Ischaemia/Reperfusion Injury (IRI) is the most common cause of acute kidney injury- a devastating clinical problem lacking any specific treatments to promote renal recovery. Macrophages (Mφ) are pleiotropic cells of the innate immune system, with roles spanning host defence, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Mφ are also known to be important mediators of renal injury in other experimental models of renal disease including transplantation, obstruction and glomerulonephritis. This work sought to examine the role of Mφ in mediating renal IRI. Conditional renal Mφ and monocyte depletion prior to experimental IRI was achieved by administering diphtheria toxin to the CD11b-DTR transgenic animal. This had no impact on either renal function or structural injury. In contrast liposomal clodronate mediated Mφ depletion provided functional and structural protection from injury. Administration of exogenous apoptotic cells also protected renal function if delivered 24h prior to IRI. Immunodeficient SCID mice exhibited a protected injury phenotype after IRI, however derived no additional protection from the administration of either liposomal clodronate or i.v. apoptotic cells. These findings suggest that the protective phenotype must involve either lymphocyte populations or circulating antibody. Preliminary work demonstrates that SCID mice lack IgM natural antibody which deposits in the kidney in the first 30 minutes after IRI. It was also demonstrated that apoptotic cells bind IgM natural antibody present within the circulation. The potential for the key antioxidant enzyme Heme oxygenase-1 (HO-1) to protect renal function was also examined in aged mice using hemearginate (HA) - a potent HO-1 inducer. Echoing epidemiological studies in humans aged mice had increased susceptibility to IRI, whilst failing to induce medullary HO-1. The main site of medullary HO-1 induction by HA was in medullary Mφ, and the protective phenotype was abolished by Mφ ablation, implicating Mφ as the key mediators of HA induced protection in renal IRI. Final studies employed adenoviral transduction to overexpress HO-1 within bone marrow derived Mφ, leading to a modified phenotype with increased IL- 10 and phagocytosis, and reduced TNFα and NO production. When these were introduced in vivo after IRI renal function was improved, potentially due to accelerated clearance of renal platelet deposition.

The endothelium plays a pivotal role in the maintenance of vascular homeostasis and its dysregulation promotes vascular complications. This thesis proposes that heme oxygenase-1 (HO-1), an anti-inflammatory enzyme with antioxidant properties, is endothelial protective factor that prevents endothelial injury induced by cisplatin or activated neutrophils. Specifically, this thesis aimed to test (i) that overexpression of HO-1 prevents cisplatin-induced endothelial injury and suppresses caspase activity; (ii) whether neutrophil-endothelial cell activation resulted in the release of soluble Flt-1 (sFlt-1) and soluble endoglin (sEng), the two anti-angiogenic factors known to induce the clinical signs of preeclampsia; (iii) whether HO-1 prevented activated neutrophils from stimulating the release of these factors from the endothelium; (iv) the relative contribution and the co-dependency of neutrophil activation and anti-angiogenic growth factors in preeclampsia where systemic endothelial dysfunction is known to occur. This thesis shows that cisplatin inhibited human umbilical vein endothelial cells (HUVEC) metabolism as measured by MTT assay and resulted in the release of placenta growth factor (PlGF). Immunoblotting confirmed that cisplatin increased cleaved caspase-3 expression in HUVEC. These effects of cisplatin were attenuated in HUVEC infected with adenovirus encoding HO-1 and the effects were exacerbated when HO-1 was silenced by siRNA. Furthermore, cisplatin stimulated PlGF release was suppressed by the overexpression of HO-1. In addition, HO-1 overexpression inhibited angiogenesis as determined by vascular endothelial growth factor-induced capillary tube formation on Matrigel coated plates. Thus these studies indicate that agents which upregulate HO-1 could increase the effectiveness and tolerability to cisplatin in cancer treatment. Although neutrophils are early contributors to endothelial cell activation, no studies have determined their contribution to the release of sFlt-1 and sEng. We therefore investigated the effect of activated neutrophils on the release of sFlt-1 and sEng in endothelial/neutrophil co-cultures and in the circulation of women with normal pregnancy and preeclampsia. LPS-mediated neutrophil activation stimulated the release of sEng but not sFlt-1 from endothelial cells in culture. In the absence of neutrophils, overexpression of HO-1 in HUVEC downregulated the release of sEng. In contrast, HO-1 overexpression failed to inhibit the release of sEng in the presence of activated neutrophils. The release of sEng by activated neutrophils-endothelial cell cocultures appears to be mediated by metalloproteinases (MMP) as the broad-spectrum MMP inhibitor (GM6001) attenuated sEng release. Clinical studies demonstrated that sEng, pro-inflammatory interleukin-6 (IL-6) and the soluble markers of neutrophil activation (α-defensins and calprotectin) were all elevated in women with preeclampsia. We identified a direct correlation between neutrophil activation and IL-6 release. However, no correlation could be established between these factors and sEng release in preeclampsia. Hence, these results provide compelling clinical evidence to show that the increase in neutrophil activation and IL-6 release during preeclampsia are unlikely to significantly contribute to the clinical signs of preeclampsia as they fail to correlate directly with the anti-angiogenic factors, which form the final common pathway to the clinical signs of preeclampsia and systemic endothelial dysfunction.

