Personne :
Labbé, Raymond

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Département chirurgie, Faculté de médecine, Université Laval
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  • Publication
    In vitro construction of a human blood vessel from cultured vascular cells : a morphologic study
    (C.V. Mosby Co., 1993-03-01) Germain, Lucie; L'Heureux, Nicolas; Labbé, Raymond; Auger, François A.
    Purpose: The purpose of this study was to create a tubular vascular model exclusively made of human cells and collagen. Methods: The blood vessel equivalent was constructed with the three following human cell types: vascular smooth muscle cells, endothelial cells, and fibroblasts. A tissuelike structure was obtained from the contraction of a tubular collagen gel (human origin) by vascular smooth muscle cells, which created a media-like structure. An adventia- like tissue was added around the media-like structure by embedding fibroblasts into a collagen gel. An endothelium was established within the tubular structure after intraluminal cell seeding. Results: Cell orientation and gel contraction were followed up over time. Vascular smooth muscle cells developed a complex tridimensional network and were oriented in a circular fashion around the tube's axis. In contrast, fibroblasts were randomly oriented. A viable, homogeneous, and well-characterized endothelium was observed. These endothelial cells showed a slightly elongated structure and were oriented parallel to this vascular equivalent axis. Conclusion: An in vitro tridimensional vascular model that exhibits some phenotypic characteristics of in vivo vascular cells could be useful in the study of events that lead to atherosclerotic plaque formations.
  • Publication
    Mechanical loading modulates the differentiation state of vascular smooth muscle cells
    (Mary Ann Liebert, 2006-11-24) Bergeron, François; Grenier, Guillaume.; Germain, Lucie; Labbé, Raymond; Auger, François A.; Guignard, Rina; Baker, Kathleen; Rémy-Zolghadri, Murielle
    The cause underlying the onset of stenosis after vascular reconstruction is not well understood. In the present study, we evaluated the effect of mechanical unloading on the differentiation state of human vascular smooth muscle cells (hVSMCs) using a tissue-engineered vascular media (TEVM). hVSMCs cultured in a mechanically loaded three-dimensional environment, known as a living tissue sheet, had a higher differentiated state than cells grown on plastic. When the living tissue sheet was detached from its support, the release of the residual stress resulted in a mechanical unloading and cells within the extracellular matrix (ECM) dedifferentiated as shown by downregulation of differentiation markers. The relaxed living tissue sheet can be rolled onto a tubular mandrel to form a TEVM. The rolling procedure resulted in the reintroduction of a mechanical load leading to a cohesive compacted tissue. During this period, cells gradually redifferentiated and aligned circumferentially to the tubular support. Our results suggest that differentiation of hVSMCs can be driven by mechanical loading and may occur simultaneously in the absence of other cell types. The extrapolation of our results to the clinical context suggests the hypothesis that hVSMCs may adopt a proliferative phenotype resulting from the mechanical unloading of explanted blood vessels during vascular reconstruction. Therefore, we propose that this mechanical unloading may play an important role in the onset of vascular graft stenosis.
  • Publication
    Adventitia contribution in vascular tone : insights from adventitia-derived cells in a tissue-engineered human blood vessel
    (Federation of American Societies for Experimental Biology, 2006-04-12) Grenier, Guillaume.; Germain, Lucie; Pouliot, Stéphanie; Labbé, Raymond; Roberge, Charles; Auger, François A.; Baker, Kathleen; D’Orléans-Juste, Pedro; Rémy-Zolghadri, Murielle; Laflamme, Karina
    Whether the adventitia component of blood vessels directly participates in the regulation of vascular tone remains to be demonstrated. We have recently developed a human tissue-engineered blood vessel comprising the three tunicae of a native blood vessel using the self-assembly approach. To investigate the role of the adventitia in the modulation of vascular tone, this tissue-engineering method was used to produce three vascular constructs from cells explanted and proliferated from donor vessel tunicae 1) an adventitia + a media, or only 2) an adventitia, or 3) a media. The vasoconstriction responses of these 3 constructs to endothelin, the most potent vasopressor known up-to-date, as well as to nonselective and selective agonists and antagonists, were compared. The adventitia contracted to endothelin-1, -2, whereas the media and the media+adventitia contracted to all three endothelins. Endothelin-induced contraction of the adventitia was dependent on ETA receptors, whereas that of the media and the adventitia+media was ETA and ETB receptor-dependent. RT-PCR studies corroborated these results. SNP induced a dose-dependent relaxation of the three tissue constructs. We also demonstrated that the endothelin-converting enzyme, responsible for the formation of the active endothelin peptides, was present and functional in the adventitia. In conclusion, this is the first direct demonstration that the adventitia has the capacity to contract and relax in response to vasoactive factors. The present study suggests that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone.—Laflamme, K., Roberge, C. J., Grenier, G., Rémy-Zolghadri, M., Pouliot, S., Baker, K., Labbé, R., D’Orléans-Juste, P., Auger, F. A., Germain, L. Adventitia contribution in vascular tone: insights from adventitia-derived cells in a tissue-engineered human blood vessel. the wall of a blood vessel is composed of three tunicae: intima, media, and adventitia (1)⤻ . The innermost tunica, known as the intima, includes a single layer of endothelial cells lining the vessel lumen and the internal elastic lamina membrane. The middle tunica, termed media, is mainly composed of vascular smooth muscle cells (VSMCs) in an extracellular matrix (ECM) and corresponds to the muscular portion of the blood vessel, whereas the tunica adventitia is mainly composed of vascular fibroblasts (VFs) and ECM. It is well accepted that the media of a blood vessel is responsible for the vasomotor tone control by contracting and relaxing in response to different hormonal factors released, for example, by the endothelial cells of the intima (2)⤻ . The adventitia, on the other hand, has long been thought to mainly serve as a structural support for the media, its main contribution to vascular compliance being controlled by autonomous perivascular innervation (1)⤻ . Interestingly, recent studies suggest that the adventitia influences vascular function (3⤻ 4⤻ 5⤻ 6⤻ 7)⤻ . Nonetheless, whether the adventitia can directly participate in the regulation of vasomotor tone of blood vessels still remains to be demonstrated. The lack of appropriate technical procedures to separate the adventitia tunica from the other components of a native blood vessel (stripping) has prevented direct investigations on the possible role of that layer in the regulation of vasomotor tone. For example, the stripping method used in these procedures can result in the injury of the media tunica and does not permit us to obtain functional adventitia isolated from a native blood vessel (6)⤻ . We have recently developed, using the self-assembly technique, a human tissue-engineered blood vessel (TEBV) composed of the layers representing the three tunicae found in a native blood vessel (8)⤻ . In the present study, we took advantage of the self-assembly method to produce three independent vascular constructs from amplified VSMCs and VFs isolated from the same human saphenous vein biopsy. The first vascular construct was composed of only an adventitia (TEVA), a second vascular construct contained only a media (TEVM), and the third contained a media and an adventitia (TEVMA). These three vascular models (TEVA, TEVM, and TEVMA) were reconstructed to investigate the role of the adventitia in the modulation of vascular tone by comparing each of these vascular construct responses to endothelin, the most powerful vasopressor agent known to date (9)⤻ . Studies in humans have demonstrated the importance of endothelin in the maintenance of vascular tone (10)⤻ and blood pressure (11)⤻ . Three endogenous isoforms of endothelin have been discovered, endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) (12)⤻ . ET binds two different receptor subtypes: endothelin A (ETA) receptors, which have a higher affinity for ET-1 and ET-2 than ET-3, and endothelin B (ETB) receptors, which have equal affinity for ET-1, ET-2, and ET-3 (13)⤻ . The endothelin receptors (ETA and ETB) implicated in the observed responses to the peptide were also investigated in our three different vascular constructs. In the present study, all of the vascular constructs tested responded to endothelin, although a heterogeneity in the response was observed. Indeed, all three vascular constructs tested contracted to ET-1 and ET-2, but only TEVMA and TEVM responded to ET-3. Furthermore, endothelin-induced contraction of TEVA was found to be dependent on the presence of ETA receptors, while both ETA and ETB receptors were present and functional on TEVMA and TEVM. Finally, the three types of vascular constructs tested also had the capacity of vasodilating in response to a relaxing agent such as sodium nitroprusside (SNP). Our results show that the adventitia may play a greater role than expected in the maintenance of vascular tone and compliance.
