Transcriptomic and epigenetic assessment of ageing female skin and fibroblasts identifies age related reduced oxidative phosphorylation is exacerbated by smoking

Skin ageing has been widely associated with the formation and presence of increasing quantities of senescent cells, the presence of which are thought to reduce cell renewal. This study aimed to identify key factors influencing fibroblast and skin aging using RNA-seq data. Key differences in study designs included known sources of biological differences (sex, age, ethnicity), experimental differences, and environmental factors known to accelerate skin ageing (smoking, UV exposure) as well as study specific batch effects which complicated the analysis. To overcome these complications samples were stratified by these factors and differential expression assessed using Salmon and CuffDiff. Stratification of female fibroblast and skin samples combined with female specific normalisation of transcriptomic and methylation data sets increased functional enrichment and consistency across studies. The results identify the importance of considering environmental factors known to increase the rate of ageing (smoking status of donors, and UV-exposure status of skin and fibroblast samples) both independently and in combination for the identification of key ageing signatures. The results identified that in old (> 65) female skin decreases in the expression of transmembrane ion transporters coincide with increased methylation of oxidoreductases, and consequently reductions in respiration. This was further evidenced in old fibroblasts from smokers which identified reductions ion homeostasis, and the transcription of mitochondrial tRNAs, that were accompanied by reduced mitochondrial fission, reduced lipid catabolism and reduced immune signalling. These changes occurred in combination with reductions in cell proliferation, adhesion, ECM organisation, cell movement, cytoskeleton organisation and circulatory system development. Middle and old aged skin without environmental stratification’s identified decreased expression of transmembrane ion transporters occurred alongside reductions in keratinisation, reduced mitochondrial fission, and this was associated with reduced metabolism (specifically carbohydrates), and consequently a reduction in the production of lipids (phospholipids for membranes and others) occured, exacerbating ion homeostasis issues at a keratinocyte level. Interestingly in skin the combined impacts of UV-exposure, smoking and ageing yielded different results, increased expression of calcium homeostasis genes, cell adhesion molecules (integrins), structural membrane constituents (loricrin, mucins, keratins and collagens), increased cornification, as well as structural cytoskeletal molecules (KRTAPs). This occurred alongside increased expression of genes involved in skin peeling (kalikriens), proliferation and differentiation, glycosylation, oxidative stress, autophagy, lactose metabolism, and lipid catabolism. Aged UV-exposed skin from smokers is on the whole more fibrous, with cells showing significant cell membrane and cytoskeletal structural changes, similar to those seen in skin cancers. Interestingly in non-UV-exposed skin from smokers most of these processes were reduced, and in within age group comparisons of smokers they were also reduced, suggesting that smoking reduced skin development and regeneration. Female specific analysis of smokers from different age groups enrichment results identified additional factors relating to tissue development, cell adhesion, vasculature development, peptide cross-linking, calcium homeostasis, cancer and senescence, leading to age related declines skin structure and function. Interestingly many diseases and infections with overlapping molecular consequences, (ER Ca2+ stress, reduced protein targeting to membranes) including human cytomegalovirus and herpes simplex virus are identified by the age only analysis, suggesting that viral infections and ageing have similar molecular consequences for cells. ### Competing Interest Statement The authors have declared no competing interest.