Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Pollman, Matthew, Committee Member ; Galis, Zorina, Committee Member ; McIntire, Larry, Committee Member ; Taylor, W Robert, Committee Member ; Jo, Hanjoong, Committee Member ; Nerem, Robert, Committee Chair.

Thesis (Ph. D.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2006. / Dr. Peter A. Lane, Committee Member ; Dr. Larry V. McIntire, Committee Member ; Dr. Ronald W. Rousseau, Committee Member ; Dr. James R. Eckman, Committee Member ; Dr. Timothy M. Wick, Committee Chair.

Cell-derived microvesicles (MVs) are naturally released into the human circulation and an increase in the concentration of certain MV populations have been observed after exercise. However, the MV appearance dynamics, the exercise-related stimuli that induce their formation and physiological relevance are poorly understood. Hence, the overall objectives of this thesis were to: 1) characterise the circulating platelet (PMV) and endothelial-derived MVs (EMVs) responses during exercise and recovery, as well as their arteriovenous dynamics, 2) investigate the potential role of haemodynamic forces on MVs formation in vivo by vascular shear stress manipulations, and 3) explore the putative proliferative, chemotactic and angiogenic potential of exercise-derived MVs upon human vascular endothelial cells in vitro. Chapter 5 of this thesis describes the time-course of MV appearance in response to prolonged cycling, and demonstrates that intravascular [PMV] increases during and after exercise performed in the heavy intensity domain, whereas [EMV] remains unaltered. Moreover, [PMV] during exercise was related to estimates of vascular shear stress and plasma noradrenaline levels. Results from chapter 6 revealed that PMVs increased in the arterial circulation during passive heat stress, and in the arterial as well as venous circulation during short duration very heavy exercise engaging either a large or small muscle mass. The increases in [PMV] were not directly linked to local changes in vascular shear stress through heat stress and exercise, indicating a systemic PMV response. Finally, chapter 7 revealed that exercise-derived MVs supported endothelial proliferation and migration, while displaying pro-angiogenic potential in vitro. In conclusion, results of this thesis provide original information about MV dynamics, by demonstrating that PMV increase systemically in the circulation not only after but during exercise involving a small and large muscle mass. This MV response seems to be modulated by exercise intensity, and is only partially linked to levels of vascular shear stress. Moreover, circulating MVs produced during exercise present stimulatory angiogenic and mitogenic effects upon endothelial cells in vitro, suggesting a novel potential link between vascular adaptation and exercise training.

Os efeitos vasculares da associação de estrogênios e testosterona, utilizada para tratamento do distúrbio de desejo sexual hipoativo na pós-menopausa ainda não estão elucidados. O objetivo do trabalho foi avaliar as respostas vasculares ao tratamento de ratas espontaneamente hipertensas ovariectomizadas (SHR-OVX) com o conjugado estrogênio equino (CEE) associado ou não a cipionato de testosterona (CEE+T). O tratamento de SHR-OVX com CEE promove melhora da função endotelial na aorta por mecanismos que envolvem a redução da geração de espécies reativas de oxigênio (EROs) e aumento da expressão proteica de enzimas antioxidantes. O tratamento com CEE+T inibe os efeitos vasculoprotetores do CEE sobre o endotélio, aumentando a geração de EROs e diminuindo a expressão da enzima óxido nítrico sintase. A maior geração de EROs na aorta do grupo CEE+T parece depender da ativação de receptores AT1 de angiotensina II, da maior ação de fator inflamatório o 20-HETE e da ativação da enzima NADPH oxidase. / The vascular effects of estrogens and testosterone association, used for hypoactive sexual desire disorder treatment in postmenopausal women, have not been elucidated. The aim of this study was to evaluate the vascular effects of female ovariectomized (OVX) SHR treatment with conjugated equine estrogen (CEE) associated or not with testosterone cypionate (CEE+T). Our data shows that treatment of OVX-SHR with CEE improved endothelial function reducing reactive oxygen species (ROS) generation and increasing some antioxidant cellular mechanisms. Treatment with CEE+T blunted the vascular effects of CEE, increasing ROS generation and reducing eNOS expression. The increased ROS in CEE+T rats aorta seems to involve in angiotensin II-AT1 activation, 20-HETE action and NADPH oxidase activation.

