Based on a growing body of evidence,1-4 we believe that geranylgeranylacetone (GGA), a safe and widely available drug without serious side effects, has potential benefits for the treatment of ischemia/reperfusion injury clinically similar to those seen with hyperthermia. Whole-body hyperthermia 24 hours before the onset of myocardial ischemia protected the heart against ischemia/reperfusion injury, which was caused mainly by the proportional induction of heat shock protein 72 (HSP72).1,2 Our previous studies have shown that oral administration of GGA, an antiulcer drug, induces the expression of HSP72, which protects against ischemia/reperfusion injury in the rat heart.3,4 More recently, GGA-induced HSP72 expression was found to provide protective effects against delayed cerebral vasospasm after rat subarachnoid hemorrhage.5 Therefore, it is very important to investigate the role of endothelial function in the GGA-induced protection in the cardiovascular system.
It is widely accepted that endogenous nitric oxide (NO) produced by endothelial-type NO synthase (eNOS) in the endothelial cells plays an important role in protecting against myocardial ischemia/reperfusion injury.6 However, endothelial dysfunction was rapidly developed within the first minute following initiation of reperfusion.7 In isolated rat hearts subjected to global ischemia followed by reperfusion, eNOS activity was remarkably impaired, resulting in the reduced NO production.8 The role of eNOS in cardioprotection is supported by the recent observations that the infarct size is larger and the postischemic myocardial functional recovery is lower in mice lacking the eNOS gene than in wild-type mice,9-11 and that administration of exogenous NO gas or NO donors reduces the infarct size in experimental ischemia/reperfusion model.12,13 Phosphatidylinositol 3 (PI3) kinase and Akt has been reported to regulate the eNOS activity.14-16 In addition, Rho/Rho kinase-mediated pathway has been found to play an important role in vascular spasm.17 Furthermore, adrenomedullin and endothelin-1, other two endogenous peptides were raised after acute myocardial infarction and were involved in the protection against ischemia/reperfusion injury.18,19 However, the links between these pathways and the cardioprotective effects of HSP72 induced by GGA remain unexplored. We hypothesized that GGA improved the postischemic myocardial functional recovery via preventing the ischemia/reperfusion-induced endothelial dysfunction. To test this hypothesis, we compared the acetylcholine (ACh)-induced coronary vasodilation in GGA and vehicle (CON) groups and examined the effects of NO, adrenomedullin, endothelin-1, PI3 kinase, and Rho kinase on GGA-induced protection in isolated Langendorff perfused rat hearts. Our results showed that GGA attenuated the ischemia/reperfusion-induced coronary endothelial dysfunction, which might contribute to its cardioprotective effect. The PI3 kinase and/or Rho kinase pathways might be involved in this process. However, adrenomedullin and endothelin-1 were not associated with the GGA-induced cardioprotection.
Procedures complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, 1996).
GGA was provided by Eisai Co, Ltd. LY294002 (PI3 kinase inhibitor) and Y27632 (Rho kinase inhibitor) were purchased from Promega and Calbiochem, respectively. Other drugs were purchased from Sigma or Wako chemical companies. The nitrate/nitrite colorimetric assay kit (for indirect measurement of NO production), adrenomedullin EIA kit, and endothelin-1 assay kit were purchased from Funakoshi, Phoenix, and Peninsula, respectively.
Isolated Perfused Heart Experiments
Adult male Sprague-Dawley rats (220-250 g body weight) were given either oral GGA (an emulsion with 5% arabic gum and 0.008% tocopherol, at a dose of 200 mg/kg, GGA group) or vehicle (CON group), and 24 hours later the rats were heparinized (500 IU/kg, IP) and anesthetized with sodium pentobarbital (50 mg/kg, IP). Each heart was excised and immediately arrested in ice-cold Krebs-Henseleit (KH) buffer (in mmol/L: NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11; pH 7.4). The aorta was quickly cannulated and perfused retrogradely by the Langendorff apparatus with KH buffer equilibrated with a 95% O2-5% CO2 gas mixture at 36.5°C at a constant perfusion pressure of 75 mm Hg. A water-filled latex balloon was inserted through the mitral orifice into the left ventricle (LV), and the LV end-diastolic pressure (LVEDP) was adjusted to 0-5 mm Hg. Pacing electrodes were attached to the aorta and right ventricular apex, and the heart was paced at 300 beats/min using an electrical stimulator (SEN-7203, Nihon Kohden). We found that when we applied constant perfusion pressure, the coronary perfusion was reduced by ischemia/reperfusion, in association with an increase of coronary resistance. This reduction in coronary perfusion in each heart would be inappropriate for the assessment of CPP changes in response to ACh and SNP. To overcome this problem, during the initial 10 minutes of constant-pressure perfusion, the perfusion flow rate was determined for each heart, which was then perfused at the determined rate with a microtube pump. The heart was covered with water-jacketed glassware, and the relative humidity was maintained at 90% or more.
