losartan-potassium and Retinal-Neovascularization

Proliferative diabetic retinopathy (PDR), characterized by pathologic retinal angiogenesis, is a major cause of blindness in the USA and globally. Treatments targeting vascular endothelial growth factor (VEGF) have emerged as a beneficial part of the therapeutic armamentarium for this condition, highlighting the utility of identifying and targeting specific pathogenic molecules. There continues to be active research into the molecular players regulating retinal angiogenesis, including pro-angiogenic factors, anti-angiogenic factors, and integrins and matrix proteinases. New insights have been especially prominent regarding molecules which regulate specialized endothelial cells called tip cells, which play a lead role in endothelial sprouting. Together, these research efforts are uncovering new, important molecular regulators of retinal angiogenesis, which provide fertile areas for therapeutic exploration. This review discusses potential molecular targets, with an emphasis towards newer targets.

Topics: Angiogenesis Inhibitors; Blindness; Diabetic Retinopathy; Disease Progression; Erythropoietin; Female; Humans; Hyperglycemia; Macular Edema; Male; Retinal Neovascularization; Vascular Endothelial Growth Factor A

Exogenous erythropoietin (EPO) is being considered for tissue protection and angiogenesis in retinal vascular diseases. However, studies are limited by insufficient tools to address signaling effects through the EPO receptor (EPOR). We used a humanized mouse model of hypoactive EPOR signaling to test the hypothesis that EPOR signaling supports angiogenesis in retinovascular diseases.. Humanized Knockin EPOR mice (hWtEPOR) with hypoactive EPOR signaling were compared to littermate wild-type mice (WT). Postnatal day (p)7 mice of each genotype were exposed to 75% oxygen for five days, followed by 21% oxygen in the oxygen-induced retinopathy model (OIR) and compared to room-air (RA)-raised pups. At time points after OIR, pups were sacrificed, and flat-mounted, lectin-stained retinas were analyzed for central avascular area or intravitreal neovascular area (IVNV). Flash-frozen retinas were analyzed for angiogenic protein (Epo, VEGF, p-Stat3) and gene (Vegfa, Kdr, Epo, Hif1α, Hif2α) expression levels.. In OIR, hWtEPOR mice had increased AVA compared with WT at p8, p12, and p17, but there was no difference in IVNV between hWtEPOR and WT mice at p17. Although VEGF and p-STAT3 proteins were increased in WT at p17 OIR, there were no differences in retinal angiogenic factor expression levels between hWtEPOR and WT OIR at p17 despite similar areas of IVNV.. Our data support the hypothesis that EPOR signaling was associated with regrowth of vascularization following oxygen-induced capillary dropout and played a role in intravitreal angiogenesis. Additional study of EPOR signaling regulation on other angiogenic factor pathways may be considered.

Topics: Animals; Blotting, Western; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neovascularization, Pathologic; Oxygen; Real-Time Polymerase Chain Reaction; Receptors, Erythropoietin; Retina; Retinal Neovascularization; Signal Transduction; Vascular Endothelial Growth Factor A

Hypoxia induces angiogenesis while hyperoxia promotes vasoregression in the retina. We investigated herein the effect of prolonged hyperoxia on retinal angiogenesis and the underlying mechanism in an oxygen-induced retinopathy (OIR) model.. Vascular morphology was quantified in whole-mount retina from the mice subjected to the conventional OIR model (c-OIR) or the OIR model with prolonged hyperoxia (p-OIR). Expressions of genes related to angiogenesis were determined by real-time PCR.. p-OIR retinas showed few intraretinal neovascular tufts at the border of avascular zones, lacking preretinal neovascularization, whereas c-OIR retinas had numerous preretinal neovascularizations. p-OIR retinas demonstrated outgrowth of capillaries in the deep layers despite persistent hyperoxia and possess a larger avascular zone compared with the c-OIR retinas. The capillaries in the p-OIR retinas were well-formed in contrast to those in the c-OIR retinas. p-OIR retinas expressed significantly higher TNFα (∼4 fold) than c-OIR retinas. The expression of vascular endothelial growth factor, Erythropoietin, Angiopoietin 1 and 2 remained unchanged.. Our data demonstrate that TNFα transcription is increased in hyperoxia-promoted retinal angiogenesis, implicating it, in association with low VEGF levels, as a possible proponent in retinal angiogenesis under hyperoxia.

