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 ... -  《-》

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 ... -  《-》

Hypoxic preconditioning protects photoreceptors against light damage independently of hypoxia inducible transcription factors in rods.

Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors.

Kast B ，Schori C ，Grimm C 《-》

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 ... -  《-》

Retina-specific activation of a sustained hypoxia-like response leads to severe retinal degeneration and loss of vision.

Loss of vision and blindness in human patients is often caused by the degeneration of neuronal cells in the retina. In mouse models, photoreceptors can be protected from death by hypoxic preconditioning. Preconditioning in low oxygen stabilizes and activates hypoxia inducible transcription factors (HIFs), which play a major role in the hypoxic response of tissues including the retina. We show that a tissue-specific knockdown of von Hippel-Lindau protein (VHL) activated HIF transcription factors in normoxic conditions in the retina. Sustained activation of HIF1 and HIF2 was accompanied by persisting embryonic vasculatures in the posterior eye and the iris. Embryonic vessels persisted into adulthood and led to a severely abnormal mature vessel system with vessels penetrating the photoreceptor layer in adult mice. The sustained hypoxia-like response also activated the leukemia inhibitory factor (LIF)-controlled endogenous molecular cell survival pathway. However, this was not sufficient to protect the retina against massive cell death in all retinal layers of adult mice. Caspases 1, 3 and 8 were upregulated during the degeneration as were several VHL target genes connected to the extracellular matrix. Misregulation of these genes may influence retinal structure and may therefore facilitate growth of vessels into the photoreceptor layer. Thus, an early and sustained activation of a hypoxia-like response in retinal cells leads to abnormal vasculature and severe retinal degeneration in the adult mouse retina.

Lange C ，Caprara C ，Tanimoto N ，Beck S ，Huber G ，Samardzija M ，Seeliger M ，Grimm C ... -  《-》