Background - Healthy perivascular adipose tissue (PVAT) exerts an anticontractile effect in response to various vasoconstrictor agonists and this is lost in obesity. A recent study reported that bariatric surgery reverses the damaging effects of obesity on PVAT function. However, PVAT function has not been previously characterised following weight loss induced by caloric restriction, which is often the first line treatment for obesity. This study investigated the mechanisms by which PVAT modulates vascular tone in health and how these are altered in animal models of obesity and diet-induced weight loss. Methods - Male Sprague Dawley rats were fed a 45% fat diet ad libitum for 16 weeks to induce obesity, they were then randomly allocated into two groups; obese rats maintained on the 45% fat diet and weight loss rats that were subjected to 50% caloric restriction for a further four weeks. A weight maintenance group was also established where a cohort of weight loss rats were provided with 70 kcal/day for a further four weeks at the end of the caloric restriction period. A control group was also provided with a 10% fat diet during the 20 week period. The effect of PVAT on the contractility of isolated mesenteric arteries in response to norepinephrine in the presence of pharmacological tools was investigated by wire myography Changes in the PVAT environment were assessed also using western blotting, immunohistochemistry and assays of secretion. Results - PVAT from healthy control rats elicited an anticontractile effect in response to norepinephrine through release of relaxing factors, one of which was nitric oxide. The anticontractile effect was abolished in diet-induced obesity through a mechanism involving local inflammation and reduced nitric oxide bioavailability within PVAT. In addition, reduced KCNQ expression and enhanced COX activation contributed to the loss of anticontractility. Four-week caloric restriction did not restore PVAT anticontractile capacity. However, sustained weight loss led to restoration of PVAT anticontractile function associated with restoration of adipocyte size, reduced inflammation and increased nitric oxide synthase expression. Sustained weight loss and the restoration of PVAT function was associated with reversal of obesity-induced hypertension and normalisation of adipokine levels, including leptin and insulin. Conclusions - Sustained weight loss reverses obesity-induced PVAT damage through a mechanism involving reduced inflammation and reduced nitric oxide bioavailability. Reduced KCNQ activity and enhanced COX activation also contribute to the obesity-associated loss of PVAT anticontractile effect. These data reveal the beneficial effects of weight loss induced by dietary restriction on PVAT function and identify several potential targets for the treatment of PVAT dysfunction associated with obesity.

Background: Maternal obesity pre-programme offspring to develop obesity and associated cardiovascular disease later in life. In health, perivascular adipose tissue (PVAT) reduces vascular contractility, an effect lost in obesity and during pregnancy. However, neither the effect of obesity during pregnancy on PVAT function in the mothers nor the possible epigenetic effect in the offspring is known. This study sought to identify detrimental vascular changes in post-partum dams and their offspring resulting from maternal obesity. Methods: Six-eight week old female Sprague-Dawley rats were fed a 10% fat (control) or 45% fat diet (HFD) for 12 weeks before mating, throughout pregnancy and during lactation. Offspring received the control diet until sacrifice at 12 (12wo) or 24 (24wo) weeks of age. PVAT-denuded (with or without exogenous PVAT) and PVAT-intact mesenteric arteries from mothers and pups were mounted on a wire myograph and vascular contractility to thromboxane A2 agonist (U46619) and norepinephrine was assessed in the presence of pharmacological tools. Western blotting, immunoprecipitation and an AMPK activity assay were used to detect any changes in the PVAT environment. Results: Offspring of obese mothers were overweight, mildly hypertensive and insulin resistant. Contractions in PVAT-denuded arteries from HFD dams and their offspring were reduced by a mechanism involving increased protein O-GlcNAcylation. PVAT exerted an anti-contractile effect in vessels from control offspring and their mothers through the release of relaxant factors, which included nitric oxide (NO). The anti-contractile effect of PVAT was lost in HFD offspring due to reduced NO bioavailability and increased O-GlcNAcylation, which lead to decreased AMPK activity within PVAT. However, simultaneous AMPK activation within PVAT partially restored the anti-contractile capability in HFD offspring. Reduced NO bioavailability also lead to PVAT dysfunction in HFD mothers. Conclusions: Elevated insulin levels in the HFD offspring may lead to enhanced glucose uptake and increased protein O-GlcNAcylation, which contributes to the PVAT dysfunction in HFD offspring. The PVAT dysfunction, which is associated with reduced NO bioavailability in HFD mothers and their offspring may be the result of reduced AMPK phosphorylation of nitric oxide synthase within PVAT.

