Passage state affects arachidonic acid content and eicosanoid release in porcine aortic endothelial cells.

Abstract

Porcine aortic endothelial cells were cultured through four passages from primary cultures. The arachidonic acid content of individual phospholipid classes and the release of 6-keto-prostaglandin F la and 15-hydroxyeicosatetraenoic acid in response to 1 /tM ionophore A23187 were assayed at each passage. The content of arachidonic acid in phosphatidylinositol and diacyl phosphatidylethanolamine remained constant at passage 1 but declined at passage 2 by approximately 29% and at passage 4 by approximately 59%. The release of 6-keto-prostaglandin F la was also unchanged at passage 1 but decreased by 60% at passage 2 and by 82% from its original value at passage 4. In contrast, the arachidonic acid content of diacyl phosphatidylcholine and of alkenyl phosphatidylethanolamine decreased with each passage, by 34% at passage 1, 59% at passage 2, 71% at passage 3, and 76% of the original value at passage 4. Stimulation with arachidonic acid reversed the passage effect. The release of 15-hydroxyeicosatetraenoic acid decreased by 82% at passage 1 and diminished to a 97% decrement from the original value by passage 4. When stimulated with arachidonic acid, 15-hydroxyeicosatetraenoic acid steadily decreased by approximately 70% at passages 3 and 4. The data indicate that passage state strikingly and nonuniformly affects phospholipid class arachidonic acid content and eicosanoid release in response to agonist stimulation. (Arteriosclerosis and Thrombosis 1991;11:167-173) E ndothelial cells possess the ability to release metabolites of arachidonic acid, including prostacyclin (PGI 2 ) and 15-hydroxyeicosatetraenoic acid (15-HETE). PGI 2 , a product of the cyclooxygenase pathway, is a powerful vasodilator and a potent inhibitor of platelet aggregation. Its release provides at least one mechanism for maintaining a nonthrombogenic vascular surface. 15-HETE is the monohydroxylated metabolite of arachidonic acid produced by 15-lipoxygenase. The role of 15-HETE as a vascular autacoid has not been fully established, but it does modulate cellular lipid metabolism, influencing phospholipase A 2 activity, 1 leukotriene synthesis, 2 and PGI 2 synthesis.

Effect of elevated glucose on cyclic GMP and eicosanoids produced by porcine aortic endothelium.

“…In cultured endothelial cells it has been demonstrated that arachidonic acid content can decrease as much as 66% over the first four passages, and this can markedly decrease eicosanoid production. 24 This factor alone could explain the difference found in our cells, which were grown for only two passages. Also, tissues from diabetics and cells grown for several passages with serum, unlike our cells, which underwent a brief exposure to elevated glucose in PSS, are exposed to insulin, which can itself influence eicosanoid release.…”

Induction of Monocyte Binding to Endothelial Cells by MM-LDL

“…The low level of prostaglandin release most likely relates to the fact that these cells were used at passage 8 as others have shown a steady loss of cyclooxygenase products in passaged EC. 24 In the cytoplasm most of the oxidized product within the cells was 12-HETE with lower levels of 11,12-EET. It should be noted that although the product identified as 11,12-EET migrates with authentic 11,12-EET in our HPLC system, it has not been chemically identified as 11,12-EET.…”

Effects of long-term serial cell passaging on cell spreading, migration, and cell-surface ultrastructures of cultured vascular endothelial cells

“…For example, reduced proliferative capacity, increased oxidative stress, activation of the NFj-B and p53 signaling pathways (Lee et al 2010), reduced nitric oxide production and surfactant protein D (SP-D) expression (Lee et al 2009) were detected in porcine coronary arterial endothelial cells after multiple passaging in vitro. Enhanced apoptosis of porcine pulmonary artery endothelial cells (Zhang et al 2002) and declined arachidonic acid content and eicosanoid release in porcine aortic endothelial cells in response to agonist stimulation (Brown and Deykin 1991) were determined as well. The effects of cell passaging on endothelin-1 and prostaglandin F2a production of bovine luteal endothelial cells (Acosta et al 2007) and on the responsiveness of bovine aortic endothelial cells to orbital shear stress (Kudo et al 2005) were also evaluated.…”