TRPV1 Involvement in Inflammatory Tissue Fibrosis in Mice
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.043
ISSN1525-2191
AutoresYuka Okada, Peter S. Reinach, Kumi Shirai, Ai Kitano, Winston W.‐Y. Kao, Kathleen C. Flanders, Masayasu Miyajima, Hongshan Liu, Jianhua Zhang, Shizuya Saika,
Tópico(s)Pain Mechanisms and Treatments
ResumoWe examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1−/− (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow–derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor β 1 (TGFβ1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFβ1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing. We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1−/− (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow–derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor β 1 (TGFβ1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFβ1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing. The cornea is an avascular transparent tissue located at the outermost part of the eye. It must remain transparent to properly refract light for normal vision. Ocular trauma resulting from a corneal alkali burn is a serious clinical problem and may cause severe and permanent visual impairment by inducing tissue inflammation, fibrosis, and scarring, leading to subsequent corneal opacification.1Brodovsky S.C. McCarty C.A. Snibson G. Loughan M. Sullivan L. Daniell M. Taylar H.R. Management of alkali burns: an 11-year retrospective review.Ophthalmology. 2000; 107: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar The acute corneal injury sequence after alkali burn includes inflammation and degradation of the matrix of the epithelial basement membrane and stroma.2Ishizaki M. Zhu G. Hasebe T. Shafer S.S. Kao W.W.-Y. Expression of collagen I, smooth muscle a-actin, and vimentin during the healing of alkali-burned and lacerated corneas.Invest Ophthalmol Vis Sci. 1993; 34: 3320-3328PubMed Google Scholar, 3Saika S. Kobata S. Hashizume N. Okada Y. Yamanaka O. Epithelial basement membrane in alkali-burned corneas in rats Immunohistochemical study.Cornea. 1993; 12: 383-390Crossref PubMed Scopus (52) Google Scholar, 4Saika S. Uenoyama K. Hiroi K. Tanioka H. Takase K. Hikita M. Ascorbic acid phosphate ester and wound healing in rabbit corneal alkali burns: epithelial basement membrane and stroma.Graefes Arch Clin Exp Ophthalmol. 1993; 231: 221-227Crossref PubMed Scopus (43) Google Scholar Influx of inflammatory cells [ie, macrophages and/or polymorphonuclear leukocytes (PMNs)], activation of corneal fibroblasts (keratocytes), formation of myofibroblasts, and subsequent tissue scarring are all involved in the wound healing response in an alkali-burned cornea.2Ishizaki M. Zhu G. Hasebe T. Shafer S.S. Kao W.W.-Y. Expression of collagen I, smooth muscle a-actin, and vimentin during the healing of alkali-burned and lacerated corneas.Invest Ophthalmol Vis Sci. 1993; 34: 3320-3328PubMed Google Scholar, 3Saika S. Kobata S. Hashizume N. Okada Y. Yamanaka O. Epithelial basement membrane in alkali-burned corneas in rats Immunohistochemical study.Cornea. 1993; 12: 383-390Crossref PubMed Scopus (52) Google Scholar Keratocyte activation results in myofibroblast transdifferentiation and tissue contraction with increased extracellular matrix expression.5Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2008; 3: 349-363Crossref Scopus (3228) Google Scholar Despite aggressive treatment of severe injury with anti-inflammatory drugs and surgery, vision restoration often fails.1Brodovsky S.C. McCarty C.A. Snibson G. Loughan M. Sullivan L. Daniell M. Taylar H.R. Management of alkali burns: an 11-year retrospective review.Ophthalmology. 2000; 107: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 6Sridhar M.S. Bansal A.K. Sangwan V.S. Rao G.N. Amniotic membrane transplantation in acute chemical and thermal injury.Am J Ophthalmol. 