Dexamethasone Inhibits Interleukin-1β-Induced Corneal Neovascularization
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.070172
ISSN1525-2191
AutoresShintaro Nakao, Yasuaki Hata, Muneki Miura, Kousuke Noda, Yusuke Kimura, S. Kawahara, Takeshi Kita, Toshio Hisatomi, Toru Nakazawa, Yiping Jin, M. Reza Dana, Michihiko Kuwano, Mayumi Ono, Tatsuro Ishibashi, Ali Hafezi‐Moghadam,
Tópico(s)Corneal surgery and disorders
ResumoDexamethasone, a synthetic corticosteroid, is widely used as a potent anti-inflammatory drug in various diseases including corneal angiogenesis. However, dexamethasone's impact on interleukin (IL)-1β-dependent inflammatory angiogenesis is unknown. Here, we show that dexamethasone inhibits IL-1β-induced neovascularization and the expression of the angiogenesis-related factors, vascular endothelial growth factor-A, KC, and prostaglandin E2 in the mouse cornea 2 days after IL-1β implantation. IL-1β caused IκB-α phosphorylation in corneal stromal cells but not in infiltrated CD11b+ cells 2 days after IL-1β implantation. In contrast, both cell types were positive for phosphorylated IκB-α 4 days after IL-1β implantation. Dexamethasone significantly inhibited IκB-α phosphorylation 2 and 4 days after IL-1β implantation. Furthermore, dexamethasone inhibited IL-1β-induced expression of vascular endothelial growth factor-A, KC, and prostaglandin E2, and signaling of nuclear factor (NF)-κB in corneal fibroblasts in vitro. A selective NF-κB inhibitor attenuated IL-1β-induced corneal angiogenesis. These findings suggest that NF-κB activation in the corneal stromal cells is an important early event during IL-1β-induced corneal angiogenesis and that dexamethasone inhibits IL-1β-induced angiogenesis partially via blocking NF-κB signaling. Dexamethasone, a synthetic corticosteroid, is widely used as a potent anti-inflammatory drug in various diseases including corneal angiogenesis. However, dexamethasone's impact on interleukin (IL)-1β-dependent inflammatory angiogenesis is unknown. Here, we show that dexamethasone inhibits IL-1β-induced neovascularization and the expression of the angiogenesis-related factors, vascular endothelial growth factor-A, KC, and prostaglandin E2 in the mouse cornea 2 days after IL-1β implantation. IL-1β caused IκB-α phosphorylation in corneal stromal cells but not in infiltrated CD11b+ cells 2 days after IL-1β implantation. In contrast, both cell types were positive for phosphorylated IκB-α 4 days after IL-1β implantation. Dexamethasone significantly inhibited IκB-α phosphorylation 2 and 4 days after IL-1β implantation. Furthermore, dexamethasone inhibited IL-1β-induced expression of vascular endothelial growth factor-A, KC, and prostaglandin E2, and signaling of nuclear factor (NF)-κB in corneal fibroblasts in vitro. A selective NF-κB inhibitor attenuated IL-1β-induced corneal angiogenesis. These findings suggest that NF-κB activation in the corneal stromal cells is an important early event during IL-1β-induced corneal angiogenesis and that dexamethasone inhibits IL-1β-induced angiogenesis partially via blocking NF-κB signaling. Dexamethasone, a synthetic corticosteroid analogue, is a potent anti-inflammatory drug that is used in the treatment of various immune and inflammatory diseases, including those of the eye. Folkman and Ingber1Folkman J Ingber DE Angiostatic steroids. Method of discovery and mechanism of action.Ann Surg. 1987; 206: 374-383Crossref PubMed Scopus (255) Google Scholar reported the anti-angiogenic function of a class of steroids, including dexamethasone, which they named the angiostatic steroids. The anti-angiogenic effect of dexamethasone has been confirmed in various animal models.2Ishibashi T Miki K Sorgente N Patterson R Ryan SJ Effects of intravitreal administration of steroids on experimental subretinal neovascularization in the subhuman primate.Arch Ophthalmol. 1985; 103: 708-711Crossref PubMed Scopus (185) Google Scholar, 3Gross J Azizkhan RG Biswas C Bruns RR Hsieh DS Folkman J Inhibition of tumor growth, vascularization, and collagenolysis in the rabbit cornea by medroxyprogesterone.Proc Natl Acad Sci USA. 1981; 78: 1176-1180Crossref PubMed Scopus (117) Google Scholar For instance, Ishibashi and colleagues2Ishibashi T Miki K Sorgente N Patterson R Ryan SJ Effects of intravitreal administration of steroids on experimental subretinal neovascularization in the subhuman primate.Arch Ophthalmol. 1985; 103: 708-711Crossref PubMed Scopus (185) Google Scholar showed that dexamethasone reduces laser-induced subretinal neovascularization in monkey. Furthermore, dexamethasone inhibits cauterization-induced corneal neovascularization.4Proia AD Hirakata A McInnes JS Scroggs MW Parikh I The effect of angiostatic steroids and β-cyclodextrin tetradecasulfate on corneal neovascularization in the rat.Exp Eye Res. 1993; 57: 693-698Crossref PubMed Scopus (42) Google Scholar Despite the widespread use of dexamethasone and other steroids in the clinical practice, little is known about the detailed mechanisms by which these molecules exert their anti-angiogenic effects in vivo.5Longenecker JP Kilty LA Johnson LK Glucocorticoid influence on growth of vascular wall cells in culture.J Cell Physiol. 