Diversas evidências comprovam que a obesidade está associada a alterações estruturais e funcionais do coração em modelos humanos e animais. Outros estudos recentes também demonstram que a obesidade humana está associada com alterações na função e na estrutura vascular, especialmente em grandes e médias artérias. Estudos epidemiológicos têm confirmado que a obesidade é um fator de risco significativo para o aparecimento de proteinúria e de doença renal terminal em uma população normal. Com o objetivo de determinar as alterações morfológicas relacionadas ao remodelamento cardíaco, vascular e renal em um modelo experimental de obesidade induzida pelo glutamato monossódico (MSG) e os efeitos da metformina sobre estes achados, foram estudados 25 ratos divididos em cinco grupos: controle com 16 e 22 semanas (CON-16 e CON-22); obeso com 16 e 22 semanas (MSG-16 e MSG-22) e obeso + metformina (MET-22) 300mg/Kg/dia por via oral. A caracterização da resistência à insulina foi feita através da medida da insulina plasmática e cálculo do índice de HOMA-IR. As análises morfológicas e quantificação do colágeno miocárdico foram feitos pelo sistema de imagem Image Pro Plus analysis. A pressão arterial sistólica foi levemente maior no grupo MSG-22, adquirindo significância estatística quando comparada com o grupo MSG-16 (1222 vs 1082 mmHg, p<0,05). Por outro lado, o grupo MET-22 mostrou níveis mais baixos de pressão arterial (1181 mmHg), sem alcançar diferença significativa. No grupo de animais obesos, foi observado aumento na relação média-lumen com 16 semanas (39,93,7 vs 30,22,0 %, p<0,05) e com 22 semanas (39,81,3 vs 29,51,2%, p<0,05), que foi reduzida com o uso da metformina (31,50,9%). O depósito de colágeno na área perivascular no ventrículo esquerdo foi significativamente maior no grupo MSG-22 (1,390,06 vs 0,830,06 % no CON-22, p<0,01), sendo atenuado pela metformina (1,020,04%). No rim, a área seccional transversa das arteríolas intrarrenais foi semelhante entre os grupos (18,52,2 no CON-16; 19,93,7 no MSG-16; 18,93,1 no CON-22; 21,81,5 no MSG-22; 20,21,4 no MET-22). Foi observado aumento da área glomerular no grupo MSG-22 (141,34,5 vs 129,50,5 m2), mas sem significância estatística. Em conclusão, nos ratos com obesidade induzida pelo MSG, com resistência à insulina, as alterações cardíacas foram mais proeminentes do que as alterações renais. No coração foram observados sinais de remodelamento vascular hipertrófico nas pequenas artérias intramiocárdicas e evidências de fibrose miocárdica mais proeminente na área perivascular, alterações que foram, pelo menos parcialmente, atenuadas com o uso de metformina durante seis semanas, mostrando que esta droga pode ser benéfica na prevenção de complicações cardíacas, vasculares e renais associadas com a obesidade. / Many evidences show that obesity is associated to structural and functional changes in the heart of human and animal models. Recent studies also show that human obesity is associated with vascular structural and functional modifications, specially at large and medium-sized arteries. Epidemiological studies have confirmed that obesity is a significant risk factor for the development of proteinuria and end-stage renal disease in a normal population. With the objective to determinate morphological changes related to cardiac, vascular and renal remodeling in an experimental model of monosodium glutamate (MSG)-induced obesity and the effect of metformin at this finding. Twenty five rats were studied and divided into five groups: control with 16 e 22 weeks (CON-16 and CON-22); obese with 16 and 22 weeks (MSG-16 e MSG-22), and obese + metformin (MET-22) 300mg/Kg/day per oral. The characterization of insulin resistance was done through measurement of plasma insulin and calculation of HOMA-IR index. The morphological analysis and the quantification of myocardial collagen were carried out by Image Pro Plus analysis system. The systolic blood pressure was slightly higher in MSG-22 group, reaching statistical significance when compared to MSG-16 group (1222 vs 1082 mmHg, p<0.05). On the other hand, the MET-22 group demonstrated lower blood pressure levels (1181 mmHg), without reaching statistical difference. The obese animals presented increase in media-to-lumen ratio with 16 weeks (39.93.7 vs 30.22.0 %, p<0.05) and with 22 weeks (39.81.3 vs 29.51.2%, p<0.05), which was reduced with use of metformin (31.50.9%). The collagen deposition in perivascular area of left ventricle was significantly greater in MSG-22 group (1.390.06 vs 0.830.06 % in CON-22, p<0.01), and attenuated by metformin (1.020.04%). In the kidney, the media cross-sectional area of intrarenal arterioles was similar among the groups (18.52.2 in CON-16; 19.93.7 in MSG-16; 18.93.1 in CON-22; 21.81.5 in MSG-22; 20.21.4 in MET-22). An increase of glomerular area was observed in MSG-22 group (141.34.5 vs 129.50.5 m2), but without statistical significance. In conclusion, rats with MSG-induced obesity and insulin resistance presented more pronounced cardiac changes than renal alterations. In the heart, there were evidences of hypertrophic vascular remodeling were observed in intramyocardial small arteries and perivascular fibrosis. These findings were, at least partially, attenuated by metformin for six weeks, suggesting that this drug may be beneficial for prevention of cardiac, vascular and renal complications associated with obesity.

