Inhibition of Tumor Necrosis Factor-α Improves Physiological Angiogenesis and Reduces Pathological Neovascularization in Ischemic Retinopathy
2005; Elsevier BV; Volume: 166; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)62284-5
ISSN1525-2191
AutoresTom A. Gardiner, David S. Gibson, Tanyth E. de Gooyer, Vidal F. de la Cruz, Denise McDonald, Alan W. Stitt,
Tópico(s)Neonatal and fetal brain pathology
ResumoThe present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-α can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-α−/− and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-α−/− and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNF-α−/− and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-α significantly improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy. The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-α can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-α−/− and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-α−/− and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNF-α−/− and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-α significantly improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy. Angiogenesis is associated with two major paradigms of causation: inflammation and hypoxia. Although the central players, such as vascular endothelial growth factor (VEGF), are common to each of these models,1McCourt M Wang JH Sookhai S Redmond HP Proinflammatory mediators stimulate neutrophil-directed angiogenesis.Arch Surg. 1999; 134: 1322-1331Crossref Scopus (209) Google Scholar, 2Semenza GL Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance.J Clin Invest. 2001; 108: 39-40Crossref PubMed Scopus (146) Google Scholar it is clear that some molecules characteristically associated with angiogenesis in either hypoxia or inflammation may be important to both in certain circumstances. In particular, the roles of the inflammatory cytokines, especially tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-8, are emphasized in inflammation-associated angiogenesis3Leibovich SJ Polverini PJ Shepard HM Wiseman DM Shively V Nuseir N Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha.Nature. 1987; 329: 630-632Crossref PubMed Scopus (986) Google Scholar, 4Frater-Schroder M Risau W Hallmann R Gautschi P Bohlen P Tumor necrosis factor type alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo.Proc Natl Acad Sci USA. 1987; 84: 5277-5281Crossref PubMed Scopus (600) Google Scholar, 5BenEzra D Hemo I Maftzir G In vivo angiogenic activity of interleukins.Arch Ophthalmol. 1990; 108: 573-576Crossref PubMed Scopus (87) Google Scholar, 6Norrby K Interleukin-1-alpha and de novo mammalian angiogenesis.Microvasc Res. 1997; 54: 58-64Crossref PubMed Scopus (21) Google Scholar, 7Koch AE Polverini PJ Kunkel SL Harlow LA DiPietro LA Elner VM Elner SG Strieter RM Interleukin-8 as a macrophage-derived mediator of angiogenesis.Science. 1992; 258: 1798-1801Crossref PubMed Scopus (1874) Google Scholar although their importance in noninflammatory angiogenesis is also becoming evident.8Koolwijk P van Erck MG de Vree WJ Vermeer MA Weich HA Hanemaaijer R van Hinsbergh VW Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity.J Cell Biol. 1996; 132: 1177-1188Crossref PubMed Scopus (264) Google Scholar There is also considerable evidence that hypoxia-inducible factor, the chief trans-acting transcription factor for VEGF may represent an important link between angiogenesis occurring during hypoxia or in inflammation.9Stiehl DP Jelkmann W Wenger RH Hellwig-Burgel T Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway.FEBS Lett. 2002; 512: 157-162Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 10Sandau KB Zhou J Kietzmann T Brune B Regulation of the hypoxia-inducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis factor-alpha in contrast to desferroxamine and phenylarsine oxide.J Biol Chem. 2001; 276: 39805-39811Crossref PubMed Scopus (191) Google Scholar In addition to hypoxia, hypoxia-inducible factor has been shown to be regulated by cytokines, even under normoxic conditions.11Hellwig-Burgel T Rutkowski K Metzen E Fandrey J Jelkmann W Interleukin-1beta and tumor necrosis factor-alpha stimulate DNA binding of hypoxia-inducible factor-1.Blood. 