oxalylglycine and Inflammation

Cells in the human body need oxygen to function and survive, and severe deprivation of oxygen, as occurs in ischaemic heart disease and stroke, is a major cause of mortality. Nevertheless, other organisms, such as the fossorial mole rat or diving seals, have acquired the ability to survive in conditions of limited oxygen supply. Hypoxia tolerance also allows the heart to survive chronic oxygen shortage, and ischaemic preconditioning protects tissues against lethal hypoxia. The recent discovery of a new family of oxygen sensors--including prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3)--has yielded exciting novel insights into how cells sense oxygen and keep oxygen supply and consumption in balance. Advances in understanding of the role of these oxygen sensors in hypoxia tolerance, ischaemic preconditioning and inflammation are creating new opportunities for pharmacological interventions for ischaemic and inflammatory diseases.

Topics: Amino Acids, Dicarboxylic; Angiogenesis Inducing Agents; Animals; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Inflammation; Ischemia; Ischemic Preconditioning; Myocardial Contraction; Oxygen Consumption; Procollagen-Proline Dioxygenase

Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2 haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immunomodulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favorable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.

Topics: Abdomen; Amino Acids, Dicarboxylic; Anastomosis, Surgical; Anastomotic Leak; Animals; Caco-2 Cells; Collagen; Colon; Female; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Inflammation; Ischemia; Macrophages; Male; Mice; Prolyl Hydroxylases; RNA, Messenger; Sepsis; Wound Healing

When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway.. Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development.. Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway.. These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.

Topics: Amino Acids, Dicarboxylic; Animals; Cell Movement; Cytokines; Dermatitis, Allergic Contact; Eosinophils; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Mice; Mixed Function Oxygenases; Neutrophils

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1β-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1β. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.

Topics: Amino Acids, Dicarboxylic; Cell Death; Cells, Cultured; HEK293 Cells; Humans; Inflammation; Mixed Function Oxygenases; Monocytes; Prolyl-Hydroxylase Inhibitors

Gentamicin nephrotoxicity is one of the most common causes of acute kidney injury (AKI). Hypoxia-inducible factor (HIF) is effective in protecting the kidney from ischemic and toxic injury. Increased expression of HIF-1α mRNA has been reported in rats with gentamicin-induced renal injury. We hypothesizd that we could study the role of HIF in gentamicin-induced AKI by modulating HIF activity. In this study, we investigated whether HIF activation had protective effects on gentamicin-induced renal tubule cell injury. Gentamicin-induced AKI was established in male Sprague-Dawley rats. Cobalt was continuously infused into the rats to activate HIF. HK-2 cells were pre-treated with cobalt or dimethyloxalylglycine (DMOG) to activate HIF and were then exposed to gentamicin. Cobalt or DMOG significantly increased HIF-1α expression in rat kidneys and HK-2 cells. In HK-2 cells, HIF inhibited gentamicin-induced reactive oxygen species (ROS) formation. HIF also protected these cells from apoptosis by reducing caspase-3 activity and the amount of cleaved caspase-3, and -9 proteins. Increased expression of HIF-1α reduced the number of gentamicin-induced apoptotic cells in rat kidneys and HK-2 cells. HIF activation improved the creatinine clearance and proteinuria in gentamicin-induced AKI. HIF activation also ameliorated the extent of histologic injury and reduced macrophage infiltration into the tubulointerstitium. In gentamicin-induced AKI, the activation of HIF by cobalt or DMOG attenuated renal dysfunction, proteinuria, and structural damage through a reduction of oxidative stress, inflammation, and apoptosis in renal tubular epithelial cells.

