Activin A in the Regulation of Corneal Neovascularization and Vascular Endothelial Growth Factor Expression
2004; Elsevier BV; Volume: 164; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63216-6
ISSN1525-2191
AutoresVassiliki Poulaki, Nicholas Mitsiades, Friedrich E. Kruse, Sven Radetzky, Eirini Iliaki, Bernd Kirchhof, Antonia M. Joussen,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoActivin A, a dimeric glycoprotein that belongs to the transforming growth factor-β superfamily, governs cellular differentiation in a wide variety of models and has been implicated in the regulation of angiogenesis. We examined the role of activin A and its downstream signaling pathway in a murine model of inflammatory corneal neovascularization induced by mechanical injury (debridement), and in vitro in corneal epithelial cells. Activin A expression increased steadily from day 2 until day 8 after mechanical debridement in vivo, paralleling vascular endothelial growth factor (VEGF) expression. Administration of recombinant activin A in mice increased the area of neovascularization, VEGF expression, and the kinase activities of p38 and p42/44 MAPKs after mechanical debridement. Systemic inhibition of activin A in vivo with a neutralizing antibody reduced the area of neovascularization, VEGF expression, and p38 and p42/44 MAPK activity, whereas administration of an isotype-matched control antibody had no effect. In vitro treatment with activin A increased VEGF secretion, as well as p38 and p42/44 MAPK activity in corneal epithelial cells, whereas concurrent administration of specific inhibitors of p38 or p42/44 MAPK abolished the stimulatory effect of activin A on VEGF production. We conclude that activin A stimulates inflammatory corneal angiogenesis by increasing VEGF levels through a p38 and p42/44 MAPK-dependent mechanism. Activin A, a dimeric glycoprotein that belongs to the transforming growth factor-β superfamily, governs cellular differentiation in a wide variety of models and has been implicated in the regulation of angiogenesis. We examined the role of activin A and its downstream signaling pathway in a murine model of inflammatory corneal neovascularization induced by mechanical injury (debridement), and in vitro in corneal epithelial cells. Activin A expression increased steadily from day 2 until day 8 after mechanical debridement in vivo, paralleling vascular endothelial growth factor (VEGF) expression. Administration of recombinant activin A in mice increased the area of neovascularization, VEGF expression, and the kinase activities of p38 and p42/44 MAPKs after mechanical debridement. Systemic inhibition of activin A in vivo with a neutralizing antibody reduced the area of neovascularization, VEGF expression, and p38 and p42/44 MAPK activity, whereas administration of an isotype-matched control antibody had no effect. In vitro treatment with activin A increased VEGF secretion, as well as p38 and p42/44 MAPK activity in corneal epithelial cells, whereas concurrent administration of specific inhibitors of p38 or p42/44 MAPK abolished the stimulatory effect of activin A on VEGF production. We conclude that activin A stimulates inflammatory corneal angiogenesis by increasing VEGF levels through a p38 and p42/44 MAPK-dependent mechanism. Corneal neovascularization is a disabling condition that results in loss of the immunological privilege of the cornea and ultimately in visual impairment. It is a common manifestation of inflammatory, infectious, and traumatic diseases of the cornea and the limbal stem cell barrier. Although laser treatment and surgical intervention offer potential therapeutic options, corneal neovascularization still remains a therapeutic puzzle because, in many occasions, cornea avascularity and transparency are not restored. The potential benefits of controlling angiogenesis have been demonstrated in experimental models for various untreatable ocular conditions that involve neovascularization.1Danesi R Agen C Benelli U Paolo AD Nardini D Bocci G Basolo F Campagni A Tacca MD Inhibition of experimental angiogenesis by the somatostatin analogue octreotide acetate (SMS 201-995).Clin Cancer Res. 1997; 3: 265-272PubMed Google Scholar, 2Fotsis T Pepper M Adlercreutz H Fleischmann G Hase T Montesano R Schweigerer L Genistein, a dietary-derived inhibitor of in vitro angiogenesis.Proc Natl Acad Sci USA. 1993; 90: 2690-2694Crossref PubMed Scopus (736) Google Scholar, 3Joussen AM Kruse FE Volcker HE Kirchhof B Topical application of methotrexate for inhibition of corneal angiogenesis.Graefes Arch Clin Exp Ophthalmol. 