Heme oxygenase (HO) is an enzyme that facilitates the oxidative breakdown of free heme into equi-molar concentrations of carbon monoxide (CO), the bile pigment biliverdin IX and free iron. These products have immuno-modulatory and antioxidative properties, which may be useful in the treatment of diseases characterised by low-grade inflammation and oxidative stress, such as insulin resistance and hyperglycaemia in type 2 diabetes. In fact, HO-1 protein levels and carbon monoxide generation are down-regulated in murine models of obesity and type 2 diabetes. Two independent research teams have reported that pharmacological induction of HO activity by protoporphyrin-based compounds, such as hemin and cobalt (III) protoporphyrin IX chloride (CoPP), exerts anti-diabetic effects, including protection from weight gain, systemic inflammation and peripheral insulin resistance, in various experimental models of type 2 diabetes. However, the relative insolubility and instability of hemin in solution and the multiple side-effects of CoPP, including weight loss, preclude their use for the treatment of patients in clinic. Heme arginate (HA) is a stable and soluble composition of hemin and L-arginine (LA) in a solution containing propylene glycol, ethanol and water. Furthermore, HA is licensed for the treatment of acute porphyria in several European countries. Therefore, HA may potentially be used in clinical trials. The current PhD thesis tests the hypothesis that the heme component of HA ameliorates hyperglycaemia via induction of HO activity in the leptin receptor deficient db/db (db/db) mouse model of type 2 diabetes. A preliminary in vivo study demonstrates that the heme but not the LA component of HA exerts an anti-hyperglycaemic effect in db/db mice. In a separate in vivo study, concomitant treatment of HA with stannous (IV) mesoporphyrin IX dichloride (SM), an inhibitor of HO activity, further improves the glycaemic control despite complete abrogation of the HA-mediated increase in HO activity in db/db mice. This result is in contrast to the above stated hypothesis, and demonstrates that the antihyperglycaemic effect of HA is due to a HO activity independent mechanism. Furthermore, the ameliorative effect of HA and HA+SM treatment on hyperglycaemia in db/db mice coincides with a gain in body and visceral fat weight, a reduction in islet β-cell inflammation and the preservation of islet β-cell function. Subsequent in vitro experiments demonstrate that HA exerts anti-inflammatory effects by a HO activity independent mechanism in pro-inflammatory in vitro models such as in cytokine mix-stimulated MIN6 β-cells and in classically activated bone marrow derived macrophages (BMDMs). In conclusion, the current thesis demonstrates the novel finding that the heme component of HA can exert anti-inflammatory and anti-diabetic effects via a HO activity independent mechanism. Future work should focus on studies to test the hypothesis that the interaction of heme with the nuclear receptor Rev-erb-α is responsible for the anti-inflammatory and anti-diabetic effects of HA.

