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The brain requires over one-fifth of the total body oxygen demand for normal functioning. At high altitude (HA), the lower atmospheric oxygen pressure inevitably challenges the brain, affecting voluntary spatial attention, cognitive processing, and attention speed after short-term, long-term, or lifespan exposure. Molecular responses to HA are controlled mainly by hypoxia-inducible factors. This review aims to summarize the cellular, metabolic, and functional alterations in the brain at HA with a focus on the role of hypoxia-inducible factors in controlling the hypoxic ventilatory response, neuronal survival, metabolism, neurogenesis, synaptogenesis, and plasticity., International Journal of Molecular Sciences, 24 (12), ISSN:1422-0067

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53flox mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53flox mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients.

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high levels of interleukin-6 and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels who do not respond to erythropoietin.

Expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role, by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB, at normoxia, upregulated the expression of cell cyclins and increased cell survival while it prevented apoptosis in MCF7 cells. Also, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, loss of MB enhanced partial epithelial to mesenchymal transition, thus augmenting the migratory and invasive cell behavior. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration

The expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB at normoxia upregulated the expression of cell cyclins and increased cell survival, while it prevented apoptosis in MCF7 cells. Additionally, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, the loss of MB enhanced the partial epithelial to mesenchymal transition, thus, augmenting the migratory and invasive behavior of cells. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Erythropoietin (EPO) improves neuronal mitochondrial function and cognition in adults after brain injury and in those afflicted by psychiatric disorders. However, the influence of EPO on mitochondria and cognition during development remains unexplored. We previously observed that EPO stimulates hippocampal-specific neuronal maturation and synaptogenesis early in postnatal development in mice. Here we show that EPO promotes mitochondrial respiration in developing postnatal hippocampus by increasing mitochondrial content and enhancing cellular respiratory potential. Ultrastructurally, mitochondria profiles and total vesicle content were greater in presynaptic axon terminals, suggesting that EPO enhances oxidative metabolism and synaptic transmission capabilities. Behavioural tests of hippocampus-dependent memory at early adulthood, showed that EPO improves spatial and short-term memory. Collectively, we identify a role for EPO in the murine postnatal hippocampus by promoting mitochondrial function throughout early postnatal development, which corresponds to enhanced cognition by early adulthood., Robert Jacobs, Mostafa Aboouf, et al. examined the effect of erythropoietin (EPO) in hippocampal mitochondrial function and memory in two mouse models: one overexpressing EPO in the brain, and juvenile mice treated during three days with a high dose of intraperitoneal EPO. Their results suggest that erythropoietin in the neonatal brain may impact spatial memory by increasing mitochondrial content.

Erythropoietin (EPO) boosts exercise performance through increase in oxygen transport capacity following regular administration of EPO but preclinical study results suggest that single high dose of EPO also may improve exercise capacity. Twenty-nine healthy subjects (14 males/15 females; age: 25 ± 3 years) were included in a randomized, double-blind, placebo-controlled crossover study to assess peak work load and cardiopulmonary variables during submaximal and maximal cycling tests following a single dose of 60.000 IU of recombinant erythropoietin (EPO) or placebo (PLA). Submaximal exercise at 40%/60% of peak work load revealed no main effect of EPO on oxygen uptake (27.9 ± 8.7 ml min–1⋅kg–1/ 37.1 ± 13.2 ml min–1⋅kg–1) versus PLA (25.2 ± 3.7 ml min–1⋅kg–1/ 33.1 ± 5.3 ml min–1⋅kg–1) condition (p = 0.447/p = 0.756). During maximal exercise peak work load (PLA: 3.5 ± 0.6 W⋅kg–1 vs. EPO: 3.5 ± 0.6 W kg–1, p = 0.892) and peak oxygen uptake (PLA: 45.1 ± 10.4 ml⋅min–1 kg–1 vs. EPO: 46.1 ± 14.2 ml⋅min–1 kg–1, p = 0.344) reached comparable values in the two treatment conditions. Other cardiopulmonary variables (ventilation, cardiac output, heart rate) also reached similar levels in the two treatment conditions. An interaction effect was found between treatment condition and sex resulting in higher peak oxygen consumption (p = 0.048) and ventilation (p = 0.044) in EPO-treated males. In conclusion, in a carefully conducted study using placebo-controlled design the present data failed to support the hypothesis that a single high dose of EPO has a measurable impact on work capacity in healthy subjects., Frontiers in Physiology, 11, ISSN:1664-042X

