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 6

Induction of NOS2 expression in distal endothelia of Cre;Nos3fl/fl mice occurs between P30 and P90.

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Induction of NOS2 expression in distal endothelia of Cre;Nos3fl/fl mice ...
(A) In Cre;Nos3fl/fl mice at P30, NOS2 expression (green) was not observed in either the SC region (*) or DV region (arrows). DVs appeared to be normal in size, and IBA1+ macrophages (red) were distributed evenly throughout the outflow pathway. Yellow arrows point to DVs, which are shown costained with CD31 (magenta) in the inset. (B) At P90, enlarged DVs were observed and were coincident with NOS2 staining (green), resembling the staining pattern of the endothelial marker CD31 (magenta). Image shown is enlarged version of the corresponding image in Figure 5G. IBA1+ macrophages (red) were more abundant in the DV region (yellow box). >: open iridocorneal angle; asterisks: SC; arrows: DV. Scale bars: 50 μm. Both NOS2 and IBA1 antibodies were raised in rabbit, so sequential sections were used for IHC. (C) Schematic summary showing that Nos3 was gradually deleted over time in these mice (pink) from P10 to P90. At P90, Nos3 was completely deleted, so that these mice resembled global KO mice. Between P60 and P90, NOS2 expression was obvious in the endothelia of the DVs, and macrophages were more abundant in the region and surrounded the endothelial DVs (green). We hypothesize that when a certain threshold of Nos3 deletion has been reached (red box), macrophages infiltrate and NOS2 is induced in the DVs, which enlarge to compensate for loss of NOS2 in SC and DVs.