The 3 PUFA, EPA and DHA, down-regulated human endothelial adhesion molecules in the presence of an inflammatory stimulus, DHA exerted more pronounced effects than EPA. The 6 PUFA, in contrast, exerted limited control over endothelial adhesion molecule expression under the conditions employed in this study. This indicates that the specific structure of the 3 PUFA, possibly combined with their degree of unsaturation, may be critical to their regulation of endothelial function. The antioxidant, quercetin, down-regulated some cytokine-induced endothelial adhesion molecules. In addition, quercetin acted synergistically with EPA and AA to decrease TNF--induced ICAM-1 or E-selectin protein, respectively. This may reflect regulation of eicosanoid production from these PUFA by quercetin, since this antioxidant inhibits enzymes critical to these metabolic pathways (i.e. cyclooxygenase and lipoxygenase). Hence, quercetin may mediate its inhibitory effects independently of its antioxidant properties. In contrast, -tocopheryl acetate up-regulated IL-1-induced E-selectin proteins levels. This antioxidant also, at least partially, blocked most of the inhibitory actions of the PUFA investigated. Recent data in the literature indicate that PUFA may exert their inhibitory effects on endothelial function through their oxidised derivatives. This provides a putative pathway via which -tocopheryl acetate may block the PUFA effects. The strength and type of inflammatory stimulus determined the sensitivity of the activated endothelial cells to each of the agents investigated. Inhibition of leukocyte adhesion to activated HUVEC, in response to EPA, DHA, or AA in the presence of quercetin, demonstrated a direct effect of these combined agents on endothelial function. The individual agents did not significantly reduce leukocyte adhesion.

nÃo hà / A Alpinia zerumbet à uma planta comumente encontrada na regiÃo do nordeste do Brasil. Essa planta à conhecida popularmente por colÃnia e à muito utilizada na medicina popular por suas propriedades diurÃticas e anti-hipertensivas. O objetivo do presente estudo foi investigar se o extrato aquoso das folhas de A. zerumbet (EAAz) apresentava atividade vasodilatadora em aorta de rato prÃ-contraÃda com fenilefrina e, elucidar o seu mecanismo de aÃÃo. Ratos machos Wistar (250 a 300 g), provenientes do biotÃrio da UFC, foram sacrificados por deslocamento cervical e a aorta torÃcica removida e dissecada. Os anÃis da aorta (4 a 5 mm) foram montados em cÃmeras orgÃnicas, contendo soluÃÃo de Krebs e aerados com carbogÃnio a 37ÂC, para as medidas de variaÃÃes na tensÃo isomÃtrica. O endotÃlio dos anÃis da aorta foi removido mecanicamente para estudar o seu envolvimento no efeito vasodilatador do EAAz. Para estudar o envolvimento do Ãxido nÃtrico (NO), GMPc, prostanÃides, canais de potÃssio ativados por Ca2+, espÃcies reativas do oxigÃnio e a ativaÃÃo das enzimas quinases Src e PI3 quinase, as preparaÃÃes foram tratadas, respectivamente, com L-NAME (100 ÂM), ODQ (10 ÂM), indometacina (10 ÂM), caribdotoxina (100 nM) mais apamina (100 nM), MnTMPyP (100 ÂM), catalase (500 U/ml), PEG-catalase (500 U/ml), SOD (500 U/ml) e PP2 (20 ÂM), Wortmannin (300 nM). O efeito vasodilatador do EAAz (0,05; 0,15; 0,5; 1,5; 15 e 50 Âg/mL), acetilcolina (10-8 - 10-5 M) e nitroprussiato de sÃdio (10-8 M) foram avaliados em aorta de ratos prÃ-contraÃda com fenilefrina (10-8 - 3x10-8 M), antes e apÃs tratamento com os inibidores especÃficos. A vasodilataÃÃo induzida pelo EAAz mostrou-se dependente do endotÃlio e mediada pelo NO via GMPc jà que o relaxamento vascular foi abolido na presenÃa do Ãster metÃlico de NG-nitro-l-arginina e do ODQ. A vasodilataÃÃo tambÃm foi reduzida pelo MnTMPyP (permeante celular mimÃtico da SOD), polietilenoglicol catalase, PP2 (inibidor da quinase Src) e Wortmannin (inibidor da fosfoinositideo 3-quinases). A via de produÃÃo de EROs induzida pelo EAAz foi inibida significativamente pela aÃÃo do MnTMPyP e PEG-catalase. Ocorreu tambÃm a inibiÃÃo da via EROs/PI3 quinase/Src quinase que estÃo envolvidas na ativaÃÃo da eNOS. O EAAz causou um relaxamento dependente do endotÃlio via NO/GMPc com o possÃvel envolvimento de EROs e das Src quinase e fosfoinositideo 3-quinase dependente de Akt. Dessa forma, pode-se concluir que o relaxamento vascular induzido pelo EAAz poderia explicar o tradicional uso do chà das folhas de A. zerumbet para o tratamento da hipertensÃo arterial. / The Alpinia zerumbet is a plant commonly found in the northeastern region of Brazil. This plant is popularly known as colony and is widely used in folk medicine for its diuretic and antihypertensive properties. The aim of this study was to investigate whether the aqueous extract of leaves of A. zerumbet (EAAz) had vasodilator activity in rat aorta pre-contracted with phenylephrine and to elucidate its mechanism of action. Male Wistar rats (250-300 g) from the vivarium of UFC, were killed by cervical dislocation and the thoracic