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...
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.
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.
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...
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.
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.
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.
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.
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...
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.
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.
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...
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.
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.
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.
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.
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Papers by Osamu Yamanaka