Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

Purpose. The repair of the corneal epithelium is modulated by matrix metalloproteinases. The present study examined the effects of recombinant (r-) tissue inhibitors of metalloprotein ases-1 and -2 (TIMP-1 and -2) on the proliferation of cultured epithelial cells from rabbit cornea, and on the spreading of a sheet of squamous epithelium of rabbit cornea placed in an organ culture system. Methods. DNA synthesis of the cells, with or without r-TIMPs, was determined by an immunoassay for BrdU incorporation. Cell proliferation was assayed by measuring MTT mitochondrial activity. Epithelial spreading was evaluated by culturing small corneal blocks for 24 h in the presence or absence of each agent. Cryosections were prepared and the epithelial growth on the cut stromal surface was measured. Results. Each agent, r-TIMP-1 (at 50 and 100 ng/ml) and r-TIMP-2 (at 50 ng/ml) significantly enhanced the DNA synthesis and MTT activity of the corneal epithelial cells in vitro. Relative to the untreated control cells, DNA synthesis was increased 2.4-fold by r-TIMP-1 and 2.3-fold by r-TIMP-2. r-TIMP-1 (at 100 and 200 ng/ml) and r-TIMP-2 (at 10 and 50 ng/ml) each significantly enhanced the spreading of the corneal epithelium. Relative to the untreated control tissue, spreading of the epithelial sheet was increased 1.7-fold by r-TIMP-1 and 1.4-fold by r-TIMP-2. Higher concentrations of r-TIMP-1 and r-TIMP-2 did not further enhance either the DNA synthesis of the cultured cells or the spreading of the epithelium. Conclusions. Exogenous TIMPs enhanced the spreading of the corneal epithelium and proliferation of cultured corneal epithelial cells. Findings suggest that endogenous TIMPs may influence the healing of corneal epithelium in vivo.

METHODS. An in vitro model of angiogenesis was used to examine the roles of TNF on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor 1 (TGF 1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNF -null (Tnf ) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription– polymerase chain reaction.

PURPOSE To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in lens epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS Two days post-burn of the ocular surface, lens epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type lens cells. The majority of myofibroblast-type lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the lens epithelium. CONCLUSIONS Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the lens epithelium in this cataract model.

Smads2 and 3 transduce signals of TGF-β from the cell surface to the nucleus. We used mice with a targeted deletion of Smad3 to study the specific contributions of this signaling pathway to pathogenic effects of TGF-β. Focusing on models involving epithelial-to-mesenchymal transition (EMT), including injury to the lens and retina of the eye and to the kidney, we have found that loss of Smad3 blocks EMT and attenuates development of fibrotic sequelae. Smad3 also plays a critical role in both the tumor suppressor and pro-metastatic effects of TGF-β in carcinogenesis. These observations suggest that development of small molecule inhibitors of Smad3 might have clinical application in treatment of fibrotic diseases as well as late stage cancers.

Purpose : We examined the role of interleukin-7 (IL-7) in modulation of production of extracellular matrix (ECM), immunolocalization of Smads, and cell migration and expressions of transforming growth factor-beta (TGF-beta) in cultured human subconjunctival fibroblasts. IL-7 is capable of inducing Smad7, an inhibitory Smad that interferes with TGF-beta/Smad signal. Methods : The effects of IL-7 on ECM production, immunolocalization of Smads, type I collagen, fibronectin, alpha -smooth muscle actin (alpha -SMA), and cell migration were examined in human subconjunctival fibroblast culture with or without TGF-beta 1. ECM production, such as type I collagen and fibronectin, was measured by immunoassay or real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cell migration was examined using an in vitro wound model in monolayer cultures. We also examined the effects of IL-7, PKC inhibitor, and STAT inhibitor on the expressions of TGF-beta 1 and type I collagen alpha 1 chain (col1A1) m-RNA by using real-time RT-PCR. Results : IL-7 reduced the ECM production much more markedly in the cells treated with TGF-1 beta than in the control fibroblasts. TGF-beta 1 strongly showed immunolocalization of phospho-Smad2, and IL-7 also showed immunolocalization of Smad7 in the nuclei. The immunoreactivities of alpha -SMA and fibronectin were weaker in the presence of IL-7 than in the control cells. IL-7 also delayed defect closure in the monolayer cell sheets, and the delay was recovered by exogenous type I collagen or fibronectin. Each of IL-7, BIS I, or AGS 490 reduced the mRNA expressions of TGF-beta 1 and col1A1. Conclusions : These findings indicate that IL-7 is involved in ECM production in the subconjunctival fibroblasts activated by exogenous TGF-beta 1, suggesting that administration of IL-7 can be a novel therapeutic strategy in preventing undesirable bleb scar formation during healing after filtration surgery.

We have examined the effect of adenovirus-mediated expression of bone morphogenic protein-7 (BMP-7) and inhibitors of differentiation 2 and 3 (Id2 and Id3) on injury-induced epithelial-to-mesenchymal transition (EMT) of lens epithelium in mice. Id2 and Id3 are known to be upregulated by BMP-7 and to antagonize Smad2/3 signaling. The Cre-LoxP system adenoviral gene transfer was used. Three microliters of adenoviral solution (2 x 10(7) PFU/mul) were injected into the right lens of adult male C57BL/6 mice (n = 144) at the time of capsular injury induced using a hypodermic needle under both general and topical anesthesia. A mixture of Cre-adenovirus (Cre-Ad) and vector encoding mBMP-7, mId2, or mId3 was administered in a test group. Control lenses were treated with Cre-Ad alone. After healing intervals of 5 or 10 days, the animals were killed and then we performed histological processes or RNA extraction from the lens. RT-PCR, real-time RT-PCR, and immunohistochemistry showed expression of each introduced gene in the lens. Exogenous BMP-7 upregulated expression of Id2 and Id3 in injured lenses, and gene introduction of Id2 or Id3 also upregulated BMP-7 expression. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of the lens epithelial cells as evaluated by histology and expression patterns of alpha-smooth muscle actin and collagens in association with reduction of Smad2 COOH-terminal phosphorylation. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of lens epithelial cells and subsequent sealing of the capsular break with fibrous tissue in mice.

Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-alpha, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-alpha signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-alpha. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-alpha gradient serving as a morphogen of eyelid morphogenesis.