<p>A comprehensive understanding of the mechanisms regulating vascular structure and function may assist in designing effective strategies to decrease cardiovascular disease risk. The current studies were designed to investigate a) relationships between collagen markers and arterial stiffness and markers of vasoconstriction and inflammation and endothelial function in humans with a wide range of vascular health, including overweight women, elderly healthy men, individuals with coronary artery disease, individuals with spinal cord injury and young healthy men and b) changes in arterial structure and function and circulating serum markers of type I collagen synthesis and degradation, vasoconstriction and inflammation in overweight pre-menopausal women before and after a 16- week diet and exercise intervention. Resting brachial artery flow mediated dilation (FMD), upper limb and/or central pulse wave velocity (PWV_c-r and PWV_c-f) and carotid artery distensibility were assessed at baseline in all groups and, in the overweight population, after the 16-week intervention. Pro-collagen type I C-peptide (PIP), C-telopeptide of type I collagen (CTX), markers of collagen synthesis and degradation respectively, endothelin-1 (ET-1) a vasoconstrictor and interleukin-6 (IL-6) an inflammatory marker were measured. In the spectrum of vascular health, a negative relationship exists between collagen markers and central PWV (CTX–PWV_c-f: r = -0.41, p = 0.001 and PIP – PWV_c-f: r = -0.32, p = 0.01) and a positive relationship between markers and carotid distensibility (CTX: r = 0.59, pc-r increased over time in the overweight population (FMD pre: 4.1 ± 0.5 % vs. post: 6.9 ± 0.7 %, pc-r pre: 8.1 ± 0.3 m/s vs. post: 8.9 ± 0.3 m/s, p</p> / Doctor of Philosophy (PhD)

Obstructive sleep apnea syndrome (OSAS), a chronic respiratory disorder affecting as many as 1 in 5 adults, is associated with repetitive collapse of the upper airway during sleep and results in fragmented sleep and intermittent periods of hypoxia and hypercapnia. If left untreated, OSAS increases the risk for hypertension, insulin resistance, metabolic syndrome (MetS) in a manner that is independent of obesity in mid-adulthood. However, it is still unknown if evidence of these relationships is apparent in young adults with OSAS who are otherwise healthy and free of other chronic comorbidities. Objectives: To determine if functional and biochemical evidence of insulin resistance, MetS, and vascular endothelial dysfunction (VED) exists in young, overweight men with OSAS and if the combined effects of obesity and OSAS augments the evidence of chronic disease pathogenesis beyond the effects of obesity alone. Subjects: Subjects were 12 overweight men with OSAS (age = 22.8 ± 0.8; BMI = 32.4 ± 1.0; apnea-hypopnea index (AHI) = 25.4 ± 5.4), 17 overweight men without OSAS (age = 22.5 ± 0.7; BMI = 31.6 ± 1.1; AHI = 2.2 ± 0.3), and 18 normal weight men without OSAS (age = 21.1 ± 0.5; BMI = 22.4 ± 0.4; AHI = 1.9 ± 0.3). Methods: Subjects were evaluated for OSAS using an unsupervised, portable polysomnography test. Total fat and central abdominal fat (CAF) were assessed using dual energy x-ray absorptiometry (DEXA). Fasting blood samples were used to quantify biochemical markers for insulin resistance (glucose, insulin, adiponectin, IL-6, and TNF-á) and endothelial dysfunction (CRP, VEGF, and VEGFR2) using ELISA, RIA, and flow cytometry. MetS was defined according to Adult Treatment Panel III (ATP III) clinical standards. Triglycerides, HDL cholesterol, and glucose were measured using a commercial lipid panel. Resting blood pressure was obtained manually via auscultation. VED was measured via strain gauge plethysmography, with endothelium-dependent vasodilatation being assessed from forearm reactive hyperemia after a 5-minute period of upper arm occlusion. Statistics: One-way ANOVA was used to determine group differences in variables. Two-way ANOVA was used to evaluate group x time interactions during the 2-minute recovery period following upper arm occlusion. Pearson partial correlation was used to assess relationships between continuous variables, with analyses being controlled for CAF or OSAS severity. Spearman correlation was used to assess relationships between number of MetS components present and both indices of adiposity and OSAS severity. Stepwise multiple linear regression analysis was used to determine significant predictors of OSAS severity, insulin resistance, components of the MetS, and endothelial dysfunction. Results: Overweight subjects with OSAS had more CAF, higher fasting triglycerides, and lower serum adiponectin concentrations than both overweight and normal weight non-apneic controls. Furthermore, fasting triglycerides were directly correlated to OSAS severity, even after the influence of central abdominal fat was removed. OSAS severity was an independent predictor of triglyceride levels, and vice versa. Insulin resistance, leptin, insulin, and CRP were all higher in overweight subjects than controls, but no further differences were attributable to severity of OSAS. No differences in IL-6, TNF-á, ADMA, and expression of VEGFR2 were noted between any groups. No group or group x time interaction differences existed in regards to postocclusive reactive hyperemia responses. Conclusions: Young men with OSAS exhibit several unique anthropometric and biochemical abnormalities that may indicate early pathogenesis of or increased risk for future development for cardiovascular and metabolic disorders. Identification and treatment of OSAS at this age may be critical to prevent the onset and progression of these chronic disorders. / Ph. D.

