Editorial: Spotlight on nitric oxide: integrative approaches to study NO and RNS in physiology and disease

vascular tone adjustments during exercise or hypoxia (4). Disrup6ons in cofactor availability or excessive scavenging by endothelial hemoglobin α-subunits exemplify avenues leading to endothelial dysfunc6on and vascular diseases (5)(6)(7). Not to men6on its cri6cal dependence on Ca 2+ for ac6va6on (8). Inducible NOS (iNOS) represents a Ca 2+ -insensi6ve form of the enzyme that is typically absent at baseline but robustly induced in diverse cells, par6cularly macrophages, during inflammatory s6muli (9,10). Transcrip6onally regulated via inflammatory mediators such as NF-κB and IRF1, iNOS generates highoutput NO over sustained periods, underpinning cytotoxic an6microbial defenses and contribu6ng to tumoricidal ac6vity (11). Regulated also at post-transla6onal levels including proteasomal degrada6on and aggresome-mediated sequestra6on (12), iNOS ac6vity dynamically balances host defense with limi6ng collateral damage. Its pivotal roles in systemic inflamma6on and sepsis have inspired pharmacological inves6ga6ons targe6ng iNOS for therapeu6c modula6on across infec6ous and chronic inflammatory diseases (13).Neuronal NOS (nNOS), in contrast, is cons6tu6vely expressed and 6ghtly regulated by intracellular calcium fluctua6ons binding calmodulin, reflec6ng its key roles in neural signaling, synap6c plas6city, and neuromodula6on. Beyond the central nervous system, nNOS par6cipates in skeletal muscle contrac6lity and vascular tone modula6on, bridging neural and cardiovascular physiology (14)(15)(16). The divergent calcium-dependence and temporal NO release profiles embody the func6onal spectrum of NOS enzymes tailored to diverse biological impera6ves. A key aspect of this spectrum is the flux rate of NO produc6on, as it appears for physiological NO signaling, it needs to be made in the right place at the right 6me and at the right rate. This founda6onal knowledge anchors the broader implica6ons addressed by the research topic and contextualizes the diverse roles NO plays across 6ssues.One compelling theme is the intersec6on of NO with cellular metabolism and immunity, as evidenced by contemporary studies of macrophage immunometabolism (17)(18)(19). NO's concentra6ondependent effects orchestrate diverse cellular outcomes, from low-level signaling to pathological states, necessita6ng precise modula6on and context-aware inves6ga6on. For example, the metabolic reprogramming of inflammatory macrophages illustrates NO's dual regulatory roles. NO-derived electrophilic nitroalkenes, such as nitro-oleic acid (NO2-OA), sculpt macrophage polariza6on by repressing inflammatory cytokine produc6on and reshaping metabolic fluxes. By reducing accumula6on of TCA cycle intermediates succinate and itaconate, metabolites known to stabilize pro-inflammatory Hypoxia Inducible factor (HIF)1α and drive cytokine secre6on, these nitroalkenes unveil non-canonical pathways through which NO deriva6ves resolve inflamma6on. This metabolic rewiring extends to altering cellular reliance on glutamine, redirec6ng it towards glutathione synthesis, thereby enhancing an6oxidant defenses. The study from Stevenson et al. applies advanced liquid chromatography-high-resolu6on mass spectrometry (LC-HRMS) alongside stable isotope ( 13 C) tracing of both glucose and glutamine to dissect central carbon fluxes in macrophages upon NO2-OA treatment, directly comparing them with 1400W (a standard NOS2 inhibitor) and oleic acid controls. The work also quan6fies forma6on of NO2-OAglutathione (GS-OA-NO2) adducts via targeted mass spectrometry, giving insight into cellular thiol alkyla6on as a direct metabolic and signaling event. Importantly, forma6on and quan6ta6on of GS-OA-NO2 adducts reveal a direct covalent interac6on of NO2-OA with cellular GSH pools, connec6ng lipidderived electrophiles to an6oxidant defense in inflammatory seangs. Beyond mechanis6c insights, the authors' findings hold promise for clinical transla6on, with nitroalkene-based compounds under phase 2 inves6ga6on for chronic pulmonary inflamma6on and broader inflammatory diseases, sugges6ng