La radiothérapie constitue l’un des principaux traitements pour l’éradication des cancers. Cependant, elle présente un risque d’effets secondaires aux tissus sains environnant la tumeur. Dans ce processus, le système vasculaire et plus particulièrement l’endothélium jouent un rôle clé. Les cellules endothéliales activées favorisent le recrutement chronique des thrombocytes et des leucocytes, contribuant ainsi aux effets secondaires. D’autre part, dans les maladies inflammatoires, les glycanes exprimés à la surface des cellules endothéliales sont modifiés et influencent le recrutement des cellules immunitaires. Dans cette étude, nous avons évalué la modification des glycanes endothéliaux en réponse à une irradiation à forte dose, et étudié les effets fonctionnels de ces modifications sur le recrutement des leucocytes en utilisant un modèle de cellules endothéliales (HUVECs) in vitro. Nos résultats apportent les premières preuves d’une modification du glycome des cellules endothéliales en réponse à l’irradiation. Les N-glycanes hautement mannosylés, les O-glycanes et les motifs sialylées sont surexprimés. Parallèlement, le glycocalyx endothélial semble subir une dégradation. Nous avons évalué l’effet fonctionnel des modifications glycanique des cellules endothéliales irradiées sur l’adhésion d’une lignée de monocyte (THP-1). Nos résultats montrent que l’adhésion radio-induite est en partie due à la surexpression endothéliale des N-glycanes hautement mannosylés. Nous avons aussi évalué le glycome sur un modèle de souris irradiées et sur des pièces opératoires de patients traités par radiothérapies. Nos résultats de transcriptomiques sur la souris suggèrent l’existence de modifications glycaniques radio-induites in vivo. L’intégration de la composante glycanique permet de porter un regard nouveau sur le continuum d’évènement qui conduit aux lésions tissulaires radio-induites. A l’avenir, l’étude du glycome pourrait ouvrir de nouvelles pistes thérapeutiques pour une meilleure prise en charge des effets secondaires de la radiothérapie. / Radiotherapy is one of the main treatments against cancers. However, it presents a risk of adverse effects for the normal tissues surrounding the tumors. The vascular network and especially the endothelium are considered as main targets to limit normal tissue damages and prevent side effects of radiotherapy. Activated endothelial cells are involved in the chronic recruitment of thrombocytes and leukocytes, resulting in tissue complications. On the other hand, in inflammatory diseases, the glycans expressed on the surface of endothelial cells are modified and lead to immune cells recruitment. We sought to evaluate changes in endothelial glycome in a context of exposure to high dose of radiation, and studied the functional consequences on the recruitment of leukocytes. In vitro, the characterization of the glycome was performed on a primary endothelial cell model (HUVEC). Our results provide the first evidences of an endothelial modification of the glycome after exposure to ionizing radiation. We report an overexpression of high mannose N-glycans, O-glycans and syalilated motifs. At the same time, endothelial glycocalyx appeared to be damaged by exposure to radiation. Next, we evaluated these radiation-induced modifications of endothelial glycans on monocyte adhesion. We show that the radiation induced adhesion was mediated by overexpression of high mannose N-glycans. We also investigated changes in glycome in an irradiated mouse model of enteropathy and in resections of patients treated with radiotherapy. In mice, a transcriptomic study suggests changes in glycans following radiation exposure. Collectively, these findings on glycome changes provide a new perspective of the continuum of events leading to normal tissue complications. In the future, the study of the glycome should open new therapeutics opportunities for better management of tissue damages induced by radiation.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Atherosclerosis preferentially develops in vascular regions of low or disturbed flow and high spatial gradients. Endothelial cells that line the vessel walls actively participate in translating mechanical stimuli, shear stress due to fluid flow, into intracellular signals to regulate cellular activities. Atherosclerosis is a chronic disease. During its development, a cascade of inflammatory signals alters the arterial endothelial homeostatic functions. Death-associated protein (DAP) kinase and its correlated pathway have been associated with cell apoptosis, turnover, and cytoskeleton remodeling in cellular networks, ultimately leading to changes in cell motility and vascular wall permeability. DAP-kinase is also highly regulated by inflammatory triggers such as TNF-α. This thesis investigates DAP-kinase modulation due to shear stress, and the role of DAP-kinase activity in endothelial responses toward applied shear stress. Using bovine aortic endothelial cells (BAEC), DAP-kinase expression is demonstrated in both sheared (10 dynes/cm2) and static conditions. Overall DAPK expression increased with extended shearing, while the presence of phosphorylated DAPK decreased with applied shear stress, as demonstrated in Western blot analysis. In correlation, DAPK RNA expression profiles were explored to understand pre-translational behavior and to understand just how shear stress influences DAPK expression over time. There is a temporal increase in DAPK mRNA, occurring at earlier time points when compared to DAPK protein expression, displaying the precedence of mRNA expression leading to increased translation into protein. From our apoptosis assay results, shear stress reduces apoptotic and late stage/necrotic cell fractions. The exposure of shear stress potentially plays a role in inhibiting apoptosis activation and TNF-α induced death cascade. Overall, the apoptosis activity influenced by shear further exhibits a possible connection between shear stress and apoptosis inhibition. The shear stress ultimately decreases overall apoptosis, while DAPK expression is increased. Therefore, DAPK may have a function in other possible mechano-transduction cascades, when endothelial cells are exposed to constant shear. Our data suggests shear stress modulation of DAP-kinase expression and activity, and the potential crosstalk of mechano-transduction and DAPK/apoptosis pathway, may lead to further understanding the responsibility of DAPK in endothelial cell function.