Advertisement
Research Article

The COX-2/PGI2 Receptor Axis Plays an Obligatory Role in Mediating the Cardioprotection Conferred by the Late Phase of Ischemic Preconditioning

  • Yiru Guo,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Deepali Nivas Tukaye,

    Affiliation: Department of Internal Medicine, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Wen-Jian Wu,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Xiaoping Zhu,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Michael Book,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Wei Tan,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Steven P. Jones,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Gregg Rokosh,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Shuh Narumiya,

    Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan

    X
  • Qianhong Li,

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Roberto Bolli mail

    rbolli@louisville.edu

    Affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America

    X
  • Published: July 23, 2012
  • DOI: 10.1371/journal.pone.0041178

Abstract

Background

Pharmacologic studies with cyclooxygenase-2 (COX-2) inhibitors suggest that the late phase of ischemic preconditioning (PC) is mediated by COX-2. However, nonspecific effects of COX-2 inhibitors cannot be ruled out, and the selectivity of these inhibitors for COX-2 vs. COX-1 is only relative. Furthermore, the specific prostaglandin (PG) receptors responsible for the salubrious actions of COX-2-derived prostanoids remain unclear.

Objective

To determine the role of COX-2 and prostacyclin receptor (IP) in late PC by gene deletion.

Methods

COX-2 knockout (KO) mice (COX-2−/−), prostacyclin receptor KO (IP−/−) mice, and respective wildtype (WT, COX-2+/+ and IP+/+) mice underwent sham surgery or PC with six 4-min coronary occlusion (O)/4-min R cycles 24 h before a 30-min O/24 h R.

Results

There were no significant differences in infarct size (IS) between non-preconditioned (non-PC) COX-2+/+, COX-2−/−, IP+/+, and IP−/− mice, indicating that neither COX-2 nor IP modulates IS in the absence of PC. When COX-2−/− or IP−/− mice were preconditioned, IS was not reduced, indicating that the protection of late PC was completely abrogated by deletion of either the COX-2 or the IP gene. Administration of the IP selective antagonist, RO3244794 to C57BL6/J (B6) mice 30 min prior to the 30-min O had no effect on IS. When B6 mice were preconditioned 24 h prior to the 30-min O, IS was markedly reduced; however, the protection of late PC was completely abrogated by pretreatment of RO3244794.

Conclusions

This is the first study to demonstrate that targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, and that the IP, downstream of the COX-2/prostanoid pathway, is a key mediator of the late PC. These results provide unequivocal molecular genetic evidence for an essential role of the COX-2/PGI2 receptor axis in the cardioprotection afforded by the late PC.

Introduction

The cardioprotective effect afforded by late PC is a well-documented and studied phenomenon [1][6]. In the last two decades, extensive research has identified the molecular candidates involved in late PC [7]. Among the numerous identified players, nitric oxide synthase [8][19], heat shock protein [20][23], Mn-superoxide dismutase [24], [25], extracellular superoxide dismutase [26], [27], aldose reductase [28] and COX-2 [15], [18], [29][47] are candidates for pharmacological modulation with the goal of developing cardioprotective therapies.

Previous studies have shown that COX-2 mediates its effects via increasing the synthesis of prostaglandin E2 (PGE2) and prostacyclin (PGI2) [29], [36]. The identification of specific molecules involved in the late phase of PC provides a unique opportunity to develop targeted therapy to exploit the phenomenon of PC for cardioprotection.

Our current knowledge about the role of COX-2 in the late phase of PC is based on pharmacologic studies with COX-2 inhibitors [29][31], [35][38], [41], [43], [46][48]. The possible nonspecific nature of COX-2 inhibitors raises the possibility that the observed inhibition of the late phase of PC may be secondary to non-specific inhibition of other molecules including COX-1 [49]. Furthermore, the specific downstream molecules transducing the actions of COX-2/prostanoids in late PC are unclear. Earlier studies have indicated that the prostacyclin receptor, IP, confers tissue protection [50][55]. In the present study, we examined the effect on late PC of homozygous COX-2 deletion; in addition, we explored the role of the prostaglandin receptor, espicailly IP, as a downstream mediator of COX-2 in late PC using both pharmacological and genetic approaches to manipulate IP gene function. Our results demonstrate the obligatory role of COX-2 in late PC by genetically deleting COX-2, thereby unequivocally establishing COX-2 as a mediator of the late phase of PC. In addition, we demonstrate an essential role of IP in mediating the cardioprotective effects of the late phase of PC.

thumbnail

Figure 1. Experimental protocols.

Fourteen groups of mice including were studied for infarct size analysis in three phases. In Phase I (panel A), on day1, COX-2+/+ and COX-2−/− mice were subjected to either PC or sham surgery. On day 2, all mice were subjected to a 30-min LAD occlusion followed by 24 h of reperfusion. In Phase II (panel B), in addition to the day 2 protocol of Phase I, RO3244794 or vehicle was administered 30 min prior to the induction of acute MI on day 2. In Phase III (panel C), on day 1, IP+/+ and IP−/− mice were subjected either to PC or sham surgery. On day 2, all mice were subjected to a 30-min LAD occlusion followed by 24 h of reperfusion. All animals were sacrificed after 24 h of reperfusion to measure infarct size. The open square (□) indicates the reperfusion or no ischemia protocol. The solid black square (▪) indicates the occlusion protocol. (n = 6–16 each group).

doi:10.1371/journal.pone.0041178.g001

Materials and Methods

This study was performed in accordance with the guidelines and with approval of the Institutional Animal Care and Use Committee at the University of Louisville, and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, Publication No. 86-23, revised 1996).

Reagents

1. RO3244794 (R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-ph​enyl)-benzofuran-2-ylmethoxycarbonylamin​o]-propionicacid)was obtained from Roche Alto (Roche Palo Alto, CA). RO3244794 was solubilized in 0.2 M Trizma base which served as the vehicle [56], [57]; 2. Iloprost, (Cayman Chemical Co., Ann Arbor, MI); 3. Krebs-Henseleit Buffer Modified solution (Sigma-Aldrich Corp., St. Louis, MO USA); 4. TTC (Sigma-Aldrich Corp. St. Louis, MO USA); 5. Phthalo blue (Heucotech, Fairless Hill, PA).

thumbnail

Table 1. Reasons for excluding mice from study (15 groups).

doi:10.1371/journal.pone.0041178.t001

Mice

Male mice were used in this study. The COX-2 knockout (COX-2−/−) and wildtype (COX-2+/+) mice [58] were generously provided by Dr. Robert Langenbach (NIEHS, NIH, NC). Their genetic background was 129Ola/C57BL/6. RO3244794 selective IP inhibition studies were performed in male C57BL6/J (B6) mice. Heterozygous IP KO breeding pairs [59] were provided by Dr. Shuh Narumiya (Tokyo University). We used male wildtype littermates (IP+/+) as control mice and homozygous IP KO (IP−/−). PCR and Southern blot hybridization were used for genotyping.