Este estudo investigou o efeito da obesidade sobre a resposta de artérias mesentéricas a agonistas canabinóides. Ratos obesos Zucker (OZRs) apresentaram reduzido relaxamento à anandamida, aos agonistas CB1 e CB2 e à capsaicina (agonista vanilóide) comparados aos controles (LZRs). A expressão dos receptores CB1 e CB2 foi menor em OZRs. O bloqueio de canais de K+, a inibição da NOS, da COX ou do transporte de canabinóides reduziu a resposta à anandamida em LZRs. A resposta à anandamida em OZRs foi corrigida após: inibição da degradação de anandamida, ativação da via do cAMP e da AMPK, e inibição da ERK1/2. A anandamida aumentou a fosforilação da AMPK, da ACC e da eNOS em LZRs, mas reduziu em OZRs. A expressão da ERK1/2 fosforilada, maior em OZRs, foi potencializada pela anandamida. A obesidade diminui o relaxamento à anandamida por: reduzir a expressão de receptores CB1 e CB2; prejudicar respostas mediadas por receptores vanilóides; reduzir a captação e aumentar a degradação de anandamida; reduzir a ativação da AMPK e da eNOS e aumentar da ativação da ERK1/2. / This study aimed to investigate the effects of obesity on the response of mesenteric arteries to cannabinoid agonists. Obese Zucker rats (OZRs) displayed decreased relaxation to anandamide, to CB1 and CB2 agonists as well as to capsaicin (vanilloid agonist) compared to lean rats (LZRs). The CB1 and CB2 expression was decreased in OZRs. Anandamide response was decreased in LZRs after blockade of K+ channels and inhibition of NOS, COX or cannabinoid transport. Anandamide response in OZRs was corrected by: inhibition of anandamide degradation, activation of cAMP and AMPK pathway and inhibition of ERK1/2. Anandamide increased AMPK, ACC and eNOS phosphorylation in LZRs, but it reduced in OZRs. The expression of phosphorylated ERK1/2, increased in OZRs, was potentiated by anandamide. In conclusion, obesity decreases anandamide relaxation through: reduction of CB1 and CB2 receptors; impairment of signaling pathways mediated by vanilloid receptors; reduced uptake and increased degradation of anandamide; reduction of AMPK/eNOS activation and increase in ERK1/2 activation.

A obesidade pode mudar as características das células endoteliais e musculares lisas (CMLVs). Reatividade e genes inflamatórios e de marcadores de fenótipo de CMLVs foram avaliados em artérias mesentéricas de resistência (AMRs) e de aorta de camundongos alimentados com dieta hiperlipídica (OB). RNAm para citocinas pró-inflamatórias e IL-10 foi aumentado em AMRs e aorta de OB. O relaxamento não foi alterado, mas a contração foi reduzida em AMRs e aorta de OB. AMRs apresentaram redução global da contração e a aorta apresentou redução específica para agonista adrenérgico. Maior modulação negativa por NO e prostanoides vasodilatadores foi observada em aorta, mas não em AMRs. RNAm para marcador do fenótipo sintético foi aumentado em AMRs de OB. Esses resultados mostram que as células endoteliais e as CMLVs de AMRs e aorta respondem diferentemente à obesidade. Inflamação e mecanismo de contrarregulação são induzidos em AMRs e aorta, que impediria a disfunção endotelial, mas não a mudança fenotípica das CMLVs em AMRs, que então, comprometeria sua capacidade contrátil. / Obesity may change the vascular endothelial cells and smooth muscle cells (VSMC) characteristics. Vascular reactivity and inflammatory and phenotypic markers for VSMC gene expression in resistance mesenteric arteries (RMA) and aorta from mice fed with high fat diet (OB). Pro-inflammatory cytokines mRNA and IL-10 were elevated in OB\'s RMA and aorta. The relaxation wasnt altered, however a reduction in contractility was observed in OB\'s RMA and aorta. A global reduction in contractility was observed in RMA and aorta demonstrated a specific reduction to adrenergic agonist. Higher negative modulation by NO and vasodilator prostanoids were seen only in aorta. Phenotypic markers mRNA were elevated in OB\'s RMA. The results shows that endothelial cells and VSMC from RMA and aorta respond differently to obesity. Inflammation and counter regulatory mechanisms are induced in RMA and aorta of which would prevent endothelial dysfunction but not VSMC phenotypic changing from RMA, compromising the contractile ability.