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Endothelial injury and dysfunction precede the development of cardiovascular diseases. The endothelium may be regarded as the first line of defence against inflammation / obesity-induced vascular injury, therefore gaining more information on the mechanisms of injury and response to injury, as well as modulating endothelial function may be key in the prevention of cardiovascular diseases. Endothelial cells differ in structure and function, therefore endothelial heterogeneity may be relevant when investigating endothelial function and dysfunction. Understanding endothelial heterogeneity in response to pathophysiological stimuli may be of significance in the prevention of cardiovascular diseases. Oleanolic acid (OA), a plant-derived triterpenoid, has been shown to possess endothelio-protective properties; however, its role in reversing endothelial injury is poorly understood. This study investigated endothelial heterogeneity between aortic endothelial cells (AECs) and cardiac microvascular endothelial cells (CMECs) at baseline and in response to an inflammatory insult via the cytokine, tumour necrosis factor-alpha (TNF-α). An in vitro model of endothelial injury was developed by treating AECs and CMECs with 20 ng/ml TNF-α for 24 hours. Endothelial heterogeneity was investigated by comparing intracellular nitric oxide (NO) and reactive oxygen species (ROS) production, protein expression and phosphorylation, and large-scale protein expression and regulation in AECs and CMECs. The experimental techniques included flow cytometry, western blots and proteomic analyses. An ex vivo model of endothelial injury was included to investigate vascular function in aortic rings from lean and high fat diet (HFD) rats. The role of OA in reversing TNF-α-induced injury and modulating vascular function in the ex vivo model was investigated. Although baseline NO-levels were similar between AECs and CMECs, heterogeneity was observed with regards to the NO biosynthetic pathway in terms of increased eNOS expression in CMECs. Baseline ROS levels were heterogeneous between AECs and CMECs, interestingly CMECs possessed higher anti-oxidant capacity. An in vitro model of TNF-α-induced injury was confirmed by decreased NO-levels, increased ROS-levels and necrosis, up-regulation of apoptotic proteins and activation of inflammatory pathways in AECs and CMECs. Here, heterogeneity between AECs and CMECs was also observed: endothelial activation was mediated through different proteins in AECs (CD9 molecule, galectin) and CMECs (ICAM-1 and IL-36α). Apoptosis was mediated by caspase 3 in AECs and Bid in CMECs. AECs appeared to advance to a dysfunctional state shown by up-regulation of endothelin-converting enzyme and angiotensin II-converting enzyme, while CMECs maintained an activated state. OA reversed TNF-α-induced injury through restoring NO-production, decreasing ROS-production in both AECs and CMECs, and inhibiting necrosis in AECs. In the ex vivo model of injury, aortic rings from 16-week HFD rats showed a pro-contractile response to phenylephrine-induced contraction, a response that was reversed by OA. In conclusion, we demonstrated novel findings with regards to endothelial heterogeneity between AECs and CMECs under baseline and TNF-α-treated conditions. Although reduced NO-bioavailability may be the hallmark of endothelial dysfunction, signalling pathways mediating endothelial injury may differ between cell types as was shown in this study. We demonstrated that OA possess protective properties in AECs and CMECS, an observation which was translated to the ex vivo model. / AFRIKAANSE OPSOMMING: Endoteelbesering en –disfunksie gaan die ontwikkeling van kardiovaskulêre siektes vooraf. Die endoteel word as die eerste linie van verdediging teen inflammasie / vetsug-geïnduseerde vaskulêre skade beskou; dus is die ontginning van nuwe inligting betreffende die meganismes van en respons tot besering, asook die modulering van endoteelfunksie essensieël in die voorkoming van kardiovaskulêre siektes. Endoteelselle verskil t.o.v. struktuur en funksie, en dus is endoteel-heterogeniteit relevant tydens die ondersoek van endoteelfunksie en –disfunksie. ‘n Beter begrip van endoteel-heterogeniteit in die respons tot patofisiologiese stimuli kan betekenisvol tot die voorkoming van kardiovaskulêre siektes bydra. Oleanoliese suur (OA), ‘n triterpenoïed afkomstig van plante is voorheen bewys om endoteelbeskermende eienskappe te besit; die rol van OA in die omkering van endoteelbesering is egter minder bekend. Hierdie studie het endoteel-heterogeniteit tussen aorta endoteelselle (AECs) en hart mikrovaskulêre endoteeelselle (CMECs) by basislyn en in respons tot ‘n inflammatoriese besering via die sitokien, tumor nekrose faktor-alfa (TNF-α), ondersoek. ‘n In vitro model van endoteelbesering is ontwikkel deur AECs en CMECs met 20 ng/ml TNF-α vir 24 uur te behandel. Endoteel-heterogeniteit was ondersoek deur intrasellulêre stikstofoksied (NO) en reaktiewe suurstofspesies (ROS) produksie, proteïenuitdrukking en fosforilering, en grootskaalse proteïenuitdrukking en regulering in AECs en CMECs te vergelyk. Die eksperimentele tegnieke het ingesluit: vloeisitometrie, western blots en proteomika. ‘n Ex vivo model van endoteelbesering was ook