A novel specific application of pyruvate protects the mouse retina against white light damage: differential stabilization of HIF-1α and HIF-2α.

To mimic hypoxia preconditioning by a novel specific pyruvate treatment and to study its retinal protection against white light damage. Six-to-eight-week-old BALB/c mice were exposed to strong white light calculated to produce photoreceptor degeneration. Some were given injections of pyruvate in a preordained protocol because evidence exists that proves pyruvate can affect the concentration of hypoxia inducible factor (HIF). Western blotting and real-time PCR were used to determine the concentration of proteins and mRNAs in retinas. Morphology was analyzed with toluidine blue staining and was plotted using a spidergraph. A free nucleosome cell death assay was used to examine apoptosis. Retina explant cultures were used to investigate the background mechanism. Pyruvate administration stabilized hypoxia inducible factor (HIF)-1α but not HIF-2α. Expression of the downstream genes hemoxygenase-1 and erythropoietin mirrored the changes of the two HIFs, respectively. Importantly, pyruvate given not only before but also after exposure to light protected photoreceptors against apoptosis. In the retinal explant system, addition or depletion of pyruvate caused only changes of HIF-1α and prolyl hydroxylase (PHD)-2, while HIF-2α and PHD1 were not affected. However, under hypoxic conditions, HIF-2α was stabilized by pyruvate but not HIF-1α. Pyruvate evoked a hypoxia-like response under normoxic conditions and was retina-protective against strong white light. This response included stabilization of HIF-1α but not HIF-2α. This differential stabilization might be related to the distinct preference of their degrading enzyme of PHD2 and PHD1 in response to pyruvate treatment.

Ren H ，Liu NY ，Song XF ，Ma YS ，Zhai XY ... -  《-》

Increased activation of the hypoxia-inducible factor pathway in varicose veins.

Venous hypoxia has been postulated to contribute to varicose vein (VV) formation. Direct measurements of vein wall oxygen tension have previously demonstrated that the average minimum oxygen tensions were significantly lower in VVs compared with non-varicose veins (NVVs). Hypoxia-inducible factors (HIFs) are nuclear transcriptional factors that regulate the expression of several genes of oxygen homeostasis. This study aimed to investigate if hypoxia was associated with VVs by assessing the expression of HIF-1α, HIF-2α, HIF target genes, and upstream HIF regulatory enzymes in VVs and NVVs, and their regulation by hypoxia. VVs and NVVs were surgically retrieved and immediately snap-frozen or used for organ culture preparation. The relative expression of HIF-1α, HIF-2α, HIF target genes, and HIF regulatory enzymes in VVs and NVVs was analyzed with quantitative polymerase chain reaction (Q-PCR) and Western blot. VV and NVV organ ex vivo cultures were exposed to 16 hours of normoxia, hypoxia (oxygen tension 1%), or the hypoxia mimetic dimethyloxallyl glycine (DMOG) 1 mM in normoxia. The vein organ cultures were then analyzed for HIF-1α, HIF-2α, and their target gene expression with Q-PCR and Western blot. HIF-1α and HIF-2α mRNA were significantly upregulated in VVs compared with NVVs (89.8 ± 18.6 vs 10.4 ± 7.2 and 384.9 ± 209.4 vs 8.1 ± 4.2, respectively). HIF target gene mRNA expression was also significantly elevated in VVs compared with NVVs, namely glucose transporter-1 (GLUT-1; 8.7 ± 2.1 vs 1.0 ± 0.3), carbonic anhydrase-9 (CA9; 8.5 ± 2.1 vs 2.8 ± 1.2), vascular endothelial growth factor (VEGF; 7.5 ± 2.1 vs 0.9 ± 0.2), and BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3; 4.5 ± 0.7 vs 1.4 ± 0.3). The upregulation of HIF-1α, HIF-2α, and HIF target genes in VVs was also reflected at protein level. Of the HIF regulatory enzymes, the expression of prolyl-hydroxylase domain (PHD)-2 and PHD-3 was found to be elevated in VVs compared with NVVs. Exposure of VV and NVV organ cultures to hypoxia or DMOG was associated with increases in HIF-1α and HIF-2α protein and HIF target gene expression compared with normoxia only. The study concluded, we believe for the first time, an increased activation of the HIF pathway, with upregulation of the expression of HIF-1α and HIF-2α transcription factors, and HIF target genes, in VVs compared with NVVs. Exposure of VVs and NVVs to hypoxic conditions was associated with increased expression of HIF-1α and HIF-2α protein and HIF target genes. The data suggest that the HIF pathway may be associated with several pathophysiologic changes in the VV wall, and that hypoxia may be a feature contributing to VV pathogenesis.

Lim CS ，Kiriakidis S ，Paleolog EM ，Davies AH ... -  《-》

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

Thiersch M ，Lange C ，Joly S ，Heynen S ，Le YZ ，Samardzija M ，Grimm C ... -  《-》

Neuron-specific prolyl-4-hydroxylase domain 2 knockout reduces brain injury after transient cerebral ischemia.

Numerous factors involved in the adaptive response to hypoxia, including erythropoietin and vascular endothelial growth factor are transcriptionally regulated by hypoxia-inducible factors (HIFs). During normoxia, prolyl-4-hydroxylase domain (PHD) proteins hydroxylate HIF-α subunits, resulting in their degradation. We investigated the effect of neuronal deletion of PHD2, the most abundant isoform in brain, for stroke outcome. We generated neuron-specific Phd2 knockout mice and subjected animals to systemic hypoxia or transient middle cerebral artery occlusion. Infarct volume and cell death were determined by histology. HIF-1α, HIF-2α, and HIF target genes were analyzed by immunoblotting and real-time polymerase chain reaction, respectively. Neuron-specific ablation of Phd2 significantly increased protein stability of HIF-1α and HIF-2α in the forebrain and enhanced expression of the neuroprotective HIF target genes erythropoietin and vascular endothelial growth factor as well as glucose transporter and glycolysis-related enzymes under hypoxic and ischemic conditions. Mice with Phd2-deficient neurons subjected to transient cerebral ischemia exhibited a strong reduction in infarct size, and cell death of hippocampal CA1 neurons located in the peri-infarct region was dramatically reduced in these mice. Vessel density in forebrain subregions, except for caudate-putamen, was not altered in Phd2-deficient animals. Our findings denote that the endogenous adaptive response on hypoxic-ischemic insults in the brain is at least partly dependent on the activity of HIFs and identify PHD2 as the key regulator for the protective hypoxia response. The results suggest that specific inhibition of PHD2 may provide a useful therapeutic strategy to protect brain tissue from ischemic injury.

Kunze R ，Zhou W ，Veltkamp R ，Wielockx B ，Breier G ，Marti HH ... -  《-》

Normoxic activation of hypoxia-inducible factors in photoreceptors provides transient protection against light-induced retinal degeneration.

Hypoxic preconditioning activates hypoxia-inducible transcription factors (HIFs) in the retina and protects photoreceptors from light-induced retinal degeneration. The authors tested whether photoreceptor-specific activation of HIFs in normoxia is sufficient for protection. Rod-specific Vhl knockdown mice were generated using the Cre-lox system with the rod opsin promoter controlling expression of CRE recombinase to stabilize HIF transcription factors in normoxic rods. Cell death was induced by light exposure and quantified by ELISA. Rhodopsin was quantified by spectrophotometry. Gene expression was analyzed by real-time PCR, and levels of proteins were determined by Western blotting. Morphology was investigated by light microscopy and retinal function tested by ERG. The rod-specific Vhl knockdown stabilized HIF-α proteins and induced expression of HIF target genes in retinas of 10-week-old mice under normoxic conditions. Retinal morphology and function were normal. At 36 hours after exposure to excessive light, Vhl knockdowns showed significantly less photoreceptor cell death than did wild-type controls. Ten days after light exposure, however, photoreceptor degeneration in Vhl knockdowns was similar to that of control animals. Vhl knockdowns expressed Fgf2 at higher basal levels before light exposure. After light exposure, however, expression of Fgf2 was not significantly different from that of wild-type controls. Artificial activation of HIF transcription factors in normoxic photoreceptors results in an increased basal expression of Fgf2 that may contribute to a transient protection of rods against light damage. Full photoreceptor protection may require a hypoxia-like response in additional retinal cell types and/or the differential regulation of additional mechanisms.

Lange C ，Heynen SR ，Tanimoto N ，Thiersch M ，Le YZ ，Meneau I ，Seeliger MW ，Samardzija M ，Caprara C ，Grimm C ... -  《-》