Dissertations / Theses on the topic 'Endothelium and hyperpolarizing factors'

The contractile state of microcirculatory vessels is a major determinant of the blood pressure of the whole systemic circulation. Continuous bi-directional communication exists between the endothelial cells (ECs) and smooth muscle cells (SMCs) that regulates calcium (Ca2+) dynamics in these cells. This study presents theoretical approaches to understand some of the important and currently unresolved microcirculatory phenomena. Agonist induced events at local sites have been shown to spread long distances in the microcirculation. We have developed a multicellular computational model by integrating detailed single EC and SMC models with gap junction and nitric oxide (NO) coupling to understand the mechanisms behind this effect. Simulations suggest that spreading vasodilation mainly occurs through Ca2+ independent passive conduction of hyperpolarization in RMAs. Model predicts a superior role for intercellular diffusion of inositol (1,4,5)-trisphosphate (IP3) than Ca2+ in modulating the spreading response. Endothelial derived signals are initiated even during vasoconstriction of stimulated SMCs by the movement of Ca2+ and/or IP3 into the EC which provide hyperpolarizing feedback to SMCs to counter the ongoing constriction. Myoendothelial projections (MPs) present in the ECs have been recently proposed to play a role in myoendothelial feedback. We have developed two models using compartmental and 2D finite element methods to examine the role of these MPs by adding a sub compartment in the EC to simulate MP with localization of intermediate conductance calcium activated potassium channels (IKCa) and IP3 receptors (IP3R). Both models predicted IP3 mediated high Ca2+ gradients in the MP after SMC stimulation with limited global spread. This Ca2+ transient generated a hyperpolarizing feedback of ~ 2-3mV. Endothelium derived hyperpolarizing factor (EDHF) is the dominant form of endothelial control of SMC constriction in the microcirculation. A number of factors have been proposed for the role of EDHF but no single pathway is agreed upon. We have examined the potential of myoendothelial gap junctions (MEGJs) and potassium (K+) accumulation as EDHF using two models (compartmental and 2D finite element). An extra compartment is added in SMC to simulate micro domains (MD) which have NaKα2 isoform sodium potassium pumps. Simulations predict that MEGJ coupling is much stronger in producing EDHF than alone K+ accumulation. On the contrary, K+ accumulation can alter other important parameters (EC Vm, IKCa current) and inhibit its own release as well as EDHF conduction via MEGJs. The models developed in this study are essential building blocks for future models and provide important insights to the current understanding of myoendothelial feedback and EDHF.

Atherosclerosis, a chronic inflammatory disease of arteries, occurs predominantly at regions of the arterial system that are exposed to disturbed patterns of blood flow. Blood flow influences the atherosclerosis by exerting shear stress on endothelial cells (ECs). Although the signalling pathways that activate pro-inflammatory NF-κB transcription factors are well defined, the regulation and physiological significance of differential NF-κB subunit expression is poorly understood. In this thesis, we demonstrate that RelA NF-κB sub-unit expression is positively regulated in ECs via c-Jun N-terminal kinase (JNK) and the transcription factor ATF2. This pathway promoted focal arterial inflammation as genetic deletion of JNK1 reduced RelA expression and macrophage accumulation at an athero-susceptible site. Furthermore, JNK signalling to RelA is controlled by mechanical forces as en face immunostaining revealed that disturbed flow patterns (generated by a constrictive cuff) elevated RelA expression in murine carotid arteries via JNK1. Positron emission tomography and en face staining revealed that disturbed flow enhanced 18F-fluorodeoxyglucose uptake (a marker of inflammation) and accumulation of CD68-positive inflammatory cells in arteries via JNK1. We conclude that disturbed flow promotes arterial inflammation via a novel JNK-NF-κB cross-talk. The duration of RelA nuclear localisation is an important determinant of the magnitude and specificity of target gene expression. En face staining revealed that RelA rapidly accumulated in the nucleus upon LPS stimulation in ECs at both athero-protected and athero-susceptible sites. RelA was exported from the nucleus to the cytoplasm in response to prolonged stimulation in the athero-protected region but not in the athero-susceptible region. The duration of RelA nuclear localisation was suppressed by histone deacetylases which displayed higher activity at the protected site compared to the susceptible site. Overall, our findings reveal that ECs at athero-susceptible sites are primed for inflammatory activation via complementary mechanisms that enhance both the expression and the activity of NF-κB transcription factors.

Conditioned media prepared from umbilical cord (UC) segments or endothelial cells (EC) contain colony stimulating activity, Both UCCM and ECCM were partially purified by DEAE-Sepharose and ACA44 gel filtration chromatography. The molecular weights were estimated as 25,000 and 31,000 for UC-CSF and EC-CSF, respectively. UC-CSF was further fractionated by Con A Sepharose, IEF and HPLC on a hydrophobic phenyl column. The highly purified CSF stimulates human macrophage and granulocyte colony formation, indicating it is GM-CSF in nature. Characterization studies have revealed that both CSFs are heat stable at 60°C for 30 min. They are sensitive to digestion by protease and to periodate oxidation but are stable to treatment with sulfhydryl reagents. The synthesis of CSF in endothelial cells is inhibited by actinomycin D, cycloheximide and puromycin, indicating that protein and RNA synthesis are required for CSF production. Among the mitogens tested, only LPS exhibited stimulatory activity on the production of CSF. Metabolic modulators such as dibutyryl cAMP, isobutylmethylxanthine, PGE2 and lactoferrin inhibit CSF production, while PGF2 enhances CSF production.

Dendritic cells (DCs) form a key component of the immune response, as they are involved in the innate and adaptive immunity and in the process of tolerance. Under normal conditions, DCs are absent from the Central Nervous System (CNS), as the blood brain barrier (BBB) restricts their entry. However, DCs have recently been implicated in the pathogenesis of several CNS diseases. The molecular mechanisms that mediate DC trafficking across the BBB are poorly understood. The objectives of this study were to examine the role of endothelial cell adhesion molecules (eCAMs) and their ligands in the process of DC adhesion to the BBB endothelium, and to investigate the participation of DCs in human CNS diseases. To study DC adhesion, DCs were generated in vitro by culturing human blood monocytes in the presence of GM-CSF and IL- 4, and DC maturation was induced by adding inflammatory cytokines (TNF-α, IL-1β, IL-6) and PGE₂. Immature and mature DCs displayed differences in their expression of surface molecules, including eCAM ligands, by flow cytometry. Adhesion to the cerebral endothelium was investigated using an in vitro model of the BBB consisting of primary cultures of human brain microvessel endothelial cells (HBMEC). Immature or mature DCs were incubated with resting or TNF-α-activated HBMEC for up to one hour. Only a few DCs adhered to resting HBMEC, but adhesion was upregulated upon activating HBMEC (p<O.Ol). Moreover, immature DCs adhered to activated HBMEC to a greater extent compared to mature DCs (p<O.OOl). Blocking experiments indicated that the adhesion of both immature and mature DCs to HBMEC was dependent upon ICAM-1-CD18 or ICAM-2-CD18, ICAM-2-DC-SIGN, and PECAM-l PECAM-l interactions. In addition, VCAM-1-VLA-4 interactions mediated the adhesion of immature but not mature DCs to activated HBMEC. Using immunohistochemistry for DC markers, we also examined the presence of DCs in human inflammatory, infectious, and neurodegenerative diseases, stroke and tumours. The results indicate accumulation of DC SIGN—, fascin—, and MHC class Il—expressing DCs in the CNS under most pathological conditions. These findings provide further insight into the mechanisms of neuroinflammation, and highlight the role of DCs and the BBB endothelium in this process.

Thesis (Ph. D.)--West Virginia University, 2007.<br>Title from document title page. Document formatted into pages; contains xxiii, 251 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Several studies suggest that diabetes affects male and female vascular beds differently. However, the mechanisms underlying the interaction of sex and diabetes remain to be investigated. This study investigates whether there are 1) sex differences in the development of abnormal vascular responses and 2) changes in the relative contributions of endothelium-derived relaxing factors (EDRFs) in modulating vascular reactivity of mesenteric arteries taken from streptozotocin (STZ)-induced diabetic rats at early and intermediate stages of the disease (one and eight weeks, respectively). We also investigated the mesenteric expression of the mRNAs for endothelial nitric oxide synthase (eNOS) and NADPH oxidase (Nox) in STZ-induced diabetes in both sexes. Vascular responses to acetylcholine (ACh) in mesenteric arterial rings pre-contracted with phenylephrine were measured before and after pretreatment with indomethacin (cyclooxygenase inhibitor), L -NAME (NOS inhibitor), or barium chloride (K IR blocker) plus ouabain (Na + -K + ATPase inhibitor). We demonstrated that ACh-induced relaxations were significantly impaired in mesenteric arteries from both male and female diabetic rats at one and eight weeks. However, at eight weeks the extent of impairment was significantly greater in diabetic females than diabetic males. Our data also showed that in females, the levels of eNOS and Nox2- and Nox4-dependent NADPH oxidase mRNA expression and the relative importance of NO to the regulation of vascular reactivity were substantially enhanced, while the importance of endothelium-derived hyperpolarizing factor (EDHF) was significantly reduced at both one and eight weeks after the induction of diabetes. This study reveals the predisposition of female rat mesenteric arteries to vascular injury after the induction of diabetes, may be due to a shift away from a putative EDHF, initially the major vasodilatory factor, towards a greater reliance on NO, and the interaction of oxidative stress with elevated NO.

Thesis (Ph. D.)--University of Sydney, 2008.<br>Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Clinical Ophthalmology and Eye Health, Faculty of Medicine. Title from title screen (viewed Apr. 8, 2009) Includes bibliography. Also available in print form.