ingesluit deur die vaskulêre funksie in aortaringe van normale en hoë vet dieet-gevoerde (HFD) rotte te meet. Die rol van OA in die omkering van TNF-α-geïnduseerde besering en modulering van vaskulêre funksie was in hierdie model is ondersoek. Alhoewel basislyn NO-vlakke vergelykbaar was in AECs en CMECs, is heterogeniteit wel aangetoon m.b.t. die NO biosintese pad met verhoogde eNOS uitdrukking in die CMECs. Basislyn ROS-vlakke was verskillend in AECs en CMECs en die CMECs het hoër anti-oksidant kapasiteit getoon. ‘n In vitro model van TNF-α-geïnduseerde besering is bevestig met die waarneming van verlaagde NO-vlakke, verhoogde ROS-vlakke en nekrose, opregulering van apoptotiese proteïene en aktivering van inflammatoriese paaie in AECs en CMECs. Hier was heterogeniteit ook opmerkbaar: endoteelaktivering was deur verskillende proteïene in AECs (CD9 molekule, galektien) en CMECs (ICAM-1, IL-36α) bemiddel. Apotose was deur kaspase 3 in AECs en Bid in CMECs bemiddel. Dit het geblyk dat AECs tot ‘n staat van endoteeldisfunksie gevorder het met die opregulering van endotelien-omsettingsensiem en angiotensien II-omsettingsensiem, terwyl CMECs eerder ‘n geaktiveerde staat gehandhaaf het. OA het TNF-α-geïnduseerde besering omgekeer deur NO-produksie te herstel, ROS-produksie te onderdruk in beide AECs en CMECs, en nekrose te inhibeer in AECs. In die ex vivo model van besering, het aortaringe van 16-week HFD rotte ‘n pro-kontraktiele respons tot fenielefrien-geïnduseerde kontraksie getoon, wat deur OA omgekeer is. Ten slotte, nuwe bevindinge is waargeneem m.b.t. endoteel-heterogeniteit tussen AECs en CMECs onder basislyn en TNF-α-behandelde omstandighede. Alhoewel verlaagde NO-biobeskikbaarheid die waarmerk van endoteeldisfunksie is, het hierdie studie getoon dat seintransduksiepaaie wat endoteelbesering medieer verskillend is tussen seltipes. Die studie het verder ook gedemonstreer dat OA beskermende eienskappe toon in AECs en CMECs, ‘n waarneming wat ook in die ex vivo model aangetoon kon word.

La fonction de barrière de l'endothélium vasculaire est affectée par des modifications de la cadhérine des cellules endothéliales (VE-cadhérine) telles que la phosphorylation sur tyrosine dans son domaine cytoplasmique et le clivage de son domaine extracellulaire (sVE). Ce travail s'est articulé en deux parties : 1- Etude de ces modifications dans le contexte de la polyarthrite rhumatoïde (PR), et les mécanismes sous-tendus. Ce travail a permis de montrer que la VE-cadhérine est une cible du TNFα, une cytokine essentielle dans la PR, qui induit de la libération de sVE de façon dépendante de l'activité kinase de Src, suggérant l'implication d'un mécanisme de phosphorylation sur tyrosine dans ce processus. De plus, la VE-cadhérine est aussi la cible des protéases de la matrice extracellulaire telles que MMP-2. L'application de ces données fondamentales à la clinique a permis de montrer que sVE était retrouvée dans le sang de patients PR (n=63) et que son taux était corrélé à l'activité de la maladie. Ainsi, le dosage de sVE est d'intérêt dans la prise en charge des patients. 2-Etude de la phosphorylation de la VE-cadhérine dans un contexte d'angiogenèse hormono-régulée au cours du cycle ovarien chez la souris. Les résultats ont montré que le site Y685 de la VE-cadhérine est phosphorylé dans l'ovaire et l'utérus de souris de façon régulée pendant le cycle, ce qui permet de proposer ce modèle physiologique pour étudier la phosphorylation de la VE-cadhérine in vivo. L'analyse de souris knock-in Y685F de la VE-cadhérine (VE-Y685F) a montré que l'absence du site confère une perméabilité accrue dans l'ovaire et l'utérus mais aussi dans les petits capillaires de la peau. De plus, dans deux modèles d'induction d'arthrite, les souris VE-Y685F ont présenté un taux de sVE plus élevé que les contrôles. Au total, sVE et la phosphorylation de la VE-cadhérine ont un vaste champ d'application dans le traitement de la PR ainsi que pour le développement de nouvelles thérapies pouvant s'étendre à d'autres pathologies vasculaires. / The vascular endothelium barrier function is influenced by vascular endothelial cadherin (VE-cadherin) modifications such as its cytoplasmic tail tyrosine phosphorylation and its ecto-domain cleavage (sVE). The work reported herein was divided into two parts: 1- Study of VE-cadherin modifications and the mechanisms underlying these ones in the rheumatoid arthritis context. This work demonstrated that VE-cadherin is a target of TNFα, a highly important cytokine in rheumatoid arthritis (RA) pathogenesis. We found TNFα to induce sVE shedding in a Src kinase dependent manner, suggesting the involvement of phosphorylation mechanism in this process. In addition, this VE-cadherin cleavage requires matrix metalloproteinase activities such as that of MMP-2. Applying these fundamental data to clinical study showed that sVE was detected in sera of 63 RA patients and its titer was correlated with the disease activity. This finding suggests an obvious interest for assaying sVE in RA therapies. 2- Study of VE-cadherin tyrosine phosphorylation in a context of hormones-regulated angiogenesis during mouse estrous cycle. The results showed VE-cadherin phosphorylation at Y685 that was regulated along mouse estrous cycle allowing to proposing mouse estrous cycle as a physiological model for studying VE-cadherin phosphorylation in vivo. The analysis of VE-cadherin Y685F knock-in mice (VE-Y685F) showed that these mice exhibit a higher vascular permeability in uterus and ovaries and the skin small capillaries compared to wild-type animals. Moreover, VE-Y685F mice presented higher levels of soluble VE-cadherin compared to wild-type counterparts in two induced arthritis model. Altogether, sVE and VE-cadherin phosphorylation present an array of interests for RA therapies as well as developing new therapeutic tools in RA and other vascular diseases.

