impaired angiogenic response in the cornea of mice...

Impaired Angiogenic Response in the Cornea of MiceLacking Tenascin C

Takayoshi Sumioka, Norihito Fujita, Ai Kitano, Yuka Okada, and Shizuya Saika

PURPOSE. This study investigated the effects of loss of tenascinC (TNC) in the development of neovascularization in a cornealstroma in mice. Cell culture study was also conducted to clarifythe roles of TNC in the expression of vascular endothelialgrowth factor (VEGF) and transforming growth factor (TGF)�1in fibroblasts and macrophages.

METHODS. Ocular fibroblasts and macrophages from wild-type(WT) and TNC-null (KO) mice were used to study the role ofTNC in the expression of VEGF and TGF�1. The effects of theabsence of TNC on angiogenic gene expression, inflammatorycell invasion, and cornea neovascularization in the cornealstroma were then evaluated after cauterization of the center ofthe cornea in mice. Histologic, immunohistochemical, andmRNA expression analyses were performed.

RESULTS. Absence of TNC suppressed expression of VEGF andcounteracted upregulation of TGF�1 by exogenous TGF�1 inocular fibroblast culture. Such effects of the absence of TNCwere not observed in cultured macrophages. Absence of TNCattenuated expression of both VEGF and TGF�1 mRNA as wellas neovascularization into the stroma after cauterization at thecenter of the cornea in mice. Absence of TNC suppressedmacrophages, but not neutrophils, invading the cauterizedcornea.

CONCLUSIONS. TNC is involved in angiogenic gene expression inocular fibroblasts in vitro and in vivo and is required formacrophage invasion and neovascularization of injured cornealstroma. (Invest Ophthalmol Vis Sci. 2011;52:2462–2467) DOI:10.1167/iovs.10-5750

The cornea is specialized avascular and transparent tissuethat refracts light. Trauma, infection, extensive ocular dam-

age, or pathologic limbal stem deficiency induces neovascular-ization and inflammatory disorders that can potentially impairvision. Neoangiogenesis in an injured cornea is regulated in acomplex way by various growth factors that plays critical rolesin pro-fibrogenic and pro-inflammatory reactions.1–5 The majorcytokine involved in injury-induced neovascularization in-cludes vascular endothelial growth factor (VEGF).6–9 However,cell behaviors are also thought to be modulated by the scaffoldof extracellular matrix.10

Tenascin-C (TNC) is one of the wound healing-related ma-trix macromolecules that is usually transiently upregulated inan injured tissue like fibronectin. It is a disulfide-bonded hex-amer of a component composed of subunits with molecularweights in the range of 120–300 kDa. Tenascin-C is also abun-dantly detected at the invasive margin of cancer, suggesting itsrole at the progression/acquisition of invasive characteristicsby cancer cells.11,12 TNC is reportedly involved in neovascu-larization in a tissue and serum TNC level increases in angio-genesis in lung cancer.13–16 The expression patterns of TNC indiseased corneas has been reported.17–19 These reports sug-gest that TNC is expressed in response to stimuli for tissuerepair such as fibronectin.20–22 However, the regulation ofneovascularization by TNC in the healing process in cornearemains to be elucidated. To address this question in thepresent study, we used TNC-null (KO) mice to show that theabsence of tenascin C in cultured ocular fibroblasts counter-acted the acceleration of expression of angiogenic compo-nents and that cauterization-induced neovascularization in cor-neal stroma is attenuated by the suppression of expression ofangiogenic growth factors by mice lacking TNC expression.

MATERIALS AND METHODS

Experiments were approved by the DNA Recombination ExperimentCommittee and the Animal Care and Use Committee of WakayamaMedical University, and conducted in accordance with the Associationfor Research in Vision and Ophthalmology Statement for the Use ofAnimals in Ophthalmic and Vision Research.

