Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

International audienceSoft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues

The Collagen V Homotrimer [α1(V)]3 Production Is Unexpectedly Favored over the Heterotrimer [α1(V)]2α2(V) in Recombinant Expression Systems

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [α1(V)]3 homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V α-chains showed that, contrary to fibroblasts, collagen V α-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly

Analyse multi-échelles des propriétés biomécaniques de la peau de souris saine et malade

La peau est un tissu complexe composé de 3 couches : l'épiderme, le derme et l'hypoderme. Le derme est responsable de la majeure partie des propriétés mécaniques de la peau. Une modification de la composition du derme entraîne ainsi des modifications drastiques du comportement mécanique de la peau, comme dans la maladie d'Ehlers-Danlos, qui se caractérise par une hyper?élasticité des tissus. Au niveau microstructural, le derme est composé essentiellement de matrice extracellulaire, formée pour la majeure partie d'un réseau désordonné de fibres de collagène. Pour élucider le lien exact entre organisation microstructurale et propriétés mécaniques de la peau, nous réalisons des essais de traction uniaxiaux in situ sous un microscope multiphoton avec détection du signal de génération de seconde harmonique. Ceci nous permet de suivre la réponse de la microstructure du tissu au cours de l'essai mécanique. Des paramètres quantitatifs ont été développés pour caractériser à la fois la réponse mécanique macroscopique du tissu et le réarrangement du réseau de fibres de collagène sous chargement. Nous pouvons ainsi comparer le comportement multi-échelles de peau de souris saine et de peau de souris atteinte d'une mutation affectant la microstructure du derme

Etude de la réorganisation macroscopique de la peau de souris lors d'une sollicitation bi-axiale

La peau est composée en majorité de collagène et présente une microstructure très hiérarchisée qui influe sur son comportement mécanique aux différentes échelles. Pour caractériser l'influence de la microstructure sur les propriétés mécaniques, un test de traction bi-axiale couplé à une mesure macroscopique (corrélation d'images numériques) et microscopique (génération de second harmonique) a été développé . A terme, ce travail permettra de corréler les propriétés macroscopiques à la microstructures. Cette étude présente les résultats de la mesure effectuée par corrélation d'images

LE RECEPTEUR TYROSINE KINASE RET (DEREGULATION DES MECANISMES DE SIGNALISATION DANS LA MALADIE DE HIRSCHSPRUNG ET ETUDE DES VOIES DE TRANSDUCTION ACTIVEES PAR LE LIGAND GDNF)

LYON1-BU Santé (693882101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Les collagènes du derme : au-delà de leurs propriétés structurales

La matrice extracellulaire est un réseau complexe de macromolécules comme les collagènes, les protéoglycannes et l'élastine qui s'imbriquent fortement les unes aux autres et qui, par l'intermédiaire d'interactions avec les cellules environnantes, vont permettre le maintien de la cohésion tissulaire. C'est grâce à ces nombreuses connexions que se mettent en place la plupart des évènements importants pour les grands programmes cellulaires, à savoir la migration, la prolifération, la différenciation et l'apoptose. La peau, et plus particulièrement le derme, est un exemple parfait de cette grande variété de molécules dont l'organisation et la composition doivent être parfaitement maintenues afin d'assurer les propriétés biomécaniques et fonctionnelles de ce tissu. Les collagènes sont les composants les plus abondants des matrices extracellulaires et représentent une famille de protéines de 27 membres différents. Mais ce sont surtout les collagènes fibrillaires I, III et V, les collagènes FACITs XII, XIV et XVI ainsi que le collagène de type VI qui sont les plus représentés dans le derme. Ces molécules sont à présent très bien caractérisées du point de vue de leur structure, mais leur fonction demeure toujours un peu floue. De nombreux gènes ont déjà été identifiés comme étant responsables de pathologies du tissu conjonctif et plus particulièrement de la peau. Les gènes codant le collagène V sont, par exemple, la cible de nombreuses mutations conduisant au syndrome d'Ehlers-Danlos classique qui est une maladie qui atteint principalement la peau et la structure du derme. Cependant, il semble clair qu'il est très difficile d'établir un lien réel entre génotype et phénotype pas plus qu'un lien entre la localisation et la fonction, pour ces molécules. Ce sont sans doute des études in vivo, dont de nombreuses sont d'ailleurs en cours de réalisation, qui devraient apporter des réponses plus claires à toutes ces interrogations

[Dermis collagens: beyond their structural properties]

