Carbon-ion radiotherapy for locally advanced primary or postoperative recurrent epithelial carcinoma of the lacrimal gland.

PURPOSE: To evaluate the applicability of carbon ion beams for the treatment of carcinoma of the lacrimal gland with regard to normal tissue morbidity and local tumor control. METHODS AND MATERIALS: Between April 2002 and January 2011, 21 patients with locally advanced primary epithelial carcinoma of the lacrimal gland were enrolled in a Phase I/II clinical trial of carbon-ion radiotherapy (CIRT) at the National Institute of Radiological Sciences. Acute radiation toxicity was the primary endpoint of this dose-escalation study and the late toxicity, local control, and overall survival were additionally evaluated as secondary endpoints. Of the 21 subjects enrolled, all patients were followed for more than 6 months and analyzed. RESULTS: The radiation dose was increased from the initial dose of 48.0Gy equivalents (GyE)/12 fractions at 10% increments up to 52.8GyE. Of the 21 patients, five received a total dose of 48.0GyE, and 16 received a total dose of 52.8GyE. No patient developed grade 3 or higher skin toxicity. As late ocular/visual toxicity, three patients had grade 3 retinopathy and seven patients lost their vision. Among the 10 patients treated until May 2005, five patients had local recurrence, three of whom had marginal recurrence. Therefore, the margin for the CTV (clinical target volume) was set to a range according to the orbital exenteration since June 2005. After the application of the extended margin, no local recurrence has been observed. The three-year overall survival and local control rates were 82.2% and 79.0%, respectively. CONCLUSION: CIRT can be applied for primary epithelial carcinoma of the lacrimal gland, with a borderline acceptable morbidity and sufficient antitumor effect when an extended margin is adopted.

Long-term results of carbon ion radiation therapy for locally advanced or unfavorably located choroidal melanoma: usefulness of CT-based 2-port orthogonal therapy for reducing the incidence of neovascular glaucoma.

PURPOSE: To determine the long-term results of carbon ion radiation therapy (C-ion RT) in patients with choroidal melanoma, and to assess the usefulness of CT-based 2-port irradiation in reducing the risk of neovascular glaucoma (NVG). METHODS AND MATERIALS: Between January 2001 and February 2012, a total of 116 patients with locally advanced or unfavorably located choroidal melanoma received CT-based C-ion RT. Of these patients, 114 were followed up for more than 6 months and their data analyzed. The numbers of T3 and T2 patients (International Union Against Cancer [UICC], 5th edition) were 106 and 8, respectively. The total dose of C-ion RT varied from 60 to 85 GyE, with each dose given in 5 fractions. Since October 2005, 2-port therapy (51 patients) has been used in an attempt to reduce the risk of NVG. A dose-volume histogram analysis was also performed in 106 patients. RESULTS: The median follow-up was 4.6 years (range, 0.5-10.6 years). The 5-year overall survival, cause-specific survival, local control, distant metastasis-free survival, and eye retention rates were 80.4% (95% confidence interval 89.0%-71.8%), 82.2% (90.6%-73.8%), 92.8% (98.5%-87.1%), 72.1% (81.9%-62.3%), and 92.8% (98.1%-87.5%), respectively. The overall 5-year NVG incidence rate was 35.9% (25.9%-45.9%) and that of 1-port group and 2-port group were 41.6% (29.3%-54.0%) and 13.9% (3.2%-24.6%) with statistically significant difference (P<.001). The dose-volume histogram analysis showed that the average irradiated volume of the iris-ciliary body was significantly lower in the non-NVG group than in the NVG group at all dose levels, and significantly lower in the 2-port group than in the 1-port group at high dose levels. CONCLUSIONS: The long-term results of C-ion RT for choroidal melanoma are satisfactory. CT-based 2-port C-ion RT can be used to reduce the high-dose irradiated volume of the iris-ciliary body and the resulting risk of NVG.

The sensitivity and specificity of a new formula to distinguish endometrioid type endometrial carcinoma from ovarian endometrial carcinoma.

