15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo.

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10-7 M) stimulated human retinal microvessel endothelial cell migration by 42 +/- 10% (mean +/- S.E.M., p less than 0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization in vivo. Concentrations at which the in vivo effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration in vitro and neovascularization in vivo suggests that it may play a role in vasoproliferative disorders.     

Induction of Endothelial Cell Differentiation in Vitro by Fibroblast-Derived Soluble Factors

Recent studies have suggested that fibroblasts, widely distributed mesenchymal cells, not only function to sustain various organs and tissues as stroma cells but also act directly to regulate adjacent cell behavior including migration, proliferation, and differentiation. Since fibroproliferative diseases and lesions (fibroplasia) are accompanied by new capillary growth (angiogenesis), we hypothesized that fibroblasts may have direct effects on endothelial cell behavior, independent of the elaboration of extracellular matrix, that are relevant to complex process of angiogenesis. To test this hypothesis, bovine aortic endothelial cells were cocultured in collagen gels with human skin fibroblasts. This coculture system caused the endothelial cells to become spindle shaped and to organize into a capillary-like structure within the collagen gels. We found that fibroblast-conditioned medium (FCM) also induced endothelial cells initially to elongate and subsequently to organize into a capillary-like structure within collagen gels. While FCM had no significant effect on endothelial cell DNA synthesis, the soluble factor(s) in FCM increased endothelial cell motility in an in vitro wound assay and in a Boyden chamber assay. The chemoattractant(s) in FCM was alkaline (pH 9.0)—and acid (pH 3.0)—stable, relatively heat stable (stable at 60°C for 30 min, unstable at 98°C for 3 min), dithiothreitol (DTT)-sensitive, and bound to an anionic exchange resin (DEAE-cellulose). Another factor(s) stimulated endothelial cell reorganization into capillary-like structure both within a collagen gel and on a reconstituted basement membrane matrix, Matrigel. This factor(s) was alkaline (pH 9.0)—and acid (pH 3.0)—stable, heat (98°C for 3 min)stable, and DTT-sensitive and bound an anionic exchange resin (DEAE-cellulose). These in vitro results suggest that fibroblasts secrete soluble factors that can influence endothelial cell behaviors relevant to the angiogenesis process with possible implications for vascularization in fibroproliferative conditions.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI₂) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 μM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI₂ formation. The inhibition was not specific for PGI₂; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-¹⁴C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI₂ formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI₂.     

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2^−/− mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.     

12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5×10⁻⁸ − 5×10⁻⁶ M) was ineffective on melatonin release, in the presence of absence of NE (5×10⁻⁶ M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5×10⁻⁷ −5×10⁻⁵ M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occuring at 10⁻⁶ M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 uM ionophore, MC9 cells produce 270 ± 43 pmoles 5-HETE, 74 ± 40 pmoles 5,12 di HETEs and 65 ± 31 pmoles LTC₄/10⁶ cells from 37 uM exogenously added [1-¹⁴C]arachidonic acid in two minutes. 5-HETE and 5,12-di HETES, including LTB₄ were identified by GC/MS whereas LTC₄ was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD₂ and TxB₂ (8.5 ± 2.4 and 5.4 ± 1.2 pmoles/10⁶ cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca⁺² and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 ± 3% cellular histamine) and continued for 30 minutes (37 ± 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Tumor necrosis factor alpha stimulates arachidonic acid metabolism in human osteoblastic osteosarcomal cells

The effects of tumor necrosis factor alpha (TNF-α) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [³H]AA. TNF-α differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-α (10⁻⁸ M) significantly increased the cyclooxygenase pathway, with prostaglandin E₂ (PGE₂) being a major product. However, at the lowest concentration (10⁻¹⁰ M) of TNF-α, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-α was increased to 10⁻⁹ M leukotriene B₄ (LTB₄), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10⁻⁶ M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-α (10⁻⁸ M) and A23187 (10⁻⁶ M) caused an inhibitory effect on each agent-induced PGE₂ or 15-HETE production.     

