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

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

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

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

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.

Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis.

In the adult hippocampus, neuroprogenitor cells in the subgranular zone (SGZ) of the dentate gyrus give rise to newborn neuroblasts. However, only a small subset of these cells integrates into the hippocampal circuitry as mature neurons at the end of a 4 week period. Here, we show that the majority of the newborn cells undergo death by apoptosis in the first 1 to 4 days of their life, during the transition from amplifying neuroprogenitors to neuroblasts. These apoptotic newborn cells are rapidly cleared out through phagocytosis by unchallenged microglia present in the adult SGZ niche. Phagocytosis by the microglia is efficient and undeterred by increased age or inflammatory challenge. Our results suggest that the main critical period of newborn cell survival occurs within a few days of birth and reveal a new role for microglia in maintaining the homeostasis of the baseline neurogenic cascade.

Alkalinization potentiates vascular calcium deposition in an uremic milieu.

BACKGROUND: Vascular calcification is a serious complication of chronic kidney disease. Acid-base balance is a relevant, albeit somewhat forgotten factor in the regulation of calcium deposition. Hemodialysis patients undergo repeated episodes of alkaline loading from the dialysate, resulting in prolonged alkalinization. We have hypothesized that extracellular alkalinization may promote vascular calcification. METHODS: Primary cultures of vascular smooth muscle cells were induced to calcify by the phosphate donor beta-glycerophosphate, in the presence of normal or uremic sera from hemodialysis patients and at different pH conditions. The influence of sodium bicarbonate supplementation for 2 months on aorta calcification was studied in 5/6 nephrectomized uremic rats. RESULTS: Uremic serum increased vascular smooth muscle cell calcification (twofold over nonuremic human serum at day 12, p<0.001). Alkalinization of the extracellular medium also increased vascular smooth muscle cell calcification. Increasing the extracellular pH from 7.42 to 7.53 resulted in a 2.5-fold increase in calcium accumulation at day 12 (p<0.05). In vivo, arterial calcification was significantly higher in alkalinized uremic animals (aorta calcification index, uremic + sodium bicarbonate, 164 +/- 57 units, vs. uremic + vehicle, 56 +/- 14 units; p<0.01). CONCLUSIONS: Alkalinization increases vascular calcification in cultured cells and uremic rats. These data may be used to optimize dialysate composition and the degree of alkalinization in calcification-prone individuals with advanced renal disease.

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 cell protection by blockade of the JAK2 pathway.

Inhibition of the JAK2/STAT pathway has been implicated recently in cytoprotective mechanisms in both vascular smooth muscle cells and astrocytes. The advent of JAK2-specific inhibitors provides a practical tool for the study of this pathway in different cellular types. An interest in finding methods to improve endothelial cell (EC) resistance to injury led us to examine the effect of JAK2/STAT inhibition on EC protection. Furthermore, the signaling pathways involved in JAK2/STAT inhibition-related actions were examined. Our results reveal, for the first time, that blockade of JAK2 with the tyrosine kinase inhibitor AG490 strongly protects cultured EC against cell detachment-dependent death and serum deprivation and increases reseeding efficiency. Confirmation of the specificity of the effects of JAK2 inhibition was attained by finding protective effects on transfection with a dominant negative JAK2. Furthermore, AG490 blocked serum deprivation-induced phosphorylation of JAK2. In terms of mechanism, treatment with AG490 induces several relevant responses, both in monolayer and detached cells. These mechanisms include the following: 1) Increase and nuclear translocation of the active, dephosphorylated form of beta-catenin. In functional terms, this translocation is transcriptionally active, and its protective effect is further supported by the stimulation of EC cytoprotection by transfectionally induced excess of beta-catenin. 2) Increase of platelet endothelial cell adhesion molecule (PECAM)/CD31 levels. 3) Increase in total and phosphorylated AKT. 4) Increase in phosphorylated glycogen synthase kinase (GSK)3alpha/beta. The present findings imply potential practical applications of JAK2 inhibition on EC. These applications affect not only EC in the monolayer but also circulating detached cells and involve mechanistic interactions not previously described.

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.

Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma.

BACKGROUND: Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLCgamma) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2. METHODS: The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation-activated PLCgamma. RESULTS: The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase-mediated mechanism, as determined by the blockade of short-term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)-dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non-confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLCgamma with a concentration- and time-profile similar to that of [Ca(2+)](i) mobilization. CONCLUSIONS: The present study reveals that H(2)O(2) activates PLCgamma on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca(2+)](i) mobilization, shape-changing actions and damage by H(2)O(2) in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H(2)O(2) and may help to design pharmacologically protective strategies.