published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Background and objectives: Circulating endothelial progenitor cells (EPCs) play an essential role in maintaining vascular integrity and preventing endothelial dysfunction. Decreased circulating EPC levels are frequently observed in various cardiovascular risks, including aging and diabetes. Endothelial nitric oxide synthase (eNOS) and adiponectin exert their vasculo-protective effects by directly targeting the key components of the vascular system, such as endothelial cells and smooth muscle cells. Both eNOS and adiponectin have been implicated in the mobilization and in vitro functions of EPCs. However, whether and how circulating EPCs are involved in eNOS and adiponectin-mediated vascular protection remain unclear. The objective of this study is to investigate the role of circulating EPCs in eNOS and adiponectin-mediated regulation of vascular integrity after arterial injury under both physiological and pathophysiological conditions, and to elucidate the underlying mechanisms involved. Key findings: 1. Modulation of eNOS activity in vivo by replacing the serine 1176 (S1176) with an aspartate (S1176D mutation or Dki) to mimic phosphorylation or with an alanine (S1176A mutation or Aki) to render it unphosphorylatable altered reendothelialization and subsequent endothelial function after arterial injury in mice. 2. eNOS S1176D mutation increased the number of circulating EPCs and their incorporation into regenerated endothelium, whereas eNOS S1176A or knockout (KO) impaired the mobilization and reendothelializing capacity of circulating EPCs after injury. 3. eNOS S1176D elevated circulating EPCs by promoting the proliferation and differentiation of bone marrow hematopoietic stem cells (HSCs) into EPCs and by inhibiting apoptosis of circulating EPCs. 4. Adiponectin deficiency in mice resulted in progressive decrease of circulating EPCs with aging. Systemic administration of recombinant adiponectin reversed the decreased EPCs number in adiponectin KO mice. In db(-/-) diabetic mice, adiponectin deficiency further reduced circulating EPCs number and subsequent reendothelialization after injury. Rosiglitazone (Rosi), an antidiabetic drug, induced an upregulation of EPCs number and improved reendothelialization, which were partially abolished in the absence of adiponectin. 5. In cultured EPCs, adiponectin significantly inhibited high glucose-induced premature senescence, whereas its effects on proliferation and apoptosis were not evident. High glucose instigated EPCs senescence by increasing the intracellular accumulation of reactive oxygen species (ROS), activation of p38 MAPK and expression of p16INK4A, whereas all these changes could be abolished by adiponectin through adenosine monophosphate (AMP)-activated protein kinase (AMPK) and cyclic AMP (cAMP)/protein kinase A (PKA)-dependent pathways. 6. Compared to cells from db(-/-) diabetic mice, bone marrow EPCs isolated from db(-/-) plus adiponectin double KO (DKO) mice were more susceptible to high glucose-evoked senescence, which were abrogated by adiponectin in vitro. Importantly, chronic administration of adiponectin or the anti-oxidant N-acetylcysteine (NAC) prevented both aging and diabetes-associated elevation of p16INK4A and decline of circulating EPCs in DKO mice. Conclusions: Collectively, the current study demonstrates that circulating EPCs are actively involved in the vasculo-protective effects of both eNOS and adiponectin under physiological and pathological conditions. These findings enrich our knowledge of the versatile functions of eNOS and adiponectin in vascular protection and provide solid scientific evidence supporting the use of eNOS and adiponectin as possible therapeutic targets for cardiovascular diseases. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Background Nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are both important relaxing factors. Their synthesis, release and downstream signaling pathways are controlled by a number of proteins, such that alteration in the activity of these proteins may disturb vascular tone. Aim This study was aimed to investigate the role of some of the regulatory proteins in NO- and EDHF-mediated relaxations. The regulatory proteins that were examined include: 1) calcium-calmodulin dependent protein kinase II (CaMK II), 2) mitogen-activated protein kinase (MAPK), 3) adenosine monophosphate-activated protein kinase (AMPK), 4) phosphoinositide 3-kinase (PI3K) / protein kinase B (Akt) and 5) phosphoprotein phosphatase. Experimental approach Organ chamber system was used for measuring isometric tension of porcine coronary arteries. The role of the regulatory proteins was investigated by using their activators or inhibitors. In the contraction study, arterial rings without endothelium were contracted with U46619 (0.1 nM to 10 μM) or phorbol 12,13-dibutyrate (PDBu, 0.1 nM to 1 μM). In the relaxation study, arterial rings with and without endothelium were contracted with U46619 (30 or 100 nM). They were incubated with indomethacin (cyclooxygenase inhibitor, 10 μM) and TRAM-34 plus UCL1684 (intermediate- and small-conductance calcium-activated potassium channel blockers, respectively; 1 μM each) or L-NAME (NO synthase inhibitor, 30 μM) for the study of NO and EDHF components of bradykinin (0.1 nM to 10 μM)-induced relaxations. Moreover, endothelium-independent relaxations by sodium nitroprusside (SNP, exogenous NO donor, 0.1 nM to 100 μM) and diazoxide (ATP-sensitive potassium channel activator, 1 nM to 1 mM) were examined in arteries without endothelium. Key findings 1. NO and EDHF are both involved in endothelium-dependent relaxation in porcine coronary arteries, in which NO is the dominant relaxing factor. 2. KN-93 (CaMK II inhibitor, 30 μM) significantly reduced contractions to U46619 and PDBu. On the other hand, CaMK II partly involved in EDHF signaling but not in the NO-mediated relaxations. 3. Calyculin A (phosphoprotein phosphatase inhibitor, 30 nM) greatly inhibited both endothelium-dependent and –independent relaxations. 