Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response.

Recent findings have indicated a role for cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12-EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild-type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE.

Inhibition of endothelial nitric oxide synthase activity by proline-rich tyrosine kinase 2 in response to fluid shear stress and insulin.

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (approximately 50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production.

Cytochrome P450 2C9-induced angiogenesis is dependent on EphB4.

Objective: Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) are known to stimulate angiogenesis, but the mechanisms involved are incompletely understood. Because EphB4 is involved in vascular development, the aim of this study was to investigate whether, and to what extent, EphB4 is part of the signaling cascade that results in CYP2C9-mediated angiogenesis.

Methods And Results: CYP2C9 overexpression as well as stimulation with 11,12-EET (up to 48 hours) time-dependently increased EphB4 expression in endothelial cells.

Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases.

Platelet sarcoplasmic endoplasmic reticulum Ca2+-ATPase and mu-calpain activity are altered in type 2 diabetes mellitus and restored by rosiglitazone.

Background: Platelets from patients with type 2 diabetes mellitus display hyperaggregability and increased thrombogenic potential.

Methods And Results: In platelets from patients with type 2 diabetes mellitus, we found enhanced tyrosine nitration and inactivation of the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA-2), elevated platelet [Ca2+]i, and activation of mu-calpain. The tyrosine nitration of SERCA-2 and the activation of mu-calpain in vitro in platelets from healthy volunteers could be evoked in vitro by peroxynitrite.

Shear stress-induced activation of the AMP-activated protein kinase regulates FoxO1a and angiopoietin-2 in endothelial cells.

Aims: Phosphorylation of forkhead box O (FoxO) transcription factors induces their nuclear exclusion and proteosomal degradation. Here, we investigated the effect of fluid shear stress on FoxO1a in primary cultures of human endothelial cells and the kinases that regulate its phosphorylation.

Methods And Results: Shear stress (12 dynes/cm2) elicited the phosphorylation, nuclear exclusion, and degradation of FoxO1a.

NADPH oxidase plays a central role in blood-brain barrier damage in experimental stroke.

Background And Purpose: Cerebral ischemia/reperfusion is associated with reactive oxygen species (ROS) generation, and NADPH oxidases are important sources of ROS. We hypothesized that NADPH oxidases mediate blood-brain barrier (BBB) disruption and contribute to tissue damage in ischemia/reperfusion.

Methods: Ischemia was induced by filament occlusion of the middle cerebral artery in mice for 2 hours followed by reperfusion.

Epoxyeicosatrienoic acids regulate Trp channel dependent Ca2+ signaling and hyperpolarization in endothelial cells.

Objective: An initial step in endothelium-derived hyperpolarizing factor-mediated responses is endothelial cell hyperpolarization. Here we address the mechanisms by which cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) contribute to this effect in native and cultured endothelial cells.

Methods And Results: In native CYP2C-expressing endothelial cells, bradykinin elicited a Ca(2+) influx that was potentiated by the soluble epoxide hydrolase inhibitor, 1-adamantyl-3-cyclohexylurea (ACU), and attenuated by CYP inhibition.

Cytochrome P450 2C9 is involved in flow-dependent vasodilation of peripheral conduit arteries in healthy subjects and in patients with chronic heart failure.

Background: Flow-mediated dilation (FMD) of human conduit arteries is, in part, related to shear stress-induced release of endothelium-derived nitric oxide (NO). However, NO synthase inhibitors do not completely abolish this FMD-response. Recently, a cytochrome P450 (CYP) epoxygenase of the 2C family was linked to NO- and prostacyclin-independent relaxation of conduit arteries.

Nox1 mediates basic fibroblast growth factor-induced migration of vascular smooth muscle cells.

Objective: Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration.

Methods And Results: bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1.

Chemotaxis and differentiation of human adipose tissue CD34+/CD31- progenitor cells: role of stromal derived factor-1 released by adipose tissue capillary endothelial cells.

The native CD34+/CD31- cell population present in the stroma-vascular fraction of human adipose tissue (hAT) displays progenitor cell properties since they exhibit adipocyte- and endothelial cell-like phenotypes under appropriate stimuli. To analyze the signals within hAT regulating their phenotypes, the influence of hAT-derived capillary endothelial cells (CECs) was studied on the chemotaxis and differentiation of the hAT-CD34+/CD31- cells. Conditioned medium from hAT-CECs led to a strong chemotaxis of the hAT-CD34+/CD31- cells that was inhibited with pretreatments with pertussis toxin, CXCR-4 antagonist, or neutralizing antibodies.

