The study of skin aging mechanisms may be useful to understand the whole-body senescence. Therefore, epigenomic and transcriptomic analysis of aged skin from exposed and unexposed areas play important role in understanding human aging and age-related diseases. The photoaging is the major component of extrinsic aging . The management of choice is the continuous use of broad-spectrum sunscreen  and topical retinoids, particularly all-trans retinoic acid or tretinoin, despite the risk of irritative dermatitis . We have observed the increased skin roughness and deep wrinkles in photo-exposed areas, compared to non-photoexposed.
Surprisingly no difference was detected in smoothness and scaling among areas. The low density of collagen IV in upper dermis in exposed areas is coincident with the findings of Caetano et al. (2016). So far, a well-documented therapeutic strategy for reversing the integrity of dermal matrix is the prolonged use of topical tretinoin . However, the tolerability to tretinoin decreases with intrinsic aging as the skin becomes thinner and more sensitive, so other topical treatments are proposed.
Targeting age-related lipid deficiency and consequent stratum corneum dysfunction, may be a strategy in the development of therapies capable of increasing ceramides levels, improving cutaneous barrier function. Therefore, cosmeceuticals presenting efficacy comparable to retinoids in the dermis but also capable of modulating epidermis and stratum corneum are of high importance. Progresses have been made in this field based in nanotechnology and in vitro studies . However, in vivo research is complimentary and relevant.
Mercurio et al., (2016) and Carvalho et al., (2017), described the biophysical properties of photoaged skin. Our findings corroborate to their reports, regarding skin roughness and wrinkles number, which were significantly higher in photo-exposed areas as consequence of extrinsic over intrinsic aging superposition. The periorbital area was smoother than preauricular, possibly related to better use of moisturizer around the eyes. The exposed skin presented more desquamation than the unexposed, representing the cutaneous barrier damage. Surprisingly, no differences were detected in the smoothness between preauricular and gluteal areas, due to regular use of moisturizer. The significantly higher echogenicity in A1 and A2 compared to A3 was not expected but can be explained by evident elastosis present in both photo-exposed areas and dermal alterations in the unexposed skin caused by intrinsic aging, such as reduction of extracellular matrix synthesis. In addition to elastosis, the low density of collagen fibers in the upper dermis, as well as the decreased epidermal thickness in exposed areas are consistent with the description published by Caetano et al. (2016). The digital quantitative analysis of histological findings revealed reduced epidermal thickness and flattened DEJ in periorbital compared to preauricular and gluteal skin. These aspects were already reported as consequence of ROS accumulation [3, 40], in addition to intrinsic aging mechanisms and repetitive muscular movement . The papillary dermis of periorbital skin was thinner than preauricular and gluteal skin. The periorbital collagen fibers were also thinner with more irregular orientation and larger interfibrillar spaces. The level of collagen IV was lower than those of preauricular skin at DEJ. The collagen IV was considered a good marker for DEJ integrity by Raman microspectroscopy .
RNASeq data showed that cell death/survival pathways and lipid metabolism networks were enriched with a larger amount of DEGs with a predict impact by IPA analysis, suggesting they may play an important role in skin aging. As demonstrated by previous in vitro studies , we observed a down-regulation of genes related to DNA replication and repair, cell cycle regulation and apoptosis as well as up-regulation of genes related to inflammatory response in photo-exposed areas (A1 vs A2). This indicates that periorbital wrinkles seem to undergo a greater inflammatory damage than the photo-exposed area without wrinkles.
The preauricular area was free of dynamic wrinkles, therefore the main aging factor would be sun exposure. In order to verify the photodamage, we compared the activated pathways in A2 versus A3. Interestingly, our results showed an increase in lipid and aminoacids metabolism in the preauricular when compared to gluteal area. The increased expression of lipid-related genes could be explained by the inflammatory process, as discussed by Zhuang and Lyga (2014). In the epidermis there is cellular damage with oxidized lipids that are capable of causing inflammation by activating the migration of macrophages to remove cells and oxidized lipids. Thus, macrophages overloaded with oxidized lipids release cytokines and ROS, causing inflammation and long-term damage also in the dermal matrix. Therefore, some genes have increased expression to compensate for lipids damaged. Skin suffers from both intrinsic and extrinsic factors of aging, however the increased expression of lipids in exposed skin could lead to more inflammation when compared to unexposed skin.
