Dexamethasone influences FGF-induced responses in lens epithelial explants and promotes the posterior capsule coverage that is a feature of glucocorticoid-induced cataract.

Aberrant spreading of lens epithelial cells along the posterior capsule is the basis for development of glucocorticoid (GC)-induced cataract; the resulting foci of nucleated cells at the posterior pole causing disruptions to normal lens cellular architecture. In this study, rat lens epithelial explants were used to assess the effects of dexamethasone (DEX), a widely used synthetic GC, on FGF2-induced lens cell proliferation and elongation as well as the ability of lens cells to spread and cover the posterior capsule. In the presence of FGF2, DEX significantly promoted lens cell proliferation after 48 h. Cell coverage of the posterior capsule was also enhanced during 5 days culture. In contrast, cell elongation was retarded by the inclusion of DEX. In the absence of FGF2, DEX had no marked effects on any of these cellular processes. Thus, in the presence of FGF2, DEX promoted cell proliferation and posterior capsule coverage but inhibited cell elongation. These results provide insights into the molecular mechanism underlying GC-induced cataract in humans.

The MH1 domain of Smad3 interacts with Pax6 and represses autoregulation of the Pax6 P1 promoter.

Pax6 transcription is under the control of two main promoters (P0 and P1), and these are autoregulated by Pax6. Additionally, Pax6 expression is under the control of the TGFbeta superfamily, although the precise mechanisms of such regulation are not understood. The effect of TGFbeta on Pax6 expression was studied in the FHL124 lens epithelial cell line and was found to cause up to a 50% reduction in Pax6 mRNA levels within 24 h. Analysis of luciferase reporters showed that Pax6 autoregulation of the P1 promoter, and its induction of a synthetic promoter encoding six paired domain-binding sites, were significantly repressed by both an activated TGFbeta receptor and TGFbeta ligand stimulation. Subsequently, a novel Pax6 binding site in P1 was shown to be necessary for autoregulation, indicating a direct influence of Pax6 protein on P1. In transfected cells, and endogenously in FHL124 cells, Pax6 co-immunoprecipitated with Smad3 following TGFbeta receptor activation, while in GST pull-down experiments, the MH1 domain of Smad3 was observed binding the RED sub-domain of the Pax6 paired domain. Finally, in DNA adsorption assays, activated Smad3 inhibited Pax6 from binding the consensus paired domain recognition sequence. We hypothesize that the Pax6 autoregulatory loop is targeted for repression by the TGFbeta/Smad pathway, and conclude that this involves diminished paired domain DNA-binding function resulting from a ligand-dependant interaction between Pax6 and Smad3.

Wnt-frizzled signaling is part of an FGF-induced cascade that promotes lens fiber differentiation.

PURPOSE: It is well established that lens fiber differentiation depends on an FGF-initiated growth factor signaling cascade. Given that recent studies indicate Wnt-Frizzled/Planar Cell Polarity (Wnt-Fz/PCP) signaling has a role in coordinating the orientation and alignment of fibers, this study set out to investigate the relationship between this pathway and FGF-induced fiber differentiation. METHODS: Rat lens epithelial explants were cultured with FGF-2. Regulators of Wnt-Fz signaling, secreted frizzled-related protein-1 (Sfrp1), and inhibitor of Wnt production-2 (IWP-2) were applied to assess the role of this pathway in FGF-induced fiber differentiation. A TCF/Lef reporter mouse was used to assess canonical Wnt-Fz/ß-catenin signaling. RESULTS: FGF-induced fiber differentiation was accompanied by upregulation of Wnt-Fz signaling components, Fz3, Fz6, Dishevelled-2 (Dvl2), and Dishevelled-3. During differentiation, Fz and the centrosome/primary cilium translocated to the apical tip/leading edge of similarly polarized groups of cells. Addition of Sfrp1 or IWP-2 to FGF-treated explants inhibited cell elongation and reduced expression of fiber-specific markers, filensin and ß-crystallin. Expression of Wnt-Fz signaling components was also reduced and a significant reduction in the active form of Dvl2 indicated inhibition of the pathway. Analysis of the TCF/Lef reporter mouse showed no evidence of canonical Wnt-Fz/ß-catenin signaling during FGF-induced fiber differentiation. CONCLUSIONS: This study shows that Wnt-Fz signaling is a component of the FGF-initiated cascade that regulates fiber differentiation. The presence of groups of fibers with Fz and centrosome/primary cilium polarized to the leading edge of each cell is consistent with a role for noncanonical Wnt-Fz signaling in coordinating polarized behavior of differentiating fibers.