1999; 94: 1561-1567Crossref PubMed Google Scholar Oxygen-induced retinopathy (OIR) in neonatal mice12Smith LE Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'Amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar represents a model of hypoxia-driven retinal neovascularization devoid of any obvious inflammatory infiltrate. In this model the mice are exposed to hyperoxia for 5 days between postnatal day 7 (P7) and P12. As vascularization of the murine retina takes place throughout the first 2 weeks of postnatal development, the retinal vasculature at P7 is immature with only the primary inner vascular plexus in place. The hyperoxia causes vaso-obliteration of the central retinal capillary beds by two recognized mechanisms: it rapidly down-regulates VEGF production by the retinal parenchyma and renders the immature capillaries vulnerable to apoptosis13Stone J Itin A Alon T Pe'er J Gnessin H Chan-Ling T Keshet E Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia.J Neurosci. 1995; 15: 4738-4747Crossref PubMed Google Scholar, 14Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1400) Google Scholar, 15Pierce EA Foley ED Smith LE Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity.Arch Ophthalmol. 1996; 114: 1219-1228Crossref PubMed Scopus (421) Google Scholar while simultaneously inducing an oxidative insult mediated by endogenous nitric oxide production.16Brooks SE Gu X Samuel S Marcus DM Bartoli M Huang PL Caldwell RB Reduced severity of oxygen-induced retinopathy in eNOS-deficient mice.Invest Ophthalmol Vis Sci. 2001; 42: 222-228PubMed Google Scholar It is also likely that the oxidative insult is augmented by up-regulation of pigment epithelium-derived factor, which has been shown to be anti-angiogenic and proapoptotic for activated vascular endothelial cells.17Stellmach V Crawford SE Zhou W Bouck N Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor.Proc Natl Acad Sci USA. 2001; 98: 2593-2597Crossref PubMed Scopus (360) Google Scholar, 18Duh EJ Yang HS Suzuma I Miyagi M Youngman E Mori K Katai M Yan L Suzuma K West K Davarya S Tong P Gehlbach P Pearlman J Crabb JW Aiello LP Campochiaro PA Zack DJ Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth.Invest Ophthalmol Vis Sci. 2002; 43: 821-829PubMed Google Scholar, 19Dawson DW Volpert OV Gillis P Crawford SE Xu H Benedict W Bouck NP Pigment epithelium-derived factor: a potent inhibitor of angiogenesis.Science. 1999; 285: 245-248Crossref PubMed Scopus (1384) Google Scholar After return to room air at P12 the avascular regions of the central retina are rendered hypoxic with an associated liberation of angiogenic growth factors.20Simpson DA Murphy GM Bhaduri T Gardiner TA Archer DB Stitt AW Expression of the VEGF gene family during retinal vaso-obliteration and hypoxia.Biochem Biophys Res Commun. 1999; 262: 333-340Crossref PubMed Scopus (65) Google Scholar Subsequently, retinal angiogenesis occurs in two separate locations. Firstly, there is a variable revascularization of the ischemic central capillary beds. Secondly, preretinal neovascularization results in the proliferation of new vessels on the surface of the retina, within the vitreous body. The preretinal vessels are derived from viable vasculature at the margins of the ischemic central retina, from the perfused periphery and the optic disk. Neovascular complexes tend to overlie persistently ischemic areas of the central retina and are less developed in quadrants where intraretinal revascularization has occurred. This report describes how alteration of the cytokine profile during the hypoxic/angiogenic phase of OIR by semapimod (formerly known as CNI-1493), a translational inhibitor of TNF-α,21Bianchi M Bloom O Raabe T Cohen PS Chesney J Sherry B Schmidtmayerova H Calandra T Zhang X Bukrinsky M Ulrich P Cerami A Tracey KJ Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone.J Exp Med. 1996; 183: 927-936Crossref PubMed Scopus (122) Google Scholar, 22Cohen PS Nakshatri H Dennis J Caragine T Bianchi M Cerami A Tracey KJ CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.Proc Natl Acad Sci USA. 1996; 93: 3967-3971Crossref PubMed Scopus (101) Google Scholar or by ablation of the TNF-α gene may alter the balance of hypoxia and angiogenesis within the intraretinal and preretinal compartments of the eye. It also indicates fundamental differences in the needs of angiogenic vessels proliferating within differing extracellular environments. OIR was induced in C57/BL6 wild-type (WT) and TNF-α−/− mice23Korner H Cook M Riminton DS Lemckert FA Hoek RM Ledermann B Kontgen F Fazekas de St Groth B Sedgwick JD Distinct roles for lymphotoxin-alpha and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue.Eur J Immunol. 