Study of Tissue-Specific Reactive Oxygen Species Formation by Cell Membrane Microarrays for the Characterization of Bioactive Compounds

The production of reactive oxygen species (ROS) increases considerably in situations of cellular stress, inducing lipid peroxidation and multiple alterations in proteins and nucleic acids. However, sensitivity to oxidative damage varies between organs and tissues depending on the triggering process. Certain drugs used in the treatment of diverse diseases such as malaria have side effects similar to those produced by oxidative damage, although no specific study has been conducted. For this purpose, cell membrane microarrays were developed and the superoxide production evoked by the mitochondrial activity was assayed in the presence of specific inhibitors: rotenone, antimycin A and azide. Once the protocol was set up on cell membrane isolated from rat brain areas, the effect of six antimalarial drugs (atovaquone, quinidine, doxycycline, mefloquine, artemisinin, and tafenoquine) and two essential oils (Rosmarinus officinalis and Origanum majoricum) were evaluated in multiple human samples. The basal activity was different depending on the type of tissue, the liver, jejunum and adrenal gland being the ones with the highest amount of superoxide. The antimalarial drugs studied showed specific behavior according to the type of human tissue analyzed, with atovaquone and quinidine producing the highest percentage of superoxide formation, and doxycycline the lowest. In conclusion, the analysis of superoxide production evaluated in cell membranes of a collection of human tissues allowed for the characterization of the safety profile of these antimalarial drugs against toxicity mediated by oxidative stress.

Abstract 13393: Liraglutide Improves Myocardial Redox State in Humans by Regulating the Adipose Tissue-Derived Long-Chain Ceramides

Introduction: A notable reduction in major adverse cardiovascular events and cardiovascular mortality was observed in several cardiovascular studies via treatment with liraglutide, which have been suggested to be mediated through several mechanisms including inhibition of oxidative stress. Hypothesis: We hypothesised that treatment with liraglutide reduces oxidative stress via modulating circulating levels of long-chain ceramides. Methods: We measured vascular superoxide production using lucigenin-enhanced chemiluminescence and levels of circulating ceramides using LC-MS/MS in 633 participants of the Oxford Heart Vessels and Fat (OxHVF) cohort. We also measured 33 sphingolipid species (SPL) in plasma from 32 obese individuals (average BMI 33.5±2.5kg/m 2 at inclusion) participating in a randomized clinical trial (RCT) of low calorie diet and liraglutide. Results: We found a significant association between superoxide production by internal mammary arteries (IMA) and circulating levels of long-chain ceramides (C16:0, C17:0, C18:0, C18:1) (A-D) . In the RCT, all participants adhered to an 8-week low-calorie diet (800 kcal/day) (E) , during which they lost 9.9±6.0 kg of body weight exhibiting a significant reduction in BMI ( p <0.0001), which was paralleled by significant effects on the circulating levels of SPL (F) . Following this initial phase, the patients were randomised to liraglutide treatment (1.2 mg daily) or no treatment, for a period of 52 weeks. Liraglutide treatment differentially affected several SPL species compared to the control group despite no significant changes in BMI in any of the two groups (G-J) . Conclusions: In this study, we observed for the first time in human a regulatory effect of liraglutide on the circulating levels of long-chain ceramides that are shown to be associated with vascular oxidative stress suggesting a promising mechanistic link that justifies further exploration.

Characterization the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages

Background: Previous studies have demonstrated that Cytosolic phospholipase A2a (cPLA2a) is absolutely required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2a translocates to the plasma membranes of by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the oxidase cytosolic factor, p47phox in human phagocytes. Intravenous administration of an antisense against cPLA2a that significantly inhibited its expression in mouse peritoneal neutrophil and macrophages also inhibited superoxide production, in contrast to cPLA2a knockout mice that showed normal superoxide production. The aim of the present study was to determine whether there is a binding between cPLA2a-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2a in oxidase regulation also in mouse phagocytes. Methods and Results: A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2a in stimulated mouse phagocyte membranes was demonstrated by pull down experiments, although lower than that detected by human p47phox-PX domain. Substituting the amino acids Phe98, Asn99 and Gly100 to Cys98 Ser99 and Thr100 in mouse p47phox-PX domain (that are present in human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain. Conclusions: the binding between cPLA2a-C2 and p47phox-PX domains exist in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice probably due to the absence of amino acids Cys98 Ser99 and Thr100 in p47phox-PX domain that facilitate the binding to cPLA2a.