Nosso objetivo foi verificar a participação do TLR4 na pressão arterial e reatividade vascular em ratos SHR. O TLR4 está mais expresso em artérias mesentéricas de resistência de SHR com 15 semanas do que em Wistar com 15 e SHR com 5 semanas. SHR e Wistar com 15 semanas foram tratados com anti-TLR4 (1mg/dia) ou IgG (controle) por 15 dias via i.p. A expressão do TLR4, MyD88, a fosforilação da P38 e NF-kB p65, e a secreção de IL-6 foi menor nos SHR anti-TLR4 do que nos SHR IgG. Os SHR tratados com anti-TLR4 apresentaram redução na pressão arterial versus SHR IgG. A resposta máxima ao KCl e à noradrenalina (NA) foram normalizadas após o uso do anti-TLR4 no SHR, por vias dependentes de COX-1, COX-2 e do NF-kB. O uso do L-NAME diminuiu a resposta contrátil à NA no SHR IgG sendo que o anti-TLR4 melhorou essa resposta em SHR. O anti-TLR4 aumentou a expressão da eNOS e preveniu a geração de espécies reativas de oxigênio em SHR. Sugerimos que o TLR4 está associado com o aumento da pressão arterial e com a disfunção vascular presente na hipertensão arterial. / Our objective was to investigate the role of TLR4 in blood pressure and vascular reactivity in SHR. TLR4 is more expressed in mesenteric resistance arteries from SHR 15 week than in Wistar with 15 and SHR 5 weeks. Wistar and SHR with 15 weeks were treated with anti-TLR4 (1mg/dia) or IgG (control) for 15 days via ip. The expression of TLR4, MyD88, phosphorylation of p38 and NF-kB p65, and IL-6 secretion was lower in SHR TLR4 than in SHR IgG. SHR treated with anti-TLR4 had reduced blood pressure versus SHR IgG. The maximal response to KCl and noradrenaline (NA) were normalized after anti-TLR4 treatment in SHR by mechanisms dependent on COX-1, COX-2 and NF-kB. The use of L-NAME decreased the contractile response to NA in SHR IgG and anti-TLR4 improved this response in SHR. The anti-TLR4 increased eNOS expression and prevented the generation of reactive oxygen species in SHR. We suggest that the TLR4 is associated with increased blood pressure and vascular dysfunction in hypertension.

Partindo do pré-suposto que o tratamento crônico de ratos espontaneamente hipertensos (SHR) com ouabaína (OUA) amplifica a hipertensão arterial (HA) e aumenta a sensibilidade à fenilefrina em anéis de artéria caudal, buscou-se avaliar se esse tratamento modifica a reatividade vascular em artérias mesentéricas de resistência (AMR) e os mecanismos envolvidos. SHR foram tratados por 5 semanas com: veículo (CT) ou OUA (30 mg/kg/dia) ou co-tratados com ácido acetilsalicílico (AAS 100 mg/kg/dia) ou nimesulida (NID 20 mg/kg/dia). OUA aumenta a contração à noradrenalina (NOR) nas AMR dos SHR quando comparado aos CT. Na presença dos inibidores da ciclooxigenase 2 (COX-2), da sintase do tromboxano A2 (TXA2), e do antagonista do receptor TP a contração à NOR foi reduzida apenas nas AMR do grupo OUA. Os co-tratamentos com AAS e NID preveniram a potencialização da HA e a hiper-reatividade à NOR nas AMR dos SHR tratados com OUA. Conclui-se que a OUA via ativação da COX-2 aumenta a síntese de TXA2, o qual via receptor TP potencializa a contração à NOR em AMR e eleva a HA em SHR. / It is well known that chronic ouabain (OUA) treatment of spontaneously hypertensive rats (SHR) increases hypertension (HA) and the sensitivity to phenylephrine in caudal artery rings, thus, the aim of this study was to evaluate if OUA modifies vascular reactivity in mesenteric resistance (MRA) from SHR as well as the possible mechanisms involved. Male, SHR were treated for 5 weeks with: vehicle (CT) or OUA (30 mg/kg/day) or co-treated with acetylsalicylic acid (ASA 100 mg/kg/day) or nimesulide (NID 20 mg/kg/day). OUA increased norepinephrine (NOR)-induced contraction in the MRA when compared to CT. In the presence of the cyclooxygenase 2 (COX-2) and thromboxane A2 synthase (TXA2) inhibitors, and the TP-receptors antagonist NOR-induced contraction was reduced only in the MRA of OUA group. Co-treatment with ASA and NID prevented the increase in HA and the hyperreactivity to NOR in MRA of OUA treated SHR. The results suggested that OUA activates COX-2 that through TXA2/TP-receptors increases NOR-induced contraction and potentiates HA in SHR.

