Long Noncoding RNA Regulation Of Inflammation In Atherosclerosis

Atherosclerosis is a chronic inflammatory disease driven by cholesterol deposition in the artery wall. Resolution of atherosclerotic inflammation and plaque regression are important clinical goals. As long noncoding RNAs (lncRNAs) are key regulators of gene expression, there is an intense interest in understanding their contribution in atherosclerosis and their potential as therapeutic targets to induce plaque regression. To this end, we performed single cell RNA-sequencing of CD45 + immune cells isolated from aortic arch plaques of Ldlr -/- mice under conditions of plaque progression (16 weeks Western diet) or regression (16 weeks Western diet + 4 weeks apoB antisense oligonucleotide), and identified lncRNAs that were differentially expressed (DE) under these conditions. We then classified lncRNA candidates based on their proximity to protein coding genes (intergenic, antisense, intronic, bidirectional) and screened for lncRNAs acting in cis to locally regulate the expression of neighboring genes. We identified 112 DE lncRNAs with proximal protein-coding genes (<5kb) whose mRNAs were also DE in blood and plaque macrophages during atherosclerosis progression and regression, including Caslr ( C tsc a nti s ense l nc R NA) , a novel lncRNA transcribed from the opposite strand of the Cathepsin C gene ( Ctsc ) that encodes a lysosomal protease implicated in atherogenesis. The Caslr-Ctsc pair is upregulated in macrophages of progressing plaques and in response to pro-inflammatory stimuli [ i.e ., lipopolysaccharide, interleukin (IL)-6] in vitro , and conversely, downregulated during atherosclerosis regression and in response to anti-inflammatory ( i.e. , IL-4) stimuli. Loss-of-function approaches revealed that Caslr regulates Ctsc expression and protease activity, with downstream consequences on activation of pro-inflammatory NF-κB signaling and inflammasome-dependent IL-1β release. Notably, Caslr shares functional homology with a lncRNA proximal to the human CTSC gene. Collectively, our data identify Caslr as a conserved lncRNA driving expression of cathepsin C and downstream inflammatory signaling in macrophages, and suggest that, targeting this lncRNA may be beneficial in treating atherosclerosis.