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Abstract #59480 Published in IGR 16-3
eNOS-related mechanotransduction changes in aged porcine angular aqueous plexus cells
Lei Y; Stamer WD; Wu J; Sun XInvestigative Ophthalmology and Visual Science 2014; 0:
PURPOSE: To investigate effects of aging on endothelial nitric oxide synthase (eNOS) expression and signaling of angular aqueous plexus (AAP, functional equivalent to human Schlemm's canal) cells subjected to shear stress. METHOD: AAP cells were isolated differentially from porcine outflow tissues using puromycin selection. Cell aging was induced by culturing cells in hyperoxia condition (40% O2, 5% CO2) for 14 days. AAP cells grown in chamber slides were exposed to a shear stress of 8 dyns/cm2 for 24 hours. Expression of eNOS, eNOS-phospho Thr495, eNOS-phospho Ser1177 and Akt-phospho was tested by western blot analysis and immunofluoresence staining. Nitric oxide levels were measured by Griess assay. RESULTS: Compared to control, eNOS levels in aged cells was significantly reduced by 60% (p<0.05, n=6). Phosphorylation of eNOS at Ser1177, and Akt at Ser473 was 63% and 80% lower in aged cells respectively, whereas phosphorylation of the eNOS inhibition site (Thr495) increased by 6.1 fold (p<0.05, n=6). Shear stress (8 dynes/cm2 for 24 hours) increased eNOS abundance (total protein and at cell borders) and phosphorylation at Ser1177 by 1.7 and 1.8 fold respectively(p<0.05, n=6), whereas aged cells were unresponsive. In control cells exposed to shear stress, NO concentration was 1.8 fold higher than the static group (p<0.05, n=4); however aged cells were unresponsive to shear stress (4.3 ± 1.3 uM vs. 4.1 ± 1.4 uM). CONCLUSION: Aged AAP cells appear compromised in their mechanotrasduction machinery involving eNOS, the protein product of the gene, NOS3, in which polymorphisms impart a risk for the development of glaucoma .
Research Centre, Eye and ENT hospital of Fudan University, 83 Fenyang Road, Shanghai, 200031, China.
Full articleClassification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
5.3 Other (Part of: 5 Experimental glaucoma; animal models)
