Platelet-Activating Factor Acetylhydrolase (PAFAH) is a monocyte-derived phospholipase A2 that catalyzes the hydrolysis of platelet-activating factor (PAF) and has been implicated in atherosclerosis. Although PAF and other proinflammatory stimuli are postulated to induce the enzyme, mechanisms controlling PAFAH expression are largely unknown at present. We have shown that PAFAH induction in monocytes is increased in response to oxidized phospholipids. The PAFAH 5' flanking region has at least 10 putative Stat elements, and IL-6 has been shown to be downstream from the prostaglandin receptor, EP2, which has been shown to bind oxidized phospholipids, prompting the hypothesis that Stat proteins might regulate its expression. To test this hypothesis, we treated human monocytes with IL-6, a monocyte-derived cytokine that activates Stat3, IL-8, a monocyte-derived cytokine induced by Stat3, and oxidized 1-palmitoyl-2-arachidonoyl-sn-3-phosphocholine (oxPAPC), a major component of the oxidized LDL particle. Two monocyte-derived cell types, macrophages (MO) and dendritic cells (DC) were prepared from primary human monocytes. The cells were treated with various doses of IL-6, IL-8, or oxPAPC for various time frames in the absence of serum. Culture supernatants from the cytokine-treated cells were harvested and screened for PAFAH protein and activity and cell monolayers were assessed for PAFAH mRNA by quantitative real time PCR (qPCR). Cells treated with oxPAPC were further analyzed for secreted IL-6 using ELISA and activation of Stat3 using Western Blot. Both IL-6 and IL-8 induced PAFAH expression in a dose-dependent manner. Although both MO and DC responded to the cytokines, preliminary experiments suggested that induction of PAFAH is more robust in DC than MO. Cytokine-treated cells exhibited increased PAFAH activity in their culture supernatants that correlated with increased PAFAH protein levels. Treatment with oxPAPC induced IL-6 secretion and subsequent Stat3 activation in DC. Together, these data support the hypothesis that PAFAH expression is regulated by oxidized phospholipids and proinflammatory cytokines in developing atheromas.

A osteoporose é uma doença osteometabólica que atinge milhares de pessoas; e as doenças cardiovasculares são as principais causas de mortes no mundo. Estudos demonstram que pacientes com baixa densidade mineral óssea apresentam maior risco de desenvolverem doenças cardiovasculares. No entanto, os mecanismos patológicos, fisiológicos e moleculares que correlacionam a osteoporose e a aterosclerose ainda são desconhecidos. O objetivo deste estudo in vivo foi realizar avaliação histológica e imuno-histoquímica do osso fêmur de camundongos ovariectomizados e ateroscleróticos após tratamento com três drogas: doxiciclina, atorvastatina e vitamina D. Foram utilizados 36 camundongos fêmeas, como controle (linhagem C57BL/6), divididos aleatoriamente em seis grupos: Grupo I: Água; Grupo II: Etanol; Grupo III: Azeite; Grupo IV: Doxiciclina; Grupo V: Atorvastatina e Grupo VI: Vitamina D; e 36 camundongos fêmeas knockout para Apolipoproteína E (ApoE-/-) divididos aleatoriamente em seis grupos: Grupo VII: Água OVX; Grupo VIII: Etanol OVX; Grupo IX: Azeite OVX; Grupo X: Doxiciclina OVX; Grupo XI: Atorvastatina OVX e Grupo XII: Vitamina D OVX. Após indução dos quadros de osteoporose e aterosclerose foi realizado o tratamento conforme cada grupo. Decorrido o tempo experimental, os animais foram eutanasiados e o material coletado submetido ao processamento histotécnico para análise histológica, em cortes corados com Tricrômio de Masson e avaliação da expressão por imunohistoquímica de TRAP, IL-1β, TNF-α, RANKL e MMP-9. Os resultados obtidos foram submetidos à análise estatística por meio dos testes One-Way ANOVA, seguido pelo pósteste de Bonferroni (histologia e colesterol); Kruskal Wallis e Student-Newman-Keuls (imuno-histoquímica). O nível de significância adotado foi de 5%. Os resultados obtidos evidenciaram diferença significativa entre o número de células imunorreativas para MMP-9, TRAP, IL-1β, TNF-α e RANKL nos grupos VII e X; VIII e XI; demonstrando a ação da Doxiciclina e Atorvastatina sob tais moléculas. Os resultados histológicos evidenciaram a eficácia da vitamina D no tratamento da osteoporose, uma vez que promoveu aumento da área óssea / Osteoporosis is an osteometabolic disease that affects thousands of people; and cardiovascular diseases are the leading causes of death in the world. Studies have shown that patients with low bone density are at a higher risk of developing cardiovascular disease. However, the pathological, physiological and molecular mechanisms that are correlated to osteoporosis and atherosclerosis are still unknown. The present work in vivo was to perform a histological and immunohistochemical evaluation of the femoral bone of ovariectomized and atherosclerotic mice after treatment with three drugs: doxycycline, atorvastatin and vitamin D. Thirty-six female mice were used as controls (C57BL/6 strain) randomly divided into six groups: Group I: Water; Group II: Ethanol; Group III: Olive oil; Group IV: Doxycycline; Group V: Atorvastatin and Group VI: Vitamin D; and the 36 knockout mice for Apolipoprotein E (ApoE-/-) randomly divided into six groups: Group VII: Water OVX; Group VIII: Ethanol OVX; Group IX: OVX olive oil; Group X: Doxycycline OVX; Group XI: Atorvastatin OVX and Group XII: Vitamin D OVX. After induction of osteoporosis and atherosclerosis, treatment was performed according to each group. After the experimental time, the animals were euthanized and the collected material submitted to histotechnical processing for histological analysis in stained sections with Masson\'s Trichrome and evaluation of the expression by immunohistochemistry of TRAP, IL-1β, TNF-α, RANKL and MMP-9. The results were submitted to statistical analysis using One-Way ANOVA, followed by Bonferroni post-test (histology and cholesterol); Kruskal Wallis and Student-Newman-Keuls (immunohistochemistry). The level of significance was 5%. The results obtained showed a significant difference between the number of immunoreactive cells for MMP-9, TRAP, IL-1β, TNF-α and RANKL in groups VII and X; VIII and XI; demonstrating the action of Doxycycline and Atorvastatin on such molecules. The histological results evidenced the efficacy of vitamin D in the treatment of osteoporosis, since it promoted increased bone area

The principle product of each NOS is nitric oxide. However, under conditions of substrate and cofactor deficiency the enzymes directly catalyze superoxide formation. Considering this alternative chemistry of each NOS, the effects of each single enzyme on key events of atherosclerosis are difficult to predict. Here, we evaluate nitric oxide and superoxide production by all three NOS isoforms in atherosclerosis. ESR measurements of circulating and vascular wall nitric oxide production showed significantly reduced nitric oxide levels in apoE/eNOS double knockout (dko) and apoE/iNOS dko animals but not in apoE/nNOS dko animals suggesting that eNOS and iNOS majorly contribute to vascular nitric oxide production in atherosclerosis. Pharmacological inhibition and genetic deletion of eNOS and iNOS reduced vascular superoxide production suggesting that eNOS and iNOS are uncoupled in atherosclerotic vessels. Though genetic deletion of nNOS did not alter superoxide production, acute inhibition of nNOS showed that nNOS contributes significantly to superoxide production. In conclusion, uncoupling of eNOS occurs in apoE ko atherosclerosis but eNOS mediated superoxide production does not outweigh the protective effects of eNOS mediated nitric oxide production. We show that although nNOS is not a major contributor of the vascular nitric oxide formation, it prevents atherosclerosis development. Acute inhibition of nNOS showed a significant reduction of superoxide formation suggesting that nNOS is uncoupled. The exact mechanism of action of nNOS in atheroprotection is yet to be elucidated. Genetic deletion of iNOS reduced NADPH oxidase activity. Thus, iNOS has both direct and indirect proatherosclerotic effects, as it directly generates both nitric oxide and superoxide simultaneously resulting in peroxynitrite formation and indirectly modulates NADPH oxidase activity. We hypothesize that eNOS is coupled in the disease free regions of the vessel and contributes to nitric oxide generation whereas in the diseased region of the vessel it is uncoupled to produce superoxide (Figure 16). nNOS expressed in the smooth muscle cells of the plaque contributes to the local superoxide generation. iNOS expressed in smooth muscle cells and leukocytes of the plaque generates superoxide and nitric oxide simultaneously to produce the strong oxidant peroxynitrite. / Stickstoffmonoxid (NO) ist das prinzipielle Produkt aller Stickstoffmonoxid-Synthasen (NOS). Im Falle eines Mangels an Substrat (L-arginin) und Kofaktoren (Tetrahydrobiopterin, BH4) katalysieren die NOS-Enzyme direkt Superoxid (O2-). Diese Veränderung in der Radikalproduktion wird auch als Entkopplung der NOS bezeichnet. Die alternative Produktion von NO oder O2- durch die NOS bedingen, dass eine Voraussage über die Schlüsselfunktion der einzelnen Enzyme in der Entstehung der Atherosklerose schwierig ist. In unserer Studie evaluieren wir die Produktion von NO sowie O2- in atherosklerotischen Läsionen von apoE ko Mäusen und apoE/NOS doppel knockout (dko) Mäusen denen jeweils eine NOS-Isoform fehlt. Elektronen Spin Resonanz (ESR) Messungen konnten eine signifikante Reduktion sowohl des zirkulierenden, als auch der Gefäßwand eigenen Produktion von NO in apoE/eNOS dko und apoE/iNOS dko Mäusen zeigen, nicht jedoch in apoE/nNOS dko Mäusen. Dies lässt darauf schließen, dass eNOS und iNOS den hauptsächlichen Anteil der vaskulären NO-Produktion in atherosklerotischen Läsionen bewerkstelligen. Die pharmakologische Inhibierung wie auch die genetische Deletion von eNOS und iNOS führten ebenfalls zu einer reduzierten vaskulären O2- produktion, was die partielle Entkopplung beider Enzyme in atherosklerotisch veränderten Gefäßen nahe legt. Obwohl die chronische genetische Deletion von nNOS in apoE/nNOS dko die O2- Produktion nicht verändert, zeigte sich bei der akuten pharmakologischen Inhibierung von nNOS (durch L-NAANG) eine maßgebliche Beteiligung von nNOS an der O2- produktion in apoE ko Mäusen. Schlussfolgernd lässt sich sagen, dass in atherosklerotischen Gefäßen von apoE ko Tieren eine Entkopplung von eNOS statt findet, diese