Topics: Angiopoietin-1; Angiopoietin-2; Animals; Erythropoietin; Fluorescent Antibody Technique; Gene Expression Regulation; Hyperoxia; Hypoxia; Mice, Inbred C57BL; Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) was originally described for its antiapoptotic effects on erythroid progenitor cells in bone marrow. In recent years, however, EPO has also been shown to be cytoprotective in several tissues, including the retina. There, exogenous application of EPO was reported to exert neuro- and vasoprotection in several models of retinal injury. EPO and the erythropoietin receptor (EPOR) are expressed in the retina, but the role of endogenous EPO-EPOR signaling in this tissue remains elusive. Here, we investigated the consequences for cell physiology and survival when EpoR is ablated in rod photoreceptors or in the peripheral retina.. Two mouse lines were generated harboring a cyclization recombinase (CRE)-mediated knockdown of EpoR in rod photoreceptors (EpoR(flox/flox);Opn-Cre) or in a heterogeneous cell population of the retinal periphery (EpoR(flox/flox);α-Cre). The function of the retina was measured with electroretinography. Retinal morphology was analyzed in tissue sections. The vasculature of the retina was investigated on flatmount preparations, cryosections, and fluorescein angiography. Retinal nuclear layers were isolated by laser capture microdissection to test for EpoR expression. Gene expression analysis was performed with semiquantitative real-time PCR. To test if the absence of EPOR potentially increases retinal susceptibility to hypoxic stress, the knockdown mice were exposed to hypoxia.. Newborn mice had lower retinal expression levels of EpoR and soluble EpoR (sEpoR) than the adult wild-type mice. In the adult mice, the EpoR transcripts were elevated in the inner retinal layers, while expression in the photoreceptors was low. CRE-mediated deletion in the EpoR(flox/flox);Opn-Cre mice led to a decrease in EpoR mRNA expression in the outer nuclear layer. A significant decrease in EpoR expression was measured in the retina of the EpoR(flox/flox);α-Cre mice, accompanied by a strong and significant decrease in sEpoR expression. Analysis of the retinal morphology in the two knockdown lines did not reveal any developmental defects or signs of accelerated degeneration in the senescent tissue. Similarly, retinal function was not altered under scotopic and photopic conditions. In addition, EpoR knockdown had no influence on cell viability under acute hypoxic conditions. Retinal angiogenesis and vasculature were normal in the absence of EPOR. However, expression of some EPOR-signaling target genes was significantly altered in the retinas of the EpoR(flox/flox);α-Cre mice.. Our data suggest that expression of EPOR in rod photoreceptors, Müller cells, and amacrine, horizontal, and ganglion cells of the peripheral retina is not required for the maturation, function, and survival of these cells in aging tissue. Based on the expression of the EPOR-signaling target genes, we postulate that expression of soluble EPOR in the retina may modulate endogenous EPO-EPOR signaling.

Topics: Animals; Cell Hypoxia; Cell Survival; Cellular Senescence; Erythropoietin; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation; Gene Knockdown Techniques; Integrases; Mice; Protein Isoforms; Real-Time Polymerase Chain Reaction; Receptors, Erythropoietin; Retinal Neovascularization; Retinal Rod Photoreceptor Cells; Signal Transduction

Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy.. Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression.. An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system.

