Orientador: Elenice Deffune / Coorientador: Marcone Lima Sobreira / Banca: Regina Moura / Banca: Edivaldo Edner Joviliano / Resumo: O cultivo de células tronco (CT) e a descoberta de técnicas que promovem a diferenciação tecidual apontam para novos domínios da medicina: a terapia celular e a engenharia de tecidos (ET). As limitações da medicina atual encontram na ET uma nova possibilidade terapêutica, com tratamentos regenerativos ou substitutivos para os tecidos danificados. Dentre as limitações médicas encontram-se as doenças arteriais obstrutivas periféricas (DAOP). A DAOP está presente em 5% da população e cerca de 20 a 30% dos casos podem evoluir para amputação do membro inferior acometido, mesmo com os tratamentos hoje existentes. A ET apresenta-se como uma ferramenta promissora para a resolução dos problemas cardiovasculares, particularmente para os que afetam as artérias de pequeno diâmetro (<5 mm), sendo factível a produção de enxertos vasculares autólogos, sob a ótica da medicina personalizada. O objetivo deste trabalho foi construir de um modelo experimental de vaso sanguíneo para enxerto, utilizando técnicas de ET para a diferenciação de células tronco mesenquimais de tecido adiposo (CTMta) em células endoteliais. Inúmeras etapas metodológicas foram necessárias: identificação da melhor forma de descelularizar as veias de animais doadores para confecção do arcabouço (scaffold), onde foram comparados 3 diferentes protocolos utilizando os detergentes Triton X-100, deoxicolato de sódio (DS) e dodecil sulfato de sódio (SDS); obtenção, expansão e caracterização por citometria de fluxo com anti-CD90 de CTMta; comprovação da adesão das CTMta sobre o scaffold utilizando imunofluorescência e o kit Qtracker; estabelecimento do índice de apoptose das CTMta sobre o scaffold descelularizado por eventual resíduo químico; análise histológica por HE e realização de imunohistoquímica com... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Stem cells (SC) cultivation and the development of new techniques to promote tissue differentiation evolved to new medicine fields of research: cell therapy and tissue engineering (TE). The current limitations in medicine rely on TE for new therapies as they provide regeneration and substitution of the damaged tissue. Within therapeutic limitations are the peripheral arterial obstruction diseases (PAD). PAD affects 5% of population and about 20-30% of cases may progress to amputation of the limb, even if the treatments that exist today. TE is a promising tool for cardiovascular diseases, especially those where the vessel is a small caliber artery (<5 mm), as it is possible to recreate the structure of the vessel with autologous vascular grafts from the perspective of personalized medicine. This research aimed to propose the creation of an experimental blood vessel using TE techniques and adipose-derived stem cell differentiation into endothelium. Several techniques were required: to gather enough veins and remove its cells, using three different protocols with Triton X 100, sodium deoxycholate (SD) and sodium dodecil sulfate (SDS); to obtain, cultivate and characterize adipose-derived stem cells with the CD90 marker and flow citometry; to prove the cells could adhere to the scaffold using immunofluorescence and Qtracker kit; to establish the cell apoptosis on the scaffold for possible chemical residue; histology and immunohistochemistry on the cell seeded scaffold using the fascin marker. Were obtained 90 jugular and cava vein segments and were decellularized, withing 11 different protocols. The best protocols were 2h of 1% SD, 1h of 2% SDS and 2h of 1% SDS. The protocols where triton were used gave no good results. There was no relevant difference between the apoptosis on the best... (Complete abstract click electronic access below) / Mestre

Background: Myocardial infarction (MI), also known as heart attack, is the leading cause of morbidity and mortality among the heart diseases spectrum. It results from an insufficient supply of blood to the heart muscles. According to the United States (U.S.) Centers for Disease Control and Prevention (CDC), about 610 000 people die of heart disease in the U.S. every year. Myocardial infarction contributes 370 000 of these deaths annually. Every 40 seconds, someone in the U.S. experience heart attack. This burden is disproportionately distributed within the U.S. population. The rate of heart disease in Central Appalachia is 249 per 100 000, 42% higher than the national rate. Exploring further within the region, rural areas experience higher heart disease mortality rates; 27% higher than the region’s metro counties. According to 2018 America Health Ranking, the prevalence of heart attack in Tennessee is 5.9%, compared to the 4.9% nationwide, with the majority of the burden seen among adults aged ≥65 years and with a 1:1.8 female to male ratio. Patients with heart disease often have other comorbid conditions such as peripheral arterial disease (PAD), hypertension, diabetes, dyslipidemias, which contribute immensely to this chronic condition. Therefore, the aim of this study is to explore the association between cardiovascular comorbidities such as diabetes mellitus, hypertension and dyslipidemia, and