OxLDL induces IL-1β release from human EC and VSMC via different caspase-1 dependent mechanisms.

Atherosclerosis is characterised by disturbed blood flow due to abnormal lipid and cell accumulation within arterial layers. Modified cholesterol forms such as oxidised low-density lipoprotein (oxLDL) enter cells altering their phenotype, triggering over exuberant repair and arterial occlusion, myocardial infarction or stroke. We hypothesised that oxLDL enters vascular wall cells and induces IL-1β secretion, potentially via a caspase-1/NLRP3 mechanism. Human coronary artery endothelial cells (HCAEC) and smooth muscle cells (VSMC), isolated from different donors, were cultured and stimulated (primed) with pro-inflammatory cytokines TNFα and IL-1α (10 ng/ml each, for 48 hours), followed by incubation with human oxLDL (10-50ug/ml) for - up to 6 hours. Inhibitors of Caspase-1 (YVAD), NLRP3 (MCC950) and Gasdermin D (Disulfiram) were added 1h before oxLDL. Cell lysates and culture supernatants were collected and analysed for IL-1β using ELISA. Microscopy imaging showed oxLDL entered stimulated cells and formed particles. OxLDL at 20 and 50 ug/ml induced the maximum release of IL-1β from stimulated HCASMCs and HCAECs respectively compared to control. Inhibition of either NLRP3, Caspase-1 or Gasdermin D significantly reduced the release of IL-1β (4-fold, p<0.0001; 14-fold, p<0.0001, 1.5-fold, p<0.0003 respectively) in HCAEC. In contrast, in HCASMCs, only Caspase-1 inhibition reduced release of IL-1β (2.1-fold, P<0.0001). HCAECs and HCASMCs elicited release of IL-1β in response to the same stimulus via different mechanisms. In HCAECs, released IL-1β potentially exits via a GSDMD-induced membrane pore. These data suggest that caspase-1 or gasdermin D inhibition are likely to be effective vessel wall cell specific strategies for the reduction of atherosclerosis.