We have recently demonstrated that heme oxygenase is expressed in both healing wounds and in pressure ulcers. Heme oxygenase has been shown to have important cytoprotective functions in myocardial ischemia-reperfusion injury and organ allograft survival. The cytoprotective effects of heme oxygenase are multifactorial. Besides reducing levels of pro-oxidant heme, heme oxygenase products (bilirubin, carbon monoxide, and iron) have been demonstrated to possess anti-oxidant, anti-inflammatory, anti-apoptotic, and anti-proliferative properties. These properties make heme oxygenase an attractive therapeutic target for the prevention and treatment of chronic wounds. The purpose of this study was two-fold: evaluate the effects of carbon monoxide (CO) on the expression of heme oxygenase (HO-1) in dermal fibroblasts, and determine and begin to investigate the mechanisms responsible for CO-induction of HO-1. The ability of a second-generation carbon monoxide donating molecule-tricarbonyldichlororuthenium (II) dimer (CORM-2) to induce HO-1 protein expression in dermal fibroblasts was examined. Western blotting techniques were utilized to determine HO-1 expression. CORM-2 (100-300uM) induced maximum expression of HO-1. The maximum response to CORM-2 occurred between 12 and 20 hours. Inhibition of MAPK, PI3-K, JNK pathways showed no changes in HO-1 expression. Likewise inhibition of cGMP, a known pathway for CO, had no effect on protein expression suggesting that HO-1 expression by CORM-2 works by an alternate pathway. In conclusion the ability of CO, a product of heme degradation, to induce HO-1 in dermal fibroblasts may serve as a mechanism to amplify HO-1 expression in stressed tissues and may serve as the basis for a novel therapeutic approach for treating chronic wounds.

Iron plays a potential role in oxidative stress-mediated injuries and pathologies e.g. rheumatoid arthritis (RA). Four decades ago it was suggested that iron may have a crucial role in the progression of inflammation in RA. Indeed, free radicals generated by iron can cause damage to lipids, proteins, carbohydrates, and DNA. It is this destructive process that is believed to occur in rheumatoid joints. However, none had differentiated between the role of iron in both acute and chronic phases of the disease and the origin of this 'labile' iron. Since RA cells are chronically exposed to oxidative stress, we have therefore chosen Jurkat cells to be our cell model. We used the parental (J16) cell line was used to mimic the acute phase of oxidative stress and the H2O2-resistant (HJ16) cells to mimic the chronic phase. By using hydrogen peroxide (H2O2) as the oxidising agent, we aim to study the role of iron in acute and chronic phase of oxidative stress and to know its origin. In the present study, we found that both antioxidants and H2O2-induced labile iron are modulated when cells are chronically exposed to H2O2. HJ16 cells contain higher total intracellular glutathione levels and glutathione peroxidase activity than J16 cells while the superoxide dismutase and catalase activity are similar. Haem oxygenase-1 (HO-1) was not detectable nor was it induced in these cell lines; HO-2 on the other hand was expressed but not induced. Although they had the same ‘basal’ LIP and L-Ft levels, J16 cells contain more than 7-fold higher H-Ft levels than in HJ16 cells. It was also found that H2O2-induced labile iron is directly correlated with necrotic cell death. These results are consistent with the conclusion that both antioxidant defence mechanism and labile iron status are modulated in cells chronically exposed to H2O2. We have also shown that the ‘basal’ and ‘H2O2-induced’ NFκB activation was higher in the HJ16 cells. We have also provided a link between labile iron release, lysosomal membrane damage and the ensuing necrotic cell death following H2O2 treatment.

Orientador: Valéria Cristina Sandrim / Resumo: A pré-eclâmpsia (PE) é a principal causa de mortalidade e morbidade entre as gestantes no Brasil e em vários países. A fisiopatologia desta doença é complexa e envolve vários processos. Um destes, amplamente validado na literatura, relaciona-se a um status oxidativo, onde há prevalência de produção de radicais livres e/ou redução da atividade antioxidante. Apesar destas evidências, a suplementação clínica com antioxidantes (vitamina C e E) não se demonstrou promissora em PE. Recentemente, vem sendo explorado como terapia em várias doenças, a ativação de um fator de transcrição, o NRF2 (do inglês - nuclear factor, erythroid 2-like 2), que atua induzindo a transcrição de diversos genes que promovem a proteção celular através da codificação de proteínas com atividade desintoxicante e antioxidante. Entre elas a heme oxigenase (HO-1) é a mais estudada, pois apresenta efeitos antiapoptóticos, antioxidantes e citoprotetor. Além disso, o aumento do estresse oxidativo na PE pode potencialmente reduzir a biodisponibilidade de óxido nítrico (NO) que pode ser modulado por alguns polimorfismos localizados no gene da óxido nítrico sintase (NOS3). Notavelmente, os haplótipos formados pela combinação de polimorfismos foram associados a diferentes subgrupos de resposta à terapia anti-hipertensiva em PE. No presente estudo, comparamos as distribuições dos polimorfismos localizados nos genes NFE2L2 rs35652124 (C/T) e no gene HMOX1 rs2071746 (A/T) em gestantes com PE que respondem à metildopa ou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Preeclampsia (PE) is the leading cause of mortality and morbidity among pregnant women in Brazil and several countries. Its pathophysiology is complex and involves several processes, including the oxidative stress (increase of free radicals and/or decrease of antioxidant defense). Although evidences, clinical supplementation with vitamins (C and E) was not promising in preeclampsia. Currently, therapeutically the activation of transcription factor, NRF2 (nuclear factor, erythroid 2-like 2), has been explored in several diseases. This factor promote cytoprotection by activates the transcription of several antioxidant and detoxification proteins. Hemeoxigenase-1 (HO-1) is the most studied, because has antiapoptotic, anti-inflammatory and cytoprotection activities. In addition, the increased oxidative stress in PE can potentially reduce the bioavailability of nitric oxide (NO) which may impaired by some SNPs on endothelial nitric oxide synthase (NOS3) gene. Notably, haplotypes formed by the combination of polymorphisms of were associated with different subgroups of response to antihypertensive therapy in PE. Therefore, in the present study we compared the distributions of rs35652124 (C/T) NFE2L2 and rs2071746 (A/T) HMOX1 polymorphisms in PE patients who respond to methyldopa or antihypertensive therapy with those found in PE patients who do not respond to methyldopa or total antihypertensive therapy. We also examined whether NFE2L2 and HMOX1 polymorphisms affect plasma HO-1 leve... (Complete abstract click electronic access below) / Mestre

Heme Oxygenase (HO) is an enzyme universally found in animals, plants and microbes. In plants, the role of heme oxygenase in the synthesis of the phytochrome chromophore is well recognized and has been extensively studied; however its role in regulating reactive oxygen species (ROS) in plants is just beginning to be explored, particularly in legumes. Legumes interact with Rhizobium bacteria to form symbiotic nitrogen fixing nodules. ROS plays an important role in the development of roots as well as symbiotic nodules. In the model legume Medicago truncatula, ROS in the root is regulated in part by the LATD/NIP gene. The M. truncatula giraffe mutant has a deletion that removes the entire HO coding sequence. We have found that the M. truncatula GIRAFFE HO regulates expression of some of the LATD/NIP-regulated ROS genes such as RESPRATORY BURST OXIDASE HOMOLOG C (RBOHC) and a cell wall peroxidase (cwPRX2) in seedlings. This means that the wild-type function of GIRAFFE is to up-regulate expression of RBOHC and cwPRX2 in roots, in contrast to LATD/NIP, which down-regulates them. We also found that LATD/NIP and GIRAFFE do not regulate expression of each other in seedlings. Given that the highest expression of GIRAFFE HO is in a senescing nodule, we tested the expression of ROS-regulatory enzymes in senescing nodules. We found that GIRAFFE up-regulates expression of RBOHC during nitrate-induced nodule senescence. At present, with changing climatic conditions and exposure to various environmental stresses that can alter ROS homeostasis, characterizing the role of GIRAFFE in the antioxidant machinery of legumes can be useful in improving crop productivity and for enhancing soil fertility.

