NADP Oxidase Research Articles

Abstract P3010: Angptl4 Reduces Atherosclerosis And Promotes Fibrous Cap Stability By Targeting Klf4 In Vascular Smooth Muscle Cells

Background: Atherosclerosis is vascular disease of chronic inflammation, and we recently showed that angiopoietin-like 4 (ANGPTL4) promote cardiac repair by suppressing pathological inflammation. Given the contribution of inflammation to atherosclerosis, we assessed the effect of ANGPTL4 on atherosclerosis development. Methods: We injected recombinant ANGPTL4 protein or PBS to atherosclerotic Apoe-/- mice twice a week fed a high-fat diet for 8 weeks, and analyzed atherosclerotic lesion size, inflammation, and plaque stability. The effect of ANGPTL4 on vascular smooth muscle cells (VSMCs) was studied, and plasma levels of ANGPTL4 in patients were measured. Results: ANGPTL4 treatment reduced the atherosclerotic plaque size and vascular inflammation. In the atherosclerotic lesions, the number of α-SMA(+)MYH11(+) cells was higher, while the number of CD68(+)cells was lower in the ANGPTL4 group than in the vehicle-treated group. Moreover, α-SMA-expressing fibrous cap was significantly thicker in the ANGPTL4 group than in the PBS group. Then, we isolated VSMCs from atherosclerotic aortas, and found that CD68 and Krüppel-like factor 4 (KLF4) were significantly upregulated in the PBS group. As KLF4 is a phenotypic modulator and an inducer of NADP oxidase 1 (Nox1) in VSMCs, the effect of ANGPTL4 on VSMCs was assessed. Cholesterol-induced upregulations of Nox1 and KLF4 were substantially inhibited by ANGPTL4 treatment. Then, we analyzed the circulating ANGPTL4 levels of the cardiovascular disease patients, and found the positive correlation between high ANGPTL4 and low incidence of vascular events. Conclusions: Our results showed that ANGPTL4 inhibited atherogenesis by increasing the fibrous cap thickness. KLF4 and Nox1 were suggested as targets of ANGPTL4 in VSMCs to confer plaque stability by attenuating phenotypic changes of VSMCs toward inflammatory macrophage-like cells. Taken together, circulating ANGPTL4 may be associated with regulation of vascular events.

Endoplasmic reticulum-targeting delivery of curcumin alters reactive oxygen species production and phenotypes in activated macrophages

Abstract Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that organelle-targeted delivery of sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitor curcumin with the endoplasmic reticulum (ER)-targeting peptide decorated PLGA nanoparticles (NPs) rescues mutant H338Y-gp91phox protein maturation of a particular type of CGD with a CybbC1024T mutation, leading to ER retention of the mutant protein. However, how the rescuing of mutant H338Y-gp91 phox protein with ER-targeting curcumin-PLGA NPs on macrophages functions and differentiation are still unknown. In this study, we analyzed gp91phox maturation and macrophage M1/M2 polarization in bone marrow-derived macrophages (BMDMs) from wild-type (WT), Cybb−/−, and CybbC1024T transgenic Cybb−/−mice. First, we found that the levels of CD86 and CD206 expression with lipopolysaccharide+interferon-γ (LPS+IFN-γ) stimulation were similar in WT and CybbC1024T transgenic Cybb−/−BMDMs and CD206 expression was lowered in Cybb−/−mice when compared with WT and CybbC1024T transgenic Cybb−/−BMDMs. Second, we found that CD206 levels after LPS+IFN-γ stimulation were enhanced by free curcumin, curcumin-PLGA NPs and ER-targeting curcumin-PLGA NPs treatment in CybbC1024T transgenic Cybb−/−BMDMs, but not in WT and Cybb−/− BMDMs. Meanwhile, we found that ER-targeting curcumin-PLGA NPs enhanced gp91phox glycosylation in CybbC1024T transgenic Cybb−/−BMDMs. These findings may indicate that both the expression and maturation of gp91phox are crucial for macrophage M1/M2 polarization. Gp91phox maturation may be important for M2 phenotypes in activated macrophages.

Targeted Delivery of Curcumin Rescues Endoplasmic Reticulum-Retained Mutant NOX2 Protein and Avoids Leukocyte Apoptosis.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitors could be used to rescue mutant H338Y-gp91phox protein of a particular type of CGD with a CybbC1024T mutation, leading to endoplasmic reticulum (ER) retention of the mutant protein. In this study, we developed a novel mouse model with the CybbC1024T mutation on a Cybb knockout background and investigated the therapeutic effects of ER-targeted delivery of the SERCA inhibitor, curcumin, with poly(lactic-coglycolic acid) (PLGA) nanoparticles (NPs). We found that PLGA encapsulation improved the efficacy of curcumin as a SERCA inhibitor to induce ER calcium release. ER-targeting curcumin-loaded PLGA NPs reduced and delayed extracellular calcium entry and protected the cells from mitochondrial damage and apoptosis. In vivo studies showed that ER-targeting curcumin-loaded PLGA NPs treatment enhanced neutrophil gp91phox expression, ROS production and peritoneal bacterial clearance ability of the CybbC1024T transgenic Cybb -/- mice. Our findings indicate that ER-targeted delivery of curcumin not only rescues ER-retained H338Y-gp91phox protein, and hence leukocyte function, but also enhances the bioavailability and reduces cytotoxicity. Modulation of ER function by using organelle-targeted NPs may be a promising strategy to improve the therapeutic potential of curcumin as a treatment for CGD.

