Inhibition of development of laser-induced choroidal neovascularization with suppression of infiltration of macrophages in Smad3-null mice.

We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-ß1 (TGFß1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8-12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFß1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFß1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFß1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFß1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.

Impaired cornea wound healing in a tenascin C-deficient mouse model.

We investigated the effects of loss of tenascin C on the healing of the stroma using incision-injured mice corneas. Tenascin C was upregulated in the stroma following incision injury to the cornea. Wild-type (WT) and tenascin C-null (knockout (KO)) mice on a C57BL/6 background were used. Cell culture experiments were also conducted to determine the effects of the lack of tenascin C on fibrogenic gene expression in ocular fibroblasts. Histology, immunohistochemistry and real-time reverse transcription PCR were employed to evaluate the healing process in the stroma. The difference in the incidence of wound closure was statistically analyzed in hematoxylin and eosin-stained samples between WT and KO mice in addition to qualitative observation. Healing of incision injury in corneal stroma was delayed, with less appearance of myofibroblasts, less invasion of macrophages and reduction in expression of collagen Iα1, fibronectin and transforming growth factor ß1 (TGFß1) in KO mice compared with WT mice. In vitro experiments showed that the loss of tenascin C counteracted TGFß1 acceleration of mRNA expression of TGFß1, and of collagen Iα1 and of myofibroblast conversion in ocular fibroblasts. These results indicate that tenascin C modulates wound healing-related fibrogenic gene expression in ocular fibroblasts and is required for primary healing of the corneal stroma.

A patient with corneal epithelial disorder that developed after administration of a latanoprost generic, but not a brand-name drug, eye drop.

Background. We report a patient who developed corneal epithelial disorder repeatedly after changing the prescription from Xalatan eye drops (Pfizer Inc.) to Latanoprost eye drops (Kaken Pharmaceutical Co., Ltd.), both containing 0.005% latanoprost. Case Report. An 88-year-old male with glaucoma had been treated with Timoptol eye drops and Xalatan eye drops for a few years. While he stayed in a health care facility for the elderly, Xalatan eye drops was changed to Latanoprost eye drops usage, and eye pain developed on the day of this change. On the next day, he visited our department, and corneal epithelial disorder was observed. The drops were discontinued, and the corneal epithelial disorder healed after 2 days. Twenty days after the first consultation, Xalatan eye drops and Latanoprost eye drops were resumed by a physician of internal medicine in the health care facility, but eye pain developed again. After discontinuation of the two drugs, Xalatan eye drops usage was resumed the next day, but no corneal epithelial disorder was observed thereafter. Conclusions. This clinical history strongly suggested the association between a generic drug, Latanoprost eye drops, and the development of corneal epithelial disorder.

Endogenous osteopontin involvement in laser-induced choroidal neovascularization in mice.

PURPOSE: To evaluate the effects of the lack of osteopontin (OPN) and the administration of anti-OPN antibody on inflammation and vascular endothelial growth factor (VEGF) expression in choroidal tissue and on the development of choroidal neovascularization (CNV) after retinal photocoagulation in mice. METHODS: CNV was induced in one eye each of 20 C57BL/6-background OPN-deficient mice or 20 wild-type littermates. In another series of experiments, CNV was induced in 40 C57BL/6 mice treated with intraperitoneal administration of 400 µg anti-OPN (SLAYGLR) neutralizing antibody or control IgG. Four laser spots were prepared in each eye. At day 14, the size of the CNV was evaluated by high-resolution angiography with fluorescein isothiocyanate (FITC)-dextran. Six wild-type or six knockout mice also received photocoagulation and processed for histology. mRNA expression of OPN, VEGF, and F4/80 macrophage antigen in laser-irradiated choroidal tissues was analyzed at day 3 in wild-type or knockout mice as well as in wild-type mice treated with anti-OPN antibody or control antibody. RESULTS: Photocoagulation upregulated OPN expression in choroidal tissue. Histology did not uncover the effects of the lack of OPN on the healing of laser injury in choroid. The lack of OPN or systemic administration of anti-OPN antibody suppressed mRNA expression of VEGF and macrophage invasion in choroidal tissue. FITC-dextran angiography showed that lacking OPN or systemic anti-OPN antibody reduced the size of laser-induced CNV. CONCLUSIONS: OPN is upregulated in laser-irradiated choroidal tissue. Endogenous OPN is required for macrophage inflammation and VEGF expression in choroidal tissue and for CNV development after retinal photocoagulation in mice.

Impaired angiogenic response in the cornea of mice lacking tenascin C.

PURPOSE: This study investigated the effects of loss of tenascin C (TNC) in the development of neovascularization in a corneal stroma in mice. Cell culture study was also conducted to clarify the roles of TNC in the expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)ß1 in fibroblasts and macrophages. METHODS: Ocular fibroblasts and macrophages from wild-type (WT) and TNC-null (KO) mice were used to study the role of TNC in the expression of VEGF and TGFß1. The effects of the absence of TNC on angiogenic gene expression, inflammatory cell invasion, and cornea neovascularization in the corneal stroma were then evaluated after cauterization of the center of the cornea in mice. Histologic, immunohistochemical, and mRNA expression analyses were performed. RESULTS: Absence of TNC suppressed expression of VEGF and counteracted upregulation of TGFß1 by exogenous TGFß1 in ocular fibroblast culture. Such effects of the absence of TNC were not observed in cultured macrophages. Absence of TNC attenuated expression of both VEGF and TGFß1 mRNA as well as neovascularization into the stroma after cauterization at the center of the cornea in mice. Absence of TNC suppressed macrophages, but not neutrophils, invading the cauterized cornea. CONCLUSIONS: TNC is involved in angiogenic gene expression in ocular fibroblasts in vitro and in vivo and is required for macrophage invasion and neovascularization of injured corneal stroma.

Suppression of injury-induced epithelial-mesenchymal transition in a mouse lens epithelium lacking tenascin-C.

PURPOSE: To investigate the role of tenascin-C in epithelial-mesenchymal transition (EMT) of the lens epithelium during wound healing in mice. Tenascin-C is a component of the extracellular matrix in patients having post-operative capsular opacification. METHODS: The crystalline lens was injured by needle puncture in tenascin-C null (KO, n=56) and wild-type (WT, n=56) mice in a C57BL/6 background. The animals were killed at day 2, 5, or 10 post-injury. Immunohistochemistry was employed to detect alpha-smooth muscle actin (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), phospho-Smad2, phospho-adducin, and phospho-myosin light chain 9 (MLC9). The expression levels of phospho-adducin and phospho-MLC9 were used as markers for the activation of protein kinase C and Rho kinase, respectively. RESULTS: The expression of tenascin-C was upregulated in WT lens epithelial cells adjacent to the capsular break at day 5. The results showed that injury-induced EMT of the mouse lens epithelium, as evaluated by histology and the expression patterns of alphaSMA and fibronectin, was attenuated in the absence of tenascin-C. Upregulation of TGFbeta1 expression in the epithelium was also inhibited, and loss of tenascin-C attenuated the phosphorylation of Smad2 and adducin in epithelial cells adjacent to the capsular break. The expression of phospho-adducin was suppressed, while the expression level of phospho-MLC9 was unchanged, in the healing epithelium in the absence of tenascin C. CONCLUSIONS: Tenascin-C is required for injury-induced EMT in the mouse lens epithelium. The mechanism behind this might involve impaired activation of cytoplasmic signaling cascades; i.e., TGFbeta/Smad and protein kinase C-adducing signaling, in the absence of tenascin-C.

Impaired angiogenic response in the corneas of mice lacking osteopontin.

PURPOSE: To investigate the effects of loss of osteopontin (OPN) in the development of neovascularization in corneal stroma in mice. Cell culture study was also conducted to clarify the effects of OPN in transforming growth factor (TGF) beta1-driven cell signaling and expression of vascular endothelial growth factor (VEGF). METHODS: Ocular fibroblasts from wild-type and OPN-null mice were used to study the role of OPN in TGFbeta1 signal and VEGF expression. The effect of the absence of OPN on corneal neovascularization was evaluated in mice. RESULTS: In ocular fibroblast culture, loss of OPN attenuated TGFbeta1 signals (Smad3 and p38) and reduced expression of VEGF. Loss of OPN attenuated neovascularization in corneal stroma in mice. CONCLUSIONS: OPN is involved in VEGF expression in cultured fibroblasts and is required for neovascularization in corneal stroma in vivo.

Expression of cyclooxygenase-2 in corneal cells after photorefractive keratectomy and laser in situ keratomileusis in rabbits.

PURPOSE: To compare the expression pattern of cyclooxygenase-2 (COX-2) in rabbit corneal cells after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) with the same refractive correction. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Thirty adult albino rabbits were used in the study. Photorefractive keratectomy or LASIK was performed in 1 eye of each animal for the same refractive correction. Each animal was killed after healing intervals up to 6 months. Paraffin sections of the cornea were processed for immunohistochemistry for COX-2 and NFkappaB (p65). RESULTS: After PRK, the central and peripheral corneal epithelia up-regulated COX-2 at 3 days; the central epithelium was positive at 4 weeks. Central and peripheral epithelia returned to negative 3 months later. After LASIK, the central epithelium on the corneal flap up-regulated COX-2 at 1 and 2 weeks; it returned to negative at 4 weeks. The peripheral epithelium was labeled with the antibody. Keratocytes around the stromal incision between the flap and the stromal bed up-regulated COX-2 and returned to negative at 3 months. COX-1 was not detected immunohistochemically in corneal tissue during the healing intervals after both procedures. Nuclear factor kappaB was detected in the cytoplasm and nuclei of migrating corneal epithelial cells 1 day after PRK, was positive in the cytoplasm at 3 days and negative in cytoplasm and nuclei at week and later. CONCLUSIONS: Migrating injured epithelium expressed COX-2 until week 4 during post-PRK healing. Central uninjured epithelium as well as stromal keratocytes expressed COX-2 from 3 days to 2 weeks after LASIK. Uninjured peripheral epithelium also expressed COX-2 at 4 weeks. Activation of stromal keratocytes may induce expression of COX-2 in overlying uninjured epithelium via the inflammatory cytokine(s)/NFkappaB pathway.

Topical exposure of mitomycin C reduces opacification of the residual anterior lens capsule and lenticular regeneration after vitrectomy and lensectomy in rabbits.

PURPOSE: To examine the effect of topical administration of mitomycin C (MMC) on the opacification of residual anterior lens capsule in a vitrectomized/lensectomized eye. METHODS: Adult Japanese albino rabbits ( n=8) received two-port vitrectomy and lensectomy with or without topical administration of MMC (0.4 mg/ml) to the residual anterior lens capsule in one eye under both general and topical anesthesia. Four eyes after vitrectomy and lensectomy were exposed to MMC for 1 min and washed out with balanced salt solution. After 1 month, the anterior segment of each globe was observed with EAS-1000 in vivo under general anesthesia and then the enucleated globes were observed through the Miyake view. Histological examination was performed. RESULTS: During healing intervals in eyes without MMC exposure, regenerated lens structure of Soemmering's ring or fibrous tissue was formed in peripheral or central areas of the residual capsule, respectively. Both the anterior lens capsule opacification and lens fiber regeneration were statistically significantly reduced by MMC topical exposure. CONCLUSION: Lens epithelial cells produce regenerated lenticular structure and fibrous tissue on the residual capsule following vitrectomy and lensectomy in rabbits. Exposure of the residual anterior capsule to MMC may be effective in reducing both its opacification and lens fiber regeneration after vitrectomy/lensectomy.