15-hydroxy-5-8-11-13-eicosatetraenoic-acid and Hypertension--Pulmonary

Pulmonary arterial hypertension (PAH) is a rare disease with a complex aetiology characterized by elevated pulmonary artery resistance, which leads to right heart ventricular afterload and ultimately progressing to right ventricular failure and often death. In addition to other factors, metabolites of arachidonic acid cascade play an important role in the pulmonary vasculature, and disruption of signaling pathways of arachidonic acid plays a central role in the pathogenesis of PAH. 15-Lipoxygenase (15-LO) is upregulated in pulmonary artery endothelial cells and smooth muscle cells of PAH patients, and its metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) in particular seems to play a central role in the contractile machinery, and in the initiation and propagation of cell proliferation via its effects on signal pathways, mitogens, and cell cycle components. Here, we focus on our important research into the role played by 15-LO/15-HETE, which promotes a proliferative, antiapoptotic, and vasoconstrictive physiological milieu leading to hypoxic pulmonary hypertension.

Topics: Animals; Apoptosis; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Muscle, Smooth, Vascular; Vasoconstriction

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased mean pulmonary arterial pressure. Elevated plasma and lung concentrations of oxidized lipids, including 15-hydroxyeicosatetraenoic acid (15-HETE), have been demonstrated in patients with PAH and animal models. We previously demonstrated that feeding mice with 15-HETE is sufficient to induce pulmonary hypertension, but the mechanisms remain unknown. RNA sequencing data from the mouse lungs on 15-HETE diet revealed significant activation of pathways involved in both antigen processing and presentation and T cell-mediated cytotoxicity. Analysis of human microarray from patients with PAH also identified activation of identical pathways compared with controls. We show that in both 15-HETE-fed mice and patients with PAH, expression of the immunoproteasome subunit 5 is significantly increased, which was concomitant with an increase in the number of CD8/CD69 (cluster of differentiation 8 / cluster of differentiation 69) double-positive cells, as well as pulmonary arterial endothelial cell apoptosis in mice. Human pulmonary arterial endothelial cells cultured with 15-HETE were more prone to apoptosis when exposed to CD8 cells. Cultured intestinal epithelial cells secreted more oxidized lipids in response to 15-HETE, which is consistent with accumulation of circulating oxidized lipids in 15-HETE-fed mice. Administration of an apoA-I (apolipoprotein A-I) mimetic peptide, Tg6F (transgenic 6F), which is known to prevent accumulation of circulating oxidized lipids, not only inhibited pulmonary arterial endothelial cell apoptosis but also prevented and rescued 15-HETE-induced pulmonary hypertension in mice. In conclusion, our results suggest that (1) 15-HETE diet induces pulmonary hypertension by a mechanism that involves oxidized lipid-mediated T cell-dependent pulmonary arterial endothelial cell apoptosis and (2) Tg6F administration may be a novel therapy for treating PAH.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Endothelial Cells; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Immunologic Factors; Immunoproteins; Lipid Metabolism; Mice; Peptides; Proteasome Endopeptidase Complex; Pulmonary Artery; T-Lymphocytes

Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are the predominant gelatinases in the developing lung. Studies have shown that the expression of MMP-2 and MMP-9 is upregulated in hypoxic fibroblasts, 15-hydroxyeicosatetraenoic acid (15-HETE) regulated fibroblasts migration via modulating MMP-2 or MMP-9, and that hypoxia/15-HETE is a predominant contributor to the development of pulmonary arterial hypertension (PAH) through increased angiogenesis. However, the roles of MMP-2 and MMP-9 in pulmonary arterial endothelial cells (PAECs) angiogenesis as well as the molecular mechanism of hypoxia-regulated MMP-2 and MMP-9 expression have not been identified. The aim of this study was to investigate the role of MMP-2 and MMP-9 in PAEC proliferation and vascular angiogenesis and to determine the effects of hypoxia-induced 15-HETE on the expression of MMP-2 and MMP-9. Western blot, immunofluorescence, and real-time PCR were used to measure the expression of MMP-2 and MMP-9 in hypoxic PAECs. Immunohistochemical staining, flow cytometry, and tube formation as well as cell proliferation, viability, scratch-wound, and Boyden chamber migration assays were used to identify the roles and relationships between MMP-2, MMP-9, and 15-HETE in hypoxic PAECs. We found that hypoxia increased MMP-2 and MMP-9 expression in pulmonary artery endothelium both in vivo and in vitro in a time-dependent pattern. Moreover, administration of the MMP-2 and MMP-9 inhibitor MMI-166 significantly reversed hypoxia-induced increases in right ventricular systemic pressure (RVSP), right ventricular function, and thickening of the tunica media. Furthermore, up-regulation of MMP-2 and MMP-9 expression was induced by 15-HETE, which regulates PAEC proliferation, migration, and cell cycle transition that eventually leads to angiogenesis. Our study demonstrated that hypoxia increases the expression of MMP-2 and MMP-9 through the 15-lipoxygenase/15-HETE pathway, and that MMP-2 and MMP-9 promote PAEC angiogenesis. These findings suggest that MMP-2 and MMP-9 may serve as new potential therapeutic targets for the treatment of PAH.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Blood Pressure; Cell Hypoxia; Cell Movement; Cell Proliferation; Endothelial Cells; Gene Expression Regulation, Developmental; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Neovascularization, Pathologic; Pulmonary Artery; Sulfonamides; Tunica Media

Our previous studies have shown that 15-LO2/15-HETE induced by hypoxia played an important role in pulmonary arterial hypertension (PH). However, the transportations of 15-LO2/15-HETE among the cells remain elusive. In this study, we investigated the specific involvement of 15-LO2-containing exosomes in the overproliferation of pulmonary artery endothelial cells (PAECs) induced by hypoxia and the underlying mechanism. In vitro, 15-LO2 was abundantly expressed and enriched in exosomes secreted from hypoxic PAECs, which subsequently activated the STAT3 signaling pathway, resulting in a robust increase in PAECs proliferation. In vivo treatment with the exosomes inhibitor GW4869 protected the pulmonary vascular homeostasis from dysfunctional and abnormal remodeling. Moreover, 15-LO2 was ubiquitinated under hypoxia, and further inhibition of the ubiquitin-proteasome system significantly suppressed PAECs proliferation, suggesting that ubiquitination of 15-LO2 may contribute to its sorting into exosomes. Overall, these findings indicate a previously unrecognized effect of exosomes and the cargo 15-LO2 in pulmonary vascular homeostasis on the pathogenesis of PH.

Topics: Aniline Compounds; Animals; Arachidonate 15-Lipoxygenase; Benzylidene Compounds; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelial Cells; Exosomes; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; Proteasome Endopeptidase Complex; Pulmonary Artery; Signal Transduction; Ubiquitin; Ubiquitination

Topics: Hematopoietic Stem Cells; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia

We previously reported that 15-lipoxygenase (15-LO) and its metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) were up-regulated in pulmonary arterial cells from both pulmonary artery hypertension patients and hypoxic rats and that these factors mediated the progression of pulmonary hypertension (PH) by affecting the proliferation and apoptosis of pulmonary arterial (PA) cells. However, the underlying mechanisms of the remodeling induced by 15-HETE have remained unclear. As reactive oxygen species (ROS) and 15-LO are both induced by hypoxia, it is possible that ROS are involved in the events of hypoxia-induced 15-LO expression that lead to PH. We employed immunohistochemistry, tube formation assays, bromodeoxyuridine (BrdU) incorporation assays, and cell cycle analyses to explore the role of ROS in the process of 15-HETE-mediated hypoxic pulmonary hypertension (HPH). We found that exogenous 15-HETE facilitated the generation of ROS and that this effect was mainly localized to mitochondria. In particular, the mitochondrial electron transport chain and nicotinamide-adenine dinucleotide phosphate oxidase 4 (Nox4) were responsible for the significant 15-HETE-stimulated increase in ROS production. Moreover, ROS induced by 15-HETE stimulated endothelial cell (EC) migration and promoted pulmonary artery smooth muscle cell (PASMC) proliferation under hypoxia via the p38 MAPK pathway. These results indicated that 15-HETE-regulated ROS mediated hypoxia-induced pulmonary vascular remodeling (PVR) via the p38 MAPK pathway.