Abdominal aortic aneurysm (AAA) is characterized by a bulge in the abdominal aorta. AAA development is mostly asymptomatic, but such a bulge may suddenly rupture, which is associated with a high mortality rate. Unfortunately, there is no medication that can prevent AAA from expanding or rupturing. Therefore, patients with detected AAA are monitored until treatment indication, such as maximum AAA diameter of 55 mm or expansion rate of 1 cm/year. Models of AAA development may help to understand the disease progression and to inform decision-making on a patient-specific basis. AAA growth and remodeling (G&amp;R) models are rather complex, and before the challenge is undertaken, sound clinical validation is required. In Paper A, an existing thick-walled model of growth and remodeling of one layer of an AAA slice has been extended to a two-layered model, which better reflects the layered structure of the vessel wall. A parameter study was performed to investigate the influence of mechanical properties and G&amp;R parameters of such a model on the aneurysm growth. In Paper B, the model from Paper A was extended to an organ level model of AAA growth. Furthermore, the model was incorporated into a Fluid-Solid-Growth (FSG) framework. A patient-specific geometry of the abdominal aorta is used to illustrate the model capabilities. In Paper C, the evolution of the patient-specific biomechanical characteristics of the AAA was investigated. Four patients with five to eight Computed Tomography-Angiography (CT-A) scans at different time points were analyzed. Several non-trivial statistical correlations were found between the analyzed parameters. In Paper D, the effect of different growth kinematics on AAA growth was investigated. The transverse isotropic in-thickness growth was the most suitable AAA growth assumption, while fully isotropic growth and transverse isotropic in-plane growth produced unrealistic results. In addition, modeling of the tissue volume change improved the wall thickness prediction, but still overestimated thinning of the wall during aneurysm expansion. / Bukaortaaneurysm (AAA) kännetecknas av en utbuktning hos aortaväggen i buken. Tillväxt av en AAA är oftast asymtomatisk, men en sådan utbuktning kan plö̈tsligt brista, vilket har hög dödlighet. Tyvärr finns det inga mediciner som kan förhindra AAA från att expandera eller brista. Patienter med upptä̈ckt AAA hålls därför under uppsikt tills operationskrav är uppnådda, såsom maximal AAA-diameter på 55 mm eller expansionstakt på 1 cm/år. Modeller för AAA-tillväxt kan bidra till att öka förståelsen för sjukdomsförloppet och till att förbättra beslutsunderlaget på en patientspecifik basis. AAA modeller för tillväxt och strukturförändring (G&amp;R) är ganska komplicerade och innan man tar sig an denna utmaning krävs de god klinisk validering. I Artikel A har en befintlig tjockväggig modell för tillväxt av ett skikt av en AAA-skiva utö̈kats till en två-skiktsmodell. Denna modell återspeglar bättre den skiktade strukturen hos kärlväggen. Genom en parameterstudie undersö̈ktes påverkan av mekaniska egenskaper och G&amp;R-parametrar hos en sådan modell för AAA-tillväxt. I Artikel B utvidgades modellen från Artikel A till en organnivå-modell för AAA-tillväxt. Vidare inkorporerades modellen i ett “Fluid–Solid–Growth” (FSG) ramverk. En patientspecifik geometri hos bukaortan användes för att illustrera möjligheterna med modellen. I Artikel C undersöktes utvecklingen av patientspecifika biomekaniska egenskaper hos AAA. Fyra patienter som skannats fem till åtta gånger med “Computed Tomography-Angiography” (CT-A) vid olika tillfällen analyserades. Flera icke triviala statistiska samband konstaterades mellan de analyserade parametrarna. I Artikel D undersöktes effekten av olika tillväxt-kinematik för AAA tillväxt. En modell med transversellt-isotrop-i-tjockleken-tillväxt var den bäst lämpade för AAA tillväxt, medans antagandet om fullt-isotrop-tillväxt och transversellt-isotrop-i-planet-tillväxt producerade orimliga resultat. Dessutom gav modellering av vävnadsvolymsförändring ett förbättrat väggtjockleks resultat men en fortsatt överskattning av väggförtunningen under AAA-expansionen. / <p>QC 20161201</p>

Résumé Introduction L’amlodipine et l’atorvastatine offrent des avantages thérapeutiques au-delà de leur indication primaire, soit la réduction de la pression artérielle et des lipides sanguins, respectivement. L’amlodipine induit l’apoptose des cellules de muscle lisse vasculaire (CMLV) in vivo, contribuant à la régression de l'hypertrophie aortique chez le rat spontanément hypertendu (SHR). L'atorvastatine induit l’apoptose des CMLV in vitro, un effet proportionnel à la dose. Toutefois, cet effet reste à être démontré in vivo. Nous postulons que l’atorvastatine induira la régression de l’hypertrophie aortique via l’apoptose des CMLV chez le SHR, et que la combinaison de l’amlodipine et de l’atorvastatine aura un effet synergique sur la régression de l’hypertrophie aortique via l’apoptose des CMLV chez le SHR. Méthodologie L’amlodipine et l’atorvastatine ont été administrées à des SHR âgés de 11 semaines durant trois ou six semaines, individuellement ou en combinaison. Les points principaux à l'étude étaient le remodelage vasculaire et la pression artérielle. La fragmentation et le contenu en ADN, le stress oxydant, le taux de cholestérol et les niveaux de nitrates ont aussi été mesurés. Résultats Lorsque l’atorvastatine a été administrée seule, une diminution significative du stress oxydant et de la pression artérielle a été observée après trois et six semaines de traitement, respectivement. Par contre, aucune différence n’a pu être décelée quant au remodelage vasculaire. L'amlodipine a réduit la pression artérielle et l'hypertrophie aortique de façon dépendante de la dose. Une diminution significative de l'hyperplasie a été détectée après trois semaines de traitement avec la combinaison, et après six semaines avec une faible dose d'amlodipine. Conclusion Nos résultats ne supportent pas l'hypothèse que l'atorvastatine induit l'apoptose des CMLV in vivo. Par contre, lorsque combinée à l'amlodipine, elle pourrait ajouter un bénéfice supplémentaire au niveau de la réduction de l'hyperplasie aortique. / Abstract Background and purpose Antihypertensive drugs such as the calcium channel blocker (CCB) amlodipine and cholesterol lowering agents such as statins exhibit pleiotropic effects. Amlodipine reduces aortic hypertrophy and hyperplasia in spontaneously hypertensive rat (SHR) by inducing a transient wave of apoptosis. Atorvastatin induces apoptosis of vascular smooth muscle cell (VSMC) in vitro, independently of cholesterol synthesis, an effect that remains to be shown in vivo. The present studies were designed to test the hypothesis that atorvastatin can induce vascular remodeling by VSMC apoptosis in vivo in SHR, and to test whether combined therapy with low dose amlodipine would provide synergistic effects on regression of aortic hypertrophy. Experimental approach 11-week old SHR were given atorvastatin and amlodipine, alone or in combination, for three or six weeks. Primary end-points were vascular remodeling and blood pressure. Secondary end-points included DNA fragmentation and content in the aorta, oxidative stress, cholesterol and serum total nitrite and nitrate (NOx) concentrations. Key results Treatment with atorvastatin did not modify vascular structure, although it significantly reduced oxidative stress after three weeks and blood pressure after six weeks. Amlodipine dose-dependently reduced blood pressure and aortic hypertrophy. Significant reduction of cellular hyperplasia was reached after 6 weeks with a low dose of amlodipine alone or after 3 weeks when atorvastatin was combined with low dose amlodipine. Conclusions and implications Our results do not support the notion that atorvastatin induces VSMC apoptosis in vivo, although the data suggest a possible interaction with amlodipine in reducing VSMC hyperplasia in the hypertensive aorta.