Receptores ativados por protease (PARs) pertencem à família de GPCRs. Desses, PAR-1, PAR-3 e PAR-4 são ativados por trombina, e PAR-2 por tripsina. Como as proteases, peptídeos sintéticos (PARs-AP) também ativam esses receptores. Estudamos o papel dos PARs na reatividade vascular de Wistar e SHR. In vitro, PAR-1 AP, promoveu maior vasoconstrição em aorta com endotélio (E+) de SHR vs Wistar. PAR-2 AP promoveu vasodilatação similar em aorta E+ de SHR e Wistar, enquanto PAR-4 AP e peptídeos reversos não causaram efeito. In vivo/in situ PAR-1 e PAR-2 AP mostraram intensa vasomotilidade em arteríolas mesentéricas. A expressão gênica de PAR-1 está aumentada em aorta e arteríolas de SHR, mas a expressão protéica está aumentada apenas em arteríolas. Demonstramos ainda que a vasoconstrição induzida por PAR-1 AP, é dependente de Ca++ e da liberação de Ang II, ET-1 e O2- pelo endotélio. Assim, sugerimos que PAR-1 pode ser um alvo terapêutico para novos antihipertensivos com efeito antitrombótico, já que este receptor também tem sido envolvido em eventos romboembólicos. / Protease activated receptors are a new GPCRs family. The PAR-1, PAR-3 and PAR-4 are activated by thrombin and PAR-2 by tripsin. Like proteases, synthetic peptides (PARs-AP) can also activate those receptors. We studied the role of PARs in vascular reactivity of Wistar and SHR. In vitro, PAR-1 promoted higher vasoconstriction to PAR-1 AP in SHR aorta with endothelium (E+) than the Wistar ones. PAR-2 AP produced similar vasodilation in Wistar and SHR aorta E+, while neither PAR-4 nor reverse peptides presented any effect. In vivo/in situ PAR-1 and PAR-2 showed an intensive vasomotion in mesenteric vessels. PAR-1 gene expression was increased in SHR aorta and arterioles, while the protein expression was increased only in the arterioles. We have also shown that the vasoconstriction induced by PAR-1 AP, is Ca++-dependent and Ang II, ET-1 and O2- release from endothelium. Thus, we suggest that PAR-1 might represent a therapeutic target to new antihypertensive drugs with antithrombotic effect, since this receptor has been involved in thromboembolics events.

A esclerodermia sistêmica (ES) é caracterizada por fibrose da pele e órgãos internos, ativação imunológica e vasculopatia. Os modelos animais de ES sofrem com a falta de uma prova definitiva para o envolvimento vascular observado na doença humana. Um novo modelo induzido por colágeno do tipo V (COLV) reproduz muitas características da ES como fibrose e fenômenos imunológicos. No entanto, o estudo da vasculopatia ainda não foi abordado. O objetivo desse estudo foi investigar as alterações estruturais e funcionais das células endoteliais das artérias pulmonares do modelo de ES induzido pelo COLV, assim como determinar a doença pulmonar com ênfase especial no depósito de colágeno e síntese de RNAm de colágenos. Coelhos fêmeas Nova Zelândia (n = 8) foram imunizados com COLV humano emulsificado em adjuvante de Freund ou apenas com adjuvante de Freund (controle; n = 8). Após 7, 75 e 210 dias, os animais foram sacrificados e os pulmões foram analisados por microscopia eletrônica, hematoxilina e eosina, e marcações especiais incluindo imunomarcação para neovascularização (CD31), apoptose de células endoteliais (induzida pela caspase-3) e atividade endotelial (endotelina-1 e VEGF). A resposta vascular a acetilcolina (Ach) foi estudada em anéis de artéria pulmonar isolada. Para determinar o conteúdo de colágeno, expressão RNAm dos colágenos I, III e V e quantidade de colágeno (hidroxiprolina aminoácido específico), os pulmões foram submetidos a imunofluorescência, PCR em tempo real e análise bioquímica. Foi observado que os coelhos imunizados com COLV apresentaram um aumento progressivo de apoptose e atividade das células endoteliais a partir de 7 dias quando comparados aos coelhos controle (p 0,01). Em contrapartida, apenas aos 210 dias os coelhos imunizados com COLV apresentaram aumento significativo na neovascularização quando comparados com o grupo controle (p < 0,001). Coincidente com esses achados, a microscopia eletrônica mostrou alterações das células endoteliais caracterizada por apoptose, alterações degenerativas das organelas e tumefação citoplasmática. As células endoteliais pareciam estar destacadas da membrana basal. Os coelhos imunizados com COLV de 210 dias necessitaram de uma dose maior de Ach do que os controles para alcançar o relaxamento máximo de 50% (EC50) dos anéis de artéria pulmonar (p = 0,02). Por fim, foi observado que os coelhos de 210 dias imunizados com COLV apresentaram uma intensa expressão de COLV no interstício broncovascular, acompanhado de um aumento na expressão do RNAm (p < 0,001) quando comparado ao grupo controle. Os coelhos imunizados com COLV apresentaram uma maior quantidade de conteúdo de colágeno, quando comparados com os controles, nos diferentes tempos estudados (p < 0,01). Este estudo fornece evidência para a disfunção