We designed 3 protocols for the present study (Fig. 1): (A) cardiac performance, (B) endothelial function, and (C) concentrations of NO, adrenomedullin, and endothelin-1.
LV pressure was monitored with a pressure transducer to obtain the peak positive and negative first derivatives (dP/dtmax and dP/dtmin). The LV developed pressure (LVDP) was calculated as the difference between the LV systolic and diastolic pressures. Coronary perfusion pressure (CPP) was defined as the hydraulic pressure measured at the level of aortic cannulation. LVDP, LV dP/dtmax, LV dP/dtmin, LVEDP, and CPP were continuously recorded on a polygraph recorder (WS-681G, Nihon Kohden), and the data were stored on a PCM data recorder (RD-111T, TEAC) for later analysis. After 30 minutes of equilibration, the preparations were subjected to 30 minutes of low-flow (10% of the baseline flow) global ischemia followed by 30 minutes of reperfusion.20 During the reperfusion period, some hearts transiently developed ventricular fibrillation (VF). The hearts exhibiting long-lasting VF (>30 seconds) were excluded from study. In GGA and CON groups, the preparations were also performed with or without NG-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor, 10−4 mol/L) to evaluate the effect of NO (Fig. 1A).
Endothelial function was evaluated by comparing endothelium-dependent coronary vasodilation with endothelium-independent coronary vasodilation (Fig. 1B). After stabilization for 15 minutes, ACh (10−6 mol/L) was added to the perfusate for 5 minutes (endothelium-dependent coronary vasodilation). Following 10 minutes of washout, sodium nitroprusside (SNP, 3 × 10−5 mol/L) was added to perfusate for 5 minutes (endothelium-independent coronary vasodilation). Following another 10-minute washout, preparations were subjected to 30 minutes of low-flow (10% of the baseline flow) global ischemia followed by 30 minutes of reperfusion, and then ACh and SNP were sequentially added to the perfusate in the same manner as before ischemia/reperfusion. Reduction of CPP in response to ACh was defined as endothelium-dependent coronary vasodilation, whereas that in response to SNP was defined as endothelium-independent coronary vasodilation. The percentage change of CPP was calculated as:
where CPPb is the CPP before giving ACh or SNP; CPPa is the CPP after giving ACh or SNP. The effects of concomitant perfusion with LY294002 (3 × 10−6 mol/L) or Y27632 (10−6 mol/L) were also investigated.
In this protocol (Fig. 1C), the coronary effluents were collected during the basal, ischemia, and reperfusion periods, respectively, for measuring the amounts of both nitrate and nitrite as an estimate of coronary NO production.21 At physiological oxygen tension, NO in the coronary circulation is rapidly oxidized with an estimated half-life of 0.1 second, becoming virtually undetectable in plasma.22,23 Therefore, the concentrations of nitrate and nitrite in the coronary effluent were measured by the Griess reaction to estimate the total amount of NO.21 In brief, 80 μL of coronary effluent was mixed with 10 μL of the enzyme cofactor and 10 μL of the nitrate reductase, and the plate was covered and incubated for 60 minutes at room temperature. Then, Griess reagents R1 (50 μL) and R2 (50 μL) were added to the plate and incubated for 10 minutes at room temperature. Finally, absorbance was measured at 540 nm,22 and the nitrate and nitrite concentrations were determined by comparison with sodium nitrite standards. Each experiment was repeated 3 times and averaged. The effects of concomitant perfusion with LY294002 (3 × 10−6 mol/L) or Y27632 (10−6 mol/L) were also investigated.
Measurements of Adrenomedullin and Endothelin-1
From the collected coronary effluents, the concentrations of adrenomedullin and endothelin-1 were measured as previously described.24,25 Briefly, adrenomedullin peptide was extracted with 1% trifluoacetic acid and incubated with antiserum and bionylated peptide at room temperature for 2 hours. After addition of streptavidin-horseradish peroxidase solution, the sample was incubated for 1 hour. Thereafter, substrate solution was added and was incubated for 1 hour. Reaction was terminated with 2 N HCl and measured by EIA assay kit (Peninsula). For endothelin-1 measurement, the collected effluent was added in a solution containing 0.5% hexadecyl trimethyl ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 minutes at 4000 g at 4°C. An aliquot (100 μL) of the supernatant was then assayed and compared with a standard curve constructed with 0-250 pg/ml rat endothelin-1. Endothelin-1 was measured by EIA assay kit (Peninsula).
