Abstract Body: Background: Atherosclerosis-associated Peripheral Artery Disease (PAD) is a leading cause of cardiovascular mortality, driven by SMC dedifferentiation into proliferative, migratory and synthetic phenotypes, contributing to vascular stiffness and fibrosis. Despite its role in numerous vascular disorders, the mechanisms regulating SMC dedifferentiation remain unclear. Therefore, targeting SMC transition can be a powerful therapeutic strategy to attenuate vascular dysfunction.
Methods: We used single nucleus RNA (snRNA) and ATAC (snATAC) sequencing performed in n=20 femoral artery segments from controls and patients with diagnosed PAD. Immunofluorescence analyses of “early” and “advanced” PAD plaques confirmed the spatial localization of identified target. Using well-characterized small-molecule inhibitor of the identified target, we performed functional assays in primary smooth muscle cells (SMCs). To confirm our findings in vitro, we also leveraged a mouse model of peripheral vascular injury, the hind limb ischemia (HLI) model.
Results: We identified a PAD-enriched SMC subpopulation characterized by enhanced extracellular matrix remodeling and mechanosensitive genes. Differential expression of PAD versus controls highlighted Cathepsin B (CTSB), a lysosomal protease involved in the proteolysis and remodeling of the extracellular matrix. Using human PAD tissues we found increased expression of CTSB during plaque progression from early to advanced stages. Using a selective inhibitor of CTSB activity, we observed reduced Platelet Derived Growth Factor (PDGF)ββ-induced SMC proliferation and migration. We further employed stiff (4 kPa) and soft (100 kPa) extracellular matrices to model vascular mechanical stress, and found that CTSB inhibition preserved the SMC phenotype, which is typically lost in stiff matrices. To investigate the effects in vivo, we employed the HLI model in wild-type mice. Following induction of HLI, animals were treated with the CTSB inhibitor (4 mg/kg, intramuscular) or vehicle for 7, 14, or 21 days. Laser Doppler imaging performed at each time point revealed improved blood flow recovery in CTSB inhibitor–treated animals versus controls, indicating enhanced perfusion and vascular recovery following CTSB inhibition.
Conclusions: Our analysis identified PAD-associated SMC states and pathways establishing a valuable resource for the identification of new therapeutic strategies to reduce the progression of vascular dysfunction in human PAD.