The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

No description available.

L’apoptose des cellules endothéliales (CE) représente un évènement initial dans le développement de plusieurs pathologies fibrotiques telles que le rejet chronique d’allogreffe et la sclérose systémique. Nous avons démontré que les médiateurs issus des CE apoptotiques entraîne la différenciation myofibroblastique et la résistance à l’apoptose, deux mécanismes centraux à la fibrogénèse. L’activation de PI3K (phospatidylinositol-3 kinase) caractérise ces deux mécanismes. Un fragment C-terminal du perlécan (LG3) produit par les CE apoptotiques inhibe l’apoptose des fibroblastes. Les objectifs de ce travail étaient de : 1. définir les récepteurs et la signalisation impliqués dans la réponse anti-apoptotique et 2. caractériser les médiateurs fibrogéniques responsables de la différenciation myofibroblastique. En ce qui a trait à la réponse anti-apoptotique, l’inhibition des intégrines 21 ou des kinases de la famille Src (SFK) chez les fibroblastes prévient la résistance à l’apoptose et la phosphorylation d’Akt normalement induites par le milieu conditionné par des CE apoptotiques (SSC) ou le LG3. Ces résultats suggèrent que le LG3 produit par les CE apoptotiques initie un état de résistance à l’apoptose chez les fibroblastes par des voies α2β1integrines/SFK/PI3K dépendantes. Le LG3 n’induit cependant pas la différenciation myofibroblastique. Nous avons donc caractérisé le milieu SSC de façon à identifier les médiateurs responsables de la différenciation myofibroblastique. Les milieux conditionnés par des CE apoptotiques et non-apoptotiques (respectivement SSC et SSC-ZVAD) ont été analysés comparativement par chromatographie liquide bi-dimensionnelle, immunobuvardage et spectrométrie de masse. Le connective tissue growth factor (CTGF) est le seul facteur fibrogénique connu augmenté dans le milieu SSC. L’inhibition de la caspase-3 chez les CE prévient la relâche de CTGF. Au niveau du fibroblaste, l’inhibition de SFK ou de Pyk2 (proline-rich tyrosine kinase-2) prévient la différenciation myofibroblastique induite par le SSC ou le CTGF in vitro. L’anticorps neutralisant contre le TGF- (Transforming growth factor beta) n’est pas en mesure de bloquer la différenciation myofibroblastique induite par le SSC ou le CTGF. Des injections quotidiennes sous-cutanées de SSC chez la souris C3H pour 3 semaines entraîne une augmentation de l’épaisseur de la peau et des niveaux protéiques d’SMA, de vimentine et de collagène I. Cette réponse fibrogénique est réduite chez les souris qui ont reçu le SSC-ZVAD ou le SSC immunodéplété de son CTGF. Ces résultats apportent de nouvelles issues mécanistiques au niveau de la réponse fibrogénique activée par la mort des CE. L’activation des caspases chez les CE apoptotiques entraîne la production de LG3 et de CTGF qui, à leur tour, activent des voies de signalisation pro-fibrotiques SFK/PI3K dépendantes chez les fibroblastes, et ce indépendamment du TGF-. / Apoptosis of endothelial cells (EC) is an early event in various fibrotic diseases including chronic allograft vasculopathy and systemic sclerosis. We showed previously that mediators released by apoptotic EC activate myofibroblast differentiation and resistance to apoptosis, two mechanisms pivotal to fibrogenesis. PI3K (phospatidylinositol-3 kinase) activation was found to be central to these two mechanisms. A C-terminal fragment of perlecan (LG3) produced by apoptotic EC was found to inhibit apoptosis of fibroblasts. The aims of the present project were : 1. to define the receptors and pathways implicated in this anti-apoptotic response and 2. to characterize the fibrogenic mediators implicated in myofibroblast differentiation. Concerning the anti-apoptotic response, the inhibition of 21 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or LG3 prevented resistance to apoptosis and was associated with decreased levels of Akt phosphorylation. Neutralizing Src family kinases (SFK) activity in fibroblasts produced the same effects. These results suggest that LG3 produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an α2β1integrin/SFK/PI3K dependent pathway. LG3 did not induce myofibroblast differentiation. We went on to identify which mediators present in SSC are implicated in myofibroblast differentiation. Media conditioned by apoptotic and non-apoptotic EC (respectively SSC and SSC-ZVAD) were analyzed comparatively by 2-dimension liquid chromatography, western blotting and mass spectrometry. Connective tissue growth factor (CTGF) was the only known fibrogenic factor increased in SSC. Caspase-3 silencing of EC demonstrated that CTGF is released by apoptotic EC downstream of caspase-3 activation. In fibroblasts, blocking the activation of SFK or silencing the proline-rich tyrosine kinase 2 (Pyk2) blocked myofibroblast differentiation triggered by either SSC or recombinant CTGF in vitro. Exposure to a pan-transforming growth factor (TGF-β) neutralizing antibody failed to attenuate myofibroblast differentiation in fibroblasts exposed to either SSC or CTGF. Subcutaneous injection of mouse SSC to C3H mice daily for three weeks led to increased skin thickness, increased protein levels of αSMA, vimentin and collagen I. This fibrogenic response was blunted in mice injected with either SSC-ZVAD or SSC immunodepleted of CTGF. These results bring new mechanistic insights into the fibrogenic pathways activated by EC death. Caspase activation in apoptotic EC triggers the production of LG3 and CTGF which in turn activate SFK/PI3K dependant pathways in fibroblasts thus activating a TGF-β-independent fibrogenic response.