Topics: Animals; Arachidonate 15-Lipoxygenase; Cattle; Cells, Cultured; Female; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Lung; NADPH Oxidase 4; NADPH Oxidases; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Wistar; Reactive Oxygen Species; Signal Transduction; Vascular Remodeling

Pulmonary arterial hypertension (PAH) is a disease characterized by thickening of pulmonary artery walls, elevated pulmonary vascular resistance, pulmonary vascular thrombotic lesions, and right heart failure. Recent studies suggest that 15-lipoxygenase (15-LO)/15-hydroxyeicosatetraenoic acid (15-HETE) play an important role in PAH, acting on arterial walls. Here, we show evidence for the action of the 15-LO/15-HETE signaling in the pulmonary vascular thrombotic lesions in the experimental PAH models. Platelet deposition was augmented in rats exposed to hypoxia and Sugen 5416, which were both prevented by nordihydroguaiaretic acid (NDGA), a 15-LO inhibitor. Chronic hypoxic resulted in the platelet deposition specifically in pulmonary vasculature, which was reversed by 15-LO inhibitor. The 15-LO pathway mediated in the endothelial dysfunction induced by hypoxia in vivo. Meanwhile, 15-HETE positively regulated the generation of IL-6 and monocyte chemoattractant protein-1 (MCP-1). The coagulation and platelet activation induced by hypoxia were reversed by 15-LO inhibitor NDGA or the MCP-1 inhibitor synthesis inhibitor bindarit in rats. The 15-LO/15-HETE signaling promoted the coagulation and platelet activation, which was suppressed by MCP-1 inhibition. These results therefore suggest that 15-LO/15-HETE signaling plays a role in platelet activation and pulmonary vascular thrombosis in PAH, involving MCP-1.

Topics: Animals; Arachidonate 15-Lipoxygenase; Blood Platelets; Cells, Cultured; Chemokine CCL2; Cytokines; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Indazoles; Lipoxygenase Inhibitors; Male; Masoprocol; Platelet Activation; Propionates; Pulmonary Artery; Random Allocation; Rats; Rats, Wistar; RNA Interference; RNA, Small Interfering; Signal Transduction; Thrombosis; Vascular Resistance

Pulmonary arterial hypertension is a chronic lung disease associated with severe pulmonary vascular changes. A pathogenic role of oxidized lipids such as hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids is well established in vascular disease. Apolipoprotein A-I mimetic peptides, including 4F, have been reported to reduce levels of these oxidized lipids and improve vascular disease. However, the role of oxidized lipids in the progression of pulmonary arterial hypertension and the therapeutic action of 4F in pulmonary arterial hypertension are not well established.. We studied 2 different rodent models of pulmonary hypertension (PH): a monocrotaline rat model and a hypoxia mouse model. Plasma levels of hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids were significantly elevated in PH. 4F treatment reduced these levels and rescued preexisting PH in both models. MicroRNA analysis revealed that microRNA-193-3p (miR193) was significantly downregulated in the lung tissue and serum from both patients with pulmonary arterial hypertension and rodents with PH. In vivo miR193 overexpression in the lungs rescued preexisting PH and resulted in downregulation of lipoxygenases and insulin-like growth factor-1 receptor. 4F restored PH-induced miR193 expression via transcription factor retinoid X receptor α.. These studies establish the importance of microRNAs as downstream effectors of an apolipoprotein A-I mimetic peptide in the rescue of PH and suggest that treatment with apolipoprotein A-I mimetic peptides or miR193 may have therapeutic value.