Na presente dissertação objetivou-se avaliar a reatividade vascular e a repercussão do pré-tratamento com sinvastatina (SINV, 20mg/kg, p.o.), sob esse parâmetro, em artéria mesentérica superior (AMS) e pulmonar (AP) de ratos Wistar submetidos a 45 minutos de isquemia e 2 horas de reperfusão intestinal (iIR). Nas artérias avaliou-se a função dependente e independente do endotélio e a contração induzida pela fenilefrina agonista seletivo <font face=\"Symbol\">a1-adrenérgico, em preparações com e sem endotélio e na presença ou ausência de inibidores da NOS e da iNOS (L-NAME e 1400W, respectivamente). Pode-se concluir que a iIR modificou a reatividade vascular em AP, porém não em AMS. Na artéria pulmonar, a iIR causou disfunção endotelial por meio de ativação da iNOS, a qual levou à diminuição da resposta vasodilatadora dependente do endotélio e à hiporreatividade contrátil. Além disso, confirmando a segunda hipótese, uma única dose de sinvastatina antes da iIR previne as alterações de reatividade vascular, o que demonstra o efeito benéfico desse fármaco nessa doença. / This work aimed to evaluate the vascular reactivity and the effect of pre-treatment with simvastatin (SIMV, 20mg/kg, p.o.), under this parameter, in the superior mesenteric artery (SMA) and pulmonary artery (PA) of rats subjected to 45 minutes of ischemia and 2 hours of reperfusion (iIR). In these arteries, it was evaluated the endothelium-dependent and -independent function and the contraction induced by phenylephrine the selective <font face=\"Symbol\">a1-adrenergic agonist, in preparations with and without endothelium and at the presence or absence of non-selective NOS and of iNOS inhibitors (L-NAME and 1400W, respectively). It can be concluded that the iIR modified vascular reactivity in PA, but not in SMA. In the pulmonary artery, the iIR caused endothelial dysfunction via activation of iNOS, which led to decreased endothelium-dependent vasodilator response and to contractile hyporeactivity. Moreover, confirming the second hypothesis, a single dose of simvastatin prior to iIR prevents changes in vascular reactivity, which shows the beneficial effects of this drug in this disease.

Abstract Chlamydia pneumoniae is a common human pathogen worldwide, which causes both upper and lower respiratory tract infections. In addition, C. pneumoniae infections have been associated with atherosclerosis and other chronic diseases, and successful treatment and eradication of the organism from tissues would therefore be desirable. The purpose of this study was to assess the effects of C. pneumoniae inoculations on the development of chronic infection and atherosclerotic changes in normocholesterolemic, wild-type mice. We also aimed to elucidate the effects of antibiotic and other treatments on the eradication of chlamydia and on the reduction of the pathologic sequelae induced by these infections. Female C57BL/6J mice were fed either normal chow when assessing the effects of acute infection, or a diet supplemented with 0.2% cholesterol when evaluating the atherosclerotic changes. Primary or repeated inoculations with C. pneumoniae isolate K7 were given to the mice intranasally, and the effects of treatments with telithromycin, levofloxacin and erythromycin antimicrobial agents and with the phenolic compounds quercetin, luteolin and octyl gallate were evaluated. The following methods were used to measure infection and treatment effects and the presence of chlamydia in tissue: chlamydia culture, PCR and RT-PCR methods, histology of lung, heart and aortic tissue, serologic methods and measurements of aortic contractility responses. Repeated C. pneumoniae inoculations induced persistent chlamydial DNA and inflammation in lung tissue and development of mouse Hsp60 autoantibodies. Infection was shown to influence aortic endothelial function, and repeated inoculations significantly increased subendothelial lipid accumulation in the aortic sinus area. A flavonoid, luteolin, was shown to effectively decrease the chlamydial load and inflammatory reactions in lung tissue. All antimicrobial agents eradicated the presence of viable chlamydia effectively; however, PCR positivity persisted in lung tissue despite the treatments. Only immediate treatment after each inoculation was able to decrease aortic sinus lipid accumulation. In conclusion, these data support the role of C. pneumoniae in promoting atherosclerotic development via autoimmune responses and also via direct effects on aortic tissue. Conventional antimicrobial treatments may not effectively eradicate persistent infection, and further studies are warranted to seek for alternative treatment options.