HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.     

Glaucoma-induced degeneration of retinal ganglion cells prevented by hypoxic preconditioning: a model of glaucoma tolerance

Like all cells, neurons adapt to stress by transient alterations in phenotype, an epigenetic response that forms the basis for preconditioning against acute ischemic injury in the central nervous system. We recently showed that a modified repetitive hypoxic preconditioning (RHP) regimen significantly extends the window of ischemic tolerance to acute retinal ischemic injury from days to months. The present study was undertaken to determine if this uniquely protracted neuroprotective phenotype would also confer resistance to glaucomatous neurodegeneration. Retinal ganglion cell death at somatic and axonal levels was assessed after both 3 and 10 wks of sustained intraocular hypertension in an adult mouse model of inducible, open-angle glaucoma, with or without RHP before intraocular pressure elevation. Loss of brn3-positive ganglion cell soma after 3 wks of experimental glaucoma, along with increases in several apoptotic endpoints, were all significantly and robustly attenuated in mice subjected to RHP. Soma protection by RHP was also confirmed after 10 wks of intraocular hypertension by brn3 and SMI32 immunostaining. In addition, quantification of axon density in the postlaminar optic nerve documented robust preservation in RHP-treated mice, and neurofilament immunostaining also revealed preconditioning-induced improvements in axon integrity/survival in both retina and optic nerve after 10 wks of experimental glaucoma. This uniquely protracted period of phenotypic change, established in retinal ganglion cells by the activation of latent antiapoptotic, prosurvival mechanisms at both somatic and axonal levels, reflects a novel form of inducible neuronal plasticity that may provide innovative therapeutic targets for preventing and treating glaucoma and other neurodegenerative diseases.     

Podocytes protect glomerular endothelial cells from hypoxic injury via deSUMOylation of HIF-1α signaling

Hypoxia can cause severe tubulointerstitial injury and peritubular capillary loss. However, hypoxia-induced injury in glomerular capillaries is far milder than tubulointerstitium, but the reason for this difference is unclear. We hypothesized that the phenomenon is due to the protective crosstalk among intrinsic glomerular cells. To mimic the microenvironment and investigate the crosstalk process temporally, we established co-culture models of glomerular endothelial cells (GEnCs) with podocytes or with mesangial cells. We found that podocytes rather than mesangial cells prevented GEnCs from injury and hypoxia-induced apoptosis and promoted migration and angiogenesis of GEnCs under hypoxic conditions. We then identified that increased activation of the hypoxia inducible factor 1α (HIF-1α) pathway as the major mechanism enabling podocytes to protect GEnCs against hypoxia. HIF-1α stabilization during hypoxia is known to be dependent on SUMO-specific protease 1 (SENP1)-mediated deSUMOylate modifications. Therefore, we further targeted deSUMOylation, regulated by SENP1, by short hairpin RNA (shRNA) knockdown of SENP1 mRNA in vitro and measured expression of HIF-1α and its downstream gene VEGF in hypoxic podocytes. Our results showed that SENP1 was essential for HIF-1α deSUMOylation in podocytes. The blockade of deSUMOylation by SENP1 shRNA successfully abolished the activation of HIF-1α signaling and consequently suppressed the protective effects of podocytes on GEnCs. In conclusion, we demonstrate for the first time that hypoxia may promote HIF-1α stabilization and activation by increasing SENP1 expression in podocytes, which induce GEnCs survival and angiogenesis to resist hypoxia. Thus, deSUMOylation of HIF-1α signaling is a potentially novel therapeutic target for treating hypoxic renal disorders.     

Ginkgolides mimic the effects of hypoxic preconditioning to protect C6 cells against ischemic injury by up-regulation of hypoxia-inducible factor-1 alpha and erythropoietin

Hypoxic preconditioning can play a significant neuroprotective role. However, it has not been employed clinically because of safety concerns. To find a safer preconditioning stimulus that is both practical and effective, we investigated whether ginkgolides are capable of preconditioning as hypoxia to protect C6 cells against ischemic injury. We demonstrated that both ginkgolides (37.5microg/mL) and hypoxia (1% O(2) for 16h) can significantly increase cell viabilities and expression of phosphorylated glycogen synthase kinase (p-GSK), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) in ischemic cells. The inhibitors of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3'-kinase (PI3K) significantly but not completely reduced the enhanced expression of these proteins and cell viabilities induced by ginkgolides and hypoxic preconditioning. These indicated that ginkgolides could mimic hypoxic preconditioning by increasing expression of HIF-1alpha as well as its target protein EPO and that the ginkgolides and hypoxic preconditioning role might be partly mediated by the activation of the p42/p44-mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT/glycogen synthase kinase 3beta pathways. The similar tendency in the changes of protein expression, cell viabilities and responses to MAPK or PI3K inhibitors of the cells treated with ginkgolides and hypoxia suggests that ginkgolides and hypoxic preconditioning might operate by similar mechanisms. The findings also imply that ginkgolides might have the potential for clinical use to prevent injury in high-risk conditions.     