Topics: Animals; Cell Proliferation; Cells, Cultured; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Muscle, Smooth, Vascular; Peptides; Rats; Rats, Sprague-Dawley; Receptor, IGF Type 1; Retinoid X Receptor alpha

Our previous studies have proved that hypoxia enhances the 15-lipoxygenase (15-LO) expression and increases endogenous 15-hydroxyeicosatetraenoic acid (15-HETE) production to promote pulmonary vascular remodeling and angiogenesis, while the mechanisms of how hypoxia regulates 15-LO expression in endothelium is still unknown. As placenta growth factor (PlGF) promotes pathological angiogenesis by acting on the growth, migration and survival of endothelial cells, there may be some connections between PlGF and 15-LO in hypoxia induced endothelial cells proliferation. In this study, we performed immunohistochemistry, pulmonary artery endothelial cells migration and bromodeoxyuridine incorporation to determine the role of PlGF in pulmonary remodeling induced by hypoxia. Our results showed that hypoxia up-regulated PlGF expression, which was mediated by 15-LO/15-HETE pathway. Furthermore, we found that PlGF had a positive feedback regulation with 15-LO expression and 15-HETE generation. The interaction in hypoxia between 15-HETE and PlGF created a PlGF-15-LO-15-HETE loop, leading to endothelial dysfunction. Thus, these findings suggest a new therapeutic agent in combination with the blockade of PlGF as well as 15-LO in hypoxic pulmonary hypertension.

Topics: Animals; Arachidonate 15-Lipoxygenase; Cattle; Cell Cycle Proteins; Cell Hypoxia; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelial Cells; Enzyme Induction; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Male; Neovascularization, Physiologic; Placenta Growth Factor; Pregnancy Proteins; Protein Transport; Pulmonary Artery; Rats; Rats, Wistar; Transcriptional Activation

Pulmonary hypertension (PH) is characterized with pulmonary vasoconstriction and vascular remodeling mediated by 15-lipoxygenase (15-LO)/15-hydroxyeicosatetraenoic acid (15-HETE) according to our previous studies. Meanwhile, telomerase reverse transcriptase (TERT) activity is highly correlated with vascular injury and remodeling, suggesting that TERT may be an essential determinant in the development of PH. The aim of this study was to determine the contribution and molecular mechanisms of TERT in the pathogenesis of PH.. We measured the right ventricular systolic pressure (RVSP) and ventricular weight, analyzed morphometric change of the pulmonary vessels in the hypoxia or monocrotaline treated rats. Bromodeoxyuridine incorporation, transwell assay and flow cytometry in pulmonary smooth muscle cells were performed to investigate the roles and relationship of TERT and 15-LO/15-HETE in PH. We revealed that the expression of TERT was increased in pulmonary vasculature of patients with PH and in the monocrotaline or hypoxia rat model of PH. The up-regulation of TERT was associated with experimental elevated RVSP and pulmonary vascular remodeling. Coimmunoprecipitation experiments identified TERT as a novel interacting partner of 15-LO-2. TERT and 15-LO-2 augmented protein expression of each other. In addition, the proliferation, migration and cell-cycle transition from G0/G1 phase to S phase induced by hypoxia were inhibited by TERT knockdown, which were rescued by 15-HETE addition.. These results demonstrate that TERT regulates pulmonary vascular remodeling. TERT and 15-LO-2 form a positive feedback loop and together promote proliferation and migration of pulmonary artery smooth muscle cells, creating a self-amplifying circuit which propels pulmonary hypertension.

