Since integrity and functions of vascular endothelial cells are greatly affected by shear stress, a variety of in vitro systems to subject endothelial cells under precisely controlled fluid conditions has been developed. Complicated designs of the conventional flow devices, however, have impeded such implementation. In the present study, we designed and developed a novel parallel-plate flow chamber (PPFC). It consists of multiple layers of different materials to adjust the required geometries of the chamber and provide a wide span of biomedical research applications. Because the chamber stacks separate layers to constitute the flow channel, different pieces can be easily removed or replaced. Moreover, the multilayer design only requires 2D cutting, which is easier and faster to manufacture. It is also capable of accepting up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. Furthermore, it minimizes the pressure loss across the chamber while maximizing the effective area of endothelial cells up to 96 cm2. Results from mathematical analysis and dye injection experiments showed that a uniform magnitude of shear stress is applied throughout the entire surface of endothelial cells. In addition, the morphological changes and attenuated gene expression of pro-inflammatory mediators were observed in endothelial cells exposed to the physiologically relevant shear stress. These findings indicate that our newly designed PPFC can provide a better in vitro system for versatile applications of biomedical research. The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and can cause more severe damage than prolonged ischemia. The injuries caused by cessation and reperfusion of blood flow are closely related to the inflammatory reactions involving in endothelium-leukocyte cascade responding to a shear stress exerted by the flow. Shear stress is also known to play an important role in human chronic diseases including atherosclerosis, neurological disorders, and cancer metastasis. Therefore, it is important to investigate the transmission of mechanical stimuli such as shear stress to various complex endothelial cell signaling pathways which process as a whole is often referred as mechanotransduction. Shear stress-mediated signaling pathways have been known to trigger endothelial cell responses and contribute to the pathophysiology of human vascular diseases. The present study was designed to apply the novel PPFC to biomedical research, especially ischemia/reperfusion injury. The changes in mRNA and protein expression of inflammatory mediators in endothelial cells were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RBE4 and HMEC-1 cells were either maintained in continuous laminar flow condition (Normal Flow) or subjected to 1 h of flow cessation followed by reperfusion of flow (Ischemia/Reperfusion) for 24 h. Ischemia/Reperfusion significantly up-regulated expression of inflammatory mediators, such as IL-6, MCP-1, ICAM-1, VCAM-1, and E-selectin, in microvascular endothelial cells. Furthermore, antioxidant pyrrolidine dithiocarbamate (PDTC) significantly attenuated ischemia/reperfusion-induced overexpression of pro-inflammatory mediators. These data indicates that our newly designed PPFC provide a better in vitro system for versatile applications of biomedical research. / Master of Science

Innate immune and inflammatory responses contribute to myocardial and cerebral ischemia/reperfusion (I/R) injury. Toll-like receptors (TLRs) play a critical role in the induction of innate immune and inflammatory responses via activation of nuclear factor kappa B (NF-κB). We have shown that activation of NF-κB contributes to myocardial and cerebral I/R injury. Indeed, inhibition of TLR4-mediated NF-κB activation significantly decreased myocardial and cerebral I/R injury via activation of PI3K/Akt signaling. PI3K/Akt signaling is an important pathway in regulating cellular survival and inflammatory responses. Therefore, an important question is how to differentially modulate PI3K/Akt signaling and TLR/NF-κB-mediated signaling pathway during I/R injury? We demonstrated that pretreatment of mice with Pam3CSK4, a specific TLR2 ligand, significantly decreased cerebral I/R injury and improved neuronal functional recovery. Importantly, therapeutic administration of Pam3CSK4 also markedly decreased cerebral I/R injury. The mechanisms involved suppression of NF-κB binding activity and activation of PI3K/Akt signaling. We also demonstrated that CpG-ODN, a specific TLR9 ligand, induced protection against cerebral I/R injury via activation of PI3K/Akt signaling. Our findings were consistent with our previous reports showing that administration of Pam3CSK4 or CpG-ODN protected against myocardial I/R injury via a PI3K/Akt-dependent mechanism. In addition, we demonstrated for the first time that TLR3 located in endosomes played a deleterious role in myocardial I/R injury via activation of NF-κB. To investigate how to activate PI3K/Akt signaling during I/R injury, we examined the role of microRNA (miRs) in regulating PI3K/Akt signaling during myocardial ischemic injury. We discovered that Pam3CSK4 or CpG-ODN treatment significantly increased the expression of miR-130a in the myocardium, while myocardial infarction markedly decreased the levels of miR-130a in the myocardium. The data indicated that miR-130a served a protective role in myocardial ischemic injury. Indeed, we demonstrated for the first time that increased expression of miR-130a significantly attenuated cardiac dysfunction and promoted angiogenesis after myocardial infarction. The mechanisms involved activation of PI3K/Akt signaling via targeting PTEN expression by microRNA-130a. This dissertation discovers novel mechanisms of cerebral and myocardial ischemic injury and provides solid basis for developing new approaches for the treatment and management of stroke and heart attack patients.

Phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. We hypothesized that SIL-induced protection may be mediated through activation of SIRT1, an enzyme which deacetylates proteins involved in cellular stress response. Adult male ICR mice were treated with SIL (0.7mg/kg ip), Resveratrol (RSV) (5mg/kg ip) (positive control), or saline (0.2 ml ip). The hearts were harvested 24 h later and homogenized for SIRT1 activity analysis. Both SIL and RSV increased cardiac SIRT1 activity (P<0.001) as compared to Saline. Adult mouse ventricular cardiomyocytes pre-treated with either SIL or RSV (1µM) in vitro also upregulated SIRT1 activity (P<0.05). SIL also reduced infarct size following 30 min. ischemia and 24 h reperfusion in vivo. Sirtinol (5mg/kg in 10% DMSO, ip), a SIRT1 inhibitor abolished the infarct-limiting effect of SIL and RSV (P<0.001). In conclusion, activation of SIRT1 by SIL plays an essential role in cardioprotection against I-R injury.

Administration of small interfering RNA (siRNA) specific for prolyl-4 hydroxylase-2 (PHD2) results in PHD2 inhibition, Hypoxia Inducible Factor-I (HIF-1) activation, and cardioprotection versus Ischemia Reperfusion (IR). This study observes the effects of siRNA-mediated PHD2 inhibition on the distribution of cardioprotective proteins by immunofluorescence and basic histology. Fifteen mice were divided into 5 groups: PHD2 Control, Non-Targeting scramble (NTS) Control, IR Control, PHD2 IR, and NTS IR. Histologically, tissue damage was reduced dramatically in the PHD2 IR group compared to the NTS IR and IR control groups. From confocal images, total fluorescent pixels and intensities were quantified. The PHD2 IR group yielded the highest pixel quantity and intensity for HIF-1 and possessed increased pixels and intensity for Inducible Nitric Oxide Synthase, another cardioprotective protein. These results further demonstrate the cardioprotection and HIF-1 activation conferred by PHD2 siRNA administration and supports its role as a potential therapy to alleviate cardiac IR injury.

Cardiac injury is increased following ischemia-reperfusion. Mitochondria are the “effector organelles” that are damaged during ischemia (ISC) when there is no blood flow. Resumption of metabolism by damaged mitochondria during reperfusion (REP) results in increased cell injury. Current therapeutic interventions to pre-condition and post-condition the heart during ISC are ineffective during certain conditions like aging and diabetes due to defects in the signaling cascades. In contrast, mitochondrial-based strategies are effective in protecting the heart during ISC-REP. Hence direct therapeutic targeting of dysfunctional mitochondria will provide the potential to bypass the upstream signaling defects and intervene directly upon the effector organelle. Novel mitochondrial-targeted therapy relies on understanding the sites in the electron transport chain (ETC) that are damaged by ISC and produce cell-injury during REP. This project identifies a novel pathological role of cytochrome c in depleting cardiolipin during ischemia after which the mitochondria are in a defective condition that leads to additional cell death during reperfusion. During ischemia oxidants from complex III oxidize cytochrome c, forming a peroxidase, which causes oxidative damage and depletion of cardiolipin. Depletion of cardiolipin disrupts normal physiology and augments cell death. Identification of the innovative pathobiology during ISC-REP recognizes a novel therapeutic target, cytochrome c peroxidase, which can be a focal point for new therapeutic interventions to decrease cardiac injury. In order to maintain homeostatis, living organisms have the methionine sulfoxide reductase system, which reduce both free and protein bound Met(O) back to methionine (Met) in the presence of thioredoxin. Oxidized Trx is inactive and unable to bind to ASK1 thereby activating ASK1 and causing cell death via p38/JNK pathways thereby contributing to the pathogenesis of myocardial ISC-REP injury. In this study we have shown that inhibition of ASK1 protects the heart during REP via the modulation of mitochondria that sustained damage during ISC. The mitochondrial-based mechanism of cardioprotection with ASK1 inhibition enhanced the functional integrity of the inner mitochondrial membrane retaining cytochrome c thereby decreasing cell death. This therapeutic intervention is a key step to achieve the ultimate goal to improve clinical outcomes in patients that suffer an acute myocardial infarction.

