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1Division of Cardiology and 2Department of Biochemistry and Molecular Genetics, University of Texas Medical Branch, Galveston, Texas
Submitted 23 January 2006 ; accepted in final form 28 March 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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infarct size; thiazolidinediones; statins; cyclooxygenase-2; cytosolic phospholipase A2; nitric oxide synthase; protein kinase Akt; phosphatase and tensin homologue deleted on chromosome 10, anti-Src homology 2-containing inositol phosphatase-2
(PPAR-) agonist activity, reduces the composite endpoint of all-cause mortality, nonfatal myocardial infarction, and stroke in patients with Type 2 diabetes mellitus who have a high risk for macrovascular events (21). In addition to decreasing insulin resistance and 
-cell dysfunction (47), increasing serum HDL cholesterol and decreasing serum triglycerides levels (25), improving the procoagulant state and endothelial dysfunction in diabetes, and reducing "nontraditional" inflammatory cytokines (12), thiazolidinediones have been shown to reduce myocardial infarct size (IS) in the rat (35, 61, 66, 67). However, the specific underlying mechanisms for myocardial protection by thiazolidinediones are still unknown. Knowledge of the exact cardioprotective mechanisms is particularly important because another PPAR agonist, muraglitazar, which targets both PPAR- and PPAR-
, has been shown to increase the incidence of the composite end point of death, major adverse cardiovascular events (myocardial infarction, stroke, and transient ischemic attack), and heart failure (43). Other studies have shown that pretreatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduces myocardial IS in the rat (6, 7, 22, 41, 48, 58).
Currently, many patients with diabetes are receiving concomitant therapy with statins and hypoglycemic agents. We have shown that, in rats, glyburide, a sulfonylurea agent that is commonly used by diabetic patients, abrogates the IS-limiting effect of statins by blocking ATP-sensitive K+ channels (58). It is yet unknown whether there is an interaction between thiazolidinediones and statins that affects myocardial protection. The combination may have additive, or even synergistic effects, as have been shown for sildenafil and atorvastatin (ATV) (48). However, some of the described effects of thiazolidinediones may potentially interfere with the IS-limiting effects of statins, and thus the combination of thiazolidinediones with statins may result in attenuation of the protective effect. Pio and rosiglitazone increase the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), leading to inactivation of phosphatidylinositol 3-kinase (PI3K) activity and reduced phosphorylation of protein kinase Akt (38). Phosphorylation of Akt is a crucial step in mediating the protective effect of statins (22, 62). Mensah et al. (41) have recently reported that, after 1 wk of treatment with ATV, PTEN expression is increased and myocardial protection is no longer apparent (41). Early activation of PTEN may blunt the protective effect of statins. In addition, thiazolidinediones have been shown to inhibit inducible nitric oxide (NO) synthase (iNOS) (17, 19, 40) and cyclooxygenase-2 (COX-2) (19, 40) expression, both of which are crucial for the myocardial IS-limiting effects of statins (1, 7). Alternatively, both thiazolidinediones (11, 15, 31) and statins (1, 22, 37, 48, 51) increase the activity of calcium-dependent NOS (cNOS), which also participates in mediating protection, and thus may have potential additive effects.
We assessed 1) whether 3-day pretreatment with Pio limits IS; 2) whether this protective effect is mediated by endothelial NOS (eNOS), iNOS, and/or COX-2 upregulation; and 3) whether there are interactions between Pio and ATV that affect myocardial protection, leading to either augmentation or diminution of the protective effect.
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METHODS |
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All animals received humane care in compliance with The Guide for the Care and Use of Laboratory Animals, published by National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Experiments were conducted on male Sprague-Dawley rats. The study was approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee.
Materials
SC-58125, SC-560, NOS-activity kit, and ELISA kits for 6-keto-PGF1 COX-2 and phospholipase A2 (PLA2) activity were purchased from Cayman Chemical (Ann Arbor, MI). Monoclonal anti-cytosolic PLA2 (cPLA2) and polyclonal anti-Src homology (SH) 2-containing inositol phosphatase-2 (SHIP2) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-COX-2 and polyclonal anti-iNOS antibodies from Cayman Chemical; polyclonal anti Ser1177 phosphorylated-eNOS (P-eNOS) antibodies from Cell Signaling (Beverly, MA); polyclonal anti-Ser473 phosphorylated Akt (P-Akt) and monoclonal anti-PTEN antibodies from R & D (Minneapolis, MN); and monoclonal anti--actin antibody from Sigma (St. Louis, MO). N-(3-malemidylpropionyl) biocytin was purchased from Molecular Probes (Eugene, OR).