Suite à un infarctus du myocarde, la formation d’une cicatrice, nommée fibrose de réparation, représente un processus adaptatif et essentiel empêchant la rupture du myocarde. La cicatrice est constituée de myofibroblastes, de cellules vasculaires, de fibres sympathiques ainsi que de cellules souches neuronales cardiaques exprimant la nestine. Une perturbation au niveau de ces constituants cellulaires résulte en une formation maladaptative de la cicatrice et éventuellement, une diminution de la fonction cardiaque. La compréhension des événements cellulaires ainsi que les mécanismes sous-jacents participant à cette fibrose est alors d’une importance primordiale. Cette thèse est axée sur l’identification du rôle du système sympathique et des cellules souches neuronales cardiaques exprimant la nestine dans la formation de la cicatrice ainsi que leur interaction potentielle. Nos travaux examinent l’hypothèse que les cellules souches neuronales exprimant la nestine sont endogènes au cœur et que suite à un dommage ischémique, elles contribuent à la réponse angiogénique et à la réinnervation sympathique du tissu lésé. Les cellules souches neuronales exprimant la nestine sont retrouvées dans les cœurs de différentes espèces incluant le cœur infarci humain. Elles sont résidentes dans le cœur, proviennent de la crête neurale lors du développement et sont intercalées entre les cardiomyocytes n’exprimant pas la nestine. Suite à leur isolation de cœurs infarcis de rats, les cellules souches neuronales cardiaques prolifèrent sous forme de neurosphères et, dans des conditions appropriées in vitro, se différencient en neurones exprimant le neurofilament-M. Suite à un infarctus du myocarde, les niveaux de l’ARNm de nestine sont significativement augmentés au niveau de la région infarcie et non-infarcie. Nos résultats suggèrent que cette augmentation de l’expression de nestine dans la cicatrice reflète en partie la migration des cellules souches neuronales cardiaques exprimant la nestine de la région non-infarcie vers la région infarcie. Lors de la fibrose de réparation, ces cellules représentent un substrat cellulaire pour la formation de nouveaux vaisseaux et contribuent aussi à la croissance des fibres sympathiques dans la région infarcie. Finalement, nous démontrons que la formation de la cicatrice est associée à une innervation sympathique de la région infarcie et péri-infarcie. De plus, les fibres sympathiques présentes dans la région infarcie sont observées à proximité de vaisseaux de petits calibres. Ces données suggèrent indirectement que l’innervation de la cicatrice par les fibres sympathiques peut jouer un rôle dans la réponse angiogénique suite à un infarctus du myocarde. Suite à l’administration du corticostéroïde dexaméthasone, nous détectons un amincissement de la cicatrice, associé à une réduction significative des fibres sympathiques exprimant le neurofilament-M dans la région infarcie et péri-infarcie. La diminution de la densité de ces fibres par le dexaméthasone peut être reliée à une diminution de la prolifération des myofibroblastes et de la production de l’ARNm du facteur neurotrophique nerve growth factor. / GENERAL ABSTRACT Following myocardial infarction, scar formation represents an adaptive response required to heal the damaged myocardium and prevent cardiac rupture. Infarct healing requires the coordinated action of scar myofibroblasts, angiogenic cells, sympathetic fibres and nestin positive cardiac neural stem cells. A perturbation of one or more of the aforementioned events could lead to inadequate scar healing and further worsening of ventricular function. A better understanding of the cellular events and the underlying mechanisms involved in scar formation is of a primordial importance. The focus of the following studies consists of elucidating the role of the sympathetic