Shen et al. (2016) demonstrated that UV genetic signature of skin was similar to squamous cell carcinoma. After UVB irradiation of human keratinocytes, they found 401 DEGs; the alterations were UVB-dose dependent and persisted for 21 days. Downregulated genes were related to DNA replication and repair, cell cycle regulation, chromatin dynamic, and apoptosis. Additionally, upregulated genes were linked to inflammatory response and adaptive immunity. The decrease of epidermal lipids, such as triglycerides and free fatty acids, as well as the down regulation of genes related to lipid synthesis in aged skin was reported by Kim et al. (2010). The authors explained their findings by the increased expression of MMP-1, which is an inhibitor of lipid synthesis. They also suggested that moisturizer formulations similar to lipid composition of skin surface could restore the cutaneous barrier. The reduced expression of innate immunity markers in various cells during the aging process was more pronounced in sebaceous glands, pointing out a role of sebum in immune response which is compromised in elderly . Authors concluded that the down-regulation of genes related to lipid metabolism could be explained by the inclusion of menopaused women who present reduced hormonal stimulation of sebaceous glands and keratinocytes, as reported by Calleja-Agius et al. (2007). However, our results were opposite, as several pathways related to lipid metabolism were predicted to be activate in photo-exposed skin without wrinkles. We could postulate this finding as a defense response to restore cutaneous barrier. The Genotype-Tissue Expression (GTEx) compared samples from photo-protected suprapubic and photo-damaged lower leg areas from individuals of both genders, aged from 20s to 70s and skin tissue samples were obtained by rapid autopsy from donors; they created a database useful for comparison in skin genomic studies . Cho et al. (2018) analyzed RNA sequencing data from GTEx in order to identify transcriptomic changes of aged skin. The main results were the up-regulation of DEGs of epidermal differentiation complex component, vasculature development and matrix metalloproteinase in UV exposed area. The down-regulated DEGs included IL6 and IL33, involved in wound heading, and several histones. In contrast to our results, genes related to lipid metabolism were down-regulated in photo-damaged skin. This difference may be explained by the following reasons: GTEx used exposed skin from lower leg which is less damaged than the face; the samples were obtained from corpse and our study was in vivo; GTEx study population was from other geographic region, included a wide age range and both genders.
Kimball et al. (2018) demonstrated divergent molecular processes along lifetime in skin affected by intrinsic (buttocks) and extrinsic (face and dorsal forearm) aging by analyzing epidermal gene expression. They included Caucasian females from six decades of age (20 to 74 yo.) who appear younger than their chronologic age. The pathways related to oxidative stress, energy metabolism, senescence, and epidermal barrier were accelerated in the 60s and 70s. Interestingly, gene expression patterns in women who were younger-appearing were similar to those in younger women, suggesting the major role of extrinsic aging.
A limitation of in vivo studies is the difficulty to separate epidermis and dermis to provide conclusive insights into intrinsic and extrinsic aging related gene expression in different skin levels and the respective cells. In vitro molecular analysis of cultured normal human dermal fibroblasts (NHDFs) isolated from intrinsically aged human skin from young subjects compared to middle aged and elderly donors have detected 998 proteins. From those, 70 were named skin aging-associated secreted proteins (SAASP) as they exhibited age-dependent secretion pattern. Their comparison with the senescence-associated secretory phenotype (SASP) revealed common aspects related to matrix degradation and proinflammatory processes. On the other hand, 27 proteins, involved in metabolism and adherence junction, were specific for intrinsic aged skin dermal fibroblasts and possibly related to specific process. Fibroblast from female breast skin, aged 20–30 and 60–70 years and keratinocytes from young foreskin were used to obtain a reconstructed human skin (RHS). Differences in gene expression including extracellular matrix proteins, growth factors and IL-6 were detected. The influence of fibroblasts in epidermal differentiation and stratum corneum lipid production was evident. Compared to young foreskin the stratum corneum was thicker in adult and aged RHS; E-cadherin levels were reduced and filaggrin expression was increased in adult and aged RHS; the amount of barrier lipids were also increased in adult and aged RHS.
It is extremely difficult to compare results from in vivo genomic studies as multiple parameters may lead to differences in study population. However, coincident DEGs detected represent a relevant contribution to the knowledge of skin aging, in particular the role of environmental aggressors along with the study of networks predicted to be impacted according to gene expression profile. Our results add additional information about the role of sun exposure on skin aging process as well as help identify potential new targets for its control.