Drugs and Pretreatment
Protocol 1. Rats received 3-day pretreatment with 1) Pio 10 mg·kg–1·day–1; 2) ATV 10 mg·kg–1·day–1; 3) ATV 10 mg·kg–1·day–1 and Pio 10 mg·kg–1·day–1; or 4) water (control). Medications were suspended in 1 ml of water and given once a day by oral gavage. Rats underwent either coronary artery ligation (IS protocol), or the hearts were explanted without being subjected to ischemia for enzyme activity determination and for immunoblotting.
Protocol 2. An additional group of rats received oral 10 mg Pio·kg–1·day–1 for 3 days and then intravenous SC-58125 (COX-2 inhibitor, 5 mg/kg) or SC-560 (COX-1 inhibitor, 2.5 mg/kg). Fifteen minutes after the intravenous administration of the inhibitors, the rats underwent coronary artery ligation (IS protocol).
IS Surgical Protocol
The rat model of myocardial ischemia-reperfusion injury has been described in detail (6, 7, 48, 58). On the 4th day, the rats were anesthetized with intraperitoneal injection of ketamine (60 mg/kg) and xylazine (6 mg/kg), intubated, and ventilated (fractionated inspired O2 content = 30%). The rectal temperature was monitored, and body temperature was maintained between 36.7° and 37.3°C throughout the experiment. The left carotid artery was exposed and cannulated with a pressure wire (Rat Mikro-Tip BP System) connected to PowerLab data acquisition system (ADInstruments, Colorado Springs, CO). The chest was opened, and the left coronary artery was encircled with a suture and ligated for 30 min. Isoflurane (1–2.5% titrated to effect) was added after the beginning of ischemia to maintain anesthesia. The snare was then released, and myocardial reperfusion was verified by change in the color of the myocardium. Subcutaneous 0.1 mg/kg buprenorphine was administered, the chest was closed, and the rats were recovered from anesthesia. Four hours after reperfusion, the rats were reanesthetized, the coronary artery was reoccluded, 1.5 ml of Evan’s blue dye 3% was injected into the right ventricle, and the rats were killed under deep anesthesia.
Heart rate and mean blood pressure were noted at baseline (10 min after completion of surgery), just before coronary artery occlusion; at 25 min of ischemia; and at 20 min of reperfusion.
Determination of Area at Risk and IS
Hearts were excised, and the left ventricle was sliced transversely into six sections. Slices were incubated for 10 min at 37°C in 1% buffered (pH = 7.4) 2,3,5-triphenyltetrazolium chloride (TTC), fixed in a 10% formaldehyde, and photographed to identify the ischemic area at risk (AR) (uncolored by the blue dye), the IS (unstained by TTC), and the nonischemic zones (colored by blue dye). The area of AR and IS in each slice was determined by planimetry, converted into percentages of the whole for each slice, and multiplied by the weight of the slice, and the results were summed to obtain the weight of the myocardial AR and IS (6, 7, 48, 58).
NOS Activity
Myocardial samples were homogenized in a buffer [25 mM Tris·HCl (pH 7.4), 1 mM EDTA, and 1 mM EGTA] and centrifuged at 10,000 g for 15 min. The supernatant, containing the soluble enzyme iNOS, and the pellet, containing the membrane-bound eNOS and neuronal NOS (cNOS), were separated. The pellet was resuspended in homogenization buffer. NOS activity was determined by measuring the conversion of L-[14C]arginine to L-[14C]citrulline using a commercial kit (Cayman Chemical). For assessing cNOS activity, CaCl2 was added to the samples. For assessing calcium-independent (ciNOS) activity, CaCl2 was omitted from the solution. NOS activity was defined as counts per minute (48).
