Coral G. Chamberlain

TGF-β1 induces lens cells to accumulate α-smooth muscle actin, a marker for subcapsular cataracts

Spindle-shaped myofibroblast-like cells, which contain α-smooth muscle actin, have been described in anterior subcapsular cataract and after-cataract. In a previous study in this laboratory, it was shown that transforming growth factor-β (TGFβ) induces the formation of spindle-shaped cells in lens epithelial explants. The aim of this investigation was to determine whether these TGFβ-induced spindle-shaped cells contain α-smooth muscle actin. Lens epithelial explants were prepared from 21-day-old rats and cultured with either TGFβ1 or basic FGF alone, a combination of both growth factors, or without added growth factors. After three days, cellular changes were monitored by phase contrast microscopy, localisation of filamentous actin with rhodamine-phalloidin, and immunolocalisation and immunoblotting of α-smooth muscle actin.TGFβ induced rapid cell elongation and formation of characteristic spindle-shaped cells in lens epithelial explants in the presence or absence of FGF. These cells contained α-smooth mu...

Induction of Lens Fibre Differentiation by Acidic and Basic Fibroblast Growth Factor (FGF)

Explants of epithelial cells from newborn rat lenses undergo changes characteristic of fibre differentiation when cultured with neural retina or retina-conditioned medium. Here we show that similar changes occur when acidic and basic fibroblast growth factor (FGF) are used instead of retina-conditioned medium. When cultured without FGF, epithelial explants contained negligible amounts of beta-crystallin, a lens protein found only in fibre cells. However, at saturating concentrations of FGF, about 20 micrograms beta-crystallin was produced per explant in 5 days. The response was dose-dependent, half maximal response requiring 55 and 290 ng/ml of basic and acidic FGF, respectively. FGF also stimulated cell proliferation and cell migration. All three responses to basic FGF were blocked by an antibody specific for basic FGF. The concentration of FGF required to produce a maximal response was lower for cell proliferation and migration than for beta-crystallin accumulation. The results suggest a possible role for FGF in the control of events in lens development.

Evidence that fibroblast growth factor promotes lens fibre differentiation

Lens epithelial cells from newborn rats undergo changes characteristic of fibre differentiation when cultured with rat neural retina or with a soluble mitogenic factor present in calf retina-conditioned medium. Mitogens have been isolated from retina in other laboratories, but have not previously been shown to promote fibre differentiation in mammalian lens. We prepared eye-derived growth factors I and II and alpha- and beta-retina-derived growth factors from bovine retinas. These factors all promoted lens fibre differentiation in our culture system, as assessed by morphological changes and the appearance of fibre cell-specific crystallins. There is now strong evidence that these retina-derived factors are identical to the acidic and basic forms of fibroblast growth factor (FGF), which is present in a variety of tissues. We found that acidic and basic FGF from rat brain also promoted lens fibre differentiation, suggesting that FGF is the factor from the retina responsible for inducing lens fibre differentiation.

Growth factors in the eye.

In this review we report the distribution and functional significance of growth factors in the eye. Representatives of the major growth factor families are found in the eye: fibroblast growth factor, insulin and insulin-like growth factor, transforming growth factor-beta, platelet-derived growth factor, nerve growth factor, epidermal growth factor and colony-stimulating factor. There are numerous examples of their actions on ocular tissues in vitro and in some cases in vivo. The findings presented clearly illustrate that a growth factor can elicit different responses depending on the context of its action; the cell type involved, the concentration of the growth factor and the presence or absence of other growth factors can all influence the cellular response both quantitatively and qualitatively. The results of these studies in the eye are of general significance to our understanding of the role of growth factors in biological processes.

TGFβ induces morphological and molecular changes similar to human anterior subcapsular cataract

Background: Transforming growth factor β (TGFβ) 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 TGFβ1 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 TGFβ2. 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 TGFβ 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 TGFβ.

The role of fibroblast growth factor in eye lens development.

In this review we have presented evidence that FGF plays an important role in inducing events in lens morphogenesis and growth. Our studies show that FGF stimulates lens epithelial cells in explants to proliferate, migrate, and differentiate into fibers at low, medium, and high concentrations, respectively. This has some important implications for understanding the behavior of lens cells in the eye. The fact that aFGF is detected in the equatorial region of the lens where cells are actively proliferating, possibly migrating, and differentiating into fibers suggests that these events may be under autocrine control in vivo, at least to some extent. Because FGF is also present in the ciliary and iridial region of retina and in the vitreous, paracrine control may also be involved. Cell proliferation, fiber differentiation, and (possibly) cell migration occur in characteristic spatial patterns that are related to distinct compartments of the lens. We suggest that cells in the germinative zone receive only a low level of FGF stimulation arising from the cells themselves and possibly also from the ciliary and iridial regions of the retina but, whatever the source, this is only sufficient to stimulate proliferation. Lens epithelial cells that migrate or are displaced into the transitional zone below the lens equator receive some FGF from these sources but in addition receive a strong stimulus from the high level of FGF in the vitreous; thus, fiber differentiation is induced. Cells at the junction between these two zones may receive an intermediate level of FGF stimulation, sufficient to induce cell migration. In essence, we are proposing that, in the eye, FGF acts as a lens morphogen in the sense that different levels of FGF stimulation elicit different lens cell responses. Hence its characteristic distribution in the eye establishes lens polarity and growth patterns. Since FGF has an inductive effect on lens cells from mature age animals, we also propose that this specific distribution of FGF in the eye is also important for maintenance of a normal lens throughout life. Finally the synergistic effects of insulin/IGF on the FGF-induced responses highlight the importance of considering the distribution of members of the insulin/IGF family of molecules in vivo. Mechanisms that control levels of both the FGF and insulin/IGF families of factors in the eye are probably of crucial importance in the formation and maintenance of a normal lens.

Fibre differentiation and polarity in the mammalian lens: a key role for FGF

Abstract The mammalian lens exhibits characteristic antero-posterior patterns of cellular proliferation, movement and fibre differentiation. Based on our findings that fibroblast growth factor (FGF) induces proliferation, migration and differentiation in a similar sequence as its concentration is increased, we put forward the hypothesis that normal lens morphology with its antero-posterior patterns of cellular behaviour is determined by an antero-posterior gradient of FGF stimulation. Support for this hypothesis is now available from a wide range of studies, including: studies of the distribution of FGF and its mRNA in the eye and FGF activity in ocular media and the lens; studies of FGF receptor and mRNA expression in the lens; and studies of transgenic mice with altered patterns of FGF expression, which exhibit abnormal patterns of differentiation. Furthermore, gross abnormalities in lenses of transgenic mice that express a dominant-negative FGF receptor provide strong evidence that FGF is involved in the differentiation and maintenance of lens fibre cells in situ. Other biological molecules that may modulate the effects of FGF on lens cells in the normal lens have also been investigated, including capsule heparan sulphate proteoglycans, insulin and IGF and TGFβ. In addition, a potential role for TGFβ in the aetiology of cataracts has been identified. It is now clear that there are many possible mechanisms by which the behaviour of lens cells may be regulated to ensure normal growth and maintenance of polarity in the mammalian lens. Although several growth factors are probably involved, a key role for FGF has emerged.

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- f 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 TGFß‐induced cataract

Background: Studies in our laboratory have shown that transforming growth factor beta (TGFß) induces rodent lens epidielial 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 TGFß in our rodent models.

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.