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Role of Periostin and Interleukin-4 in Recurrence of Pterygia

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Role of Periostin and Interleukin-4 in Recurrence of Pterygia Cornea Role of Periostin and Interleukin-4 in Recurrence of Pterygia Chuan-Hui Kuo,1 Dai Miyazaki,1 Keiko Yakura,1 Kaoru Araki-Sasaki,2 and Yoshitsugu Inoue1 PURPOSE. To identify the candidate genes for pterygia recur- mitomycin treatment, radiation, and amniotic membrane trans- rence from a pterygia transcriptome and to analyze their tran- plantation, recurrences still occur.1 scriptional regulation and functional relationships. We have determined the minimum gene set that would ETHODS accurately differentiate primary from recurrent pterygia using a M . Transcriptional networks for pterygia recurrence 2 were constructed using network analysis that was applied to mathematical classification algorithm. We found that only 184 genes that showed a significant twofold change in the three genes—periostin, tissue inhibitor of metalloproteinases-2 (TIMP-2), and l-3-phosphoserine phosphatase homolog whole genome. Of the identified recurrence-related candidate 2 genes in the major networks, periostin and IL-4 were analyzed (PSPHL)—differentiate primary from recurrent pterygia. We have also reported on the numerous transcripts that are for transcriptional relationships using pterygia-derived fibro- 2 blasts. Immunohistochemical analysis was used to study ptery- related to pterygia recurrence. However, the functional in- gia tissue. Effector candidate molecule for recurrence periostin volvement in the genome or their significance in recurrence was analyzed for cell adhesive function. remains speculative. We have used knowledge-based analysis to understand the global interactions of the genes in the con- RESULTS. The pterygia transcriptome was divided into four text of their function and regulation. To simplify the complex major biological networks with high significance scores (P Ͻ Ϫ17 biological phenomena played by genes, network analysis has 10 ). The classifier with the highest accuracy using the been reported to be a very efficient method.3 We have applied support vector machine algorithm was periostin, which was the Ingenuity Pathways Knowledge Base (Ingenuity Systems, successfully linked to the network of cell cycle, connective Redwood City, CA, http://www.ingenuity.com), a biological tissue development and function, and cell morphology. Analy- network database created from known protein-protein interac- ses using pterygia-derived fibroblasts showed that periostin tions and the activation of signaling pathways, and found that was required for cell adhesion that was mediated by a pre- the complex phenomena of pterygia recurrences can be simply sumed pterygia-related extracellular matrix protein, fibronec- summarized into four highly significant molecular networks. In tin. Periostin was found to be transcriptionally induced by IL-4. this analyses, periostin and TIMP-2, which we reported as The IL-4-stimulated periostin induction was suppressed by significant classifier genes in the support vector machine MAP kinase/ERK kinase 1 inhibitor, indicating an involvement (SVM)-based classification algorithm,2 were statistically linked of the MAP kinase pathway. Pathologically, IL-4 was transcrip- to the generated networks. Previously, TIMP-2 was shown to tionally elevated in recurrent pterygia tissue and was localized be involved in tissue remodeling processes of pterygia.1 Al- to perivascular tissues and endothelial cells in the stroma of the though periostin showed the highest classification accuracies subconjunctiva of pterygia. of primary and recurrent pterygia,2 its functional characteris- CONCLUSIONS. Periostin is induced by IL-4 and is involved in the tics are not yet well understood, and its involvement in the fibronectin-mediated pterygia fibroblast adhesion. These find- pathogenesis of pterygia has not been reported. ings indicate that periostin probably promotes the recurrence Thus, the purpose of this study was to determine the mo- of pterygia. (Invest Ophthalmol Vis Sci. 2010;51:139–143) lecular function of periostin in pterygia pathology and the DOI:10.1167/iovs.09-4022 causative transcriptional event for periostin induction based on the generated networks. We shall show the function of peri- pterygium is an excessive fibrovascular proliferation of ostin in the recurrence of pterygia and relate it to the role of Adegenerated bulbar conjunctival tissue on the ocular sur- IL-4 as its inductive signal. face. At present, surgery is the only method for treating ptery- gia, and recurrences are a major complication of the surgical treatment of pterygia. Although numerous approaches have been attempted to reduce recurrences, such as adjunctive MATERIALS AND METHODS Patient Backgrounds and Specimen Collection From the 1Division of Ophthalmology and Visual Science, Tottori Pterygia heads were collected from 21 eyes of 21 Asian patients University Faculty of Medicine, Tottori, Japan; and 2Ideta Eye Hospital, undergoing pterygium excision with conjunctival autografting at the Kumamoto, Japan. Tottori University Hospital (Yonago, Japan) and the Ideta Eye Hospital Supported by Grant-in-Aid for Scientific Research (C) 21592258 (Kumamoto, Japan). Of these, 11 eyes of 11 patients showed primary from the Ministry of Education, Culture, Sports, Science and Technol- pterygia and 10 eyes of 10 patients showed recurrent pterygia. Patients ogy. were 11 men and 10 women whose mean age was 68 Ϯ 2 years (range, Submitted for publication May 21, 2009; revised July 1, 2009; 52–85 years). Patients with primary cicatricial ocular surface disease, accepted July 21, 2009. history of ocular allergic symptoms, and inflammatory ocular surface Disclosure: C.-H. Kuo, None; D. Miyazaki, None; K. Yakura, None; K. Araki-Sasaki, None; Y. Inoue, None disease of unknown etiology were excluded. The study protocol con- Corresponding author: Dai Miyazaki, Division of Ophthalmology formed to the tenets of the Declaration of Helsinki, and the procedures and Visual Science, Tottori University Faculty of Medicine, 36-1 Nishi- used were approved by the Tottori University Ethics Committee. cho, Yonago Tottori 683-8504, Japan; [email protected]. Signed informed consent was obtained from all patients. Investigative Ophthalmology & Visual Science, January 2010, Vol. 51, No. 1 Copyright © Association for Research in Vision and Ophthalmology 139 Downloaded from iovs.arvojournals.org on 09/29/2021 140 Kuo et al. IOVS, January 2010, Vol. 51, No. 1 Transcriptional Analysis Molecular Probes Inc., Eugene, OR) according to the manufacturer’s protocol. Briefly, the solution (CyQUANT GR; Molecular Probes Inc.) Pterygia head tissues collected from primary and recurrence cases was added to each well, and the fluorescence of the samples was were transcriptionally analyzed using an oligo microarray (AceGene, measured at 520 nm with excitation at 480 nm fluorescence micro- Hitachi, Japan) corresponding to 30,336 human genes.2 Genes that plate reader (FL600; BioTek Instruments, Winooski, VT). Cell counts were differentially induced or suppressed in the primary and recurrent were calculated based on the calibration curve of fluorescence of pterygia cases were extracted from the whole genome using t tests. diluted cell suspension. This set of genes was analyzed using a bioinformatics approach. To gain insight into the transcriptional network of molecular events Evaluation of Periostin Expression by in terms of the mechanism of recurrence, we applied commercially available analysis software (Ingenuity Pathway Analysis 3.0, based on Flow Cytometry the Ingenuity Pathways Knowledge Base; Ingenuity Systems, Redwood Pterygia fibroblasts were cultured with recombinant IL-4 (PeproTech, City, CA; http://www.ingenuity.com). The resultant networks were Rocky Hill, NJ) at the selected concentration for 48 hours. To inhibit evaluated by the significance score, which is expressed as the negative MAP kinase activity, a highly selective inhibitor of MAP kinase/ERK logarithm of that P-value. The obtained score in each instance indi- kinase1 (MEK1), PD98059 (10 ␮M; New England Biolabs, Beverly, MA), cated the likelihood that the assembly of a set of focus genes in a was added to the media. Then the serum-starved fibroblasts were network could be explained by random chance alone. collected by enzyme treatment (Accutase; Innovative Cell Technolo- gies) and incubated for 20 minutes at 37°C in a tube rotator. The Real-Time RT-PCR fibroblasts were stained by incubation with antiperiostin antibody (1 ␮g/mL) or control IgG in 0.1% BSA/PBS for 30 minutes at 4°C. After Total RNA was isolated from the treated fibroblasts using a purification kit washing, the cells were incubated with Alexa Fluor 488-antirabbit IgG (RNeasy Mini Kit; Qiagen, Hilden, Germany) according to the manufac- (Molecular Probes, Inc.) for 20 minutes at 4°C. The expression of turer’s protocol. One microgram of RNA samples was reverse transcribed periostin on the surfaces of the fibroblasts was analyzed (FACSCalibur; using random hexamers and superscript III (Invitrogen, Carlsbad, CA). Becton-Dickinson, San Jose, CA). The cDNAs were amplified and quantified (LightCycler; Roche, Mann- heim, Germany) with a one-step quantification of RNA kit (QuantiTect Statistical Analysis SYBR Green PCR kit; Qiagen). Sequences of the used real-time PCR primer pairs were as follows: IL-4: forward, 5Ј-CACCGAGTTGACCGTAACAG-3Ј; Data are presented as the mean Ϯ SEM. Statistical analyses were reverse, 5Ј- GCCCTGCAGAAGGTTTCC-3Ј; periostin: forward, 5Ј-TTGAG- performed by unpaired Student’s t-tests (two tailed) or ANOVA analy- ACGCTGGAAGGAAAT-3Ј; reverse, 5Ј-AGATCCGTGAAGGTGGTTTG-3Ј; sis. IL-13:
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