2000; 130: 124-137Abstract Full Text Full Text PDF Scopus (93) Google Scholar, 7Meller D. Pires R.T. Mack R.J. Figueiredo F. Heiligenhaus Park W.C. Prabhasawat P. John T. McLeod S.D. Steuhl K.P. Tseng S.C. Amniotic membrane transplantation for acute chemical or thermal burns.Ophthalmology. 2000; 107: 980-989Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar This limitation is the basis for efforts to develop new and more effective prevention/treatment strategies. Transient receptor potential (TRP) channels are polymodal receptors that are activated by a host of stimuli to mediate sensory transduction. The TRP superfamily is composed of 28 different genes that are subdivided into seven different subfamilies (TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, and TRPV).8Pedersen S.F. Owsianik G. Nilius B. TRP channels: an overview.Cell Calcium. 2005; 38: 233-252Crossref PubMed Scopus (622) Google Scholar Each of them possesses variable cation permeability. They are activated by multiple endogenous and external stimuli.9Ramsey I.S. Delling M. Clapham D.E. An introduction to TRP channels.Annu Rev Physiol. 2006; 68: 619-647Crossref PubMed Scopus (1250) Google Scholar, 10Owsianik G. Talavera K. Voets T. Nilius B. Permeation and selectivity of TRP channels.Annu Rev Physiol. 2006; 68: 685-717Crossref PubMed Scopus (428) Google Scholar They could be activated by the following: i) direct ligand binding, ii) depletion of intracellular Ca2+ store and Ca2+/calmodulin-dependent activation, and iii) indirect activation by osmotic stress, temperature variation, pheromones, taste, and mechanical as well as other stimuli. The capsaicin receptor, TRPV1, is a nocioceptor and one of the isoforms belonging to the seven-member TRPV subfamily. It elicits responses to a variety of diverse noxious stimuli that include various ligand-like agents and a plethora of seemingly unrelated stimuli such as chemical irritants, inflammatory mediators, tissue-damaging stimuli, a decline in pH (<6.0), moderate heat (≥43°C), and hypertonic challenges. All of them lead to nocioceptions and evoke pain in human beings and pain-related behaviors in animals.11Montell C. Birnbaumer L. Flockerzi V. The TRP channels, a remarkably functional family.Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 12Ciura S. Bourque C.W. Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for normal thirst responses to systemic hyperosmolality.J Neurosci. 2006; 26: 9069-9075Crossref PubMed Scopus (215) Google Scholar, 13Steen K.H. Reeh P.W. Anton F. Handwerker H.O. Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro.J Neurosci. 1992; 12: 86-95Crossref PubMed Google Scholar, 14García-Hirschfeld J. López-Briones L.G. Belmonte C. Valdeolmillos M. Intracellular free calcium responses to protons and capsaicin in cultured trigeminal neurons.Neuroscience. 1995; 67: 235-243Crossref PubMed Scopus (41) Google Scholar TRPV1 is a cationic nonselective channel whose activation leads to increases in Ca2+ influx through a highly permeable cation channel, and has an outward-rectifying current–voltage relationship.15Caterina M.J. Schumacher M. Tominaga M. Rosen T.A. Levine J.D. Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (7218) Google Scholar TRPV1 activation causes release of tachykinin neuropeptides [eg, substance P (SP), neurokinin A, and calcitonin gene-related peptide] from sensory nerves, eliciting neurogenic inflammation in the surrounding area. Studies using mice lacking TRPV1 have shown that TRPV1 is essential for the development of heat hyperalgesia in response to tissue inflammation.16Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Petersen-Zeitz K.R. Kolzenburg M. Basbaum A.I. Julius D. Impaired nocioception and pain sensation in mice lacking the capsaicin receptor.Science. 