Shizuya Saika

Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

Role of Lumican in the Corneal Epithelium during Wound Healing

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis,e.g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum −/− mice was significantly delayed compared with Lum +/− mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.

TGFβ pathobiology in the eye

Transforming growth factor β (TGFβ), a multifunctional growth factor, is one of the most important ligands involved in the regulation of cell behavior in ocular tissues in physiological or pathological processes of development or tissue repair, although various other growth factors are also involved. Increased activity of this ligand may induce unfavorable inflammatory responses and tissue fibrosis. In mammals, three isoforms of TGFβ, that is, β1, β2, and β3, are known. Although all three TGFβ isoforms and their receptors are present in ocular tissues, lack of TGFβ2, but not TGFβ1 or TGFβ3, perturbs embryonic morphogenesis of the eyes in mice. Smads2/3 are key signaling molecules downstream of cell surface receptors for TGFβ or activin. Upon TGF binding to the respective TGF receptor, Smads2/3 are phosphorylated by the receptor kinase at the C-terminus, form a complex with Smad4 and translocate to the nucleus for activation of TGFβ gene targets. Moreover, mitogen-activated protein kinase, c-Jun N-terminal kinase, and p38 modulate Smad signals directly via Smad linker phosphorylation or indirectly via pathway crosstalk. Smad signals may therefore be a critical threrapeutic target in the treatment of ocular disorders related to fibrosis as in other systemic fibrotic diseases. The present paper reviews recent progress concerning the roles of TGFβ signaling in the pathology of the eye.

Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial–mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-β (TGF-β) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-β and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of α-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-β signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Relationship between posterior capsule opacification and intraocular lens biocompatibility

The type of healing process that occurs in response to cataract surgery and intraocular lens (IOL) implantation is dependent on a complex set of variables. Their interactions determine whether or not optical clarity is restored as a result of this procedure. In this process, wound healing entails cells undergoing either epithelial-mesenchymal transition, resulting in the generation of fibroblastic cells and accumulation of extracellular matrix, or lenticular structure formation. Such desperate cellular behaviors are regulated by the localized release of different cytokines, including transforming growth factor beta and fibroblast growth factors, which can result in post-operative capsular opacification. Other factors affecting the biological and mechanical outcome of IOL implantation are its composition, surface properties and shape.

Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial–mesenchymal transition of lens epithelium in mice

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFβ/activin signaling, on injury-induced epithelial–mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3u2009μl of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2u2009h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and α-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFβ1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.

TGF-beta signal transduction in corneal wound healing as a therapeutic target.

Abstract This article reviews recent progress in research on the role of Smad signaling in corneal wound healing. Smad2 and Smad3 are key signaling molecules downstream of the cell surface receptor of transforming growth factor-β (TGF-β) or activin. On ligand binding to the receptor, Smads2/3 undergo phosphorylation, form complexes with Smad4, and thence convey signaling. TGF-β isoforms have been detected in corneal epithelium and are also deposited in wounded stroma, suggesting their participation in the wound-healing process in corneal tissue. Human or mouse uninjured healthy corneal epithelium shows nuclear accumulation of Smads3/4, indicating active Smad signaling in this tissue. Migrating corneal epithelium lacks nuclear Smad accumulation with up-regulation of Smad7, but p38MAPK is activated. Organ-culture experiments show that p38MAPK activation depends on endogenous TGF-β and that activation of p38MAPK results in cell proliferation cessation with a reduction of Erk activation and acceleration of cell migration in healing corneal epithelium. These findings indicate that during healing of corneal epithelial defects, endogenous TGF-β activates p38MAPK for cell migration and suppression of cell proliferation and up-regulates Smad7 for inhibition of Smad2 and Smad3 signaling, resulting in rapid initial resurfacing of the epithelium. Such involvement of p38MAPK in cell migration has been reported in many cell types and observed in keratocyte culture. Possible benefits of preserving non-Smad cascades in treating problems in corneal wound healing by manipulating TGF-β signals have been suggested.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases of fibrous humans lens capsules with intraocular lenses

Purpose. We located immunohistochemically the matrix metalloproteinases (MMP) -1, -2, -3 and -9 and the tissue inhibitors of matrix metalloproteinases (TIMP) -1 and -2 in the fibrous capsule of patients with intraocular lenses (IOLs). Methods. During vitreoretinal surgery in 10 patients we obtained post-cataract surgery lens capsules with or without an IOL. The mean interval between the previous cataract operation and the extraction of the specimens was 35.2 months (range: 2-120 months). Circular sections of the anterior capsule with lens epithelial cells (LECs) were also obtained during cataract surgery. Specimens were processed for immunohistochemical identification of MMPs and TIMPs by light microscopy. Results. While all the members of MMPs and TIMPs were not detected in the normal anterior capsules, they were detected in the ECM and/or LECs on the lens capsules extracted within 18 months after IOL implantations in all of the 4 patients, but were not observed in specimens obtained 18 months or longer postoperatively. In LECs of 1 capsule specimen 10 years postoperatively, MMP-1, but not other MMPs and TIMPs, was detected. Conclusions. MMPs and TIMPs were detected in the ECM and/or LECs on post-cataract surgery capsules. These proteins may be remodeling the newly deposited ECM and regulating LEC behavior on residual lens capsules in the early phase of healing after cataract surgery.

Growth factor deposition in anterior subcapsular cataract

Purpose: To characterize immunohistochemically the distribution of growth factors and extracellular matrix (ECM) components in an anterior subcapsular cataract (ASC) and to determine the role of growth factors in the development of ASC. Setting: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. Methods: During cataract surgery in 22 patients, anterior capsules with an ASC were obtained. Sections of each specimen were immunostained with a panel of antibodies against ECM components, growth factors, cytoskeletal components, and signal transduction‐related molecules. Results: Collagen types I, V, and VI; fibronectin; fibrillin‐1; and latent transforming growth factor β binding protein‐1 (LTBP‐1) were localized to the ECM in ASC tissues. Collagen IV was localized to the ECM and the capsule. Lens epithelial cells (LECs) were positive for α‐smooth muscle actin (αSMA). Lens epithelial cells and ECM stained for transforming growth factor β2 (TGFβ2) and TGFβ3 in all samples, but TGFβ1 latency‐associated peptide (TGFβ1‐LAP) were detected in some samples. Fibroblast growth factor‐2 (FGF‐2) and hepatocyte growth factor‐α (HGF‐α) were localized to the ECM. Lens epithelial cells with nuclear staining for Erk‐1, the mitogen‐activated protein kinase (MAP kinase) cascade‐related molecule, and Smad3, 1 of the Smad family members involving TGFβ signaling, were detected. Conclusions: Matrix components (ie, collagen types, fibronectin, fibrillin‐1), as well as growth factors such as TGFβ1‐LAP, TGFβ2, TGFβ3, FGF‐2, and HGF‐α, were detected in ASC. Fibrillin‐1 might serve as a repository for TGFβs. These growth factors may modulate the phenotypic alteration and behavior of LECs. The MAP kinase cascade and TGFβ signaling are both activated in LECs in ASC.

Biological Compatibility of Polymethyl Methacrylate, Hydrophilic Acrylic and Hydrophobic Acrylic Intraocular Lenses

Purpose: Extensive clinical investigations of the biocompatibility of different intraocular lenses (IOLs) have been made in an effort to optimize the outcome of modern cataract surgery. The aim of this study was to add animal eye experimental implantation data regarding cellular reaction on the anterior surface of IOLs. Methods: Thirteen adult albino rabbits had phacoemulsification/aspiration of the crystalline lens followed by implantation of a posterior chamber IOL in each eye. Three types of IOLs were studied: Hydroview® (Bausch & Lomb; n = 7), Acrysof® (Alcon, USA; n = 7), and polymethyl methacrylate (PMMA; HOYA, Japan; n = 7). The animals were killed by intravenous pentobarbital 1, 4, or 8 weeks later. The IOLs were explanted and stained with hematoxylin and eosin, and observed under a light microscope. The shape of mouse ascites-induced macrophages on the anterior surface of the three different IOL types (Hydroview, PMMA, and Acrysof) was studied after 24 h of oven culture. Results: Hydrophilic acrylic IOLs showed the highest affinity for lens epithelial cell (LEC) outgrowth, and the lowest and slowest maturation rate reaction of macrophages. PMMA IOLs showed the lowest affinity for LEC outgrowth, and the highest reaction of macrophages. Hydrophobic acrylic IOLs showed intermediate results both regarding LECs and macrophages. Conclusions: Results suggest that IOL biomaterial properties are the key factor that influences the quantity of monocytes/macrophages as well as the process of their maturation/senescence. LEC outgrowth is influenced both by the biomaterial of IOLs and by the monocyte/macrophage reaction.