Adaptation to chronic hypoxia increases myocardial resistance to acute ischemia/reperfusion (I/R) injury, similarly to application of exogenous erythropoietin (EPO). Nevertheless, it is not known if EPO induced by chronic hypoxia plays a role in its cardioprotective mechanism. The aim of this study was to find out if protective effect of exogenous EPO adds up to protection offered by chronic hypoxia. Adult male mice (ICR) were adapted to intermittent hypobaric hypoxia 8 hours per day, 5 days per week for 5 weeks. The degree of hypoxia corresponded to 7000 metres. Control animals were housed for the same time in normoxic environment. Resistance to I/R injury was assessed according to size of myocardial infarction induced by 45-min global ischemia and 1-h reperfusion of the heart in vitro. Animals were treated 24 h before the experiment with 200 or 5000 U/kg EPO. Treatment with 200 U/kg EPO was sufficient to significantly limit infarct size in normoxic animals (33,56 ± 2,93 % vs. 25,71 ± 2,29 %). Hypoxic adaptation decreased infarct area to 23,49 ± 2,30%, but additive effect of EPO in hypoxic group was not detected. The results indicate that exogenous EPO employs the same cardioprotective mechanisms as adaptation to chronic intermittent hypoxia. Preliminary results indicate that repeated application of EPO...

Les flavonoïdes sont des composés naturels appartenant à la famille des polyphénols, qui sont étudiés depuis de nombreuses années pour leur caractère antioxydant ainsi que leurs propriétés thérapeutique. La mise en évidence, pour certaines d’entre eux, d’une activité vasorelaxante variant en fonction de leur structure, en fait un modèle d’étude de choix pour la prévention des maladies cardiovasculaires. Le 3’,4’-dihydroxyflavonol (DiOHF) est le dérivé le plus décrit pour son fort potentiel en terme de propriétés cardioprotectrices et antioxydantes et est considéré comme un composé d’intérêt dans le traitement anti-ischémique, bien que sa faible solubilité dans l’eau soit un facteur limitant pour son utilisation. Dans ce contexte, notre premier objectif a été de synthétiser une trentaine de dérivés du DiOHF, fonctionnalisés par un groupement hydrophile, tout en préservant ses propriétés antioxydantes et pour certaines des structures obtenues, ses propriétés vasorelaxantes. Pour cela, nous avons développé une méthode de synthèse rapide utilisant les irradiations micro-onde, qui nous a permis d’optimiser les rendements, de réduire les temps de réaction et qui ne conduit à aucune formation de sous-produits. Notre second objectif a été de déterminer l’activité antioxydante de ces dérivés flavonoïques par des tests mettant en œuvre des réactions de compétition avec des radicaux libres. Faisant suite à ce premier screening, des études biologiques chez le rat ont été réalisées sur les composés montrant les meilleures performances. A partir d’essais in vitro et in vivo nous avons démontré, l’innocuité de ces dérivés flavonols, et le fort impact des modifications de structure sur l’activité vasorelaxante et antioxydante, ainsi que sur la récupération fonctionnelle et métabolique consécutive à une ischémie/reperfusion myocardique. Un autre champ d’action de ces flavonols hydrosolubles pourrait être d’explorer leur utilisation dans le traitement du diabète de type 2. Pour cela nous avons greffé deux motifs antidiabétiques, correspondant à des molécules actives déjà utilisées dans le traitement de l’insulinorésistance, sur les dérivés flavonols les plus cardioprotecteurs et/ou vasorelaxants de notre série, ainsi que sur des dérivés de l’acide cinnamique. Ces composés ont été testés pour leur activité antioxydante, laissant supposer un potentiel significativement supérieur aux analogues antidiabétiques non fonctionnalisés, en termes de protection antioxydante et fonctionnelle. / Flavonoids are natural polyphenolic compounds, and epidemiological and pharmacological studies have shown that flavonoids intake is associated with many beneficial effects. An important example is the 3’,4’-dihydroxyflavonol (DiOHF) which showed interesting therapeutic properties in vitro so it could be considered as an important lead compound to the treatment of cardiovascular diseases. However is poor water solubility prevents it use as a drug. Starting from this