This study aims to explore the effects of arachidonic acid lipoxygenase metabolism in vascular calcification. We used 5/6 nephrectomy and high-phosphorus feeding to establish a model of vascular calcification in mice. Six weeks after nephrectomy surgery, vascular calcium content was measured, and Alizarin Red S and Von Kossa staining were applied to detect calcium deposition in aortic arch. Control aortas and calcified aortas were collected for mass spectrometry detection of arachidonic acid metabolites, and active molecules in lipoxygenase pathway were analyzed. Real-time quantitative PCR was used to detect changes in the expression of lipoxygenase in calcified aortas. Lipoxygenase inhibitor was used to clarify the effect of lipoxygenase metabolic pathways on vascular calcification. The results showed that 6 weeks after nephrectomy surgery, the aortic calcium content of the surgery group was significantly higher than that of the sham group (P < 0.05). Alizarin Red S staining and Von Kossa staining showed obvious calcium deposition in aortic arch from surgery group, indicating formation of vascular calcification. Nine arachidonic acid lipoxygenase metabolites were quantitated using liquid chromatography/mass spectrometry (LC-MS) analysis. The content of multiple metabolites (12-HETE, 11-HETE, 15-HETE, etc.) was significantly increased in calcified aortas, and the most abundant and up-regulated metabolite was 12-HETE. Furthermore, we examined the mRNA levels of metabolic enzymes that produce 12-HETE in calcified blood vessels and found the expression of arachidonate lipoxygenase-15 (Alox15) was increased. Blocking Alox15/12-HETE by Alox15 specific inhibitor PD146176 significantly decreased the plasma 12-HETE content, promoted calcium deposition in aortic arch and increased vascular calcium content. These results suggest that the metabolism of arachidonic acid lipoxygenase is activated in calcified aorta, and the Alox15/12-HETE signaling pathway may play a protective role in vascular calcification.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Animals ; Arachidonate 12-Lipoxygenase ; Arachidonate 15-Lipoxygenase/metabolism* ; Arachidonic Acid ; Hydroxyeicosatetraenoic Acids ; Lipoxygenase/metabolism* ; Mice ; Signal Transduction ; Vascular Calcification

Arachidonic acids (AA) widely exist in multiple organs and can be metabolized into small lipid molecules with strong biological functions through several pathways. Among them, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), which are produced by cytochrome P450 enzymes, have attracted a lot of attentions, especially in vascular homeostasis. The regulation of vascular function is the foundation of vascular homeostasis, which is mainly achieved by manipulating the vascular structure and biological function. In the past 30 years, the roles of EETs and 20-HETE in the regulation of vascular function have been widely explored. In this review, we discussed the effects of EETs and 20-HETE on angiogenesis and vascular inflammation, respectively. Generally, EETs can dilate blood vessels and inhibit vascular inflammation, while 20-HETE can induce vasoconstriction and vascular inflammation. Interestingly, both EETs and 20-HETE can promote angiogenesis. In addition, the roles of EETs and 20-HETE in several vascular diseases, such as hypertension and cardiac ischemia, were discussed. Finally, the therapeutic perspectives of EETs and 20-HETE for vascular diseases were also summarized.
Arachidonic Acid ; Arachidonic Acids ; Cytochrome P-450 Enzyme System ; Humans ; Hydroxyeicosatetraenoic Acids ; Hypertension ; Vasoconstriction

Pulmonary arterial hypertension (PAH) is a rare disease with a complex aetiology characterized by elevated pulmonary artery resistance, which leads to progressive right ventricular failure and ultimately death. The aberrant metabolism of arachidonic acid in the pulmonary vasculature plays a central role in the pathogenesis of PAH. The levels of 15-lipoxygenase (15-LO) and 15-hydroxyeicosatetraenoic acid (15-HETE) are elevated in the pulmonary arterial endothelial cells (PAECs), pulmonary smooth muscle cells (PASMCs) and fibroblasts of PAH patients. Under hypoxia condition, 15-LO/15-HETE induces pulmonary artery contraction, promotes the proliferation of PAECs and PASMCs, inhibits apoptosis of PASMCs, promotes fibrosis of pulmonary vessels, and then leads to the occurrence of PAH. Here, we review the research progress on the relationship between 15-LO/15-HETE and hypoxic PAH, in order to clarify the significance of 15-LO/15-HETE in hypoxic PAH.
Arachidonate 15-Lipoxygenase ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; Humans ; Hydroxyeicosatetraenoic Acids ; Hypoxia ; Myocytes, Smooth Muscle ; Pulmonary Arterial Hypertension ; Pulmonary Artery

