Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity.

Cyclosporin A is an immunosuppressant drug widely used in solid organ transplantation, but it has nephrotoxic properties that promote oxidative stress. The JAK2/STAT pathway has been implicated in both cell protection and cell injury; therefore, we determined a role of JAK2 in oxidative stress-mediated renal cell injury using pathophysiologically relevant oxidative challenges. The AG490 JAK2 inhibitor and overexpression of a dominant negative JAK2 protein protected endothelial and renal epithelial cells in culture against peroxide, superoxide anion and cyclosporin A induced cell death while reducing intracellular oxidation in cells challenged with peroxide and cyclosporin A. The decrease in Bcl2 expression and caspase 3 activation, induced by oxidative stress, was prevented by AG490. In mouse models of ischemia/reperfusion and cyclosporin A nephrotoxicity, AG490 decreased peritubular capillary and tubular cell injury. Our study shows that JAK2 inhibition is a promising renoprotective strategy defending endothelial and tubular cells from cyclosporin A- and oxidative stress-induced death.

Mechanisms of endothelial response to oxidative aggression: protective role of autologous VEGF and induction of VEGFR2 by H2O2.

The defense mechanisms of endothelial cells (EC) against reactive oxygen species (ROS) are insufficiently characterized. We have addressed the hypothesis that vascular endothelial growth factor (VEGF) and its receptors are relevant elements in this response. Cell viability, VEGF and VEGF receptor (VEGFR1 and VEGFR2) expression, and transcription factor activation were studied on transient exposure of monolayer EC to H2O2. Wild-type and mutant inhibitors of kappaBalpha (IkappaBalpha) constructions were used to further assess the role of NF-kappaB in the induction of VEGFR2 expression. A concentration of H2O2>or=60 microM elicited clear-cut damaging effects on EC, whereas lower concentrations (2-4 microM) were cytoprotective. The cytoprotective effect was shifted to an EC-damaging pattern by means of specific VEGF blockade, therefore revealing a major role of autologous VEGF. Exposure to H2O2 increased VEGF and VEGFR2 mRNA and protein in EC, without affecting VEGFR1 expression. Also, H2O2 challenge was accompanied by increased NF-kappaB, activator protein-1, and specific protein-1 nuclear binding. A role of NF-kappaB as the mediator of the H2O2 induction of VEGFR2 mRNA expression was supported by inhibition by the ROS scavenger pyrrolidine dithiocarbamate and by the blocking effect of transfected IkappaBalpha. Exposure to exogenous VEGF also increased VEGFR2 and induced NF-kappaB in EC. In summary, autologous VEGF is instrumental for EC protection induced by low concentrations of ROS. ROS induce expression not only of VEGF but also of VEGFR2. VEGFR2 increase by ROS is mainly driven through a NF-kappaB-dependent pathway.

Role of endogenous vascular endothelial growth factor in tubular cell protection against acute cyclosporine toxicity.

BACKGROUND: Recent studies have shown that exogenous administration of vascular endothelial growth factor (VEGF) is protective against cyclosporine A (CsA) renal toxicity. No data are available, however, on the possible role of endogenous VEGF. Our objective was to examine whether endogenous VEGF has a significant role in the renal response against CsA toxicity. METHODS: In vivo, we used high-dose (50-150 mg/kg/day) CsA +/- specific goat anti-mouse VEGF blocking monoclonal antibody (alpha-VEGF) in mice. In vitro, we exposed mouse tubular cells (MCT) to CsA +/- alpha-VEGF. RESULTS: alpha-VEGF markedly enhanced CsA renal toxicity, inducing severe tubular damage and increased blood urea nitrogen. In animals treated with CsA + alpha-VEGF, damage progressed to generalized tubular injury (histology) and apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling) with associated anemia and reticulocytosis (18 days of treatment). CsA + alpha-VEGF treatments strikingly increased tubular VEGF and Bcl-xL proteins. In vitro, autocrine production of VEGF by MCT was identified by Western blot. Of specific interest, CsA toxicity in MCT increased significantly in the presence of alpha-VEGF. CONCLUSIONS: Endogenous VEGF has a relevant role in the renal tubular defense against CsA toxicity. Blockade of the VEGF effect by alpha-VEGF results in clear-cut intensification of the tubular injury and appearance of regenerative anemia in the CsA + alpha-VEGF-treated animals. The occurrence of both in vivo and in vitro effects of VEGF blockade provides evidence of a direct protective effect of VEGF on the tubular cell.