pulmonar do endotélio vascular em coelhos com ES induzida pelo COLV. As alterações observadas, nesse estudo, reforçam o papel do endotélio como evento patogênico primário na ES. Desta forma, o modelo ES induzido pelo COLV pode fornecer informações importantes sobre a patogênese da doença humana / The hallmarks of systemic sclerosis (SSc) are fibrosis of skin and internal organs, immune activity and vasculopathy. Animal models of SSc suffer from lack of a definitive evidence for vascular involvement observed in human disease. A novel model induced by collagen type V (COLV) reproduces many features of SSc, however vascular study has not been addressed. The aim of the present study was therefore evaluate pulmonary arteries for structural and functional alterations in endothelial cells in the COLV-induced SSc model, as well as determine the lung disease with special emphasis on collagen deposition and mRNA collagen synthesis. Female New Zealand rabbits (n = 8) were immunized with human COLV/Freund´s adjuvant or Freund´s adjuvant alone (control; n = 8). After 7, 75 and 210 days, the animals were sacrificed and the lungs were examined by electron microscopy, hematoxylin&eosin and special stains including immunostaining for neovascularization (CD31), endothelial cells apoptosis (caspase-3 induced) and endothelial activity (endothelin-1 and VEGF). Vascular response to acetylcholine (Ach) on isolated pulmonary artery rings was evaluated. To determine collagen content, mRNA expressions of COL I, III and V, and the quantity of collagen-specific amino acid hydroxyproline, the lungs were submitted to immunofluorescence, real-time qPCR and biochemical examination. The COLV rabbits demonstrated an endothelial cells apoptosis and activity compared to control rabbits starting at day-7 (p 0.01). A significant increase in neovascularization was observed only in the COLV-rabbits at day-210 (p < 0,001). Coincident with these findings, the electron microscopy revealed extensive endothelial cells abnormalities characterized by apoptosis, degenerative organelle changes and cytoplasmic tumefaction. Endothelial cells appeared to be detached from the basement membrane. Ach dose required to achieve 50% maximum relaxation (EC50) of pulmonary artery rings was increased in COLV rabbits at day-210 (p = 0.02). The content of hydroxyproline was increased in COLV rabbits compared with that observed in control rabbits, at the different days studied (p < 0,01). Ultimately, the COLV rabbits at 210-day showed an intense expression of COLV in the bronchovascular interstitium, followed by a markedly up-regulation of COLV mRNA (p < 0.001) as compared to controls. This study provides evidence for pulmonary vascular endothelial cell dysfunction in rabbits with COLV-induced SSc. The findings of this study reinforce the role of endothelial cells as a primary pathogenic event in SSc. The COLV model may provide insight into the pathogenesis of human disease

A relativa raridade de casos graves de malária no Brasil sugere que os isolados locais de Plasmodium falciparum tenham menor virulência que os parasitas africanos e asiáticos. Este trabalho investigou os padrões de aderência de sete isolados de P. falciparum, provenientes de uma área da Amazônia brasileira em que a malária grave é rara, a dois receptores do endotélio vascular, CD36 e ICAM-1. Também analisamos a resposta de anticorpos de indivíduos locais contra oitos antígenos de superfície, incluindo isolados de campo e a cepa 3D7. Mostramos que: (a) de modo geral, os isolados locais de P. falciparum expressam VSAs capazes de aderir tanto a ICAM-1 quanto a CD36; (b) detectamos anticorpos contra antígenos apresentados por isolados de campo e pela cepa 3D7 entre moradores de uma área endêmica próxima à origem dos isolados; (c) vimos que alguns dos soros testados foram capazes de bloquear a adesão de hemácias parasitadas a ICAM-1 e CD36, in vitro; (d) detectamos uma baixa prevalência do alelo S (hemoglobina S) na população de estudo. / The relative rarity of severe malaria in Brazil suggests that the local Plasmodium falciparum isolates are less virulent than African and Asian parasites. This work investigated adherence patterns to CD36 and ICAM-1, two receptors of the vascular endothelium, in P. falciparum isolates from an area of the Brazilian Amazonia, where severe malaria is rare. We also analyzed, in the same area, the antibody responses of people against eight VSAs. We found that: (a) local P. falciparum isolates express VSAs capable to adhere to both receptors, CD36 and ICAM-1; (b) we detected antibodies against VSAs in a human population exposed to malaria, expressed from local parasites and the 3D7 control; (c) we found in vitro that some sera contained naturally acquired antibodies which blocked the adherence of the parasitized RBCs to ICAM-1 and CD36; (d) we detected a low frequency of the S allele (hemoglobin S) in the study population.

