Differential mRNA expression of seven genes involved in cholesterol metabolism and transport in the liver of atherosclerosis-susceptible and -resistant Japanese quail strains.

BACKGROUND: Two atherosclerosis-susceptible and -resistant Japanese quail (Coturnix japonica) strains obtained by divergent selection are commonly used as models to study atherosclerosis, but no genetic characterization of their phenotypic differences has been reported so far. Our objective was to examine possible differences in the expression of genes involved in cholesterol metabolism and transport in the liver between these two strains and to evaluate the value of this model to analyze the gene system affecting cholesterol metabolism and transport. METHODS: A factorial study with both strains (atherosclerosis-susceptible versus atherosclerosis-resistant) and two diets (control versus cholesterol) was carried out. The mRNA concentrations of four genes involved in cholesterol biosynthesis (HMGCR, FDFT1, SQLE and DHCR7) and three genes in cholesterol transport (ABCG5, ABCG8 and APOA1) were assayed using real-time quantitative PCR. Plasma lipids were also assayed. RESULTS: Expression of ABCG5 (control diet) and ABCG8 (regardless of dietary treatment) and expression of HMGCR, FDFT1 and SQLE (regardless of dietary treatment) were significantly higher in the atherosclerosis-resistant than in the atherosclerosis-susceptible strain. Plasma triglyceride and LDL levels, and LDL/HDL ratio were significantly higher in the atherosclerosis-susceptible than in the atherosclerosis-resistant strain fed the cholesterol diet. In the atherosclerosis-susceptible strain, ABCG5 expression regressed significantly and positively on plasma LDL level, whereas DHCR7 and SQLE expression regressed significantly and negatively on plasma triglyceride level. CONCLUSIONS: Our results provide support for the hypothesis that the atherosclerosis-resistant strain metabolizes and excretes cholesterol faster than the atherosclerosis-susceptible strain. We have also demonstrated that these quail strains are a useful model to study cholesterol metabolism and transport in relation with atherosclerosis.

15-F(2t)-isoprostane exacerbates myocardial ischemia-reperfusion injury of isolated rat hearts.

Reactive oxygen species induce formation of 15-F(2t)-isoprostane (15-F(2t)-IsoP), a specific marker of in vivo lipid peroxidation, which is increased after myocardial ischemia and during the subsequent reperfusion. 15-F(2t)-IsoP possesses potent bioactivity under pathophysiological conditions. However, it remains unknown whether 15-F(2t)-IsoP, by itself, can influence myocardial ischemia-reperfusion injury (IRI). Adult rat hearts were perfused by the Langendorff technique with Krebs-Henseleit (KH) solution at a constant flow rate of 10 ml/min. 15-F(2t)-IsoP (100 nM), SQ-29548 (1 microM, SQ), a thromboxane receptor antagonist that can abolish the vasoconstrictor effect of 15-F(2t)-IsoP, 15-F(2t)-IsoP + SQ in KH, or KH alone (vehicle control) was applied for 10 min before induction of 40 min of global ischemia followed by 60 min of reperfusion. During ischemia, saline (control), 15-F(2t)-IsoP, 15-F(2t)-IsoP + SQ, or SQ in saline was perfused through the aorta at 60 microl/min. 15-F(2t)-IsoP, 15-F(2t)-IsoP + SQ, or SQ in KH was infused during the first 15 min of reperfusion. Coronary effluent endothelin-1 concentrations were significantly higher in the group treated with 15-F(2t)-IsoP than in the control group during ischemia and also in the later phase of reperfusion (P < 0.05). Infusion of 15-F(2t)-IsoP increased release of cardiac-specific creatine kinase, reduced cardiac contractility during reperfusion, and increased myocardial infarct size relative to the control group. SQ abolished the deleterious effects of 15-F(2t)-IsoP. 15-F(2t)-IsoP exacerbates myocardial IRI and may, therefore, act as a mediator of IRI. 15-F(2t)-IsoP-induced endothelin-1 production during cardiac reperfusion may represent a mechanism underlying the deleterious actions of 15-F(2t)-IsoP.

Application of high-dose propofol during ischemia improves postischemic function of rat hearts: effects on tissue antioxidant capacity.

