No description available.

Les cellules et la matrice extracellulaire composant les tissus humains ont une architecture tridimensionnelle organisée leur conférant des propriétés propres à leur physiologie. La plupart des substituts créés par génie tissulaire ne possèdent pas cette organisation architecturale leur permettant d'être physiologiquement comparable aux tissus à remplacer. Cela s'avère particulièrement crucial lors de la conception de substituts vasculaires où les propriétés mécaniques ont une importance primordiale afin de résister à la pression artérielle, mais aussi la compliance nécessaire pour ne pas induire une athérosclérose postimplantation due à une trop grande rigidité. De plus, les présents modèles vasculaires conçus par génie tissulaire n'intègrent pas la microcirculation dans les parois vasculaires. Les vasa vasorum, capillaires de l'adventice, servent à nourrir et oxygéner les parois des vaisseaux sanguins et ont probablement un rôle clé dans l'intégration dans les tissus avoisinants des substituts vasculaires. À ces fins, nous avons conçu un biomatériau pouvant être microstructuré afin de reproduire une topographie similaire à celle composant le micro environnement des tissus. Les polymères thermoplastiques permettent la production à grande échelle de substrats avec une nanotopographie de surface permettant l'alignement des cellules et de la matrice extracellulaire. Nous avons fait la démonstration que ces substrats permettent l'auto-assemblage tridimensionnel des cellules et de la matrice extracellulaire suivant un angle précis, lequel correspond à la même organisation physiologique intrinsèque du tissu et varie en fonction du type cellulaire en culture. Pour les cellules musculaires lisses vasculaires composant la média, nous avons montré que cette organisation se traduit par une augmentation de plus de 100% en résistance mécanique. D'autre part, une nouvelle technique de culture cellulaire pour les adventices vasculaires a permis de créer un réseau de capillaires in vitro s'apparentant au vasa vasorum. Ces substituts vasculaires ont été implantés en sous-cutané dans un modèle animal permettant de démontrer la rapidité d'intégration des tissus pré-vascularisés (48h) en comparaison avec des tissus non-vascularisés (141). Ces améliorations aux substituts vasculaires leurs confèrent une importante ressemblance anatomique et physiologique avec les vaisseaux utilisés notamment pour les pontages des artères soit coronaires ou périphériques, les rapprochant ainsi d'une utilisation en clinique.

À l'heure actuelle, l'obésité est une pathologie qui prend de l'ampleur. Elle est associée à maintes pathologies, dont la résistance à l'insuline et la dysfonction endothéliale. Selon les lits vasculaires, les impacts de ces phénomènes peuvent avoir des conséquences physiologiques importantes. Précédemment, nous avons démontré qu'il y avait une augmentation de 30 à 50% de la perméabilité endothéliale dans le muscle strié du modèle de rat Zucker obèse. Pour sa part, le tissu adipeux est unique de par ses fonctions métaboliques, endocrines et sa microcirculation. Trois facteurs impliqués dans le contrôle de la microcirculation et de la perméabilité vasculaire dans ce tissu nous ont intéressés: le facteur de croissance de l'endothélium vasculaire, la NO synthétase endothéliale et l'angiotensine II via son récepteur de type 1. Tout d'abord, le VEGF est reconnu pour augmenter l'angiogénèse et la perméabilité vasculaire. La eNOS est une enzyme qui produit le NO à partir de la L-arginine. En dehors de son rôle classique de vasorelaxation, le NO stimule l'expression du VEGF et parallèlement, le VEGF active la production de NO: il y a donc synergie entre ces deux facteurs. L'angiotensine II est un peptide qui engendre une forte vasoconstriction, via son récepteur AT[indice inférieur 1]R. Notre hypothèse de recherche est que la microcirculation du tissu adipeux possède un rôle primordial dans l'obésité, étant donné que celui-ci tient une place importante sur le plan métabolique et endocrinien. Nos objectifs ont consisté à évaluer s'il y avait des changements dans la perméabilité endothéliale chez le rat Zucker obèse par la méthode du Bleu d'Evans, par des mesures de l'expression des trois facteurs étudiés (Western et qRT-PCR) et par l'observation en microscopie électronique des caractéristiques morphologiques des cellules endothéliales, dans les divers types de tissus adipeux (sous-cutané, rétropéritonéal et épididymique). Nos résultats ont démontré qu'il existe une grande hétérogénéité dans la perméabilité des divers sous-types de tissu adipeux. Contrairement aux autres organes, cette perméabilité est majoritairement à la baisse. De plus, nos expériences ont permis d'observer plusieurs changements significatifs dans l'expression protéique de la eNOS, de l'AT[indice inférieur 1]R et du VEGF A et ceci, principalement dans le tissu adipeux sous-cutané. Les changements protéiques de ces trois facteurs ne corrèlent pas exactement avec les changements de perméabilité observés avec le Bleu d'Evans, ce qui nous permet d'affirmer que la régulation de la perméabilité vasculaire est régulée par une myriade d'autres facteurs. D'autre part, les résultats préliminaires obtenus en microscopie électronique ne nous ont pas permis d'identifier de changements marquants au niveau de la morphologie des cellules endothéliales. Ceci suggère que les changements de perméabilité ne sont pas la conséquence de changements morphologiques, mais plutôt fonctionnels et en relation avec différents facteurs vasoactifs. En somme, nos résultats ont permis d'établir qu'il y a une grande hétérogénéité entre les divers sous-types de tissu adipeux, au point de vue de la perméabilité et de l'expression de certains facteurs vasoactifs. De plus, ces expériences nous ont démontré que le contrôle de la perméabilité vasculaire est un phénomène très complexe qui implique une multitude de phénomènes. La compréhension de ces derniers [phénomènes] dans le tissu adipeux pourrait permettre l'élaboration de traitements ayant la capacité de limiter l'expansion de ce tissu en diminuant sa perméabilité vasculaire, ceci afin de restreindre les effets néfastes liés à l'obésité.