1999; 237: 920-927Crossref PubMed Scopus (76) Google Scholar These studies have highlighted the pivotal role of vascular endothelial growth factor (VEGF) in regulating endothelial cell growth and neovascularization. VEGF refers to a family of angiogenic and vascular permeability-enhancing peptides derived from alternatively spliced mRNAs.4Tischer E Mitchell R Hartman T Silva M Gospodarowicz D Fiddes JC Abraham JA The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.J Biol Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar VEGF bioactivity is primarily mediated via two high-affinity cognate receptors, KDR/Flk-1 and Flt-1.5Senger DR Galli SJ Dvorak AM Perruzzi CA Harvey VS Dvorak HF Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science. 1983; 219: 983-985Crossref PubMed Scopus (3443) Google Scholar, 6Ferrara N Houck K Jakeman L Leung DW Molecular and biological properties of the vascular endothelial growth factor family of proteins.Endocr Rev. 1992; 13: 18-32Crossref PubMed Scopus (1567) Google Scholar Recently, it was suggested that VEGF plays an important role in corneal neovascularization because exogenous VEGF stimulates this process7Moromizato Y Stechschulte S Miyamoto K Murata T Tsujikawa A Joussen AM Adamis AP CD18 and ICAM-1-dependent corneal neovascularization and inflammation after limbal injury.Am J Pathol. 2000; 157: 1277-1281Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar and a neutralizing anti-VEGF antibody inhibits it.8Amano S Rohan R Kuroki M Tolentino M Adamis AP Requirement for vascular endothelial growth factor in wound- and inflammation-related corneal neovascularization.Invest Ophthalmol Vis Sci. 1998; 39: 18-22PubMed Google Scholar We have previously reported that VEGF expression is up-regulated in scraped corneas during the course of corneal neovascularization. Understanding the molecular mechanisms regulating this forced expression will help identify potential therapeutic candidates for the treatment or even prevention of corneal neovascularization. Activins represent a distinct group of the transforming growth factor (TGF)-β superfamily, that comprise one α and three β chains (βA, βB, and βC).9Vale W Rivier C Hsueh A Campen C Meunier H Bicsak T Vaughan J Corrigan A Bardin W Sawchenko P et al.Chemical and biological characterization of the inhibin family of protein hormones.Recent Prog Horm Res. 1988; 44: 1-34PubMed Google Scholar, 10Mather JP Moore A Li RH Activins, inhibins, and follistatins: further thoughts on a growing family of regulators.Proc Soc Exp Biol Med. 1997; 215: 209-222Crossref PubMed Scopus (248) Google Scholar The bioactive molecule activin A consists of two monomeric βA chains linked by disulfide bonds.10Mather JP Moore A Li RH Activins, inhibins, and follistatins: further thoughts on a growing family of regulators.Proc Soc Exp Biol Med. 1997; 215: 209-222Crossref PubMed Scopus (248) Google Scholar The biological effects of activins are mediated via signaling through two families (type I and II) of transmembranous serine-threonine kinase receptors.11Attisano L Wrana JL Cheifetz S Massague J Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors.Cell. 1992; 68: 97-108Abstract Full Text PDF PubMed Scopus (459) Google Scholar, 12Massague J Andres J Attisano L Cheifetz S Lopez-Casillas F Ohtsuki M Wrana JL TGF-beta receptors.Mol Reprod Dev. 1992; 32: 99-104Crossref PubMed Scopus (110) Google Scholar, 13Attisano L Wrana JL Montalvo E Massague J Activation of signalling by the activin receptor complex.Mol Cell Biol. 1996; 16: 1066-1073Crossref PubMed Scopus (289) Google Scholar, 14Mathews LS Vale WW Expression cloning of an activin receptor, a predicted transmembrane serine kinase.Cell. 1991; 65: 973-982Abstract Full Text PDF PubMed Scopus (681) Google Scholar, 15ten Dijke P Yamashita H Ichijo H Franzen P Laiho M Miyazono K Heldin CH Characterization of type I receptors for transforming growth factor-beta and activin.Science. 1994; 264: 101-104Crossref PubMed Scopus (512) Google Scholar After ligand binding, a heterodimeric complex is formed by a type I and a type II receptor that initiates phosphorylation of the type I receptor and activation of downstream signaling cascades involving the Sma and Mad (mothers against decapentaplegic)-related protein (Smad).16Kretzschmar M Massague J SMADs: mediators and regulators of TGF-beta signaling.Curr Opin Genet Dev. 1998; 8: 103-111Crossref PubMed Scopus (434) Google Scholar One of the most important functions of activin A is the regulation of cell differentiation. Activin A controls several aspects of hematopoiesis17Eto Y Tsuji T Takezawa M Takano S Yokogawa Y Shibai H Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1.Biochem Biophys Res Commun. 1987; 142: 1095-1103Crossref PubMed Scopus (382) Google Scholar, 18Murata M Onomichi K Eto Y Shibai H Muramatsu M Expression of erythroid differentiation factor (EDF) in Chinese hamster ovary cells.Biochem Biophys Res Commun. 1988; 151: 230-235Crossref PubMed Scopus (43) Google Scholar, 19Shiozaki M Kosaka M Eto Y Activin A: a commitment factor in erythroid differentiation.Biochem Biophys Res Commun. 1998; 242: 631-635Crossref PubMed Scopus (33) Google Scholar and regulates cell differentiation in the ovary, placenta, prostate, and testis.10Mather JP Moore A Li RH Activins, inhibins, and follistatins: further thoughts on a growing family of regulators.Proc Soc Exp Biol Med. 1997; 215: 209-222Crossref PubMed Scopus (248) Google Scholar, 20Mather JP Woodruff TK Krummen LA Paracrine regulation of reproductive function by inhibin and activin.Proc Soc Exp Biol Med. 1992; 201: 1-15Crossref PubMed Scopus (164) Google Scholar During embryogenesis, it is instrumental for axis development and organogenesis in a variety of species.21Gurdon JB Harger P Mitchell A Lemaire P Activin signalling and response to a morphogen gradient.Nature. 1994; 371: 487-492Crossref PubMed Scopus (306) Google Scholar In adults, activins function as hormone-like feedback regulators in the reproductive system.22Vale W Rivier J Vaughan J McClintock R Corrigan A Woo W Karr D Spiess J Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid.Nature. 1986; 321: 776-779Crossref PubMed Scopus (1067) Google Scholar Furthermore, the presence of activin has been related to wound healing. In the eye, members of the activin family have been discovered in retinal pigment epithelium.23Jaffe GJ Harrison CE Lui GM Roberts WL Goldsmith PC Mesiano S Jaffe RB Activin expression by cultured human retinal pigment epithelial cells.Invest Ophthalmol Vis Sci. 1994; 35: 2924-2931PubMed Google Scholar Activin A and its receptors were recently described in preretinal membranes from eyes with ischemic and nonischemic vitreoretinal proliferative diseases, a result that agrees with a role of activin A in neovascularization.24Yamamoto T Takeuchi S Suzuki K Yamashita H Expression and possible roles of activin A in proliferative vitreoretinal diseases.Jpn J Ophthalmol. 2000; 44: 221-226Crossref PubMed Scopus (9) Google Scholar We have recently found that VEGF and activin A expression is co-ordinated in fibrovascular membranes from patients with age-related macular degeneration (manuscript submitted). Because we have also shown that genes encoding for the βA chain as well as several activin receptors are transcribed in the cornea,25You L Kruse FE Differential effect of activin A and BMP-7 on myofibroblast differentiation and the role of the Smad signaling pathway.Invest Ophthalmol Vis Sci. 2002; 43: 72-81PubMed Google Scholar we investigated the role of activin A in corneal angiogenesis and the regulation of VEGF expression. Human corneal epithelial cells (passage 2; Cascade Biologics, Portland, OR) were maintained in tissue culture media according to the manufacturer's instructions. Cells were plated into six-well plastic dishes and used for experiments when they reached 80% confluence. Fresh serum-free media were placed on the cells 12 hours before experiments. The p38 kinase inhibitor SB 202190 or the MAPK inhibitor PD 098059 (Calbiochem, La Jolla, CA) or vehicle (dimethyl sulfoxide), were added to the cells at a concentration of 50 μmol/L, followed 1 hour later by activin A (100 nmol/L) for an additional 12 hours. Each experimental condition was prepared in triplicate, and the experiments were performed at least three times with reproducible results. Representative experiments are shown in the figures. C57 BL/6 mice, weighting 20 to 25 g were purchased from Jackson Laboratories (Bar Harbor, ME). All animal experiments followed the guidelines of the Association for Research in Vision and Ophthalmology and were approved by the Animal Care and Use Committee of Cologne, Germany. All surgical procedures were performed under general anesthesia [xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (35 mg/kg) i.m.]. To monitor systemic side effects of the treatment, body weight and temperature were measured on every observation day. Animals were kept in groups of 10 and fed regular lab chow and water ad libitum. A 12-hour day and night cycle was maintained. Under intramuscular general anesthesia with xylazine (10 mg/kg; Bayer, Leverkusen, Germany) and ketamine hydrochloride (150 mg/kg; Phoenix, MO) and additional topical application of lidocaine (Alcon, CA), inflammatory neovascularization was induced by application of 2 μl of 0.15 mmol/L NaOH to the central cornea of each mouse. The mice were randomly divided into three groups that received treatment with vehicle, systemic treatment with a neutralizing polyclonal antibody against activin A, an isotype-matched control antibody, or with recombinant activin A (R&D Systems, Minneapolis, MN). Each group consisted of 10 animals unless otherwise specified (20 corneas per group in total). The corneal epithelium was subsequently scraped off with a blunt von Graefe's knife. The limbal areas were gently massaged over 360° for 3 minutes. To prevent infection, eyes were treated with antibiotic ointment (neomycin sulfate, 3.5 I.E./mg; bacitracin, 0.3 I.E./mg; and polymyxine B sulfate, 7.5 I.E./mg, Polyspectran; Alcon, Germany) after surgery. Each set of experiments was repeated three times. For visualization of endothelial cells and pericytes, immunostaining for CD31 was performed. Corneas were carefully dissected and rinsed in phosphate-buffered saline (PBS). To allow penetration of the antibodies and flattening of the tissue, the corneal epithelium and endothelium were whipped off and four peripheral incisions were made. Corneas were then fixed in ice-cold acetone for 20 minutes and after subsequent washes in PBS transferred to the antibody solution and incubated overnight at 4°C. Phycoerythrin-coupled anti-mouse CD31 (BD Biosciences, Franklin Lakes, NJ) was used in a dilution of 1:500. After further washing on PBS, corneas were mounted with anti-fading agent and analyzed by fluorescence microscopy. Images of the CD31-stained corneas were captured using a CD-330 charge-coupled device camera (Dage-MIT; Improvision, Inc., Heidelberg, Germany) attached to a Zeiss microscope (Zeiss, Oberkochem, Germany). The images were captured on an Apple G4 Computer (Apple, Cupertino, CA) and analyzed using Openlab software (Improvision Inc.). The images were resolved at 624 × 480 pixels and converted to tagged information file format (.tiff) files. The neovascularization was quantified by setting a threshold level of fluorescence, above which only vessels were captured. The entire mounted cornea was analyzed to minimize sampling bias. The total corneal surface was outlined using the innermost vessel of the limbal arcade as the border. The total vascularized area was then normalized to the total corneal area and the percentage of the cornea covered by vessels was calculated. All quantifications and calculations were performed in a masked manner. The delivery of the drug with osmotic pumps, instead of simple intraperitoneal injections, is necessary to achieve steady levels of each drug in the circulation, avoid peak levels caused by every day injections, and limit the chance of toxicities. One week after the scraping of their corneas, the mice were deeply anesthetized, and osmotic pumps (Alzet, Salt Lake City, UT) containing either the vehicle (PBS), or 20 μg of a neutralizing rat/human/mouse polyclonal antibody against activin A (R&D Systems, Minneapolis, MN), or 20 μg of isotype-matched control antibody (R&D Systems), or 250 μg of recombinant activin A (R&D Systems), each diluted in 200 μl of PBS, were inserted into the peritoneal space. In detail, the abdominal skin was shaved, scrubbed with betadine, and wiped with alcohol. A small incision of 15-mm in length was made within the midline, through the skin and muscles, to enter the peritoneal cavity. Then the pumps were inserted into the peritoneal space floating freely without attachment to a certain structure. Each pump was 1.3 cm long and 6 mm in diameter. The wound was closed with separate suturing of the muscle layer and the skin. Mice were scraped as described above and treated with recombinant activin A, or the neutralizing antibody against activin A (seven animals in each group), or the isotype-matched control antibody, or vehicle (eight animals), and sacrificed on days 2, 4, or 8 after treatment. Corneas were dissected and placed in 60 μl of lysis buffer (20 mmol/L imidazole HCl, 10 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L EGTA, 1% Triton, 10 mmol/L NaF, 1 mmol/L Na molybdate, 1 mmol/L EDTA, pH 6.8) supplemented with a protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN) followed by mechanical homogenization. The lysate was cleared of debris by centrifugation at 14,000 rpm for 15 minutes (4°C), and the supernatant was collected. Total protein was determined using a commercial assay (bicinchoninic acid kit; Bio-Rad, Hercules, CA). VEGF and activin A levels were determined using enzyme-linked immunosorbent assay according to the manufacturer's instructions (R&D Systems) and normalized to total protein. P38 MAP kinase activity was analyzed in whole retinal tissue using a commercially available enzyme-linked immunosorbent assay based method (Assay Designs, Inc.). Briefly, retinal tissue was homogenized in lysis buffer containing 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L Na3O4, 1 μg/ml leupeptin, 1 mmol/L phenylmethyl sulfonyl fluoride. The lysates were cleared by centrifugation and protein was quantified with the bicinchoninic acid assay (Biorad). Corneal lysates, or recombinant phopsho-p38 MAPK standards (provided by the manufacturer), were subsequently incubated with a monoclonal antibody against the phosphorylated (activated) form of p38 MAPK (Assay Designs), immobilized on a microtiter 96-well plate for 1 hour at room temperature on a plate shaker at 500 rpm. After washes with a Tris-buffered saline-based solution (provided by the manufacturer), the plate was incubated with a rabbit polyclonal antibody against phopsho-p38 for 1 hour at room temperature on a plate shaker at 500 rpm. This antibody binds to the phopsho-p38 bound on the plate. After the incubation, the excess antibody was removed with repetitive washes with the Tris-buffered saline-based solution and the plate was incubated with a donkey anti-rabbit IgG conjugated with horseradish peroxidase that binds to the polyclonal phospho-p38 antibody. After a short incubation for 1 hour at room temperature as above, and subsequent washes, the peroxidase reaction was developed and measured at 450 nmol/L with a reference wavelength at 570 nmol/L. The measured optical density is directly proportional to the concentration of phospho-p38 in either the standards or the samples. A standard curve was plotted for the standards and the concentration of phopsho-p38 of each of the samples was determined by interpolation. P42/44 MAP kinase activity was analyzed in whole retinal tissue using a commercially available enzyme-linked immunosorbent assay based method (Assay Designs, Inc.). Briefly, retinal tissue was homogenized in lysis buffer containing 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L Na3O4, 1 μg/ml leupeptin, 1 mmol/L phenylmethyl sulfonyl fluoride. The lysates were cleared by centrifugation and protein was quantified with the bicinchoninic acid assay (Biorad). Corneal lysates, or recombinant phopsho-p42/44 MAPK standards (provided by the manufacturer), were subsequently incubated with a monoclonal antibody against the phosphorylated (activated) form of p42/44 MAPK (Assay Designs), immobilized on a microtiter 96-well plate, for 1 hour at