Inflammation is increasingly linked to disease progression, particularly in cancer, where elevated levels of inducible nitric oxide synthase (iNOS or NOS2), driven by tumor inflammation, is correlated with aggressive tumors and poor outcomes. Measuring nitric oxide levels in tumor cells is hampered by the reactive nature of the molecule and generally inferred through indirect measurement of reaction products such as nitrate and nitrite. Here, we adapt the oxyhemoglobin detection method to tissue culture and examine nitric oxide production in tumor cells in response to inflammatory cytokines. Our assay provides real-time nitric oxide measurement, is highly sensitive, linear for at least an hour, inexpensive, and easy to implement. We show that triple negative breast and colorectal cancer cells respond to interferon gamma (IFNγ), interleukin 1-β (IL1-β) and tumor necrosis factor α (TNFα) to generate surprisingly high levels of NOS2 protein and nitric oxide, as high as seen in activated macrophages for fighting infection. NO detection levels reach 1.3 pmol NO/min/μg total cellular protein. The assay is readily adapted to assessing IC50 values for NOS2 inhibition, inhibition rates, and inhibition persistence. Using triple negative breast cancer cell line 4T1, a syngeneic murine tumor model, we estimate an IC₅₀ = 3.4 μM for NOS2-specific inhibitor 1400W, which displays a low nanomolar binding constant to isolated protein. Inhibition is rapid (<10 min) and persists for at least an hour. These results highlight the importance of nitric oxide production in the tumor and provide a means for developing new therapeutic strategies.

2025-06-01·Free Radical Biology and Medicine

Nitric oxide mediates negative feedback on the TXNIP/NLRP3 inflammasome pathway to prevent retinal neurovascular unit dysfunction in early diabetic retinopathy

Article

作者: Zheng, Weidong ; Huang, Libin ; Yang, Li ; Yao, Yao ; Xie, Maosong ; Zheng, Xuedong

Diabetic retinopathy (DR) is a leading cause of vision impairment in working-age adults, and is driven by complex neurovascular dysfunction. This study aimed to elucidate whether nitric oxide (NO) can modulate the TXNIP/NLRP3 inflammasome pathway and mitigate retinal neurovascular unit (NVU) damage during early DR. In an in vitro co-culture system, silencing TXNIP or NLRP3 in retinal microglia (RMG) significantly upregulated glial cell-derived neurotrophic factor (GDNF) and downregulated inducible nitric oxide synthase (iNOS) expression in retinal ganglion cells (RGC). Moreover, it resulted in decreased iNOS and vascular endothelial growth factor A (VEGFA) levels and enhanced the expression of tight junction proteins (Occludin and ZO-1) in retinal microvascular endothelial cells (RMEC), while also reducing NO release and inhibiting RMEC tube formation. Treatment with S-nitroso-N-acetyl penicillamine (SNAP), an NO donor, significantly downregulated TXNIP/NLRP3 inflammasome signaling in RMG, decreased RGC apoptosis, and inhibited tube formation in RMEC. It also upregulated GDNF, suppressed iNOS in RGC, decreased VEGFA, and improved tight junction proteins in RMEC. Treatment with 1400W, an iNOS inhibitor, resulted in decreased NO concentration and increased IL-1β levels in the co-culture supernatant, without significantly affecting iNOS expression in RGC or RMEC. In an early DR rat model, Electroretinogram (ERG), Optical Coherence Tomography (OCT), Fluorescein Angiography (FFA), Evans blue assays, Immunofluorescence staining, and TUNEL staining confirmed that sodium nitroprusside (SNP), NO donor administration mitigated retinal neural and vascular dysfunction, and preserved retinal NVU integrity. Concurrently, SNP treatment reduced IL-1β expression and increased GDNF and Occludin levels in the early DR retina. Genetic Association Database (GAD) enrichment analysis and protein-protein interaction (PPI) network validation indicated that NO functions as a downstream mediator of the TXNIP/NLRP3 inflammasome pathway and exhibits a strong association with DR. These findings suggest that NO mediates negative feedback in the TXNIP/NLRP3 inflammasome pathway to exert protective effects against retinal NVU dysfunction in early DR, thereby offering potential therapeutic strategies for early intervention in DR.

2025-04-01·Journal of Clinical Investigation

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

Article

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

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.

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