jedoch zu keinem Ausgleich der protektiven Effekte der eNOS vermittelten NO-Produktion führt. Unsere Ergebnisse in apoE/nNOS dko Mäusen zeigen eine atheroprotektive Rolle der nNOS, die sich nicht allein durch eine lokale, vaskuläre NO-Produktion durch das Enzym erklären lässt. Wir postulieren weitere systemisch atheroprotektive Eigenschaften der nNOS. Die signifikante Reduktion der Superoxidproduktion durch eine akute Inhibierung der nNOS weist auf eine Entkopplung der nNOS hin. Der exakte Wirkungsmechansimus von nNOS in der Atheroskleroseprävention ist weiterhin noch zu eruieren. Die genetische Deletion von iNOS führt zu einer reduzierten Aktivität der NADPH-Oxidase. Demnach sind für iNOS direkte sowie indirekte atherosklerosefördernde Effekte anzunehmen, da sie auf direktem Wege gleichzeitig NO und O2- produziert, was in einer Peroxynitritbildung resultiert. Wir stellen die Hypothese auf, dass eNOS in den läsionsfreien Gefäßregionen gekoppelt ist und dort seine atheroprotektiven Effekte durch die NO-Produktion vermittelt, während die eNOS in atherosklerotischen Läsionen entkoppelt vorliegt und hier O2- produziert (Fig. 16). iNOS, welches vor allem in den Plaques, in glatten Muskelzellen und Leukozyten zu finden ist, produziert gleichzeitig hohe Konzentrationen von O2- und NO, die als gemeinsames Endprodukt das stark oxidierende Peroxynitrit ergeben und die von uns dokumentierte proatherosklerotische Wirkung der iNOS vermittelt.

Lipid peroxidation is believed to play a key role in the onset and progression of degenerative disease. Interestingly, although cholesterol is the most abundant lipid in the human body, our understanding of its autoxidation and subsequent decomposition is relatively limited. In fact, until recently, cholesterol-7-hydroperoxide was accepted as the only primary product of cholesterol autoxidation in organic solution, however, our group exhibited that the 4-, 5-, and 6-hydroperoxides are also formed. Although this work facilitated thorough investigation of the complexities of both H-atom abstraction and addition in cholesterol autoxidation in organic solution, it did not account for the dynamic environment of a cell membrane. Herein, we report on the product distribution of these primary autoxidation products in lipid bilayers and how antioxidant supplementation, H-bonding interactions, and concentration of polyunsaturated fatty acid (PUFA) substrate influence both the product distribution and efficiency of autoxidation. Indeed, not only does H-bonding of the 3β-OH of cholesterol appear to shut-down C4 H-atom abstraction, the absence of kinetic chol-5α-OOH product is likely due to the poor potency of α- tocopherol (α-TOH), also as a result of H-bonding with phosphate head group of lipid membrane phospholipids. Therefore, within a lipid membrane the 7-hydroperoxide products predominate, consistent with literature precedent, however the factors involved are more complex than previously understood. Moreover, with the authentic cholesterol hydroperoxides in hand, we sought to determine if the different regioisomers exhibit different cytotoxicity. Glutathione peroxidases (GPXs) are cytoprotective enzymes that reduce harmful hydroperoxides to benign alcohols in vivo. Using RSL3, a small-molecule inhibitor for GPX4, we were able to sensitize mammalian cells to ferroptotic cell death via administration of our exogenously prepared chol-OOHs. Surprisingly, we found that the toxicities of each of 7α-OOH, 6β-OOH and 5α-OOH were only marginally augmented by RSL3 treatment, suggesting that they do not substantially sensitize cells to ferroptosis, perhaps because their decomposition to lipid peroxidation chain-initiating species (i.e. alkoxyl radicals) is not particularly efficient. Instead their cytotoxicities may derive from other mechanisms, such as the induction of apoptosis. This inspired our investigation of the fate of lipid hydroperoxides in vivo, namely the secondary products of the predominant 7-hydroperoxide species. Acid-catalyzed Hock fragmentation, known for the industrial synthesis of phenol and acetone from cumene or implication in the generation of 4-hydroxynonenal (4-HNE), of 5α- and 6β-OOH has been shown by our group to produce highly electrophilic secosterol species; we sought to investigate the same decomposition mechanism for 7α-OOH in light of our investigations in the lipid membrane. Interestingly, we found that Hock fragmentation of 7α-OOH does not exhibit products resulting from the anticipated O-vinyl oxocarbenium intermediate, rather, the mechanism appears to funnel through an α-epoxy carbenium to produce unprecedented A-ring cleavage and epoxide products. Herein, we describe our thorough analysis of this chol-7α-OOH Hock fragmentation and attempts to investigate the presence of these products in biological samples, similar to previous analyses of similar products in atherosclerotic plaque extracts. The products isolated and characterized through this work have provided new mechanistic insight with regards to the primary and secondary oxidation products of cholesterol in vivo; through further development of these findings, we hope to provide a better understanding of the implications of cholesterol oxidation in the pathogenesis of atherosclerosis.

A lipoproteína de baixa densidade eletronegativa (LDL-) é um importante antígeno envolvido na patogênese da aterosclerose. A LDL(-) provoca resposta inflamatória e imunológica, levando à produção de autoanticorpos anti-LDL(-) e a formação subsequente de imunocomplexos (IC-LDL-), os quais também contribuem com o processo aterosclerótico. Diante disso, este trabalho teve como objetivo o desenvolvimento e validação de imunoensaios para quantificação plasmática de LDL(-), anti-LDL(-) e IC-LDL(-), assim como o desenvolvimento de ferramentas aplicáveis em um biossensor para LDL(-). Após a padronização de cada ELISA, foram avaliadas as características de desempenho dos métodos: limites de detecção (LD) e quantificação (LQ), precisão intra e inter-ensaios, exatidão, linearidade de diluição e interferentes. Os LD e LQ do ELISA para LDL(-) foram 0,423 mg/L e 0,517 mg/L de LDL(-), respectivamente. As concentrações plasmáticas de LDL(-) apresentaram linearidade quando os plasmas foram diluídos 1:1000, 1:2000, 1:4000 e 1:8000. Os LD e LQ do ELISA para anti-LDL(-) foram 0,0028 mg/L e 0,0032 mg/L de anti-LDL(-), respectivamente. Os plasmas apresentaram linearidade na diluição quando diluídos 1:100, 1:200, 1:400 e 1:800. Os LD e LQ do ELISA para IC-LDL(-) foram 0,023 g/L e 0,034 g/L de IC-LDL(-), respectivamente. Os plasmas apresentaram linearidade quando diluídos 1:12,5, 1:25, 1:50 e 1:100. Os três ELISAs apresentaram precisão intra e inter-ensaios e recuperação dentro dos limites requeridos para imunoensaios. Para o desenvolvimento de um biossensor para LDL(-), uma proteína recombinante, denominada GFP5-scFv, foi expressa em bactérias Escherichia coli da linhagem BL21(DE3). Para obtenção dessa proteína foi realizada a inserção da sequência de DNA de um fragmento variável de cadeia única (scFv) anti-LDL(-) em um vetor bacteriano com a sequência de DNA da proteína verde fluorescente (GFP5). Dessa forma, a GFP5-scFv é fluorescente e tem afinidade pela LDL(-). Também foram sintetizadas nanopartículas de ouro, as quais podem ser eficientemente utilizadas na supressão da emissão da fluorescência de GFP5-scFv. Portanto, os imunoensaios validados e os aplicativos desenvolvidos para o biossensor são ferramentas que tem potencial para serem utilizadas na avaliação da patogênese da aterosclerose. / The electronegative low-density lipoprotein (LDL) is an important antigen involved in the pathogenesis of atherosclerosis. The LDL(-) causes inflammatory and immune response, leading to production of autoantibodies anti-LDL(-) and the subsequent formation of immune complexes (IC-LDL), which also contribute to the atherosclerotic process. Thus, this study aimed to develop and validate immunoassays for quantification of plasma LDL(-), anti-LDL(-) and LDL-IC(-) as well as developing tools applicable to a biosensor for LDL(-). After the standardization of each ELISA, were evaluated the performance characteristics of methods: limits of detection (LD) and quantification (LQ), intra- and inter-assays precision, accuracy, linearity of dilution and interferences. The LD and LQ of LDL(-) ELISA were 0.423 mg/L and 0.517 mg/L of LDL(-), respectively. Plasmas showed linearity when diluted 1:1000, 1:2000, 1:4000 and 1:8000. The LD and LQ of anti-LDL(-) ELISA were 0.0028 mg/L and 0.0032 mg/L of anti-LDL(-), respectively. Plasmas showed linearity when diluted 1:100, 1:200, 1:400 and 1:800. The LD and LQ of IC-LDL(-) ELISA were 0.023 g/L and 0.034 g/L of LDL-IC(-), respectively. Plasma showed linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. The three ELISAs showed good intra- and inter-assays precision and recovery within the limits required for immunoassays. To develop a biosensor for LDL(-), a recombinant protein, called GFP5-scFv was expressed in Escherichia coli BL21(DE3) strain. The obtainment of this protein was performed inserting the DNA sequence of an anti-LDL(-) single chain variable fragment (scFv) in a bacterial vector with a DNA sequence of green fluorescent protein (GFP5). Thus, the scFv-GFP5 is fluorescent and has affinity for LDL(-). Were also synthesized gold nanoparticles, which can be efficiently used for quenching the fluorescence emission of GFP5-scFv. Therefore, immunoassays validated and developed applications for the biosensor are tools that have potential to be used in evaluating the pathogenesis of atherosclerosis.

