Personne :
Proulx, Maryse

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Proulx
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Maryse
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Département de biochimie et de microbiologie, Faculté des sciences et de génie, Université Laval
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Voici les éléments 1 - 7 sur 7
  • Publication
    Accès libre
    Étude des effets de l'expression forcée de PAX5 sur les cellules issues de myélome multiple
    (2009) Proulx, Maryse; Jung, Daniel; Darveau, André
    Le myélome multiple est un cancer incurable caractérisé par une accumulation de plasmocytes malins dans la moelle osseuse. Récemment, nous avons découvert que l'expression forcée du facteur de transcription PAX5 (paired box protein 5) dans des lignées cellulaires humaines de myélome multiple induisait une forte diminution de la viabilité cellulaire. Des viabilités de près de 50% ont été observées dans les lignées cellulaires de myélome multiple RPMI-8226 et U266 aux jours quatre et sept respectivement après l'infection de ces cellules par un vecteur adenoviral codant pour PAX5. Nous avons constaté que la mortalité était due à l'apoptose induite par une baisse de MCL-1 (myeloid cell leukemia-1). La spécificité de cet effet a été démontrée grâce à des lignées cellulaires de leucémie à plasmocytes, lignées cellulaires dont la viabilité ne variait pas suite à l'expression forcée de PAX5, suggérant ainsi une nouvelle avenue de traitement du myélome multiple.
  • Publication
    Accès libre
    Creating capillary networks within human engineered tissues: impact of adipocytes and their secretory products
    (Elsevier, 2014-09-30) Vincent, Caroline; Proulx, Maryse; Aubin, Kim; Fradette, Julie; Mayrand, Dominique
    The development of tissue-engineered substitutes of substantial volume is closely associated with the need to ensure rapid vascularization upon grafting. Strategies promoting angiogenesis include the in vitro formation of capillary-like networks within engineered substitutes. We generated both connective and adipose tissues based on a cell sheet technology using human adipose-derived stromal cells. This study evaluates the morphology and extent of the capillary networks that developed upon seeding of human microvascular endothelial cells during tissue production. We posited that adipocyte presence/secretory products could modulate the resulting capillary network when compared to connective substitutes. Analyses including confocal imaging of CD31-labeled capillary-like networks indicated slight differences in their morphological appearance. However, the total volume occupied by the networks as well as the frequency distribution of the structure’s volumes were similar between connective and adipose tissues. The average diameter of the capillary structures tended to be 20% higher in reconstructed adipose tissues. Quantification of pro-angiogenic molecules in conditioned media showed greater amounts of leptin (15×), angiopoietin-1 (3.4×) and HGF (1.7×) secreted from adipose than connective tissues at the time of endothelial cell seeding. However, this difference was attenuated during the following coculture period in endothelial cell-containing media, correlating with the minor differences noted between the networks. Taken together, we developed a protocol allowing reconstruction of both connective and adipose tissues featuring well-developed capillary networks in vitro. We performed a detailed characterization of the network architecture within engineered tissues that is relevant for graft assessment before implantation as well as for in vitro screening of angiogenic modulators using three-dimensional models.
  • Publication
    Accès libre
    Characterization of in vitro engineered human adipose tissues : relevant adipokine secretion and impact of TNF-α
    (Public Library of Science, 2015-09-14) Roy, Alphonse; Proulx, Maryse; Côté, Jean-François; Safoine, Meryem; Aubin, Kim; Audet-Casgrain, Marie-Alice; Fradette, Julie; Têtu, Félix-André
    Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells.
  • Publication
    Accès libre
    Magnetic resonance imaging of human tissue-engineered adipose substitutes
    (Mary Ann Liebert, Inc, 2015-02-23) Audet, Pierre; Proulx, Maryse; Auger, Michèle; Fortin, Marc-André; Aubin, Kim; Lagueux, Jean; Fradette, Julie
    Adipose tissue (AT) substitutes are being developed to answer the strong demand in reconstructive surgery. To facilitate the validation of their functional performance in vivo, and to avoid resorting to excessive number of animals, it is crucial at this stage to develop biomedical imaging methodologies, enabling the follow-up of reconstructed AT substitutes. Until now, biomedical imaging of AT substitutes has scarcely been reported in the literature. Therefore, the optimal parameters enabling good resolution, appropriate contrast, and graft delineation, as well as blood perfusion validation, must be studied and reported. In this study, human adipose substitutes produced from adipose-derived stem/stromal cells using the self-assembly approach of tissue engineering were implanted into athymic mice. The fate of the reconstructed AT substitutes implanted in vivo was successfully followed by magnetic resonance imaging (MRI), which is the imaging modality of choice for visualizing soft ATs. T1-weighted images allowed clear delineation of the grafts, followed by volume integration. The magnetic resonance (MR) signal of reconstructed AT was studied in vitro by proton nuclear magnetic resonance (1H-NMR). This confirmed the presence of a strong triglyceride peak of short longitudinal proton relaxation time (T1) values (200±53 ms) in reconstructed AT substitutes (total T1=813±76 ms), which establishes a clear signal difference between adjacent muscle, connective tissue, and native fat (total T1 ∼300 ms). Graft volume retention was followed up to 6 weeks after implantation, revealing a gradual resorption rate averaging at 44% of initial substitute's volume. In addition, vascular perfusion measured by dynamic contrast-enhanced-MRI confirmed the graft's vascularization postimplantation (14 and 21 days after grafting). Histological analysis of the grafted tissues revealed the persistence of numerous adipocytes without evidence of cysts or tissue necrosis. This study describes the in vivo grafting of human adipose substitutes devoid of exogenous matrix components, and for the first time, the optimal parameters necessary to achieve efficient MRI visualization of grafted tissue-engineered adipose substitutes.