IsdG and IsdI are non-canonical heme oxygenases (HO) from Staphylococcus aureus that catalyze the oxidative cleavage of heme to give novel organic products (staphylobilins) and iron as a nutrient for the pathogen. Comparison of the reported equilibrium dissociation constant (Kd) values for heme from IsdG and IsdI compared to the reported concentration of the labile heme pool called into question whether these enzymes are competent HOs in vivo. We took advantage of a second-sphere Trp whose fluorescence is quenched upon heme binding, which led to Kd values 2-3 orders of magnitude smaller than reported in the literature. Importantly, these Kd values were on the same order of magnitude as human HO, precluding design of a competitive inhibitor as an effective therapeutic. Based upon the kinetic and equilibrium data, and the finding that the half-life of IsdG is increased 2.5-fold by the presence of heme, we proposed IsdG is the main HO involved in iron acquisition which motivated further characterization of IsdG. IsdG-catalyzed heme catabolism proceeds through ferric-peroxoheme and meso-hydroxyheme intermediates en route to staphylobilin. A second-sphere Asn is known to be critical for enzymatic function, but its role in heme cleavage was unknown. Site-directed mutagenesis was employed to probe the role of Asn using ferric-azidoheme and ferric-cyanoheme as models of the putative ferric-peroxoheme intermediate. An optical spectroscopic study established that a hydrogen-bond between Asn and the iron-ligating (α) atom of the distal ligand perturbs the heme electronic structure. Density functional theory (DFT) suggested this hydrogen-bond triggers rotation of the distal ligand, which was corroborated by circular dichroism (CD), and delocalizes spin density onto the meso carbons. Electron paramagnetic resonance (EPR) revealed the Asn hydrogen-bond increases the Fe 3dxy character in the singly occupied molecular orbital (SOMO), a mechanism that can increase spin density on the meso carbons. Finally, the Asn hydrogen-bond moves the meso carbon resonances downfield in the 13C nuclear magnetic resonance (NMR) spectrum, consistent with excess spin density, confirming a DFT-predicted, Asn-induced spin delocalization. These results suggest IsdG funnels the reactivity of ferric-peroxoheme toward heme hydroxylation through an Asn-dependent bridged transition state, circumventing production of reactive, uncontrolled intermediates.

The endothelium is important for the regulation of vascular tone. In diseases like hypertension, the endothelial cells become dysfunctional. This dysfunction is characterized by nitric oxide (NO) deficiency, impairment of endothelium-dependent hyperpolarization (EDH) and the overwhelming production of endothelium-derived contracting factor (EDCF). Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, producing carbon monoxide(CO), bilirubin and free iron. Up-regulation of the inducible isoform (HO-1) of the enzyme lowers blood pressure in animals. The purpose of the present study was to investigate whether or not up-regulation of HO-1by the pharmacological agent hemin improves endothelial function in arteries of spontaneously hypertensive rats(SHR). Twenty four hours after intraperitoneal injection of hemin (50mg/kg) in 36 weeks old SHR, the expression and activity of HO-1 were augmented, in both the endothelium and vascular smooth muscle. Hemin-treatment potentiated endothelium-dependent relaxations to the muscarinic agonist acetylcholine in both the aorta and the mesenteric artery, whereas the HO inhibitor protoporphyrin IX zinc (II) (ZnPP; 30 mg/kg) prevented the beneficial effect of hemin, suggesting that HO-1 induction improves endothelial function. Hemin-treatment did not augment acetylcholine-induced NO-mediated relaxations, and did not alter the expression level of either phosphorylated eNOS (Ser1177) or total eNOS, suggesting that the improvement of endothelial function by HO-1 induction cannot be attributed to an increased bioavailability of NO. In the mesenteric arteries, hemin treatment potentiated acetylcholine-evoked EDH-mediated relaxations in the presence of L-NAME and indomethacin. The IKCa channel blocker TRAM-34andthe Na+-K+-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34-and ouabain-sensitive relaxations were enhanced by hemin-treatment. K+-induced ouabain-sensitive relaxations and the expression of Na+-K+-ATPase were increased by hemin-treatment. Taken in conjunction, these observations imply that the improved EDH-mediated relaxations by HO-1 induction is due to an improvement of IKCa-Na+-K+-ATPase pathway. Treatment with an antioxidant apocynin (50mg/kg) showed a similar effect as hemin, and the combined treatment with hemin and apocynin did not cause a greater improvement. In vitro treatment with bilirubin, enhanced EDH responses and K+-induced ouabain-sensitive relaxations. These observations suggest that the effect of HO-1 induction on EDH-mediated relaxations is possibly due to its antioxidant properties and the production of bilirubin. In the aortae, hemin-treatment reduced endothelium-dependent contractions in response to acetylcholineor to a calcium ionophoreA23187. Production of reactive oxygen species (ROS) was suppressed by hemin-treatment, judging from the results of 2’,7’-dichlorodihydrofluoresein diacetate staining, dihydroethidium staining and lucigenin chemiluminescence, which was attributed to the decreased expressions of NADPH oxidase-2 (Nox2) and cyclooxygenase-1(COX-1). The production of prostacyclin was decreased, which was explained by a lower expression of COX-1. Contractions to vasoconstrictor concentrations of prostacyclin and its mimetic iloprost were attenuated, suggesting that the responsiveness of thromboxane-prostanoid receptors (TP receptors) to prostacyclin was decreased by hemin-treatment. The effects of HO-1 on the suppressed production of ROS and prostacyclin, and the decreased responsiveness of TP receptors, contribute to its inhibitory role on EDCF-mediated response. Thus, up-regulation of HO-1 improves endothelial function in the SHR by potentiating EDH response and impairing EDCF. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Inorganic phosphate (Pi) plays a critical role in many biological structures and processes. However, Pi typically occurs at low concentrations, particularly in marine environments. In comparison, naturally occurring organophosphonates, which are characterized by a stable carbon-phosphorus (CP) bond, are frequently present at higher concentrations. Accordingly, bacteria have evolved different mechanisms for cleaving the CP bond of organophosphonates to liberate Pi for metabolic use. Two prominent enzyme pathways for catabolic cleavage of a CP bond are examined in this thesis. The first, called CP-lyase, is encoded by the phn operon that consists of 14 genes (phnCDEFGHIJKLMNOP). CP-lyase has long been of interest for its ability to degrade a wide array of organophosphonates through a homolytic CP bond cleaving reaction. A soluble protein complex consisting of PhnGHIJK was isolated from E. coli, suggesting that protein-protein interactions are important for CP bond cleavage. Intermediates of organophosphonate catabolism by E. coli CP-lyase were also detected and isolated, including -D-ribosyl-1,2-cyclic phosphate and N-acetylated aminoalkylphosphonates, 2-N-acetamidoethylphosphonate and 5’-phospho--D-ribosyl-1’-alkylphosphonates. The former compound was shown to be converted by the phosphodiesterase PhnP to -D-ribosyl-1-phosphate. It was also shown that PhnO is an aminoalkylphosphonate N-acetyl transferase and that N-acetylation by this enzyme is necessary for CP bond cleavage of 1-aminoalklyphosphonates. These results demonstrated that in addition to forming protein complexes, CP-lyase also comprises a catabolic pathway, with ribosylation of organophosphonates playing a key part in setting up the CP bond cleaving reaction. The second pathway examined in this thesis is comprised of marine bacterial enzymes PhnY and PhnZ and is specific for 2-aminoethylphosphonate. PhnY was shown to be an -ketoglutarate / Fe(II) dependent dioxygenase that hydroxylates the -carbon of 2-aminoethylphosphonate to form (R)-2-amino-1-hydroxyethylphosphonate. PhnZ was shown to be a novel Fe(II) dependent oxygenase that converts (R)-2-amino-1-hydroxyethylphosphonate to glycine and Pi. Site directed mutagenesis, kinetic analysis, reactions with substrate analogues, and X-ray crystallography examined the roles of active site residues and the di-iron active site. Additionally, a unique induced-fit mechanism was discovered which appears to synchronize substrate binding with activation of molecular oxygen. Overall these results show that PhnZ represents a new mechanism for catabolic cleavage of a CP bond. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-09-13 16:24:17.261