Previously, it was reported that the morphological changes (pseudopod projection) that circulating neutrophils adopt due to cell activation raises peripheral vascular resistance by disrupting microvascular rheology. Studies utilized murine muscle preparations to link neutrophil pseudopod formation to cell activation and a viscous impact on hemodynamic resistance. But because of the complexity associated with the organization of the vasculature and microvasculature in tissues, it was unclear whether the effects of neutrophil activation on hemodynamic resistance were associated with the macro-/micro- circulation. This research describes an in vitro analysis using viscometry and microvascular network mimics (microporous membranes) to assess the rheological impact of pseudopods on capillary-like flow. Suspensions of neutrophil-like HL-60 promyelocytes (dHL60’s) and human neutrophils, stimulated with 10 nM fMLP were used, with/without hematocrit. Stimulation of dHL60s or human neutrophils with fMLP altered their flow behavior, which was detected as an increase in solution viscosity. Addition of hematocrit negated the effect of neutrophil activation on suspension viscosity. Moreover, cell activation increased the resistance of microporous membranes to flow of neutrophil suspensions with addition of hematocrit exacerbating this effect. Combined, the results of this study provided evidence that activated neutrophils influence microscale flow resistance via a rheological impact.

A miocardiopatia chagásica constitui uma doença prevalente e de alta morbimortalidade no Brasil. Embora seu agente causador, o Trypanosoma cruzi, já tenha sido identificado, os mecanismos fisiopatológicos e a predileção do acometimento por determinados segmentos do miocárdio ainda não foram totalmente esclarecidos. Dentre os mecanismos envolvidos nos distúrbios da microcirculação, inclui-se a hipótese na qual o acometimento preferencial das regiões apical e posterolateral do VE deva-se à presença de áreas de fluxo arterial fronteiriço (\"watershed\") entre as artérias que irrigam as paredes do VE, locais estes com maior predisposição a sofrer processo de isquemia. Ainda que plausível, esta hipótese ainda não foi testada frente a razoável variabilidade da distribuição dos vasos arteriais coronários, o que implica em variações na presença ou não de regiões de \"watershed\" em cada paciente. Objetivo: Estudar a correlação topográfica entre as regiões de \"watershed\" com as alterações da mobilidade ventricular e defeitos de perfusão miocárdica no ventrículo esquerdo. Casuística e Métodos: Realizamos um estudo descritivo com análise de 63 pacientes portadores de cardiopatia chagásica crônica que possuíam cineangiocoronariografia sem lesões obstrutivas significativas e fração de ejeção igual ou superior a 40%. Utilizando a cineangiocoronariografia definimos as regiões passíveis de \"watershed\" baseando-nos na distribuição anatômica das artérias coronárias, tendo como critérios a dominância, a extensão e os limites das artérias coronárias direita e esquerda na irrigação das paredes do VE. Em seguida verificamos se as alterações de mobilidade parietal na ventriculografia e/ou os defeitos de perfusão segmentar na cintilografia de perfusão miocárdica coincidiam com estas regiões. Utilizamos Teste Exato de Fischer para a análise estatística dos resultados. Resultados: Na ventriculografia por contraste as frequências dealterações de mobilidade para as paredes apical, inferior e posterolateral nos pacientes na presença e ausência de respectiva região de WS correspondente foram: 76,2% vs 68,3%, 46,3% vs 38,1% e 54,6% vs 42,9%. Os valores de p obtidos foram respectivamente 0,51; 0,59 e 0,69. Considerando todas as regiões onde o WS estava presente 55,6% apresentavam alteração da mobilidade, em compensação 56,5% das regiões onde o WS estava ausente também apresentavam alteração da mobilidade, com o valor de p = 1,0. Na cintilografia de perfusão miocárdica as frequências de defeitos de captação para os segmentos apicais, inferiores médio e apical, laterais e inferior basal na presença e ausência de respectiva região de WS correspondente foram: 41,2% vs 57,1%, 67,8% vs 47,2%, 50% vs 66,6% e 33,1% vs 50%. Os valores de p obtidos foram respectivamente 0,36; 0,21; 1,0 e 1,0. Considerando todas as regiões onde o WS estava presente 53,3% apresentavam defeitos de captação, em compensação 52,2% das regiões onde o WS estava ausente também apresentavam defeitos de captação com o valor de p = 1,0. Conclusão: No estudo apresentado não se comprovou que as alterações da mobilidade parietal ou os defeitos de perfusão nos segmentos do ventrículo esquerdo ocorram nas regiões de \"watershed\". Portanto nossos resultados não apoiam a hipótese de que as regiões de \"watershed\" contribua para o mecanismo de lesão miocárdica na cardiopatia chagásica crônica. / Chagasic cardiomyopathy is a prevalent disease with high morbidity and mortality in Brazil. Although its causative agent, Trypanosoma cruzi, has already been identified, pathophysiological mechanisms and the predilection for involvement of certain segments of the myocardium have not yet been fully elucidated. Among the mechanisms involved in microcirculation disorders, the hypothesis is included in which the preferential affection of the apical and posterolateral regions of the LV is due to the presence of areas of bordered arterial flow (\"watershed\") between the arteries that irrigate the walls of the LV, these places are more likely to undergo ischemia. Although plausible, this hypothesis has not yet been tested against the reasonable variability of coronary artery distribution, which implies variations in the presence or absence of watershed regions in each patient. Objective: To study the topographic correlation between watershed regions with the ventricular mobility abnormalities and myocardial perfusion defects in the left ventricle. Methods: We conducted a descriptive study with 63 patients with chronic chagasic cardiopathy who had coronary angiography without significant obstructive lesions and ejection fraction equal to or greater than 40%. Using the cineangiocoronariography, we defined watershed regions based on the anatomical distribution of the coronary arteries, taking as criteria the dominance, extension and limits of the right and left coronary arteries in the irrigation of the LV walls. Next, we verified whether the parietal mobility abnormalities in ventriculography and / or segmental perfusion defects in myocardial perfusion scintigraphy coincided with these regions. We used Fisher\'s exact test for the statistical analysis of the results. Results: In contrast ventriculography, the frequencies of mobility abnormalities for the apical, inferior and posterolateral walls in patients in the presence and absence of a corresponding WS region were: 76.2% vs 68.3%, 46.3% vs 38 , 1% and 54.6% vs 42.9%. The values of p obtained were respectively 0.51; 0.59 and 0.69. Considering all the regions where WS was present, 55.6% had mobility alterations; in contrast, 56.5% of the regions where WS was absent alsopresented mobility alterations with p value = 1.0. In myocardial perfusion scintigraphy, the frequencies of uptake defects for the apical, lower mid and apical, lateral and lower basal segments in the presence and absence of the corresponding WS region were: 41.2% vs 57.1%, 67.8 % vs 47.2%, 50% vs 66.6% and 33.1% vs 50%. The values of p obtained were respectively 0.36; 0.21; 1.0 and 1.0. Considering all the regions where the WS was present, 53.3% presented perfusional defects, in compensation 52.2% of the regions where WS was absent also had perfusional defects with p value = 1.0. Conclusion: In the presented study it was not verified that the alterations of the parietal mobility or the perfusion defects in the segments of the left ventricle occur in the watershed regions. Therefore our results do not support the hypothesis that watershed regions contribute to the mechanism of myocardial injury in chronic Chagas\' heart disease.