Topics: Adaptor Protein Complex 1; Aldosterone; Animals; Animals, Newborn; Aquaporin 4; Body Weight; Cells, Cultured; Diet, Sodium-Restricted; Disease Models, Animal; Drinking Behavior; Ependymoglial Cells; Epithelial Sodium Channels; Erythropoietin; Gliosis; Hematocrit; Ion Transport; Ischemia; Kidney Glomerulus; MAP Kinase Signaling System; Microglia; Phosphorylation; Potassium Channels, Inwardly Rectifying; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Retinal Ganglion Cells; Retinal Neovascularization; Retinopathy of Prematurity; Sodium; Sodium Chloride, Dietary; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Topics: Abdominal Neoplasms; Adolescent; Adult; Basic Helix-Loop-Helix Transcription Factors; Child; DNA Mutational Analysis; Erythropoietin; Exudates and Transudates; Female; Fibrosis; Fluorescein Angiography; Humans; Male; Optic Disk; Pancreatic Neoplasms; Paraganglioma; Polycythemia; Retinal Neovascularization; Somatostatinoma

This study aimed to investigate the effect and potential mechanisms of exogenous administration of recombinant human erythropoietin (rhEPO) on retinal angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Postnatal day 7 (P7) mice (n=132) were randomly assigned to one of six groups: Control group (n=22), OIR group (n=22), OIR + vehicle control group (n=22), OIR + rhEPO 10 IU group (n=22), OIR + rhEPO 50 IU group (n=22), and OIR + rhEPO 100 IU group (n=22). OIR was induced by exposing mice to 75±2% O2 for five days, followed by exposure to room air for a further five days. Animals in groups 3-6 (the OIR + vehicle control group and OIR + rhEPO 10 IU, 50 IU, and 100 IU groups) received an intraperitoneal injection of saline, or rhEPO 10 IU, 50 IU and 100 IU, respectively, which were administered daily from P7-P12. Immunofluorescent and hematoxylin-eosin staining were used to detect retinal neovascularization (RNV) in retinal whole mounts. Quantitative polymerase chain reaction and western blot analysis were used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS). RNV occurred in the OIR groups and was accompanied by dilated, twisted and occluded blood vessels. rhEPO treatment resulted in an increase in the number of newly formed and severely dilated vessels. rhEPO increased RNV in a dose-dependent manner, which was accompanied by an increase in the messenger RNA and protein expression of VEGF, eNOS and nNOS. Thus, exogenous use of rhEPO promotes the RNV in a mouse model of OIR and is accompanied by increased expression levels of VEGF, eNOS and nNOS.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Humans; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Oxygen; Recombinant Proteins; Retina; Retinal Neovascularization; RNA, Messenger; Vascular Endothelial Growth Factor A

Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker.. Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006-2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls.. Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9).. The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.

Topics: Aged; Angiopoietin-1; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; Prospective Studies; Retinal Neovascularization; Retinal Perforations; Tomography, Optical Coherence; Transforming Growth Factor beta1; Up-Regulation; Vascular Endothelial Growth Factor A; Vesicular Transport Proteins; Vitrectomy; Vitreous Body

Retinal angiogenesis is a major cause of blindness in ischemic retinopathies including diabetic retinopathy and retinopathy of prematurity. Integrin αvβ3 is a promising therapeutic target for ocular angiogenesis, modulating the pro-angiogenic actions of multiple growth factors. In this study, we sought to determine the effects of the integrin αvβ3 antagonist tetra-iodothyroacetic acid (tetrac) on the angiogenic actions of VEGF and erythropoietin (EPO) in cultured human retinal endothelial cells. In addition, we investigated the effect of tetrac and a nanoparticulate formulation of tetrac on retinal angiogenesis in vivo, in the mouse oxygen-induced retinopathy (OIR) model. Tetrac inhibitory activity was evaluated in human retinal endothelial cells treated with VEGF and/or EPO. Endothelial cell proliferation, migration, and tube formation were assessed, in addition to phosphorylation of ERK1/2. For the studies of the oxygen-induced retinopathy model, C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12, and then returned to room air. Tetrac and tetrac-nanoparticle (tetrac-NP) were administered at P12 and P15 by either intraperitoneal or intravitreal injection. Retinal neovascularization was quantitated at P18. Tetrac significantly inhibited pro-angiogenic effects of VEGF and/or EPO on retinal endothelial cells, indicating that the angiogenic effects of both growth factors are dependent on integrin αvβ3. Retinal neovascularization in the OIR model was significantly inhibited by both tetrac and tetrac-NP. These results indicate that the integrin αvβ3 antagonist, tetrac, is an effective inhibitor of retinal angiogenesis. The ability of tetrac to inhibit the pro-angiogenic effect of both VEGF and EPO on retinal endothelial cells suggests that tetrac (and antagonism of integrin αvβ3) is a viable therapeutic strategy for proliferative diabetic retinopathy.