White adipocytes surround almost all blood vessels in the human body. It was thought previously that these cells merely provide mechanical support for the adjacent small vessels and are little more than fat storage units. Recent studies have identified these cells as metabolic and vasoactive engines that produce and secrete molecules that can affect the function of their adjacent small vessels. The adipocytes and a number of other cell types (including inflammatory cells) surrounding the vessels are collectively termed the PeriVascular Adipose Tissue (PVAT). Work from our group has shown previously that, in health, PVAT conveys a vasorelaxant effect on adjacent small arteries and that this effect is not observed in obesity thus the vessels must exist at an elevated level of basal tone. It is plausible that increased basal vessel constriction can explain the elevated blood pressure amongst the obese population and a better understanding of the obesity-induced PVAT damage may lead to clues to a new approach in the treatment of the condition which burdens its sufferers with a greater cardivascular risk profile. In this thesis we have studied individuals with morbid obesity at baseline and six months following surgery and observed that PVAT function following dramatic weight loss restores the PVAT vasorelaxant effect close to that observed in lean patients. Moreover, we have concluded that inflammation plays a significant role in this process and indeed using protocols with antioxidant enzymes we were able to restore the damaged PVAT function at baseline. We have have shown also that in health, PVAT vasorelaxant function is independent of the endothelium, and that obesity-induced PVAT damage and its reversal following weight loss and ex-vivo anti-oxidant treatment are both independent of the endothelium and at least in part due to nitric oxide bioavailability. Finally, we have observed that in sleep apnoea, which often coexists with morbid obesity and hypertension, there is a greater degree of PVAT inflammation.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Nitric oxide (NO) is an important chemical messenger in the cardiovascular system. Despite considerable progress in this field, there remains an on-going need for affordable and user-friendly NO measurement techniques. Therefore, in this study we aimed to develop and characterise NO-detection techniques not previously used in our laboratory, and, in addition, characterise an ex vivo method to measure the functional effects of the endothelium and NO production in the vasculature. Methods: Three different NO-detection techniques were compared: (i) Amperometric NO sensors. Here, NO-increasing effects of known NO synthase (NOS) activators were investigated (insulin, acetylcholine and biosynthetic human insulin). Three different NO sensors were evaluated on cultured endothelial cells and aortic tissue. Putative NOincreasing effects of shear stress were also investigated; (ii) Nitrite (NO2 -) + nitrate (NO3 -) sensors. Here, I aimed to measure NO release from cultured endothelial cells; (iii) Colorimetric NO2 - measurement assay with the Griess reagent. Here, NO2 - production by endothelial cells was measured with a plate reader. In the second part of the study an organ bath - isometric tension technique was established to measure endothelium-dependent function of aortic rings. Functional differences in aortic rings isolated from diet-induced obese rats compared to lean rats were investigated. Ring contraction was induced with phenylephrine and relaxation with acetylcholine. These investigations were further supported by western blot analyses of selected critical proteins. Lastly, the effects of perivascular adipose tissue (PVAT) on contraction and relaxation were investigated in endothelium-containing or denuded aortic ring segments. Results: Although some success was achieved with the amperometric sensors regarding calibration, any experimental results obtained were difficult to repeat due to instability of the sensors. With the NO2 -/NO3 - sensor we were not able to carry out any planned experiments due to failure to properly calibrate and standardise the sensors. Success was achieved with the Griess method. All the drugs used as positive controls (DEA/NO, fenofibrate, oleanolic acid and IL-1ß) proved to be potent inducers of NO2 - release from endothelial cells. Interestingly, the isometric tension studies showed a higher % relaxation in high fat (HF) diet aortic rings compared to those from lean animals. Western