6-keto-PGF1 and PLA2 Activity
Myocardial samples of the anterior wall of the left ventricle were rinsed in PBS solution (pH 7.4) containing 0.16 mg/ml heparin to remove red blood cells and clots, homogenized in cold PBS (pH 7.4), and centrifuged at 10,000 g for 10 min at 4°C. The supernatants were collected and stored on ice. Measurement of 6-keto-PGF1, the stable metabolite of prostacyclin, and PLA2 activity were made using immunoassay assay kits (Cayman Chemical).
COX-2 Activity
Myocardial samples of the anterior wall of the left ventricle were perfused and rinsed with 0.05 M Tris buffer, pH 7.4, containing 0.16 mg/ml heparin to remove any red blood cells and clots. Samples were homogenized in 5–10 ml of cold buffer (0.1 M Tris·HCl, pH 7.8, containing 1 mM EDTA) per gram tissue and centrifuged at 10,000 g for 15 min at 4°C. The supernatant was collected and stored on ice. The COX activity assay kit (Cayman Chemical) measures the peroxidase activity of COX, assayed colorimetrically by monitoring the appearance of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine at 590 nm. Each myocardial sample was tested in triplicate (the first without an inhibitor; the second with DuP-697, a specific COX-2 inhibitor; and the third with SC-560, a specific COX-1 inhibitor). Per the manufacturer instructions, COX-1 activity was calculated as the difference between total COX activity in the first sample and the activity in the sample with SC-560 (the COX-1 inhibitor). COX-2 activity was calculated as the difference between total COX activity in the first sample and the activity in the sample with DuP-697 (the specific COX-2 inhibitor).
Western Immunoblotting
Determinations of PTEN, SHIP2, P-Akt, eNOS, P-eNOS, iNOS, cPLA2, and COX-2 expression were performed in samples taken from the left ventricles of rats (6 rats in each group). The hearts were rapidly explanted, rinsed in cold PBS (pH 7.4) containing 0.16 mg/ml heparin to remove red blood cells and clots, frozen in liquid nitrogen, and stored at –80°C.
Tissue samples were homogenized in buffer A [25 mM Tris·HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 25 mM NaF, 1 mM Na3VO4, and 1% protease inhibitor cocktail (P8340; Sigma)] and centrifuged at 10,000 g for 15 min at 4°C. The supernatants were collected as cytosolic fraction. The pellets were then incubated on ice in buffer B (buffer A plus 1% Triton X-100) for 2 h and centrifuged for 12 min at 4°C. The resulting supernatants were collected as membranous fractions and combined with the cytosolic fraction. The expression of the proteins (cytosolic + membranous protein) was assessed by standard SDS/PAGE Western immunoblotting (7). Protein (50 µg) was loaded for each sample, except that, for immunoblotting of COX-2 in the sham and Pio group, we loaded 300 µg protein per sample. The protein signals were quantified by an image-scanning densitometer, and the strength of each protein signal was normalized to the corresponding -actin stain signal. Data are expressed as percent of the expression in the control group.
Statistical Analysis
Data are presented as means (SD). The significance level is 0.05. Body weight, left ventricular weight, the size of the AR and IS, enzyme activity, and protein expression were compared by using analysis of variance (ANOVA). The differences in heart rate and mean blood pressure were compared using two-way repeated-measures ANOVA with Holm-Sidak multiple comparison procedures. Values of P <0.05 were considered statistically significant.
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RESULTS |
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Protocol 1. Fifty-five rats were included in the protocol. Overall, there were 16 rats in the sham-treated group (four rats were excluded: three died during the reperfusion period, and one did not exhibit evidence of ischemia). There were 13 rats in the Pio group (one died during ischemia). There were 13 rats in the ATV group (one was excluded because of lack of reperfusion). There were 13 rats in the Pio + ATV group (one was excluded because of lack of reperfusion). There were no differences in body weight, left ventricular weight, and AR among the groups (Table 1). IS (percentage of the AR) was significantly reduced in the Pio (by 56%), ATV (by 58%), and Pio + ATV (by 81%) groups compared with the sham-treated group (Fig. 1). IS in the Pio + ATV group was significantly smaller than in the sham-treated, Pio-alone, and ATV-alone groups. There was no significant difference in IS between the Pio and ATV groups.