1997; 27: 2600-2609Crossref PubMed Scopus (268) Google Scholar (on a C57/BL6 genetic background) (Banton & Kingman, Hull, UK) as described by Smith and colleagues.12Smith LE Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'Amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar Briefly, neonatal mice and their nursing dams were exposed to 75% oxygen (PRO-OX 110 chamber oxygen controller used; Biospherix Ltd., Redfield, NY) between postnatal day 7 (P7) and P12 producing vaso-obliteration and cessation of vascular development in the capillary beds of the central retina. Return of the animals to room air at P12 renders the central retina ischemic and hypoxic, and results in preretinal neovascularization between P15 and P21 with maximal neovascularization at P17. In each experiment two P12 animals received an intravenous injection of fluorescein-dextran (2 × 106 MW) (Sigma-Aldrich Ltd., Gillingham, UK) and were sacrificed immediately on return to room air. Retinal flat-mount angiograms were then prepared to confirm consistent vaso-obliteration of the central capillary beds. As the C57/BL6 dams showed inconsistent care and nutrition of the pups when returned to room air, at P12 all C57/BL6 and TNF-α−/− pups were cross-fostered to nursing Swiss dams that had not been exposed to hyperoxia. Multiple timed matings were prepared for both WT and TNF-α−/− mice and subjected to OIR. WT neonates were divided into two equal groups; one being treated with semapimod (Cytokine PharmaSciences, Inc., King of Prussia, PA) (5 mg/kg) by daily intraperitoneal injection between P12 and P17 and the other OIR control group treated with vehicle (2.5% mannitol in water) alone. In all cases there was a minimum of 10 pups/group. At P17, semapimod-treated mice, controls, and TNF-α−/− mice previously exposed to hyperoxia were terminally anesthetized and perfused with fluorescein-dextran via the left ventricle. On enucleation, both eyes were fixed in 4% paraformaldehyde for 4 hours at 4°C, one for subsequent wax histology and the other for flat-mount angiography or fluorescent lectin histochemical staining with Griffonia simplicifolia isolectin B4 (Figure 1). Each experiment was conducted three times. Configuration of the microvascular tree was assessed in retinal flat-mounts using an image analysis-based evaluation of 1) avascular regions, 2) areas of intraretinal perfusion, and 3) preretinal neovascularization (Figure 1). Whole retinas fluorescently stained with biotin-labeled isolectin B4 and Alexa-488 streptavidin (described below and illustrated in Figure 1) were analyzed in a blinded manner by a skilled investigator using the Lucia image analysis system (supplied by Nikon, UK). The three categories described above were outlined and quantified in each quadrant of the retina as fractions of the total area of retina analyzed. Images were acquired using a Nikon Eclipse E400 fluorescence microscope at ×40 magnification using a Nikon DXM 1200 digital camera via Nikon image-capture software (at 600 dpi resolution) and stored as TIFF files. Five separate fields were required to cover the four retinal quadrants and the optic disk within each flat-mount. The files were opened in LUCIA G (version 4.71) image analysis software (obtained from Nikon), which allows pixel calibration in μm for each microscope lens magnification. A binary overlay was prepared from each image and total retinal areas, in μm2, were assessed by interactively drawing a line around the edge of individual retinal quadrants. Ischemic (nonvascularized) or neovascularized areas were similarly outlined and their area fractions of the total retinal area in each image calculated by the software. Often the greater fluorescent intensity of the neovascular tissue could be outlined and assessed completely automatically by setting a suitable threshold of image intensity for capture in the binary image, sufficient to outline only the neovascular tufts. Statistical analysis was performed using a one-way analysis of variance and a