blot data showed downregulation of eNOS activation and iNOS expression in obese groups, which was suggestive of endothelial dysfunction. Interestingly, proteins associated with oxidative stress (p22phox and nitrotyrosine) were downregulated in obese groups. The presence of PVAT exerted anti-contractile effects on the rings from HF rats, however in denuded aortic rings, PVAT showed a significant pro-contractile response in both lean and HF groups. PVAT also exerted anti-relaxation effects in aortic rings from both lean and HF rats. Conclusion: We managed to successfully establish two new techniques for our laboratory (Griess method and the organ bath – isometric tension method) which can complement the more established techniques in our laboratory in order to aid us in future vascular research. Finally, the isometric tension technique used in the obese rat studies generated interesting data, which further assisted in characterising the dietinduced obesity rat model in our laboratory. / AFRIKAANSE OPSOMMING: Inleiding: Stikstofoksied (NO) is ‘n belangrike chemiese boodskapper in die kardiovaskulêre sisteem. Ondanks vordering in die veld, bestaan daar ‘n aangaande behoefte aan bekostigbare en gebruikersvriendelike NO-metingstegnieke. Gevolglik het ons in hierdie studie daarna gemik om NO-metingstegnieke wat nie vantevore in ons laboratorium beskikbaar was nie, te ontwikkel en karakteriseer. Verder het ons ten doel gehad om ‘n ex vivo model te karakteriseer om die funksionele effekte van vaskulêre endoteel en NO produksie te meet. Metodes: Drie verskillende NO-metingstegnieke was ondersoek: (i) Amperometriese NO sensors. Hier het ons die verhogende effekte op NO van bekende aktiveerders van NO sintetase (NOS) ondersoek (Insulien, asetielcholien en biosintetiese menslike insulien). Drie verskillende NO-sensors was ge-evalueer in gekultuurde endoteelselle en aortaweefsel. Die vermeende NO verhogende effekte van die wrywingskragte opgewek deur laminere vloei (“shear stress”) is ook ondersoek. (ii) Nitriet (NO2 -) + nitraat (NO3 -) sensors. Hier het ons beplan om NO-vrystelling deur gekultuurde endoteelselle te meet. (iii) Kolorimetriese meting van NO2 - met die Griess reagens. Hier het ons m.b.v. ‘n mikroplaat leser die NO2 - - vrystelling deur endoteelselle gemeet. In die tweede deel van die studie het ons ‘n orgaan bad–isometriese spanningstegniek opgestel om endoteelafhanklike funksie van aortaringe te meet. Funksionele verskille in aortaringe van vetsugtige rotte is vergelyk met kontrole rotte. Ringkontraksie is met fenielefrien geïnduseer en verslapping met asetielcholien. Hierdie ondersoeke is verder ondersteun deur Western blot analises van sleutelproteïene in die aortaweefsel. Laastens het ons die effekte van perivaskulêre vetweefsel (PVAT) op kontraksie en verslapping in aortaringe met of sonder intakte endoteel ondersoek. Resultate: Alhoewel ‘n mate van sukses behaal was met die kalibrasie van die amperometriese sensors, was eksperimentele resultate moeilik om te herhaal a.g.v. sensor-onstabiliteit. Geen eksperimente kon met die NO2 -/NO3 - sensors uitgevoer word nie weens ‘n onvermoë om ordentlike kalibrasie en standardisering uit te voer. Ons het egter wel sukses behaal met die Griess-metode. Al die middels wat as positiewe kontroles gebruik was (DEA/NO, fenofibraat, oleanoliese suur and IL-1ß) het geblyk kragtige induseerders van NO2 - produksie vanaf endoteelselle te wees. Die isometriese spanningsstudies het ‘n hoer % verslapping getoon in die hoë vet (HF) dieet aortaringe in vergelyking met die kontroles. Western blot data het ‘n afregulering van eNOS en iNOS getoon in die HF diere, wat aanduidend is van endoteel disfunksie, terwyl proteïene geassosieer met oksidatiewe stress (p22phox en nitrotirosien) afgereguleer was in die HF groep. Die aanwesigheid van PVAT het ‘n anti-kontraktiele effek gehad op die ringe van die HF groep. Toe die endoteel egter verwyder was, het PVAT in beide kontrole en HF ringe ‘n beduidende pro-kontraktiele effek gehad. Verder het PVAT ook anti-verslappingseffekte op aortaringe beide kontrole en HF rotte uitgeoefen. Gevolgtrekking: Ons het daarin geslaag om twee nuwe tegnieke vir ons laboratorium suksesvol te vestig (Griess metode en die orgaanbad-isometriese spanningstegniek) wat in die toekoms die meer gevestigde tegnieke in ons laboratorium kan komplementeer. Laastens het die isometriese spanningstegniek wat in die dieetstudies gebruik is, data opgelewer wat ons verder sal help om die vetsug model in ons laboratorium te karakteriseer.