system and cardiac neural stem cells during scar healing and their potential interaction. We tested the hypothesis that nestin positive neural stem cells are endogenous to the heart, contribute to angiogenesis and sympathetic innervation of the infarcted myocardium following ischemic injury. Nestin positive cardiac neural stem cells are found in a number of species including the infarcted human heart. Nestin positive cardiac neural stem cells represent a resident population in the heart, are derived from the neural crest and detected intercalated between nestin negative cardiac myocytes. Following their isolation from the infarcted rat heart, neural stem cells proliferate as a neurosphere and under appropriate in vitro conditions differentiate to a neurofilament-M immunoreactive neuron. Following myocardial infarction, nestin mRNA levels are significantly elevated in the viable left ventricle and infarct region. Our data further suggests that the increased expression of nestin in the infarct region reflects in part the migration of these neural stem cells from the viable myocardium. During cardiac wound healing, neural stem cells may represent a novel substrate for de novo blood vessel formation and further contribute to sympathetic fibre growth and innervation of the infarct region. Lastly, we demonstrate that scar formation and healing is associated with sympathetic fibre sprouting of the peri-infarct/infarct region. In addition, sympathetic fibres in the infarct region were detected in close proximity to small calibre blood vessels. These latter data indirectly suggest that innervating sympathetic fibres may play a role in angiogenesis during cardiac wound healing. Following the administration of the corticosteroid dexamethasone inadequate scar healing was observed and associated with a significant reduction of neurofilament-M immunoreactive fibres in the peri-infarct/infarct region. The loss of sympathetic fibre sprouting in the scar may be related to a dexamethasone-mediated suppression of myofibroblast growth and the concomitant reduction of nerve growth factor mRNA expression.

L’apoptose des cellules endothéliales (CE) représente un évènement initial dans le développement de plusieurs pathologies fibrotiques telles que le rejet chronique d’allogreffe et la sclérose systémique. Nous avons démontré que les médiateurs issus des CE apoptotiques entraîne la différenciation myofibroblastique et la résistance à l’apoptose, deux mécanismes centraux à la fibrogénèse. L’activation de PI3K (phospatidylinositol-3 kinase) caractérise ces deux mécanismes. Un fragment C-terminal du perlécan (LG3) produit par les CE apoptotiques inhibe l’apoptose des fibroblastes. Les objectifs de ce travail étaient de : 1. définir les récepteurs et la signalisation impliqués dans la réponse anti-apoptotique et 2. caractériser les médiateurs fibrogéniques responsables de la différenciation myofibroblastique. En ce qui a trait à la réponse anti-apoptotique, l’inhibition des intégrines 21 ou des kinases de la famille Src (SFK) chez les fibroblastes prévient la résistance à l’apoptose et la phosphorylation d’Akt normalement induites par le milieu conditionné par des CE apoptotiques (SSC) ou le LG3. Ces résultats suggèrent que le LG3 produit par les CE apoptotiques initie un état de résistance à l’apoptose chez les fibroblastes par des voies α2β1integrines/SFK/PI3K dépendantes. Le LG3 n’induit cependant pas la différenciation myofibroblastique. Nous avons donc caractérisé le milieu SSC de façon à identifier les médiateurs responsables de la différenciation myofibroblastique. Les milieux conditionnés par des CE apoptotiques et non-apoptotiques (respectivement SSC et SSC-ZVAD) ont été analysés comparativement par chromatographie liquide bi-dimensionnelle, immunobuvardage et spectrométrie de masse. Le connective