Hypoxia-induced upregulation of pigment epithelium-derived factor by retinal glial (Müller) cells

Neuronal degeneration and aberrant neovascularization are common problems of ischemic retinopathies. Pigment epithelium-derived factor (PEDF), a neuroprotective protein and an inhibitor of angiogenesis, is produced by retinal glial (Müller) cells and can counterbalance elevated levels of vascular endothelial growth factor (VEGF), the expression of which is regulated primarily by hypoxia-inducible factor (HIF)-1. In an approach to mimic transient ischemia in vitro, primary Müller cells were cultured under transient and strong hypoxia (0.2% O(2) ), followed by reoxygenation at 2.5% O(2) , and molecular mechanisms that might contribute to changes in the intraretinal PEDF level were determined. Hypoxic conditions caused an increasing expression of HIF-1α and led to upregulation of both PEDF and VEGF. Treatment of the cells with synthetic HIF-1α blockers or neutralization of VEGF binding to VEGF receptors (VEGFR-1 and-2) suppressed hypoxia-induced PEDF upregulation. Furthermore, the presence of CoCl(2) (a hypoxia mimetic) induced an accumulation of elevated HIF-1α protein in the nucleus and an upregulation of PEDF expression in Müller cells. Increasing PEDF expression was attenuated when HIF-1α levels were suppressed using HIF-1α small interfering RNA (siRNA). On the other hand, siRNA-mediated depletion of PEDF facilitated HIF-1α upregulation caused by CoCl(2) and resulted in increasing VEGF mRNA and protein levels. These results demonstrate that VEGF and PEDF may be unidirectionally regulated in hypoxia through HIF-1α activation, with upregulation of PEDF, which may occur in a VEGF-dependent manner. However, endogenously produced PEDF seems to be an inherent control element of HIF-1α expression in Müller cells, indicating an important feedback mechanism for limiting upregulation of VEGF.     

Adaptation to moderate hypoxia protects cortical neurons against ischemia-reperfusion injury and excitotoxicity independently of HIF-1α

Continuous exposure of cultured cortical neurons to moderate hypoxia (1% O(2)) elevates cellular accumulation of hypoxia-inducible factor-1α (HIF-1α) and improves basal survival of cultured cortical neurons. We examined the effects of adaptation to moderate hypoxia on the vulnerability of cultured neurons to the acute injury of simulated ischemia-reperfusion. Cortical neurons cultured continuously in 1% O(2) were markedly protected against simulated ischemia-reperfusion, with protection persisting through 72h after ischemia. Neurons from 1% O(2) conditions were also highly resistant to glutamate-induced NMDA receptor-dependent excitotoxic injury, despite expression of NMDA receptors at levels not significantly changed from controls. Inhibition of prolyl hydroxylase, mimicking cellular signaling effects of hypoxia including HIF-1α stabilization, also protected neurons against simulated ischemia-reperfusion injury. Nevertheless, genetic deletion of HIF-1α expression did not diminish the protection of neurons adapted to 1% O(2) from excitotoxicity or ischemia-reperfusion injury, nor did it prevent the protective effect of prolyl hydroxylase inhibition. We conclude that chronic exposure to moderate hypoxia, through HIF-1α-independent mechanisms, produces strong protective effects against excitotoxic and ischemia-reperfusion related injury.     

Transient exposure of rat pups to hyperoxia at normobaric and hyperbaric pressures does not cause retinopathy of prematurity

We have shown that hyperoxia reduces brain damage in a rat model of hypoxia-ischemia. The purpose of this study was to examine the possibility of hyperoxia in inducing vision-threatening retinopathy. Two different experiments were conducted in this study. PART 1: seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O2 at 37 degrees C). Pups were treated with 100% oxygen at 1 ATA, 1.5 ATA, and 3.0 ATA for a duration of 1 h. PART 2: Newborn rat pups were exposed to 100% oxygen at 1, 1.5, or 3.0 ATA for 1 h, the same treatment protocol used for brain protection after hypoxia-ischemia. Retinopathy was evaluated by the degree of neovascularization (measuring retinal vascular density), by the structural abnormalities (histology) in the retina, and by the expression of hypoxia-hyperoxia sensitive proteins including hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) at 24 h, 1, 2, and 10 weeks after hyperoxia exposure. Hyperoxic treatment at all pressures administered significantly reduced the hypoxia-ischemic-induced reduction in brain weight. Retinal vascular density measurements revealed no signs of neovascularization after hyperoxia exposure. There were also no abnormalities in the structure of the retina and no changes in the protein expression of HIF-1alpha and VEGF following hyperoxia exposure. Exposure to hyperoxia for 1 h at normobaric or hyperbaric pressures did not result in the structural changes or abnormal vascularization that is associated with retinopathy of prematurity, suggesting that hyperoxia is a safe treatment for hypoxic newborn infants.     