Previous studies have shown that reactive oxygen species mediated lipid peroxidation in patients undergoing cardiac surgery occurs primarily during cardiopulmonary bypass. We examined whether application of a high concentration of propofol during ischemia could effectively enhance postischemic myocardial functional recovery in the setting of global ischemia and reperfusion in an isolated heart preparation. Hearts were subjected to 40 min of global ischemia followed by 90 min of reperfusion. During ischemia, propofol (12 microg/mL in saline) was perfused through the aorta at 60 microL/min. We found that application of high-concentration propofol during ischemia combined with low-concentration propofol (1.2 microg/mL) administered before ischemia and during reperfusion significantly improved postischemic myocardial functional recovery without depressing cardiac mechanics before ischemia, as is seen when high-concentration propofol was applied prior to ischemia and during reperfusion. The functional enhancement is associated with increased heart tissue antioxidant capacity and reduced lipid peroxidation. We conclude that high-concentration propofol application during ischemia could be a potential therapeutic and anesthetic strategy for patients with preexisting myocardial dysfunction.

Protective role of heme oxygenase in the blood vessel wall during atherogenesis.

Several lines of evidence suggest that antioxidant processes and (or) endogenous antioxidants inhibit proatherogenic events in the blood vessel wall. Heme oxygenase (HO), which catabolizes heme to biliverdin, carbon monoxide, and catalytic iron, has been shown to have such antioxidative properties. The HO-1 isoform of heme oxygenase is ubiquitous and can be increased several fold by stimuli that induce cellular oxidative stress. Products of the HO reaction have important effects: carbon monoxide is a potent vasodilator, which is thought to play a role in modulation of vascular tone; biliverdin and its by-product bilirubin are potent antioxidants. Although HO induction results in an increase in catalytic free iron release, the enhancement of intracellular ferritin protein through HO-1 has been reported to decrease the cytotoxic effects of iron. Oxidized LDL has been shown to increase HO-1 expression in endothelial and smooth muscle cell cultures, and during atherogenesis. Further evidence of HO-1 expression associated with atherogenesis has been demonstrated in human, murine and rabbit atherosclerotic lesions. Moreover, genetic models of HO deficiency suggest that the actions of HO-1 are important in modulating the severity of atherosclerosis. Recent experiments in gene therapy using the HO gene suggest that interventions aimed at HO in the vessel wall could provide a novel therapeutic approach for the treatment or prevention of atherosclerotic disease.

Effects of oxidant-induced injury on heme oxygenase and glutathione in cultured aortic endothelial cells from atherosclerosis-susceptible and -resistant Japanese quail.

Recent studies on cultured aortic endothelial cells (AECs) from atherosclerosis-susceptible (SUS) and -resistant (RES) strains of Japanese quail suggest that differences in atherosclerosis susceptibility between RES and SUS may be due to differences in endothelial heme oxygenase (HO) and antioxidant components. We have now investigated the effects of oxidant-induced injury on HO and glutathione (GSH) in AECs from SUS and RES quail. We report that cultured AECs from SUS and RES birds differ in their response to oxidative stress. AECs from the SUS strain cells are more susceptible than those from the RES strain to oxidative stress induced by tert-butylhydroperoxide, as judged by lower HO activity, HO-1 expression, ferritin and GSH levels. Aortic endothelial cells from SUS birds also showed higher levels of catalytic iron, TBARS production and LDH release compared with RES cells, indicating that SUS AECs are more susceptible to oxidative stress than cells from the resistant strain. Furthermore, independently of genetic status, AECs from old birds have higher TBARS and lower levels of HSP70 induction than AECs from younger birds, suggesting that aging is associated with a decreased ability of AECs to respond to oxidative stress, and this may be relevant to the permissive effect of aging on the process of atherogenesis. Our results indicate that genetic factors and endogenous antioxidant systems in the blood vessel wall may be important in determining the susceptibility of vascular cells to oxidative stress and atherosclerotic plaque formation.

Alterations in aortic antioxidant components in an experimental model of atherosclerosis: a time-course study.

Antioxidant component alterations in the aorta during atherogenesis were examined in atherosclerosis-susceptible (SUS) Japanese quail fed a cholesterol-supplemented (0.5% w/w) diet. Birds fed a non-supplemented diet provided information on the effects of aging on endogenous antioxidants. One hundred adult SUS males were used. Birds were sacrificed after 0, 4, 8 and 12 weeks on the diets and were examined for plaque development and corresponding antioxidant component alterations in aorta and myocardium. With aging, superoxide dismutase (SOD) activity was increased in both tissues, whereas aortic glutathione peroxidase (GPx) activity and myocardial glutathione reductase (GRd) activity decreased. Myocardial ascorbate levels increased with aging, with a reciprocal decrease in myocardial tocopherol levels. Following 4 weeks of cholesterol supplementation, aortic GRd decreased, SOD activity increased, but activities of GPx and catalase were unchanged. This same qualitative pattern of antioxidant enzyme changes was also found in myocardium. Thus, although aortic antioxidant enzyme changes produced by cholesterol feeding and aging showed some similarities, the early phase of atherogenesis does not simply reflect accelerated aging. In the late stages of atherogenesis, SOD activity returned to baseline, but other antioxidant enzymes remained unaltered from levels characterizing the early phase of lesion development. There was no detectable functional coupling between changes in GPx and GRd, nor between SOD (which produces hydrogen peroxide) and GPx or catalase (which utilize hydrogen peroxide as substrate). Previously reported alterations in erythrocyte antioxidant enzyme components during atherogenesis in quail were not predictive of changes in the corresponding enzymes in the aorta and myocardium.