tissue growth factor (CTGF) est le seul facteur fibrogénique connu augmenté dans le milieu SSC. L’inhibition de la caspase-3 chez les CE prévient la relâche de CTGF. Au niveau du fibroblaste, l’inhibition de SFK ou de Pyk2 (proline-rich tyrosine kinase-2) prévient la différenciation myofibroblastique induite par le SSC ou le CTGF in vitro. L’anticorps neutralisant contre le TGF- (Transforming growth factor beta) n’est pas en mesure de bloquer la différenciation myofibroblastique induite par le SSC ou le CTGF. Des injections quotidiennes sous-cutanées de SSC chez la souris C3H pour 3 semaines entraîne une augmentation de l’épaisseur de la peau et des niveaux protéiques d’SMA, de vimentine et de collagène I. Cette réponse fibrogénique est réduite chez les souris qui ont reçu le SSC-ZVAD ou le SSC immunodéplété de son CTGF. Ces résultats apportent de nouvelles issues mécanistiques au niveau de la réponse fibrogénique activée par la mort des CE. L’activation des caspases chez les CE apoptotiques entraîne la production de LG3 et de CTGF qui, à leur tour, activent des voies de signalisation pro-fibrotiques SFK/PI3K dépendantes chez les fibroblastes, et ce indépendamment du TGF-. / Apoptosis of endothelial cells (EC) is an early event in various fibrotic diseases including chronic allograft vasculopathy and systemic sclerosis. We showed previously that mediators released by apoptotic EC activate myofibroblast differentiation and resistance to apoptosis, two mechanisms pivotal to fibrogenesis. PI3K (phospatidylinositol-3 kinase) activation was found to be central to these two mechanisms. A C-terminal fragment of perlecan (LG3) produced by apoptotic EC was found to inhibit apoptosis of fibroblasts. The aims of the present project were : 1. to define the receptors and pathways implicated in this anti-apoptotic response and 2. to characterize the fibrogenic mediators implicated in myofibroblast differentiation. Concerning the anti-apoptotic response, the inhibition of 21 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or LG3 prevented resistance to apoptosis and was associated with decreased levels of Akt phosphorylation. Neutralizing Src family kinases (SFK) activity in fibroblasts produced the same effects. These results suggest that LG3 produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an α2β1integrin/SFK/PI3K dependent pathway. LG3 did not induce myofibroblast differentiation. We went on to identify which mediators present in SSC are implicated in myofibroblast differentiation. Media conditioned by apoptotic and non-apoptotic EC (respectively SSC and SSC-ZVAD) were analyzed comparatively by 2-dimension liquid chromatography, western blotting and mass spectrometry. Connective tissue growth factor (CTGF) was the only known fibrogenic factor increased in SSC. Caspase-3 silencing of EC demonstrated that CTGF is released by apoptotic EC downstream of caspase-3 activation. In fibroblasts, blocking the activation of SFK or silencing the proline-rich tyrosine kinase 2 (Pyk2) blocked myofibroblast differentiation triggered by either SSC or recombinant CTGF in vitro. Exposure to a pan-transforming growth factor (TGF-β) neutralizing antibody failed to attenuate myofibroblast differentiation in fibroblasts exposed to either SSC or CTGF. Subcutaneous injection of mouse SSC to C3H mice daily for three weeks led to increased skin thickness, increased protein levels of αSMA, vimentin and collagen I. This fibrogenic response was blunted in mice injected with either SSC-ZVAD or SSC immunodepleted of CTGF. These results bring new mechanistic insights into the fibrogenic pathways activated by EC death. Caspase activation in apoptotic EC triggers the production of LG3 and CTGF which in turn activate SFK/PI3K dependant pathways in fibroblasts thus activating a TGF-β-independent fibrogenic response.

