Debora Zamora

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.

Bioenergetic Profiles Diverge During Macrophage Polarization: Implications for the Interpretation of 18F-FDG PET Imaging of Atherosclerosis

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 18F-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 MLPS) or alternative (M2) polarization, respectively. Glucose uptake was measured using 3H-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 3H-deoxyglucose uptake. Enhanced glucose uptake by MLPS 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 MLPS, 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 18F-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.

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 H2O2 formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, platelet-derived growth factor B, and RANTES. Both H2O2 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. Conclusion—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.

Intracelluar pH (pHi) Measurements in the In Vitro Tadpole Brainstem:Direct Correlations between Changes in pHi and Ventilation

Central respiratory chemoreceptors measure pH in the brain stem and are an integral part of the neural circuitry that modulates respiratory rhythmogenesis, specifically in response to hypercapnic acidosis. Central respiratory chemore- ceptor membrane potential and/or action potential firing rate are altered in response to changes in intracellular pH (pHi), which changes with the hydration of CO2 in both the extracellular and intracellular space, however the role of cellular changes in chemoreceptor properties on respiratory motor output has been difficult to identify. We studied whole nerve respiratory activity while simultaneously visualizing pHi dynamics using the pH-sensitive dye, BCECF, in the spontane- ously active in vitro tadpole brainstem. The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neu- ronsunder physiological conditions, where mammalian preparations do not. An ammonium prepulse was used to selec- tively induce a decrease in pHi. Our results show intracellular pH is regulated differently in cells located in chemosensi- tive and non-chemosensitive regions of the tadpole brainstem during hypercapnia. We were also able to show an inverse correlation between pHi in cells located in chemosensitive regions of the tadpole brainstem and whole nerve respiratory- related activity. Using this approach, the microenvironment of individual cells may be manipulated while monitoring real time changes in pHi, neuronal activity and ventilatory-related activity to elucidate the role of a variety of signals in elicit- ing changes in ventilation.

Employing a pH Sensitive Fluorophore to Measure Intracellular pH in the In Vitro Brainstem Preparation of Rana catesbeiana~!2009-05-06~!2009-10-29~!2009-12-24~!

We developed an in vitro tadpole brainstem preparation in order to investigate the development of central respiratory chemoreception and rhythmogenesis. pH sensitive fluorescent dyes have been utilized to record intracellular pH (pHi) optically in central respiratory chemoreceptive regions in mammals. Our goal in this study was to develop the ability to record pHi optically while simultaneously recording respiratory motor output in the superfused tadpole brainstem preparation. We developed a dye-loading protocol that demonstrated our ability to adequately load the majority of brainstem neurons. The presence of the dye was not disruptive to ongoing respiratory rhythmogenesis or the respiratory response to central respiratory chemoreceptor stimulation. The tadpole brainstem is an excellent model to study the development of the neural control of respiration, as it is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. Validating of the use of the pH sensitive dyes to record pHi optically in central respiratory chemoreceptors in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors.