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 2

Reduced Nos3/NOS3 expression was observed in SC and distal vessels with age in Cre;Nos3fl/fl mice.

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Reduced Nos3/NOS3 expression was observed in SC and distal vessels with ...
(A) Schematic showing Nos3 deletion in the absence of tamoxifen due to a leaky promoter (indicated by faucet). (B) Representative images of RNAscope labeling at each age are shown. Staining for nuclei (DAPI) is in blue. >: open iridocorneal angle; arrows: DV; asterisks: SC. Images are representative of n = 3–6 eyes, 5–10 sections per eye for each time point. (C) A significant decrease in Nos3 signal intensity in SC was observed in Cre;Nos3fl/fl mice compared to C57BL/6J mice from P10 to P90 (P < 0.0001). Each data point represents a single unpaired eye. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; 1-way ANOVA with Tukey’s multiple-comparison test. (D) Significant loss of Nos3 in DVs paralleled that in SC in Cre;Nos3fl/fl mice by P90 compared with C57BL/6J mice at P90 (P < 0.0001). This loss of Nos3 in Cre;Nos3fl/fl mice at P90 was no different from in Nos3-KO mice at P90 (P > 0.9999). (E) Representative images of NOS3 expression at each age. (F) Relative NOS3 signal intensity in SC of Cre;Nos3fl/fl mice showed a steady loss of NOS3 in SC from P10 to P90, with a significant difference from C57BL/6J mice at P90 (P = 0.0003) but no difference from Nos3-KO mice at P90 (P = 0.9943). (G) NOS3 signal intensity with age in the DVs looked like the SC region. (H) Real-time PCR of eye and lung showed Nos3 RNA expression in Cre;Nos3fl/fl mice resembling that in Nos3-KO mice, compared with C57BL/6J mice, at P90. (I) Schematic showing the expression patterns of Nos3 mRNA, NOS3 protein, SC/TM development, and Cre-ERT activity with age in Cre;Nos3fl/fl mice.