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Roberge, Charles

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Roberge

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Charles

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Université Laval. Département de chirurgie

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ncf10923368

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  • PublicationRestreint
    Tissue-engineered human vascular media produced in vitro by the self-assembly approach present functional properties similar to those of their native blood vessels
    (Mary Ann Liebert, Inc, 2006-09-12) Germain, Lucie; Pouliot, Stéphanie; Roberge, Charles; D’Orléans-Juste, Pedro; Auger, François A.; Laflamme, Karina
    We have developed a tissue-engineering approach for the production of a completely biological blood vessel from cultured human cells. In the present study, we took advantage of this tissue-engineering method to demonstrate that it can be used to reproduce the subtle differences in the expression of receptors present on the media of native human blood vessels. Indeed, a small percentage (3 of 18) of native human umbilical cord veins (HUCVs) responded to endothelin, the most powerful vasopressor agent known to date, via both endothelin A (ETA) and endothelin B (ETB) receptor activation. In contrast, most HUCVs tested responded toETviaETA receptor activation only. Tissue-engineered vascular media (TEVM) were next reconstructed by using vascular smooth muscle cells (VSMCs) isolated and cultured from HUCVs expressing both ETA and ETB receptors to determine the functional integrity of our TEVM model. The reconstructed TEVM presents an endothelin response similar to that of respective HUCVs from which VSMCs were isolated. Reverse transcriptase polymerase chain reaction on TEVM reconstructed in vitro correlated these vasocontractile profiles by showing the presence of messenger RNA for both ETA and ETB receptors. Taken together with recently published results on TEVM expressing only ETA receptor, these results show that our reconstructed TEVM present a similar ET response profile as the blood vessel from which the VSMCs were isolated and cultured. These findings indicate that subtle differences, such as receptor expression, are preserved in the reconstructed tissue. Therefore, our TEVM offers a valuable human in vitro model with which to study the functionality of human blood vessels, such as their vasoactive response, or to perform pharmacologic studies.
  • PublicationRestreint
    Restoration of the transepithelial potential within tissue-engineered human skin in vitro and during the wound healing process in vivo
    (Mary Ann Liebert, 2010-08-05) Plante, Michel; Germain, Lucie; Roberge, Charles; Auger, François A.; Gauvin, Robert; Dubé, Jean; Goulet, Daniel; Rochette-Drouin, Olivier; Bourdages, Michel; Lévesque, Philippe; Moulin, Véronique
    Normal human epidermis possesses a transepithelial potential (TEP) that varies in different parts of the body (10–60 mV). The role of TEP in normal epidermis is not yet identified; but after skin injury, TEP disruption induces an endogenous direct current electric field (100–200 mV/mm) directed toward the middle of the wound. This endogenous electric field could be implicated in the wound healing process by attracting cells, thus facilitating reepithelialization. However, little is known on the restoration of the TEP during human skin formation and wound healing. In this study, the variations in TEP and Na+/K+ ATPase pump expression during the formation of the epithelium were investigated in vitro using human tissue-engineered human skin (TES) reconstituted by tissue engineering and in vivo with a porcine wound healing model. Results showed that TEP undergoes ascending and decreasing phases during epithelium formation in TES as well as during wound repair within TES. Similar results were observed during in vivo reepithelialization of wounds. The ascending and decreasing TEP values were correlated with changes in the expression of Na+/K+ ATPase pump. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Taken together, these results suggest that the variations in the expression of Na+/K+ ATPase pump over time and across epidermis would be a determinant parameter of the TEP, dictating a cationic transport during the formation and restoration of the epidermis. Therefore, this study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process. This might have important future medical applications regarding the treatment of chronic wound healing.
  • PublicationAccès libre
    Electric potential across epidermis and its role during wound healing can be studied by using an in vitro reconstructed human skin
    (S-N Publications, 2012-02-28) Plante, Michel; Germain, Lucie; Roberge, Charles; Auger, François A.; Gauvin, Robert; Dubé, Jean; Rochette-Drouin, Olivier; Goulet, Daniel; Lévesque, Philippe; Bourdages, Michel; Moulin, Véronique
    Background : After human epidermis wounding, transepithelial potential (TEP) present in nonlesional epidermis decreases and induces an endogenous direct current epithelial electric field (EEF) that could be implicated in the wound re-epithelialization. Some studies suggest that exogenous electric stimulation of wounds can stimulate healing, although the mechanisms remain to be determined. The Problem : Little is known concerning the exact action of the EEF during healing. The mechanism responsible for TEP and EEF is unknown due to the lack of an in vitro model to study this phenomenon. Basic Science Advances : We carried out studies by using a wound created in a human tissue-engineered skin and determined that TEP undergoes ascending and decreasing phases during the epithelium formation. The in vitro TEP measurements over time in the wound were corroborated with histological changes and with in vivo TEP variations during porcine skin wound healing. The expression of a crucial element implicated in Na+ transport, Na+/K+ ATPase pumps, was also evaluated at the same time points during the re-epithelialization process. The ascending and decreasing TEP values were correlated with changes in the expression of these pumps. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Further, inhibition of the pump activity induced a significant decrease of the TEP and of the re-epithelization rate. Clinical Care Relevance : A better comprehension of the role of EEF could have important future medical applications regarding the treatment of chronic wound healing. Conclusion : This study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process.
