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

Abstract

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.

Fig. 1

Schematic representation of our current understanding of the cellular mechanisms whereby COX-2 is upregulated by ischemic PC and participates in cardioprotection. A sublethal ischemic stress (ischemic PC) activates a complex signal transduction cascade that includes PKC (specifically, the ε isoform), PTKs (specifically, Src and/ or Lck kinases), and probably other as-yet-unknown kinases, leading to phosphorylation of IκBα and mobilization of the transcription factor NF-κB. In addition, ischemic PC activates the non-receptor tyrosine kinases JAK1 and JAK2 with subsequent tyrosine phosphorylation and activation of the transcription factors STAT1 and STAT3. Other, as yet unknown, transcription factors are most likely involved as well. The promoter of both the iNOS and the COX-2 genes contains cognate sequences for NF-κB and STAT1/STAT3. Binding of NF-κB and STAT1/STAT3 to these promoters results in a coordinated transcriptional activation of the iNOS and COX-2 genes with synthesis of new iNOS and COX-2 proteins. The activity of newly-synthetized COX-2 protein requires iNOS-dependent NO generation whereas the activity of iNOS does not require COX-2-dependent prostanoid generation. Thus, COX-2 is downstream of iNOS in the pathophysiological cascade of late PC. iNOS-derived NO can protect the myocardium from recurrent ischemia both via direct actions and via activation of COX-2-dependent synthesis of cardioprotective prostanoids. Among the products of COX-2, PGE₂ and/ or PGI₂ appear to be the most likely effectors of cytoprotection. A similar upregulation of COX-2 can be elicited pharmacologically by δ-opioid receptor agonists but not by adenosine A₁ or A₃ receptor agonists.

Fig. 2

Effect of ischemic PC on COX-2 mRNA expression in rabbit myocardium. Tissue samples were obtained from the anterior and posterior LV wall of control rabbits and from the ischemic-reperfused and nonischemic regions of rabbits that underwent ischemic PC with six 4-min coronary occlusion /4-min reperfusion cycles and were euthanized 1 h, 3 h, or 24 h later. (A) Representative RPA gel; (B) densitometric analysis of COX-2 mRNA signals. Each COX-2 signal was normalized to the GAPDH signal from the same sample to control for RNA loading. The normalized values are expressed as percentage of the signal in the anterior LV wall of control hearts. Data are means±S.E.M. (Reproduced with permission of the National Academy of Sciences from Shinmura et al. [46]).

Fig. 3

Effect of ischemic PC on the expression of COX-2 protein in rabbit myocardium. Tissue samples were obtained as described in the legend to in Fig. 2 from control rabbits and from rabbits that underwent ischemic PC 24 h earlier. (A) COX-2 immunoreactivity in the membranous fraction increased markedly in the ischemic-reperfused region 24 h after ischemic PC. Robust expression of COX-2 was observed in rabbit kidney and in murine macrophages stimulated with interferon-γ and lipopolysaccharide (positive controls). (B) Densitometric analysis of COX-2 signals in the membranous fraction. In all samples, the densitometric measurements of COX-2 immunoreactivity were expressed as a percentage of the average value measured in the anterior LV wall of control rabbits. Data are means±S.E.M. (Reproduced with permission of the National Academy of Sciences from Shinmura et al. [46]).

Fig. 4

Effect of ischemic PC on the myocardial content of PGE₂, PGF_2α, and 6-keto-PGF_1α (measured by EIA). In rabbits that underwent ischemic PC 24 h earlier, the levels of PGE₂ and 6-keto-PGF_1α in the ischemic-reperfused region increased markedly vs. control rabbits; the levels of PGF_2α were higher than in the nonischemic region of the same group but did not differ significantly from controls. The increase in PGE₂, PGF_2α, and 6-keto-PGF_1α was completely abrogated when rabbits were given NS-398 or celecoxib 24 h after ischemic PC (40 min before euthanasia). The myocardial content of PGE₂ and 6-keto-PGF_1α in the nonischemic region was similar in all groups, indicating that the COX-2 inhibitors did not affect constitutive production of these eicosanoids by COX-1. Data are means±S.E.M. (Reproduced with permission of the National Academy of Sciences from Shinmura et al. [46]).

Fig. 5

Total deficit of WTh after the sixth reperfusion (a measure of the severity of myocardial stunning) on days 1, 2, and 3 in groups VII (control, n = 10), VIII (DMSO on day 2, n = 7), IX (NS-398 on day 1, n = 9), X (NS-398 on day 2, n = 10), XI (celecoxib on day 1, n = 9) and XII (celecoxib on day 2, n = 10). The total deficit of WTh was measured in arbitrary units, as described in the text. Data are means±S.E.M. (Reproduced with permission of the National Academy of Sciences from Shinmura et al. [46]).

Fig. 6

Myocardial infarct size in groups XIII (control, n = 10), XIV (PC, n = 9), XV(PC+DMSO, n = 10), XVI (PC+NS-398, n = 8), XVII (NS-398, n = 9), XVIII (PC+celecoxib, n = 8), and XIX (celecoxib, n = 9). Infarct size is expressed as a percentage of the region at risk of infarction. Solid circles represent means±S.E.M. (Reproduced with permission of the National Academy of Sciences from Shinmura et al. [46]).

Fig. 7

Infarct size, expressed as a percent of the risk region, in the six groups of mice. Open circles indicate individual measurements, solid circles represent means±S.E.M. (Reproduced with permission of Steinkopff Verlag from Guo et al. [52]).