Enhancement of the HIF-1α/15-LO/15-HETE Axis Promotes Hypoxia-Induced Endothelial Proliferation in Preeclamptic Pregnancy

Preeclampsia (PE) is an extremely serious condition in pregnant women and the leading cause of maternal and fetal morbidity and mortality. Despite active research, the etiological factors of this disorder remain elusive. The increased release of 15-hydroxyeicosatetraenoic acid (15-HETE) in the placenta of preeclamptic patients has been studied, but its exact role in PE pathogenesis remains unknown. Mounting evidence shows that PE is associated with placental hypoxia, impaired placental angiogenesis, and endothelial dysfunction. In this study, we confirmed the upregulated expression of hypoxia-inducible factor 1α (HIF-1α) and 15-lipoxygenase-1/2 (15-LO-1/2) in patients with PE. Production of the arachidonic acid metabolite, 15-HETE, also increased in the preeclamptic placenta, which suggests enhanced activation of the HIF-1α–15-LO–15-HETE axis. Furthermore, this study is the first to show that the umbilical cord of preeclamptic women contains significantly higher serum concentrations of 15-HETE than that of healthy pregnant women. The results also show that expression of 15-LO-1/2 is upregulated in both human umbilical vein endothelial cells (HUVECs) collected from preeclamptic women and in those cultured under hypoxic conditions. Exogenous 15-HETE promotes the migration of HUVECs and in vitro tube formation and promotes cell cycle progression from the G0/G1 phase to the G2/M + S phase, whereas the 15-LO inhibitor, NDGA, suppresses these effects. The HIF-1α/15-LO/15-HETE pathway is therefore significantly associated within the pathology of PE.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. 1.¹⁴C linoleic acid was converted to 1.¹⁴C hydroxy octadecadienoic acids (HODES) at about the same rate with which 1.¹⁴C 12-HETE was produced from 1.¹⁴C arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by GC-MS. The production of HODES by intact washed platelets was inhibited by indomethacin (IC50:5×10⁻⁷M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10⁻³M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10⁻⁵M) and baicalein (10⁻⁵M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Jasmonic acid inhibition of in vitro shoot organogenesis in Pinus radiata cotyledons

The effect of jasmonic acid (JA) onde novo shoot formation in excised cotyledons of radiata pine (Pinus radiata D. Don), was examined. JA had no effect on shoot-forming (SF) tissues at concentrations up to 10⁻⁶ mol · L⁻¹. At concentrations greater than that, JA caused a reduction in the number of shoots formed, as well as the lengths of the shoots and fresh and dry masses of the tissues. Reciprocal transfer of excised radiata pine cotyledons from a SF medium with 10⁻⁵ mol · L⁻¹ JA to a SF medium without JA and vice versa showed that any exposure of the cotyledons to JA either during the shoot induction phase (days 0-21) or the shoot development phase (beyond day 21) led to a reduction in shoot formation. However, the JA effect was significantly less if the cotyledons were not exposed to JA during the initial 10 days in culture; indicating that the JA effect was mainly during shoot primordia formation and the subsequent development into shoots.     

Activation of ERK pathway is required for 15-HETE-induced angiogenesis in human umbilical vascular endothelial cells

Angiogenesis plays a critical role in the progression of cardiovascular disease, retinal ischemia, or tumorigenesis. The imbalance of endothelial cell proliferation and apoptosis disturbs the establishment of the vasculogenesis, which is affected by several arachidonic acid metabolites. 15-Hydroxyeicosatetraenoic acid (15-HETE) is one of the metabolites. However, the underlying mechanisms of angiogenesis induced by 15-HETE in human umbilical vascular endothelial cells (HUVECs) are still poorly understood. Since extracellular signal-regulated kinase (ERK) is a critical regulator of cell proliferation, there may be a crosstalk between 15-HETE-regulating angiogenic process and ERK-proliferative effect in HUVECs. To test this hypothesis, we study the effect of 15-HETE on cell proliferation, angiogenesis, and apoptosis using cell viability measurement, cell cycle analysis, western blot, scratch–wound, tube formation assay, and nuclear morphology determination. We found that 15-HETE promoted HUVEC angiogenesis, which were mediated by ERK. Moreover, 15-HETE-induced proliferation and cell cycle transition from the G₀/G₁ phase to the G₂/M?+?S phase. All these effects were reversed after blocking ERK with PD98059 (an ERK inhibitor). In addition, HUVEC apoptosis was relieved by 15-HETE through the ERK pathway. Thus, ERK is necessary for the effects of 15-HETE in the regulation of HUVEC angiogenesis, which may be a novel potential target for the treatment of angiogenesis-related diseases.     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