Ischemic and hypoxic preconditioning protect cardiac muscles via intracellular ROS signaling

Oxidative stress can cause extensive damage to cardiac tissue under reperfusion conditions. However, preconditioning the myocardium may diminish these negative effects and alleviate reperfusion injury. There are a variety of preconditioning therapies, such as ischemic preconditioning (IPC) and hypoxic preconditioning (HPC), each targeting specific channels, receptors, and/or intracellular molecules. Ischemic preconditioning involves brief periods of ischemia followed by brief periods of reperfusion, thus strengthening the cardiac resistance for a longer period of ischemia. IPC involves complex mechanisms, some of which are still not completely understood today. Nevertheless, many studies have already established models of IPC. In addition, similar to IPC, HPC has also been recognized as preventing reperfusion injury. Reactive oxygen species (ROS) are known mediators of IPC and HPC. Particularly, mitochondria-generated ROS initiate activity of several beneficial preconditioning pathways. The role of ROS is paradoxical; low levels of ROS are key factors in signaling IPC/HPC, but high levels of ROS can contribute to increased oxidative stress on cardiomyocytes. Therefore, it is important to determine the molecular mechanism of IPC and HPC to avoid excessive accumulation of ROS to prevent cardiac injury. In this review, we will outline IPC and HPC, explaining the putative role of ROS in both pathways. We will also discuss preconditioning efficacy in certain conditions such as exercise and how the aging myocardium responds to preconditioning therapies.     

Prevention of HIF-1 activation and iNOS gene targeting by low-dose cadmium results in loss of myocardial hypoxic preconditioning in the rat

This study aimed to underline the interaction between hypoxia-inducible factor-1 (HIF-1) and the inducible nitric oxide synthase (iNOS) gene in vivo and their contribution to the delayed myocardial preconditioning induced by acute intermittent hypoxia (IH) in the rat using chromatin immunoprecipitation and pharmacological inhibition by low-dose cadmium. Langendorff-perfused hearts of Wistar rats exposed to normoxia or IH 24 h earlier were submitted to global ischemia and reperfusion. Effects of iNOS inhibition by aminoguanidine (100 microM) before ischemia or of low-dose injection of cadmium chloride (1 mg/kg) before normoxia or IH were tested. Myocardial HIF-1 and iNOS quantification and in vivo chromatin immunoprecipitation of HIF-1 bound to the iNOS gene promoter were performed. IH-induced delayed cardioprotection resulted in an improvement in coronary flow and functional recovery at reperfusion and a decrease in infarct size. Myocardial HIF-1 activity was increased with resulting targeting of the iNOS gene. Aminoguanidine abolished the cardioprotective effects of IH. Cadmium chloride treatment before IH prevented myocardial HIF-1 activation (72.3 +/- 4.0 vs. 42.1 +/- 9.7 arbitrary units after cadmium chloride; P < 0.05), targeting of the iNOS gene, iNOS expression, and preconditioning (infarct size: 15.9 +/- 5.6 vs. 30.1 +/- 5.4% after cadmium chloride; P < 0.05). This study is the first to demonstrate the interaction of HIF-1 with the myocardial iNOS gene in situ after hypoxic preconditioning. Prevention of HIF-1 activation and iNOS gene targeting by a single low dose of cadmium abolished the delayed cardioprotective effects, bringing insight into the cardiovascular consequences of cadmium exposure.     