the prevalence of heart attack among patients with PAD in rural Central Appalachia. Methods: We used a cross-sectional data of patients diagnosed with PAD in the Central Appalachian region. A total of 13455 patients with PAD were recruited using ICD 9 and 10 search terms for PAD from the electronic medical records (EMR) system between January 1, 2008, and April 30, 2018. Descriptive statistics of the variables were extracted. The association between the comorbidities, including hypertension, diabetes, dyslipidemia, body mass index(BMI) and the prevalence of MI was determined using a binomial logistic regression model. All analysis was done using IBM SPSS statistics 25. Results: Of the total 13455 patients with PAD, 3045 had MI (37.7% female and 62.3% male) with a mean age of 69±10.5years. While 93% had hypertension, 56% had diabetes. For the lipids, the mean of HDL, Cholesterol, and LDL among participants with a history of MI is 40.99mg/dl±13, 156.32mg/dl±45, 82.08mg/dl±36.35 respectively. The results of binomial logistic regression with stratification based on gender shows that female patients with diabetes had 86% increased odds of MI [OR: 1.858, C.I: 1.308-2.638, p-value=0.001), and for female hypertensives, 4.51 times increased odds of MI was found (C.I: 1.576-12.895, p-value=0.005). The male diabetics and hypertensives showed a similarly increased odds of MI with (OR 1.138, C.I: 0.870-1.489 p-value=0.345) and (OR 3.697C.I: 1.559-8.736, p-value=0.003) respectively. No significant association was found among the various lipid profiles examined. Conclusion: The results showed that female PAD patients with hypertension and diabetes have a significantly increased likelihood of having MI. In contrast, male with PAD also showed increased likelihood (although to a lesser degree) of MI in those with hypertension, but not those with diabetes. These findings underscore the importance of a proactive approach to preventive care and adequate control among PAD patients with diabetes and hypertension in a bid to curbing the morbidity and mortality associated with myocardial infarction among residents in Central Appalachia.

Objectives: Peripheral arterial disease (PAD) is a common and often overlooked entity responsible for considerable morbidity and mortality. Recent evidence suggests that nontraditional risk factors such as vitamin D may contribute to atherosclerosis. We hypothesized that vitamin D status was associated with cardiovascular risk factors and that vitamin D deficiency (25(OH)D <20 ng/mL) enhanced the risk of amputation. Design: We reviewed medical records of 1435 veterans between 2000 and 2008 in Tennessee via retrospective chart analysis using correlations, logistic regressions, t tests, and χ2 analyses. Results: Vitamin D status was significantly and inversely correlated with body mass index (BMI), glucose, and triglyceride values. Hypertension and diabetes but not smoking also emerged as significantly associated. Of the sample population, 5.2% (n = 75) had an amputation performed. Those individuals who were vitamin D deficient had a significantly higher amputation rate (6.7%) compared with patients who were nondeficient (4.2%). BMI, triglyceride, total cholesterol, hypertension, and diabetes were found to account for 5.7% of the variation in amputation status. Vitamin D concentration and deficiency status accounted for a nonsignificant amount of additional variance. Conclusions: We conclude that vitamin D deficiency is closely linked to increased adiposity, triglyceride, and glucose measurements. Vitamin D deficiency was associated with an increased amputation risk in veterans with PAD and appears to mediate its effects through traditional risk factors.

Background: Peripheral arterial disease (PAD) affects over 8 million people in the United States and has been found to be associated with an increased incidence of coronary and carotid artery disease. The ankle brachial index (ABI) measurement is a highly specific noninvasive screening and diagnostic test for PAD, but is rarely performed in primary care office settings. This study sought to determine the actual performance time involved in completing an ABI in a primary care office. Methods: Data were collected by one provider on a convenience sample of women who met the inclusion criteria. The time was recorded at the beginning and upon the completion of the ABI procedure for each patient. Analysis of the time data was completed and barriers to performing the ABI were recorded by the provider. Results: The average time to complete an ABI was 5 minutes, with a range of 3-11 minutes. In 83.8% of patient encounters, the ABI procedure took less than 6 minutes to complete. Barriers identified by the provider included the additional time needed to explain the test and assist patients into the proper testing position. Conclusions: The actual performance time for an ABI in a primary care setting takes an average of 5 minutes, but additional time may be required for patient preparation and education. With proper scheduling and training, the ABI can be completed in a timely manner. The ABI is an important screening/diagnostic test that can be performed in primary care and potentially impact patient treatment plans.

