Lipidomic changes in a novel sepsis outcome-based analysis reveals potent pro-inflammatory and pro-resolving signaling lipids.

The purpose of this study was to investigate changes in the lipidome of patients with sepsis to identify signaling lipids associated with poor outcomes that could be linked to future therapies. Adult patients with sepsis were enrolled within 24h of sepsis recognition. Patients meeting Sepsis-3 criteria were enrolled from the emergency department or intensive care unit and blood samples were obtained. Clinical data were collected and outcomes of rapid recovery, chronic critical illness (CCI), or early death were adjudicated by clinicians. Lipidomic analysis was performed on two platforms, the Sciex™ 5500 device to perform a lipidomic screen of 1450 lipid species and a targeted signaling lipid panel using liquid-chromatography tandem mass spectrometry. For the lipidomic screen, there were 274 patients with sepsis: 192 with rapid recovery, 47 with CCI, and 35 with early deaths. CCI and early death patients were grouped together for analysis. Fatty acid (FA) 12:0 was decreased in CCI/early death, whereas FA 17:0 and 20:1 were elevated in CCI/early death, compared to rapid recovery patients. For the signaling lipid panel analysis, there were 262 patients with sepsis: 189 with rapid recovery, 45 with CCI, and 28 with early death. Pro-inflammatory signaling lipids from ω-6 poly-unsaturated fatty acids (PUFAs), including 15-hydroxyeicosatetraenoic (HETE), 12-HETE, and 11-HETE (oxidation products of arachidonic acid [AA]) were elevated in CCI/early death patients compared to rapid recovery. The pro-resolving lipid mediator from ω-3 PUFAs, 14(S)-hydroxy docosahexaenoic acid (14S-HDHA), was also elevated in CCI/early death compared to rapid recovery. Signaling lipids of the AA pathway were elevated in poor-outcome patients with sepsis and may serve as targets for future therapies

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Paraoxonase 2 (PON2) in Cardiovascular Disease (CVD): The Role of PON2 in Acute Myocardial Ischemia-Reperfusion Injury and Diet-Induced Obesity

Paraoxonase 2 (PON2) is a ubiquitously expressed antioxidant, anti-inflammatory protein localized in the inner mitochondrial membrane, where it protects against mitochondrial dysfunction and oxidative stress, among other protective properties. Human PON2 polymorphisms, particularly human PON2 Ser311Cys, has been implicated in the development of coronary artery disease, including myocardial infarction and ischemic stroke. Therefore, in this thesis, I explored the cardioprotective capacity of PON2 against myocardial infarction, specifically acute myocardial ischemia-reperfusion injury (IRI). My hypothesis is that PON2 protects against myocardial IRI in cardiomyocytes by modulating mitochondrial dysfunction and oxidative stress, particularly mitigating mitochondrial lipotoxicity (lipid peroxidation). I employed in vitro (rat ventricular cardiomyocyte cell line, H9c2 cells), ex vivo (cardiomyocytes derived from PON2-deficient mice hearts), and in vivo (PON2-deficient mice) models, subjecting them to cardiac IRI, to test my hypothesis. I determined that PON2 protects against myocardial IRI in cardiomyocytes by reducing mitochondrial dysfunction (stabilizing mitochondrial membrane potential and improving calcium retention capacity) and oxidative stress (reducing mitochondrial reactive oxygen species) via the reperfusion injury salvage kinase pathway (increasing phosphorylated glycogen synthase kinase-3β). Furthermore, there is an increase in mitochondrial lipid peroxidation in PON2-deficient mice subjected to myocardial IRI, with an increase in oxidized phosphatidylcholine species and eicosanoids (12, 15- hydroxyeicosatetraeonic acid), and a decrease in prostanoid production. These injuries are rescued by PON2 overexpression. Also, I observe an increase in cytoplasmic phospholipase A2 in the hearts of PON2-def mice subjected to myocardial IRI, serving as a potential mechanism by which PON2 modulates the increase in unesterified eicosanoid production. In order to further examine whether PON2 deficiency, exclusively in cardiomyocytes, is sufficient to induce increased myocardial IRI, I generated cardiomyocyte-specific PON2-KO mice. Lastly, I investigated the protective role of a novel drug, HSG4112, against cardiovascular risk factor, diet-induced obesity. I found that HSG4112 protects against diet-induced obesity by enhancing lactonase/arylesterase activity, reducing body weight and fat mass, and increasing lean (muscle) mass. Nevertheless, the protective effects of HSG4112 are significantly reduced in PON2-def mice, suggesting that PON2 is at least partly involved as a mode of action for HSG4112’s protective capacity

Vutiglabridin Modulates Paraoxonase 1 and Ameliorates Diet-Induced Obesity in Hyperlipidemic Mice

Vutiglabridin is a clinical-stage synthetic small molecule that is being developed for the treatment of obesity and its target proteins have not been fully identified. Paraoxonase-1 (PON1) is an HDL-associated plasma enzyme that hydrolyzes diverse substrates including oxidized low-density lipoprotein (LDL). Furthermore, PON1 harbors anti-inflammatory and antioxidant capacities and has been implicated as a potential therapeutic target for treating various metabolic diseases. In this study, we performed a non-biased target deconvolution of vutiglabridin using Nematic Protein Organisation Technique (NPOT) and identified PON1 as an interacting protein. We examined this interaction in detail and demonstrate that vutiglabridin binds to PON1 with high affinity and protects PON1 against oxidative damage. Vutiglabridin treatment significantly increased plasma PON1 levels and enzyme activity but not PON1 mRNA in wild-type C57BL/6J mice, suggesting that vutiglabridin modulates PON1 post-transcriptionally. We further investigated the effects of vutiglabridin in obese and hyperlipidemic LDLR−/− mice and found that it significantly increases plasma PON1 levels, while decreasing body weight, total fat mass, and plasma cholesterol levels. Overall, our results demonstrate that PON1 is a direct, interacting target of vutiglabridin, and that the modulation of PON1 by vutiglabridin may provide benefits for the treatment of hyperlipidemia and obesity

