Osamu Yamanaka - Academia.edu (original) (raw)
Papers by Osamu Yamanaka
The American Journal of Pathology, 2005
PURPOSE Smad7 is a molecule that blocks the Smad2/3 signal. Herein, we examined the effects of Sm... more 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 epith...
Cell and Tissue Research
The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repa... more The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor β1 (TGFβ1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFβ1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFβ-induced granulation tissue formation.
The Ocular Surface
To examine effects of alkali injury of the ocular surface on meibomian gland pathology in mice. T... more To examine effects of alkali injury of the ocular surface on meibomian gland pathology in mice. Three μL of 1 N NaOH were applied under general anesthesia to the right eye of 10-week-old BALB/c (n = 54) mice to produce a total ocular surface alkali burn. The meibomian gland morphology was examined at days 1, 2, 5, 10, and 20 by stereomicroscopy and non-contact infrared meibography. Mice were then sacrificed and eyelids processed for histology with hematoxylin-eosin and immunohistochemistry for ELOVL4, PPARγ, myeloperoxidase (a neutrophil marker) and F4/80 macrophage antigen, as well as TUNEL staining. Another set of specimens was processed for cryosectioning and Oil red O staining. Alkali injury to the ocular surface produced cellular apoptosis, infiltration of neutrophils and macrophages, degeneration of the meibomian gland, and ductal dilation. Inflammation in and destruction of acunal stricture seemed more prominent in the lower eyelid, while duct dilation was more frequently observed in the upper eyelid during healing. Surviving acinar cells were labeled for ELOVL4 and PPARγ. Oil red O staining showed that the substance in the dilated duct contained predominantly neutral lipid. Alkali injury to the ocular surface results in damage and destruction of the eyelid meibomian glands. The pattern of the tissue damage differs between glands of the upper and lower eyelids.
Current Eye Research, Jul 1, 2009
Purpose. We investigated the effects of OPC-15161 on the growth of cultured human Tenon's capsule... more Purpose. We investigated the effects of OPC-15161 on the growth of cultured human Tenon's capsule fibroblasts (TCFs), as well as on the production of type I procollagen, fibronectin, and laminin. These effects were examined in the presence or absence of TGF- 1. Methods. Cell proliferation was assayed by counting cell number and assay of DNA synthesis. Cytotoxicity was determined by the MTT method. Matrix components were assayed by enzyme immunoassay of material in the medium and in the cell lysate with or without OPC-15161. Total protein content was determined. Cellular ultrastructure was also evaluated. Results. Treatment with OPC-15161 (up to 100.0 g ml-1) significantly reduced the proliferation and DNA synthesis of TCFs. No significant decrease in MTT values was observed in confluent TCF cultures with OPC-15161 (up to 100.0 g ml Ϫ 1). TGF- 1 enhanced the TCF production of procollagen I and fibronectin. OPC-15161 significantly decreased the procollagen I content in both the medium, in the cell lysate of TGF- 1-stimulated cells, and fibronectin content in the lysate. OPC-15161 did not affect the laminin or total protein content, either with or without TGF- 1. No ultrastructural evidence of cytotoxicity was observed. Conclusions. OPC-15161 inhibited the proliferation of TCFs, and reduced their production of procollagen I and fibronectin in the presence of TGF- 1 without evidence of cytotoxicity. OPC-15161 may be useful in inhibiting the excessive fibrosis produced in the wound in response to filtering surgery. Curr. Eye Res. 17:933-940, 1998.
While transformation of epithelial cells to a motile form is the first step in wound healing of t... more While transformation of epithelial cells to a motile form is the first step in wound healing of the corneal epithelium, the migratory mechanism in these cells is not fully understood. We studied the expression of proto-oncogene mRNAs: c-fos; c-jun; fos B; jun B; jun D in injured corneal epithelium using in situ hybridization. Moreover, we examined immunolocalization of c-Fos and c-Jun protein products to elucidate the transcriptional activation prior to the onset of migration in corneal epithelium. An epithelial defect was made on one cornea of 60 Wistar rats. The affected eye was enucleated immediately (within 5 min) or was allowed to heal for 15, 30, 60, 90, 120 and 180 min. Frozen sections were processed for in situ hybridization with c-fos, c-jun, fos B, jun B and jun D mRNAs or were stained with anti-c-fos and anti-c-jun antibodies. Fifteen min after the epithelial ablation, weak signals for c-fos and c-jun mRNAs were detected in the corneal epithelium surrounding the wound. These signals reached a peak 30 to 60 min after ablation, but were no longer evident at 120 min. Immunoreactivities for these proteins were also detected in the same area at 60 to 120 min after the epithelial ablation. Fos B mRNA was detected in the same region at 30 min after the ablation, and reached its peak after 30 to 60 min, but was no longer evident at 120 min. Jun B mRNA was detected in the epithelium around the defect 60 min after the ablation, later than the other proto-oncogenes, and reached its peak after 90 min. The message for jun D was detected in normal epithelium, and was not affected by wounding. These findings indicate that transcriptional activation of epithelial cells is initiated in the early phase after epithelial ablation, before the cells start to migrate, and that these proto-oncogene products may play important roles in wound healing in corneal epithelium. The time lag of the peak of expression of these proto-oncogenes in this process.