Patients with Peripheral Arterial Disease have a reduced capacity for exercise, the exact causes of which are poorly understood. This thesis investigated alternative testing procedures that aim to provide a more complete and precise description of the exercise capacities of these patients. Furthermore, the potential roles of gastrocnemius muscle fibre morphometry, capillary supply and glycogen stores in the exercise tolerance of PAD patients were studied. Study one aimed to determine the effect of test repetition on maximal exercise performance and test-to-test variability in PAD patients using an incremental treadmill walking test (T) (n=5), an incremental cycle test (C) (n=5), and incremental endurance (PF-endurance) and maximal strength (PF-strength) plantar flexion tests (n=5). Tests were conducted once per week for eight weeks. Performance was stable on the T (~530 s) and C (~500 s) tests across the eight weeks. Test-to-test variance on T decreased from 16%CV (CV: coefficient of variation) to 6%CV (p=.21,NS), and from ~8%CV to 2%CV on C (p<.05) over the eight week period. Variance of peak gas exchange variables tended to decrease with performance variance on both tests; however, other physiological variables, and the associated variance levels, were stable throughout the study. PF strength (635-712N) gradually increased over the initial 2-3 weeks (p<.05) which was accompanied by a reduction in variance from ~8%CV to ~3%CV (p<.05). Similarly, PF endurance increased over the first two weeks (~32,000 to 41500 N.s-1) while variance of this measure fell from ~21%CV to ~10%CV (p<.05) over the study duration. It is concluded that the implementation of familiarisation sessions leads to a reduction in whole body and local calf muscular performance variance in patients with PAD. Using a randomised crossover design, study two aimed to compare performance and the physiological and symptomatic responses between a T test and a C test in 16 patients with PAD. Peak exercise time on C (690 s) was greater than that on T (495 s); however the two were significantly correlated (n=16, r=.69, p<.05). Peak HR (120 bpm), VO2 (~1.22 l.min-1) and rate pressure product (~20') did not differ between the two tests, nor did the post exercise ankle pressure (T: 56; C: 61 mmHg). In two subjects with lower back pain during C, the ankle pressure of their "worst" limbs failed to fall by >10mmHg. Performance on both the T and C tests was closely related to the onset of leg symptoms; however the site of pain during C was much more variable than during T. Incremental cycle testing would overcome some of the limitations of treadmill testing (e.g. measurement of mechanical work), and it appears to be an acceptable alternative for measuring the exercise capacity and physiological exercise responses in known claudicants. Use of cycle ergometry for the diagnosis of PAD requires testing in the general population. Study three aimed to compare whole body (T test and C test) and local calf muscular (PF strength and endurance) exercise performance between 16 PAD patients (age: 63 ± 2; BMI: 25.9 ± 1.1) and 13 healthy, sedentary control (CON) subjects (age: 62 ± 1; BMI: 25.9 ± 0.4), and to describe relationships between the whole body and local calf muscular exercise capacities within the two groups. Furthermore, this study aimed to compare several histochemical characteristics of the medial gastrocnemius muscle fibres between PAD and CON, and to establish whether these factors were related to the exercise capacities of both groups. Maximal performance on T was 59% lower in the PAD group compared with the CON group, as was performance on C (50%), PF strength (25%), and PF endurance (58%). Compared with CON, PAD patients had a lower estimated calf muscle mass and a slight reduction (10%) in muscle fibre size (p=.14, NS). They also had a lower proportion of type I fibres (PAD: 49%; CON: 62%) that was offset by a greater proportion of type IIA fibres (PAD: 27%; CON: 16%), and a reduction in the capillary contacts per muscle fibre (PAD: 1.63; CON: 2.12) compared with CON. When expressed relative to fibre area there were no differences in capillarisation between PAD and CON; however this index was significantly related to resting and post exercise ABI in the PAD patients. There were no differences in the mixed muscle [glycogen], nor the optical density of glycogen in the individual fibres, between the two groups. PF endurance was poorly predictive of walking performance, and did not correlate with any of the morphological variables in both groups. Calf muscle mass correlated with PF strength (r=.59 - .62), and strength was correlated with T performance (r= .61 - .63) in both groups. In the PAD patients, T performance was correlated with the cross sectional area (n=12, r=.72, p<.05), capillary contacts (n=10, r=.81, p<.05) and glycogen density (n=9, r=.81, p<.05) of type I fibres. This study confirms that a reduction in calf strength, which appears to be mediated through muscle atrophy, plays some role in the reduced exercise capacity of claudicants. While both fibre area and capillary supply seem to be of relevance to the exercise capacity of PAD patients, these two factors are closely linked and further research is required to establish the determinants, and relative importance of both. An important, and possibly limiting role of carbohydrate oxidisation in PAD patients is supported by the strong relationship between type I glycogen stores and whole body exercise capacity.