2000; 288 (306–303)Crossref PubMed Scopus (2961) Google Scholar, 17Davis J.B. Gray J. Gunthorpe M.J. Hatcher J.P. Davey P.T. Overend P. Harries M.H. Latcham J. Clapham C. Atkinson K. Hughes S.A. Rance K. Grau E. Harper A.J. Pugh P.L. Roger D.C. Blingham S. Randall A. Sheardown S.A. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia.Nature. 2000; 405: 183-187Crossref PubMed Scopus (1509) Google Scholar The present study was undertaken to elucidate the role of corneal alkali burn–induced TRPV1 activation in eliciting inflammation and scarring during wound healing. The results show that loss of TRPV1 expression or blockage of its activation suppressed severe and persistent corneal inflammation and fibrosis/scarring, resulting in marked improvement in the restoration of tissue transparency. Experimental protocols and the use of experimental mice were approved by the DNA Recombination Experiment Committee and the Animal Care and Use Committee of Wakayama Medical University and conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Intact or alkali-burned mouse corneas were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 24 hours, embedded in paraffin, and then processed for histology. Paraffin sections (5-μm thick) were deparaffinized, rehydrated, and subjected to immunohistochemistry (IHC) for TRPV1. The rabbit polyclonal anti-TRPV1 antibody (1:500; Neuromics, Edina, MN) was diluted in PBS. A total of 3 μL of 1 N NaOH solution was applied to the right eye of 6- to 8-week-old TRPV1-null (KO) (n = 88) mice or wild-type (WT) (n = 94) mice under general anesthesia to produce an ocular surface alkali burn.18Saika S. Miyamoto T. Yamanaka O. Kato T. Ohnishi Y. Flanders K.C. Ikeda K. Nakajima Y. Kao WW- Y. Sato M. Muragaki Y. Ooshima A. Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-B, in treatment of corneal alkali burns in mice.Am J Pathol. 2005; 166: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Saika S. Ikeda K. Yamanaka O. Flanders K.C. Okada Y. Miyamoto T. Kitano A. Ooshima A. Nakajima Y. Ohnishi Y. Kao W.W. Loss of tumor necrosis factor a potentiates transforming growth factor b-mediated pathogenic tissue response during wound healing.Am J Pathol. 2006; 168: 1848-1860Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Ofloxacin ointment was administered topically twice a week to reduce the risk of bacterial infection. The eyes with obvious bacterial infection were excluded from the study. Eye globe diameters were measured from photographs obtained under a microscope. The corneal tissue then was processed for histology, IHC, Western blotting, or quantitative RT-PCR (qRT-PCR) on days 1, 2, 5, 10, and 20 after alkali burn. Reciprocal bone marrow transplantation (BMT) was performed. Briefly, BM cells were obtained by flushing the tibia and femur of experimental TRPV1 KO and WT mice with PBS. A total of 2 × 106 WT BM cells were transplanted via tail vein infusion into recipient mice that had received whole-body irradiation of 12 Gy before BMT (from WT mice to KO mice or vice versa). The mice were subjected to alkali burn on the right eyes 3 weeks after BMT, as described earlier. Ten days later, the experimental mice were sacrificed and excised corneas were subjected to histology and IHC examination. Repopulation of transplanted BM was confirmed by RT-PCR detection of TRPV1 mRNA in the spleens of transplanted mice (not shown). To assess the percentage of macrophages derived from the transplanted BM in total macrophages in an alkali-burned, healing, corneal stroma with inflammation, we used a transgenic mouse with green fluorescent protein (GFP) expression (Riken, Tokyo, Japan). TRPV1−/−/GFP+/+ and TRPV1+/+/GFP+/+ mice were used as BM donors, and the recipient was a WT or a KO mouse. Three weeks after the BMT procedure, the cornea was affected by an alkali exposure as described earlier. Cryosections were cut and processed for F4/80 IHC (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) 10 days after the alkali treatment. After binding of tetramethyl rhodamine isothiocyanate (TRITC)-labeled secondary antibodies (1:200; Dako Cytomation, Carpinteria, CA), the specimens were observed under a microscope followed by mounting with VectaShield (Vector Laboratories, Burlingame, CA) for nuclear DAPI staining. We determined if the KO phenotype is reproduced by intraperitoneal injection into WT mice (n = 36) after a corneal alkali burn of one of two different TRPV1 antagonists. These antagonists [SB366791 (0.5 mg/kg)20Varga A. Németh J. Szabó A. McDougall J.J. Zhang C. Elekes K. Pintér E. Szolcsányi J. Helyes Z. Effects of the novel TRPV1 receptor antagonist SB366791 in vitro and in vivo in rat.Neurosci Lett. 2005; 385: 137-142Crossref PubMed Scopus (106) Google Scholar and JYL1421 (2.0 mg/kg)21Jakab B. Helyes Z. Varga A. Bölcskei K. Szabó A. Sándor K. Elekes K. Börzsei R. Keszthelyi D. Pintér E. Petho G. Németh J. Szolcsányi J. Pharmacological characterization of the TRPV1 receptor antagonist JYL1421 (SC0030) in vitro and in vivo in the rat.Eur J Pharmacol. 2005; 517: 35-44Crossref PubMed Scopus (43) Google Scholar] or their vehicle were administered daily until euthanasia. Ofloxacin ointment was administered topically twice a week to reduce the risk of bacterial infection. Infected eyes were excluded from the study. Eyes then were processed for histology or IHC at days 5, 10, and 20 after alkali burn. Paraffin sections (5 μm) were processed for H&E staining and IHC as previously reported.19Saika S. Ikeda K. Yamanaka O. Flanders K.C. Okada Y. Miyamoto T. Kitano A. Ooshima A. Nakajima Y. Ohnishi Y. Kao W.W. Loss of tumor necrosis factor a potentiates transforming growth factor b-mediated pathogenic tissue response during wound healing.Am J Pathol. 2006; 168: 1848-1860Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar The following antibodies were diluted in PBS: rabbit polyclonal anti-TRPV1 antibody (1:500; Neuromics), and mouse monoclonal anti–α smooth muscle actin (α-SMA) antibody (1:200; Neomarker, Fremont, CA). The presence of monocytes/macrophages was examined by using rat monoclonal F4/80 antimacrophage antigen antibody. Neutrophil presence was examined by using rabbit polyclonal myeloperoxidase (MPO) antibody (1:200; Neomarker). IHC for transforming growth factor β 1 (TGFβ1) was performed as previously reported.18Saika S. Miyamoto T. Yamanaka O. Kato T. Ohnishi Y. Flanders K.C. Ikeda K. Nakajima Y. Kao WW- Y. Sato M. Muragaki Y. Ooshima A. Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-B, in treatment of corneal alkali burns in mice.Am J Pathol. 2005; 166: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Flanders K.C. Ludecke G. Engels S. Cissel D.S. Roberts A.B. Kondaiah P. Lafyatis R. Sporn M.B. Unsicker K. Localization and actions of transforming growth factor-bs in the embryonic nervous system.Development. 1991; 113: 183-191PubMed Google Scholar The antibody used here detects only the active form of TGFβ1, but does not react with the latent form. Negative control staining was performed by omission of each primary antibody and did not yield specific staining (not shown). To semiquantify the expression level of F4/80, α-SMA, and fibronectin we also conducted Western blotting as previously reported.23Saika S. Ikeda K. Yamanaka O. Flanders K.C. Ohnishi Y. Nakajima Y. Muragaki Y. Ooshima A. Adenoviral gene transfer of BMP-7 Id2 or Id3 suppresses injury-induced epithelial-mesenchymal transition of lens epithelium in mice.Am J Physiol Cell Physiol. 2006; 290: C282-C289Crossref PubMed Scopus (73) Google Scholar, 24Saika S. Yamanaka O. Okada Y. Tanaka S. Miyamoto T. Sumioka T. Kitano A. Shirai K. Ikeda K. TGF beta in fibroproliferative diseases in the eye.Front Biosci (Schol Ed). 