Ageing tissue is characterised by an alteration in mechanical properties, with tissues becoming increasingly fragile, stiff and less resilient over time. This can lead to a loss of function in tissues which undergo a high degree of stretch and recoil, such as the skin, blood vessels and lungs. These changes are attributed to remodelling of the dermal extra cellular matrix (ECM) proteins which endow tissue with its mechanical properties. Principally the fibrillar collagens provide tensile strength, complemented by the elastic fibres which confer resilience and allow tissues to recoil following deformation. Intriguingly, at the time of menopause, which sees the cessation of the majority of circulating estrogen, skin undergoes accelerated ageing. This strongly suggests a direct link between circulating estrogen and youthful skin. Using an ovariectomised (Ovx) mouse model of human menopause the link between ageing, estrogen deficiency and the dermal ECM proteins was explored. Mechanical testing revealed a significant reduction in the tensile strength, Young’s modulus and stress relaxation time of estrogen deprived tissue, indicating postmenopausal skin would be weaker, more lax and less able to withstand sustained force. Corresponding histological analysis highlighted the elastic fibres are dramatically reduced by estrogen deprivation with seemingly little effects on the fibrillar collagen abundance or alignment. Direct comparison between Ovx and age, reveals age affects mechanical properties in a completely opposing way, with aged tissue higher in tensile strength, Young’s modulus and stress relaxation time compared to control and Ovx. Proteomic analysis by mass spectrometry confirmed elastic fibres to be highly sensitive to estrogen levels. Additionally small leucine rich proteoglycans (SLRP’s) were also significantly reduced by estrogen deficiency, which could affect collagen fibrillogenesis and leave tissue weaker and more susceptible to damage. Despite the opposing effects on mechanical properties, ageing and estrogen deficiency had comparable effects on ECM abundance, suggesting the amount of ECM is not a predictor of mechanical properties; however disparity may be in part due to increased advanced glycation end product crosslinking. Further investigation suggests the mechanism for elastic fibre degradation may be via significant subcutaneous adipose hypertrophy and/or increased gelatinase activity. Both estrogen replacement and stimulation of estrogen receptor α with the agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (PPT) were found to prevent elastic fibre degradation and adipose hypertrophy. Additionally these treatments were also found to induce key elastic fibre proteins in both the mouse model and cultured human dermal fibroblasts. A deeper understanding of estrogen mediated ECM remodelling offers opportunities for targeted pharmacological intervention to slow the effects of menopause and ageing.

The nuclear hormone receptor (NHR) liver X receptor β (LXRβ) has been highlighted as a possible candidate for involvement in ageing by several recent findings. LXRβ is the closest human homologue to the longevity-associated gene daf-12 in the nematode worm and LXRβ haplotypes have been associated with longevity at old ages in a longitudinal human genetic study. Whilst LXRβ is primarily responsible for mediating the effects of LXR oxysterol ligands throughout most of the body, LXRβ is the primary mediator of these effects uniquely in the skin. In this thesis studies are presented on the expression of LXRβ mRNA and protein in human skin, comparing young vs intrinsically (chronologically) aged skin, photoprotected vs photoaged (dueto ultraviolet radiation exposure) skin and untreated vs retinoid-treated photoaged skin, retinoid treatment being a primary clinical intervention for photoageing. In situ hybridisation and quantitative polymerase chain reaction (qPCR) were used to identify LXRβ mRNA and immunofluorescence was used to identify LXRβ protein. These comparisons revealed that both the mRNA and protein expression of LXRβ are highly stable throughout the ageing, photoageing and retinoid treatment of human skin. Previous authors have identified overlap between microarray gene expression datasets in the LXRβ-/- mouse and in normal human skin ageing. In these studies comparisons of different microarray datasets have been conducted with the finding that LXR agonist treatment of mice produces gene regulation patterns with significant overlap to that seen in both ageing and calorie restriction in mice (binomial test; p<0.001). Furthermore, when considering the genes commonly regulated in LXR agonist treatment and ageing, 73% of these genes are regulated in opposite directions. Conversely, when considering genes commonly regulated in LXR agonist treatment and calorie restriction, 70% of these genes are regulated in the same direction. These findings suggest that LXR agonists have possible benefit as ageing therapies, perhaps due to stimulating a calorie restriction-like response. Further work would be necessary to confirm these properties of LXR agonists and to define the roles of LXRβ in the ageing and normal function of human skin.

