|Molecular Vision 2003;
Received 16 July 2003 | Accepted 8 December 2003 | Published 15 December 2003
Exacerbation of TGF-β-induced cataract by FGF-2 in cultured rat lenses
Anna Cerra, Kylie J. Mansfield,
Coral G. Chamberlain
Department of Anatomy and Histology and Institute for Biomedical Research (F13), The University of Sydney, New South Wales, Australia
Correspondence to: Coral G. Chamberlain, Department of Anatomy and
Histology (F13), University of Sydney, Sydney, New South Wales,
Australia 2006; Phone: 61-2-9351-5169; FAX: 61-2-9351-2817; email:
Dr. Mansfield is currently at the Department of Physiology and Pharmacology, University of New South Wales, Sydney, New South Wales, Australia 2052
Purpose: Culturing rat lenses with transforming growth factor-beta (TGFβ) 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 TGFβ-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 (α-smooth muscle actin, fibronectin, and type I collagen), or by measuring DNA accumulation in the epithelial region.
Results: Supplementing TGFβ 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 TGFβ 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 TGFβ alone.
Conclusions: FGF has been identified as a factor capable of exacerbating the cataractogenic effects of TGFβ. Thus FGF inhibitors, as well as TGFβ inhibitors, have the potential to protect the lens against TGFβ-induced cataractous changes.
Cataract is emerging as a major global public health issue because of increasing life expectancy and the strong link between cataract and aging. While many predisposing factors have been identified, elucidating the cellular events underlying the development of human cataracts is an ongoing challenge. There is now substantial evidence that members of the transforming growth factor-beta (TGFβ) family, already implicated in many human diseases , induce aberrant changes in lens cells [2-14] that mimic events in the development of human subcapsular cataracts [15,16] and posterior capsule opacification (PCO) , a condition that occurs post-cataract surgery. These changes include formation of spindle-shaped cells accompanied by wrinkling of the lens capsule, accumulation of extracellular matrix (ECM) and cell death associated with nuclear pyknosis and other features of apoptosis. TGFβ is present in and near the lens in situ, predominantly as the TGFβ-2 isoform and in latent form, and its activity appears to be tightly regulated under normal conditions . All three mammalian isoforms induce cataractous changes in lens cells in vitro, TGFβ-2 and TGFβ-3 being more potent than TGFβ-1 .
Whole rat lenses cultured with TGFβ develop anterior opacities corresponding to fibrotic plaques that are analogous to human anterior subcapsular cataract [4,6,7,10]. Furthermore, in rat models in vivo and in vitro, TGFβ promotes an epithelial-mesenchymal transition that results in the synthesis of proteins such as α-smooth muscle actin, type I collagen, fibronectin, and tenascin [3,4,6,9-12]. These are found in human and canine anterior subcapsular cataract [18-21] but not in the normal lens. The findings of these studies in the rat have been confirmed and extended using a variety of mouse, rabbit, bovine, canine, and human models, both in vitro and in vivo [12,14,22,23]. Thus there is a marked consistency between mammalian species in terms of the changes associated with subcapsular cataract development and related effects of TGFβ on lens cells.
Members of the FGF family (FGF) are considered to play a significant role in establishing and maintaining normal lens structure and function [24-26]. They alone are known to have the capacity to initiate the differentiation of lens epithelial cells into fiber cells, both in vitro and in vivo. FGF-1 and the more potent isoform FGF-2 are continuously present in the normal lens environment . Several recent studies indicate that TGFβ and another TGFβ superfamily member, bone morphogenetic protein, may be involved in certain aspects of normal lens fiber cell differentiation [27-29]. However, exposure to active TGFβ at concentrations and/or under conditions that lead to cataractous changes clearly represents an abnormal event.
In general, when cells are exposed to both TGFβ and FGF, there may be either a reduction of biological activity or an additive or synergistic enhancement, depending on the cellular system and the culture conditions . Opposing effects of TGFβ and FGF and various interactions between TGFβ and FGF have been noted previously in studies of explanted or cultured lens epithelial cells [2,3,11,31-33]. For example, FGF and TGFβ, respectively, increase and decrease proliferation of cultured lens cells , while TGFβ inhibits FGF-induced αA-crystallin promoter activity in lens explants .
The present study has revealed that FGF exacerbates the cataractous changes induced by TGFβ in cultured rat lenses. This phenomenon occurs in lenses from young rats (3-5 weeks old) and adult rats. FGF is effective both at the relatively high doses generally required to induce fiber differentiation in lens epithelial cells  and at lower doses. Responses to TGFβ and FGF, administered alone or in combination, were assessed by monitoring lens opacification directly, by routine histology and immunolocalization of α-smooth muscle actin, fibronectin, and type I collagen, or by measuring accumulation of DNA in the epithelial region. An investigation of the effects on TGFβ-induced cataract of poly(4-styrenesulfonic acid) (PSS), a known inhibitor of FGF , and suramin, which inhibits a range of growth factors including FGF and TGFβ , was also included in this study.
Recombinant human FGF-2 and PDGF-AA (PeproTech, Rock Hill, NJ) and TGFβ-2 (active form; Genzyme Cambridge, MA) were reconstituted and stored according to the manufacturers' instructions. PSS was obtained from Polysciences (Warrington, PA; molecular weight 70,000), suramin from Sigma (St Louis, MO).
Whole lens cultures
All experimental procedures conformed to the Institute for Laboratory and Animal Research (Guide for the Care and Use of Laboratory Animals). Culture experiments were carried out using lenses from young and adult rats. Initially, young lenses were obtained from 21-25 day old (weanling) Wistar rats. Subsequently, 4-5 week old (immature) rats were used, as these are less susceptible to damage during setting up of cultures. Lenses from weanling and immature rats showed similar patterns of response. Adult lenses were obtained from 6-9-month-old female rats. Whole lenses were removed as described previously  and placed in 4 ml (young rats) or 5 ml (adults) of serum-free M199 culture medium containing bovine serum albumin and antibiotics  and maintained at 37 °C in 5% CO2/air. About 2 h after dissection of the last lens, medium was changed and TGFβ and FGF were added alone or in combination, as indicated (10-20 μl aliquots). Lenses were cultured for 4-5 days (young rats) or 8 days (adults) with replacement of medium every two days. TGFβ-induced opacification developed more slowly in lenses from adult rats . For young rats, these additions were made on day 0 only. For adults, FGF was re-added on day 2 because adult lens cells are less responsive to FGF than those from young rats . Lenses cultured without growth factors served as controls. In some experiments, PSS or suramin was included on day 0 at final concentrations of 0.2 μM and 50-200 μM respectively, based on concentrations shown to be effective in inhibiting FGF in other studies in vitro [34,35].
Doses of 0.5-3 ng/ml TGFβ were used in experiments involving FGF as indicated, depending on factors such as the batch of TGFβ and the age/sex of the rat. In each of these experiments, the aim was to provide the lenses with a barely cataractogenic TGFβ stimulus in the absence of FGF. In some experiments, relatively high doses of FGF were used (20-30 ng/ml), shown previously to be capable of inducing lens epithelial cells to undergo fiber differentiation . In others, lower doses that favor proliferation were used (2.5-10 ng/ml) . PDGF-AA was used in place of FGF at a final concentration of 10 ng/ml. For assessment of PSS or suramin inhibition, 1.2 ng/ml TGFβ was used to generate a modest, readily detectable opacification response in the absence of the inhibitor.
Lenses were monitored daily for opacification and photographed using a dissecting microscope (Wild, Heerbrug, Switzerland) adapted to provide dark field illumination. At the end of the culture period, representative lenses were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Alternatively, whole mounts were prepared as follows. The epithelial region, which included transitional cells in the equatorial region, was peeled from the fiber mass and pinned out in a culture dish cell surface uppermost, using a dissecting microscope, as described previously for the preparation of epithelial explants . The explants were fixed in Carnoy's fixative (3:1, ethanol:acetic acid) and stored in 70% ethanol. Fixed epithelia were either photographed using a DMIL inverted microscope with Integrated Modulation Contrast and a DC200 digital camera (Leica Microsystems, Wetzlar, Germany), processed for scanning electron microscopy (SEM), or used for immunolocalization as whole mounts or after embedding in paraffin. Alternatively, the freshly collected epithelial region was transferred to 50 μl distilled water and frozen for assay of DNA.
