Kazuo Ichihara

All hydrophobic HMG-CoA reductase inhibitors induce apoptotic death in rat pulmonary vein endothelial cells

3-Hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors (statins) are effective in patients with hypercholesterolemia to reduce risk of cardiovascular diseases, because of not only their lowering cholesterol effects but also their pleiotropic effects, such as improvement of endothelial cell dysfunction. On the other hand, statins prevent cell proliferation of various cells, including endothelial cells. We examined effects of all statins available at present on the viability of cultured rat pulmonary vein endothelial cells. Lovastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin, which are hydrophobic statins, markedly reduced cell viability associated with DNA fragmentation, DNA laddering and activation of caspase-3, suggesting apoptotic cell death. Pravastatin, which is a hydrophilic statin, however, did not induce cell apoptosis. Apoptosis induced by hydrophobic statins was associated with activation of apoptosis-related intracellular signal transduction systems; attenuation of localization of RhoA to the membrane, induction of Rac1, and increase in phosphorylation of c-Jun N-terminal kinase and c-Jun. Endothelial cell apoptosis is underlying the improvement of the endothelial dysfunction with hydrophobic statins.

Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes

We investigated the effect of fluvastatin sodium (fluvastatin) and pravastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, on the formation of thiobarbituric acid reactive substances both in vivo and in vitro in rat liver microsomes and on active oxygen species. Oral administration of fluvastatin at low doses (3.13 and 6.25 mg/kg) inhibited the formation of thiobarbituric acid reactive substances in rat liver microsomes, but high doses (12.5 and 25 mg/kg) did not change the formation of thiobarbituric acid reactive substances. Fluvastatin at any dose used had no effect on the content of cytochrome P-450 and the activity of NADPH-cytochrome P-450 reductase. In in vitro experiments, concentrations of fluvastatin ranging from 1 x 10(-6) - 1 x 10(-4) M markedly inhibited NADPH-dependent lipid peroxidation in liver microsomes, but pravastatin weakly inhibited lipid peroxidation. The order of magnitude of inhibition of each drug on in vitro lipid peroxidation was butylated hydroxytoluene > probucol > or = fluvastatin > pravastatin. Moreover, fluvastatin chemically scavenged active oxygen species such as hydroxyl radicals and superoxide anion generated by the Fenton reaction and by the xanthine-xanthine oxidase system, respectively, but pravastatin showed no scavenging of superoxide anion. These results indicate that the suppression of in vivo and in vitro lipid peroxidation in liver microsomes may be, at least in part, due to the scavenging by fluvastatin of free radicals.

Effects of diltiazem and propranolol on irreversibility of ischemic cardiac function and metabolism in the isolated perfused rat heart.

Ischemia was induced by lowering the after-load pressure of the perfused working rat heart, and continued until the heart was reperfused by raising the after-load. After ischemia, the following changes were observed: decreases in the pressure-rate product (peak aortic pressure × heart rate) and coronary flow; depletion of adenosine triphosphate and creatine phosphate; and accumulation of lactate. When the heart was exposed to ischemia for more than 20 min, reperfusion of the ischemic heart could not restore the pressure-rate product and the tissue adenosine triphosphate completely, suggesting that irreversible ischemic damage occurred. Diltiazem (2.41 × 10−6, 1.21 × 10−5, and 2.41 × 10−5 M) or propranolol (1.69 × 10−5 and 3.38 × 10−5 M) was provided for the heart 5 min before the onset of ischemia. In the presence of diltiazem or propranolol, the levels of adenosine triphosphate and creatine phosphate were preserved even after 20 min of ischemia. Reperfusion with the normal perfusate after 20 min of ischemia with the buffer containing diltiazem or propranolol recovered the pressure-rate product that had been decreased by ischemia, depending on the concentration of diltiazem or propanolol provided. These results indicate that diltiazem, as well as propranolol, delays the onset of irreversible ischemic damage of the heart, suggesting their protective effects on the ischemic myocardium.

Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischaemic dog hearts.

