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1.
J Biol Chem ; 291(14): 7242-55, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26846851

ABSTRACT

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 Diverset(TM)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 µmin 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 IC50values. 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.


Subject(s)
Angiogenesis Inhibitors , Drug Discovery , Phenols , Quinolines , Retinal Neovascularization/drug therapy , Signal Transduction/drug effects , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacokinetics , Angiogenesis Inhibitors/pharmacology , Animals , Animals, Genetically Modified , Cell Line , Diabetic Retinopathy/drug therapy , Diabetic Retinopathy/metabolism , Humans , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Mice , Phenols/chemistry , Phenols/pharmacokinetics , Phenols/pharmacology , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Zebrafish
2.
Diabetologia ; 58(5): 1129-37, 2015 May.
Article in English | MEDLINE | ID: mdl-25687235

ABSTRACT

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy. METHODS: Diabetes was induced in wild-type (WT) and Rage (-/-) mice (also known as Ager (-/-) mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. RESULTS: Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage (-/-) mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage (-/-) mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05-0.001). Rage (-/-) mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. CONCLUSIONS/INTERPRETATION: Rage (-/-) in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/metabolism , Receptor for Advanced Glycation End Products/metabolism , Retina/metabolism , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/pathology , Lactoylglutathione Lyase/metabolism , Leukostasis/metabolism , Leukostasis/pathology , Male , Mice , Mice, Knockout , Microvessels/metabolism , Microvessels/pathology , Pyruvaldehyde/metabolism , Retina/pathology
3.
PLoS One ; 9(2): e89548, 2014.
Article in English | MEDLINE | ID: mdl-24586862

ABSTRACT

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.


Subject(s)
Choroidal Neovascularization/immunology , Neovascularization, Pathologic/immunology , Receptors, Immunologic/physiology , Retina/immunology , S100 Calcium Binding Protein beta Subunit/metabolism , Animals , Blotting, Western , Cells, Cultured , Chemotaxis , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Disease Models, Animal , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptor for Advanced Glycation End Products , Retina/metabolism , Retina/pathology , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium Binding Protein beta Subunit/genetics
4.
Br J Pharmacol ; 166(1): 327-38, 2012 May.
Article in English | MEDLINE | ID: mdl-22035179

ABSTRACT

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. EXPERIMENTAL APPROACH: Cumulative relaxation responses to obestatin peptides were assessed in rat isolated aorta and mesenteric artery (n≥ 8) in the presence and absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC). KEY RESULTS: Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1-23) were greater than those to obestatin(1-10) and obestatin(11-23). Obestatin(1-23)-induced relaxation was attenuated by endothelial denudation, l-NAME (NOS inhibitor), high extracellular K(+) , GDP-ß-S (G-protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK(Ca) blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked GPCR, PI3K/PKB, Ca(2+) -dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K(+) . Supporting data from BAEC indicated that nitrite production, intracellular Ca(2+) and PKB phosphorylation were increased after exposure to obestatin(1-23). Relaxations to obestatin(1-23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarizing factors (EDHFs) and combined SK(Ca) /IK(Ca) blockade, suggesting that EDHF-mediated pathways were not involved. CONCLUSIONS AND IMPLICATIONS: Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterized by endothelial dysfunction and cardiovascular complications.


Subject(s)
Endothelium, Vascular/drug effects , Nitric Oxide/metabolism , Peptide Hormones/metabolism , Vasodilation/drug effects , Animals , Aorta/drug effects , Aorta/metabolism , Calcium/metabolism , Cattle , Cells, Cultured , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/metabolism , Peptide Hormones/administration & dosage , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects
5.
Diabetes ; 60(11): 2995-3005, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21911748

ABSTRACT

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.


Subject(s)
Diabetic Retinopathy/drug therapy , Erythropoietin/chemistry , Nerve Degeneration/prevention & control , Neuroglia/drug effects , Peptide Fragments/therapeutic use , Retinal Degeneration/prevention & control , Retinal Vessels/drug effects , Animals , Animals, Newborn , Apoptosis/drug effects , Cytokines/genetics , Cytokines/metabolism , DNA Damage/drug effects , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Gene Expression Regulation/drug effects , Ischemia/drug therapy , Ischemia/metabolism , Ischemia/pathology , Male , Mice , Mice, Inbred C57BL , Neuroglia/pathology , Peptide Fragments/adverse effects , Peptide Fragments/chemistry , Protein Interaction Domains and Motifs , Random Allocation , Rats , Rats, Sprague-Dawley , Retina/drug effects , Retina/metabolism , Retina/pathology , Retinal Vessels/pathology
6.
PLoS One ; 5(7): e11870, 2010 Jul 29.
Article in English | MEDLINE | ID: mdl-20686695

ABSTRACT

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 TNFalpha 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.


Subject(s)
Hematinics/therapeutic use , Ischemia/complications , Neovascularization, Pathologic/drug therapy , Retinal Diseases/drug therapy , Animals , Cells, Cultured , Chromatography, High Pressure Liquid , Disease Models, Animal , Erythropoietin/therapeutic use , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Neovascularization, Pathologic/etiology , Recombinant Proteins , Retina/pathology , Retina/ultrastructure , Retinal Diseases/etiology , Reverse Transcriptase Polymerase Chain Reaction
7.
Fertil Steril ; 87(5): 1064-70, 2007 May.
Article in English | MEDLINE | ID: mdl-17335822

ABSTRACT

OBJECTIVE: To determine whether sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction. DESIGN: Laboratory analysis of sperm motility after exposure to sildenafil citrate using computer-assisted semen analysis and acrosome reaction by fluorescein isothiocyanate-labeled peanut agglutinin staining. SETTING: An assisted reproductive technology (ART) unit. PATIENT(S): Fifty-seven male patients. INTERVENTION(S): Sperm were divided into 90% (those with the best fertilizing potential used in assisted conception) and 45% (the poorer population) fractions by density centrifugation and incubated with sildenafil citrate (0.67 muM) at 37 degrees C for up to 180 minutes. MAIN OUTCOME MEASURE(S): Both the number and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Furthermore, samples revealed that these effects were consistent in the 90% and 45% populations of sperm. In both populations, sildenafil also caused a significant increase in the proportion of acrosome-reacted sperm-22.1% compared with 11.8% in the control group of the good quality fraction and 16.6% compared with 9.4% in the control group of the poorer quality fraction. CONCLUSION(S): The use of sildenafil citrate may adversely affect male fertility.


Subject(s)
Acrosome Reaction/drug effects , Piperazines/pharmacology , Sperm Motility/drug effects , Sulfones/pharmacology , Acrosome Reaction/physiology , Adult , Humans , Infertility, Male/chemically induced , Male , Piperazines/adverse effects , Purines/adverse effects , Purines/pharmacology , Sildenafil Citrate , Sperm Motility/physiology , Sulfones/adverse effects
8.
Fertil Steril ; 87(4): 834-41, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17241627

ABSTRACT

OBJECTIVE: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy. DESIGN: Testicular biopsies from vasectomized (n = 26) and fertile men (n = 46), were milked to calculate sperm/gram and also formalin-fixed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay. SETTING: An ART unit. PATIENT(S): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies. MAIN OUTCOME MEASURE(S): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining. RESULT(S): Sperm yields from men vasectomized >5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased significantly in vasectomized men compared to fertile men. CONCLUSION(S): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas-FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation.


Subject(s)
Apoptosis , Sperm Count , Vasectomy , Adult , Biopsy , DNA Fragmentation , Fas Ligand Protein/analysis , Fas Ligand Protein/physiology , Humans , Male , Middle Aged , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-2/physiology , Reactive Oxygen Species/metabolism , Sertoli Cells/cytology , Testis , bcl-2-Associated X Protein/analysis , bcl-2-Associated X Protein/physiology , fas Receptor/analysis , fas Receptor/physiology
9.
Fertil Steril ; 82(5): 1443-5, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15533376

ABSTRACT

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4-hour and 24-hour incubations and after cryopreservation with the effect is intensified by post-thaw incubation. Testicular sperm for use in intracytoplasmic sperm injection (ICSI) should be injected without delay.


Subject(s)
DNA Fragmentation , Oligospermia/genetics , Spermatozoa , Testis , Cell Culture Techniques , Cryopreservation , Humans , Male , Time Factors
10.
Fertil Steril ; 81 Suppl 1: 767-74, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15019807

ABSTRACT

OBJECTIVE: To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm. DESIGN: Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay. SETTING: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S) AND INTERVENTION(S): Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies. MAIN OUTCOME MEASURE(S): Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment. RESULT(S): The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile men's sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage. CONCLUSION(S): Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.


Subject(s)
DNA Fragmentation , DNA/metabolism , Ejaculation , Spermatozoa/physiology , fas Receptor/metabolism , Apoptosis , Blotting, Western , Case-Control Studies , Comet Assay , Flow Cytometry , Fluorescent Antibody Technique , Humans , Infertility, Male/metabolism , Male , Semen , Sperm Motility , Spermatozoa/cytology , Spermatozoa/metabolism , Staining and Labeling
11.
Regul Pept ; 115(1): 1-10, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12873792

ABSTRACT

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.


Subject(s)
Chromogranins/metabolism , Chromogranins/pharmacology , Neoplasm Proteins/metabolism , Animals , Chromatography , Chromogranin A , Estrous Cycle , Female , Follicle Stimulating Hormone/metabolism , Immunohistochemistry , Luteinizing Hormone/metabolism , Male , Pituitary Gland, Anterior , Protein Processing, Post-Translational , Radioimmunoassay , Rats , Rats, Wistar
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