acase@tulane.edu / An emerging need in the microcirculation research is the development of biomimetic angiogenesis models that recapitulate the complexity of a real tissue. Angiogenesis, defined as the growth of new vessels from pre-existing vessels, involves multiple cell types, such as endothelial and perivascular cells, in a multi-system setting since blood vessel networks are usually accompanied by lymphatic and nervous systems. Therefore, a need exists for a model of angiogenesis from intact microvascular networks that more closely reflects an in vivo scenario for the investigation of underlying mechanisms and the pre-clinical development of therapies. While other approaches have proven useful in identifying mechanistic signaling information, they are often limited in their complexity and capability to mimic physiologically relevant scenarios in one way or another and do not fully recapitulate the in vivo scenario. The first aim of this study was to demonstrate the ability for time-lapse comparisons of microvascular networks in angiogenesis scenarios to investigate the fate of vascular islands and investigate the endothelial cell plasticity. We developed a time-lapse angiogenesis model based on our previously introduced rat mesentery model. We demonstrated that time-lapse rat mesentery culture model is a powerful tool to study multi-cell, multi-system dynamics in microvascular networks. For the second aim of this study, we used the method developed in aim one to establish rat mesentery culture model as a novel anti-angiogenic drug screening tool. Using time-lapse model enabled tissue-specific comparisons before and after drug treatment to investigate its effects on entire microvascular networks. Validation of this method for anti-angiogenic drug testing was demonstrated using known angiogenesis inhibitor. Next, we showcased a potential application of the model for evaluating unknown effects of drug repositioning based on FDA-approved drug combinations. The results demonstrated the ability to identify concentration-dependent effects in an intact network scenario. The objective of the third aim was to showcase the capability of the rat mesentery culture model to study stem cell fate. We developed a protocol to deliver mesenchymal stem cells to mesentery tissues and culture for a period of time in a controlled environment. We confirmed the perivascular location of a subset of stem cells within capillaries, with morphologies resembling pericytes, and expressing pericyte markers. We also demonstrated that tracking stem cells within the microvascular networks is possible using the rat mesentery culture model. Furthermore, we reported a high variability in perivascular incorporation among cells from different donors. This work establishes for the first time, to the best of our knowledge, an ex vivo model to look at microvascular networks before and after growth. We confirmed, for the first time, vascular island incorporation as a new mode of angiogenesis using a novel method for time-lapse imaging of microvascular networks ex vivo. The results also establish this method for drug testing and stem cell tracking in a microvascular setting. / 1 / Mohammad Sadegh Azimi

Bibliography: leaves 157-177.

