Martina H. Lundberg

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 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 F1&agr; in all cells. Conclusion—Cyclooxygenase-1 is expressed in endothelial cells cultured under chronic shear stress of high or low directionality.

Blockade of the purinergic P2Y12 receptor greatly increases the platelet inhibitory actions of nitric oxide

Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y12 receptors, strongly amplifies aggregation. Platelet P2Y12 receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y12 receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y12 receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y12, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y12 receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.

LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

COX-2 Protects against Atherosclerosis Independently of Local Vascular Prostacyclin: Identification of COX-2 Associated Pathways Implicate Rgl1 and Lymphocyte Networks

Cyxlo-oxygenase (COX)-2 inhibitors, including traditional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with increased cardiovascular side effects, including myocardial infarction. We and others have shown that COX-1 and not COX-2 drives vascular prostacyclin in the healthy cardiovascular system, re-opening the question of how COX-2 might regulate cardiovascular health. In diseased, atherosclerotic vessels, the relative contribution of COX-2 to prostacyclin formation is not clear. Here we have used apoE−/−/COX-2−/− mice to show that, whilst COX-2 profoundly limits atherosclerosis, this protection is independent of local prostacyclin release. These data further illustrate the need to look for new explanations, targets and pathways to define the COX/NSAID/cardiovascular risk axis. Gene expression profiles in tissues from apoE−/−/COX-2−/− mice showed increased lymphocyte pathways that were validated by showing increased T-lymphocytes in plaques and elevated plasma Th1-type cytokines. In addition, we identified a novel target gene, rgl1, whose expression was strongly reduced by COX-2 deletion across all examined tissues. This study is the first to demonstrate that COX-2 protects vessels against atherosclerotic lesions independently of local vascular prostacyclin and uses systems biology approaches to identify new mechanisms relevant to development of next generation NSAIDs.

Shape and Compliance of Endothelial Cells after Shear Stress In Vitro or from Different Aortic Regions: Scanning Ion Conductance Microscopy Study

Objective To measure the elongation and compliance of endothelial cells subjected to different patterns of shear stress in vitro, and to compare these parameters with the elongation and compliance of endothelial cells from different regions of the intact aorta. Materials and Methods Porcine aortic endothelial cells were cultured for 6 days under static conditions or on an orbital shaker. The shaker generated a wave of medium, inducing pulsatile shear stress with a preferred orientation at the edge of the well or steadier shear stress with changing orientation at its centre. The topography and compliance of these cells and cells from the inner and outer curvature of ex vivo porcine aortic arches were measured by scanning ion conductance microscopy (SICM). Results Cells cultured under oriented shear stress were more elongated and less compliant than cells grown under static conditions or under shear stress with no preferred orientation. Cells from the outer curvature of the aorta were more elongated and less compliant than cells from the inner curvature. Conclusion The elongation and compliance of cultured endothelial cells vary according to the pattern of applied shear stress, and are inversely correlated. A similar inverse correlation occurs in the aortic arch, with variation between regions thought to experience different haemodynamic stresses.

Effects of high flavanol dark chocolate on cardiovascular function and platelet aggregation.

Regular consumption of chocolate and cocoa products has been linked to reduced cardiovascular mortality. This study compared the effects of high flavanol dark chocolate (HFDC; 1064mg flavanols/day for 6weeks) and low flavanol dark chocolate (LFDC; 88mg flavanols/day for 6weeks) on blood pressure, heart rate, vascular function and platelet aggregation in men with pre-hypertension or mild hypertension. Vascular function was assessed by pulse wave analysis using radial artery applanation tonometry in combination with inhaled salbutamol (0.4mg) to assess changes due to endothelium-dependent vasodilatation. HFDC did not significantly reduce blood pressure compared to baseline or LFDC. Heart rate was increased by LFDC compared to baseline, but not by HFDC. Vascular responses to salbutamol tended to be greater after HFDC. Platelet aggregation induced by collagen or the thromboxane analogue U46619 was unchanged after LFDC or HFDC, whereas both chocolates reduced responses to ADP and the thrombin receptor activator peptide, SFLLRNamide (TRAP6), relative to baseline. Pre-incubation of platelets with theobromine also attenuated platelet aggregation induced by ADP or TRAP6. We conclude that consumption of HFDC confers modest improvements in cardiovascular function. Platelet aggregation is modulated by a flavanol-independent mechanism that is likely due to theobromine.

P2Y12 receptor blockade synergizes strongly with nitric oxide and prostacyclin to inhibit platelet activation

Aims In vivo platelet function is a product of intrinsic platelet reactivity, modifiable by dual antiplatelet therapy (DAPT), and the extrinsic inhibitory endothelial mediators, nitric oxide (NO) and prostacyclin (PGI2), that are powerfully potentiated by P2Y12 receptor blockade. This implies that for individual patients endothelial mediator production is an important determinant of DAPT effectiveness. Here, we have investigated this idea using platelets taken from healthy volunteers treated with anti‐platelet drugs. Methods Three groups of male volunteers (n = 8) received either prasugrel (10 mg), aspirin (75 mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet reactivity in the presence of diethylammonium (Z)‐1‐(N,N‐diethylamino)diazen‐1‐ium‐1,2‐diolate (DEA/NONOate) and PGI2 was studied before and following treatment. Results Ex vivo, PGI2 and/or DEA/NONOate had little inhibitory effect on TRAP‐6‐induced platelet reactivity in control conditions. However, in the presence of DAPT, combination of DEA/NONOate + PGI2 reduced platelet aggregation (74 ± 3% to 19 ± 6%, P < 0.05). In vitro studies showed even partial (25%) P2Y12 receptor blockade produced a significant (67 ± 2% to 39 ± 10%, P < 0.05) inhibition when DEA/NONOate + PGI2 was present. Conclusions We have demonstrated that PGI2 and NO synergize with P2Y12 receptor antagonists to produce powerful platelet inhibition. Furthermore, even with submaximal P2Y12 blockade the presence of PGI2 and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial mediator in vivo modulation of P2Y12 inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to predict thrombotic outcomes better and adjust therapy to prevent adverse outcomes in individual patients.

Reduced endothelial dependent vasodilation in vessels from TLR4 mice is associated with increased superoxide generation

Highlights ► The absence of TLR4 is associated with reduced endothelial cell function in mesenteric arteries. ► TLR4−/− mouse arteries have reduced acetylcholine responses. ► TLR4−/− have increased superoxide levels when stimulated with acetylcholine. ► An increase in superoxide generation reduces the activity of endothelial derived nitric oxide.