Ian Michael Wormstone

The aging human lens: structure, growth, and physiological behaviour

The aging human lens has been the subject of intense research over the past 20 years, for a number of quite disparate reasons. The fact that the incidence of cataract rises exponentially with age after 50 years1 provides the driving influence for much of the effort, but the unique accessibility, homogeneity, and basic simplicity of structure of the organ itself makes it a fruitful system for fundamental studies of tissue growth, development, and differentiation.2-4 Images of the whole human lens in vivo have been available for detailed analysis since the introduction of the quantitative slit lamp (or Scheimpflug) camera (Fig 1). This has been invaluable in providing an understanding of the changes in shape and optical properties both of the ‘normal’ and cataractous aging lens.5-7 The lens is also accessible as an in vitro system of study through the provision of donor eyes for corneal transplant and general research. Since the lens has no direct blood supply, it survives well both in the globe itself and in organ culture media.8-10 Whole cataractous lenses were also once routinely available before the advent of extracapsular cataract extraction (ECCE) with intraocular lens implantation and in the past there have been combined slit lamp and in vitro studies which have correlated changes in light scatter and absorbance with specific alterations in ion and protein levels (Fig 1 and Marcantonio et al 11 and Hockwin et al 12). Figure 1 Images of normal human lenses (A, B, and C), posterior polar cataract (D, E, F), and pure nuclear cataract (G, H, I). Note that slit lamp camera images (A, D, G) all have a scattering reflect artefact (small white rectangle). The normal subject (A) was 40 years of age and the accompanying in vitro grid photographs (B) and polarising images (C) were …

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.

TGFβ/Smad4-dependent and -independent regulation of human lens epithelial cells.

PURPOSE Transforming growth factor (TGF)-beta can mediate fibrotic responses via Smad4-dependent and -independent signaling pathways. TGFbeta-induced transdifferentiation of lens epithelial cells to myofibroblasts and matrix contraction contribute to posterior capsule opacification (PCO) after cataract surgery. The primary objective of the study was to determine the importance of Smad4 in TGFbeta2-induced transdifferentiation, matrix contraction, and Smad signaling by human lens epithelial cells. METHODS The human lens cell line FHL 124 was used. Smad4 knockdown was achieved by using siRNA technology and efficiency tested at the message and protein level by real-time PCR and Western blots, respectively. Smad4 and Smad2/3 nuclear distribution after TGFbeta2 stimulation (10 ng/mL) was determined by immunocytochemistry. Gene expression of transdifferentiation markers, alpha-smooth muscle actin and fibronectin and the inhibitory Smad, Smad7, in the presence and absence of TGFbeta2 (10 ng/mL) was determined with real-time PCR. TGFbeta2-induced contraction was assessed with a patch contraction assay. Suspended bead array technology was used to determine pERK, pP38, and pJNK levels in response to TGFbeta2 over a 2-hour time course. RESULTS SiRNA targeted against Smad4, successfully reduced expression of Smad4 message and protein after both the initial transfection period and removal of transfection conditions. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 after siSmad4 treatment relative to control siRNA in both the presence and absence of 10 ng/mL TGFbeta2. Smad4 knockdown cells exhibited TGFbeta2-induced Smad2/3 nuclear translocation which did not differ significantly from Smad4-expressing cells. Real-time PCR analysis showed significant induction of alphaSMA and fibronectin transcript in the Smad4-expressing cell line (control siRNA group). These inductions were suppressed in Smad4-knockdown cells. TGFbeta-induced mSmad7 expression in FHL 124 cells was unaffected by Smad4 knockdown. Smad4-expressing cells did not exhibit a significant contractile response after 24 hours of culture in the presence or absence of 10 ng/mL TGFbeta2. However, Smad4-knockdown cells demonstrated a significant reduction in patch area (i.e., contraction), after TGFbeta2 treatment. Bead array analysis showed that TGFbeta2 significantly increased pERK and pP38 levels relative to unstimulated control. No significant change was observed with pJNK. CONCLUSIONS Smad4 is critical for the expression of transdifferentiation markers, alphaSMA, and fibronectin; in contrast, Smad4 signaling is not essential for TGFbeta-induced Smad7 expression. Smad4 does not appear to be necessary for TGFbeta-induced matrix contraction. TGFbeta does activate Smad-independent pathways in human lens epithelial cells. Therefore, TGFbeta2 regulation of transdifferentiation and matrix contraction appears to be mediated by distinct TGFbeta signaling pathways.

Calcium cell signalling and cataract: role of the endoplasmic reticulum

The lens possesses an impressive array of G-protein receptors that are coupled to the release of intracellular calcium. They include members of the muscarinic, adrenergic and purinergic families and activation of the former has been implicated in cataract for some time. There are several possible mechanisms whereby activation of such receptors could give rise to cataract. A prolonged increase in intracellular calcium would be expected to activate proteases such as calpain and so could induce unscheduled and irreversible breakdown of important structural proteins. It has recently been shown that activation of G-protein receptors also modulates lens cell growth, and any interference with the highly controlled pattern of cell growth and development within the lens is also likely to have catastrophic consequences. If the calcium store is totally inactivated in lens cells, for example by exposure to thapsigargin, then growth ceases. This finding provides a means of inhibiting the lens cell growth which leads to posterior capsular opacification (PCO). For example, it has been shown that thapsigargin-coated intraocular lenses totally inhibit lens cell growth within cultured capsular bags, and if this technology could be transferred to the clinic then it could provide a simple and relatively inexpensive means of preventing PCO.

Sulforaphane Can Protect Lens Cells Against Oxidative Stress: Implications for Cataract Prevention

PURPOSE Protecting the lens against oxidative stress is of great importance in delaying the onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide cytoprotection against oxidative stress. We therefore tested the ability of SFN to perform this role in lens cells and establish its ability to delay the onset of cataract. METHODS The human lens epithelial cell line FHL124 and whole porcine lens culture systems were used. The ApoTox-Glo Triplex Assay was used to assess FHL124 cell survival, cytotoxicity, and apoptosis. The MTS assay was used to assess cell populations. To determine levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess level of gene expression, an Illumina whole-genome HT-12 v4 beadchip was used. Protein expression was determined by Western blot and immunocytochemistry. RESULTS Exposures of 30 μM H2O2 to FHL124 cells caused a reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24-hour pretreatment with 1 μM SFN. In addition, 1 μM SFN significantly reduced H2O2-induced DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole-genome HT-12 v4 beadchip microarray data revealed eight genes upregulated following 24-hour exposure to 1- and 2-μM SFN, which included NQO1 and TXNRD1. This pattern was confirmed at the protein level. Nrf2 translocated to the nucleus in response to 0.5- to 2.0-μM SFN exposure CONCLUSIONS The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.

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.

Phacoemulsification versus extracapsular cataract extraction: a comparative study of cell survival and growth on the human capsular bag in vitro

AIMS/BACKGROUND Phacoemulsification is rapidly replacing conventional extracapsular cataract extraction (ECCE) as the method of choice for cataract surgery in the Western world. However, posterior capsule opacification (PCO) still remains the major postoperative complication, affecting 20–50% of patients, and results from persistent cell growth of epithelial cells remaining after surgery. This study aimed to compare cell survival and growth on capsular bags following ECCE and phacoemulsification surgery using an established human capsular bag culture system. METHODS Sham ECCE and phacoemulsification cataract operations were performed on pairs of human donor eyes. Capsular bags were dissected free, pinned flat on a petri dish, and incubated with Eagle’s minimum essential medium (EMEM) alone or EMEM supplemented with 10% fetal calf serum (FCS). Ongoing observations were made using phase contrast microscopy. RESULTS Cell growth was observed across the posterior capsule of all preparations studied. It was found that there was no significant difference in the rate of cell growth on the posterior capsule with the two extraction methods, such that 50% confluency was achieved in 7.0 (SD 1.8) (n=7) days for ECCE and 7.43 (2.1) (n=7) days for phacoemulsification surgery. The physical changes to the capsule as a result of cell growth, such as wrinkling and capsular tensioning, were also seen in both groups. CONCLUSIONS Cell survival and growth is dependent on the donor, rather than the surgical technique performed. There is no significant difference between phacoemulsification and ECCE surgery on the rate and nature of cell growth on the posterior capsule in vitro.

Age-Related Differences in Signaling Efficiency of Human Lens Cells Underpin Differential Wound Healing Response Rates following Cataract Surgery

PURPOSE Cataract surgery is blighted by posterior capsule opacification (PCO), which is more severe and frequent in the young than the elderly (>60 years). Our aim was to understand the biological basis for these age-related differences in PCO/wound healing rates. METHODS Human capsular bags were prepared by cataract surgery on donor lenses (young [<40 years] and elderly [>60 years] groups) and maintained in serum-free Eagles minimum essential medium. Cell growth was determined using the MTS assay. Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) levels were determined using ELISA. Protein synthesis rates were elucidated by 35S-methionine incorporation. U0126, SB203580, and SP600125 were used to disrupt ERK-, p38-, and JNK-mediated signaling, respectively. Level of total and phospho-ERK, -c-jun, -P38, and -JNK plus cytokines were detected using a BIOPLEX array system. RESULTS Following a 2-day culture period, significant decreases in IL-1β and IL-6, and increases in IL-10, IL-12, IL-13, and VEGF in the >60 years group were observed compared with their younger counterparts. Capsular bags (cells and capsule) from aged donors contained greater than or equal levels of HGF and FGF than younger counterparts and had greater rates of protein synthesis. Inhibition of ERK, p38, and JNK signaling significantly suppressed cell coverage on the posterior capsule. pERK, p-c-jun, p-p38, and pJNK were consistently lower in aged cell populations; total signaling protein expression was unaffected by age. Serum stimulation increased pERK, p-c-jun, and pJNK levels in cells of all ages; p-p38 was significantly increased in the >60 years group only. CONCLUSIONS Ligand availability to cells is not a limiting factor as we age, but the ability to convert this resource into signaling activity is. We therefore propose that overall signaling efficiency is reduced as a function of age, which consequently limits wound-healing response rates after injury.

ULTRAVIOLET IRRADIATION (UVB) INTERRUPTS CALCIUM CELL SIGNALING IN LENS EPITHELIAL CELLS

A preliminary study was undertaken to establish whether low-dose UV irradiation (UVB) affects calcium cell signaling in rabbit lens epithelia. In a suspension of lens epithelial cells (line NN1003A), changes in intracellular Ca2+ were measured by Fura-2 fluorescence in response to exogenously added ATP. The cellular response to ATP, referred to as the calcium signal, is characterized by a brief increase and subsequent decrease in cytosolic Ca2+ levels. Ultraviolet B irradiation (1.8-9 mJ/cm2) was found to reduce the magnitude of the Ca2+ signal in a dose-dependent manner. A 5 min UVB exposure (9 mJ/cm2) completely altered the biphasic nature of the calcium signal, causing only an immediate and steady rise in cytosol Ca2+ levels. Lower fluences of UVB irradiation (2 min exposure times or 3.6 mJ/cm2) induced a 50% reduction in the calcium signal. When irradiated cells were returned to culture for 3 h after irradiation, calcium signals induced by ATP were normal. In view of the photooxidative nature of UVB irradiation, the oxidative state of cells was assessed by measuring glutathione (GSH) levels. Ultraviolet B irradiation caused a rapid 20% decline in GSH levels that returned to near-control values after a 3 h postirradiation incubation. The results of this study indicate that fluences lower than previously found to be cataractogenic can perturb calcium cell signaling in cultured lens epithelial cells.

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.