Hemodynamic Pilot Study

To verify the specificity and dosage of specific IP antagonist RO3244794, we monitored arterial blood pressure during the administration of the specific IP agonist, iloprost (30 µg/kg, iv) with either vehicle or RO3244794 to see whether the hypotensive effect induced by iloprost could be prevented. This study was also conducted using IP−/− mice. In selected pilot studies, a catheter was inserted into the carotid artery for measurement of blood pressure (DTXTM pressure transducer, Viggo-Spectramed, Oxnard, CA). Surface leads were placed subcutaneously to obtain the ECG, which was recorded throughout the experiments on a thermal array chart recorder (Gould TA6000) [1], [9], [30], [60].

Preconditioning (PC) and Myocardial Infarction in vivo Protocols

The murine model of late PC has been previously described in detail [1], [9], [17], [30], [61], [62]. Briefly, on day 1, mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p), intubated, and ventilated with room air supplemented with oxygen at a rate of 105 strokes/min and with a tidal volume of 0.3±0.1 ml using a mouse ventilator (MiniVent 845, Hugo Sachs Elektronik, Hugstetten, Germany). These respiratory settings were found to result in optimal values of arterial pH, PO2, and PCO2 [1], [9], [17], [30], [62][66]. Body temperature was carefully monitored with a rectal probe and maintained as close as possible to 37.0°C. To prevent blood pressure drops, blood from a donor mouse was transfused at a dose of 40 mL/kg IV in three divided equal volume boluses. The chest was opened through a midline sternotomy with the aid of a dissecting microscope and a microcoagulator. An 8-0-nylon suture was passed under the mid-left anterior descending (LAD) coronary artery and a nontraumatic balloon occluder was applied on the artery. Ischemic PC was elicited by a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles (Figs. 1A, 1B and 1C). On day 2, mice were reanesthetized with sodium pentobarbital (60 mg/kg i.p.). The chest was reopened. The same 8-0-nylon suture and nontraumatic balloon occluder were used. Infarction was produced by a 30-min coronary occlusion and followed by 24 hours reperfusion (Figs. 1A, 1B and 1C). Ischemia was confirmed by noting ST elevation on ECG and blanching of the LV. After the coronary occlusion/reperfusion procedures, the chest was closed in layers and mice were allowed to recover [1], [9][11], [13][18], [20], [30], [40], [61][80].

thumbnail

Table 2. Size of left ventricle, risk region, and infarction in Phase I study.

doi:10.1371/journal.pone.0041178.t002
thumbnail

Table 3. Size of left ventricle, risk region, and infarction in Phase II study.

doi:10.1371/journal.pone.0041178.t003
thumbnail

Table 4. Size of left ventricle, risk region, and infarction in Phase III study.

doi:10.1371/journal.pone.0041178.t004
thumbnail

Table 5. Rectal temperature and heart rate on the day of the 30-min coronary occlusion in Phase I study.

doi:10.1371/journal.pone.0041178.t005
thumbnail

Table 6. Rectal temperature and heart rate on the day of the 30-min coronary occlusion in Phase II study.

doi:10.1371/journal.pone.0041178.t006
thumbnail

Table 7. Rectal temperature and heart rate on the day of the 30-min coronary occlusion in Phase III study.

doi:10.1371/journal.pone.0041178.t007

In vitro Tissue Staining

At the conclusion of the study, the heart was excised and perfused with Krebs-Henseleit solution through an aortic cannula. To delineate infarcted from viable myocardium, the heart was perfused with 1% TTC in phosphate buffer. To delineate the occluded/reperfused bed, the coronary artery was tied at the site of the previous occlusion and the aortic root was perfused with 10% phthalo blue dye. As a result of this procedure, the region at risk was identified by the absence of blue dye, whereas the rest of the LV was stained dark blue. The left ventricle was cut into 5–7 transverse slices, which were fixed in 10% neutral buffered formaldehyde, weighed, and photographed under a microscope [1], [9][11], [13][18], [20], [30], [40], [61][80].

Infarct Size (IS) Measurement

Areas identified as infarct, at-risk, and nonischemic based on tissue staining were measured by computerized videoplanimetry and from these measurements infarct size was calculated as a percentage of the region at risk [1], [9][11], [13][18], [20], [30], [40], [61][80].

Kidney and Liver Function Measurements

We collected the blood samples from the COX-2 knockout and wildtype mice before harvesting the mouse heart and sent to a commercial company to test the liver and renal function.

Statistical Analysis

Data are reported as means ± SEM. Data analysis was performed using the SigmaStat software. Statistical comparisons were performed with one-way ANOVA followed by unpaired Student’s t-tests [9], [17], [30], [64].

thumbnail

Table 8. Liver profile of COX2 KO and WT mice.

doi:10.1371/journal.pone.0041178.t008
thumbnail

Table 9. Renal profile of COX2 KO and WT mice.

doi:10.1371/journal.pone.0041178.t009

Results

Exclusions

A total of 211 mice were used for these experiments. Twenty-six mice died (Table 1); thus, total mortality was 12.3% (Table 1). Seventeen mice (8%) were excluded because of severe bleeding during surgery (2 mice), technical problem (12 mice, including malfunction of the ventilation system, damage to the coronary vessels, balloon malfunction) or inadequate postmortem staining (3 mice). One hundred and sixty-eight mice successfully completed the entire protocol and were included in the study (Table 1).

General Characteristics, Heart Rate and Temperature

The mice used in the various groups had similar heart-to-body weight ratios. There were no significant differences in age, body weight, and risk region among groups (Tables 2, 3, and 4). Heart rate and rectal temperature before the 30-min coronary occlusion (pre-occlusion), at 5, 15 and 30 min into the occlusion, and at 5, 15 and 30 min after reperfusion in all groups are shown in Tables 5, 6, and 7. Heart rate, a fundamental physiological parameter that may impact infarct size, was similar in all the groups. Within the same group, heart rate did not differ significantly at any time-point before and during the 30-min occlusion or the ensuing reperfusion. By experimental design, rectal temperature, another potential determinant of infarct size, remained within a narrow physiologic range (36.8–37.2°C) in all groups (Tables 5, 6, and 7).