Artrite reumatóide é uma doença auto-imune que apresenta acentuado quadro inflamatório e proliferação celular o que, provavelmente, determina a alta prevalência de doenças cardiovasculares quando comparados a população mundial. A mortalidade e a morbidade conseqüentes das doenças cardiovasculares estão 2 vezes aumentadas em pacientes com artrite reumatóide e um dos principais fatores de risco relacionados ao desenvolvimento da aterosclerose é a dislipidemia. Esse importante fator de risco vem sendo associado à artrite reumatóide e as concentrações plasmáticas de lípides são constantemente avaliadas, já que se encontra bem estabelecido a relação entre dislipidemia e alta incidência de doença cardiovascular. No entanto, o verdadeiro impacto das alterações lipídicas na artrite reumatóide não é bem conhecido, já que os resultados de perfil lipídico são contraditórios. Alterações nas concentrações plasmáticas de lípides não necessariamente acompanham distúrbios no metabolismo das lipoproteínas plasmáticas. O objetivo do presente estudo foi avaliar aspectos do metabolismo da LDL e da HDL, em pacientes com artrite reumatóide. Nesse sentido, foi avaliada a cinética plasmática de uma nanoemulsão lipídica artificial com comportamento metabólico semelhante ao da LDL em 30 pacientes com artrite reumatóide divididos em 2 grupos de acordo com a atividade da doença, alta atividade (n=14) e remissão (n=16) e 30 indivíduos controle. A nanoemulsão marcada com éster de colesterol 14EC (EC-14C) e colesterol livre 3H (CL-3H) foi injetada endovenosamente após 12 horas de jejum. As amostras de sangue foram coletadas em tempos pré-determinados (5 min, 1, 2, 4, 6, 8 e 24 horas) após a injeção, para determinação das curvas de decaimento plasmático e da taxa fracional de remoção (TFR) dos lípides marcados, por análise compartimental. As TFR-EC-14C e TFR-CL-3H foram maiores no grupo AR quando comparado ao grupo controle (49%, p<0,05 e 44%, p<0,05, respectivamente), não havendo diferença entre os subgrupos de artrite reumatóide. No grupo artrite reumatóide e em seus subgrupos, as concentrações de HDL-C e apo E foram maiores quando comparados ao grupo controle (33%, p<0,0001 e 20%, p<0,01, respectivamente), enquanto os níveis de apo B foram menores na artrite reumatóide quando comparados ao grupo controle (16%, p<0,05). A transferência de colesterol esterificado radioativo da nanoemulsão para a HDL foi menor na artrite reumatóide, comparando-se com o grupo controle. A transferência dos outros lípides foi similar nos dois grupos. A HDL dos pacientes com artrite reumatóide foi menor do que a dos controles. Esses resultados podem contribuir com a melhor compreensão de possíveis mecanismos relacionados a uma maior incidência de doenças cardiovasculares em pacientes com artrite reumatóide / Mortality and morbidity, as a consequence of cardiovascular diseases, is twice as high in patients with rheumatoid arthritis than in the general worldwide population. This autoimmune disease has predominant inflammatory and cell proliferation background probably explains the high prevalence of cardiovascular disease. Dyslipidemias are important risk factors for cardiovascular disease. This study investigated the link between RA and plasma lipids as a predisposition to this high cardiovascular disease incidence. However, the impact of lipids on cardiovascular risk in rheumatoid arthritis is unclear. So much so, that lipid profiles in patients with rheumatoid arthritis in published studies is contradictory. The events of intravascular lipoprotein metabolism do not necessarily produce altered levels of plasma lipids. In an attempt to unravel novel dysfunctional mechanisms that could trigger pro-atherogenic processes beyond the concentration of the plasma lipids, plasma clearance of a lipidic nanoemulsion that resembles the LDL metabolic behavior were investigated in rheumatoid arthritis patients and compared to control subjects without the disease. 30 patients with rheumatoid arthritis divided into 2 groups according to disease activity, high activity (n=14) and remission (n=16), and 30 controls were studied. A nanoemulsion labeled with 14C-cholesteryl esther (14C-CE) and 3H-free cholesterol (3H-FC) were endovenously injected after which blood samples were collected at pre-determined periods (5 min, 1, 2, 4, 6, 8 and 24 hours), in order to determine the radioactivity of the plasma decay curves and calculate the fractional clearance rate (FCR) of the labeled lipids for compartmental analysis. In the rheumatoid arthritis group and subgroups the HDL-C and apo E concentration were higher when compared to control group (33%, p<0,0001 e 20%, p<0,01, respectively) while apo B concentration was lower (16%, p<0,05). The 14-CE-FCR and 3H-FC-FCR were greater in rheumatoid arthritis group and subgroups when compared to controls (49%, p<0,05 e 44%, p<0,05, respectively). There were no differences between the rheumatoid arthritis subgroups. Therefore, rheumatoid arthritis accelerates the LDL plasma removal, as indicated by a higher 14-CE-FCR and 3H-FC-FCR. The transfer of other lipids was also similar in both groups. The HDL of the rheumatoid arthritis patients was lower than that of the control group. These results could clarify possible mechanisms that can be related to a higher cardiovascular incidence in patients with rheumatoid arthritis