Topics: Acute Kidney Injury; Amino Acids, Dicarboxylic; Animals; Apoptosis; Caspase 3; Caspase 9; Cells, Cultured; Cobalt; Gentamicins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Kidney Tubules; Male; Oxidative Stress; Random Allocation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

This study investigates whether deoxy-2-[18F]fluoro-d-glucose (FDG) micro-positron emission tomography (μPET)/computed tomography (CT) can serve as a tool for monitoring of the commonly used dextran sodium sulfate (DSS)-induced murine model of inflammatory bowel disease (IBD).. DSS-colitis was induced in Sv129 mice. In a first experiment, four animals were serially scanned with CT and FDG-μPET on days 0, 3, 7, 11, and 14. The ratio of the mean voxel count of the PET images in the colon and the brain was compared with the histological inflammation score and the colonic myeloperoxidase levels. A second experiment was performed to investigate whether FDG-μPET was able to detect differences in inflammation between two DSS-treated groups, one receiving placebo (n = 4) and one receiving dimethyloxalylglycine (DMOG) (n = 4), a compound that protects against DSS-induced colitis.. The progression of the colonic/brain FDG-signal ratio (over days 0-14) agreed with the predicted histological inflammation score, obtained from a parallel DSS-experiment. Moreover, the quantification of normalized colonic FDG-activity at the final timepoint (day 14) showed an excellent correlation with both the MPO levels (Spearman's rho = 1) and the histological inflammation score (Spearman's rho = 0.949) of the scanned mice. The protective action of DMOG in DSS colitis was clearly demonstrated with FDG-μPET/CT (normalized colonic FDG-activity DMOG versus placebo: P < 0.05).. FDG-μPET-CT is a feasible and reliable noninvasive method to monitor murine DSS-induced colitis. The implementation of this technique in this widely used IBD model opens a new window for pathophysiological research and high-throughput screening of potential therapeutic compounds in preclinical IBD research.

Topics: Amino Acids, Dicarboxylic; Animals; Colitis; Dextran Sulfate; Disease Models, Animal; Female; Image Processing, Computer-Assisted; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Positron-Emission Tomography; Tomography, X-Ray Computed

Ischemia/Reperfusion injury and hemolysis are characterized by erythrocyte lysis and release of free heme into the microcirculation. Following substantial erythrocyte lysis, heme overwhelms circulatory heme-binding protein networks rapidly forming hemin, the oxidized form of iron protoporphyrin IX. Hemin's role in modulating inflammatory responses in microvascular endothelium (MVEC) remains ill-defined. We studied the impact of hemin exposure on human MVEC interleukin-8 (IL-8) expression. Hemin significantly up-regulated MVEC IL-8 secretion and was associated with cellular iron loading. Hemin-induced IL-8 up-regulation was significantly attenuated by increasing environmental serum concentrations. As well, hemin-induced IL-8 secretion was significantly reduced in a concentration-dependent fashion following pyrrolidine dithiocarbamate exposure, suggesting that induction occurred via an oxidant-sensitive mechanism. Interestingly, transfection studies revealed that oxidant-driven transcription factors NF-kappaB and AP-1 played no role in hemin-induced IL-8 transcription. In studies employing actinomycin D, hemin was found to dramatically lengthen IL-8 mRNA half-life. Of major importance in the current report was the finding that hypoxia inducible factor-1 (HIF-1), a powerful transcription factor mediating tissue responses to hypoxia, potently regulated hemin-induced IL-8 secretion in human MVEC. Activation of HIF-1 via the prolyl hydroxylase inhibitor dimethyloxalylglycine attenuated hemin-induced IL-8 secretion. These studies were confirmed via DNA-directed siRNA silencing of HIF-1alpha. In conclusion, hemin induces a serum protein-sensitive pro-inflammatory phenotype in MVEC via an oxidant-sensitive mechanism that is powerfully regulated by HIF-1.

Topics: Amino Acids, Dicarboxylic; Antioxidants; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Inhibitors; Hemin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-8; Iron; Microcirculation; Procollagen-Proline Dioxygenase; Pyrrolidines; RNA Stability; RNA, Messenger; RNA, Small Interfering; Serum; Signal Transduction; Thiocarbamates; Time Factors; Transcription, Genetic