Glutathione and catalase suppress TGFbeta-induced cataract-related changes in cultured rat lenses and lens epithelial explants.

PURPOSE: The damaging effects of oxidative stress and transforming growth factor-beta (TGFbeta)-induced transdifferentiation of lens epithelial cells have both been implicated independently in the etiology of cataract. The aim of this study was to investigate whether the presence of antioxidant systems in the lens influences the ability of lens epithelial cells to respond to TGFbeta. METHODS: Whole lenses from young rats were cultured with or without TGFbeta in the presence or absence of reduced glutathione (GSH). Lens epithelial explants from weanling rats were used to investigate the effects of GSH and catalase on TGFbeta-induced cataract-related changes. Lenses were monitored for opacification for three to four days, photographed, and then processed for routine histology. Explants were assessed by phase contrast microscopy, enzyme-linked immunosorbent assay (ELISA) of alpha-smooth muscle actin (alphaSMA), and/or immunolocalization of alphaSMA and Pax6, markers for transdifferentiation and normal lens epithelial phenotype, respectively. RESULTS: In cultured lenses, GSH strongly suppressed TGFbeta-induced opacification and subcapsular plaque formation. In explants, both GSH and catalase suppressed changes typically associated with TGFbeta-induced transdifferentiation including wrinkling of the lens capsule, cell-surface blebbing, apoptotic cell loss, induction of alphaSMA, and loss of Pax6 expression. CONCLUSIONS: This study suggests that antioxidant systems present in the normal lens, which protect the epithelium against the damaging effects of reactive oxygen species, may also serve to protect it against the potentially cataractogenic effects of TGFbeta. Taken together with other recent studies, it also raises the possibility that TGFbeta may induce cataract-related changes in lens epithelial cells via release of hydrogen peroxide.

Nitric oxide, a survival factor for lens epithelial cells.

PURPOSE: Nitric oxide (NO) is capable of promoting either cell death or cell survival depending on cell type and experimental conditions. In this study, the possible effects of NO on the viability of lens epithelial cells were investigated in an explant model used previously to identify cellular changes associated with posterior capsule opacification following cataract surgery. METHODS: Rat lens epithelial explants prepared from weanling rats were cultured in a serum-free medium for five days with or without the addition of the nitric oxide synthase inhibitor, L-N(omega)-nitro-L-arginine methyl ester (L-NAME), using the inactive enantiomer D-NAME as a control. Alternatively, explants were cultured for nine days with or without the NO donor, sodium nitroprusside. Explants were assessed morphologically and immunohistochemically or by determining DNA content. RESULTS: In the presence of L-NAME but not in controls, progressive rounding up and detachment of cells from the lens capsule occurred, leading to extensive cell loss. Affected cells showed apoptosis-like cell-surface blebbing and nuclear fragmentation. Conversely, inclusion of sodium nitroprusside suppressed the morphological changes and spontaneous cell loss that occurred when sparsely covered explants were cultured for nine days, increased cell coverage fourfold during that period, and prevented the expression of the transdifferentiation markers alpha-smooth muscle actin and fibronectin. In addition, whereas L-NAME exacerbated cell loss induced by culturing with 50 pg/ml transforming growth factor-beta2, sodium nitroprusside offered protection. CONCLUSIONS: This study points to a previously unidentified role for NO as an endogenously produced survival factor for lens epithelial cells, raising the possibility of using NO deprivation as a means of removing residual lens cells following cataract surgery and thereby preventing posterior capsule opacification.

Aging-related changes in astrocytes in the rat retina: imbalance between cell proliferation and cell death reduces astrocyte availability.

The aim of this study was to investigate changes in astrocyte density, morphology, proliferation and apoptosis occurring in the central nervous system during physiological aging. Astrocytes in retinal whole-mount preparations from Wistar rats aged 3 (young adult) to 25 months (aged) were investigated qualitatively and quantitatively following immunofluorohistochemistry. Glial fibrillary acidic protein (GFAP), S100 and Pax2 were used to identify astrocytes, and blood vessels were localized using Griffonia simplicifolia isolectin B4. Cell proliferation was assessed by bromodeoxyuridine incorporation and cell death by TUNEL-labelling and immunolocalization of the apoptosis markers active caspase 3 and endonuclease G. The density and total number of parenchymal astrocytes in the retina increased between 3 and 9 months of age but decreased markedly between 9 and 12 months. Proliferation of astrocytes was detected at 3 months but virtually ceased beyond that age, whereas the proportion of astrocytes that were TUNEL positive and relative expression of active caspase 3 and endonuclease G increased progressively with aging. In addition, in aged retinas astrocytes exhibited gliosis-like morphology and loss of Pax2 reactivity. A small population of Pax2(+)/GFAP(-) cells was detected in both young adult and aged retinas. The reduction in the availability of astrocytes in aged retinas and other aging-related changes reported here may have a significant impact on the ability of astrocytes to maintain homeostasis and support neuronal function in old age.

MHC class II expression by beta2 integrin (CD18)-positive microglia, macrophages and macrophage-like cells in rabbit retina.

The aim of this study was to investigate the developmental expression of major histocompatibility complex class II (MHCII) by microglia and macrophages and their relationship to blood vessels in the retina, a representative tissue of the central nervous system. Such information is crucial to understanding the role of these cells in immune surveillance. Wholemount preparations of retinas from late embryonic, postnatal and adult rabbits were subjected to three-colour fluorescence microscopy using beta2 integrin (CD18) and MHCII antibodies and biotinylated Griffonia simplicifolia B4 isolectin labelling of blood vessels. CD18+ cells consistently exhibited characteristics of macrophages or microglia in the vascularized and non-vascularized regions of the retina, respectively. At all ages, MHCII was expressed by a high proportion of cells in the vascularized region, which contained macrophage-like 'parenchymal cells' as well as typical perivascular macrophages. MHCII expression by ramified microglia, first detected on postnatal day 30, was lower in the peripheral retina and intermediate in the avascular region of the myelinated streak. The observed localization of MHCII+ cells in relation to blood vessels and location-dependent differences in MHCII expression point to the possibility that these cells may be distributed strategically within the retina to provide multiple lines of defence against immune challenge arriving via the retinal vasculature.

Differing effects of dexamethasone and diclofenac on posterior capsule opacification-like changes in a rat lens explant model.

Posterior capsular opacification (PCO) arises from lens cells that remain associated with the lens capsule after cataract surgery and subsequently become abnormal, proliferate and migrate into the visual pathway. In this study, a rat lens explant model was used to assess the effects of the prototype steroidal and non-steroidal anti-inflammatory drugs, dexamethasone (DEX) and diclofenac (DIC), on epithelial cells undergoing PCO-like changes. Such drugs are widely used at the time of cataract surgery. TGFbeta2 and FGF-2 were added sequentially and explants were cultured for up to 30 days, with or without addition of DEX or DIC at a clinically relevant concentration. Without DEX or DIC, explants became multilayered and cells tended to retract into PCO-like plaques. Inclusion of DEX, but not DIC, resulted in transient formation of needle-like cells, enhanced cell coverage, and the retention a monolayer of migratory cells surrounding PCO-like plaques. With or without drug addition, most cells became aberrant, as indicated by loss of Pax6 expression and the presence of PCO markers alpha-smooth muscle actin and type I collagen; however, DEX and DIC both strongly enhanced type I collagen accumulation. Furthermore, DEX enhanced cell coverage in explants treated with TGFbeta alone. Thus the behaviour of lens cells was significantly and differentially affected by the presence of DEX and DIC, highlighting the possibility that drugs used to control inflammation after cataract surgery, and the clinician's choice of drugs, may influence PCO development.

Posterior capsule opacification-like changes in rat lens explants cultured with TGFbeta and FGF: effects of cell coverage and regional differences.

Following cataract surgery, many patients suffer secondary loss of vision because of posterior capsule opacification (PCO), which arises when residual lens epithelial cells become aberrant and migrate into the light path. Transforming growth factor-beta (TGFbeta)-induced transdifferentiation of lens cells appears to play a key role in this process. Fibroblast growth factor (FGF) may also play a role by promoting the survival of TGFbeta-affected cells and influencing their subsequent behaviour. In the present study, the effects of two different TGFbeta and FGF treatment regimes were compared in rat lens epithelial explants with either low or high initial cell coverage. Explants treated with 50 pg ml(-1) TGFbeta2 and 20 ng ml(-1) FGF-2 sequentially (day 0, day 1) or simultaneously (day 0), then cultured for up to 30 days with FGF, were assessed by light microscopy and immunolocalisation of markers for transdifferentiation (alpha-smooth muscle actin (alphaSMA) and type I collagen) or lens epithelial phenotype (Pax6) and fibre differentiation (beta-crystallin). By day 4, most cells had lost Pax6 reactivity, alphaSMA reactivity was evident, and there were differences between growth factor treatment groups, low and high initial cell coverage explants, and peripheral and central regions of explants. On day 30 of culture, all explants were well populated with cells, irrespective of treatment and initial cell coverage, and exhibited diverse PCO-like morphological changes, with expression of transdifferentiation markers and beta-crystallin in virtually all cells. Such overall resilience to variations in conditions may contribute to the insidious nature of PCO, while factors related to observed early differences between groups may contribute to PCO pleiomorphism.

Laminin-binding integrins in rat lens morphogenesis and their regulation during fibre differentiation.

Mammalian lens development involves cell-cell and cell-ECM interactions. As integrins are a major family of cell adhesion molecules, we examined the expression patterns of several integrin subunits (alpha3A, alpha3B, alpha6A, alpha6B, beta1 and beta4) during rat lens development. RT-PCR, in situ hybridisation, immunofluorescence and immunoblotting were used to investigate expression of integrin subunits during lens development and differentiation. RT-PCR showed expression of alpha3A, alpha6A, alpha6B and beta1A but not alpha3B or beta4 subunits in postnatal rat lenses. Each subunit displayed distinct spatio-temporal expression patterns. beta1 integrin was expressed in both epithelium and fibres. alpha3A subunit expression was restricted to the epithelium; expression ceased abruptly at the lens equator. Expression of the alpha6A subunit increased during fibre differentiation, whereas alpha6B expression was predominantly associated with epithelial cells during lens development. In lens epithelial explants, FGF induced some of the changes in integrin expression that are characteristic of fibre differentiation in vivo. One notable exception was the inability of FGF to reproduce the distinctive down-regulation of the alpha3 isoform that is associated with initiation of elongation in vivo. Interestingly, vitreous treatment was able to reproduce this shift in alpha3 expression indicating that another factor(s), in addition to FGF, may be required for full and complete transition from an epithelial cell to a fibre cell. Integrin subunit expression therefore appears to be highly regulated during lens development and fibre differentiation with evidence of major changes in alpha3 and alpha6 isoform expression. These results indicate that integrins may play important roles in development and growth of the lens. How specific integrin subunits influence the behaviour of cells in different developmental compartments of the lens remains to be determined.

Effects of dexamethasone on posterior capsule opacification-like changes in a rat lens explant model.

