Endothelial nitric oxide synthase-related mechanotransduction changes in aged porcine angular aqueous plexus cells.

Journal Article (Journal Article)

PURPOSE: To investigate effects of aging on endothelial nitric oxide synthase (eNOS) expression and signaling in angular aqueous plexus (AAP) (functional equivalent to human Schlemm's canal) cells subjected to shear stress. METHODS: The AAP cells were isolated differentially from porcine outflow tissues using puromycin selection. Cell aging was induced by culturing cells in hyperoxia condition (40% oxygen and 5% carbon dioxide) for 14 days. The AAP cells grown in chamber slides were exposed to a shear stress of 8 dynes/cm(2) for 24 hours. Expression of eNOS, eNOS-phospho Thr495, eNOS-phospho Ser1177, and Akt-phospho was tested by Western blot analysis and immunofluorescence staining. Nitric oxide (NO) levels were measured using the Griess assay. RESULTS: Compared with control, eNOS levels in aged cells were 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/cm(2) for 24 hours) increased eNOS abundance (total protein and at cell borders) and phosphorylation at Ser1177 by 1.7-fold and 1.8-fold, respectively (P < 0.05; n = 6), whereas aged cells were unresponsive. In control cells exposed to shear stress, the NO concentration was 1.8-fold higher than in the static group (P < 0.05; n = 4); however, aged cells were unresponsive to shear stress (mean ± SD, 4.3 ± 1.3 vs. 4.1 ± 1.4 μM). CONCLUSIONS: Aged AAP cells appear compromised in their mechanotransduction machinery involving eNOS, the protein product of the gene, NOS3, polymorphisms of which impart a risk for the development of glaucoma.

Full Text

Duke Authors

Cited Authors

  • Lei, Y; Stamer, WD; Wu, J; Sun, X

Published Date

  • November 6, 2014

Published In

Volume / Issue

  • 55 / 12

Start / End Page

  • 8402 - 8408

PubMed ID

  • 25377220

Pubmed Central ID

  • PMC4541478

Electronic International Standard Serial Number (EISSN)

  • 1552-5783

Digital Object Identifier (DOI)

  • 10.1167/iovs.14-14992


  • eng

Conference Location

  • United States