2009; 1: 376-390Crossref PubMed Google Scholar In brief, the corneas were harvested in Sigma Mammalian Tissue Lysis buffer (Sigma-Aldrich, St. Louis, MO) (50 μL/4 corneas) or the cells were harvested in Sigma-Aldrich Mammalian Cell Lysis buffer (100 μL/dish) and processed for SDS-PAGE and Western blotting for F4/80 (clone A3-1, 1:1000; BMA Biomedicals, August, Switzerland), α-SMA (1:2000; Neomarker), and fibronectin (1:500; Santa Cruz Biotechnology) as previously reported.23Saika S. Ikeda K. Yamanaka O. Flanders K.C. Ohnishi Y. Nakajima Y. Muragaki Y. Ooshima A. Adenoviral gene transfer of BMP-7 Id2 or Id3 suppresses injury-induced epithelial-mesenchymal transition of lens epithelium in mice.Am J Physiol Cell Physiol. 2006; 290: C282-C289Crossref PubMed Scopus (73) Google Scholar, 24Saika S. Yamanaka O. Okada Y. Tanaka S. Miyamoto T. Sumioka T. Kitano A. Shirai K. Ikeda K. TGF beta in fibroproliferative diseases in the eye.Front Biosci (Schol Ed). 2009; 1: 376-390Crossref PubMed Google Scholar The membrane then was stripped and restained for β-actin. Total RNA was extracted from corneal tissue excised from 4 burned mouse eyes using a Sigma RNA extraction kit (St. Louis, MO) according to the manufacturer's protocol and processed for qRT-PCR. The corneas were processed for total RNA extraction and qRT-PCR for collagen Ia1, α-SMA, F4/80, MPO, TGFβ1, vascular endothelial growth factor, monocyte/macrophage-chemoattractant protein-1 (MCP-1), IL-6, and SP.23Saika S. Ikeda K. Yamanaka O. Flanders K.C. Ohnishi Y. Nakajima Y. Muragaki Y. Ooshima A. Adenoviral gene transfer of BMP-7 Id2 or Id3 suppresses injury-induced epithelial-mesenchymal transition of lens epithelium in mice.Am J Physiol Cell Physiol. 2006; 290: C282-C289Crossref PubMed Scopus (73) Google Scholar qRT-PCR using the TaqMan one-step RT-PCR master mix reagents kit and the Applied Biosystems Prism 7300 (Applied Biosystems, Foster City, CA) were used. Primers and oligonucleotide probes used are listed in Table 1 and were designed according to the cDNA sequences in the GenBank database, using Primers Express software (Applied Biosystems, Foster City, CA). Data at each time point were analyzed for significance by using the Mann–Whitney U test.Table 1Primers and Oligonucleotide Probes UsedPrimerOligonucleotide Probeα-SMAMm01204962_ghF4/80Mm00802524_mlMPOMm01298422_glCollagen 1a1Mm00801666_glMCP-1Mm9999056_mlVEGFMm01281447_mlSPMm01166996_mlIL-6Mm01210732_glGAPDHMm03302249_glα-SMA, α-smooth muscle actin; MPO, myeloperoxidase; MCP-1, monocyte chemoattractant protein-1; VEGF, vascular endothelial growth factor; SP, substance P; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Open table in a new tab α-SMA, α-smooth muscle actin; MPO, myeloperoxidase; MCP-1, monocyte chemoattractant protein-1; VEGF, vascular endothelial growth factor; SP, substance P; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Mouse macrophages were obtained from the peritoneal cavity using a glycogen stimulation method.19Saika S. Ikeda K. Yamanaka O. Flanders K.C. Okada Y. Miyamoto T. Kitano A. Ooshima A. Nakajima Y. Ohnishi Y. Kao W.W. Loss of tumor necrosis factor a potentiates transforming growth factor b-mediated pathogenic tissue response during wound healing.Am J Pathol. 