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Two randomised controlled trials were conducted to explore the relationship between the consumption of alcoholic beverages and cardiovascular disease risk factors. Study 1 was primarily designed to test the hypothesis that the cardio-protective effect of light alcohol could be mediated, in part, via improvements in endothelial function. Study 1 was also designed to explore the effect of alcohol on both traditional risk factors for cardiovascular disease, such as changes in lipid profile, haemostatic factors and blood pressure, and novel risk factors such as homocysteine, markers of inflammation and oxidative stress. The experimental design of this study also allowed us to determine whether reducing alcohol intake in these moderate-to-heavy drinkers could improvement insulin sensitivity, a component of the metabolic syndrome. In this group of sixteen healthy middle-aged men with a history of moderate to heavy alcohol intake of seven standard drinks per day, reducing intake down to approximately one standard drink per day for four weeks had no beneficial effects on conduit vessel endothelial function as assessed by post-ischaemic brachial artery flow-mediated dilatation, nor were there any detectable changes in soluble E-selectin, endothelin-1 and von Willebrand Factor, which are considered biomarkers of endothelial activation. As this study did not investigate the effect of alcohol on endothelial function in resistance vessels, it cannot exclude the possibility that alcohol may affect endothelial cells resident in that vascular bed. This study does show and confirm, however, that the relationship between alcohol and risk factors for cardiovascular disease is an extremely complex one. On the one hand it demonstrated that alcohol was potentially harmful, increasing blood pressure, plasma F2-isoprostane (oxidative stress), and homocysteine. On the other hand it showed that increasing alcohol intake led to significant reductions in two (i.e. fibrinogen and IL-6) of five inflammatory markers, in addition to improving the HDL-cholesterol profile of these subjects. Although the effects of alcohol on blood pressure, fibrinogen and HDL-cholesterol are not in themselves new, they support our choice of study design and strengthen the argument in favour of accepting the more novel findings of this study, specifically, the lack of effect on endothelial function and insulin sensitivity, and the harmful effect of alcohol in increasing oxidative stress and homocysteine. Study 2 was primarily designed to test the hypothesis that the consumption of red wine may confer greater cardio-protection than beer via improvements in endothelial function. Simultaneously, the study was also designed to determine whether drinking red wine for 4-weeks would have different effects than beer on either traditional risk factors for cardiovascular disease (i.e. blood pressure and lipid profile) or the more novel risk factors, homocysteine and oxidative stress. Using a randomised controlled cross-over study design, Study 2 provides evidence that the regular daily consumption of 4 standard drinks of either beer or red wine does not alter endothelial function, as measured by post-ischaemic flow-mediated vasodilatation of the brachial artery in healthy middle-aged men, nor was there evidence of any beneficial effect of de-alcoholised red wine on brachial artery response. As compliance with drinking protocol was confirmed with increased serum γ-GT and HDL during red wine and beer periods, and increased 24-hr urinary excretion of 4OMGA during red wine and de-alcoholised red wine periods, we are confident that there was excellent compliance with the beverage treatments. Study 2 also provides the first evidence from a carefully controlled intervention study that both red wine and beer elevate blood pressure to a similar degree, with no detectable difference in the magnitude of either treatment. As with endothelial function, there was also no evidence of any beneficial effect of de-alcoholised red wine on blood pressure. In addition, although post hoc analysis found evidence that alcohol increased both plasma homocysteine and urinary excretion of F2-isoprostane and endothelin-1, there was no apparent protective effect conferred from either red wine or de-alcoholised red wine on these cardiovascular risk markers. The results from this study cannot disprove the hypothesis that red wine is more beneficial for cardiovascular health; however, they suggest that if red wine has properties beyond those of beer to confer protection, they are not via any interactions with the nitric oxide regulatory function of the endothelium in conduit vessels nor are they via moderation of the vasopressor, homocysteine-raising, and oxidative stress effects of alcohol. The interpretation of the findings from both intervention studies and their place in the context of our current understanding of the role that alcoholic beverages play in the development and/or prevention of cardiovascular disease are explored in this thesis.

In systemic sclerosis (SSc), interstitial lung disease (ILD) and engagement of the vascular system lead to increased morbidity and mortality. The aim of this thesis was to elucidate, in a consecutively included cohort of SSc (limited and diffuse) patients (n = 33), the T cell cytokine profile driving the disease in ILD and to explore the role of matrix metalloproteinase 9 (MMP-9) and its inhibitor: tissue inhibitor of metalloproteinase 1 (TIMP-1) in the extracellular matrix (ECM) degrading process leading to fibrous scarring and honey combing. Moreover, to characterize the role of nitric oxide (NO) in vascular engagement. Peripheral arterial changes cause Raynaud’s phenomenon and digital ulcers. Nitric oxide (NO) a main inducer of vasodilation is produced by endothelial nitric oxide synthase (eNOS) in response to changes in blood flow or by inflammatory cytokine inducible (i) NOS. In the vascular smooth muscle cell (VSMC) NO activates guanylate cyclase to produce cGMP, causing relaxation. We showed elevated plasma nitrate, a degradation product of NO, and increased urinary excretion of nitrate and cGMP. Plasma nitrate correlated with elevated levels of endothelial adhesion molecules: endothelial (E) selectin and vascular adhesion molecule 1, indicating that the activated endothelium is the site of NO synthesis by iNOS. Endothelial staining for E-selectin and the finding of iNOS and eNOS in SSc skin biopsies supported this notion. In SSc increased vascular stiffness may limit the NO vasodilatory effects. We found normal endothelium-dependent (i.e. flow mediated (FMD%)) and endothelium-independent (i.e. nitroglycerin-induced (NTG%)) vasodilation in the brachial artery. Radial arterial wall stiffness measured as maximum increase in pulse pressure (dP/dtmax) was increased. FMD% and especially NTG% correlated negatively and dP/dtmax positively to measures of endothelial inflammation: plasma- nitrate and adhesion molecule levels. Thus inflammatory vascular wall changes may interfere with dilation as may the presence of nitrate tolerance. We found elevated alveolar MMP-9 in both its pro- and active form in ILD. The levels correlated to decline in lung capacity, pointing at a causal relation. We suggest that neutrophils secrete MMP-9, which may degrade collagen IV, (the main constituent of basal membranes), collagen V, gelatins, proteoglycans and elastin. MMP-9 activity is partly regulated by the binding of pro- and active form to TIMP-1. Alveolar TIMP-1, which even stimulates fibroblast ECM synthesis, was increased independent of ILD. The inflammatory process in ILD is orchestrated by activated T helper (h) lymphocytes. We found a mixed Th1/Th2 reaction in SSc alveolar T cells expressing messenger for interferon gamma (Th1), IL-6 and IL-10 (both Th2). No particular cytokine mRNA profile distinguished alveolar T cells in ILD. Neutrophils invaded the bronchial epithelium, which seemed otherwise inert as levels of inflammatory cytokine sensitive transcription factors and their nuclear translocation tended to be low. The neutrophil recruitment pathway is uncertain as chemoattractants and endothelial adhesion molecules were normally expressed. In conclusion, MMP-9 probably causes degradation of lung tissue in ILD and may represent a future therapeutic target. Alveolar T cells show a mixed Th1/Th2 cytokine profile independent of ILD. Neutrophils invade the bronchial epithelium. Activated endothelium produces increased amounts of NO and adhesion molecules and the level of activation influences brachial arterial FMD% and NTG% and radial arterial compliance. Nitrate tolerance may be present.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.<br>ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model.<br>AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