Histology and immunolocalization
For SEM, samples were dehydrated with 100% ethanol, critical-point dried, gold-coated, and viewed using a Philips XL30 scanning electron microscope. Otherwise, serial sagittal 6 μm sections of paraffin-embedded lenses (or epithelial preparations) were cut from the equatorial to the central region and every fifth slide was stained with hematoxylin and eosin (H&E). Stained sections close to the central region of the lens were photographed and adjacent sections were used for immunolocalization or for picro-Sirius red staining of collagen. Immunolocalization of α-smooth muscle actin and fibronectin was carried out using a double-labelling technique based on previous methods [4,12,37]. Briefly, samples were blocked with 3% goat serum, then 3% rabbit serum, followed by overnight incubation with mouse α-smooth muscle actin antibody (clone 1A4, 1:400; Sigma) and a 90 min incubation with goat anti-rat fibronectin (1:100; Calbiochem, La Jolla, CA). Alexa 488-conjugated anti-mouse and Alexa 546-conjugated anti-goat secondary antibodies (1:400; Molecular Probes, Eugene, OR) were used. For immunolocalization of type I collagen and β-crystallin, the rabbit serum blocking step was omitted, and anti-fibronectin was replaced with rabbit anti-rat type I collagen (1:50; Chemicon, Temecula, CA) or anti-β-crystallin (1:100)  visualised with a Cy3-conjugated anti-rabbit antibody (1:400; Zymed, San Francisco, CA). Phosphate-buffered saline supplemented with 0.1% bovine serum albumin (PBS-BSA) was used to dilute the antibodies and to rinse the samples briefly between antibody applications. Fixed epithelial whole mounts were rehydrated with PBS-BSA and permeabilised with 0.5% Tween 20 for 10 min prior to immunolocalization. All samples were counterstained with Hoechst dye (catalog number 33342; Sigma) and mounted using Vectashield (Vector Laboratorie, Burlingame, CA). Under these conditions, immunoreactivity was absent from controls in which antibodies were replaced with corresponding non-immune IgGs.
After dilution with an equal volume of 2X cell culture lysis reagent (Promega, Madison, WI) and incubation at 37 °C for 30 min, samples were diluted further with distilled water and assayed by the PicoGreen dsDNA method (Molecular Probes) using a FLUOstar Galaxy plate reader (BMG Labtechnologies, Offenburg, Germany).
Data from DNA analyses were analysed by Student's t test, or by one-way ANOVA with comparison of selected treatment groups and Bonferroni correction for multiple comparisons, using GraphPad Prism (GraphPad Software, San Diego, CA). Fisher's exact test was used for comparing numbers of lenses with and without opacities in various treatment groups.
Effect of FGF on TGFβ-induced lens opacification: Young rats
Culturing lenses from weanling rats with TGFβ at 3 ng/ml resulted in the formation of distinct anterior opacities over a four day culture period (Figure 1A). Initial patchy clouding of the lens anterior, usually detectable by day 2 of culture, was followed by a process of "condensation" leading to the formation of discrete opacities surrounded by relatively clear areas . In contrast, opacities did not form in any lenses cultured in control medium only, whether lenses were from weanlings (Figure 1B) or from immature or adult rats (not shown).
An experiment in which TGFβ was used at a lower, weakly cataractogenic dose is shown in Figure 1C,D. Two of four lenses cultured with TGFβ alone developed small discrete opacities (Figure 1C), the rest exhibited clouding of the anterior only. Inclusion of 20 ng/ml FGF with TGFβ resulted in a marked increase in opacification in all lenses (Figure 1D). Numerous opacities were distributed over the lens anterior. These were exceptionally large in the peripheral region above the lens equator (Figure 1D).
Comparable enhancement of TGFβ-induced opacification by 20 ng/ml FGF was observed in lenses from immature male and female rats (not shown; TGFβ concentration, 0.8 ng/ml). Marked enhancement of opacification was also observed under the latter conditions when FGF was used at relatively low concentrations (Figure 1E,F). However, plaques were more peripherally located and appeared to be only partially condensed, especially at the lower concentration.
No opacities were detected when lenses were cultured with 20 ng/ml FGF alone, although the anterior tended to develop a hazy, "dusty" appearance by day 4 of culture over the entire anterior of weanling lenses but confined to the peripheral region above the equator in lenses from immature rats (not shown).
Effect of FGF on other TGFβ-induced cataractous changes: Young rats
Whole mounts of cells and plaques adhering to the anterior capsule were prepared from some lenses and investigated by SEM. Alternatively, whole mounts were paraffin-embedded and assessed histologically and for the presence of markers of fibrotic change.