Topics: Animals; Arachidonate 15-Lipoxygenase; Blood Pressure; Cell Cycle; Cell Proliferation; Cells, Cultured; Feedback, Physiological; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Male; Monocrotaline; Myocytes, Smooth Muscle; Protein Binding; Pulmonary Artery; Rats; Rats, Wistar; Signal Transduction; Telomerase

We previously reported that 15-lipoxygenase (15-LO) induced by hypoxia catalyzed the conversion of arachidonic acid (AA) into 15-hydroxyeicosatetraenoic acid (15-HETE), which plays an essential role in the development of hypoxic pulmonary arterial hypertension (HPH). However, the mechanisms by which hypoxia up-regulated 15-LO are still unclear. Heme oxygenase-1 (HO-1), an oxygen-dependent enzyme regulating vascular tone and cell proliferation, was implicated in HPH and was promoted by hypoxia. Therefore, the present study was carried out to determine whether hypoxia induced the expression of 15-LO via the HO-1 pathway. To test this hypothesis, we studied the role of HO-1 in HPH and 15-LO/15-HETE expression We found increased right ventricular systolic pressure and pulmonary arteries (PAs) reactivity to vasoconstrictors as well as intima-to-media ratio of PAs in HO-1 overexpressing transgenic mice. Moreover, HO-1 up-regulated 15-LO transcription and translation as well as 15-HETE in both transgenic mice and cultured pulmonary arterial smooth muscle cells (PASMCs). Results from immunoprecipitation and immunocytochemistry showed the interaction and colocalization of HO-1 and 15-LO. Together, these data suggest that HO-1 is an important upstream mediator in the hypoxia-induced 15-LO up-regulation during HPH. Unveiling the relevance of HO-1 signaling in PHP provides attractive treatment targets for HPH.

Topics: Animals; Arachidonate 15-Lipoxygenase; Cell Growth Processes; Cells, Cultured; Female; Heme Oxygenase-1; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Rats; RNA, Messenger; Tissue Distribution; Up-Regulation

We have found that 15-hydroxyeicosatetraenoic acid (15-HETE) induced by hypoxia was an important mediator in the regulation of hypoxic pulmonary hypertension, including the pulmonary vasoconstriction and remodeling. However, the underlying mechanisms of the remodeling induced by 15-HETE are poorly understood. In this study, we performed immunohistochemistry, pulmonary artery endothelial cells migration and tube formation, pulmonary artery smooth muscle cells bromodeoxyuridine incorporation, and cell cycle analysis to determine the role of 15-HETE in hypoxia-induced pulmonary vascular remodeling. We found that hypoxia induced pulmonary vascular medial hypertrophy and intimal endothelial cells migration and angiogenesis, which were mediated by 15-HETE. Moreover, 15-HETE regulated the cell cycle progression and made more smooth muscle cells from the G(0)/G(1) phase to the G(2)/M+S phase and enhanced the microtubule formation in cell nucleus. In addition, we found that the Rho-kinase pathway was involved in 15-HETE-induced endothelial cells tube formation and migration and smooth muscle cell proliferation. Together, these results show that 15-HETE mediates hypoxia-induced pulmonary vascular remodeling and stimulates angiogenesis via the Rho-kinase pathway.

Topics: Animals; Arachidonate 15-Lipoxygenase; Cattle; Cell Cycle; Cell Movement; Cell Proliferation; Cells, Cultured; Chick Embryo; Chorioallantoic Membrane; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Male; Models, Animal; Muscle, Smooth, Vascular; Neovascularization, Pathologic; Pulmonary Artery; Rats; Rats, Wistar; rho-Associated Kinases; Signal Transduction

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.

Topics: Animals; Cells, Cultured; Female; Hydroxyeicosatetraenoic Acids; Hypertension, Pulmonary; Hypoxia; Male; Muscle, Smooth, Vascular; Pulmonary Artery; Rats; Rats, Wistar; RNA, Messenger; Shaw Potassium Channels; Vasoconstriction