Activation of Signaling Cascades by Weak Extremely Low Frequency Electromagnetic Fields

Background/Aims: Results from recent studies suggest that extremely low frequency magnetic fields (ELF-MF) interfere with intracellular signaling pathways related to proliferative control. The mitogen-activated protein kinases (MAPKs), central signaling components that regulate essentially all stimulated cellular processes, include the extracellular signal-regulated kinases 1/2 (ERK1/2) that are extremely sensitive to extracellular cues. Anti-phospho-ERK antibodies serve as a readout for ERK1/2 activation and are able to detect minute changes in ERK stimulation. The objective of this study was to explore whether activation of ERK1/2 and other signaling cascades can be used as a readout for responses of a variety of cell types, both transformed and non-transformed, to ELF-MF. Methods: We applied ELF-MF at various field strengths and time periods to eight different cell types with an exposure system housed in a tissue culture incubator and followed the phosphorylation of MAPKs and Akt by western blotting. Results: We found that the phosphorylation of ERK1/2 is increased in response to ELF-MF. However, the phosphorylation of ERK1/2 is likely too low to induce ELF-MF-dependent proliferation or oncogenic transformation. The p38 MAPK was very slightly phosphorylated, but JNK or Akt were not. The effect on ERK1/2 was detected for exposures to ELF-MF strengths as low as 0.15 µT and was maximal at ∼10 µT. We also show that ERK1/2 phosphorylation is blocked by the flavoprotein inhibitor diphenyleneiodonium, indicating that the response to ELF-MF may be exerted via NADP oxidase similar to the phosphorylation of ERK1/2 in response to microwave radiation. Conclusions: Our results further indicate that cells are responsive to ELF-MF at field strengths much lower than previously suspected and that the effect may be mediated by NADP oxidase. However, the small increase in ERK1/2 phosphorylation is probably insufficient to affect proliferation and oncogenic transformation. Therefore, the results cannot be regarded as proof of the involvement of ELF-MF in cancer in general or childhood leukemia in particular.

Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca $^{2+}_{\text {cyt}})$ concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8g and μg during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca $^{2+}_{\text {cyt}}$ level during the hypergravity phases of a parabola but a significant increase of the Ca $^{2+}_{\text {cyt}}$ concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca $^{2+}_{\text {cyt}}$ response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca $^{2+}_{\text {cyt}}$ changes entirely. We thus conclude that the significant increase in Ca $^{2+}_{\text {cyt}}$ under microgravity is largely due to extracellular Ca2+ sources.

Role of defective autophagia and the intestinal flora in Crohn disease

The precise mechanisms underlying the development of Crohn disease (CD) remain controversial, but sufficient data have been collected to suggest that an uncontrolled immune response within the intestinal mucosa leads to inflammation in a genetically susceptible host. Although lack of mucosal regulatory T cells causes colitis in humans and experimental rodents, patients with CD have more rather than less regulatory activity in the intestine, apparently excluding defects in tolerance as the cause of CD. Genome-wide association studies have identified many gene variants that confer susceptibility and which seem associated to diminished functioning of especially innate immunity. In apparent agreement, CD patients are impaired with respect to innate immune responses and controlling bacterial flora in the intestine. Furthermore, severe genetic deficiencies in innate immunity, like e.g., lack of NADP oxidase activity or diminished function of the Wiskott Aldrich syndrome protein are associated with colitis in mice and men, and are often mistakenly diagnosed as CD. Thus we favor the view that the primary defect in CD is a lack in innate immunity, causing second tier immunological defenses to combat otherwise easily controlled bacterial breaches of the mucosal barrier.

The Third ISN Special Neurochemistry Conference – 8th International Meeting on Brain Energy Metabolism –‘Neurodegeneration and Regeneration’

The Third ISN Special Neurochemistry Conference – 8th International Meeting on Brain Energy Metabolism –‘Neurodegeneration and Regeneration’

Lipoxygenase-dependent superoxide release in skeletal muscle

Superoxide anion radical (O(2)(*-)) is released from skeletal muscle at rest and is particularly elevated during conditions of heat stress (42 degrees C). Previous studies have shown that in isolated rat diaphragm O(2)(*-) release is not dependent on mitochondrial electron transport, reduced NADP oxidase activity, or the integrity of membrane anion channels. This study hypothesized that O(2)(*-) release, as measured by cytochrome c reduction, is linked to metabolism of arachidonic acid. Phospholipase A(2) inhibition with manoalide significantly decreased O(2)(*-) release. In downstream pathways, neither the blockage of cyclooxygenase with indomethacin nor the inhibition of cytochrome P-450-dependent monooxygenase with SKF-525A decreased O(2)(*-) release. However, lipoxygenase (LOX) inhibition with general LOX blockers 5,8,11,14-eicosatetraynoic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate greatly attenuated the signal. Furthermore, the specific 5-LOX inhibitor diethylcarbamazine also significantly decreased O(2)(*-) release. Immunohistochemistry localized 5- and 12-LOX to the cytosol and sarcolemma of muscle cells. Confocal studies, using the O(2)(*-)-sensitive fluorescent indicator hydroethidine, demonstrated that LOX inhibition had no significant influence on intracellular O(2)(*-) formation. When compared with the cytochrome c results, this indicates that intra- and extracellular O(2)(*-) must arise from different sources. These data show for the first time that arachidonic acid metabolism through LOX activity, is a major source of extracellular O(2)(*-) release in skeletal muscle.