VEGF Expression in TNC-Deficient OcularFibroblasts and Macrophages

To examine the effect of endogenous TNC on VEGF expression inocular fibroblasts, we conducted a real-time reverse transcription-polymerase chain reaction (RT-PCR) using the previously reportedTaqMan probes.3,4 The cells were obtained from C57BL/6wild-type(WT) and TNC-KO mice as previously reported with a minor mod-ification.23 We enucleated eyeballs from postnatal day 2 mice afterCO2 asphaxia and obtained eyeshells by removing intraocular tis-sues. The tissues were minced and explanted for the outgrowth ofocular fibroblasts. These cells were positive for vimentin and colla-gen type I (data not shown). The cells were grown to confluence ina 60 mm culture dish and were then treated with serum-free me-dium for 24 hours, followed by incubation in serum-free medium orwith transforming growth factor (TGF)�1 at 1.0 ng/mL for 24 hours.Six wells were prepared for each culture condition. RNA wasextracted and processed for real-time RT-PCR for mRNAs of VEGFand TGF�1. Data were analyzed by unpaired t-test.

Mouse macrophages were obtained from the peritoneal cavity aspreviously reported,24 and allowed to adhere to 60-mm plastic dishesfor 24 hours The cells were then treated with serum-free medium for24 hours, followed by incubation in serum-free medium or with TGF�1at 1.0 ng/mL for 24 hours. RNA was extracted and processed forreal-time RT-PCR for mRNAs of VEGF and TGF�1. Data were analyzedby unpaired t-test.

From the Department of Ophthalmology, Wakayama Medical Uni-versity, 811-1 Kimiidera, Wakayama, 641-0012, Japan.

Supported by a grant from the Ministry of Education, Science,Sports and Culture of Japan (C19592036 to SS, C40433362 to TS), 19Wakayama Medical Award for Young Researchers Mitsui Life SocialWelfare Foundation, Mochida Memorial Foundation, Takeda ScienceFoundation and Uehara Foundation (to S. S.).

Submitted for publication April 21, 2010; revised September 6 and14 and October 6 and 13, 2010; accepted October 14, 2010.

Disclosure: T Sumioka, None; N. Fujita, None; A. Kitano, None;Y. Okada, None; S. Saika, None

Corresponding author: Takayoshi Sumioka, MD, PhD, Departmentof Ophthalmology, Wakayama Medical University, 811-1 Kimiidera,Wakayama, 641-0012, Japan; [email protected].

Cornea

Investigative Ophthalmology & Visual Science, April 2011, Vol. 52, No. 52462 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.

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Induction of Stromal Neovascularization byCauterization of the Central Cornea in Mice

We then performed an in vivo experiment using WT or KO mice.There were no differences in histologic findings in the cornea betweenWT and KO mice (data not shown). Corneal neovascularization fromthe limbal vessels was induced by cauterization of the central cornea ofthe eye using a disposable tool (OPTEMP; Alcon, Fort Worth, TX) aspreviously reported.8 One eye in each WT and KO mouse (n � 15 ofeach genotype) was treated and mice were killed on days 3, 7, and14. The eye was then enucleated and processed for cryo (n � 12 ineach genotype) and paraffin sectioning (n � 3 in each genotype).Corneas of each genotype were examined histologically and immu-nohistochemically.

To examine the expression of angiogenic growth factors andinflammatory cell markers in vivo, centrally cauterized corneas (n �6 in each of WT or KO group) were excised on days 3 and 7. TotalRNA was extracted from cauterized tissue and processed for real-time RT-PCR for VEGF, TGF�1, F4/80 macrophage antigen, andmyeloperoxidase (MPO) as previously reported. Primers for mRNAsof TGF�1 (Mm03024053 mL), F4/80 (Mm00802524 mL), and MPO(Mm01298422 gL) were purchased from Applied Biosystems (Taq-Man; Foster City, CA). Data were statistically analyzed by employingANOVA.