Yes / Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate) to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30%) and angiogenesis (~40%) compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp.), effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.

O endotélio vascular desempenha um papel central no controle do tônus vascular pela liberação de fatores relaxantes derivados do endotélio (EDRFs) e fatores contráteis derivados do endotélio (EDCFs). O óxido nítrico (NO) é um dos mais importantes mediadores da vasodilatação, sua produção é catalisada pelas enzimas NO-sintases (NOS). A eNOS é constitutiva e depende do Ca2+ para ser ativada. A fosfolipase A2 (cPLA2), também é dependente de Ca2+, esta enzima é responsável pela conversão de fosfolipídeos de membrana a ácido araquidônico, o precursor de prostanóides, tais como prostaciclina (PGI2) e tromboxano (TXA2). A disfunção endotelial está relacionada com uma menor biodisponibilidade de NO e maior produção de EDCFs e está presente em várias doenças cardiovasculares, como hipertensão arterial. Embora esta disfunção seja multifatorial, o aumento da produção de espécies reativas de oxigênio (EROs) parece contribuir de forma considerável. O aumento na [Ca2+]c está relacionado com o aumento na produção de EROs e liberação de EDCFs. Em nosso estudo, utilizamos o ionóforo de cálcio A23187 para avaliar as alterações na sinalização celular decorrentes da mobilização de cálcio, independente da ativação de receptores, que ocorrem na hipertensão arterial. O objetivo deste trabalho foi estudar as respostas vasculares desencadeadas pelo aumento da [Ca2+]c promovido pelo A23187 em aorta de ratos normotensos (2R) e hipertensos renais (2R-1C). Verificamos que o A23187 induz efeito vasodilatador dependente da produção de NO em aortas de ratos 2R e 2R-1C, pois o relaxamento foi abolido na presença do inibidor da NOS (L-NAME), bem como em aortas sem endotélio. Em aortas de ratos 2R-1C, mas não em aortas de ratos 2R, verificamos a participação da PGI2 no efeito vasodilatador induzido pelo A23187. A produção de PGI2 está aumentada em aortas de ratos 2R-1C em comparação com aortas de ratos 2R, o que indica que esse prostanóide está ativando receptores TP, induzindo contração. O A23187 induziu efeito contrátil de forma independente do endotélio em aorta de ratos 2R. Entretanto, em aortas com endotélio, de ratos 2R-1C o efeito contrátil está prejudicado. O efeito anti-contrátil em aortas de ratos 2R-1C é devido à produção de NO, que está aumentada a ponto de impedir a contração induzida pelo A23187. Verificamos que a contração induzida pelo A23187 é dependente da produção dos prostanóides contráteis, TXA2 e PGI2, que ativam os receptores TP, pois quando utilizamos o inibidor da ciclooxigenase (ibuprofeno) o efeito contrátil foi atenuado. Além disso, quando utilizamos o antagonista dos receptores TP (SQ29548) o efeito foi completamente abolido. O estímulo com A23187 aumentou a produção de TXA2 em aorta de ratos 2R e 2R-1C. Porém, a produção em aorta de ratos 2R-1C foi maior do que aquela observada em aorta de ratos 2R. Observamos que a catalase atenuou a resposta contrátil induzida pelo A23187, demonstrando que o peróxido de hidrogênio modula positivamente a contração induzida pelo A23187. / The vascular endothelium plays a pivotal role in the vascular tone due to the release of relaxing factors (EDRFs) and contractile factors (EDCFs). Nitric oxide (NO) is one of the most important EDRFs involved in the vasodilation and it is produced by the NO-synthases (NOS). eNOS is a constitutive isoform and its activity is dependent of the transient calcium. Besides eNOS, other important enzymes are modulated by Ca2+ such as phospholipase A2 (cPLA2). This enzyme converts membrane phospholipids to araquidonic acid, responsible for the formation of the prostanoids prostaciclin (PGI2) and thromboxane (TXA2). Endothelial dysfunction is related to the decreased NO bioavailability and increased production of EDCFs. It is present in several cardiovascular disorders like hypertension. Endothelial dysfunction is a multifactorial proccess that is also caused by the increased production of reactive oxygen species (ROS). Increased cytosolic calcium concentration ([Ca2+]c) is related to augmented ROS and EDCFs production. In the present study, we have used the calcium ionophore A23187 in order to evaluate the altered cellular signaling caused by [Ca2+]c in a receptor activation-independent way that occurs in hypertension. This work aimed to study the vascular responses stimulated by A23187 in normotensive rat (2K) aorta and in renal hypertensive (2K-1C) rat aorta. We have verified that A23187 induces vasodilator effect dependent on the production of NO in 2K and 2K- 1C rat aortas. The vascular relaxation was abolished by the non-selective NOS inhibitor (L-NAME) and by the endothelium removal. In 2K-1C but not in 2K rat aortas, PGI2 contributes to He vasodilator effect induced by A23187. PGI2 production is greater in 2K-1C than in 2K rat aortas, which suggests that PGI2 activates TP receptors inducing contraction. The contractile effect of A23187 is endotheliumdependent in 2K rat aorta. However, in 2K-1C intact-endothelium aortas, the contractile effect of A23187 is impaired. The anti-contractile effect is due to increased NO production that inhibits the contractile response to A23187. The contractile response induced by A23187 is dependent of the prostanoid production like TXA2 and PGI2 that activate TP receptors because this response is inhibited by the cyclooxygenase inhibitor (ibuprofen). In addition, this effect was abolished by the TP receptor antagonist (SQ29548). TXA2 production was stimulated with A23187 in 2K and 2K-1C rat aorta, which was greater in 2K-1C than in 2K rat aorta. We have also observed that catalase blunted the contractile response induced by A23187. These results suggest that hydrogen peroxide positively modulates A23187-induced contractile response.