O consumo crônico de etanol é um importante fator de risco no desenvolvimento de doenças cardiovasculares induzindo elevação da pressão arterial, inflamação, disfunção vascular e aumento do estresse oxidativo em vários tecidos. Além disso, o consumo crônico de etanol induz aumento dos níveis de Fator de necrose tumoral-? (TNF-?). O tecido adiposo perivascular (Perivascular adipose tissue - PVAT) é reconhecido como uma importante fonte de adipocinas e citocinas pró-inflamatórias e esse tecido está envolvido na fisiopatologia de diferentes doenças cardiovasculares. A hipótese deste trabalho é a de que o consumo crônico de etanol estimule a produção de TNF-? no tecido vascular, que por sua vez, induzirá um aumento na produção de espécies reativas de oxigênio (ERO), redução da biodisponibilidade do óxido nítrico (NO), inflamação vascular, prejuízo na função do PVAT, alteração da reatividade vascular e aumento da pressão arterial. Portanto, o objetivo deste estudo foi investigar a participação do TNF-? nas disfunções vasculares induzidas pelo consumo de etanol e avaliar o papel do PVAT em tais danos. O trabalho mostra que o consumo crônico de etanol por 12 semanas induziu aumento da pressão arterial sistólica (PAS) em camundongos machos C57BL/6 (Wild Type -WT) e este aumento foi menor nos animais nocautes para os receptores R1 do TNF-? (TNFR1-/-). O tratamento com etanol não alterou o relaxamento vascular induzido por acetilcolina e nitroprussiato de sódio (NPS). O consumo de etanol induziu aumento da geração de ânion superóxido (O2-), aumento das espécies reativas ao ácido tiobarbitúrico (TBARS) e redução dos níveis de peróxido de hidrogênio (H2O2) em aortas com e sem PVAT (PVAT+ e PVAT-, respectivamente) de animais WT, mas não em camundongos TNFR1-/-. Houve aumento da atividade da catalase (CAT) e da superóxido dismutase (SOD) em aorta PVAT- e PVAT+ e redução dos níveis plasmáticos da glutationa reduzida (GSH) em animais WT, mas não em TNFR1-/-, após o consumo crônico de etanol. Não houve alteração na atividade da glutationa peroxidase (GPx) na aorta dos grupos estudados após o consumo de etanol. Observou-se redução dos níveis de nitrato/nitrito (NOx) em aortas de animais WT após o consumo de etanol, porém não em camundongos TNFR1-/-. O consumo de etanol gerou aumento tecidual das citocinas TNF-? e IL-6 e aumento da atividade da mieloperoxidase (MPO), evidenciando uma exacerbada inflamação vascular e indicando migração de neutrófilos para o tecido aórtico. Tais alterações não foram observadas em animais TNFR1-/-. Os resultados mostram pela primeira vez a participação do TNF-? no aumento da pressão arterial, no aumento do estresse oxidativo e nos prejuízos vasculares induzidos pelo consumo crônico de etanol. O tecido adiposo perivascular não apresentou qualquer efeito benéfico nas alterações supracitadas. / Chronic ethanol consumption is an important risk factor in the development of cardiovascular diseases, inducing increased blood pressure, inflammation, vascular dysfunction and enhanced oxidative stress in several tissues. Furthermore, chronic ethanol consumption induces the production of Tumor necrosis factor-? (TNF-?). Perivascular adipose tissue (PVAT) is known as an important source of adipokines and proinflammatory cytokines. This tissue is involved in the pathophysiology of different cardiovascular diseases. The hypothesis of this work is that the chronic ethanol consumption stimulates the production of TNF-?, which in turn, will induce an increase in reactive oxygen species (ROS) generation, nitric oxide (NO) reduction, vascular inflammation, impairment of PVAT function, alterations of vascular reactivity and increased blood pressure. Therefore, the aim of this study was to investigate the role of TNF-? in chronic ethanol consumption-induced vascular dysfunctions and to evaluate the role of PVAT in such damages. This study demonstrated that chronic ethanol consumption for 12 weeks induced an increase in systolic blood pressure (SBP) in C57BL/6 mice (wild type-WT) and this increase was blunted in TNF-? receptor 1 knockout mice (TNFR1-/-). There was no change in vascular relaxation induced by acetylcholine and sodium nitroprusside (NPS). Ethanol consumption increased the superoxide anion (O2-) generation, thiobarbituric acid reactive species (TBARS) and reduction of hydrogen peroxide (H2O2) levels in aorta without and with PVAT (PVAT- and PVAT+, respectively) from WT animals, but not from TNFR1-/- mice. There was an increase in catalase (CAT) and superoxide dismutase (SOD) activities in aorta PVAT- and PVAT+, decrease on plasma reduced-glutathione (GSH) levels from ethanol-treated WT but not in TNFR1-/-. Ethanol consumption did not change glutathione peroxidase (GPx) activity in any group. Nitrate/nitrite (NOx) aortic levels were decreased in WT animals, but not in TNFR1-/- after chronic ethanol consumption. Ethanol consumption increased TNF-?, IL-6 cytokines and myeloperoxidase activity (MPO) which suggest a strong vascular inflammation and migration of neutrophils into the aortic tissue. Such changes were not observed in TNFR1-/- mice. The results show for the first time the participation of TNF-? in the increase of blood pressure, increase of oxidative stress and vascular dysfunction induced by the chronic ethanol consumption. The perivascular adipose tissue had no beneficial effect on these changes.