4. PD98059 (MAPK inhibitor, 30 μM) significantly potentiated bradykinin-induced relaxation that was mediated by EDHF but not that mediated by NO. On the other hand, it potentiated SNP-induced but not diazoxide-induced endothelium-independent relaxations. 5. AMPK and Akt do not play a role in regulating vascular tone as compound C (AMPK inhibitor, 30 μM), AICAR (AMPK activator, 1 mM) and wortmannin (PI3K inhibitor, 100 nM) did not affect contractions to U46619 and PDBu, and relaxations to bradykinin, SNP and diazoxide in porcine coronary arteries. Conclusions and implications Different regulatory proteins (CaMKII, MAPK, AMPK, Akt, phosphoprotein phosphatase) have different effects on the regulation of vascular tone. While the present study has the limitation of using pharmacological agents at only one concentration to examine the role of these proteins, it still produces scientific information for the development of therapeutic agents. In considering CaMK II, MAPK and phosphoprotein phosphatase as potential therapeutic targets, the vascular effects (which can be therapeutic or adverse) of the compounds acting on these proteins should be taken into account. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Nitrifier denitrification is a poorly quantified microbial process leading to emissions of N2O from soils. Nitrous oxide emissions, particularly from agricultural soils, are currently being targeted for reduction due to the contribution of this gas to anthropogenic climate change. Measurements specific to nitrifier-denitrification are hampered by poor culturability of many of the strains involved, and the inability of single isotope labelling methods to distinguish it from denitrification carried out by other organisms, dual-labelling approaches and evidence form pure cultures suggest that its contribution to nitrous oxide emissions may be large. Environmental conditions favouring the pathway are unknown, leading to difficulties in mitigation or modelling. In this thesis data from both dual-labelling isotopic techniques and isotopomer measurements of the nitrous oxide emitted are used to determine whether isotopomer techniques can quantify nitrifier-denitrification in situ, and the conditions under which nitrous oxide emissions from the pathway are increased are investigated. Data is also presented on site preference (isotopomer ratio) from ammonia oxidation in several Nitrosospira strains for which this has previously not been measured. The capacity of Nitrosospira strains in pure culture to reduce N2O to N2, an environmentally neutral product of the nitrogen cycle, are investigated. Site preference results from this research suggest that nitrifier denitrification cannot be distinguished from heterotrophic denitrification by site preference, indicating that previously published data stating proportional outputs of N2O from dentrification may overestimate heterotrophic contributions. Several Nitrosospira strains are found to be capable of a reduction step from N2O to N2 in pure culture conditions. Nitrifier denitrification is found to respond to environmental factors of soil N-level, pH and rainfall events, and changes in site preference also occur under these conditions. Site preference is linked to microbial phylogeny for the strains of Nitrosospira tested, indicating a possible effect of enzyme structure above pathway level determination.

This dissertation focuses on chemical and biological evaluation of diazeniumdiolate based nitrogen oxide releasing prodrugs. Three projects are described. i. Synthesis and biological evaluation of a series of new nitroxyl (HNO) releasing non-steroidal antiinflammatory drugs (NSAIDs) and comparison to related nitric oxide (NO) releasing NSAIDs A series of HNO releasing isopropylamine-based diazeniumdiolate adducts of NSAIDs and the NO releasing diethylamine diazeniumdiolate counterpart were synthesized. The aspirin derivatives were evaluated for antiinflammatory, cardioprotective and anticancer effects. Both prodrugs demonstrated similar antiinflammatory properties to aspirin but significantly lower gastrointestinal ulceration, which is a common side effect of aspirin. The HNO adduct also improved cardiac contractility. The chemotherapeutic potential of the prodrugs was assessed in vitro and in vivo. Both the prodrugs inhibited growth of cultured carcinoma cells without inducing cytotoxicity towards non-tumorogenic cell lines. The higher cytotoxicity of the HNO adduct was in part due to increased production of reactive nitrogen and oxygen species leading to oxidative damage to DNA, inhibition of glyceraldehydes-3-phosphate dehydrogenase and upregulation of signaling pathways leading to caspase-3 mediated induction of apoptosis. The NO adduct is a promising candidate for reduction of metastasis by increasing E-cadherin levels, which influences cellular adhesion. Both derivatives showed significantly reduced angiogenesis in cultured cells and tumor volume in nude mice. ii. Synthesis and characterization of primary amine based cyclic amine diazeniumdiolates and comparison to their acetoxy methyl ester derivatives. A series of HNO releasing cyclic amine diazeniumdiolates were synthesized to expand upon the few examples of primary amine diazeniumdiolates. An ester derivative of cyclopentylamine NONOate was also synthesized, to increase decomposition half-life and to improve HNO production and better cellular uptake. This modification increased its cytotoxicity compared to ionic NONOates. iii. Evaluation of mechanism of action of JS-K. JS-K (O²-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1-yl]-diazeniumdiolate) has previously been found to be highly cytotoxic in many cancer cell lines compared to ionic diazeniumdiolates. Thus, the role of NO in cytotoxicity of JS-K was explored. A low intracellular NO flux in combination with a lack of any effect on cyclic guanosine monophosphate (cGMP) dependent pathway suggests that NO is not directly responsible for the cytotoxicity of JS-K.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Chemistry / Master / Master of Philosophy