Endothelial nitric oxide synthase-related mechanotransduction changes in aged porcine angular aqueous plexus cells.
OBJECTIVE
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.
Research Centre, Eye and ENT Hospital, Shanghai Medical College, Fudan University.
Yuan
Y
Lei
Department of Ophthalmology, Duke University.
William Daniel
WD
Stamer
Research Centre, Eye and ENT Hospital, Shanghai Medical College, Fudan University Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University.
Jihong
J
Wu
Key Laboratory of Myopia, Ministry of Health (Fudan University) Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Shanghai Medical College, Fudan University State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University.
Xinghuai
X
Sun
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D003315:Cornea / Q000201:enzymology / Q000473:pathology*; D004195:Disease Models, Animal; D005901:Glaucoma / Q000201:enzymology* / Q000473:pathology; D008856:Microscopy, Fluorescence; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis*; D015398:Signal Transduction; D013314:Stress, Mechanical; D013552:Swine
D052250:Nitric Oxide Synthase Type III
Schlemm's canal; aging; eNOS