Abstract Body (Do not enter title and authors here): Background: Over 1 million people undergo coronary artery bypass grafting annually worldwide with approximately 20,000 procedures performed annually in the UK. The most commonly used conduit, the long saphenous vein (LSV) suffers 50% occlusion within 10 years post-surgery due to the development of vein graft disease (VGD). VGD occurs as the result of abnormal migration and proliferation of smooth muscle cells leading to intimal hyperplasia, accompanied by TGFβ1-mediated progressive fibrosis. The Ca2+-activated K+ channel KCa3.1 (encoded by KCNN4) known to regulate TGFβ1-dependent pro-fibrotic activity has previously not been assessed as a novel target for the prevention of VGD and it’s the focus of the study.

Hypothesis: K_Ca3.1-dependent cell processes are one of the common denominators in VGD and blocking K_Ca3.1 (using senicapoc) might serve as a therapeutic candidate for the prevention of VGD.

Methods: HUVECs and surplus LSV segments were subjected to high acute shear stress (HSS) and acute arterial haemodynamics (12dyn/cm²) respectively using an in vitro or ex-vivo model perfusion bioreactor. Changes in marker expression at the RNA and protein level were evaluated using spatial scRNAseq, qRT-PCR and immunofluorescence.

Results: The exposure of HUVECs to HSS upregulated expression of phosphorylated NF-κB, MAPK-P38 and SMAD at 45 mins and pro-inflammatory mediators such as MCP-1, IL-8 as well as KCa3.1 at 4 hrs time point. These markers were significantly suppressed with senicapoc pre-treatment. Data from the first-in-man spatial RNA sequencing in vein segments exposed to acute arterial haemodynamics demonstrated the activation of pro-inflammatory and endothelial-mesenchymal transition (EndMT) mediators including CXCL8, CCL2, TWIST2 as well as KCa3.1. Network analysis for KCa3.1 showed a direct connection to different genes including EMP3, PELZO1, PANX1, IL1B, IL6 and TNFα. This non-targeted sequencing data was validated using qRT-PCR in LSV. The exposure of LSV to acute arterial haemodynamics was associated with significant upregulation of KCa3.1, MCP-1 and IL-8 mRNA at 4 h and the upregulation of KCa3.1 mRNA was also associated with increased protein expression. These molecular changes were significantly modulated when LSV were pre-treated with senicapoc.

Conclusion: The expression of K_Ca3.1 is upregulated in LSV following acute arterial haemodynamics and targeting KCa3.1 using senicapoc can reduce VGD by modulating inflammation and EndMT.