Dietary fat has long been implicated in the etiology of metabolic and cardiovascular disease, and both the amount of fat and the fatty acid composition of the diet play a role in disease progression. Although national health organizations have set guidelines for the recommended intake of dietary fats, questions remain regarding the optimal dietary lipid profile for maintaining health and improving disease conditions. Whether certain types of fatty acids from plant-based oils can improve metabolic and vascular disease has been studied and debated, but not fully determined. In this study, we investigated the role of dietary fatty acids from plant-based oils, and examined their effects on metabolic and vascular disease parameters. Obese fa/fa Zucker rats were fed a diet containing flaxseed oil, which resulted in smaller adipocytes and decreased adipose tissue T-cell infiltration. Obese-prone Sprague Dawley rats were fed high-fat diets with different proportions of mono- and polyunsaturated fats. Changes were observed in adipose tissue levels of fatty acid synthase, adiponectin and fatty acid receptors GPR41 and GPR43, but other metabolic and inflammatory mediators in adipose tissue and serum remained stable. A systematic review and meta-analysis on the impact of n3 fatty acids on major cardiovascular endpoints showed that little evidence exists to support their role in peripheral arterial disease. Then again, very few studies on this topic have been conducted. To address this research gap, a clinical trial was designed to investigate the effects of a dietary intervention on blood vessel properties in people with peripheral arterial disease. Participants in the Canola-PAD Study consumed 25 g/day of canola oil or a Western diet oil mixture as part of their usual diet for 8 weeks. Although the intervention altered phospholipid fatty acids, vascular function, the lipid profile and inflammatory markers stayed relatively stable. Overall, this research demonstrates that dietary fatty acids from plant-based oils can be immunomodulatory, but at the physiological doses tested they are not potent mediators of functional changes in obesity or vascular physiology. / October 2015

3-nitrotyrosine (3NT), a stable end product arising from the interaction of proteins and reactive nitrogen species such as peroxynitrite, is produced during periods of oxidative stress. 3NT is, therefore, of interest as a potential biomarker in a variety of disease states where oxidative stress is known to be involved in the pathology, for example intermittent claudication. The aim of this thesis was to develop sensitive and specific immunoassays to assess the levels of 3NT in plasma samples from claudicants and to investigate the protein nitration profile. Clinical data and plasma samples were collected from claudicant (n=33) and control (n=6) subjects. Analysis of data confirmed the difficulty of using parameters such as ankle brachial index (ABI) in diagnosis, supporting the need for investigations into potential biomarkers. Development of indirect and competitive ELISAs using electrochemically nitrated bovine serum albumin as the standard revealed that the detection of 3NT was dependent on the antibody being able to access the 3NT-residues within the protein. Various denaturing conditions and different types of microtitre plate were utilised during development. Initially the presence of 3NT in claudicant or control whole plasma samples could only be detected using dot blot immunodetection. Affinity purification techniques for the fractionation of the plasma proteins were therefore applied. Subsequently, 3NT-containing plasma proteins were found to be present in all of the claudicant and control samples using the developed competitive ELISA. Proteomic analysis of the 3NT-affinity purified samples, using MALDI-MS and LC-ESI-MS/MS, confirmed the presence of human serum albumin, serotransferrin and apolipoprotein A1 and A2 precursors within those protein bands staining immunopositive for 3NT on SDS-PAGE gels. The identification of apolipoprotein A1 within 3NT-immunopositive bands confirms previous reports suggesting the oxidative modification of HDL may contribute to the link between inflammation and the pathology of atherosclerosis.