Tese de doutoramento em Ciências Farmacêuticas (Farmacologia e Farmacoterapia) apresentada à Faculdade de Farmácia da Universidade de Coimbra<br>Coronary artery disease represents a major health problem worldwide, for which coronary bypass surgery remains a standard of care. In this context, the internal thoracic artery (ITA), also commonly known as internal mammary artery, has long been recognized the best vessel graft. The biology and structure of this special vessel graft has attracted a lot of attention due to its advantages compared to other arterial (e.g. radial artery, right gastroepiploic artery) and venous (i.e. saphenous vein) grafts, namely lower incidence of atherosclerosis, better graft patency and lower incidence of vasospasm. Despite the clear advantages of ITA, several studies have shown both functional impairment and presence of preatherosclerotic lesions, such as intimal and/or medial thickening, medial fibrosis, among others, in the presence of certain cardiovascular risk factors. Although several risk factors have been associated with these functional and structural changes, diverse results have been observed in previous literature. The heterogeneity of the populations included in those studies are probably a major reason for the disparity of results. In this context, we aimed at studying the vasoactivity and histomorphology of left ITA in the presence of multiple cardiovascular risk factors, concomitant diseases and diverse types of medication. In terms of vasoactivity, ITA rings generally presented a contraction to potassium chloride (KCl) and noradrenaline (NA). Regarding endothelium-dependent vasodilation induced by acetylcholine (ACh), a significant impairment was observed in average. Additionally, the vasodilation induced by sodium nitroprusside (SNP) showed a generally preserved endothelium-independent vasodilation. Inhibition of endothelial production of nitric oxide with Nω-monomethyl-L-arginine (L-NMMA), an endothelial nitric oxide synthase (eNOS) inhibitor, produced a marked decrease in maximal relaxation to ACh, as expected. Interestingly, this eNOS inhibition also produced a dose-dependent decrease in maximal contraction to NA. In addition to other associations, age, peripheral vascular disease, recent acute myocardial infarction (MI) history and nitrate medication influenced both the contractile response and the endothelium-(in)dependent vasodilation of the left ITA. Thus, results showed that left ITA vasoactivity may be influenced not only by cardiovascular risk factors but also by medication. The contractile stimulation with NA produced a rhythmic oscillation in ring tension in several patients, a behaviour nowadays known as vasomotion. In this work, we showed that increasing age and female sex are associated with higher risk of this phenomenon, whereas recent MI history is associated with lower risk. Regarding left ITA histomorphology, 37% of the population presented intimal hyperplasia, according to an intima/media ratio (IMR) higher than 0.25. Although no overt atherosclerotic lesion was detected, all samples presented a mild degree of adventitial calcification, as most samples also presented mild medial and/or intimal calcifications. In addition, internal elastic lamina breaks were detected in all samples, suggesting some degeneration of the vascular wall. The maximal intimal width was influenced by several variables, namely age and MI history. Medial width at maximal intimal width was most influenced by β-blocker medication in the total population. Moreover, aging, recent smoking and arterial hypertension were factors associated with increased IMR, which has been proposed as the best histomorphometric parameter in this type of experiment. Also, aging and female sex were associated with intimal hyperplasia defined as IMR higher than 0.25. In addition, we also studied the potential influence of histomorphometric characteristics on the vasoactivity of left ITA. The construction of multivariable linear regression models showed that the sensitivity to KCl is influenced by medial width at maximal intimal width and that the sensitivity to ACh was influenced by maximal intimal width. These results suggest that the structural characteristics might have a more important role in the vessel functional response of what has been recognized. In conclusion, this work contributes to a better scientific understanding of the function and structure of a vessel traditionally seen as atherosclerosis-free, the left ITA, in patients undergoing coronary bypass surgery.<br>A doença arterial coronária representa atualmente um considerável problema de saúde pública, para a qual a cirurgia de bypass coronário permanece como o padrão de cuidados. Neste contexto, a artéria torácica interna (ITA) esquerda, também comumente conhecida como artéria mamária interna esquerda, tem sido desde há muito reconhecida como o conduto de excelência. A biologia e estrutura deste vaso de enxerto especial tem captado a atenção da comunidade científica devido às suas vantagens em relação a outros enxertos arteriais (por exemplo, a artéria radial ou a artéria gastroepiploica direita) e venosos (em particular, a veia safena), nomeadamente: menor incidência de aterosclerose, melhor patência do enxerto e menor incidência de vasospasmo. Apesar das claras vantagens da ITA esquerda, vários estudos têm demonstrado quer alterações funcionais quer lesões pre-ateroscleróticas, tais como espessamento da túnica íntima e/ou da túnica média, fibrose da túnica média, entre outros, na presença de determinados fatores de risco cardiovascular. A associação entre alguns fatores de risco e estas alterações funcionais e estruturais encontra-se já reportada, apesar de alguma literatura ser contraditória. A heterogeneidade das populações incluídas nestes estudos é provavelmente uma das principais razões para a discrepância dos resultados. Assim, estudámos a vasoatividade e histomorfologia da ITA esquerda na presença de múltiplos fatores de risco cardiovasculares, doenças concomitantes e medicação com diversos grupos farmacológicos. Relativamente à vasoatividade, os aneis de ITA apresentaram geralmente uma contração ao cloreto de potássio (KCl) e à noradrenalina (NA). Quanto à vasodilatação dependente do endotélio induzida pela acetilcolina (ACh), foram observadas alterações significativas. Adicionalmente, foi demonstrada uma vasodilatação independente do endotélio, induzida pelo nitroprussiato de sódio (SNP), geralmente preservada. A inibição da produção endotelial de monóxido de azoto (ou óxido nítrico) com recurso ao inibidor da sintase endotelial do monóxido de azoto (eNOS), Nω-monometil-L-arginina (L-NMMA), produziu uma diminuição marcada do relaxamento máximo à ACh, tal como esperado. Curiosamente, a inibição da eNOS promoveu também um decréscimo dose-dependente da contração máxima à NA. Além de outras associações, a idade, a doença vascular periférica, a história recente de enfarte agudo do miocárdio (MI) e a medicação com nitratos influenciaram quer a resposta contráctil quer a vasodilatação dependente ou independente do endotélio da ITA esquerda. Assim, os resultados demonstraram que a vasoatividade da ITA esquerda é passível de ser influenciada não só por fatores de risco mas também pela medicação. A estimulação contráctil com NA produziu uma oscilação rítmica da tensão do anel em diversos doentes, um comportamento hoje em dia conhecido como vasomotion. Neste estudo, demonstrámos que o aumento da idade e o sexo feminino estão associados a um maior risco deste fenómeno, enquanto a história recente de MI está associada a um menor risco. Em relação à histomorfologia da ITA esquerda, 37% da população incluída neste trabalho apresentou hiperplasia da íntima, definida por uma razão íntima/média (IMR) superior a 0,25. Embora nenhuma lesão aterosclerótica evidente tenha sido detetada, todas as amostras apresentaram um grau ligeiro de calcificação na adventícia, enquanto a maioria das amostras apresentou igualmente um grau ligeiro de calcificação na média e/ou íntima. Em adição, foram detectadas quebras na lâmina elástica interna em todas as amostras, o que sugere alguma degeneração da parede vascular. O parâmetro espessura máxima da íntima foi essencialmente influenciado pelas variáveis idade e história de MI. A espessura da média à espessura máxima da íntima foi maioritariamente influenciada pela medicação com β-bloqueantes na população total. O aumento da idade, o tabagismo recente e a hipertensão arterial foram fatores associados ao aumento da IMR, o qual tem sido proposto como o melhor parâmetro histomorfométrico neste tipo de ensaio. Além disso, o aumento da idade e o sexo feminino encontraram-se associados a hiperplasia da íntima definida por um IMR superior a 0,25. Adicionalmente, analisámos a influência das características histomorfométricas na vasoatividade da ITA esquerda. A construção de modelos multivariados de regressão linear demonstrou que a sensibilidade ao KCl foi influenciada pela espessura da média à espessura máxima da íntima e que a sensibilidade à ACh foi influenciada pela máxima espessura da íntima. Estes resultados sugerem que as características estruturais poderão ter um papel mais importante na resposta funcional vascular do que lhe tem sido reconhecido. Consideramos que este trabalho contribui para um melhor conhecimento científico da função e estrutura vascular de um vaso tradicionalmente visto como livre de aterosclerose, a ITA esquerda, em doentes submetidos a cirurgia de bypass coronário.

Made available in DSpace on 2014-10-09T12:51:58Z (GMT). No. of bitstreams: 0<br>Made available in DSpace on 2014-10-09T14:07:22Z (GMT). No. of bitstreams: 0<br>Tese (Doutoramento)<br>IPEN/T<br>Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Bien que la lutte contre le tabagisme passif se soit récemment accentuée, la fumée de tabac demeure un des principaux polluants atmosphériques d’intérieur. Le tabagisme passif génère des réactions cardiovasculaires néfastes qui, lors d’expositions répétées, augmentent le risque de mortalité cardiovasculaire. Le but des travaux réalisés a été de repenser la toxicité cardiovasculaire aiguë de la pollution de l’air par la fumée de tabac, en analysant plus particulièrement le rôle joué par la nicotine sur les déterminants endothéliaux et sympathiques du tonus vasculaire.<p>Nous avons tout d’abord caractérisé, chez des volontaires sains non-fumeurs, les réactions vasculaires provoquées par une heure d’inhalation passive de fumée de tabac ou de fumée non tabagique.1 Nos résultats démontrent que le tabagisme passif provoque une réflexion plus précoce de l’onde de pouls au niveau aortique ainsi qu’une perte de la capacité vasodilatatrice microvasculaire dépendante de l’endothélium. De plus, nous avons observé que ces réactions sont spécifiques à la fumée de tabac, et perdurent plusieurs dizaines de minutes après l’arrêt de l’exposition. Le stimulus nicotinique exerce un rôle prédominant dans les changements de réflexion d’onde de pouls. Cependant, l’interprétation du rôle joué par la nicotine dans la toxicité endothéliale du tabagisme passif est limitée in vivo par les effets de la nicotine sur d’autres déterminants du tonus vasculaire, tel que le système orthosympathique. <p>Nous avons dès lors spécifiquement comparé, à l’aide d’un modèle d’aorte isolée de rats, les effets d’extraits de fumée de tabac, de fumée non tabagique et de nicotine pure sur la fonction endothéliale et la production radicalaire.2 Chaque type de fumée a entraîné une augmentation similaire de la production radicalaire au sein des vaisseaux, mais seul l’extrait tabagique a altéré la relaxation vasculaire dépendante de l’endothélium. Dans les mêmes conditions, la nicotine pure a respecté l’intégrité fonctionnelle de l’endothélium, ce qui permet d’exclure son implication dans les effets délétères aigus du tabagisme passif sur l’endothélium vasculaire. <p>Nous avons ensuite déterminé, par des mesures directes du trafic nerveux autonome sur une population de volontaires sains non fumeurs, les effets directs de la nicotine sur le contrôle chémoréflexe périphérique du système orthosympathique.3 Nos résultats démontrent que des taux de nicotine similaires à ceux générés par une heure d’exposition au tabagisme passif augmentent la sensibilité d’une importante boucle réflexe participant à l’homéostasie du système nerveux autonome. <p>Enfin, malgré tous ces effets délétères de la nicotine observés chez le non-fumeur, nous avons établi que la perfusion myocardique du patient à risque coronaire élevé n’est toutefois pas altérée par une prise de nicotine sublinguale.4 <p>Les effets cardiovasculaires du tabagisme passif sont rapides, spécifiques, et réunissent des conditions de stimulation orthosympathique et de dysfonction endothéliale potentiellement néfastes pour la perfusion coronaire. Les effets sympathicomimétiques de la nicotine représentent l’axe prédominant de la toxicité cardiovasculaire aiguë du tabagisme passif. Toutefois, la nicotine pure n’altèrant pas la perfusion myocardique du patient à risque coronaire, son utilisation peut donc être encouragée dans l’aide au sevrage tabagique. <p><br>Doctorat en Sciences médicales<br>info:eu-repo/semantics/nonPublished

Objectives: To date little is known of the interaction between diabetes and sex hormones in the vasculature. A number of studies suggest that premenopausal diabetic women loose their gender based cardiovascular protection. However, there is insufficient evidence to explain the mechanism underlying the loss of this gender based cardioprotection in premenopausal diabetic women. The objectives of this study were to investigate whether there is a gender difference in the aortic endothelial function in · streptozotocin (STZ, 58 mg/kg, iv)-induced diabetic rats, and the potential role of superoxide and cyclooxygenase (COX) metabolites in diabetes-induced vascular dysfunction.

A doença cardiovascular aterosclerótica é a principal causa de morte no hemisfério ocidental, portanto também no Brasil. A detecção não invasiva da aterosclerose é fundamental para prevenção. Objetivos: Correlacionar os fatores de risco (escore de Framingham), o perfil lipídico, a PCR-us, a espessura da íntima-média da carótida, a função endotelial, o índice tornozelo-braquial e o escore de cálcio pela tomografia computadorizada, com a extensão da doença coronariana determinada pelo índice de Friesinger, na cinecoronariografia. Casuística e métodos: Foram estudados 100 pacientes de ambos os sexos, com idade de 55,1±10,7 sendo 55% homens e 45% mulheres. Não foram incluídos pacientes com síndrome coronariana aguda, insuficiência renal dialítica, doença do colágeno e câncer. Todos se submeteram a avaliação clínica, laboratorial (glicemia, perfil lipídico e PCR-us), função endotelial da artéria braquial e ultrasonografia da artéria carótida por ultra-som de alta resolução, índice tornozelo-braquial e tomografia computadorizada coronária para determinação do escore de cálcio. Foram calculados o colesterol não HDL-c e a relação TG/HDL-c. Todos os pacientes foram submetidos à cinecoronariografia por indicação do médico assistente. Foram considerados normais pacientes sem lesão obstrutiva na cinecoronariografia. Resultados: Pela análise univariada, escore de cálcio (529,5 ± 930,9) um HDL-c (45,9±15,5), relação TG/HDL (5,5±9,2) e IMT (0,77±0,22) mostraram correlação significativa com o índice de Friesinger. Já por análise multivariada somente o escore de cálcio, relação TG/HDL-c aumentada e HDL-c baixo correlacionaram-se significativamente com a extensão da DAC. A PCR-s (3,4±3,5), LDL-c (122,8±51,5) e DMF (4,8±5,7) não se correlacionaram com o índice de Friensinger. Conclusão: Assim, é possível ter uma idéia aproximada da presença/extensão da DAC usando métodos não invasivos, especialmente escore de cálcio, relação TG/HDL-c e HDL-c baixo.<br>Background: Atherosclerotic cardiovascular disease is the leading cause of death in the western hemisphere including Brazil. Non-invasive detection of atherosclerosis is critical to prevention. Objectives: We correlated the risk factors (score of Framingham), lipid profile, PCR-us, carotid intima-media thickness, endothelial function, ankle-brachial index and calcium score by computed tomography with the extent of coronary disease determined by the Friesinger index, by coronary angiography. Methods: We studied 100 patients of both sexes, aged from 55.1 ± 10.7, 55% men and 45% women. Patients with acute coronary syndrome, renal dialitic, collagen disease and cancer were not included. All were submitted to clinical evaluation, laboratory (blood glucose, lipid profile and hs-CRP), endothelial function of brachial artery and ultra-sonography of carotid artery by high-resolution ultra-sound, ankle-brachial index and computed tomography for coronary determination of calcium score. We calculated the cholesterol not HDL-c and TG/HDL-c ratio. All patients were submitted to coronary angiography for indication by attending physician. We considered normal patients without obstructive lesion in coronary angiography. Results: By univariate analysis, calcium score, HDL-c, TG/HDL ratio and IMT showed significant correlation with Friesinger index. However, multivariate analysis only calcium score, increased TG/HDL-c and low HDL-c correlated significantly with the extension of the CAD. The hs-CRP, LDL-c and FMD, did not correlate Friensinger index. Conclusion: Thus, it is possible to have an approximate idea of the presence/extension of CAD by non-invasive methods, especially calcium score, TG/HDL-C ratio and HDL-c.

Peptides play a critical role in human physiology and harbour a huge potential as therapeutic agents. In this thesis, new peptides have been discovered as ligands for the epidermal growth factor (EGF) and as new BBB-shuttles, using phage display and chemical synthesis of peptides and proteins. EGF is overexpressed in several cancers, inducing the proliferation and survival of these cells. By inhibiting EGF, we will prevent the activation of the receptor and, consequently, its negative effects. Moreover, phage display is a powerful tool to identify peptide ligands. However, only L-peptides can be displayed, which are protease unstable. Hence, mirror image phage display was developed to identify D-peptides, more stable in front of proteases. In this methodology, the selection is carried out against the mirror image of the original target and, after the panning selection and the synthesis of the enantiomer of the ligand, D-peptides are obtained. In this regard, the enantiomer of EGF was synthesised using a combination of solid-phase peptide synthesis and native chemical ligation. After the panning of two phage display peptide libraries against the immobilised protein, nine sequences were selected and synthesised with D-amino acids. Three of these peptides have high affinities for EGF and are stable in serum proteases for more than 24h. Most potential drugs for the treatment of central nervous system disorders (such as brain cancer and Alzheimer’s disease) do not cross the blood-brain barrier (BBB). Much effort has been devoted to the discovery of BBB-shuttle peptides – entities that have the capacity to carry cargoes across the BBB. The sources of BBB-shuttle peptides are diverse, ranging from the mimicry of endogenous proteins to the use of phage display. Phage display has been applied against a human BBB cellular model which consists in the co-culture of human brain capillary endothelial cells and bovine pericytes. From the screening of a phage display library containing random 12-amino acid sequences, SGVYKVAYDWQH (SGV) was selected. Validation studies were performed confirming that SGV is able to increase the uptake of a model protein in endothelial cells. When a BBB-shuttle is conjugated to a cargo, the ratio BBB-shuttle:cargo can range from 1:1 to 400:1, depending on the cargo. However, there are cases where the modification of the cargo with one copy of the BBB-shuttle is not sufficient to promote its passage through the BBB. More copies can be introduced, but a mixture of ratios of BBB-shuttle:cargo conjugates may be obtained. In those cases, it may be interesting to use multivalent BBB-shuttles, where more than one copy of the shuttle is attached to a core, which is linked to the cargo at one position. In this regard, branched dimerisation of a known BBB-shuttle was explored to enhance BBB permeability of model proteins. The results obtained with THRre peptide as the BBB-shuttle suggest that the mild improvement in permeability may not compensate the increased synthetic effort that these constructs represent. Moreover, chlorotoxin (CTX), a disulphide-rich peptide found in the venom of a scorpion, is reported to be able to enter the brain and bind specifically to tumour tissue. However, CTX is a relatively large peptide (36 amino acids) to be used as a BBB-shuttle, where we may need to scale it up or modify it. In this regard, we designed minimised versions of CTX (MiniCTXs) and evaluated their BBB properties, as well as their toxicity and stability. The results from these experiments produced two peptides as good BBB-shuttles, with similar stability, cellular uptake and BBB permeability than the best BBB-shuttles.<br>En aquesta tesi doctoral, s’han descobert nous pèptids com a lligands del factor de creixement endotelial (EGF) i com a noves llançadores peptídiques, usant les tècniques de phage display i la síntesi química de pèptids. EGF es troba sobre-expressat en diversos càncers i, mitjançant la seva inhibició, la proliferació d’aquestes cèl·lules es podria reduir. Amb phage display es poden trobar aquests lligands, però els fags només poden expressar pèptids L, els quals són degradats per proteases. En el mirror image phage display, la selecció es duu a terme contra la imatge especular de la diana original i, després de la selecció i la síntesi dels enantiòmers dels millors candidats, s’obtenen pèptids D com a lligands. En aquest context, l’enantiòmer de EGF s’ha sintetitzat usant una combinació de síntesi en fase sòlida i lligació química nativa. Després del procés de selecció amb dos biblioteques de fags contra la proteïna immobilitzada, nou seqüencies s’han seleccionat i sintetitzat amb aminoàcids D. Tres d’aquests pèptids presenten altes estabilitats i afinitats per EGF. La majoria de fàrmacs pel tractament de malalties del sistema nerviós central no creuen la barrera hematoencefàlica. Una alternativa és l’ús de llançadores, que tenen la capacitat de transportar fàrmacs al cervell. La metodologia de phage display s’ha aplicat en un model humà de la barrera hematoencefàlica i, després del cribratge, la seqüència SGVYKVAYDWQH (SGV) s’ha seleccionat. Estudis de validació s’han dut a terme confirmant que SGV és capaç d’incrementar el transport d’una proteïna en cèl·lules endotelials. Hi ha casos en que la modificació del fàrmac amb una sola llançadora no és suficient per creuar la barrera hematoencefàlica. En aquest sentit, s’ha explorat la dimerització d’una llançadora (THRre) per incrementar el transport de proteïnes. Els resultats obtinguts suggereixen un moderat increment en el transport, el qual pot no compensar l’esforç sintètic requerit. La clorotoxina (CTX), pèptid trobat en el verí d’un escorpí, i versions reduïdes d’aquesta (MiniCTXs), s’han estudiat com a nova font de llançadores. Les propietats d’aquests pèptids de creuar la barrera hematoencefàlica han estat avaluades. Al final d’aquest procés, dos MiniCTXs han estat seleccionades, amb uns valors de transport semblants a les millors llançadores.

