Aging impairs cutaneous healing through miR-133 mediated inhibition of regenerative pathways and increased cell senescence

Summary: Abstract Body: The skin serves a critical role in the protection of our body from the outside world, yet our ability to restore this barrier after injury greatly declines with age. Despite understanding the pathways influencing cutaneous regeneration, there is still a knowledge gap in how these factors change with age and whether they can be targeted therapeutically. To answer this, we used a wound-induced hair neogenesis (WIHN) mouse model to quantify regeneration. WIHN is a phenomenon where adult mammals undergo embryonic-like regeneration in large cutaneous wounds via the development of neogenic hair follicles (HFs) and functional, non-scarred skin. Young (4-week) and aged (91-week) C57BL/6J mice were given full-thickness dorsal cutaneous wounds of 1.25x1.25cm and 1.5x1.5cm, respectively. Aged mice took twice as long to re-epithelialize wounds (p<0.0005) and had a 20-fold decrease in neogenic HFs (p<0.0005). mRNA sequencing and qPCR of re-epithelialized wounds showed that young mice had an increase in pro-regenerative markers LGR6, Wnt7B, SHH, and TLR3 (p<0.005 for all). Conversely, aged mice had a 7-fold increase in senescence marker p53 (p <0.001) and an 3-fold increase in p16 (p<0.0005). miRNA sequencing revealed a 16-fold (p<0.0005) increase of miR-133 in aged mice. Treatment of human keratinocytes with miR-133 partially phenocopied aging through reduced expression of regenerative markers LGR6 (p<0.005), Axin2 (p<0.05), and TLR3 (p<0.0005), while increasing expression of senescence markers p53 (p<0.0005) and p16 (p<0.0005). Treatment of young mice with miR-133 resulted in a 5-fold decrease in neogenic HF formation (p<0.0005) as well as a decrease in the expression of LGR6 (p<0.05), Wnt7B (p<0.005) and an increase in p16 (p<0.05). Inhibition of miR-133 in aged mice resulted in a 3-fold increase (p<0.005) in neogenic HFs.These results elucidate the molecular pathways of wound healing that change with aging and provide a potential therapeutic target through miR-133 to improve the regenerative capacity of the skin. Christian Hopkins¹, Kevin Y. Yang¹, Seray Er¹, Jennifer Lee¹, Sashank Reddy¹ 1. Plastic and Reconstructive Surgery, Johns Hopkins Medicine, Baltimore, MD, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing