Carmel McVicar

Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.

Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy

OBJECTIVE Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy. RESEARCH DESIGN AND METHODS After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1–30 μg/kg pHBSP or control peptide). RESULTS pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01–0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose. CONCLUSIONS Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products

Aims/hypothesisThe impact of AGEs and advanced lipoxidation end-products (ALEs) on neuronal and Müller glial dysfunction in the diabetic retina is not well understood. We therefore sought to identify dysfunction of the retinal Müller glia during diabetes and to determine whether inhibition of AGEs/ALEs can prevent it.MethodsSprague–Dawley rats were divided into three groups: (1) non-diabetic; (2) untreated streptozotocin-induced diabetic; and (3) diabetic treated with the AGE/ALE inhibitor pyridoxamine for the duration of diabetes. Rats were killed and their retinas were evaluated for neuroglial pathology.ResultsAGEs and ALEs accumulated at higher levels in diabetic retinas than in controls (p < 0.001). AGE/ALE immunoreactivity was significantly diminished by pyridoxamine treatment of diabetic rats. Diabetes was also associated with the up-regulation of the oxidative stress marker haemoxygenase-1 and the induction of glial fibrillary acidic protein production in Müller glia (p < 0.001). Pyridoxamine treatment of diabetic rats had a significant beneficial effect on both variables (p < 0.001). Diabetes also significantly altered the normal localisation of the potassium inwardly rectifying channel Kir4.1 and the water channel aquaporin 4 to the Müller glia end-feet interacting with retinal capillaries. These abnormalities were prevented by pyridoxamine treatment.Conclusions/interpretationWhile it is established that AGE/ALE formation in the retina during diabetes is linked to microvascular dysfunction, this study suggests that these pathogenic adducts also play a role in Müller glial dysfunction.

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 10(5) molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 10(5) molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium‐dependent NO signalling

BACKGROUND AND PURPOSE Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

RAGE regulates immune cell infiltration and angiogenesis in choroidal neovascularization

Purpose RAGE regulates pro-inflammatory responses in diverse cells and tissues. This study has investigated if RAGE plays a role in immune cell mobilization and choroidal neovascular pathology that is associated with the neovascular form of age-related macular degeneration (nvAMD). Methods RAGE null (RAGE−/−) mice and age-matched wild type (WT) control mice underwent laser photocoagulation to generate choroidal neovascularization (CNV) lesions which were then analyzed for morphology, S100B immunoreactivity and inflammatory cell infiltration. The chemotactic ability of bone marrow derived macrophages (BMDMs) towards S100B was investigated. Results RAGE expression was significantly increased in the retina during CNV of WT mice (p<0.001). RAGE−/− mice exhibited significantly reduced CNV lesion size when compared to WT controls (p<0.05). S100B mRNA was upregulated in the lasered WT retina but not RAGE−/− retina and S100B immunoreactivity was present within CNV lesions although levels were less when RAGE−/− mice were compared to WT controls. Activated microglia in lesions were considerably less abundant in RAGE−/− mice when compared to WT counterparts (p<0.001). A dose dependent chemotactic migration was observed in BMDMs from WT mice (p<0.05–0.01) but this was not apparent in cells isolated from RAGE−/− mice. Conclusions RAGE-S100B interactions appear to play an important role in CNV lesion formation by regulating pro-inflammatory and angiogenic responses. This study highlights the role of RAGE in inflammation-mediated outer retinal pathology.

Differential modulation of angiogenesis by erythropoiesis-stimulating agents in a mouse model of ischaemic retinopathy.

Background Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models. Methodology/Principal Findings The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1–20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNFα and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001). Conclusions This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs.

Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge DiversetTM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μm in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1–2) at micromolar IC50 values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.

Chromogranin A proteolysis to generate β-granin and WE-14 in the adenohypophysis during the rat oestrous cycle

Immunohistochemical analysis of the male and female rat adenohypophysis revealed that chromogranin A (CgA), beta-granin and WE-14 immunostaining was localised to follicle stimulating hormone (FSH) producing cells, while luteinizing hormone (LH) producing cells exhibited chromogranin A and beta-granin immunostaining. The intensity of chromogranin A, beta-granin and WE-14 immunostaining exhibited variation during the oestrous cycle; weak immunostaining was observed during proestrous and oestrous, corresponding with the lowest cellular concentration of luteinizing and follicle stimulating hormone. Chromogranin A and beta-granin immunostaining were similar in both the male and female (at dioestrous), however, a larger number of more intense WE-14 immunopositive cells were evident in the male adenohypophysis relative to the female at any stage of the cycle. The tissue and plasma concentrations of beta-granin and WE-14 immunoreactivity fluctuated throughout the oestrous cycle. Maximum and minimum beta-granin and WE-14 tissue concentration counterpoised the latent maximum and minimum plasma concentration. Chromatographic analysis of adenohypophysis extracts revealed the degree of chromogranin A proteolysis throughout the oestrous cycle; in contrast, plasma profiles consistently possessed a large chromogranin A-like immunoreactant. This data suggests that chromogranin A biosynthesis, proteolysis and the secretion of its derived peptides parallels that of the gonadotroph hormones throughout the oestrous cycle.

Inhibition or deletion of 11β-HSD1 does not increase angiogenesis in ischemic retinopathy.

Diabetes & Metabolism - In Press.Proof corrected by the author Available online since jeudi 12 janvier 2017