Abstract Body: INTRODUCTION
Chronic limb-threatening ischemia (CLTI), the most severe form of peripheral arterial disease (PAD), is characterized by persistent ischemia, inflammation, and microvascular dysfunction. There are no therapies for CLTI. We previously identified the long non-coding RNA LINC02397 as consistently upregulated in gastrocnemius muscle from CLTI patients compared with matched controls by RNA sequencing, which was subsequently validated by RT-PCR. Expression of LINC02397 was also confirmed in monocytes and macrophages.
HYPOTHESIS
We hypothesized that targeted knockdown of LINC02397 can attenuate inflammatory paracrine effects from macrophages.
METHODS
Functional studies were conducted in human THP1 cells under ischemia-mimicking (hypoxia and serum starvation, HSS) and inflammatory stimulation with lipopolysaccharide (LPS), with or without LINC02397 knockdown. Endothelial proliferation and permeability were assessed in human umbilical vein endothelial cells (HUVECs) treated with conditioned media from monocytes/macrophages under HSS conditions. Biotin-labeled LINC02397 RNA pulldown followed by mass spectrometry was performed to identify LINC02397-interacting proteins, with key interactions validated by Western blot. RNA immunoprecipitation (RIP) was used to identify ILF3-regulated target mRNAs.
RESULTS
RNA sequencing and pathway analysis demonstrated that the inflammatory response pathway was the most significantly downregulated pathway following LINC02397 knockdown in LPS-treated THP1 cells. Conditioned medium from LPS-polarized THP1 cells impaired endothelial cell proliferation and increased endothelial permeability under HSS conditions; these effects were significantly alleviated when LINC02397 was downregulated in THP1 cells.. Proteomic analysis comparing biotin-labeled sense and antisense LINC02397 RNA pulldowns identified ILF2 and ILF3 as the most differentially enriched interacting proteins, with associated enrichment of PRKRA and PKR. Western blot analysis of RNA pulldown samples confirmed preferential enrichment of ILF3 with sense LINC02397. RIP further demonstrated that the pro-inflammatory gene IL1B and the matrix-remodeling gene MMP9, both of which were downregulated following LINC02397 knockdown, are regulated by ILF3.
CONCLUSION
LINC02397 is a CLTI-associated lncRNA that modulates macrophage inflammatory signaling and influences endothelial function, highlighting its potential as a knockdown-based therapeutic target.