1982; 113: 197-202Crossref PubMed Scopus (43) Google Scholar The cornea, a transparent and avascular tissue, encompasses extracellular matrix, keratocytes,6West-Mays JA Dwivedi DJ The keratocyte: corneal stromal cell with variable repair phenotypes.Int J Biochem Cell Biol. 2006; 38: 1625-1631Crossref PubMed Scopus (261) Google Scholar and leukocytes.7Nakamura T Ishikawa F Sonoda KH Hisatomi T Qiao H Yamada J Fukata M Ishibashi T Harada M Kinoshita S Characterization and distribution of bone marrow-derived cells in mouse cornea.Invest Ophthalmol Vis Sci. 2005; 46: 497-503Crossref PubMed Scopus (101) Google Scholar Corneal neovascularization occurs in a number of corneal disorders and causes significant loss of visual acuity. In corneal diseases, a number of cytokines and growth factors are up-regulated and induce infiltration of neutrophils, macrophages, and lymphocytes.8Wilson SE Mohan RR Mohan RR Ambrosio Jr, R Hong J Lee J The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells.Prog Retin Eye Res. 2001; 20: 625-637Crossref PubMed Scopus (510) Google Scholar Infiltration of inflammatory cells is often accompanied by an angiogenic response. However, the mechanistic role of inflammatory cells in corneal angiogenesis is only beginning to be understood. Corneal stromal cells, known as keratocytes or corneal fibroblasts, are normally quiescent but can readily respond to injury and transit into activated phenotypes under pathological conditions.6West-Mays JA Dwivedi DJ The keratocyte: corneal stromal cell with variable repair phenotypes.Int J Biochem Cell Biol. 2006; 38: 1625-1631Crossref PubMed Scopus (261) Google Scholar After corneal injury, corneal stromal cells are activated and migrate to the site of injury.9Fini ME Stramer BM How the cornea heals: cornea-specific repair mechanisms affecting surgical outcomes.Cornea. 2005; 24: S2-S11Crossref PubMed Scopus (198) Google Scholar In fibroblast growth factor-2-implanted corneas, stromal cells but not leukocytes express vascular endothelial growth factor (VEGF).10Chang LK Garcia-Cardena G Farnebo F Fannon M Chen EJ Butterfield C Moses MA Mulligan RC Folkman J Kaipainen A Dose-dependent response of FGF-2 for lymphangiogenesis.Proc Natl Acad Sci USA. 2004; 101: 11658-11663Crossref PubMed Scopus (241) Google Scholar However, it is unknown whether corneal stromal cells contribute to inflammation-induced neovascularization. Interleukin (IL)-1β, a multipotent cytokine, is critically involved in the acute inflammatory response, activation of inflammatory and antigen-presenting cells, chemotaxis, up-regulation of adhesion molecules and costimulatory factors on cells, and neovascularization.11Dinarello CA Biologic basis for interleukin-1 in disease.Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar In the eye, IL-1 activity has been correlated with corneal neovascularization.12Dana MR Zhu SN Yamada J Topical modulation of interleukin-1 activity in corneal neovascularization.Cornea. 1998; 17: 403-409Crossref PubMed Scopus (109) Google Scholar, 13BenEzra D Hemo I Maftzir G In vivo angiogenic activity of interleukins.Arch Ophthalmol. 1990; 108: 573-576Crossref PubMed Scopus (87) Google Scholar The expression of IL-1β in the cornea is increased in various corneal diseases including chemical burns14Fukuda M Mishima H Otori T Detection of interleukin-1β in the tear fluid of patients with corneal disease with or without conjunctival involvement.Jpn J Ophthalmol. 1997; 41: 63-66Crossref PubMed Scopus (30) Google Scholar and herpetic stromal keratitis.15Staats HF Lausch RN Cytokine expression in vivo during murine herpetic stromal keratitis. Effect of protective antibody therapy.J Immunol. 1993; 151: 277-283PubMed Google Scholar, 16Biswas PS Banerjee K Kim B Rouse BT Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis.J Immunol. 2004; 172: 3736-3744PubMed Google Scholar, 17Biswas PS Banerjee K Kim B Kinchington PR Rouse BT Role of inflammatory cytokine-induced cyclooxygenase 2 in the ocular immunopathologic disease herpetic stromal keratitis.J Virol. 2005; 79: 10589-10600Crossref PubMed Scopus (31) Google Scholar Previously, we reported that IL-1β causes corneal angiogenesis by inducing VEGF-A, COX-2/prostanoids, and CXC chemokines (such as KC and MIP-2).18Kuwano T Nakao S Yamamoto H Tsuneyoshi M Yamamoto T Kuwano M Ono M Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis.FASEB J. 2004; 18: 300-310Crossref PubMed Scopus (248) Google Scholar, 19Nakao S Kuwano T Tsutsumi-Miyahara C Ueda S Kimura YN Hamano S Sonoda KH Saijo Y Nukiwa T Strieter RM Ishibashi T Kuwano M Ono M Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1β-induced neovascularization and tumor growth.J Clin Invest. 