aorta removed and dissected. The aortic rings (4-5 mm) were mounted in organ cameras, and containing Krebs solution aerated with carbogen at 37  C to measure changes in isometric tension. The endothelium of the aortic rings was removed mechanically to study their involvement in the vasodilator effect of EAAz. To study the involvement of nitric oxide (NO), cGMP, prostanoid, potassium channels activated by Ca2+, reactive oxygen species and activation of enzymes kinases Src and PI3 kinase, the preparations were treated respectively with L-NAME (100 ÂM ), ODQ (10 ÂM), indomethacin (10 ÂM), carybdotoxin (100 nM) plus apamin (100 nM), MnTMPyP (100 ÂM), catalase (500 U / ml), PEG-catalase (500 U/ ml), SOD (500 U / ml) and PP2 (20 ÂM), wortmannin (300 nM). The vasodilatory effect of EAAz (0.05, 0.15, 0.5, 1.5, 15 and 50 Âg / mL), acetylcholine (10-8 - 10-5 M) and sodium nitroprusside (10-8 M ) were evaluated in the aorta of rats pre-contracted with phenylephrine (10-8 - 3x10-8 M) before and after treatment with the specific inhibitors. EAAz induced vasodilation was dependent and endothelium mediated by NO via cGMP as the vascular relaxation was abolished in the presence of the methyl ester of NG-nitro-L-arginine and ODQ. Vasodilation was also reduced by MnTMPyP (mimetic cellular permeant SOD), catalase polyethylene glycol, PP2 (Src kinase inhibitor) and wortmannin (inhibitor of phosphoinositide 3-kinase). The route of production of ROS induced EAAz was significantly prevented by the action of MnTMPyP and PEG-catalase. There was also the inhibition of the kinase ROS/PI3 / Src kinase that are involved in the activation of the eNOS. The EAAz caused an endothelium-dependent relaxation via NO / cGMP with the possible involvement of ROS and Src kinase and phosphoinositide 3-kinase-dependent Akt. Taking us to the conclusion that the vascular relaxation induced by EAAz could explain the traditional use of the tea leaves of A. zerumbet for the treatment of hypertension.

Doctor of Philosophy / This thesis describes work done at the Institute of Dental Research in Sydney between February of 1986 and January 1990. The broad subject of the work is the role of vascular endothelial cells (ECs) in chronic inflammation. Periodontitis has been used as an example of chronic inflammatory disease, and provides the focus for this study of endothelial biology. In Chapter 1, aspects of the endothelial literature which provide relevant background information for work described in later chapters are reviewed. In Chapter 2, literature relating to aetiology and pathogenesis of chronic inflammatory periodontal disease is discussed. To maintain relevance of literature reviews to experimental work, each subsequent chapter contains a small literature review of material relating to the subject of the specific chapter. Early laboratory work is described in Chapter 3, and consisted of a morphological survey of the vascular changes occurring in gingival tissues with development of chronic periodontitis. Expansion of the vasculature and appearance of phenotypically specialised high endothelial cells (HECs), were associated with progression of the disease. Vessels with HECs and had a similar appearance to those known to be responsible for lymphocyte recirculation described in lymphoid tissues and chronic inflammatory sites. In the course of performing this survey, a perivascular hyaline material was noted surrounding capillaries close to the bacterial plaque irritant. The incidence, distribution, extent, ultrastructre and immuo-histochemistry of this material was more closely investigated, and the possible pathogenesis and significance of the material discussed in Chapter 4. In Chapter 5, the ultrastructural, histochemical and functional properties of gingival HECs are described, and compared with the well characterised HECs of rat lymph nodes. It was found that periodontal vessels were very similar to those in rat lymph nodes, with the exception however, that the gingival vessels appeared to exchange polymorphonuclear leukocytes almost exclusively, while vessels with HECs in lymph nodes and other locations are known as sites of lymphocyte recirculation. This observation indicated that the function of HECs requires further investigation, with particular regard to the synthetic activity of the cells. HECs were consistently alkaline phosphatise (AP) negative. The negative association between leukocyte emigration and AP activity (APA), as well as evidence in the literature illustrating both the wide substrate specificity of this enzyme and the importance of phosphorylation in the control of protein activation, suggested that AP could play a role in regulating leukocyte emigration. A pre-requisite for the investigation of this possibility, is the identification of a rich source of the identical iso-enzyme of AP to what is present in ECs. In Chapter 6, the sensitivity of endothelial AP to a panel of inhibitors is compared with that of a number of tissues