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      Expression of Tissue Inhibitor of Metalloproteinase-4 in Normal Human Corneal Cells and Experimental Corneal Neovascularization


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          Purpose: To study the expression of TIMP-4 in cultured corneal cells and in corneal neovascularization. Methods: Human limbo-corneal epithelial cells, fibroblasts, and endothelial cells were cultured in serum-free, PMA- or basic fibroblast growth factor (bFGF)-treated condition. Neovascularization in rat cornea was induced by suturing. The expression of TIMP-4 was examined by immunohistochemistry, Western blot and RT-PCR. Results: TIMP-4 was constitutively expressed in cultured human corneal cells. The expression was only mildly enhanced after mitogen treatment. TIMP-4 immunoreactivity was predominantly expressed in normal rat corneal epithelium, and also in ingrowing blood vessels following suturing, which persisted up to day 28. Increased staining in corneal epithelium and blood vessels were also noted in vascularized human corneas. Conclusions: TIMP-4 is expressed in the cornea, which may play a role in modulating extracellular matrix remodeling associated with corneal wound healing and angiogenesis.

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          Most cited references10

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          Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4.

          The tissue inhibitors of metalloproteinases (TIMPs) constitute a family of proteins, of which three members have so far been described. Using the expressed sequence tag sequencing approach, we have identified a novel TIMP-related cDNA fragment and subsequently cloned a fourth human TIMP (TIMP-4) from a human heart cDNA library. The open reading frame encodes a 224-amino acid precursor including a 29-residue secretion signal. The predicted structure of the new protein shares 37% sequence identity with TIMP-1 and 51% identity with TIMP-2 and -3. The protein has a predicted isoelectric point of 7.34. The open reading frame-directed expression of TIMP-4 protein in MDA-MB-435 human breast cancer cells showed metalloproteinase inhibitory activity on reverse zymography. By Northern analysis, only the adult heart showed abundant TIMP-4 transcripts with a 1. 4-kilobase predominant transcript band; very low levels of the transcripts were detected in the kidney, placenta, colon, and testes, and no transcripts were detected in the liver, brain, lung, thymus, and spleen. This unique expression pattern suggests that TIMP-4 may function in a tissue-specific fashion in extracellular matrix homeostasis.
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            Topical modulation of interleukin-1 activity in corneal neovascularization.

            To determine whether inflammatory corneal neovascularization (CNV) is associated with interleukin-1 (IL-1) activity and if so, to assess the efficacy of topical interleukin-1 receptor antagonist (IL-1ra) to suppress CNV. Inflammatory CNV was induced on day 0 by placement of paracentral intrastromal sutures in BALB/c murine eyes. Quantification of IL-1alpha and -beta cytokine levels was done by a sandwich enzyme-linked immunosorbent assay (ELISA) on the supernatants of incubated corneas excised at specified time points after induction of CNV (n = 6 per time point studied). To study suppression of CNV by IL-1ra, animals were divided into treatment subgroups that received topical 20 mg/ml of IL-1ra mixed in 0.2% sodium hyaluronate (n = 28) or placebo (vehicle) alone (n = 22) 3 times daily during days 0-35. Other groups of animals received placebo for 1 (n = 10) or 2 (n = 14) weeks before being switched and retained on IL-1ra. Neovascularization was assessed biomicroscopically and graded by using a standardized scheme. Induction of CNV stimulus was associated with a significant surge in the expression of both IL-1alpha (p < 0.001) and IL-1beta (p < 0.001) as early as 2 h after the stimulus, which peaked at 24 h, before decreasing substantially in the case of IL-1beta and returning to basal levels by day 7. Topical application of IL-1ra led to a significant suppression of CNV for the duration of therapy only if initiated early after induction of the neovascular stimulus. Initiation of therapy 1 week after CNV induction was associated only with a transient suppression in the angiogenic response. Our data strongly implicate IL-1 as a critical mediator in the early phase of CNV and suggest that IL-1ra can be an effective modality in suppressing CNV if initiated sufficiently early after the inflammatory neovascular stimulus.
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              Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4).

              TIMP-4, a novel human tissue inhibitor of metalloproteinase, was identified and cloned (Greene, J., Wang, M., Raymond, L. A., Liu, Y. E., Rosen, C., and Shi, Y. E. (1996) J. Biol. Chem. 271, 30375-30380). In this report, the production and characterization of recombinant TIMP-4 (rTIMP4p) are described. rTIMP4p, expressed in baculovirus-infected insect cells, was purified to homogeneity by a combination of cation exchange, hydrophobic, and size-exclusion chromatographies. The purified protein migrated as a single 23-kDa band in SDS-polyacrylamide gel electrophoresis and in Western blot using a specific anti-TIMP-4 antibody. Inhibition of matrix metalloproteinase (MMP) activities by rTIMP4p was demonstrated in five MMPs. Enzymatic kinetic studies revealed IC50 values (concentration at 50% inhibition) of 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Purified rTIMP4p demonstrated a strong inhibitory effect on the invasion of human breast cancer cells across reconstituted basement membranes. Thus, TIMP-4 is a new enzymatic inhibitor in MMP-mediated extracellular matrix degradation and may have therapeutic potential in treating cancer malignant progression.

                Author and article information

                Ophthalmic Res
                Ophthalmic Research
                S. Karger AG
                August 2003
                20 June 2003
                : 35
                : 4
                : 199-207
                aDepartment of Ophthalmology, Chang-Gung Memorial Hospital, Taoyuan, Taiwan; bOcular Cell and Gene Therapy Laboratory, Department of Ophthalmology, University of California, San Francisco, Calif., USA; cExelixis, South San Francisco, Calif., USA; dMaryknoll Hospital, and ePusan National University, Pusan, Korea; fDepartment of Physiology, Chang-Gung University, Taoyuan, Taiwan
                71171 Ophthalmic Res 2003;35:199–207
                © 2003 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                : 13 July 2002
                : 04 March 2003
                Page count
                Figures: 5, References: 40, Pages: 9
                Original Paper

                Vision sciences,Ophthalmology & Optometry,Pathology
                Corneal neovascularization,Inflammation,Tissue inhibitors of metalloproteinase-4


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