2005; 115: 2979-2991Crossref PubMed Scopus (243) Google Scholar Furthermore, IL-1β induces infiltration of various inflammatory cells, including neutrophils and macrophages into the cornea.19Nakao S Kuwano T Tsutsumi-Miyahara C Ueda S Kimura YN Hamano S Sonoda KH Saijo Y Nukiwa T Strieter RM Ishibashi T Kuwano M Ono M Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1β-induced neovascularization and tumor growth.J Clin Invest. 2005; 115: 2979-2991Crossref PubMed Scopus (243) Google Scholar We demonstrated that infiltration of the CD11b+ inflammatory cells, a major source of angiogenic factors, is important in IL-1β-induced corneal neovascularization.19Nakao S Kuwano T Tsutsumi-Miyahara C Ueda S Kimura YN Hamano S Sonoda KH Saijo Y Nukiwa T Strieter RM Ishibashi T Kuwano M Ono M Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1β-induced neovascularization and tumor growth.J Clin Invest. 2005; 115: 2979-2991Crossref PubMed Scopus (243) Google Scholar Dexamethasone blocks the transcription of inflammatory proteins by prohibiting the activity of the transcription factor nuclear factor (NF)-κB.20Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids—new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2238) Google Scholar NF-κB plays an important role in IL-1β-related inflammatory diseases, including various corneal diseases.21Saika S Miyamoto T Yamanaka O Kato T Ohnishi Y Flanders KC Ikeda K Nakajima Y Kao WW Sato M Muragaki Y Ooshima A Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-κB, in treatment of corneal alkali burns in mice.Am J Pathol. 2005; 166: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 22Lu Y Fukuda K Liu Y Kumagai N Nishida T Dexamethasone inhibition of IL-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.Invest Ophthalmol Vis Sci. 2004; 45: 2998-3004Crossref PubMed Scopus (46) Google Scholar Blockade of NF-κB reduces corneal epithelial defects during healing in a model of corneal injury.21Saika S Miyamoto T Yamanaka O Kato T Ohnishi Y Flanders KC Ikeda K Nakajima Y Kao WW Sato M Muragaki Y Ooshima A Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-κB, in treatment of corneal alkali burns in mice.Am J Pathol. 2005; 166: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar However, it is unknown whether dexamethasone has an impact on IL-1β-dependent angiogenesis. In this work we investigate dexamethasone's potential in inhibiting IL-1β-induced angiogenesis and candidate mechanisms both in vivo and in vitro. All animal experiments were approved by the Committee on the Ethics of Animal Experiments at the Kyushu University Graduate School of Medical Sciences and the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Male BALB/c mice, 6 to 10 weeks old, were purchased from Kyudo (Saga, Japan) and Taconic (Hudson, NY). BALB/c mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). Hydron pellets (0.3 μl) containing 30 ng of human IL-1β (201-LB; R&D Systems, Minneapolis, MN) were prepared and implanted into the corneas. Pellets were positioned at 1 mm to the corneal limbus. Implanted eyes were treated with Levofloxacin eye drops (Santen Pharmaceuticals, Osaka, Japan) to prevent infection. Dexamethasone (5 mg/kg) (D2915; Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally daily, starting 1 day before (−1) and continued until the 5th day after implantation. A peptide inhibitor of NF-κB, SN50 (P-600; Biomol International, Plymouth Meeting, PA), or the control peptide SN50M (P-601; Biomol International) was applied topically to IL-1β-implanted eyes twice a day from days −1 to 5. Two, 4, and 6 days after implantation, digital images of the corneal vessels were obtained and recorded using Viewfinder 3.0 (Pixera, San Jose, CA) or OpenLab software, version 2.2.5 (Improvision Inc., Lexington, MA) with standardized illumination and contrast and were saved to disks. The quantitative analysis of neovascularization in the mouse corneas was performed using Scion Image software (version 4.0.2; Scion Corp., Frederick, MD). On days 2 and 4 after IL-1β implantation, five corneas including limbal vessel were harvested, pooled, and dissected with microscissors. The tissues were then treated twice at 37°C for 30 minutes with 0.5 mg/ml collagenase type D (Boehringer-Mannheim, Indianapolis, IN) in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% fetal calf serum (Life Technologies, Inc.), 10 mg/ml gentamicin, 50 μmol/L 2-mercaptoethanol, and 5 mg/ml HEPES buffer. The supernatants were collected, passed through a stainless-steel mesh sieve, and washed three times. Infiltrated cells into the cornea were stained with phycoerythrin-conjugated anti-CD11b mAb (1:50, RM2804; Caltag Laboratory, Burlingame, CA) for 30 minutes on ice. After washing with phosphate-buffered saline (PBS) twice, flow cytometry was performed using