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)β. 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 E 2 . 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, PPARβ and guanylyl cyclase pathways in vessels and platelets, is discussed.

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.

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. (Circ Res. 2012;110:1423–1434.)

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-ketoprostaglandin F 1␣ in all cells. Key Words: eicosanoids Ⅲ endothelium Ⅲ prostacyclin Ⅲ cyclooxygenase Ⅲ shear stress E ndothelial cells line the luminal surface of blood vessels and are continuously exposed to hemodynamic shear stress. The level of shear stress that cells experience varies from region to region within the vasculature. In areas of high laminar shear stress, endothelial cells are elongated, aligned, and protected from inflammation. In areas of low, oscillatory shear stress, endothelial cells are randomly orientated and susceptible to inflammation. Areas of low shear stress are thought to be atheroprone, whereas areas of high shear stress are thought to be atheroprotected. [1][2][3] Cultured endothelial cells are routinely studied under static conditions, where they appear nonaligned, with a cobblestone morphology. 4,5 It is increasingly recognized that endothelial cells grown under static conditions may not be representative of endothelial cells in the body. 6,7 In addition, evidence suggests that endothelial endocrine function and expression of key enzymes, including cyclooxygenase (COX), is also regulated by shear stress. 8 COX is present in 2 isoforms: COX-1 and COX-2. Generally, COX-1 is expressed constitutively, whereas COX-2 is induced at sites of inflammation. 9 In endothelial cells, COX-1 activity results predominantly in the production of the antithrombotic hormone prostacyclin. 10 COX-1 and COX-2 are the targets for nonsteroidal antiinflammatory drugs (NSAIDs). They have attracted much media attention since the association of COX-2-selective NSAIDs with adverse cardiovascular events, 11,12 although the mechanism behind this association remains unclear. One leading hypothesis is that COX-2-selective drugs reduce the production of the cardioprotective hormone prostacyclin, 13,14 which, in susceptible individuals, increases the risk of arterial thrombosis. Prostacyclin is formed mainly by endothelial cells, which express high levels of COX. It has previously been shown that COX-1 predominates over COX-2 in endothelial cells cultured under static conditions, 15-17 which raises the question of how COX-2-s...

BackgroundPulmonary 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 FindingsHere we show that the PPARβ/δ 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 PPARβ/δ 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/SignificanceThese observations are the first to show a therapeutic benefit of ‘PPARβ/δ’ agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.

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.—Harrington, L. S., Lucas, R., McMaster, S. K., Moreno, L., Scadding, G., Warner, T. D., Mitchell, J. A. COX-1, and not COX-2 activity, regulates airway function: relevance to aspirin-sensitive asthma.

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, a, b methylene ATP, induces vasodilation of rat isolated mesenteric arteries and that P2X 1 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 a, b methylene ATP induced a constriction followed by a vasodilation. The dilator effects of either ATP or a, b 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 a, b 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, P2X 1 receptor immunoreactivity was localised to the endothelial, smooth muscle and adventitial layers of mesenteric vessels. P2X 1 protein migrated as a primary band at around 50-60 kDa in vascular tissue. 6 These results show for the first time that P2X 1 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.

BackgroundNociceptive input during early development can produce somatosensory memory that influences future pain response. Hind-paw incision during the 1st postnatal week in the rat enhances re-incision hyperalgesia in adulthood. We now evaluate its modulation by neonatal analgesia.MethodsNeonatal rats [Postnatal Day 3 (P3)] received saline, intrathecal morphine 0.1 mg kg−1 (IT), subcutaneous morphine 1 mg kg−1 (SC), or sciatic levobupivacaine block (LA) before and after plantar hind-paw incision (three×2 hourly injections). Six weeks later, behavioural thresholds and electromyography (EMG) measures of re-incision hyperalgesia were compared with an age-matched adult-only incision (IN) group. Morphine effects on spontaneous (conditioned place preference) and evoked (EMG sensitivity) pain after adult incision were compared with prior neonatal incision and saline or morphine groups. The acute neonatal effects of incision and analgesia on behavioural hyperalgesia at P3 were also evaluated.ResultsAdult re-incision hyperalgesia was not prevented by neonatal peri-incision morphine (saline, IT, and SC groups > IN; P<0.05–0.01). Neonatal sciatic block, but not morphine, prevented the enhanced re-incision reflex sensitivity in adulthood (LA < saline and morphine groups, P<0.01; LA vs IN, not significant). Morphine efficacy in adulthood was altered after morphine alone in the neonatal period, but not when administered with neonatal incision. Morphine prevented the acute incision-induced hyperalgesia in neonatal rats, but only sciatic block had a preventive analgesic effect at 24 h.ConclusionsLong-term effects after neonatal injury highlight the need for preventive strategies. Despite effective analgesia at the time of neonatal incision, morphine as a sole analgesic did not alter the somatosensory memory of early-life surgical injury.