L’artériopathie oblitérante des membres inférieurs (AOMI) est une affection vasculaire obstructive athéroscléreuse. Elle touche 20% des plus de 70 ans. L’ischémie critique des membres inférieurs (ICMI) est le stade ultime et nécessite une revascularisation. La thérapie cellulaire (TC) tente de promouvoir l’angiogenèse chez les patients en impasse thérapeutique. Les cellules souches mésenchymateuses (MSCs) sont un bon candidat, car elles associent des propriétés angiogéniques et immunomodulatrices. L’objectif de cette thèse a été 1), d’évaluer dans un modèle murin d’ischémie de la patte, l’efficacité de deux types de MSCs: les MSCs indifférenciées et les MSCs « endothelial like » (MELs) en les comparant aux cellules dérivées de la moelle osseuse; 2) d’analyser le membre ischémié par spectroscopie Raman.Les MELs et les MSCs ont induit une restauration complète du flux et une amélioration fonctionnelle du membre. Aucune nécrose n’a été signalée après administration des MELs. Histologiquement, les MELs ont induit les taux les plus élevés de néoangiogenèse et de régénération musculaire. Les acquisitions spectrales ont révélé que la spectroscopie Raman peut différencier un membre ischémié d'un membre sain et indique une augmentation du ratio lipide/protide au cours de l’ischémie.Notre étude indique que les MELs de patients ICMI rétablissent le flux sanguin et permettent la réparation musculaire. La spectroscopie Raman peut être utilisée pour évaluer l'ischémie chez les patients atteints d’ICMI. / Peripheral artery disease (PAD) is an atherosclerotic obstructive disease affecting lower limbs arteries. It affects nearly 20% of over 70s. Critical limb ischemia (CLI) is the ultimate stage and requires revascularization. Cell therapy (CT) has been proposed for patients with CLI. Clinical trials were encouraging but failed to establish efficacy. Mesenchymal stem cells (MSCs) may be a better option as they combine angiogenic and immunomodulatory properties. MSCs can be obtained from BMCs of CLI-patients. The aim of this study was first to evaluate, in a murine hindlimb ischemia model, the efficacy of two types of MSCs: undifferentiated mesenchymal stem cells (MSCs) and “endothelial like” MSCs (MELs) in comparison with currently used BMCs. Secondly, the objective was to perform a non-invasive analysis of ischemic limb using Raman Spectroscopy. MELs and MSCs induced complete perfusion restoration whereas BMCs did not. The complete flow recovery was significantly earlier with MELs in comparison with MSCs. Both MSCs and MELs improved functionality more efficiently than BMCs. Interestingly, complete limb salvage was observed in the MELs treated group exclusively. In muscles, MELs induced the highest rate of neoangiogenesis and the best muscle repair as shown by the presence of regenerated myofibers. Spectral acquisitions revealed that Raman spectroscopy can discriminate ischemic limb from healthy limb and can grade ischemia over time.Our study brings evidence that MELs obtained from CLI-patients can restore blood flow and provide muscle repair. Moreover Raman spectroscopy could be used clinically to assess ischemia in CLI-patients.

O aumento da morbimortalidade cardiovascular nos indivíduos com doença arterial periférica (DAP) e claudicação intermitente (CI) se associa a alterações hemodinâmicas, autonômicas, endoteliais, inflamatórias e de estresse oxidativo, que são inerentes ao desenvolvimento da própria doença. O treinamento de caminhada (TC) pode atenuar os processos fisiopatológicos que cursam com a doença, o que precisa ser melhor investigado. Por outro lado, a execução da caminhada até a dor máxima nesses indivíduos provoca episódios de isquemia, que geram alterações nesses processos e promovem sobrecarga cardiovascular. É possível que o TC possa atenuar essas respostas após o esforço máximo, o que também foi pouco investigado. Dessa forma, o objetivo do presente estudo foi verificar, em indivíduos com DAP e CI, o efeito de um TC sobre a função e regulação cardiovasculares, bem como sobre marcadores locais (músculo) e sistêmicos (sangue) de função endotelial, estresse oxidativo e inflamação, avaliados em repouso e após uma caminhada até a dor máxima de claudicação. Para tanto, 32 homens com DAP e CI foram divididos aleatoriamente em dois grupos: TC (n = 16, 2 sessões/sem, 15 séries de 2 min de caminhada na frequência cardíaca do limiar de dor intercaladas com 2 min de pausa passiva) e