Endothelial cell–specific postnatal deletion of Nos3 preserves intraocular pressure homeostasis via macrophage recruitment and NOS2 upregulation
Ruth A. Kelly, … , Darryl R. Overby, W. Daniel Stamer
Ruth A. Kelly, … , Darryl R. Overby, W. Daniel Stamer
Published February 11, 2025
Citation Information: J Clin Invest. 2025;135(7):e183440. https://doi.org/10.1172/JCI183440.
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Research Article Immunology Ophthalmology Vascular biology

Endothelial cell–specific postnatal deletion of Nos3 preserves intraocular pressure homeostasis via macrophage recruitment and NOS2 upregulation

Abstract

Polymorphisms in Nos3 increase risk for glaucoma, the leading cause of irreversible blindness worldwide. A key modifiable risk factor for glaucoma is intraocular pressure (IOP), which is regulated by NO — a product of nitric oxide synthase 3 (encoded by Nos3) — in Schlemm’s canal of the conventional outflow pathway. We studied the effects of a conditional, endothelial cell–specific postnatal deletion of Nos3 (Endo-SclCre-ERT;Nos3fl/fl) on tissues of the outflow pathway. We observed that Cre-ERT expression spontaneously and gradually increased with time in vascular endothelia including in Schlemm’s canal, beginning at P10, with complete Nos3 deletion occurring around P90. Whereas outflow resistance was reduced in global Nos3-KO mice, outflow resistance and IOP in Endo-SclCre-ERT;Nos3fl/fl mice were normal. We observed — coincident with Nos3 deletion — recruitment of macrophages to and induction of both ELAM1 and NOS2 expression by endothelia in the distal portion of the outflow pathway, which increased vessel diameter. These adjustments reduced outflow resistance to maintain IOP in these Endo-SclCre-ERT;Nos3fl/fl mice. Selective inhibition of iNOS by 1400W resulted in narrowing of distal vessels and IOP elevation. Together, the results emphasize the pliability of the outflow system and the importance of NO signaling in IOP control, and imply an substantial role for macrophages in IOP homeostasis.

Authors

Ruth A. Kelly, Megan S. Kuhn, Ester Reina-Torres, Revathi Balasubramanian, Kristin M. Perkumas, Guorong Li, Takamune Takahashi, Simon W.M. John, Michael H. Elliott, Darryl R. Overby, W. Daniel Stamer

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Figure 1

Spontaneous Cre activity in endothelial cells of SC and the DV in 2 different reporter mouse strains.

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Spontaneous Cre activity in endothelial cells of SC and the DV in 2 diff...
(A) Schematic showing localization of tissues of the conventional outflow pathway in cross section of mouse eye. The left panel shows a magnified view of conventional outflow tissues in the right panel (indicated by the box). (B) Schematic showing both R26R and Ai9 tdTomato reporter mouse strains crossed with Cre;Nos3fl/fl mice to give R26R/Cre;Nos3fl mice and Ai9/Cre;Nos3fl mice, respectively. X-gal staining was carried out on tissue from R26R/Cre;Nos3fl mice to identify LacZ expression (blue), indicating Cre activity; and RFP indicates Cre activity in Ai9/Cre;Nos3fl mice. (C) LacZ expression (blue), indicating Cre-ERT activity, was apparent in endothelial cells of SC (*) and the surrounding DV (arrows) in R26R/Cre;Nos3fl mice. Staining was also present in the CB, which also contains blood vessels. There was no blue stain present in the P8 mice examined. Light blue staining appeared at P10 and intensified with age until P90. Images shown are representative of n = 8 eyes, 10–15 sections per eye from each quadrant (from both male and females) from each age group. (D) Semiquantitative scoring of X-gal staining, which was carried out by 2 trained observers masked to the experimental protocol, showed increased stain intensity with age. (E) Flat-mount image showing RFP (red) expression along the entire SC and DV of Ai9/Cre;Nos3fl mice at P30. Image represents n = 6 eyes (from both males and females). (F) Cross-section images showing RFP (red) in endothelial cells of the SC and the DV, following the same pattern as the endothelial cell marker CD31 (green). Images represent n = 8 eyes, 10–15 sections per eye from each quadrant (from both male and females). Scale bars: 50 μm in all images. R, retina. Asterisk: SC; arrows: DV.