PURPOSE: Many patients whose sight is initially restored by cataract surgery eventually suffer secondary loss of vision because of posterior capsule opacification (PCO; after-cataract), a condition in which lens epithelial cells left behind at surgery become aberrant and migrate into the light path. The aim of this study was to determine whether dexamethasone (DEX), an anti-inflammatory agent widely used before and after cataract surgery, influences the behavior of lens cells under conditions relevant to PCO development. METHODS: An established rat PCO model was used in which explanted epithelial cells attached to the lens capsule are exposed sequentially to TGFbeta2 and FGF-2. Cultures with or without DEX (100 nM), and appropriate controls, were maintained for up to 30 days and assessed by light and scanning electron microscopy or immunolocalization of PCO markers (alpha-smooth muscle actin or fibronectin) or a marker for lens epithelial cell phenotype (Pax-6). RESULTS: In the absence of DEX, explants become multilayered and plaques that express PCO markers form. Cells tend to gather up into the plaques, leaving the surrounding lens capsule denuded. Changes in lens cell behavior with addition of DEX included rapid formation of long, needle-like cells, less extracellular matrix deposited on explant surface, and plaques surrounded by a monolayer of migratory cells. Immunolocalization confirmed that the latter were not normal lens epithelial cells. CONCLUSIONS: Lens cell behavior in this PCO model was significantly affected by inclusion of DEX, highlighting the possibility that its use as an anti-inflammatory at the time of cataract surgery may influence PCO development.

FGF-2 counteracts loss of TGFbeta affected cells from rat lens explants: implications for PCO (after cataract).

PURPOSE: While cataract surgery initially benefits most patients, many suffer secondary loss of vision because of posterior capsule opacification (PCO). Lens epithelial cells left behind at surgery become aberrant and migrate into the light path. TGF-beta (TGFbeta) appears to play a key role in this process by inducing the cells to undergo an epithelial-mesenchymal transition. Paradoxically, it also typically induces them to undergo apoptotic death. The present study was undertaken to investigate the hypothesis that FGF plays a role in PCO formation by promoting the survival of abnormal cells with PCO-like characteristics. METHODS: Rat lens epithelial explants were cultured for one day with TGFbeta2 (25-100 pg/ml) then in control medium with or without FGF-2 (5-100 ng/ml) for up to 31 days, with assessment by light and scanning electron microscopy and immunolocalization. RESULTS: Survival of TGFbeta treated cells was promoted by FGF-2 but not by EGF, PDGF, IGF, or HGF. In the absence of FGF virtually all cells were lost from explants within 5 days. However, when FGF was included cells remained viable throughout culture. These cells, which no longer expressed the lens epithelial marker Pax6, exhibited immunoreactivity for non-lens cell proteins associated with PCO (alpha-smooth muscle actin, type I collagen, and fibronectin) and also beta-crystallin. FGF inclusion also promoted ECM production, multilayering, and plaque formation, features of PCO known to contribute to visual loss. CONCLUSIONS: This study points to a key role for FGF in the etiology of PCO and suggests that FGF inhibitors may be useful in preventing PCO.

Exacerbation of TGF-beta-induced cataract by FGF-2 in cultured rat lenses.

PURPOSE: Culturing rat lenses with transforming growth factor-beta (TGFbeta) results in the formation of anterior, opaque subcapsular plaques which exhibit many of the features of human subcapsular cataract. The present study was undertaken to determine whether this process is influenced by the presence of fibroblast growth factor (FGF), a normal component of the lens environment in situ. METHODS: Rat lenses were cultured for 4-8 days with TGFbeta-2, alone or in combination with FGF-2, PDGF-AA, or the growth factor inhibitors poly(4-styrenesulfonic acid) (PSS) and suramin. Responses were assessed by monitoring opacification, by routine histology and immunolocalization of markers for fibrotic change (a-smooth muscle actin, fibronectin, and type I collagen), or by measuring DNA accumulation in the epithelial region. RESULTS: Supplementing TGFbeta at a barely cataractogenic dose with 2.5-30 ng/ml FGF-2 resulted in a very strong opacification response. The exceptionally large plaques that formed were similar histologically to those induced by TGFbeta alone at higher concentrations and showed immunoreactivity for all markers. PDGF at a concentration equivalent to FGF in terms of proliferative potential did not demonstrate this effect. Addition of either PSS or suramin reduced the opacification response induced by a cataractogenic dose of TGFbeta alone. CONCLUSIONS: FGF has been identified as a factor capable of exacerbating the cataractogenic effects of TGFbeta. Thus FGF inhibitors, as well as TGFbeta inhibitors, have the potential to protect the lens against TGFbeta-induced cataractous changes.