La peau est un excellent modèle d’étude du vieillissement général. En plus de facteurs environnementaux, les facteurs génétiques jouent un rôle majeur dans le vieillissement cutané. Dans le cadre de ma thèse, j’ai eu accès à une cohorte exceptionnelle de 502 femmes caucasiennes très bien caractérisées sur le plan cutané, pour effectuer deux études d’association « génome-entier ». La première étude a montré le rôle joué par le système immunitaire, et en particulier le gène HLA‑C, dans la sévérité des lentigines du visage. La seconde a mis en évidence une association entre le gène H2AFY2 et la sévérité de l’affaissement de la paupière supérieure. La recherche de voies de signalisation biologiques associées à différents indicateurs du vieillissement cutané a souligné le rôle de la mélanogénèse et des mécanismes de réparation de l’ADN.Ces résultats ouvrent de nouvelles perspectives dans la compréhension des mécanismes inhérents au vieillissement cutané et général. / The skin is an excellent model to study general ageing. In addition to environmental factors, genetic factors play a key role in skin ageing mechanisms. During my PhD, I have had access to a unique cohort of 502 Caucasian women very-well characterized regarding their facial features to perform two genome-wide association studies. The first one pointed to the role of the immune system, and especially the HLA‑C gene, in the severity of facial lentigines. The second one identified an association between the H2AFY2 gene and the severity of superior eyelid drooping. I also looked for associations between biological pathways and several skin ageing indicators which underlined the role of the melanogenesis and several mechanisms of DNA repair.Overall, these results lead to new insights in the understanding of the molecular mechanisms underlying skin and global ageing.

Background. The development of non-invasive imaging techniques has been stimulated by the shortcomings of histopathology. Currently the only valid diagnostic technique in dermatology is skin biopsy which remains a painful, invasive intervention for the patient. Moreover, this approach is not always convenient for monitoring and follow-up of a skin disease. Optical imaging technologies could solve these shortcomings as they are fast, precise, repeatable and painless. There are four established non-invasive skin imaging techniques used in daily practice: dermoscopy, high-frequency ultrasound, reflectance confocal microscopy (RCM) and conventional optical coherence tomography (C-OCT). In imaging there is a trade-off between resolution and penetration depth. The former permits the visualization of cells, if the resolution is at least 3 µm. The latter enables the recognition of patterns and structures in deeper layers of the skin if the penetration depth is deeper than 150 µm. New non-invasive techniques using infrared light sources have been developed recently. The technique used in this work is a high-definition optical coherence tomography (HD-OCT).Objectives. The overall aims of this thesis were the feasibility of HD-OCT to visualize in/ex vivo, in real time and in 3-D the cellular and structural morphology of the skin, secondly the assessment of the capability of this technology to measure in vivo and real time the cutaneous optical properties, and finally the determination of the contribution of this technique to the non-invasive near-infrared imaging technologies. Five specific objectives have been established: i) could cells be observed in their 3-D microenvironment in normal and diseased skin, ii) could we describe morphologic features of cells and structures in normal and diseased skin (m_HD-OCT), iii) could these morphologic features be quantified by optical property analysis (o_HD-OCT), iv) was it possible to perform accurate thickness measurements in normal and diseased skin, and finally v) what was the diagnostic potential of this technique?Methodology. HD-OCT uses a combination of parallel time-domain interferometry, high power tungsten lamp (with Gaussian filter, very low lateral coherence and ultra-high bandwidth (1300 nm +/- 100 nm)), and last but not least, full field illumination with real time focus tracking. A constant homogeneous resolution of 3 µm resolution in all three dimensions is obtained up to a depth of 570 µm. Hence, the system is capable of capturing real time full 3-D images. Moreover, the in vivo assessment of optical properties of the skin is only applicable to OCT when operating in focus-tracking mode, which is the case for HD-OCT. The means to obtain answers to the five specific questions were the comparison of en face HD-OCT images with RCM and HD-OCT cross-sectional images with histopathology and C-OCT. Results. At least 160 line pares were observed by imaging a high resolution phantom with HD-OCT. This suggested a 3 µm lateral resolution. The presence of cells such as keratinocytes, melanocytes, inflammatory cells, fibroblasts and melanophages in their 3-D cutaneous microenvironment in vivo as well as ex vivo has been demonstrated .A qualitative description of structures and patterns in normal and diseased skin could be performed by HD-OCT. Clear structural changes of the epidermis, dermo-epidermal junction, papillary dermis and reticular dermis related to intrinsic skin ageing could be observed. Lobulated structures, surrounded by stretched stromal fibers and arborizing vessels, could be demonstrated in nodular basal cell carcinoma (BCC). The o_HD-OCT of normal and diseased skin could be assessed in vivo. This approach permitted the quantitative assessment of the OCT signal attenuation profiles of normal healthy skin, actinic keratosis (AK) and squamous cell carcinoma (SCC). Differences in signal attenuation profiles could be demonstrated between these three groups. These differences were also observed between BCC subtypes. The slope of the exponential attenuation of the signal in the upper part of the epidermis was very high in benign nevi. The more malignant the lesion the lower the slope. Thickness measurements of epidermis and papillary dermis could be performed by m_HD-OCT, based on a cross-sectional images and their corresponding en face image. More accurate measurements of epidermal and papillary dermal thickness could be performed based on the optical analysis of a skin volume by o_HD-OCT. The diagnostic potential of HD-OCT in comparison with dermoscopy, RCM and C-OCT could be assessed regarding i) melanoma, ii) BCC differentiation from BCC imitators and BCC sub-differentiation and iii) SCC differentiation from AK. A much higher diagnostic potential could be demonstrated for o_HD-OCT in comparison with m_HD-OCT concerning melanoma detection. The diagnostic potential of HD-OCT to discriminate BCC from clinical BCC imitators was moderate. However, HD-OCT seemed to have high potential in sub-differentiation of BCC subtypes: i) it seemed to be the best technique to include and exclude a superficial BCC, ii) the technique appeared to be the best approach to exclude nodular BCC, and iii) HD-OCT looked to be the best technique to include an infiltrative BCC. Finally, HD-OCT has proven to be a powerful method to discriminate AK from SCC.Conclusions. HD-OCT is able to capture real time 3-D imaging with a sufficiently high optical resolution and penetration depth to allow the visualization of cells in and ex vivo in their micro-architectural context. At the same time, HD-OCT permits the recognition of patterns and structures in a sufficiently large volume of skin (1.5 mm³). HD-OCT closes therefore the gap between RCM with a high resolution but low penetration depth and C-OCT with a low resolution but high penetration depth. Moreover, HD-OCT permits, in contrast to RCM and C-OCT, the real time in vivo analysis of optical properties of the skin. HD-OCT seems to be a promising tool for early diagnosis of melanoma, BCC sub-differentiation and differentiation between SCC and AK.Future perspectives. Multicenter validation studies are needed to determine the diagnostic performance of this promising new technology, especially in other clinical settings combining both morphological and optical property analysis. This combined analysis could be a valuable method not only for diagnosis, monitoring and therapeutic guidance of dermatologic diseases but it could also be helpful in the management of non-dermatologic conditions such as diabetic micro-angiopathy, infantile cystinosis or even osteoporosis. / Doctorat en Sciences médicales (Santé Publique) / info:eu-repo/semantics/nonPublished