J.W. McAvoy

Transforming Growth Factor-β-Induced Epithelial-Mesenchymal Transition in the Lens: A Model for Cataract Formation

The vertebrate lens has a distinct polarity and structure that are regulated by growth factors resident in the ocular media. Fibroblast growth factors, in concert with other growth factors, are key regulators of lens fiber cell differentiation. While members of the transforming growth factor (TGFβ) superfamily have also been implicated to play a role in lens fiber differentiation, inappropriate TGFβ signaling in the anterior lens epithelial cells results in an epithelial-mesenchymal transition (EMT) that bears morphological and molecular resemblance to forms of human cataract, including anterior subcapsular (ASC) and posterior capsule opacification (PCO; also known as secondary cataract or after-cataract), which occurs after cataract surgery. Numerous in vitro and in vivo studies indicate that this TGFβ-induced EMT is part of a wound healing response in lens epithelial cells and is characterized by induced expression of numerous extracellular matrix proteins (laminin, collagens I, III, tenascin, fibronectin, proteoglycans), intermediate filaments (desmin, α-smooth muscle actin) and various integrins (α2, α5, α7B), as well as the loss of epithelial genes [Pax6, Cx43, CP49, α-crystallin, E-cadherin, zonula occludens-1 protein (ZO-1)]. The signaling pathways involved in initiating the EMT seem to primarily involve the Smad-dependent pathway, whereby TGFβ binding to specific high affinity cell surface receptors activates the receptor-Smad/Smad4 complex. Recent studies implicate other factors [such as fibroblast growth factor (FGFs), hepatocyte growth factor, integrins], present in the lens and ocular environment, in the pathogenesis of ASC and PCO. For example, FGF signaling can augment many of the effects of TGFβ, and integrin signaling, possibly via ILK, appears to mediate some of the morphological features of EMT initiated by TGFβ. Increasing attention is now being directed at the network of signaling pathways that effect the EMT in lens epithelial cells, with the aim of identifying potential therapeutic targets to inhibit cataract, particularly PCO, which remains a significant clinical problem in ophthalmology.

Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition.

Posterior capsule opacification (PCO) is a common complication of cataract surgery caused by epithelial mesenchymal transition (EMT) and aberrant lens cell growth. One path to prevention depends on maintaining the quiescent lens epithelial phenotype. Here we report that lithium chloride (LiCl) is a potent stabilizer of the lens epithelial phenotype. In lens epithelial explants (controls), at low cell density, cells readily depolarized, spread out, and proliferated. By contrast, in the presence of LiCl, cells did not spread out or exhibit migratory behaviour. Using concentrations of 1–30 mM LiCl we also showed that cell proliferation is inhibited in a dose‐dependent manner. Confocal microscopy and immunohistochemistry for ZO‐1 and E‐cadherin showed that LiCl treatment maintained tight junctions at the apical margins of cells. Taken together with measurements of cell heights, this showed that the cells in LiCl‐treated explants maintained the apical baso‐lateral polarity and cobblestone‐like packing that is characteristic of lens epithelial cells in vivo. Significantly, the effects of LiCl also extended to blocking the potent EMT/cataract‐promoting effects of transforming growth factor β (TGFβ) on lens epithelial cells. In TGFβ‐treated explants, cells progressively dissociated from one another, taking on various elongated spindle shapes and strongly expressing α‐smooth muscle actin (α‐SMA). These features are characteristic of PCO. In both rat and human capsulorhexis explants, LiCl treatment effectively blocked the accumulation of α‐SMA and maintained the cells in a polarized, adherent, cobblestone‐packed monolayer. These findings highlight the feasibility of applying molecular strategies to stabilize lens epithelial cells and prevent aberrant differentiation and growth that leads to cataract. Copyright

TGFβ promotes Wnt expression during cataract development

TGFbeta induces lens epithelial cells to undergo epithelial mesenchymal transition (EMT) and many changes with characteristics of fibrosis including posterior capsular opacification (PCO). Consequently much effort is directed at trying to block the damaging effects of TGFbeta in the lens. To do this effectively it is important to know the key signaling pathways regulated by TGFbeta that lead to EMT and PCO. Given that Wnt signaling is involved in TGFbeta-induced EMT in other systems, this study set out to determine if Wnt signaling has a role in regulating this process in the lens. Using RT-PCR, in situ hybridization and immunolocalization this study clearly shows that Wnts 5a, 5b, 7b, 8a, 8b and their Frizzled receptors are upregulated in association with TGFbeta-induced EMT and cataract development. Both rat in vitro and mouse in vivo cataract models show similar profiles for the Wnt and Frizzled mRNAs and proteins that were assessed. Currently it is not clear if the canonical beta-catenin/TCF signaling pathway, or a non-canonical pathway, is activated in this context. Overall, the results from the current study indicate that Wnt signaling is involved in TGFbeta-induced EMT and development of fibrotic plaques in the lens.

