Evidence for 15-HETE synthesis by human umbilical vein endothelial cells.

Abstract

Incubation of cultured human umbilical vein endothelial cells with -4C]-arachidonic acid, followed by RP-HPLC analysis, resulted in the appearance of two principal radioactive products besides 6-keto-PGF,. The first peak was HHT, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by GC/MS and was shown to be the lipoxygenase product 1 5-HETE. Stimulation of endothelial cells with thrombin enhanced 15-HETE synthesis from arachidonate. Subsequent experiments showed that 5-HETE and 12-HETE were also synthesized by endothelial cells, but no evidence of leukotriene synthesis was found. Incubation of the 15-HETE precursor 1 5-HPETE with endothelial cells resulted in the formation of four distinct ultraviolet light-absorbing peaks. Ultraviolet and GC/MS analysis showed these peaks to be 8, 15-diHETEs that differed only in their hydroxyl configuration and cis-trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14,15-LTA4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-LTA4. Circulation 72, No. 4, 708-712, 1985. PG12* is the principal prostaglandin biosynthesized by cultured human umbilical vein endothelial cells in response to thrombin, histamine, A-23 187, trypsin, and ATP. LTC4 and LTD4 were shown to stimulate PGI2 biosynthesis in human endothelial cells, and agonist-specific desensitization to LTC4 was also observed.6. 7 Although leukotrienes are known to stimulate prostaglandin biosynthesis in endothelial cells, no physical evidence of arachidonate lipoxygenation by endothelial cells has appeared. In this report we present evidence that human umbilical vein endothelial cells do contain a 15-lipoxygenase.

Elevated glucose alters eicosanoid release from porcine aortic endothelial cells.

“…The data shown in Fig. 4 and Tables I and II demonstrate that porcine endothelial cells produce both 5-and 15-HETE, as has been previously indicated for human umbilical endothelial cells (5,6). The decreased net release of arachidonic acid was evident after 5 min and was striking after 30 min.…”

“…Both elevated platelet TXA2 and diminished vascular prostacyclin (PGI2) synthesis have been reported (1,2). In addition to cyclooxygenase-derived products, both platelets and endothelial cells produce hydroxyeicosatetraenoic acids (HETEs),1 metabolized by the lipoxygenase pathway of arachidonic acid oxygenation (3)(4)(5)(6)(7). Several studies have suggested that these HETEs may influence the production of PGI2 (7,8).…”

Expression of mRNA for Phospholipase A₂, Cyclooxygenases, and Lipoxygenases in Cultured Human Umbilical Vascular Endothelial and Smooth Muscle Cells and in Biopsies from Umbilical Arteries and Veins

“…Thus, whether or not a constitutive production of LTA 4 and the other leucotrienes occur in these cells is controversial at present. In aortic and other arterial EC from various animals, 5-, 12-, 15-HETE and LTB 4 have been identified, demonstrating a constitutive expression of 5-, 12-and 15-LOX activity in EC at these locations [14,15,37,38]. However, the presence of 5-LOX in HUVEC has not been reported.…”

Metabolic interactions between eicosanoids in blood and vascular cells

“…In addition to lymphocytes, erythrocytes represent another example of cells virtually devoid of dioxygenase activity (Schrier, 1967;Kobayashi & Levine, 1983), which are however able to generate lipid mediators through cell-cell interactions. Further metabolism of LTA4 into peptidoleukotriene, LTC4 [(5S) -hydroxy -(6R) -S-glutathionyl -7,9,11,14eicosatetraenoic acid], by vascular endothelium, which is unable to produce LTA4 itself (Gorman et al, 1985;Miller et al, 1985), has been described (Feinmark & Cannon, 1986). Similar conversion has been observed in platelets, which also fail to synthesize LTA4 (Maclouf & Murphy, 1988).…”