Tukey-Kramer post test for multiple comparisons; a P value ≤0.05 was considered significant. In a parallel experiment semapimod-treated mice and OIR controls were sacrificed at P13, P15, or P17 and the retinas immediately removed for Western analysis. The disrupted tissue samples of neural retina from each group of animals (n = 8) were pooled, centrifuged at 15,000 rpm for 15 minutes to remove cell debris, and the soluble protein extract in the supernatant was harvested for subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of protein in the samples was estimated using a BCA protein assay kit (Pierce, Rockford, IL) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed under both denaturing and nondenaturing conditions. After transfer to a nitrocellulose membrane (Bio-Rad, Hercules, CA) and blocking with 5% nonfat milk the blot was probed with anti-TNF-α goat polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), iNOS rabbit polyclonal antibody (AbCam, Cambridge, UK), or β-actin rabbit polyclonal antibody (AbCam) in Tris-buffered saline-Tween for 2 hours. After extensive washing in Tris-buffered saline-Tween an anti-goat secondary antibody-horseradish peroxidase conjugate (DAKO, AS, Glostrup, Denmark) was added to the membrane at a dilution of 1:2500 for 45 minutes. As a loading control, β-actin was probed with a 1:5000 dilution of a monoclonal antibody (mAb) (Sigma-Aldrich Ltd.) for 1 hour, washed as before, and detected with an appropriate second Ab-horseradish peroxidase conjugate (Santa Cruz Biotechnology Inc.). Immunoreactivity was detected with Supersignal West Pico chemiluminescent substrate (Pierce) on Hyperfilm (Amersham Biosciences UK Ltd., Chalfont St. Giles, UK). Band densitometry was performed using LabWorks v4.08 software on images acquired by AutoChemi system (charge-coupled device camera and autorad transillumination) (both from UVP Inc., Upland, CA). Statistical analysis was conducted using a one-way analysis of variance and data were considered significant at 95% (P < 0.05). For determination of retina hypoxia, further groups at P13 and P17 (n = 8) of hyperoxia-exposed mice (WT and TNF-α−/−) received an intraperitoneal injection of the bio-reductive drug pimonidazole24Arteel GE Thurman RG Yates JM Raleigh JA Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver.Br J Cancer. 1995; 72: 889-895Crossref PubMed Scopus (253) Google Scholar (HP; Chemicon Europe Ltd., Chandlers Ford, UK) at 60 mg/kg 3 hours before sacrifice to assess retinal hypoxia in semapimod-treated WT and TNF-α−/− animals compared to OIR controls. For this, one eye from each animal was fixed in 4% paraformaldehyde for 4 hours and then washed extensively throughout a 4-hour period after which the anterior segment lens complexes were removed and the posterior eye cups relaxed by placing four radial cuts from the retinal periphery to points within 1 mm from the optic disk. The specimens were then given an overnight soak in 96-well plates at 4°C in phosphate-buffered saline (PBS) containing 0.5% Triton X-100 to permeabilize the tissue, and 5% normal goat serum (Sigma-Aldrich Ltd.) to block nonspecific binding of the primary antibody. For visualization of the HP-protein adducts an anti-HP mouse monoclonal antibody (HP-mAb; Chemicon Europe Ltd.) or a rabbit polyclonal antibody (gift from Dr. James A. Raleigh, University of North Carolina School of Medicine, Chapel Hill, NC) were used at a dilution of 1:100 for 4 hours at 37°C and 100% humidity. Each immunoreagent incubation was followed by extensive washing in the permeabilization buffer (8× changes) throughout a further 4 hours at 37°C and an additional block with 5% normal goat serum for 2 hours at 37°C. A 1:500 dilution of a goat anti-mouse polyclonal antibody labeled with Alexa-488 (Molecular Probes Europe BV, Leiden, The Netherlands) was used for secondary detection; the staining time and subsequent washing were as used for the primary antibody. Both retinas of two OIR mice were used for immunocytochemical controls: two were stained with the second antibody only and two with 5% mouse serum (Vector Laboratories Ltd., Peterborough, UK) in place of the primary antibody. If nuclear staining was required, retinas were incubated in 5 nmol/L propidium iodide in permeabilization buffer for 10 minutes at 37°C. In