room temperature on a plate shaker at 500 rpm. After washes with a Tris-buffered saline-based solution (provided by the manufacturer), the plate was incubated with a rabbit polyclonal antibody against phopsho-p42/44 for 1 hour at room temperature on a plate shaker at 500 rpm. This antibody binds to the phopsho-p42/44 bound on the plate. After the incubation, the excess antibody was removed with repetitive washes with the Tris-buffered saline-based solution and the plate was incubated with a goat anti-rabbit IgG conjugated with horseradish peroxidase that binds to the polyclonal phospho-p42/44 antibody. After a short incubation for 1 hour at room temperature as above, and subsequent washes, the peroxidase reaction was developed and measured at 450 nmol/L with a reference wavelength at 570 nmol/L. The measured optical density is directly proportional to the concentration of phospho-p42/44 in either the standards or the samples. A standard curve was plotted for the standards and the concentration of phopsho-p42/44 of each of the samples was determined by interpolation. To analyze the differences between treated and control eyes, as well as within the treatment groups, an unpaired t-test with two-tailed P value or analysis of variance (for multiple comparisons) was used. Results are presented as mean ± SD. We have previously shown that activin A expression correlates with VEGF expression in fibrovascular epiretinal membranes from patients with age-related macular degeneration (AM Jaussen and V Poulaki, submitted). To investigate the role of activin A in the regulation of VEGF expression, we used corneal epithelial cells in vitro. Treatment of corneal epithelial cells with activin A up-regulated VEGF levels (1.58 ± 0.099 versus 0.62 ± 0.046 pg/mg of total protein in vehicle-treated cells, P < 0.001; Figure 1). As we have previously shown, mitogen-associated kinases such as p38 and p42/44MAPK can regulate VEGF expression.26Poulaki V Qin W Joussen AM Hurlbut P Wiegand SJ Rudge J Yancopoulos GD Adamis AP Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1alpha and VEGF.J Clin Invest. 2002; 109: 805-815Crossref PubMed Scopus (244) Google Scholar Therefore, we investigated the role of the above kinases in the activin-induced VEGF up-regulation. P38 and p42/44 MAPK inhibition reduced activin-induced VEGF up-regulation in corneal epithelial cells (0.803 ± 0.104 and 0.97 ± 0.12 pg/mg of total protein, respectively, versus 0.158 ± 0.099 pg/mg of total protein for cells treated with activin alone, P < 0.001; Figure 1). Activin A stimulated both p38 (22.5 ± 7.2 in activin A treated versus 8.26 ± 2.65 in vehicle-treated cells) and p42/44 activity (29.5 ± 6.89 in activin A treated versus 3.58 ± 0.68 in control-treated cells, P < 0.001 in both cases; Figure 2).Figure 2Activin A up-regulates both p38 and p42/44 MAPK activity in corneal epithelial cells. Cells were treated with activin A and p38 (A) and p42/44 (B) MAPK activity was measured as described in the Materials and Methods section. Bars represent the 450-nm reading that corresponds to the p38 or p42/44 MAPK activity (mean ± SD). Treatment with activin A up-regulated both p38 and p42/44 MAPK activity.View Large Image Figure ViewerDownload (PPT) We have previously shown that VEGF levels increase during the course of neovascularization in the cornea scrape model. Because recombinant activin A increases production of VEGF in our in vitro model, we investigated whether systemic activin A levels increase during the course of neovascularization in vivo. In agreement with our previous findings, VEGF levels increased from 3.36 ± 0.74 pg/mg of total protein on day 0, to 9.05 ± 0.92 pg/mg of total protein on day 2 (P < 0.005), to 12.162 ± 0.93 pg/mg of total protein on day 4 (P < 0.005), to 14.34 ± 0.65 pg/mg of total protein on day 8 (P < 0.005) after the scraping (Figure 3A). Activin A levels paralleled the increases in VEGF levels from 0.13 ± 0.01 pg/mg of total protein on day 0, to 0.25 ± 0.01 μg/mg of total protein on day 2 (P < 0.001), to 0.33 ± 0.02 μg/mg of total protein on day 4 (P < 0.005), to 0.37 ± 0.01 μg/mg of total protein on day 8 (P < 0.001) (Figure 3B). Administration of recombinant activin A increased VEGF levels (13.93 ± 2.46 versus 7.74 ± 1.39 pg/mg of total protein on day 2, P < 0.001, 15.36 ± 1.62 versus 12.67 ± 1.45 pg/mg of total protein on day 4, P < 0.05 and 16.31 ± 1.22 versus 14.44 ± 1.21 pg/mg of total protein on day 8, P < 0.02 in the animals treated with the recombinant activin A, versus the vehicle, respectively) in the cornea scrape model. Inhibition of endogenous activin A via the administration of a neutralizing antibody against activin A reduced VEGF levels (5.31 ± 0.92 versus 8.03 ± 1.99 pg/mg of total protein on day 2, P < 0.001; 7.2 ± 2.4 versus 12.87 ± 1.86 pg/mg of total protein, P < 0.05 on day 4; and 8.24 ± 0.67 versus 13.7 ± 1.37 pg/mg of total protein on day 8, P < 0.01) (Figure 4 in the animals treated with the neutralizing antibody against activin versus the isotype-matched control antibody, respectively). In agreement with our previous report,27Joussen AM Beecken WD Moromizato Y Schwartz A Kirchhof B Poulaki V Inhibition of inflammatory corneal angiogenesis by TNP-470.Invest Ophthalmol Vis Sci. 2001; 42: 2510-2516PubMed Google Scholar the percentage of vascularized corneal surface increased on day 12 after the scraping to 32.16 ± 6.79%, whereas unscraped corneas are not vascularized (therefore the percentage of vascularized corneal area is ∼0). Administration of recombinant activin A significantly enhanced neovascularization (increase to 50.22 ± 4.76%, versus 32.16 ± 6.79% in the vehicle-treated mice P < 0.0001) (Figure 5). Administration of a neutralizing antibody against activin A and not of an isotype-matched control antibody, suppressed the increase of neovascularization in the scrape murine model (increase by only 18.62 ± 9.47%, versus 35.5 ± 6.78% in the isotype-matched control antibody-treated animals; P < 0.000001) (Figure 5). We have previously demonstrated that p42/44 and p38 MAPK regulate VEGF expression during the course of diabetes.26Poulaki V Qin W Joussen AM Hurlbut P Wiegand SJ Rudge J Yancopoulos GD Adamis AP Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1alpha and VEGF.J Clin Invest. 2002; 109: 805-815Crossref PubMed Scopus (244) Google Scholar To investigate the role of the MAPK signaling pathway during corneal neovascularization and the effect of activin A on their enzymatic activity, we measured the activity of p38 and p42/44. P42/44 activity increased during neovascularization (22.96 ± 4.25 μg/mg of total protein on day 8 versus 7.53 ± 3.3 μg/mg of total protein on day 0, P < 0.005; Figure 5A), as did p38 MAPK activity (83.6 ± 13.86 μg/mg of total protein on day 8 versus 41.7 ± 4.87 on day 0, P < 0.005; Figure 5B). Administration of recombinant activin A significantly increased both p42/44 MAPK activity (32.5 ± 4.8 μg/mg of total protein on day 7 in mice treated with recombinant activin A versus 22.96 ± 4.25 μg/mg of total protein, in the vehicle-treated mice, P < 0.001; Figure 5A) and p38 MAPK activity (140.2 ± 14.12 μg/mg of total protein on day 8 in mice treated with recombinant activin versus 83.6 ± 13.86 μg/mg of total protein in the vehicle-treated mice, P < 0.01; Figure 6B). Administration of activin A-neutralizing antibody and not the isotype-matched control antibody, reduced both p42/44 MAPK (11.22 ± 2.35 μg/mg of total protein on day 8 in mice treated with the neutralizing anti-activin A antibody versus 26.27 ± 5.38 in the isotype-matched control antibody-treated mice, P < 0.001, Figure 5A) and p38 MAPK activity (53.94 ± 6.43 μg/mg of total protein on day 8 in mice treated with the neutralizing anti-activin A antibody versus 98.06 ± 15.35 μg/mg of total protein isotype-matched control antibody-treated mice, P < 0.005; Figure 5B). Corneal neovascularization is a sight-threatening complication of severe insults to the cornea, such as chemical burns and corneal infections. It is characterized by an ingrowth of neovessels originating from the limbus and is often accompanied by an inflammatory response. VEGF is an important factor in this process because it stimulates corneal neovascularization7Moromizato Y Stechschulte S Miyamoto K Murata T Tsujikawa A Joussen AM Adamis AP CD18 and ICAM-1-dependent corneal neovascularization and inflammation after limbal injur
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