Cellular Inflammation in Models of Acute Gout

<p>Gout is a common form of inflammatory arthritis that is caused by the precipitation of monosodium urate crystals (MSU) in the joints. The acute form of gout is associated with sudden painful inflammatory episodes characterised by a large infiltration of neutrophils. The involvement of monocytes and macrophages is also recognised to be important, although the exact roles of monocytes and macrophages in gout need to be clarified. The overall objective of this thesis was to investigate the contributions of neutrophils, monocytes and macrophages to acute gouty inflammation. To determine whether the presence of other cells may be affecting neutrophil activation in gout inflammation, human blood neutrophils were stimulated with MSU as a purified population and in a mixed white blood cell population. The half-life of neutrophils in culture increased from 10h in purified neutrophil cultures to >24h in mixed cell cultures. The increase in viability was associated with large increases in cytokine production (TNF[alpha], IL-1[Beta], IL-6, IL-8) in mixed cell cultures. Exposure of neutrophils to media conditioned by MSU-stimulated mononuclear cells improved both neutrophil viability and stimulated the IL-8 production from neutrophils to a greater extent than direct contact with MSU. Exposure to conditioned media also primed MSU-stimulated neutrophil superoxide responses. High superoxide production was also observed when serum was lowered to <10%. These results indicate that neutrophil activation in gout largely occurs via soluble factors present in the pro-inflammatory environment, rather than by direct contact with MSU alone. Polygodial, a compound derived from the plant, Horopito (Pseudowintera colorata), and a number of structurally related sesquiterpene dialdehydes were tested for their potential to suppress neutrophil activation in gout. Polygodial inhibited MSU- and PMAstimulated neutrophil superoxide production in vitro with IC50 values of 0.78 [micron] and 0.16 [micron] respectively. This activity was largely dependent on the dialdehyde functionality but was enhanced by the presence of ring hydroxyl groups. The removal of hydroxyl groups was also associated with an increased cytoxicity. Polygodial and two other sesquiterpene dialdehyde compounds inhibited neutrophil superoxide production in an MSU model of acute gout, while Polygodial and a second compound also inhibited neutrophil recruitment. These studies confirm Polygodial and two structurally related sesquiterpene dialdehyde as potential anti-inflammatory compounds for potential use in gout. To confirm whether monocytes and/or macrophages were important in the onset of gout, resident macrophages and MSU-recruited monocytes were studied in a peritoneal model of MSU-induced inflammation. Gr-1+,7/4- monocytes were recruited to MSU-induced inflammation within 4h, however high cytokine production (IL-1[beta], TNF[alpha], IL-6, MCP-1) in the peritoneum preceded peak monocyte infiltration. Infiltrating monocytes did not produce high amounts of IL-6 and TNF[alpha], nor were they able to produce proinflammatory cytokines when re-stimulated with MSU ex vivo. However, resident macrophages exhibited production of IL-1[beta], TNF[alpha] and IL-6 following exposure to MSU ex vivo. Depletion of macrophages in vivo by clodronate liposomes led to a reduced recruitment of neutrophils and a lowered production of IL-1[beta] and IL-6 without affecting the recruitment of monocytes. These data identify macrophages, rather than monocytes, as key cells in initiationing and driving of inflammation in acute gout. The lack of responses from MSU-stimulated monocytes led to the question of what function, if any, was being exhibited by monocytes during MSU-induced inflammation. Therefore, the phenotype and corresponding function of monocytes was profiled over the course of MSU-induced inflammation. Newly recruited monocytes were small in size and expressed high levels of Gr-1 and 7/4, low levels of F4/80 and CD80. Over 48h the expression of Gr-1 and 7/4 was lost, while the expression of CD80 increased. These changes were associated with an increase in cell size, phagocytic capacity, and the production of pro-inflammatory cytokines in response to MSU. Changes in phenotype indicated differentiation into immature and then resident-like macrophages, and this was confirmed by PKH26-labelling infiltrating monocytes, which acquired an F4/80hi, resident macrophage phenotype after 3 to 5 days. Differentiation into macrophages was associated with an increased uptake of apoptotic neutrophils both in vivo and following ex vivo incubation. The phagocytosis of apoptotic neutrophils was associated with a reduced production of IL-1[beta] following stimulation with MSU ex vivo. These results indicate that MSU-recruited monocytes are not pro-inflammatory cells in gout, rather, they differentiate into resident macrophages over 3 to 5 days, and may over time aid in the resolution of gouty inflammation through the clearance of apoptotic neutrophils, a process that also suppresses further inflammatory responses. Together, the results of this thesis indicate that macrophages are key pro-inflammatory cells in gout contributing to neutrophil recruitment and cytokine production; that recruited neutrophils are activated by soluble mediators produced by MSU-activated cells, as well as by low serum environments; and that recruited monocytes are capable of both mediating the resolution of MSU-induced inflammation and replenishing the local resident macrophage population. These results provide a more clear model of the cellular events that occur during acute gout in humans that to date have been limited by a lack of in vivo-based studies.</p>

Cellular Inflammation in Models of Acute Gout

<p>Gout is a common form of inflammatory arthritis that is caused by the precipitation of monosodium urate crystals (MSU) in the joints. The acute form of gout is associated with sudden painful inflammatory episodes characterised by a large infiltration of neutrophils. The involvement of monocytes and macrophages is also recognised to be important, although the exact roles of monocytes and macrophages in gout need to be clarified. The overall objective of this thesis was to investigate the contributions of neutrophils, monocytes and macrophages to acute gouty inflammation. To determine whether the presence of other cells may be affecting neutrophil activation in gout inflammation, human blood neutrophils were stimulated with MSU as a purified population and in a mixed white blood cell population. The half-life of neutrophils in culture increased from 10h in purified neutrophil cultures to >24h in mixed cell cultures. The increase in viability was associated with large increases in cytokine production (TNF[alpha], IL-1[Beta], IL-6, IL-8) in mixed cell cultures. Exposure of neutrophils to media conditioned by MSU-stimulated mononuclear cells improved both neutrophil viability and stimulated the IL-8 production from neutrophils to a greater extent than direct contact with MSU. Exposure to conditioned media also primed MSU-stimulated neutrophil superoxide responses. High superoxide production was also observed when serum was lowered to <10%. These results indicate that neutrophil activation in gout largely occurs via soluble factors present in the pro-inflammatory environment, rather than by direct contact with MSU alone. Polygodial, a compound derived from the plant, Horopito (Pseudowintera colorata), and a number of structurally related sesquiterpene dialdehydes were tested for their potential to suppress neutrophil activation in gout. Polygodial inhibited MSU- and PMAstimulated neutrophil superoxide production in vitro with IC50 values of 0.78 [micron] and 0.16 [micron] respectively. This activity was largely dependent on the dialdehyde functionality but was enhanced by the presence of ring hydroxyl groups. The removal of hydroxyl groups was also associated with an increased cytoxicity. Polygodial and two other sesquiterpene dialdehyde compounds inhibited neutrophil superoxide production in an MSU model of acute gout, while Polygodial and a second compound also inhibited neutrophil recruitment. These studies confirm Polygodial and two structurally related sesquiterpene dialdehyde as potential anti-inflammatory compounds for potential use in gout. To confirm whether monocytes and/or macrophages were important in the onset of gout, resident macrophages and MSU-recruited monocytes were studied in a peritoneal model of MSU-induced inflammation. Gr-1+,7/4- monocytes were recruited to MSU-induced inflammation within 4h, however high cytokine production (IL-1[beta], TNF[alpha], IL-6, MCP-1) in the peritoneum preceded peak monocyte infiltration. Infiltrating monocytes did not produce high amounts of IL-6 and TNF[alpha], nor were they able to produce proinflammatory cytokines when re-stimulated with MSU ex vivo. However, resident macrophages exhibited production of IL-1[beta], TNF[alpha] and IL-6 following exposure to MSU ex vivo. Depletion of macrophages in vivo by clodronate liposomes led to a reduced recruitment of neutrophils and a lowered production of IL-1[beta] and IL-6 without affecting the recruitment of monocytes. These data identify macrophages, rather than monocytes, as key cells in initiationing and driving of inflammation in acute gout. The lack of responses from MSU-stimulated monocytes led to the question of what function, if any, was being exhibited by monocytes during MSU-induced inflammation. Therefore, the phenotype and corresponding function of monocytes was profiled over the course of MSU-induced inflammation. Newly recruited monocytes were small in size and expressed high levels of Gr-1 and 7/4, low levels of F4/80 and CD80. Over 48h the expression of Gr-1 and 7/4 was lost, while the expression of CD80 increased. These changes were associated with an increase in cell size, phagocytic capacity, and the production of pro-inflammatory cytokines in response to MSU. Changes in phenotype indicated differentiation into immature and then resident-like macrophages, and this was confirmed by PKH26-labelling infiltrating monocytes, which acquired an F4/80hi, resident macrophage phenotype after 3 to 5 days. Differentiation into macrophages was associated with an increased uptake of apoptotic neutrophils both in vivo and following ex vivo incubation. The phagocytosis of apoptotic neutrophils was associated with a reduced production of IL-1[beta] following stimulation with MSU ex vivo. These results indicate that MSU-recruited monocytes are not pro-inflammatory cells in gout, rather, they differentiate into resident macrophages over 3 to 5 days, and may over time aid in the resolution of gouty inflammation through the clearance of apoptotic neutrophils, a process that also suppresses further inflammatory responses. Together, the results of this thesis indicate that macrophages are key pro-inflammatory cells in gout contributing to neutrophil recruitment and cytokine production; that recruited neutrophils are activated by soluble mediators produced by MSU-activated cells, as well as by low serum environments; and that recruited monocytes are capable of both mediating the resolution of MSU-induced inflammation and replenishing the local resident macrophage population. These results provide a more clear model of the cellular events that occur during acute gout in humans that to date have been limited by a lack of in vivo-based studies.</p>

