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 4

Arterioles and venules enlarge between P30 and P90 in Cre;Nos3fl/fl mice.

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Arterioles and venules enlarge between P30 and P90 in Cre;Nos3fl/fl mice...
(A) Representative confocal images of maximum-intensity projection images (taken from Z-stacks) shows limbal vasculature from Cre;Nos3fl/fl mice at P30 and P90, C57BL/6J mice at P90, and Nos3-KO mice at P90. Immunofluorescence labeling of CD31 (gray) stains all vasculature (left column) and α-SMA (red) stains all vessels except capillaries (middle column). Merged stains are shown in the right column. Enlargement of both arterioles (A) and venules (V) was visible in outflow tissues of Cre;Nos3fl/fl mice at P90 compared with C57BL/6J mice. C, capillary. Scale bar: 50 μm. Box-and-whisker plots display summary data (+ representing the mean) showing that (B) arteriole diameter was significantly larger in Cre;Nos3fl/fl mice at P90 (n = 7) compared with both C57BL/6J (n = 6) and Nos3-KO mice, both at P90 (n = 6) (P = 0.0185 and P = 0.0238, respectively; 1-way ANOVA with Tukey’s multiple-comparison test). (C) Similarly, venule diameter was significantly larger in Cre;Nos3fl/fl mice at P90 mice compared with C57BL/6J mice (P = 0.0074), but not Nos3-KO mice or Cre;Nos3fl/fl mice at P30 (n = 8) (P = 0.5407 and P = 0.3756, respectively). (D) Capillary diameter was significantly larger in Cre;Nos3fl/fl mice at P30 compared with C57BL/6J animals (P = 0.02). Each individual data point represents the average vessel measurement per mouse eye. *P < 0.05 and **P < 0.01; 1-way ANOVA with Tukey’s multiple-comparison test.