12-HETE is a metabolite of arachidonic acid (AA). AA is normally present in membrane phospholipids. The exposure to different stimuli can trigger the release of AA through the activity of phospholipase A₂ (PLA₂) by cells. An important metabolic pathway which utilizes AA as its substrate is 12-Lipoxygenase (12-LOX), resulting in the formation of 12-HETE. 12-HETE plays an important role in many diseases such as cancer, diabetes, hypertension, and participates in the pathogenesis of inflammation and oxidative stress and other pathological processes.Current research shows that it participates in metamorphism and exudation in the process of inflammation. This review is aimed at summarizing its role in inflammation and oxidative stress, with improved understanding of 12-HETE.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Arachidonic Acid ; Humans ; Inflammation ; Oxidative Stress

Atopic dermatitis, one of the most important skin diseases, is characterized by both skin barrier impairment and immunological abnormalities. Although several studies have demonstrated the significant relationship between atopic dermatitis and immunological abnormalities, the role of hydroxyeicosatetraenoic acids (HETE) in atopic dermatitis remains unknown. To develop chiral methods for characterization of 12-HETE enantiomers in a 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model and evaluate the effects of 12-HETE on atopic dermatitis, BALB/c mice were treated with either DNCB or acetone/olive oil (AOO) to induce atopic dermatitis, after which 12(R)- and 12(S)-HETEs in the plasma, skin, spleen, and lymph nodes were quantified by chiral liquid chromatography-tandem mass spectrometry. 12(R)- and 12(S)-HETEs in biological samples of DNCB-induced atopic dermatitis mice increased significantly compared with the AOO group, reflecting the involvement of 12(R)- and 12(S)-HETEs in atopic dermatitis. These findings indicate that 12(R)- and 12(S)-HETEs could be a useful guide for understanding the pathogenesis of atopic dermatitis.
Animals ; Biomarkers/blood/metabolism ; *Chromatography, Liquid ; Dermatitis, Atopic/*chemically induced ; Dinitrochlorobenzene/adverse effects ; Female ; Humans ; Hydroxyeicosatetraenoic Acids/blood/*metabolism ; Irritants/adverse effects ; Mice ; Mice, Inbred BALB C ; Models, Animal ; *Tandem Mass Spectrometry

Skin exposure to low-dose ultraviolet B (UVB) light up-regulates the expression of matrix metalloproteinase-1 (MMP-1), thus contributing to premature skin aging (photo-aging). Although cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been associated with UVB-induced signaling to MMP expression, very little are known about the roles of lipoxygenases and their products, especially leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), in MMP-1 expression in skin keratinocytes. In the present study, we demonstrate that BLT2, a cell surface receptor for LTB4 and 12(S)-HETE, plays a critical role in UVB-mediated MMP-1 upregulation in human HaCaT keratinocytes. Moreover, our results demonstrated that BLT2-mediated MMP-1 upregulation occurs through a signaling pathway dependent on reactive oxygen species (ROS) production and the subsequent stimulation of ERK. Blockage of BLT2 via siRNA knockdown or with the BLT2-antagonist LY255283 completely abolished the up-regulated expression of MMP-1 induced by low-dose UVB irradiation. Finally, when HaCaT cells were transiently transfected with a BLT2 expression plasmid, MMP-1 expression was significantly enhanced, along with ERK phosphorylation, suggesting that BLT2 overexpression alone is sufficient for MMP-1 up-regulation. Together, our results suggest that the BLT2-ROS-ERK-linked cascade is a novel signaling mechanism for MMP-1 upregulation in low-dose UVB-irradiated keratinocytes and thus potentially contributes to photo-aging.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/biosynthesis ; Cell Line ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Keratinocytes/metabolism/*radiation effects ; Leukotriene B4/biosynthesis ; Matrix Metalloproteinase 1/*biosynthesis ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*physiology ; Signal Transduction ; Ultraviolet Rays/*adverse effects