Extracellular release of mitochondrial DNA is triggered by cigarette smoke and is detected in COPD

Chronic obstructive pulmonary disease (COPD) is characterized by continuous and irreversible inflammation frequently caused by persistent exposure to toxic inhalants such as cigarette smoke (CS). CS may trigger mitochondrial DNA (mtDNA) extrusion into the cytosol, extracellular space, or foster its transfer by extracellular vesicles (EVs). The present study aimed to elucidate whether mtDNA is released upon CS exposure and in COPD. We measured cell-free mtDNA (cf-mtDNA) in the plasma of former smokers affected by COPD, in the serum of mice that developed CS-induced emphysema, and in the extracellular milieu of human bronchial epithelial cells exposed to cigarette smoke extract (CSE). Further, we characterized cells exposed to sublethal and lethal doses of CSE by measuring mitochondrial membrane potential and dynamics, superoxide production and oxidative stress, cell cycle progression, and cytokine expression. Patients with COPD and mice that developed emphysema showed increased levels of cf-mtDNA. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased superoxide production and oxidative damage, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles. The release of mtDNA into the extracellular milieu occurred concomitantly with increased expression of DNase III, DNA-sensing receptors (cGAS, NLRP3), proinflammatory cytokines (IL-1B, IL-6, IL-8, IL-18, CXCL2), and markers of senescence (p16, p21). Exposure to a lethal dose of CSE preferentially induced mtDNA and nuclear DNA release in cell debris. Our findings demonstrate that CS-induced stress triggers mtDNA release and is associated with COPD, supporting cf-mtDNA as a novel signaling response to CS exposure.