Yau Ho Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 131-144). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENTS --- p.II / ENGLISH ABSTRACT --- p.III / CHINESE ABSTRACT --- p.VI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Oxidative Stress --- p.1 / Chapter 1.1.1 --- Historical Background of reactive oxygen/nitrogen species --- p.1 / Chapter 1.1.2 --- What is Oxidative Stress? --- p.3 / Chapter 1.1.3 --- Reactive Oxygen Species (ROS) --- p.4 / Chapter 1.1.3.1 --- Superoxide anion (02-) --- p.4 / Chapter 1.1.3.2 --- Hydrogen peroxide (H202) --- p.5 / Chapter 1.1.3.3 --- Hydroxyl radical --- p.6 / Chapter 1.1.3.4 --- Nitric oxide (NO) --- p.7 / Chapter 1.2 --- Cardiovascular System --- p.8 / Chapter 1.2.1 --- Enzymatic and Non-enzymatic Sources of ROS in Cardiovascular System --- p.8 / Chapter 1.2.1.1 --- NADPH oxidase --- p.8 / Chapter 1.2.1.2 --- Hypoxanthine-Xanthine oxidase (HX-XO) --- p.9 / Chapter 1.2.1.3 --- Nitric oxide synthase (NOS) --- p.10 / Chapter 1.2.1.4 --- Mitochondrial electron transport chain (ETC) --- p.11 / Chapter 1.2.1.5 --- Cyclooxygenase --- p.11 / Chapter 1.2.1.6 --- Lipoxygenae --- p.12 / Chapter 1.2.1.7 --- Endoplasmic reticulum --- p.12 / Chapter 1.2.2 --- ROS/RNS Scavenging Systems --- p.13 / Chapter 1.2.2.1 --- Superoxide dismutase (SOD) --- p.13 / Chapter 1.2.2.2 --- Catalase --- p.14 / Chapter 1.2.2.3 --- Glutathione peroxidase --- p.15 / Chapter 1.2.2.4 --- Non-enzymatic antioxidants --- p.15 / Chapter 1.2.3 --- Factors that stimulate ROS production in cardiovascular system --- p.18 / Chapter 1.2.3.1 --- Oxygen tension --- p.18 / Chapter 1.2.3.2 --- "Flow, Shear, and Stretch as an initial stimulus for endothelial oxidant signalling" --- p.18 / Chapter 1.2.3.3 --- Activation of rennin-angiotensin system promote oxidative stress in cardiovascular system --- p.19 / Chapter 1.2.3.4 --- Regulation of vascular ROS production by vasoactive substances --- p.19 / Chapter 1.2.4 --- Regulation of vascular tone in Cardiovascular System by ROS/RNS --- p.20 / Chapter 1.2.4.1 --- Regulation of vascular tone --- p.20 / Chapter 1.2.5 --- Pathophysiological Effects of ROS --- p.23 / Chapter 1.2.5.1 --- Cellular injury by lipid peroxidation --- p.23 / Chapter 1.2.5.2 --- Role of ROS in immune defence --- p.23 / Chapter 1.2.5.3 --- Redox regulation of cell adhesion --- p.24 / Chapter 1.2.6 --- Evidences from Clinical Studies of Oxidative Stress-Related Vascular Diseases --- p.25 / Chapter 1.2.6.1 --- Hyperlipidaemia --- p.25 / Chapter 1.2.6.2 --- Hypertension --- p.25 / Chapter 1.2.6.3 --- Chronic heart failure (CHF) --- p.26 / Chapter 1.2.6.4 --- Chronic renal failure (CRF) --- p.26 / Chapter 1.2.6.5 --- Atherosclerosis --- p.27 / Chapter 1.2.6.6 --- Ischemia/reperfusion (I/R) injury --- p.27 / Chapter 1.2.7 --- Role of Vascular Endothelium in Oxidative Stress --- p.29 / Chapter 1.2.8 --- Role of Ca in oxidative stress in cardiovascular system --- p.29 / Chapter 1.2.8.1 --- Calcium Signaling in Vascular Endothelial Cells --- p.30 / Chapter 1.2.9 --- ROS effect on endothelial Ca2+ --- p.31 / Chapter 1.2.9.1 --- Multiple targets of ROS on intracellular Ca2+ mobilization --- p.32 / Chapter 1.2.9.2 --- Reports of H202-induced Ca2+ release in various cell types --- p.33 / Chapter 1.2.9.3 --- Reported effects of H202 on agonist-induced Ca2+ signal --- p.34 / Chapter 1.2.9.4 --- Differences between macrovessels and microvessels --- p.34 / Chapter 1.3 --- TRP Channel --- p.41 / Chapter 1.3.1 --- Discovery of Drosophila TRP --- p.41 / Chapter 1.3.2 --- Mammalian TRP subfamily --- p.41 / Chapter 1.3.3 --- General topology of TRP channel --- p.42 / Chapter 1.3.4 --- Interactions of oxidative stress with TRP channels --- p.44 / Chapter 1.3.5 --- The role of TRPC3 and TRPC4 in oxidative stress --- p.44 / Chapter 1.3.6 --- TRPM subfamily --- p.44 / Chapter 1.3.6.1 --- Expression of TRPM2 --- p.45 / Chapter 1.3.6.2 --- Dual Role of TRPM´2ؤChannel and Enzyme --- p.45 / Chapter 1.3.6.3 --- Regulatory mechanisms of TRPM2 --- p.46 / Chapter 1.3.6.3.1 --- ADP-ribose (ADPR) directly regulating --- p.46 / Chapter 1.3.6.3.2 --- NAD regulating --- p.46 / Chapter 1.3.6.3.3 --- Oxidative stress regulating independent of ADPR or NAD --- p.47 / Chapter 1.4 --- Cell Death Induced by Oxidative Stress --- p.48 / Chapter 1.4.1 --- Redox status as a factor to determine cell death --- p.48 / Chapter 1.4.2 --- Role of TRPM2 in oxidative stress-induced cell death --- p.48 / Chapter 1.5 --- Aims of the Study --- p.49 / Chapter Chapter 2: --- Materials and Methods --- p.50 / Chapter 2.1 --- Functional Characterization of TRPM2 by Antisense Technique --- p.50 / Chapter 2.1.