  • PublicationRestreint
    Regulation of skin collagen metabolism in vitro using a pulsed 660 nm LED light source: clinical correlation with a single-blinded study.
    (2009-07-09) Barolet, Daniel; Germain, Lucie; Roberge, Charles; Boucher, Annie; Auger, François A.
    It has been reported that skin aging is associated with a downregulation in collagen synthesis and an elevation in matrix metalloproteinase (MMP) expression. This study investigated the potential of light-emitting diode (LED) treatments with a 660 nm sequentially pulsed illumination formula in the photobiomodulation of these molecules. Histological and biochemical changes were first evaluated in a tissue-engineered Human Reconstructed Skin (HRS) model after 11 sham or LED light treatments. LED effects were then assessed in aged/photoaged individuals in a split-face single-blinded study. Results yielded a mean percent difference between LED-treated and non-LED-treated HRS of 31% in levels of type-1 procollagen and of -18% in MMP-1. No histological changes were observed. Furthermore, profilometry quantification revealed that more than 90% of individuals showed a reduction in rhytid depth and surface roughness, and, via a blinded clinical assessment, that 87% experienced a reduction in the Fitzpatrick wrinkling severity score after 12 LED treatments. No adverse events or downtime were reported. Our study showed that LED therapy reversed collagen downregulation and MMP-1 upregulation. This could explain the improvements in skin appearance observed in LED-treated individuals. These findings suggest that LED at 660 nm is a safe and effective collagen-enhancement strategy.
  • PublicationRestreint
    Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries
    (Mary Ann Liebert, 2010-02-16) Germain, Lucie; Roberge, Charles; Rochon, Marie-Hélène; Tomasetig, Florence; Auger, François A.; Baker, Kathleen; Fortin, Véronique; Berthod, François; Fradette, Julie
    The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal keratinocytes from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 μm2) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 μm2), but the addition of keratinocyte-conditioned media or exogenous vascular endothelial growth factor induced their normalization toward a smaller size. Vascular endothelial growth factor neutralization inhibited the effect of keratinocyte-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro.
  • PublicationRestreint
    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.
  • PublicationRestreint
    Human keratinocytes respond to direct current stimulation by increasing intracellular calcium : preferential response of poorly differentiated cells
    (Liss, 2012-02-24) Plante, Michel; Germain, Lucie; Roberge, Charles; Auger, François A.; Gauvin, Robert; Dubé, Jean; Goulet, Daniel; Rochette-Drouin, Olivier; Bourdages, Michel; Lévesque, Philippe; Moulin, Véronique
    A direct current (DC) endogenous electric field (EF) is induced in the wound following skin injury. It is potentially implicated in the wound healing process by attracting cells and altering their phenotypes as indicated by the response to an EF of keratinocytes cultured as individual cells. To better define the signalization induced by a direct current electric field (DCEF) in human keratinocytes, we took advantage of an in vitro model more representative of the in vivo situation since it promotes cell–cell interactions and stratification. Human keratinocytes were grown into colonies. Their exposure to a DCEF of physiological intensity induced an increase of intracellular calcium. This variation of intracellular calcium resulted from an extracellular calcium influx and was mediated, at least in part, by the L-type voltage-gated calcium channel. The increase in intracellular calcium in response to a DCEF was however not observed in all the cells composing the colonies. The intracellular calcium increase was only detected in keratinocytes that didn't express involucrin, a marker of differentiated cells. These results indicate that DCEF is able to induce a specific calcium response in poorly differentiated keratinocytes. This study brings a new perspective for the understanding of the signaling mechanism of endogenous EF in reepithelialization, a critical process during skin wound healing.
  • PublicationRestreint
    Tissue-engineered human vascular media with a functional endothelin system.
    (American Heart Association etc, 2005-02-01) Labonté, Julie; Germain, Lucie; Pouliot, Stéphanie; Roberge, Charles; D’Orléans-Juste, Pedro; Auger, François A.; Laflamme, Karina
    Background— Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor’s hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel. Methods and Results— Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ETA receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ETA but not the ETB receptor was present in these human tissue–engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media. Conclusions— Our results suggest that the media component of our tissue-engineered blood vessel has the potential of controlling vascular resistance via the presence of functional endothelin ETA receptors and endothelin-converting enzyme. endothelinmuscle contractionmyocytes, smooth m