L’hypertrophie du ventricule gauche (HVG) est un processus adaptif et compensatoire qui se développe conséquemment à l’hypertension artérielle pour s’opposer à l’élévation chronique de la pression artérielle. L’HVG est caractérisée par une hypertrophie des cardiomyocytes suite à l’augmentation de la synthèse d’ADN, une prolifération des fibroblastes, une augmentation du dépôt de collagène et une altération de la matrice extracellulaire (MEC). Ces changements génèrent des troubles de relaxation et mènent au dysfonctionnement diastolique, ce qui diminue la performance cardiaque. La suractivité du système nerveux sympathique (SNS) joue un rôle essentiel dans le développement de l’hypertension artérielle et de l’HVG à cause de la libération excessive des catécholamines et de leurs effets sur la sécrétion des cytokines pro-inflammatoires et sur les différentes voies de signalisation hypertrophiques et prolifératives. Le traitement antihypertenseur avec de la moxonidine, un composé sympatholytique d’action centrale, permet une régression de l’HVG suite à une réduction soutenue de la synthèse d'ADN et d’une stimulation transitoire de la fragmentation de l'ADN qui se produit au début du traitement. En raison de l’interaction entre l’HVG, les cytokines inflammatoires, le SNS et leurs effets sur les protéines de signalisation hypertrophiques, l’objectif de cette étude est de détecter dans un modèle animal d’hypertension artérielle et d’HVG, les différentes voies de signalisation associées à la régression de l’HVG et à la performance cardiaque. Des rats spontanément hypertendus (SHR, 12 semaines) ont reçu de la moxonidine à 0, 100 et 400 µg/kg/h, pour une période de 1 et 4 semaines, via des mini-pompes osmotiques implantées d’une façon sous-cutanée. Après 4 semaines de traitement, la performance cardiaque a été mesurée par écho-doppler. Les rats ont ensuite été euthanasiés, le sang a été recueilli pour mesurer les concentrations des cytokines plasmatiques et les cœurs ont été prélevés pour la détermination histologique du dépôt de collagène et de l'expression des protéines de signalisation dans le ventricule gauche. Le traitement de 4 semaines n’a eu aucun effet sur les paramètres systoliques mais a permis d’améliorer les paramètres diastoliques ainsi que la performance cardiaque globale. Par rapport au véhicule, la moxonidine (400 µg/kg/h) a permis d’augmenter transitoirement la concentration plasmatique de l’IL-1β après une semaine et de réduire la masse ventriculaire gauche. De même, on a observé une diminution du dépôt de collagène et des concentrations plasmatiques des cytokines IL-6 et TNF-α, ainsi qu’une diminution de la phosphorylation de p38 et d’Akt dans le ventricule gauche après 1 et 4 semaines de traitement, et cela avec une réduction de la pression artérielle et de la fréquence cardiaque. Fait intéressant, les effets anti-hypertrophiques, anti-fibrotiques et anti-inflammatoires de la moxonidine ont pu être observés avec la dose sous-hypotensive (100 µg/kg/h). Ces résultats suggèrent des effets cardiovasculaires bénéfiques de la moxonidine associés à une amélioration de la performance cardiaque, une régulation de l'inflammation en diminuant les niveaux plasmatiques des cytokines pro-inflammatoires ainsi qu’en inhibant la MAPK p38 et Akt, et nous permettent de suggérer que, outre l'inhibition du SNS, moxonidine peut agir sur des sites périphériques. / Left ventricular hypertrophy (LVH) is an adaptive and compensatory process that develops in hypertension to oppose the chronic elevation of blood pressure. LVH is characterized by hypertrophy of cardiomyocytes following the increase in DNA synthesis, proliferation of fibroblasts, increased collagen deposition and alteration of the extracellular matrix (ECM). These changes generate relaxation and diastolic dysfunction which reduced cardiac performance. The overactivity of the sympathetic nervous system plays an essential role in the development of hypertension and left ventricular hypertrophy pathogenesis due to the excessive release of catecholamines and norepinephrine spillover and their effects on the secretion of pro-inflammatory cytokines and hypertrophic signaling pathways. Antihypertensive treatment with moxonidine, a centrally acting sympatholytic imidazoline compound, results in prevention of left ventricular hypertrophy, resulting from a sustained reduction of DNA synthesis and transient stimulation of DNA fragmentation that occur early after treatment. Due to the interaction between LVH, inflammatory cytokines, the SNS and their effects on hypertrophic signaling proteins, the objective of this study is to detect in an animal model of hypertension and LVH, the different signaling pathways associated with regression of LVH and cardiac performance. Spontaneously hypertensive rats (SHR, 12 weeks old) received moxonidine at 0, 100 and 400 µg/kg/h, for 1 and 4 weeks, via subcutaneously implanted osmotic minipumps. After 4 weeks of treatment, cardiac performance was measured by echo-Doppler. Then the rats were euthanized, blood was collected for measurement of plasma cytokines and hearts for histologic determination of collagen deposition and for measurement of left ventricular expression of downstream signaling proteins. Treatment for 4 weeks had no effect on systolic parameters but improved diastolic parameters and global cardiac performance. Compared to vehicle, moxonidine (400 µg/kg/h) transiently increased plasma IL-1β after 1 week and reduced left ventricular mass. Similarly, there was a decrease in collagen deposition and plasma concentrations of IL-6 and TNF-α, and decreased phosphorylation of p38 and Akt in the left ventricle after 1 and 4 weeks treatment, in association with reduced blood pressure and heart rate. Interestingly, the anti-hypertrophic, anti-fibrotic, and anti-inflammatory effects of moxonidine were observed with a sub-hypotensive dose (100µg/kg/h). These results suggest the beneficial cardiovascular effects of moxonidine associated with improved cardiac performance, regulation of inflammation by decreasing pro-inflammatory plasma levels, inhibition of p38 MAPK and Akt, and allow us to suggest that besides inhibiting the SNS, moxonidine may act on peripheral sites.