Molecular Vision, Dec 11, 2008
To understand the role of TGF-β related signals in the repair of a corneal endothelium defect and... more To understand the role of TGF-β related signals in the repair of a corneal endothelium defect and also to evaluate the therapeutic effect of Smad7 gene transfer on injury induced fibrosis of the corneal endothelium in rats. Methods: (1) Japanese albino rabbits (n=108) were used. Blocks of central cornea (4×4 mm) were prepared. After partially scraping the endothelium to produce a defect, the blocks were organ cultured for 24 h in the presence of either exogenous growth factors, transforming growth factor β (TGF-β)-neutralizing antibody, or inhibitors of each TGF-β related signal. Endothelium repair was assayed under light microscopy. (2) Adult Wistar rats (n=62) were then used. Smad7 expressing adenoviral vector (Smad7-Ad) or non-functioning control vector (Cre-Ad) was administered to the anterior chamber of an eye. The cornea was burned with topical 1 N NaOH (10 μl) three days later. After specific intervals, the eye was histologically observed. Results: (1) The endothelial layer that elongated toward the defect lacked proliferation after 24 h in organ culture. Endogenous TGF-β was required for endothelium defect repair. Inhibition of p38 and Erk but not c-Jun NH2-terminal kinase (JNK) and ALK5 signal (Smad) retarded such cell spreading. (2) Adenoviral Smad7 overexpression suppressed fibrogenic reaction of the endothelium of an alkali-burned cornea as evaluated by immunohistochemistry for phospho-Smad2, collagen I, and α-smooth muscle actin, a marker for endothelial-mesenchymal transition (EnMT), and by electron microscopy. Conclusions: Inhibition of Smad and JNK signals do not affect corneal endothelium defect repair. Inhibition of Smad suppresses fibrogenic reaction via EnMT of corneal endothelium in vivo.
Journal of Cataract and Refractive Surgery, Sep 30, 1998
Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their lo... more Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their localization in opacified human posterior capsules and in lens epithelial cells (LECs) was assayed.
Frontiers in bioscience (Scholar edition), 2009
Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathog... more Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathogenesis of fibrotic diseases in the eye. Such ocular fibrotic diseases include scarring in the cornea and conjunctiva, fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, excess scarring the tissue around the extraocular muscles in the strabismus surgery and proliferative vitreoretinopathy. In the proliferative stage of diabetic retinopathy, fibrogenic reaction causes tractional retinal detachment in association with contraction of the tissue. A myofibroblast, the major cellular component in the fibrotic lesions, is derived from both mesenchymal cells (in cornea and conjunctiva) and epithelial cell types (lens or retinal pigment epithelium or corneal endothelium) through epithelial-mesenchymal transition (EMT). The myofibroblasts cause excess accumulation of fibrogenic extracellular matrix with resultant tissue contraction and impaired functions. Altho...
Nippon Ganka Gakkai zasshi, 2005
Cell proliferation and related cellular behavior in ocular neoplastic disease and in the healing ... more Cell proliferation and related cellular behavior in ocular neoplastic disease and in the healing process in ocular surgery or post-injury management, as well as new treatment strategy were investigated. The roles of growth factors and their signal transduction pathways were studied. Cell proliferation-related signals were found to be activated to a greater extent in malignant ocular tumors than in benign tumor cells regardless of the similarity of simple histological findings. Suppression of cell proliferation-related signals can be a new treatment for ocular neoplastic diseases. The causes of complications associated with tissue repair response include acceleration of cell proliferation and extracellular matrix expression and cellular phenotypic alteration, i. e., epithelial-mesenchymal transition. These cellular activities can be controlled by modulation of growth factor signaling by employing such strategy including gene introduction.