International audienceThe extracellular matrix is a complex network composed of macromolecules such as collagens, proteoglycans and elastin that strongly interact with each other and with cells to maintain the structural integrity of many tissues. These interactions also sustain important cell programs such as migration, proliferation, differentiation and apoptosis. The skin, and more specifically the dermis, contains an extreme diversity of macromolecules that reflects the importance of the composition and organization of the matrix components in providing physical properties and function of the tissues. The most abundant matrix components are the collagens that form a super-family of 27 different members which are divided into different subgroups. The fibrillar collagens, types I, III and V, the FACIT collagens, types XII, XIV and XVI, and collagen VI are all expressed in the collagen-rich dermis. Although the structural features of these collagens are now well characterized, their functions remain elusive. Mutations in human collagen genes give rise to numerous connective tissue diseases including dermis disorders. For example, clinical manifestations in the classical Elhers-Danlos syndrome caused by collagen V gene mutations occur predominantly in the dermis. However, the genotype-phenotype relationship is not clearly established as well as the relation between the distribution and the function of the collagens in dermis. There is no doubt that the ongoing and future work using in vivo approaches will provide new cues regarding the function of collagens in dermis.The extracellular matrix is a complex network composed of macromolecules such as collagens, proteoglycans and elastin that strongly interact with each other and with cells to maintain the structural integrity of many tissues. These interactions also sustain important cell programs such as migration, proliferation, differentiation and apoptosis. The skin, and more specifically the dermis, contains an extreme diversity of macromolecules that reflects the importance of the composition and organization of the matrix components in providing physical properties and function of the tissues. The most abundant matrix components are the collagens that form a super-family of 27 different members which are divided into different subgroups. The fibrillar collagens, types I, III and V, the FACIT collagens, types XII, XIV and XVI, and collagen VI are all expressed in the collagen-rich dermis. Although the structural features of these collagens are now well characterized, their functions remain elusive. Mutations in human collagen genes give rise to numerous connective tissue diseases including dermis disorders. For example, clinical manifestations in the classical Elhers-Danlos syndrome caused by collagen V gene mutations occur predominantly in the dermis. However, the genotype-phenotype relationship is not clearly established as well as the relation between the distribution and the function of the collagens in dermis. There is no doubt that the ongoing and future work using in vivo approaches will provide new cues regarding the function of collagens in dermis

Quantification of OXPHOS gene transcripts during muscle cell differentiation in patients with mitochondrial myopathies.

International audienceThe transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns-Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase beta transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies.The transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns-Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase beta transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies

Caspase cleavage of the transcription factor FLI-1 during preB leukemic cell death.

International audienceProgrammed cell death (apoptosis) is a complex phenomenon that is mediated in mammals mainly via the selective cleavage of intracellular proteins by the large family of cysteine aspartate protease caspases. Apoptosis is tightly regulated by the competitive effect of numerous proteins displaying either pro-apoptotic or anti-apoptotic activity. The ETS-family transcription factor FLI-1, frequently associated with malignant transformation, has been shown to display anti-apoptotic activity in several cell types including avian erythroblasts, mouse fibroblasts or lymphoid cells. We show here that apoptosis of murine preB leukemic cells is accompanied with the specific cleavage of FLI-1 by a caspase-like activity. We also demonstrate that the two isoforms of FLI-1 are indeed cleaved at three conserved sites by caspase 3 in vitro. The conservation of these cleavage sites among species suggests that the caspase cleavage of the anti-apoptotic transcription factor FLI-1 may represent a critical step to ensure irreversible cell death.Programmed cell death (apoptosis) is a complex phenomenon that is mediated in mammals mainly via the selective cleavage of intracellular proteins by the large family of cysteine aspartate protease caspases. Apoptosis is tightly regulated by the competitive effect of numerous proteins displaying either pro-apoptotic or anti-apoptotic activity. The ETS-family transcription factor FLI-1, frequently associated with malignant transformation, has been shown to display anti-apoptotic activity in several cell types including avian erythroblasts, mouse fibroblasts or lymphoid cells. We show here that apoptosis of murine preB leukemic cells is accompanied with the specific cleavage of FLI-1 by a caspase-like activity. We also demonstrate that the two isoforms of FLI-1 are indeed cleaved at three conserved sites by caspase 3 in vitro. The conservation of these cleavage sites among species suggests that the caspase cleavage of the anti-apoptotic transcription factor FLI-1 may represent a critical step to ensure irreversible cell death