nonfluorescein-dextran-perfused eyes the retinal vasculature was visualized by reaction with biotinylated isolectin B4 from G. simplicifolia (Sigma-Aldrich Ltd.) at 50 ng/ml followed by streptavidin-Alexa 568 (Molecular Probes Europe BV). Retinal flat-mounts were washed extensively and mounted in Vectasheild (Vector Laboratories Ltd.). Images were acquired using an Olympus BX60 fluorescence microscope (Olympus UK Ltd., London, UK) fitted with a MicroRadiance confocal-scanning laser microscope (Bio-Rad). Unfixed neural retina of WT (semapimod-treated and untreated) and TNF-α−/− mice (n = 6) were dissected and subjected to ultrasonic disruption in RIPA buffer. For quantification of HP-protein binding, retinal samples from P13 and P17 mice were prepared for a HP competitive enzyme-linked immunosorbent assay (ELISA) methodology. Briefly, 100 μl of sample or standard in duplicate were serially diluted in PBS/5% Tween 20 in a 96-well polystyrene assay plate (Corning Inc., Corning, NY) and incubated with 25 μl of rabbit polyclonal antibody against HP (1:3300 in PBS/5% Tween; kindly donated by Dr. James A. Raleigh, University of North Carolina School of Medicine, Chapel Hill, NC) for 1 hour at 37°C. The contents of each well was then transferred to NUNC C96 Maxisorp plates that had been precoated with solid phase antigen (1:5000 in carbonate buffer, pH 9.6) overnight at 4°C and blocked with 1% gelatin for 1 hour. The competition between solid phase and soluble antigens proceeded for 1 hour at 37°C, after which the wells were washed 4 × 5minutes with PBS/5% Tween, and 100 μl of 1:2000 alkaline-phosphatase goat anti-rabbit IgG (Sigma-Aldrich) was added to each well. Plates were then washed 4 × 5minutes with PBS/5% Tween, and 100 μl of 1 mg/ml of the alkaline-phosphatase substrate, 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich) dissolved in 10% diethanolamine buffer (pH 9.6) was added to each well. The color development of the subsequent reaction was measured at 405 nm every 5 minutes for 2 hours on a Safire microplate reader (Tecan Instruments) and reaction kinetics analyzed using Magellan 3 software. Sample HP-1 binding was determined by comparison to a Lineweaver-Burk enzyme kinetic standard curve, and is expressed as a proportion of sample protein concentration. The solid-phase antigen used in this assay was HP, which had been reductively bound to bovine serum albumin.25Raleigh JA La Dine JK Cline JM Thrall DE An enzyme-linked immunosorbent assay for hypoxia marker binding in tumours.Br J Cancer. 1994; 69: 66-71Crossref PubMed Scopus (27) Google Scholar Statistical analysis was conducted using the Student's t-test and a P value <0.05 was considered significant. Western analysis for retinal TNF-α expression demonstrated a reduction in non-OIR mice from P13 to P17 (Figure 2). On densitometric quantification of retinal extracts from OIR-controls, there was a significant increase in TNF-α expression at P15 and P17 compared to the P13 time point (P < 0.05) (Figure 2). In semapimod-treated mice subjected to OIR, there was a significant reduction in retinal TNF-α expression at both P15 and P17 when compared to OIR controls (∼50%; P < 0.05) (Figure 2). When neonatal mice were returned to room air after hyperoxia (P12) the established closure and nondevelopment of the central capillary beds rendered the central retina ischemic/hypoxic (Figure 3A). A further 5 days in room air produced some intraretinal vascular recovery with revascularization from the perfused retinal periphery, especially in semapimod-treated or TNF-α−/− mice (Figure 3B). Neovascular complexes originating at the perfused periphery and optic disk were seen to invade the vitreous body and proliferate on the retinal surface overlying the hypoxic neuropile (Figure 3, C and D). At P13, which represents a full 24 hours of ischemic hypoxia in the central retina, treatment with semapimod caused an increase in the area of avascular/ischemic retina in comparison to OIR controls (P < 0.01) (Figure 4A); a similar trend was noted in the TNF-α−/− mice and reflected in the overall level of retinal hypoxia as quantified by competitive ELISA analysis of HP adducts (Figure 4B). However, by P17 the situation was reversed. Compared to OIR controls at P17, there was a significant decrease in area of avascular (ischemic) retina in