A sepse é uma disfunção orgânica causada por uma resposta do hospedeiro à infecção desregulada, com risco de morte. Quando o tratamento da sepse não é efetivo, o quadro pode progredir para hipotensão severa. O tecido adiposo perivascular (perivascular adipose tissue - PVAT) é reconhecido como um elemento regulador na biologia vascular, com implicações na fisiopatologia de doenças cardiovasculares. No entanto, em relação à sepse, pouco se sabe acerca dos efeitos desta sobre a ação modulatória que o PVAT exerce no tônus vascular. Dessa maneira a hipótese do presente trabalho foi a de que a sepse poderia aumentar o efeito anti-contrátil do PVAT. Portanto, o objetivo do trabalho foi avaliar o efeito da sepse experimental na ação modulatória que o PVAT exerce sobre tônus vascular e os possíveis mecanismos envolvidos nessa resposta. Para isso foram utilizados ratos Wistar Hannover com idade entre 60 e 70 dias (270 a 300g). Os ratos foram distribuídos aleatoriamente em 2 grupos: 1) Grupo Sham: foi realizada apenas uma laparotomia sem os procedimentos de ligadura e punção do ceco; 2) Grupo CLP (Cecal Ligation and Puncture) : a sepse letal foi induzida utilizando o modelo CLP no qual foi realizada uma laparotomia para exposição do ceco, onde foi feito uma ligadura e 20 punções intermediárias entre a ligadura e a ponta do ceco com agulha de calibre 18 gauge (G). Os animais foram anestesiados com quetamina/xilasina (80/10 mg/kg, i.p.) e mortos 6 h após a indução da sepse. A aorta torácica foi coletada para realização das análises bioquímicas e funcionais. A sepse letal reduziu a taxa de sobrevida, a pressão arterial média (PAM), não alterou os níveis de leucócitos e neutrófilos, mas aumentou a contagem de bactérias no sangue e no lavado peritoneal, aumentou os níveis plasmáticos de nitrato, uréia e CK-MB. A sepse diminuiu a contração induzida pela fenilefrina e serotonina nas aortas PVAT(-)/Endo(+) ou Endo (-) do grupo CLP, quando comparada ao Sham. Porém nas artérias PVAT(+)/Endo(+) ou Endo (-), o CLP induziu redução mais pronunciada da contração induzida tanto pela fenilefrina, quanto pela serotonina. O aumento do efeito anti-contrátil do PVAT na condição séptica não foi encontrado nas artérias após a incubação com L-NAME, 7-nitroindazol, 1400W, A779, carboxy-PTIO, ODQ, apamina, RO11384552 e indometacina. Tiron, catalase, 4-aminopiridina, glibenclamida e caribdotoxina não alteraram a contração induzida pela fenilefrina no grupo CLP. A sepse aumentou a concentração de H2O2 na aorta, mas não afetou a concentração no PVAT. Aumento dos níveis de ânion superóxido (O2-) e prostaglandina (PG)I2 foram detectados tanto na aorta, quanto no PVAT do grupo CLP. A sepse não alterou os níveis de PGE2 ou angiotensina (1-7) na aorta ou PVAT. Portanto, a sepse letal induzida por CLP aumenta a ação anti-contrátil do PVAT por um mecanismo que envolve a produção de NO pelas enzimas iNOS e nNOS, a participação de canais para KCa de baixa condutância e ativação da enzima guanilato ciclase solúvel. Além disso, sugere-se o envolvimento da PGI2 e angiotensina (1-7) na hiporresponsividade vascular mediada pelo PVAT durante a sepse / Sepsis is an organic dysfunction caused by an unregulated host response to lifethreatening infection. When treatment of sepsis is ineffective, the condition may progress to severe hypotension that is drug-irresponsive. Perivascular adipose tissue (PVAT) is recognized as a regulatory element in vascular biology that is implicated in the pathophysiology of cardiovascular diseases. However, little is known about the effects of sepsis in the modulatory action of PVAT. Thus, the hypothesis of the present study was that sepsis could increase the anti-contractile effect of PVAT. Therefore, the objective of the study was to evaluate the effect of experimental (lethal) sepsis in the modulatory action that PVAT exerts on vascular tone and the possible mechanisms underlying this response. With this purpose, male Wistar Hannover rats (250-300g) were divided in 2 groups: 1) Sham: the cecum was exteriorized without ligation and puncture; 2) CLP: lethal sepsis was induced using the cecal ligation and puncture (CLP) model. The thoracic aorta was isolated 6 h after sepsis for functional and biochemical assays. Lethal sepsis reduced survival rate, mean arterial pressure (MAP), did not alter leukocytes and neutrophils migration, but increased bacterial count in the blood and peritoneal cavity, increased plasma levels of nitrate, urea and CK-MB. We found that in aortas PVAT(-)/Endo(+) or Endo(-), sepsis decreased the contraction induced by phenylephrine or serotonin, when compared to sham. In PVAT(+)/Endo(+) or Endo (-) arteries sepsis induced a more pronounced reduction of phenylephrine-induced contraction. Sepsis-induced increase of anti-contractile action of PVAT was not found after incubation of arteries with L-NAME, 7-nitroindazole, 1400W, A779, carboxyPTIO , ODQ, apamin, indomethacin and RO1138452. Tiron, catalase, charybdotoxin, 4- aminopyridine and glibenclamide did not alter phenylephrine-induced contraction in the CLP group. Sepsis increased H2O2 concentration in the aorta, but did not affect H2O2 concentration in PVAT. Increased levels of superoxide anion (O2-) and prostaglandin (PG) I2 were detected in both aorta and PVAT. Sepsis did not alter the levels of PGE2 or angiotensin (1-7) in the aorta or PVAT. Conclusion: Lethal sepsis increases the anticontractile action of PVAT by a mechanism that involves the production of nitric oxide (NO) by iNOS and nNOS, the participation of calcium-dependent K+ channel of low conductance and activation of the enzyme soluble guanylate cyclase. Angiotensin (1-7) and PGI2 also contribute to the increased anti-contractile effect displayed by PVAT during sepsis. Financial Support: CAPES

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Perivascular adipose (PVAT) has been hypothesized to influence arterial health, where an excess can lead to pathogenesis of atherosclerosis and other arterial pathologies. A novel assessment of carotid PVAT is the use of carotid extra media thickness (EMT) ultrasonography. Currently, there is a lack of research to demonstrate the relationship between carotid EMT and existing measures of arterial health, notably, central pulse wave velocity, and carotid distensibility and intimal media thickness. In the current cross sectional study, 81 participants of younger recreationally active (ages 23.2 ± 2.5 years), younger sedentary (ages 26.4 ± 7.2 years), older healthy (ages 70.3 ± 5.4 years) and older adults with coronary artery disease (CAD) (ages 67.9 ± 8.7 years) were recruited. Resting measures of central arterial stiffness was examined through the assessment of aPWV, while measures of local carotid stiffness were examined through carotid distensibility. Aortic PWV was calculated using an accepted direct distance method (80% of carotid to femoral direct distance) and time difference between the feet of the carotid and femoral waveforms. Carotid intima-media thickness (IMT), a measure of the inner arterial walls, and carotid extra media thickness (EMT), a measure of carotid PVAT, were assessed through B-mode ultrasound images and a semi-automated edge tracking software. Carotid EMT, IMT, and aPWV were significantly greater in older adults than in younger adults (p < 0.05). No difference in carotid EMT was found between younger recreationally active (0.47 ± .08 mm) and sedentary adults (0.46 ± .06 mm). There were also no differences in carotid EMT between the older healthy (0.58 ± .06 mm) and older adults with CAD (0.54 ± 0.08 mm). Carotid EMT was also significantly correlated with age (r =0 .500), waist circumference (r = 0.521), aPWV (r =0.431), carotid distensibility (r = -0.364 and IMT (r = 0.404). Despite significant correlations, carotid EMT was not an independent predictor of aPWV, carotid distensibility and IMT. Because of the lack of predictive power in measures of arterial stiffness and carotid IMT, there is a potential that carotid EMT may be an independent vascular disease marker. Future investigations should involve carotid EMT in longitudinal studies to evaluate the potential marker for a more comprehensive cardiovascular risk assessment. / Thesis / Master of Science (MSc)

