Influence of CD4+/CD25+ regulatory T cells on atherogenesis in patients with end-stage kidney disease.

Atherosclerosis, which is influenced by both traditional and nontraditional cardiovascular risk factors and has been characterized as an inflammatory process, is considered to be the main cause of the elevated cardiovascular risk associated with chronic kidney disease. The inflammatory component of atherosclerosis can be separated into an innate immune response involving monocytes and macrophages that respond to the excessive uptake of lipoproteins and an adaptive immune response that involves antigen-specific T cells. Concurrent with the influx of immune cells to the site of atherosclerotic lesion, the role of the adaptive immune response gradually increases. One of those cells are represented by the CD4+/CD25+ Tregs, which play indispensable roles in the maintenance of natural self-tolerance and negative control of pathological, as well as physiological, immune responses. Altered self-antigens such as oxidized LDLs may induce the development of CD4+/CD25+ Tregs with atheroprotective properties. However, atherosclerosis may be promoted by an imbalance between regulatory and pathogenic immunity that may be represented by the low expression of the forkhead box transcription factor (Foxp3) in CD4+/CD25+ Tregs. Such a defect may break immunologic tolerance and alter both specific and bystander immune suppression, leading to exacerbation of plaque development. Patients with end-stage kidney disease (ESKD) display a cellular immune dysfunction and accelerated atherosclerosis. Uremic solutes that accumulate during ESKD may be involved in these processes. In patients with ESKD and especially in those that are chronically hemodialyzed, oxidative stress induced by oxidized LDLs may increase CD4+/CD25+ Treg sensitivity to Fas-mediated apoptosis as a consequence of specific dysregulation of IL-2 expression. This review will focus on the current state of knowledge regarding the influence of CD4+/CD25+ Tregs on atherogenesis in patients with ESKD, and the potential effect of statins on the circulating number and the functional properties of these cells.

Regulation of neurotrophic factors and energy metabolism by antidepressants in astrocytes.

There is growing evidence that astrocytes are involved in the neuropathology of major depression. In particular, decreases in glial cell density observed in the cerebral cortex of individuals with major depressive disorder are accompanied by a reduction of several astrocytic markers suggesting that astrocyte dysfunction may contribute to the pathophysiology of major depression. In rodents, glial loss in the prefrontal cortex is sufficient to induce depressive-like behaviors and antidepressant treatment prevents the stress-induced reduction of astrocyte number in the hippocampus. Collectively, these data support the existence of a link between astrocyte loss or dysfunction, depressive-like behavior and antidepressant treatment. Astrocytes are increasingly recognized to play important roles in neuronal development, neurotransmission, synaptic plasticity and maintenance of brain homeostasis. It is also well established that astrocytes provide trophic, structural, and metabolic support to neurons. In this article, we review evidence that antidepressants regulate energy metabolism and neurotrophic factor expression with particular emphasis on studies in astrocytes. These observations support a role for astrocytes as new targets for antidepressants. The contribution of changes in astrocyte glucose metabolism and neurotrophic factor expression to the therapeutic effects of antidepressants remains to be established.

Inhibition of voltage-gated Na(+) currents in sensory neurones by the sea anemone toxin APETx2.

BACKGROUND AND PURPOSE: APETx2, a toxin from the sea anemone Anthropleura elegantissima, inhibits acid-sensing ion channel 3 (ASIC3)-containing homo- and heterotrimeric channels with IC(50) values < 100 nM and 0.1-2 µM respectively. ASIC3 channels mediate acute acid-induced and inflammatory pain response and APETx2 has been used as a selective pharmacological tool in animal studies. Toxins from sea anemones also modulate voltage-gated Na(+) channel (Na(v) ) function. Here we tested the effects of APETx2 on Na(v) function in sensory neurones.¦EXPERIMENTAL APPROACH: Effects of APETx2 on Na(v) function were studied in rat dorsal root ganglion (DRG) neurones by whole-cell patch clamp.¦KEY RESULTS: APETx2 inhibited the tetrodotoxin (TTX)-resistant Na(v) 1.8 currents of DRG neurones (IC(50) , 2.6 µM). TTX-sensitive currents were less inhibited. The inhibition of Na(v) 1.8 currents was due to a rightward shift in the voltage dependence of activation and a reduction of the maximal macroscopic conductance. The inhibition of Na(v) 1.8 currents by APETx2 was confirmed with cloned channels expressed in Xenopus oocytes. In current-clamp experiments in DRG neurones, the number of action potentials induced by injection of a current ramp was reduced by APETx2.¦CONCLUSIONS AND IMPLICATIONS: APETx2 inhibited Na(v) 1.8 channels, in addition to ASIC3 channels, at concentrations used in in vivo studies. The limited specificity of this toxin should be taken into account when using APETx2 as a pharmacological tool. Its dual action will be an advantage for the use of APETx2 or its derivatives as analgesic drugs.