Ophthalmic Research, 1995
To investigate the changes in the corneal epithelial basement membrane following an alkali burn, ... more To investigate the changes in the corneal epithelial basement membrane following an alkali burn, we examined the immunolocalization of type IV collagen and laminin in the eye of the guinea pig burned with alkali. The burn damaged the corneal, limbal and conjunctival epithelium. After regeneration, basement membrane was interrupted, as indicated by laminin immunoreactivity. Type IV collagen immunoreactivity was transiently expressed in the early healing phase in the epithelial derived from both the cornea and conjunctiva, but was not seen in the normal corneal epithelial basement membrane. Later in the healing process, following transdifferentiation of the conjunctival epithelium into a cornea-like epithelium, its type IV collagen immunoreactivity was weaker than that in the basement membrane of the nontransdifferentiated epithelium. Conjunctival transdifferentiation during healing may have led to transient development of type IV collagen immunoreactivity.
Ophthalmic Research, 1993
Recruitment of keratocytes into injured corneal stroma, and secretion of proteins including colla... more Recruitment of keratocytes into injured corneal stroma, and secretion of proteins including collagen in the cells are essential for wound healing of the corneal stroma. We examined the effect of a proline analog, cis-hydroxyproline, on the adhesion, migration and growth of rabbit keratocytes in vitro. This agent decreased the plating efficiency, migration and growth of the keratocytes in a dose-dependent manner. Reduction in these cellular activities may reflect altered functions of pericellular proteins such as collagen. Further studies are needed to determine which specific protein is involved.
Journal of Cataract & Refractive Surgery, 1998
Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their lo... more Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their localization in opacified human posterior capsules and in lens epithelial cells (LECs) was assayed.
Japanese Journal of Ophthalmology, 2002
To investigate the nature of capsular opacification after cataract-intraocular lens (IOL) surgery... more To investigate the nature of capsular opacification after cataract-intraocular lens (IOL) surgery in rabbit eyes, we immunohistochemically located extracellular matrix components in lens capsules after the surgery using light microscopy. The study was conducted also to compare the extracellular matrix components in rabbit capsules with those previously reported in the human eye. Methods : Twenty-seven eyes of 17 Japanese albino rabbits were lensectomized by phacoemulsification, and IOLs were implanted. Using immunohistochemical methods, the lens capsules were examined immediately after surgery, and 1, 2, 4, and 8 weeks after surgery. Results: In all cases at each time point, the edge of the anterior capsulotomy had contracted and was found to adhere to the inner surface of the posterior capsule, with both IOL haptics remaining in the capsular bag. Collagen types I and III were detected around the adhesion between the anterior capsulotomy edge and posterior capsule during all stages of healing and also observed on the central posterior capsules 1 or more weeks after surgery. Immunoreactivity for cellular fibronectin was seen around the adhesion between the anterior capsulotomy edge and posterior capsule during all stages of healing. It was also detected on the posterior capsules 2 and 4 weeks after surgery, but disappeared 8 weeks after surgery. Conclusion: Extracellular matrix components such as collagen types I and III and cellular fibronectin were expressed inside the residual lens capsular bag. Cellular fibronectin may play a role in the early wound healing process in the postoperative posterior capsule because the immunoreactivity in the central posterior capsule disappears in the later phase of healing.
Investigative Ophthalmology & Visual Science, 2013
PURPOSE. To investigate the effects of gene ablation of epiplakin on the homeostasis of corneal e... more PURPOSE. To investigate the effects of gene ablation of epiplakin on the homeostasis of corneal epithelium in mice. METHODS. Light and transmission electron microscopic histology, immunohistochemistry, and real-time RT-PCR were carried out to evaluate the effects of the loss of epiplakin on structure and gene expression of cell-cell adhesion-related components in mice. Integrity against mechanical intervention and wound-healing response of corneal epithelium were also tested. RESULTS. Epiplakin protein was detected in the cells of the basal layer of corneal epithelium. Morphologically basal-like cells were observed in the suprabasal layer of adult epiplakin-null corneal epithelium, suggesting an impaired intraepithelial cell differentiation. Such abnormality was not detected in mice before the age of postnatal day 14. Epiplakin-deficient epithelium exhibits fragility against mechanical intervention as compared with wild-type epithelium. Although cell proliferation is suppressed, migration-dependent wound healing is promoted in epiplakin-null epithelium. E-cadherin expression was suppressed by the loss of epiplakin in the epithelium. CONCLUSIONS. Lacking epiplakin affects cell differentiation of the corneal epithelium, as well as its proliferation activity and its structural integrity. The mechanism of acceleration of cell migration in the epiplakin-null corneal epithelium is to be further investigated, although suppression of expression of E-cadherin might be included.