Phase I: Role of COX-2−/− in Late PC in vivo

These studies were conducted in male mice, 19–23 wk old, weighing 28–32 g (Table 2). On day 1, mice were subjected to either the PC protocol or sham surgery. On day 2, all mice were subjected to a 30-min coronary occlusion and 24 h of reperfusion (Fig. 1A). Mortality was significantly greater in the KO group (Table 1), possibly because COX-2−/− mice suffered from renal and liver abnormalities (the data are shown in Tables 8 and 9). Heart slices demonstrating the postmortem staining of representative hearts for each group are shown in Figure 2A.

In non-PC COX-2+/+ controls (Table 2 and Fig. 3A, group I), infarct size averaged 59.5±2.8% of the risk region. In PC COX-2+/+ controls (Table 2 and Fig. 3A, group II), infarct size was significantly reduced to 34.0±3.7%; p<0.05, indicating the cardioprotective infarct-sparing effect conferred by late PC. In non-PC mice homozygous for a null COX-2 allele (COX-2−/−) (Table 2 and Fig. 3A, group III), infarct size (62.0±2.2%) was similar to COX-2+/+ non-PC controls, indicating that COX-2 does not affect infarct size in the absence of PC. In contrast, COX-2−/− mice in the PC (Table 2 and Fig. 3A, group IV) group had a similar infarct size (59.8±3.0%) to non-PC COX-2+/+ and COX-2−/− mice, indicating that deletion of COX-2 abolished the cardioprotection afforded by late PC. These results show that COX-2 does not affect infarct size in naïve conditions (no PC) and that targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, providing unequivocal molecular genetic evidence for an obligatory role of COX-2 in late PC.

Phase II: Role of IP in Late PC

The PGI2 receptor, IP, is known to be a specific transducer of PGI2 signaling in immunomodulation. We hypothesized that IP is a downstream mediator in COX-2 mediated late PC. We tested our hypothesis by inhibiting IP with the selective IP inhibitor RO3244794 and by using IP−/− mice.

thumbnail

Figure 2. Representative examples of a heart from each group.

The infarcted region was delineated by perfusing the aortic root with 2,3,5-triphenyltetrazolium chloride (TTC); the region at risk was delineated by perfusing the aortic root with phthalo blue after tying the previously occluded artery. As a result of this procedure, the nonischemic portion of the left ventricle (LV) was stained dark blue and viable tissue within the region at risk was stained bright red (TTC positive), whereas infarcted tissue was light yellow or white (TTC negative). Phase I (panel A). Non-preconditioned COX-2+/+ and COX-2−/− mice have similarly large infarct sizes. PC 24 h prior to MI results in a significant reduction in infarct size in COX-2+/+ but not COX-2−/− mice. Phase II (panel B). Non-preconditioned B6 mice in naïve, vehicle-treated, and RO3244794-treated groups have similar infarct sizes. PC results in significantly smaller infarct sizes in naïve and vehicle-treated mice but not in RO3244794-treated mice. Phase III (panel C). Non-preconditioned IP+/+ and IP−/− mice have similar infarct sizes. PC results in a significant reduction in infarct size in IP+/+ but not IP−/− mice. Scale at bottom is in mm. Note the large, confluent areas of infarction spanning most of the thickness of the LV wall, with thin rims of viable subendocardial tissue. This pattern was characteristic of all 7 nonpreconditioned groups (groups I, III, V–VII, XI and XIII) and all 3 PC groups (PC in COX-2−/− [group IV] and IP−/− mice [group XIV] or pretreated with the RO compound [group X]). In contrast, mice subjected to the PC protocol exhibited small, sporadic areas of infarction, a pattern that was characteristic of all 3 PC alone WT mice (groups II, VIII and XIII) and of the mice with PC + vehicle (group IX).

doi:10.1371/journal.pone.0041178.g002
thumbnail

Figure 3. Myocardial infarct size in groups I–XIV.

Infarct size is expressed as a percentage of the region at risk of infarction. Data are expressed as means ± SEM. Phase I (panel A). COX-2−/− mice did not exhibit the infarct-sparing effects of late PC. Phase II (panel B). RO3244794-treated mice did not exhibit the infarct-sparing effects of late PC. Phase III (panel C). IP−/− mice did not exhibit the infarct-sparing effects of late PC. (*) Marks a significant infarct size reduction in preconditioned mice compared with non-PC mice; P<0.05. ○, Individual mice; •, mean ± SE for respective group.

doi:10.1371/journal.pone.0041178.g003
thumbnail

Figure 4. Pilot study.

Effect of RO3244794 and IP−/− on iloprost-induced hypotension. Heart rate and mean arterial blood pressure (MAP) are shown as the changes of percentage of baselines in Figs. 4B and 4C, respectively. Data are expressed as means ± SEM. A) Experimental protocol for hemodynamic studies. B) Effect on heart rate (HR). There was no statistic significant difference in HR among the three groups (including the absolutely numbers). C) Effect on arterial blood pressure. Iloprost resulted in a significant drop in main arterial pressure (MAP); pretreatment with RO3244794 abolished the effect of iloprost on MAP, and iloprost had no effect on MAP in IP−/− mice.

doi:10.1371/journal.pone.0041178.g004

Pilot Studies

To confirm the specificity of this compound for IP receptors and to select the dose, we determined whether the specific IP antagonist (RO3244794) or IP deletion can attenuate the hypotensive effect induced by an IP agonist (iloprost).

Mice were assigned to three groups (Fig. 4A). Iloprost (a PGI2 analog) was administered intraperitoneally at a high dose of 30 µg/kg to IP+/+ mice 30 min after RO3244794 (group A) or vehicle (group B). The same dose of iloprost was injected into IP−/− mice (group C). Iloprost injection to vehicle-pretreated animals resulted in a slight increase in heart rate (Fig. 4B) and a pronounced drop in mean arterial pressure (MAP, Fig. 4C), a normal response to iloprost. Administering iloprost to IP−/− mice did not affect the MAP. Similarly, in, RO3244794-treated mice, iloprost failed to reduce MAP. These data indicate that RO3244794, at the doses used here, effectively inhibits the PGI2 effect on MAP. RO3244794 did not alter baseline MAP and heart rate, indicating that the drug in the doses used does not have significant hemodynamic side effects.

Selective IP Inhibition with RO3244974 Abolishes the Infarct-sparing Effect of Late PC in vivo

Male C57BL/6J (B6) mice, 9–13wk old; weighing 24–31 g, were used to test whether selective pharmacological inhibition of IP abrogates late PC. RO3244794 (10 mg/kg) or vehicle (7 ml/kg) was administered intraperitoneally 30 min before the 30-min occlusion. Representative examples of postmortem staining are shown in Figure 2B.

