Vascular adhesion molecule 1+ fibro-adipogenic progenitors mark fatty infiltration in chronic limb-threatening ischemia

Background

Skeletal muscle health and function are critical determinants of clinical outcomes in peripheral arterial disease. Chronic limb-threatening ischemia (CLTI), the most severe clinical manifestation of peripheral arterial disease, is associated with a 1-year amputation rate of 25%. In patients with CLTI, myosteatosis—the ectopic deposition of adipocytes—is independently associated with amputation. The mechanisms responsible for myosteatosis in patients with CLTI remain unknown. In this study, we aim to identify both the causal cellular population and the molecular mechanisms in patients with CLTI that promote myosteatosis.

Methods

To identify a candidate causal cell type and putative signaling axis that promotes myosteatosis, we performed single cell transcriptomic and chromatin accessibility profiling of ischemic muscle in a preclinical CLTI model. To assess the adipogenic potential for candidate subpopulations, we used an in vitro adipogenesis assay; myosteatosis was determined by Oil Red O (ORO), perilipin, and peroxisome proliferator-activated receptor gamma (PPAR-γ) staining. To determine the necessity of candidate transcriptional and epigenetic regulators, we used a small interfering RNA (siRNA). Finally, to assess the clinical significance of our findings, we used a publicly available human CLTI single cell RNA-sequencing dataset.

Results

Bulk-RNA sequencings and ORO staining reveal myosteatosis as a hallmark feature of the CLTI limb. Bioinformatic analyses reveal vascular adhesion molecule 1 (Vcam1)⁺ fibro-adipogenic progenitors (FAPs) to be a proadipogenic cluster. Vcam1⁺ FAPs display increased adipogenic potential compared with Vcam1⁻ FAPs (ORO staining, P < .001; perilipin staining, P < .01; PPAR-γ staining, P < .05). Analyses of bulk and single cell RNA-sequencing datasets identify Sfrp1 as a regulator of Vcam1⁺ FAP adipogenic differentiation. In vitro inhibition of Sfrp1 with a siRNA demonstrated impaired Vcam1⁺ FAP adipogenic differentiation. Single cell ATAC sequencing identifies Nr3c1 as a candidate transcription factor that regulates Vcam1⁺ FAP adipogenic differentiation. In vitro inhibition of Nr3c1 with a siRNA demonstrated decreased Sfrp1 expression (P < .01) and impaired adipogenic differentiation (ORO staining, P < .01; perilipin staining, P < .05; PPAR-γ, P < .001). Single cell transcriptomic profiling of paired nonischemic and ischemic muscle specimens from patients with CLTI displayed enriched gene expression of Vcam1 (P = 5.24e⁻¹⁶⁶; log2FC = 0.89), Sfrp1 (P = 0; log2FC = 1.49) and Nr3c1 (P = .047; log2FC = 0.050) in ischemic CLTI muscle tissues. Altogether, these data reveal a candidate signaling axis, Nr3c1-Sfrp1, that regulates the differentiation of Vcam1⁺ FAPs into adipocytes.

Conclusions

Collectively, our results identify a pro-adipogenic FAP subpopulation in patients with CLTI and provide a potential therapeutic target for myosteatosis in patients with CLTI.

Clinical Relevance

Myosteatosis, the pathological accumulation of fat within skeletal muscle, is increasingly recognized as a critical determinant of adverse clinical outcomes in peripheral arterial disease (PAD). Current therapies for PAD focus on revascularization and risk factor modification, but do not directly target myopathy. There is a critical need for regenerative and cellular therapies to restore muscle integrity and function. This study addresses this need by identifying a candidate causal cellular population, Vcam1⁺ fibroadipogenic progenitors, and a signaling axis, Nr3c1-Sfrp1, that promote myosteatosis in the ischemic limb.