Thèse réalisée en cotutelle avec Dre Christine Des Rosiers / Le processus de l’athérosclérose est associé à des changements vasculaires structuraux et mécaniques dont la rigidification carotidienne et aortique. Ce phénomème est bien connu et contraste avec l’augmentation paradoxale de la distensibilité cérébrovasculaire observée dans les artères cérébrales exposées aux facteurs de risque cardiovasculaire, tels que l’hypertension. L’impact de l’athérosclérose sur le remodelage, la compliance et la fonction des artères cérébrales est inconnu. En ciblant l’endothélium, l’athérosclérose induit une dysfonction endothéliale cérébrale sévère qui interfère avec le contrôle du débit sanguin cérébral et ultimement avec les fonctions cognitives. Dans les artères cérébrales, le remodelage de la paroi artérielle est toujours accompagné d’une perte des fonctions vasodilatatrices, ce qui suggère que ces deux évènements sont au cœur d’un cercle vicieux. Nos études visent à vérifier l’hypothèse selon laquelle le remodelage de la paroi est déterminé par la fonction endothéliale au niveau cérébrovasculaire alors qu’au niveau de la carotide, le stress mécanique du pouls sanguin régule les propriétés structurales et biomécaniques. Afin de vérifier cette hypothèse, dans une première étude, nous avons sélectionné trois interventions thérapeutiques aux mécanismes d’action différents qui modulent la fonction endothéliale indirectement en diminuant le stress mécanique exercé sur la paroi via une diminution de la fréquence cardiaque. Suite à un traitement chronique de trois mois chez la souris athérosclérotique, LDLr-/-; hApoB-100+/+, l’efficacité de l’ivabradine, du métoprolol et de l’exercice physique volontaire dans la prévention de l’augmentation de la compliance cérébrovasculaire s’est avérée proportionnelle à l’étendue des bénéfices sur la fonction endothéliale. La rigidification carotidienne n’a été prévenue que par les interventions qui réduisent vraiment la fréquence cardiaque, c’est-à-dire l’ivabradine et le métoprolol. Dans une deuxième étude, nous avons confirmé nos résultats en utilisant un traitement antioxydant dans le but de cibler plus directement l’endothélium. La catéchine ne réduit pas la fréquence cardiaque, mais elle est reconnue pour protéger l’endothélium cérébral en neutralisant le stress oxydant. Ainsi, la carotide est restée rigide alors que le remodelage cérébral a été prévenu. Une technique d’imagerie novatrice, la tomographie par cohérence optique, nous a permis de valider nos observations in vivo et de proposer que la catéchine prévient l’hypoperfusion du cerveau en protégeant la fonction endothéliale et l’intégrité de la paroi vasculaire cérébrale. Finalement, les deux études identifient la métalloprotéinase de type 9 comme un joueur potentiellement impliqué dans l’augmentation de la compliance cérébrovasculaire. Nos études démontrent que les changements structuraux et biomécaniques affectant la paroi des artères cérébrales sont indubitablement dépendants de l’endothélium alors que dans la carotide, le stress mécanique est le paramètre le plus déterminant. Somme toute, en protégeant indirectement l’endothélium cérébral on empêche les processus de remodelage, telle que l’activation de la métalloprotéinase de type 9. De nombreuses études ont suggéré l’implication des dysfonctions cérébrovasculaires dans la maladie d’Alzheimer. En effet, les affections vasculaires qui compromettent chroniquement le débit sanguin cérébral, telles la dysfonction endothéliale et la réduction de la lumière artérielle, vont entraîner un déficit métabolique des neurones à l’origine de la neurodégénérescence. Les traitements préventifs cardioprotecteurs, tels que l’ivabradine, l’exercice physique et la catéchine améliorent la fonction endothéliale, la structure et la biomécanique des artères cérébrales, et pourraient donc prévenir l’hypoperfusion chronique du cerveau et le déclin cognitif dans l’athérosclérose. / Large artery stiffness and endothelial dysfunction are markers of atherosclerosis. Stiffening of the carotid arteries contrast with the paradoxical increase in distensibility of cerebral arteries that was reported in the presence of risk factors for cardiovascular diseases, such as hypertension. However, our knowledge concerning the influence of atherosclerosis on cerebrovascular compliance and structure remains incomplete. By targeting the endothelium, atherosclerosis induces a severe cerebral endothelial dysfunction affecting chronically the cerebral blood flow and potentially leading to cognitive dysfunctions. Few studies have shown that the paradoxical increase in cerebrovascular distensibility is consistently reported in animal model of risk factors for cardiovascular diseases exhibiting a cerebral endothelial dysfunction. That being said, we hypothesized that the compliance and structure of cerebral arteries is essentially controlled by the endothelium. To validate our hypothesis, in a first study, we selected three distinct therapeutic approaches that modulated the cerebral endothelial function and the mechanical stress imposed to the vascular wall by lowering heart rate in a mouse model of atherosclerosis, LDLr-/-; hApoB-100+/+ during three months. Ivabradine, metroprolol and voluntary physical training protected, with different efficiencies, the cerebral flow-mediated dilation and this was reflected by a prevention, or not, of the increase in compliance. A 13.5 % heart rate reduction with ivabradine and metoprolol limited carotid artery stiffening. Voluntary physical training did not induce an overall reduction of heart rate explaining the lack of effect on carotid mechanics and suggesting that carotids compliance is more influenced by the mechanical stress imposed to the vascular wall by the cardiac cycle. In a second study, we confirmed our previous findings using a diatery approach that targeted more directly the endothelium, the polyphenol antioxidant catechin. Catechin was previously proven, by us and others, to reverse endothelial dysfunction, reduce inflammation and neutralize reactive oxygen species in diverse vascular beds from animal models of atherosclerosis. Accordingly, we found that catechin prevents adverse cerebral wall remodeling but, again, without a significant heart rate reduction, carotids remained stiff. We also integrated a new live imaging technology allowing us to confirm our findings in vivo and to demonstrate that endothelial, structural and mechanical protection by catechin can result in an improvement of basal cerebral blood flow. Finally, both studies identified metalloproteinase -9 as a potential player in the process leading the weakening of the cerebral artery walls. Taken together, our studies highlight that structural and biomechanical alterations are genuinely triggered by endothelial dysfunction. In carotids, mechanicals stress seems to be the main factor controlling remodeling. In essence, indirect protection of the endothelium impedes in cerebral vessels the remodeling processes, such as the activation of metalloproteinase -9. Numerous studies have revealed that vascular, especially cerebral endothelial dysfunction is implicated in the pathogenesis of Alzheimer’s disease. When brain perfusion is compromised, the suboptimal energy delivery causes neuronal death. Deleterious cerebrovascular outcomes that promote the impairment of vasodilation and the encroachment of the lumen will limit cerebral blood flow in a chronic manner. Chronic treatment with ivabradine, voluntary physical training and catechin preserved the endothelial function, the structure and the mechanics of cerebral arteries, which guarantees a closer management of cerebral blow flow in atherosclerotic mice and a reduce propensity to develop cognitive deficiency.