La microcirculation cutanée a été proposée comme modèle d'étude de la dysfonction microvasculaire globale dans les maladies cardiovasculaires. Par ailleurs, elle est spécifiquement atteinte dans le syndrome de Raynaud, qui est une ischémie paroxystique des extrémités déclenchée notamment par le froid. L'exploration de la fonction microvasculaire cutanée suscite donc un réel intérêt, mais les méthodes d'étude souffrent d'une hétérogénéité importante, et leur variabilité intra-individuelle est mal connue. La première partie de ce travail fait la synthèse des différentes méthodes d'étude la fonction microvasculaire cutanée, et rapporte les résultats de deux études consacrées à leur reproductibilité. Nous avons dans une seconde partie étudié grâce à ces tests la réactivité microvasculaire cutanée dans le syndrome de Raynaud, et mis en évidence des anomalies chez ces patients, notamment du contrôle neuro-vasculaire. La dernière partie de cette thèse est consacrée à l'étude d'approches pharmacologiques ciblées sur les anomalies de la microcirculation cutanée identifiées chez les patients. Nous avons évalué l'effet du sildenafil, un inhibiteur de la phosphodiesterase-5, sur le flux sanguin digital et montré son effet vasodilatateur lors d'un refroidissement local dans le syndrome de Raynaud. Enfin, nous avons étudiés chez l'animal et chez l'homme l'iontophorèse de vasodilatateurs, une approche innovante d'administration locale de médicaments pour augmenter le flux sanguin cutané.

Aging is associated with increases in regional and systemic vascular resistance and arterial blood pressure. One possible mechanism through which these age-associated alterations occur is enhanced vasoconstrictor responsiveness, or alterations in the structural properties of the resistance vasculature. We hypothesized that stiffness and vasoconstriction would be greater in skeletal muscle arterioles from old rats, and that endurance exercise training would ameliorate the associated with aging alterations. METHODS: Young sedentary (YS; 4 months), old sedentary (OS; 24 months), young trained (YT) and old trained (OT) male Fischer 344 rats were used. Training modality was treadmill exercise at 15 m/min up a 15o incline, 5 days/wk for 12wks. Skeletal muscle first-order arterioles were isolated for in vitro experimentation. Intraluminal diameter was measured in response to the cumulative addition of endothelin-1, norepinephrine, KCl, and isoproterenol. Stiffness was measure by examining the arterioles' stress and strain relation to increased luminal pressure in Ca++ free solution. RESULTS: Skeletal muscle arterioles had augmented vasoconstriction to endothelin-1 and norepinephrine. Adrenergic vasodilation was diminished in aged rat arterioles. Stiffness increased with age. Exercise training ameliorated the age-associated changes in stiffness and norepinephrine vasoconstriction. Exercise training did not alter endothelin-1 vasoconstriction or adrenergic vasodilation. CONCLUSIONS: These findings suggest that enhanced vascular sensitivity to vasoconstrictors and increased arteriole stiffness may play a role in the increase in skeletal muscle and systemic vascular resistance and, thus, contribute to the elevated blood pressure which occurs in aging humans. These results also demonstrate some of the cardioprotective effects of exercise training.

Aging is associated with increases in regional and systemic vascular resistance and arterial blood pressure. One possible mechanism through which these age-associated alterations occur is enhanced vasoconstrictor responsiveness, or alterations in the structural properties of the resistance vasculature. We hypothesized that stiffness and vasoconstriction would be greater in skeletal muscle arterioles from old rats, and that endurance exercise training would ameliorate the associated with aging alterations. METHODS: Young sedentary (YS; 4 months), old sedentary (OS; 24 months), young trained (YT) and old trained (OT) male Fischer 344 rats were used. Training modality was treadmill exercise at 15 m/min up a 15o incline, 5 days/wk for 12wks. Skeletal muscle first-order arterioles were isolated for in vitro experimentation. Intraluminal diameter was measured in response to the cumulative addition of endothelin-1, norepinephrine, KCl, and isoproterenol. Stiffness was measure by examining the arterioles' stress and strain relation to increased luminal pressure in Ca++ free solution. RESULTS: Skeletal muscle arterioles had augmented vasoconstriction to endothelin-1 and norepinephrine. Adrenergic vasodilation was diminished in aged rat arterioles. Stiffness increased with age. Exercise training ameliorated the age-associated changes in stiffness and norepinephrine vasoconstriction. Exercise training did not alter endothelin-1 vasoconstriction or adrenergic vasodilation. CONCLUSIONS: These findings suggest that enhanced vascular sensitivity to vasoconstrictors and increased arteriole stiffness may play a role in the increase in skeletal muscle and systemic vascular resistance and, thus, contribute to the elevated blood pressure which occurs in aging humans. These results also demonstrate some of the cardioprotective effects of exercise training.