Cyclooxygenase-1, not cyclooxygenase-2, is responsible for physiological production of prostacyclin in the cardiovascular system.

Prostacyclin is an antithrombotic hormone produced by the endothelium, whose production is dependent on cyclooxygenase (COX) enzymes of which two isoforms exist. It is widely believed that COX-2 drives prostacyclin production and that this explains the cardiovascular toxicity associated with COX-2 inhibition, yet the evidence for this relies on indirect evidence from urinary metabolites. Here we have used a range of experimental approaches to explore which isoform drives the production of prostacyclin in vitro and in vivo. Our data show unequivocally that under physiological conditions it is COX-1 and not COX-2 that drives prostacyclin production in the cardiovascular system, and that urinary metabolites do not reflect prostacyclin production in the systemic circulation. With the idea that COX-2 in endothelium drives prostacyclin production in healthy individuals removed, we must seek new answers to why COX-2 inhibitors increase the risk of cardiovascular events to move forward with drug discovery and to enable more informed prescribing advice.

Role of RhoB in the regulation of pulmonary endothelial and smooth muscle cell responses to hypoxia.

RATIONALE: RhoA and Rho kinase contribute to pulmonary vasoconstriction and vascular remodeling in pulmonary hypertension. RhoB, a protein homologous to RhoA and activated by hypoxia, regulates neoplastic growth and vasoconstriction but its role in the regulation of pulmonary vascular function is not known. OBJECTIVE: To determine the role of RhoB in pulmonary endothelial and smooth muscle cell responses to hypoxia and in pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension. METHODS AND RESULTS: Hypoxia increased expression and activity of RhoB in human pulmonary artery endothelial and smooth muscle cells, coincidental with activation of RhoA. Hypoxia or adenoviral overexpression of constitutively activated RhoB increased actomyosin contractility, induced endothelial permeability, and promoted cell growth; dominant negative RhoB or manumycin, a farnesyltransferase inhibitor that targets the vascular function of RhoB, inhibited the effects of hypoxia. Coordinated activation of RhoA and RhoB maximized the hypoxia-induced stress fiber formation caused by RhoB/mammalian homolog of Drosophila diaphanous-induced actin polymerization and RhoA/Rho kinase-induced phosphorylation of myosin light chain on Ser19. Notably, RhoB was specifically required for hypoxia-induced factor-1α stabilization and for hypoxia- and platelet-derived growth factor-induced cell proliferation and migration. RhoB deficiency in mice markedly attenuated development of chronic hypoxia-induced pulmonary hypertension, despite compensatory expression of RhoA in the lung. CONCLUSIONS: RhoB mediates adaptational changes to acute hypoxia in the vasculature, but its continual activation by chronic hypoxia can accentuate vascular remodeling to promote development of pulmonary hypertension. RhoB is a potential target for novel approaches (eg, farnesyltransferase inhibitors) aimed at regulating pulmonary vascular tone and structure.

Reduced endothelial dependent vasodilation in vessels from TLR4(-/-) mice is associated with increased superoxide generation.

Toll like receptor (TLR)4 is a pattern recognition receptor expressed in endothelial and other cells, responsible for the sensing of endotoxin and host derived ligands. Our group has shown previously that the absence of TLR4 is associated with reduced endothelial dependent vasodilator responses and left heart hypertrophy in animal models. However, the mechanism behind reduced endothelial cell function in TLR4(-/-) mice is not known. We have used en face confocal imaging of mesenteric arteries from mice deficient in the TLR4 receptor stained with dihydroethidium (DHE) to measure superoxide production. Using the isometric wire myograph, mesenteric artery vasodilator responses to acetylcholine and MnCl(2) (a superoxide dismutase mimetic) were measured. Mesenteric arteries from TLR4(-/-) mice had a reduced endothelial dependent relaxant response and increased superoxide levels when stimulated with acetylcholine. Increased levels of superoxide, as detected by DHE staining, were seen in vessels from TLR4(-/-) mice, which were reduced to control levels in the presence of MnCl(2). Our observations suggest that loss of TLR4 increases superoxide generation which reduces the biological activity of endothelial derived nitric oxide and thereby explains the endothelial dysfunction and associated cardiovascular phenotype in TLR4(-/-) mice. These data implicate a novel cardio-protective role for TLR4 in vascular homeostasis.

Role of shear stress in endothelial cell morphology and expression of cyclooxygenase isoforms.

OBJECTIVE: The goal of this study was to examine the effect of chronic heterogeneous shear stress, applied using an orbital shaker, on endothelial cell morphology and the expression of cyclooxygenases 1 and 2. METHODS AND RESULTS: Porcine aortic endothelial cells were plated on fibronectin-coated Transwell plates. Cells were cultured for up to 7 days either under static conditions or on an orbital shaker that generated a wave of medium inducing shear stress over the cells. Cells were fixed and stained for the endothelial surface marker CD31 or cyclooxygenases 1 and 2. En face confocal microscopy and scanning ion conductance microscopy were used to show that endothelial cells were randomly oriented at the center of the well, aligned with shear stress nearer the periphery, and expressed cyclooxygenase-1 under all conditions. Lipopolysaccharide induced cyclooxygenase-2 and the production of 6-keto-prostaglandin F(1α) in all cells. CONCLUSIONS: Cyclooxygenase-1 is expressed in endothelial cells cultured under chronic shear stress of high or low directionality.

The PPARbeta/delta agonist GW0742 relaxes pulmonary vessels and limits right heart hypertrophy in rats with hypoxia-induced pulmonary hypertension.

BACKGROUND: Pulmonary vascular diseases are increasingly recognised as important clinical conditions. Pulmonary hypertension associated with a range of aetiologies is difficult to treat and associated with progressive morbidity and mortality. Current therapies for pulmonary hypertension include phosphodiesterase type 5 inhibitors, endothelin receptor antagonists, or prostacyclin mimetics. However, none of these provide a cure and the clinical benefits of these drugs individually decline over time. There is, therefore, an urgent need to identify new treatment strategies for pulmonary hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the PPARbeta/delta agonist GW0742 induces vasorelaxation in systemic and pulmonary vessels. Using tissue from genetically modified mice, we show that the dilator effects of GW0742 are independent of the target receptor PPARbeta/delta or cell surface prostacyclin (IP) receptors. In aortic tissue, vascular relaxant effects of GW0742 were not associated with increases in cGMP, cAMP or hyperpolarisation, but were attributed to inhibition of RhoA activity. In a rat model of hypoxia-induced pulmonary hypertension, daily oral dosing of animals with GW0742 (30 mg/kg) for 3 weeks significantly reduced the associated right heart hypertrophy and right ventricular systolic pressure. GW0742 had no effect on vascular remodelling induced by hypoxia in this model. CONCLUSIONS/SIGNIFICANCE: These observations are the first to show a therapeutic benefit of 'PPARbeta/delta' agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.

COX-1, and not COX-2 activity, regulates airway function: relevance to aspirin-sensitive asthma.

Cyclooxygenase (COX) -1 and COX-2 are expressed in airway cells, where their activities influence functions such as airway hyperreactivity. Clinical data show that mixed COX-1/COX-2 inhibitors such as aspirin, but not COX-2 selective inhibitors such as rofecoxib, induce bronchoconstriction and asthma in sensitive individuals. This anomaly has not yet been explained. Here, we have used tissue from genetically modified mice lacking functional COX-1 (COX-1(-/-)), as well as airway tissue from "aspirin-sensitive" and control patients to address this issue. Bronchi from wild-type mice contained predominantly COX-1 immunoreactivity and contracted in vitro in response to acetylcholine and U46619. Bronchi from COX-1(-/-) mice were hyperresponsive to bronchoconstrictors. Inhibitors of COX (naproxen, diclofenac, or ibuprofen) increased bronchoconstriction in tissue from wild-type but not from COX-1(-/-) mice. Cells cultured from aspirin-sensitive or control human donors contained similar levels of COX-1 and COX-2 immunoreactivity. COX activity in cells from aspirin-sensitive or tolerant patients was inhibited by aspirin, SC560, which blocks COX-1 selectively, but not by rofecoxib, which is a selective inhibitor of COX-2. These observations show that despite the presence of COX-2, COX-1 is functionally predominant in the airways and explains clinical observations relating to drug specificity in patients with aspirin-sensitive asthma.

Role of nitric oxide and prostacyclin as vasoactive hormones released by the endothelium.