SEM confirmed that inclusion of FGF with TGFβ caused a marked enhancement of plaque formation in weanling lenses (Figure 2A,B). It also gave rise to differences in the nature of the plaques that formed and in the morphology of the cells (compare Figure 2A,C,E with Figure 2B,D,F). In addition, cellular surfaces were often coated with a meshwork of ECM in lenses cultured with TGFβ plus FGF. A region with a particularly heavy deposit is shown in Figure 2F. In contrast, with TGFβ alone a fine meshwork of ECM-like material was observed only occasionally (not shown). In the latter case, this was restricted to non-elongated cells, which in some other regions appeared to be detaching from the lens capsule.
Plaques were absent from control lenses (no additions) and epithelial cells were present in a loosely packed cobblestone array (Figure 2G). No plaques were present in lenses cultured with FGF alone, but cells in the epithelial region showed distinctive changes including cell enlargement and formation of tongue- or flap-like processes (Figure 2H). These changes, which are consistent with early fiber differentiation , were observed also in lenses cultured with TGFβ plus FGF, but they were limited to occasional patches of cells (not shown).
Lenses from rats of all ages cultured without growth factors retained normal morphology. The fiber mass was covered anteriorly by a continuous monolayer of closely packed epithelial cells (not shown). When lenses from weanling (or immature) rats were cultured with a low dose of TGFβ alone, most of the lens epithelium remained as a monolayer (Figure 3A), but occasional patches of multilayering (2-3 cells thick) were observed. In the lens depicted in Figure 3A, a distinct plaque was present at the anterior pole (Figure 3B). This was associated with slight wrinkling of the lens capsule. When FGF was included with TGFβ, generalized multilayering of the epithelium occurred (2-7 cells thick), numerous large, deep plaques were present, and strong wrinkling of the capsule was observed (Figure 3C). With FGF alone, the epithelium remained as a monolayer (Figure 3D) but was slightly thicker than the normal epithelium.
Plaques induced by culturing lenses with TGFβ alone expressed markers for fibrosis found in human anterior subcapsular cataracts, α-smooth muscle actin, type I collagen, and fibronectin as reported previously [4,6,10,12]. The large plaques induced when TGFβ was supplemented with FGF also expressed these markers, as did cells in multilayered regions of the epithelium (Figure 4A-C; fibronectin not shown). Sections from corresponding lenses cultured without addition of growth factors or with FGF alone generally exhibited negligible reactivity for these markers (Figure 4D-I). However, occasional patches of weak reactivity for α-smooth muscle actin were observed in no growth factor controls (Figure 4E) but not in FGF controls (Figure 4H). Analogous suppression of basal α-smooth muscle expression by FGF has been noted previously in lens epithelial explants from rats of this age . TGFβ/FGF-induced plaques also contained clusters of pyknotic nuclei (Figure 4A) and Sirius red staining revealed abnormal deposits of ECM (not shown). These are also features of cataractous plaques induced by TGFβ alone [4,6].
The extensive multilayering and plaque formation observed in lenses cultured with TGFβ and TGFβ/FGF suggested that cell proliferation occurred under these conditions. This was confirmed by DNA analysis. Culturing lenses from immature rats with TGFβ at 0.8 ng/ml led to a significant increase in the DNA content of the epithelial region of the lens, from 170±50 ng in controls to 510±190 ng in lenses cultured with TGFβ (mean±SEM; n=13-15; p<0.001). Supplementing TGFβ with FGF at 20 or 2.5 ng/ml resulted in a further overall increase to 760±230 ng DNA per epithelial region (n=15-16; p=0.003), with no significant difference between the effects of FGF at the two concentrations tested.
Effect of FGF on TGFβ-induced cataractous change: Adult rats
Because of the known link between aging and the incidence of human cataract, adult rats were included in the present study to ascertain whether FGF-induced exacerbation of cataractous changes occurs in these lenses as in lenses from young rats. It was important to address this issue because epithelial cells in adult rat lenses are known to be less responsive than those from young rats in terms of their ability to undergo FGF-induced lens fiber differentiation . The lenses of adult female rats were used. Their lenses are relatively resistant to the cataractogenic effects of TGFβ in the absence of FGF .
It was found that FGF also exacerbated TGFβ-induced opacification in lenses from adult rats. TGFβ alone (3 ng/ml) induced only a weak opacification response, with formation of a few small opacities in the lens anterior. Culturing lens with TGFβ and FGF (30 ng/ml, re-added on day 2) resulted in the entire lens anterior becoming heavily clouded but without formation of discrete opacities at any stage of culture, the response being greater towards the lens periphery (not shown). Clouding of this intensity was not observed in lenses cultured with TGFβ or FGF alone at any age. The clouding was associated with the formation of a thickened cellular layer (4-12 cells deep) over virtually the whole of the lens anterior with no evidence of discrete plaque formation. Multilayering was most extensive in the more peripheral regions, as depicted in Figure 4J. Elongated nuclei were present, especially adjacent to the fiber mass. In corresponding lenses cultured with TGFβ alone under these conditions, several small plaques with associated thickening of the cell layer formed in the central region and at the lens equator but generally the cell layer was only 1-2 cells deep (not shown).
The thick cellular layer induced by TGFβ and FGF showed reactivity for α-smooth muscle actin and fibronectin. In the peripheral region where multilayering was maximal, these markers tended to be localized predominantly in layers of cells close to the capsule (Figure 4J-L). Elsewhere, reactivity was more uniform. Many cells also exhibited reactivity for type I collagen (not shown). In contrast, β-crystallin, which is a marker for FGF-induced lens fiber differentiation , was detected only in the lens fiber cell mass (not shown). In corresponding lenses cultured with TGFβ alone, reactivity for α-smooth muscle actin and fibronectin was detectable in plaques but not in the cell layer that covered most of the lens anterior (not shown).
Comparison of effects of PDGF and FGF on TGFβ-induced opacification
Lenses from immature rats were cultured with a low dose of TGFβ (0.5 ng/ml) without further additions or with either FGF or PDGF at a concentration of 10 ng/ml. For each of these growth factors, the latter dose has been shown to induce near maximal proliferation of normal rat lens cells in explants [39,40]. TGFβ alone induced a few small, weak opacities in the anterior of some lenses over the 4 day culture period (Figure 5A). Inclusion of PDGF with TGFβ appeared to have no effect on the opacification response. With or without PDGF three of four lenses showed a weak response (Figure 5B) while the other remained clear. In contrast, when FGF was included with TGFβ instead of PDGF, a marked exacerbation of opacification occurred in all four lenses, especially in the region just above the lens equator (Figure 5C).
Effect of inhibitors on TGFβ-induced cataractous changes
As the lens itself is a source of FGF , it is possible that endogenous FGF contributes to the opacification response when lenses are exposed to TGFβ alone. Because of this and the marked exacerbating effect of even low concentrations of exogenous FGF noted above, inhibitors known to be capable of inhibiting FGF were tested for efficacy in suppressing TGFβ-induced cataract. Lenses from immature rats were cultured with a cataractogenic dose of TGFβ, alone or with 0.2 μM PSS. Inclusion of PSS resulted in a significant reduction in the number of lenses that exhibited TGFβ-induced opacities by day 4 of culture (Table 1). PSS itself had no effect on lens transparency. With TGFβ alone, plaques and spindle cells formed in the central epithelial region of the lens (Figure 6A,C). In contrast, when PSS was included with TGFβ, plaques and spindle cells were not readily distinguishable and most cells were present in a cobblestone array (Figure 6B,D) as in the normal lens epithelium. Immunoreactivity for α-smooth muscle actin was readily detectable in the plaques induced by TGFβ alone, but undetectable when PSS was included with TGFβ (not shown).
Including suramin at 100-200 μM completely inhibited TGFβ-induced plaque formation (Table 1, Figure 7A,B), while at 50 μM there was partial inhibition. The corresponding increase in lens epithelial DNA was also substantially blocked by suramin (p<0.05, Figure 8). In controls, the epithelium retained the monolayered morphology typical of the normal lens (Figure 7C). However, the epithelium of lenses cultured with TGFβ plus suramin consisted of two layers of cells in most regions (Figure 7B). A similar bilayer of cells was observed in about one third of the epithelium of a lens cultured with 200 μM suramin alone, which otherwise retained normal morphology (not shown). Also, with or without TGFβ, suramin induced a streaky whitening of the anterior and posterior lens capsule, especially in the polar regions and at higher concentrations. Typical localization of α-smooth muscle actin and fibronectin was detected in lenses cultured with TGFβ but reactivity was undetectable in lenses cultured with TGFβ plus suramin and in controls cultured with or without suramin (not shown).
There is now considerable evidence that, in humans and other mammals, TGFβ plays a role in the etiology of anterior subcapsular cataract (see Introduction). TGFβ-1 and TGFβ-2 are expressed by lens epithelial cells in situ , together with TGFβ type I and II receptors . TGFβ-2 is also present in the ocular media that bathe the lens mainly in latent form [41,42], but it may become activated under certain conditions . Average concentrations of active TGFβ-2 ranging from 0.2-2.5 ng/ml have been reported for aqueous collected at the time of cataract surgery from otherwise normal human eyes and levels may be even higher when cataract is associated with other diseases [41-44]. As lens cells appear to require stimulation by TGFβ (or related molecules) at certain stage(s) in the normal lens fiber differentiation process [27-29], its activity must be tightly regulated under normal conditions. Some ways in which lens cells may be protected from inappropriate TGFβ stimulation have been elucidated [5,6] but other mechanisms may exist.
Lens cells are also exposed to FGF in situ [24,45]. FGFs play a unique role in initiating the differentiation of lens epithelial cells into fiber cells near the lens equator , a process that continues throughout life. The ciliary body, retina, lens capsule, and the lens cells themselves are all potential sources of FGF, which is also present in the aqueous and vitreous, the ocular media that bathe the lens (reviewed in [24,46]). Little is known about absolute concentrations of the various FGF isoforms available to lens cells. Bovine vitreous contains considerably more than 1 ng/ml FGF-2, with much less in the aqueous . However, in certain regions, for example, near the equator where the lens lies close to the FGF-rich ciliary body, local concentrations probably exceed average values. Concentrations of 30-50 ng/ml FGF-2 have been reported in human vitreous from patients with diabetic retinopathy .
The present study in a cultured rat lens model has revealed that the presence of FGF exacerbates the cataractogenic effects of TGFβ. Relevant data are summarized in Table 2. This effect of FGF was exemplified by the formation of extremely large, deep, opaque plaques in lenses from young rats and a generalized deep multilayering of the epithelial region in lenses from adults under the culture conditions used, which differed for young and adult rats. For young rats, a concomitant increase in the number of cells in the epithelial region was confirmed by DNA assays. Note that the distribution of plaque formation differs in this rat model from human anterior subcapsular cataract, in which a single large anterior polar opacity forms. This probably reflects the fact that in the rat model the entire lens epithelium is exposed to effective doses of TGFβ, a situation that does not necessarily prevail in situ (discussed in ).
Clearly, enhanced cell proliferation contributes to the phenomenon reported here but it is not known whether FGF acts alone or in concert with TGFβ to achieve this effect. FGF has been shown to additively or synergistically enhance TGFβ-induced proliferation of many cell types . Interestingly, PDGF did not exacerbate TGFβ-induced cataract (Figure 5) when added at a dose comparable to that of FGF in terms of its proliferative effects on normal rat lens epithelial cells in explants . Note that the majority of the cells that accumulated in response to TGFβ/FGF treatment were not normal lens cells, as evidenced by their expression of α-smooth muscle actin, type I collagen, or fibronectin.
Whereas FGF is a potent mitogen for normal lens epithelial cells [24,40], TGFβ has been shown to induce apoptosis [2,5,13], and apoptotic cells have been found in human anterior subcapsular cataract and PCO . Thus, potentially, FGF may contribute to the outcome observed in this lens study not only by stimulating cell proliferation but also by suppressing TGFβ-induced apoptosis. A recent study in lens epithelial explants is consistent with the latter suggestion. It was found that addition of FGF-2, but not PDGF or a range of other growth factors tested, promoted the survival of TGFβ-affected cells (unpublished). Without addition of FGF, exposure of explants to even low doses of TGFβ results in apoptotic changes that lead to loss of virtually all cells within a few days .
Of particular interest in the present context is a study of newborn mice by Shinozaki et al. . They showed that subcutaneous injection with TGFβ led to transient induction of granulation tissue, whereas simultaneous injection of TGFβ and FGF but not FGF alone caused a marked fibrotic response which persisted for at least two weeks. The fibrosis-exacerbating effect of FGF was not mimicked by PDGF or epidermal growth factor. The ECM-like deposits and abundant accumulation of ECM proteins, such as fibronectin and type I collagen, observed in TGFβ/FGF-treated lenses (Figure 2F and Figure 4C), are consistent with a comparable role for FGF in promoting a fibrotic response in the lens. In the mouse model , FGF appears to exert its effect via induction of connective tissue growth factor . Elevated expression of connective tissue growth factor occurs in human anterior subcapsular cataract , which has been likened to a fibrotic wound healing response [12,24], but it is not known whether FGF influences connective tissue growth factor synthesis in the rat lens model described here.
