Abstract
BackgroundNonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. However, the mechanisms underlying these effects are still unknown. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca2+i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs).ResultsMyristic (MA), palmitic (PA), stearic (SA), oleic (OA) and linoleic acid (LA) rapidly increased Ca2+i. The calcium response to all tested NEFAs showed an extracellular calcium dependence and only the LA response was significantly inhibited until the intracellular calcium was chelated. The EC50 values for MA and LA were 125 μM and 37 μM, respectively, and the MA and LA effects were dependent on calcium release from the endoplasmic reticulum stores and on the L-type calcium channels. Only the calcium response to MA was significantly reduced by GW1100, a selective G-protein-coupled free fatty acid receptor (GPR40) antagonist. We also detected a functional FFAR1/GPR40 protein in BUVECs by using western blotting and the FFAR1/GPR40 agonist TAK-875. Only LA increased the cellular nitric oxide levels in a calcium-dependent manner. LA stimulation but not MA stimulation increased ICAM-1 and IL-8-expression in BUVECs. This effect was inhibited by GW1100, an antagonist of FFAR1/GPR40, but not by U-73122, a phospholipase C inhibitor.ConclusionsThese findings strongly suggest that each individual NEFA stimulates endothelial cells in a different way, with clearly different effects on intracellular calcium mobilization, NO production, and IL-8 and ICAM-1 expression in primary BUVECs. These findings not only extend our understanding of NEFA-mediated diseases in ruminants, but also provide new insight into the different molecular mechanisms involved during endothelial cell activation by NEFAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0654-3) contains supplementary material, which is available to authorized users.
Highlights
Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells
Acute treatment with NEFAs does not affect the viability of primary bovine umbilical vein endothelial cells (BUVECs) Cells exposed to 300 μM linoleic acid (LA), palmitic acid (PA), oleic acid (OA), myristic acid (MA), or stearic acid (SA) showed no significant difference in the propidium iodide signal for 15 min when compared with untreated cells
We demonstrated that 0.3 mM ethylene glycol-bis-(2-aminoethyl)-N (EGTA), 50 μM BAPTA-AM or each NEFA plus EGTA or BAPTA did not affect the viability of BUVECs
Summary
Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca2+i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs). The levels of nonesterified fatty acids (NEFAs) in bovine plasma increase during transition periods, such as after calving and during early lactation [1]. During these stages, a negative energy balance is generated, which is mainly characterized by an increased release of NEFAs from adipose tissues into the plasma [2]. The effects of NEFAs on bovine leukocytes and endothelial cells have been examined previously [1, 7, 8], the exact molecular mechanisms are still poorly understood
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