All data are expressed as mean ± SEM. Comparison between 2 groups was performed by unpaired Student t tests, and among multiple groups was performed by 2-way ANOVA analysis followed by the Bonferroni-Dunn test. A value of P < 0.05 was regarded as significant.
There was no significant difference in the hemodynamic parameters at basal condition between the CON group and the GGA group (Table 1). Perfusion with L-NAME did not cause any significant change in these parameters (data not shown).
The change in LVDP, dP/dt, and LVEDP during both ischemia and reperfusion in CON, GGA, CON-L-NAME, and GGA-L-NAME groups is shown in Figure 2. In all 4 groups, low-flow ischemia quickly decreased LVDP, LV dP/dtmax, and LV dP/dtmin and gradually increased LVEDP. There was no difference in all parameters among the 4 groups (Fig. 2). On reperfusion, GGA significantly increased the recovery of LVDP, dP/dtmax, and dP/dtmin but suppressed the increase of LVEDP, and L-NAME (10−4 M) markedly inhibited the effect of GGA (P < 0.05, Fig. 2). However, L-NAME had no effect in CON group.
Coronary Endothelial Function
The response of CPP to ACh or SNP, before and after ischemia/reperfusion, is depicted in Figure 3. Before ischemia/reperfusion, GGA did not affect the CPP changes in response to ACh. LY294002 and Y27632 had no effect on the CPP in response to ACh or SNP in CON and GGA groups (Fig. 3, top). After ischemia/reperfusion, there was a significant reduction of the percentage change of CPP in response to ACh in both groups. However, the percentage change of CPP was greater in the GGA group than that in the CON group (P < 0.05). In addition, LY294002 abolished the effect of GGA on the ACh-induced vasodilation but had no effect in the CON group. Conversely, Y27632 markedly augmented the ACh-induced vasodilation in the CON group but had no effect in the GGA group.
GGA did not affect the CPP changes in response to SNP before and after ischemia/reperfusion, compared with that in the CON group (Fig. 3, bottom). LY294002 and Y27632 had no effect in the CPP changes in response to SNP before or after ischemia/reperfusion in CON and GGA groups.
In addition, ACh- or SNP-induced coronary vasodilation after ischemia/reperfusion was not affected by the perfusion with ACh followed by SNP before ischemia (Fig. 3), which confirmed that no preconditioning phenomenon occurred when the applications of ACh and SNP were performed before ischemia.
Production of NO
Figure 4 summarizes NO production in the coronary effluent at stabilization, ischemia, and reperfusion. At stabilization, there was no significant difference in the NO production among the 8 groups. During both ischemia and reperfusion, however, GGA significantly enhanced NO production (P < 0.05). Y27632 alone also increased NO production, but no additive effect was found after cotreatment with GGA and Y27632. In addition, L-NAME and LY294002 alone did not affect NO production, but they inhibited the effect of GGA on NO production (Fig. 4).
Adrenomedullin and Endothelin-1
The concentrations of adrenomedullin and endothelin-1 in the coronary effluent at basal, ischemia, and reperfusion conditions are depicted in Figure 5. The concentration of adrenomedullin at basal condition was lower in the GGA group than in the CON group, but at both ischemia and reperfusion, there was no significant difference in the concentration of adrenomedullin between CON and GGA groups (Fig. 5, left).
The concentration of endothelin-1 was also measured (Fig. 5, right). There was no difference in the concentration of endothelin-1 in basal condition, ischemia, and reperfusion between CON and GGA groups.
There are several important findings in the present study. (1) The NOS inhibitor L-NAME inhibits the GGA-induced improvement in LV functional recovery during reperfusion; (2) after ischemia/reperfusion, the impairment of the ACh-induced coronary vasodilation is prevented by GGA; (3) the amount of NO in coronary effluent during both ischemia and reperfusion is increased by GGA; and L-NAME abolishes the effect of GGA; (4) PI3 kinase inhibitor LY294002 inhibits the effect of GGA on the preservation of ACh-induced vasodilation and NO production, whereas Rho kinase inhibitor Y27632 alone augments the ACh-induced vasodilation and NO production but it does not potentiate the effect of GGA, and (5) GGA does not affect the concentrations of adrenomedullin and endothelin-1 during both ischemia and reperfusion, compared with that in CON group.
A lot of studies have shown that induction of HSP in vivo protects against a number of acute insults such as ischemia, reperfusion, and oxidative injury.26-29 We have previously shown that oral administration of GGA induces the expression of HSP72 in the rat heart, thus protecting it against ischemia/reperfusion injury.3,4 In the same animal model and with the same dose of GGA in the present study, the NO production was enhanced during ischemia and reperfusion. Fike et al30 found that HSP90 antagonist geldanamycin abolished ACh-induced dilation in control piglet pulmonary arteries, which was consistent with the reduced NO production and/or bioavailability by inhibition of HSP90 chaperone function and subsequent NOS uncoupling. Together with these observations, it is reasonable to assume that the attenuated endothelial damage induced by overexpression of HSP72, at least in part, contributes to the cardioprotective effect of GGA. However, the present study does not clarify the mechanism responsible for the HSP72-mediated NO production, neither may it remain clear whether NO conversely modulate HSP72 expression.
Bravo et al31 reported that the isolated rat kidney treated with L-NAME for 3 weeks induced 8 to 20 time increases in HSP72 expression in the cortex, suggesting that NOS inhibition resulted in the increase in HSP72 expression. However, Swiecki et al32 found that HSP72 expression was markedly decreased in intestine and liver from L-NNA-treated rats. Therefore, further studies are necessary to clarify these unresolved issues.
Rokutan et al also reported a link between GGA and NO production using cultured guinea pig gastric pit cells.33 In their experiments, GGA increased the release of NO degeneration products, NO2−, and NO3−. The [3H]glucosamine uptake was completely inhibited by the nonselective NOS inhibitor, NG-nitro-L-arginine and NG-monomethyl-L-arginine. GGA did not up-regulate the inducible NOS (iNOS) and eNOS mRNA nor induce protein expression. In contrast, GGA induced neuronal NOS (nNOS) overexpression. From these results, they concluded that GGA stimulated mucin synthesis at least in part through an NO-dependent pathway, leading to an increase in the integrity of the gastric mucosa.33 In our previous study, in which the whole heart was used for Western blot analysis, oral administration of GGA did not induce expression of eNOS, iNOS, or nNOS.3 In the present study, L-NAME abolished the GGA-induced improvement in postischemic LV function, and the GGA-treated heart showed the enhanced production of NO. In addition, GGA preserved the ACh-induced (endothelium-dependent) vasodilation after ischemia/reperfusion. Based on these results, we could assume that the GGA-induced functional preservation of the coronary endothelial cells was involved in the cardioprotection induced by GGA. Here, a caution should be needed because there was distinct difference in method for GGA administration between the 2 studies. Rokutan et al33 added GGA in the culture medium for 48 hours, whereas we orally administered single doses of GGA. Although we used GGA as a trigger to increase HSP72 expression and NO production, other signaling pathways could be involved in animal cardioprotection when GGA was continuously administrated. In fact, Rokutan et al suggested that beside HSP72 expression, GGA stimulates multiple pathways leading to cytoprotection, including stimulation of hexamine production and mediation of endogenous prostaglandins.33 Further study is needed to explore the precise mechanism of the cardioprotection induced by GGA.
We mainly emphasized the protective role of NOS/NO in the present study. However, some studies have shown that NOS/NO also has the detrimental effects.34,35 Poon et al36 reported that iNOS expressed in isolated cardiomyocytes regulated the response to β-adrenergic stimulation during sepsis. However, as the neutrophils migrated in proximity to myocytes, iNOS became essential for the ability of neutrophils to damage myocytes.36 Recently a new hypothesis has been formulated that eNOS and nNOS, producing a small amount of NO, can cause the beneficial role, whereas high levels of NO produced by iNOS can induce tissue damage.36 Because we do not know which isomer is responsible for the production of NO in the present study, further work is needed to clarify whether the GGA-induced NO production causes the myocardial injury.