In non-preconditioned untreated controls (Table 3 and Fig. 3B, group V), infarct size averaged 63.3±2.2% of the risk region. In preconditioned untreated controls (Fig. 3B, group VIII), infarct size was significantly reduced to 33.5±3.5% (p<0.05), indicating the cardioprotective infarct-sparing effect conferred by late PC. In non-preconditioned mice treated with the selective IP inhibitor RO3244794 (Table 3 and Fig. 3B, group VII), infarct size (68.4±1.2%) was similar to untreated non-preconditioned controls, indicating that IP does not confer cardioprotective effects in the absence of PC. In preconditioned mice treated with RO3244794 (Table 3 and Fig. 3B, group X), infarct size (63.8±4.5%) was similar to non-preconditioned untreated controls and RO3244794-treated mice, indicating that inhibition of IP abolishes the cardioprotection offered by late PC. To determine whether the RO3244794 vehicle (0.2 M Trizma base) had any biological effects, non-preconditioned and preconditioned mice were treated with vehicle in the same amount as required for RO3244794 delivery. The infarct size of non-preconditioned vehicle-treated mice (65.7±3.2%; Table 3 and Fig. 3B, group VI) was very similar to non-preconditioned untreated controls (group V). In contrast, treating preconditioned mice with vehicle (Table 3 and Figure 3B, group IX) resulted in a significant reduction in infarct size (32.3±4.5%; p<0.05) comparable to that seen in preconditioned untreated mice (group VIII). These results indicate that selective IP inhibition by RO3244794 results in abolition of the infarct-sparing effect of late PC, implying a prominent role of IP in transducing the signals mediating late PC.

Phase III: Deletion of IP Blocks the Cardioprotective Infarct-sparing Effect of Late PC in vivo

To corroborate the pharmacologic studies in phase II, in phase III we performed studies using genetic ablation of IP. We tested if targeted disruption of the IP gene abrogates late PC in male mice, 20–21 wks old; weighing 25–30 g. Mortality was not significantly different among the four groups. Representative examples of postmortem staining are shown in Figure 2C.

In non-preconditioned IP+/+ controls (Table 4 and Fig. 3C, group XI), infarct size averaged 50.7±2.7% of the risk region. In preconditioned IP+/+ controls (Table 4 and Fig. 3C, group XII), infarct size was markedly reduced to 38.9±2.6% (p<0.05). In non-preconditioned mice homozygous for the null IP allele (IP−/−) (Table 4 and Fig. 3C, group XIII), infarct size (52.9±2.1%) was similar to IP+/+ non-preconditioned controls, confirming that IP does not confer cardioprotective effects in the absence of PC. In contrast, when IP−/− mice were preconditioned (Table 4 and Fig. 3C, group XIV), infarct size (52.4±3.7) was similar to non-preconditioned IP+/+ and IP−/− mice. These results indicate that the IP receptor does not modulate myocardial ischemia/reperfusion injury at baseline and that targeted disruption of the IP gene completely abrogates the infarct-sparing effect of late PC, providing, for the first time, molecular genetic evidence for an obligatory role of IP in the cardioprotection conferred by late PC.

Discussion

Over the last 20 years, considerable efforts have been directed towards better understanding of the molecular interplay involved in the process of PC. The cardioprotective effects of PC are manifest in two phases [7], [81][83], an early phase starting few minutes after the ischemic stimulus lasting for 2–4 h and a late phase starting about 12–24 h after the stimulus and lasting for 24–72 h [7], [81][83]. The late phase of PC is mediated by pathways involving modulation of gene transcription, producing relatively long lasting effects [6], [7], [24], [81]. A number of candidate genes have been identified that can mediate this long lasting late phase of PC [7], [9], [26], [60], [84][86]. Understanding the molecular basis of PC may provide targets for developing drugs that can reproduce the cardioprotective effects conferred by the late phase of PC with minimal side effects.

Clinical evidence of increased cardiovascular mortality following use of COX-2 inhibitors has brought COX-2 into focus as a cardioprotective molecule [87][94]; however, even before this evidence started to appear, we showed for the first time the cardioprotective effects of COX-2 and its involvement in the late phase of PC [29], [30], [33], [35][37], [40]. We demonstrated upregulation of cardiac COX-2 mRNA/protein and PGE2/6-keto-PGF levels in a rabbit model [35] and a mouse model [32] of late PC. We further demonstrated that the infarct-sparing effect of late PC was abolished by COX-2 inhibitors (NS-398 and celecoxib) administered 24 h after PC [29], [30]. Thus far, the experimental evidence supporting the role of COX-2 in late PC has been based on the observations that: 1) COX-2 and prostanoids are upregulated in animal models in which the infarct-sparing effects of late PC are evident [29], [32] and, 2) pharmacologic COX-2 inhibitors abolish late PC [29], [30]. These data are limited by the possible nonspecific effects of COX-2 inhibitors. Therefore, in this study, we have assessed the role of COX-2 in late PC by using COX-2−/− mice. The abrogation of late PC in COX-2−/− mice provides conclusive, unequivocal proof of the role of COX-2 in mediating the late phase of PC.

COX-2−/− mice may have poor survival secondary to the key role played by COX-2 in maintenance of hemodynamics, immunity and other vital functions. Understanding the molecules downstream of COX-2 is important if this pathway is to be exploited for therapeutic purposes. Although it appears that COX-2 probably mediates its cardioprotective effects via upregulation of PGI2 and/or PGE2 [29], the downstream signal transduction pathways mediating late PC via COX-2-derived prostanoids are unknown. Studies have pointed to prostacyclin (PGI2) [36], [71] and PGE2 [71] as the main prostanoids involved in cardioprotective effects during ischemia/reperfusion myocardial injury. A previous study from our group has shown that 6-keto-PGF1α, a stable metabolite of PGI2, is upregulated in opioid-induced late phase PC [41]. In the same study it was shown that COX-2 inhibition resulted in abolition of the infarct-sparing effect of opioid-induced late PC. This study suggests that coupling of COX-2 and PGI2 is the most likely mechanism mediating the cardioprotective effects of late PC. Given this evidence, we hypothesized that the PGI2 receptor, IP, is a key mediator, downstream from COX-2/prostanoids, of the late phase PC. Our experiments show that late PC was abolished by selective IP inhibition by RO3244794 and that IP−/− mice lack the infarct-sparing effect of late PC. This is the first study to establish the obligatory role of IP as a mediator of late PC.

