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MD2 blockade prevents modified LDL-induced retinal injury in diabetes by suppressing NADPH oxidase-4 interaction with Toll-like receptor-4.

ABSTRACT: Modified LDL-induced inflammation and oxidative stress are involved in the pathogenesis of diabetic retinopathy. Recent studies have also shown that modified LDL activates Toll-like receptor 4 (TLR4) to mediate retinal injury. However, the mechanism by which modified LDL activates TLR4 and the potential role of the TLR4 coreceptor myeloid differentiation protein 2 (MD2) are not known. In this study, we inhibited MD2 with the chalcone derivatives L2H17 and L6H21 and showed that MD2 blockade protected retinal Müller cells against highly oxidized glycated-LDL (HOG-LDL)-induced oxidative stress, inflammation, and apoptosis. MD2 inhibition reduced oxidative stress by suppressing NADPH oxidase-4 (NOX4). Importantly, HOG-LDL activated TLR4 and increased the interaction between NOX4 and TLR4. MD2 was required for the activation of these pathways, as inhibiting MD2 prevented the association of NOX4 with TLR4 and reduced NOX4-mediated reactive oxygen species production and TLR4-mediated inflammatory factor production. Furthermore, treatment of diabetic mice with L2H17 significantly reduced LDL extravasation in the retina and prevented retinal dysfunction and apoptosis by suppressing the TLR4/MD2 pathway. Our findings provide evidence that MD2 plays a critical role in mediating modified LDL-induced cell injury in the retina and suggest that targeting MD2 may be a potential therapeutic strategy.

SUBMITTER: Chen H

PROVIDER: S-EPMC8102522 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Endothelial AIP1 Regulates Vascular Remodeling by Suppressing NADPH Oxidase-2.

Project description:Objective: AIP1 expression is downregulated in human atherosclerotic plaques and global deletion of AIP1 in mice exacerbates atherosclerosis in ApoE-KO mouse models. However, the direct role of AIP1 in endothelium, vascular remodeling and associated vascular diseases has not been determined. Approach and Results: We used endothelial cell (EC)-specific AIP1-deficient (AIP1-ECKO) mice to define the role of AIP1 in vascular remodeling and intima-media thickening in a mouse carotid artery ligation model characterized by both neointimal hyperplasia and inward vessel remodeling. Compared to WT littermates, AIP1-ECKO mice had 2.2-fold larger intima area and 4.4-fold thicker intima as measured by intima/media ratio in arteries with more proliferating vascular smooth muscle cells (VSMCs) at week 2-4 post-injury. Increased reactive oxygen species (ROS) in endothelium at early time points induced inflammation and vessel dysfunction in AIP1-ECKO prior to VSMC accumulations. Moreover, knockdown of AIP1 in human EC enhanced ROS generation which was attenuated by co-silencing of NOX2. Mechanistically, AIP1 via its proline-rich region binds to the SH3 domain of cytosolic subunit p47phox to disrupt formation of an active NOX2 complex, attenuating ROS production. Conclusion: Our study supports that AIP1 regulates vascular remodeling with intima-media thickening by suppressing endothelial NOX2-dependent oxidative stress. Highlights: •In a carotid ligation model, endothelial cell (EC)-specific AIP1-deficient (AIP1-ECKO) mice had much larger media area, thicker vessel wall and augmented neointima formation.•Increased production of reactive oxygen species in vascular EC at early time points concomitant with vessel dysfunction in AIP1-ECKO.•AIP1 via its proline-rich region binds to the SH3 domain of cytosolic subunit p47phox to disrupt formation of an active NOX2 complex, attenuating ROS production.

| S-EPMC5921534 | biostudies-literature

Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4-MD2 complex.

Project description:Reactive oxygen species (ROS) contribute to the development of non-alcoholic fatty liver disease. ROS generation by infiltrating macrophages involves multiple mechanisms, including Toll-like receptor 4 (TLR4)-mediated NADPH oxidase (NOX) activation. Here, we show that palmitate-stimulated CD11b+F4/80low hepatic infiltrating macrophages, but not CD11b+F4/80high Kupffer cells, generate ROS via dynamin-mediated endocytosis of TLR4 and NOX2, independently from MyD88 and TRIF. We demonstrate that differently from LPS-mediated dimerization of the TLR4-MD2 complex, palmitate binds a monomeric TLR4-MD2 complex that triggers endocytosis, ROS generation and increases pro-interleukin-1β expression in macrophages. Palmitate-induced ROS generation in human CD68lowCD14high macrophages is strongly suppressed by inhibition of dynamin. Furthermore, Nox2-deficient mice are protected against high-fat diet-induced hepatic steatosis and insulin resistance. Therefore, endocytosis of TLR4 and NOX2 into macrophages might be a novel therapeutic target for non-alcoholic fatty liver disease.

| S-EPMC5740170 | biostudies-literature

Selective targeting of MD2 attenuates intestinal inflammation and prevents neonatal necrotizing enterocolitis by suppressing TLR4 signaling.