  • Publication
    Accès libre
    Tissus adipeux humains reconstruits par génie tissulaire : applications in vitro et in vivo
    (2018) Proulx, Maryse; Fradette, Julie
    Le tissu adipeux blanc est l’un des tissus les plus abondants du corps humain et il constitue sa plus grande réserve énergétique. Il est également reconnu en tant qu’organe sécrétoire et endocrine pouvant agir à distance et influencer plusieurs processus biologiques en plus d’être une source de cellules stromales/souches mésenchymateuses multipotentes. De nombreux patients souffrent de déficits en tissus adipeux à la suite d’une résection de tumeur, d’un accident, de brûlures graves, de maladies congénitales, etc. Le génie tissulaire représente une alternative prometteuse aux approches traditionnelles pour répondre à la forte demande en substituts adipeux et combler les déficits en chirurgie de reconstruction. Afin de déterminer la performance in vivo de substituts adipeux reconstruits par génie tissulaire et d’optimiser les études précliniques, il est crucial de développer des méthodes d’imagerie biomédicale permettant la délinéation des greffons ainsi que l’analyse de leur volume et de leur perfusion. Pour ce faire, des tissus adipeux humains reconstruits (hrATs, Human reconstructed adipose tissues) possédant des adipocytes matures et fonctionnels ainsi qu’une matrice extracellulaire naturelle ont été produits par la technique d’auto-assemblage à partir de cellules stromales/souches du tissu adipeux, puis ont été implantés chez la souris athymique. Nous avons émis l’hypothèse que l’imagerie par résonance magnétique (IRM) permettrait une analyse améliorée de la performance in vivo de hrATs en favorisant le suivi non invasif et longitudinal du volume et de la perfusion sanguine des greffons après l’implantation. En effet, l’IRM en pondération T1 a permis la délinéation des greffons et la détermination de leur volume. Le maintien du volume a été évalué jusqu’à six semaines après l’implantation, révélant une résorption graduelle atteignant 44 % du volume initial. Puis, leur perfusion a été confirmée après 14 et 21 jours. La persistance d’une grande quantité d’adipocytes et d’une riche matrice extracellulaire a également été confirmée. Ces travaux décrivent, pour la première fois, les paramètres optimaux d’IRM pour visualiser de façon efficace des substituts adipeux humains reconstruits par génie tissulaire et implantés in vivo. De plus, assurer la vascularisation rapide de substituts reconstruits par génie tissulaire lors d’une greffe permettrait de favoriser leur survie. Nous avons alors émis l’hypothèse que l’incorporation d’une composante endothéliale aux hrATs serait avantageuse pour les tissus et leur vascularisation globale. Trois jours après l’implantation chez la souris athymique, un réseau étendu de capillaires humains a été observé à l’intérieur des greffons enrichis de cellules endothéliales, puis l’anastomose de ce réseau avec celui de l’hôte a été détectée. Le réseau humain a persisté au moins 14 jours après la greffe en plus de contenir des globules rouges. L’analyse de la vascularisation globale des greffons indique un avantage potentiel de l’ajout d’une composante endothéliale. L’évaluation de l’impact de cette approche sur la vascularisation de tissus plus épais permettra le développement et l’utilisation de substituts volumineux pour combler les déficits adipeux en chirurgie de reconstruction. Enfin, l’inflammation peut être présente à la suite d’une greffe tissulaire. Il est donc important d’étudier ses impacts sur les tissus reconstruits et leurs diverses composantes. Nous avons utilisé le modèle de hrATs contenant des cellules endothéliales organisées en un réseau préformé afin de déterminer l’impact d’un environnement inflammatoire sur le réseau de capillaires dans un contexte adipeux tridimensionnel. Notre hypothèse est que la présence prolongée d’un microenvironnement inflammatoire modélisé in vitro, par l’incubation de hrATs avec le facteur de nécrose tumorale (TNF) et l’interleukine-1β (IL- 1β), modulerait leur profil de sécrétion et aurait une influence néfaste sur leur réseau de capillaires. L’analyse des milieux conditionnés a confirmé la réactivité des tissus. Une désorganisation caractérisée par une diminution de l’étendue et de la complexité du réseau a été observée après une exposition chronique de trois et six jours en présence de TNF et d’IL-1β et serait attribuable entre autres à l’apoptose. Le modèle de hrATs permettra d’élaborer des stratégies visant la préservation de l’intégrité du réseau de capillaires et de développer des substituts adipeux bien adaptés pour faire face à des conditions inflammatoires in vivo. En résumé, ces données contribuent à l’avancement du génie tissulaire adipeux grâce à l’optimisation de l’IRM pour les greffons adipeux reconstruits, mais également par le développement de hrATs possédant une performance in vivo avantageuse en plus de constituer des modèles d’études in vitro tridimensionnels pertinents.
