The growth of the human lens is linear from age 10 to 90. Thus, lens epithelial cells divide and differentiate into lens fibers at a constant rate during adulthood. Any defects that arise during fiber formation will remain in the lens. We postulate that the unusual growth properties of the lens contribute to cataract formation, a disease that usually occurs later in life. If the growth rate of the lens could be reduced, the onset of cataracts might be delayed and fewer cataracts would reach clinical significance. To test these hypotheses we must be able to control the growth of the lens. However, the factor responsible for the growth of the lens in vivo has not been identified. We have recently identified a lens growth activity in chicken embryo serum. Several well-characterized growth factors did not replace this activity. We will use three approaches to describe and characterize this activity, including: purification by microbore liquid chromatography, the assay of new, purified growth factors as they become available and the identification of all tyrosine kinase growth factor receptors on lens epithelial cells, using PCR and limited DNA sequencing. These studies will identify, for the first time, a factor that controls lens growth in vivo. This laboratory has previously shown that lens fiber cell elongation is driven by an increase in cell volume. Increased cell volume is initially caused by potassium accumulation and the accompanying influx of water. A candidate for the potassium channel that controls this process has recently been identified. In collaboration with Dr. James Rae, we will test whether this is the channel that is regulated during fiber formation. If it is, we will identify the factors that control its activity. Our recent data suggest that high levels of cyclic AMP increase potassium efflux and prevent fiber cell elongation. We will measure cAMP levels, then examine the effects of cAMP agonists and antagonists on ion channels with patch clamp methods. If a regulated lens potassium channel is identified, it will be cloned and its mRNA expressed in Xenopus oocytes, where more extensive studies of channel regulation will be possible. We showed that lens fiber formation in chicken embryos is caused by exposure of lens epithelial cells to IGF-1 in the vitreous humor. In collaboration with two other labs, we recently identified binding proteins for IGF-1 in chicken vitreous humor. The kinds and amounts of these proteins were different in serum and vitreous humor. We will describe the distribution of IGF binding proteins and their mRNAs in embryonic eyes. Vitreous humor will be depleted of IGF binding proteins to test whether these molecules augment or suppress IGF activity. We will also determine the distribution of IGF-1 mRNA in the eye by in situ hybridization and the polymerase chain reaction. Recent studies led us to question the widely-held view that lens epithelial cells must be directly coupled to underlying lens fibers. To resolve this issue, we will use confocal microscopy to examine the pathways by which small molecules enter the extracellular spaces of the lens, measure the rate of transfer of small molecules between epithelial cells and fibers and determine how the lens epithelium maintains the ionic composition of the fibers.
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