The endothelium lines the luminal surface of every blood vessel, allowing it contact with circulating blood elements, as well as the underlying vascular smooth muscle layer. In healthy vessels, the endothelium expresses constitutive forms of nitric oxide synthase (NOSIII) and cyclo-oxygenase (COX-1), which produce the vasoactive hormones NO and prostacyclin, respectively. Both NO and prostacyclin relax blood vessels and inhibit platelet activation. The actions of prostacyclin are mediated by cell surface prostacyclin (IP) receptors and/or intracellular peroxisome proliferator-activated receptors (PPAR) beta. The actions of NO are mediated predominately by activation of intracellular guanylyl cyclase, leading to the formation of cGMP. In platelets, the actions of NO and prostacyclin are synergistic, but in vessels their actions are additive. In diseased vessels, inducible forms of NOS (NOSII) and cyclo-oxygeanse (COX-2) are expressed in vascular smooth muscle, resulting in the release of large amounts of NO, prostacyclin and prostaglandin E2. The relative contribution of NOSII and COX-2 to vascular inflammation is still debated, but is likely to result in both protective and damaging responses. The relative contribution of constitutive forms of NOS and COX, as well as interactions between IP, PPAR beta and guanylyl cyclase pathways in vessels and platelets, is discussed.

Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels.

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10(-6) mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10(-3) mol/l) but not TRAM-34 (10(-6) mol/l) plus apamin (5 x 10(-7) mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII(-/-) mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A(2) inhibitor AACOCF(3), suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.

Homeostatic role of Toll-like receptor 4 in the endothelium and heart.

Toll-like receptor 4 (TLR4) is a pattern recognition receptor for lipopolysaccharide from Gram negative bacteria and thus is integral to the innate immune response in mammals. In addition, TLR4 is associated with atherosclerosis in murine models. The current study shows that blood vessels from TLR4(-/-) mice have an intact endothelial layer and comparable expression of nitric oxide synthase 3 protein. However, endothelium-dependent dilation in response to acetylcholine in vessels from TLR4(-/-) mice is greatly reduced. By contrast, endothelium-independent smooth muscle dilation in response to sodium nitroprusside in vessels from TLR4(-/-) mice remains intact. Furthermore, this study shows that hearts from TLR4(-/-) mice display signs of left ventricular dilation. In contrast to results in vessels from TLR4(-/-) mice, endothelium-dependent responses to acetylcholine in vessels from TLR2(-/-) mice remain intact. These observations illustrate a novel role for TLR4 in the homeostatic control of a functional endothelium and, thereby, cardiovascular health.

Not so EEZE: the 'EDHF' antagonist 14, 15 epoxyeicosa-5(Z)-enoic acid has vasodilator properties in mesenteric arteries.

P-450 metabolites, including the epoxyeicosatrienoic acids, are likely candidates for endothelial derived hyperpolarising factor (EDHF). In the present study, we confirm that the stable analogue 11-nonyloxyundec-8(Z)-enoic acid is a vasodilator of murine vessels. However, we also show that the 'epoxyeicosatrienoic acid receptor' antagonist 14,15 EEZE similarly dilates murine vessels contracted with U46619, prostaglandin F2alpha or methoxamine, but not with endothelin-1 or potassium. We suggest that 14,15 EEZE is a partial agonist for the epoxyeicosatrienoic acids/EDHF receptor. These results illustrate an important pharmacological property of this antagonists, which is being increasingly used to study the nature of EDHF.

Novel role for P2X receptor activation in endothelium-dependent vasodilation.

1 ATP is an important vasoactive mediator, which acts via two receptor classes: P2X and P2Y. Activation of P2X receptors has traditionally been associated with the well-characterised vasoconstrictor properties of ATP. 2 In the current study, we have shown that the P2X(1 & 3) receptor ligand, alpha, beta methylene ATP, induces vasodilation of rat isolated mesenteric arteries and that P2X1 receptors are abundantly expressed in the endothelium of these vessels. 3 Second-order rat mesenteric arteries were mounted in myographs and vasomotor responses recorded. Both ATP and alpha, beta methylene ATP induced a constriction followed by a vasodilation. The dilator effects of either ATP or alpha, beta methylene ATP were slower in onset than those induced by acetylcholine. By contrast, the traditional vasodilator P2Y ligand, ADP, induced vasodilation without contraction. 4 Vasodilation induced by alpha, beta methylene ATP was endothelial dependent, but was not affected by treatment of the vessels with L-NAME plus indomethacin alone. Dilation was, however, partially inhibited by the combination of apamin plus charybdotoxin and blocked by treating vessels with all four drugs. 5 Using confocal microscopy, P2X1 receptor immunoreactivity was localised to the endothelial, smooth muscle and adventitial layers of mesenteric vessels. P2X1 protein migrated as a primary band at around 50-60 kDa in vascular tissue. 6 These results show for the first time that P2X1 receptors are expressed on the endothelium and that a selective ligand of this receptor results in vasoconstriction followed by vasodilation. These observations have important implications for our understanding of the role of purines in biological responses.