低氧及低氧预处理时心肌细胞HIF-1α与凋亡相关蛋白表达的变化

目的:观察低氧时心肌细胞HIF-1α表达变化与凋亡相关蛋白表达关系.方法:采用体外心肌细胞培养的方法,将原代培养4～6 d的大鼠乳鼠心肌细胞随机分为对照组、低氧组与低氧预处理组.低氧预处理组在低氧培养箱中通入1%O2、5%CO2、94%N2的低氧混合气体,每天低氧12 h,低氧5 d,第6 d与急性低氧组一同放入0%O2、5%CO2、95%N2的低氧培养箱中进行低氧暴露.低氧48 h后,通过Western blot方法分别检测心肌细胞中HIF-1α、Bcl-2、P53及Bax的表达变化.结果:常氧时细胞不表达HIF-1α,低氧可增加HIF-1的表达,低氧预处理后,能降低HIF-1α的表达.低氧时,Bax的表达变化大致与此相同.p53在低氧时的变化也与其相同,但低氧预处理后似乎没有明显的改变.Bcl-2在低氧时表达下降,低氧预处理后可增加其表达.结论:HIF-1α的表达可协同Bcl-2家族凋亡相关蛋白的表达,在低氧导致的心肌细胞凋亡中发挥重要作用.     

Primary amines protect against retinal degeneration in mouse models of retinopathies

Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore 11-cis-retinal and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomerized product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing Food and Drug Administration (FDA)-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by MS. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that shows features of Stargardt's disease and age-related retinal degeneration.     

低氧预适应训练在急进高海拔高原部队中的应用研究

目的：在高海拔高原部队验证低氧预适应降低急性高原反应、改善体能的效果,并观察效果的时间保留规律。方法：拟进驻4300m高原新兵利用低氧呼吸器进行低氧预适应训练,每天上、下午各1h,连续5d,分别于训练结束后第2天和第6天进入高原,观测受试者的急性高原反应症状和体能。结果：低氧预适应训练可显著降低急性高原病的患病率;训练结束后第2天进入高原者VO2 max和PWC170显著降低,而第6天进入高原者,VO2max无明显改变。结论：进驻高海拔高原前进行为期5天的低氧预适应训练可显著降低急性高原病的患病率,训练效果至少可保留5天。     

Folic Acid Exerts Post-Ischemic Neuroprotection In Vitro Through HIF-1α Stabilization

The constant failure of single-target drug therapies for ischemic stroke necessitates the development of novel pleiotropic pharmacological treatment approaches, to effectively combat the aftermath of this devastating disorder. The major objective of our study involves a multi-target drug repurposing strategy to stabilize hypoxia-inducible factor-1 α (HIF-1α) via a structure-based screening approach to simultaneously inhibit its regulatory proteins, PHD2, FIH, and pVHL. Out of 1424 Food and Drug Administration (FDA)-approved drugs that were screened, folic acid (FA) emerged as the top hit and its binding potential to PHD2, FIH, and pVHL was further verified by re-docking, molecular dynamics (MD) simulation and by Drug Affinity Responsive Target Stability (DARTS) assay. HIF-1α stabilization by FA was demonstrated by the nuclear translocation and increased green fluorescence emission of HIF-1α using HIF1α-GFPSpark tag vector. Further, FA treatment enhanced the cell survival following oxygen glucose deprivation and its neuroprotective mechanism was elucidated by measuring the expression of BAX, NFE2L2, VEGF, and EPO genes in a time-dependent manner (5 and 11 h following FA treatment). VEGF and EPO expressions were significantly increased by 5.41- and 1.35-folds, respectively, whereas BAX expression reduced by 4-fold at 11 h post-FA treatment. NFE2L2 expression was elevated (1.65-fold) at 5 h with no major difference at 11 h post-FA treatment. The chicken chorioallantoic membrane (CAM) assay demonstrated the pro-angiogenic potential of FA as evidenced by an increased blood vessel density and branching. The present study elucidates for the first time that the post-ischemic neuroprotection exerted by FA may be attributed to its HIF-1α stabilization and pro-angiogenic properties.     

Hyperthermic preconditioning protects astrocytes from ischemia/reperfusion injury by up-regulation of HIF-1 alpha expression and binding activity

It has been demonstrated that hypoxia-inducible factor-1 alpha (HIF-1 alpha) mediates ischemic tolerance induced by hypoxia/ischemia or pharmacological preconditioning. In addition, preconditioning stimuli can be cross-tolerant, safeguarding against other types of injury. We therefore hypothesized HIF-1 alpha might also be associated with ischemic tolerance induced by hyperthermic preconditioning. In the present study, we demonstrated for the first time that 6 h of hyperthermia (38 °C or 40 °C) could induce a characteristic “reactive” morphology and a significant increase in the expression of bystin in astrocytes. We also showed that pre-treatment with 6 h of hyperthermia resulted in a significant increase in cell viability and a remarkable decrease in lactate dehydrogenase (LDH) release and apoptosis development in the astrocytes that were exposed to 24 h of ischemia and a subsequent 24 h of reperfusion. Analysis of mechanisms showed that hyperthermia could lead to a significant increase in HIF-1 alpha expression and also the HIF-1 binding activity in the ischemia/reperfusion astrocytes. The data provide evidence to our hypothesis that the up-regulation of HIF-1 alpha is associated with the protective effects of hyperthermic preconditioning on astrocytes against ischemia/reperfusion injury.     