The effects of COX-2 selective and non-selective NSAIDs on the initiation and progression of atherosclerosis in ApoE-/- mice.

In this study, we investigated the effects of prolonged administration of the selective COX-2 inhibitors celecoxib and rofecoxib and the non-selective NSAID naproxen on the initiation and progression of atherosclerosis. ApoE(-/-) mice, as well as corresponding wild-type mice, were fed either a normal chow or a high fat Western diet with or without addition of the respective drugs over a period of 16 weeks. Thereafter, aortic lesion size, plasma lipid levels, and COX-2 expression in the plaques were determined.

Fluid shear stress and NO decrease the activity of the hydroxy-methylglutaryl coenzyme A reductase in endothelial cells via the AMP-activated protein kinase and FoxO1.

The rate-limiting enzyme for cholesterol synthesis, the hydroxy-methylglutaryl coenzyme A reductase (HCR), is phosphorylated by the AMP-activated protein kinase (AMPK). As shear stress activates the AMPK in endothelial cells, we determined whether it affects HCR activity and subsequent HCR-dependent signaling. Shear stress (12 dynes cm(-2)) rapidly increased the phosphorylation and activity (6.

Inactivation of extracellular superoxide dismutase contributes to the development of high-volume hypertension.

Objectives: Extracellular superoxide dismutase (ecSOD) lowers superoxide anions and maintains vascular nitric oxide level. We studied the function of ecSOD in high-volume hypertension induced by the 1-kidney-1-clip model in wild-type, ecSOD-/- mice, and endothelial nitric oxide synthase (eNOS)-/- mice.

Methods And Results: The 1-kidney-1-clip model resulted in impaired endothelium-dependent relaxation and hypertension and vascular oxidative stress in wild-type and ecSOD-/- mice.

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet.

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis.

Noxa1 is a central component of the smooth muscle NADPH oxidase in mice.

NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC.

Cytochrome P450 epoxygenase gene function in hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling.

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase.

Endothelium-derived epoxyeicosatrienoic acids and vascular function.

Epoxyeicosatrienoic acids (EETs) are epoxides of arachidonic acid generated by cytochrome P450 (CYP) epoxygenases. The activation of CYP epoxygenases in endothelial cells is an important step in the NO and prostacyclin-independent vasodilatation of several vascular beds, and EETs have been identified as an endothelium-derived hyperpolarizing factor. However, EETs also exert membrane potential-independent effects and modulate several signaling cascades that affect endothelial cell proliferation and angiogenesis.

Angiotensin-converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells.

The binding of angiotensin-converting enzyme (ACE) inhibitors to ACE initiates a signaling cascade that involves the phosphorylation of the enzyme on Ser1270 as well as activation of the c-Jun NH2-terminal kinase (JNK) and leads to alterations in gene expression. To clarify how ACE inhibitors activate this pathway, we determined their effect on the ability of the enzyme to dimerize and the role of ACE dimerization in the initiation of the ACE signaling cascade. In endothelial cells, ACE was detected as a monomer as well as a dimer in native gel electrophoresis and dimerization/oligomerization was confirmed using the split-ubiquitin assay in yeast.

The tissue renin-angiotensin system and intracellular signalling.

Purpose Of Review: The renin-angiotensin system is not what it was, or for that matter not necessarily where we thought it should be. For example, there is a novel angiotensin I-metabolizing enzyme that generates angiotensin 1-7 rather than angiotensin II. Moreover, we are slowly realizing the importance of local rather than circulating angiotensin II.

Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis.

Recent studies suggest that cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the role of CYP 2C8/9-derived EETs in the process of angiogenesis under hypoxic conditions. In human endothelial cells, hypoxia enhanced the activity of the CYP 2C9 promoter, increased the expression of CYP 2C mRNA and protein and augmented 11,12-EET production.

Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress.

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2.

Signaling via the angiotensin-converting enzyme results in the phosphorylation of the nonmuscle myosin heavy chain IIA.

The phosphorylation of the short C-terminal cytoplasmic domain of the somatic angiotensin-converting enzyme (ACE) is involved in the regulation of enzyme shedding. We determined whether the phosphorylation of the cytoplasmic domain of ACE (ACEct) on Ser1270 regulates the cleavage/secretion of the enzyme by affecting its association with other proteins. ACE was associated with beta-actin and the nonmuscle myosin heavy chain IIA (MYH9) in endothelial cells, as determined by coimmunoprecipitation experiments as well as an ACEct affinity column.

Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS.