Abstract Body: Background: Atherosclerosis-associated Peripheral Artery Disease (PAD) is a leading cause of cardiovascular mortality, driven by SMC dedifferentiation into proliferave and migratory phenotypes. Despite its role in numerous vascular disorders, the mechanisms regulating SMC dedifferentiation remain unclear, limiting effective treatment development.
Methods: Using single nucleus RNA and ATAC sequencing in femoral arteries from controls (n=9) and patients with PAD (n=11), we elucidated the epigenetic and transcriptional changes characterizing PAD to identify potential disease targets. Immunofluorescence (IF) of “early” and “advanced” PAD plaques confirmed the spatial localization of the identified target. Using well-characterized small-molecule modulators of the identified target, we performed functional assays in primary smooth muscle cells (SMCs) to evaluate the impact of target modulation on SMC phenotypic transition. To confirm these findings in vivo, a mouse model of hind limb ischemia (HLI), was used to examine the effects of target inhibition on vascular function.
Results: From our integrative multi-omics analysis, we identified a disease-enriched dedifferentiated SMC subpopulation characterized by enhanced extracellular matrix (ECM) remodeling, named SMC-ECM. Differential expression of genes in PAD versus control ECM-SMCs
pinpointed Cathepsin B (CTSB), a lysosomal protease involved in the ECM proteolysis. IF analysis of human PAD plaques revealed co-localization of CTSB with ACTA2, a key SMC marker. Moreover, CTSB expression increased with plaque progression from early to advanced stages. Using a selective inhibitor of CTSB activity (CA-074me, 0.1 μM), we observed reduced Platelet Derived Growth Factor (PDGF)ββ-induced SMC proliferation and migration. To further investigate effects of CTSB inhibition in vivo, we employed the HLI model in 10-weeks old wild-type mice. Following induction of HLI, animals were treated with the CTSB inhibitor (4 mg/kg, intramuscular) or vehicle control for 7, 14, or 21 days. Laser Doppler imaging performed at each time point revealed improved blood flow recovery in CTSB inhibitor-treated animals compared with controls, indicating enhanced perfusion and functional vascular recovery.
Conclusions: Our analysis identified PAD-associated SMC states establishing a valuable resource for the investigation of new therapeutic strategies to reduce the progression of vascular dysfunction and atherosclerosis in human PAD.