OxLDL induces the release of IL-1β from primed human endothelial and smooth muscle cells via different caspase -1-dependent mechanisms

Abstract

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.

MALT1 accelerates proatherogenic vascular smooth muscle cell growth, invasion and synthetic phenotype switching via nuclear factor‑κB signaling‑dependent way

“…It has been reported that oxLDL, one of the major members of lipidemia, is a main culprit of atherosclerosis and is involved in the formation, progression and rupture of atherosclerotic lesions (22). In addition, oxLDL is widely used in preclinical studies to mimic atherosclerotic conditions (23)(24)(25). In the current study, oxLDL was also used to establish a proatherogenic VSMC model.…”

“How to Release or Not Release, That Is the Question.” A Review of Interleukin‐1 Cellular Release Mechanisms in Vascular Inflammation

“… 53 We have shown recently that for primed coronary artery ECs incubated for 6 hours with oxLDL, IL‐1β was released via a nonvesicular canonical caspase‐1, NLRP3‐dependent mechanism. 54 Interestingly, primed VSMC treated with oxidized LDL (for just 2 hours) also released IL‐1 to a similar extent, but through a caspase‐1, non‐NLRP3‐dependent pathway. 54 This contrast with the mechanisms of IL‐1β release was also shown by calcium phosphate deposits, which stimulate IL‐1β release from human VSMCs by activation of SYK, caspase‐1 cleavage, and release in part through exosomes.…”

“… 54 Interestingly, primed VSMC treated with oxidized LDL (for just 2 hours) also released IL‐1 to a similar extent, but through a caspase‐1, non‐NLRP3‐dependent pathway. 54 This contrast with the mechanisms of IL‐1β release was also shown by calcium phosphate deposits, which stimulate IL‐1β release from human VSMCs by activation of SYK, caspase‐1 cleavage, and release in part through exosomes. 55 Exosomes also seem to be responsible for IL‐1β release in primed human coronary artery ECs after neutrophil elastase treatment.…”