RÉSUMÉ GÉNÉRAL Suite à un infarctus du myocarde, la formation d’une cicatrice, nommée fibrose de réparation, représente un processus adaptatif et essentiel empêchant la rupture du myocarde. La cicatrice est constituée de myofibroblastes, de cellules vasculaires, de fibres sympathiques ainsi que de cellules souches neuronales cardiaques exprimant la nestine. Une perturbation au niveau de ces constituants cellulaires résulte en une formation maladaptative de la cicatrice et éventuellement, une diminution de la fonction cardiaque. La compréhension des événements cellulaires ainsi que les mécanismes sous-jacents participant à cette fibrose est alors d’une importance primordiale. Cette thèse est axée sur l’identification du rôle du système sympathique et des cellules souches neuronales cardiaques exprimant la nestine dans la formation de la cicatrice ainsi que leur interaction potentielle. Nos travaux examinent l’hypothèse que les cellules souches neuronales exprimant la nestine sont endogènes au cœur et que suite à un dommage ischémique, elles contribuent à la réponse angiogénique et à la réinnervation sympathique du tissu lésé. Les cellules souches neuronales exprimant la nestine sont retrouvées dans les cœurs de différentes espèces incluant le cœur infarci humain. Elles sont résidentes dans le cœur, proviennent de la crête neurale lors du développement et sont intercalées entre les cardiomyocytes n’exprimant pas la nestine. Suite à leur isolation de cœurs infarcis de rats, les cellules souches neuronales cardiaques prolifèrent sous forme de neurosphères et, dans des conditions appropriées in vitro, se différencient en neurones exprimant le neurofilament-M. Suite à un infarctus du myocarde, les niveaux de l’ARNm de nestine sont significativement augmentés au niveau de la région infarcie et non-infarcie. Nos résultats suggèrent que cette augmentation de l’expression de nestine dans la cicatrice reflète en partie la migration des cellules souches neuronales cardiaques exprimant la nestine de la région non-infarcie vers la région infarcie. Lors de la fibrose de réparation, ces cellules représentent un substrat cellulaire pour la formation de nouveaux vaisseaux et contribuent aussi à la croissance des fibres sympathiques dans la région infarcie. Finalement, nous démontrons que la formation de la cicatrice est associée à une innervation sympathique de la région infarcie et péri-infarcie. De plus, les fibres sympathiques présentes dans la région infarcie sont observées à proximité de vaisseaux de petits calibres. Ces données suggèrent indirectement que l’innervation de la cicatrice par les fibres sympathiques peut jouer un rôle dans la réponse angiogénique suite à un infarctus du myocarde. Suite à l’administration du corticostéroïde dexaméthasone, nous détectons un amincissement de la cicatrice, associé à une réduction significative des fibres sympathiques exprimant le neurofilament-M dans la région infarcie et péri-infarcie. La diminution de la densité de ces fibres par le dexaméthasone peut être reliée à une diminution de la prolifération des myofibroblastes et de la production de l’ARNm du facteur neurotrophique nerve growth factor. / GENERAL ABSTRACT Following myocardial infarction, scar formation represents an adaptive response required to heal the damaged myocardium and prevent cardiac rupture. Infarct healing requires the coordinated action of scar myofibroblasts, angiogenic cells, sympathetic fibres and nestin positive cardiac neural stem cells. A perturbation of one or more of the aforementioned events could lead to inadequate scar healing and further worsening of ventricular function. A better understanding of the cellular events and the underlying mechanisms involved in scar formation is of a primordial importance. The focus of the following studies consists of elucidating the role of the sympathetic system and cardiac neural stem cells during scar healing and their potential interaction. We tested the hypothesis that nestin positive neural stem cells are endogenous to the heart, contribute to angiogenesis and sympathetic innervation of the infarcted myocardium following ischemic injury. Nestin positive cardiac neural stem cells are found in a number of species including the infarcted human heart. Nestin positive cardiac neural stem cells represent a resident population in the heart, are derived from the neural crest and detected intercalated between nestin negative cardiac myocytes. Following their isolation from the infarcted rat heart, neural stem cells proliferate as a neurosphere and under appropriate in vitro conditions differentiate to a neurofilament-M immunoreactive neuron. Following myocardial infarction, nestin mRNA levels are significantly elevated in the viable left ventricle and infarct region. Our data further suggests that the increased expression of nestin in the infarct region reflects in part the migration of these neural stem cells from the viable myocardium. During cardiac wound healing, neural stem cells may represent a novel substrate for de novo blood vessel formation and further contribute to sympathetic fibre growth and innervation of the infarct region. Lastly, we demonstrate that scar formation and healing is associated with sympathetic fibre sprouting of the peri-infarct/infarct region. In addition, sympathetic fibres in the infarct region were detected in close proximity to small calibre blood vessels. These latter data indirectly suggest that innervating sympathetic fibres may play a role in angiogenesis during cardiac wound healing. Following the administration of the corticosteroid dexamethasone inadequate scar healing was observed and associated with a significant reduction of neurofilament-M immunoreactive fibres in the peri-infarct/infarct region. The loss of sympathetic fibre sprouting in the scar may be related to a dexamethasone-mediated suppression of myofibroblast growth and the concomitant reduction of nerve growth factor mRNA expression.

No description available.