both TNF-α−/− mice and WT animals treated with semapimod (P < 0.001) (Figure 5A). This was accompanied by a corresponding and significant increase (P < 0.001) in intraretinal vascularization (normal vascularization, Figure 5B) and a decrease in preretinal neovascularization (P < 0.01 to 0.001) (Figure 5C) in semapimod-treated and TNF-α−/− mice when compared to WT OIR control animals. In all of the retinal vascular parameters analyzed, there was no significant difference between semapimod treatment and deletion of TNF-α (Figure 5). ELISA quantification of HP adducts at P17 proved unreliable because some HP deposits in the vascular endothelial cells during postmortem hypoxia and the large increase in the overall quantity of retinal vessels at P17 compared to P13 meant that this discrepancy became significant. Interpretation of HP ELISA data at P17 was further complicated by the fact, as noted below in immunofluorescent staining, that the preretinal neovascular complexes provided sufficient oxygenation to prevent the reduction of HP in the underlying neural retina.Figure 4A: TNF-α modulates hyperoxia-induced retinal vascular closure. The areas of avascular retina were quantified in retinal flat-mounts at P13. When WT OIR mice were treated with semapimod there was an increase in the area of avascular retina in comparison to OIR WT controls. This pattern of avascular retina was also evident in TNF-α−/− mice subjected to OIR. Data are the mean ± SD for n = 3 independent experiments (*, P < 0.05). B: Retinal hypoxia is increased with semapimod or absence of TNF-α. Competitive HP ELISA demonstrates increased HP immunoreactivity in retinal lysates from P13 mice for both semapimod-treated mice and TNF-α−/− mice. Data are expressed as the mean concentration per g of total protein (μmol/L/g) ± SD for n = 6 (**, P < 0.01; n = 3 independent experiments).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5TNF-α inhibition or deletion reduces retinal neovascularization with concomitant reduction of ischemia and promotion of normal retinal vascularization. Quantitative analysis was performed on the entire retinal microvasculature in flat-mounts at P17. Semapimod treatment and absence of TNF-α resulted in a significant decrease in retinal ischemia (A), an increase in normal (intraretinal) vascularization (B), and a decrease in preretinal neovascularization (C) when compared to WT controls. Data are the mean ± SD for n = 3 independent experiments. **, P < 0.01; ***, P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunofluorescent localization of the hypoxia marker pimonidazole (HP) showed deposition of protein adducts within the nonperfused central retina distal from the traversing major blood vessels (Figure 6, A and B). Interestingly, no HP staining was detected in nonperfused retina underlying preretinal neovascularization complexes (Figure 6C) indicating that the underlying inner retina was receiving significant oxygenation from the overlying neovascularization to prevent the reductive metabolism of HP and deposition of stainable drug-protein adducts. HP was deposited predominantly within the cytoplasm of neurons in the ganglion cell layer (Figure 6D) reflecting both their metabolic activity and distal location to the nearest source of oxygen in the choroid; the stained cells probably included both ganglion and displaced amacrine cells. Positive staining for HP was also detected in some horizontally orientated cell processes in the inner plexiform layer but not in the outer retina beyond the inner nuclear layer (data not shown). It was of interest that HP did not appear to deposit in the retinal astrocytes, located between the ganglion cell layer and the internal limiting membrane. Double immunostaining of the astrocytes for HP and glial fibrillary acidic protein revealed no co-localization (data not shown). Positive staining of the blood vessels by the HP monoclonal Ab was initially thought to be because of detection of mouse IgG in the blood column by the secondary Ab as it was also present in second Ab only controls, however, subsequent staining with a rabbit polyclonal Ab for HP also showed positive, although reduced, staining in the vessels, confirming reductive metabolism of HP because of postmortem hypoxia. N
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