The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival. INTRODUCTIONCell survival requires the continued exposure to specific survival factors. In a classic study (Ishizaki et al., 1993), lens epithelial cells were shown to survive in culture, in the absence of cell-cell contact, as long as the right growth factor cues were available. These factors could be provided exogenously either in the form of conditioned media or in serum supplement (Ishizaki et al., 1993), leading to the widely accepted general hypothesis that cells are programmed to die unless the appropriate signals are received.The recent proposal of the extracellular matrix (ECM) reservoir hypothesis suggests that the ECM itself can act as a reservoir for growth and survival factors (Bergers et al., 2000;Mott and Werb, 2004) that are released via the action of various matrix metalloproteinases (MMPs; reviewed in McCawley and Matrisian, 2001;Mott and Werb, 2004). MMPs have been shown to release fibroblast growth factor (FGF)-2 (MMP-1, -3; Whitelock et al., 1996) and fibroblast growth factor receptor (FGFR)-1 (MMP-2; Levi et al., 1996), activate transforming growth factor (TGF)-␤1/2 (Yu and Stamenkovic, 2000), and to both activate and release insulin-like growth factor (IGF)-1 (MMP-1, -2, -3, and -9;Fowlkes et al., 1994; and MMP-2, -3, and -7;Imai et al., 1997). MMPs are therefore an essential component in this proposed role for the ECM in cell survival and cell proliferation (Ishizuya-Oka et al., 2000;Wiseman et al., 2003). From such data, we can see that the ECM reservoir hypothesis requires three essential components: an ECM, growth factors sequestered by the ECM, and the presence of MMPs to release the growth factors from the ECM. This hypothesis changes our perception of the ECM as it can no longer be considered to solely provide a physical support or appropriate cues for integrin signaling (Hynes, 1992;Adams and Watt, 1993), but is also a source of cell proliferation and survival factors (Klagsbrun, 1990;Taipale and Keski-Oja, 1997;Bergers et al., 2000;...

We present a mathematical (ordered pull-through; OPT) model of the cell-density profile for the mammalian lens epithelium together with new experimental data. The model is based upon dimensionless parameters, an important criterion for inter-species comparisons where lens sizes can vary greatly (e.g. bovine (approx. 18 mm); mouse (approx. 2 mm)) and confirms that mammalian lenses scale with size. The validated model includes two parameters: β/α, which is the ratio of the proliferation rate in the peripheral and in the central region of the lens; and γGZ, a dimensionless pull-through parameter that accounts for the cell transition and exit from the epithelium into the lens body. Best-fit values were determined for mouse, rat, rabbit, bovine and human lens epithelia. The OPT model accounts for the peak in cell density at the periphery of the lens epithelium, a region where cell proliferation is concentrated and reaches a maximum coincident with the germinative zone. The β/α ratio correlates with the measured FGF-2 gradient, a morphogen critical to lens cell survival, proliferation and differentiation. As proliferation declines with age, the OPT model predicted age-dependent changes in cell-density profiles, which we observed in mouse and human lenses.

Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/μm2; equator: 8.2 ± 1.9 particles/μm2; posterior pole: 4 ± 0.9 particles/μm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/μm2; equator: 5 ± 2 particles/μm2; posterior pole: 1.9 ± 0.7 particles/μm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface.

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