Julie Ann Eldred

The lens as a model for fibrotic disease

Fibrosis affects multiple organs and is associated with hyperproliferation, cell transdifferentiation, matrix modification and contraction. It is therefore essential to discover the key drivers of fibrotic events, which in turn will facilitate the development of appropriate therapeutic strategies. The lens is an elegant experimental model to study the processes that give rise to fibrosis. The molecular and cellular organization of the lens is well defined and consequently modifications associated with fibrosis can be clearly assessed. Moreover, the avascular and non-innervated properties of the lens allow effective in vitro studies to be employed that complement in vivo systems and relate to clinical data. Using the lens as a model for fibrosis has direct relevance to millions affected by lens disorders, but also serves as a valuable experimental tool to understand fibrosis per se.

TGF -Induced Contraction Is Not Promoted by Fibronectin-Fibronectin Receptor Interaction, or SMA Expression

PURPOSE Transforming growth factor (TGF)-beta is a potent inducer of both transdifferentiation and contraction, which are regarded as critical processes that underpin tissue fibrosis. Consequently, transdifferentiation is believed to drive TGFbeta-mediated contraction. This study was conducted to determine the relationship between transdifferentiation of human lens epithelial cells and matrix contraction. METHODS Real-time PCR was used to investigate gene expression of transdifferentiation markers in the human lens cell line FHL 124 and native lens epithelia. Contraction was assessed with a patch-contraction assay, whereby all areas covered by cells were measured with imaging techniques after fixation and cell staining with Coomassie blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To prevent fibronectin-fibronectin receptor interaction 100 microM RGDS peptide was used. Suppression of TGFbeta-induced alphaSMA expression was mediated by siRNA technology. RESULTS Real-time PCR analysis showed 10 ng/mL TGF-beta1 or -beta2 significantly increased expression of alphaSMA, fibronectin, and alpha5beta1 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGFbeta and RGDS showed a marked increase in the rate of contraction relative to TGF-beta alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and alphaSMA protein when transfected with siRNA. alphaSMA knockdown did not prevent TGFbeta-induced contraction. CONCLUSIONS A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFbeta-induced matrix contraction.

Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens.

Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H(2)O(2) led to reduced cell viability and increased apoptosis. In response to 30 μM H(2)O(2), levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H(2)O(2). Application of 10 and 30 μM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H(2)O(2) induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H(2)O(2) induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.

MMP2 Activity is Critical for TGFβ2-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 (TGFβ) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFβ-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 TGFβ2 (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 TGFβ2-induced matrix contraction. Active TGFβ2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 μM), MMP2 siRNA, and MMP2 neutralizing antibody (4 μg/mL). TGFβ2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 μg/mL). CONCLUSIONS MMP2 plays a critical role in TGFβ2-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.

Effect of total lens epithelial cell destruction on intraocular lens fixation in the human capsular bag

Purpose To evaluate the effect of complete destruction of lens epithelial cells (LECs) in the capsular bag on intraocular lens (IOL) stability. Setting School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. Design Comparative evaluation. Methods An in vitro organ culture model using the bag–zonule–ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and fiber extraction were performed, and an Acrysof IOL was implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining it in 6 mL Eagle minimum essential medium supplemented with 5% v/v fetal calf serum and 10 ng/mL transforming growth factor‐&bgr;2 for 3 weeks or more. One bag of each pair was treated with 1 &mgr;M thapsigargin to destroy all LECs. Observations of LEC growth were captured by phase‐contrast microscopy, IOL stability by video microscopy, and endpoint analysis through scanning electron microscopy and immunocytochemistry. Results The LECs in control capsular bags migrated centrally, closing the bag and fixating the IOL between the anterior and posterior capsules, as seen clinically. These events were not observed in the thapsigargin‐treated group. After a period of controlled orbital movement, the IOL in the control group stabilized quicker than in the treated bags. There was no IOL rotation in the bag; however, the IOLs in the treated group rocked with axial movement. Conclusions The LECs appeared to aid stabilization of current IOL designs in the capsular bag. The results have clinical implications for IOL design and for strategies to prevent posterior capsule opacification. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target

Secondary visual loss occurs in millions of patients due to a wound-healing response, known as posterior capsule opacification (PCO), following cataract surgery. An intraocular lens (IOL) is implanted into residual lens tissue, known as the capsular bag, following cataract removal. Standard IOLs allow the anterior and posterior capsules to become physically connected. This places pressure on the IOL and improves contact with the underlying posterior capsule. New open bag IOL designs separate the anterior capsule and posterior capsules and further reduce PCO incidence. It is hypothesised that this results from reduced cytokine availability due to greater irrigation of the bag. We therefore explored the role of growth factor restriction on PCO using human lens cell and tissue culture models. We demonstrate that cytokine dilution, by increasing medium volume, significantly reduced cell coverage in both closed and open capsular bag models. This coincided with reduced cell density and myofibroblast formation. A screen of 27 cytokines identified nine candidates whose expression profile correlated with growth. In particular, VEGF was found to regulate cell survival, growth and myofibroblast formation. VEGF provides a therapeutic target to further manage PCO development and will yield best results when used in conjunction with open bag IOL designs.

Fibrotic modifications of the lens epithelium

Maintaining lens integrity is a lifetime ambition of the lens. However, if this integrity is breached through an altered ocular environment or physical disruption, such as cataract surgery, then fibrotic modifications to the lens can occur that are ultimately detrimental to vision. Fibrotic changes involve hyperproliferation, transdifferentiation from an epithelial to a myofibroblast phenotype, matrix deposition and matrix contraction. Fibrotic conditions of the lens include anterior subcapsular cataract and posterior capsule opacification, which affect millions. In this chapter we will discuss the regulatory mechanisms that facilitate fibrotic events, which will involve the intimate relationship between growth factors (especially transforming growth factor beta—TGF-β), signal transduction pathways and the extracellular matrix.