Vascular adhesion molecule 1+ fibro-adipogenic progenitors mark fatty infiltration in chronic limb-threatening ischemia
Bibliographic record
Abstract
Background Skeletal muscle health and function are critical determinants of clinical outcomes in peripheral arterial disease (PAD). Chronic limb threatening ischemia (CLTI), the most severe clinical manifestation of peripheral arterial disease (PAD), is associated with a one-year amputation rate of 25%. In CLTI patients, myosteatosis, the ectopic deposition of adipocytes, is independently associated with amputation. The mechanisms responsible for myosteatosis in CLTI patients remain unknown. In this study, we aim to identify both the causal cellular population and the molecular mechanisms in CLTI patients 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 sub-populations, we used an in-vitro adipogenesis assay; myosteatosis was determined by Oil Red O (ORO), perilipin, and Pparγ 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 utilized a publicly available human CLTI single cell RNA-sequencing (scRNA-seq) dataset. Results Bulk-RNA sequencings and Oil red O (ORO) staining reveal myosteatosis as a hallmark feature of the CLTI limb. Bioinformatic analyses reveal Vcam1+ fibro-adipogenic progenitors (FAPs) to be a pro-adipogenic cluster. Vcam1+ FAPs display increased adipogenic potential compared to Vcam1- FAPs (ORO staining, p<0.001; perilipin staining, p<0.01; Ppary staining, p<0.05). Analyses of bulk and scRNA-seq 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 (scATAC-seq) 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<0.01) and impaired adipogenic differentiation (ORO staining, p<0.01; perilipin staining, p<0.05; Ppary, p<0.001). Single-cell transcriptomic profiling of paired non-ischemic and ischemic muscle specimens from CLTI patients displayed enriched gene expression of Vcam1 (p = 5.24e -166 , log2FC=0.89), Sfrp1 (p = 0, log2FC=1.49) and Nr3c1 (p = 0.047, 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 CLTI patients and provide a potential therapeutic target for myosteatosis in CLTI patients.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.002 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".