O diabetes mellitus (DM) está associado a diversas complicações micro e macrovasculares diretamente relacionadas a doenças cardiovasculares. A prolongada exposição à hiperglicemia e a resistência a insulina são considerados os principais fatores envolvidos nestas complicações, as quais são exacerbadas pela disfunção endotelial. Mediadores inflamatórios contribuem potencialmente para o desenvolvimento de disfunção endotelial pela geração de espécies reativas de oxigênio (EROs) que, por sua vez, estimulam a transcrição de fatores pró- inflamatórios. Receptores específicos, como os NLRs (NOD-like receptors, receptores do tipo NOD) contribuem para instalação de processo inflamatório pela ativação do complexo inflamassoma. Este regula a ativação da caspase-1 e o processamento proteolítico dos precursores pró-IL-1? e pró-IL-18 nas citocinas maduras. Diversos mediadores podem ativar o inflamassoma NLRP3 como, por exemplo, EROs e DNA mitocondrial. Pouco é conhecido sobre o envolvimento de receptores NLRP3 e DNA mitocondrial na disfunção endotelial associada ao diabetes. Testamos a hipótese que a deficiência genética do receptor NLRP3 confere resistência à ativação de processo inflamatório na vasculatura de animais com diabetes tipo 1 (DM1) e, ainda, que DNA mitocondrial contribui para a ativação vascular do inflamassoma NLRP3 e para disfunção endotelial. Foram utilizados camundongos C57Bl/6 e deficientes para NLRP3, os quais foram tratados com veículo ou submetidos a protocolo para indução de DM1 com estreptozotocina. Parâmetros vasculares funcionais foram determinados em artérias mesentéricas de resistência. Células de músculo liso vascular (CMLV) e endoteliais foram utilizadas para avaliação da ativação do inflamassoma NLRP3 por DNA mitocondrial. A geração de EROs foi avaliada pela fluorescência para o dihidroetídio e pela quimiluminescência para lucigenina. A ativação de caspase-1 e IL-1? foi avaliada por western blot e o influxo de cálcio, por fluorescência. DNA mitocondrial foi avaliado pela expressão gênica de componentes da mitocôndria. O diabetes reduziu a vasodilatação dependente de endotélio, o que não ocorreu em artérias de animais deficientes de NLRP3. Animais diabéticos apresentaram aumento da expressão vascular do receptor NLRP3, da ativação de caspase-1 eIL-1? e da geração de EROs e peróxido de hidrogênio no leito mesentérico, eventos que ocorreram em menor intensidade em camundongos deficientes de NLRP3. Houve redução na expressão proteica vascular de Nox4 (NADPH oxidase 4), bem como na expressão gênica da molécula de adesão celular vascular-1 (VCAM-1, vascular cell adhesion molecule-1) e molécula de adesão intercelular-1 (ICAM-1, intercellular adhesion molecule-1) em animais deficientes de NLRP3. Houve aumento da liberação de DNA mitocondrial citosólico no pâncreas de animais diabéticos. A incubação com o DNA mitocondrial extraído do pâncreas de animais diabéticos promoveu ativação do inflamassoma em CMLV provenientes de animais C57Bl/6, mas não em CMLV provenientes de animais deficientes de NLRP3. Esta ativação foi associada ao aumento de EROs e influxo de cálcio. Essa mesma ativação também foi observada em células endoteliais. DNA mitocondrial de camundongos diabéticos também reduziu a dilatação dependente do endotélio em artérias mesentéricas, o que foi associado à geração de EROs e ativação do inflamassoma NLRP3. Pacientes diabéticos apresentaram aumento do DNA mitocondrial circulante e ativação de caspase-1 e IL-1? no soro. Os resultados demonstram que o DNA mitocondrial pancreático de animais diabéticos promove ativação, em CMLV e células endoteliais, do inflamassoma NLRP3 através do aumento no influxo de cálcio e da geração de EROs, contribuindo para o processo de disfunção endotelial. A deficiência de NLRP3 protege os animais diabéticos contra os danos vasculares inflamatórios e disfunção endotelial. / Diabetes mellitus (DM) is associated with several micro and macrovascular complications directly related to cardiovascular diseases. Prolonged exposure to hyperglycemia and insulin resistance are considered the main factors involved in these complications, which are exacerbated by endothelial dysfunction. Inflammatory mediators potentially contribute to the development of endothelial dysfunction by the generation of reactive oxygen species (ROS), which, in turn, stimulate the transcription of pro-inflammatory factors. Specific receptors such as NLRs (NOD-like receptors) contribute to the onset of inflammatory processes by the activation of a multiprotein complex called inflammasome. The NLRP3 inflammasome regulates the activation of caspase-1 and the proteolytic processing of pro-IL-1? and pro-IL-18 precursors into mature cytokines. Several mediators, such as ROS and mitochondrial DNA activate the NLRP3 inflammasome. Considering that it is not clear whether NLRP3 and mitochondrial