Rôle et prévention du « stress carbonyle » au cours du vieillissement de la peau

Le vieillissement est un phénomène naturel, lent et complexe, caractérisé par une perte progressive des fonctions physiologiques et des capacités physiques et mentales des êtres vivants, en particulier chez l'homme. Le vieillissement est au cœur des préoccupations de chaque société, de par ses conséquences biologiques, psychologiques et sociétales. Une meilleure connaissance des processus physiologiques impliqués dans le vieillissement permettrait de mettre en place des approches thérapeutiques permettant de ralentir l'apparition de certains effets du vieillissement. Sur un plan biologique, le stress oxydant joue un rôle important dans les mécanismes impliqués dans le vieillissement tissulaire et cellulaire. Parmi les conséquences du stress oxydant, la peroxydation des acides gras polyinsaturés génère des produits d'oxydation lipidique, de type aldéhyde, qui s'accumulent dans les tissus âgés et photoexposés. Ces aldéhydes forment des adduits sur les protéines tissulaires, ce qui entraine une perte progressive de leur propriétés (stress carbonyle). L'implication du stress carbonyle dans le mécanisme du vieillissement tissulaire n'est pas clarifiée, mais pourrait participer aux processus de dégénérescence. Notre travail de thèse a été axé sur l'implication du stress carbonyle dans le mécanisme du vieillissement physiologique et du photovieillissement de la peau. Dans la première partie de ce travail nous montrons que l'exposition chronique de souris hairless à des UVA (modèle animal de vieillissement cutané accéléré), s'accompagne d'une accumulation de matériel élastotique (élastine anormale), en partie modifié par les aldéhydes (4-hydroxynonénal ou 4-HNE, acroléine). Le taux de ces aldéhydes, normalement très peu présents dans la peau non exposée, est fortement augmenté après exposition aux UVA. L'application chronique d'un piégeur de carbonyle, la L-carnosine, utilisée à 1% dans du propylène-glycol, permet de réverser l'accumulation des aldéhydes et de restaurer un matériel élastique normal dans les peaux. Nous avons mis en évidence que l'élastine soluble ou insoluble modifiée in vitro par l'acroléine n'est progressivement plus dégradée par l'élastase, ce qui peut contribuer à l'accumulation de matériel élastotique dans le derme. La carnosine permet de réverser in vitro et in vivo la modification de l'élastine par l'acroléine. Ces résultats nous permettent de confirmer le rôle important des produits de peroxydation lipidique dans le photovieillissement. Dans la deuxième partie de ce travail, nous étudions l'impact du stress carbonyle sur la signalisation du TGF-β dans le cadre du vieillissement chronologique. Nous avons montré, que le vieillissement chronologique conduit à une augmentation de 4-HNE, dans la peau et les artères. Cet aldéhyde altère le renouvellement d'élastine induite par le TGF-β, en modifiant et activant la voie EGFR/ERK/TGIF. Enfin des résultats précédemment publiés par le laboratoire ainsi que d'autres expérimentations nous ont permis de montrer que le 4-HNE, inhibe la prolifération des fibroblastes, les cellules responsables de la synthèse de matrice extracellulaire. L'ensemble de nos résultats nous permet de dire que le vieillissement physiologique et le photovieillissement s'accompagnent de la génération de produits de peroxydation lipidique. Ces aldéhydes agissent sur la matrice extracellulaire en inhibant d'une part la prolifération des fibroblastes, en formant des adduits sur l'élastine empêchant ainsi sa dégradation et favorisant son accumulation sous forme fragmentée, et en inhibant son renouvellement par l'activation de la voie EGFR/ERK/TGIF. L'ensemble de ces mécanismes conduit à une perte de fonction progressive de l'élastine participant aux effets classiquement observés au cours du vieillissement.Aging is a complex and multifactorial process characterized by physical, psychological, and social changes. The place of aging in all human societies is very important because it reflects the biological, cultural and societal conventions as well, while it is also the largest known risk factor for most human diseases. The aging process is characterized by a progressive loss of biological functions and abilities to manage metabolic, functional and esthetic changes. These changes may result from both intrinsic genetically progammed processes and from environmental factors. Among these extrinsic factors involved in aging, the continuous exposure to ultraviolet solar radiations generates the characteristic modifications of the skin, also called photoaging. Because of the general importance of aging, it is important to investigate and understand the physiological mechanisms involved in this natural process, in order to develop new approaches allowing to slow down the onset of some aging's effects. Oxidative stress plays an important role in the mechanisms involved in cellular and tissular aging. Among the consequences of oxidative stress, lipid peroxidation of polyunsaturated fatty acids generates lipid oxidation products, aldehydes, which accumulate in aged and photoexposed tissues. These aldehydes form adducts on proteins, which causes a progressive loss of their properties (carbonyl stress). The involvement of carbonyl stress in aging is not well clarified, but could contribute to the process of cell degeneration. This thesis is focused on the role of carbonyl stress in some aspects of physiological and skin photoaging. In the first part of this work we show that the chronic exposure of hairless mice to UVA radiations, generates an accumulation of elastotic material (abnormal elastin), partly modified by aldehydes (4-hydroxynonenal, acrolein). The presence of these aldehydes, in the unexposed skin is usually low, but it is greatly increased upon exposure to UVA. We show that elastin modified in vitro by acrolein becomes progressively resistant to leukocyte elastase, which could contribute to the accumulation of elastotic material in the dermis. A daily topical application of a carbonyl scavenger, L-carnosine, used at 1% in propylene glycol, protects against the accumulation of aldehydes on elastin, and restores a normal elastic material in skins. We show that L-carnosine also reverses in vitro the modification of elastin by acrolein. These results highlight a role for aldehydes in photoaging. In the second part of this work, we show that carbonyl stress (4-HNE) exerts an inhibitory effect on the elastogenic activity of TGF-ß signaling, leading to tropoelastin synthesis. This effect involves the formation of 4-HNE adducts on the EGF receptor (EGFR) which, once activated, stimulates the EGFR/ERK/TGIF signaling which inhibits the TGF-ß-induced expression of tropoelastin messengers. That chronological aging in C57/BL6 mice, is correlated with a strong increase in 4-HNE adducts formation in vessels and in unexposed skin, as well as an overexpression and increased modification of EGFR by 4-HNE. Furthermore, we show that 4-HNE inhibits the proliferation of fibroblasts, which secrete the extracellular matrix in the dermis, via a modification of both EGFR and the PDGF receptor. All these results indicate that lipid peroxidation products are generated during physiological aging and are strongly increased during photoaging. These aldehydes alter the composition of the extracellular matrix in the skin i/ by inhibiting the proliferation of fibroblasts, ii/by forming adducts on elastin, thereby preventing its degradation by elastase, that contributes to the accumulation of elastotic material, and iii/ by inhibiting elastin renewal via the activation of the EGFR/ERK/TGIF pathway, which inhibits TGF-β signaling. All of these effects may contribute to the progressive loss of elastin properties, a key-event in the process of aging