Immunohistochemistry

Immunohistochemistry was performed as previously reported to de-tect stromal neovascularization and cytokines. New vessels or macro-phages were detected by immuno-detection of CD31 antigen, F4/80 (amacrophage marker) or myeloperoxidase (MPO, a marker for a neu-trophil leukocytes). Cryosections (7 �m thick) were fixed in coldacetone and processed for immunohistochemistry with rat monoclonalanti-CD31 (PECAM) antibody as previously reported.9,25 Statistical anal-ysis of CD31-labeled neovascularization data were performed by

Tukey-Kramer’s test and P � 0.05 was considered significant. Paraffinsections (5 �m thick) were used for detection of F4/80 antigen, MPO,VEGF, and activated form of TGF�1 as previously reported.3,4 Antibodycomplexation with the peroxidase-conjugated secondary antibody wasvisualized with 3,3�-diaminobenzidine reaction.

RESULTS

Effects of the Absence of TNC on Expressionof Angiogenic Cytokines in Fibroblastsand Macrophages

A cell culture experiment was conducted to determinewhether endogenous TNC affects the expressions of VEGF andTGF�1 in the two major wound healing–related cellular com-ponents, fibroblasts and macrophages. Adding recombinantTGF�1 at 1.0 ng/mL did not affect the expression level of VEGFmRNA in both WT and KO fibroblasts. Loss of TNC, however,significantly reduced its expression level in fibroblasts underthe conditions with and without exogenous TGF�1 (Fig. 1a).Adding TGF�1 to the culture upregulated mRNA expression ofTGF�1 in WT fibroblasts, but not in KO cells. Loss of TNCsignificantly reduced its expression level in fibroblasts culturedwith exogenous TGF�1 (Fig. 1b). Neither the addition of ex-ogenous TGF�1 nor the absence of TNC demonstrated signif-icant effects on mRNA expression in cultured macrophages(Figs. 1c, 1d).

Neovascularization in Corneal Stroma

CD31 immunostaining was performed in cryosections. In WTmouse corneas, formation of CD31-labeled neovascularizationfrom the limbus in the corneal stroma was detected in theperipheral cornea on day 3 (Fig. 2a, between black and white

FIGURE 1. Expression of vascularendothelial growth factor (VEGF) incultured mouse ocular fibroblasts (a,b) and cultured mouse macrophages(c, d). VEGF expression was lower intenascin C (TNC)-null (KO) fibro-blasts than in wild-type (WT) cellsregardless of the presence or ab-sence of exogenous transforminggrowth factor �1 (TGF�1). Absenceof TNC did not affect the expressionof VEGF mRNA in cultured macro-phages.

IOVS, April 2011, Vol. 52, No. 5 Tenascin C and Corneal Neovascularization 2463


arrowheads). The length of such neovascularization in cornealstroma peaked on day 7 and then began to decline on day 14.The length of neovascularization was less in KO mice com-pared with that in WT mice on day 3 and day 7, but not on day14 (Fig. 2b).

Expression of Angiogenic Components inCentrally Cauterized Cornea

Expressions of both VEGF and TGF�1 mRNA were both signif-icantly lower in KO cornea compared with those in WT corneaon day 3, but such differences were not detected on day 7(Figs. 3a, 3b).

To detect protein expression of VEGF and TGF�1, weadditionally performed immunohistochemistry. VEGF proteinwas immunohistochemically detected in tissue. Expression pat-tern of VEGF was similar to that of F4/80 macrophagic antigen(as shown below), mainly being detected in cells beneath theepithelium. The number of labeled cells observed by immuno-detection seemed greater in WT cornea than in KO cornea onday 3 (Figs. 4a, 4b). On days 7 and 14, the protein expressionlevel seemed lower than that on day 3. At these timepoints, theexpression level in WT tissues seemed similar to that in KOtissue (Figs. 4c–f). As for the expression pattern of the activeform of TGF�1, activated TGF�1 was not detected at theimmunohistochemical level, although mRNA was detected asdescribed above (Figs. 4g, 4h).