DNA contribute to diabetes-associated endothelial dysfunction, we hypothesized that the genetic deficiency of the NLRP3 confers resistance to vascular inflammatory processes in animals with type 1 diabetes (T1D) and that mitochondrial DNA contributes to vascular activation of NLRP3 inflammasome and endothelial dysfunction. C57B1/6 and NLRP3 knockout mice were treated with vehicle or streptozotocin to induce T1D. Functional vascular parameters were determined in resistance mesenteric arteries. Cultured vascular smooth muscle cells (VSMC) and endothelial cells were used to determine NLRP3 inflammasome activation by mitochondrial DNA. ROS generation was evaluated by dihydroethidium fluorescence and by chemiluminescence for lucigenin. Caspase-1 and IL-1? activation was evaluated by western blot. Calcium influx was determined by fluorescence and mitochondrial DNA by mRNA expression of mitochondrial components. Diabetes reduced endothelium-dependent vasodilation in C57B1/6, but not in NLRP3 knockout mice. Diabetic mice presented increased vascular NLRP3 receptor expression, increased caspase-1 and IL-1? activation, as well as ROS and hydrogen peroxide generation, events that were mildly observed in NLRP3 knockout mice. There was a reduction in the vascular protein expression of Nox4 (NADPH oxidase 4) as wellas in the gene expression of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) in NLRP3 knockout animals. There was an increase in cytosolic mitochondrial DNA release in pancreas from diabetic animals. Mitochondrial DNA from the pancreas of diabetic mice induced NLRP3 inflammasome activation in VSMC from C57B1/6 mice, but not in VSMC from NLRP3 knockout mice. This activation was associated with increased levels of ROS and calcium influx and was also detected in endothelial cells. Mitochondrial DNA from diabetic mice also decreased endothelium-dependent dilation in mesenteric arteries, which was associated with ROS generation and NLRP3 inflammasome activation. Diabetic patients exhibited increased serum mitochondrial DNA and caspase-1 and IL-1? activation. The results demonstrate that pancreatic mitochondrial DNA from diabetic mice activates the NLRP3 inflammasome in VSMC and endothelial cells by increasing calcium influx and ROS generation, contributing to endothelial dysfunction. NLRP3 deficiency prevents diabetes-related vascular inflammatory damage and endothelial dysfunction.

O endotélio vascular é responsável por várias funções como o controle do tônus vascular pela produção e/ou liberação de substâncias vasoconstritoras (EDCFs) e relaxantes (EDRFs). Na hipertensão arterial ocorre disfunção endotelial caracterizada pelo desequilíbrio entre EDCFs e EDRFs. Vários autores sugerem que essas alterações são decorrentes do aumento nas concentrações de espécies reativas de oxigênio (EROs). As EROs podem afetar a sinalização, produção e/ou biodisponibilidade do óxido nítrico (NO), principal EDRF, assim como aumentar os níveis de prostanóides como prostaglandinas e tromboxanos, EDCFs produtos da COX. O principal alvo para o NO é a ativação da guanilil ciclase solúvel (GCs) no musculo liso vascular causando vasorelaxamento. No modelo de hipertensão arterial dois rins um clipe (2R-1C), ocorre aumento nos níveis de EROs e ativação da NADH/NADPH-oxidase, principal enzima produtora de EROs em células endoteliais. Em nosso estudo, utilizamos o composto NCX2121, que é estruturalmente formado por um doador de NO e inibidor da COX (indometacina). O estudo teve por objetivo caracterizar farmacologicamente a resposta relaxante do NCX2121 na aorta de ratos hipertensos 2R-1C e investigar a contribuição do endotélio vascular e das EROs para essa resposta. Verificamos que o composto NCX 2121 produz relaxamento da aorta de ratos 2R e 2R-1C, que é reduzido pela remoção do endotélio e inibição da enzima NO-Sintase (NOS). O relaxamento do composto NCX 2121 deve ser promovido pelo NO, uma vez que em aortas sem endotélio esse relaxamento foi abolido pelo ODQ. Porém, foi apenas reduzido em aortas com endotélio, isoladas de ratos normotensos (2R) e não foi alterado nas aortas com endotélio, isoladas de ratos 2R-1C. O NCX 2121 não alterou a fosforilação dos sítios de ativação ou inibição da eNOS. O NO não foi detectado em solução por análise amperométrica. O composto NCX2121 aumentou a concentração citosólica de NO, medida pela sonda fluorescente sensível a NO (DAF-2DA), por microscopia confocal. Na aorta de ratos 2R-1C, o relaxamento estimulado com o composto NCX2121 foi inibido pelas EROs e os níveis de EROs em células endoteliais isoladas, foi reduzido pelo composto NCX2121. O composto NCX 2121 reduziu os níveis de tromboxano na aorta de ratos 2R e 2R-1C. Os nossos resultados demonstram que o composto NCX2121 promove relaxamento pela liberação intracelular de NO e inibição da COX por reduzir a produção de prostanóides vasoconstrictores como o tromboxano. O composto NCX2121 não interfere com a ativação da NOS, mas reduz as EROs nas células endoteliais. / The vascular endothelium plays multiple roles on the tone control by the production and/or release of contractile factors (EDCFs) and relaxing factors (EDRFs). There is an imbalance between EDCFs and EDRFs in hypertension that is defined by endothelial dysfunction. In accordance to several authors, these alterations are due to increased production of reactive oxygen species (ROS). The ROS can affect the nitric oxide (NO) signaling, production and bioavailability that is the major EDRF. ROS can also increase the levels of prostaglandins and thromboxane (TX) that are EDCFs products of COX. The main target for NO is the activation of soluble guanylyl-cyclase (sGC) in the vascular smooth muscle cells causing vasorelaxation. In renal hypertensive rats (2K-1C), there is an increased production of ROS by NADH-NADPH-oxidase in the rat aorta endothelial cells. In the present study we used the compound NCX2121, in which chemical structure there is a NO donor and a non-selective COX inhibitor indomethacin. This study aimed to pharmacologically characterize the NCX2121 relaxing effect in 2K-1C rat aorta, and to investigate the contribution of the endothelial factors and ROS for this response. We verified that the relaxation-induced by NCX2121 was impaired by the endothelium removal and NO-synthase (NOS) inhibition. The relaxation induced by NCX2121 is due to NO, since sGC inhibition by ODQ completely abolished its effect in denuded endothelium 2K-1C rat aorta. However, in intact endothelium normotensive 2K rat aorta, the relaxing effect of NCX2121 was only partially inhibited whereas in 2K-1C it was not changed. NCX2121 did not change the phosphorylation sites of activation or inhibition of NOS. NO was not detected by amperometry in the organ bath during the relaxation induced by NCX2121, but it was measured in the cell cytoplasm by confocal microscopy. The vasorelaxation was inhibited by ROS, and NCX2121 decreased the ROS in isolated endothelial cells. NCX2121 reduced TX in 2K and 2K-1C rat aortas. Therefore, our results indicate that the compound NCX2121 induces relaxation by intracellular NO release and COX inhibition by the reduced production of contractile prostanoids such as TX. The compound NCX2121 does not modulate NOS, but it decreases ROS in the endothelial cells.

O diabetes mellitus (DM) está associado a diversas complicações micro e macrovasculares diretamente relacionadas a doenças cardiovasculares. A prolongada exposição à hiperglicemia e a resistência a insulina são considerados os principais fatores envolvidos nestas complicações, as quais são exacerbadas pela disfunção endotelial. Mediadores inflamatórios contribuem potencialmente para o desenvolvimento de disfunção endotelial pela geração de espécies reativas de oxigênio (EROs) que, por sua vez, estimulam a transcrição de fatores pró- inflamatórios. Receptores específicos, como os NLRs (NOD-like receptors, receptores do tipo NOD) contribuem para instalação de processo inflamatório pela ativação do complexo inflamassoma. Este regula a ativação da caspase-1 e o processamento proteolítico dos precursores pró-IL-1? e pró-IL-18 nas citocinas maduras. Diversos mediadores podem ativar o inflamassoma NLRP3 como, por exemplo, EROs e DNA mitocondrial. Pouco é conhecido sobre o envolvimento de receptores NLRP3 e DNA mitocondrial na disfunção endotelial associada ao diabetes. Testamos a hipótese que a deficiência genética do receptor NLRP3 confere resistência à ativação de processo inflamatório na vasculatura de animais com diabetes tipo 1 (DM1) e, ainda, que DNA mitocondrial contribui para a ativação vascular do inflamassoma NLRP3 e para disfunção endotelial. Foram utilizados camundongos C57Bl/6 e deficientes para NLRP3, os quais foram tratados com veículo ou submetidos a protocolo para indução de DM1 com estreptozotocina. Parâmetros vasculares funcionais foram determinados em artérias mesentéricas de resistência. Células de músculo liso vascular (CMLV) e endoteliais foram utilizadas para avaliação da ativação do inflamassoma NLRP3 por DNA mitocondrial. A geração de EROs foi avaliada pela fluorescência para o dihidroetídio e pela quimiluminescência para lucigenina. A ativação de caspase-1 e IL-1? foi avaliada por western blot e o influxo de cálcio, por fluorescência. DNA mitocondrial foi avaliado pela expressão gênica de componentes da mitocôndria. O diabetes reduziu a vasodilatação dependente de endotélio, o que não ocorreu em artérias de animais deficientes de NLRP3. Animais diabéticos apresentaram aumento da expressão vascular do receptor NLRP3, da ativação de caspase-1 eIL-1? e da geração de EROs e peróxido de hidrogênio no leito mesentérico, eventos que ocorreram em menor intensidade em camundongos