Timothy D. Warner

Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic

The beneficial actions of nonsteroidal anti‐inflammatory drugs (NSAIDs) have been linked to their ability to inhibit inducible COX‐2 at sites of inflammation, and their side effects (e.g., gastric damage) to inhibition of constitutive COX‐1. Selective inhibitors of COX‐2, such as celecoxib, etoricoxib, lumiracoxib, rofecoxib, and valdecoxib have been developed and the greatest recent growth in our knowledge in this area has been come from the clinical use of these compounds. Although clinical data indicate that COX‐2 selectivity is associated with a reduction in severe gastrointestinal events, they also reveal there are roles for constitutive COX‐2 within tissues such as the brain, kidney, pancreas, intestine, and blood vessels. We now better understand the roles of COX‐1 and COX‐2 in functions as disparate as the perception of pain and the progression of cancers. Clinical use of COX‐2‐selective compounds has ignited strong debates regarding potential side effects, most notably those within the cardiovascular system such as myocardial infarctions, strokes, and elevation in blood pressure. This review will discuss how the latest studies help us understand the roles of COX‐1 and COX‐2 and what clinically proven benefits the newer generation of COX‐2‐selective inhibitors offer.—Warner, T. D., Mitchell, J. A. Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic. FASEB J. 18, 790–804 (2004)

Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids

1 Lipopolysaccharide (LPS) co‐induces nitric oxide synthase (iNOS) and cyclo‐oxygenase (COX‐2) in J774.2 macrophages. Here we have used LPS‐activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX‐2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX‐2 activity was assessed by measuring the formation of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α) by radioimmunoassay. Western blot analysis was used to determine the expression of COX‐2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS‐treated rats.

Endothelin-1 and Endothelin-3 Release EDRF from Isolated Perfused Arterial Vessels of the Rat and Rabbit

Summary We have previously shown that porcine endothelin (ET-1) releases endothelium-derived relaxing factor (EDRF) in the rat isolated perfused mesentery. Here we show that both ET-1 (1–100 pmol) and rat endothelin (ET-3, 1–300 pmol) release EDRF in this preparation and that ET-1 releases EDRF from the luminally perfused aorta of the rabbit. Furthermore, we confirm that, as a pressor agent, ET-1 is greater than 10 times more potent than ET-3. Vasodilatations in the rat isolated perfused mesentery in response to ET-1 and ET-3 were due to the release of EDRF since they were inhibited by removal of the endothelium, methylene blue (100

Cyclo‐oxygenase‐2: pharmacology, physiology, biochemistry and relevance to NSAID therapy

mUM), or hemoglobin (30

Cyclooxygenase-3 (COX-3): Filling in the gaps toward a COX continuum?

mUM). ET-3 was more selective than ET-1 as a vasodilator because ET-1 induced vasodilatations were limited and in the higher doses overwhelmed by concurrent vasoconstrictions. Release of EDRF from the rabbit aorta in response to ET-1 but not to other agonists (acetylcholine, substance P, or adenosine diphosphate) was potentiated by infusion of potassium chloride (3 mM). Bay K 8644 failed to release EDRF in either system or to constrict the nondepolarized rat mesentery. Thus, both ET-1 and ET-3 release EDRF by activation of receptors or channels that differ from dihydropyridine-sensitive calcium channels.

COX isoforms in the cardiovascular system: understanding the activities of non-steroidal anti-inflammatory drugs

Cyclo‐oxygenase is expressed in cells in two distinct isoforms. Cyclo‐oxygenase‐1 is present constitutively whilst cyclo‐oxygenase‐2 is expressed primarily after inflammatory insult. The activity of cyclo‐oxygenase‐1 and ‐2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo‐oxygenase include the nonsteroidal anti‐inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo‐oxygenase‐2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo‐oxygenase‐1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo‐oxygenase‐2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo‐oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimers disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

Activation of PPARβ/δ Induces Endothelial Cell Proliferation and Angiogenesis

Humans have been using nonsteroid antiinflammatory drugs (NSAIDs) in various forms for more than 3,500 years (1). They are still our favorite medicines. Estimates vary, but it appears, for instance, that each year we consume around 40,000 metric tons of aspirin, equating to about 120 billion aspirin tablets (300 mg is a standard size). In addition, dozens of other NSAIDs and NSAID formulations are available and enthusiastically consumed in most countries. However, despite this long history and large volume of use, we still have an incomplete understanding of how the NSAIDs achieve their actions. Most recently, molecular biology, together with pharmacology, has brought the greatest steps forward in knowledge. It is in this vein that Dan Simmons group report the discovery of a novel cyclooxygenase (COX) enzyme variant that could be the target of acetaminophen and other analgesic/antipyretic drugs (2). After 3,500 years, the first real progress in our understanding of the mechanism of the NSAIDs came 30 years ago.

Circulating MicroRNAs as Novel Biomarkers for Platelet Activation

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of prostanoids by the enzyme cyclooxygenase (COX). Work in the past 15 years has shown that COX exists in two forms: COX1, which is largely associated with physiological functions, and COX2, which is largely associated with pathological functions. Heated debate followed the introduction of selective COX2 inhibitors around 5 years ago: do these drugs offer any advantages over the traditional NSAIDs they were meant to replace, particularly in regard to gastrointestinal and cardiovascular side effects? Here we discuss the evidence and the latest recommendations for the use of selective inhibitors of COX2 as well as the traditional NSAIDs.

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF

Objective—The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-&bgr;/&dgr; in endothelial cells remains unclear. Interestingly, the selective PPAR&bgr;/&dgr; ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPAR&bgr;/&dgr; in endothelial cell proliferation and angiogenesis. Methods and Results—Western blot analysis indicated PPAR&bgr;/&dgr; was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPAR&bgr;/&dgr; target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPAR&bgr;/&dgr;. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation. Conclusions—PPAR&bgr;/&dgr; is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

Sputum and plasma endothelin-1 levels in exacerbations of chronic obstructive pulmonary disease

Rationale: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyze changes in plasma miRNAs in response to antiplatelet therapy. Methods and Results: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33), who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.