Peter Bishop Lecture: growth factors in lens development and cataract: key roles for fibroblast growth factor and TGF-beta.

Growth factors play key roles in influencing cell behaviour and cell fates during development. Studies in our laboratory indicate that members of the fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β) growth factor families have central roles in lens developmental biology. The first section of this brief review presents evidence that indicates a role for FGF in normal lens development. The second section describes the varied effects of TGF-β on lens cells. This includes the induction of changes characteristic of certain types of cataract as well as evidence for a role in lens fibre cell maturation and/or survival.

The spatial relationship between presumptive lens and optic vesicle/cup during early eye morphogenesis in the rat

Mesodermal cells are pushed aside as the optic vesicle approaches the ectoderm but some remain sandwiched between the two tissues. Some of these sandwiched cells degenerate, others persist at least until the lens pit stage. During the period of close association between the optic vesicle/cup and presumptive lens there are cytoplasmic processes extending from both tissues. The processes are detected early on the 12th day before the lens placode forms. Processes are abundant between lens placode and optic vesicle. They are also detected between lens pit and optic cup but are not as common as between placode and vesicle. Cytoplasmic processes are not detected after 13 days when the space between the tissues has widened to form primary vitreous. The processes may be necessary for adequate communication between the tissues during the early period of their interaction. Coated pits and vesicles are commonly found in the processes. It is generally accepted that coated pits and vesicles are involved in receptor-mediated endocytosis, therefore it is suggested that they may be involved in the uptake of inducer molecules from the optic vesicle/cup. Coated pits and vesicles are also abundant at 13 days in the basal cytoplasm of the cells in the posterior part of the lens vesicle which form the primary fibre cells. In addition to the cytoplasmic processes there is a network of fibrils in the interspace. These are associated with the cytoplasmic processes and the basal surfaces of the presumptive lens and optic vesicle/cup. These are probably important for the co-ordinated invagination of lens placode and optic vesicle to form lens pit and optic cup, respectively. The vitreous arises from the space between presumptive lens and optic vesicle. There is a noticeable build-up of basal lamina material as development proceeds. Some of this extends into the interspace and is intimately associated with clumps of amorphous material. Thus at least some of the earliest vitreous constitutents are laid down by the epithelial cells lining the interspace.

Cytoplasmic processes interconnect lens placode and optic vesicle during eye morphogenesis

At 11 days of embryonic development of the rat there is a close association between presumptive lens ectoderm and optic vesicle. A space of 5·7±0·9 μm which separates the two tissues at this stage of development contains cytoplasmic processes and fibrils. The fibrils are probably mostly collagenous since some are seen to extend from the basel laminae and others have characteristic collagen banding patterns. The cytoplasmic processes have thick and thin components. The thin processes commonly arise from thick processes and are more numerous. In some cases the processes have been shown to bridge the interspace and make contact with the adjacent tissue.

Cell lineage analysis of lens epithelial cells induced to differentiate into fibres

In explant cultures, lens epithelial cells grown in unsupplemented medium retain a morphology and packing arrangement similar to that found in the lens in vivo. In this culture system the epithelial cells can be induced to differentiate into fibres by the addition of retina conditioned medium (RCM). RCM also stimulates cell division. In order to trace the fibre cell lineage and to examine the relationship between cell division and fibre differentiation, single epithelial cells in explants from neonatal rat lenses were labelled with fluorescein-isothiocyanate-dextran. Explants were sub-divided into nine squares and one cell per square was injected with fluorescent label via a microcapillary. By marking the positions of labelled cells at 24-hr intervals for 6 days it was shown that most of the epithelial cells moved laterally within the explant. On average, cells in control explants moved about 20 microns day-1. Cells did not move in any particular direction within the explants and often changed direction. RCM stimulated a dramatic increase in migratory activity. There was about a four-fold increase in migratory activity in the first 24-hr interval, then, even with continued exposure to RCM, this activity quickly dropped over the next 2 days to the same levels as found in controls. As in controls, the cells moved in no particular direction and often changed direction. The observation that RCM stimulates cell migration in this explant system raises the possibility of an important role for active cell migration in the lens in situ. After 6 days culture the dimensions of labelled cells were measured using an image analyser. The areas of cells in controls fell within a narrow range from about 60- to 200 microns2. In contrast, explants grown in RCM had a wide range of cell areas from about 120- to 1500 microns2 and a large proportion of the cells showed some degree of elongation. In explants grown in RCM, 27.3% of labelled cells divided and half of these divisions were during the first 24 hr of culture. Overall there were about 9% more divisions recorded in RCM-treated than in control explants. An analysis of sizes of cells after 6 days of culture showed that whether or not cells divided after addition of RCM they showed very similar frequencies of cell sizes. Therefore, proliferating cells in the explants appear to be as capable of elongating and differentiating into fibres as the non-proliferating cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of an inductive interaction between lens and neural retina in rats of different ages