UPLC-TOF-MS for plant metabolomics: a sequential approach for wound marker analysis in Arabidopsis thaliana.

The model plant Arabidopsis thaliana was studied for the search of new metabolites involved in wound signalling. Diverse LC approaches were considered in terms of efficiency and analysis time and a 7-min gradient on a UPLC-TOF-MS system with a short column was chosen for metabolite fingerprinting. This screening step was designed to allow the comparison of a high number of samples over a wide range of time points after stress induction in positive and negative ionisation modes. Thanks to data treatment, clear discrimination was obtained, providing lists of potential stress-induced ions. In a second step, the fingerprinting conditions were transferred to longer column, providing a higher peak capacity able to demonstrate the presence of isomers among the highlighted compounds.

Study of the physiological role of RasGAP cleavage

Résume Les caspases sont un groupe de protéases à cystéine qui s?activent lors de l'apoptose. Leur activation induit le clivage de nombreuses cibles intracellulaires, conduisant à l'activation de voies pro-apoptotiques et finalement au démantèlement des cellules. Cependant, des caspases ont été décrites dans de nombreux autres processus indépendants de l'apoptose, notamment dans la physiologie des cellules hématopoïétiques, des cellules musculaires, des cellules de la peau et des neurones. Comment est-ce que les cellules réconcilient-elles ces deux fonctions distinctes? Une partie de la réponse réside dans la nature des substrats qu'elles clivent. Certains substrats, une fois clivées, deviennent anti-apoptotiques. RasGAP est une cible des caspases et contient deux sites spécifiques de clivage par les caspases. Lorsque le niveau d?activité des caspases est faible le clivage de RasGAP produit un fragment N-terminal qui active un signal antiapoptotique, relayé par la voie de Ras/PI3K/Akt. Lorsque le niveau d?activité des caspases est plus élevé le fragment RasGAP N-terminal est à nouveau clivé, perdant de ce fait ses propriétés anti-apoptotiques. Dans cette étude, nous avons mis en évidence que l'activation de la voie Ras/PI3K/Akt induite par le fragment RasGAP N-terminal dépend de RasGAP lui-même. Par ailleurs, dans le but d?étudier l?importance du clivage de RasGAP dans un contexte physiologique, nous avons développé un modèle animal exprimant une gêne mutée de RasGAP de sorte que la protéine est devenu insensible a l?action de caspases. Les données préliminaires obtenues montrent que le clivage de RasGAP n'est pas indispensable pour le développement et l?homéostasie chez la souris. Finalement, nous avons développé une souris transgénique surexprimant le fragment de RasGAP N-terminal dans les cellules ß du pancréas. Les animaux obtenus ne montrent pas de symptômes dans les conditions basales bien qu?ils soient plus résistants au diabète induit expérimentalement. Ces résultats montrent que la surexpression du fragment N-terminal de RasGAP protége efficacement les cellules ß du pancréas de l?apoptose induite par le stress sans pourtant affecter d?autres paramètres physiologiques des Ilot de Langerhans.<br/><br/>Caspases are a series of proteases that are activated during apoptosis. Their activation causes the cleavage of numerous intracellular targets, which leads to cell dismantling and activation of pro-apoptotic pathways. Caspases have been found to be involved in the physiology of numerous cell types including haematopoietic cells, muscle cells, skin cells and neurons. How cells conciliate these two opposite functions? Part of the answer lies in the nature of the substrates they cleave. Some substrates become anti-apoptotic once cleaved by caspases. RasGAP is a caspase substrate that possesses two conserved caspase-cleavage sites. At low caspase activity, RasGAP is first cleaved and the generated N-terminal fragment activates a potent anti-apoptotic signal, mediated by the Ras/PI3K/Akt pathway. At higher caspase activity, the N-terminal fragment is further cleaved thereby losing its anti-apoptotic properties. In the present study we show that the activation of the Ras/PI3K/Akt pathway mediated by RasGAP N-terminal fragment is dependent on RasGAP itself. Moreover, to study the role of RasGAP cleavage in a physiological model, we have developed a knock-in mouse model expressing a RasGAP mutant that is not cleavable by caspases. Preliminary data shows that RasGAP cleavage is not required for normal development and homeostasis in mice. Finally, we have developed a transgenic mouse model overexpressing RasGAP N-terminal fragment in the ß-cell of the pancreas. In basal conditions, these mice show no difference with their wt counterparts. However, they are protected against experimentally induced diabetes. These results indicate that fragment N can protect ? cells from stress-induced apoptosis without affecting other physiological parameters of the Islets.

HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease.

Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth.

Psychosocial Quality-of-Life, Lifestyle and Adiposity: A Longitudinal Study in Pre-schoolers (Ballabeina Study).

PURPOSE: In obesity prevention, understanding psychosocial influences in early life is pivotal. Reviews reported contradictory results and a lack of longitudinal studies focusing on underlying lifestyle factors. This study tested whether psychosocial Quality-Of-Life (QOL) was associated with pre-schoolers' lifestyle and adiposity changes over one school year and whether lifestyle moderated the latter. It was hypothesised that QOL might not impact adiposity in everybody but that this might depend on preceding lifestyle. METHOD: Longitudinal data from 291 Swiss pre-schoolers (initially 3.9-6.3 years) was available. The following measures were used in longitudinal regressions: psychosocial QOL by PedsQL, adiposity (BMI z-score, waist, fat%), diet (food frequency), sedentary time and accelerometer-based activity. RESULTS: Concerning lifestyle, low psychosocial QOL was only related to unfavourable changes in diet (less fruit β = 0.21 and more fat intake β = -0.28) and lower physical activity (β = 0.21). Longitudinal QOL-adiposity relations appeared only after moderation by lifestyle factors (beta-range 0.13-0.67). Low psychosocial QOL was associated with increased adiposity in children with an unhealthy diet intake or high sedentary time. By contrast, low psychosocial QOL was associated with decreasing adiposity in high fruit consumers or more physically active pre-schoolers. CONCLUSION: Results emphasise the need for testing moderation in the QOL-adiposity relation. An unhealthy diet can be a vulnerability factor and high physical activity a protective factor in QOL-related adiposity. Consequently, QOL and lifestyle should be targeted concurrently in multi-factorial obesity prevention. The environment should be an 'activity encouraging, healthy food zone' that minimises opportunities for stress-induced eating. In addition, appropriate stress coping skills should be acquired.

Angiotensin II receptor blockade prevents acute renal sodium retention induced by low levels of orthostatic stress.

BACKGROUND: Depending on its magnitude, lower body negative pressure (LBNP) has been shown to induce a progressive activation of neurohormonal, renal tubular, and renal hemodynamic responses, thereby mimicking the renal responses observed in clinical conditions characterized by a low effective arterial volume such as congestive heart failure. Our objective was to evaluate the impact of angiotensin II receptor blockade with candesartan on the renal hemodynamic and urinary excretory responses to a progressive orthostatic stress in normal subjects. METHODS: Twenty healthy men were submitted to three levels of LBNP (0, -10, and -20 mbar or 0, -7.5, and -15 mm Hg) for 1 hour according to a crossover design with a minimum of 2 days between each level of LBNP. Ten subjects were randomly allocated to receive a placebo and ten others were treated with candesartan 16 mg orally for 10 days before and during the three levels of LBNP. Systemic and renal hemodynamics, renal sodium excretions, and the hormonal response were measured hourly before, during, and for 2 hours after LBNP. RESULTS: During placebo, LBNP induced no change in systemic and renal hemodynamics, but sodium excretion decreased dose dependently with higher levels of LBNP. At -20 mbar, cumulative 3-hour sodium balance was negative at -2.3 +/- 2.3 mmol (mean +/- SEM). With candesartan, mean blood pressure decreased (76 +/- 1 mm Hg vs. 83 +/- 3 mm Hg, candesartan vs. placebo, P < 0.05) and renal plasma flow increased (858 +/- 52 mL/min vs. 639 +/- 36 mL/min, candesartan vs. placebo, P < 0.05). Glomerular filtration rate (GFR) was not significantly higher with candesartan (127 +/- 7 mL/min in placebo vs. 144 +/- 12 mL/min in candesartan). No significant decrease in sodium and water excretion was found during LBNP in candesartan-treated subjects. At -20 mbar, the 3-hour cumulative sodium excretion was + 4.6 +/- 1.4 mmol in the candesartan group (P= 0.02 vs. placebo). CONCLUSION: Selective blockade of angiotensin II type 1 (AT1) receptors with candesartan increases renal blood flow and prevents the antinatriuresis during sustained lower body negative pressure despite a modest decrease in blood pressure. These results thus provide interesting insights into potential benefits of AT1 receptor blockade in sodium-retaining states such as congestive heart failure.

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy.

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-kappa B and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38 alpha) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-alpha, markers of fibrosis (transforming growth factor-beta, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-kappa B activation, and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis. (J Am Coll Cardiol 2010;56:2115-25) (C) 2010 by the American College of Cardiology Foundation.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Urate-induced acute renal failure and chronic inflammation in liver-specific Glut9 knockout mice.