Heme oxygenase and antioxidant status in cultured aortic endothelial cells isolated from atherosclerosis-susceptible and -resistant Japanese quail.

We have investigated heme oxygenase (HO) and antioxidant status in the novel isolation and characterization of aortic endothelial cells (AECs) from a random bred wild-type strain (WILD) and selectively bred atherosclerosis-susceptible (SUS) and -resistant (RES) strains of Japanese quail. Cultured AECs expressed acetylated LDL, and were probed with endothelial and smooth muscle cell specific antibodies to confirm purity of culture. Subconfluent monolayers of RES AECs had higher HO activity than SUS AECs. At confluence, HO activity levels were similar among strains. However, RES AECs had higher HO-1 protein than WILD and SUS cells. Although ferritin protein levels were similar among the three strains, catalytic iron was higher in SUS AECs than WILD and RES cells. Glutathione levels were highest in SUS cells, intermediate in WILD, and lowest in RES, while glutathione reductase was higher in WILD and RES AECs than SUS AECs. We suggest that differences in atherosclerosis susceptibility between RES and SUS may be due, at least in part, to differences in endothelial HO and antioxidant components.

Propofol enhances ischemic tolerance of middle-aged rat hearts: effects on 15-F(2t)-isoprostane formation and tissue antioxidant capacity.

OBJECTIVE: Experimental study has shown that myocardial ischemic tolerance is reduced during middle-age. We investigated the effect of propofol on ischemic tolerance of middle-aged rat hearts. METHODS: Hearts of young adult (10 weeks old, Y) and middle-aged rats (20 weeks old, M) were assigned to propofol (P-Y, P-M) and control (C-Y, C-M) groups (n=6 each). Hearts were perfused using a Langendorff preparation with Krebs-Henseleit solution (KH) at constant flow rates. We applied propofol (P-Y, P-M) for 10 min at 12 microg/ml before inducing 40 min global ischemia. During ischemia, saline (C-Y, C-M) or propofol (P-Y, P-M) in saline was perfused through the aorta at 60 microl/min. Propofol in KH was perfused at 12 microg/ml for the first 15 min of reperfusion and subsequently reduced to 5 microg/ml in propofol treatment groups. Coronary effluent was assayed for 15-F(2t)-isoprostane after equilibration, during ischemia (T(1)) and at 0.5 (T(2)) and 5 (T(3)) min of reperfusion. After 90 min of reperfusion (T(4)), hearts were harvested to assess tissue antioxidant capacity. RESULTS: In P-Y, we observed an increased latency to ischemic-contracture and a significantly reduced contracture after 35 min ischemia compared to control groups. No ischemic contracture was observed in P-M. There were significantly lower 15-F(2t)-isoprostane levels in P-M and P-Y than in C-M and C-Y at T(1). At T(4), the recovery of left ventricular developed pressure in P-M was greater than in P-Y (P<0.05); both were greater than in C-M and C-Y. CONCLUSION: Propofol enhanced ischemic tolerance of middle-aged hearts, primarily by inhibiting lipid peroxidation.

Dose-dependent protection of cardiac function by propofol during ischemia and early reperfusion in rats: effects on 15-F2t-isoprostane formation.

