Collapsed Fibroblasts in Aged Human Skin in vivo - A Critical Driver of Dermal Aging

Skin aging is characterized by structural and functional disruptions of the collagen-rich extracellular matrix (ECM). The impact of these ECM alterations on the morphology and function of dermal fibroblasts, the cells primarily responsible for ECM homeostasis, remains incompletely understood. To address this issue, we used quantitative three-dimensional (3D) multiphoton fluorescence microscopy with second-harmonic generation to determine the structure of fibrillar collagen bundles and the morphology of fibroblasts in young and aged human skin. 3D imaging reveals that, in contrast to the young dermis, the ECM in aged dermis exhibits significant deterioration, associated with reduced fibroblast attachment and a contracted, "collapsed" fibroblast morphology. This altered morphology is accompanied by altered function, as indicated by single-cell RNA sequencing and multiplex spatial in situ transcriptomics. Notably, two of the major pathways in fibroblasts that regulate ECM homeostasis, TGF-β/Smad and YAP/TAZ, are impaired, as is expression of ECM-related genes. Primary human fibroblast culture models with a collapsed morphology exhibit similar impairments in TGF-β/Smad and YAP/TAZ signaling and ECM homeostasis. Remarkably, restoring fibroblast spreading fully normalizes disrupted collagen homeostasis. These findings highlight the importance of "outside-in" adaptation of dermal fibroblasts to age-related ECM deterioration and emphasize the role of the extracellular microenvironment in cellular aging.