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Ser473-phosphorylated Akt. There were significant differences in P-Akt expression among groups (P < 0.001). The expression of P-Akt in the Pio group was 142% of sham-treated group (P = not significant). In contrast, P-Akt expression in the ATV group was 440% compared with the sham-treated group. In the combination group, P-Akt was increased by 374% (Fig. 2). These data suggest that there is no correlation between P-Akt levels and the magnitude of myocardial protection. The magnitude of IS limitation was comparable between the Pio and ATV groups, despite a significant difference in P-Akt augmentation. Moreover, in the Pio + ATV group, there was less augmentation of P-Akt than in the ATV-alone group, despite significantly smaller IS. Thus it is plausible that Akt activation could not entirely explain the protective effect, especially of Pio. Alternatively, only a small increase in P-Akt may be needed to confer protection; further increase in P-Akt does not increase the protective effect. However, if this is true, it is unclear why IS was smaller in the Pio + ATV group than in the Pio and ATV groups.
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25-fold; P < 0.001 vs. sham). In the same blot, we were not able to detect increased COX-2 expression in the Pio-alone group. Therefore, we repeated the experiment for sham and Pio groups (n = 8 in each group); with a larger protein load in each sample (see METHODS) and longer film exposure, we were able to detect a significant increase in the Pio-alone group (3.4-fold; P = 0.007 vs. sham), although of a much smaller magnitude than seen with ATV (Fig. 5). Pio did not blunt COX-2 expression by ATV in the Pio + ATV group (25-fold) compared with the sham-treated group (Fig. 8). Thus there is no linear correlation between augmentation of COX-2 expression and IS limitation by Pio, ATV, and their combination.
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cNOS activity significantly increased in the ATV and Pio + ATV groups (Fig. 9). Pio alone caused a much smaller increase in cNOS activity, although the difference was significant compared with the sham-treated group (P = 0.012).
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Myocardial content of 6-keto-PGF1 was increased by Pio and, to a greater extent, by ATV. Myocardial content of 6-keto-PGF1 in the Pio + ATV group was significantly higher than in the sham-treated and the Pio-alone group, but the difference between the Pio + ATV and ATV-alone groups was not significant (Fig. 11). These results suggest that there is a correlation between myocardial 6-keto-PGF1 levels and the magnitude of IS limitation.
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DISCUSSION |
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Thiazolidinediones and Myocardial Protection
Intravenous administration of rosiglitazone at 1 and 3 mg/kg reduces IS by 30% and 37%, respectively (67), whereas a 7-day pretreatment with oral rosiglitazone at 3 mg·kg–1·day–1 reduces IS by 24% (67). Rosiglitazone reduces the accumulation of neutrophils and macrophages in the ischemic heart and blunts the ischemia-related induction of CD11b/CD18, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) and the downregulation of L-selectin (67). Eight-day pretreatment with rosiglitazone at 3 mg·kg–1·day–1 reduces both apoptosis and necrosis in Male Zucker diabetic fatty rats (66). This protective effect is diminished with wortmannin, suggesting that the phosphatidylinositol 3-kinase/Akt pathway is mediating the protective effect (66). In an in vivo rat model, using 25 min of ischemia and 2 h of reperfusion, the intravenous administration of rosiglitazone (0.3, 1, or 3 mg/kg), ciglitazone (0.3 mg/kg), or Pio (0.3 or 1 mg/kg) 30 min before myocardial ischemia reduced IS. Of note is that ciglitazone at 1 mg/kg did not reduce IS (61). Pretreatment with Pio at 3 mg·kg–1·day–1 for 7 days reduced IS and the number of infiltrating macrophages in the ischemic region in rats subjected to 30 min of ischemia followed by 24 h of reperfusion (35). The expression of ICAM-1 and MCP-1 mRNA in the ischemic-reperfused region after 24 h of reperfusion was significantly lower in the Pio-treated rats (35). Recently, Wynne et al. (65) used the Langendorff perfused rat heart model to show that pretreatment with Pio reduces IS and that adding LY-294002 or wortmannin, both PI3K inhibitors, before ischemia blocks the protective effect. Thus most published studies support an anti-inflammatory mechanism for myocardial protection by thiazolidinediones, although two studies did suggest that activation of PI3K/Akt may mediate the protective effect.