Expression of Inflammatory Cell Invasion inCentrally Cauterized Cornea

Immunohistochemistry detected F4/80-labeled macrophagesin centrally cauterized corneas (Fig. 5). On day 3, F4/80-posi-tive cells were observed in the full-thickness stroma of the WTcentral cornea of the cauterization area. At the same timepoint,F4/80-positive cells were detected in the area beneath theepithelium, but not in the deep stroma, in a KO mouse. Ondays 7 and 14, F4/80-positive macrophages were seen beneaththe epithelium in a WT cornea, while there were very few suchcells in a KO cornea (Fig. 5). These immunohistochemicalfindings suggest that the absence of TNC attenuated recruit-ment of macrophages in the injured corneal stroma. A fewMPO-positive cells were observed beneath the epithelium in aWT cornea, but not in a KO cornea, on day 3. On days 7 and14, MPO-labeled cells were no longer detected in WT and KOtissues (Fig. 6).

To quantify the invasion of macrophages and neutrophilesin tissue we further conducted real-time RT-PCR to clarifymRNA expressions of F4/80 and MPO, as well as mRNA ex-pressions of VEGF and TGF�1. Expression of F4/80 was sup-pressed by the loss of TNC on day 3, but not on day 7 (Fig. 7a).Expression of MPO was not significantly affected by the loss ofTNC (Fig. 7b).

FIGURE 2. (a) CD31-immunostaining in corneas of wild type (WT) andtenascin C (TNC)-null (KO) mice. Cauterization-induced stromal neovascular-ization from the limbal area was positively stained. (aA, aC, aE) Findings ofWT mice; (aB, aD, aF) corneas of KO mice; (aA, aB) at day 3; (aC, aD) at day7; (aE, aF) at day 14. Black arrows: the point of corneo-limbal border; whitearrows: the leading edge of neovascularization. Bar, 100 �m. (b) Length ofthe stromal neovascularization as indicated by the distance between thelimbus (black arrows in a) and the leading edge of the neovascularization(white arrows in a) at each timepoint (day 3, n � 12; day 7, n � 12; day 14,n � 12 for each genotype). The length of stromal neovascularization wassignificantly shorter in tenascin C (TNC)-null (KO) mice than in wild type(WT) mice on days 3 and 7. * P � 0.05, ** P � 0.01.

FIGURE 3. mRNA expression of an-giogenic growth factors in centrally-cauterized cornea. Expression of vas-cular endothelial growth factor (a)and transforming growth factor �1(TGF�1) (b) in a centrally cauterizedcornea was lower in a tenascin-null(KO) mouse than in a wild-type (WT)mouse on day 3. * P � 0.05; ** P �0.01 significant by ANOVA test.

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DISCUSSION

The present in vitro experiments first showed that endogenousTNC is required for angiogenic gene expression in culturedocular fibroblasts and the development of neovascularizationin mouse corneal stroma.

The loss of TNC in cultured fibroblasts indeed suppressesexpression of major angiogenic cytokines (i.e., VEGF andTGF�1). Although it could be postulated that binding of TNCto its specific receptors such as integrins activates cytoplasmicsignaling that facilitates crosstalk between TGF�-derived sig-nals, further detailed study is to be conducted to uncover themechanism underlying the phenomenon. In macrophages ex-pression of VEGF and TGF�1 were not markedly affected bythe loss of TNC in vitro.

Our real-time RT-PCR then showed that loss of TNC atten-uates mRNA expression of angiogenic growth factors (i.e.,VEGF and TGF�1) and suppresses injury-induced neovascular-ization in mouse corneal stroma on day 3. Macrophages areknown to be one of the major components expressing angio-genic cytokines/growth factors in an injured tissue. Our immu-nohistochemistry suggested that invasion of macrophages inthe cauterized corneal stroma might be attenuated by the lossof TNC. This hypothesis was confirmed by the analysis ofmRNA expression; expression of F4/80 mRNA was suppressedby the loss of TNC on day 3, but not on day 7. Althoughexpression of angiogenic growth factors (i.e., VEGF andTGF�1) in a cultured macrophage is not significantly affectedby the loss of TNC in vitro, the lower number of macrophagesin the stroma of KO mice on day 3 could explain the decreasedTGF�1 and VEGF in expression levels centrally cauterizedcornea specimens from KO mice. Immunohistochemical detec-tion of VEGF protein further supports this theory; localizationof VEGF-labeled cells was similar to that of macrophages.TGF�1 protein expression might be below the level of immu-nohistochemical detection, although real-time RT-PCR clearlyshowed greater expression in WT tissue. There was no differ-