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le cancer colorectal représente la troisième forme de cancer la plus fréquente au Canada. Malgré les récents développements, aucun traitement curatif n’est disponible pour les patients diagnostiqués à un stade avancé de la maladie. Plusieurs évidences supportent un rôle important des prostaglandines dans cette maladie. En effet, une surexpression des récepteurs de type EP et FP est remarquée. Ces derniers ainsi que les molécules de signalisations intracellulaires qu’ils activent représentent donc de nouvelles cibles pour traiter ce cancer. Nous et d’autres groupes avons démontré que les protéines G monomériques sont des petits interrupteurs moléculaires importants dans la signalisation intracellulaire engagée par les récepteurs dans des cellules saines mais qu’un dérèglement de celles-ci est associé au cancer. L’objectif principal de ce projet de recherche vise donc à identifier les voies de signalisations par lesquelles le récepteur FP contribue aux capacités invasives des cellules tumorales d’origine colorectale. Notre hypothèse est que la protéine ARF6, une des 6 isoformes des ARFs, et la protéine RhoA, agissent pour coordonner l’activation des voies de signalisations associées à la migration et l’invasion cellulaire. Nos résultats ont indiqué que la stimulation des cellules HEK293 exprimant de façon stable le récepteur FP (HA-FP) ainsi que les cellules SW480, une lignée invasive de cancer colorectal, par le PGF2α augmentait les niveaux d’activation d’ARF6 mais également de la protéine RhoA. De plus, d’autres médiateurs et intermédiaires associés à la réorganisation du cytosquelette comme la cofiline et la chaine légère de la myosine (MLC) ont été hautement phosphorylés suite à la stimulation par la prostaglandine. Ces observations sont associées avec une augmentation des fibres de stress dans les cellules. Nous avons tenté de déterminer si l’inhibition de ces protéines G affectait la capacité du PGF2α à activer ces intermédiaires de signalisation ainsi que certains effets biologiques. Ainsi, nos expériences contribueront à identifier de nouvelles cibles thérapeutiques potentielles pour le traitement du cancer colorectal. / Colorectal cancer is the third most common form of cancer in Canada. Despite recent developments, no curative treatment is available for patients diagnosed at an advanced stage of the disease. Several evidences support an important role of prostaglandins in this disease. Indeed, overexpression of EP-like and FP receptors is noticed. These and intracellular signaling molecules that are activated following receptor stimulation, represent new targets for treating cancer. We and others have demonstrated that monomeric G proteins are important molecular switches coordinating intracellular signaling cascades initiated by receptors in normal cells but that disruption thereof is associated with cancer. The main objective of this research project aims to identify the signaling pathways by which the FP receptor contributes to the invasive capacity of tumor cells of colorectal origin. Our hypothesis is that the proteins ARF6, one of the 6 ARF isoforms, and RhoA protein, act to coordinate the activation of signaling pathways associated with cell migration and invasion. Our results show that stimulation of HEK293 cells that stably express the FP receptor (HA-FP) and SW480 cells, a lineage of invasive colorectal cancer that endogenously express FP receptor, by PGF2α, increased the level of activation of ARF6, but also that of RhoA. In addition, other mediators and intermediates associated with cytoskeleton reorganization such as cofilin and the myosin light chain (MLC) were found highly phosphorylated following stimulation by prostaglandin PGF2α. These observations are associated with an increase of stress fibers in cells. Finally, we have examinated whether inhibition of ARF6 and RhoA, affected the ability of PGF2α to activate these effectors and some biological effects. Thus, our results will contribute to identify new potential therapeutic targets for the treatment of colorectal cancer.

Dans cette thèse nous avons étudié quatre aspects fondamentaux de l’interaction ADN-protéine, notamment : l’affinité, la spécificité, l’accessibilité et la cinétique. En particulier, nous avons adressé les questions suivantes : comment un dimer du facteur de transcription NF-kB reconnait spécifiquement sa séquence d’ADN-cible, quelle est la rapidité de ces interactions, comment NF-kB interagit avec son site de fixation dans le contexte de la chromatine? Récemment, la spécificité de l’interaction NF-kB – ADN a reçu une attention particulière après l’observation que NF-kB peut se lier à des séquences qui n’entrent pas dans la classification de ses motifs « consensus ». Nous avons étudié la spécificité d’interaction de sept de ces motifs avec quatre dimers de NF-kB. Nos résultats montrent que le homo-dimer p50 sont les moins discriminatives et peuvent s’associer spécifiquement avec ces sept séquences. Par contre, les homo-dimers RelA se sont révélés hautement discriminatives ne pouvant pas s’associer spécifiquement avec ces séquences. Pour mesurer l’affinité de l’interaction nous avons utilisés deux méthodes distinctes : le traditionnel gel de retard (EMSA) et une nouvelle technique – « l’empreinte » au laser UV. Nos résultats montrent que le deuxième approche est plus approprié pour la mesure des constantes spécifiques de dissociation.Pour étudier la dynamique de l’interaction NF-kB – ADN, nous avons couplé l’empreinte au laser UV avec un appareil de mélange-rapide à façon. Cette combinaison nous a permis d’atteindre une résolution spatiale d’un nucléotide et temporaire de quelques millisecondes. Nous avons montré que l’homo-dimer p50 s’associe avec sa séquence-cible (MHC) H2 en suivant une cinétique à 2 pas. Le premier, de durée ~100 ms, reflète une reconnaissance initiale rapide, tandis que le deuxième, de durée ~1s, reflète une stabilisation lente du complexe. Nos expériences suggèrent aussi que les séquences voisines du site de reconnaissance jouent aussi un rôle dans la stabilisation du complexe.Finalement, nous avons étudié aussi l’accessibilité du nucléosome pour le NF-kB. Nos données in vitro montre que l’invasion spécifique de l’ADN à l’intérieure du nucléosome par NF-kB nécessite une perturbation majeure de la structure du nucléosome telle que l’éviction d’au moins un dimer d’histones H2A-H2B. / In this thesis we have attempted to study four basic aspects of DNA-protein interactions: Affinity, specificity, accessibility and kinetics. With NF-kB as our model transcription factor, we wanted to investigate how a particular dimer recognizes a specific binding sequence? How fast are these interactions? And finally, how does the NF-kB interact with it binding site in the chromatin context? Specificity of NF-kB-DNA interactions has recently come into focus after it was shown that these dimers can bind to the sequences which do not fall into the NF-kB general consensus motif. We studied seven such sequences for their specificity for four NF-kB dimers. Our results show that p50 homodimers are least discriminative and can bind specifically to all these sequences. While as, RelA homodimers were highly discriminative and did not bind to most of these nontraditional sequences. We used two different methods to measure binding affinities: traditional gel mobility shift assay (EMSA) and a novel technique called as UV laser footprinting. Our results show that UV laser footprinting is the better method to determine the binding constants.For studying the dynamics of NF-kB-DNA binding, we combined UV laser footprinting with stopped flow device. This combination, not only give us one base pair resolution but also milli-second time resolution. Using p50 homodimers as a model transcription factor, we showed that the binding of this factor follows a two-step mechanism. First step involves the fast recognition of the sequence and second step follows a slower kinetics most likely for the stabilization of the complex. Our experiments suggest that flanking sequences play a role in the recognition and stabilization process of the complex formation.Finally, we also studied the accessibility of nucleosomes to NF-kB. Our in vitro data sheds light on the in vivo requirements for the alterations in chromatin structure necessary for the productive binding of NF-kB. These include either a removal of H2A-H2B dimers from the nucleosome and/or chromatin remodeler induced relocation of the histone octamer.Our data sheds light on the in vivo requirements for the alterations in chromatin structure necessary for the productive binding of NF-kB. We hypothesize that some factors like PU.1 might be able to target the chromatin remodeling/dimer eviction machinery to particular nucleosomes and lead to productive binding of NF-kB.

