Dhanasekaran A, Gruenloh SK, Buonaccorsi JN, Zhang R, Gross GJ, Falck JR, Patel PK, Jacobs ER, Medhora M. Multiple antiapoptotic targets of the PI3K/Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia.

oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time-and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47 phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47 phox and tyrosine phosphorylation of p47 phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species PRODUCTS OF CYTOCHROME P-450 (CYP) -hydroxylases (including CYP4 isoforms) mediate key physiological functions including autoregulation of blood flow, tubuloglomerular feedback, Na ϩ reabsorption in the kidney, and relaxation of pulmonary arterioles (29,38). Our studies have focused on the role of CYP4 and its arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), in pulmonary vascular function and biology. 20-HETE is the CYP -hydroxylation product of AA cleaved from membrane phospholipid sources. Enzymes of the CYP4A, -4B, and -4F families catalyze the -hydroxylation of fatty acids, and several isoforms in these families produce 20-HETE when incubated with AA. For example, rat CYP4A1, -4A2, and -4A3 catalyze AA -and -1-hydroxylations with the highest catalytic efficiency accruing to CYP4A1 (35). Although CYP4A2 and CYP4A3 exhibit an additional arachidonate 11,12-epoxidation activity, CYP4A1 operates solely as an -hydroxylase. Most investigators suggest that CYP4 isoforms constitute the major source of 20-HETE synthesis in extrahepatic tissues, including the lung (35, 38). Accordingly, we have investigated the effects of CYP4 product, 20-HETE, in our studies of this system in pulmonary vascular biology.We have identified a unique role f...

A polyphasic study was undertaken to clarify the taxonomic position of endospore-forming strains 433-D9, 433-E17 and 121-X1. BOX-PCR-generated fingerprints indicated that they may be members of a single species. 16S rRNA gene sequence similarity demonstrated that a representative of this group, 433-D9, is affiliated closely with Bacillus arvi DSM 16317T (100 %), Bacillus arenosi DSM 16319T (99.8 %) and Bacillus neidei NRRL BD-87T (97.1 %). Sequence similarities revealed Bacillus pycnus NRRL NRS-1691T and several Kurthia species as the next nearest relatives. DNA–DNA hybridization results showed that strain 433-D9 is a member of B. arvi. Detection of l-Lys–d-Asp-based peptidoglycan in strain 433-D9, B. arvi DSM 16317T and B. arenosi DSM 16319T was in agreement with their close relationship, but differentiated these strains from B. neidei NRRL BD-87T and B. pycnus NRRL NRS-1691T, for which l-Lys–d-Glu was reported. A similar quinone system was detected in strains 433-D9, 433-E17, 121-X1, B. arvi DSM 16317T, B. arenosi DSM 16319T and B. neidei NRRL BD-87T. This system, unusual for bacilli, consisted of the major compound menaquinone MK-8 (69–80 %) and moderate amounts of MK-7 (19–30 %). This observation was in contrast to the predominance of MK-7 of the closest relative B. pycnus NRRL NRS-1691T, as also reported for representatives of the closely related non-endospore-forming genus Kurthia. Strains 433-D9, B. arvi DSM 16317T and B. arenosi DSM 16319T exhibited homogeneous and discriminative polar lipid profiles and fatty acid profiles consisting of major acids i-C15 : 0 and ai-C15 : 0 and moderate amounts of i-C17 : 1 ω10c and i-C17 : 1 I/ai-C17 : 1 B that discriminated them from closely related strains such as B. neidei NRRL BD-87T. On the basis of clear-cut discriminative chemotaxonomic markers, we propose strains 433-D9, 433-E17 and 121-X1, B. arvi DSM 16317T, B. arenosi DSM 16319T and B. neidei NRRL BD-87T to be reclassified within a separate genus. For this new taxon, we propose the name Viridibacillus gen. nov., and we propose the reclassification of Bacillus arvi, Bacillus arenosi and Bacillus neidei as Viridibacillus arvi gen. nov., comb. nov. (the type species of Viridibacillus, with the type strain DSM 16317T =LMG 22165T), Viridibacillus arenosi comb. nov. (type strain DSM 16319T =LMG 22166T) and Viridibacillus neidei comb. nov. (type strain NRRL BD-87T =DSM 15031T =JCM 11077T).

Hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P-450 product present in vascular smooth muscle and uniquely located in the vascular endothelium of pulmonary arteries (PAs). 20-HETE enhances reactive oxygen species (ROS) production of bovine PA endothelial cells (BPAECs) in an NADPH oxidase-dependent manner and is postulated to promote angiogenesis via activation of this pathway in systemic vascular beds. We tested the capacity of 20-HETE or a stable analog of this compound, 20-hydroxy-eicosa-5(Z),14(Z)-dienoic acid, to enhance survival and protect against apoptosis in BPAECs stressed with serum starvation. 20-HETE produced a concentration-dependent increase in numbers of starved BPAECs and increased 5-bromo-2Ј-deoxyuridine incorporation. Caspase-3 activity, nuclear fragmentation studies, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays supported protection from apoptosis and enhanced survival of starved BPAECs treated with a single application of 20-HETE. Protection from apoptosis depended on intact NADPH oxidase, phosphatidylinositol 3 (PI3)-kinase, and ROS production. 20-HETE-stimulated ROS generation by BPAECs was blocked by inhibition of PI3-kinase or Akt activity. These data suggest 20-HETEassociated protection from apoptosis in BPAECs required activation of PI3-kinase and Akt and generation of ROS. 20-HETE also protected against apoptosis in BPAECs stressed by lipopolysaccharide, and in mouse PAs exposed to hypoxia reoxygenation ex vivo. In summary, 20-HETE may afford a survival advantage to BPAECs through activation of prosurvival PI3-kinase and Akt pathways, NADPH oxidase activation, and NADPH oxidase-derived superoxide.reduced nicotinamide adenine dinucleotide phosphatase oxidase; reactive oxygen species; phosphatidylinositol 3-kinase; Akt; hypoxia CYTOCHROME P-450 (CYP) ENZYMES can metabolize arachidonic acid into numerous eicosanoids, with the relative abundance dependent on the tissue and species (21). The major products in most tissues are the -hydroxylated metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) and regio-and stereo-specific epoxyeicosatrienoic acids. 20-HETE, a -hydroxylation product of arachidonic acid catalyzed by CYP4A, is a paracrine and autocrine mediator of numerous cellular processes (20,29,35). It is produced in vascular smooth muscle, renal, cerebral, pulmonary, mesenteric, and skeletal muscle beds and acts on the microvasculature and kidney tubules (11,18,22,33,38).Our laboratory has recently reported that 20-HETE/CYP4 enhances reactive oxygen species (ROS) production in bovine pulmonary artery (PA) endothelial cells (BPAECs) in a manner that is associated with activation of NADPH oxidase (24). 20-HETE has been linked to ROS production in other vascular beds (19), with 20-HETE-stimulated production of ROS being reported to exert either positive or negative effects in a tissueand concentration-specific manner (e.g., Refs. 9, 17). In addition, NADPH oxidase is proposed to have a key role in growth and migration of human coronary endot...