O exercício físico regular contribui para prevenção e redução da aterosclerose, em grande parte, por melhorar o perfil lipídico e o transporte reverso de colesterol (TRC). O TRC é um sistema antiaterogênico que promove a remoção do excesso de colesterol de macrófagos pelas apo A-I e HDL e seu transporte ao fígado, com eliminação de colesterol na bile e fezes. Em camundongos selvagens e transgênicos para proteína de transferência de colesterol esterificado (CETP-tg), o treinamento físico aumentou a transferência de colesterol radioativo de macrófagos para o plasma, fígado e fezes e o conteúdo dos receptores B-E e SR-BI no fígado. Em leucócitos, hepatócitos e enterócitos, o exercício físico aumentou a expressão do receptor de HDL, ABCA-1. Entretanto, não é claro se o exercício físico modula o fluxo de lípides em macrófagos, o que seria determinante para a primeira etapa do TRC. Assim sendo, avaliou-se, em camundongos selvagens e CETP-tg, o efeito do treinamento físico aeróbio sobre: 1) a expressão de genes envolvidos no fluxo de lípides, modulação da resposta inflamatória, vasodilatadora e antioxidante: Pparg (PPAR?), Nr1h3 (LXRalfa), Nr1h2 (LXRbeta), Abca1 (ABCA-1), Abcg1 (ABCG-1), Scarb1 (SR-BI), Cd36 (CD-36), Olr1 (LOX-1), Ccl2 (MCP-1), Tnf (TNFalfa), Il6 (IL-6), Il10 (IL10), Nos3 (eNOS) e Cat (Catalase) na parede arterial e em macrófagos peritoneais; 2) o efluxo de 14C-colesterol de macrófagos peritoneais para HDL2 e apo A-I e 3) a captação de 3H-colesteril oleoil éter-LDL (3H-COE-LDL) acetilada por macrófagos peritoneais. Camundongos machos com 12 semanas de idade, recebendo dieta padrão e água ad libitum, foram aleatoriamente divididos em grupo sedentário e treinado. O treinamento físico foi realizado em esteira, 15m/min, 30 min/dia, 5 vezes/semana, durante 6 semanas. Arco aórtico e macrófagos da cavidade peritoneal foram isolados dos animais sedentários e treinados, imediatamente (0 h) e após 48 h da última sessão de exercício. A expressão de genes foi avaliada por RT-PCR e o efluxo de colesterol, por meio da sobrecarga de macrófagos peritoneais com LDL acetilada e 14C-colesterol, seguindo-se incubação com apo A-I ou HDL2. A captação de LDL foi determinada pela incubação de macrófagos com 3H-COE-LDL acetilada. Não foram observadas alterações sistemáticas na expressão de genes envolvidos no fluxo de lípides em macrófagos e na aorta comparando-se animais sedentários e treinados, selvagens ou CETP-tg. De modo semelhante, não houve diferença no efluxo de colesterol celular e na captação de LDL. Em conclusão, não foram evidenciadas alterações em macrófagos peritoneais e na parede arterial frente ao treinamento físico aeróbio que possam contribuir para o TRC em modelo experimental de camundongos não dislipidêmicos, sem intervenção farmacológica ou alimentar. Sendo assim, o efeito do treinamento físico na melhora do transporte reverso de colesterol, observado em estudos anteriores, deve ser consequente à sua ação sistêmica sobre mediadores deste transporte e expressão de receptores no fígado e intestino / Regular physical exercise prevents and reduces atherosclerosis mainly by improving lipid profile and reverse cholesterol transport (RCT). RCT is an antiatherogenic system that promotes excess cholesterol removal from macrophages by apo A-I and HDL and its transport to the liver. Then, cholesterol can be secreted into bile and excreted in feces. In wild type and cholesteryl ester transfer protein transgenic (CETP-tg) mice exercise training increased the transfer of 14C-cholesterol from macrophages to plasma, liver and feces and elevated SR-BI and B-E receptor content in the liver. In leucocytes, hepatocytes and enterocytes, physical exercise increased mRNA of HDL receptor, ABCA-1. Nonetheless, it is not clear if exercise can modulate lipid flux in macrophages that can be important for the first phase of the RCT. It was analyzed in wild type and CETP-tg mice the effect of aerobic exercise training in: 1) the expression of genes involved in lipid flux, inflammation, oxidation and vasodilation: Pparg (PPAR?), Nr1h3 (LXRalfa), Nr1h2 (LXRbeta), Abca1 (ABCA-1), Abcg1 (ABCG-1), Scarb1 (SR-BI), Cd36 (CD-36), Olr1 (LOX-1), Ccl2 (MCP-1), Tnf (TNFalfa), Il6 (IL-6), Il10 (IL10), Nos3 (eNOS) and Cat (Catalase) in arterial wall and peritoneal macrophages; 2) the apo A-I and HDL2-mediated cholesterol efflux from macrophages and 3) the uptake of 3H-cholesteryl oleoyl ether- acetylated LDL (3H-COE-LDL) by macrophages. Twelve week old male mice fed a chow diet and water ad libitum were randomly assigned to sedentary and trained groups. Exercise training was performed in a treadmill (15m/min, 30 min/day, 5 times/week, during 6 weeks). Aortic arch and peritoneal macrophages were isolated from sedentary and trained animals immediately (time 0) and 48 h after the last exercise session. Gene expression was analyzed by RT-PCR and cholesterol efflux mediated by apo A-I or HDL2 after macrophage overloading with acetylated LDL and 14C-cholesterol. LDL uptake by macrophages was determined by incubation with 3H-COE-acetylated LDL. There were no systematic changes in the expression of macrophages and aortic genes comparing sedentary and trained wild type or CETP-tg mice. Similarly, there were no changes in cholesterol efflux and LDL uptake by macrophages. In conclusion, it was not found alteration in gene expression and cholesterol flux in macrophages and arterial wall that can contribute to the RCT in experimental model of non-dyslipidemic mice without pharmacological or dietary interventions. Therefore, the benefits of aerobic training in improving RCT, observed in previews studies, should be consequent to its systemic action on mediators of this transport and on the expression of hepatic and intestinal receptors