  • Publication
    Accès libre
    Short-term post-implantation dynamics of in vitro engineered human microvascularized adipose tissues
    (Elsevier Science Publishers, 2018-10-01) Boisvert, Annie; Vincent, Caroline; Proulx, Maryse; Aubin, Kim; Trottier, Valérie; Fradette, Julie; Mayrand, Dominique
    Engineered adipose tissues are developed for their use as substitutes for tissue replacement in reconstructive surgery. To ensure a timely perfusion of the grafted substitutes, different strategies can be used such as the incorporation of an endothelial component. In this study, we engineered human adipose tissue substitutes comprising of functional adipocytes as well as a natural extracellular matrix using the self-assembly approach, without the use of exogenous scaffolding elements. Human microvascular endothelial cells (hMVECs) were incorporated during tissue production in vitro and we hypothesized that their presence would favor the early connection with the host vascular network translating into functional enhancement after implantation into nude mice in comparison to the substitutes that were not enriched in hMVECs. In vitro, no significant differences were observed between the substitutes in terms of histological aspects. After implantation, both groups presented numerous adipocytes and an abundant matrix in addition to the presence of host capillaries within the grafts. The substitutes thickness and volume were not significantly different between groups over the short-term time course of 14 days (d). For the microvascularized adipose tissues, human CD31 staining revealed a human capillary network connecting with the host microvasculature as early as 3 d after grafting. The detection of murine red blood cells within human CD31+ structures confirmed the functionality of the human capillary network. By analyzing the extent of the global vascularization achieved, a tendency towards increased total capillary network surface and volume was revealed for prevascularized tissues over 14 d. Therefore, applying this strategy on thicker reconstructed adipose tissues with rate-limiting oxygen diffusion might procure added benefits and prove useful to provide voluminous substitutes for patients suffering from adipose tissue loss or defects.
  • Publication
    Accès libre
    Impact of TNF and IL-1β on capillary networks within engineered human adipose tissues
    (Royal Society of Chemistry, 2016-04-27) Proulx, Maryse; Maux, Amandine; Safoine, Meryem; Aubin, Kim; Fradette, Julie; Mayrand, Dominique
    Inflammation is a normal phase of the wound healing process, which likely occurs following tissue transplantation. For reconstructive surgery purposes, engineered adipose tissues represent promising alternatives to autologous fat grafts. It is therefore important to study the impact of an inflammatory microenvironment on the cellular functions of the different cell types comprised within matrix-rich reconstructed tissues. In this study, human reconstructed adipose tissues (hrATs) featuring a preformed capillary network formed by microvascular endothelial cells (hMVECs) were produced from adipose-derived stem/stromal cells (ASCs) by the self-assembly approach of tissue engineering. We hypothesized that a prolonged inflammatory context, mediated by tumor necrosis factor (TNF) and interleukin-1β (IL-1β), would impact hrATs' secretory profile and mediate detrimental effects on the microvascular network in vitro. Analysis of conditioned media established tissue responsiveness through the increased secretion of monocyte chemoattractant protein-1 (up to 23 fold), interleukin-6 (up to 69 fold) and angiopoietin-1 (up to 2.7 fold) after 3 and 6 days of cytokine exposure, along with a significant reduction in adiponectin secretion. Imaging of the preformed capillary network within the hrATs revealed increased disorganization in the presence of TNF/IL-1β, featuring a less extended and less ramified network with apoptotic hMVECs in the remaining capillary structures. These results indicate that a prolonged inflammatory context can be deleterious to the capillary network featured by in vitro engineered tissues. Strategies aiming at preserving the integrity of the vascular network will help develop substitutes that are better suited to face inflammatory conditions upon grafting.