Introduction: Vascular smooth muscle cells (VSMCs) retain extraordinary plasticity throughout adulthood to maintain arterial homeostasis. Aging results in in VSMC dysregulation that is marked by loss of contractile markers and pathological extracellular matrix (ECM) remodeling. Lysyl oxidase-like 2 (Loxl2/LOXL2) is an ECM-crosslinking enzyme that is critical in the development and maintenance of cardiovascular tissue. Nonetheless, pathological remodeling by LOXL2 is implicated in numerous fibrotic diseases, which has made it a promising therapeutic target. Thus, we first sought to temporally resolve the age-related changes in LOXL2 crosslinking activity and its impacts on vascular mechanics. We then leveraged these results to assess the therapeutic potential of Loxl2/LOXL2 inhibition in vitro as well as in vivo using clinically relevant metrics. 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 were then subjected to tensile testing to gauge bulk tissue stiffness. Mouse aortic smooth muscle cells (maSMCs) were isolated from each group and probed for key ECM transcripts with or without the addition of TGF-β1. Fluorescent collagen substrates were then used to assess type I collagen (Col-I) degradation and incorporation in 2D cell culture. Cells were further treated with LOXL2 inhibitor to measure their impacts on Col-I turnover. Finally, a floxed/Loxl2 mouse model was generated to test the impact of Loxl2 deletion during middle-age (~6mo). These inducible Loxl2 KO animals were further aged (to 18mo) and subjected to in vivo cardiovascular assessment (BP and PWV) as well tensile testing to quantify changes in hemodynamic profile and arterial mechanics. Results: Aortic tissue shows a steady increase in stiffness beginning at early middle-age. Decellularized tissue shows a stepwise increase during the middle-age (6mo-9mo) transition. These increases are mediated by dysregulated cellular behavior, including increased expression of Loxl2 in older animals. There was age-dependent sensitization to TGF-β1, resulting in increased expression of ECM constituents and crosslinkers in older cells. Collagen integration by maSMCS peaks at 9mo, while degradation progressively decreases with age. Treatment with LOXL2 inhibitor limits collagen integration in maSMCs of all age groups. Temporal deletion of Loxl2/LOXL2 (in 6mo mice) was protective against arterial stiffening, as 18mo KO animals had more compliant aortas and lower BP than their WT counterparts. 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. Increased cellular expression of Loxl2 during this period suggest that is a primary driver of this development. Given than aged maSMCs exposed to TGF-β1 become more fibrotic than younger counterparts, we hypothesize and alterations in this signaling pathway play a crucial role in arterial stiffening, though further research is needed. Most notably, Loxl2/LOXL2 deletion in middle-aged mice effectively protected against increased blood pressure and tissue stiffness in aged animals. Together, these data identify the target (Loxl2) and timing (middle-age) for a therapy that mitigates age-related arterial stiffening. This abstract was presented at the American Physiology Summit 2026 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.
Brady et al. (Fri,) studied this question.