a new class of endogenous-inspired therapeu6cs targe6ng immune-metabolic communica6on.Further interlacing NO with disease biology, NO's modula6on of the human epidermal growth factor receptor (HER/ErbB) family underscores the complexity of oncogenic signaling nuances modulated by nitrosa6ve signals. Either by direct S-nitrosa6on or by increasing phosphoryla6on, NO can influence receptor dimeriza6on, downstream MAPK, PI3K-Akt, and JAK-STAT signaling cascades central to cellular prolifera6on, differen6a6on, and survival pathways. These molecular modifica6ons not only contribute to tumor progression but also poten6ally alter responses to targeted therapies. For instance, the ability of NO to modulate EGFR and HER2 phosphoryla6on states correlates with both enhanced tumor aggressiveness and therapeu6c resistance, emphasizing the transla6onal need for combinatorial approaches integra6ng NO pathway modula6on alongside HER-targeted regimens. The work by O'Neill et al. demonstrates that S-nitrosa6on of EGFR at C498 directly ac6vates the receptor and downstream oncogenic signaling. This process occurs with NO concentra6on thresholds that have been defined for EGFR ac6va6on in breast cancer. It shows that aberrant iNOS expression and altered S-nitrosa6on in HER2driven breast cancer can confer resistance to trastuzumab therapy. This work men6ons recent clinical trial designs combining NOS inhibitors (e.g., L-NMMA) and HER-targeted agents in breast cancer. Moreover, the work exposes gaps in knowledge pertaining to lesser-studied HER family members and their nitrosa6ve post-transla6onal modifica6on, emphasizing fer6le ground for future research in redox-oncogenic interplay.NO biosynthesis extends beyond canonical NOS-dependent mechanisms, par6cularly evident in skeletal muscle physiology. Upanan et al. inves6gate compensatory nitrate-nitrite reduc6ve pathways which provide alterna6ve NO sources under condi6ons of nNOS deficiency or altered enzyma6c ac6vity. Dietary nitrate supplementa6on upregulates nitrate transporters (sialin and CLC1) and the nitrate reductase enzyme XOR, markedly increasing muscle nitrate reserves and maintaining NO signaling cri6cal for mitochondrial func6on and muscle performance. The authors show that protein and mRNA levels of eNOS or iNOS and eNOS ac6va6on are not upregulated, indica6ng NO synthase-independent compensatory mechanisms. Interes6ngly, muscle, but not liver, of nNOS-deficient mice exhibits pronounced induc6on of the nitrate-nitrite reduc6ve pathway genes/proteins in response to nitrate overload, pinpoin6ng 6ssue-specific adapta6on in NO genera6on. Finally, nitrate supplementa6on does not increase inflammatory IL-6 expression, dissocia6ng the nitrate-nitrite pathway from overt inflammatory signaling in muscle. These adap6ve mechanisms, more robustly present in the muscle than the liver, support muscle contrac6le func6on in gene6c or disease states related to NOS impairment, laying groundwork for nutri6onal or pharmacological exploita6on aimed at enhancing endogenous NO availability and mi6ga6ng muscular dystrophies or metabolic disorders.Cardiovascular studies further highlight NOS isoform-specific effects, notably within the context of erythropoie6n (EPO)-mediated cardiac responses. Lee et al. deploy a mul6-strain mouse model approach to dissect 6ssue-specific and NOS isoform contribu6ons to EPO-induced heart effects. They demonstrate that while acute EPO administra6on promotes cardiovascular protec6on, chronic high-dose EPO produces deleterious cardiac dysfunc6on dependent on nNOS ac6vity and on EPO receptor expression in non-erythroid 6ssues. This dichotomy is revealed in transgenic mouse models where nNOS knockout confers cardioprotec6on from EPO-induced adverse effects despite preserved erythropoiesis. Interes6ngly, EPO treatment in nNOS-knockout mice improves glucose and insulin tolerance, revealing a separa6on between metabolic and cardiac EPO ac6ons and new evidence for nNOS-specific metabolic regula6on under EPO challenge. Akt phosphoryla6on (a key pro-survival and remodeling pathway) and Nox4 (a major source of cardiac reac6ve oxygen species) are upregulated