Akt Phosphorylation and Myocardial Protection
The PI3K pathway plays an important role in regulating numerous biological processes, including survival, proliferation, adhesion, migration, insulin activity, and cell activation, through the generation of the potent second messenger PI-3,4,5-trisphosphate (56). PI-3,4,5-trisphosphate promotes Akt activation via phosphorylation; thus initiating a pro-survival pathway (4). Statins also induce Akt phosphorylation (22, 37, 41, 51). Blocking PI3K with wortmannin prevents Akt phosphorylation and blocks the IS-limiting effects of statins (22, 51) and Pio (64). Here we show that ATV increased Akt phosphorylation by 440%. In contrast, Pio caused a minor but not significant increase in myocardial P-Akt expression (142%) as compared with the sham-treated group. The small P-Akt increase present in the Pio group was not sufficient to induce a detectable change in P-eNOS levels, and the increase in cNOS activity in the Pio group was also less than that in the ATV group. Moreover, despite the significant difference in P-Akt expression in the Pio and ATV groups, the IS-limiting effects of these drugs were comparable. This suggests that the IS-limiting effect of Pio is probably independent of Akt phosphorylation.
There are conflicting reports on the effect of thiazolidinediones on Akt phosphorylation. Some studies (2, 15, 42) have shown that troglitazone increases P-Akt. However, there are reports that Pio (15, 32, 38), ciglitazone (24), and rosiglitazone (5, 38, 55) suppress Akt phosphorylation. Barthel et al. (3) showed that, at low concentrations, troglitazone enhances Akt phosphorylation, whereas at high concentrations, Akt phosphorylation is inhibited. Alternatively, Pio increases P-Akt levels in adipose biopsies of patients with insulin resistance (27). These observations are consistent with the biphasic nature of stress response mechanisms and tissue-specific nature of stress responses.
NOS Expression and Activity
Several studies (22, 37, 51) have suggested that statins increase the activity of eNOS by either increasing its expression or augmenting its phosphorylation at Ser1177. eNOS has a crucial role in mediating the IS-limiting effects of statins; for example, statins are not protective in eNOS knockout mice (36, 52). We have previously shown that 3-day pretreatment with ATV augments the activity of cNOS by increasing the expression of P-eNOS, but not total eNOS or neuronal NOS, in the rat (7, 48). In the present study, we confirmed that ATV increases P-eNOS expression and cNOS activity.
In contrast, Pio did not affect P-eNOS expression when given alone and did not change P-eNOS expression by ATV in the combination treatment group. cNOS activity was mildly increased by Pio; however, the magnitude of this increase was much smaller than with ATV. Previous studies (11, 31, 50) have suggested that PPAR- agonists increase NO production by eNOS, without affecting total eNOS expression. A recent study (31) has suggested that ciglitazone increases the bioavailability of NO, given its antioxidant effects, which are mediated by increased expression of Cu/Zn-superoxide dismutase and suppression of NADPH oxidase. However, in this study, P-eNOS expression was not measured. On the other hand, Cho et al. (15) demonstrated that troglitazone increased cNOS activity in bovine aortic endothelial cells by augmenting eNOS phosphorylation at Ser1179 by Akt, without altering total eNOS expression. Thus our results are in accordance with previous studies showing that Pio may cause a mild increase in eNOS activity; however, in contrast to Cho, we did not detect a change in P-eNOS levels in our in vivo model.
iNOS is essential for mediating the cardioprotective effects of late ischemic preconditioning (54, 68), opioid agonists (26), and sildenafil (48). Previously, we have shown that iNOS activation is mandatory for mediating the protective effect of ATV in the rat (1). In addition, simvastatin failed to reduce IS in iNOS knockout mice (52). In the present study, we confirmed that ATV increased iNOS expression and calcium-independent NOS activity. However, Pio did not affect iNOS expression and activity. In contrast, several investigators have suggested that PPAR- agonists, including ciglitazone (17), Pio (18), and rosiglitazone (18, 19), reduce iNOS expression and activity in various experimental models.