Embora a grande maioria dos pacientes com asma tenha controle com o padrão de tratamento com corticosteroides, 5 a 10% destes asmáticos ainda desenvolvem algumas formas graves da doença e são considerados pacientes difíceis de controlar. A ativação do NFkB contribui para a manutenção e desenvolvimento de inflamação pulmonar crônica na fisiopatologia da asma. O éster fenetilico do ácido cafeico (CAPE), um componente ativo de própolis das colmeias de abelhas, é conhecido por ter propriedades antiinflamatórias e imunomoduladoras. É conhecido como um potente e um inibidor específico da ativação de NFk B. Nós anteriormente mostramos que Y27632, um inibidor de Rho quinase, atenuou a resposta pulmonar das vias aéreas, inflamação e remodelamento. Para o nosso conhecimento, tratamentos com os inibidores de NFk B ou Rho-quinase em comparação com o uso de corticosteroides não foram previamente investigados em um modelo animal de inflamação pulmonar crônica alérgica. MÉTODOS: Trinta camundongos BALB/C machos (20 a 25g) foram divididos em cinco grupos (n=6 em cada grupo): SAL (instilado com solução salina), OVA (expostos à ovalbumina), OVA-CAPE (expostos à ovalbumina e tratados com CAPE), OVA-RHO (expostos à ovalbumina e tratados com inibidor de Rho), OVA-CORT (expostos à ovalbumina e tratados com corticosteróide). Os animais foram sensibilizados para ovalbumina durante 28 dias e os controles receberam solução salina. A administração de corticosteróide (1mg/Kg/dia) e CAPE (10mg/Kg/dia) foi feita por via intraperitoneal e Y-27632 (10 mg/kg) administrado por via intranasal, todos nos dias 22, 24, 26 e 28 do protocolo experimental. Vinte e quatro horas após a conclusão do protocolo experimental, os animais foram submetidos a uma avaliação da curva de resposta à dose à metacolina, consideramos as respostas máximas da Resistência ao Sistema Respiratório (Rrs) e Elastância (Ers) às 24 horas após o último desafio com ovalbumina (respostas basais e máximas após a metacolina desafio 3, 30 e 300mg/mL). Posteriormente, os pulmões foram removidos e a análise histológica foi realizada usando morfometria. RESULTADOS: não houve diferenças nos valores basais de todos os grupos. expostos à ovalbumina Houve um aumento em Rrs e Ers após o desafio da metacolina em comparação com o grupo SAL (p < 0,05). No grupo exposto ao OVA, os tratamentos com CAPE, CORT e RHO reduziram a resposta máxima de Ers em relação ao OVA (p < 0,05). Em relação ao tratamento com somente CAPE atenuou a resposta máxima de Rrs em relação ao OVA (p < 0,05). Houve diminuição nos eosinófilos, conteúdo de fibras de colágeno, células positivas para iNOS, MMP-9, TIMP-1 em grupos OVA-CAPE, OVA-CORT e OVA-RHO em comparação com os animais sensibilizados (grupo OVA) (p < 0,05). CONCLUSÃO: a inibição de NFkB e Rho-quinase contribuiu para o controle da hiperresponsividade, inflamação, processo de remodelamento da matriz extracelular e ativação do estresse oxidativo. Embora os inibidores de NFkB e Rho-quinase sejam uma alternativa ao tratamento da asma, são necessários mais estudos / Although the great majority of asthma patients obtain control with the gold standard treatment using corticosteroids, 5 to 10% of these asthmatics still develop some severe forms of the disease, called difficult to control patients. Activation of NFk B contributes to the maintenance and development of chronic lung inflammation in pathophysiology of asthma. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have anti-inflammatory and immunomodulatory properties. It is known as a potent and a specific inhibitor of NFk B activation. We previously have shown that Y27632, a Rho-quinase inhibitor, attenuated airway lung responsiveness, inflammation and remodeling. To our knowledge, treatments with the NFk B or Rho-kinase inhibitors compared to the use of corticosteroids have not been previously investigated in an animal model of chronic allergic lung inflammation. METHODS: Thirty male BALB/c mice (20 to 25g) were divided into five groups (n=6 in each group): SAL (saline-instilled), OVA (exposed-ovalbumin), OVA-CAPE (exposedovalbumin and treated with CAPE), OVA-RHO (exposed-ovalbumin and treated with Rho inhibitor), OVA-CORT (exposed-ovalbumin and treated with corticosteroid). The BALB/c mice were sensitized to ovalbumin for 28 days and controls received saline. The administration of corticosteroid (1mg/Kg/day) and CAPE (10mg/Kg/day) was made intraperitoneally and Y-27632 (10mg/kg) was intranasally, all on days 22, 24, 26 and 28 of the experimental protocol. Twentyfour hours after completion of the experimental protocol, animals were subject to an evaluation of dose response curve to methacholine. We considered the maximal responses of respiratory system resistance (Rrs) and elastance (Ers) 24 hours after the last challenge with ovalbumin (baseline and maximal responses after metacholine challenge 3, 30 and 300mg/mL). Afterwards, lungs were removed and histological analysis was performed using morphometry. RESULTS: There were no differences on baseline values of all groups. Ovalbumin-exposed mice had an increase in Rrs and Ers after methacholine challenge compared to SAL group (p < 0.05). In OVA-exposed groups, the treatments with CAPE, CORT and RHO reduced maximal response of %Ers compared to OVA (p < 0.05). In relation to %Rrs only CAPE treatment attenuated the maximal response compared to OVA (p < 0.05). There was a decrease in eosinophils, collagen fibers content, iNOS, MMP-9, TIMP-1 positive cells in OVACAPE, OVA-CORT and OVA-RHO groups compared to only sensitized animals (OVA group) (p < 0.05). CONCLUSION: NFkB and Rho-quinase inhibition contributes to the control of hyperresponsiveness, inflammation, extracellular matrix remodeling process and oxidative stress activation. Although NFkB and Rho-quinase inhibitors were an alternative to asthma treatment, more studies are needed