Activation of Coronary Arteriolar PKCβ2 Impairs Endothelial NO-Mediated Vasodilation: Role of JNK/Rho Kinase Signaling and Xanthine Oxidase Activation

Protein kinase C (PKC) activation can evoke vasoconstriction and contribute to coronary disease. However, it is unclear whether PKC activation, without activating the contractile machinery, can lead to coronary arteriolar dysfunction. The vasoconstriction induced by the PKC activator phorbol 12,13-dibutyrate (PDBu) was examined in isolated porcine coronary arterioles. The PDBu-evoked vasoconstriction was sensitive to a broad-spectrum PKC inhibitor but not affected by inhibiting PKCβ2 or Rho kinase. After exposure of the vessels to a sub-vasomotor concentration of PDBu (1 nmol/L, 60 min), the endothelium-dependent nitric oxide (NO)-mediated dilations in response to serotonin and adenosine were compromised but the dilation induced by the NO donor sodium nitroprusside was unaltered. PDBu elevated superoxide production, which was blocked by the superoxide scavenger Tempol. The impaired NO-mediated vasodilations were reversed by Tempol or inhibition of PKCβ2, xanthine oxidase, c-Jun N-terminal kinase (JNK) and Rho kinase but were not affected by a hydrogen peroxide scavenger or inhibitors of NAD(P)H oxidase and p38 kinase. The PKCβ2 protein was detected in the arteriolar wall and co-localized with endothelial NO synthase. In conclusion, activation of PKCβ2 appears to compromise NO-mediated vasodilation via Rho kinase-mediated JNK signaling and superoxide production from xanthine oxidase, independent of the activation of the smooth muscle contractile machinery.

Abstract MP08: New Function Of Sox6 In Renovascular Hypertension Induced Oxidative Stress And Kidney Damage Through Renin, Prorenin And The (pro)renin Receptor Expression Control.

Introduction: During renovascular hypertension induced by renal artery stenosis (RAStenosis), smooth muscle cells are recruited to produce and release renin compensating for the decrease in renal perfusion. In renovascular hypertension, renin expression increases causing a surge in angiotensin II, oxidative stress and kidney damage. Here we present a new function of the transcription factor Sox6 in kidney injury and oxidative damage during RAStenosis. Methods: We used a mouse model in which Sox6 is knockout specifically in renin expressing cells (Ren1d Cre /Sox6 fl/fl -Sox6-KO). We developed a modified 2-kidney 1-clip (2K1C) Goldblatt model to induce RAStenosis. We measured superoxide production (pmol/mg pr) in kidney using high-performance liquid chromatography in Sox6-KO and Ren1d Cre /Sox6 wt/wt (Sox6-WT) littermates. Renin, prorenin, (pro)renin receptor (PRR) and N-GAL expressions were measured using Western blot using β-actin as housekeeping gene. Western blot band density analysis was performed using ImageJ. Kidney injury was determined by measuring creatinine clearance. Results: We found that Sox6-KO mice exhibit lower expression of renin (0.36 vs. 0.45, SEM= 0.11 n= 11-15, p<0.01), and prorenin (1.25 vs. 2.07, SEM=0.30, n=11-15, p<0.05) and mice are protected against renovascular hypertension (SBP, 114.5 vs. 131.1, SEM=5.31 n=13-15, p<0.01), and kidney injury measuring N-GAL expression (1.3 vs. 2.7, SEM=0.38, n=6-8, p<0.01) and creatinine clearance (2372 vs. 1341, SEM=342.3, n=4-5, p<0.05). Furthermore, we found that the levels of superoxide were significantly lower in Sox6-KO mice compared to Sox6-WT littermates (218 vs 47, SEM=50.6, N=7, P<0.01). The expression of PRR was significantly lower in Sox6-KO compared to Sox6-WT during RAStenosis (1.23 vs. 0.72, SEM=0.33, n=2-5, p<0.01). Conclusions: Our results show that Sox6 depletion in renin expressing cells inhibits the increase in renovascular hypertension and prevents the increase in renin, prorenin, PRR, and N-GAL expression as well as superoxide production, preserving creatinine clearance during RAStenosis. These results suggest that Sox6 has a new function in hypertension and kidney injury induced by RAS.

Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.