FIGURE 4. Immunohistochemical detection of angiogenic growth fac-tors in centrally-cauterized cornea. Expression of VEGF protein wasdetected in the cells beneath the epithelium. The number of labeledcells observed by immunodetection seemed greater in WT cornea (a)than in KO cornea (b) on day 3. On days 7 and 14, the proteinexpression level seemed less than that on day 3. At these timepointsthe expression levels in WT tissues seemed similar to those in KOtissue (c–f). There was no apparent TGF�1 protein-immunoreactivityon day 3 (g, h) or at the other timepints (not shown) in either WT orKO cornea. Bar, 50 �m.

FIGURE 5. Immunohistochemical de-tection of F4/80-labeled cells (i.e.,macrophages in the tissue). (A) Onday 3, F4/80-positive cells were ob-served in the full-thickness stroma inthe central cauterization area of theWT cornea. (B) At the same time-point, F4/80-positive cells were de-tected in the area beneath the epithe-lium, but not in the deep stroma, in aKO mouse. On days 7 and 14, F4/80-positive macrophages were seen be-neath the epithelium in a WT cornea(C, E), while such cells were veryfew in KO cornea (D, F). Arrows:F4/80-labeled macrophages.



ence in the expression level of MPO neutrophil between WTand KO corneas on either day 3 or day 7, although immuno-histochemistry detected a few neutrophil in WT cornea on day3. Both TGF�1 and VEGF are chemoattractants for macro-phages. Because expression of TGF�1 and VEGF in culturedocular fibroblasts was suppressed in the absence of TNC,initiation of macrophage invasion by resident cells in the cor-neal stroma might be attenuated in KO tissue on tissue cauter-ization.

TNC is reportedly involved in the persistence of inflamma-tion in various tissue. For example, TNC is expressed in areasof inflammation and tissue damage in inflamed rheumatoidjoints. Furthermore, TNC-deficient mice show rapid resolutionof joint inflammation. Another report showed that TNC mayplay a critical role in regulating traffic of macrophages cells inhuman malignant tumor.26–28 Angiogenic effects similar tothose of TNC were reported by another extracellular matrixcomponent, osteopontin; absence of osteopontin impairs theangiogenic responses by myocardium or cornea, as well as theprocess of tumor progression. Osteopontin upregulates tumornecrosis factor, interleukin (IL)-1b, -6, and -8 in associationwith p38 phosphorylation in human macrophages.29

Roles of TNC in neovascularization were also investigated inother fields; in a malignant brain tumor, glioblastoma, perivas-cular deposition of TNC plays a role in angiogenesis and tumorcell proliferation.30 Inflammation, neovascularization, and sub-sequent tissue fibrosis are key phenomena occurring in tissuesafter inflammatory diseases and in the healing process post-injury.4,29,31,32 TNC is reported to be involved in the persis-tence of tissue inflammation in other tissues in addition to itsrole in the development of neovascularization. For example,zymosan-induced synovitis (joint inflammation) is less severe inKO mice than in WT mice.33 TNC utilizes specific integrinreceptors to promote cell reactions against external stimuli.Our unpublished data from immunohistochemical analysis ofhealing corneas from tenascin C-deficient mice and wild typeC57/BL6 mice did not show any difference in alpha v or beta 6subunit expression in stromal cells.

In conclusion, endogenous TNC modulates the expressionof angiogenic cytokines and is involved in the development ofcorneal neovascularization. To overcome injury induced cor-neal neovascularization, a better understanding is still requiredof the complex mechanism modulating this response.

