IMPACT OF AGING ON MAMMALIAN INTRA- AND EXTRA-CELLULAR ENVIRONMENTS

Aging is a physiological process characterized by a gradual decline in cell, tissue, and organ functions. Although a significant amount of information is available on the senescence-related processes that take place in the cellular compartment, little is known about the role of the extracellular matrix (ECM) and its changes. Given the close relationship between the ECM and the cells residing within it, as well as the well-known ability of cells to interact with the surrounding matrix, it can be hypothesized that age-related modifications in the extracellular microenvironment might compromise cell behaviour, affecting tissue homeostasis and causing organ dysfunctions. This thesis has the specific aims to: -study the role of the ECM in cell differentiation. -identify age-related ECM changes. -generate age-specific ECM-based bio-scaffolds. -assess whether age-related ECM modifications may influence cell behaviour. -take advantage of ECM-derived biomechanical cues to develop/support novel rejuvenating strategies. I adopted the porcine ovary as experimental model, based on its complex microanatomy and heterogenous architecture, and used a decellularization protocol previously developed in the Laboratory hosting my PhD activities to generate ECM-based bio-scaffolds. These were then repopulated with different sets of cells, namely a) freshly isolated porcine ovarian cells, b) high-plasticity cells and c) cells previously rejuvenated with soluble factors. The obtained results demonstrate that aging directly impact on the ovarian ECM that shows significant changes in its composition and organization with collagen, glycosaminoglycans, and laminins significantly incremented, and elastin, as well as fibronectin, decreased. This is accompanied by a dynamic response in the expression levels of key ECM and protease-related genes, suggesting a direct impact of aging on the transcriptional machinery. The ECM-based 3D bio-scaffolds generated preserved the structural changes occurring in vivo demonstrating their ability to act as a powerful high predictive in vitro model for reproductive aging and its prevention. When ECM-based scaffolds were repopulated, they were able to properly drive the differentiation, fate and viability of all cells tested. In particular, aged cells previously rejuvenated with miRNA or young cell-derived factors stably maintained the newly acquired phenotype when engrafted onto young ECM-based scaffolds. This suggests that an adequate young environment is able to stabilize miRNA and EV-mediated rejuvenation and implies a synergistic interaction among molecular effectors and ECM-derived bio-mechanical stimuli. Altogether, these data indicate that the experimental model here developed may represent a useful tool to finely dissect the several biochemical and biomechanical cues driving tissue and organ aging.