Evolution of Cell-ECM Crosstalk and Biomechanics During Arterial Aging

Introduction: Depending on surrounding cells, substrate mechanics, and biochemical signaling, vascular cell types – including smooth muscle cells (VSMC) – modulate their behavior to maintain short- and long-term vascular homeostasis. Aging perturbs this homeostasis, resulting in SMC dysfunction marked by pathological ECM remodeling and loss of contractile protein expression. The overall goal of this project is to obtain an integrated understanding of the molecular links between functional cellular behavior and mechanics of the vasculature and its key constituents (i.e. cells and ECM). Given that these constituents define overall in vivo stiffness, this will enable us to identify effective targets and ages for clinical intervention. Materials and Methods: Male C57Bl6/J mice aged 3mo, 6mo, 9mo, or greater than 18mo were assessed for in vivo cardiovascular function via blood pressure (BP) and pulse wave velocity (PWV). Intact and decellularized aortic segments from each age group were then subject to tensile testing to gauge bulk tissue stiffness. Mouse aortic smooth muscle cells (maSMCs) were isolated from each group and characterized for mechanical and phenotypic profile. Cell mechanics were measured using magnetic twisting cytometry/spontaneous nanoscale tracer motions (MTC/SNTM) and atomic force microscopy (AFM). Fluorescent collagen substrates were used to assess type I collagen (Col-I) integration and degradation in 2D cell culture. Cells of each age were further treated with MMP inhibitor, LOXL2 inhibitor or TGF-β1 to measure the impacts on Col-I turnover, as well as expression of relevant matricellular proteins and their associated transcripts. Results: Aortic tissue shows a steady increase in stiffness beginning at early middle-age. The aged vasculature is also more brittle, failing at lower strain values. These changes in bulk tissue mechanics are mediated by dysregulated cellular behavior, including increased expression of matrix crosslinking proteins (such as TG2) and increased cell stiffness. Collagen integration by isolated maSMCS peaks at 9mo, while collagen degradation progressively decreases with age. Treatment with LOXL2 inhibitor effectively limits collagen integration in all age groups. MMP inhibitor treatment causes a marked decrease in Col-I cleavage, though this effect is muted in the oldest maSMCs which show impaired Col-I cleavage even in the absence of MMPi. TGF-β1 treatment increases expression of stress fibers, indicating a transition to a contractile phenotype. Conclusions: There is a progressive decrease in enzymatic cleavage of Col-I by resident maSMCs with age. Col-I integration, however, is at its highest during middle-age. This implies that bulk tissue stiffening in old age is driven in large part by accumulation of crosslinked collagens that are unable to be cleared. Early in life however, changes in inherent cellular stiffness and/or cell-ECM adhesion are likely drivers of short-term tissue stiffening. Together, these data suggest EMA/middle-age is the most suitable time for clinical intervention, given that this is when cellular dysregulation begins to produce discernable increases to aortic stiffness. Financial and academic support has been provided by the NASEM Ford Foundation Fellowship, the Porter Physiology Development Fellowship and NHBLI grant R01HL148112 01 (L.S.). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.