1 Effects of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors, pravastatin and simvastatin, on the myocardial level of coenzyme Q10, and on mitochrondrial respiration were examined in dogs 2 Either vehicle (control), pravastatin (4 mg kg−1day−1), or simvastatin (2 mg kg−1day−1) was administered orally for 3 weeks. First, the myocardial tissue level of coenzyme Q10 was determined in the 3 groups. Second, ischaemia was induced by ligating the left anterior descending coronary artery (LAD) in anaesthetized open chest dogs, pretreated with the inhibitors. After 30 min of ischaemia, nonischaemic and ischaemic myocardium were removed from the left circumflex and LAD regions, respectively, and immediately used for isolation of mitochondria. The mitochondrial respiration was determined by polarography, with glutamate and succinate used as substrates 3 Simvastatin significantly decreased the myocardial level of coenzyme Q10, but pravastatin did not 4 Ischaemia decreased the mitochondrial respiratory control index (RCI) in both groups. Significant differences in RCI between nonischaemic and ischaemic myocardium were observed in the control and simvastatin‐treated groups 5 Only in the simvastatin‐treated group did ischaemia significantly decrease the ADP/O ratio, determined with succinate 6 The present results indicate that simvastatin but not pravastatin may cause worsening of the myocardial mitochondrial respiration during ischaemia, probably because of reduction of the myocardial coenzyme Q10 level.

Disparity between angiographic regression and clinical event rates with hydrophobic statins

Statins are effective at lowering blood cholesterol concentration, and are given to patients with hypercholesterolaemia or hyperlipidaemia. Reduction of cholesterol concentration in serum can delay, or even regress, atherosclerotic lesions, leading to diminution of risk of cardiovascular disease. Results of studies have shown that the beneficial effects of statins depend not only on lowering cholesterol but also on many other factors. Production of mevalonic acid, a precursor of various essential substances for cell function (figure), is inhibited by hydrophobic statins. Thus these drugs might cause unexpected adverse effects in various tissues including the heart.

Rebound recovery of myocardial creatine phosphate with reperfusion after ischemia

It has been accepted that both mechanical function and tissue level of creatine phosphate (CP) of the heart decrease after interruption of myocardial perfusion (ischemia of the heart). A strange but interesting fact is that the myocardial CP level, that has been decreased by ischemia, recovers to a level higher than that of the preischemic level after reperfusion of the ischemic myocardium (rebound recovery or overshoot phenomenon1-6), even when mechanical function of the heart does not recover completely; i.e., although the CP store is refilled as energy conservation, the mechanical function of the reperfused heart does not recover. The mechanism of this phenomenon, however, remains obscure. We therefore reexamined the phenomenon, and found a plausible and reasonable interpretation-the rebound recovery of the CP level is due to the decrease of mechanical function of the heart during reperfusion after ischemia. Hearts removed from male Sprague-Dawley rats weighing 300 to 350 gm were perfused by the working heart technique with Krebs-Henseleit bicarbonate buffer containing 11 mmol/L glucose.7 Ischemia was induced by lowering the afterload pressure from 60 to 0 mm Hg.6 Under these conditions of ischemia, coronary flow was not exactly 0 ml/min but was less than 0.2 ml/min (ischemic perfusion). In some experiments, the buffer containing high concentration of KC1 (20 mmol/L), propranolol (10 mg/L), or diltiazem (10 mg/L), or that was being cooled at loo or 20” C was used as a perfusion solution for a period of ischemic perfusion in order to protect the heart against ischemic injury. After 20 minutes of ischemia, hearts were reperfused by again raising the afterload pressure to 60 mm Hg. Ischemia produced the heart arrest with a marked decrease in the tissue levels of CP and adenosine

Lipophilic Hmg-coa Reductase Inhibitors Increase Myocardial Stunning in Dogs

Pretreatment of dogs with simvastatin, a lipophilic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, increases myocardial contractile dysfunction during reperfusion after ischemia (stunning), with reduction of tissue adenosine triphosphate (ATP). This was thought to be a consequence of prevention of ubiquinone biosynthesis by the lipophilic inhibitor in the myocardial cell. We examined whether other lipophilic HMG-CoA reductase inhibitors also influence myocardial stunning in dogs. Vehicle, atorvastatin (2 mg/ kg/day), fluvastatin (4 mg/kg/day), or cerivastatin (40 microg/kg/ day) was orally administered for 3 weeks. Hydrophilic pravastatin (4 mg/kg/day) also was given. After 3 weeks, pentobarbital-anesthetized dogs were subjected to 15-min left anterior descending coronary artery occlusion followed by 2-h reperfusion. Myocardial segment function was determined by sonomicrometry. Tissue levels of ATP were determined in 2-h reperfused hearts. All inhibitors significantly decreased serum cholesterol level. The three lipophilic inhibitors resulted in a worsening of segment function in the reperfused myocardium, as compared with the vehicle group. The levels of ATP in the atorvastatin, fluvastatin, and cerivastatin groups were significantly lower than that in the vehicle group. These results confirm that lipophilic HMG-CoA reductase inhibitors enhance myocardial stunning in association with ATP reduction after ischemia and reperfusion.