the FACSCaliber system (Becton-Dickinson, Mountain View, CA). Four corneas with or without dexamethasone treatment were harvested at the indicated time points after pellet implantation. The corneas were pooled in 200 μl of media, dissected with scissors, extracted with 200 μl of Triton X-100 buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 1% Triton X-100, and 10% glycerol containing 1 mmol/L phenylmethyl sulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmol/L sodium orthovanadate), and centrifuged. The supernatants were used in ELISA kits (R&D Systems) for mouse KC (MKC00B), mouse MIP-2 (MM200), mouse VEGF-A (MMV00), and prostaglandin E2 (PGE2; DE0100). Mice were sacrificed under deep anesthesia with pentobarbital sodium (60 mg/kg i.p.). The eyes were harvested, snap-frozen in optimal cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan), and 10-μm sections were prepared, air-dried, and fixed in ice-cold acetone for 10 minutes. The sections were blocked with 3% skim milk and stained with anti-phospho-IκB-α (1:100, no. 9246; Cell Signaling, Beverly, MA) and anti-CD11b mAb (1:100, 550282; BD Pharmingen, San Diego, CA). After an overnight incubation, sections were washed and stained for 20 minutes with secondary antibodies (Abs) (Chemicon International, Temecula, CA), fluorescein isothiocyanate-conjugated goat anti-rat (1:100, AP136F), Cy5-conjugated donkey anti-rabbit (1:100, AP182S), and Cy5-conjugated goat anti-mouse (1:100, AP181S). Immortalized keratocytes from corneal stroma of C57BL/6 WT mice (MK/T-1 cells)23Gendron RL Liu CY Paradis H Adams LC Kao WW MK/T-1, an immortalized fibroblast cell line derived using cultures of mouse corneal stroma.Mol Vis. 2001; 7: 107-113PubMed Google Scholar were grown in low-glucose Dulbecco's minimum essential medium (no. 11885-084; Life Technologies, Inc.), supplemented with 10% fetal bovine serum at 37°C in 5% CO2. After culture for 12 hours in serum-free medium with or without the 100 nmol/L dexamethasone, MK/T-1 cells were stimulated with 1 ng/ml IL-1β for 15 minutes (whole cell lysates) or 30 minutes (nuclear extracts) at 37°C. After rinsing with ice-cold PBS, the cells were lysed in a mammalian cell lysis kit (MCL1; Sigma Chemical Co.). Nuclear extracts were prepared with a nuclear extract kit (no. 40010; Active Motif, Carlsbad, CA). Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon membranes (Millipore, Bedford, MA). Blots were incubated with anti-IκB-α (1:1000, no. 9242; Cell Signaling), anti-phospho-IκB-α (1:1000, no. 9241; Cell Signaling), anti-phospho-NF-κB p65 (1:1000, no. 3033; Cell Signaling), anti-NF-κB p65 (1:1000, no. 3034; Cell Signaling), or anti-β-tubulin (1:1000, ab11308; Abcam, Cambridge, UK) and visualized with a secondary antibody coupled to horseradish peroxidase (Amersham, Arlington Heights, IL) and enhanced chemiluminescence system. After culture for 12 hours in serum-free medium with or without the 100 nmol/L dexamethasone, MK/T-1 cells were stimulated with 1 ng/ml IL-1β for 30 minutes at 37°C. After preparation of whole cell lysates, the activity of NF-κB p65 transcription in MK/T-1 cells was measured using the Transfactor NF-κB p65 colorimetric kit (631930; Clontech, Palo Alto, CA), which allows identification of DNA-protein interactions. The concentrations of VEGF, KC, and PGE2 in the conditioned media from MK/T-1 cells were measured using ELISA kits as described previously.24Ueda S Basaki Y Yoshie M Ogawa K Sakisaka S Kuwano M Ono M PTEN/Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib.Cancer Res. 2006; 66: 5346-5353Crossref PubMed Scopus (64) Google Scholar In brief, MK/T-1 cells were seeded in 24-well dishes at 2.5 × 104 cells in a 2-ml volume per well, and when subconfluent, the medium was replaced with serum-free medium for 24 hours, with or without 100 nmol/L dexamethasone, with or without 1 ng/ml IL-1β at 37°C. Comparisons were evaluated by the two-tailed unpaired Student's t-test. N-numbers per group were as indicated. Data are presented as mean ± SD. The differences between the groups were considered statistically significant for values of P < 0.05. To investigate whether dexamethasone affects IL-1β-induced angiogenesis, we implanted IL-1β-containing pellets into the corneas of BALB/c mice, treated them with different doses of dexamethasone or vehicle, and quantified the amount of angiogenesis 6 days after implantation. Dexamethasone inhibited IL-1β-induced angiogenesis in a dose-dependent manner (Figure 1, A and B). Five mg/kg dexamethasone completely blocked IL-1β-induced angiogenesis on day 6 (Figure 1, A and B). Furthermore, dexamethasone inhibited IL-1β-induced corneal angiogenesis by 77.5, 78.2, and 75.7% on day 2 (P < 8 × 10−8, n = 6 and 8), day 4 (P < 9 × 10−6, n = 6 and 8), and day 6 (P < 3 × 10−8, n = 6 and 8), respectively (Figure 1, C and D), suggesting that dexamethasone has an impact on IL-1β-induced corneal angiogenesis from an early stage. To investigate whether dexamethasone changes the expression of angiogenesis-related factors, such as VEGF-A25Loureiro RM D'Amore PA Transcriptional regulation of vascular endothelial growth factor in cancer.Cytokine Growth Factor Rev. 2005; 16: 77-89Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar and CXC chemokines26Strieter RM Burdick MD Mestas J Gomperts B Keane MP Belperio JA Cancer CXC chemokine networks and tumour angiogenesis.Eur J Cancer. 