We examined the effects of propofol (2,6-diisopropylphenol) on functional recovery and 15-F2t-isoprostane generation during ischemia-reperfusion in Langendorff-perfused rat hearts. Before the induction of 40 min of global ischemia, hearts were perfused (10 min) with propofol at 5 (lo-P) or 12 microg/mL (hi-P) in saline or with saline only (control). During ischemia, saline, lo-P, or hi-P was perfused through the aorta at 60 microL/min. During the first 15 min of reperfusion, propofol (5 or 12 microg/mL) was continued, followed by perfusion with 5 microg/mL propofol for 75 min in both propofol-treated groups. After 90 min of reperfusion (Rep-90), heart tissues were harvested for assessment of antioxidant status. In hi-P, we observed increased latency to and greater reduction of ischemic contracture relative to the lo-P or control groups. 15-F2t-Isoprostane concentrations increased during ischemia and were significantly lower in hi-P and lo-P than in control (P < 0.01). At Rep-90, myocardial functional recovery was greater in both propofol-treated groups relative to control, and it correlated positively with tissue antioxidant capacity preservation. Tissue antioxidant capacity was better preserved in hi-P than in lo-P treatment (P < 0.05). We conclude that oxidant injury occurs during ischemia and reperfusion, and propofol provides dose-dependent protection primarily by enhancing tissue antioxidant capacity and reducing lipid peroxidation.

Role of platelet activating factor in cardiac dysfunction, apoptosis and nitric oxide synthase mRNA expression in the ischemic-reperfused rabbit heart.

BACKGROUND: The role of platelet activating factor (PAF) and nitric oxide in myocardial ischemia-reperfusion (MIR) injury and the interrelationship of the two mediators is poorly understood. The contribution of PAF to apoptosis during MIR has not been studied. OBJECTIVES: To determine the contribution of PAF to ex vivo cardiac dysfunction during the initial 5 h of postischemia reperfusion, to determine the contribution of PAF to inducible nitric oxide synthase (NOS) and endothelial NOS mRNA expression during MIR, and to determine whether PAF contributes to apoptosis during MIR. METHODS: Isolated blood-perfused rabbit hearts underwent 30 min of global ischemia and 5 h reperfusion. Animals were divided into four groups, which received either PAF antagonist TCV-309 or vehicle before ischemia, or were sham operated (heart perfusion only), or were control (no heart perfusion). RESULTS: Administration of the PAF antagonist significantly improved myocardial contractility (614 mmHg/s versus 308 mmHg/s, positive dP/dt, P<0.0001) and coronary vascular flow rate (5.5 mL/min versus 3.9 mL/min, P<0.01) during reperfusion compared with untreated animals (values at 5 h reperfusion). Treatment with PAF antagonist significantly increased mRNA expression of endothelial NOS (2.8 versus 1.3 ratio, P<0.05) compared with the untreated group. PAF antagonist reduced procaspase-3 cleavage (66 versus 108 ratio, P<0.05) and DNA fragmentation (8.2 versus 11.0 positive cells per field) compared with untreated animals. CONCLUSIONS: PAF antagonism with TCV-309 protected against myocardial contractile depression and coronary vasoconstriction during the initial 5 h reperfusion. PAF may play a role in the regulation of endothelial NOS mRNA expression and contribute to apoptosis during ischemia-reperfusion in the heart.

Tissue antioxidant capacity during anesthesia: propofol enhances in vivo red cell and tissue antioxidant capacity in a rat model.

UNLABELLED: The effects of anesthesia on ischemia-reperfusion injury are of considerable scientific and clinical interest. We examined the effects of propofol (known to possess antioxidant activity) and halothane (devoid of antioxidant activity in vitro) on tissue and red blood cell (RBC) antioxidant capacity. Adult male Wistar rats were anesthetized with halothane 0.5%-1.0% (n = 7), propofol 500 microg x kg(-1) x min(-1) with halothane 0.25%-0.5% (small-dose propofol; n = 9), or propofol 2000 microg x kg(-1) x min(-1) (large-dose propofol; n = 8) for 45 min. Blood and tissue samples of liver, kidney, heart, and lung were then harvested for in vitro exposure to a peroxidizing agent. Red cell malondialdehyde and tissue thiobarbituric acid reactive substances were determined spectrophotometrically. Antioxidant capacities of blood and tissues in the Large-Dose Propofol group, and of blood and all tissues except lung in the Small-Dose Propofol group, were increased significantly compared with halothane (P < 0.003). The increases in tissue antioxidant capacities varied in their magnitude: RBC > liver > kidney > heart > lung. There was a high correlation between changes in RBC susceptibility to oxidative damage and corresponding changes in tissues. These findings demonstrate that large-dose propofol significantly enhances tissue antioxidant capacity, and RBC antioxidant capacity can serve as a functional measure of tissue activity, in vivo. IMPLICATIONS: We designed this study to investigate the antioxidant effects of propofol in various tissues in a rat model. Pretreatment of animals with propofol led to a reduction in the susceptibility to an in vitro oxidative stress of five different tissues investigated, demonstrating the drug's ability to limit oxidative injury. This may have future application in limiting organ dysfunction after periods of tissue ischemia (which results in oxidative damage).