a Laboratoire de Pharmacologie, Faculté de Médecine de Grenoble, HP2 EA 3745, France
b Department of Cardiology, Grenoble University Hospital, France
* Correspondence to: Laboratoire de Pharmacologie, Faculté de Médecine de Grenoble, HP2 EA 3745, 38700 La Tronche, France. Tel.: +33 4 76 76 92 60; fax: +33 4 76 76 92 62 (E-mail: jean-luc.cracowski{at}ujf-grenoble.fr).
Arachidonic acid is an essential unsaturated fatty acid and is the most abundant in cell membranes. Its metabolism leads to the formation of the well known prostaglandins and thromboxanes, which are implicated in the modulation of vascular tone and growth and play an important role on the blood-vessel interface. The discovery of their pharmacological activity led to the development of some potent drugs such as the prostacyclin analogues, while thromboxane A2-receptor antagonists are currently under development. While research on arachidonic acid metabolites focused for decades on the enzymatic pathway, Morrow and Roberts 1 described in 1990 another pathway of arachidonic acid metabolism, i.e., a free radical pathway, leading to a large series of compounds termed isoprostanes.
A first level of complexity is that, unlike the enzymatic formation of prostaglandins, F2-isoprostanes derive from a non-specific free radical attack of arachidonic acid, i.e., that four different series of compounds, called regioisomers, differing in the nature of their side chains, are formed (Fig. 1). The second level of complexity is that eight isomers may be formed among each of these four regioisomers, i.e., 64 different F2-isoprostanes are formed. The third level of complexity is man-made: three different nomenclatures co-exist2. Given the potential number of compounds, this can lead to a nightmare for the non-specialist, and a puzzle for novices to determine which is which. For clarity, the nomenclature validated by the International Union of Pure and Applied Chemistry3 should be used. The fourth level of complexity is that F2-isoprostanes are only one family of the myriad of compounds produced through free radical peroxidation of arachidonic acid, including two recent families of compounds termed isoketals and isofurans (Fig. 1)4.
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15-F2t-IsoP is released in conditions associated with free radical generation, such as cardiac ischaemiareperfusion, and is the most sensitive and specific biomarker of lipid peroxidation in vascular disorders11. Several authors have tried to answer an important question: are these compounds merely biomarkers, or do they act as mediators in pathological conditions12,13? As we do not possess a pharmacological agent that may specifically inhibit the biological effects of 15-F2t-IsoP, the answer cannot be straightforward. Basal plasma concentrations of 15-F2t-IsoP have been found to range from approximately 1x1010 to 5x1010 moll1 in plasma samples. These concentrations are unlikely to induce a systemic vasoactive effect considering the EC50 values of 15-F2t-IsoP observed in vitro5 However, F2-isoprostanes are released at the site of free radical injury and then diluted in the circulation and therefore local concentrations might be sufficiently high to induce regional vasoconstriction. Indeed, the concentrations of 15-F2t-IsoP and 5-F2t-IsoP were increased markedly (from basal levels) in the coronary sinus and plasma following coronary angioplasty14,15. 15-F2t-IsoP concentrations were in the nanomolar range, and thus unlikely to contribute to epicardial coronary artery vasoconstriction16. However, the concentration of 15-F2t-IsoP might be sufficiently high to induce a vasoconstriction in the microcirculation because the potency of 15-F2t-IsoP seems to be higher in these arteries compared with conductance arteries17. Considering 5-F2t-IsoP, this compound does not possess any vasomotor activity18,19, and is therefore unlikely to play a pathogenic role on the coronary vasculature.
No specific inhibition of 15-F2t-IsoP or other isoprostane vascular effects can currently be achieved. However, TP receptor antagonists but not aspirin, are effective in the inhibition of atherosclerosis in apo E-KO (knock-out) mice, showing that TP receptor blockade by S18886 is effective by a mechanism independent of platelet-derived thromboxane A220, whereas isoprostane suppression with vitamin E retards atherogenesis in the same animal model21. Similarly, TP receptor antagonism by L670596, but not COX-2 inhibition, prevented pulmonary hypertension and endothelin-1 upregulation in 60% O2-mediated pulmonary hypertension in newborns rats22. In addition to these animal data, a study showed that in patients suffering from coronary artery disease, S18886 (a TP receptor antagonist) improved acetylcholine-induced and flow-mediated vasodilation in patients treated with aspirin23. An hypothesis is that endogenous TP receptor activation induced by 15-F2t-IsoP or other isoprostanes may be involved in the COX-independent effects of TP receptor antagonists24. However, because TP receptors share other endogenous ligands such as prostaglandin H2 or HETEs, such data give strength to the hypothesis that isoprostanes are involved in the vascular physiology and pathogenesis, but does not enable a definitive conclusion.
Given that many isomeric isoprostanes exist, one should not focus only on 15-F2t-IsoP. Other isoprostanes possess potent pharmacological activity, the most potent ones being the isoprostanes from the E series. For example, 15-E2t-IsoP is 10 times more potent than 15-F2t-IsoP in systemic as well as pulmonary vessels25,26. Furthermore, in a recent report Hou et al.,19 showed that many other regioisomers, including those from the 12-series, constricted retinal and brain microvessels. These regioisomers are likely to be produced under the same conditions as 15-F2t-IsoP, but no data is currently available concerning their production in cardiac ischaemiaperfusion. Finally, other potential physiological mediators of the isoprostane pathway include the isothromboxanes27 and the receptor independent effects of isoketals, that rapidly adduct to membrane proteins28.
Isoprostanes are currently the more valuable biomarkers of lipid peroxidation11. They have been measured in biological fluids such as urine, plasma, exhaled breath condensate, bronchoalveolar lavage fluid, bile, cerebrospinal, seminal and pericardial fluids. They were also detectable in normal tissues, including umbilical cords29. Their measurement in biological fluids has prompted clinical investigations on the pathophysiological role of lipid peroxidation in cardiovascular diseases (see 30,31). Among the biological fluids available, most studies were performed on urine because of the non-invasiveness of the procedure and the lack of artefactual generation. A strong link between lipid peroxidation and vascular diseases associated with ischaemiareperfusion, atherosclerosis and inflammation has been suggested by the elevated levels of lipid peroxidation observed in such diseases.
In addition to being a pathophysiological marker, the quantification of F2-isoprostanes might represent a prognostic marker. F2-isoprostane levels consistently correlate to the severity of heart failure32,33,34, and correlate to the haemodynamic response to NO in pulmonary hypertension35. Furthermore, Schwedhelm et al., showed in a recent case-control study that urinary 15-F2t-IsoP was a strong independent concentration-dependent risk marker of coronary heart disease36. There are currently no published clinical studies aimed at testing isoprostanes as a long-term prognostic marker, with strong endpoints such as mortality or morbidity, but at least two cohort studies are currently on-going.
In conclusion, we are just beginning to explore the wilderness of arachidonic acid metabolism leading to isoprostane formation. While the available data provides fuel for discussion concerning the potential implication of 15-F2t-IsoP in vascular disorders, the unknown physiological and pathological significance of the myriad of compounds produced from arachidonic acid in conditions of enhanced free radical generation, observed in many vascular diseases, leaves scope for many more investigations.
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