Ken Shinmura

Discovery of a new function of cyclooxygenase (COX)-2: COX-2 is a cardioprotective protein that alleviates ischemia/reperfusion injury and mediates the late phase of preconditioning

More than 10 years after its discovery, the function of cyclooxygenase-2 (COX-2) in the cardiovascular system remains largely an enigma. Many scholars have assumed that the allegedly detrimental effects of COX-2 in other systems (e.g. proinflammatory actions and tumorigenesis) signify a detrimental role of this protein in cardiovascular homeostasis as well. This view, however, is ill-founded. Recent studies have demonstrated that ischemic preconditioning (PC) upregulates the expression and activity of COX-2 in the heart, and that this increase in COX-2 activity mediates the protective effects of the late phase of PC against both myocardial stunning and myocardial infarction. An obligatory role of COX-2 has been observed in the setting of late PC induced not only by ischemia but also by delta-opioid agonists and physical exercise, supporting the view that the recruitment of this protein is a central mechanism whereby the heart protects itself from ischemia. The beneficial actions of COX-2 appear to be mediated by the synthesis of PGE(2) and/or PGI(2). Since inhibition of iNOS in preconditioned myocardium blocks COX-2 activity whereas inhibition of COX-2 does not affect iNOS activity, COX-2 appears to be downstream of iNOS in the protective pathway of late PC. The results of these studies challenge the widely accepted paradigm that views COX-2 activity as detrimental. The discovery that COX-2 plays an indispensable role in the anti-stunning and anti-infarct effects of late PC demonstrates that the recruitment of this protein is a fundamental mechanism whereby the heart adapts to stress, thereby revealing a novel, hitherto unappreciated cardioprotective function of COX-2. From a practical standpoint, the recognition that COX-2 is an obligatory co-mediator (together with iNOS) of the protection afforded by late PC has implications for the clinical use of COX-2 selective inhibitors as well as nonselective COX inhibitors. For example, the possibility that inhibition of COX-2 activity may augment myocardial cell death by obliterating the innate defensive response of the heart against ischemia/reperfusion injury needs to be considered and is the object of much current debate. Furthermore, the concept that the COX-2 byproducts, PGE(2) and/or PGI(2), play a necessary role in late PC provides a basis for novel therapeutic strategies designed to enhance the biosynthesis of these cytoprotective prostanoids in the ischemic myocardium. From a conceptual standpoint, the COX-2 hypothesis of late PC expands our understanding of the function of this enzyme in the cardiovascular system and impels a critical reassessment of current thinking regarding the biologic significance of COX-2.

Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning

Cyclooxygenase-2 (COX-2) is known to mediate the cardioprotective effects of the late phase of ischemic preconditioning (PC); however, the signaling pathways involved in COX-2 induction following ischemic PC are unknown. In addition, although inducible nitric oxide synthase (iNOS) has been identified as a co-mediator of late PC together with COX-2, the interaction between iNOS and COX-2 in the heart is unknown. Using conscious rabbits, we found that the induction of COX-2 expression 24 hours after ischemic PC was blocked by pretreatment with inhibitors of protein kinase C (PKC), Src protein tyrosine kinases (PTKs), and nuclear factor-&kgr;B (NF-&kgr;B) but not by inhibitors of NOS or scavengers of reactive oxygen species (ROS). The selective iNOS inhibitors SMT and 1400W, given 24 hours after PC, abrogated the increase in myocardial prostaglandin E2 (PGE2) and 6-keto-PGF1&agr;, whereas the selective soluble guanylate cyclase inhibitor ODQ had no effect. COX-2 selective inhibitors (celecoxib and NS-398) did not affect iNOS activity. These results demonstrate that (i) ischemic PC upregulates cardiac COX-2 via PKC-, Src PTK-, and NF-&kgr;B–dependent signaling pathways, whereas generation of NO and ROS is not necessary, and (ii) the activity of newly synthesized COX-2 following PC requires iNOS-derived NO whereas iNOS activity is independent of COX-2–derived prostanoids, indicating that COX-2 is located downstream of iNOS in the protective pathway of late PC. The data also indicate that iNOS modulates COX-2 activity via cGMP-independent mechanisms. To our knowledge, this is the first demonstration that iNOS-derived NO drives prostanoid synthesis by COX-2 in the heart. NO-mediated activation of COX-2 may be a heretofore unrecognized mechanism by which NO exerts its salubrious effects in the late phase of PC.

Aldose Reductase Is an Obligatory Mediator of the Late Phase of Ischemic Preconditioning

Abstract— Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been shown to metabolize toxic aldehydes generated by lipid peroxidation, suggesting that it may serve as an antioxidant defense. To investigate its role in the late phase of ischemic preconditioning (PC), conscious rabbits underwent 6 cycles of 4-minute coronary occlusion/4-minute reperfusion. Twenty-four hours later, there was a marked increase in AR protein and activity and in the myocardial content of sorbitol, a unique product of AR catalysis. Pretreatment with N&ohgr;-nitro-l-arginine, a nitric oxide synthase inhibitor, or chelerythrine, a protein kinase C inhibitor (both given at doses that block late PC in this model), prevented the increase in AR protein 24 hours later, demonstrating that ischemic PC upregulates AR via nitric oxide- and protein kinase C-dependent signaling pathways. The AR-selective inhibitors tolrestat and sorbinil prevented AR-mediated accumulation of sorbitol and abrogated the infarct-sparing effect of late PC, demonstrating that enhanced AR activity is necessary for this cardioprotective phenomenon to occur. Inhibition of AR did not affect infarct size or the myocardial accumulation of the lipid peroxidation product 4-hydroxy trans-2-nonenal (HNE) in nonpreconditioned rabbits. The accumulation of HNE was inhibited by late PC, and this effect was abrogated by sorbinil. Taken together, these results establish AR as an essential mediator of late PC. Furthermore, the data suggest that myocardial ischemia/reperfusion injury is due in part to the generation of lipid peroxidation products and that late PC diminishes this source of injury by upregulating AR.

Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice

Abstract Recent studies have demonstrated that cyclooxygenase-2 (COX-2) is an essential mediator of the cardioprotective effects of the late phase of ischemic preconditioning (PC) in rabbits. The goal of this study was to determine whether COX-2 also plays an essential role in late PC in the mouse. B6129F2/J mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. Administration of the COX-2 selective inhibitor, NS-398, 30 min prior to the 30-min occlusion (5 mg/kg i.p.) had no appreciable effect on infarct size compared with untreated controls (58.8 ± 2.1%, vs. 58.8 ± 4.3% of the risk region, respectively). When mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h prior to the 30-min occlusion, infarct size was markedly reduced (19.3 ± 3.4%), indicating a late PC effect. The protective effect of late PC was completely abrogated by administration of NS-398 30 min before the 30-min coronary occlusion (67.7 ± 3.0%), but not by administration of vehicle alone (23.6 ± 3.7%). These results indicate that COX-2 mediates the late phase of ischemic PC in the mouse and imply that the role of this enzyme in cardioprotection is not species-specific.

Role of Cyclic Guanosine Monophosphate in Late Preconditioning in Conscious Rabbits

Background—Although NO has been shown to serve both as the trigger and the mediator of the late phase of ischemic preconditioning (PC), it is unknown whether NO acts via activation of soluble guanylate cyclase (sGC). The objective of this study was to investigate the role of sGC in late PC in conscious rabbits using the selective sGC inhibitor 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Methods and Results—A total of 172 conscious rabbits were used. When nonpreconditioned rabbits were subjected to a sequence of 4-minute coronary occlusion/4-minute reperfusion cycles, myocardial cyclic guanosine monophosphate (cGMP) levels increased significantly at the end of the third and sixth occlusions. In rabbits preconditioned 24 hours earlier (on day 1) with six occlusion/reperfusion cycles, myocardial cGMP levels on day 2 were significantly higher than in nonpreconditioned rabbits even before ischemia but did not increase further during a second sequence of 4-minute occlusion/reperfusion cycles. Administration of ODQ before the six occlusion/reperfusion cycles on day 1 did not prevent the development of late PC against either stunning or infarction on day 2. In contrast, administration of ODQ on day 2 completely ablated the late PC effect against both stunning and infarction. Conclusions—These results indicate that enhanced synthesis of cGMP by sGC is not necessary for ischemia to trigger a late PC effect but is required for the protection to become manifest 24 hours later. This implies that NO participates in late PC via two distinct mechanisms; ie, it triggers late PC on day 1 via a cGMP-independent mechanism and it mediates late PC on day 2 via a cGMP-dependent mechanism.

Effect of aspirin on late preconditioning against myocardial stunning in conscious rabbits

OBJECTIVES The goal of this study was to investigate the effect of three different doses of acetylsalicylic acid (aspirin) (ASA) on the late phase of ischemic preconditioning (PC) against myocardial stunning. BACKGROUND Although recent evidence indicates that the late phase of ischemic PC is mediated by cyclooxygenase-2 (COX-2), the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit COX-2 activity on late PC has not been evaluated; ASA is the most widely used NSAID. Therefore, we determined whether ASA impedes the development of late PC. METHODS Conscious rabbits underwent a protocol consisting of three days of six 4-min coronary occlusion/4-min reperfusion cycles. RESULTS Neither 5 mg/kg nor 10 mg/kg x 3 of ASA interfered with the protective effects of late PC against stunning. In contrast, the late PC effect was completely abrogated by 25 mg/kg of ASA. Low-dose (5 mg/kg) ASA effectively inhibited platelet aggregation but did not prevent the increase in COX-2 activity, whereas the highest dose (25 mg/kg) completely blocked COX-2 activity. CONCLUSIONS The administration of ASA either at antithrombotic doses (5 mg/kg), which are widely used to prevent cardiovascular events in patients, or at analgesic/antipyretic doses (10 mg/kg) does not interfere with the cardioprotective effects of late PC against myocardial stunning. In contrast, high doses of ASA (25 mg/kg), which are used as antirheumatic therapy, abrogate both COX-2 activity and late PC, suggesting that nonselective doses of NSAIDs should be used with caution in patients with atherosclerotic cardiovascular disease because they may deprive the heart of its innate defensive response.