The effects of a diet rich in fish oil on human neutrophils: Identification of leukotriene B5 as a metabolite

Diets that are enriched with fish oil have been shown to alter arachidonic acid metabolism via the cyclooxygenase pathway. Recently it has been shown that one of the major component fatty acids of fish oil, eicosapentaenoate (EPA), is a substrate for the leukotriene B (LTB) pathway when added exogenously to human neutrophils . We fed a diet that contained 8–10 gm/day of EPA to four human subjects for three weeks and compared the arachidonate metabolism of their neutrophils to the same functions while the subjects were on their usual diet. The fish oil-supplementation increased neutrophil EPA content from undetectable levels to 7.4 ± 2.4% (p<0.01, expressed as % of total fatty acid), and decreased arachidonate from 15.4 ± 2.3% to 12.8 ± 2.3% (p<0.05). Leukotriene B₅ was identified as a metabolite during the fish oil-diet by its chromatographic profile and mass spectrum. During the experimental diet LTB₄ decreased from 160 ± 37 ng/10⁷ neutrophils to 120 ± 12 (p<0.05), and LTB₅ increased from 0 to 39 ± 9 ng/10⁷ neutrophils (p<0.005). The diet had no effect on neutrophil aggregation or adherence to nylon fibers.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

Vascular Endothelial Tight Junctions and Barrier Function Are Disrupted by 15(S)-Hydroxyeicosatetraenoic Acid Partly via Protein Kinase C?-mediated Zona Occludens-1 Phosphorylation at Threonine 770/772

Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCϵ-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCϵ and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO^−/− mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction.     

Arachidonylethanolamide (anandamide) binds with low affinity to dihydropyridine binding sites in brain membranes

The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [³H]PN-200. AEA competed for [³H]PN-200 binding with a K₁ of 40 ± 4 μM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K₁ of AEA (48 ± 6 μM). Arachidonic acid had a slightly higher K₁ (120 ± 11 μM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-γ-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [³H]PN-200 binding at concentrations as high as 10 μM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.     

Differential involvement of TGF-beta1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1–10 µg/ml) and inhibition at high concentrations (25–100 µg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5–25 µg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to αvβ3 integrin. TGF-β1 (0.1–200ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-β1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-β1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-β1, whereas the effects on fibroblast and tumour cell migration are TGF-β1-independent.     

Ferulic acid augments angiogenesis via VEGF,PDGF and HIF-1α

Therapeutic angiogenesis is critical to wound healing and ischemic diseases such as myocardial infarction and stroke. For development of therapeutic agents, a search for new angiogenic agents is the key. Ferulic acid, a phytochemical found in many fruits and vegetables, exhibits a broad range of therapeutic effects on human diseases, including diabetes and cancer. This study investigated the augmenting effect of ferulic acid on angiogenesis through functional modulation of endothelial cells. Through endothelial cell migration and tube formation assays, ferulic acid (10^?6–10^?4 M) was found to induce significant angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro without cytotoxicity. With chorioallantoic membrane assay, ferulic acid (10^?6–10^?5 M) was also found to promote neovascularization in vivo. Using Western blot analysis and quantitative real-time polymerase chain reaction, we found that ferulic acid increased vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression in HUVECs. Furthermore, the amounts of hypoxic-induced factor (HIF) 1α mRNA and protein, the major regulator of VEGF and PDGF, also showed up-regulation by ferulic acid. Electrophoretic migration shift assay showed that the binding activity of HIF-1α was also enhanced with ferulic acid treatment of HUVECs. Moreover, inhibitors of extracellular-signal-regulated kinase 1/2 and phosphoinositide-3 kinase (PI3K) abolished the binding activity of HIF-1α and the subsequent activation of VEGF and PDGF production by ferulic acid. Thus, both mitogen-activated protein kinase and PI3K pathways were involved in the angiogenic effects of ferulic acid. Taken together, ferulic acid serves as an angiogenic agent to augment angiogenesis both in vitro and in vivo. This effect might be observed through the modulation of VEGF, PDGF and HIF-1α.