We have reported that hypoxia increases the activation of 15-lipoxygenase (15-LO), which converts arachidonic acid (AA) into 15-hydroxyeicosatetraenoic acid (15-HETE) in small pulmonary arteries (PAs). Through inhibition of Kv channels, 15-HETE causes more robust concentration-dependent contraction of PA rings from the hypoxic compared to the normoxic controls. However, the subtypes of Kv channels inhibited by 15-HETE are incompletely understood. The aim of the present study was to identify the contribution of Kv3.4 channel in the process of pulmonary vasoconstriction induced by 15-HETE using the tension studies of PA rings from rat with Kv3.4 channel blocker in tissue bath; to explore the role of vascular endothelium in15-HETE-induced pulmonary vasoconstriction through denuded endothelia of PA rings; and to define the downregulation of 15-HETE on the expression of Kv3.4 channel in cultured pulmonary artery smooth muscle cells (PASMCs) with RT-PCR and Western blot. In the present study, healthy Wistar rats were divided randomly into two groups: Group A with normal oxygen supply and group B with hypoxia. Six days later, the rats were killed. Pulmonary artery rings were prepared for organ bath experiments. Firstly, different concentrations of 15-HETE (10~1 000 nmol/L) were added to the Krebs solution. The isometric tension was recorded using a four-channel force-displacement transducer. Then Kv3.4 channel blocker, 100 nmol/L BDS-I, was added, followed by adding 1 mumol/L 15-HETE, and the isometric tension was recorded. Furthermore, RT-PCR and Western blot were employed to identify the influence of 15-HETE on the expression of Kv3.4 channel in cultured rat PASMCs.The results showed the PA tension was significantly increased both in groups A and B by 15-HETE in a concentration-dependent manner (P<0.05), especially in group B (P<0.05 compared to control); denuded endothelia enhanced 15-HETE concentration-related constrictions in rat PA rings; Kv3.4 channel blocker, BDS-I, significantly decreased the PA ring constriction induced by 15-HETE (P<0.05); the expressions of Kv3.4 mRNA and protein in rat PASMCs were significantly downregulated by 15-HETE (P<0.05). Based on all the information above, we conclude that Kv3.4 channel is involved in vasoconstriction induced by 15-HETE in rat PAs.
Animals ; Cells, Cultured ; Female ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypertension, Pulmonary ; physiopathology ; Hypoxia ; physiopathology ; Male ; Muscle, Smooth, Vascular ; cytology ; pathology ; Pulmonary Artery ; cytology ; physiopathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Shaw Potassium Channels ; antagonists & inhibitors ; genetics ; metabolism ; Vasoconstriction ; drug effects

AIMTo observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.

METHODSIn the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.

RESULTSBlocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.

CONCLUSIONThe results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Animals ; Cell Hypoxia ; Cells, Cultured ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypoxia ; physiopathology ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Pulmonary Artery ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Shab Potassium Channels ; biosynthesis ; genetics ; Vasoconstriction ; drug effects

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.
Animals ; Cattle ; Down-Regulation ; drug effects ; Endothelium, Vascular ; cytology ; drug effects ; enzymology ; Hydroxyeicosatetraenoic Acids ; pharmacology ; In Vitro Techniques ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Pulmonary Artery ; cytology ; enzymology ; physiology ; Rats ; Rats, Wistar

Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.
Animals ; Flavonoids ; pharmacology ; Hydroxyeicosatetraenoic Acids ; antagonists & inhibitors ; pharmacology ; Hypoxia ; physiopathology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; Pulmonary Artery ; cytology ; drug effects ; physiopathology ; Rats ; Rats, Wistar ; Vasoconstriction ; drug effects