Curso de Extensão de Leitura Crítica de Artigos Científicos: relato de experiência

Resumo: objetivou-se relatar a experiência do Curso de Extensão de Leitura Crítica de Artigos Científicos. O relato foi elaborado a partir do projeto de extensão que visou a construção de um espaço para discussão sobre a leitura de artigos científicos. O curso envolveu a participação de estudantes universitários e profissionais da saúde. Foram realizados sete encontros, sendo três presenciais e quatro virtuais. Nos encontros presenciais, oportunizou-se a discussão de um texto, enviado previamente para que os participantes pudessem fazer as leituras. Nos encontros virtuais, foram utilizados disparadores para discussão por meio de um grupo formado em uma rede social

Elastin aging and lipid oxidation products in human aorta

Vascular aging is associated with structural and functional modifications of the arteries, and by an increase in arterial wall thickening in the intima and the media, mainly resulting from structural modifications of the extracellular matrix (ECM) components. Among the factors known to accumulate with aging, advanced lipid peroxidation end products (ALEs) are a hallmark of oxidative stress-associated diseases such as atherosclerosis. Aldehydes generated from the peroxidation of polyunsaturated fatty acids (PUFA), (4-hydroxynonenal, malondialdehyde, acrolein), form adducts on cellular proteins, leading to a progressive protein dysfunction with consequences in the pathophysiology of vascular aging. The contribution of these aldehydes to ECM modification is not known. This study was carried out to investigate whether aldehyde-adducts are detected in the intima and media in human aorta, whether their level is increased in vascular aging, and whether elastin fibers are a target of aldehyde-adduct formation. Immunohistological and confocal immunofluorescence studies indicate that 4-HNE-histidine-adducts accumulate in an age-related manner in the intima, media and adventitia layers of human aortas, and are mainly expressed in smooth muscle cells. In contrast, even if the structure of elastin fiber is strongly altered in the aged vessels, our results show that elastin is not or very poorly modified by 4-HNE. These data indicate a complex role for lipid peroxidation and in particular for 4-HNE in elastin homeostasis, in the vascular wall remodeling during aging and atherosclerosis development

Role of protein kinase C δ in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells.