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.1.2 --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.51 / Chapter 2.1.3 --- "Ligation of TRPM2 Genes into Mammalian Vector, pcDNA3" --- p.52 / Chapter 2.1.4 --- Transformation for the Desired Clones --- p.52 / Chapter 2.1.5 --- Plasmid DNA Preparation for Transfection --- p.53 / Chapter 2.1.6 --- Confirmation of the Clones --- p.53 / Chapter 2.1.6.1 --- Restriction Enzymes Strategy --- p.53 / Chapter 2.1.6.2 --- Polymerase Chain Reaction (PCR) Check --- p.54 / Chapter 2.1.6.3 --- Automated Sequencing --- p.55 / Chapter 2.2 --- Establishing Stable Cell Lines --- p.56 / Chapter 2.2.1 --- Cell Culture --- p.56 / Chapter 2.2.2 --- Geneticin Selection --- p.57 / Chapter 2.3 --- Expression of TRPM2 in Transfected and non-Transfected H5V Cells --- p.57 / Chapter 2.3.1 --- Protein Sample Preparation --- p.57 / Chapter 2.3.2 --- Western Blot Analysis --- p.58 / Chapter 2.3.3 --- Protein Expression Analysis --- p.59 / Chapter 2.4 --- "Immunolocalization of TRPM2 in Human Heart, Cerebral Artery, Renal, Hippocampus and Liver" --- p.59 / Chapter 2.4.1 --- Paraffin Section Preparation --- p.59 / Chapter 2.4.2 --- Immunohistochemistry --- p.60 / Chapter 2.5 --- [Ca2+ ］i Measurement in Confocal Microscopy --- p.62 / Chapter 2.5.1 --- Cytosolic Ca2+ measurement --- p.62 / Chapter 2.5.2 --- Measuring the Ca2+ in the Internal Calcium Stores --- p.63 / Chapter 2.5.3 --- Data Analysis --- p.64 / Chapter 2.6 --- Examining Cell Death Induced by H2O2 by DAPI Staining --- p.65 / Chapter 2.6.1 --- DAPI Staining --- p.65 / Chapter Chapter 3: --- Results --- p.66 / Chapter 3.1 --- Superoxide Anion-Induced [Ca 2+]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.66 / Chapter 3.1.1 --- Superoxide Anion-induced [Ca2+ ]i Rise --- p.66 / Chapter 3.1.2 --- Effect of Catalase on the Superoxide Anion-induced [Ca2+]i］］ Rise --- p.66 / Chapter 3.1.3 --- IP3R inhibitor Inhibits Superoxide anion-induced [Ca 2+]i Rise --- p.67 / Chapter 3.1.4 --- Effect of Phospholipase A2 Inhibitor on Superoxide anion- induced [Ca2+]i Rise --- p.67 / Chapter 3.1.5 --- Effect of Hydroxyl Radical Scavenger on Superoxide Anion- induced [Ca2+]i Rise --- p.68 / Chapter 3.2 --- Hydrogen Peroxide-induced Ca2+ Entry in Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.1 --- Hydrogen Peroxide Induces [Ca2 +]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.2 --- Hydrogen Peroxide Induces [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.74 / Chapter 3.2.3 --- Hydrogen Peroxide Induces [Ca 2+]i rise in a Extracellular Ca + Concentration Dependent Manner --- p.77 / Chapter 3.3 --- Hydrogen Peroxide Reduces Agonist-induced [Ca2+]i rise --- p.79 / Chapter 3.3.1 --- Hydrogen Peroxide Reduces ATP-induced [Ca2+ ]i rise in a H2O2 Concentration Dependent Manner --- p.79 / Chapter 3.3.2 --- Hydrogen Peroxide Reduces ATP-induced [Ca 2+]i rise in a H2O2 Incubation Time Dependent Manner --- p.79 / Chapter 3.3.3 --- Hydrogen Peroxide Reduces the ATP-induced Intracellular Ca2+ Release --- p.80 / Chapter 3.3.4 --- XeC Inhibited H202-induced [Ca2+]i rise --- p.80 / Chapter 3.3.5 --- Hydrogen Peroxide Partially Depletes Internal Ca2+ Stores --- p.81 / Chapter 3.4 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.82 / Chapter 3.4.1 --- Effect of Phospholipase C Inhibitor on H202-induced [Ca2 +]i rise --- p.82 / Chapter 3.4.2 --- Effect of Phospholipase A2 Inhibitor on H202-induced [Ca 2+]i rise --- p.83 / Chapter 3.4.3 --- Effect of hydroxyl radical scavenger on H2O2-induced [Ca 2+]i rise --- p.83 / Chapter 3.5 --- Functional Role of TRPM2 Channel in H202-induced [Ca2+]i Rise in H5V Cells --- p.92 / Chapter 3.5.1 --- Expression of TRPM2 and the Effect of TRPM2 Antisense Construct on TRPM2 Protein Expression --- p.92 / Chapter 3.5.2 --- Effect of Antisense TRPM2 on H202-induced Ca2+ Entry --- p.94 / Chapter 3.6 --- H202-induced Cell Death --- p.101 / Chapter 3.7 --- Expression Pattern of TRPM2 Channel in Vascular System --- p.104 / Chapter 3.7.1 --- Immunolocalization of TRPM2 in Human Cerebral Arteries --- p.104 / Chapter 3.7.2 --- Immunolocalization of TRPM2 in Human Cardiac Muscles --- p.105 / Chapter 3.7.3 --- Immunolocalization of TRPM2 in Human Kidney --- p.105 / Chapter Chapter 4: --- Discussion --- p.113 / Chapter 4.1 --- Oxidative modification of Ca2+ homeostasis --- p.113 / Chapter 4.2 --- Pathophysiological effects of ROS on endothelium --- p.113 / Chapter 4.3 --- Effects of ROS on microvascular endothelial Ca2+ reported by other investigators --- p.115 / Chapter 4.4 --- Studies of the effect of HX-XO on cytosolic [Ca2+]i --- p.116 / Chapter 4.4.1 --- Role of 0´2Ø- and H202 in HX-XO-induced [Ca2+]i elevation --- p.116 / Chapter 4.4.2 --- IP3R involvement in HX-XO-evoked Ca + movements in H5V cells --- p.118 / Chapter 4.4.3 --- PLA2 involvement in HX-XO experiment --- p.119 / Chapter 4.5 --- Studies of the effect of direct H202 application on cytosolic [Ca2+]i --- p.120 / Chapter 4.5.1 --- Hydrogen Peroxide Induced [Ca2 +]i rise in a Extracellular Ca2 + Concentration Dependent Manner --- p.120 / Chapter 4.5.2 --- Hydrogen Peroxide Induced [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.121 / Chapter 4.6 --- Effect of H202 on ATP-induced Ca2+ response --- p.121 / Chapter 4.6.1 --- H202 inhibited ATP-induced Ca2+ release in a concentration and time dependent manner --- p.121 / Chapter 4.6.2 --- IP3R involvement and store depletion in H202 experiment --- p.123 / Chapter 4.7 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.124 / Chapter 4.7.1 --- PLC involvement in H2O2 experiment --- p.124 / Chapter 4.7.2 --- PLA2 involvement in H2O2 experiment --- p.125 / Chapter 4.7.3 --- Hydroxyl radical did not involve in H2O2 experiment --- p.125 / Chapter 4.8 --- Functional Studies of TRPM2 --- p.127 / Chapter 4.8.1 --- Expression of TRPM2 in H5V on protein level --- p.127 / Chapter 4.8.2 --- TRPM2 involvement in the Ca2+ signalling in response to H2O2 in H5V cells --- p.127 / Chapter 4.9 --- H202 concentration in my projec´tؤphysiological or pathological? --- p.128 / Chapter 4.10. --- H20´2ؤTRPM´2ؤCell death --- p.129 / Chapter 4.11 --- Expression of TRPM2 in human blood vessels and other tissues --- p.130 / References --- p.131