2006; 42: 768-778Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar (KC and MIP-2), we quantified these factors in corneas of mice during IL-1β-induced angiogenesis. VEGF-A, KC, and MIP-2 levels were significantly increased in IL-1β-implanted corneas compared with those of controls on day 2 (P < 0.05, n = 3) (Figure 2, A–C). VEGF-A and KC protein levels were significantly reduced by dexamethasone on day 2 (P = 0.004 and 0.03; n = 3 and 4, respectively) (Figure 2, A and B). However, dexamethasone did not significantly affect MIP-2 protein levels in IL-1β-implanted corneas (P = 0.8, n = 3 and 4) (Figure 2C). Interestingly, dexamethasone increased KC protein levels in IL-1β-implanted corneas on day 4 (P = 0.03, n = 4). These data show that dexamethasone inhibits IL-1β-induced VEGF-A and KC but not MIP-2 expression 2 days after implantation. Furthermore, to examine whether dexamethasone impacts IL-1β-induced PGE2 expression, we quantified its concentration in corneal extracts by ELISA. Dexamethasone treatment showed a significant decrease in PGE2 levels 2 (P = 0.04, n = 3) and 4 days (P = 0.004, n = 7 and 8) after IL-1β implantation (Figure 2D). To examine the effect of dexamethasone on IL-1β-induced infiltration of inflammatory cells, we quantified the number of CD11b+ cells in corneas of implanted animals using flow cytometry and histology. Corneas from mice treated with dexamethasone or vehicle control were harvested, and the percentage of CD11b+ cells were determined by FACScan. The percentage of the infiltrating CD11b+ cells was increased to 55 ± 10.9% and 28.1 ± 9.9% on days 2 and 4 after IL-1β implantation, respectively (Figure 3A). In comparison, the percentage of infiltrating CD11b+ cells in dexamethasone-treated mice was 51.3 ± 6.9% and 33.3 ± 5.8% on days 2 and 4 after IL-1β implantation, respectively (Figure 3A). There was no statistical difference between the results in the dexamethasone-treated and untreated IL-1β-implanted mice (P = 0.6, n = 4 on day 2; and P = 0.2, n = 7 and 8 on day 4). To analyze the effect of dexamethasone on the number of infiltrated CD11b+ cells into corneas, we next performed immunostaining for CD11b. On day 2, in corneas of dexamethasone-treated mice significantly less CD11b+ cells were found when compared with those of control mice (P < 0.05, n = 5), whereas there was no significant difference between the number of CD11b+ cells in dexamethasone-treated and untreated control (P = 0.8, n = 4 and 5). These data indicate that 5 mg/kg dexamethasone inhibits IL-1β-induced infiltration of CD11b+ into cornea on day 2 but not day 4. To elucidate the molecular events underlying the inhibition of IL-1β-induced angiogenesis by dexamethasone, we examined whether dexamethasone affects NF-κB signaling in IL-1β-implanted corneas. We performed immunohistochemistry with Abs against CD11b and phosphorylated IκB-α, a key signaling molecule upstream of NF-κB. Surprisingly, IκB-α phosphorylation was observed mainly in stromal cells (94 ± 29.3 cells/field at ×200) but not in the CD11b+ cells (13 ± 4.69 cells/field) in IL-1β-implanted corneas on day 2 (Figure 4, A and B). On day 4, most (91.9 ± 6.7%, n = 8) of CD11b+ cells (38.8 ± 14.0 cells/field) as well as stromal cells (49.5 ± 10.9 cells/field) were positive for phosphorylated IκB-α (Figure 4, A and B). In dexamethasone-treated mice, we observed infiltration of CD11b+ cells in corneas both on day 2 and day 4. On day 2, phosphorylated IκB-α cells were not observed in dexamethasone-treated mice (Figure 4A). On day 4, some of CD11b+ cells were stained with Ab against phosphorylated IκB-α, whereas CD11b− stromal cells were negative for phosphorylated IκB-α (Figure 4, A and C). These results show that IL-1β induces IκB-α phosphorylation mainly in stromal cells but not infiltrated CD11b+ cells on day 2 (Figure 4B, P < 6 × 10−6, n = 6) and in both CD11b+ cells and stromal cells on day 4 (Figure 4C) and that dexamethasone inhibits NF-κB signaling predominantly in the CD11b− stromal cells (Figure 4C). To investigate the impact of dexamethasone on IL-1β-induced expression of various angiogenesis-related factors and its relation to NF-κB signaling, we cultured corneal stromal fibroblasts, MK/T1, and treated them with dexamethasone or control and measured in these cells the concentration of various angiogenic factors and NF-κB activity. Dexamethasone significantly inhibited IL-1β-induced production