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K. S. Kompass, Y.-B. Shui, D. C. Beebe; Oxygen Regulates Lens Cell Proliferation and Circadian Gene Expression in vivo in an Age-Dependent Manner. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1906.
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The lens grows continuously throughout life. Lens size is a risk factor for nuclear and cortical cataracts. We showed that the slowing of lens growth that normally occurs with age depends on maintaining the normal hypoxic environment around the lens. Exposing rodent lenses to elevated oxygen increased lens cell proliferation and lens size in older, but not young animals (Shui and Beebe, IOVS in press). We report microarray analyses to identify the genes that are differentially regulated by oxygen in young and old lens epithelial cells in vivo.
One- or eight-month-old mice were exposed to room air (21% O2) or 60% O2 in the late afternoon and lens epithelia dissected 24 hours later. Peripheral epithelial cells were isolated from epithelial explants with a trephine, RNA was extracted by standard methods and used to interrogate Illumina microarrays containing ~46,000 probes. Triplicate biological replicates were analyzed for each age and treatment (12 samples). Microarray data were normalized and analyzed to identify transcripts that were significantly increased or decreased in an age- and/or oxygen-dependent manner.
Numerous transcripts were differentially regulated by age and/or oxygen exposure. At both ages, exposing the lens to increased oxygen caused a significant decrease in the accumulation of 17 transcripts that are known to increase in hypoxia, due to the action of the transcription factor, HIF-1. These included Vegfa, Car9, Mif, Pdk1, and several RNAs encoding glycolyic enzymes. The transcripts most significantly differentially regulated by oxygen in young and old lenses included genes that function in the core circadian pacemaker (clock). These included the transcription factors Bhlhb2, Dbp, Clock, Per2, Per3, Bmal1 and Nr1d1.
Previous studies identified a diurnal rhythm in lens cell mitosis (Von Sallmann and Grimes, Invest. Ophthalmol. 1966). Oxygen levels regulate lens cell proliferation, circadian gene expression and genes that are normally controlled by HIF-1. We are testing whether an endogenous circadian clock controls lens cell division and whether HIF-1α regulates lens cell proliferation by altering the circadian cycle.
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