International audienceDuring atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture

4-Hydroxynonenal impairs transforming growth factor-β1-induced elastin synthesis via epidermal growth factor receptor activation in human and murine fibroblasts

International audienceElastin is a long-lived protein and a key component of connective tissues. The tissular elastin content decreases during chronological aging, and the mechanisms underlying its slow repair are not known. Lipid oxidation products that accumulate in aged tissues may generate protein dysfunction. We hypothesized that 4-hydroxynonenal (4-HNE), a highly reactive α,β-aldehydic product generated from polyunsaturated fatty acid peroxidation, could contribute to inhibiting elastin repair by antagonizing the elastogenic signaling of transforming growth factor-β1 (TGF-β1) in skin fibroblasts. We report that a low 4-HNE concentration (2µmol/L) inhibits the upregulation of tropoelastin expression stimulated by TGF-β1 in human and murine fibroblasts. The study of signaling pathways potentially involved in the regulation of elastin expression showed that 4-HNE did not block the phosphorylation of Smad3, an early step of TGF-β1 signaling, but inhibited the nuclear translocation of Smad2. Concomitantly, 4-HNE modified and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR) and subsequently ERK1/2 activation, leading to the phosphorylation/stabilization of the Smad transcriptional corepressor TGIF, which antagonizes TGF-β1 signaling. Inhibitors of EGFR (AG1478) and MEK/ERK (PD98059), and EGFR-specific siRNAs, reversed the inhibitory effect of 4-HNE on TGF-β1-induced nuclear translocation of Smad2 and tropoelastin synthesis. In vivo studies on aortas from aged C57BL/6 mice showed that EGFR is modified by 4-HNE, in correlation with an increased 4-HNE-adduct accumulation and decreased elastin content. Altogether, these data suggest that 4-HNE inhibits the elastogenic activity of TGF-β1, by modifying and activating the EGFR/ERK/TGIF pathway, which may contribute to altering elastin repair in chronological aging and oxidative stress-associated aging processes

Protein disulfide isomerase modification and inhibition contribute to ER stress and apoptosis induced by oxidized low density lipoproteins.

International audienceAIMS: Protein disulfide isomerase (PDI) is an abundant endoplasmic reticulum (ER)-resident chaperone and oxidoreductase that catalyzes formation and rearrangement (isomerization) of disulfide bonds, thereby participating in protein folding. PDI modification by nitrosative stress is known to increase protein misfolding, ER stress, and neuronal apoptosis. As LDL oxidation and ER stress may play a role in atherogenesis, this work was designed to investigate whether PDI was inactivated by oxLDLs, thereby participating in oxLDL-induced ER stress and apoptosis. RESULTS: Preincubation of human endothelial HMEC-1 and of macrophagic U937 cells with toxic concentration of oxLDLs induced PDI inhibition and modification, as assessed by 4-HNE-PDI adducts formation. PDI inhibition by bacitracin potentiated ER stress (increased mRNA expression of CHOP and sXBP1) and apoptosis induced by oxLDLs. In contrast, increased PDI activity by overexpression of an active wild-type PDI was associated with reduced oxLDL-induced ER stress and toxicity, whereas the overexpression of a mutant inactive form was not protective. These effects on PDI were mimicked by exogenous 4-HNE and prevented by the carbonyl-scavengers N-acetylcysteine and pyridoxamine, which reduced CHOP expression and toxicity by oxLDLs. Interestingly, 4-HNE-modified PDI was detected in the lipid-rich areas of human advanced atherosclerotic lesions. Innovation and CONCLUSIONS: PDI modification by oxLDLs or by reactive carbonyls inhibits its enzymatic activity and potentiates both ER stress and apoptosis by oxLDLs. PDI modification by lipid peroxidation products in atherosclerotic lesions suggests that a loss of function of PDI may occur in vivo, and may contribute to local ER stress, apoptosis, and plaque progression