Investigative Ophthalmology & Visual Science, 2004
Graefe's Archive for Clinical and Experimental Ophthalmology, 2003
We previously demonstrated that the AP-1 components c-fos/c-jun are up-regulated in healing rat c... more We previously demonstrated that the AP-1 components c-fos/c-jun are up-regulated in healing rat corneal epithelium in a relatively early phase following epithelial débridement, implicating the AP-1 function in the initiation of cell movement. To explore this hypothesis, we examined the effect of lack of c-Fos and c-Jun protein expressions on the spreading of corneal epithelium and in situ in organ culture. Antisense-oligonucleotide (AS) c-fos-null mice were used for this purpose. A rectangular piece of corneal tissue (2 x2 mm) was obtained from each eye of recently killed adult C57BL/6 mice and was incubated for 11 h in culture medium with 8 microM c-fos AS or c-jun AS probe. Sense probes were used for negative control. A rectangular section of corneal tissue was also obtained from each eye of c-fos(-/-), c-fos(+/-) and c-fos(+/+) mice and was organ-cultured for 11 h. The length of the path of epithelial spreading on stromal cut surface (both sides) was measured in hematoxylin-eosin-stained specimens. Data were analyzed by unpaired Student's t-test. Addition of c-fos AS to the medium decreased the length of epithelial spread to 40.36% of that in the control with the S probe. Addition of c-jun AS decreased the length of epithelial spreading rate to 42.71% of control with S probe. Lacking c-Fos decreased the epithelial spreading to 17.73% of control data from c-fos(+/-) and c-fos(+/+) mice. AP-1 (c-Fos/c-Jun) is required for the corneal epithelial spreading.
Graefe's Archive for Clinical and Experimental Ophthalmology, 2003
We examined the effects of interferon-gamma (IFN-gamma) on protein production of extracellular ma... more We examined the effects of interferon-gamma (IFN-gamma) on protein production of extracellular matrix (ECM) components in cultured human subconjunctival fibroblasts or those stimulated by exogenous transforming growth factor beta1 (TGFbeta1). IFN-gamma reportedly up-regulates Smad7, an inhibitory mediator of TGFbeta-Smad signaling, and blocks TGFbeta effects. Proliferation and migration as well as the ultrastructure of these cells were examined in the presence and absence of IFN-gamma. Cell migration was examined using an in vitro wound healing model in monolayer fibroblast cultures. The results showed that IFN-gamma reduced ECM production in normal subconjunctival fibroblasts, as well as in those treated with TGFbeta1, below the control levels. IFN-gamma had no effect on cell proliferation and fibroblast ultrastructure. On the other hand, IFN-gamma delayed defect closure in monolayer cell sheets in a dose-dependent manner. Immunohistochemistry also revealed that the addition of IFN-gamma attenuated the translocation of Smads2/4 into the nuclei of TGFbeta1-treated subconjunctival fibroblasts. These findings suggest that IFN-gamma may be clinically effective in attenuating excessive ECM accumulation in conjunctiva after ocular surgery and in the presence of inflammatory ocular surface disorder. IFN-gamma modulates the Smads2/4 pathway of TGFbeta1 signal transduction toward the up-regulation of ECM components.
Graefe's Archive for Clinical and Experimental Ophthalmology, 1995
Background: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen produc... more Background: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen production, and decreases collagen production in vitro. We investigated the in vitro effects of minoxidil on behavior such as proliferation and migration of rabbit subconjunctival fibroblasts (SCFs). The ultrastructural effect of the drug on SCFs was also examined. • Methods: Proliferation of SCFs and closure of the defect produced in monolayer cultures in the presence or absence of minoxidil was studied. The ultrastructure of SCFs treated with minoxidil was also examined. • Results: Minoxidil inhibited SCF proliferation and the closure of the defect produced in monolayer cell sheets. Ultrastructural observations revealed extensive areas of irregularly dilated endoplasmic reticulum in cells treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors, in the cisternae of endoplasmic reticulum. • Conclusions: The results indicated that minoxidil attenuated cellular activities of SCFs such as proliferation and migration in vitro. The exact mechanism of the inhibitory effects of minoxidil on these cellular activities is unknown. The findings suggest that the drug might help to prevent bleb scarring after glaucoma filtering surgery.
Graefe's Archive for Clinical and Experimental Ophthalmology, 1993
We investigated the origin of fibronectin (FN) on five posterior and four anterior chamber explan... more We investigated the origin of fibronectin (FN) on five posterior and four anterior chamber explanted intraocular lenses (IOLs) using immunohistochemical methods. Cellular deposits (assumed to be macrophages) and fibrous or membrane-like proteinaceous deposits on the IOLs showed immunoreactivity to an antibody against cellular FN. These proteinaceous deposits were believed to be products of the cells that adhered to the IOLs.
The American Journal of Pathology, 2005
PURPOSE Smad7 is a molecule that blocks the Smad2/3 signal. Herein, we examined the effects of Sm... more 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 epith...
Cell and Tissue Research
The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repa... more The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor β1 (TGFβ1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFβ1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFβ-induced granulation tissue formation.
The Ocular Surface
To examine effects of alkali injury of the ocular surface on meibomian gland pathology in mice. T... more To examine effects of alkali injury of the ocular surface on meibomian gland pathology in mice. Three μL of 1 N NaOH were applied under general anesthesia to the right eye of 10-week-old BALB/c (n = 54) mice to produce a total ocular surface alkali burn. The meibomian gland morphology was examined at days 1, 2, 5, 10, and 20 by stereomicroscopy and non-contact infrared meibography. Mice were then sacrificed and eyelids processed for histology with hematoxylin-eosin and immunohistochemistry for ELOVL4, PPARγ, myeloperoxidase (a neutrophil marker) and F4/80 macrophage antigen, as well as TUNEL staining. Another set of specimens was processed for cryosectioning and Oil red O staining. Alkali injury to the ocular surface produced cellular apoptosis, infiltration of neutrophils and macrophages, degeneration of the meibomian gland, and ductal dilation. Inflammation in and destruction of acunal stricture seemed more prominent in the lower eyelid, while duct dilation was more frequently observed in the upper eyelid during healing. Surviving acinar cells were labeled for ELOVL4 and PPARγ. Oil red O staining showed that the substance in the dilated duct contained predominantly neutral lipid. Alkali injury to the ocular surface results in damage and destruction of the eyelid meibomian glands. The pattern of the tissue damage differs between glands of the upper and lower eyelids.
Current Eye Research, Jul 1, 2009
Purpose. We investigated the effects of OPC-15161 on the growth of cultured human Tenon's capsule... more Purpose. We investigated the effects of OPC-15161 on the growth of cultured human Tenon's capsule fibroblasts (TCFs), as well as on the production of type I procollagen, fibronectin, and laminin. These effects were examined in the presence or absence of TGF- 1. Methods. Cell proliferation was assayed by counting cell number and assay of DNA synthesis. Cytotoxicity was determined by the MTT method. Matrix components were assayed by enzyme immunoassay of material in the medium and in the cell lysate with or without OPC-15161. Total protein content was determined. Cellular ultrastructure was also evaluated. Results. Treatment with OPC-15161 (up to 100.0 g ml-1) significantly reduced the proliferation and DNA synthesis of TCFs. No significant decrease in MTT values was observed in confluent TCF cultures with OPC-15161 (up to 100.0 g ml Ϫ 1). TGF- 1 enhanced the TCF production of procollagen I and fibronectin. OPC-15161 significantly decreased the procollagen I content in both the medium, in the cell lysate of TGF- 1-stimulated cells, and fibronectin content in the lysate. OPC-15161 did not affect the laminin or total protein content, either with or without TGF- 1. No ultrastructural evidence of cytotoxicity was observed. Conclusions. OPC-15161 inhibited the proliferation of TCFs, and reduced their production of procollagen I and fibronectin in the presence of TGF- 1 without evidence of cytotoxicity. OPC-15161 may be useful in inhibiting the excessive fibrosis produced in the wound in response to filtering surgery. Curr. Eye Res. 17:933-940, 1998.
While transformation of epithelial cells to a motile form is the first step in wound healing of t... more While transformation of epithelial cells to a motile form is the first step in wound healing of the corneal epithelium, the migratory mechanism in these cells is not fully understood. We studied the expression of proto-oncogene mRNAs: c-fos; c-jun; fos B; jun B; jun D in injured corneal epithelium using in situ hybridization. Moreover, we examined immunolocalization of c-Fos and c-Jun protein products to elucidate the transcriptional activation prior to the onset of migration in corneal epithelium. An epithelial defect was made on one cornea of 60 Wistar rats. The affected eye was enucleated immediately (within 5 min) or was allowed to heal for 15, 30, 60, 90, 120 and 180 min. Frozen sections were processed for in situ hybridization with c-fos, c-jun, fos B, jun B and jun D mRNAs or were stained with anti-c-fos and anti-c-jun antibodies. Fifteen min after the epithelial ablation, weak signals for c-fos and c-jun mRNAs were detected in the corneal epithelium surrounding the wound. These signals reached a peak 30 to 60 min after ablation, but were no longer evident at 120 min. Immunoreactivities for these proteins were also detected in the same area at 60 to 120 min after the epithelial ablation. Fos B mRNA was detected in the same region at 30 min after the ablation, and reached its peak after 30 to 60 min, but was no longer evident at 120 min. Jun B mRNA was detected in the epithelium around the defect 60 min after the ablation, later than the other proto-oncogenes, and reached its peak after 90 min. The message for jun D was detected in normal epithelium, and was not affected by wounding. These findings indicate that transcriptional activation of epithelial cells is initiated in the early phase after epithelial ablation, before the cells start to migrate, and that these proto-oncogene products may play important roles in wound healing in corneal epithelium. The time lag of the peak of expression of these proto-oncogenes in this process.
Molecular Vision, Dec 11, 2008
To understand the role of TGF-β related signals in the repair of a corneal endothelium defect and... more To understand the role of TGF-β related signals in the repair of a corneal endothelium defect and also to evaluate the therapeutic effect of Smad7 gene transfer on injury induced fibrosis of the corneal endothelium in rats. Methods: (1) Japanese albino rabbits (n=108) were used. Blocks of central cornea (4×4 mm) were prepared. After partially scraping the endothelium to produce a defect, the blocks were organ cultured for 24 h in the presence of either exogenous growth factors, transforming growth factor β (TGF-β)-neutralizing antibody, or inhibitors of each TGF-β related signal. Endothelium repair was assayed under light microscopy. (2) Adult Wistar rats (n=62) were then used. Smad7 expressing adenoviral vector (Smad7-Ad) or non-functioning control vector (Cre-Ad) was administered to the anterior chamber of an eye. The cornea was burned with topical 1 N NaOH (10 μl) three days later. After specific intervals, the eye was histologically observed. Results: (1) The endothelial layer that elongated toward the defect lacked proliferation after 24 h in organ culture. Endogenous TGF-β was required for endothelium defect repair. Inhibition of p38 and Erk but not c-Jun NH2-terminal kinase (JNK) and ALK5 signal (Smad) retarded such cell spreading. (2) Adenoviral Smad7 overexpression suppressed fibrogenic reaction of the endothelium of an alkali-burned cornea as evaluated by immunohistochemistry for phospho-Smad2, collagen I, and α-smooth muscle actin, a marker for endothelial-mesenchymal transition (EnMT), and by electron microscopy. Conclusions: Inhibition of Smad and JNK signals do not affect corneal endothelium defect repair. Inhibition of Smad suppresses fibrogenic reaction via EnMT of corneal endothelium in vivo.
Journal of Cataract and Refractive Surgery, Sep 30, 1998
Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their lo... more Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their localization in opacified human posterior capsules and in lens epithelial cells (LECs) was assayed.
Frontiers in bioscience (Scholar edition), 2009
Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathog... more Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathogenesis of fibrotic diseases in the eye. Such ocular fibrotic diseases include scarring in the cornea and conjunctiva, fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, excess scarring the tissue around the extraocular muscles in the strabismus surgery and proliferative vitreoretinopathy. In the proliferative stage of diabetic retinopathy, fibrogenic reaction causes tractional retinal detachment in association with contraction of the tissue. A myofibroblast, the major cellular component in the fibrotic lesions, is derived from both mesenchymal cells (in cornea and conjunctiva) and epithelial cell types (lens or retinal pigment epithelium or corneal endothelium) through epithelial-mesenchymal transition (EMT). The myofibroblasts cause excess accumulation of fibrogenic extracellular matrix with resultant tissue contraction and impaired functions. Altho...
Nippon Ganka Gakkai zasshi, 2005
Cell proliferation and related cellular behavior in ocular neoplastic disease and in the healing ... more Cell proliferation and related cellular behavior in ocular neoplastic disease and in the healing process in ocular surgery or post-injury management, as well as new treatment strategy were investigated. The roles of growth factors and their signal transduction pathways were studied. Cell proliferation-related signals were found to be activated to a greater extent in malignant ocular tumors than in benign tumor cells regardless of the similarity of simple histological findings. Suppression of cell proliferation-related signals can be a new treatment for ocular neoplastic diseases. The causes of complications associated with tissue repair response include acceleration of cell proliferation and extracellular matrix expression and cellular phenotypic alteration, i. e., epithelial-mesenchymal transition. These cellular activities can be controlled by modulation of growth factor signaling by employing such strategy including gene introduction.
Ophthalmic Research, 1995
To investigate the changes in the corneal epithelial basement membrane following an alkali burn, ... more To investigate the changes in the corneal epithelial basement membrane following an alkali burn, we examined the immunolocalization of type IV collagen and laminin in the eye of the guinea pig burned with alkali. The burn damaged the corneal, limbal and conjunctival epithelium. After regeneration, basement membrane was interrupted, as indicated by laminin immunoreactivity. Type IV collagen immunoreactivity was transiently expressed in the early healing phase in the epithelial derived from both the cornea and conjunctiva, but was not seen in the normal corneal epithelial basement membrane. Later in the healing process, following transdifferentiation of the conjunctival epithelium into a cornea-like epithelium, its type IV collagen immunoreactivity was weaker than that in the basement membrane of the nontransdifferentiated epithelium. Conjunctival transdifferentiation during healing may have led to transient development of type IV collagen immunoreactivity.
Ophthalmic Research, 1993
Recruitment of keratocytes into injured corneal stroma, and secretion of proteins including colla... more Recruitment of keratocytes into injured corneal stroma, and secretion of proteins including collagen in the cells are essential for wound healing of the corneal stroma. We examined the effect of a proline analog, cis-hydroxyproline, on the adhesion, migration and growth of rabbit keratocytes in vitro. This agent decreased the plating efficiency, migration and growth of the keratocytes in a dose-dependent manner. Reduction in these cellular activities may reflect altered functions of pericellular proteins such as collagen. Further studies are needed to determine which specific protein is involved.
Journal of Cataract & Refractive Surgery, 1998
Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their lo... more Purpose: To investigate the role of hyaluronan and its receptor CD44 in capsular repair, their localization in opacified human posterior capsules and in lens epithelial cells (LECs) was assayed.
Japanese Journal of Ophthalmology, 2002
To investigate the nature of capsular opacification after cataract-intraocular lens (IOL) surgery... more To investigate the nature of capsular opacification after cataract-intraocular lens (IOL) surgery in rabbit eyes, we immunohistochemically located extracellular matrix components in lens capsules after the surgery using light microscopy. The study was conducted also to compare the extracellular matrix components in rabbit capsules with those previously reported in the human eye. Methods : Twenty-seven eyes of 17 Japanese albino rabbits were lensectomized by phacoemulsification, and IOLs were implanted. Using immunohistochemical methods, the lens capsules were examined immediately after surgery, and 1, 2, 4, and 8 weeks after surgery. Results: In all cases at each time point, the edge of the anterior capsulotomy had contracted and was found to adhere to the inner surface of the posterior capsule, with both IOL haptics remaining in the capsular bag. Collagen types I and III were detected around the adhesion between the anterior capsulotomy edge and posterior capsule during all stages of healing and also observed on the central posterior capsules 1 or more weeks after surgery. Immunoreactivity for cellular fibronectin was seen around the adhesion between the anterior capsulotomy edge and posterior capsule during all stages of healing. It was also detected on the posterior capsules 2 and 4 weeks after surgery, but disappeared 8 weeks after surgery. Conclusion: Extracellular matrix components such as collagen types I and III and cellular fibronectin were expressed inside the residual lens capsular bag. Cellular fibronectin may play a role in the early wound healing process in the postoperative posterior capsule because the immunoreactivity in the central posterior capsule disappears in the later phase of healing.
Investigative Ophthalmology & Visual Science, 2013
PURPOSE. To investigate the effects of gene ablation of epiplakin on the homeostasis of corneal e... more PURPOSE. To investigate the effects of gene ablation of epiplakin on the homeostasis of corneal epithelium in mice. METHODS. Light and transmission electron microscopic histology, immunohistochemistry, and real-time RT-PCR were carried out to evaluate the effects of the loss of epiplakin on structure and gene expression of cell-cell adhesion-related components in mice. Integrity against mechanical intervention and wound-healing response of corneal epithelium were also tested. RESULTS. Epiplakin protein was detected in the cells of the basal layer of corneal epithelium. Morphologically basal-like cells were observed in the suprabasal layer of adult epiplakin-null corneal epithelium, suggesting an impaired intraepithelial cell differentiation. Such abnormality was not detected in mice before the age of postnatal day 14. Epiplakin-deficient epithelium exhibits fragility against mechanical intervention as compared with wild-type epithelium. Although cell proliferation is suppressed, migration-dependent wound healing is promoted in epiplakin-null epithelium. E-cadherin expression was suppressed by the loss of epiplakin in the epithelium. CONCLUSIONS. Lacking epiplakin affects cell differentiation of the corneal epithelium, as well as its proliferation activity and its structural integrity. The mechanism of acceleration of cell migration in the epiplakin-null corneal epithelium is to be further investigated, although suppression of expression of E-cadherin might be included.
Investigative Ophthalmology & Visual Science, 2004
Graefe's Archive for Clinical and Experimental Ophthalmology, 2003
We previously demonstrated that the AP-1 components c-fos/c-jun are up-regulated in healing rat c... more We previously demonstrated that the AP-1 components c-fos/c-jun are up-regulated in healing rat corneal epithelium in a relatively early phase following epithelial débridement, implicating the AP-1 function in the initiation of cell movement. To explore this hypothesis, we examined the effect of lack of c-Fos and c-Jun protein expressions on the spreading of corneal epithelium and in situ in organ culture. Antisense-oligonucleotide (AS) c-fos-null mice were used for this purpose. A rectangular piece of corneal tissue (2 x2 mm) was obtained from each eye of recently killed adult C57BL/6 mice and was incubated for 11 h in culture medium with 8 microM c-fos AS or c-jun AS probe. Sense probes were used for negative control. A rectangular section of corneal tissue was also obtained from each eye of c-fos(-/-), c-fos(+/-) and c-fos(+/+) mice and was organ-cultured for 11 h. The length of the path of epithelial spreading on stromal cut surface (both sides) was measured in hematoxylin-eosin-stained specimens. Data were analyzed by unpaired Student's t-test. Addition of c-fos AS to the medium decreased the length of epithelial spread to 40.36% of that in the control with the S probe. Addition of c-jun AS decreased the length of epithelial spreading rate to 42.71% of control with S probe. Lacking c-Fos decreased the epithelial spreading to 17.73% of control data from c-fos(+/-) and c-fos(+/+) mice. AP-1 (c-Fos/c-Jun) is required for the corneal epithelial spreading.
Graefe's Archive for Clinical and Experimental Ophthalmology, 2003
We examined the effects of interferon-gamma (IFN-gamma) on protein production of extracellular ma... more We examined the effects of interferon-gamma (IFN-gamma) on protein production of extracellular matrix (ECM) components in cultured human subconjunctival fibroblasts or those stimulated by exogenous transforming growth factor beta1 (TGFbeta1). IFN-gamma reportedly up-regulates Smad7, an inhibitory mediator of TGFbeta-Smad signaling, and blocks TGFbeta effects. Proliferation and migration as well as the ultrastructure of these cells were examined in the presence and absence of IFN-gamma. Cell migration was examined using an in vitro wound healing model in monolayer fibroblast cultures. The results showed that IFN-gamma reduced ECM production in normal subconjunctival fibroblasts, as well as in those treated with TGFbeta1, below the control levels. IFN-gamma had no effect on cell proliferation and fibroblast ultrastructure. On the other hand, IFN-gamma delayed defect closure in monolayer cell sheets in a dose-dependent manner. Immunohistochemistry also revealed that the addition of IFN-gamma attenuated the translocation of Smads2/4 into the nuclei of TGFbeta1-treated subconjunctival fibroblasts. These findings suggest that IFN-gamma may be clinically effective in attenuating excessive ECM accumulation in conjunctiva after ocular surgery and in the presence of inflammatory ocular surface disorder. IFN-gamma modulates the Smads2/4 pathway of TGFbeta1 signal transduction toward the up-regulation of ECM components.
Graefe's Archive for Clinical and Experimental Ophthalmology, 1995
Background: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen produc... more Background: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen production, and decreases collagen production in vitro. We investigated the in vitro effects of minoxidil on behavior such as proliferation and migration of rabbit subconjunctival fibroblasts (SCFs). The ultrastructural effect of the drug on SCFs was also examined. • Methods: Proliferation of SCFs and closure of the defect produced in monolayer cultures in the presence or absence of minoxidil was studied. The ultrastructure of SCFs treated with minoxidil was also examined. • Results: Minoxidil inhibited SCF proliferation and the closure of the defect produced in monolayer cell sheets. Ultrastructural observations revealed extensive areas of irregularly dilated endoplasmic reticulum in cells treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors, in the cisternae of endoplasmic reticulum. • Conclusions: The results indicated that minoxidil attenuated cellular activities of SCFs such as proliferation and migration in vitro. The exact mechanism of the inhibitory effects of minoxidil on these cellular activities is unknown. The findings suggest that the drug might help to prevent bleb scarring after glaucoma filtering surgery.
Graefe's Archive for Clinical and Experimental Ophthalmology, 1993
We investigated the origin of fibronectin (FN) on five posterior and four anterior chamber explan... more We investigated the origin of fibronectin (FN) on five posterior and four anterior chamber explanted intraocular lenses (IOLs) using immunohistochemical methods. Cellular deposits (assumed to be macrophages) and fibrous or membrane-like proteinaceous deposits on the IOLs showed immunoreactivity to an antibody against cellular FN. These proteinaceous deposits were believed to be products of the cells that adhered to the IOLs.