It is increasingly evident that mitochondria are crucial in regulating neurodevelopment, brain function and cognition. Erythropoietin (EPO) has been shown to improve mitochondrial function and cognition following brain damage and in patients with neurological disorders. However, potential EPO-mediated influence(s) on hippocampal mitochondrial function during postnatal development and it corresponds to enhanced cognition is unknown. Here we show in mice, that EPO receptors (EpoR)s express postnatally in the CA1 pyramidal cells of the hippocampus reaching a zenith at puberty (postnatal (P) age 21). Constitutive neuronal EPO overexpression increases hippocampal Erk1/2 and AKT phosphorylation along with increases in cellular respiration and mitochondrial content by the third postnatal week of development. Indices of cellular oxidant balance do not appear altered by higher respiratory potentials and greater mitochondrial content. Finally, EPO overexpression also enhances hippocampal-dependent learning and memory at early adulthood (P60). Collectively, this data identifies a novel function for EPO signaling, promoting improvements in hippocampal-specific mitochondrial function and cognition during postnatal development and early adulthood.

The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO2). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the “browning” BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection. © Copyright © 2020 Ruetten, Steinmetz, Thiersch, Kik, Vaughan, Altamura, Muckenthaler and Gassmann.

Thiersch, Markus, and Erik R. Swenson. High altitude and cancer mortality. High Alt Med Biol 19:116-123, 2018.-Humans living at high altitude (HA) are exposed to chronic (hypobaric) hypoxia. Despite the permanent stress of hypoxic exposure, humans populating HA areas have reduced cancer mortality over a broad spectrum of cancer types. In fact, the majority of the physiological adaptive processes at HA occurring in response to hypoxia might be the driving force for reduced cancer mortality at HA. In this review, we summarize epidemiological and animal studies that compare cancer incidence and cancer mortality between HA and low altitude or between hypoxia and normoxia, respectively. We discuss the potential role of oxygen-independent and oxygen-dependent mechanisms that might contribute to reduced cancer mortality at HA. Reactive oxygen species and their detoxification as well as the hypoxia-inducible factors are especially promising targets and may be related to why cancer mortality is reduced at HA. In addition, we briefly discuss two aspects with a proven impact on tumorigenesis, namely the immune system and tumor surveillance as well as HA-induced metabolic changes. Further animal and clinical studies are clearly needed to explain why cancer mortality is reduced at HA and to decide whether HA or hypoxia-based therapeutic approaches could be implemented for cancer treatment. However, exposure to HA activates multiple adaptive mechanisms (oxygen independent and oxygen dependent) sharing common pathways as well as activating counteracting pathways, which complicate the identification of specific HA-induced mechanisms of tumor suppression.

Breast cancer today is typically seen as malignancy with longer survival due to early-detection diagnostics, yet the cancer remains by far the most common tumor type in adult females. Recently, our group co-discovered myoglobin (MB) to be expressed in luminal cells of healthy and cancerous breast epithelia of human and mice. In human patients, MB positivity emerged as hallmark of the luminal subtype and was directly correlated with estrogen receptor alpha positivity and hence a significantly better prognosis. Cancerous cells’ MB also appears to interact with the known tumor suppressor p53. However, if and how myoglobin itself restricts mammary tumorigenesis is completely unclear. To understand how MB exerts its alleged tumor-suppressive effects and if it impacts response of mammary tumors to chemo- and/or radiotherapy, we examine the molecular role of MB in breast tumor formation and progression through a) in vitro studies (MCF7 breast cancer BrCa cell clones; wildtype (wt) controls versus CRISPR/Cas9-engineered clones of a MB and/or p53 knockout (MBko/p53ko), b) in vivo mouse models to obtain spontaneously forming breast tumors, with or without MB, in a p53 deficient background. Comparing MCF7 BrCa MBko and p53ko clones vs. their corresponding wildtype counterpart supports our hypothesis of a feed forward loop between MB and p53 proteins, besides interfering with estrogen receptor expression. In addition to that, loss of MB was noticed to exert p53-independent upregulation on cell cyclins with more S phase cell population. That was reflected by a significant increase in survival of the MBko cells, in presence or absence of p53. Hypoxic MBko cells exhibited a partial epithelial to mesenchymal transition and hence a more migratory phenotype, relative to MBwt controls. Additionally, MB seems to stimulate apoptosis of the normoxic cancer cells. On the other hand, murine tumors were found to phenocopy human BrCa in the extent of their MB expression. Interestingly, MB-devoid tumors showed a significantly higher proliferation index and fat accumulation. Further studies will look into the clinical significance of that. In conclusion, MB seems to occupy unknown roles in cancer cells beyond or different from its classical O2 storage/transport functions. Unraveling these novel roles in governing tumor development and virulence will hopefully provide innovative strategies for future breast cancer interventions. Citation Format: Mostafa A. Aboouf, Julia Armbruster, Franco Guscetti, Markus Thiersch, Max Gassmann, Thomas A. Gorr. The role of myoglobin in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-17.

Exposure to hypobaric hypoxia at high altitude (above 2500 m asl) causes cognitive impairment, mostly attributed to changes in brain perfusion and consequently neuronal death. Enriched environment and voluntary exercise has been shown to improve cognitive function, to enhance brain microvasculature and neurogenesis, and to be neuroprotective. Here we show that high-altitude exposure (3540 m asl) of Long Evans rats during early adulthood (P48-P59) increases brain microvasculature and neurogenesis but impairs spatial and visual memory along with an increase in neuronal apoptosis. We tested whether enriched environment including a running wheel for voluntary exercise (EE) can prevent cognitive impairment at high-altitude and whether apoptosis is prevented. We found that EE retained spatial and visual memory at high altitude, and prevented neuronal apoptosis. Further, we tested whether vascular endothelial growth factor (VEGF) signaling is required for the EE-mediated recovery of spatial and visual memory and the reduction in apoptosis. Pharmacological inhibition of VEGF signaling by oral application of a tyrosine kinase inhibitor (Vandetanib) prevented the recovery of spatial and visual memory in animals housed in EE, along with an increase in apoptosis and a reduction in neurogenesis. Surprisingly, inhibition of VEGF signaling also caused impairment in spatial memory in EE-housed animals reared at low altitude, affecting mainly dentate gyrus microvasculature but not neurogenesis. We conclude that EE-mediated VEGF signaling is neuroprotective and essential for the maintenance of cognition and neurogenesis during high-altitude exposure, and for the maintenance of spatial memory at low altitude. Finally, our data also underlines the potential risk of cognitive impairment and disturbed high altitude adaption from the use of VEGF-signaling inhibitors for therapeutic purposes. This research was supported by the Swiss National Science Foundation [Marie Heim-Vogtlin (MHV) - SNF grant PMPDP3_145480], the Institute of Veterinary Physiology and the Institute of Pharmacology and Toxicology at the University of Zurich, the Institute of Anatomy at the University of Freiburg, and the Institute of Neuroscience at the University of Basque, Spain.

Residency at high altitude (HA) demands adaptation to challenging environmental conditions with hypobaric hypoxia being the most important one. Epidemiological and experimental data suggest that chronic exposure to HA reduces cancer mortality and lowers prevalence of metabolic disorders like diabetes and obesity implying that adaption to HA modifies a broad spectrum of physiological, metabolic and cellular programs with a generally beneficial outcome for humans. However, the complexity of multiple, potentially tumor-suppressive pathways at HA impedes the understanding of mechanisms leading to reduced cancer mortality. Many adaptive processes at HA are tightly interconnected and thus it cannot be ruled out that the entirety or at least some of the HA-related alterations act in concert to reduce cancer mortality. In this review we discuss tumor formation as a concept of competition between healthy and cancer cells with improved fitness - and therefore higher competitiveness - of healthy cells at high altitude. We discuss HA-related changes in glucose, lipid and iron metabolism that may have an impact on tumorigenesis. Additionally, we discuss two parameters with a strong impact on tumorigenesis, namely drug metabolism and physical activity, to underpin their potential contribution to HA-dependent reduced cancer mortality. Future studies are needed to unravel why cancer mortality is reduced at HA and how this knowledge might be used to prevent and to treat cancer patients.

Purpose 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. Methods 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. Results 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. Conclusions 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.