Estudos envolvendo cultura de células, animais e humanos tem sido extensivamente aplicados no monitoramento da aterosclerose, visando seu controle ou mesmo reversão. Entre os modelos animais atualmente utilizados, tem-se observado uma resposta adequada à indução de dislipidemia e inflamação, porém elevada resistência à indução de estresse oxidativo. Uma alternativa para superar essa limitação seria a ingestão de dietas contendo ácidos graxos oxidados. Entretanto, os estudos que investigaram o efeito do consumo de peróxidos e produtos secundários da oxidação lipídica na progressão da aterosclerose têm mostrado resultados inconclusivos. Portanto, o objetivo deste estudo foi de alterar um modelo animal de referência para desenvolvimento de aterosclerose, visando elevar a variação de biomarcadores de estresse oxidativo, através do consumo crônico de ácidos graxos poli-insaturados parcialmente oxidados, em concentrações naturalmente presentes na dieta humana. Camundongos \"knockout\" para receptores LDL (C57BL/6), foram divididos em 4 grupos experimentais. Todos os grupos receberam uma dieta hiperlipídica modificada composta por 30% de lipídios durante 90 dias. Três níveis de oxidação foram induzidos no óleo de linhaça, representando neste estudo um nível baixo, moderado e elevado, caracterizados por uma concentração de hidroperóxidos de 2,47 ± 0,02 (LOW), 3,87 ± 0,04 (MED) e 4,69 ± 0,04 meq LOOH/Kg (HIGH), respectivamente. O grupo que recebeu a ração LOW pode ser considerado o grupo controle negativo, pois o óleo utilizado na formulação de sua ração não sofreu o processo de oxidação. O quarto grupo recebeu a dieta LOW, além da indução de diabetes mellitus do tipo 1 através de estreptozotocina no inicio do estudo sendo esse o grupo controle positivo (CONT+). O óleo de linhaça pré-oxidado utilizado no preparo das rações foi mantido a 4ºC. A peletização das rações alterou a concentração dos marcadores primários e secundários, porém de forma proporcional, mantendo assim a diferença entre os três níveis. O grupo CONT+ apresentou menor ganho de peso que os demais. Nenhuma diferença foi observada entre os grupos em relação ao perfil lipoproteico. Os grupos CONT+ e HIGH apresentaram menor concentração de ácidos graxos no fígado, em especial de ácidos graxos poli-insaturados. Igualmente, ambos os grupos CONT+ e HIGH apresentaram maior concentração de MDA hepático que os grupos MED e LOW, sugerindo um aumento do estresse oxidativo decorrente da ingestão de ácidos graxos poli-insaturados parcialmente oxidados. O grupo HIGH também apresentou um aumento da espessura da parede vascular e do tamanho do lúmen da aorta. Este perfil de resultados sugere que a ingestão crônica de ácidos graxos poli-insaturados parcialmente oxidados contribui para o aumento do estresse oxidativo em camundongos LDLr (-/-), promovendo um aumento na concentração de MDA hepático similar àquele obtido com modelos que utilizam formas mais agressivas de indução. Considerando-se a tendência de aumento no consumo de ácidos graxos poli-insaturados Omega 3, os resultados deste estudo realizados com o óleo de linhaça apontam para a necessidade controle da estabilidade oxidativa desses óleos durante seu processamento e armazenamento, visto que níveis moderados de oxidação já poderiam apresentar potencial aterogênico. / Studies involving cell culture, animals and humans have been extensively applied in the monitoring of atherosclerosis, aiming its control or even this reversal. Among the animal models, it has been observed an appropriate response to the induction of dyslipidemia and inflammation, but a high resistance to oxidative stress induction. An alternative to overcome this limitation would be by the intake of diets containing oxidized fatty acids. However, studies that investigated the effect of consumption of peroxides and secondary products of lipid oxidation in the progression of atherosclerosis have shown controversial results. Therefore, the aim of this study was to alter an animal model for the development of atherosclerosis, aiming to raise the variation in biomarkers of oxidative stress through chronic consumption of polyunsaturated fatty acids partially oxidized under concentrations naturally present in the human diet. LDL receptor \"knockout\" mice (C57BL / 6) were divided into 4 experimental groups. All groups received a modified fat diet containing 30% fat for 90 days. Three levels of oxidation were induced in flaxseed oil, representing low, moderate and high levels, characterized by a hydroperoxide concentration of 2.47 ± 0.02 (LOW), 3.87 ± 0.04 (MED) and 4.69 ± 0.04 LOOHs meq / kg (HIGH), respectively. The fourth group received the LOW diet, beyond diabetes mellitus type 1 through induction by streptozotocin at baseline (CONT +). The pre-oxidized flaxseed oil used to prepare the diets was maintained at 4 °C. The pelletization changed the concentration of primary and secondary products, but proportionally, maintaining the difference between the three levels. CONT + group showed less weight gain than the others. No differences were observed between groups in relation to the lipoprotein profile. The CONT + and HIGH groups showed lower concentration of fatty acids in the liver, especially polyunsaturated fatty acids. In addition both CONT+ and HIGH groups showed higher concentrations MDA in the liver than MED and LOW groups, suggesting increased oxidative stress resulting from the intake of polyunsaturated fatty acids partially oxidized. The HIGH group also showed an increase in the thickness of the vascular wall and the size of the aorta lumen. This profile suggests that the chronic ingestion of partially oxidized polyunsaturated fatty acids contributes to increase oxidative stress in mice LDLr (-/-) , similar to that obtained with models using more aggressive forms of induction. Considering the trend of increased consumption of polyunsaturated fatty acids Omega 3, the results of this study conducted with flaxseed oil highlight to the need of controlling the oxidative stability of these oils during processing and storage, since moderate oxidation levels already present atherogenic potential.