Potential roles for presenilin-1 in oxygen sensing and in glial-specific gene expression

The integral membrane glycoprotein presenilin-1 (PS1), in concert with beta-secretase and beta amyloid precursor protein (betaAPP), orchestrate cleavage of betaAPP into amyloidogenic Abeta peptides. To gain further insight into PS1 function, we undertook a gene expression profiling study that interrogated the expression of 12,000 genes in the forebrain of PS1-hypomorphic mice that exhibit highly attenuated PS1 activity. Using stringent RNA screening, DNA array and Northern assay, we report significant down-regulation in the expression of betaAPP and hypoxia inducible factor-1 alpha (HIF-1alpha), and a marked up-regulation in the expression of glial-specific markers that include S100beta protein, glycerol phosphate dehydrogenase, and glial fibrillary acidic protein. These data suggest potential roles for PS1 in the cellular response to hypoxia and glial-specific gene expression.     

Long-term effects of acute and of chronic hypoxia on behavior and on hippocampal histology in the developing brain

Ten-day-old rat pups (P10) subjected to acute hypoxia (down to 4% O2) had as adults increased aggression (handling test), memory impairment (water maze test), and decreased CA1 cell counts. Pups subjected to chronic hypoxia (10% O2 from P0 to P21) had increased aggression, hyperactivity (open-field test), and decreased CA1 cell counts. Chronic hypoxia with superimposed acute hypoxia resulted in consequences that were not different from those of chronic hypoxia.     

Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes.

The brain's adaptive response to ischemic preconditioning (IPC) is mediated in part via hypoxia inducible factor (HIF)-responsive genes. We previously showed that IPC induces cytochrome P450 2C11 expression in the brain, associated with protection from stroke. Cytochrome P450 2C11 is an arachidonic acid (AA) epoxygenase expressed in astrocytes, which metabolizes AA to epoxyeicosatrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1alpha, and that the P450 epoxygenase pathway contributes to enhanced astrocyte tolerance to ischemia-like injury induced by oxygen-glucose deprivation (OGD). Primary cultured astrocytes were incubated under normoxic or hypoxic conditions for 1, 3, 6, 24, or 48 h, and protein levels of P450 2C11 and HIF-1alpha were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1alpha to 2C11 promoter was evaluated using electrophoretic mobility shift assay (EMSA) with 2C11 promoter DNA containing putative HIF-binding sites. Levels of 2C11 mRNA and protein were significantly increased starting at 3 and 6 h of hypoxia, respectively. The increase in 2C11 expression was preceded by an increase in HIF-1alpha protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1alpha in nuclear extracts from astrocytes. HPC and EETs reduced astrocyte cell death, and P450 epoxygenase inhibition prevented protection by HPC. We conclude that HPC induces tolerance in astrocytes, at least in part, via HIF-1alpha-linked upregulation of P450 2C11.     

Protective effects of sesamin and sesamolin on hypoxic neuronal and PC12 cells

Reactive oxygen species (ROS) are important mediators of a variety of pathological processes, including inflammation and ischemic injury. The neuroprotective effects of sesame antioxidants, sesamin and sesamolin, against hypoxia or H2O2-induced cell injury were evaluated by cell viability or lactate dehydrogenase (LDH) activity. Sesamin and sesamolin reduced LDH release of PC12 cells under hypoxia or H2O2-stress in a dose-dependent manner. Dichlorofluorescein (DCF)-sensitive ROS production was induced in PC12 cells by hypoxia or H2O2-stress but was diminished in the presence of sesamin and sesamolin. We evaluated further the role of mitogen-activated protein kinases (MAPKs) and caspase-3 in hypoxia-induced PC12 cell death. Extracellular signal-regulated protein kinase (ERK) 1, c-jun N-terminal kinase (JNK), and p38 MAPKs of signaling pathways were activated during hypoxia. We found that the inhibition of MAPKs and caspase-3 by sesamin and sesamolin correlated well with the reduction in LDH release under hypoxia. Furthermore, the hypoxia-induced apoptotic-like cell death in cultured cortical cells as detected by a fluorescent DNA binding dye was reduced significantly by sesamin and sesamolin. Taken together, these results suggest that the protective effect of sesamin and sesamolin on hypoxic neuronal and PC12 cells might be related to suppression of ROS generation and MAPK activation.