RPE65 is a retinal pigment epithelial protein essential for the regeneration of 11-cis-retinal, the chromophore of cone and rod visual pigments. Mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber's congenital amaurosis to autosomal recessive retinitis pigmentosa. One of the most frequent missense mutations is an amino acid substitution at position 91 (R91W). Affected patients have useful cone vision in the first decade of life, but progressively lose sight during adolescence. We generated R91W knock-in mice to understand the mechanism of retinal degeneration caused by this aberrant Rpe65 variant. We found that in contrast to Rpe65 null mice, low but substantial levels of both RPE65 and 11-cis-retinal were present. Whereas rod function was impaired already in young animals, cone function was less affected. Rhodopsin metabolism and photoreceptor morphology were disturbed, leading to a progressive loss of photoreceptor cells and retinal function. Thus, the consequences of the R91W mutation are clearly distinguishable from an Rpe65 null mutation as evidenced by the production of 11-cis-retinal and rhodopsin as well as by less severe morphological and functional disturbances at early age. Taken together, the pathology in R91W knock-in mice mimics many aspects of the corresponding human blinding disease. Therefore, this mouse mutant provides a valuable animal model to test therapeutic concepts for patients affected by RPE65 missense mutations.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly- l -lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro . Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α−ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo . Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells – precursors for mature blood vessels – during wound healing. We show that – depending on the selection of the therapeutic target gene – PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health.

Purpose HIF1A is one of the major transcription factors that regulate tissue response to low oxygen tension. It controls expression of a large number of genes involved in cell survival, proliferation, angiogenesis, and other cellular processes. HIF1A is present at increased levels in the early postnatal retina. In this study its potential function during postnatal development of the mouse retina and retinal vasculature was analyzed. Methods A mouse line was generated with a Cre-mediated Hif1a knockdown in the peripheral retina. Retinal morphology and vasculature were analyzed in sections and flat mount preparations. Gene and protein expression were determined by real-time PCR and Western blot analysis. Results The Cre-mediated knockdown caused a significant reduction in Hif1a gene expression and HIF1A protein levels in the early postnatal retina. Retinal morphology was normal but the Hif1a knockdown prevented the formation of the intermediate vascular plexus in the peripheral retina. The primary plexus and the outer plexus were less affected. The Hif1a knockdown did not affect expression of such angiogenesis-related genes as vascular endothelial growth factor (Vegf) but strongly induced expression of erythropoietin (Epo). At the protein level, EPAS1 (HIF2A) was stabilized in the Hif1a knockdown mice. Conclusions The results suggest that HIF1A may be directly or indirectly required for normal development of the retinal vasculature, especially of the intermediate plexus. EPO but not VEGF may play a significant role in the development of this phenotype. HIF1A may not be the main factor that regulates Vegf expression during retinal development in the mouse.

Retinal degeneration causes the induction of a leukemia inhibitory factor (LIF)-controlled survival pathway which includes Janus kinase/signal transducer and activator of transcription signaling. Lack of LIF prevents activation of this signaling cascade and accelerates disease progression leading to a fast loss of photoreceptor cells. In this study, we show that expression of Janus kinase 3 (Jak3), but not of the other members of the family of Janus kinases, is induced in four different models of retinal degeneration and that LIF is essential and sufficient to activate Jak3 gene expression. We also show that the induction of Jak3 and Lif may not depend directly on cell death but rather on the retinal stress during photoreceptor degeneration. However, despite its dependence on LIF, JAK3 is not essential for LIF-mediated photoreceptor protection or gene expression. Also, absence of JAK3 in knockout mice did not affect immune-related responses in the degenerating retina. JAK3 may therefore play a different, yet unknown, role in the retinal response to photoreceptor injury.

Retinal degenerative diseases are a major cause of severe visual impairment or blindness in humans. To develop therapeutic strategies it is of particular importance to understand the molecular mechanisms taking place during the progression of the disease. Genes and proteins of the Janus kinase/Signal Transducer and Activator of Transcription (Jak/STAT) signaling pathway have been shown to play an important role in models of retinal degeneration (RD). Here we investigated the expression of additional genes involved in the Jak/STAT pathway in an induced (light exposure) and an inherited (rd1 mouse) model of RD. We show that STAT mRNAs as well as the Jak2/shp-1 pathway are differentially regulated in the two models. In contrast, we show that Jak3 mRNA is upregulated in both, the light damaged and the degenerative retina of the rd1 mouse. This common answer to probably different apoptotic stimuli suggests a prominent role for Jak3 in the damaged retina and could therefore be interesting for further investigations.

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.

RPE65 is a retinoid isomerase required for the production of 11-cis-retinal, the chromophore of both cone and rod visual pigments. We recently established an R91W knock-in mouse strain as homologous animal model for patients afflicted by this mutation in RPE65. These mice have impaired vision and can only synthesize minute amounts of 11-cis-retinal. Here, we investigated the consequences of this chromophore insufficiency on cone function and pathophysiology. We found that the R91W mutation caused cone opsin mislocalization and progressive geographic cone atrophy. Remnant visual function was mostly mediated by rods. Ablation of rod opsin corrected the localization of cone opsin and improved cone retinal function. Thus, our analyses indicate that under conditions of limited chromophore supply rods and cones compete for 11-cis-retinal that derives from regeneration pathway(s) which are reliant on RPE65. Due to their higher number and the instability of cone opsin, rods are privileged under this condition while cones suffer chromophore deficiency and degenerate. These findings reinforce the notion that in patients any effective gene therapy with RPE65 needs to target the cone-rich macula directly to locally restore the cones' chromophore supply outside the reach of rods.

Background Retinal degeneration is a main cause of blindness in humans. Neuroprotective therapies may be used to rescue retinal cells and preserve vision. Hypoxic preconditioning stabilizes the transcription factor HIF-1α in the retina and strongly protects photoreceptors in an animal model of light-induced retinal degeneration. To address the molecular mechanisms of the protection, we analyzed the transcriptome of the hypoxic retina using microarrays and real-time PCR. Results Hypoxic exposure induced a marked alteration in the retinal transcriptome with significantly different expression levels of 431 genes immediately after hypoxic exposure. The normal expression profile was restored within 16 hours of reoxygenation. Among the differentially regulated genes, several candidates for neuroprotection were identified like metallothionein-1 and -2, the HIF-1 target gene adrenomedullin and the gene encoding the antioxidative and cytoprotective enzyme paraoxonase 1 which was previously not known to be a hypoxia responsive gene in the retina. The strongly upregulated cyclin dependent kinase inhibitor p21 was excluded from being essential for neuroprotection. Conclusion Our data suggest that neuroprotection after hypoxic preconditioning is the result of the differential expression of a multitude of genes which may act in concert to protect visual cells against a toxic insult.

PURPOSE. Caspase-1 gene expression has been reported to be upregulated during light-induced retinal degeneration and to be reduced after neuroprotective treatments. Thus, caspase-1 may be proapoptotic in the retina. To test directly the role of caspase-1 in photoreceptor apoptosis, three mouse models were analyzed for retinal degeneration in the presence or absence of caspase-1. METHODS. Photoreceptor apoptosis was monitored in one model of induced (exposure to light) and in two models of inherited (rd1, VPP) retinal degeneration. Retinal degeneration was assessed qualitatively by light microscopy and quantitatively by the determination of free nucleosomes with ELISA or by rhodopsin measurements. Gene expression and protein levels were assessed by real-time RT-PCR and by Western blot analysis, respectively. RESULTS. Levels of caspase-1 proenzyme increased in all models of retinal degeneration concomitantly with the onset of cell death. Maturation or classic activity of caspase-1 was not detected in the retina. Ablation of caspase-1 was protective in the model of adRP (VPP mouse), but not in the two other models. Ablation of interleukin-1 receptor type 1 was without effect. Expression of monocyte chemoattractant protein (MCP)-1 increased in the model protected by caspase-1 ablation. CONCLUSIONS. Increased retinal expression of caspase-1 proenzyme may be a common marker for photoreceptor degeneration. The differential effects of caspase-1 ablation suggests a modulatory role of caspase-1 for photoreceptor apoptosis in some but not all models. Such a modulatory activity may involve a caspase-1 function different from the classic activation of interleukin-1 .( Invest Ophthalmol Vis Sci. 2006;47: 5181–5190) DOI:10.1167/iovs.06-0556

Retinal degeneration is a major cause of severe visual impairment or blindness. Understanding the underlying molecular mechanisms is a prerequisite to develop therapeutic approaches for human patients. We show in three mouse models that induced and inherited retinal degeneration induces LIF and CLC as members of the interleukin (IL)-6 family of proteins, activates proteins of the Jak-STAT signaling pathway, and up-regulates suppressors of cytokine signaling as a negative feedback loop. Inhibition of Jak2 leads to protection of photoreceptors in a model of induced but not in a model of inherited retinal degeneration. Differential activation of Akt suggests alternative pathways for cell death and/or survival in different models. Proteins induced during photoreceptor degeneration are not mainly expressed in photoreceptors but in cells of other retinal layers. This suggests a model in which photoreceptor injury is signaled to cells of the inner retina, which in turn initiate a response either to support viability or accelerate death of injured cells.