Topics: Angiogenesis Inhibitors; Animals; Blotting, Western; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Humans; Integrin alphaVbeta3; Intravitreal Injections; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oxygen; Phosphorylation; Retinal Neovascularization; Thyroxine; Vascular Endothelial Growth Factor A

Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV.

Topics: Animals; Animals, Newborn; Caspase 3; Down-Regulation; Erythropoietin; Janus Kinases; Oxygen; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Signal Transduction; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A

To examine vitreous succinate levels from proliferative diabetic retinopathy (PDR) patients and ascertain their association with PDR activity.. Comparative case series.. A total of 81 eyes of 72 PDR patients were divided into active PDR (22 eyes), quiescent PDR (21 eyes), and active PDR with intravitreal bevacizumab injection (38 eyes). Twenty epiretinal membrane (ERM) patients (21 eyes) served as controls.. Mean vitreous succinate levels were 1.27 μM in ERM and 2.20 μM in PDR, with the differences statistically significant (P = .03). When comparing mean vitreous succinate levels (active PDR: 3.32 μM; quiescent PDR: 1.02 μM; active PDR with intravitreal bevacizumab injection: 1.20 μM), significant differences were found between active and quiescent PDR (P < .01) and between active PDR and active PDR with intravitreal bevacizumab injection (P < .01). Even though succinate levels were low, retinopathy activities were very high in patients with active PDR with intravitreal bevacizumab injection. Mean vitreous vascular endothelial growth factor (VEGF) levels (active PDR: 1696 pg/mL; quiescent PDR: 110 pg/mL; active PDR with intravitreal bevacizumab injection: n.d.) were similar to previous reports. Mean vitreous erythropoietin levels (active PDR: 703 mIU/mL; quiescent PDR: 305 mIU/mL; active PDR with intravitreal bevacizumab injection: 1562 mIU/mL) suggested very high retinopathy activities in patients with active PDR with intravitreal bevacizumab injection.. Succinate, like VEGF, may be an angiogenic factor that is induced by ischemia in PDR. Although succinate is reported to promote VEGF expression, VEGF inhibition decreases succinate. Thus, VEGF, via a positive feedback mechanism, may regulate succinate.

Topics: Aged; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Aqueous Humor; Bevacizumab; Chromatography, High Pressure Liquid; Diabetic Retinopathy; Epiretinal Membrane; Erythropoietin; Female; Humans; Intravitreal Injections; Male; Mass Spectrometry; Middle Aged; Retinal Neovascularization; Succinic Acid; Vascular Endothelial Growth Factor A; Vitreous Body

Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy.

Topics: Animals; Apoptosis; Cell Hypoxia; Disease Models, Animal; Electroretinography; Erythropoietin; Ischemia; Kidney; Mice; Mice, Transgenic; Oxygen; Recombinant Proteins; Retina; Retinal Neovascularization; Retinal Neurons; Retinal Vessels; Up-Regulation

Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects.. Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups.. None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor.. Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of β-adrenergic receptors is an appropriate therapeutic approach for treating ROP.

Topics: Administration, Oral; Adrenergic beta-Antagonists; Angiopoietin-1; Angiopoietin-2; Animals; Animals, Newborn; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Profiling; Humans; In Vitro Techniques; Infant, Newborn; Injections, Intraperitoneal; Injections, Subcutaneous; Mice; Oxygen; Propranolol; Receptors, Adrenergic, beta; Receptors, Vascular Endothelial Growth Factor; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA; Up-Regulation; Vascular Endothelial Growth Factor A

Doping with erythropoietic proteins such as erythropoietin (EPO) is a serious issue in sport. There is little information on the possible ophthalmologic alterations followed by frequent EPO abuse in athletes. EPO is a potent retinal angiogenic factor and is capable of stimulating retinal angiogenesis and neovascularization in the presence of ischemia. Systemic and intravitreal EPO concentrations are highly correlated. A linkage between EPO doping and retinal proliferation is possible and further studies are warranted. Gathering and analyzing data on retinal findings from these athletes, either retrospectively or prospectively might yield preliminary information to support the safety of those athletes. Implications of this hypothesis cover other kinds of neovascularizations and angiogenesis.

Topics: Athletes; Doping in Sports; Erythropoietin; Humans; Retinal Neovascularization; Risk Factors; Tissue Distribution

We determined whether the concentrations of VEGF, erythropoietin, and endostatin in the vitreous are altered after vitrectomy in patient with proliferative diabetic retinopathy (PDR).. We measured the levels of VEGF, erythropoietin, and endostatin by sandwich ELISA in vitreous samples collected from 38 eyes of 33 patients with PDR before pars plana vitrectomy (without IOL implantation) and the same 38 eyes during IOL implantation 3.1 to 25.7 (mean 6.7) months after the initial vitrectomy.. The mean vitreous levels of VEGF (964.5 pg/mL) and erythropoietin (1359.5 pg/mL) in the samples collected before vitrectomy were significantly higher in patients with PDR than in the control patients (0.68 and 70.7 pg/mL, respectively; P < 0.01). The levels of VEGF (292.5 pg/mL) and erythropoietin (557.9 pg/mL) in the samples from eyes with PDR collected at the time of IOL implantation were significantly lower than those collected before vitrectomy (P < 0.01). In contrast, the changes in the level of endostatin were not significant after vitrectomy. The VEGF and erythropoietin levels in the vitreous fluid from patients with PDR were correlated inversely with the interval between the initial vitrectomy and the time of the IOL implantation.. The significant decrease in the intravitreal concentration of VEGF and erythropoietin, and an absence of a significant change in the endostatin indicated a shift in the antiangiogenic balance in the vitreous of patients with PDR after successful vitrectomy.

Topics: Adult; Aged; Diabetic Retinopathy; Endostatins; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Humans; Laser Coagulation; Lens Implantation, Intraocular; Male; Middle Aged; Retinal Neovascularization; Vascular Endothelial Growth Factor A; Vitrectomy; Vitreous Body

Prolyl hydroxylases (PHDs) are oxygen sensors that stabilize hypoxia-inducible factors (HIFs) to induce proinflammatory, vasopermeability, and proapoptotic factors. These may be potential targets to reduce the complications of ischemic retinopathies.. Oxygen-induced ischemic retinopathy (OIR) was generated as a model for retinopathy of prematurity (ROP) by placing 7-day-old mice in 75% oxygen for 5 days and returning them to the relative hypoxia of room air for 5 days. Neovascularization (NV) and avascular areas were assessed on retinal flat-mounts by image analysis. Blood-retinal barrier breakdown was assessed using ³H-mannitol as a tracer. Apoptosis was detected with TUNEL staining. HIF-1α and VEGF were quantified using Western blot analysis and ELISA.. PHD1-deficient mice demonstrated reduced hyperoxia-associated vascular obliteration during oxygen-induced ischemic retinopathy. This was associated with subsequent reduced avascularity, vascular leakage, and pathologic NV during the hypoxic phase, which could be accounted for by a reduced expression of HIF-1α and VEGF. Apoptosis in the retina was also reduced in PHD1-depleted mice after 2 days in hyperoxia.. PHD1 deficiency is associated with a reduction of ischemia-induced retinal NV. The regulatory mechanism in this model appears to be: PHD1 depletion prevents HIF-1α degradation in hyperoxia, which induces VEGF, thus preventing hyperoxia-related vessel loss. Without a vessel deficiency, there would not be relative hypoxia when the mice are returned to room air and there would be no need to initiate angiogenesis signaling. Blocking PHD1 may be beneficial for ischemic retinopathies and inflammatory and neurodegenerative disorders.