controle (CO, n =16, 2 sessões/semana, 30 min alongamento). No início e ao final do estudo, os indivíduos realizaram uma caminhada máxima e as seguintes avaliações foram realizadas pré e pós-caminhada: função cardiovascular (pressão arterial - PA, frequência cardíaca - FC, duplo produto - DP); regulação autonômica cardiovascular (variabilidade da FC e da PA e sensibilidade barorreflexa - SBR); função endotelial (óxido nítrico sanguíneo - NO e óxido nítrico sintase muscular - eNOS); estresse oxidativo (catalase - CAT, superóxido dismutase - SOD, peroxidação lipídica - LPO no sangue e no músculo); e inflamação (interleucina-6 - IL-6, proteína C-reativa - PCr, fator de necrose tumoral alfa - TNF-alfa, moléculas de adesão intercelular - ICAM, moléculas de adesão vascular - VCAM no sangue e no músculo). Os dados foram avaliados pela ANOVA de 2 fatores, empregando-se o teste de post-hoc de Newman-Keuls e adotando-se P<0,05 como significante. No repouso, o TC diminuiu a sobrecarga cardiovascular (PA sistólica, PA média, FC e DP) e o balanço simpatovagal cardíaco; aumentou a SBR, a biodisponibilidade de NO, a eNOS e a defesa antioxidante (SOD e CAT no sangue; SOD no músculo), além de reduzir o perfil inflamatório (PCr, ICAM e VCAM no sangue; IL-6 e PCr no músculo) (todos, p<0,05). Em relação à resposta à caminhada máxima, o TC: 1) não modificou o aumento da sobrecarga cardiovascular ao esforço, mas diminuiu a sobrecarga absoluta após o exercício (PA sistólica, PA média e DP); 2) diminuiu a resposta do NO sanguíneo e da eNOS muscular, sem alterar os valores absolutos atingidos após o exercício; 3) não modificou a resposta e os valores absolutos pós-exercício da capacidade antioxidante (SOD e CAT) e do estresse oxidativo (LPO) sistêmicos e locais, mas impediu o aumento da LPO pós-exercício observado no grupo CO; e 4) aumentou a resposta inflamatória sistêmica e local ao exercício (TNF-alfa, ICAM e VCAM no sangue e IL-6, PCr e VCAM no músculo) com manutenção da inflamação sistêmica pós-exercício e redução da inflamação local (VCAM). Em conclusão, em homens com DAP e CI, o TC melhora a modulação autonômica e a função cardiovascular, aumenta a biodisponibilidade de NO e diminui o estresse oxidativo e a inflamação tanto sistêmicos quanto locais. Além disso, o TC, de modo geral, não altera ou mesmo reduz as respostas desses marcadores após uma caminhada até a dor máxima de claudicação / The increase in cardiovascular morbimortality in individuals with peripheral artery disease (PAD) and intermittent claudication (IC) is associated with alterations in cardiovascular function, cardiac autonomic modulation, endothelial function, oxidative stress and inflammation, which are processes inherent to the disease development. Walking training (WT) may attenuate these pathophysiological processes, however, knowledge about these effects of WT is scarce and controversial. On the other hand, in these individuals, a bout of walking promotes ischemic episodes that may exacerbate these processes, leading to cardiovascular overload. WT might attenuate these post-walking responses; however, these effects were also poorly studied. Thus, the aim of the present study was to evaluate, in individuals with PAD and IC, the effects of WT on cardiovascular autonomic modulation and function as well as on blood and muscle markers of endothelial function, oxidative stress and inflammation assessed at rest and after a walking until maximal leg pain. Thirty-two men with PAD and IC were randomly allocated in two groups: WT (n = 16, 2 sessions/week, 15 bouts of 2 min walking at an intensity corresponding to the heart rate of the pain threshold interspersed with 2 min of passive pause) and control (CO, n =16, 2 sessions/week, 30 min of stretching). At the beginning and end of the study, the subjects underwent a maximal walking and the following evaluations were done pre and post-exercise: cardiovascular function (blood pressure - BP, heart rate - HR, rate pressure product - RPP); cardiovascular autonomic modulation (HR and BP variabilities and baroreflex sensitivity - BRS); endothelial function (blood nitric oxide - NO and muscle nitric oxide synthase - eNOS); oxidative stress (catalase - CAT, superoxide dismutase - SOD, lipid peroxidation - LPO measured in blood and muscle); and inflammation (interleukin-6 - IL-6, C-reactive protein - CRP, tumor necrosis factor alpha - TNF-alpha, intercellular adhesion molecules - ICAM, vascular adhesion molecules - VCAM measured in blood and muscle). Data were evaluated by 2-way ANOVA, and Newman-Keuls test was used as a post-hoc. P <0.05 was set as significant. At rest, WT decreased cardiovascular overload (systolic BP, mean BP, HR and RPP) and sympathovagal balance; increased BRS, blood NO, muscle eNOS and antioxidant defence (blood SOD and CAT, and muscle SOD), besides decreasing inflammatory markers (blood CRP, ICAM and VCAM and muscle IL-6 and CRP). Concerning the response after maximal walking, WT: 1) did not change cardiovascular overload increase after the effort, but reduced the absolute post-exercise overload (systolic BP, mean BP and RPP); 2) decreased blood NO and muscle eNOS responses without changing the absolute values achieved after the exercise; 3) did not change systemic and local antioxidant (SOD and CAT) and oxidative stress (LPO) responses as well as post-exercise absolute values; but mitigated the increase in postexercise oxidative stress observed in the CO group; and 4) increased systemic and local inflammatory responses (blood TNF-alpha, ICAM e VCAM and muscle IL-6, PCr e VCAM), but did not change post-exercise absolute systemic inflammation and decreased post-exercise absolute local inflammation (VCAM). In conclusion, in men with PAD and IC, WT improves cardiovascular function and autonomic modulation, increases NO bioavailability and decreases systemic and local oxidative stress and inflammation. In addition, in general, WT does not alter or even reduces these processes responses after a walking until maximal claudication pain

O aumento da morbimortalidade cardiovascular nos indivíduos com doença arterial periférica (DAP) e claudicação intermitente (CI) se associa a alterações hemodinâmicas, autonômicas, endoteliais, inflamatórias e de estresse oxidativo, que são inerentes ao desenvolvimento da própria doença. O treinamento de caminhada (TC) pode atenuar os processos fisiopatológicos que cursam com a doença, o que precisa ser melhor investigado. Por outro lado, a execução da caminhada até a dor máxima nesses indivíduos provoca episódios de isquemia, que geram alterações nesses processos e promovem sobrecarga cardiovascular. É possível que o TC possa atenuar essas respostas após o esforço máximo, o que também foi pouco investigado. Dessa forma, o objetivo do presente estudo foi verificar, em indivíduos com DAP e CI, o efeito de um TC sobre a função e regulação cardiovasculares, bem como sobre marcadores locais (músculo) e sistêmicos (sangue) de função endotelial, estresse oxidativo e inflamação, avaliados em repouso e após uma caminhada até a dor máxima de claudicação. Para tanto, 32 homens com DAP e CI foram divididos aleatoriamente em dois grupos: TC (n = 16, 2 sessões/sem, 15 séries de 2 min de caminhada na frequência cardíaca do limiar de dor intercaladas com 2 min de pausa passiva) e controle (CO, n =16, 2 sessões/semana, 30 min alongamento). No início e ao final do estudo, os indivíduos realizaram uma caminhada máxima e as seguintes avaliações foram realizadas pré e pós-caminhada: função cardiovascular (pressão arterial - PA, frequência cardíaca - FC, duplo produto - DP); regulação autonômica cardiovascular (variabilidade da FC e da PA e sensibilidade barorreflexa - SBR); função endotelial (óxido nítrico sanguíneo - NO e óxido nítrico sintase muscular - eNOS); estresse oxidativo (catalase - CAT, superóxido dismutase - SOD, peroxidação lipídica - LPO no sangue e no músculo); e inflamação (interleucina-6 - IL-6, proteína C-reativa - PCr, fator de necrose tumoral alfa - TNF-alfa, moléculas de adesão intercelular - ICAM, moléculas de adesão vascular - VCAM no sangue e no músculo). Os dados foram avaliados pela ANOVA de 2 fatores, empregando-se o teste de post-hoc de Newman-Keuls e adotando-se P<0,05 como significante. No repouso, o TC diminuiu a sobrecarga cardiovascular (PA sistólica, PA média, FC e DP) e o balanço simpatovagal cardíaco; aumentou a SBR, a biodisponibilidade de NO, a eNOS e a defesa antioxidante (SOD e CAT no sangue; SOD no músculo), além de reduzir o perfil inflamatório (PCr, ICAM e VCAM no sangue; IL-6 e PCr no músculo) (todos, p<0,05). Em relação à resposta à caminhada máxima, o TC: 1) não modificou o aumento da sobrecarga cardiovascular ao esforço, mas diminuiu a sobrecarga absoluta após o exercício (PA sistólica, PA média e DP); 2) diminuiu a resposta do NO sanguíneo e da eNOS muscular, sem alterar os valores absolutos