A obesidade desencadeia mudanças estruturais e funcionais no tecido adiposo perivascular (PVAT), levando a um desequilíbrio em favor de substâncias vasoconstritoras e pró- inflamatórias, bem como alterações em suas vias de sinalização no vaso. Um importante mecanismo proposto para explicar a perda do efeito anticontrátil do PVAT na obesidade é o estresse oxidativo. Espécies reativas de oxigênio (EROs) possuem papel importante na modulação da função vascular mediada pelo PVAT. Considerando que a mitocôndria representa fonte potencial de EROs nas células, o presente estudo testou a hipótese que a disfunção mitocondrial no PVAT está envolvida na perda do efeito anticontrátil do PVAT em modelo experimental de obesidade. Este estudo avaliou se a matriz mitocondrial nas células que compõem o tecido adiposo periaórtico representa fonte importante de EROs, e se as mesmas contribuem para as alterações na regulação, pelo PVAT, da reatividade vascular. Nosso estudo demonstrou que animais obesos apresentaram disfunção vascular e perda do efeito anticontrátil do PVAT. O estresse oxidativo está envolvido na disfunção do PVAT, com participação significativa da mitocôndria na geração de EROs, capazes de modular a reatividade vascular. A obesidade favoreceu a disfunção mitocondrial, reduzindo o consumo de oxigênio. Estes eventos favoreceram o aumento na geração de peróxido de hidrogênio mitocondrial no PVAT, o qual prejudica a ação anticontrátil deste tecido por ser ativador direto da via de contração RhoA/Rho cinase / Obesity promotes structural and functional changes in the perivascular adipose tissue (PVAT), favoring the release of vasoconstrictor and proinflammatory substances, as well as altering the vascular signaling pathways activated by PVAT-derived factors. Oxidative stress is an important mechanism proposed to explain the loss of anticontractile effects of the PVAT in obesity. Reactive oxygen species (ROS) play an important role in the modulatory effects of PVAT on vascular function. Considering that mitochondria are a potential source of ROS in the cells, the present study tested the hypothesis that mitochondrial dysfunction leads to the loss of the anticontractile effects of PVAT in obesity. We evaluated whether the mitochondrial matrix of the cells that make up the periaortic fat tissue constitute a major source of ROS, and if mROS contribute to defective regulation of vascular reactivity by the PVAT. Our study shows that obese animals exhibit vascular dysfunction and loss of anticontractile effects of PVAT. Oxidative stress is involved in PVAT dysfunction, with a significant contribution of mitochondria to ROS generation. Obesity promotes mitochondrial dysfunction, reducing oxygen consumption. These events increase the generation of mitochondrial hydrogen peroxide in the PVAT, which impairs the anticontractile effects of this tissue via direct activation of the RhoA / Rho kinase pathway

A obesidade desencadeia mudanças estruturais e funcionais no tecido adiposo perivascular (PVAT), levando a um desequilíbrio em favor de substâncias vasoconstritoras e pró- inflamatórias, bem como alterações em suas vias de sinalização no vaso. Um importante mecanismo proposto para explicar a perda do efeito anticontrátil do PVAT na obesidade é o estresse oxidativo. Espécies reativas de oxigênio (EROs) possuem papel importante na modulação da função vascular mediada pelo PVAT. Considerando que a mitocôndria representa fonte potencial de EROs nas células, o presente estudo testou a hipótese que a disfunção mitocondrial no PVAT está envolvida na perda do efeito anticontrátil do PVAT em modelo experimental de obesidade. Este estudo avaliou se a matriz mitocondrial nas células que compõem o tecido adiposo periaórtico representa fonte importante de EROs, e se as mesmas contribuem para as alterações na regulação, pelo PVAT, da reatividade vascular. Nosso estudo demonstrou que animais obesos apresentaram disfunção vascular e perda do efeito anticontrátil do PVAT. O estresse oxidativo está envolvido na disfunção do PVAT, com participação significativa da mitocôndria na geração de EROs, capazes de modular a reatividade vascular. A obesidade favoreceu a disfunção mitocondrial, reduzindo o consumo de oxigênio. Estes eventos favoreceram o aumento na geração de peróxido de hidrogênio mitocondrial no PVAT, o qual prejudica a ação anticontrátil deste tecido por ser ativador direto da via de contração RhoA/Rho cinase / Obesity promotes structural and functional changes in the perivascular adipose tissue (PVAT), favoring the release of vasoconstrictor and proinflammatory substances, as well as altering the vascular signaling pathways activated by PVAT-derived factors. Oxidative stress is an important mechanism proposed to explain the loss of anticontractile effects of the PVAT in obesity. Reactive oxygen species (ROS) play an important role in the modulatory effects of PVAT on vascular function. Considering that mitochondria are a potential source of ROS in the cells, the present study tested the hypothesis that mitochondrial dysfunction leads to the loss of the anticontractile effects of PVAT in obesity. We evaluated whether the mitochondrial matrix of the cells that make up the periaortic fat tissue constitute a major source of ROS, and if mROS contribute to defective regulation of vascular reactivity by the PVAT. Our study shows that obese animals exhibit vascular dysfunction and loss of anticontractile effects of PVAT. Oxidative stress is involved in PVAT dysfunction, with a significant contribution of mitochondria to ROS generation. Obesity promotes mitochondrial dysfunction, reducing oxygen consumption. These events increase the generation of mitochondrial hydrogen peroxide in the PVAT, which impairs the anticontractile effects of this tissue via direct activation of the RhoA / Rho kinase pathway

O tecido adiposo perivascular (PVAT) da aorta torácica (AT) possui ação anticontrátil (AC). O PVAT da AT e das artérias mesentéricas de resistência (AM) possuem diferentes características. Na obesidade ocorre expansão do PVAT. Nós avaliamos a modulação da contração pelo PVAT da AT e AM em camundongos controles (CT) e obesos (OB) e a participação do sistema renina-angiotensina (SRA), por meio do tratamento com antagonista do receptor AT1 (BRA). PVAT da AT e AM apresentaram ação AC. Ação AC do PVAT das AM, mas não da TA, foi abolida no grupo OB. BRA resgatou a ação AC do PVAT das AM no grupo OB, que foi abolida pelo antagonismo do receptor AT2 e pela inibição da óxido nítrico (NO) sintase (NOS). Em AM, a expressão dos receptores AT1 e AT2 não foi modificada e da NOS endotelial foi aumentada em AM e reduzida no PVAT da AM no grupo OB. BRA aumentou a expressão da eNOS no PVAT das AM nos dois grupos. Assim, concluímos que a obesidade induz disfunção do PVAT de AM e há envolvimento do SRA. BRA corrige a função do PVAT de AM por mecanismo dependente do receptor AT2 e NO. / The perivascular adipose tissue (PVAT) of thoracic aorta (TA) has an anticontractile (AC) action. TA and resistance mesenteric arteries (MA) PVAT have different characteristics. Expansion of PVAT occurs in obesity. We evaluated the modulation of contraction by PVAT of TA and MA in control (CT) and obese (OB) mice and the participation of the renin-angiotensin system (RAS), by treating mice with AT1 receptor antagonist (ARB). PVAT of both TA and MA showed an AC action. The AC action of MA PVAT, but of TA PVAT, was abolished in the OB group. ARB recovered the AC action of MA PVAT in OB group, which was abolished by both AT2 receptor antagonism and nitric oxide (NO) synthase (NOS) inhibition. In MA, the expression of AT1 and AT2 receptors was not changed and the expression of eNOS was increased in MA and reduced in MA PVAT of OB group. ARB increased the expression of eNOS in MA PVAT in both CT and OB groups. In conclusion, obesity induced MA PVAT dysfunction, in which RAS is involved. ARB recovered the MA PVAT function by mechanisms that depend on the AT2 receptor and NO.

Heart failure (HF) induced by aortic pressure over-load is associated with increased coronary artery stiffness. Perivascular adipose tissue (PVAT) and advanced glycation end products (AGE) both promote arterial stiffness. However, the mechanisms by which coronary PVAT promotes arterial stiffness and the efficacy of exercise to prevent coronary stiffness are unknown. The present study hypothesized both chronic continuous and interval exercise training would prevent coronary artery stiffness associated with inhibition of PVAT secreted AGE. Yucatan mininature swine were divided into four groups: control-sedentary (CON), aortic-banded sedentary heart failure (HF), aortic-banded HF continuous exercise trained (HF+CONT), and aortic-banded HF interval exercise trained (HF+IT). Coronary artery stiffness was assessed by ex vivo mechanical testing and coronary artery elastin, collagen and AGE-related proteins were assessed by immunohistochemistry. HF promoted coronary artery stiffness with reduced elastin content and greater AGE accumulation which was prevented by chronic continuous and interval exercise training. HF PVAT secreted higher AGE compared with CON and was prevented in the HF+CONT and HF+IT groups. Young healthy mouse aortas cultured in HF PVAT conditioned media had increased stiffness, lower elastin content and AGE accumulation compared with CON, which was prevented by PVAT from the HF+CONT and HF+IT groups. HF coronary PVAT secreted greater interleukin-6 (IL-6) and IL-8 compared to CON which was prevented by both continuous and interval exercise training regimens. We conclude chronic continuous and interval exercise is a potential therapeutic strategy to prevent coronary artery stiffness via inhibition of PVAT-derived AGE secretion in a pre-clinical mini-swine model of pressure overload-induced HF.

Abnormal vascular smooth muscle cell (VSMC) proliferation is thought to play an important role in the pathogenesis of atherosclerosis. Adipocytes produce several paracrine bioactive substances that can affect VSMC growth and migration. Our study focused on the ability of epicardial adipocytes to produce bioactive substances together with studying of direct effect of these substances on the VSMC proliferation rate. The gene expression of human cytokines (IL-6, IL-8, IL-18, RANTES and MCP-1) and adipokines (leptin and adiponectin) was measured in primary cell lines of epicardial and visceral adipocytes, both in undifferentiated and mature statuses. Moreover, adipokine production (IL-6, IL-8, MCP-1, VEGF and adiponectin) in conditioned media obtained from above mentioned primary cell cultures of adipocytes was measured by a Luminex assay. The VSMC proliferation rate was measured after co-culturing with CM obtained from primary cell cultures of adipocytes. The epicardial preadipocytes showed an increased expression of IL-8 (3,25-fold, p<0,05) compared with visceral preadipocytes. The expression of the adiponectin in epicardial preadipocytes was markedly decreased in comparison of the expression in visceral preadipocytes (p< 0,0001). Moreover, the gene expression was dependent on the differentiation...

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