CYP2J34 is expressed, but not regulated by toll-like receptor (TLR) ligands in porcine peripheral blood mononuclear and vascular smooth muscle cells.

Michael Davies1, Emma Milford1, Helen Slater1, Scott Thomson1, Gillian Cockerill2, Dirk Werling1, David Bishop-Bailey1. 1Royal Veterinary College, London, UK, 2St George's University London, London, UK

Epoxygenases are specific CYP450 enzymes that convert fatty acids into epoxy-oxylipins e.g. arachidonic acid in to epoxy-eicosatrienoic acids (EETs) (1). Epoxygenase products are considered protective in cardiovascular disease. We and others have shown a role for CYP2J2 in vascular and inflammatory cells. However, CYP450 in particular show a great deal of species variability. Pigs are a commonly used large animal model to study cardiovascular disease. We have therefore examined the expression and regulation of CYP2J34 (the homologue of CYP2J2) in porcine vascular smooth muscle cells and peripheral blood mononuclear cells (PBMCs).

Vascular smooth muscle from carotid vessels (pCASMC) were grown by explant (2). Porcine blood was collected by venupuncture and collected in to citrate. Human leukocyte cones were obtained from the NHS Blood Bank. pPBMCs were isolated by density centrifugation and cultured in RPMI media supplemented with FCS (10%) and antibiotics (1%). pCASMC, or pPBMCs (passage 2-3) were either left unstimulated or stimulated with the TLR4 agonist LPS (10ng/ml) or a panel or TL ligands for 4h, and mRNA levels for CYP2J34, TNFα and the housekeeping gene β-actin measured by qRT-PCR.

CYP2J34 mRNA was expressed in pCASMC (Fig 1) and pPBMCs (Fig 2). In contrast to human PBMCs, where LPS (100ng/ml) induces CYP2J2 (9±3 fold ; p<0.05 one sample test), LPS did not induce CYP2J34 in either pCASMC or pPBMCs. LPS was active in porcine cells as TNFα mRNA was strongly induced in pCASMC (Figure 1; p<0.05 one sample t-test; data are mean±sem from n=3 experiments). Similar to LPS, no other TLR ligand tested TLR1/2, PAM3 100ng/ml, TLR2, HKLM at 108 cells/ml, TLR3, Poly I:C and Poly I:C LMW at 1μg/ml, TLR4, LPS at 100ng/ml, TLR5, flagellin, 0.01ng/ml, TLR6 FSL-1 0.100ng/ml, TLR7 imiquimod 250ng/ml or TLR8 ssRNA 250ng/ml) significantly effected CYP2J34 expression in pPBMCs (Figure 2); data are mean±sem from n=4 animals.

Here we show for the first time that pCASMC and pPBMC contain the epoxygenase CYP2J34, the porcine homologue of human CYP2J2. Unlike CYP2J2, CYP2J34 does not appear to be induced by LPS (TLR-4) or other TLR ligands.

1. Bishop-Bailey D et al., (2014) Annu Rev Nutr. 34: 261-79.

2. Bishop-Bailey D et al., (1998) Arterioscler Thromb Vasc Biol. 18:1655-61.