deficientes de NLRP3. Houve redução na expressão proteica vascular de Nox4 (NADPH oxidase 4), bem como na expressão gênica da molécula de adesão celular vascular-1 (VCAM-1, vascular cell adhesion molecule-1) e molécula de adesão intercelular-1 (ICAM-1, intercellular adhesion molecule-1) em animais deficientes de NLRP3. Houve aumento da liberação de DNA mitocondrial citosólico no pâncreas de animais diabéticos. A incubação com o DNA mitocondrial extraído do pâncreas de animais diabéticos promoveu ativação do inflamassoma em CMLV provenientes de animais C57Bl/6, mas não em CMLV provenientes de animais deficientes de NLRP3. Esta ativação foi associada ao aumento de EROs e influxo de cálcio. Essa mesma ativação também foi observada em células endoteliais. DNA mitocondrial de camundongos diabéticos também reduziu a dilatação dependente do endotélio em artérias mesentéricas, o que foi associado à geração de EROs e ativação do inflamassoma NLRP3. Pacientes diabéticos apresentaram aumento do DNA mitocondrial circulante e ativação de caspase-1 e IL-1? no soro. Os resultados demonstram que o DNA mitocondrial pancreático de animais diabéticos promove ativação, em CMLV e células endoteliais, do inflamassoma NLRP3 através do aumento no influxo de cálcio e da geração de EROs, contribuindo para o processo de disfunção endotelial. A deficiência de NLRP3 protege os animais diabéticos contra os danos vasculares inflamatórios e disfunção endotelial. / Diabetes mellitus (DM) is associated with several micro and macrovascular complications directly related to cardiovascular diseases. Prolonged exposure to hyperglycemia and insulin resistance are considered the main factors involved in these complications, which are exacerbated by endothelial dysfunction. Inflammatory mediators potentially contribute to the development of endothelial dysfunction by the generation of reactive oxygen species (ROS), which, in turn, stimulate the transcription of pro-inflammatory factors. Specific receptors such as NLRs (NOD-like receptors) contribute to the onset of inflammatory processes by the activation of a multiprotein complex called inflammasome. The NLRP3 inflammasome regulates the activation of caspase-1 and the proteolytic processing of pro-IL-1? and pro-IL-18 precursors into mature cytokines. Several mediators, such as ROS and mitochondrial DNA activate the NLRP3 inflammasome. Considering that it is not clear whether NLRP3 and mitochondrial DNA contribute to diabetes-associated endothelial dysfunction, we hypothesized that the genetic deficiency of the NLRP3 confers resistance to vascular inflammatory processes in animals with type 1 diabetes (T1D) and that mitochondrial DNA contributes to vascular activation of NLRP3 inflammasome and endothelial dysfunction. C57B1/6 and NLRP3 knockout mice were treated with vehicle or streptozotocin to induce T1D. Functional vascular parameters were determined in resistance mesenteric arteries. Cultured vascular smooth muscle cells (VSMC) and endothelial cells were used to determine NLRP3 inflammasome activation by mitochondrial DNA. ROS generation was evaluated by dihydroethidium fluorescence and by chemiluminescence for lucigenin. Caspase-1 and IL-1? activation was evaluated by western blot. Calcium influx was determined by fluorescence and mitochondrial DNA by mRNA expression of mitochondrial components. Diabetes reduced endothelium-dependent vasodilation in C57B1/6, but not in NLRP3 knockout mice. Diabetic mice presented increased vascular NLRP3 receptor expression, increased caspase-1 and IL-1? activation, as well as ROS and hydrogen peroxide generation, events that were mildly observed in NLRP3 knockout mice. There was a reduction in the vascular protein expression of Nox4 (NADPH oxidase 4) as wellas in the gene expression of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) in NLRP3 knockout animals. There was an increase in cytosolic mitochondrial DNA release in pancreas from diabetic animals. Mitochondrial DNA from the pancreas of diabetic mice induced NLRP3 inflammasome activation in VSMC from C57B1/6 mice, but not in VSMC from NLRP3 knockout mice. This activation was associated with increased levels of ROS and calcium influx and was also detected in endothelial cells. Mitochondrial DNA from diabetic mice also decreased endothelium-dependent dilation in mesenteric arteries, which was associated with ROS generation and NLRP3 inflammasome activation. Diabetic patients exhibited increased serum mitochondrial DNA and caspase-1 and IL-1? activation. The results demonstrate that pancreatic mitochondrial DNA from diabetic mice activates the NLRP3 inflammasome in VSMC and endothelial cells by increasing calcium influx and ROS generation, contributing to endothelial dysfunction. NLRP3 deficiency prevents diabetes-related vascular inflammatory damage and endothelial dysfunction.

Introduction: Obstructive sleep apnoea (OSA) is common and is caused by repetitive obstruction of the upper airway during sleep. OSA is associated with increased cardiovascular morbidity and mortality and is an independent risk factor for hypertension. The immediate physiological effects of OSA include intermittent hypoxia, repeated arousal from sleep and intra-thoracic pressure swings. The resulting activation of the sympathetic nervous system, systemic inflammation and oxidative stress may result in increased arterial stiffness and endothelial dysfunction, potentially explaining any causal link between OSA and cardiovascular disease (CVD). Continuous positive airway pressure (CPAP) therapy improves excessive daytime sleepiness (EDS) and in non-randomised studies, reduces cardiovascular mortality. Prior to starting this study, there was a limited amount of evidence suggesting that CPAP therapy improved arterial stiffness and endothelial function, but the effects in subjects without pre-existing CVD were unclear. Aims: i) to determine whether CPAP therapy has an effect upon measures of arterial stiffness and endothelial function in patients with OSA, in the absence of known CVD. ii) To compare arterial stiffness and endothelial function in a subset of patients with OSAHS (defined as OSA and EDS), with a group of well-matched control subjects. Methods: Fifty three patients with OSA, defined as an apnoea/hypopnoea index of ≥15, and without known CVD, entered a double-blind placebo-controlled crossover trial of 12 weeks CPAP therapy, of whom forty three completed the study protocol. Sham CPAP was used in the placebo arm of the study and vascular assessments were made at baseline and after each arm of the study. Arterial stiffness was determined by measuring aortic distensibility using cardiovascular magnetic resonance imaging and by measuring the augmentation index (AIx) and aortic pulse wave velocity (PWV) by applanation tonometry. Endothelial function was assessed non-invasively by measuring vascular reactivity after administration of salbutamol and glyceryl trinitrate. In a subset of twenty patients with OSAHS, arterial stiffness and endothelial function at baseline were compared to readings obtained from healthy control subjects, matched on a one-to-one basis for age, sex and BMI. Results: Patients with OSAHS (n=20) had increased arterial stiffness [AIx 19.3(10.9) vs. 12.6(10.2) %; p=0.017] and impaired endothelial function, measured as the change in AIx following salbutamol [-4.3(3.2) vs. -8.0(4.9) %; p=0.02] compared to controls. Twelve weeks of CPAP therapy had no significant effect upon any measure of arterial stiffness or endothelial function in patients with OSA (n=43). A trend towards a reduction in AIx following CPAP therapy was seen, but this was non-significant. There was a reduction in systolic blood pressure following CPAP therapy [126(12) vs. 129(14) mmHg]. Sub group analysis showed CPAP to have no effect on arterial stiffness or endothelial function in patients with EDS or in patients using CPAP for ≥4 hours per night. Conclusions: This study demonstrates that even in the absence of known CVD, patients with OSAHS have evidence of increased arterial stiffness and impaired endothelial function. However, in patients with OSA, free from CVD, CPAP therapy did not lead to an improvement in any measure of arterial stiffness or endothelial function after 12 weeks.

L’hypertension artérielle pulmonaire (HTAP) est une maladie grave caractérisée par une obstruction progressive des petites artères pulmonaires, conduisant à une augmentation progressive des résistances vasculaires pulmonaires (RVP) et à terme à une défaillance cardiaque droite et au décès du patient. La vasoconstriction, le remodelage vasculaire pulmonaire, la thrombose in situ et la dysfonction endothéliale sont autant de facteurs qui contribuent au développement et à la progression de la maladie. Ces travaux de doctorat ont visé à préciser et mieux comprendre le phénotype anormal de la cellule endothéliale (CE) pulmonaire de patients HTAP afin d’identifier de nouvelles cibles thérapeutiques adéquates pour corriger et restaurer la dysfonction endothéliale associée à l’hypertension pulmonaire. Plusieurs perturbations fonctionnelles liées à des anomalies moléculaires ont pu être identifiées. Les CE HTAPi sont caractérisées par une prolifération, une migration, une survie et une réponse excessive à différents facteurs de croissance : PDGF, EGF, et FGF2. Parmi les anomalies moléculaires à l’origine de ces perturbations fonctionnelles, nous avons retrouvé une production et une libération anormale de FGF2, de MMP2, de MIF, d’IL-6 et de MCP-1, mais aussi une activité constitutive de la MAPK, une surexpression des facteurs anti-apoptotiques BCL2 et BCL-xL, de FGFR2, de p130cas et de CD74. Cette meilleure compréhension de la CE-HTAPi a déjà permis d’identifier plusieurs cibles qui pourraient être évaluées et à aussi permis de soulever un certain nombre de questions qui requiert des études plus poussées. / Pulmonary arterial hypertension (PAH) is a severe disease characterized by progressive obstruction of small pulmonary arteries, leading to a progressive increase in pulmonary vascular resistance and to right heart failure and death. Vasoconstriction, pulmonary vascular remodeling, in situ thrombosis and endothelial dysfunction are all factors that contribute to the development and progression of the disease. These work aimed to clarify and better understand the abnormal phenotype of the pulmonary endothelial cell (P-EC) of PAH patients to identify new therapeutic targets to correct and restore the endothelial dysfunction associated with pulmonary hypertension. Several functional disturbances related to molecular abnormalities have been identified. The IPAH-P-EC is characterized by proliferation, migration, survival and an excessive response to various growth factors: PDGF, EGF, and FGF2. Among the molecular abnormalities responsible for these functional disturbances, we found an abnormal production and release of FGF2, MMP2, MIF, IL-6 and MCP-1, but also a constitutive activity of MAPK, overexpression of anti-apoptotic factors BCL2 and BCL-xL, of FGFR2, of p130Cas and CD74. This better understanding of the EC-IPAH has already identified several targets that could be evaluated and also helped raise a number of issues requiring further study.

L’hypertension pulmonaire (HP) est définie par une élévation de la pression artérielle pulmonaire moyenne (PAPm) au-delà de 25 mm de mercure (Hg) au repos en raison de l’augmentation progressive et soutenue des résistances vasculaires pulmonaires, menant à l'insuffisance cardiaque droite. La dysfonction endothéliale pulmonaire associée à l'HTAP est maintenant considérée comme un mécanisme pathogénique clé qui pourrait être préjudiciable à la fois pour la susceptibilité et le développement du remodelage vasculaire pulmonaire. Au niveau des cellules endothéliales (CE), la fixation du facteur inhibiteur de la migration des macrophages (MIF), un des plus anciens médiateurs immunologiques connus, sur le CD74 va initier une cascade de signalisation intracellulaire clef pour la prolifération, la survie cellulaire et la production de différents facteurs inflammatoires. C’est pourquoi, ces travaux de doctorat ont visés à : 1) Etudier l’importance de la voie MIF/CD74 dans l'acquisition/maintien d’un phénotype pro-inflammatoire des CE pulmonaires dans l'HTAP ; 2) Tester l’efficacité de nouveaux antagonistes de MIF, synthétisés et brevetés par la société MIFCARE, contre ce phénotype endothélial et le développement d’HP expérimentales.Nos données mettent en lumière le rôle critique de la voie MIF/CD74 pour le phénotype aberrant des CE pulmonaires HTAP et soulignent son importance comme nouvelle cible thérapeutique prometteuse pour lutter contre le remodelage vasculaire pulmonaire. Cette meilleure compréhension du rôle de la voie MIF/CD74 dans le phénotype endothélial aberrant, nous a permis l’identification d’une nouvelle molécule à forte affinité, administrable par voie orale, capable de ralentir la progression d’HP expérimentales (brevet européen en soumission). Cependant, des études plus poussées, en cours de réalisation, sont encore nécessaires avant de pouvoir transférer ces connaissances vers une utilisation clinique de ces nouveaux candidats « médicaments ». / Pulmonary arterial hypertension (PAH) is a severe progressive cardiopulmonary disorder characterized by vascular proliferation and remodeling of the small pulmonary arteries. These can lead to a progressive increase in pulmonary vascular resistance and ultimately to right ventricular failure and death. Pulmonary endothelial dysfunction and pro-inflammatory phenotype associated with PAH are now considered as a key pathogenic mechanism that could be detrimental to both the susceptibility and development of the pulmonary vascular remodeling.In pulmonary endothelial cells (EC), the binding of the immune mediator MIF (Macrophage Migration Inhibitory Factor), to its receptor CD74 initiates an intracellular signaling cascade leading to cell proliferation, cell survival and the secretion of various inflammatory mediators. Therefore, the present work seeks to: (1) Determine the importance of the MIF/CD74 signaling pathway in the acquisition of an abnormal pro-inflammatory EC phenotype in PAH; (2) Test the efficacies of MIF inhibitors, synthesized and patented by MIFCARE, on this abnormal pro-inflammatory EC phenotype and on the development of experimental pulmonary hypertension (PH).Our data highlight the critical role of the MIF/CD74 axis in the endothelial dysfunction and pro-inflammatory phenotype of pulmonary EC in PAH. In addition, our data emphasize its importance as a promising new therapeutic target to prevent the pulmonary vascular remodeling associated to this disorder. Furthermore, we were successful in identifying an agent from a novel class of MIF antagonists optimized for in vivo use that have the ability to partially reverse established PH in rats and to partially inhibit the pro-inflammatory EC phenotype observed in PAH.Collectively, we demonstrated the importance of the MIF/CD74 axis and that its inhibition with MIF antagonist agents could represent a promising strategy for the treatment of PAH (under patent). However, further studies are still needed before transferring this knowledge to clinical use of these new candidates.