PTEN and SHIP2
PI-3,4,5-trisphosphate levels are negatively controlled by several phosphatases, including the ubiquitously expressed PTEN (which hydrolyzes PI-3,4,5-trisphosphate to PI-4,5-bisphosphate) and SHIP2 [which hydrolyzes PI-3,4,5-trisphosphate to PI-3,4-bisphosphate (56)]. Mensah et al. (41) have shown that myocardial expression of PTEN was increased after 7 and 14 days of treatment with ATV at 20 mg·kg–1·day–1, although after a 3-day pretreatment, PTEN expression was unchanged. The IS-limiting effect of ATV at 20 mg·kg–1·day–1, apparent after 1 and 3 days, was lost after 7 and 14 days of treatment, suggesting that PTEN prevented activation of Akt and, hence, blunted the IS-limiting effect of ATV (41). Here, our finding that 3-day ATV treatment did not affect PTEN expression is in agreement with Mensah et al.’s (41) finding.
Previous studies (14, 23, 29, 38, 46, 63) have shown that thiazolidinediones increase the expression of PTEN and downregulate PI3K activity. In our study, Pio increased myocardial PTEN expression. Both ATV and Pio increased the expression of SHIP2. Previous studies (30) have also shown that rosiglitazone decreases SHIP2 overexpression in skeletal muscles and fat tissue of diabetic db/db mice. Nevertheless, in the present study, the increased expression of SHIP2 and PTEN did not prevent the induction of Akt and Ser1177 eNOS phosphorylation by ATV and the IS-limiting effect of Pio alone, ATV alone, or Pio and ATV in combination. Because activation of Akt occurs rapidly after statins are introduced (37), we cannot exclude that Akt phosphorylation occurs before the increase in PTEN and SHIP2 expression and that, with longer pretreatment periods (i.e., 1–2 wk), PTEN and/or SHIP2 may eventually block Akt phosphorylation and thus the protective effect of ATV, Pio, or the combination. Alternatively, it might be that the activation of both SHIP2 and PTEN is reactive to the increase in PI3K activity and consequently, PI-3,4,5-trisphosphate levels, because it has recently been shown that prolonged activation of P-Akt may have deleterious effects (44). However, there is a net increase in PI-3,4,5-trisphosphate levels and, therefore, in Akt phosphorylation, as shown in the present study. The effect of Pio and ATV on SHIP2 and PTEN, especially with long-term treatment, should be further investigated.
Myocardial Protection and PGI2 Production
Late ischemic preconditioning causes an increase in myocardial concentrations of PGE2 and 6-keto-PGF1, with only marginal increases in PGF2 (53). Intravenous PGI2, given before ischemia, reduces IS (59, 65). It has been reported that administration of COX-2 inhibitors before an infarction abrogates the IS-limiting effects of late ischemic preconditioning (10, 54).
Statins and Prostaglandin Production
There have been only sparse and conflicting data on the effects of statins on COX-2 regulation. Both mevastatin and lovastatin increase COX-2 content and 6-keto-PGF1 concentration in human aortic smooth muscle cells (20). COX-2 levels also increase in interleukin-1-stimulated mesangial cells after incubation with cerivastatin (9). On the other hand, three other studies have shown opposite results. Fluvastatin and simvastatin treatment have been reported to decrease the concentrations of COX-2 mRNA and protein levels in human umbilical vein endothelial cells (34). ATV also decreases the expression of COX-2 mRNA and protein in macrophages and smooth muscle cells of hypercholesterolemic rabbits (28). Finally, simvastatin decreases COX-2 expression in human carotid artery plaques (16). The present study confirms our previous findings that ATV increases the myocardial expression and activity of COX-2 in the rat myocardium (1, 7). Thus it seems that statins suppress COX-2 expression in inflammation models and atherosclerotic plaque but increase COX-2 expression in the normal myocardium.
We have previously reported that selective COX-2 inhibitors, given either orally together with ATV (7) or intravenously just before ischemia (1), abrogated the IS-limiting effect of ATV, suggesting that COX-2 products are also mediating the IS-limiting effects of statins. In both studies, when given alone, COX-2 inhibitors did not affect IS. In the same study, we have shown that SC-560, a selective COX-1 inhibitor, does not block the IS-limiting effect of ATV and does not affect IS when administered alone (1).
Thiazolidinediones and Prostaglandin Production
Here, we show that Pio increased myocardial 6-keto-PGF1 levels. Pio increased cPLA2 expression and activity. Pio induced a mild increase in COX-2 activity, which was not associated with any detectable changes in COX-2 expression.