Dong Yingying.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 99-125).<br>Abstracts in English and Chinese.<br>Declaration --- p.i<br>Acknowledgement --- p.ii<br>Publication list --- p.iii<br>Abstract (English) --- p.ix<br>Abstract (Chinese) --- p.xii<br>Abbreviations --- p.xiv<br>List of figures / tables --- p.xvi<br>Chapter Chapter 1. --- General Introduction<br>Chapter 1.1 --- The role of endothelium in regulating vascular tone --- p.1<br>Chapter 1.1.1 --- Nitric oxide (NO) --- p.2<br>Chapter 1.1.2 --- Endothelium-derived hyperpolarizing factor (EDHF) --- p.7<br>Chapter 1.1.3 --- Prostacyclin (PGI2) --- p.20<br>Chapter 1.2 --- EDHF-mediated endothelial function in coronary circulation --- p.22<br>Chapter 1.2.1 --- Role of EDHF in coronary microarteries --- p.23<br>Chapter 1.2.2 --- Role of EDHF in cardiac veins --- p.24<br>Chapter 1.3 --- Effect of ischemia-reperfusion on endothelial function in coronary circulation --- p.25<br>Chapter 1.3.1 --- Ischemia-reperfusion injury --- p.26<br>Chapter 1.3.2 --- Effect of ischemia-reperfusion on endothelial function in coronary microarteries --- p.28<br>Chapter 1.3.3 --- Effect of ischemia-reperfusion on endothelial function in cardiac veins --- p.29<br>Chapter 1.4 --- Alteration of endothelial function during cardiac surgery<br>Chapter 1.4.1 --- Cardioplegia and organ preservation solutions --- p.31<br>Chapter 1.4.2 --- Combined effects of hypoxia-reoxygenation and ST solution on endothelial function in coronary microarteries/cardiac veins --- p.34<br>Chapter 1.4.3 --- Effect of nicorandil on endothelial function --- p.34<br>Chapter Chapter 2. --- Materials and Methods --- p.37<br>Chapter 2.1 --- Isometric force study in micro arteries/veins --- p.37<br>Chapter 2.1.1 --- Preparation of vessels --- p.37<br>Chapter 2.1.1.1 --- Preparation of porcine coronary microarteries --- p.37<br>Chapter 2.1.1.2 --- Preparation of porcine cardiac veins --- p.37<br>Chapter 2.1.2 --- Technique of setting up --- p.39<br>Chapter 2.1.2.1 --- Mounting of microvessels --- p.39<br>Chapter 2.1.2.2 --- Normalization procedure for microvessels --- p.39<br>Chapter 2.1.3 --- EDHF-mediated vasorelaxation --- p.40<br>Chapter 2.1.3.1 --- Precontraction and stimuli of EDHF --- p.40<br>Chapter 2.1.3.2. --- “Truéحresponse of EDHF --- p.40<br>Chapter 2.1.4 --- Data acquisition and analysis --- p.41<br>Chapter 2.2 --- Hypoxia and reoxygenation --- p.41<br>Chapter 2.2.1 --- Calibration of 02-special electrode --- p.41<br>Chapter 2.2.2 --- Measurement of --- p.02<br>Chapter 2.3 --- Statistical analysis --- p.42<br>Chapter 2.4 --- Chemicals --- p.43<br>Chapter Chapter 3. --- Hypoxia-Reoxygenation in Coronary Microarteries: Combined Effect with St Thomas Cardioplegia and Temperature on the Endothelium- derived Hyperpolarizing Factor and Protective Effect of Nicorandil --- p.44<br>Chapter 3.1 --- Abstract --- p.44<br>Chapter 3.2 --- Introduction --- p.45<br>Chapter 3.3 --- Experimental design and analysis --- p.47<br>Chapter 3.3.1 --- Vessel Preparation --- p.47<br>Chapter 3.3.2 --- Normalization --- p.48<br>Chapter 3.3.3 --- Hypoxia --- p.48<br>Chapter 3.3.4 --- Effect of H-R on EDHF-mediated relaxation in coronary microarteries --- p.49<br>Chapter 3.3.5 --- Combined effects ofH-R and ST solution on EDHF-mediated relaxation in coronary microarteries --- p.49<br>Chapter 3.3.6 --- Effect of addition of nicorandil Krebs or ST solution under H-R on EDHF-mediated relaxation in coronary microarteries --- p.49<br>Chapter 3.3.7 --- Data analysis --- p.50<br>Chapter 3.4 --- Results --- p.51<br>Chapter 3.4.1 --- Resting force --- p.51<br>Chapter 3.4.2 --- U46619-induced contraction force --- p.51<br>Chapter 3.4.3 --- Partial pressure of oxygen in hypoxia --- p.51<br>Chapter 3.4.4 --- EDHF-mediated relaxation in coronary microarteries --- p.51<br>Chapter 3.4.4.1 --- Effect of H-R --- p.51<br>Chapter 3.4.4.2 --- Combined effects ofH-R and ST solution on EDHF-mediated relaxation --- p.52<br>Chapter 3.4.4.3 --- Effects of addition of nicorandil to Krebs or ST solution under H-R on EDHF-mediated relaxation --- p.52<br>Chapter 3.5 --- Discussion --- p.53<br>Chapter 3.5.1 --- EDHF-mediated relaxation after exposure to H-R --- p.53<br>Chapter 3.5.2 --- EDHF-mediated relaxation after H-R in ST solution at different temperature --- p.54<br>Chapter 3.5.3 --- Effect of addition of nicorandil to Krebs or ST solution during H-R on EDHF-mediated relaxation --- p.55<br>Chapter 3.5.4 --- Clinical implications --- p.56<br>Chapter Chapter 4. --- Hypoxia-Reoxygenation in Cardiac Microveins: Combined Effect with Cardioplegia and Temperature on the Endothelial Function --- p.68<br>Chapter 4.1 --- Abstract --- p.68<br>Chapter 4.2 --- Introduction --- p.69<br>Chapter 4.3 --- Experimental design and analysis --- p.73<br>Chapter 4.3.1 --- Vessel Preparation --- p.73<br>Chapter 4.3.2 --- Normalization --- p.73<br>Chapter 4.3.3 --- Hypoxia --- p.73<br>Chapter 4.3.4 --- Effect of H-R on EDHF-mediated relaxation in cardiac micro veins --- p.74<br>Chapter 4.3.5 --- Combined effects of H-R and ST solution on EDHF-mediated relaxation in cardiac microveins --- p.74<br>Chapter 4.3.6 --- Data analysis --- p.75<br>Chapter 4.4 --- Results --- p.75<br>Chapter 4.4.1 --- Resting force --- p.75<br>Chapter 4.4.2 --- U46619-induced contraction force --- p.76<br>Chapter 4.4.3 --- Partial pressure of oxygen in hypoxia --- p.76<br>Chapter 4.4.4 --- EDHF-mediated relaxation after H-R in Krebs solution at 37°C --- p.76<br>Chapter 4.4.5 --- EDHF-mediated relaxation after exposure to H-R in ST solution at different temperatures --- p.77<br>Chapter 4.5 --- Discussion --- p.78<br>Chapter 4.5.1 --- Effect of H-R on EDHF-mediated relaxation --- p.78<br>Chapter 4.5.2 --- Combined effects of H-R with ST solution on EDHF-mediated relaxation --- p.80<br>Chapter 4.5.3 --- Clinical implications<br>Chapter Chapter 5. --- General Discussion --- p.89<br>Chapter 5.1 --- EDHF-mediated endothelial function in porcine coronary circulation --- p.89<br>Chapter 5.1.1 --- EDHF in porcine coronary microarteries --- p.92<br>Chapter 5.1.2 --- EDHF in porcine cardiac veins --- p.90<br>Chapter 5.2 --- Alteration of EDHF-mediated function after exposure to H-R --- p.91<br>Chapter 5.2.1 --- In coronary microarteries --- p.91<br>Chapter 5.2.2 --- In cardiac veins --- p.92<br>Chapter 5.3 --- Alteration of EDHF-mediated function after exposure to ST solution under H-R --- p.92<br>Chapter 5.3.1 --- In coronary microarteries --- p.93<br>Chapter 5.3.2 --- In cardiac veins --- p.93<br>Chapter 5.4 --- EDHF-mediated function in nicorandil-supplemented ST solution under H-R in coronary microarteries --- p.93<br>Chapter 5.5 --- Clinical implications<br>Chapter 5.5.1 --- H-R injury --- p.94<br>Chapter 5.5.2 --- H-R injury and cardioplegic solution --- p.95<br>Chapter 5.5.2 --- Nicorandil-supplementation in cardioplegic solution --- p.95<br>Chapter 5.6 --- Limitation of the study --- p.96<br>Chapter 5.7 --- Future investigations --- p.96<br>Chapter 5.8 --- Conclusions --- p.97<br>References --- p.99

Yang Qin.<br>"June 2003."<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2003.<br>Includes bibliographical references (p. 168-207).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.<br>Abstracts in English and Chinese.

博士<br>東海大學<br>生命科學系<br>100<br>In diabetic population, more than 65% of death is related to cardiovascular diseases. Reduced myocardial blood flow is commonly observed in diabetic patients without obstructive atherosclerosis. This indicates that microvascular complications, especially the impairment of arteriolar dilation in resistant vessels, are involved in the diabetic vascular dysfunction. Endothelium-derived hyperpolarizing factor (EDHF) is an important substance released from the endothelium that regulates vasomotor function, but its role in diabetes/hyperglycemia-induced vascular impairment remains unclear. In the coronary circulation, the EDHF pathway is mediated by the cytochrome P450 epoxygenase (CYP) and its derivative epoxyeicosatrienoic acids (EETs). The present study aimed at determining the mechanistic effect of high glucose (HG) on EDHF/EET-mediated arteriolar dilation. Porcine coronary arterioles were isolated and pressurized for vasomotor study. Cultured porcine coronary artery endothelial cells (ECs) were used for molecular and biochemical analysis. Bradykinin (BK), a potent vasodilator, was used to generate endothelium-dependent vasodilation before and after high glucose (HG, 25 mM vs. 5 mM in control) treatment. We found that incubation of coronary arterioles with HG impaired vasodilation to BK but not to sodium nitroprusside (endothelium-independent vasodilator). In the presence of potassium channel inhibitor targeting EDHF-mediated vasodilation, the NO component of arteriolar dilation in response to BK was reduced by HG incubation. In the presence of inhibitors of NO synthase and cyclooxygenase, the EDHF-mediated arteriolar dilation was reduced by HG incubation. The inhibitory effect of HG was prevented by treating the vessels with superoxide scavenger Tempol. In cultured coronary artery endothelial cells (ECs), the NO production in response to BK stimulation was reduced by HG. HG also reduced endothelial EET production as well as CYP activity. The superoxide production was elevated in ECs after HG incubation. Pretreatment with superoxide scavenger Tempol before HG incubation abolished the increase of cellular superoxide and prevented the decrease of CYP activity. Collectively, our results suggest that, in addition to the NO-mediated pathway, HG impairs the EET/EDHF-mediated vasodilation in coronary arterioles by inhibiting CYP activity in coronary ECs via elevated level of superoxide. The present study provides further information on the mechanisms of hyperglycemia-induced vascular dysfunction.