OBJECTIVES: Endometrioid type adenocarcinoma sometimes occupies both endometrium and ovary and in some cases the origin cannot be determined. STUDY DESIGN: In this study, we established a formula to distinguish ovarian endometrioid cancer (EOC) from endometrioid type endometrial cancer (EEC), based on our previous report of cyclin and KI67 expression pattern by immunohistochemistry of 36 EECc and 37 OECc by the logistic regression. We calculated the diagnostic accuracy using 92 test samples retrospectively and finally could diagnose the origin of 16 cases in whom endometrioid type adenocarcinoma arose in both ovary and endometrium and could be determined by Scully's criteria, and 15 cases in whom endometrioid type adenocarcinoma arose in both ovary and endometrium and Scully's criteria were not useful retrospectively. RESULTS: The estimated formula is as follows: Logit(Prob(EOC))=-1.1437-0.0853 CNA+0.0423 CNB+0.173 CND1+0.0129 CNE+0.0224 CNF+0.0508 KI67, where Prob(EOC) is the probability that a clinical sample is EOC. If Prob(EOC) is larger than 0.5, the diagnosis is ovarian cancer; if less than 0.5 it is endometrial cancer. Finally, using the formula, 37 of 48 EECs (77.1%) and 33 of 44 EOCs (75.0%) were correctly classified, with an accuracy of 76.1% (p<0.0001), retrospectively. In 12 of the 16 cases (75%) who could be determined by Scully's criteria, the origin determined by Scully's criteria was concordant with the origin determined by the formula retrospectively. In the other 15 cases, 12 cases were judged as ovary/ovary, 2 cases were judged as uterus/uterus and 1 case was judged as uterus/ovary. CONCLUSION: The formula we established was thought to be useful to distinguish the origin of the cases in whom endometrioid type adenocarcinoma arises in both ovary and endometrium.

Case of atypical fibroxanthoma in the palpebral conjunctiva.

BACKGROUND: We report a case of atypical fibroxanthoma that developed in the palpebral conjunctiva. CASE: A 94-year-old woman had a hemorrhagic tumor in the right lower palpebral conjunctiva that was resected, and adjunctive cryotherapy was applied to the surgical bed. OBSERVATIONS: The tumor was bleeding and appeared as a pale red, elastic but firm nodule approximately 15x16x8 mm in size. It was composed mainly of fibroblast-like cells and pleomorphic histiocyte-like cells. A storiform pattern was observed in the fibroblast-like cells. The tumor stained positive for vimentin, CD68, and CD10, weakly for CD74 and CD99, and was negative for keratin (wide), KL-1, alpha-fetoprotein, myoglobin, S-100, alpha-smooth muscle actin, desmin, leukocyte common antigen, and glial fibrillary acidic protein immunohistochemically. The MIB-1 index was about 10%. From these findings, we diagnosed the tumor as an atypical fibroxanthoma. There has been no recurrence in the 2 years since the resection. CONCLUSIONS: An atypical fibroxanthoma in the palpebral conjunctiva is very rare. The clinical presentation and histological and immunohistochemical studies are helpful in distinguishing between an atypical fibroxanthoma and a malignant fibrous histiocytoma.

Connective tissue growth factor modulates extracellular matrix production in human subconjunctival fibroblasts and their proliferation and migration in vitro.

PURPOSE: We examined the role of connective tissue growth factor (CTGF) in transforming growth factor beta1 (TGFbeta1)-related behavior in cultured human subconjunctival fibroblasts (SCFs), protein production, mRNA expression of CTGF and type I collagen alpha1 chain (colIA1), and cell proliferation and migration. TGFbeta1 is the major factor involved in bleb scarring following filtration surgery. METHODS: An antisense deoxynucleotide (antisense) (5 microM) for CTGF mRNA was used to block endogenous CTGF expression. Effects of antisense on extracellular matrix (ECM) production and immunolocalization, mRNA expression, and cell proliferation and migration were examined in human SCF cultures with or without TGFbeta1 (5 ng/ml). Cell migration was examined in an in vitro wound model of monolayer fibroblast cultures. RESULTS: CTGF antisense reduced mRNA expression of CTGF and colIA1 and production of the ECM components type I collagen, and fibronectin much more markedly in cells treated with TGFbeta1 compared with control fibroblasts, and it inhibited the proliferation of cultured SCFs to 71.9% of that of controls after 13 days of culture. CTGF antisense also delayed defect closure in monolayer cell sheets. In the culture, the defect was closed by TGFbeta1 by 36 h, whereas 7.0% of the defect remained at 48 h in the antisense-treated culture. CONCLUSIONS: These findings indicate that CTGF is involved in ECM production in SCFs activated by exogenous TGFbeta1 in vitro. Inhibition of CTGF expression may be effective in preventing undesirable scar formation during healing following filtration surgery.

Inhibition of p38MAP kinase suppresses fibrogenic reaction in conjunctiva in mice.

PURPOSE: To examine the effects of blocking p38 mitogen-activated protein kinase (MAPK) on post-injury conjunctival scarring in mice. Its effects on the behaviors of cultured subconjunctival fibroblasts were also investigated. METHODS: An in vivo study was conducted using an adenoviral vector carrying a dominant-negative (DN)-p38MAPK gene. A circumferential incision was made in the equatorial conjunctiva by scissors in the right eye of generally anesthetized adult C57BL/6 mice. DN-p38MAPK-expressing adenoviral vector was topically applied. The left control eye received non-functioning adenoviral vector. At 2, 5, and 7 days (each, n=22) the eyes were processed for histological or immunohistochemical examination to evaluate the tissue scarring. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of p38MAPK inhibitor on the proliferation, migration, and fibrogenic gene/protein expression of cultured human fibroblasts were also studied. RESULTS: The in vivo DN-p38MAPK gene introduction blocked the phospho-p38 expression with reduction of myofibroblast generation and suppression of mRNA expression of connective tissue growth factor (CTGF) and monocyte/macrophage chemoattractant protein-1 (MCP-1) in the mouse-injured conjunctiva. Blocking p38MAPK signal in the fibroblasts by a chemical inhibitor counteracted TGFbeta1's enhancement of expressions of type-I collagen, fibronectin, and CTGF. It also retarded cell migration, but cell proliferation was unchanged. CONCLUSIONS: Inhibiting p38MAPK signal impairs the fibrogenic reaction induced by the subconjunctival fibroblasts in vivo and in vitro, suggesting its potential effectiveness in preventing excessive scarring following glaucoma filtering surgery.

Effect of overexpression of PPARgamma on the healing process of corneal alkali burn in mice.

Wound healing involves both local cells and inflammatory cells. Alkali burn of ocular surface tissue is a serious clinical problem often leading to permanent visual impairment resulting from ulceration, scarring and neovascularization during healing. Behaviors of corneal cells and inflammatory cells are orchestrated by growth factor signaling networks that have not been fully uncovered. Here we showed that adenoviral gene introduction of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits activation of ocular fibroblasts and macrophages in vitro and also induced anti-inflammatory and anti-fibrogenic responses in an alkali-burned mouse cornea. PPARgamma overexpression suppressed upregulation of inflammation/scarring-related growth factors and matrix metalloproteinases (MMPs) in macrophages. It also suppressed expression of such growth factors and collagen Ialpha2 and myofibroblast generation upon exposure to TGFbeta1. Exogenous PPARgamma did not alter phosphorylation of Smad2, but inhibited its nuclear translocation. PPARgamma overexpression enhanced proliferation of corneal epithelial cells, but not of fibroblasts in vitro. Epithelial cell expression of MMP-2/-9 and TGFbeta1 and its migration were suppressed by PPARgamma overexpression. In vivo experiments showed that PPARgamma gene introduction suppressed monocytes/macrophages invasion and suppressed the generation of myofibroblasts, as well as upregulation of cytokines/growth factors and MMPs in a healing cornea. In vivo re-epitheliazation with basement membrane reconstruction in the healing, burned, cornea was accelerated by PPARgamma-Ad expression, although PPARgamma overexpression was considered to be unfavorable for cell migration. Together, these data suggest that overexpression of PPARgamma may represent an effective new strategy for treatment of ocular surface burns.

Endogenous TNFalpha suppression of neovascularization in corneal stroma in mice.

PURPOSE: To examine the role of tumor necrosis factor alpha (TNFalpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro. METHODS: An in vitro model of angiogenesis was used to examine the roles of TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) 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. RESULTS: HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice. CONCLUSIONS: In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.

Effect of Smad7 gene overexpression on transforming growth factor beta-induced retinal pigment fibrosis in a proliferative vitreoretinopathy mouse model.