Topics: Animals; Animals, Newborn; Apoptosis; Blood-Retinal Barrier; Blotting, Western; Capillary Permeability; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; In Situ Nick-End Labeling; Infant, Newborn; Mice; Mice, Knockout; Oxygen; Procollagen-Proline Dioxygenase; Reperfusion Injury; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) plays a critical role in the vascular system and exhibits angiogenic activity in endothelial cells (ECs) such as stimulation of cell proliferation, migration and tube formation in vitro. EPO is the major regulator of cell proliferation and differentiation of erythroid precursors and there by preventing the apoptosis. Pigment epithelial derived factor (PEDF) is a potent anti-angiogenic factor whose effects are partially mediated through the induction of EC apoptosis. The mechanism of EPO and PEDF in retinal neovascularization has not been well documented yet. The effect of EPO and PEDF on cell proliferation was determined by MTT assay. In vitro wound-scratch assay was performed to study the migration of ECs and in vitro tube formation was assessed by the on-gel assay system using gelatin. Inhibitor assay was carried out using LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Further, PI3K/Akt activity was assessed by transient transfection assay using constitutively active (CA) and dominant negative (DN) Akt mutants. Dextran permeability assay was performed to determine the vascular permeability. We report that EPO stimulates EC proliferation, migration, tube formation and permeability whereas PEDF inhibits the EPO-induced ECs proliferation and permeability. Over expression of DN Akt blocked EPO stimulation of proliferation and permeability, while over expression of CA Akt rescues the inhibitory effect of PEDF on proliferation and permeability. These results demonstrate that PEDF may inhibit the EPO-induced proliferation and permeability via PI3K/Akt-dependent pathway.

Topics: Angiogenesis Inhibitors; Animals; Capillary Permeability; Cell Culture Techniques; Cell Movement; Cell Proliferation; Chromones; Dextrans; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Eye Proteins; Goats; Morpholines; Nerve Growth Factors; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Retinal Neovascularization; Retinal Vessels; Serpins; Transfection

Erythropoietin (EPO), an oxygen-regulated hormone stimulating erythrocyte production, was recently found to be critical for retinal angiogenesis. EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy. The authors investigated the inhibition of retinal EPO mRNA expression with RNA interference as a potential strategy to suppress retinal neovascularization and to prevent proliferative retinopathy.. The authors used a mouse model of oxygen-induced retinopathy. Retinal EPO and Epo receptor (EpoR) expression during retinopathy development were quantified with real-time RT-PCR in whole retina and on laser-captured retinal vessels and neuronal layers. Retinal hypoxia was assessed with an oxygen-sensitive hypoxyprobe. A small interference RNA (siRNA) targeting EPO or control negative siRNA was injected intravitreally at postnatal (P) day 12, P14, and P15 during the hypoxic phase, and the effect on neovascularization was evaluated in retinal flatmounts at P17.. Retinal EPO mRNA expression in total retina was suppressed during the initial phase of vessel loss in retinopathy and was significantly elevated during the hypoxia-induced proliferative phase in all three neuronal layers in the retina, corresponding to an increased level of retinal hypoxia. EpoR mRNA expression levels also increased during the second neovascular phase, specifically in hypoxia-induced neovascular vessels. Intravitreous injection of EPO siRNA effectively inhibited approximately 60% of retinal EPO mRNA expression and significantly suppressed retinal neovascularization by approximately 40%.. Inhibiting EPO mRNA expression with siRNA is effective in suppressing retinal neovascularization, suggesting EPO siRNA is a potentially useful pharmaceutical intervention for treating proliferative retinopathy.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Humans; Infant, Newborn; Injections; Mice; Microscopy, Confocal; Oxygen; Receptors, Erythropoietin; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Vitreous Body

To observe the effect of inhibition of retinal neovascularization by small-interference RNA (siRNA) targeting erythropoietin (EPO).. Three siRNAs against EPO were designed and synthesized. Then they were transfected to NIH/3T3 cells by liposomes. RT-PCR and Western blot were used to evaluate the efficacy of siRNA in attenuating EPO expression in NIH/3T3 cells. One-week-old C57BL/6J mice were exposed to 75 +/- 2% oxygen for 5 days, then they were returned to room air to induce retinal neovascularization. The siRNA type shown as most powerful in reducing EPO expression in vitro was intravitreally injected in the treatment group. Retinal neovascularization was evaluated by angiography with injection of fluorescein-dextran and quantification of neovascular proliferative retinopathy after 5 days in room air. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of siRNA could affect the expression of EPO in murine retinas.. Among the 3 designed siRNAs (named siEPO1-3), siEPO2 is the most efficient in inhibiting EPO expression. In this murine model of oxygen-induced retinopathy, retinal neovascularization in the eyes with siEPO2 injection was significantly reduced compared with that of the contralateral control eyes. Similarly, histological analysis indicates that the number of neovascular nuclei protruding into the vitreous cavity was decreased compared to the control eyes. Furthermore, the expression of EPO in the retinas injected with siEPO2 was dramatically decreased.. siRNA against EPO could inhibit experimental retinal neovascularization by reducing EPO expression in the retinas of mice. It may provide a powerful and novel therapeutic tool for ischemia-induced retinal diseases.

Topics: Animals; Animals, Newborn; Base Sequence; Blotting, Western; Cell Culture Techniques; Dextrans; Disease Models, Animal; Erythropoietin; Fluorescein Angiography; Fluoresceins; Gene Expression Regulation; Humans; Infant, Newborn; Injections; Mice; Mice, Inbred C57BL; Molecular Sequence Data; NIH 3T3 Cells; Oxygen; Retinal Neovascularization; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Transfection; Vitreous Body

Topics: Erythropoietin; Humans; Infant, Newborn; Retinal Neovascularization; Retinopathy of Prematurity; Vascular Endothelial Growth Factor A; Vitreous Body

Retinal neovascularization in diabetes has been thought to follow the release of local angiogenic factors in the retina. We hypothesize that neovascularization of diabetic retinopathy is a systemic vasculogenesis rather than a local angiogenesis. Thus, we evaluate the concentrations of circulating endothelial progenitor cells (EPCs) and stem cell modulation factors such as vascular endothelial growth factor (VEGF), erythropoietin (Epo), and substance p (SP) in the peripheral blood of diabetic retinopathy patients.. We studied 15 normal controls and 45 type II diabetic patients (no DR group (n=15), NPDR group (n=15), and PDR group (n=15)). We measured circulating CD34+mononuclear cells (CD34+MNCs), c-Kit+mononuclear cells (c-Kit+MNCs) by flow cytometry. VEGF, Epo, and SP in the peripheral blood were measured by ELISA.. The circulating CD34+MNCs and c-Kit+MNCs increased in the NPDR and PDR groups compared with the control group (P<0.01). Serum level of VEGF increased in the NPDR and PDR groups compared with the control group (P<0.05). The level of Epo elevated exclusively in the no DR group compared with the other three groups (P<0.01). Circulating SP level increased in the NPDR and PDR groups compared with the control group (P<0.05). SP and CD34+MNCs were shown to have increased correlation according to the diabetic retinopathy in the NPDR and PDR groups (r=0.440, P<0.05 and r=0.460, P<0.05, respectively).. The present study is the first to demonstrate that CD34+MNCs, c-Kit+MNCs and their modulator are elevated in diabetic retinopathy patients. Therefore, it is possible that circulating EPCs and serum Epo, VEGF, and SP may be involved in the progression of diabetic retinopathy.

