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We previously reported that laminar flow activates peroxisome proliferator-activated receptor ␥ (PPAR␥) in vascular endothelial cells in a ligand-dependent manner that involves phospholipase A2 and cytochrome P450 epoxygenases. In this study, we investigated whether epoxyeicosatrienoic acids (EETs), the catalytic products of cytochrome P450 epoxygenases, are PPAR␥ ligands. Competition and direct binding assays revealed that EETs bind to the ligandbinding domain of PPAR␥ with Kd in the M range. In the presence of adamantyl-ureido-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, EETs increased PPAR␥ transcription activity in endothelial cells and 3T3-L1 preadipocytes. Inclusion of AUDA in the perfusing media enhanced, but overexpression of sEH reduced, the laminar flow-induced PPAR␥ activity. Furthermore, laminar flow augmented cellular levels of EETs but decreased sEH at the levels of mRNA, protein, and activity. Blocking PPAR␥ by GW9662 abolished the EET͞AUDA-mediated antiinflammatory effect, which indicates that PPAR␥ is an effector of EETs. endothelial cells ͉ shear stressA therosclerosis preferentially localizes in branches and curved regions of the arterial tree, where the blood flow is disturbed. In contrast, the straight parts of vessels exposed to nondisturbed laminar flow have few lesions (1). The focal distribution of atherosclerotic lesions has been proposed to be related to the proinflammatory effect of disturbed flow imposed on the endothelium vs. the antiinflammatory effect of laminar flow. In vitro studies using flow channels with cultured endothelial cells (ECs) revealed that disturbed flow induces a number of molecules involved in inflammation, including chemoattractants, adhesion molecules, and cytokines (2, 3). However, prolonged exposure to laminar flow suppresses the cytokine-stimulated or oxidized low-density lipoprotein (LDL)-stimulated inflammatory response in ECs (4).Recent studies showed that the nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) is involved in antiinflammatory effects in the artery wall (5, 6). The activation of PPAR␥ in cultured ECs suppresses the NF-B-mediated expression of molecules such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and endothelin 1 that are involved in the inflammatory response (7,8). Troglitazone, a synthetic PPAR␥ ligand, attenuates the formation of lesions in both apolipoprotein E-and low-density lipoprotein receptordeficient mice (7, 9), due in part to the reduction of monocytes͞ macrophages homing to the plaques.We previously demonstrated that laminar flow activates PPAR␥ in a ligand-dependent manner, which exerts an antiinflammatory effect in ECs. Furthermore, we showed that such induction of PPAR␥ ligands involves phospholipase A2 and cytochrome P450 epoxygenases (CYPs) (10). Epoxyeicosatrienoic acids (EETs), the main products of arachidonic acid catalyzed by CYPs, have been reported to dilate coronary arteries by hyperpolarizing vascular smooth muscles (11) and to exe...

Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 mumol/L) produced 75 +/- 9% and 52 +/- 4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 mumol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET- and 14,15-DHET-induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET-labeled endothelial cells to the Ca2+ ionophore A23187 (2 mumol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.

Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross-rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context.

The role of reactive oxygen species, such as superoxide anions (O 2) and hydrogen peroxide (H 2 O 2), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H 2 O 2 , rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (AdCat) or a control gene,-galactosidase (AdLacZ). Infection with AdCat resulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of AdCat, cellular catalase activity was increased by 50-to 100-fold, and intracellular H 2 O 2 concentration was reduced, as compared with control. Infection with AdCat reduced [ 3 H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.370.09 disintegrations per minute per cell [AdLacZ] versus 0.220.08 disintegrations per minute per cell [AdCat], P0.05). Five days after infection with 100 MOI of AdCat, cell numbers were reduced as compared with noninfected or AdLacZ-infected cells (157 7808413 [AdCat], P0.05 versus 233 7003032 [noninfected] or 222 4105332 [AdLacZ]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of AdCat as compared with control. Infection with AdCat resulted in induction of cyclooxygenase (COX)-2, and treatment with a COX-2 inhibitor overcame the AdCat-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2-dependent mechanism. Our results suggest that endogenously produced H 2 O 2 importantly modulates survival and proliferation of vascular smooth muscle cells. (Circ Res. 1999;85:524-533.) Key Words: catalase apoptosis vascular smooth muscle cell cell proliferation hydrogen peroxide P roliferation of vascular smooth muscle cells is an important contributing factor in the pathophysiology of hyper-tension and atherosclerosis, as well as in coronary artery restenosis after angioplasty and stent placement. 1,2 However, the factors that induce proliferation of vascular smooth muscle cells, which normally exist in the arterial wall in a state of quiescence, are unknown. 3 Recently, it has been reported that reactive oxygen species, such as superoxide anions (O 2) and hydrogen peroxide (H 2 O 2), are capable of stimulating vascular smooth muscle cell proliferation. 4,5 These oxidants were shown to be rapidly produced by smooth muscle cells after exposure to platelet-derived growth factor or angiotensin II, factors that stimulate smooth muscle cell growth. 5,6 In addition, the production of reactive oxygen species in the blood vessel wall is enhanced in experimental models of hypercholesterolemia, hypertension, diabetes, and balloon injury to the coronary arteries. 7-10 Moreover, in angiotensin II-induced hypertension, excess free radical production was predominantly localized...

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Epoxyeicosatrienoic acids (EETs) are potent vasodilators derived from cytochrome P-450 metabolism of arachidonic acid. The rapid conversion of EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs) has been proposed as a process whereby EETs are rendered biologically inactive. However, the vascular metabolism of EETs and the vasoactivities of EET metabolites have not been extensively studied. Accordingly, 11,12-EET metabolism was characterized in porcine aortic smooth muscle cells. The cells converted [3H]11,12-EET to 11,12-DHET and to a newly identified metabolite, 7,8-dihydroxy-hexadecadienoic acid (DHHD). 11,12-DHET accumulation in the medium reached a maximum in 2 to 4 hours and then declined, whereas 7,8-DHHD accumulation increased continuously and exceeded the amount of 11,12-DHET by 8 hours. [3H]11,12-EET conversion to radiolabeled 7,8-DHHD was reduced in the presence of unlabeled 11,12-DHET, indicating that 11,12-DHET is an intermediate in the conversion of 11,12-EET to 7,8-DHHD. This is consistent with a pathway whereby 11,12-EET is converted by an epoxide hydrolase to 11,12-DHET, which then undergoes two beta-oxidations to form 7,8-DHHD. In porcine coronary artery rings contracted with a thromboxane mimetic, 11,12-DHET produced relaxation similar in magnitude to that produced by 11,12-EET (77% versus 64% relaxation at 5 mumol/L, respectively). 7,8-DHHD also produced vasorelaxation. Thus, the vasoactivity of 11,12-EET is not eliminated by conversion to 11,12-DHET and 7,8-DHHD. These results suggest that 11,12-DHET and its metabolite, 7,8-DHHD, may contribute to the regulation of vascular tone in the porcine coronary artery and possibly other vascular tissues.

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