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 3

Cre;Nos3fl/fl mice at P90 have open angles and normal IOPs due to healthy outflow facility, despite loss of Nos3 in outflow vessels.

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Cre;Nos3fl/fl mice at P90 have open angles and normal IOPs due to healt...
(A) IOP measurements are displayed in box and whisker plots, where the box represents the interquartile range, the horizontal line in the box represents the median, the plus symbol in the box shows the mean, and the whiskers show the minimum and maximum values. Mean IOP measurements of 15.7, 14.7, and 18.6 mmHg were observed in C57BL/6J (n = 11 eyes), Cre;Nos3fl/fl (n = 11 eyes), and Nos3-KO mice (n = 10 eyes), respectively. While IOP of Cre;Nos3fl/fl mice was not different from that of C57BL/6J mice, Nos3-KO mice showed significantly elevated IOP compared with both Cre;Nos3fl/fl mice (P < 0.0001) and C57BL/6J mice (P = 0.0027). (B) Mean facility values of 1.6, 1.9, and 1.1 nL/min/mmHg were determined in C57BL/6J mice (n = 10 eyes), Cre;Nos3fl/fl mice (n = 13 eyes), and Nos3-KO mice (n = 11 eyes), respectively. Cre;Nos3fl/fl mice showed a significant increase in outflow facility compared with Nos3-KO mice (P = 0.0002). (C) Representative images of angle structures from methylene blue–stained sections were used to analyze gross morphological differences between strains at P90. Distal vessels (arrows) in Cre;Nos3fl/fl mice appeared enlarged compared with those in both C57BL/6J and KO mice. The red arrow points to a GV along the inner wall of SC. Asterisk: SC, arrows: DV; and CB: ciliary body. Scale bar: 50 μm. (D) Representative OCT images of living mice of each strain at P90 showing open iridocorneal angles (n = 2 eyes), with no significant difference in the size of SC lumen (E) or the number of GVs per SC (F) between strains. Nos3-KO mice had fewer GVs per SC compared with both C57BL/6J and Cre;Nos3fl/fl mice, but not to a significant degree. (G) Distal vessels (arrows) in Cre;Nos3fl/fl mice were significantly larger compared with those in C57BL/6J mice (P < 0.05). Each individual data point represents a single unpaired, eye and statistical analysis was carried out using 1-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.