Zou Wei.<br>Thesis submitted in: December 2001.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.<br>Includes bibliographical references (leaves 98-119).<br>Abstracts in English and Chinese.<br>Declaration --- p.i<br>Acknowledgements --- p.ii<br>Publication lists --- p.iii<br>Abstract --- p.ix<br>Abbreviations --- p.xiii<br>List of tables and figures --- p.xiv<br>Chapter Chapter 1: --- General Introduction --- p.1<br>Chapter 1.1. --- Endothelium-dependent relaxation in coronary and pulmonary circulation --- p.1<br>Chapter 1.1.1. --- Endothelium-derived relaxing factors --- p.2<br>Chapter 1.1.1.1. --- Nitric Oxide --- p.3<br>Chapter 1.1.1.2. --- PGI2 --- p.5<br>Chapter 1.1.1.3. --- EDHF --- p.6<br>Chapter 1.1.2. --- EDHF in coronary and pulmonary circulation --- p.8<br>Chapter 1.1.2.1. --- EDHF in coronary circulation --- p.8<br>Chapter 1.1.2.2. --- EDHF in pulmonary circulation --- p.9<br>Chapter 1.2. --- Effect of hyperkalemia on EDHF-mediated relaxation --- p.10<br>Chapter 1.3. --- Organ Preservation Solutions --- p.13<br>Chapter 1.3.1. --- Euro-Collins solution --- p.14<br>Chapter 1.3.2. --- University of Wisconsin solution --- p.15<br>Chapter Chapter 2: --- Objectives and research approaches --- p.16<br>Chapter 2.1. --- Objectives --- p.16<br>Chapter 2.1.1. --- "Endothelium-dependent relaxation resistant to INDO, L-NNA, and HbO in porcine and pulmonary coronary micro-arteries" --- p.16<br>Chapter 2.1.2. --- "EET11,12 and EDHF-mediated function in porcine coronary micro-arteries" --- p.17<br>Chapter 2.1.3. --- "Comparison of EC or UW solution on endothelium-dependent relaxation resistant to INDO, l-NNA, and HbO in porcine pulmonary arteries" --- p.17<br>Chapter 2.2. --- Research approaches --- p.18<br>Chapter 2.2.1. --- "Endothelium-dependence of the relaxation by BK or EET11,12" --- p.18<br>Chapter 2.2.2. --- Effect of hypothermic storage with EC and UW solution on EDHF-related relaxation --- p.18<br>Chapter 2.2.3. --- Time-dependent alteration of endothelium-dependent relaxation in pulmonary micro-arteries by EC and UW solution --- p.19<br>Chapter 2.2.4. --- Effect of HbO in endothelium-dependent relaxation --- p.19<br>Chapter Chapter 3: --- Material and Methods --- p.21<br>Chapter 3.1. --- General Methods --- p.21<br>Chapter 3.1.1. --- Porcine heart and lung collection and transportion<br>Chapter 3.1.2. --- Myograph --- p.21<br>Chapter 3.1.3. --- Myosight --- p.24<br>Chapter 3.1.4. --- Anatomizing blood vessel --- p.24<br>Chapter 3.1.5. --- Mounting --- p.24<br>Chapter 3.1.6 --- Normalization --- p.26<br>Chapter 3.1.6.1. --- Normalization of coronary micro-artery --- p.27<br>Chapter 3.1.6.2. --- Normalization of pulmonary micro-artery --- p.28<br>Chapter 3.1.7. --- Precontraction --- p.30<br>Chapter 3.1.8. --- Endothelium-dependent relaxation --- p.31<br>Chapter 3.2. --- Coronary artery studies --- p.32<br>Chapter 3.2.1. --- Porcine heart harvest and anatomy --- p.32<br>Chapter 3.2.2. --- Characteristic of histology of porcine coronary micro-artery --- p.32<br>Chapter 3.3. --- Pulmonary artery studies --- p.35<br>Chapter 3.3.1. --- Porcine lung harvest and anatomy --- p.35<br>Chapter 3.3.2. --- Characteristic of histology of porcine pulmonary micro- artery --- p.36<br>Chapter 3.4. --- Drugs --- p.41<br>Chapter 3.4.1. --- Drugs --- p.41<br>Chapter 3.4.2. --- Preparation of oxyhemoglobin solution --- p.41<br>Chapter 3.5. --- Statistical Analysis --- p.42<br>Chapter 3.5.1. --- Calculation of EC50 --- p.42<br>Chapter 3.5.2. --- Statistical analysis --- p.42<br>Chapter Chapter 4: --- "Epoxyeicosatrienoic Acids (EET11,12) May Partially Restore EDHF-Mediated Function in Coronary Micro-Arteries" --- p.43<br>Chapter 4.1. --- Abstract --- p.43<br>Chapter 4.2. --- Introduction --- p.44<br>Chapter 4.3. --- Experimental Protocol --- p.45<br>Chapter 4.3.1. --- Precontraction --- p.45<br>Chapter 4.3.2. --- "EDHF-mediated (INDO, L-NNA, and HbO-resistant) relaxation" --- p.45<br>Chapter 4.3.3. --- "EET11,12-mediated relaxation after exposure to hyperkalemia" --- p.46<br>Chapter 4.3.4. --- "The effect of incubation with EET11,12 on the BK-induced, EDHF-mediated relaxation" --- p.46<br>Chapter 4.4. --- Results --- p.47<br>Chapter 4.4.1. --- Resting force --- p.47<br>Chapter 4.4.2. --- HbO and U46619-induced contraction force --- p.48<br>Chapter 4.4.3. --- "EET11,12-induced relaxation in coronary micro-arteries after exposure to hyperkalemia" --- p.49<br>Chapter 4.4.4. --- "The EDHF-mediated relaxation to BK resistant to INDO, l- NNA,and HbO" --- p.51<br>Chapter 4.4.4.1. --- Incubated in either hyperkalemic solution (K+ 20mmol/L) or Krebs' solution (control) --- p.51<br>Chapter 4.4.4.2. --- "Incubated in either hyperkalemic solution (K+ 20mmol/L) plus EET11,12 or Krebs' solution (control)" --- p.53<br>Chapter 4.5. --- Discussion --- p.57<br>Chapter 4.5.1. --- EDHF plays an important role in the coronary micro-arteries --- p.57<br>Chapter 4.5.2. --- "EDHF-mediated (INDO, l-NNA, and HbO-resistant) relaxation in the coronary micro-arteries" --- p.58<br>Chapter 4.5.3. --- "EET11,12 may partially mimic the EDHF-mediated relaxation in the porcine coronary micro-artery" --- p.59<br>Chapter 4.5.4. --- "Effect of EET11,12 added in hyperkalemia may partially restore the EDHF-mediated relaxation in the porcine coronary micro-arteries" --- p.59<br>Chapter Chapter 5: --- Impaired EDHF-Mediated Relaxationin Porcine Pulmonary Micro-arteries by Cold Store with Euro-Collin's and University of Wisconsin Solution --- p.61<br>Chapter 5.1. --- Abstract --- p.61<br>Chapter 5.2. --- Introduction --- p.62<br>Chapter 5.3. --- Experimental Protocol --- p.64<br>Chapter 5.3.1. --- Precontraction --- p.64<br>Chapter 5.3.2. --- "Role of EDHF-mediated (INDO, L-NNA and HbO-resistant) relaxation in porcine pulmonary micro-arteries by BK orA23187" --- p.64<br>Chapter 5.3.3. --- Effect of hyperkalemia or preservation solutions (EC or UW) on the EDHF-mediated relaxation by BK or A23187 --- p.65<br>Chapter 5.3.3.1. --- The effect of hyperkalemia --- p.65<br>Chapter 5.3.3.2. --- Effect of EC solution on the EDHF-mediated relaxation --- p.65<br>Chapter 5.3.3.3. --- Effect of UW solution on the EDHF-mediated relaxation --- p.66<br>Chapter 5.3.3.4. --- The effect of UW and EC solutions on the contractility of the pulmonary micro-artery --- p.66<br>Chapter 5.4. --- Results --- p.66<br>Chapter 5.4.1. --- Resting force --- p.66<br>Chapter 5.4.2. --- U46619-induced contraction force --- p.67<br>Chapter 5.4.3. --- Role of EDHF-mediated relaxation induced by BK or A23187 --- p.67<br>Chapter 5.4.4. --- The effect of hyperkalemia --- p.71<br>Chapter 5.4.5. --- Effect of EC solution on the EDHF-mediated relaxation --- p.72<br>Chapter 5.4.6. --- Effect of UW solution on the EDHF-mediated relaxation --- p.73<br>Chapter 5.4.7. --- The effect of UW and EC solution on the contractility of the pulmonary micro-artery --- p.73<br>Chapter 5.5. --- Discussion --- p.77<br>Chapter 5.5.1. --- EDHF-mediated endothelial function exists in the pulmonary micro-circulation --- p.77<br>Chapter 5.5.2. --- Hyperkalemia exposure reduces EDHF-related relaxation and possible mechanism --- p.78<br>Chapter 5.5.3. --- The effect of EC and UW solutions on the EDHF-media relaxation in the pulmonary micro-arteries --- p.79<br>Chapter Chapter 6: --- General Discussion --- p.82<br>Chapter 6.1. --- Endothelium-dependent vasodilators: BK and A23187 --- p.82<br>Chapter 6.2. --- EDHF in porcine coronary and pulmonary micro-arteries --- p.84<br>Chapter 6.2.1. --- EDHF in porcine coronary micro-arteries --- p.84<br>Chapter 6.2.2. --- EDHF in porcine pulmonary micro-arteries --- p.87<br>Chapter 6.2.3. --- Vascular stretch and release of endothelium-derived vasodilators --- p.87<br>Chapter 6.2.4. --- "EET11,12" --- p.88<br>Chapter 6.3. --- "Endothelium-dependent relaxation resistant to INDO, L- NNA, and HbO in porcine coronary and pulmonary microcirculation" --- p.89<br>Chapter 6.4. --- "Alteration of endothelium-dependent relaxation resistant to INDO, l-NNA, and HbO after exposure to hyperkalemia" --- p.90<br>Chapter 6.5. --- "Alteration of endothelium-dependent contraction resistant to INDO, L-NNA, and HbO after exposure to EC or UW solutions" --- p.91<br>Chapter 6.6. --- Clinical implications --- p.92<br>Chapter 6.7. --- Limitations --- p.93<br>Chapter 6.7.1. --- Common limitations --- p.93<br>Chapter 6.7.2. --- Limitation of in vitro study --- p.93<br>Chapter 6.8. --- Future work --- p.94<br>Chapter Chapter 7: --- Conclusion --- p.96<br>References --- p.98<br>Appendies<br>"Wei Zou, Qin Yang, Anthony PC Yim, & Guo-Wei He Epoxyeicosatrienoic acids (EET11,12) may partially restore endothelium- derived hyperpolarizing factor-mediated function in coronary micro- arteries. Annals of Thoracic Surgery. 2001; 72(12): 1970~1976."