Influence of platelet-derived growth factor on lens epithelial cell proliferation and differentiation.

The expression pattern of platelet-derived growth factor (PDGF) and its receptor suggest a role in lens cell proliferation. PDGF is strongly expressed in the iris and ciliary body, situated opposite the proliferative cells of the lens epithelium which express the PDGF-alpha receptor. In this study, using lens epithelial explant cultures, we report that PDGF can induce a dose and time dependent increase in lens cell DNA synthesis. Culturing lens explants with both PDGF and FGF (a mitogen and differentiation factor for lens cells) resulted in responses greater than those induced by either growth factor alone. PDGF did not induce any changes typical of fibre differentiation; however, in combination with FGF it potentiated the fibre differentiating activity of FGF. Results obtained in this study support previous indications that PDGF has an important role in regulating lens cell proliferation. In addition, PDGF may have a role in potentiating FGF-induced lens fibre differentiation in vivo.

Apoptosis is a feature of TGF beta-induced cataract.

BACKGROUND: Studies in our laboratory have shown that transforming growth factor beta (TGF beta) induces rodent lens epithelial cells to undergo aberrant growth and differentiation that reproduces morphological and molecular features of human anterior subcapsular cataract and posterior capsule opacification. In addition, features of apoptosis have been described in some forms of human cataract. In the present study we investigated apoptotic changes induced by TGF beta in our rodent models. METHODS: Lens epithelial explants and whole lenses from rats were cultured with TGF beta. Morphological analysis and TUNEL were used to detect apoptotic changes. Transgenic mice expressing a self-activating form of human TGF beta 1 in the lens were included in the analysis. RESULTS: TGF beta-induced cell loss in epithelial explants coincided with increased numbers of pyknotic nuclei. Some of these nuclei were TUNEL-positive. Studies on lenses cultured with TGF beta and lenses from transgenic mice showed that the subcapsular plaques that developed contained pyknotic nuclei and that many of these were TUNEL-positive. DISCUSSION: This study shows that cells develop morphological and molecular features of apoptosis in TGF beta-induced cataract models. This confirms that apoptosis can be included as another TGF beta-induced cellular change that mimics events in human cataract.

TGFbeta induces morphological and molecular changes similar to human anterior subcapsular cataract.

BACKGROUND: Transforming growth factor beta (TGFbeta) has been shown to induce subcapsular plaques in cultured rat lenses as well as in lenses of transgenic mice. In the present study the authors have extended their analysis of these cataract models to determine how closely they mimic human cataract. In particular, they studied the maturation of cataract in the transgenic model to determine if it develops similar features as previously described for anterior subcapsular cataract (ASC) in humans. Furthermore, they investigated whether both of these animal models express the range of molecular markers that have now been shown to be present in human ASC. METHODS: Histology and periodic acid Schiff staining were used to study the development and maturation of subcapsular plaques in transgenic mice overexpressing TGFbeta1 in the lens. Immunolabelling methods were used to identify the molecular markers for ASC in both the transgenic mouse model and in rat lenses cultured with TGFbeta2. RESULTS: Histological analysis showed that the subcapsular plaques that develop in adult transgenic mouse lenses bear a striking similarity to mature human ASC, including the formation of a new epithelial-like layer extending between the subcapsular plaque and the underlying fibre mass. All known molecular markers for human ASC were induced in both rodent models, including collagen types I and III, tenascin, and fibronectin. They also identified the presence of desmin in these plaques, a putative novel marker for human cataract. CONCLUSIONS: In both transgenic mouse and rat lens culture models TGFbeta induces markers similar to those found in human ASC. Atypical expression of these cataract markers is also characteristic of posterior capsular opacification (PCO). The molecular markers expressed are typical of a myofibroblastic/fibroblastic phenotype and suggest that a common feature of ASC and PCO may be induction of an epithelial-mesenchymal transition by TGFbeta.