FIGURE 6. Immunohistochemicaldetection of myeloperoxidase-la-beled (MPO) cells (i.e., neutrophilsin the tissue). A few MPO-positivecells were observed beneath theepithelium in WT cornea (a), butnot in KO cornea (b), on day 3. Ondays 7 and 14, MPO-labeled cellswere no longer detected in WT orKO tissues (c–f). Bar, 50 �m.

FIGURE 7. Relative mRNA expres-sion levels of F4/80 antigen and my-eloperoxidase (MPO) in the affectedcornea. Expression of F4/80 (a), amarker for macrophages, was alsolower in KO cornea than in WT cor-nea on day 3. There was no differ-ence in the expression levels of my-eloperoxidase (MPO), a marker for apolymorphonuclear leukocytes, be-tween WT and KO corneas on days 3and 7 (b). P � 0.05 by ANOVA test.

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References

1. Brodovsky SC, McCarty CA, Snibson G, et al. Management of alkaliburns: an 11-year retrospective review. Ophthalmology. 2000;107:1829–1835.

2. Meller D, Pires RT, Mack RJ, et al. Amniotic membrane transplan-tation for acute chemical or thermal burns. Ophthalmology. 2000;107:980–989.

3. Saika S, Ikeda K, Yamanaka O, et al. Expression of Smad7 in mouseeyes accelerates healing of corneal tissue after exposure to alkali.Am J Pathol. 2005;166:1405–1418.

4. Saika S, Miyamoto T, Yamanaka O, et al. Therapeutic effect oftopical administration of SN50, an inhibitor of nuclear factor-kappaB, in treatment of corneal alkali burns in mice. Am J Pathol.2005;166:1393–1403.

5. Saika S, Yamanaka O, Sumioka T, et al. Fibrotic disorders in theeye: targets of gene therapy. Prog Retin Eye Res. 2008;27:177–196.

6. Azar DT. Corneal angiogenic privilege: angiogenic and antiangio-genic factors in corneal avascularity, vasculogenesis, and woundhealing (an American Ophthalmological Society thesis). Trans AmOphthalmol Soc. 2006;104:264–302.

7. Papathanassiou M, Theodossiadis PG, Liarakos VS, Rouvas A,Giamarellos-Bourboulis EJ, Vergados IA. Inhibition of corneal neo-vascularization by subconjunctival bevacizumab in an animalmodel. Am J Ophthalmol. 2008;145:424–431.

8. Saika S. TGFb pathobiology in the eye. Lab Invest. 2006;86:106–115.

9. Fujita S, Saika S, Kao WW, et al. Endogenous TNFalpha suppressionof neovascularization in corneal stroma in mice. Invest Ophthal-mol Vis Sci. 2007;48:3051–3055.

10. Francis ME, Uriel S, Brey EM. Endothelial cell-matrix interactions inneovascularization. Tissue Eng Part B Rev. 2008;14:19–32.

11. Midwood KS, Orend G. The role of tenascin-C in tissue injury andtumorigenesis. J Cell Commun Signal. 2009 Oct 17. [Epub aheadof print.]

12. Shrestha P, Sakamoto F, Takagi H, et al. Enhanced tenascin immu-noreactivity in leukoplakia and squamous cell carcinoma of theoral cavity: an immunohistochemical study. Eur J Cancer B OralOncol. 1994;30:132–137.

13. Ballard VL, Sharma A, Duignan I, et al. Vascular tenascin-C regu-lates cardiac endothelial phenotype and neovascularization.FASEB J. 2006;20:717–719.

14. Ishiwata T, Takahashi K, Shimanuki Y, et al. Serum tenascin-C as apotential predictive marker of angiogenesis in non-small cell lungcancer. Anticancer Res. 2005;25:489–495.

15. Saito Y, Shiota Y, Nishisaka M, et al. Inhibition of angiogenesis bya tenascin-c peptide which is capable of activating beta1-integrins.Biol Pharm Bull. 2008;31:1003–1007.