Pseudomonas putida KT2440 is often used as a model to investigate toxicity mechanisms and adaptation to hazardous chemicals in bacteria. The objective of this paper was to test the impact of the chlorophenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and their metabolites 2,4-dichlorophenol (DCP) and 3,5-dichlorocatechol (DCC), on protein expression patterns and physiological parameters. Both approaches showed that DCC has a different mode of action and induces different responses than DCPP, 2,4-D and DCP. DCC was the most toxic compound and was active as an uncoupler of oxidative phosphorylation. It repressed the synthesis of ferric uptake regulator (Fur)-dependent proteins, e.g. fumarase C and L-ornithine N5-oxygenase, which are involved in oxidative stress response and iron uptake. DCPP, 2,4-D and DCP were less toxic than DCC. They disturbed oxidative phosphorylation to a lesser extent by a yet unknown mechanism. Furthermore, they repressed enzymes of energy-consuming biosynthetic pathways and induced membrane transporters for organic substrates. A TolC homologue component of multidrug resistance transporters was found to be induced, which is probably involved in the removal of lipophilic compounds from membranes.

Recombinant erythropoietin (Epo) is an effective anti-anemic agent in most cancer patients and improves their quality of life. Yet, concerns of disease progression and reduced survival in recombinant human Epo (rhuEpo)-treated patients have been raised by Phase III clinical trial data showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Epo has pleiotropic actions and its receptor, EpoR, is expressed by many different cell types outside the erythroid compartment. It was thus proposed that a major possible mechanism for this potentially harmful effect of Epo in cancer patients is the activation of EpoRs on cancer cells. The original clinical studies have been criticized because they deployed polyclonal antibodies later shown to lack specificity for EpoR. Furthermore, multiple isoforms of EpoR caused by differential splicing have been reported, but only at the RNA level, in different cancer cell lines. Investigations into whether these spliced versions actually result in abnormal EpoR forms at the protein level, alter Epo responsiveness and have an impact on tumor progression in vivo, have been hampered by a lack of well characterized monoclonal antibodies. The ‘EpoCan’ Consortium, funded by the EU, is directed to promote improved pathological testing of EpoR in patient samples, leading to safer clinical use of rHuEpo and other Erythropoietic Stimulating Agents (ESAs). To date, 25 murine and rat monoclonal antibodies have been produced against the EpoR, using novel genetic and traditional peptide immunization protocols. Of these antibodies, several were found to specifically recognize the receptor in various assays including Western blot (WB), immunoprecipitation (IP), immunofluorescence (IF), flow cytometry (FACS) and immunohistochemistry (IHC). Table 1 lists the antibodies that were selected for further analysis, and their experimental applications. The antibodies were tested on EpoR transfected cells (HEK 293 cells and COS cells) and Epo-dependent UT7 cells which endogenously express EpoR. In addition the antibodies were tested on non-erythroid cells viz. the non-small cell lung carcinoma A549, and breast cancer MDA-MB 231 cell lines. Specificity of the antibodies towards the EpoR in these two latter cell lines was ensured by the lack of reactivity with the corresponding EpoR silenced cells.Table 1Anti EpoR Antibodies - immunizing antigens and applicationsImmunogenSubclone nameSystematic nameIsotypeEpitope locationApplicationsPeptideBCO-3H2-D3GM1201rIgG2bhEpoR cytoplasmic domainWB, IP, IF, IHCPeptideBCO-4B5-C9GM1202rIgG2ah/mEpoR cytoplasmic domainWB, IF, IHCDNAVP-2E8-B6GM1204mIgG1hEpoR extracellular domainIP, IF, FACSDNAVP-4D8-C4GM1205mIgG1hEpoR extracellular domainIP, IF, FACSDNABBQ-9C4-D8GM1206rIgG2ah/mEpoR extracellular domainFACSDNABBQ-10E10-F2GM1207rIgG2ah/mEpoR extracellular domainWB Immunohistochemical analysis was performed on a panel of human non-small cell lung carcinoma sections. Two rat monoclonal antibodies, BCO-3H2-D3 which recognizes full length EpoR and BCO-4B5-C9 which recognizes full length and truncated isoforms, have proved valuable in comparative immunohistochemical and Western blot studies in a range of human tumors and cell lines. These well characterized monoclonal antibodies will enable a careful dissection of EpoR function in cancer cells and the detection of EpoR isoforms in tumor tissue. In the longer term such studies should allow clinicians to balance the benefits and risks of ESA treatment in cancer. THIS WORK WAS SUPPORTED BY THE FP7-HEALTH EUROPEAN COMMISSION EPOCAN GRANT (282551). Disclosures: Grunert: Aldevron Freiburg GmBH: Employment. Thompson:Aldevron Freiburg GmbH: Employment.

PURPOSE: During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. METHODS: Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. RESULTS: Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. CONCLUSIONS: Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.