Zhang Rongzhen.<br>"July 2004."<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (p. 130-176).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.<br>Abstracts in English and Chinese.

Anesthetics cause profound alterations in respiratory and cardiovascular systems. Our experiments demonstrated that different anesthetics caused different changes in blood pressure regulating components. The role of particular BP regulating systems was disclosed by their selective inhibition - sympathetic nervous system blocked by pentolinium (peripheral ganglionic blockade), renin-angiotensin system by captopril (angiotensin converting enzyme blocker) and nitric oxide production by L-NAME (nitric oxide synthase blocker). Components of blood pressure regulating mechanisms in conscious normotensive Wistar rats and spontaneously hypertensive rats were compared with four different groups of anesthetized rats by pentobarbital, ketamine-xylazine, chloralose-urethane and isoflurane. Each anesthesia caused different hemodynamic changes. If hemodynamic conditions should be similar to conscious rats, the most suitable anesthetic is pentobarbital. L-serine-induced effects represent endothelium-derived hyperpolarizing factor (EDHF)-mediated response, which is a type of endothelium-dependent regulation of vascular tone, independent of nitric oxide and prostacyclin production. Pronounced L-serine effects on blood pressure were shown in NO-deficient type of hypertension. Our study demonstrated its pronounced effects in...

The inner lining of blood vessels, the endothelium, once considered only to serve barrier and metabolic functions, also produces mediators which actively influence the underlying vascular smooth muscle. Because the endothelium normally produces vasorelaxant factors, such as endothelium-derived relaxing factor (EDRF), and vasocontracting factors, such as endothelin and thromboxane A$\sb2$ (TXA$\sb2$), the abnormal production or action of these factors may underlie vascular diseases including pulmonary and systemic hypertension, reperfusion injury, atherosclerosis, and endotoxic shock The pulmonary vascular bed exhibits an enormous capacity for adaptation to local stimuli, and the regulation of the pulmonary circulation is probably localized. Therefore, in this project it was hypothesized that local factors, particularly EDRF, could play an important role in the regulation of the pulmonary circulation. The role of these factors in the regulation of the pulmonary circulation was studied under conditions of controlled blood flow and constant left atrial pressure in isolated perfused rat lungs and in vivo in the intact-chest cat The EDRF released in response to acetylcholine and other stimuli is now widely believed to be nitric oxide (NO) or a labile nitroso compound. Like NO, EDRF activates soluble guanylate cyclase and stimulates increases in cyclic guanosine 3$\sp\prime$,5$\sp\prime$-cyclic monophosphate (cGMP) levels in vascular smooth muscle cells. In the present study, the NO synthesis inhibitor N$\sp\Omega$-nitro-L-arginine methyl ester (L-NAME) and the guanlyate cyclase inhibitor methylene blue (MB) increased pulmonary vascular resistance and selectively inhibited pulmonary vasodilator responses to exogenous acetylcholine, substance P, the cGMP-specific phosphodiesterase inhibitor Zaprinast; and to acetylcholine released endogenously. L-NAME selectively enhanced the pulmonary vasoconstrictor response to serotonin in the intact-chest cat and enhanced hypoxic pulmonary vasoconstriction in the isolated rat lung These data suggest that the basal production of endothelium-derived NO (EDNO) maintains the pulmonary vascular bed in a dilated state and is the major stimulus for the activation of soluble guanylate cyclase in the pulmonary vascular bed. Moreover, EDNO release and cGMP formation appear to play an important role in mediating vasodilator responses to endogenous and exogenous stimuli and in limiting responses to vasoconstrictor stimuli in the lung<br>acase@tulane.edu

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology.<br>Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11866

Yeung Lam.<br>Thesis submitted in: December 2003.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (leaves 134-150).<br>Abstracts in English and Chinese.<br>ABSTRACT --- p.I<br>摘要 --- p.III<br>ACKNOWLEDGMENT --- p.V<br>Chapter 1. --- Introduction --- p.1<br>Chapter 1.1 --- General review of angiogenesis --- p.1<br>Chapter 1.2 --- Vascular endothelial growth factors (VEGFs) --- p.2<br>Chapter 1.2.1 --- VEGF-A --- p.2<br>Chapter 1.2.2 --- P1GF --- p.4<br>Chapter 1.2.3 --- VEGF-B --- p.5<br>Chapter 1.2.4 --- VEGF-C and VEGF-D --- p.6<br>Chapter 1.3 --- VEGF receptors (VEGFRs) --- p.9<br>Chapter 1.3.1 --- VEGFR-1 (or flt-1) --- p.9<br>Chapter 1.3.2 --- VEGFR-2 ( or flk-1) --- p.10<br>Chapter 1.3.3 --- VEGFR-3 ( or flt-4) --- p.11<br>Chapter 1.4 --- Hormonal regulation of VEGFs by LH/hCG --- p.14<br>Chapter 1.5 --- General review of the testis --- p.17<br>Chapter 1.5.1 --- Structure and function of the testis --- p.17<br>Chapter 1.5.2 --- Testicular vasculature --- p.18<br>Chapter 1.5.3 --- Testicular angiogenesis --- p.19<br>Chapter 1.6 --- Localization of VEGF and VEGF receptors in the testis --- p.20<br>Chapter 1.7 --- Aims of the present study --- p.21<br>Chapter 2. --- Materials and methods --- p.23<br>Chapter 2.1 --- Animals --- p.23<br>Chapter 2.1.1 --- Depletion of Leydig cell --- p.23<br>Chapter 2.1.2 --- Suppression of Leydig cell and stimulation by hCG --- p.24<br>Chapter 2.1.3 --- Collection of tissue --- p.25<br>Chapter 2.2 --- Preparation of primary cells from rat testes --- p.27<br>Chapter 2.2.1 --- Sertoli cell preparation --- p.27<br>Chapter 2.2.2 --- Germ cell preparation --- p.29<br>Chapter 2.2.3 --- Interstitial cell and Leydig cell preparation --- p.30<br>Chapter 2.3 --- Cell cultures --- p.32<br>Chapter 2.3.1 --- Reagents and cell lines --- p.32<br>Chapter 2.3.2 --- "Mouse Leydig cell line, TM3 and Sertoli cell line, TM4" --- p.33<br>Chapter 2.3.3 --- "Mouse tumor Leydig cell line, MLTC-1" --- p.34<br>Chapter 2.3.4 --- "Rat tumor Leydig cell line, R2C" --- p.34<br>Chapter 2.3.5 --- "Rat tumor Leydig cell line, LC540" --- p.35<br>Chapter 2.4 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.35<br>Chapter 2.4.1 --- Extraction of total RNA --- p.35<br>Chapter 2.4.2 --- Quantitation of total RNA --- p.37<br>Chapter 2.4.3 --- RT-PCR --- p.37<br>Chapter 2.4.4 --- Purification and authentication of PCR products --- p.47<br>Chapter 2.5 --- Immunohistochemical staining --- p.48<br>Chapter 2.5.1 --- Perfusion and processing of testes for histological sections --- p.48<br>Chapter 2.5.2 --- Immunohistochemical staining of tissue sections --- p.50<br>Chapter 2.6 --- Western immunoblotting --- p.52<br>Chapter 2.6.1 --- Extraction and quantitation of total protein --- p.52<br>Chapter 2.6.2 --- SDS-PAGE --- p.53<br>Chapter 2.6.3 --- Immunoblotting --- p.55<br>Chapter 2.7 --- Statistical analyses --- p.57<br>Chapter 3. --- Results --- p.58<br>Chapter 3.1 --- Expression and localization of VEGFs in the rat testis --- p.58<br>Chapter 3.1.1 --- VEGF-A --- p.58<br>Chapter 3.1.2 --- VEGF-B --- p.64<br>Chapter 3.1.3 --- VEGF-C --- p.69<br>Chapter 3.1.4 --- VEGF-D --- p.73<br>Chapter 3.1.5 --- P1GF --- p.77<br>Chapter 3.2 --- Effect of Leydig cell depletion on VEGFs expression in the rat testis --- p.81<br>Chapter 3.2.1 --- Effect on VEGF-A --- p.81<br>Chapter 3.2.2 --- Effect on VEGF-B --- p.82<br>Chapter 3.2.3 --- Effect on VEGF-C --- p.88<br>Chapter 3.2.4 --- Effect on VEGF-D --- p.91<br>Chapter 3.2.5 --- Effect on P1GF --- p.94<br>Chapter 3.3 --- Effect of Leydig cell suppression and hCG stimulation on VEGFs expression in the rat testis --- p.97<br>Chapter 3.3.1 --- Effect on VEGF-A --- p.97<br>Chapter 3.3.2 --- Effect on VEGF-B --- p.107<br>Chapter 3.3.3 --- Effect on VEGF-C --- p.113<br>Chapter 3.3.4 --- Effect on VEGF-D --- p.119<br>Chapter 4. --- Discussion --- p.126<br>Chapter 5. --- References --- p.134

碩士<br>國防醫學院<br>解剖學研究所<br>84<br>In addition to well-characterized growth promoters or growth inhibitors, vasoconstrictors such as endothelin-11 ( ET-1 ) has been shown to promote VSMC growth, whereas vasodilators such as prostacyclin ( PGI2 ) has been described as growth inhibitor. According to our previous studies, directly adding vasoactive compounds to cultured VSMC has no obvious growth-regulatory effect on VSMC. In this study, the different combination of rat aortic endothelial cells ( EC ) and SMC from SHR and WKY rats were cocultured to maintain the effects of some compounds with extremely short half-life secreted from endothelium. After coculture, the effects of endothelium on SMC proliferation were studied. Using the 3H-thymidine incorporation test, we found that the SHR EC can enhance SHR SMC proliferation; however, it has no obvious growth- promoting effect on WKY SMC. on the other hand, WKY EC has no obvious growth promoting effect on both WKY and SHR SMC. Furthermore, the coculture medium of all groups were collected for detecting the concentration of ET-1, thromboxane A2 ( Tx A2 ), and PGI2 by enzyme immunoassay ( EIA ). According to the results, we know that the SHR SMC release more ET-1 compared with WKY SMC. In addition, the SHR EC tend to release more TxA2 and less PGI2 than WKY EC. Taken together, these results indicate that the different secretory ability of vasoactive factors in SHR and WKY may cause different growth- promoting effect on SMC. Furthermore, the EC and SMC may react synergetically to promote SHR SMC proliferation, but the growth- promoting effect of SMC itself is more important than the stimulation of endothelium - released vasoactive factors.