  • Publication
    Prospective study on the treatment of lower-extremity chronic venous and mixed ulcers using tissue-engineered skin substitute made by the self-assembly approach
    (Lippincott Williams & Wilkins, 2013-09-01) Beaudoin-Cloutier, Chanel; Germain, Lucie; Larouche, Danielle; Labbé, Raymond; Rochon, Marie-Hélène; Roy, Michel A.; Genest, Hervé; Soucy, Jacques; Dubé, Nathalie; Auger, François A.; Ospina, Carlos E.; Arsenault, Frédéric; Rodrigue, Bertrand; Boa, Olivier; Moulin, Véronique
    BACKGROUND: Despite present optimal standard treatment of lower-extremity ulceration, a high incidence of recurrence and treatment failure is observed. The objective of this project was to evaluate the effect of a self-assembled skin substitute (SASS) made by tissue engineering as a temporary cutaneous dressing in the treatment of hard-to-heal chronic ulcers. PATIENTS AND METHODS: The prospective uncontrolled case study includes patients suffering from venous or mixed ulcers lasting more than 6 months and unresponsive to compression therapy, with an Ankle Brachial Index greater than 0.5. Compression therapy was combined with the weekly application of SASS, produced from the patient’s own skin cells, until healing. A weekly follow-up recorded wound size, skin aspect, pain, drainage, and percentage of wound healing. Photographs were also taken to assess ulcer evolution. RESULTS: Fourteen ulcers present on 5 patients were treated. A mean of 6.7 SASS depositions by ulcer was required for healing. Two ulcers developed a minor wound infection, which was treated with oral antibiotics; another 2 ulcers recurred, and 1 healed with a second course of treatment, whereas 1 ulcer had a small recurrence treated with local wound care. CONCLUSION: The authors’ study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy.
  • Publication
    A completely biological tissue-engineered human blood vessel
    (FASEB Journal, 1998-01-01) Germain, Lucie; L'Heureux, Nicolas; Labbé, Raymond; Auger, François A.; Pâquet, Stéphanie
    Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.
  • Publication
    Défis et perspectives de la médecine régénératrice cardiovasculaire
    (John Libbey Eurotext, 2008-01-01) Germain, Lucie; D’Orléans-Juste, Pedro; Labbé, Raymond; Auger, François A.
    Le présent chapitre sera consacré aux diverses méthodes de génie tissulaire ayant trait à la reconstruction des vaisseaux sanguins (in vitro) avec une visée clinique (in vivo). Toutefois, nous dédierons quelques lignes à l’utilisation de ces substituts vasculaires comme modèle in vitre pour des études parfois très pointues et complexes, dans les applications suivantes : physiologie, pathophysiologie, pharmacologie et toxicologie. Ainsi, un tour d’horizon non exhaustif des travaux de la reconstruction vasculaire au plan mondial s’accompagnera de notre expérience unique au Laboratoire d’Organogenèse EXpérimentale (LOEX). En effet, notre groupe est l’un des rares, sinon le seul, groupes de recherche à effectuer en parallèle des travaux en génie tissulaire tant sur les microvaisseaux (capillaires) que les artères de petit calibre (s 5 mm) [5, 6]. Ces deux aspects vasculaires du génie tissulaire répondent à deux impératifs cliniques. En premier lieu, les micro-vaisseaux permettent d’entrevoir une vascularisation préimplantatoire des organes reconstruits. Ainsi, les espoirs de survie de divers substituts seraient grandement améliorés puisqu'il s’agit là d’une des principales pierres d’achoppement de ce domaine. En second lieu, la création de vaisseaux cultivés de petits calibres répond à un besoin clinique, tels des pontages cardiaques et infrapoplités où les prothèses synthétiques sont inutilisables en raison d’une fréquence plus élevée de thrombose. Ainsi, le cahier de charge de ces substituts vasculaires (SV) obtenus par génie tissulaire est très exigeant comme démontre le Tableau I. Enfin, notons, dans un registre entièrement différent, que notre programme de recherche sur la reconstruction des valves cardiaques se poursuit actuellement [7].
  • Publication
    Tissue-Engineered Vascular Adventitia with Vasa Vasorum Improves Graft Integration and Vascularization Through Inosculation
    (2010-05-05) Guillemette, Maxime.; Perron, Cindy; Germain, Lucie; Labbé, Raymond; Auger, François A.; Gauvin, Robert
    Tissue-engineered blood vessel is one of the most promising living substitutes for coronary and peripheral artery bypass graft surgery. However, one of the main limitations in tissue engineering is vascularization of the construct before implantation. Such a vascularization could play an important role in graft perfusion and host integration of tissue-engineered vascular adventitia. Using our self-assembly approach, we developed a method to vascularize tissue-engineered blood vessel constructs by coculturing endothelial cells in a fibroblast-laden tissue sheet. After subcutaneous implantation, enhancement of graft integration within the surrounding environment was noted after 48 h and an important improvement in blood circulation of the grafted tissue at 1 week postimplantation. The distinctive branching structure of end arteries characterizing the in vivo adventitial vasa vasorum has also been observed in long-term postimplantation follow-up. After a 90-day implantation period, hybrid vessels containing human and mouse endothelial cells were still perfused. Characterization of the mechanical properties of both control and vascularized adventitia demonstrated that the ultimate tensile strength, modulus, and failure strain were in the same order of magnitude of a pig coronary artery. The addition of a vasa vasorum to the tissue-engineered adventitia did not influence the burst pressure of these constructs. Hence, the present results indicate a promising answer to the many challenges associated with the in vitro vascularization and in vivo integration of many different tissue-engineered substitutes.
  • Publication
    Polyphenols modulate calcium-independent mechanisms in human arterial tissue-engineered vascular media
    (Mosby, 2007-09-30) Germain, Lucie; Diebolt, Myriam; Labbé, Raymond; Auger, François A.; Laflamme, Karina; Andriantsitohaina, Ramaroson
    Background: In the present study, an arterial tissue-engineered vascular media (TEVM) was produced from cultured human smooth muscle cells of the umbilical artery and we took advantage of this model to evaluate the regulation of contraction and the signalling pathways of polyphenols in arteries. Methods: Cultured human smooth muscle cells of the umbilical artery were used to produce arterial TEVMs. Contraction experiments were performed to determine intracellular targets involved in the modulation of contraction by polyphenols extract from red wine, Provinols (SEPPIC Groupe Air Liquide, Paris, France). Results: Smooth muscle cells in arterial TEVM displayed a differentiated phenotype as demonstrated by the expression of alpha-smooth muscle actin, a vascular smooth muscle-specific marker, and tissue contraction in response to vasoconstrictor and vasodilator agents. Contractions caused by histamine were associated with an increase in [Ca(2+)](i) and a Ca(2+)-independent signalling pathway. The latter pathway involved mechanisms sensitive to protein kinase C, myosin light chain kinase, and Rho-associated protein kinase inhibitors. The regulation of contraction induced by Provinols shows that treatment of arterial TEVM with this compound significantly decreased histamine-induced contraction. This effect was associated with the inhibition of the Rho-associated protein kinase pathway and the decrease in alpha-smooth muscle actin expression. Conclusion: The use of arterial TEVM, brings new insights into the mechanisms by which polyphenols regulate vascular contraction in the human artery.
  • Publication
    Isolation and culture of the three vascular cell types from a small vein biopsy sample
    (Tissue Culture Association, 2003-04-18) Grenier, Guillaume.; Germain, Lucie; Labbé, Raymond; Auger, François A.; Guignard, Rina; Rémy-Zolghadri, Murielle; Bergeron, François
    The availability of small-diameter blood vessels remains a significant problem in vascular reconstruction. In small-diameter blood vessels, synthetic grafts resulted in low patency; the addition of endothelial cells (EC) has clearly improved this parameter, thereby proving the important contribution of the cellular component to the functionality of any construct. Because the optimal source of cells should be autologous, the adaptation of existing methods for the isolation of all the vascular cell types present in a single and small biopsy sample, thus reducing patient’s morbidity, is a first step toward future clinical applications of any newly developed tissue-engineered blood vessel. This study describes such a cell-harvesting procedure from vein biopsy samples of canine and human origin. For this purpose, we combined preexisting mechanical methods for the isolation of the three vascular cell types: EC by scraping of the endothelium using a scalpel blade, vascular smooth muscle cells (VSMC), and perivascular fibroblasts according to the explant method. Once in culture, cells rapidly grew with the high level of enrichment. The morphological, phenotypical, and functional expected criteria were maintained: EC formed cobblestone colonies, expressed the von Willebrand factor, and incorporated acetylated low-density lipoprotein (LDL); VSMC were elongated and contracted when challenged by vasoactive agents; perivascular fibroblasts formed a mechanically resistant structure. Thus, we demonstrated that an appropriate combination of preexisting harvesting methods is suitable to isolate simultaneously the vascular cell types present in a single biopsy sample. Their functional characteristics indicated that they were suitable for the cellularization of synthetic prosthesis or the reconstruction of functional multicellular autologous organs by tissue engineering.
  • Publication
    Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication
    (John Wiley & Sons, Inc, 2016-04-28) Guillemette, Maxime.; Tondreau, Maxime; Laterreur, Véronique; Germain, Lucie; Miville-Godin, Caroline; Ruel, Jean; Mounier, Maxence; Tremblay, Catherine; Labbé, Raymond; Bourget, Jean-Michel; Veres, Teodor; Auger, François A.; Gauvin, Robert
    In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs.