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 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

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 (β-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 17β-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

Role of the receptor for advanced glycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetes in mice

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, HbA and retinal levels of methylglyoxal (MG) were evaluated up to 24\ua0weeks 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\ua0weeks (p < 0.01) but not after 24\ua0weeks. 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

Epoetin delta modulates intra-retinal neovascularisation.

<p>The retina in Epoetin delta-treated mice often demonstrated lectin-stained “atypical vessels” (A). This is a dismounted flatmount imaged immediately prior to cryobedding. A representative of the serial sections of the flat-mounts is illustrated in Fig. (B). The area within the white box of Fig. 7B is more clearly illustrated in Fig. 7B Here it is clear that the “atypical vessels” are located inside the retina with lectin stained green and GFAP red (C). Using electron microscopy it is evident that the neovascular tissue and glial cells were on both sides of the internal limiting membrane (green arrows) (D). The pre-retinal vessels often had pericyte covering (P) and multiple glial associations (G) although both intra- and preretinal vessels had well-formed lumens (L). In the middle of the ischaemic regions there were exclusively intra-retinal vessels in the nerve fibre layer and sometimes between the Inner nuclear layer (INL) and outer nuclear layer (ONL) that were perfused with red blood cells in the lumen (RBC) (E). Epoetin delta treatment at high dose significantly increased “atypical”, intra-retinal vascularisation in OIR (<i>*p<0.05</i>) (F). Low dose displayed no difference in the “atypical”, intra-retinal vascularisation of the entire retina. Darbepoetin Alfa and Epoetin Beta reduced the intra-retinal vascularisation of the retina (p<0.05) (n = 7). (Error bars  =  Standard Error of the Mean).</p

Epoetin delta stimulates haematopoetic stem cells in bone marrow and infiltration into the retinal vasculature.

<p>Sca-1 is a marker for haematopoetic stem cells (HSCs) and this was significantly enhanced in the marrow of mice treated with epoetin delta, both at P17 (A) and also P23 (B). (n = 5/group) ***p<0.001 (Error bars  =  Standard Error of the Mean). Sca-1 immunoreactivity in retinal flat-mounts demonstrated that this HSC marker was present in cells localised to intra (C) and pre-retinal blood vessels (D). Assessment of the retinal vasculature penetrating the ischaemia retina at P23 demonstrated that these vessels contained many Sca-1 positive cells (E). As with P17, these cells were also in clusters of pre-retinal neovessels at P23 (F).</p

VEGF dose response and prevention of EPO-induced angiogenesis by blockade of VEGF bioavailability.

<p>(A) In primary HDMEC cells VEGF was used as a positive control and the anti-VEGF drug Lucentis reduced the angiogenic effect of Epoetin Delta (ED) at 100 IU/ml in tubules grown from primary HDMEC cells. It had little effect on 1 and 20 IU/ml. (B) Darbepoetin Alfa (DA) was considerably more angiogenic than at the 1 and 20 IU/ml doses (p<0.001) although 100 IU failed to induce angiogenesis. (C) Only Epoetin Beta (EB) at 100 IU/ml dose was pro-angiogenic. (n = 6) (Error bars  =  Standard Error of the Mean) * = p<0.05.</p