It has previously been shown that the effect of FGF on normal lens epithelial cells is dose dependent . In rat lens epithelial explants, proliferation and migration are induced by relatively low concentrations of FGF-2, but induction of fiber differentiation requires concentrations of 10 ng/ml or more . In the normal lens in situ, only the epithelial cells near the lens equator receive a strong enough FGF stimulus to initiate fiber differentiation .
In the present study, a low non-fiber-differentiating dose of FGF-2 (2.5 ng/ml [31,50]) was found to be sufficient to enhance TGFβ-induced opacification. However, in lenses from young rats, at this low dose "condensation" of regions of opacification into large, discrete plaques appeared incomplete and the response occurred mainly in the cells near the lens equator, which are more responsive to the fiber differentiating effects of FGF [36,51]. Furthermore in TGFβ/FGF-treated lenses from adult lenses, in which epithelial cells are generally less responsive to the fiber-differentiating effects of FGF than those from younger animals [36,51], discrete plaques did not form at any stage despite a relatively high FGF stimulus, prolonged culture, and the accumulation of extensive multilayers of abnormal cells in the epithelial region. The present study thus raises the possibility that the process of condensation into plaques requires a relatively high level of FGF stimulation, as does fiber differentiation.
FGF has been shown to influence cataract-related effects of TGFβ in several previous studies. For example, in lens epithelial explants from weanling rats FGF appears to suppress TGFβ-induced α-smooth muscle actin expression . Furthermore, in bovine lens epithelial cells cultured in a collagen gel FGF inhibits TGFβ-induced increases in both α-smooth muscle actin expression and collagen gel contraction . Gel contraction is presumably due to the induction of contractile myofibroblastic cells which express α-smooth muscle actin. Thus in the latter two studies [3,32] FGF was found to suppress one particular aspect of TGFβ-induced cataractogenesis, the induction of myofibroblasts. In contrast, in the present study in which TGFβ-induced cataractogenesis was assessed mainly in terms of lens opacification and fibrotic plaque formation, FGF was found to have an exacerbating effect. Note that plaques in rat lenses cultured with TGFβ plus FGF (Figure 4) or TGFβ alone  contain both cells that are strongly reactive for α-smooth muscle actin and cells that are not, especially deep within the plaques or multilayered regions. In addition, two morphologically distinct cell types were observed by SEM in lenses cultured with TGFβ with an apparent association between plaque formation and channels of non-spindle-like cells (Figure 2).
The fact that FGF is present in the lens itself (see above) raises the possibility that endogenous FGF contributes to cataract development when intact lenses are exposed to TGFβ in vitro. Consistent with this suggestion, inclusion of PSS, a heparin mimetic which disrupts binding of FGF to its receptors and is a potent inhibitor of FGF-2 , suppressed both TGFβ-induced opacification and expression of α-smooth muscle actin. However, as the specificity of PSS for FGF has not been established  the possibility that it is acting by inhibiting some other heparin-binding growth factor cannot be excluded.
The polyanion suramin, which has been shown to inhibit FGF, TGFβ, and several other growth factors [35,52], was also effective in preventing TGFβ-induced opacification and other cataractous changes. However, the observed secondary effects of suramin on lens cellular architecture and lens capsule transparency suggest it would not be suitable for use as a therapeutic agent in the eye. More potent derivatives of suramin, currently under investigation in other therapeutic contexts , may be worth investigating in relation to cataract prevention.
In summary, inclusion of FGF, but not PDGF, in rat lens cultures enhances the ability of TGFβ to induce opaque subcapsular plaques with features of human subcapsular cataract, while PSS and suramin counter the cataractogenic effects of TGFβ. This study reveals that FGF inhibitors, as well as TGFβ inhibitors, may prove useful for the prevention of TGFβ-related forms of cataract, paving the way for new therapeutic approaches.
This project was supported by a grant from the National Health and Medical Research Council of Australia and by iCare Pty Ltd. The authors also thank Yongjuan Chen for skilled research assistance, Roland Smith and Clive Jeffrey for advice and assistance with figure preparation, Jane Radford, Department of Pathology, University of Sydney, for carrying out Sirius red staining and the Electron Microscope Unit, University of Sydney, for the use of its facilities.
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