Plasma urate levels are higher in humans than rodents (240-360 vs. â^¼30 μM) because humans lack the liver enzyme uricase. High uricemia in humans may protect against oxidative stress, but hyperuricemia also associates with the metabolic syndrome, and urate and uric acid can crystallize to cause gout and renal dysfunctions. Thus, hyperuricemic animal models to study urate-induced pathologies are needed. We recently generated mice with liver-specific ablation of Glut9, a urate transporter providing access of urate to uricase (LG9KO mice). LG9KO mice had moderately high uricemia (â^¼120 μM). To further increase their uricemia, here we gavaged LG9KO mice for 3 days with inosine, a urate precursor; this treatment was applied in both chow- and high-fat-fed mice. In chow-fed LG9KO mice, uricemia peaked at 300 μM 2 h after the first gavage and normalized 24 h after the last gavage. In contrast, in high-fat-fed LG9KO mice, uricemia further rose to 500 μM. Plasma creatinine strongly increased, indicating acute renal failure. Kidneys showed tubule dilation, macrophage infiltration, and urate and uric acid crystals, associated with a more acidic urine. Six weeks after inosine gavage, plasma urate and creatinine had normalized. However, renal inflammation, fibrosis, and organ remodeling had developed despite the disappearance of urate and uric acid crystals. Thus, hyperuricemia and high-fat diet feeding combined to induce acute renal failure. Furthermore, a sterile inflammation caused by the initial crystal-induced lesions developed despite the disappearance of urate and uric acid crystals.

Induction of cardiac Angptl4 by dietary fatty acids is mediated by peroxisome proliferator-activated receptor beta/delta and protects against fatty acid-induced oxidative stress.

RATIONALE: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart. OBJECTIVE: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences. METHODS AND RESULTS: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding. CONCLUSIONS: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.

Neuroendocrine, metabolic, and immune functions during the acute phase response of inflammatory stress in monosodium L-glutamate-damaged, hyperadipose male rat.

In rats, neonatal treatment with monosodium L-glutamate (MSG) induces several metabolic and neuroendocrine abnormalities, which result in hyperadiposity. No data exist, however, regarding neuroendocrine, immune and metabolic responses to acute endotoxemia in the MSG-damaged rat. We studied the consequences of MSG treatment during the acute phase response of inflammatory stress. Neonatal male rats were treated with MSG or vehicle (controls, CTR) and studied at age 90 days. Pituitary, adrenal, adipo-insular axis, immune, metabolic and gonadal functions were explored before and up to 5 h after single sub-lethal i.p. injection of bacterial lipopolysaccharide (LPS; 150 microg/kg). Our results showed that, during the acute phase response of inflammatory stress in MSG rats: (1) the corticotrope-adrenal, leptin, insulin and triglyceride responses were higher than in CTR rats, (2) pro-inflammatory (TNFalpha) cytokine response was impaired and anti-inflammatory (IL-10) cytokine response was normal, and (3) changes in peripheral estradiol and testosterone levels after LPS varied as in CTR rats. These data indicate that metabolic and neroendocrine-immune functions are altered in MSG-damaged rats. Our study also suggests that the enhanced corticotrope-corticoadrenal activity in MSG animals could be responsible, at least in part, for the immune and metabolic derangements characterizing hypothalamic obesity.

Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice.

The relationship between metabolism and reactive oxygen species (ROS) production by the mitochondria has often been (wrongly) viewed as straightforward, with increased metabolism leading to higher generation of pro-oxidants. Insights into mitochondrial functioning show that oxygen consumption is principally coupled with either energy conversion as ATP or as heat, depending on whether the ATP-synthase or the mitochondrial uncoupling protein 1 (UCP1) is driving respiration. However, these two processes might greatly differ in terms of oxidative costs. We used a cold challenge to investigate the oxidative stress consequences of an increased metabolism achieved either by the activation of an uncoupled mechanism (i.e. UCP1 activity) in the brown adipose tissue (BAT) of wild-type mice or by ATP-dependent muscular shivering thermogenesis in mice deficient for UCP1. Although both mouse strains increased their metabolism by more than twofold when acclimatised for 4 weeks to moderate cold (12°C), only mice deficient for UCP1 suffered from elevated levels of oxidative stress. When exposed to cold, mice deficient for UCP1 showed an increase of 20.2% in plasmatic reactive oxygen metabolites, 81.8% in muscular oxidized glutathione and 47.1% in muscular protein carbonyls. In contrast, there was no evidence of elevated levels of oxidative stress in the plasma, muscles or BAT of wild-type mice exposed to cold despite a drastic increase in BAT activity. Our study demonstrates differing oxidative costs linked to the functioning of two highly metabolically active organs during thermogenesis, and advises careful consideration of mitochondrial functioning when investigating the links between metabolism and oxidative stress.

Nitrosative stress and corneal transplant endothelial cell death during acute graft rejection.

BACKGROUND: Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death. METHODS: The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count. RESULTS: Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection. CONCLUSION: Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.