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C. K. Chan, L. N. Pham, J. Zhou, Y. Ding, C. Spee, S. J. Ryan, D. R. Hinton; Differential Expression of Caspases and Fas/FasL in Mouse Strain-Dependent Hyperoxia-Induced Vessel Regression. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2976.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated the heterogeneity of oxygen-induced retinal vessel regression.
Using a mouse model of hyperoxia-induced vessel regression, strain-dependent vaso-obliteration was quantified among multiple strains of mice in flat-mount retinal specimens under darkfield microscopy and apoptosis ELISA. Co-localization of isolectin B4 and TUNEL staining allowed identification of endothelial specific apoptosis. Strain-dependent variations in ocular posterior pole gene expression of caspase-3, -8, and -9, and Fas and Fas ligand (FasL) were determined at 0-120 h hyperoxia by quantitative real time RT-PCR. Caspase activities were confirmed by substrate cleavage assays. Indirect ELISA of Fas/FasL protein expression correlated with mRNA expression.
The following rank order was determined for mouse strain-dependent hyperoxia-induced retinal vessel regression: CD-1 > C57BL/6J, DBA > A/J, 129S3/SvIM; DBA > AKR > 129S3/SvIM (Kruskall-Wallis p < 0.003). TUNEL staining revealed that hyperoxia-induced retinal vessel regression occurs via endothelial apoptosis. Following 24h and 120h hyperoxia, apoptosis ELISA was increased 1.6- and 1.3-fold in posterior poles of C57BL/6J mice compared with 129S3/SvIM strain (p<0.0001). Throughout hyperoxia, a 1.4 to1.9-fold increase in caspase-3 mRNA was observed in C57BL/6J mice compared to 129S3/SvIM (p < 0.0007). At 12 and 24 h hyperoxia, peak inter-strain differences of 1.5- and 1.3-fold increased caspase-9 expression respectively were also observed in C57BL/6J mice compared to 129S3/SvIM strain (p < 0.05). At 24 h hyperoxia, caspase activity between inbred strains correlated with mRNA expression. Fas mRNA expression increased from 72 h through 120 h hyperoxia reaching peak 2.5- and 3.5-fold increased levels in C57BL/6J and 129S3/SvIM mice respectively compared to that of 0 h hyperoxia (p < 0.01). In contrast, throughout hyperoxia, 129S3/SvIM mice expressed 1.7- to 2.4-fold increased FasL mRNA levels compared to C57BL/6J strain (p < 0.003). Using indirect protein ELISA at 72 h hyperoxia, while membrane-bound FasL protein was similarly expressed between C57BL/6J and 129S3/SvIM animals, soluble FasL protein was increased 1.9-fold in 129S3/SvIM mice compared to C57BL/6J animals at 72 h hyperoxia (p < 0.0008).
Strain-dependent expression of apoptotic factors influences heterogeneity in vessel regression.
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