of VEGF-A, KC, and PGE2 by MK/T1 cells (P = 0.02, 0.008, and 0.04, respectively; n = 6) (Figure 5, A–C). To understand how dexamethasone modulates IL-1β-induced angiogenesis, we next examined the effect of dexamethasone on IL-1β-dependent NF-κB-p65 activity in MK/T1 cells. Dexamethasone significantly inhibited IL-1β-induced DNA-binding activity and nuclear localization of NF-κB-p65 in MK/T1 cells (P = 0.03, n = 6) (Figure 5D). Furthermore, Western blots using nuclear extracts from dexamethasone- and vehicle control-treated MK/T1 cells revealed that dexamethasone inhibited IL-1β-induced translocation of NF-κB-p65 into the nucleus (Figure 5E). To detect the signaling molecule that is targeted by dexamethasone, we performed immunoblot analysis of whole cell lysates with antibodies against NF-κB signaling molecules. Dexamethasone inhibited IL-1β-induced IκB-α degradation as well as NF-κB phosphorylation (Figure 5E). These data indicate that dexamethasone inhibits IL-1β-induced NF-κB signaling through blockade of IκB-α degradation in corneal stromal cells. To examine the role of NF-κB in the IL-1β-induced angiogenesis in the mouse cornea, we treated the animals with the specific NF-κB inhibitor peptide SN50 (n = 6) or the control peptide SN50M (n = 5) and quantified their corneal angiogenesis after IL-1β implantation. SN50 significantly reduced IL-1β-induced angiogenesis on day 6, whereas the animals treated with the control SN50M peptide showed regular levels of corneal angiogenesis (P = 0.002) (Figure 6, A and B). Dexamethasone potently suppresses the immunity and is commonly used in the treatment of a wide variety of immune and inflammatory diseases.20Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids—new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2238) Google Scholar IL-1β, an inflammatory cytokine, is up-regulated in various corneal diseases.14Fukuda M Mishima H Otori T Detection of interleukin-1β in the tear fluid of patients with corneal disease with or without conjunctival involvement.Jpn J Ophthalmol. 1997; 41: 63-66Crossref PubMed Scopus (30) Google Scholar, 15Staats HF Lausch RN Cytokine expression in vivo during murine herpetic stromal keratitis. Effect of protective antibody therapy.J Immunol. 1993; 151: 277-283PubMed Google Scholar, 16Biswas PS Banerjee K Kim B Rouse BT Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis.J Immunol. 2004; 172: 3736-3744PubMed Google Scholar, 17Biswas PS Banerjee K Kim B Kinchington PR Rouse BT Role of inflammatory cytokine-induced cyclooxygenase 2 in the ocular immunopathologic disease herpetic stromal keratitis.J Virol. 2005; 79: 10589-10600Crossref PubMed Scopus (31) Google Scholar Recently, we showed that IL-1β induces corneal neovascularization via induction of various angiogenesis-related factors including VEGF, CXC chemokines, and COX-2/prostanoids.18Kuwano T Nakao S Yamamoto H Tsuneyoshi M Yamamoto T Kuwano M Ono M Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis.FASEB J. 2004; 18: 300-310Crossref PubMed Scopus (248) Google Scholar, 19Nakao S Kuwano T Tsutsumi-Miyahara C Ueda S Kimura YN Hamano S Sonoda KH Saijo Y Nukiwa T Strieter RM Ishibashi T Kuwano M Ono M Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1β-induced neovascularization and tumor growth.J Clin Invest. 2005; 115: 2979-2991Crossref PubMed Scopus (243) Google Scholar In this study, we demonstrate that dexamethasone inhibits IL-1β-dependent corneal neovascularization partly through regulation of NF-κB signaling and inhibition of VEGF, CXC chemokines, and PGE2 production in corneal stromal fibroblast (Figure 7). Corticosteroids inhibit inflammation through various different pathways.20Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids—new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2238) Google Scholar For instance, corticosteroid-induced MAPK phosphatase 1 dephosphorylates and inactivates Jun N-terminal kinase, thereby inhibiting c-Jun-mediated transcription.20Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids—new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2238) Google Scholar Corticosteroid-glucocorticoid receptor complex also interacts with NF-κB to block its transcription activity.20Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids—new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2238) Google Scholar Recent work suggests that glucocorticoids also have rapid nongenomic effects on inflammation. Hafezi-Moghadam and colleagues27Hafezi-Moghadam A Simoncini T Yang Z Limbourg FP Plumier JC Rebsamen MC Hsieh CM Chui DS Thomas KL Prorock AJ Laubach VE Moskowitz MA French BA Ley K Liao JK Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase.Nat Med. 2002; 8: 473-479Crossref PubMed Scopus (474) Google Scholar reported that high-dose corticosteroids exert cardiovascular protection through nontranscriptional mechanisms involving rapid activation of endothelial nitric oxide synthase. In this study we examined the effect of dexamethasone on NF-κB signaling and demonstrated that dexamethasone mainly inhibited NF-κB activation. However, whether the angiostatic effects of dexamethasone involves nontranscriptional mechanisms remains to be investigated. We first demonstrated that IL-1β induces NF-κB signaling in the mouse cornea during neovascularization. In the corneal alkali burns model, NF-κB is activated in the corneal epithelial and stromal cells.21Saika S Miyamoto T Yamanaka O Kato T Ohnishi Y Flanders KC Ikeda K Nakajima Y Kao WW Sato M Muragaki Y Ooshima A Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-κB, in treatment of corneal alkali burns in mice.Am J Pathol. 