atingidos após o exercício; 3) não modificou a resposta e os valores absolutos pós-exercício da capacidade antioxidante (SOD e CAT) e do estresse oxidativo (LPO) sistêmicos e locais, mas impediu o aumento da LPO pós-exercício observado no grupo CO; e 4) aumentou a resposta inflamatória sistêmica e local ao exercício (TNF-alfa, ICAM e VCAM no sangue e IL-6, PCr e VCAM no músculo) com manutenção da inflamação sistêmica pós-exercício e redução da inflamação local (VCAM). Em conclusão, em homens com DAP e CI, o TC melhora a modulação autonômica e a função cardiovascular, aumenta a biodisponibilidade de NO e diminui o estresse oxidativo e a inflamação tanto sistêmicos quanto locais. Além disso, o TC, de modo geral, não altera ou mesmo reduz as respostas desses marcadores após uma caminhada até a dor máxima de claudicação / The increase in cardiovascular morbimortality in individuals with peripheral artery disease (PAD) and intermittent claudication (IC) is associated with alterations in cardiovascular function, cardiac autonomic modulation, endothelial function, oxidative stress and inflammation, which are processes inherent to the disease development. Walking training (WT) may attenuate these pathophysiological processes, however, knowledge about these effects of WT is scarce and controversial. On the other hand, in these individuals, a bout of walking promotes ischemic episodes that may exacerbate these processes, leading to cardiovascular overload. WT might attenuate these post-walking responses; however, these effects were also poorly studied. Thus, the aim of the present study was to evaluate, in individuals with PAD and IC, the effects of WT on cardiovascular autonomic modulation and function as well as on blood and muscle markers of endothelial function, oxidative stress and inflammation assessed at rest and after a walking until maximal leg pain. Thirty-two men with PAD and IC were randomly allocated in two groups: WT (n = 16, 2 sessions/week, 15 bouts of 2 min walking at an intensity corresponding to the heart rate of the pain threshold interspersed with 2 min of passive pause) and control (CO, n =16, 2 sessions/week, 30 min of stretching). At the beginning and end of the study, the subjects underwent a maximal walking and the following evaluations were done pre and post-exercise: cardiovascular function (blood pressure - BP, heart rate - HR, rate pressure product - RPP); cardiovascular autonomic modulation (HR and BP variabilities and baroreflex sensitivity - BRS); endothelial function (blood nitric oxide - NO and muscle nitric oxide synthase - eNOS); oxidative stress (catalase - CAT, superoxide dismutase - SOD, lipid peroxidation - LPO measured in blood and muscle); and inflammation (interleukin-6 - IL-6, C-reactive protein - CRP, tumor necrosis factor alpha - TNF-alpha, intercellular adhesion molecules - ICAM, vascular adhesion molecules - VCAM measured in blood and muscle). Data were evaluated by 2-way ANOVA, and Newman-Keuls test was used as a post-hoc. P <0.05 was set as significant. At rest, WT decreased cardiovascular overload (systolic BP, mean BP, HR and RPP) and sympathovagal balance; increased BRS, blood NO, muscle eNOS and antioxidant defence (blood SOD and CAT, and muscle SOD), besides decreasing inflammatory markers (blood CRP, ICAM and VCAM and muscle IL-6 and CRP). Concerning the response after maximal walking, WT: 1) did not change cardiovascular overload increase after the effort, but reduced the absolute post-exercise overload (systolic BP, mean BP and RPP); 2) decreased blood NO and muscle eNOS responses without changing the absolute values achieved after the exercise; 3) did not change systemic and local antioxidant (SOD and CAT) and oxidative stress (LPO) responses as well as post-exercise absolute values; but mitigated the increase in postexercise oxidative stress observed in the CO group; and 4) increased systemic and local inflammatory responses (blood TNF-alpha, ICAM e VCAM and muscle IL-6, PCr e VCAM), but did not change post-exercise absolute systemic inflammation and decreased post-exercise absolute local inflammation (VCAM). In conclusion, in men with PAD and IC, WT improves cardiovascular function and autonomic modulation, increases NO bioavailability and decreases systemic and local oxidative stress and inflammation. In addition, in general, WT does not alter or even reduces these processes responses after a walking until maximal claudication pain