Schaller, Melinda S.

Background: Peripheral arterial disease (PAD) is a manifestation of systemic atherosclerosis characterized by chronic inflammation and immune dysregulation. Single-cell RNA sequencing (scRNA-seq) studies of human atherosclerotic plaques have identified distinct transcriptional programs within plaque-associated immune cells. We sought to determine whether transcriptional features described within these plaque-resident cells can be identified in peripheral blood mononuclear cells (PBMCs). Methods: PBMCs from patients with PAD (n = 4) and healthy controls (HC) (n = 5) were profiled using scRNA-seq using the Parse Biosciences platform, yielding approximately 135,000 high-quality cells. Data analysis was conducted in R, using Seurat for quality control, dimensionality reduction and clustering. Cell-type annotation was performed using Azimuth reference-based mapping. Plaque-associated inflammatory and metabolic gene programs were curated from published human plaque single-cell atlases. Results: Gene expression differences between HC and PAD cells were visualized using UMAP plots. CD14+ mononuclear phagocytes (MNPs) exhibited the most prominent and consistent transcriptional differences between HC and PAD samples (Figure 1). Within CD14+ MNPs, genes involved in lipid handling and macrophage metabolic function demonstrated a higher median percentage of expressing cells in PAD compared with HC (Figure 2). Further analysis of AQP9 and CD36, genes implicated in macrophage metabolic activation and lipid uptake in atherosclerotic plaques, with z-score normalization and subject-level averaging revealed modest but consistent increases in relative expression in PAD across subjects. These findings support the relevance of these circulating transcriptional shifts to PAD pathophysiology. Conclusions: Circulating CD14+ MNPs in PAD have detectable transcriptional features consistent with potential engagement of plaque-associated myeloid activation programs. In PAD, increased expression of CD36, which encodes the immune cell scavenger receptor for oxidized LDL, and AQP9, associated with unstable plaques and the endothelial-to-mesenchymal transition, suggest altered immunometabolism states in circulating MNPs. Although effect sizes are modest, they are consistent across subjects and statistically robust. These findings highlight PBMC scRNA-seq as a minimally invasive approach to interrogate systemic immune programs relevant to PAD pathobiology.

Introduction: Atherosclerotic arterial disease is the leading cause of death throughout the world. One of the most advanced forms of atherosclerosis, peripheral arterial disease (PAD), affects over 8.5 million people in the United States and over 230 million globally. This disease begins from an inflammatory lesion, known as the fatty streak, formed by mononuclear phagocytes (MNPs), a broad cell type classification that includes monocytes, dendritic cells, and macrophages. Development of more efficacious treatments for PAD requires that we better understand the cellular and molecular processes by which MNPs form these inflammatory lesions. Methods: To compare gene expression of MNPs, we performed scRNAseq on PBMCs from an initial cohort of 4 patients with PAD, mild claudicants to minor tissue loss (Rutherford 1-5), and 5 healthy individuals, defined as those without any chronic diseases and a hsCRP <2.0 mg/L. We conducted single cell RNA library generation using Parse Bio and sequenced to a depth of 70k reads per cell and downstream bioinformatic and differentially gene expression analysis via R and Seurat. Results: Gene expression differences in healthy versus PAD cells were visualized on a UMAP plot. Aligning our cells to the human PBMC Cell Atlas, we found that the cell type with the greatest transcriptional change (healthy vs. PAD) was CD14 + MNPs. Next, we screened for differentially expressed genes within this CD14 + MNP cluster between healthy and PAD subjects and identified 854 upregulated and 730 downregulated genes (log 2 fold change ≥ 0.25 and p < 0.05, Welch’s t-test). Two of the most significantly upregulated genes in PAD CD14 + MNPs are FAM20A and TMTC1. FAM20A encodes a psuedokinase important in calcium homeostasis as well as bone mineralization and that is upregulated in advanced atherosclerotic plaques. TMTC1 encodes for an endoplasmic reticulum protein also involved in calcium homeostasis, vascular homeostasis, and endothelial cadherin egress. Finally, one gene we found downregulated in PAD is CD83, a molecule implicated in resolution of inflammation, that when downregulated promotes an inflammatory state. Conclusion: Based on these findings we hypothesize that in PAD there exists a distinct transcriptional program along the CD14 + MNP cell type lineage that is pro-inflammatory and that may play a role in disease progression and response to intervention.