In the Phase I study, there was no significant difference in infarct size in non-preconditioned COX-2−/− mice compared with non-preconditioned COX-2+/+ mice, indicating that COX-2-dependent signaling does not modulate ischemia-reperfusion injury in the basal (non-preconditioned) state (Table 2 and Fig. 3A). The result is internally consistent and corroborated with our previous findings which we tested the effect on the infarct size with COX-2 inhibitors in naïve rabbits [29] and mice [30] in vivo. Although, this result is contrary to that of Camitta et al (Circulation 2001), who reported that COX-2−/− mice exhibited a significantly larger infarct size compared to COX-2+/+ [95]. We think that: 1) the models were different (Langendorff setting vs. in vivo) between these two studies; 2) the duration of LAD occlusion in the Camitta study was shorter (20 min vs. 30 min) than our study; 3) the duration of reperfusion in the Camitta study was also shorter (40 min vs. 24 hours) than our study. It is possible that COX-2 signaling may play different role in modulating injury with different durations of ischemia and reperfusion. In the Phase III study, there was no significant difference in infarct size in non-preconditioned IP−/− mice compared with non-preconditioned IP+/+ mice, indicating that IP-dependent signaling does not modulate ischemia-reperfusion injury in the basal (non-preconditioned) state (Table 4 and Fig. 3C). The same result was also confirmed in the phase II study of pretreatment of IP antagonist, RO3244794 in the naïve mice (Table 3 and Fig. 3B). This result is contrary to that of Xiao et al (Circulation 2001), who reported that IP−/− mice exhibited a significantly larger infarct size compared to IP+/+. We do not have an obvious explanation for this discrepancy; however, the duration of LAD occlusion in the Xiao study was longer (60 min vs. 30 min) than our study. It is possible that IP signaling may become important in modulating injury with longer durations of ischemia.

The combination of pharmacological and genetic evidence strongly supports our hypothesis that IP is a key downstream molecular mediator of late PC in the COX-2/prostanoid pathway. Additionally, our lab and other investigators have shown that the transcription factor STAT3 plays a key role in late PC by upregulating the expression of cardioprotective proteins such as iNOS, COX-2, HO1, and anti-apoptotic factors [7], [42], [96]. Recent studies in human erythroleukemia cells have shown that IP mediates STAT3 activation by stimulating STAT3 Tyr(705) and Ser(727) phosphorylation [97]. Thus, it appears that IP not only mediates signal transduction for COX-2 but also may act as a facilitator for feedback enhancement of multiple pathways mediating the late phase of PC. This receptor is therefore emerging as an important player in the pathophysiology of late PC.

The prostanoid receptors are a family of cell surface 7-transmembrane-domain G-protein coupled receptor (GPCR) classified into five subtypes [98]. The human IP receptor stimulates downstream activation primarily coupled to Gαs-adenylyl cyclase but also has been shown to act through Gq-mediated phospholipase C (PLC) activation [97]. We currently have a good understanding of the structure of IP based on homology modeling with the thromboxane A2 (TP) receptor and the cellular processing of IP from transcription to trafficking [99]. The already existing structural [100], [101] and biochemical knowledge of IP should facilitate strategies for pharmacological modulation of IP for therapeutic purposes.

Identifying selective and specific IP agonists would be an appealing pharmacological approach to mimic the late phase of PC. For example, targeted drug screening strategies may lead to the discovery of selective IP agonists that could mimic the cardioprotective effects of late PC.

In conclusion, the present results advance our understanding of the intricate process of late PC. To the best of our knowledge, this is the first study to demonstrate the obligatory role of COX-2 in late PC by using a genetic approach. This is also the first study to demonstrate, using genetic and pharmacological evidence, the obligatory role of IP in this process. Finally, we have shown that selective IP modulation for cardioprotection is feasible, suggesting that it has the potential to be exploited as a therapeutic target.

Acknowledgments

We thank Dr. Robert Langenbach (National Institute of Environmental Health Sciences in Research Triangle Park, N.C.) for kindly providing the COX-2−/− and COX-2+/+ mice. We also thank Dr. Mary-Frances Jett (Roche Palo Alto, Palo Alto, CA) for kindly providing RO3244794.

Author Contributions

Conceived and designed the experiments: YG RB. Performed the experiments: YG WJW XZ WT GR. Analyzed the data: YG WJW WT XZ MB SPJ QL. Contributed reagents/materials/analysis tools: YG SPJ GR SN. Wrote the paper: YG DNT SPJ RB.

References

  1. 1. Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, et al. (1998) Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol 275: H1375–1387.
  2. 2. Ovize M, Kloner RA, Hale SL, Przyklenk K (1992) Coronary cyclic flow variations “precondition” ischemic myocardium. Circulation 85: 779–789.
  3. 3. Bolli R (1996) The early and late phases of preconditioning against myocardial stunning and the essential role of oxyradicals in the late phase: an overview. Basic Res Cardiol 91: 57–63.
  4. 4. Qiu Y, Tang XL, Park SW, Sun JZ, Kalya A, et al. (1997) The early and late phases of ischemic preconditioning: a comparative analysis of their effects on infarct size, myocardial stunning, and arrhythmias in conscious pigs undergoing a 40-minute coronary occlusion. Circ Res 80: 730–742.
  5. 5. Stein AB, Tang XL, Guo Y, Xuan YT, Dawn B, et al. (2004) Delayed adaptation of the heart to stress: late preconditioning. Stroke 35: 2676–2679.
  6. 6. Bolli R, Manchikalapudi S, Tang XL, Takano H, Qiu Y, et al. (1997) The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase. Evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning. Circ Res 81: 1094–1107.
  7. 7. Bolli R (2000) The late phase of preconditioning. Circ Res 87: 972–983.
  8. 8. Jones WK, Flaherty MP, Tang XL, Takano H, Qiu Y, et al. (1999) Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism. J Mol Cell Cardiol 31: 1469–1481.
  9. 9. Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, et al. (1999) The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci U S A 96: 11507–11512.
  10. 10. Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, et al. (2007) Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway. Circulation 116: 535–544.
  11. 11. West MB, Rokosh G, Obal D, Velayutham M, Xuan YT, et al. (2008) Cardiac myocyte-specific expression of inducible nitric oxide synthase protects against ischemia/reperfusion injury by preventing mitochondrial permeability transition. Circulation 118: 1970–1978.
  12. 12. Wang Y, Guo Y, Zhang SX, Wu WJ, Wang J, et al. (2002) Ischemic preconditioning upregulates inducible nitric oxide synthase in cardiac myocyte. J Mol Cell Cardiol 34: 5–15.
  13. 13. Li Q, Guo Y, Wu WJ, Ou Q, Zhu X, et al. (2011) Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences. Basic Res Cardiol 106: 1355–1366.
  14. 14. Li Q, Guo Y, Tan W, Stein AB, Dawn B, et al. (2006) Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences. Am J Physiol Heart Circ Physiol 290: H584–589.
  15. 15. Li Q, Guo Y, Tan W, Ou Q, Wu WJ, et al. (2007) Cardioprotection afforded by inducible nitric oxide synthase gene therapy is mediated by cyclooxygenase-2 via a nuclear factor-kappaB dependent pathway. Circulation 116: 1577–1584.
  16. 16. Li Q, Guo Y, Ou Q, Cui C, Wu WJ, et al. (2009) Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway. Circulation 120: 1222–1230.
  17. 17. Guo Y, Sanganalmath SK, Wu W, Zhu X, Huang Y, et al. (2012) Identification of inducible nitric oxide synthase in peripheral blood cells as a mediator of myocardial ischemia/reperfusion injury. Basic Res Cardiol 107: 253.
  18. 18. Dawn B, Xuan YT, Guo Y, Rezazadeh A, Stein AB, et al. (2004) IL-6 plays an obligatory role in late preconditioning via JAK-STAT signaling and upregulation of iNOS and COX-2. Cardiovasc Res 64: 61–71.
  19. 19. Heusch G, Boengler K, Schulz R (2008) Cardioprotection: nitric oxide, protein kinases, and mitochondria. Circulation 118: 1915–1919.
  20. 20. Benjamin IJ, Guo Y, Srinivasan S, Boudina S, Taylor RP, et al. (2007) CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice. Am J Physiol Heart Circ Physiol 293: H3201–3209.
  21. 21. Yoshida K, Maaieh MM, Shipley JB, Doloresco M, Bernardo NL, et al. (1996) Monophosphoryl lipid A induces pharmacologic ‘preconditioning’ in rabbit hearts without concomitant expression of 70-kDa heat shock protein. Mol Cell Biochem 156: 1–8.
  22. 22. Qian YZ, Bernardo NL, Nayeem MA, Chelliah J, Kukreja RC (1999) Induction of 72-kDa heat shock protein does not produce second window of ischemic preconditioning in rat heart. Am J Physiol 276: H224–234.
  23. 23. Amour J, Brzezinska AK, Weihrauch D, Billstrom AR, Zielonka J, et al. (2009) Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning. Anesthesiology 110: 317–325.
  24. 24. Tang XL, Qiu Y, Turrens JF, Sun JZ, Bolli R (1997) Late preconditioning against stunning is not mediated by increased antioxidant defenses in conscious pigs. Am J Physiol 273: H1651–1657.
  25. 25. Yamashita N, Hoshida S, Taniguchi N, Kuzuya T, Hori M (1998) A “second window of protection” occurs 24 h after ischemic preconditioning in the rat heart. J Mol Cell Cardiol 30: 1181–1189.
  26. 26. Li Q, Bolli R, Qiu Y, Tang XL, Guo Y, et al. (2001) Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation 103: 1893–1898.
  27. 27. Li Q, Bolli R, Qiu Y, Tang XL, Murphree SS, et al. (1998) Gene therapy with extracellular superoxide dismutase attenuates myocardial stunning in conscious rabbits. Circulation 98: 1438–1448.
  28. 28. Shinmura K, Bolli R, Liu SQ, Tang XL, Kodani E, et al. (2002) Aldose reductase is an obligatory mediator of the late phase of ischemic preconditioning. Circ Res 91: 240–246.
  29. 29. Shinmura K, Tang XL, Wang Y, Xuan YT, Liu SQ, et al. (2000) Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci U S A 97: 10197–10202.
  30. 30. Guo Y, Bao W, Wu WJ, Shinmura K, Tang XL, et al. (2000) Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice. Basic Res Cardiol 95: 479–484.
  31. 31. Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, et al. (2005) Role of the protein kinase C-epsilon-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation 112: 1971–1978.
  32. 32. Xuan YT, Guo Y, Zhu Y, Han H, Langenbach R, et al. (2003) Mechanism of cyclooxygenase-2 upregulation in late preconditioning. J Mol Cell Cardiol 35: 525–537.
  33. 33. Wang Y, Kodani E, Wang J, Zhang SX, Takano H, et al. (2004) Cardioprotection during the final stage of the late phase of ischemic preconditioning is mediated by neuronal NO synthase in concert with cyclooxygenase-2. Circ Res 95: 84–91.
  34. 34. Stein AB, Bolli R, Dawn B, Sanganalmath SK, Zhu Y, et al. (2012) Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium. J Mol Cell Cardiol 52: 228–236.