OBJECTIVE: To determine the effects of Smad7 gene transfer in the prevention of fibrogenic responses by the retinal pigment epithelium, a major cause of proliferative vitreoretinopathy after retinal detachment, in mice. METHODS: Retinal detachment-induced proliferative vitreoretinopathy in a mouse model. Forty-eight eyes received either an adenoviral gene transfer of Smad7 or Cre recombinase gene only. The eyes were histologically analyzed. A retinal pigment epithelial cell line, ARPE-19, was used to determine whether Smad7 gene transfection suppresses the fibrogenic response to transforming growth factor (TGF) beta2 exposure. RESULTS: The Smad7 gene transfer inhibited TGF-beta2/Smad signaling in ARPE-19 cells and expression of collagen type I and TGF-beta1 but had no effect on their basal levels. In vivo Smad7 overexpression resulted in suppression of Smad2/3 signals and of the fibrogenic response to epithelial-mesenchymal transition by the retinal pigment epithelium. CONCLUSION: Smad7 gene transfer suppresses fibrogenic responses to TGF-beta2 by retinal pigment epithelial cells in vitro and in vivo. Clinical Relevance Smad7 gene transfer might be a new strategy to prevent and treat proliferative vitreoretinopathy.

AP-1 expression in ethanol-treated corneal epithelium in vivo.

PURPOSE: To examine the expression pattern of stress-related genes, c-fos and c-jun, both the major components of activator protein-1 (AP-1), in rat corneal epithelium treated with a short-term ethanol exposure. The purpose of the current study was to examine if the ethanol exposure during laser epithelial keratomileusis (LASEK) may stimulate or damage the corneal epithelial cells. METHOD: Sixty male Wistar rats were used. Fifty microliters of 20% ethanol was placed onto a surface 2.4 mm in diameter of the central corneal epithelium for 30 s. The affected eyes, washed with saline, were then enucleated after various intervals of healing. To know the expression pattern of c-fos and c-jun mRNAs and c-Fos, c-Jun and Jun D proteins, in situ hybridization and immunohistochemistry were carried out. The expression level of c-fos and c-jun mRNAs was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Apoptotic nuclei in the tissue sections were identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. RESULTS: Thirty to 60 min after the treatment, c-fos and c-jun mRNAs were detected in the corneal epithelium. These signals were no longer evident at 90 min. c-Fos protein was detected in the corneal epithelium around the area of ethanol exposure from 60 to 120 min after the treatment, while c-Jun protein was not detected. Jun D protein was detected in control whole corneal epithelium and not affected by ethanol exposure in the periphery. The levels of c-fos and c-jun mRNAs were increased approximately 8 times at 30 min compared with the control level. TUNEL-positive apoptotic nuclei in the tissue sections were identified. CONCLUSION: Corneal epithelial cells, especially those surrounding the ethanol-exposed area, are transiently transcriptionally activated at a very early phase after the ethanol exposure. mRNA expression for c-fos is followed by protein synthesis, but that of c-jun is not followed by protein synthesis. Resistance of Jun D protein expression to ethanol suggests that it might be a candidate for an AP-1 complex with c-Fos.

Loss of osteopontin perturbs the epithelial-mesenchymal transition in an injured mouse lens epithelium.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

Carbon-ion radiotherapy for locally advanced or unfavorably located choroidal melanoma: a Phase I/II dose-escalation study.

PURPOSE: To evaluate the applicability of carbon ion beams for the treatment of choroidal melanoma with regard to normal tissue morbidity and local tumor control. METHODS AND MATERIALS: Between January 2001 and February 2006, 59 patients with locally advanced or unfavorably located choroidal melanoma were enrolled in a Phase I/II clinical trial of carbon-ion radiotherapy at the National Institute of Radiologic Sciences. The primary endpoint of this study was normal tissue morbidity, and secondary endpoints were local tumor control and patient survival. Of the 59 subjects enrolled, 57 were followed >6 months and analyzed. RESULTS: Twenty-three patients (40%) developed neovascular glaucoma, and three underwent enucleation for eye pain due to elevated intraocular pressure. Incidence of neovascular glaucoma was dependent on tumor size and site. Five patients had died at analysis, three of distant metastasis and two of concurrent disease. All but one patient, who developed marginal recurrence, were controlled locally. Six patients developed distant metastasis, five in the liver and one in the lung. Three-year overall survival, disease-free survival, and local control rates were 88.2%, 84.8%, and 97.4%, respectively. No apparent dose-response relationship was observed in either tumor control or normal tissue morbidity at the dose range applied. CONCLUSION: Carbon-ion radiotherapy can be applied to choroidal melanoma with an acceptable morbidity and sufficient antitumor effect, even with tumors of unfavorable size or site.

Risk factors for neovascular glaucoma after carbon ion radiotherapy of choroidal melanoma using dose-volume histogram analysis.

PURPOSE: To determine the risk factors for neovascular glaucoma (NVG) after carbon ion radiotherapy (C-ion RT) of choroidal melanoma. METHODS AND MATERIALS: A total of 55 patients with choroidal melanoma were treated between 2001 and 2005 with C-ion RT based on computed tomography treatment planning. All patients had a tumor of large size or one located close to the optic disk. Univariate and multivariate analyses were performed to identify the risk factors of NVG for the following parameters; gender, age, dose-volumes of the iris-ciliary body and the wall of eyeball, and irradiation of the optic disk (ODI). RESULTS: Neovascular glaucoma occurred in 23 patients and the 3-year cumulative NVG rate was 42.6 +/- 6.8% (standard error), but enucleation from NVG was performed in only three eyes. Multivariate analysis revealed that the significant risk factors for NVG were V50IC (volume irradiated > or =50 GyE to iris-ciliary body) (p = 0.002) and ODI (p = 0.036). The 3-year NVG rate for patients with V50IC > or =0.127 mL and those with V50IC <0.127 mL were 71.4 +/- 8.5% and 11.5 +/- 6.3%, respectively. The corresponding rate for the patients with and without ODI were 62.9 +/- 10.4% and 28.4 +/- 8.0%, respectively. CONCLUSION: Dose-volume histogram analysis with computed tomography indicated that V50IC and ODI were independent risk factors for NVG. An irradiation system that can reduce the dose to both the anterior segment and the optic disk might be worth adopting to investigate whether or not incidence of NVG can be decreased with it.

Cyclooxygenase 2 expression in rat corneas after ethanol exposure.

PURPOSE: To evaluate the effects of ethanol exposure of the cornea on inflammation in corneal epithelium. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: One cornea of Wistar rats (n = 60) was exposed to ethanol 20% for 30 seconds. The animals were killed 0.5, 1.0, 1.5, 2.0, 6.0, 12.0, 24.0, 48.0, or 72.0 hours or 7 days after treatment. The paraffin section or cryosection of the treated eyes was processed for histology; immunohistochemistry for cyclooxygenase 2 (COX2); p65 subunit of nuclear factor kappa B (NF-kappaB), which is the major transcription factor involved in COX2 expression; phospho-IkappaB; or in situ hybridization for COX2 mRNA. RESULTS: In the uninjured corneas, faint immunoreactivity for COX2 was detected in the basal cells of the corneal epithelium, but not in other cell layers. Cyclooxygenase 2 mRNA was not observed in the injured epithelium; it was expressed 2 hours after ethanol exposure, but not 3 hours or later after treatment. The COX2 protein was detected in the corneal epithelium throughout the epithelial layers from 3 to 72 hours, but not at 7 days. The p65 of NF-kappaB translocated to the nuclei of corneal epithelium 3 to 24 hours after treatment but was not seen in the nuclei 48 hours after treatment. Phospho-I kappaB was detected in corneal epithelium 6 hours after treatment, but not 12 hours or later. CONCLUSION: Ethanol exposure activated NF-kappaB and upregulated COX2 expression, which may cause inflammation in corneal tissue.

Genipin suppression of fibrogenic behaviors of the alpha-TN4 lens epithelial cell line.

PURPOSE: To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium. RESULTS: Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic. CONCLUSION: Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.

Gene transfer of Smad7 modulates injury-induced conjunctival wound healing in mice.

PURPOSE: Smad7 is a molecule that blocks the Smad2/3 signal. Herein, we examined the effects of Smad7 gene introduction on post-injury conjunctival wound healing in mice. Its effects on the cultured human subconjunctival fibroblasts (SCFs) were also investigated. METHODS: A circumferential incision was made in the equatorial conjunctiva by using scissors in the right eye of fully anesthetized adult C57BL/6 mice (n=72). Smad7 cDNA-expressing adenoviral vector was topically applied. The control eye received nonfunctioning adenoviral vector. After 2, 5, 7, and 30 days the eyes were processed for histological or immunohistochemical examination to evaluate wound healing of conjunctiva. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of Smad7 gene introduction on the cultured human SCFs were also studied. RESULTS: Marked Smad7 protein expression was detected in the vector-treated conjunctival epithelium and fibroblasts that coincided with green fluorescein protein expression, whereas faint endogenous Smad7 expression was observed in the control tissue. In vivo Smad7 gene introduction blocked Smad2/3 nuclear translocation with suppression of alpha-smooth muscle actin (alphaSMA) and vascular endothelial growth factor (VEGF) in fibroblasts and invasion of macrophages. Smad7 gene transfer suppressed mRNA expressions of connective tissue growth factor (CTGF), VEGF, and monocyte chemoattractant protein-1 in vivo and those of type-I collagen, alphaSMA, and CTGF in vitro. CONCLUSIONS: Smad7 gene transfer modulates injury-induced wound healing of conjunctival tissue in mice, suggesting that this strategy may be effective in preventing excessive scarring following filtration surgery. The mechanism might include suppression of activation of fibroblasts and reduction of macrophage invasion.

Treatment of eyelid epithelial neoplasm by targeting sonic hedgehog signaling: an experimental study.

PURPOSE: To evaluate the effect of cyclopamine, an inhibitor of the Sonic hedgehog (Shh) signal, on the growth of an epithelial neoplasm. METHODS: Chemically induced eyelid tumors in XPC-null mice (n=40) were treated daily with a subcutaneous injection of cyclopamine (1 mg/animal) for 7 days. The animals were killed after bromodeoxyuridine (BrdU) labeling, and the tumors were histologically examined. An in vitro study was conducted by using a squamous cell carcinoma (SCC) cell line. The SCC cells were treated with 0, 12.5, or 25.0 microg/ml recombinant Shh (rShh) and either 0 or 100 microM cyclopamine, and cell proliferation was evaluated by using an MTT assay. Cells from this cell line were also implanted subcutaneously in nude mice (n=8) to develop tumors, and the effect of cyclopamine administration was examined in the developed tumors. RESULTS: Histology showed that cyclopamine treatment suppressed BrdU incorporation and induced apoptosis in the majority of cells in tumors chemically induced in the eyelid of the XPC-null mice. Cell proliferation of the SCC cell line was enhanced by adding rShh, and this effect was abolished by adding cyclopamine. Proliferation of the SCC cell line was not affected by adding cyclopamine in the absence of rShh. On the other hand, the SCC cells expressed Shh in vivo in tumors developed in nude mice, but cyclopamine suppressed cell proliferation in the tumors, and the Shh-signaling pathway was inhibited by cyclopamine-induced apoptosis. CONCLUSIONS: Cyclopamine inhibits proliferation and induces apoptosis in epithelial tumor cells in vivo. The Shh-signaling pathway may be a potential therapeutic target for patients with eyelid tumors.

A new model of anterior subcapsular cataract: involvement of TGFbeta/Smad signaling.

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.

Interleukin-7 modulates extracellular matrix production and TGF-beta signaling in cultured human subconjunctival fibroblasts.

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-beta1. 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-beta1 and type I collagen alpha1 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-1beta than in the control fibroblasts. TGF-beta1 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-beta1 and col1A1. CONCLUSIONS: These findings indicate that IL-7 is involved in ECM production in the subconjunctival fibroblasts activated by exogenous TGF-beta1, suggesting that administration of IL-7 can be a novel therapeutic strategy in preventing undesirable bleb scar formation during healing after filtration surgery.

Loss of tumor necrosis factor alpha potentiates transforming growth factor beta-mediated pathogenic tissue response during wound healing.

Animal cornea is an avascular transparent tissue that is suitable for research on wound healing-related scarring and neovascularization. Here we show that loss of tumor necrosis factor alpha (TNFalpha) potentiates the undesirable, pathogenic response of wound healing in an alkali-burned cornea in mice. Excessive invasion of macrophages and subsequent formation of a vascularized scar tissue were much more marked in TNFalpha-null knockout (KO) mice than in wild-type mice. Such an unfavorable outcome in KO mice was abolished by Smad7 gene introduction, indicating the involvement of transforming growth factor beta or activin/Smad signaling. Bone marrow transplantation from wild-type mice normalized healing of the KO mice, suggesting the involvement of bone marrow-derived inflammatory cells in this phenomenon. Co-culture experiments showed that loss of TNFalpha in macrophages, but not in fibroblasts, augmented the fibroblast activation as determined by detection of alpha-smooth muscle actin, the hallmark of myofibroblast generation, mRNA expression of collagen Ialpha2 and connective tissue growth factor, and detection of collagen protein. TNFalpha in macrophages may be required to suppress undesirable excessive inflammation and scarring, both of which are promoted by transforming growth factor beta, and for restoration of tissue architecture in a healing alkali-burned cornea in mice.