Project description:Neonatal necrotizing enterocolitis (NEC) is an inflammatory disease that occurs in premature infants and has a high mortality rate; however, the mechanisms behind this disease remain unclear. The TLR4 signaling pathway in intestinal epithelial cells, mediated by TLR4, is important for the activation of the inflammatory storm in NEC infants. Myeloid differentiation protein 2 (MD2) is a key auxiliary component of the TLR4 signaling pathway. In this study, MD2 was found to be significantly increased in intestinal tissues of NEC patients at the acute stage. We further confirmed that MD2 was upregulated in NEC rats. MD2 inhibitor (MI) pretreatment reduced the occurrence and severity of NEC in neonatal rats, inhibited the activation of NF-κB and the release of inflammatory molecules (TNF-α and IL-6), and reduced the severity of intestinal injury. MI pretreatment significantly reduced enterocyte apoptosis while also maintaining tight junction proteins, including occludin and claudin-1, and protecting intestinal mucosal permeability in NEC rats. In addition, an NEC in vitro model was established by stimulating IEC-6 enterocytes with LPS. MD2 overexpression in IEC-6 enterocytes significantly activated NF-κB. Further, both MD2 silencing and MI pretreatment inhibited the inflammatory response. Overexpression of MD2 increased damage to the IEC-6 monolayer cell barrier, while both MD2 silencing and MI pretreatment played a protective role. In conclusion, MD2 triggers an inflammatory response through the TLR4 signaling pathway, leading to intestinal mucosal injury in NEC. In addition, MI alleviates inflammation and reduces intestinal mucosal injury caused by the inflammatory response by blocking the TLR4-MD2/NF-κB signaling axis. These results suggest that inhibiting MD2 may be an important way to prevent NEC.

| S-EPMC9663461 | biostudies-literature

Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit expression: implication for native LDL oxidation.

Project description:Shear stress regulates endothelial nitric oxide and superoxide (O2-*) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 microg/mL of native LDL were exposed to (1) pulsatile flow with a mean shear stress (tau(ave)) of 25 dyne/cm2 and (2) oscillating flow at tau(ave) of 0. After 4 hours, aliquots of culture medium were collected for high-performance liquid chromatography analyses of electronegative LDL species, described as LDL- and LDL2-. In response to oscillatory shear stress, gp91phox mRNA expression was upregulated by 2.9+/-0.3-fold, and its homologue, Nox4, by 3.9+/-0.9-fold (P<0.05, n=4), with a corresponding increase in O2-* production rate. The proportion of LDL- and LDL2- relative to static conditions increased by 67+/-17% and 30+/-7%, respectively, with the concomitant upregulation of monocyte chemoattractant protein-1 expression and increase in monocyte/BAEC binding (P<0.05, n=5). In contrast, pulsatile flow downregulated both gp91phox and Nox4 mRNA expression (by 1.8+/-0.2-fold and 3.0+/-0.12-fold, respectively), with an accompanying reduction in O2-* production, reduction in the extent of LDL modification (51+/-12% for LDL- and 30+/-7% for LDL2-), and monocyte/BAEC binding. The flow-dependent LDL oxidation is determined in part by the NADPH oxidase activity. The formation of modified LDL via O2-* production may also affect the regulation of monocyte chemoattractant protein-1 expression and monocyte/BAEC binding.

| S-EPMC4433384 | biostudies-literature

Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase.

Project description:Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H(+) along with extracellular superoxide, and the intracellular H(+) must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na(+)/H(+) exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia-reperfusion was likewise prevented by inhibiting Na(+)/H(+) exchange and by reduced expression of the Na(+)/H(+) exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na(+)/H(+) exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.

| S-EPMC3832003 | biostudies-literature

NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

Project description:Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

| S-EPMC3789746 | biostudies-literature

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages.

Project description:Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2(•-)) due to the NADPH-dependent univalent reduction of oxygen to O2(•-) by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2(•-) in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2(•-) formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2(•-) production in a dose- (IC50=4.1±1.8 μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40(phox) and p47(phox)), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47(phox) was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.

| S-EPMC3622795 | biostudies-literature

NADPH oxidase 4 modulates hepatic responses to lipopolysaccharide mediated by Toll-like receptor-4.

Project description:Chronic inflammation plays a key role in development of many liver diseases. Stimulation of Toll-like receptor 4 (TLR4) by bacterial lipopolysaccharide (LPS) initiates inflammation and promotes development of hepatocellular carcinoma and other liver diseases. NADPH oxidases contribute to LPS-induced reactive oxygen species (ROS) production and modulate TLR responses, but whether these enzymes function in TLR4 responses of hepatocytes is unknown. In the present work, we examined the role of NADPH oxidase 4 (Nox4) in LPS-induced TLR4 responses in human hepatoma cells and wildtype and Nox4-deficient mice. We found that LPS increased expression of Nox4, TNF-?, and proliferating cell nuclear antigen (PCNA). Nox4 silencing suppressed LPS-induced TNF-? and PCNA increases in human cells. The LPS-induced TNF-? increases were MyD88-dependent, and were attenuated in primary hepatocytes isolated from Nox4-deficient mice. We found that Nox4 mediated LPS-TLR4 signaling in hepatocytes via NF-?B and AP-1 pathways. Moreover, the effect of Nox4 depletion was time-dependent; following six weeks of repeated LPS stimulation in vivo, hepatic TNF-? and PCNA responses subsided in Nox4-deficient mice compared with wildtype mice. Therefore, our data suggest that Nox4 mediates LPS-TLR4 signaling in human hepatoma cells and murine hepatocytes and may contribute to the ability of LPS to stimulate liver pathology.

| S-EPMC5662726 | biostudies-literature

NADPH oxidase 4 protects against development of endothelial dysfunction and atherosclerosis in LDL receptor deficient mice.

Project description:AimsEndothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model.Methods and resultsEx vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4(-/-)/Ldlr(-/-) mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4(-/-)/Ldlr(-/-) mice compared with Ldlr(-/-) mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H2O2 production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4(-/-) mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo.ConclusionsNox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H2O2 could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions.

| S-EPMC4900759 | biostudies-literature

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration.

Project description:Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.

| S-EPMC2943442 | biostudies-literature

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