Fármacos antiproliferativos vêm sendo utilizados em procedimentos de angioplastia, onde são administrados localmente por meio de \"stents\" farmacológicos. Embora seja possível que um tratamento sistêmico apresente maior eficiência, os efeitos colaterais dos fármacos antiproliferativos disponíveis constituem uma grande limitação. Estudos recentes em nosso laboratório demonstraram que uma nanoemulsão lipídica rica em colesterol concentra-se em regiões onde a proliferação celular é maior, possivelmente devido a uma maior demanda por colesterol para a síntese de membranas celulares. Assim, consideramos a possibilidade de utilização desta nanoemulsão como veículo de fármacos antiproliferativos para o tratamento da aterosclerose. No presente trabalho, dezesseis coelhos brancos, machos, da raça New Zealand, pesando aproximadamente 3 kg, foram submetidos a uma dieta contendo 1% de colesterol durante quatro semanas e divididos em dois grupos: um grupo recebendo uma injeção endovenosa contendo apenas solução salina e outro grupo tratado com uma associação de etoposídeo e metotrexato, veiculados na nanoemulsão lipídica. A análise da morfometria macroscópica mostra que o tratamento reduz as áreas de lesão aterosclerótica em aproximadamente 84% (razão da área de lesão sobre área total 0,57±0,2 nos animais controle versus 0,089±0,05 nos animais tratados, p<0,05). Houve redução de aproximadamente 3 vezes na razão da íntima-média dos animais tratados em relação aos animais controle. Adicionalmente, observamos que o tratamento reduz a migração de macrófagos na camada íntima do arco aórtico destes animais. Houve amplificação do efeito terapêutico em função da associação destes fármacos e redução na toxicidade. Portanto, a associação destes fármacos antiproliferativos veiculados em um sistema nanoparticulado pode ser promissora para o tratamento da aterosclerose. Estudos em humanos deverão confirmar o potencial desta proposta na terapêutica cardiovascular. / Drug eluting stents have been used in angioplastic procedures to deliver antiproliferative drugs directly to arterial tissue. Presumably, a systemic treatment could be more effective than local administration, but the side-effects of available antiproliferative drugs limit this approach. In recent studies, we have shown that a cholesterol-rich nanoemulsion concentrates in sites of high cell proliferation, possibly due to a high demand of cholesterol to membrane cell synthesis. Thus, we sought to test the emerging hypothesis that our nanoemulsion could be used as a vehicle to antiproliferative drugs for systemic treatment of atherosclerosis. In the present study, sixteen New Zealand White rabbits weighing from 3.2 to 3.8 kg were submitted to a diet containing 1% cholesterol during 4 weeks and then they were split in two groups: a group treated with an intravenous injection of saline solution and a group treated with an association of etoposide and methotrexate delivered in our lipid nanoemulsion. We have shown by macroscopic morphometry that the treatment reduced the lesion areas by roughly 84% (lesion area/total area ratio 0,57±0,2 in control animals, versus 0,089±0,05 in treated animals, p<0,05). The intima-media ratio was reduced by three fold in treated animals. Furthermore, we have observed a reduction in macrophage migration to the arterial intima. The association of etoposide and methotrexate resulted in a synergistic effect without increasing the toxicity. In conclusion, the association of antiproliferative drugs delivered in a nanoemulsion is a promising approach for atherosclerosis treatment. Further studies in humans are needed to confirm the potential of this treatment in cardiovascular therapeutics.

La déstabilisation de la plaque d’athérosclérose reste de nos jours un problème majeur, malgré les progrès récents dans sa compréhension. Les neutrophiles sont des acteurs puissants de l’immunité innée capables d’altérer les plaques. Un chimio-attractant majeur des neutrophiles, le leucotriène B4, pourrait être un des contributeurs potentiels de la déstabilisation des plaques en particulier dans l’endotoxémie, elle-même associée aux accidents cardiovasculaires. L’objectif de ce travail a été de définir le rôle du leucotriène B4 dans l’attraction des neutrophiles dans la plaque au cours de l’endotoxémie et de déterminer si les neutrophiles peuvent basculer l’équilibre qui maintient les plaques stables. Nous avons montré que le recrutement des neutrophiles médié par le leucotriène B4 a un impact délétère sur la stabilité des plaques au cours de l’endotoxémie en favorisant l’apoptose et la dégradation de fibres matricielles. En conclusion, cette étude ouvre la voie vers de nouvelles approches thérapeutiques visant à cibler l’axe leucotriène-neutrophiles dans la maladie athérosclérotique. / Atherosclerotic plaque destabilisation remains an important issue, in spite of the recent advances in its comprehension. Neutrophils are powerful innate immune actors capable of altering plaques. In this context, the leukotriene B4, one of the main chemoattractants of neutrophils, has been proposed as a potential contributor to plaque destabilisation. A particular context in which these two actors are closely linked is endotoxemia, itself associated with plaque destabilisation This work was aimed at determining whether leukotriene B4 plays a role in the chemoattraction of neutrophils in plaques during endotoxemia and at assessing whether neutrophils can tip the balance which maintains plaques stable. We have herein evidenced that the recruitment of neutrophils mediated by leukotriene B4 has a deleterious impact upon plaque stability during endotoxemia by promoting apoptosis and degrading matrix fibres. In conclusion, this study paves the way to novel therapeutic approaches aimed at targeting the axis leukotriene-neutrophil in atherosclerotic disease.