Nitric oxide donors attenuate myocardial stunning in conscious rabbits

Although previous studies suggested that the protection of late preconditioning (PC) against myocardial stunning is mediated by nitric oxide (NO), direct evidence that exogenous administration of NO attenuates myocardial stunning is lacking. Furthermore, although exogenous NO administration was shown to elicit a late PC phase, it is unknown whether NO donors also induce an early PC phase. Therefore, conscious rabbits underwent two experimental stages (3 days of six 4-min occlusion/4-min reperfusion cycles each) 2 wk apart. In study I, both stages were control stages ( n = 7). In studies II and III, stage I was the control stage. On day 1of stage II, seven rabbits received infusion of nitroglycerin (NTG; 2 μg ⋅ kg-1 ⋅ min-1iv) during the ischemia-reperfusion sequence, starting 30 min before the 1st occlusion and ending 10 min after the 6th reperfusion ( study II). Another seven rabbits received infusion of NTG (2 μg ⋅ kg-1 ⋅ min-1iv) for 1 h followed by a 30-min washout interval and then underwent six 4-min occlusion/4-min reperfusion cycles ( study III). In the control stage of all three studies, recovery of wall thickening (WTh) after occlusion/reperfusion cycles was markedly enhanced on days 2 and 3 compared with day 1, indicating late PC. In study II, infusion of NTG during the occlusion/reperfusion cycles on day 1 resulted in significant and sustained enhancement in WTh recovery. A similar attenuation of stunning was observed in study IV in six rabbits given intravenous infusion of S-nitroso- N-acetylpenicillamine (SNAP) during occlusion/reperfusion cycles. The magnitude of the protection afforded by NTG and SNAP was comparable to that afforded by the late ischemic PC phase. In contrast, in study III infusion of NTG before occlusion/reperfusion cycles did not enhance WTh recovery, indicating that NTG failed to induce an early PC effect against stunning. This study demonstrates that administration of hemodynamically inactive doses of two unrelated NO donors alleviates myocardial stunning in conscious rabbits, providing direct evidence for a protective action of NO in this setting.Although previous studies suggested that the protection of late preconditioning (PC) against myocardial stunning is mediated by nitric oxide (NO), direct evidence that exogenous administration of NO attenuates myocardial stunning is lacking. Furthermore, although exogenous NO administration was shown to elicit a late PC phase, it is unknown whether NO donors also induce an early PC phase. Therefore, conscious rabbits underwent two experimental stages (3 days of six 4-min occlusion/4-min reperfusion cycles each) 2 wk apart. In study I, both stages were control stages (n = 7). In studies II and III, stage I was the control stage. On day 1 of stage II, seven rabbits received infusion of nitroglycerin (NTG; 2 microg. kg(-1). min(-1) iv) during the ischemia-reperfusion sequence, starting 30 min before the 1st occlusion and ending 10 min after the 6th reperfusion (study II). Another seven rabbits received infusion of NTG (2 microg. kg(-1). min(-1) iv) for 1 h followed by a 30-min washout interval and then underwent six 4-min occlusion/4-min reperfusion cycles (study III). In the control stage of all three studies, recovery of wall thickening (WTh) after occlusion/reperfusion cycles was markedly enhanced on days 2 and 3 compared with day 1, indicating late PC. In study II, infusion of NTG during the occlusion/reperfusion cycles on day 1 resulted in significant and sustained enhancement in WTh recovery. A similar attenuation of stunning was observed in study IV in six rabbits given intravenous infusion of S-nitroso-N-acetylpenicillamine (SNAP) during occlusion/reperfusion cycles. The magnitude of the protection afforded by NTG and SNAP was comparable to that afforded by the late ischemic PC phase. In contrast, in study III infusion of NTG before occlusion/reperfusion cycles did not enhance WTh recovery, indicating that NTG failed to induce an early PC effect against stunning. This study demonstrates that administration of hemodynamically inactive doses of two unrelated NO donors alleviates myocardial stunning in conscious rabbits, providing direct evidence for a protective action of NO in this setting.