Lens epithelial cells from neonatal rats cultured with neural retinas or neural retina-conditioned medium (RCM), undergo fibre differentiation. This is characterized by cell elongation, increased alpha-crystallin synthesis and the initiation of beta and gamma-crystallin synthesis. To determine if this tissue interaction continues in later life we developed an ELISA method to analyse patterns of alpha and beta-crystallin accumulation in epithelia from 3-day-, 10-day- and 21-day-old rats. Culture of lens epithelia with RCM resulted in the formation of multilayers of elongated fibres and the accumulation of alpha and beta-crystallins. The patterns of crystallin accumulation were essentially similar whether expressed as microgram crystallin per explant, or crystallin per DNA (ng per ng). alpha- and beta-Crystallins accumulated rapidly in explants after 2 days of culture in RCM, whereas explants grown in control medium showed no change in the crystallin levels from day 0 to day 10. Patterns of alpha- and beta-crystallin accumulation showed that there were no significant differences between the ability of lens epithelia from 3-day-, 10-day or 21-day-old rats to undergo fibre differentiation in response to RCM. Therefore we conclude that the inductive interaction between lens and neural retina is not restricted to embryonic or neonatal stages, but continues on throughout life maintaining normal patterns of fibre differentiation in the lens.

Fibrosis in the lens. Sprouty regulation of TGFβ-signaling prevents lens EMT leading to cataract.

Cataract is a common age-related condition that is caused by progressive clouding of the normally clear lens. Cataract can be effectively treated by surgery; however, like any surgery, there can be complications and the development of a secondary cataract, known as posterior capsule opacification (PCO), is the most common. PCO is caused by aberrant growth of lens epithelial cells that are left behind in the capsular bag after surgical removal of the fiber mass. An epithelial-to-mesenchymal transition (EMT) is central to fibrotic PCO and forms of fibrotic cataract, including anterior/posterior polar cataracts. Transforming growth factor β (TGFβ) has been shown to induce lens EMT and consequently research has focused on identifying ways of blocking its action. Intriguingly, recent studies in animal models have shown that EMT and cataract developed when a class of negative-feedback regulators, Sprouty (Spry)1 and Spry2, were conditionally deleted from the lens. Members of the Spry family act as general antagonists of the receptor tyrosine kinase (RTK)-mediated MAPK signaling pathway that is involved in many physiological and developmental processes. As the ERK/MAPK signaling pathway is a well established target of Spry proteins, and overexpression of Spry can block aberrant TGFβ-Smad signaling responsible for EMT and anterior subcapsular cataract, this indicates a role for the ERK/MAPK pathway in TGFβ-induced EMT. Given this and other supporting evidence, a case is made for focusing on RTK antagonists, such as Spry, for cataract prevention. In addition, and looking to the future, this review also looks at possibilities for supplanting EMT with normal fiber differentiation and thereby promoting lens regenerative processes after cataract surgery. Whilst it is now known that the epithelial to fiber differentiation process is driven by FGF, little is known about factors that coordinate the precise assembly of fibers into a functional lens. However, recent research provides key insights into an FGF-activated mechanism intrinsic to the lens that involves interactions between the Wnt-Frizzled and Jagged/Notch signaling pathways. This reciprocal epithelial-fiber cell interaction appears to be critical for the assembly and maintenance of the highly ordered three-dimensional architecture that is central to lens function. This information is fundamental to defining the specific conditions and stimuli needed to recapitulate developmental programs and promote regeneration of lens structure and function after cataract surgery.

Epithelial Explants and Their Application to Study Developmental Processes in the Lens

Publisher Summary The preparation of mammalian lens epithelial explants was originally described in brief by McAvoy and Fernon in 1984. Since that time, this model has been used extensively, and little has changed in the way the explants are prepared. Depending on what the resultant lens material is required for, much of the processing of explants can be carried out in the original tissue culture dish. If the resultant explants are required for determining protein or gene expression levels, the explants can simply be gently lifted off the base of the culture dish with fine forceps and transferred to the appropriate reagents for processing. On the contrary, if one would like to stain or immunolabel the lens cells as a wholemount, this is carried out in the dish. First and foremost, the explants will need to be fixed. Given that the explants start as a monolayer of cells that may become multilayered over the culture period, these tissues are relatively thin, so they will not require a lengthy fixation period. Twenty minutes usually suffices for aldehyde fixatives, and methanol fixation usually takes only a few minutes. Depending on the proteins to be identified, the type of fixative usually needs to be experimented with, because it may influence the labeling process, as for any other cell type. Following several rinses to wash out the fixative, tissue can be partially dehydrated and stored in 70% ethanol at 4°C until ready for use.