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels, systemic inflammation, and atherosclerosis

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr-/- mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr-/-/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr-/- mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis

Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion injury

Macrophages polarize into heterogeneous proinflammatory M1 and antiinflammatory M2 subtypes. Heme oxygenase 1 (HO-1) protects against inflammatory processes such as ischemia-reperfusion injury (IRI), organ transplantation, and atherosclerosis. To test our hypothesis that HO-1 regulates macrophage polarization and protects against IRI, we generated myeloid-specific HO-1-knockout (mHO-1-KO) and -transgenic (mHO-1-Tg) mice, with deletion or overexpression of HO-1, in various macrophage populations. Bone marrow-derived macrophages (BMDMs) from mHO-1-KO mice, treated with M1-inducing LPS or M2-inducing IL-4, exhibited increased mRNA expression of M1 (CXCL10, IL-1β, MCP1) and decreased expression of M2 (Arg1 and CD163) markers as compared with controls, while BMDMs from mHO-1-Tg mice displayed the opposite. A similar pattern was observed in the hepatic M1/M2 expression profile in a mouse model of liver IRI. mHO-1-KO mice displayed increased hepatocellular damage, serum AST/ALT levels, Suzuki's histological score of liver IRI, and neutrophil and macrophage infiltration, while mHO-1-Tg mice exhibited the opposite. In human liver transplant biopsies, subjects with higher HO-1 levels showed lower expression of M1 markers together with decreased hepatocellular damage and improved outcomes. In conclusion, myeloid HO-1 expression modulates macrophage polarization, and protects against liver IRI, at least in part by favoring an M2 phenotype

Abstract 17401: Diesel Exhaust Induces Mitochondrial Dysfunction, Hyperlipidemia and Liver Steatosis

Introduction: Air pollution associates with increased cardiovascular morbidity and mortality, partly due to induction of dyslipidemia and metabolic syndrome. Our goal was to dissect mechanisms involved. Hypothesis: Diesel exhaust exposure induces hyperlipidemia and liver steatosis by dysregulating lipid metabolism, and altering gut microbiota composition. Methods: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE and 13-HODE, lipid and carbohydrate metabolism, and gut microbiota composition. Findings were corroborated and mechanisms further assessed in culture of HepG2 hepatocytes. Results: ApoE KO mice exposed to inhaled diesel exhaust (DE, 6 hours/day, 5 days/week for 16 weeks) exhibited significantly (p< 0.05) elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and increased hepatic levels of 9-HODE and 13-HODE (oxidative products of linoleic acid) indicative of increased oxidative stress, as compared with control mice treated with filtered air (FA). DE also led to downregulation of Acad9 , which suggested decreased β-oxidation of fatty acids and decreased lipid catabolism. A direct effect of DEP exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo , this led to decreased ACAD9 expression, increased triglyceride content, and altered total, mitochondrial and ATP-linked respiration assessed by a Seahorse metabolic flux analyzer, indicative of mitochondrial dysfunction. Direct treatment of mitochondrias with DE particles affected mitochondrial complexes I and IV. Conclusions: Diesel exhaust exposure leads to dyslipidemia and liver steatosis in ApoE KO mice, likely due to mitochondrial dysfunction and decreased lipid catabolism

Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux[S]

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol

Treating the Intestine with Oral ApoA-I Mimetic Tg6F Reduces Tumor Burden in Mouse Models of Metastatic Lung Cancer.

Having demonstrated that apolipoprotein A-I (apoA-I) mimetic peptides ameliorate cancer in mouse models, we sought to determine the mechanism for the anti-tumorigenic function of these peptides. CT-26 cells (colon cancer cells that implant and grow into tumors in the lungs) were injected into wild-type BALB/c mice. The day after injection, mice were either continued on chow or switched to chow containing 0.06% of a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F). After four weeks, the number of lung tumors was significantly lower in Tg6F-fed mice. Gene expression array analyses of jejunum and lung identified Notch pathway genes significantly upregulated, whereas osteopontin (Spp1) was significantly downregulated by Tg6F in both jejunum and lung. In jejunum, Tg6F increased protein levels for Notch1, Notch2, Dll1, and Dll4. In lung, Tg6F increased protein levels for Notch1 and Dll4 and decreased Spp1. Tg6F reduced oxidized phospholipid levels (E06 immunoreactivity) and reduced 25-hydroxycholesterol (25-OHC) levels, which are known to inhibit Notch1 and induce Spp1, respectively. Notch pathway promotes anti-tumorigenic patrolling monocytes, while Spp1 facilitates pro-tumorigenic myeloid derived suppressor cells (MDSCs) formation. Tg6F-fed mice had higher numbers of patrolling monocytes in jejunum and in lung (p < 0.02), and lower plasma levels of Spp1 with reduced numbers of MDSCs in jejunum and in lung (p < 0.03). We conclude that Tg6F alters levels of specific oxidized lipids and 25-OHC to modulate Notch pathways and Spp1, which alter small intestine immune cells, leading to similar changes in lung that reduce tumor burden