Thesis (M.Sc.)--University of Alberta, 2009.<br>A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on January 17, 2010). Includes bibliographical references.

Sun, Minghui.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.<br>Includes bibliographical references (leaves 125-159).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.

Le vieillissement vasculaire est caractérisé par une dysfonction de l’endothélium. De nombreux facteurs de risque cardiovasculaire tels que l’obésité et l’hypertension prédisposent l’endothélium à un stress oxydant élevé aboutissant à une dysfonction endothéliale, celle-ci étant communément accompagnée d’une diminution de la biodisponibilité du monoxyde d’azote. Bien que la fonction endothéliale soit un déterminant majeur de la prédiction du risque cardiovasculaire des patients, son évaluation individuelle reste très limitée. En conséquence, il existe un intérêt scientifique grandissant pour la recherche de meilleurs biomarqueurs. L’Angiopoiétine like-2 (angptl2), une protéine identifiée récemment, joue un rôle pro-inflammatoire et pro-oxydant dans plusieurs désordres causés par une inflammation chronique allant de l’obésité à l’athérosclérose. L’inflammation et un stress oxydant accru ont été établis comme des mécanismes sous-jacents à l’apparition d’une dysfonction endothéliale, c’est pourquoi ce travail met l’accent sur le rôle de l’angptl2 dans la dysfonction endothéliale. Plus précisément, ce travail vise à: 1) déterminer les effets aigus de l’angptl2 sur la fonction endothéliale, 2) caractériser la fonction endothéliale et la contribution des différents facteurs relaxants dérivés de l'endothélium (EDRF) dans plusieurs lits vasculaires, et ce, dans un modèle de souris réprimant l’expression de l’angptl2 (knock-down, KD), et 3) examiner si l'absence d'expression angptl2 protège contre la dysfonction endothéliale induite par un régime riche en graisses (HFD) ou par perfusion d'angiotensine II (angII) chez la souris. Dans la première étude, l’incubation aigue avec de l’angptl2 recombinante induit une dysfonction endothéliale dans les artères fémorales isolées de souris de type sauvage (WT), probablement en raison d’une production accrue d'espèces réactives oxygénées. Les artères fémorales de souris angptl2 KD présentent une meilleure fonction endothéliale en comparaison aux souris WT, vraisemblablement par une plus grande contribution de la prostacycline dans la vasodilatation. Après 3 mois d’une diète HFD, les principaux EDRF respectifs des artères fémorales et mésentériques sont conservés uniquement dans les souris angptl2 KD. Cette préservation est associée à un meilleur profil métabolique, une moindre accumulation de triglycérides dans le foie et des adipocytes de plus petite taille. De plus, l’expression de gènes inflammatoires dans ces tissus adipeux n’est augmentée que chez les souris WT. Dans la seconde étude, l’absence d’angptl2 résulte en une production accrue de monoxyde d’azote dans les artères cérébrales isolées par rapport à celles des souris WT. La perfusion chronique d’angII provoque, seulement chez les souris WT, une dysfonction endothéliale cérébrale probablement par le biais d’une augmentation de la production d’espèces réactives oxygénées, probablement dérivé des NADPH oxydase 1 et 2, ainsi que l'augmentation des facteurs constricteurs dérivés de l’endothélium issus de la cyclo-oxygénase. En revanche, l’apocynine réduit la dilatation cérébrale chez les souris KD traitées à l’angII, ce qui suggère le recrutement potentiel d’une voie de signalisation compensatoire impliquant les NADPH oxydases et qui aurait un effet vaso-dilatateur. Ces études suggèrent fortement que l’angptl2 peut avoir un impact direct sur la fonction endothéliale par ses propriétés pro-inflammatoire et pro-oxydante. Dans une optique d’application à la pratique clinique, les niveaux sanguins d’angptl2 pourraient être un bon indicateur de la fonction endothéliale.<br>Vascular aging is characterized by changes in the endothelium. Common cardiovascular risk factors, including obesity and hypertension, predispose the endothelium to increased oxidative stress, leading to endothelial dysfunction commonly characterized by diminished nitric oxide bioavailability. Although endothelial function can be a major determinant of cardiovascular risk prediction in patients, individual testing is still limited in clinical settings and thus there is increasing scientific interest in finding better biomarkers. Angiopoietin like-2 (angptl2), a recently identified protein, is a pro-inflammatory and pro-oxidative protein involved in chronic inflammatory disorders ranging from obesity to atherosclerosis. As inflammation and increased oxidative stress are established underlying mechanisms by which endothelial dysfunction occurs, this work focuses on the role of angptl2 in endothelial dysfunction, a topic that is largely unexplored. Specifically, this work aims to 1) determine the acute effects of angptl2 on endothelial function, 2) characterize endothelial function and contribution of different endothelium-derived relaxing factors in various vascular beds in a newly generated angptl2 knock-down (KD) mouse model, and 3) examine whether the lack of angptl2 expression protects against endothelial dysfunction induced by either a high-fat diet (HFD) or angiotensin II (angII) infusion in mice. In the first study, we show that a recombinant angptl2 acutely evokes endothelial dysfunction in the femoral artery isolated from wild-type (WT) mice, likely due to increased production of reactive oxygen species. Also in the femoral artery, angptl2 KD mice display better endothelial function compared to WT, which may be a result of greater prostacyclin contribution to vasodilation. After a 3-month HFD, the main respective endothelium-derived relaxing factors in the femoral and mesenteric arteries are preserved in angptl2 KD mice only, which was associated with a better metabolic profile, such as lower total cholesterol-to-high-density lipoprotein and low-density-to-high-density lipoprotein ratios compared to WT mice. After a HFD, KD mice have less triglyceride accumulation in the liver and smaller adipocytes in their mesenteric and epididymal white adipose tissues compared to WT mice, while inflammatory gene expressions in adipose tissues increase in WT mice only. In the second study, we reveal that the lack of angptl2 in KD mice results in greater nitric oxide production compared to WT mice in their isolated cerebral arteries. Chronic infusion of pro-inflammatory and pro-oxidative angII results in cerebral endothelial dysfunction only in WT mice, which is acutely ameliorated with either N-acetylcysteine, apocynin, or indomethacin, suggesting increased reactive oxygen species, likely derived from the NADPH oxidases 1/2, and increased cyclooxygenase-derived endothelium-derived contracting factors. In contrast, apocynin reduces cerebral dilation in angII-treated KD mice, suggesting recruitment of a potential compensatory dilatory NADPH oxidase pathway. These studies are the first to explore angptl2 contribution to endothelial dysfunction in different vascular beds, and strongly suggest that angptl2 can directly impair endothelial function by its pro-inflammatory and pro-oxidative properties. Translating this to the clinical setting, expression levels of angptl2 may be an indicator of endothelial function, and lowering angptl2 levels could become a potential therapeutic approach in the treatment of chronic inflammatory disorders including cardiovascular diseases.

Indiana University-Purdue University Indianapolis (IUPUI)<br>The impact of tobacco smoking on human health is well documented. The influence of smoking on tooth loss and cardiovascular diseases was investigated in the current study via both epidemiology and in vitro studies. From analyzing the 2006 Behavioral Risk Factor Surveillance System (2006 BRFSS) database, we confirmed that smoking was significantly associated with the number of teeth lost in a dose-dependent manner and smoking cessation reduced the risk when compared to those subjects continuing to smoke. In addition, the virulence factors related to caries were compared between Streptococcus mutans and Streptococcus gordonii in response to cigarette smoking condensate (CSC) treatment. We observed that S. gordonii was more susceptible to CSC treatment than S. mutans. CSC significantly enhanced S. mutans sucrose-dependent and independent adherence. Western blot assays revealed that several bacterial surface proteins including glucosyltransferase (GTF), glucan-binding proteins and antigen I/II, were significantly upregulated for the treated S. mutans. These findings suggested that the oral environment with CSC may favor a cariogenic dominant composition, which may increase the risk for smokers to develop caries. We also found that smoking and oral health status modified each other and synergistically increased the risk of CVD and this joint effect was more pronounced among the youngest age group using the 2006 BRFSS database. To further understand the joint effect, we conducted an in vitro study to investigate bacterial attachment to fibronectin and endothelial cells in response to smoking condensate treatment. Our study clearly demonstrated CSC significantly enhanced S. mutans attachment to both soluble and immobilized fibronectin as well as endothelial cells. Furthermore, our data suggested that bacteria possessed several adhesins that bound to host tissues and endothelial cells also had multiple receptors for bacterial attachment. Among these adhesins, antigen I/II seemed essential for bacterial attachment to endothelial cells without CSC. The knowledge of bacterial attachment to host tissues in the presence of CSC may help in developing different preventive or therapeutic strategies against attachment and colonization of the host by S. mutans.