Aim: Coronary artery disease and its sequelae myocardial infarction and heart failure are the most common cause of death in humans worldwide. Chronic kidney disease (CKD) is a major risk factor for the development of atherosclerotic plaques. In CKD uremic toxins, such as Indoyxlsulfate (IS) accumulate in the blood and trigger vascular inflammation and endothelial dysfunction. Underlying molecular mechanisms how IS impairs endothelial regeneration are only incompletely understood. Latest research has identified long-non-coding RNAs (lncRNA) to be important mediators of cardiovascular disease development.

Methods: Human coronary artery endothelial cells (HCAECs) were subjected to 250µM IS for 24h. Migration, proliferation and monocyte adhesion were assessed by use of a scratch-wound assay, BrdU-Incorporation assay and a THP-1 cell adhesion experiment. For total RNA sequencing 750 ng of total RNA were used with the Next Ultra II Directional RNA Kit in a NovaSeq 6000 with a SP 200 Flowcell and a read length of 2x100bp. For a pilot cohort, patients undergoing routine coronary angiography at University Hospital of Bonn were recruited. Molecular analyses were performed from citrate plasma. For single qPCR analyses Taqman assays from ThermoFisher Scientific were used.

Results: IS injury causes activation of HCAECs leading to increased monocyte adhesion (A) and reduced migration and proliferation. Transcriptomic of analysis of HCAECs upon IS-injury revealed 363 differentially regulated transcripts (B). Functional annotation clustering by use of DAVID showed that the clusters involved in proliferation were downregulated, while inflammatory clusters were upregulated (C). Among the differentially regulated transcripts, we identified 16 lncRNAs, of which MIR503HG, MIR3142HG and HIF1A-AS3 were the most significantly regulated ones. The differential regulation was of MIR3142HG and HIF1A-AS3 was confirmed by use of single qPCR analyses (D). As the role of HIF1A-AS2/3 has already been explored in endothelial cell biology, we focused on MIR3142HG for further analyses. In a pilot cohort of 9 patients with and without CKD, we assessed peripheral blood levels of MIR3142HG (F). We found that MIR3124HG levels were inversely correlated with the estimated glomerular filtration rate (G). This indicates that patients with impaired kidney function had higher levels of MIR3124HG in the blood.

Conclusions: The lncRNA MIR3142HG is upregulated in endothelial cells after IS-injury and exhibits higher blood levels in individuals with lower glomerular filtration rate. MIR3124HG is therefore an interesting target for further investigation of CKD associated endothelial injury. Further experiments to identify potential molecular interaction partners as well as loss-of-function experiments of MIR3142HG are needed to investigate the molecular role of MIR3142HG. Furthermore, the results in our pilot cohort will need to be validated in a larger patient cohort.