Influences of pravastatin and simvastatin, HMG-CoA reductase inhibitors, on myocardial stunning in dogs.

Summary: We examined the effects of pravastatin and simvastatin, HMG-CoA reductase inhibitors, on stunned myocardium in vivo. Pravastatin and simvastatin were given orally 2 mg/kg for 3 weeks. After 3 weeks of administration, pentobarbital-anesthetized dogs were subjected to 15-min left anterior descending (LAD) coronary artery occlusion followed by 2-h reperfusion. Myocairdial segment function was determined by sonomicrometry. The tissue energy and carbohydrate metabolites were determined in the 2-h-reperfused hearts. Administration of pravastatin and simvastatin for 3 weeks decreased serum cholesterol level and blood pressure (BP). Simvastatin resulted in a worsening of segment shortening in the reperfused myocardium as compared with control and pravastatin groups. The level of ATP in the simvastatin group was significantly lower as compared with that in the control group. The other metabolite levels were not significantly altered by either pravastatin or simvastatin. These results suggest that simvastatin enhances stunning of the myocardium in association with ATP reduction after reperfusion subsequent to ischemia

Effects of sevoflurane on ischaemic myocardium in dogs.

We studied the effect of sevoflurane on ischaemic myocardium in terms of myocardial energy and carbohydrate metabolism. Mongrel dogs were anaesthetized initially with sodium pentobarbitone, and then inhaled sevoflurane at 0% (0 MAC), 2.4% (1.0 MAC) or 4.7% (2.0 MAC) of inspired concentration for 60 min. Ischaemia was then induced for 3 min by ligating the left anterior descending coronary artery. The tissue levels of energy and carbohydrate metabolites were determined before and after sevoflurane inhalation, and after 3 min of ischaemia. Sevoflurane significantly decreased systolic and diastolic blood pressures, heart rate, and rate‐pressure product in a dose dependent manner. When the animals did not inhale sevoflurane (0 MAC), ischaemia significantly decreased adenosine triphosphate and creatine phosphate levels, and produced alterations of carbohydrate metabolism. These metabolic changes induced by ischaemia were lessened by inhalation of sevoflurane. To exclude the influence of haemodynamic changes, blood pressure and heart rate were maintained during 1.0 MAC sevoflurane inhalation. Significant attenuation of ischaemia‐induced metabolic changes caused by sevoflurane was still observed in some metabolites. These results indicate that the ischaemic influences on the myocardium may be reduced by sevoflurane, and this protective effect can be explained not only by its haemodynamic effect.

Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischemic rat hearts

The aim of the present study was to examine the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on mitochondrial respiration in ischemic rat hearts, and to compare the effects between water-soluble pravastatin and lipid-soluble simvastatin. Either vehicle (0.5% carboxymethyl cellulose), pravastatin (2 or 4 mg/kg per day), or simvastatin (1 or 2 mg/kg per day) was orally administered for 3 weeks. Ischemia was induced by ligating the aorta for 60 min in anesthetized open chest rats under artificial respiration. The hearts were removed, mitochondria were isolated, and the respiration was determined by polarography using glutamate and succinate as substrates. When succinate was used as a substrate, the ADP-stimulated respiration (QO3) and ATP production per unit oxygen (ADP/O ratio) were decreased by ischemia. The decreases in QO3 and ADP/O ratio in the pravastatin- and simvastatin-treated groups appeared to be more prominent than those in the vehicle-treated group. This was especially true in the simvastatin-treated group. The ADP-limited respiration (QO4) with succinate in the vehicle-treated heart was slightly increased by ischemia, while that in the pravastatin- or simvastatin-treated hearts was decreased. In conclusion, HMG-CoA reductase inhibitors may result in worsening of myocardial mitochondrial respiration during ischemia.