Redox regulation of MAPK phosphatase 1 controls monocyte migration and macrophage recruitment.

Monocytic adhesion and chemotaxis are regulated by MAPK pathways, which in turn are controlled by redox-sensitive MAPK phosphatases (MKPs). We recently reported that metabolic disorders prime monocytes for enhanced recruitment into vascular lesions by increasing monocytes' responsiveness to chemoattractants. However, the molecular details of this proatherogenic mechanism were not known. Here we show that monocyte priming results in the S-glutathionylation and subsequent inactivation and degradation of MKP-1. Chronic exposure of human THP-1 monocytes to diabetic conditions resulted in the loss of MKP-1 protein levels, the hyperactivation of ERK and p38 in response to monocyte chemoattractant protein-1 (MCP-1), and increased monocyte adhesion and chemotaxis. Knockdown of MKP-1 mimicked the priming effects of metabolic stress, whereas MKP-1 overexpression blunted both MAPK activation and monocyte adhesion and migration induced by MCP-1. Metabolic stress promoted the S-glutathionylation of MKP-1, targeting MKP-1 for proteasomal degradation. Preventing MKP-1 S-glutathionylation in metabolically stressed monocytes by overexpressing glutaredoxin 1 protected MKP-1 from degradation and normalized monocyte adhesion and chemotaxis in response to MCP-1. Blood monocytes isolated from diabetic mice showed a 55% reduction in MKP-1 activity compared with nondiabetic mice. Hematopoietic MKP-1 deficiency in atherosclerosis-prone mice mimicked monocyte priming and dysfunction associated with metabolic disorders, increased monocyte chemotaxis in vivo, and accelerated atherosclerotic lesion formation. In conclusion, we identified MKP-1 as a central redox-sensitive regulator of monocyte adhesion and migration and showed that the loss of MKP-1 activity is a critical step in monocyte priming and the metabolic stress-induced conversion of blood monocytes into a proatherogenic phenotype.

NADPH oxidase 4 mediates monocyte priming and accelerated chemotaxis induced by metabolic stress.

OBJECTIVE: Metabolic disorders increase monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis in mice. The goal of this study was to determine the molecular mechanisms responsible for the enhanced responsiveness of monocytes to chemoattractants induced by metabolic stress. METHODS AND RESULTS: Chronic exposure of monocytes to diabetic conditions induced by human LDL plus high D-glucose concentrations (LDL+HG) promoted NADPH Oxidase 4 (Nox4) expression, increased intracellular H(2)O(2) formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, platelet-derived growth factor B, and RANTES. Both H(2)O(2) added exogenously and overexpression of Nox4 mimicked LDL+HG-induced monocyte priming, whereas Nox4 knockdown protected monocytes against metabolic stress-induced priming and accelerated chemotaxis. Exposure of monocytes to LDL+HG promoted the S-glutathionylation of actin, decreased the F-actin/G-actin ratio, and increased actin remodeling in response to MCP-1. Preventing LDL+HG-induced protein S-glutathionylation by overexpressing glutaredoxin 1 prevented monocyte priming and normalized monocyte chemotaxis in response to MCP-1. Induction of hypercholesterolemia and hyperglycemia in C57BL/6 mice promoted Nox4 expression and protein S-glutathionylation in macrophages, and increased macrophage recruitment into MCP-1-loaded Matrigel plugs implanted subcutaneous in these mice. CONCLUSIONS: By increasing actin-S-glutathionylation and remodeling, metabolic stress primes monocytes for chemoattractant-induced transmigration and recruitment to sites of vascular injury. This Nox4-dependent process provides a novel mechanism through which metabolic disorders promote atherogenesis.

Optical recording of intracellular pH in respiratory chemoreceptors.

We studied the spontaneously active in vitro tadpole brainstem and recorded whole nerve respiratory activity while simultaneously visualizing intracellular pH (pHi) dynamics using the pH-sensitive dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, AM). The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. We generated a calibration curve to correlate the emitted fluorescence of BCECF to pHi. In addition, we demonstrated that the dye loading protocol that we established labeled an adequate number of cells and did not disrupt spontaneous respiratory rhythmogenesis or the respiratory response to central chemoreceptor stimulation. Validation of the use of the pH sensitive dye BCECF in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors and allow correlation between the changes in pHi in central chemoreceptors and respiratory motor output recorded from cranial nerves.

Serotonergic modulation of respiratory rhythmogenesis and central chemoreception.

In addition to evidence supporting serotonergic modulation of respiratory rhythmogenesis, serotonergic mechanisms play a role in central respiratory chemoreception. We examined the role of serotonin 5HT1A receptors in respiratory rhythmicity and central respiratory chemosensitivity in in vitro brainstem preparations of the bullfrog tadpole, Rana catesbeiana. Spontaneous respiratory motor output was recorded from cranial nerve 7 at control bath pH (7.8) and hypercapnic bath pH (7.4) as bath concentrations of a 5HT1A receptor agonist were steadily increased from 0.5 to 25 microM. Activation of the 5HT1A receptor significantly altered the respiratory burst cycle. Significant increases in both gill and lung burst cycle were observed in response to bath application of 8-OH-DPAT; gill burst cycle in response to 8-OH-DPAT was influenced by bath pH, as gill burst cycle at bath pH 7.8 was not significantly increased at 0.5 or 5.0 microM 8-OH-DPAT. However, when the pH was reduced to 7.4 gill burst cycle was significantly increased at these same bath concentrations of 8-OH-DPAT. Gill burst amplitude was not altered in response to bath application of 8-OH-DPAT; however, lung burst amplitude was significantly decreased at 25.0 microM 8-OH-DPAT at bath pH 7.8. These data indicate that 5HT1A receptors are involved in neural respiratory rhythmogenic and chemoreceptive circuits in the bullfrog tadpole, and support the hypothesis that abnormalities in serotonergic systems may be an underlying component of Sudden Infant Death Syndrome.

Bioenergetic profiles diverge during macrophage polarization: implications for the interpretation of 18F-FDG PET imaging of atherosclerosis.

UNLABELLED: Conventional cardiovascular imaging is invaluable for the assessment of late sequelae of atherosclerosis, such as diminished perfusion reserve and luminal stenosis. Molecular imaging provides complementary information about plaque composition and ongoing biologic processes in the vessel wall, allowing the early diagnosis and risk stratification of patients. Detection of enhanced glucose uptake, using (18)F-FDG PET, has been proposed as a noninvasive approach to track macrophage activation as a critical event in the development and progression of atherosclerosis. In this study, we determined the impact of macrophage polarization on glucose metabolism and oxidative phosphorylation. METHODS: Murine peritoneal macrophages were incubated in the presence of interferon-γ (IFN-γ) plus tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), or interleukin-4 (IL-4) to induce classic (M1 and M(LPS)) or alternative (M2) polarization, respectively. Glucose uptake was measured using (3)H-deoxyglucose. Oxidative phosphorylation was evaluated using an extracellular flux analyzer. Mitochondrial DNA copy numbers were quantified by polymerase chain reaction. The expression of glucose transporter-1 (Glut-1), hexokinase-1 and -2 (Hk-1 and Hk-2, respectively), mitochondrial transcription factor-1 (Tfam), and cytochrome c oxidase subunit I (Cox-1) was determined by quantitative reverse transcription polymerase chain reaction. RESULTS: Stimulation of macrophages by LPS, but not polarization with either IFN-γ plus TNF-α (M1) or IL-4 (M2), resulted in a 2.5-fold increase in (3)H-deoxyglucose uptake. Enhanced glucose uptake by M(LPS) macrophages paralleled the overexpression of rate-limiting proteins involved in transmembrane transport and intracellular trapping of glucose--that is, Glut-1, Hk-1, and Hk-2. Alternatively polarized M2 macrophages developed a markedly higher spare respiratory capacity than both nonpolarized and classically polarized M1 macrophages. M2 polarization was associated with a 4.6-fold increase in mitochondrial content of the cells, compared with nonpolarized macrophages. The expression of Tfam, a major regulator of mitochondrial biogenesis, and Cox-1, a critical component of respiratory chain, was significantly increased in M2 polarized macrophages. CONCLUSION: Polarization of macrophages induces distinct metabolic profiles with respect to glycolysis versus oxidative phosphorylation, with alternatively polarized macrophages shifting to mitochondria as their main source of adenosine triphosphate. Only M(LPS), but not M1 or M2 polarized macrophages, showed increased glucose uptake, suggesting that glucose metabolism is regulated independent of the polarization state and macrophage polarization may not be detectable by (18)F-FDG PET.