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1 in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1 accumulation in different tumor cell lines. Hypoxia-induced HIF-1 accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F₀F₁-ATPase, prevented hypoxia-induced HIF-1 protein accumulation and had no effect on HIF-1 induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1 content was pronounced in cells exposed to hypoxia (1.5% O₂) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1 regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1 accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function. hypoxia-inducible factor 1; oxygen sensing     

Photoreceptor morphogenesis and retinal degeneration: lessons from Drosophila

Cells exhibit an amazingly wide range of different forms, and in most cases the shape of a cell is crucial for performing its specific function(s). But how does a cell obtain its particular shape during development, how can the shape be adapted to different environmental conditions, and what are the consequences if morphogenesis is impaired? An ideal cell type to study these questions is the photoreceptor cell, a photosensitive cell present in most metazoa, highly specialised to transform the energy from the light into a visual response. In the last few years, studies in the Drosophila eye have led to a considerable increase in understanding of the genetic control of photoreceptor morphogenesis; lessons, which may apply to other cell types as well. Most of the genes involved have been conserved during evolution, and mutations in several of them result in retinal degeneration, both in flies and humans. This makes the fly eye an attractive model to unravel the genetic, molecular and cell biological basis of the mechanisms that prevent retinal dystrophies.     

Neuroprotection by iron chelator against proteasome inhibitor-induced nigral degeneration

The cause of the neurodegenerative process in Parkinson's disease (PD) remains unclear, but evidence suggests that failure of the ubiquitin-proteasome system may play a major role in the pathogenesis of the disease. Iron is believed to be a key contributor to PD pathology by inducing aggregation of alpha-synuclein and by generating oxidative stress. Our present studies have shown that micro-injection of the proteasome inhibitor lactacystin into the substantia nigra (SN) of C57BL/6 mice causes significant loss of dopaminergic cells and induces intracellular inclusion body formation. We have also found that co-injection of the iron chelator desferrioxamine not only attenuates the lactacystin-induced dopamine neuron loss, but also reduces the presence of ubiquitin-positive intracellular inclusions in the SN, whereas use of iron-deficient diet has no such protective effects. These results may support that iron plays a key role in proteasome inhibitor-induced nigral pathology and that reducing iron reactivity may prevent dopaminergic neuron degeneration and reduce abnormal protein aggregation.     

Constituents of bile, bilirubin and TUDCA, protect against oxidative stress-induced retinal degeneration

Two constituents of bile, bilirubin and tauroursodeoxycholic acid (TUDCA), have antioxidant activity. However, bilirubin can also cause damage to some neurons and glial cells, particularly immature neurons. In this study, we tested the effects of bilirubin and TUDCA in two models in which oxidative stress contributes to photoreceptor cell death, prolonged light exposure and rd10+/+ mice. In albino BALB/c mice, intraperitoneal injection of 5 mg/kg of bilirubin or 500 mg/kg of TUDCA prior to exposure to 5000 lux of white light for 8 h significantly reduced loss of rod and cone function assessed by electroretinograms. Both treatments also reduced light-induced accumulation of superoxide radicals in the outer retina, rod cell death assessed by outer nuclear layer thickness, and disruption of cone inner and outer segments. In rd10+/+ mice, intraperitoneal injections of 5 or 50 mg/kg of bilirubin or 500 mg/kg of TUDCA every 3 days starting at postnatal day (P) 6, caused significant preservation of cone cell number and cone function at P50. Rods were not protected at P50, but both bilirubin and TUDCA provided modest preservation of outer nuclear layer thickness and rod function at P30. These data suggest that correlation of serum bilirubin levels with rate of vision loss in patients with retinitis pigmentosa could provide a useful strategy to test the hypothesis that cones die from oxidative damage in patients with retinitis pigmentosa. If proof-of-concept is established, manipulation of bilirubin levels and administration of TUDCA could be tested in interventional trials.