published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Hyperglycemia is associated with a higher risk for the development of cardiovascular diseases such as atherosclerosis and hypertension. Hyperglycemia-induced generation of reactive oxygen species and the endothelial dysfunction largely account for this phenomenon. Ergothioneine is a naturally occurring amino acid that is abundantly found in mushroom. Numerous benefits have been found associated with ergothioneine such as cation chelating, regulation of gene expression, improvement in immunity and bioenergetics, and of most concern its antioxidative property. The aim of this study was to investigate whether mushroom extract and synthetic ergothioneine can exert protective effect on endothelial cells against oxidative stress. Human umbilical vein endothelial cells served as the cell model. Pyrogallol, hydrogen peroxide and high glucose were used to create the oxidative stress condition in endothelial cells. Biochemical assay was used to measure the viability of the cells. It was found that only the mushroom extract could significantly reduce the cell death induced by pyrogallol. Both the mushroom extract and synthetic ergothioneine significantly decreased the cell death induced by high glucose. However, neither mushroom extract nor synthetic ergothioneine have any positive effect on hydrogen peroxide-induced cell death. These results indicated that mushroom extract and synthetic ergothioneine did exert certain level of protective effect on endothelial cells. However, this protective effect is relatively weak. Besides, it is still unclear if antioxidation is the sole mechanism accounting for the cytoprotective effect of ergothioneine. Further investigation is required to examine if other mechanisms are also involved. / 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

Not available / text