  35. 35. Shinmura K, Xuan YT, Tang XL, Kodani E, Han H, et al. (2002) Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ Res 90: 602–608.
  36. 36. Shinmura K, Nagai M, Tamaki K, Tani M, Bolli R (2002) COX-2-derived prostacyclin mediates opioid-induced late phase of preconditioning in isolated rat hearts. Am J Physiol Heart Circ Physiol 283: H2534–2543.
  37. 37. Shinmura K, Nagai M, Tamaki K, Bolli R (2004) Gender and aging do not impair opioid-induced late preconditioning in rats. Basic Res Cardiol 99: 46–55.
  38. 38. Shinmura K, Kodani E, Xuan YT, Dawn B, Tang XL, et al. (2003) Effect of aspirin on late preconditioning against myocardial stunning in conscious rabbits. J Am Coll Cardiol 41: 1183–1194.
  39. 39. Sato H, Bolli R, Rokosh GD, Bi Q, Dai S, et al. (2007) The cardioprotection of the late phase of ischemic preconditioning is enhanced by postconditioning via a COX-2-mediated mechanism in conscious rats. Am J Physiol Heart Circ Physiol 293: H2557–2564.
  40. 40. Li Q, Guo Y, Xuan YT, Lowenstein CJ, Stevenson SC, et al. (2003) Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism. Circ Res 92: 741–748.
  41. 41. Kodani E, Xuan YT, Shinmura K, Takano H, Tang XL, et al. (2002) Delta-opioid receptor-induced late preconditioning is mediated by cyclooxygenase-2 in conscious rabbits. Am J Physiol Heart Circ Physiol 283: H1943–1957.
  42. 42. Bolli R, Stein AB, Guo Y, Wang OL, Rokosh G, et al. (2011) A murine model of inducible, cardiac-specific deletion of STAT3: its use to determine the role of STAT3 in the upregulation of cardioprotective proteins by ischemic preconditioning. J Mol Cell Cardiol 50: 589–597.
  43. 43. Bolli R, Shinmura K, Tang XL, Kodani E, Xuan YT, et al. (2002) 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. Cardiovasc Res 55: 506–519.
  44. 44. Bolli R, Dawn B, Xuan YT (2003) Role of the JAK-STAT pathway in protection against myocardial ischemia/reperfusion injury. Trends Cardiovasc Med 13: 72–79.
  45. 45. Hu LF, Pan TT, Neo KL, Yong QC, Bian JS (2008) Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes. Pflugers Arch 455: 971–978.
  46. 46. Patel HH, Hsu AK, Gross GJ (2004) COX-2 and iNOS in opioid-induced delayed cardioprotection in the intact rat. Life Sci 75: 129–140.
  47. 47. Przyklenk K, Heusch G (2003) Late preconditioning against myocardial stunning. Does aspirin close the “second window” of endogenous cardioprotection? J Am Coll Cardiol 41: 1195–1197.
  48. 48. Tanaka K, Ludwig LM, Krolikowski JG, Alcindor D, Pratt PF, et al. (2004) Isoflurane produces delayed preconditioning against myocardial ischemia and reperfusion injury: role of cyclooxygenase-2. Anesthesiology 100: 525–531.
  49. 49. Gierse JK, Koboldt CM, Walker MC, Seibert K, Isakson PC (1999) Kinetic basis for selective inhibition of cyclo-oxygenases. Biochem J 339 (Pt 3): 607–614.
  50. 50. Lin H, Lin TN, Cheung WM, Nian GM, Tseng PH, et al. (2002) Cyclooxygenase-1 and bicistronic cyclooxygenase-1/prostacyclin synthase gene transfer protect against ischemic cerebral infarction. Circulation 105: 1962–1969.
  51. 51. Xiao CY, Hara A, Yuhki K, Fujino T, Ma H, et al. (2001) Roles of prostaglandin I(2) and thromboxane A(2) in cardiac ischemia-reperfusion injury: a study using mice lacking their respective receptors. Circulation 104: 2210–2215.
  52. 52. Johnson G III, Furlan LE, Aoki N, Lefer AM (1990) Endothelium and myocardial protecting actions of taprostene, a stable prostacyclin analogue, after acute myocardial ischemia and reperfusion in cats. Circ Res 66: 1362–1370.
  53. 53. Lefer AM, Ogletree ML, Smith JB, Silver MJ, Nicolaou KC, et al. (1978) Prostacyclin: a potentially valuable agent for preserving myocardial tissue in acute myocardial ischemia. Science 200: 52–54.
  54. 54. Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC (1981) Dissimilar effects of prostacyclin, prostaglandin E1, and prostaglandin E2 on myocardial infarct size after coronary occlusion in conscious dogs. Circ Res 49: 685–700.
  55. 55. Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC (1980) Dissimilar effects of PGE1 and PGE2 on myocardial infarct size after coronary occlusion in conscious dogs. Adv Prostaglandin Thromboxane Res 7: 675–677.
  56. 56. Hong TT, Huang J, Barrett TD, Lucchesi BR (2008) Effects of cyclooxygenase inhibition on canine coronary artery blood flow and thrombosis. Am J Physiol Heart Circ Physiol 294: H145–155.
  57. 57. Bley KR, Bhattacharya A, Daniels DV, Gever J, Jahangir A, et al. (2006) RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists. Br J Pharmacol 147: 335–345.
  58. 58. Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, et al. (1995) Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 83: 473–482.
  59. 59. Murata T, Ushikubi F, Matsuoka T, Hirata M, Yamasaki A, et al. (1997) Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature 388: 678–682.
  60. 60. Bolli R (2001) Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33: 1897–1918.
  61. 61. Stein AB, Guo Y, Tan W, Wu WJ, Zhu X, et al. (2005) Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction. J Mol Cell Cardiol 38: 127–134.
  62. 62. Guo Y, Stein AB, Wu WJ, Zhu X, Tan W, et al. (2005) Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and delta1-opioid receptor agonists is mediated by iNOS. Am J Physiol Heart Circ Physiol 289: H2251–2257.
  63. 63. Guo Y, Stein AB, Wu WJ, Tan W, Zhu X, et al. (2004) Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol 286: H1649–1653.
  64. 64. Guo Y, Li Q, Wu WJ, Tan W, Zhu X, et al. (2008) Endothelial nitric oxide synthase is not necessary for the early phase of ischemic preconditioning in the mouse. J Mol Cell Cardiol 44: 496–501.
  65. 65. Guo Y, Bolli R, Bao W, Wu WJ, Black RG, et al. (2001) Targeted deletion of the A3 adenosine receptor confers resistance to myocardial ischemic injury and does not prevent early preconditioning. J Mol Cell Cardiol 33: 825–830.
  66. 66. Guo Y, Tang XL, Bao W, Bolli R (2000) Measuring Ischemic injury-How to produce infarction in the mouse in vivo. The ISHR Handbook of Experimental of Laboratory Procedures (H.E.L.P.). Available: http://www.ishrworld.org/.
  67. 67. Wang GW, Guo Y, Vondriska TM, Zhang J, Zhang S, et al. (2008) Acrolein consumption exacerbates myocardial ischemic injury and blocks nitric oxide-induced PKCepsilon signaling and cardioprotection. J Mol Cell Cardiol 44: 1016–1022.