A obesidade é caracterizada por inflamação no tecido adiposo. A angiotensina II via receptor AT1 induz estresse oxidativo e inflamação vascular. O tecido adiposo perivascular (PVAT) circunda os vasos sanguíneos, possui ação parácrina na parede vascular e é classificado como marrom e branco, sendo que este último é mais propenso à inflamação. No presente estudo, avaliamos os compenentes do sistema renina-angiotensina no PVAT marrom (da aorta torácica - AT) e branco (do leito mesentérico LM) de camundongos controles (CT) e obesos (OB). O RNAm para angiotensinogênio tanto no PVAT marrom quanto no PVAT branco foi reduzido em camundongos OB. A atividade enzimática da ECA 1 foi reduzida no PVAT-marrom do grupo OB em comparação ao grupo CT e foi semelhante no PVAT branco dos dois grupos. No PVAT marrom, o conteúdo proteico do receptor AT1 (AT1R) foi semelhante nos grupos CT e OB e o receptor AT2 (AT2R) não foi detectado no PVAT dos dois grupos. No PVAT branco, o conteúdo proteico dos receptores AT1 e AT2 foi aumentado no grupo OB. A expressão de RNAm do receptor CCR2 de MCP-1 (proteína quimiotáxica de monócitos) no PVAT marrom foi semelhante nos dois grupos, mas foi aumentada no PVAT branco do grupo OB. O conteúdo proteico da MCP1 foi aumentado no PVAT branco do grupo OB em relação ao grupo CT. O tratamento de camundongos OB com antagonista de receptor AT1 de angiotensina II (losartana; 10 mg/kg) por 30 dias, não reverteu os parâmetros que caracterizam a obesidade e não diminuiu a expressão de MCP-1 no PVAT branco. Em relação aos componentes do sistema renina-angiotensina, o PVAT marrom e o PVAT branco respondem de maneira diferente à obesidade induzida por dieta hiperlipídica. / Obesity is characterized by inflammation in adipose tissue. Angiotensin II/AT1 receptor pathway induces oxidative stress and vascular inflammation. Perivascular adipose tissue surrounds blood vessels and has paracrine actions in the vascular wall. It is classified as brown and white being the last one more willing to inflammation. In the present study, we evaluated the components of renin angiotensin-system in brown (thoracic aortic - TA) and white (mesenteric bed MB) PVAT in lean (CT) as well as in obese mice (OB). RNAm content of angiotensinogen in brown and white PVAT was reduced in obese mice. ACE1 activity was lower in brown PVAT of OB group in comparison to CT group and it was similar to white PVAT in both groups. In brown PVAT, protein content of AT1 receptor (AT1R) was similar to CT and OB groups and AT2 receptor (AT2R) was not detected in both groups. In white PVAT, protein contents of AT1R and AT2R were increased in obese group. RNAm expression of CCR2 receptor from MCP1 (monocytes chemoctatic protein 1) in brown PVAT was similar to both groups; however, it was increased in white PVAT in OB group. Protein content of MCP1 was increased in white PVAT in OB group. Treatment in obese mice with angiotensin II receptor blocker (ARB) (losartan; 10 mg/kg) for 30 days did not reverse the parameters which characterize obesity and did not diminish MCP-1 expression in white PVAT. In relation to the components of renin angiotensinsystem, brown and white PVAT responded differently to high-fat diet obesity.