In the present study, concomitant perfusion with LY294002, a PI3 kinase inhibitor, abolished the preserved endothelium-dependent vasodilation and attenuated the augmented NO production in the GGA-treated heart, which was in accordance with recent studies that had shown the importance of this pathway in the attenuation of reperfusion injury.14-16 For instance, exogenous bradykinin administered to mice at the time of reperfusion limited the infarct size with concomitant phosphorylation of Akt and eNOS.15 Atorvastatin, a lipid-lowering agent, also attenuated lethal reperfusion-induced injury in mice in a manner that relied on PI3 kinase-Akt and eNOS.16 Therefore, these results suggested that the protection of the GGA-treated heart against ischemia/reperfusion injury involved PI3 kinase-Akt-eNOS signaling pathway, and the latter led to preservation of the endothelium-dependent coronary vasodilation. Alternatively, Rho kinase has been identified as an effector of the small GTP-binding protein (Rho)17 and the Rho/Rho kinase-mediated signaling pathway plays an important role in various cellular functions, including vascular smooth muscle contraction.17 Y27632 and fasudil are specific inhibitors of Rho kinase.17 Experimental and clinical studies have demonstrated that intracoronary infusion of fasudil effectively ameliorates coronary vasospasm, probably by inhibiting the hypercontractility of the vascular smooth muscle cells.37,38 With respect to endothelial cells, electron microscopy and immunofluorescence studies of the rat heart have shown that Rho kinase is localized primarily in the intracellular membranes of endothelial cells and that Y27632 blunts the change in the shape of endothelial cells induced by ischemia and reperfusion.39 In the present study, Y27632 augmented the ACh-induced vasodilation and increased NO production in CON group toward the level as seen in GGA group, which suggested the involvement of Rho kinase activation in the endothelial dysfunction and the decreased NO production following ischemia/reperfusion observed in CON hearts. Although this finding was not direct evidence, we postulated that inhibition of Rho kinase was involved in GGA-induced endothelial protection.
Adrenomedullin is a potent vasodilatory peptide originally isolated from human pheochromocytoma.40 This peptide and its mRNA were expressed in the heart,40 and the plasma and cardiac concentrations of adrenomedullin markedly increased after acute myocardial infarction.18 Accordingly, the amount of adrenomedullin in the coronary effluent was increased in response to ischemia in CON and GGA groups. Interestingly, although the baseline production of adrenomedullin in GGA group was lower than in CON group, it was markedly increased in the GGA group during ischemia toward the level shown in CON group. The significance of these findings remains unclear. Adrenomedullin induced activation of PI3 kinase/Akt in the endothelium of the rat aorta, resulting in enhancement of subsequent NO production and endothelium-dependent vasodilation.41 More recently, intravenous injection of adrenomedullin attenuated myocardial ischemia/reperfusion injury through the PI3 kinase/Akt-dependent pathway.42 Further studies are needed to clarify the role of cardiac and/or endothelial adrenomedullin production in the activation of the PI3 kinase/Akt pathway by GGA. In the present study, the amount of endothelin-1 during both ischemia and reperfusion was not increased as compared with that during basal condition. No significant difference was observed between CON and GGA-treated hearts. However, the clinical observation has shown that the plasma endothelin-1 level is raised after acute myocardial infarction and recanalization in humans.19 The heterogeneity of these results could reflect the difference of species and models.
First, a nonspecific NOS inhibitor L-NAME was employed in the present study at a concentration of 10−4 mol/L because this concentration was reported to almost completely inhibit all 3 components of NOS.43 From the data presented here, it is impossible to determine which isomer is responsible for the GGA-induced cardioprotection. Further study is needed to evaluate the relative role of eNOS, nNOS, and iNOS. Second, we used ACh, SNP, LY294002, and Y27632 only at a single concentration. ACh and SNP induced coronary artery vasodilation in a dose-dependent manner.44,45 According to these findings, 10−6 mol/L ACh and 3 × 10−5 mol/L SNP were enough to induce almost fully endothelium-dependent and endothelium-independent vasodilation, respectively. In addition, LY294002 at a concentration of 3 × 10−6 mol/L was used in the previous study to exclude the role of PI3 kinase from cardioprotective effects in mice overexpressing A1 adenosine receptors.46 Therefore, we introduced the same concentration of LY294002 in the present study. Regarding Rho kinase inhibition, Y27632 at a concentration of 10−6 mol/L was previously used to evaluate whether Rho kinase was involved in endothelial cell shape changes after ischemia/reperfusion injury.39 Therefore, the same concentration was applied in the present study. However, IC50 of LY294002 is 1.4 μM,47 and Y27632 (1 μM) has been shown to be selective at the stated concentration.48,49 Therefore, although we used these inhibitors at a single concentration, we think our conclusion from our results is reasonable. Third, the isolated Langendorff heart perfused with KH solution was used in the present study. Activation of leukocytes or platelets in response to ischemia/reperfusion also causes the myocardial dysfunction in blood-perfused heart experiments.50 Therefore, care should be taken when extrapolating the data obtained here to the clinical situation and to in vivo experiments.
In conclusion, our results indicate that GGA attenuates the endothelial dysfunction induced by ischemic insult, which may contribute, at least in part, to its cardioprotective effect. The PI3 kinase and/or Rho kinase pathways may also play an important role in this process.
The authors thank Yuhi Yamamoto and Sho Yoneda, for the excellent technical assistance.
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