for which isoenzyme has been identified. Endothelial AP was identified as the liver/bone/kidney isoenzyme. This allows the use of kidney tissue as a relevant source of AP for use in further study of the role of this enzyme in EC biology. It was clear that in order to study both the synthetic activity of HECs, as well as the role of AP in the control of leukocyte emigration, a method for obtaining high density primary cultures of HECs had to be established. Chapter 7 describes work done towards the development of such a culture system. The availability in the latter phase of the work of suitable probe for the technique of the in-situ hybridization allowed the possibility of testing the hypothesis that HECs are important cytokine producers. It was felt that this would provide some basis for the further study of those cells in-virtro. This work is described in the appendix. The general discussion in Chapter 8, summarises the work, and develops potential areas of study arising from the finding of this thesis.

La circulation des cellules, du sang vers les tissus, est guidée par des molécules exprimées spécifiquement au niveau des cellules endothéliales constituant les vaisseaux sanguins. Les molécules conférant la spécificité de la localisation sont différentes selon l'organe et le microenvironnement considérés. A partir de lignées endothéliales spécifiques d'organes, les études menées sur le rôle des chimiokines et de leurs récepteurs dans la spécificité de l'endothélium ont montré que l'activité biologique de certaines chimiokines est restrictive à une lignée endothéliale en terme de recrutement des lymphocytes et par leur capacité à augmenter la formation de vaisseaux sanguins (angiogénèse) in vitro. De plus, notre modèle d'inflammation cutanée basé sur les cellules endothéliales de la peau, a permis des études plus appliquées mettant en évidence des molécules anti-inflammatoires vis-à-vis de l'endothélium de la peau en collaboration avec la Société BioEurope du groupe Solabia.

Stroke is the third most common leading cause of death worldwide and is a major cause of serious long-term disability among adults. Platelet activation and it¡¦s interaction with leukocytes plays an important role in the pathophysiology of ischemic stroke. Anti-platelet drugs are widely used for secondary prevention after cerebral ischemia of non-cardioembolic origin and different anti-platelet drugs exert different pharmacologic effects on platelets. Several stimulations cause elevation of cytosolic Ca2+ level ([Ca2+]i), activating the secondary messenger in the platelet, and then leading to platelet activation. The majority of damage following acute stroke does not occur immediately, but rather develops gradually over the course of the following hours. Leukocytes are believed to liberate inflammatory cytokines and other neurotoxins in the ischemic brain and to promote microvascular occlusion through platelet-leukocyte-endothelium interactions in the ischemic penumbra. In this thesis, we tested the serial changes of platelet [Ca2+]i movement in patients after acute ischemic stroke. We evaluated platelet activation markers, leukocyte adhesion molecules and platelet-leukocyte interaction in patients with acute ischemic stroke. We also analyzed the relationship between these biomarkers and the clinical outcome. Furthermore, we compared the antiplatelet effect of aspirin and clopidogrel in patients after acute stroke Dysregulation of Ca2+ Movement in Platelets from Patients with Acute Ischemic Stroke Thirty-one patients with acute ischemic stroke and 27 at-risk controls were enrolled in this study. The platelet [Ca2+]i was measured using a fluorescent dye fura-2 after stimulation with arachidonic acid (AA), adenosine diphosphate (ADP), platelet-activation factor (PAF), and thrombin. The basal [Ca2+]i was higher in the stroke group than in the at-risk controls irrespective of the presence or absence of extracellular Ca2+. In the Ca2+-containing medium, both PAF and ADP, but not AA and thrombin, significantly increased the platelet [Ca2+]i in the stroke patients than in the at-risk controls. However, in the Ca2+-free medium, only PAF significantly increased the platelet [Ca2+]i in the stroke patients than in the at-risk controls. The basal [Ca2+]i and PAF-induced platelet [Ca2+]i rises were still higher in the stroke patients at the subacute stage than in the at-risk controls. Levels and Value of Platelet Activation Markers in Different Subtypes of Acute Non-Cardio-Embolic Ischemic Stroke Platelet activation markers (CD62P, CD63, and CD40L) and platelet-leukocyte interaction were measured by flow cytometry at different time points in 54 (32 small-vessel and 22 large-vessel diseases) acute ischemic stroke patients, 28 convalescent stroke patients (3 to 9 months after acute stroke), and 28 control subjects. Patients with ischemic stroke had significantly increased circulating CD62P, CD63, platelet-monocyte interaction, and