2006; 168: 1848-1860Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar In brief, 1 mL of 5% sterilized oyster glycogen (Sigma-Aldrich) was injected into the peritoneal cavity of either a WT or KO mouse. After 4 days, the peritoneal cavity was irrigated with culture medium to harvest macrophages. Approximately 90% of the cells obtained by this method were positive for F4/80. The cells in medium were allowed to adhere to 60-mm culture dishes for 6 hours, and the nonadherent cells were washed out with PBS. The RNA extracted from the adherent cells (macrophages) was analyzed by qRT-PCR for TGFβ1 mRNA. Five dishes were prepared for each condition. Data were analyzed statistically by using the nonpaired Student's t-test. The eye shells (including cornea and sclera) of WT and KO mice were minced and explanted in a 60-mm culture dish (Falcon; Fisher Scientific, Waltham, MA) on postnatal day 1 for eliciting outgrowth of ocular fibroblasts. The primary cultured cells were used directly without passage. Cells were grown to confluence and then treated with recombinant human TGFβ1 (1.0 ng/mL; R&D Systems, Minneapolis, MN) or vehicle control in the medium. Total RNA prepared from the cells was subjected to qRT-PCR to determine the expression levels of collagen Ia1, SP, IL-6, TGFβ1, vascular endothelial growth factor, MCP-1, and α-SMA expression. Five dishes were prepared for each condition. Data were analyzed statistically by analysis of variance. Another set of cultures was incubated for 24 or 48 hours with or without exogenous TGFβ1 at 1.0 ng/mL and was processed for Western blotting for fibronectin protein as previously reported.23Saika S. Ikeda K. Yamanaka O. Flanders K.C. Ohnishi Y. Nakajima Y. Muragaki Y. Ooshima A. Adenoviral gene transfer of BMP-7 Id2 or Id3 suppresses injury-induced epithelial-mesenchymal transition of lens epithelium in mice.Am J Physiol Cell Physiol. 2006; 290: C282-C289Crossref PubMed Scopus (73) Google Scholar, 24Saika S. Yamanaka O. Okada Y. Tanaka S. Miyamoto T. Sumioka T. Kitano A. Shirai K. Ikeda K. TGF beta in fibroproliferative diseases in the eye.Front Biosci (Schol Ed). 2009; 1: 376-390Crossref PubMed Google Scholar We used a co-culture model to determine whether fibrosis after an alkali burn corneal injury is caused by TRPV1 activation on corneal fibroblasts rather than infiltrating macrophages. Co-culture experiments were performed using these two cell types obtained from WT and KO mice as previously reported.19Saika S. Ikeda K. Yamanaka O. Flanders K.C. Okada Y. Miyamoto T. Kitano A. Ooshima A. Nakajima Y. Ohnishi Y. Kao W.W. Loss of tumor necrosis factor a potentiates transforming growth factor b-mediated pathogenic tissue response during wound healing.Am J Pathol. 2006; 168: 1848-1860Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar A suspension of WT or KO macrophages (2.4 × 106 cells) in culture medium supplemented with 3% fetal calf serum was added to confluent WT/KO fibroblast cultures in 60-mm dishes and further incubated for 24 hours, thereafter total RNA obtained from the cells was subjected to qRT-PCR for expression of collagen Ia1 mRNA. Four dishes were prepared as described. TRPV1 expression in the intact mouse cornea is restricted to the basal epithelial cell layer. On the other hand, TRPV1 also was detected in stromal cells of alkali-burned corneas healed for 10 days (Figure 1), suggesting that alkali burn activates stromal cell TRPV1 expression. To examine the role of TRPV1 in modulating wound healing of alkali-burned corneas, we first compared corneal haze development in the injured corneas of TRPV1 KO and WT mice. At each time point, the incidence and degree of epithelial defect/ulceration and opacification in the burned cornea were more severe in WT mice than those in TRPV1 KO mice (Figure 2A). The healing stroma was thicker in WT corneas as compared with KO corneas throughout the interval examined, suggesting the presence of more severe tissue swelling or edema in the presence of TRPV1. The eye globe diameters of alkali-burned eyes were determined after various periods of healing. WT globes have a smaller diameter at 20 days than those of KO mice (Figure 2B). This finding suggested that myofibroblast transdifferentiation is greater in the WT cornea as compared with KO corneas. To further characterize the tissue reaction to an alkali burn, we next conducted IHC and qRT-PCR to assess the variations in inflammation between WT and TRPV1 KO mice. The persistent and severe inflammation induced by alkali burn very markedly worsened the wound healing outcome in WT mice. For example, in WT mice there was a higher level of MPO (PMN marker) and F4/80 (macrophage marker) staining than that in KO mice (Figure 2C). Immunostaining with anti–α-SMA, a marker of myofibroblasts,24Saika S. Yamanaka O. Okada Y. Tanaka S. Miyamoto T. Sumioka T. Kitano A. Shirai K. Ikeda K. TGF beta in fibroproliferative diseases in the eye.Front Biosci (Schol Ed). 2009; 1: 376-390Crossref PubMed Google Scholar revealed that there was an immense increase in stromal myofibroblasts of alkali-burned corneas of WT mice at 5 to 20 days, in contrast, the majority of stromal cells of alkali-burned corneas of TRPV1 KO mice were negative for α-SMA (Figure 2C), suggesting that a much higher number of fibroblasts underwent transdifferentiation into myofibroblasts in the WT mice. We performed Western blotting for F4/80, α-SMA, and fibronectin. Expression of F4/80, α-SMA, and fibronectin was higher in WT tissue at 10 and 20 days after alkali burn (Figure 2D). Thus, we then performed qRT-PCR for mRNA expression of MPO, F4/80, and α-SMA to verify the immunostaining observations. The data confirmed that there were significantly fewer inflammatory cells in KO tissue than WT tissue at most time points, except for the presence of PMN at day 5 after alkali burn (Figure 2E). Therefore, the improved wound healing outcome in the KO mice is associated with less inflammation and myofibroblast transdifferentiation. qRT-PCR showed that lacking TRPV1 significantly suppressed the mRNA levels of IL-6, MCP-1, SP, and collagen Ia1 in the healing alkali-burned corneas at certain time point(s) throughout the wound closure interval (Figure 3). We immunostained the active form of TGFβ1 in tissue. Expression of active TGFβ1 was much more marked in a WT stroma as compared with a KO stroma at day 10 (Figure 4).Figure 4IHC of the active form of TGFβ1 in alkali-burned cornea. Expression of active TGFβ1 was much more marked in a WT stroma as compared with a KO stroma at day 10. Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There was no difference in the expression level of TGFβ1 mRNA between cultured WT and KO macrophages (Figure 5). Adding exogenous TGFβ1 up-regulated TRPV1 mRNA expression in WT ocular fibroblasts (Figure 6A). Any increase in mRNA expression levels induced by TGFβ1 was validated by showing that in KO ocular fibroblasts such effects were ablated (Figure 6, B–H). Loss of TRPV1 receptor reduced the mRNA expression level of TGFβ1 in ocular fibroblasts (Figure 6B). Expression of IL-6 mRNA was markedly up-regulated by adding exogenous TGFβ1, but such up-regulation was abolished by the loss of TRPV1 gene in the fibroblasts (Figure 6C). Expression of MCP-1 and vascular endothelial growth factor also was suppressed in ocular fibroblasts lacking TRPV1, but the expression pattern was not affected by exogenous TGFβ1 (Figure 6, D and E). There was no difference in the expression level of SP mRNA between cultured WT and KO ocular fibroblasts, and the expression pattern also was not affected by exogenous TGFβ1 (Figure 6F). Expression of the major fibrogenic markers, mRNAs of collagen Iα1 and α-SMA, was up-regulated by adding exogenous TGFβ1, but such up-regulation was abolished by the loss of TRPV1 gene in the fibroblasts (Figure 6, G and H). Western blotting also showed that fibronectin also was suppressed in ocular fibroblasts lacking TRPV1. Adding exogenous TGFβ1 up-regulated fibronectin in WT ocular fibroblasts, but such up-regulation was aboli
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