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M. L. Archibald, J. A. Murphy, W. H. Baldridge, B. C. Chauhan; Role of Endothelin-1 in Rat Optic Nerve Head Astrocyte Proliferation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2788.
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Astrocyte migration and proliferation is likely a key factor in the remodeling of the optic nerve head in glaucoma. The goal of this research was to characterize the influence of endothelin-1 (ET-1), a vasoactive peptide, and its receptors ETA and ETB on rat optic nerve head astrocyte (ONHA) proliferation.
Optic nerve head astrocytes (ONHA) were isolated from adult Brown Norway rats. Astrocyte specificity was determined immunohistochemically with positive labeling for glial fibrillary acidic protein (GFAP), and negative labeling for A2B5 (a marker for Type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP). Immunohistochemistry was also performed for ETA and ETB. To determine whether ET-1 influenced ONHA proliferation, cells were treated with ET-1 (10-6, 10-7, 10-9 or 10-11 M), or vehicle for 24, 48 or 72 hrs and assessed for proliferation using a commercial proliferation assay. To determine whether ET-1 induces proliferation through ETA or ETB receptors we investigated ONHA proliferation 48 hrs following ET-1 (at 10-7 M) exposure with 10-6 M BQ-610 (ETA antagonist) or 10-6 M BQ-788 (ETB antagonist). In all cases, comparisons were made to vehicle-only controls.
The specificity of the cultured cells as ONHA was confirmed with positive labeling for GFAP, and negative labeling for A2B5 and MBP. ONHA also expressed ETA and ETB. Cell numbers did not change significantly 24 hrs after treatment (7% increase with 10-6 M ET-1 and 13% each with the remaining concentrations relative to vehicle; n = 4/group; P = 0.45, ANOVA); however, a significant increase occurred at 48 hrs with 10-6, 10-7, 10-9 M ET-1 concentrations (18%, 33%, and 24% increase relative to vehicle, respectively; P < 0.01) but not 10-11 M (5% increase). After 72 hrs ONHA numbers increased significantly at all concentrations (88%, 77%, 99%, 97% increase relative to vehicle, for 10-6, 10-7, 10-9 and 10-11 M, respectively; P < 0.01). No significant proliferation occurred in the presence of either antagonist (2% decrease with BQ-610 and 9% decrease with BQ-788).
These findings indicate that (1) purified ONHA can be isolated from rats, (2) these cells proliferate with 48 hrs or more exposure to ET-1, and (3) this proliferation requires both ETA and ETB receptors.
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