platelet-lymphocyte interaction in the acute stage compared with the convalescent stage and control groups. Levels of CD62P and CD63 were significantly higher in the large-vessel disease group than in the small-vessel disease group, and differences in CD62P were significant even at one month. The CD40L level in the poor outcome group was significantly higher than that in the good outcome group. The Value of Leukocyte Adhesion Molecules in Patients after Ischemic Stroke We examined serially the change of PSGL-1, Mac-1, and LFA-1 expression on leukocytes by using flow cytometry at various time points in 65 acute ischemic stroke patients and 60 controls. PSGL-1 expression on neutrophils and monocytes was significantly higher from Day 1 to Day 90 after stroke as compared with control subjects. The expression of monocyte Mac-1, LFA-1, and neutrophil Mac-1 were more significantly increased on Day 1 and 7 after stroke as compared with the control subjects. Furthermore, the neutrophil PSGL-1 expression on admission was independently associated with early neurologic deterioration. Serial Changes in Platelet Activation Markers with Aspirin and Clopidogrel after Acute Ischemic Stroke We designed a prospectively randomized case-control study and 70 patients with non-cardioembolic stroke who were treated with either aspirin (100 mg/d) or clopidogrel (75 mg/d) after acute ischemic stroke were evaluated. Ischemic stroke patients had significantly increased circulating CD62P, CD63, and CD40L in the acute stage as compared to the control group. Levels of CD62P, CD63 and CD40L were more significantly reduced in the clopidogrel group than in the aspirin group in the first week after stroke. Furthermore, differences in CD62P and CD63 levels were significant even at one-month post-stroke. Conclusion Stroke patients have enhanced platelet activity and platelet-leukocyte interaction in acute and convalescent phase of ischemic stroke compared with controls. Large-vessel cerebral infarction elicits higher platelets activation than small-vessel infarction in the acute phase of stroke. The dysregulation of Ca2+ movement, through the PAF- and ADP- receptor mediated pathway, in platelets may persist up to the subacute stage of ischemic stroke. It is possible that clopidogrel, an ADP-receptor inhibitor, elicit stronger antiplatelet effect than aspirin in the acute and convalescent phases after ischemic stroke. Sustained leukocyte-endothelium interaction, as reflected by expression of Mac-1 and LFA-1 on circulating leukocytes, may cause substantial inflammatory reaction and lead to secondary injury of potentially salvageable neurons after cerebral infarction. Ischemic stroke patients presenting with a higher CD40L level on admission were associated with a 3-month poor outcome.

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

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Diabetic patients have an increased risk of developing both micro- and macrovascular complications. It is thought that the primary lesion in the genesis of these complications is at the level of the vascular endothelium. The effects of diabetes on endothelial function can be monitored in vitro using aortae isolated from streptozotocin-diabetic rats. This project found that two months of untreated diabetes resulted in a profound deficit in maximum endothelium-dependent relaxation to cumulative doses of acetylcholine in phenylepherine precontracted aortae. Treatment of rats with the antioxidants vitamin E and trientine from the induction of diabetes prevented the development of the deficit in endothelium-dependent relaxation, suggesting that increased levels of oxidative stress in diabetes contribute to abnormal endothelial function. Compensation for impaired essential fatty acid metabolism in diabetic rats with evening primrose oil partially prevented the development of defective aortic endothelium-dependent relaxation, implication this hyperglycaemia-induced metabolic disturbance in diabetic endothelial dysfunction. Diabetes is also associated with impaired penis erectile performance and importance in animals and patients. An in vitro preparation was developed to examine the effects of streptozotocin-induced diabetes on chemical stimulation of the endothelium and electrical stimulation of the autonomic nerves of rat corpus cavernosum. Both endothelium and neurally mediated relaxation responses were found to be impaired in two month untreated diabetic rats. Treatment of diabetic rats with the antioxidants α-lipoic acid and trientine prevented the formation of defective endothelium and nerve relaxation responses, thus implicating oxidative stress in the pathogenesis of these impairments. Inhibition of defective polyol pathway activity also prevented the development of reduced endothelium and nitrergic nerve responses, thus suggesting a role for this role for this well studied metabolic abnormality in impaired corpus cavernosum function in experimental diabetes.