Abstract 14080: LINC02397 in Skeletal Muscle: A Marker of, and Therapeutic Target for, Chronic Limb Threatening Ischemia

Abstract

Background: Chronic limb threatening ischemia (CLTI) is the most severe complication of peripheral arterial disease (PAD) and the leading cause of amputations for US adults. The response to reduced blood flow within ischemic muscle plays a role in determining disease outcome in PAD, but the response mechanisms have not been fully elucidated. Recently, long non-coding RNAs (lncRNAs) have been shown to regulate gene expression and modify disease. The objective of this study is to identify and characterize lncRNA that is differentially expressed in ischemic muscle in CLTI to serve as a therapeutic target in PAD. Methods: RNA sequencing (n=6/group) and whole transcriptome shotgun sequencing (WTSS n=15/group) from human muscle tissue in CLTI and healthy adults (HA) were performed to identify differentially expressed lncRNA candidates. qPCR (n=10/group) was performed on separate muscle samples to validate the findings. In-vitro experiments were conducted to examine which major cell type in muscle tissue likely expressed the candidate lncRNAs which were subsequently knocked down in cell types with the highest expression to determine function by qPCR, metabolic assay, and flow cytometry. Results: RNA-seq and WTSS analysis identified LINC02397, and qPCR confirmed LINC02397 was expressed significantly higher in CLTI patients (cT count 27.3±1.7 vs 34.1±1.4 in HA, p <0.0001). Differentiation of monocytes isolated from human PBMCs to macrophages (a process triggered by inflammatory stimuli) increased expression of LINC02397 by 2 4 , the highest expression level among major cell types in muscle. In monocytes (THP-1 cells), PMA treatment induced a macrophage-like phenotype, and LINC02397 expression increased in conditions that trigger inflammatory activation: PMA, LPS, and hypoxia serum starvation. LINC02397 knockdown significantly downregulated inflammatory cytokines IL-1β and IL-6, CD86+ cells, and glycolysis. LPS-activated monocyte media increased the proliferative capacity of endothelial cells after knockdown, suggesting potential promotion of angiogenesis. Conclusions: LINC02397 is uniquely expressed in skeletal muscle of CLTI patients and may serve as a promising therapeutic via its ability to limit inflammation and promote angiogenesis.