Ursolic acid protects diabetic mice against monocyte dysfunction and accelerated atherosclerosis.

AIMS: Accelerated atherosclerosis is a major diabetic complication initiated by the enhanced recruitment of monocytes into the vasculature. In this study, we examined the therapeutic potential of the phytonutrients ursolic acid (UA) and resveratrol (RES) in preventing monocyte recruitment and accelerated atherosclerosis. METHODS AND RESULTS: Dietary supplementation with either RES or UA (0.2%) protected against accelerated atherosclerosis induced by streptozotocin in high-fat diet-fed LDL receptor-deficient mice. However, mice that received dietary UA for 11 weeks were significantly better protected and showed a 53% reduction in lesion formation while mice fed a RES-supplemented diet showed only a 31% reduction in lesion size. Importantly, UA was also significantly more effective in preventing the appearance of proinflammatory GR-1(high) monocytes induced by these diabetic conditions and reducing monocyte recruitment into MCP-1-loaded Matrigel plugs implanted into these diabetic mice. Oxidatively stressed THP-1 monocytes mimicked the behavior of blood monocytes in diabetic mice and showed enhanced responsiveness to monocyte chemoattractant protein-1 (MCP-1) without changing MCP-1 receptor (CCR2) surface expression. Pretreatment of THP-1 monocytes with RES or UA (0.3-10µM) for 15h resulted in the dose-dependent inhibition of H(2)O(2)-accelerated chemotaxis in response to MCP-1, but with an IC(50) of 0.4µM, UA was 2.7-fold more potent than RES. CONCLUSION: Dietary UA is a potent inhibitor of monocyte dysfunction and accelerated atherosclerosis induced by diabetes. These studies identify ursolic acid as a potential therapeutic agent for the treatment of diabetic complications, including accelerated atherosclerosis, and provide a novel mechanism for the anti-atherogenic properties of ursolic acid.

Effect of prevention of lung inflation on metamorphosis and respiration in the developing bullfrog tadpole, Rana catesbeiana.

We tested the hypothesis that respiratory development would be retarded in tadpoles reared in aquaria in which a barrier prevented access to the air-water interface. To test this hypothesis, we examined swimming behavior and respiration in intact tadpoles and gill and lung respiratory activity and central chemosensory responses in an in vitro brainstem preparation. The "barrier" tadpoles had significantly lower resting gill frequencies and higher lung breath attempts than control tadpoles at the same metamorphic stage. Control tadpoles swam greater distances and spent more time in the upper one third of the aquaria, while barrier tadpoles spent significantly more time at the bottom of the aquaria. There was significantly greater mortality for barrier tadpoles compared to control animals in the earliest and latest metamorphic stages. Mean body weight was significantly greater, and metamorphic rate was reduced in barrier tadpoles. Neither control nor barrier tadpole brainstem preparations demonstrated a gill ventilatory response to CO(2); however, both control and barrier preparations possessed significant lung frequency responses to central CO(2) chemoreceptor stimulation. Bath application of the GABA(A) and glycine receptor antagonists, bicuculline and strychnine, had greater effects on control tadpole gill burst activity and produced a similar large-amplitude bursting pattern in both control and barrier tadpoles, that was insensitive to CO(2) chemoreceptor stimulation. We conclude that development of the respiratory pattern was perturbed by the barrier, but the major effect was on gill ventilation rather than lung ventilation as we had expected.