O objetivo deste trabalho foi avaliar se a proteína bifuncional de planta, CrataBL, que tem lectina e as propriedades inibidoras de enzima, modula alterações de mecânica pulmonar, inflamatórias e remodelamento induzidas por elastase intratraqueal em camundongos. Métodos: 36 camundongos C57BL6 receberam elastase (0,025 mg) por instilação intratraqueal (grupo ELA e ELA-CrataBL). Os grupos controles receberam salina (grupo SAL e SAL-CrataBL). Os camundongos foram tratados com instilação intraperitoneal de CrataBL (2mg/kg) nos dias 1, 14 e 21 após a instilação intratraqueal de elastase (grupo SAL-CrataBL e ELA-CrataBL) os animais controle receberam salina no mesmo volume. No dia 28, os camundongos foram anestesiados, ventilados mecanicamente e foram analisados a resistência e elastância do sistema respiratório (Ers e Rrs), elastância e resistência tecidual (Htis e Gtis), resistência das vias aéreas (Raw) e óxido nítrico exalado (NOex). Após, o lavado broncoalveolar (LBA) foi realizado, os pulmões foram retirados e por morfometria, e foram quantificados o intercepto linear médio (Lm), a quantidade de neutrófilos, células positivas para TNF-alfa, fibras colágenas, elásticas, células positivas para MMP-9, MMP-12, TIMP-1, eNOS e iNOS e isoprostano no parênquima pulmonar e vias aéreas. No parênquima foram avaliados os macrófagos nos septos alveolares e nas vias aéreas, foram também avaliadas as células para MUC-5. Resultados: No grupo ELA houve um aumento na Ers, Raw, Gtis, Htis, Lm, NOex, nas células totais, macrófagos, neutrófilos, eosinófilos e linfócitos no LBA em relação aos controles (p < 0,05), sendo que Raw, diminuiu também nos grupos SAL-CrataBL e ELA-CrataBL. Nos grupos tratados com CrataBL houve uma diminuição de Ers (37,0±2,2 cmH2O/L), Htis (37,9±3,5 cmH2O/ml/s), ENO (14,7±0,7 ppb), comparativamente ao grupo ELA (p < 0,05). No LBA houve atenuação de neutrófilos (0,003±0,001 104células/ml), linfócitos (0,003±0,001 104células/ml) e de Lm (54,6±6,0 mm). Complementando a avaliação, no grupo que recebeu elastase houve um aumento no número de macrófagos (22,88 +- 2,24 células/104um2), neutrófilos (1,18 +- 0,15 células/10 4um2), células positivas para TNF-ala (12,52 +- 0,42 células/104um2) no parênquima pulmonar. Nas alterações de remodelamento no parênquima pulmonar, houve um aumento da proporção de volume de fibras colágenas (11,5 +- 0,11%), elásticas (0,5 +- 0,03%), na quantidade de células positivas para MMP-9 (18,59 +- 1,87 células/104?m2), MMP-12 (20,17 +- 1,92 células/104?m2), TIMP-1 (14,42 +- 2,05 células/104um2) em comparação com os controlos (p < 0,001). No estresse oxidativo, houve um aumento de eNOS (13,15 +- 0,40 células/104um2), iNOS (10,49 +- 0,65 células/104um2) e isoprostano (18,11 =- 5,38%). O tratamento CrataBL (grupo ELA-CrataBL) reduziu no parênquima pulmonar a quantidade de macrófagos (9,58 +- 1,36 células/104um2), neutrófilos (0,75 +- 0,1 células/104um2), células positivas para TNF-alfa (10.4±0,49 células/104?m2), fibras colágenas (10,8 +- 0,13%), elásticas (0,3 +- 0,02%), a quantidade de células positivas para a MMP-9 (10,35±0,65 células/104um2), MMP-12 (14,15±0,59 células/104um2), TIMP-1 (9,89 +- 2,79 células/104um2), MUC-5 (3,56 +- 0,54 células/104um2), eNOS (6.98 +- 0.32 células/104um2) e iNOS (6,21 +- 0,42 células/104um2) e isoprostano (8,96 +- 3,08 %) em relação ao grupo ELA (p < 0,001). Nas vias aéreas também ocorreu um aumento significativo de neutrófilos (5,97 +- 1,03 células/104um2), células positivas para TNF-alfa (15,82 +- 1,03 células/104um2). Nas alterações de remodelamento pulmonar nas vias aéreas também ocorreu um aumento da proporção de volume de fibras colágenas (8,73 +- 2,59%), elásticas (2,56 +- 0,18%), na quantidade de células positivas para MMP-9 (14,86 +- 1,77 células/104um2), MMP-12 (18,56 +- 1,79 células/104um2), TIMP-1 (1,31 +- 0,12 células/104um2) e MUC-5 (7,09 +- 1,71 células/104um2) em comparação com os controlos (p < 0,001). No estresse oxidativo, houve um aumento de células positivas para eNOS (3,09 +- 0,08 células/104um2), iNOS (5,4 +- 0,3 células/104um2) e isoprostano (18,11 +- 5,38%) em comparação com os controlos (p < 0,001). O tratamento CrataBL (grupo ELA-CrataBL) reduziu nas vias aéreas a quantidade de neutrófilos (4,62 +- 0,61 células/104um2), TNF- alfa (14,30 +- 1,28 células/104um2), fibras colágenas (7,80 +- 1,37%), elásticas (1,4 +- 0,13%), a quantidade de células positivas para a MMP-9 (9,93 +- 1,39 células/104um2), MMP-12 (12,06 +- 1,15 células/104um2), TIMP-1 (0,73 +- 0,05 células/104?m2), MUC-5 (3,56 +- 0,54 células/104um2), eNOS (1,89 +- 0,16 células/104um2) e iNOS (4,3 +- 0,31 células/104um2), isoprostano (7,34 +- 2,31%) em relação ao grupo ELA (p < 0,001). Conclusão: CrataBL atenua as alterações de mecânica pulmonar, lavado bronco alveolar, responsividade inflamatória, controle do remodelamento e estresse oxidativo induzidas pela elastase. Embora mais estudos devam ser realizados, esta proteína bifuncional pode contribuir como potencial ferramenta terapêutica para o tratamento da DPOC / The aim of this study was to evaluate whether the bifunctional protein plant, CrataBL, which has lectin and enzyme inhibitory properties, modulates changes in lung mechanics, inflammatory and remodeling induced by intratracheal elastase in mice.Methods : 36 C57/Bl6 mice received elastase (0.025 mg) by intratracheal (group ELA and ELA-CrataBL). Control groups received saline (group SAL and SAL-CrataBL).The mice were treated with intraperitoneal instillation of CrataBL (2mg/kg) on days 1, 14 and 21 after intratracheal instillation of elastase (group SAL-CrataBL and ELA-CrataBL), control animals received saline in the same volume. On day 28, the mice were anesthetized and mechanically ventilated were analyzed resistance and respiratory system elastance (Ers and Rrs), elastance and tissue resistance (Htis and Gtis), airway resistance (Raw) and exhaled nitric oxide (ENO). After the bronchoalveolar lavage (BAL) was performed, the lungs were removed and morphometry were quantified and the linear intercept mean (Lm), the number of neutrophils, positive cells for TNF-alfa, collagen fibers, positive cells for MMP-9, MMP-12, TIMP-1, eNOS, iNOS and isoprostane in lung parenchyma and airways. Parenchyma was also evaluated macrophages in the alveolar septa. Airway was also evaluated MUC-5 cells. Results: In group ELA was an increase in Ers, Raw, Gtis, Htis, Lm, ENO, in total cells, macrophages, neutrophils, eosinophils and lymphocytes in BAL compared to controls (p < 0.05), and Raw, decreased in both groups SAL-CrataBL and ELA-CrataBL. In the groups treated with CrataBL there was a decrease in Ers (37.0±2.2 cmH2O/L) Htis (37 9±3.5 cmH2O/ml/s) and ENO (14.7±0.7 ppb) compared to the ELA group (p < 0.05). In BAL there was attenuation of neutrophils (0.003±0.001 104cells/ml), lymphocytes (0.003±0.001 104cells/ml) and Lm (54.6±6.0 mm). Complementing the assessment, the group that received elastase was an increase in the number of macrophages (22.88±2.24 cells/104um2), neutrophils (1.18±0.15 cells/104um2), positive TNF-alfa cells (12.52±0.42 cells/104um2) in the lung parenchyma. In remodeling changes in lung parenchyma, there was an increase in the volume ratio of collagen fibers (11.5 ± 0.11%), elastic (0.5±0.03%), the number of positive MMP-9 cells (18.59±1.87 cells/104um2), MMP-12 (20.17 ± 1.92 cells/104um2) TIMP-1 (14.42±2.05 cells/104um2) compared to controls (p < 0.001). Oxidative stress, was an increased of eNOS (13.15±0.40 cells/104um2), iNOS (10.49 ± 0.65 cells/104um2) and isoprostane (18.11±5.38%). Treatment CrataBL (ELA-CrataBL group) reduced the amount of parenchymal lung macrophages (9.58±1.36 cells/104um2), neutrophils (0.75±0.1 cells/104um2), positive TNF-alfa cells (10.4±0.49 cells/104um2), collagen (10.8±0.13%), elastic (0.3±0.02%), the number of positive MMP-9 cells (10.35±0.65 cells/104?m2), MMP-12 (14.15±0.59 cells/104um2), TIMP-1 (9.89±2.79 cells/104um2) MUC-5 (3.56±0.54 cells/104um2), eNOS (6.98±0:32 cells/104um2) and iNOS (6.21±0.42 cells/104um2) and isoprostane (8.96 ± 3.08%) compared to group ELA (p < 0.001). Airway was also a significant increase in neutrophils (5.97±1.03 cells/104um2), positive TNF-alfa cells (15.82±1.03 cells/104um2). Changes in lung airway remodeling also occurred an increase in the volume ratio of collagen fibers (8.73±2.59%), elastic (2.56±0.18%), the number of positive MMP-9 cells (14.86±1.77 cells/104um2), MMP-12 (18.56±1.79 cells/104um2) TIMP-1 (1.31±0.12 cells/104um2) and MUC-5 (7.09±1.71 cells/104um2) compared to controls (p < 0.001). Oxidative stress, an increase of eNOS (3.09 ± 0.08 cells/104um2), iNOS (5.4±0.3 cells/104um2) and isoprostane (18.11±5.38%) compared to controls (p < 0.001). Treatment CrataBL (ELA-CrataBL group) reduced the amount airway neutrophils (4.62±0.61 cells/104um2), TNF-alfa (14.30 ± 1.28 cells/104um2), collagen fibers (7 80±1.37%), elastic (1.4±0.13%), the number of positive MMP-9 cells (9.93±1.39 cells/104um2), MMP-12 (12.06±1.15), TIMP-1 (0.73±0.05 cells/104um2), MUC-5 (3.56±0.54 cells/104um2), eNOS (1.89±0,16 cells/104um2) and iNOS (4.3±0.31 cells/104um2), isoprostane (7.34±2.31%) compared to group ELA (p < 0.001). Conclusion: CrataBL attenuates changes in lung mechanics, broncho alveolar inflammatory responsiveness, control remodeling and oxidative stress induced by elastase. Although more studies should be conducted, this bifunctional protein may contribute as a potential therapeutic tool for the treatment of COPD