Durante o processo de reparação tecidual, o fator de transformação de crescimento <font face=\"symbol\">b1 (TGF-<font face=\"symbol\">b1) apresenta um importante papel na regulação da expressão da <font face=\"symbol\">a-actina de músculo liso (<font face=\"symbol\">a-AML) e portanto, na diferenciação miofibroblástica. Como os fibroblastos pulpares apresentam características peculiares, com a expressão de proteínas específicas que os diferem de fibroblastos de outros tecidos conjuntivos, o presente estudo avaliou in vitro se o TGF-<font face=\"symbol\">b1 aumenta a expressão de <font face=\"symbol\">a-AML em fibroblastos pulpares humanos comparando-os com fibroblastos de gengiva. Para tal, diferentes doses de TGF-<font face=\"symbol\">b1 (5 à 10 ng/ml) foram adicionadas às culturas de células, sendo a expressão da <font face=\"symbol\">a-AML analisada por imunofluorescência e western-blotting. Ambos os tipos celulares imunoexpressaram <font face=\"symbol\">a-AML mesmo sem o tratamento com o TGF-<font face=\"symbol\">b1, estando aumentada consideravelmente, quando o TGF-<font face=\"symbol\">b1 foi adicionado às culturas. Os resultados do presente estudo demonstraram que o TGF-<font face=\"symbol\">b1 induz a expressão de <font face=\"symbol\">a-AML, sugerindo a indução do fenótipo miofibroblástico em fibroblastos pulpares. / Transforming growth factor-beta 1 (TGF-<font face=\"symbol\">b1) has been related to induce the expression of <font face=\"symbol\">a-smooth muscle actin (<font face=\"symbol\">a-SMA) in fibroblasts during repair. Since pulpal fibroblasts seem to be somewhat different from other fibroblasts, the present study investigated in vitro whether TGF-<font face=\"symbol\">b1 enhances the expression of <font face=\"symbol\">a-SMA in human pulpal fibroblasts. TGF-<font face=\"symbol\">b1 was added in doses between 5-10 ng/ml to cultures of both dental pulp and gingiva human fibroblasts. The expression of <font face=\"symbol\">a-SMA was analyzed by immunofluorescence and western-blotting. Both cell types were immunoreactive for <font face=\"symbol\">a-SMA even without TGF-<font face=\"symbol\">b1. When TGF-<font face=\"symbol\">b1 was added to cell cultures, the expression of <font face=\"symbol\">a-SMA increased dramatically in pulpal fibroblasts, independent of the concentration used. It was confirmed by the western blot analysis. The present findings showed that TGF-<font face=\"symbol\">b1 up-regulated the expression of <font face=\"symbol\">a-SMA thus inducing pulpal fibroblasts to acquire the myofibroblast phenotype.

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.

Made available in DSpace on 2014-12-17T15:32:29Z (GMT). No. of bitstreams: 1 CassianoFWN_TESE.pdf: 3914655 bytes, checksum: c6fb9bd86bba433eb6a5a047a9337861 (MD5) Previous issue date: 2010-09-23 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The odontogenic keratocysts are distinguished from other odontogenic cystic lesions by their potentially aggressive clinical behavior and association, in some cases, with Gorlin syndrome. Studies have suggested that syndrome keratocysts, in comparison with sporadic lesions, have higher growth and infiltration capacity and higher recurrence tendency. The aim of this study was to analyze, by means of immunohistochemistry, the expressions of receptor activator of nuclear factor ?B ligand (RANKL) and osteoprotegerin (OPG), the angiogenic index (CD34) and the presence of myofibroblasts (?-SMA) in primary and recurrent sporadic keratocysts and in keratocysts associated with Gorlin syndrome. The sample was composed by 30 sporadic keratocysts (22 primary and 8 recurrent) and 22 syndrome keratocysts. In the epithelium and in the fibrous capsule of the lesions, the immunoexpression of RANKL and OPG was evaluated by determination of the percentage of positive cells, according to the following scores: 0 (less than 10% of positive cells), 1 (11% - 50% of positive cells), 2 (51% - 75% of positive cells) and 3 (more than 76% of positive cells). In addition, cases were classified according to the RANKL score/ OPG score ratio, as follows: RANKL > OPG, RANKL < OPG, and RANKL = OPG. The angiogenic index was analyzed by counting the microvessels immunoreactive to anti-CD34 antibody in 5 fields (200?). The analysis of myofibroblasts was performed by counting the cells immunoreactive to anti-?