A fully human in vitro capsular bag model to permit intraocular lens evaluation.

PURPOSE: To establish a fully human in vitro culture model with which to test the putative effects of intraocular lens (IOL) designs in preventing posterior capsule opacification (PCO) after cataract surgery. METHODS: A sham cataract operation was performed to prepare human capsular bags from donor lenses. In one capsular bag of a donor pair, an intraocular lens (PMMA round-edge IOL or acrylic IOL) was implanted while the other capsular bag remained aphakic. Bags were transferred to a Petri dish and secured anterior-face down using entomological pins. Capsular bags were maintained in Eagle's minimum essential medium supplemented with 2% human serum and 10 ng/mL TGF-ß to drive growth and matrix contraction. RESULTS: In the absence of an IOL, cells appeared within the central posterior capsule at 4.38 ± 0.26 days, whereas in the presence of a PMMA round-edge IOL or an acrylic IOL they appeared at 8 ± 0.41 days and 11 ± 0.7 days, respectively. Immunocytochemical analysis showed an accumulation of cells at the edge of the acrylic IOL and a less evident accumulation with the PMMA round-edge IOL. Moreover, matrix contraction was more prominent in the absence of an IOL but was still apparent, to a lesser degree, in the presence of a PMMA round-edge IOL. The acrylic IOL greatly suppressed matrix contraction. CONCLUSIONS: The authors have developed a fully human in vitro capsular bag system that relates well to clinical observations and permits the testing of novel intraocular lenses.

MMP2 activity is critical for TGFß2-induced matrix contraction--implications for fibrosis.

PURPOSE: The fibrotic lens disorder posterior capsule opacification (PCO) develops in millions of patients following cataract surgery. PCO characteristics are extensive extracellular matrix (ECM) production and contraction of the posterior lens capsule, resulting in light-scattering ECM modification (wrinkling). The pro-fibrotic cytokine transforming growth factor beta (TGFß) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFß-mediated PCO formation. METHODS: The human lens epithelial cell-line FHL-124 and human capsular bag models were employed. Gene expression of MMP family members was determined by oligonucleotide microarray and quantitative real-time RT-PCR. MMP2 and MT1-MMP protein levels were analyzed by ELISA and Western blotting, respectively. Matrix contraction was determined using an FHL-124 patch contraction assay; at end-point, cells were stained with Coomassie brilliant blue and area was determined using image analysis software. Cell coverage and wrinkle formation on the posterior capsule were also assessed using human capsular bag models. RESULTS: Active TGFß2 (10 ng/mL) increased gene and protein levels of MMP2 and MT1-MMP and induced matrix contraction in FHL-124 cells. Specific siRNA inhibition of MT1-MMP did not suppress TGFß2-induced matrix contraction. Active TGFß2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 µM), MMP2 siRNA, and MMP2 neutralizing antibody (4 µg/mL). TGFß2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 µg/mL). CONCLUSIONS: MMP2 plays a critical role in TGFß2-mediated matrix contraction, which appears to be independent of MT1-MMP. MMP2 inhibition provides a novel strategy for the treatment of PCO and potentially other fibrotic disorders.

Short-term exposure to transforming growth factor beta induces long-term fibrotic responses.

Transforming growth factor beta (TGFbeta), a potent inducer of cell transdifferentiation, is heavily implicated in fibrotic disorders. Following cataract surgery, aberrant cell growth across the collagenous matrix of the lens capsule leads to fibrosis, and in turn secondary visual loss, known as posterior capsule opacification (PCO). These modifications are associated with transdifferentiated cells. Following surgery, protein levels in the eye transiently increase, lasting a matter of days whereas PCO takes much longer to reach clinical significance. In the present study, a human lens culture model was employed to show that a relatively brief 2-day exposure to TGFbeta gives rise to persistent, long-term signalling events resulting 28 days later in matrix contraction and transdifferentiation. These events can be suppressed by application of the human monoclonal anti-TGFbeta2 antibody CAT-152 either simultaneously or after TGFbeta2 exposure. Radiolabel binding studies revealed the lens capsule serves as a store for TGFbeta2. Importantly, similar binding studies showed that the capsule could also serve as a reservoir for CAT-152. The data reveal the longevity of TGFbeta2 action through matrix association, but also demonstrate how early application of a TGFbeta2 antibody can overcome the detrimental TGFbeta actions leading to potential inhibition of PCO development and other fibrotic disorders.