model we have synthesized a series of new flavones functionalized by a hydrophilic group in order to increase water solubility. To obtain these derivatives we have set up a fast, simple and clean method without formation of by-products, using micro-waves irradiation to optimize yield and to decrease reaction time. Our second purpose was to investigate the antioxidant activity of the synthesized compounds by undergoing several assays. The most promising compounds were tested in biological studies, in vitro and in vivo. The results showed a significant enhancement of the post-ischemic mechanical function recovery in vivo and in vitro. Another purpose was to designed new flavonols linked to selected antidiabetic moieties already used in the treatment of insulinoresistance. It was shown that these new compounds retain the antioxidant properties of the parent flavonols, and can be of interest in the development of new diabete 2 treatments.

OBJECTIF : L’intégrité vasculaire est une pierre angulaire de la viabilité des organes solides, notamment en cas de transplantation. L’objectif de ce travail est de décrire les modifications du réseau vasculaire du cortex rénal dues à une séquence d’ischémie-reperfusion (IR) induite dans un modèle préclinique porcin d’autotransplantation rénale. MATÉRIELS ET MÉTHODES : Nous avions mis en place une technique d’analyse de la microvascularisation corticale rénale après acquisition microscanner et traitement des images de reins de porc perfusés avec un polymère de silicone radio-opaque.RÉSULTATS : L’ischémie-reperfusion rénale induit une diminution corticale du volume du segment vasculaire associée à une raréfaction des microvaisseaux inférieurs à 30 µm en particulier dans la corticale interne. Dans le cortex, une augmentation de la connectivité caractérisée par une augmentation des bifurcations et une diminution du facteur d’arborescence a été observée. L’hypercholestérolémie associée à l’ischémie-reperfusion entraine une raréfaction microvasculaire dans la corticale moyenne. Trois mois post-autotransplantation, l’augmentation de la durée de conservation des greffons induit une augmentation significative de l’épaisseur de la zone corticale, un effondrement du pourcentage de microvaisseaux. CONCLUSION : Trois mois après autotransplantation rénale, l’ischémie-reperfusion induit une raréfaction des microvaisseaux de la corticale interne. Nos résultats suggéreraient que l’altération de la fonction rénale chez des greffons pourrait être liée à une hypoxie chronique et une fibrose induite par une raréfaction microvasculaire proportionnelle à la durée de la conservation. / Purpose:Vascular integrity is a cornerstone of organ viability, particularly in cases of transplantation. The aim of this study is to describe the modifications of renal cortex vascular network due to an ischemia-reperfusion sequence in a preclinical pig kidney autotransplantation model.MATERIALS AND METHODS: Porcine kidneys were perfused with a radio-opaque silicone polymer and the cortex studied by X-ray micro-computed tomography. RESULTS: Renal ischemia-reperfusion led to decreased vascular segment volume associated with the rarefaction of vessels less than 30 µm diameter, particularly in the inner cortex. Vessels showed higher connectivity throughout the cortex. The decrease in microvasculature correlated with a deterioration of renal function, proteinuria, and tubular dysfunction, and was associated with the development of fibrous tissue. The hypercholesterolemia associated with ischemia-reperfusion promoted concomitant microvascular rarefaction for small vascular segments particularly in the middle cortex. At M3, the hypothermic preservation for 48 H of grafts compared to 24 H induced a significant increase in the cortical area thickness, associated with a drastic decrease of microvessels. CONCLUSION: Three months after renal autotransplantation, ischemia reperfusion induces a rarefaction of microvessels of the internal cortex. The hypercholesterolemia leads to a renal microvascular rarefaction which accentuates vascular remodeling due to ischemia reperfusion. Our results suggest that long preservation duration impacting the renal function in grafts could be associated with chronic hypoxia and fibrosis related to microvascular rarefaction.

