reperfusion was significantly elevated compared to both portal vein and IVC plasma GSSG. These data suggest that oxidant stress after intestinal reperfusion is reflected by elevations in plasma GSSG. The step up in plasma GSSG across the pulmonary vascular bed, a site of known oxidant injury, suggests that plasma GSSG may be a useful marker of oxidant stress in vivo, particularly with regard to the pulmonary microvasculature. This simple in vivo approach to assessing oxidant stress related to inflammatory tissue injury may have the potential to be of significant use in the clinical setting.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28648/1/0000464.pd

Pogostemon cablin as ROS Scavenger in Oxidant-induced Cell Death of Human Neuroglioma Cells

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of acute and long-term neurodegenerative diseases. This study was undertaken to examine the efficacy of Pogostemon cablin, a well-known herb in Korean traditional medicine, on ROS-induced brain cell injury. Pogostemon cablin effectively protected human neuroglioma cell line A172 against both the necrotic and apoptotic cell death induced by hydrogen peroxide (H2O2). The effect of Pogostemon cablin was dose dependent at concentrations ranging from 0.2 to 5 mg ml−1. Pogostemon cablin significantly prevented depletion of cellular ATP and activation of poly ADP-ribose polymerase induced by H2O2. The preservation of functional integrity of mitochondria upon the treatment of Pogostemon cablin was also confirmed by 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Furthermore, Pogostemon cablin significantly prevented H2O2-induced release of cytochrome c into cytosol. Determination of intracellular ROS showed that Pogostemon cablin might exert its role as a powerful scavenger of intracellular ROS. The present study suggests the beneficial effect of Pogostemon cablin on ROS-induced neuroglial cell injury. The action of Pogostemon cablin as a ROS-scavenger might underlie the mechanism

Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA

Introduction: The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood. Methods: In a close-contact co-culture model of human airway epithelial and endothelial cells cellular crosstalk was analysed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system the temporal release of mediators was analysed in the co-culture model. Results: Within 4h of challenge, double-stranded RNA induced the release of TNF-a by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-a. Conclusion: By facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signalling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation

IL8 and Cathepsin B as Melanoma Serum Biomarkers

Melanoma accounts for only a small portion of skin cancer but it is associated with high mortality. Melanoma serum biomarkers that may aid early diagnosis or guide therapy are needed clinically. However, studies of serum biomarkers have often been hampered by the serum interference that causes false readouts in immunological tests. Here we show that, after using a special buffer to eliminate the serum interference, IL-8 and cathepsin B levels were significantly elevated in melanoma patients (p < 0.05). More importantly, the combination of IL-8 and cathepsin B were also studied as a prognosis marker for melanoma mortality. Our study provides a novel approach to examine serum biomarkers

Poly[ADP-Ribose] Polymerase-1 Expression Is Related To Cold Ischemia, Acute Tubular Necrosis, and Delayed Renal Function In Kidney Transplantation

Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD) transplantation. Ischemia-reperfusion (IR) injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1) activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN)

Hyaluronan is crucial for stem cell differentiation into smooth muscle lineage

Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell-based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen-IV in differentiation medium to generate ESC-derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4-methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)-HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW-HA-stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2-induced HA synthesis and organization drives ESC-SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases