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

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

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 beta 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 alpha-smooth muscle actin (alphaSMA). Lens epithelial cells and ECM stained for transforming growth factor beta2 (TGFbeta2) and TGFbeta3 in all samples, but TGFbeta1 latency-associated peptide (TGFbeta1-LAP) were detected in some samples. Fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor-alpha (HGF-alpha) 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 TGFbeta signaling, were detected. CONCLUSIONS: Matrix components (ie, collagen types, fibronectin, fibrillin-1), as well as growth factors such as TGFbeta1-LAP, TGFbeta2, TGFbeta3, FGF-2, and HGF-alpha, were detected in ASC. Fibrillin-1 might serve as a repository for TGFbetas. These growth factors may modulate the phenotypic alteration and behavior of LECs. The MAP kinase cascade and TGFbeta signaling are both activated in LECs in ASC.

Inhibition of p38MAP kinase suppresses fibrotic reaction of retinal pigment epithelial cells.

Proliferative vitreoretinopathy (PVR) is one of the major causes of the failure of retinal detachment surgery. Its pathogenesis includes a fibrotic reaction by the retinal pigment epithelium and other retina-derived non-neural cells, leading to fixation of the detached retina. We examined the role of p38 mitogen-activated protein kinase (MAPK) in transforming growth factor (TGF)-beta2-dependent enhancement of the fibrogenic reaction in a human retinal pigment epithelial cell line, ARPE-19, and also evaluated the therapeutic efficacy of inhibiting p38MAPK by adenoviral gene transfer of dominant-negative (DN) p38MAPK in a mouse model of PVR. Exogenous TGF-beta2 activates p38MAPK in ARPE-19 cells. It also suppresses cell proliferation, but this was unaffected by addition of the p38MAPK inhibitor, SB202190. SB202190 interfered with TGF-beta2-dependent cell migration and production of collagen type I and fibronectin, but had no effect on basal levels of these activities. While SB202190 did not affect phosphorylation of the C-terminus of Smads2/3, it did suppress the transcriptional activity of Smads3/4 as indicated by a reporter gene, CAGA12-Luc. Gene transfer of DN-p38MAPK attenuated the post-retinal detachment fibrotic reaction of the retinal pigment epithelium in vivo in mice, supporting its effectiveness in preventing/treating PVR.

Accumulation of thrombospondin-1 in post-operative capsular fibrosis and its down-regulation in lens cells during lens fiber formation.

Thrombospondin-1 (TSP-1) is a glycoprotein involved in activation of latent transforming growth factor beta (TGFbeta) expression. We examined changes in its expression pattern during human capsular opacification (PCO) and anterior subcapsular cataractogenesis (ASC), as well as in a healing injured mouse lens. Its expression pattern was also compared in a mouse embryonic lens with that in an adult lens. Based on immunohistochemistry under light microscopy, TSP-1 expression and other matrix components were evident in the anterior epithelium of an uninjured human lens, whereas fiber-differentiating cells in the equator of human lens lack TSP-1 immunoreactivity. In contrast, in post-operative human lens epithelial or fibroblastic cells, there was TSP-1 immunoreactivity, whereas it decreased in fiber-differentiating cells in PCO. Matrix components accumulated on the healing capsule also labeled with anti-TSP-1 antibody like antibodies against collagen I, IV, V and laminin. In uninjured, injured mouse lens epithelial cells and its matrix, there was TSP-1 expression. Embryonic lens cells in the posterior pole, undergoing differentiation to fiber cells, began to express TSP-1 protein at embryonic day (E) 11.5 whereas anterior epithelial cells started to express it at E13.5 in association with marked expression in central fiber cells. At E16.5, TSP-1 was detected in fibers just beneath the anterior epithelium, but the fiber mass showed minimal expression. At E18.5 and post-natally day 1, lens fiber TSP-1 expression was no longer seen. On the other hand, it was evident in both intact human anterior epithelial and dispersed mouse cells. The results indicate that there is TSP-1 expression in uninjured human and mouse lens epithelial cells and their fibrous tissue. In contrast, in post-operative lens cells differentiating to fiber cells, its expression levels decline. Further study is needed to clarify the roles of TSP-1 in modulating lens cell phenotype expression.

Response of lens epithelial cells to injury: role of lumican in epithelial-mesenchymal transition.

PURPOSE: Lens epithelial cells (LECs) undergo epithelial-mesenchcymal transition (EMT) after injury and transform into myofibroblasts positive for alpha-smooth muscle actin (alphaSMA), an established marker of this process. Lumican is a keratan sulfate proteoglycan core protein. This study was conducted to examine whether human and mouse LECs express lumican after injury. To determine whether lumican may modulate EMT of LECs in response to injury or to exposure to transforming growth factor-beta2 (TGFbeta2), alphaSMA expression by the LECs was examined in lumican (Lum)-knockout mice in vivo and in organ culture. METHODS: Human postoperative capsular specimens and healing, injured mouse lenses at various intervals were immunostained for lumican or alphaSMA. alphaSMA was also immunolocalized in healing, injured lenses of Lum-knockout mice. Finally, expression of lumican and alphaSMA was examined in lenses of Lum-knockout mice incubated with TGFbeta2. RESULTS: Lumican was immunolocalized in matrix in human postoperative capsular opacification. Lumican and alphaSMA were upregulated in mouse LECs from 8 hours and day 5 after an injury, respectively. LECs accumulated adjacent to the capsular break were of epithelial shape in Lum(-/-) mice and fibroblast-like in Lum(+/-) mice during healing. alphaSMA expression by LECs was significantly delayed in Lum(-/-) mice, indicating that lumican may modulate injury-induced EMT in LECs. TGFbeta2-induced EMT appeared to be suppressed in organ-cultured lenses of Lum(-/-) mice compared with those of Lum(+/+) mice. CONCLUSIONS: Human capsular opacification contains lumican, and mouse LECs upregulate lumican and alphaSMA in response to injury. Loss of lumican perturbs EMT of mouse LECs.

Osteopontin: a component of matrix in capsular opacification and subcapsular cataract.

PURPOSE: To examine whether tissues of human capsular opacification and subcapsular cataract contain osteopontin, an adhesive matrix protein, and whether mouse lens epithelium expresses osteopontin after injury. METHODS: An immunohistochemical examination was conducted to determine whether matrices in human postoperative capsular specimens and anterior subcapsular cataract contain osteopontin. The spatial and temporal protein expression patterns of osteopontin were then determined in epithelium of a healing mouse lens after a capsular incision. RESULTS: Human lens epithelial cells in the specimens extracted at the time of vitrectomy 10 days after cataract surgery and also after longer healing intervals were labeled with an anti-osteopontin antibody, whereas uninjured lens epithelium was not. In the later healing phase, matrix of capsular opacification was positive for osteopontin. Lens cells amid anterior subcapsular cataract tissue were also positive. Osteopontin was detected in the cell surface and membrane and the cytoplasm of lens cells, as well as in the matrix. Unlike normal uninjured specimens, anterior lens capsule of some of the healing postoperative specimens and anterior subcapsular cataract specimens also faintly or weakly stained for osteopontin. Mouse lens epithelium started to express osteopontin protein at 8 hours after injury, before the cells changed their shape from epithelial cell type to fibroblast type. Expression of osteopontin lasted during the healing interval, even after the cells transformed into fibroblast-like cells. CONCLUSIONS: Extracellular matrix in human postoperative capsular opacification and anterior subcapsular cataract contains osteopontin. Epithelial cells of a mouse lens also ectopically express osteopontin in response to capsular injury.

Lens epithelial cell death after cataract surgery.

PURPOSE: To determine whether lens epithelial cells (LECs) undergo apoptosis during healing after cataract surgery to further characterize the healing process of the postoperative lens capsule. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Apoptotic cells were detected in human postoperative lens capsules using the terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling (TUNEL) method. The effect of exogenous transforming growth factor-beta2 (TGF-beta2) on mouse LEC apoptosis was also examined in an organ-culture system. RESULTS: Three of 17 postoperative specimens contained TUNEL-positive cells. In 2 lens capsules obtained earlier than 10 days, many TUNEL-positive cells, presumably apoptotic LECs, were observed beneath the residual anterior capsule. In cell multilayers in capsule opacification extracted later than 10 days, a few TUNEL-positive cells were seen in 1 specimen; most cells remained negative. In mouse lenses organ-cultured with 1.0 ng/mL TGF-beta2 for 48 hours, TUNEL-positive cells were detected beneath the lens capsule. CONCLUSIONS: Lens epithelial cells undergo apoptosis during healing after cataract surgery, especially in the early phase. Transforming growth factor-beta2 may be a factor inducing apoptosis in in vivo LECs.

Cellular fibronectin, but not collagens, disappears in the central posterior capsules during healing after lens extraction and IOL implantation in rabbits.

PURPOSE: 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.