CONTEXTE: Au cours de l'infarctus du myocarde, l'homéostasie du calcium entre leréticulum sarcoplasmique (SR), les mitochondries et le cytosol est altérée chez lescardiomyocytes (CM) et conduit à la mort cellulaire. Les canaux de fuite de calcium sontconsidérés comme des régulateurs clés de l'homéostasie calcique réticulaire. Le translocon(TLC), un composant majeur de la machine de la traduction protéique, est un important canalde fuite calcique réticulaire.METHODES: Par des moyens optiques, nous avons d'abord évalué l'organisation spatiale etla fonction du TLC dans le SR de souris adultes CM. Dans un second temps, nous avonsinterrogé si et comment la modulation pharmacologique TLC pourrait réduire les lésionsd'ischémie/reperfusion (I/R) cardiaque dans un modèle d'infarctus du myocarde de souris.RÉSULTATS: Nos données montrent que le TLC est spécifiquement localisée dans le SRlongitudinale des CM chez la souris adulte. Nous démontrons que la puromycine (activateurpharmacologique du TLC) induit une réduction partielle des réserves de calcium dans le SRlongitudinale, alors que nous n'observons aucune altération des réserves de calcium dépendantdu récepteur ryanodine dans le SR jonctionnelle. Le préconditionnement de la souris par lapuromycine, soumis à un infarctus du myocarde, diminue significativement la zone d'infarctusde près de 30,9±6,3%. Ceci est corrélé à une diminution de l'activation des protéines proapoptotiquesmitochondriales et à une augmentation d'un mécanisme de pro-survie:l'autophagie. Nous avons également démontré que le préconditionnement de la puromycinediminue la vitesse d'augmentation du calcium dans le cytosol du CM adulte pendant la duréede l'ischémie en corrélation avec la diminution de l'activation des calpains calciques.CONCLUSIONS: Dans cette étude, nous avons caractérisé le TLC comme un canal de fuitespécifiquement situé dans le compartiment longitudinale du SR dans les CM de souris adultes.Nous avons constaté que l'activation pharmacologique de la TLC avant l'infarctus dumyocarde exerce un effet de préconditionnement sur le myocarde sans altérer les réserves de calcium dépendant de la ryanodine. Dans l'ensemble, ces résultats mettent l'accent sur les connaissances actuelles sur la dualité entre le SR jonctionnelle et le SR longitudinale et ouvrent de nouvelles perspectives thérapeutiques / BACKGROUND: During myocardial infarction, alteration of calcium homeostasis between sarcoplasmic reticulum (SR), mitochondria and cytosol occurs in cardiomyocytes (CM) and leads to cell death. Calcium leak channels are thought to be key regulators of the reticular calcium homeostasis. Translocon (TLC), a major component of the translation machinery, is a major reticular calcium leak channel.METHODS: By the mean of photonics, we first assessed the spatial organization and the function of TLC in the SR of adult mouse CM. In a second time, we questioned if and how the pharmacological TLC modulation could reduce ischemia/reperfusion (I/R)-mediated heart injury in a model of mouse myocardial infarction.RESULTS: Our data show that TLC is specifically located in the longitudinal SR in adult mouse CM. We demonstrate that puromycin induces a partial reduction of calcium stores in the longitudinal SR, while we observe no alteration in the ryanodine receptor-dependent calcium stores in the junctional SR. Puromycin preconditioning of mouse subjected to myocardial infarction significantly decreases the infarct area by near 30.9±6.3%. This is correlated with a decrease in the activation of mitochondrial pro-apoptotic proteins and an increase of a pro-survival mechanism: autophagy. We further demonstrated that puromycin preconditioning decreases the rate of calcium increase in the cytosol of adult CM during the ischemia duration in correlation with the decreased activation of calcium-dependent calpains.CONCLUSIONS: In this study, we characterized TLC as a leak channel specifically located in the longitudinal SR compartment of adult mouse CM. We found that the pharmacological activation of TLC before myocardial infarction exerts a preconditioning effect on myocardium without altering the ryanodine-dependent calcium stores. Altogether, these findings emphasize the present knowledge on the duality between junctional and longitudinal SR in CM and open up new therapeutic perspectives

A gravidade da síndrome isquemia/reperfusão determina o prognóstico da lesão renal aguda (LRA). Agentes como o citrato de sildenafil (Sil) e a N-acetilcisteína (NAC) tem demonstrado efeito renoprotetor na LRA isquêmica com resultados ainda inconclusivos. Esse estudo investigou o efeito do Sil e da NAC na LRA com diferentes tempos de isquemia. Foram utilizados grupos de ratos Wistar, adultos e machos: SHAM; Isquemia 30 min (clampeamento dos pedículos renais por 30 min); Isquemia 30 + Sil (Sil 0,25 mg/kg 60 min antes da isquemia renal); Isquemia 30 + NAC (NAC 150 mg/kg antes e após a isquemia renal); Isquemia 45 (clampeamento dos pedículos renais por 45 min); Isquemia 45 + Sil e Isquemia 45 + NAC. Foram avaliadas a função renal (clearance de creatinina e fração de excreção de sódio-FENa); a lesão oxidativa (peróxidos urinários; substâncias reativas ao ácido tiobarbitúrico - TBARS; óxido nítrico - NO e tióis no tecido renal); a síntese protéica da óxido nítrico sintase induzível iNOS e da heme oxigenase-1 HO-1 (western blotting) e análise histológica renal (área intersticial relativa - AIR e lesão tubulointersticial). Os grupos 30 min tratados com Sil e NAC demonstraram melhora da função renal, redução dos índices oxidantes e NO, ausência de iNOS e presença de HO-1. Nos grupos 45 min, o Sil manteve a função renal, porém demonstrou proteção tubular e oxidante; a NAC não demonstrou efeito protetor em nenhum dos parâmetros avaliados. Quanto à histologia, apenas o Sil induziu redução da AIR e da lesão tubulointersticial nos tempos 30 e 45 min. O estudo confirmou que as características funcionais e histológicas induzidas pelo tempo de isquemia na LRA determinam as respostas às manobras farmacológicas de prevenção. A expressão HO-1 pode ser considerada um mediador de proteção renal. / Severity of ischemic/reperfusion injury syndrome determines the prognosis of acute kidney injury (AKI). Agents such as sildenafil citrate (Sil) and N-acetylcysteine (NAC) have demonstrated renoprotective effect on ischemic AKI which data is still inconclusive. This study investigated the protective action of Sil and NAC in the AKI with different time of ischemia. Adult, male, Wistar rats were divided: SHAM, Ischemia 30 min (renal pedicles clamping for 30 min), Ischemia 30 + Sil (Sil 0,25 mg/kg 60 min before renal ischemia), Ischemia 30 + NAC (NAC 150 mg/kg before and after renal ischemia), Ischemia 45 min (renal pedicles clamping for 45 min), Ischemia 45 + Sil, Ischemia 45 + NAC. Renal function (creatinine clearance and urine sodium fractional excretion - FENa); oxidative injury (urinary peroxides, thiobarbituric acid reactive substances - TBARS, nitric oxide - NO and thiols in renal tissue); expression of inducible nitric oxide synthase - iNOS and heme oxygenase-1 HO-1 (western blotting) and kidney histological analysis (fractional interstitial area - FIA and tubuleinterstitial injury) were evaluated. Sil and NAC treatment in 30 min renal ischemia induced increase in renal function, decrease in the rate of oxidation and NO levels, iNOS absence and HO-1 expression. In the Ischemia 45 groups, Sil it maintained renal function, however demonstrated tubular and oxidative protection; NAC produced no renoprotective effect on any of the parameters evaluated. Histology studies showed that, only Sil induced reduction in FIA and tubuleinterstitial injury at 30 and 45 min ischemic time. The study confirmed that the functional and histological characteristics induced by time of ischemia determine the renoprotective pharmacological agent action in the AKI. HO-1 expression can be a renal protection mediator.