2005; 166: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar In our corneal angiogenesis model, IκB-α was mainly phosphorylated in the corneal stromal cells, suggesting that NF-κB-activated corneal stromal fibroblasts play an important role in corneal inflammatory conditions, such as angiogenesis and wound healing. Dexamethasone is widely used in the treatment of corneal inflammation; however, steroid therapy in the management of some corneal diseases remains controversial because of its side effects.28Leibowitz HM Kupferman A Stewart RH Kimbrough RL Evaluation of dexamethasone acetate as a topical ophthalmic formulation.Am J Ophthalmol. 1978; 86: 418-423Abstract Full Text PDF PubMed Google Scholar, 29Raizman M Corticosteroid therapy of eye disease. Fifty years later.Arch Ophthalmol. 1996; 114: 1000-1001Crossref PubMed Scopus (82) Google Scholar Dexamethasone inhibits NF-κB but not AP-1 activity in transfected human corneal fibroblasts.30Lu Y Fukuda K Nakamura Y Kimura K Kumagai N Nishida T Inhibitory effect of triptolide on chemokine expression induced by proinflammatory cytokines in human corneal fibroblasts.Invest Ophthalmol Vis Sci. 2005; 46: 2346-2352Crossref PubMed Scopus (43) Google Scholar We show that IL-1β-induced NF-κB activation is inhibited by dexamethasone and that a selective NF-κB inhibitor diminishes inflammatory corneal angiogenesis. These findings indicate that NF-κB inhibition may be an effective therapeutic option for inflammatory corneal angiogenesis.31Olivier S Robe P Bours V Can NF-κB be a target for novel and efficient anti-cancer agents?.Biochem Pharmacol. 2006; 72: 1054-1068Crossref PubMed Scopus (116) Google Scholar, 32Miller JW Stinson WG Folkman J Regression of experimental iris neovascularization with systemic α-interferon.Ophthalmology. 1993; 100: 9-14Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 33Manna SK Mukhopadhyay A Aggarwal BB IFN-α suppresses activation of nuclear transcription factors NF-κB and activator protein 1 and potentiates TNF-induced apoptosis.J Immunol. 2000; 165: 4927-4934PubMed Google Scholar Further studies will be necessary to assess the safety and side effects for the treatment of human corneal diseases. CXC chemokines containing the ELR motif mediate angiogenesis through G protein-coupled receptor CXCR2 on endothelial cells.26Strieter RM Burdick MD Mestas J Gomperts B Keane MP Belperio JA Cancer CXC chemokine networks and tumour angiogenesis.Eur J Cancer. 2006; 42: 768-778Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 34Addison CL Daniel TO Burdick MD Liu H Ehlert JE Xue YY Buechi L Walz A Richmond A Strieter RM The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity.J Immunol. 2000; 165: 5269-5277PubMed Google Scholar Previously, we showed that CXCR2 blockade partially inhibits IL-1β-induced corneal angiogenesis.19Nakao S Kuwano T Tsutsumi-Miyahara C Ueda S Kimura YN Hamano S Sonoda KH Saijo Y Nukiwa T Strieter RM Ishibashi T Kuwano M Ono M Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1β-induced neovascularization and tumor growth.J Clin Invest. 2005; 115: 2979-2991Crossref PubMed Scopus (243) Google Scholar NF-κB is known to play an important role as a master switch in the transactivation of angiogenic CXC chemokines.26Strieter RM Burdick MD Mestas J Gomperts B Keane MP Belperio JA Cancer CXC chemokine networks and tumour angiogenesis.Eur J Cancer. 2006; 42: 768-778Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar Members of the CXC chemokines, such as KC and MIP-2, induce corneal angiogenesis34Addison CL Daniel TO Burdick MD Liu H Ehlert JE Xue YY Buechi L Walz A Richmond A Strieter RM The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity.J Immunol. 2000; 165: 5269-5277PubMed Google Scholar and have been implicated in the pathogenesis of inflammatory corneal diseases.30Lu Y Fukuda K Nakamura Y Kimura K Kumagai N Nishida T Inhibitory effect of triptolide on chemokine expression induced by proinflammatory cytokines in human corneal fibroblasts.Invest Ophthalmol Vis Sci. 2005; 46: 2346-2352Crossref PubMed Scopus (43) Google Scholar, 35Yamagami S Miyazaki D Ono SJ Dana MR Differential chemokine gene expression in corneal transplant rejection.Invest Ophthalmol Vis Sci. 1999; 40: 2892-2897PubMed Google Scholar We observed that dexamethasone inhibits IL-1β-induced NF-κB signaling and the level of KC protein but not that of MIP-2. These data suggest that the contribution of MIP-2 may be less than that of KC in IL-1β-induced corneal angiogenesis. A recent report shows that fibroblasts produce SDF-1 and promote tumor progression via SDF-1-CXCR4-mediated angiogenesis.36Orimo A Gupta PB Sgroi DC Arenzana-Seisdedos F Delaunay T Naeem R Carey VJ Richardson AL Weinberg RA Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.Cell. 2005; 121: 335-348Abstract Full Text Full Text PDF PubMed Scopus (2913) Google Scholar Another report demonstrated that human corneal fibroblasts express SDF-1 mRNA.37Bourcier T Berbar T Paquet S Rondeau N Thomas F Borderie V Laroche L Rostene W Haour F Lombet A Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts.Mol Vis. 2003; 9: 96-102PubMed Google Scholar We examined whether SDF-1 protein expression was increased in IL-1β-implanted corneas during neovascularization using ELISA. Unexpectedly, however, we did not detect SDF-1 overexpression in IL-1β-implanted corneas (data not shown), suggesting that SDF-1 may not be necessary in our IL-1β-induced corneal angiogenesis model. The IL-1 receptor, which binds both IL-1α and IL-1β, is constitutively expressed in corneal fibroblasts.38Wilson SE Lloyd SA He YG Glucocorticoid receptor and interleukin-1 receptor messenger RNA expression in corneal cells.Cornea. 1994; 13: 4-8Crossref PubMed Scopus (28) Google Scholar After corneal injury, IL-1 protein is detectable in corneal fibroblasts. IL-1 up-regulates the expression of various cytokines or enzymes by corneal fibroblasts,8Wilson SE Mohan RR Mohan RR Ambrosio Jr, R Hong J Lee J The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells.Prog Retin Eye Res. 2001; 20: 625-637Crossref PubMed Scopus (510) Google Scholar, 22Lu Y Fukuda K Liu Y Kumagai N Nishida T Dexamethasone inhibition of IL-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.Invest Ophthalmol Vis Sci. 2004; 45: 2998-3004Crossref PubMed Scopus (46) Google Scholar, 39Hong JW Liu JJ Lee JS Mohan RR Mohan RR Woods DJ He YG Wilson SE Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea.Invest Ophthalmol Vis Sci. 2001; 42: 2795-2803PubMed Google Scholar and it also contributes to wound healing. In our model, IL-1β binds to the IL-1 receptor on corneal fibroblasts, up-regulates various angiogenesis-related factors, and induces corneal neovascularization. Cancer cells are known to alter their adjacent stroma to form a permissive and supportive microenvironment by producing various growth factors and cytokines.40Mueller MM Fusenig NE Friends or foes—bipolar effects of the tumour stroma in cancer.Nat Rev Cancer. 2004; 4: 839-849Crossref PubMed Scopus (1469) Google Scholar Recent studies reported that experimentally induced genetic alterations in stromal fibroblasts cause epithelial neoplasia and invasive carcinoma.36Orimo A Gupta PB Sgroi DC Arenzana-Seisdedos F Delaunay T Naeem R Carey VJ Richardson AL Weinberg RA Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.Cell. 2005; 121: 335-348Abstract Full Text Full Text PDF PubMed Scopus (2913) Google Scholar, 41Olumi AF Grossfeld GD Hayward SW Carroll PR Tlsty TD Cunha GR Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium.Cancer Res. 1999; 59: 5002-5011PubMed Google Scholar Various angiogenesis-related factors, including CXC chemokines and growth factors, are produced in IL-1β-treated corneal stromal fibroblasts. Activation of stromal fibroblasts by IL-1β plays a central role in tumor angiogenesis as well as in corneal angiogenesis.42Saijo Y Tanaka M Miki M Usui K Suzuki T Maemondo M Hong X Tazawa R Kikuchi T Matsushima K Nukiwa T Proinflammatory cytokine IL-1β promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction.J Immunol. 2002; 169: 469-475PubMed Google Scholar To date, steroid therapy has been the standard anti-inflammatory and angiostatic treatment in the cornea.28Leibowitz HM Kupferman A Stewart RH Kimbrough RL Evaluation of dexamethasone acetate as a topical ophthalmic formulation.Am J Ophthalmol. 1978; 86: 418-423Abstract Full Text PDF PubMed Google Scholar, 29Raizman M Corticosteroid therapy of eye disease. Fifty years later.Arch Ophthalmol. 1996; 114: 1000-1001Crossref PubMed Scopus (82) Google Scholar Our results suggest that specific molecular or cellular targeting strategies, such as blockade of NF-κB or regulation of the activation status of corneal stromal cells, may offer novel approaches in the treatment of inflammatory angiogenesis in the cornea. We thank K. Watari, T. Furuta, K. Kano, M. Takahara, H. Fujii (Kyushu University) and N. Lara-Castillo (Harvard University) for technical support; and K.L. Thomas (Harvard University) for editorial support.
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