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.

BackgroundAn increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. Here, we have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion.MethodsAnimals were subjected to focal cerebral ischemia with reperfusion. To assess the role of Rcan1 after stroke, we measured infarct volume after 48 h of reperfusion in Rcan1 knockout (KO) and wild-type (WT) mice. In vitro studies were performed in astrocyte-enriched cortical primary cultures subjected to 3% oxygen (hypoxia) and glucose deprivation (HGD). Adenoviral vectors were used to analyze the effect of overexpression of Rcan1-4 protein. Protein expression was examined by immunohistochemistry and immunoblotting and expression of mRNA by quantitative real-time Reverse-Transcription Polymerase Chain Reaction (real time qRT-PCR).ResultsBrain ischemia/reperfusion (I/R) injury in vivo increased mRNA and protein expression of the calcium-inducible Rcan1 isoform (Rcan1-4). I/R-inducible expression of Rcan1 protein occurred mainly in astroglial cells, and in an in vitro model of ischemia, HGD treatment of primary murine astrocyte cultures induced Rcan1-4 mRNA and protein expression. Exogenous Rcan1-4 overexpression inhibited production of the inflammatory marker cyclo-oxygenase 2. Mice lacking Rcan1 had higher expression of inflammation associated genes, resulting in larger infarct volumes.ConclusionsOur results support a protective role for Rcan1 during the inflammatory response to stroke, and underline the importance of the glial compartment in the inflammatory reaction that takes place after ischemia. Improved understanding of non-neuronal mechanisms in ischemic injury promises novel approaches to the treatment of acute ischemic stroke.

Transcriptional regulation of vascular endothelial growth factor (VEGF) is critically dependent on hypoxia-inducible factor 1 (HIF-1). However, not only hypoxia, but selected growth factors can induce HIF-1. High levels of both VEGF and HIF-1 coexist in certain conditions, e.g. tumors. Nonetheless, the possibility that the stimulatory relationship between HIF-1 and VEGF may be bi-directional has not been addressed up to date. The present study in endothelial cells analyzed whether HIF-1 is regulated by a product of its own transcriptionally activated genes, namely, VEGF. As a main finding, VEGF-A 165 induced the increase of HIF-1␣ mRNA and HIF-1␣ protein and nuclear translocation. Autologous endothelial cell VEGF mRNA and protein were also increased upon exposure to exogenous VEGF. The signaling implication of reactive oxygen species was examined by comparison with H 2 O 2 and hypoxanthine/xanthine oxidase and by the superoxide dismutase mimetic, MnTMPyP, the Rac1-NAD(P)H oxidase complex inhibitor, apocynin, transfection of a dominant negative Rac1 mutant, and transfection of a p67phox antisense oligonucleotide. Superoxide anion, largely dependent on Rac1-NAD(P)H oxidase complex activity, was the critical signaling element. The transductional functionality of the pathway was confirmed by means of a reporter gene flanked by a transcription site-related VEGF sequence and by quantitative PCR. In summary, the present results reveal a previously undescribed action of VEGF on the expression of its own transcription factor, HIF-1, and on VEGF itself. This effect is principally mediated by superoxide anion, therefore identifying a new, potentially relevant role of reactive oxygen species in VEGF signaling.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

Increase in intracellular calcium ([Ca(2+) ]i ) is a key mediator of astrocyte signaling, important for activation of the calcineurin (CN)/nuclear factor of activated T cells (NFAT) pathway, a central mediator of inflammatory events. We analyzed the expression of matrix metalloproteinase 3 (Mmp3) in response to increases in [Ca(2+) ]i and the role of the CN/NFAT pathway in this regulation. Astrocyte Mmp3 expression was induced by overexpression of a constitutively active form of NFATc3, whereas other MMPs and tissue inhibitor of metalloproteinases (TIMP) were unaffected. Mmp3 mRNA and protein expression was also induced by calcium ionophore (Io) and 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and Mmp3 upregulation was prevented by the CN inhibitor cyclosporin A (CsA). Ca(2+) -dependent astrocyte Mmp3 expression was also inhibited by actinomycin D, and a Mmp3 promoter luciferase reporter was efficiently activated by increased [Ca(2+) ]i , indicating regulation at the transcriptional level. Furthermore, Ca(2+) /CN/NFAT dependent Mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (Ast-NSC), demonstrating that the induced Mmp3 expression occurs in astrocytes, and not microglial cells. In an in vivo stab-wound model of brain injury, MMP3 expression was detected in NFATc3-positive scar-forming astrocytes. Because [Ca(2+) ]i increase is an early event in most brain injuries, these data support an important role for Ca(2+) /CN/NFAT-induced astrocyte MMP3 expression in the early neuroinflammatory response. Understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain.

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.

Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl2 significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl2, both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects.

AG490 is a tyrphostin originally described as a Janus Activated Kinase (JAK) 2 inhibitor. AG490 also inhibits epidermal growth factor receptor (EGFR) and guanylyl cyclases (GC). More recently, AG490 was associated with oxidative stress protection in experimental acute kidney injury models. We now show that AG490 is also a strong activator of the Hypoxia Inducible Factor (HIF)-1. Under normoxic conditions HIF-1α is degraded through hydroxylation, von Hippel Lindau protein (VHL)-mediated ubiquitin tagging and proteasomal degradation. AG490 increased HIF-1α protein, but not HIF-1α mRNA levels, dose- and time-dependently in cultured endothelial, vascular smooth muscle and kidney proximal tubular epithelial cells. AG490 increased HIF-1α protein half-life, suggesting that HIF-1α protein accumulation resulted from a decreased degradation. In this regard, AG490 prevented HIF-1α hydroxylation and increased HIF-1α protein levels in human renal carcinoma cells expressing VHL, but did not further increase HIF-1α in VHL negative cells. AG490 did not prevent the proteasomal degradation of other proteins. HIF-1α was not upregulated by dominant negative JAK2constructs, tyrphostin AG9, the EGFR inhibitors erbstatin and genistein, the GC inhibitor Ly83583 or cGMP analogues. Finally, AG490 also increased HIF-1α transcriptional activity evidenced by the increased HIF-1α-dependent VEGF expression. In conclusion, AG490 is a novel HIF-1α activator that increases HIF-1α half-life and protein levels through interference with HIF-1α hydroxylation and VHL-mediated degradation. This action may contribute to the cell and tissue protective effects of AG490.