COX-2 and the vascular system: interactions with endothelial pathways and the eNOS system

Endothelial cells release protective hormones such as prostacyclin and nitric oxide involving the enzyme pathways of cyclooxygenase and nitric oxide synthase (NOS). Both prostacyclin and nitric oxide act to oppose the effects of thromboxane A2 released following the actions also of cyclooxygenase by platelets. Cyclooxygenase (cyclooxygenase-2) is also present in inflammation and is the therapeutic target for the nonsteroidal anti-inflammatory group of drugs (NSAIDs). NSAIDs are among the most popular in the world. But NSAIDs also have side effects in the gut, this is why selective cyclooxygenase-2 types of NSAID were introduced. However, now after their introduction, there is an important concern regarding the cardiovascular side effects caused by all NSAIDs that work by blocking cyclooxygenase-2. My PhD thesis has used a number of techniques to show that the constitutive isoform (cyclooxygenase-1) drives prostacyclin in blood vessels and that in the kidney knocking out cyclooxygenase-2 results in changes in genes and proteins that regulate the methylarginines ADMA and LNMMA which are NOS inhibitors. I show that in cyclooxygenase-2 knock out mice ADMA and LNMMA are increased and that eNOS responses are reduced and that the effect is reversed by the substrate L-arginine. This work suggests that ADMA could explain why NSAIDs that work by blocking cyclooxygenase-2 affects endothelial responses in an indirect way. The data also suggests that ADMA could be a biomarker and that for some people L-arginine supplements might be protective. By using a mathematical model that I devised myself I also showed that cyclooxygenase-2 knock out causes morphological changes in the endothelium that suggest that in that region the enzyme might be pro-inflammatory and that for this observation a relationship with eNOS does not seem to be involved.Open Acces

Distribution of luciferin-dependent bioluminescence in cardiovascular tissue from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Quantification of basal expression from the aortic tree, vena cava, chambers of the heart and, for comparison, brain from <i>Cox2</i>^{<i>fLuc/+</i>} mice and (b) and representative images of bioluminescence. Arteries, veins and chambers of the heart were essentially devoid of expression from the <i>Cox2</i> gene, in comparison with the brain as a reference tissue. The only exception to this was weak, but detectable, expression in the region of the aortic arch. n=3.</p

6-keto-PGF_1α production in isolated mouse aorta; measurement by enzyme immunoassay, radio immunoassay, and liquid chromatography tandem mass spectrometry (LC-MS/MS).

<p>Prostacyclin release by isolated rings of mouse aorta stimulated with Ca²⁺ ionophore A23187 (50µM), measured as the stable breakdown product 6-keto-PGF_1α, was not altered by <i>Cox2</i> gene deletion, but was reduced >10-fold by <i>Cox1</i> gene deletion. The pattern and level of 6-keto-PGF_1α accumulation was similar whether measured by (a) enzyme immunoassay, (b) radio immunoassay or (c) LC-MS/MS. Representative LC-MS/MS chromatograms show the presence or absence of 6-keto PGF_1α in all sample types (retention time 2.81 min; transition ion <i>m</i>/<i>z</i> 369>163). n=4-7. *, p<0.05 by 1-way ANOVA with Bonferonni’s post-test.</p

Distribution of luciferin-dependent bioluminescence in tissues from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Basal expression from organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mice was visualized by bioluminescent imaging of tissues dissected from <i>Cox2</i>^{<i>fLuc/+</i>} reporter mice after injection of D-luciferin in vivo (125mg/kg i.p.). (b) Imaging data are expressed as maximum luminescent emission from each tissue. Basal <i>Cox2</i> gene driven luciferase expression was present in many tissues including the vas deferens, brain, intestine, and thymus but was notably low to absent in the aorta (highlighted with red circles). Sub-division of the (c) brain, (d) intestine, (e) kidney and (f) stomach revealed regional expression patterns within each tissue. n=5.</p

Bradykinin-stimulated prostanoid accumulation in the circulation <i>in</i><i>vivo</i> in wild-type, <i>Cox1</i>^<i>-/-</i>, and <i>Cox2</i>^<i>-/-</i> mice.

<p>Accumulation of the stable prostacyclin breakdown product, 6-keto-PGF_1α in plasma after bradykinin administration (100nmol/kg i.v.) is dependent on COX-1 but not COX-2 when measured by LC-MS/MS (a). Representative LC-MS/MS chromatograms show the presence or absence of 6-keto PGF_1α in all sample types (retention time 2.81 min; transition ion <i>m</i>/<i>z</i> 369>163). Similar data were obtained for plasma levels of PGE₂ (b), 13,14-dihydro-15-keto-PGE₂ (c), PGD₂ (d), TXB₂ (e) and (f) PGF_2α. Plasma 6-keto-PGF_1α levels in all genotypes compare well with those previously published using enzyme immunoassay measurements. n=6. *, p<0.05 by 1-way ANOVA with Bonferonni’s post-hoc test.</p

COX-2-dependent prostanoid production by aorta versus other mouse tissues in <i>Cox1</i>^<i>-/-</i> mice.

<p>(a) PGE₂ formation, normalized to tissue mass, was measured by immunoassay in supernatants of Ca²⁺ ionophore A23187 (50µM)-stimulated tissue segments from <i>Cox1</i>^<i>-/-</i> mice. Cox1^-/- tissues released a variable amount of PGE₂ with low levels in the aorta (highlighted in red), and substantially higher levels in the thymus, intestines, renal medulla, brain and vas deferens. This distribution correlates well with luciferase expression in organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mouse, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069524#pone-0069524-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069524#pone-0069524-g004" target="_blank">4</a>. n=6.</p

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

This research was supported by a program grant from the Wellcome Trust (0852551Z108/Z to JAM) and NIH-NCI P50 award CA086306 (to HRH). Copyright: © 2013 Kirkby et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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, antibodybased 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 systemPeer reviewedFinal Published versio