Background: The incidence of coronary heart diseases, including atherosclerosis, increases with ageing. The factors which influence arterial function, and which may be changed with ageing, are multiple but effects of perivascular adipose tissue (PVAT) on large arteries have not previously been considered. A key role for nitric oxide (NO) in mediating the anti-contractile capacity of PVAT has been suggested. Caveolin-1 (Cav-1) modulates the production of NO in vivo by tonic inhibition of eNOS. The influence of aortic PVAT and the contribution of NO to vascular reactivity in ageing C57BL/6 mice, atherosclerotic ApoE knockout mice (ApoE-/-), Cav-1 knockout mice (Cav-1-/-) and atheroprotected ApoECav-1 double knockout mice (ApoE-/-Cav-1-/-) is unknown. Hypothesis: The influence of PVAT on vascular function is modulated by ageing and the development of atherosclerosis via NO bioavailability. Methods: Male mice were used in this study. C57BL/6 mice were obtained at 4 weeks of age and maintained on a normal rodent diet (ND) for 8, 16 or 26 weeks. ApoE-/- and Cav-1-/- mice were bred from in-house colonies and ApoE-/-Cav-1-/- mice were generated by interbreeding ApoE-/- and Cav-1-/- mice. Upon weaning, ApoE-/-, Cav-1-/- and ApoE-/-Cav-1-/- mice were maintained on either a ND or Western-type diet (WD) for 8, 16 or 26 weeks. Vascular reactivity studies on isolated aortic ring preparations were performed in the presence or absence of PVAT. The contribution of NO to the vascular reactivity of aortic PVAT was determined using pharmacological inhibition of NO synthase. Aortic PVAT was assessed for evidence of morphological and/or compositional changes associated with ageing or a WD. Results: NO mediated an anti-contractile effect of aortic PVAT in C57BL/6 mice fed a ND up to 16 weeks. The anti-contractile capacity of aortic PVAT was lost after 26 weeks on a ND and preceded endothelial dysfunction. Loss of the PVAT anti-contractile effect was accompanied by alterations in PVAT morphology and composition. Aortic PVAT from ND-fed ApoE-/- mice was dysfunctional and did not exert an anti-contractile effect. Furthermore, a WD did not alter the influence of PVAT on vascular reactivity in ApoE-/- mice and PVAT morphology and composition was unchanged. NOS inhibition did not alter the contractile responses. The aortic PVAT of ND-fed Cav-1-/- mice did not exert an anti-contractile effect and PVAT composition was unchanged with increasing age. However, after 26 weeks on a WD, aortic PVAT from Cav-1-/- mice potentiated contractions to phenylephrine and white adipocyte hypertrophy was observed. NOS inhibition revealed a pro-contractile effect of aortic PVAT from Cav-1-/- mice. Loss of Cav-1-/- conferred significant protection against the development of atherosclerosis in WD-fed ApoE-/-Cav-1-/- mice despite a proatherogenic lipid profile. Aortic PVAT from ND-fed ApoE-/-Cav-1-/- mice did not exhibit an anti-contractile capacity and PVAT morphology was unchanged with ageing. Additionally, a WD did not influence the effect of PVAT on vascular reactivity in ApoE-/-Cav-1-/- mice although white adipocyte hypertrophy was observed after 26 weeks of high fat feeding. NOS inhibition revealed a pro-contractile effect of aortic PVAT in 8-week ND-fed ApoE-/-Cav-1-/- mice. Conclusions: This work has produced novel insights into the influence of aortic PVAT and NO on vascular reactivity and the morphology of aortic PVAT in ageing C57BL/6 mice, atherosclerotic ApoE-/- mice, Cav-1-/- mice and athero-protected ApoE-/-Cav-1-/- double knockout mice. Ageing to pre-middle age in C57BL/6 mice results in a loss of the anti-contractile effect of PVAT prior to endothelial dysfunction. This is associated with altered NO bioavailability and changes to the morphology and composition of PVAT. This may reveal potential therapeutic targets to restore the anti-contractile capacity of PVAT if comparable age-related PVAT dysfunction is observed in humans. Aortic PVAT of ApoE-/- mice does not exert an anti-contractile effect which may be attributed to decreased basal eNOS activity. A WD does not alter the vascular reactivity of PVAT. In addition, aortic PVAT from Cav-1-/- mice does not exhibit an anti-contractile capacity yet it exerts a pro-contractile effect after 26 weeks on a WD. The aortic PVAT of ApoE-/-Cav-1-/- mice does not modulate vascular reactivity and this is unaltered with feeding of a WD although white adipocyte hypertrophy was observed within the PVAT. The critical role of Cav-1 in the initiation and progression of atherosclerosis is reinforced by the atheroprotected phenotype of the ApoE-/-Cav-1-/- mice even though a severely proatherogenic lipid profile is observed in both the ND and WD-fed mice. Therapeutically targeting LDL transcytosis into the arterial wall could potentially prevent or halt the development of atherosclerosis. Aortic PVAT of ND-fed Cav-1-/- and ApoE-/-Cav-1-/- mice may not be dysfunctional but unable to modulate vascular reactivity due to attenuated vasoconstrictor responses of PVAT-denuded aortic rings as a result of excess NO, although this requires further investigation.

O consumo crônico de etanol é um importante fator de risco no desenvolvimento de doenças cardiovasculares induzindo elevação da pressão arterial, inflamação, disfunção vascular e aumento do estresse oxidativo em vários tecidos. Além disso, o consumo crônico de etanol induz aumento dos níveis de Fator de necrose tumoral-? (TNF-?). O tecido adiposo perivascular (Perivascular adipose tissue - PVAT) é reconhecido como uma importante fonte de adipocinas e citocinas pró-inflamatórias e esse tecido está envolvido na fisiopatologia de diferentes doenças cardiovasculares. A hipótese deste trabalho é a de que o consumo crônico de etanol estimule a produção de TNF-? no tecido vascular, que por sua vez, induzirá um aumento na produção de espécies reativas de oxigênio (ERO), redução da biodisponibilidade do óxido nítrico (NO), inflamação vascular, prejuízo na função do PVAT, alteração da reatividade vascular e aumento da pressão arterial. Portanto, o objetivo deste estudo foi investigar a participação do TNF-? nas disfunções vasculares induzidas pelo consumo de etanol e avaliar o papel do PVAT em tais danos. O trabalho mostra que o consumo crônico de etanol por 12 semanas induziu aumento da pressão arterial sistólica (PAS) em camundongos machos C57BL/6 (Wild Type -WT) e este aumento foi menor nos animais nocautes para os receptores R1 do TNF-? (TNFR1-/-). O tratamento com etanol não alterou o relaxamento vascular induzido por acetilcolina e nitroprussiato de sódio (NPS). O consumo de etanol induziu aumento da geração de ânion superóxido (O2-), aumento das espécies reativas ao ácido tiobarbitúrico (TBARS) e redução dos níveis de peróxido de hidrogênio (H2O2) em aortas com e sem PVAT (PVAT+ e PVAT-, respectivamente) de animais WT, mas não em camundongos TNFR1-/-. Houve aumento da atividade da catalase (CAT) e da superóxido dismutase (SOD) em aorta PVAT- e PVAT+ e redução dos níveis plasmáticos da glutationa reduzida (GSH) em animais WT, mas não em TNFR1-/-, após o consumo crônico de etanol. Não houve alteração na atividade da glutationa peroxidase (GPx) na aorta dos grupos estudados após o consumo de etanol. Observou-se redução dos níveis de nitrato/nitrito (NOx) em aortas de animais WT após o consumo de etanol, porém não em camundongos TNFR1-/-. O consumo de etanol gerou aumento tecidual das citocinas TNF-? e IL-6 e aumento da atividade da mieloperoxidase (MPO), evidenciando uma exacerbada inflamação vascular e indicando migração de neutrófilos para o tecido aórtico. Tais alterações não foram observadas em animais TNFR1-/-. Os resultados mostram pela primeira vez a participação do TNF-? no aumento da pressão arterial, no aumento do estresse oxidativo e nos prejuízos vasculares induzidos pelo consumo crônico de etanol. O tecido adiposo perivascular não apresentou qualquer efeito benéfico nas alterações supracitadas. / Chronic ethanol consumption is an important risk factor in the development of cardiovascular diseases, inducing increased blood pressure, inflammation, vascular dysfunction and enhanced oxidative stress in several tissues. Furthermore, chronic ethanol consumption induces the production of Tumor necrosis factor-? (TNF-?). Perivascular adipose tissue (PVAT) is known as an important source of adipokines and proinflammatory cytokines. This tissue is involved in the pathophysiology of different cardiovascular diseases. The hypothesis of this work is that the chronic ethanol consumption stimulates the production of TNF-?, which in turn, will induce an increase in reactive oxygen species (ROS) generation, nitric oxide (NO) reduction, vascular inflammation, impairment of PVAT function, alterations of vascular reactivity and increased blood pressure. Therefore, the aim of this study was to investigate the role of TNF-? in chronic ethanol consumption-induced vascular dysfunctions and to evaluate the role of PVAT in such damages. This study demonstrated that chronic ethanol consumption for 12 weeks induced an increase in systolic blood pressure (SBP) in C57BL/6 mice (wild type-WT) and this increase was blunted in TNF-? receptor 1 knockout mice (TNFR1-/-). There was no change in vascular relaxation induced by acetylcholine and sodium nitroprusside (NPS). Ethanol consumption increased the superoxide anion (O2-) generation, thiobarbituric acid reactive species (TBARS) and reduction of hydrogen peroxide (H2O2) levels in aorta without and with PVAT (PVAT- and PVAT+, respectively) from WT animals, but not from TNFR1-/- mice. There was an increase in catalase (CAT) and superoxide dismutase (SOD) activities in aorta PVAT- and PVAT+, decrease on plasma reduced-glutathione (GSH) levels from ethanol-treated WT but not in TNFR1-/-. Ethanol consumption did not change glutathione peroxidase (GPx) activity in any group. Nitrate/nitrite (NOx) aortic levels were decreased in WT animals, but not in TNFR1-/- after chronic ethanol consumption. Ethanol consumption increased TNF-?, IL-6 cytokines and myeloperoxidase activity (MPO) which suggest a strong vascular inflammation and migration of neutrophils into the aortic tissue. Such changes were not observed in TNFR1-/- mice. The results show for the first time the participation of TNF-? in the increase of blood pressure, increase of oxidative stress and vascular dysfunction induced by the chronic ethanol consumption. The perivascular adipose tissue had no beneficial effect on these changes.

Orientador: Carlos Alan Candido Dias Junior / Resumo: INTRODUÇÃO: O tecido adiposo perivascular (PVAT) libera sob condições fisiológicas fatores de relaxamento derivados do tecido adiposo (ADRF), que possivelmente desempenham um papel importante na modulação do tônus vascular. Foi demonstrado que a inativação de canais de potássio sensíveis ao ATP (KATP) aboliu o efeito anticontratil do PVAT. Outros estudos mostraram que o sulfeto de hidrogênio (H2S) é produzido pelo PVAT devido a expressão da sua enzima precursora cistationina gamma liase (CSE) e que o H2S atua através ativação dos KATP. Além disso, o PVAT sofre uma disfunção em condições fisiopatológicas como a hipertensão. No entanto, seus efeitos ainda não são bem explorados durante a hipertensão gestacional. Portanto, nosso objetivo foi examinar o envolvimento do H2S derivado do PVAT na modulação do tônus vascular de ratas prenhes hipertensas e normotensas. MÉTODOS: Foram realizadas curvas concentração-resposta induzidas pela fenilefrina na presença e ausência de PVAT e endotélio em ratas prenhes normotensas (Norm- Preg) e hipertensas (HTN-Preg). A pressão arterial materna, os parâmetros fetais e placentários, a angiogenese e os níveis de H2S também foram avaliados. RESULTADOS: A hipertensão gestacional foi associada ao desequilíbrio angiogênico e a restrição do crescimento fetal e placentário e o PVAT não se mostrou disfuncional. Além disso, sob a formação estimulada de H2S pelo PVAT, mas não no endotélio, houve redução nas curvas concentração-resposta à fenilefrina em aor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: INTRODUCTION: Perivascular adipose tissue (PVAT) releases diffusible adipocyte- derived relaxing factors (ADRFs) under physiological conditions, which possibly play key roles to modulate the vascular tone. It was demonstrated that the inactivation of ATP-sensitive potassium channels (KATP channels) impaired the anticontractile effect of PVAT. Further studies have shown that hydrogen sulfide (H2s) is released by PVAT due to the expression of cystathionine gamma lyase (CSE) and that (H2s) acts through the activation of KATP channels. In addition, PV A T is impaired in pathophysiological situations such as hypertension. However, it is not yet well explorated during hypertension in pregnancy. Therefore, we aimed to examine the involvement of PVAT-derived H2S to modulate the vascular tone in aorta from normotensive and hypertensive pregnant rats. METHODS: Phenylephrine-induced contractions in the presence and absence of PVAT and endothelium in aortas from normotensive pregnant (Norm-Preg) and HTN-Preg rats were investigated. Maternal blood pressure, fetal-placental parameters, angiogenesis and H2S levels were also assessed. RESULTS: Hypertensive pregnancy associated with angiogenic imbalance and fetal- placental growth restrictions revealed that there is no PVAT dysfunction. Moreover, under stimulated H2S formation PVAT, but not endothelium, reduces phenylephrine-induced contractions in aortas from HTN-Preg rats. Also, H2S synthesis inhibitor abolishes anticontractile effects disp... (Complete abstract click electronic access below) / Mestre

No description available.