-SMA antibody in 10 fields (400?). The analysis of the expressions of RANKL and OPG in the epithelial lining and in the fibrous capsule did not reveal significant differences between groups (p > 0.05). Regarding the RANKL/ OPG ratio in the epithelial lining, most sporadic primary (54.5%) and syndrome lesions (59.1%) showed RANKL < OPG ratio and RANKL = OPG ratio, respectively (p > 0.05). With respect to the RANKL/ OPG ratio in the fibrous capsule, the majority of sporadic primary (81.8%) and sporadic recurrent lesions (75.0%) and most syndrome lesions (45.5%) showed RANKL = OPG ratio (p > 0.05). The mean number of microvessels was 69.2 in sporadic primary lesions, 67.6 in recurrent lesions, and 71.6 in syndrome lesions, with no significant differences between groups (p > 0.05). The mean number of myofibroblasts was 34.4 in sporadic primary lesions, 29.3 in recurrent lesions, and 33.7 in syndrome lesions, with no significant differences between groups (p > 0.05). In conclusion, the results of the present study suggest that the differences in the biological behavior between sporadic keratocysts and keratocysts associated with Gorlin syndrome may not be related to the expressions of RANKL and OPG, the RANKL/ OPG ratio, the angiogenic index or the number of myofibroblasts in these lesions / Os ceratocistos odontog?nicos se destacam em rela??o a outras les?es c?sticas odontog?nicas pelo comportamento cl?nico potencialmente agressivo e por se apresentarem associados, em alguns casos, ? s?ndrome de Gorlin. Estudos t?m sugerido que os ceratocistos sindr?micos, em compara??o ?s les?es isoladas, possuem maior capacidade de crescimento e infiltra??o e maior tend?ncia ? recorr?ncia. O objetivo do presente trabalho consistiu em analisar, por meio de imuno-histoqu?mica, as express?es do ligante do receptor ativador do fator nuclear ?B (RANKL) e da osteoprotegerina (OPG), o ?ndice angiog?nico (CD34) e a presen?a de miofibroblastos (?-SMA), em ceratocistos isolados prim?rios e recorrentes e ceratocistos associados ? s?ndrome de Gorlin. A amostra foi composta por 30 ceratocistos isolados (22 prim?rios e 8 recorrentes) e 22 ceratocistos sindr?micos. A express?o de RANKL e OPG foi avaliada no epit?lio e na c?psula fibrosa das les?es, estabelecendo-se o percentual de c?lulas imunopositivas, de acordo com os escores: 0 (? 10% das c?lulas positivas), 1 (11% - 50% das c?lulas positivas), 2 (51% - 75% das c?lulas positivas) e 3 (? 76% das c?lulas positivas). Al?m disso, os casos foram categorizados, segundo a propor??o RANKL/ OPG, em: RANKL > OPG, RANKL < OPG e RANKL = OPG. O ?ndice angiog?nico foi analisado por meio da contagem dos microvasos imunomarcados pelo anticorpo anti-CD34, em 5 campos (200?). Para a avalia??o dos miofibroblastos, foram quantificadas as c?lulas imunorreativas ao anticorpo anti-?-SMA, em 10 campos (400?). A an?lise das express?es de RANKL e OPG, no revestimento epitelial e na c?psula fibrosa, n?o revelou diferen?as significativas entre os grupos (p > 0,05). Em rela??o ? propor??o RANKL/ OPG no revestimento epitelial, grande parte das les?es isoladas prim?rias (54,5%) e sindr?micas (59,1%) exibiu propor??o RANKL < OPG e propor??o RANKL = OPG, respectivamente (p > 0,05). Em rela??o ? propor??o RANKL/ OPG na c?psula fibrosa, a maioria das les?es isoladas prim?rias (81,8%) e isoladas recorrentes (75,0%) e grande parte das les?es associadas ? s?ndrome de Gorlin (45,5%) revelaram propor??o RANKL = OPG (p > 0,05). O n?mero m?dio de microvasos foi de 69,2 nas les?es isoladas prim?rias, 67,6 nas les?es recorrentes e 71,6 nas les?es sindr?micas, sem diferen?as significativas entre os grupos (p > 0,05). A an?lise dos miofibroblastos revelou valores m?dios de 34,4 nas les?es isoladas prim?rias, 29,3 nas les?es recorrentes e 33,7 nas les?es sindr?micas, sem diferen?as significativas entre os grupos (p > 0,05). Em conclus?o, os resultados do presente estudo sugerem que as diferen?as no comportamento biol?gico entre ceratocistos isolados e associados ? s?ndrome de Gorlin podem n?o estar relacionadas ?s express?es de RANKL e OPG, ? propor??o RANKL/ OPG, ao ?ndice angiog?nico ou ? quantidade de miofibroblastos presentes nas les?es