Topics: Aged; Aged, 80 and over; Angiogenesis Inducing Agents; Antigens, CD34; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelium, Vascular; Erythropoietin; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Retinal Neovascularization; Severity of Illness Index; Stem Cells; Substance P; Vascular Endothelial Growth Factor A

Topics: Adolescent; Anemia; Anti-Inflammatory Agents; Cardiomyopathies; Epoetin Alfa; Erythropoietin; Fluorescein Angiography; Humans; Injections; Laser Coagulation; Male; Methylprednisolone; Methylprednisolone Acetate; Muscular Dystrophy, Duchenne; Recombinant Proteins; Retinal Neovascularization; Vision Disorders; Vitrectomy; Vitreous Hemorrhage

Although vascular endothelial growth factor (VEGF) is a primary mediator of retinal angiogenesis, VEGF inhibition alone is insufficient to prevent retinal neovascularization. Hence, it is postulated that there are other potent ischemia-induced angiogenic factors. Erythropoietin possesses angiogenic activity, but its potential role in ocular angiogenesis is not established.. We measured both erythropoietin and VEGF levels in the vitreous fluid of 144 patients with the use of radioimmunoassay and enzyme-linked immunosorbent assay. Vitreous proliferative potential was measured according to the growth of retinal endothelial cells in vitro and with soluble erythropoietin receptor. In addition, a murine model of ischemia-induced retinal neovascularization was used to evaluate erythropoietin expression and regulation in vivo.. The median vitreous erythropoietin level in 73 patients with proliferative diabetic retinopathy was significantly higher than that in 71 patients without diabetes (464.0 vs. 36.5 mIU per milliliter, P<0.001). The median VEGF level in patients with retinopathy was also significantly higher than that in patients without diabetes (345.0 vs. 3.9 pg per milliliter, P<0.001). Multivariate logistic-regression analyses indicated that erythropoietin and VEGF were independently associated with proliferative diabetic retinopathy and that erythropoietin was more strongly associated with the presence of proliferative diabetic retinopathy than was VEGF. Erythropoietin and VEGF gene-expression levels are up-regulated in the murine ischemic retina, and the blockade of erythropoietin inhibits retinal neovascularization in vivo and endothelial-cell proliferation in the vitreous of patients with diabetic retinopathy in vitro.. Our data suggest that erythropoietin is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy.

Topics: Animals; Case-Control Studies; Cattle; Cell Proliferation; Cells, Cultured; Diabetic Retinopathy; Dose-Response Relationship, Drug; Erythropoietin; Extracellular Matrix Proteins; Female; Humans; Logistic Models; Male; Mice; Middle Aged; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Proteins; Receptors, Erythropoietin; Retina; Retinal Neovascularization; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A; Vitreous Body

Topics: Diabetic Retinopathy; Erythropoietin; Humans; Retina; Retinal Neovascularization; Vascular Endothelial Growth Factor A

Topics: Analysis of Variance; Diabetic Retinopathy; Erythropoietin; Humans; Infant, Newborn; Infant, Very Low Birth Weight; Retinal Neovascularization; Retinopathy of Prematurity; Risk Factors

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Topics: Animals; Antibodies; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Electroretinography; Erythropoietin; Female; Gene Expression Regulation; Gene Targeting; Genetic Vectors; Helix-Loop-Helix Motifs; Humans; Hyperoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; In Situ Nick-End Labeling; Infant, Newborn; Integrases; Mice; Mice, Knockout; Retina; Retinal Neovascularization; Retinopathy of Prematurity; RNA, Messenger; Tissue Distribution; Trans-Activators; Transcription Factors; Vimentin; Viral Proteins

Topics: Adult; Erythropoietin; Female; Humans; Kidney Failure, Chronic; Recombinant Proteins; Retinal Detachment; Retinal Neovascularization; Retinitis Pigmentosa