There are conflicting data in the literature on the effect of thiazolidinedione on COX-2 expression. It has been reported that very high doses of rosiglitazone (30 µM) increase the expression of COX-2 in cultures of rat aortic vascular smooth muscle cells (8). However, at lower concentrations (10 µM), there was an increased release of prostaglandins without an increase in COX-2 expression (8). The authors have suggested that, at the lower concentrations, rosiglitazone induces PLA2, although in their study, there was no induction of type II soluble PLA2 by rosiglitazone (8). Troglitazone, but not 15d-PGJ2, induces COX-2 expression and PGE2 production in the human lung epithelial cell line A549 (45); however, this activation could not be blocked by PPAR- antagonists (45). Levine (39) has reported that ciglitazone increased the release of arachidonic acid from rat liver cells; however, the author did not explore the mechanism of this effect. Others have reported that both rosiglitazone and Pio augment prostaglandin production by increasing the availability of arachidonic acid, without affecting COX-1, COX-2, and cPLA2 expression (60). They suggested that thiazolidinediones increase the availability of arachidonic acid by preventing the reuptake of arachidonic acid by cell membranes (60). On the other hand, others have suggested that PPAR- negatively controls COX-2 expression (33). The induction of COX-2 expression by mINF- in macrophages was blunted by both troglitazone and ciglitazone (13).
In the present study, we show that Pio alone caused a smaller (3.4-fold) increase in COX-2 expression than ATV (25-fold). However, total COX and COX-2 activity were both increased by Pio, and total COX and COX-2 activity were significantly higher in the Pio + ATV group than in the ATV-alone or Pio-alone group. Recently, we have shown that ATV activates COX-2 via S-nitrosylation mediated by iNOS (1). Although the increase in COX-2 expression and activity in the Pio-treated rats was smaller than in the ATV-treated rats, the IS-limiting effect of both agents was abrogated by SC-58125, a selective COX-2 inhibitor. In contrast, despite the fact that COX-1 activity is higher than COX-2 activity in the Pio group, selective COX-1 inhibition did not blunt the protective effect of Pio, as has been previously shown for ATV (1). Thus the Pio effect may be due in part to posttranslational modification of COX-2 and not only to its synthesis regulation.
cPLA2 and Myocardial Protection
A previous study (57) suggested that PLA2 is involved in ischemic preconditioning. We have reported that ATV induced the expression and activity of cPLA2 (1, 7). Coadministration of valdecoxib, a specific COX-2 inhibitor, together with ATV for 3 days blunts the ATV-induced increased expression and activity of cPLA2 (7). In contrast, acute intravenous administration of SC-58125, another specific COX-2 inhibitor, after a 3-day pretreatment with ATV, did not affect cPLA2 expression or activity (1), suggesting that COX-2 is needed to augment the expression, but not the immediate activation, of cPLA2.
In the present study, we show that both ATV and Pio increased the expression and activity of cPLA2 and COX-2. Pio and ATV have additive effects on cPLA2 expression and activity. However, as selective COX-2 inhibition abrogated the IS-limiting effects of both Pio and ATV (1, 7), it seems that cPLA2 does not have an independent effect, rather, the arachidonic acid, released by cPLA2, is converted to protective prostaglandins by COX-2.
In conclusion, the IS-limiting effects of both Pio and ATV involve prostaglandin production by COX-2. However, the upstream steps differ. ATV induces eNOS phosphorylation, iNOS, cPLA2, and COX-2 expression, whereas Pio induces mainly the expression and activity of cPLA2. Although the activity of COX-2 is increased in Pio-treated rats, there is a relatively small increase in COX-2 expression. The effect of Pio and ATV is additive. The efficacy of this combination in myocardial protection should be further investigated in the clinical setting.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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  M.-H. Huang, V. Nguyen, Y. Wu, S. Rastogi, C. Y. Lui, Y. Birnbaum, H.-Q. Wang, D. L. Ware, M. Chauhan, N. Garg, et al. Reducing ischaemia/reperfusion injury through {delta}-opioid-regulated intrinsic cardiac adrenergic cells: adrenopeptidergic co-signalling Cardiovasc Res, December 1, 2009; 84(3): 452 - 460. [Abstract] [Full Text] [PDF]
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 S. Yasuda, H. Kobayashi, M. Iwasa, I. Kawamura, S. Sumi, B. Narentuoya, T. Yamaki, H. Ushikoshi, K. Nishigaki, K. Nagashima, et al. Antidiabetic drug pioglitazone protects the heart via activation of PPAR-{gamma} receptors, PI3-kinase, Akt, and eNOS pathway in a rabbit model of myocardial infarction Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1558 - H1565. [Abstract] [Full Text] [PDF]
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Y. Ye, Y. Lin, S. Manickavasagam, J. R. Perez-Polo, B. C. Tieu, and Y. Birnbaum Pioglitazone protects the myocardium against ischemia-reperfusion injury in eNOS and iNOS knockout mice Am J Physiol Heart Circ Physiol, December 1, 2008; 295(6): H2436 - H2446. [Abstract] [Full Text] [PDF]
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 L. Gao, W. Wang, and I. H. Zucker Simvastatin Inhibits Central Sympathetic Outflow in Heart Failure by a Nitric-Oxide Synthase Mechanism J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 278 - 285. [Abstract] [Full Text] [PDF]
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Y. Ye, J. D. Martinez, R. J. Perez-Polo, Y. Lin, B. F. Uretsky, and Y. Birnbaum The role of eNOS, iNOS, and NF-{kappa}B in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H343 - H351. [Abstract] [Full Text] [PDF]
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 Y.-J. Yang, H.-Y. Qian, J. Huang, Y.-J. Geng, R.-L. Gao, K.-F. Dou, G.-S. Yang, J.-J. Li, R. Shen, Z.-X. He, et al. Atorvastatin treatment improves survival and effects of implanted mesenchymal stem cells in post-infarct swine hearts Eur. Heart J., June 2, 2008; 29(12): 1578 - 1590. [Abstract] [Full Text] [PDF]
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 Y. Birnbaum, Y. Lin, Y. Ye, R. Merla, J. R. Perez-Polo, and B. F. Uretsky Pretreatment With High-Dose Statin, But Not Low-Dose Statin, Ezetimibe, or the Combination of Low-Dose Statin and Ezetimibe, Limits Infarct Size in the Rat Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2008; 13(1): 72 - 79. [Abstract] [PDF]
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 E. Erdmann Review: Statin plus thiazolidinedione use in patients with diabetes at high cardiovascular risk The British Journal of Diabetes & Vascular Disease, September 1, 2007; 7(5): 211 - 216. [Abstract] [PDF]
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R. Merla, Y. Ye, Y. Lin, S. Manickavasagam, M.-H. Huang, R. J. Perez-Polo, B. F. Uretsky, and Y. Birnbaum The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1918 - H1928. [Abstract] [Full Text] [PDF]
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M.-H. Huang, H.-Q. Wang, W. R. Roeske, Y. Birnbaum, Y. Wu, N.-P. Yang, Y. Lin, Y. Ye, D. J. McAdoo, M. G. Hughes, et al. Mediating {delta}-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H376 - H384. [Abstract] [Full Text] [PDF]
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Y. Ye, Y. Lin, R. Perez-Polo, M.-H. Huang, M. G. Hughes, D. J. McAdoo, S. Manickavasagam, B. F. Uretsky, and Y. Birnbaum Enhanced cardioprotection against ischemia-reperfusion injury with a dipyridamole and low-dose atorvastatin combination Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H813 - H818. [Abstract] [Full Text] [PDF]
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G. Salinas, U. C. Rangasetty, B. F. Uretsky, and Y. Birnbaum The Cycloxygenase 2 (COX-2) Story: It's Time to Explain, Not Inflame Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2007; 12(2): 98 - 111. [Abstract] [PDF]
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Y. Birnbaum, Y. Lin, Y. Ye, J. D. Martinez, M.-H. Huang, C. Y. Lui, J. R Perez-Polo, and B. F. Uretsky Aspirin before reperfusion blunts the infarct size limiting effect of atorvastatin Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2891 - H2897. [Abstract] [Full Text] [PDF]
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P < 0.001 vs. Pio + ATV; 
P < 0.001 vs. ATV.
P = 0.002 vs. control group; #P < 0.001 vs. ATV.