16. Wallner K, Li C, Shah PK, et al. Tenascin-C is expressed in macro-phage-rich human coronary atherosclerotic plaque. Circulation.1999;99:1284–1289.

17. Ljubimov AV, Saghizadeh M, Pytela R, et al. Increased expressionof tenascin-C-binding epithelial integrins in human bullous kera-topathy corneas. J Histochem Cytochem. 2001;49:1341–1350.

18. Wachtlin J, Langenbeck K, Schrunder S, et al. Immunohistology ofcorneal wound healing after photorefractive keratectomy and laserin situ keratomileusis. J Refract Surg. 1999;15:451–458.

19. Akhtar S, Bron AJ, Hawksworth NR, et al. Ultrastructural morphol-ogy and expression of proteoglycans, betaig-h3, tenascin-C, fibril-lin-1, and fibronectin in bullous keratopathy. Br J Ophthalmol.2001;85:720–731.

20. Matsuda A, Yoshiki A, Tagawa Y, et al. Corneal wound healing intenascin knockout mouse. Invest Ophthalmol Vis Sci. 1999;40:1071–1080.

21. Herold-Mende C, Mueller MM, Bonsanto MM, et al. Clinical impactand functional aspects of tenascin-C expression during gliomaprogression. Int J Cancer. 2002;20:362–369.

22. Hirata E, Arakawa Y, Shirahata M, et al. Endogenous tenascin-Cenhances glioblastoma invasion with reactive change of surround-ing brain tissue. Cancer Sci. 2009;100:1451–1459.

23. Saika S, Ikeda K, Yamanaka O, et al. Loss of tumor necrosis factora potentiates transforming growth factor b-mediated pathogenictissue response during wound healing. Am J Pathol. 2006;168:1848–1860.

24. Cambridge GW, Parsons JF, Friend JV, et al. Some effects oflignocaine on cultured mouse peritoneal macrophages. AgentsActions. 1985;16:548–551.

25. Kitano A, Saika S, Yamanaka O, et al. Emodin suppression of ocularsurface inflammatory reaction. Invest Ophthalmol Vis Sci. 2007;48:5013–5022.

26. Goh FG, Piccinini AM, Krausgruber T, et al. Transcriptional regu-lation of the endogenous danger signal tenascin-C: a novel auto-crine loop in inflammation. J Immunol. 2010;184:2655–2662.

27. Midwood K, Sacre S, Piccinini AM, et al. Tenascin-C is an endog-enous activator of Toll-like receptor 4 that is essential for main-taining inflammation in arthritic joint disease. Nat Med. 2009;15:774–780.

28. Kulla A, Liigant A, Piirsoo A, et al. Tenascin expression patternsand cells of monocyte lineage: relationship in human gliomas. ModPathol. 2000;13:56–67.

29. Naldini A, Leali D, Pucci A, et al. Cutting edge: IL-1beta mediatesthe proangiogenic activity of osteopontin-activated human mono-cytes. J Immunol. 2006;177:4267–4270.

30. Behrem S, Zarkovic K, Eskinja N, et al. Distribution pattern oftenascin-C in glioblastoma: correlation with angiogenesis and tu-mor cell proliferation. Pathol Oncol Res. 2005;11:229–235.

31. Sato T, Nakai T, Tamura N, et al. Osteopontin/Eta-1 upregulated inCrohn’s disease regulates the Th1 immune response. Gut. 2005;54:1254–1262.

32. Tamaoki M, Imanaka-Yoshida K, Yokoyama K, et al. Tenascin-Cregulates recruitment of myofibroblasts during tissue repair aftermyocardial injury. Am J Pathol. 2005;167:71–80.

33. Midwood K, Sacre S, Piccinini AM, et al. Tenascin-C is an endog-enous activator of Toll-like receptor 4 that is essential for main-taining inflammation in arthritic joint disease. Nat Med. 2009;15:774–780.



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