by EPO in wild-type but not nNOS-knockout mice, reinforcing nNOS's role as mediator of cardiac maladapta6on under chronic EPO. The underlying modula6on of heart failure-associated gene networks and signaling pathways like ERK and Akt elucidate the nuanced interplay between NOS isoforms and cardiac remodeling. This supports a future where isoform-specific NO modula6on op6mizes therapeu6c benefits while minimizing risks. Moreover, the iden6fica6on of nNOS as essen6al to EPO-induced cardiac injury reveals a poten6al therapeu6c target to decouple the hematopoie6c benefits of EPO from its cardiovascular side effects and opens explora6on of nNOS-linked protein S-nitrosyla6on in EPO biology.Lewis et al. introduce technological advances to exploit endogenous NO pools, such as the use of infrared (IR) light to induce rapid, sustained vasodila6on via NO release from endothelial stores and ac6va6on of soluble guanylyl cyclase pathways. This is cri6cal work, since it has long been known that light can mediate vasodila6on, but the wavelengths involved have been thought to be in the UV-range. The authors u6lize a custom setup for delivering precise IR light pulses (at 1,460 nm, a wavelength not previously explored for direct vascular effects) to live, isolated rat occipital arteries mounted in wire myographs, allowing direct spa6al targe6ng and real-6me tension recording. The work combines pharmacological manipula6on (endothelial denuda6on and use of ODQ, a selec6ve sGC inhibitor) with IR s6mula6on, rigorously isola6ng the contribu6on of endothelium and NO/sGC/cGMP signaling to IRinduced vasodila6on. This photobiomodula6on effect, prominently seen in rat occipital arteries, implicates pre-formed S-nitrosothiol pools and endothelial vesicular release as mechanisms. The sustained post-irradia6on vasodila6on suggests long-lived intracellular signaling cascades including second messengers (cGMP, Ca 2+ ) and protein nitrosa6on/phosphoryla6on contribute to vascular tone regula6on beyond immediate s6muli. The reported vasodilatory effects of IR persist indeed well beyond the exposure period, sugges6ng that short flashes of light can sustain improved vascular func6on longer than tradi6onal pharmacologic interven6ons. These findings hold clinical promise for non-invasive vascular and neural disorder treatments, with IR light offering precise temporal and spa6al control of NO-mediated vasoreac6vity, posi6oning these energy-based therapies to restore targeted blood flow in disease contexts such as hypertension and diabetes. Lastly, the segment-specific vascular responses revealed may inform future precision vascular interven6ons or neuromodula6on strategies using IR light, especially in 6ssues or organs with unique endothelial architecture. This integra6ve collec6on affirms NO and RNS as nodal regulators spanning metabolism, immunity, oncogenesis, and 6ssue physiology, with interdependencies shaped by concentra6on, compartmentaliza6on, and temporal dynamics. Transla6onally, these insights predict promising avenues toward redox-targeted therapies, dietary modula6on, and biophysical interven6ons. Looking forward, several trajectories may be taken: the refinement of spa6al-temporal NO imaging to resolve subcellular signaling gradients; dissec6ng redox-driven post-transla6onal modifica6ons in disease contexts; exploi6ng isoform-specific NOS modula6on; and harnessing endogenous NO metabolite pools for innova6ve therapies. Advancements in measurement technologies, bioinforma6c integra6on, and mul6-omics promise to unravel NO-related signaling networks with greater resolu6on, fueling precision medicine approaches tailored to redox biology mechanisms. Ul6mately, this body of work elevates NO from a mere diffusible mediator to a master integrator of physiological homeostasis and disease modula6on, offering a framework for bridging molecular discovery and clinical transforma6ve poten6al. The collec6ve findings announce an era where NO-centric strategies reshape the landscape of inflamma6on, cancer, cardiovascular, and metabolic therapeu6cs, fulfilling the vision of transla6ng elegant redox chemistry into robust clinical impact.