Based on the results aforementioned, I went on in the second part of the study to examine the impact of aging on EDCF-mediated contractions - the alterations of COX-2-mediated endothelium-dependent contractions and the associated release of prostaglandin(s) in the aortae from aged (>18 month-old) hamsters. Endothelium-dependent contractions in the presence of NG-nitro-L-arginine methyl ester (L-NAME) were significantly greater in the aortae from aged hamsters and contractions could also be observed without L-NAME, which were sensitive to COX-2 inhibitors and TP receptor antagonists. The levels of COX-2 expression, the release of PGF2alpha and vascular sensitivity to PGF 2alpha were augmented in aortae of aged hamsters. The present results indicate a positive impact of aging on COX-2-derived PGF2alpha-mediated endothelium-dependent contractions. / In the first part of the study, I investigated whether COX-2 participated in the occurrence of endothelium-dependent contractions in the aortae from young (-3 month-old) hamsters and identified the most possible EDCF. Endothelium-dependent contractions were elicited by acetylcholine and abolished by COX-2 inhibitors (NS-398, DuP-697 and celecoxib) and thromboxane-prostanoid (TP) receptor antagonists (S 18886, L-655,240 and GR 32191), but not by COX-1 inhibitors (valeryl salicylate and sc 560). RT-PCR and Western blot analysis using aortae with and without endothelium revealed that the COX-2 expression was localized mainly in the endothelium. Levels of prostangladin F2alpha (PGF2alpha ) and prostacyclin (PGI2) increased in response to acetylcholine and the release of both prostaglandins was inhibited by COX-2 but not COX-1 inhibitors. Exogenous PGF2alpha but not PGI2 caused contractions at a concentration that corresponded to the amount released endogenously. The release of PGF2alpha was not affected by the presence of nitric oxide (NO). The results of the present study suggest that a novel constitutive role of COX-2 in endothelium-dependent contractions, with its metabolites PGF2alpha acting as a physiological EDCF in the young hamster aortae. / In the third part of the study, I investigated the relationship and the intracellular signaling cascades linking two pro-inflammatory factors Ang II and COX-2, and tested whether COX-2 mediated the Ang II-induced vascular pathogenesis. Eight hour-incubation with 100 nmol/L Ang II resulted in maximal COX-2 expression in primary rat endothelial cells and it was inhibited by losartan and RNA synthesis inhibitor (actinomycin-D). Inhibitors of either p38 MAPK or ERK1/2 (respectively SB 202190 and PD 98059) decreased the COX-2 expression, and co-treatment with both inhibitors caused an additive effect, suggesting a joint mediation through both kinases. Protein kinase C (PKC) inhibitor (GF109203X), and particularly, the specific PKCdelta inhibitor (rottlerin), prevented Ang II-induced phosphorylation of ERK1/2 and COX-2 expression, indicating an upstream regulation of ERK1/2 by PKC delta. A pivotal role of PKCdelta in Ang II-induced COX-2 expression was further supported by a similar stimulatory effect of PKC activator, signified by the Ang II-stimulated translocation of PKCdelta to the membrane and confirmed by its phosphorylation (Tyr311). Small interfering RNA targeting PKCdelta (siPKCdelta) diminished COX-2 expression, which was abrogated in siPKCdelta-treated cells treated with SB 202190, confirming the parallel pathways of PKC delta-ERK1/2 and p38 MAPK. Aortae and renal arteries from Ang II-infused rats exhibited an increased endothelial COX-2 expression and impaired acetylcholine-induced relaxation that was normalized by celecoxib. Human mesenteric arteries incubated with Ang II demonstrated elevated endothelial COX-2 and MCP-1 expressions, of which the former was inhibited by SB 202190 plus rottlerin and the latter prevented by COX-2 inhibitor celecoxib. Renal arteries from hypertensive or diabetic patients revealed an exaggerated expression of COX-2 and MCP-1 in the endothelium. The present novel findings indicate that the activation of PKCdelta-ERK1/2 and p38 MAPK is critical in Ang II-induced COX-2 up-regulation in endothelial cells, and identify a COX-2-dependent pro-atherosclerotic cytokine MCP-1. (Abstract shortened by UMI.) / Wong, Siu Ling. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 192-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.