  68. 68. Li Q, Guo Y, Ou Q, Wu WJ, Chen N, et al. (2011) Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences. Basic Res Cardiol 106: 1367–1377.
  69. 69. Hu X, Dai S, Wu WJ, Tan W, Zhu X, et al. (2007) Stromal cell derived factor-1 alpha confers protection against myocardial ischemia/reperfusion injury: role of the cardiac stromal cell derived factor-1 alpha CXCR4 axis. Circulation 116: 654–663.
  70. 70. Guo Y, Xuan YT, Wu WJ, Zhu X, Zhu Y, et al. (2002) Role of the JAK-STAT pathway and cyclooxygenase-2 in exercise-induced late preconditioning. Circulation 106: II–313. (1571).
  71. 71. Guo Y, Xuan YT, Wu WJ, Zhu X, Tan W, et al. (2003) Heme oxygenase-1 mediates three different types of late preconditioning. Circulation 108: IV–93. (435).
  72. 72. Guo Y, Xuan YT, Wu WJ, Li QH, Tang XL, et al. (2001) Nitric oxide plays a dual role in exercise-induced late preconditioning. Circulation 104: II–60. (288).
  73. 73. Guo Y, Wu WJ, Zhu XP, Li QH, Tang XL, et al. (2001) Exercise-induced late preconditioning is triggered by generation of nitric oxide. J Mol Cell Cardiol 33: A41.
  74. 74. Guo Y, Wu WJ, Zhu X, Tan W, Bolli R (2003) The development of the early phase of ischemic preconditioning in mice is not strain dependent. J Mol Cell Cardiol 35: A47. (P110).
  75. 75. Guo Y, Bao W, Wu WJ, Tang XL, Bolli R (1999) Nitric oxide donors induce late preconditioning against myocardial infarction in mice. J Mol Cell Cardiol 31: A11. (A-19).
  76. 76. Guo Y, Bao W, Tang XL, Wu WJ, Takano H, et al. (2000) Pharmacological preconditioning (PC) with adenosine A1 and opioid δ1 receptor agonists is iNOS-dependent. Circulation 102: II–121. (582).
  77. 77. Guo Y, Bao W, Tang XL, Wu WJ, Takano H, et al. (2000) Activation of adenosine A1 and δ1 opioid receptors induces late preconditioning in mice. J Mol Cell Cardiol 32: A51. (H-54).
  78. 78. Flaherty MP, Guo Y, Tiwari S, Rezazadeh A, Hunt G, et al. (2008) The role of TNF-alpha receptors p55 and p75 in acute myocardial ischemia/reperfusion injury and late preconditioning. J Mol Cell Cardiol 45: 735–741.
  79. 79. Dawn B, Guo Y, Rezazadeh A, Wang OL, Stein AB, et al. (2004) Tumor necrosis factor-alpha does not modulate ischemia/reperfusion injury in naive myocardium but is essential for the development of late preconditioning. J Mol Cell Cardiol 37: 51–61.
  80. 80. Black RG, Guo Y, Ge ZD, Murphree SS, Prabhu SD, et al. (2002) Gene dosage-dependent effects of cardiac-specific overexpression of the A3 adenosine receptor. Circ Res 91: 165–172.
  81. 81. Bolli R, Marban E (1999) Molecular and cellular mechanisms of myocardial stunning. Physiol Rev 79: 609–634.
  82. 82. Baxter GF (1997) Ischaemic preconditioning of myocardium. Ann Med 29: 345–352.
  83. 83. Gres P, Schulz R, Jansen J, Umschlag C, Heusch G (2002) Involvement of endogenous prostaglandins in ischemic preconditioning in pigs. Cardiovasc Res 55: 626–632.
  84. 84. Bolli R, Dawn B, Xuan YT (2001) Emerging role of the JAK-STAT pathway as a mechanism of protection against ischemia/reperfusion injury. J Mol Cell Cardiol 33: 1893–1896.
  85. 85. Angeloni C, Motori E, Fabbri D, Malaguti M, Leoncini E, et al. (2011) H2O2 preconditioning modulates phase II enzymes through p38 MAPK and PI3K/Akt activation. Am J Physiol Heart Circ Physiol 300: H2196–2205.
  86. 86. Hausenloy DJ, Yellon DM (2010) The second window of preconditioning (SWOP) where are we now? Cardiovasc Drugs Ther 24: 235–254.
  87. 87. McGettigan P, Henry D (2006) Cardiovascular risk and inhibition of cyclooxygenase: a systematic review of the observational studies of selective and nonselective inhibitors of cyclooxygenase 2. JAMA 296: 1633–1644.
  88. 88. Caldwell B, Aldington S, Weatherall M, Shirtcliffe P, Beasley R (2006) Risk of cardiovascular events and celecoxib: a systematic review and meta-analysis. J R Soc Med 99: 132–140.
  89. 89. Krotz F, Schiele TM, Klauss V, Sohn HY (2005) Selective COX-2 inhibitors and risk of myocardial infarction. J Vasc Res 42: 312–324.
  90. 90. Emery P, Moore A, Hawkey C (2005) Increased risk of cardiovascular events with coxibs and NSAIDs. Lancet 365: 1538.
  91. 91. Juni P, Nartey L, Reichenbach S, Sterchi R, Dieppe PA, et al. (2004) Risk of cardiovascular events and rofecoxib: cumulative meta-analysis. Lancet 364: 2021–2029.
  92. 92. Mukherjee D, Nissen SE, Topol EJ (2001) Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 286: 954–959.
  93. 93. Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, et al. (2000) Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med 343: 1520–1528, 1522 p following 1528.
  94. 94. Kerr DJ, Dunn JA, Langman MJ, Smith JL, Midgley RS, et al. (2007) Rofecoxib and cardiovascular adverse events in adjuvant treatment of colorectal cancer. N Engl J Med 357: 360–369.
  95. 95. Camitta MG, Gabel SA, Chulada P, Bradbury JA, Langenbach R, et al. (2001) Cyclooxygenase-1 and -2 knockout mice demonstrate increased cardiac ischemia/reperfusion injury but are protected by acute preconditioning. Circulation 104: 2453–2458.
  96. 96. Heusch G, Musiolik J, Gedik N, Skyschally A (2011) Mitochondrial STAT3 activation and cardioprotection by ischemic postconditioning in pigs with regional myocardial ischemia/reperfusion. Circ Res 109: 1302–1308.
  97. 97. Lo RK, Liu AM, Wise H, Wong YH (2008) Prostacyclin receptor-induced STAT3 phosphorylation in human erythroleukemia cells is mediated via Galpha(s) and Galpha(16) hybrid signaling. Cell Signal 20: 2095–2106.
  98. 98. Woodward DF, Jones RL, Narumiya S (2010) International union of basic and clinical pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years of progress. Pharmacol Rev 63: 471–538.
  99. 99. Wilson SJ, Dowling JK, Zhao L, Carnish E, Smyth EM (2007) Regulation of thromboxane receptor trafficking through the prostacyclin receptor in vascular smooth muscle cells: role of receptor heterodimerization. Arterioscler Thromb Vasc Biol 27: 290–296.
  100. 100. Ruan CH, Wu J, Ruan KH (2005) A strategy using NMR peptide structures of thromboxane A2 receptor as templates to construct ligand-recognition pocket of prostacyclin receptor. BMC Biochem 6: 23.
  101. 101. Stitham J, Arehart E, Gleim SR, Li N, Douville K, et al. (2007) New insights into human prostacyclin receptor structure and function through natural and synthetic mutations of transmembrane charged residues. Br J Pharmacol 152: 513–522.