Huirong Liu

Antioxidative, Antinitrative, and Vasculoprotective Effects of a Peroxisome Proliferator–Activated Receptor-γ Agonist in Hypercholesterolemia

Background—Peroxisome proliferator–activated receptor (PPAR) signaling pathways have been reported to exert anti-inflammatory effects and attenuate atherosclerosis formation. However, the mechanisms responsible for their anti-inflammatory and antiatherosclerotic effects remain largely unknown. The present study tested the hypothesis that a PPAR&ggr; agonist may exert significant endothelial protection by antioxidative and antinitrative effects. Methods and Results—Male New Zealand White rabbits were randomized to receive a normal (control) or a high-cholesterol diet and treated with vehicle or rosiglitazone (a PPAR&ggr; agonist) 3 mg · kg−1 · d−1 for 5 weeks beginning 3 weeks after the high-cholesterol diet. At the end of 8 weeks of a high-cholesterol diet, the rabbits were killed, and the carotid arteries were isolated. Bioactive nitric oxide was determined functionally (endothelium-dependent vasodilatation) and biochemically (the phosphorylation of vasodilator-stimulated phosphoprotein, or P-VASP). Vascular superoxide production, PPAR&ggr;, gp91phox, and inducible nitric oxide synthase (iNOS) expression, and vascular ONOO− formation were determined. Hypercholesterolemia caused severe endothelial dysfunction and reduced P-VASP, despite a marked increase in iNOS expression and total NOx production. Treatment with rosiglitazone enhanced PPAR&ggr; expression, improved endothelium-dependent vasodilatation, preserved P-VASP, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation. Conclusions—The PPAR&ggr; agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrative stresses. The endothelial protective effects of PPAR&ggr; agonists may reduce leukocyte accumulation in vascular walls and contribute to their antiatherosclerotic effect.

Role of Omi/HtrA2 in Apoptotic Cell Death After Myocardial Ischemia and Reperfusion

Background—Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death. However, the role of Omi/HtrA2 in the apoptotic cell death that occurs in vivo under pathological conditions remains unknown. The present study was designed to investigate whether Omi/HtrA2 plays an important role in postischemic myocardial apoptosis. Methods and Results—Male adult mice were subjected to 30 minutes of myocardial ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, 10 minutes before reperfusion. Myocardial ischemia/reperfusion significantly increased cytosolic Omi/HtrA2 content and markedly increased apoptosis. Treatment with ucf-101 exerted significant cardioprotective effects, as evidenced by less terminal dUTP nick end-labeling staining, a lower incidence of DNA ladder fragmentation, and smaller infarct size. Furthermore, treatment with ucf-101 before reperfusion attenuated X-linked inhibitor of apoptosis protein degradation and inhibited caspase-9 and caspase-3 activities. Conclusion—Taken together, these results demonstrate for the first time that ischemia/reperfusion results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it promotes cardiomyocyte apoptosis via a protease activity–dependent, caspase-mediated pathway.

Autoantibody against AT1 receptor from preeclamptic patients induces vasoconstriction through angiotensin receptor activation.

Preeclampsia is a serious pathologic complication during pregnancy, and its pathogenesis remains poorly understood. Recent studies have demonstrated that autoantibodies against angiotensin II type 1A receptor (AT1-AA) are present in women with preeclampsia. However, their role in the development of hypertension in preeclamptic patients has never been previously investigated. The present study was designed to determine whether AT1-AA isolated from the sera of preeclamptic patients causes vascular constriction and, if so, to further investigate the cellular receptors that mediate their vasoactivity. Blood samples were collected from 49 pregnant women (preeclampsia = 31, control = 18) and AT1-AA was detected using enzyme-linked immunosorbent assay. Vasoconstrictive effect of purified IgG from the sera of either preeclamptic patients or normal pregnant women was determined in isolated rat thoracic aorta, arteriae cerebri media and coronary artery. Compared with normal pregnant women, frequency of AT1-AA positive samples was markedly increased in preeclamptic patients (80.7 vs. 5.6%, P < 0.01). In isolated thoracic aortic rings, middle cerebral artery and coronary artery segments, AT1-AA induced vasoconstriction in a concentration-dependent fashion (P < 0.01). The vasoconstrictive effect of AT1-AA was completely blocked by losartan, an AT1-receptor antagonist. These data demonstrate that the AT1-AA causes significant vascular constriction in large conduit vessel as well as small resistant vessels though activation of the AT1 receptor. These results suggest that overproduction of AT1-AA is a novel risk factor in pregnant women and may play a causative role in the development of hypertension and vascular injury in preeclamptic patients.

Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion

Polymorphonuclear leukocyte (PMN) accumulation/activation has been implicated as a primary mechanism underlying MI/R injury. Recent studies have demonstrated that PMNs express inducible nitric oxide synthase (iNOS) and produce toxic reactive nitrogen species (RNS). However, the role of iNOS-derived reactive nitrogen species and resultant nitrative stress in PMN-induced cardiomyocyte apoptosis after MI/R remains unclear. Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. Animals were randomized to receive one of the following treatments: MI/R+vehicle; MI/R+L-arginine; PMN depletion followed by MI/R+vehicle; PMN depletion followed by MI/R+L-arginine; MI/R+1400 W; MI/R+1400 W+L-arginine and MI/R+ FeTMPyP. Ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis were determined. PMN depletion virtually abolished ischemia/reperfusion- induced PMN accumulation, attenuated ischemic/reperfusion-induced and L-arginine-enhanced nitrative stress, and reduced ischemic/reperfusion-induced and L-arginine-enhanced cardiomyocyte apoptosis (P values all <0.01). Pre-treatment with 1400 W, a highly selective iNOS inhibitor, had no effect on PMN accumulation in the ischemic/reperfused tissue. However, this treatment reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis to an extent that is comparable as that seen in PMN depletion group. Treatment with FeTMPyP, a peroxynitrite decomposition catalyst, had no effect on either PMN accumulation or total NO production. However, treatment with this ONOO− decomposition catalyst also reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis (P values all <0.01). These results demonstrated that ischemic/reperfusion stimulated PMN accumulation may result in cardiomyocyte injury by an iNOS-derived nitric oxide initiated and peroxynitrite-mediated mechanism. Therapeutic interventions that block PMN accumulation, inhibit iNOS activity or scavenge peroxynitrite may reduce nitrative stress and attenuate tissue injury.

Rosiglitazone inhibits hypercholesterolaemia-induced myeloperoxidase upregulation—a novel mechanism for the cardioprotective effects of PPAR agonists

AIMS Hypercholesterolaemia and myeloperoxidase (MPO) overexpression are two well-recognized risk factors for ischaemic heart disease. Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists have recently been shown to reduce ischaemic heart injury in hypercholesterolaemic animals. However, whether PPARgamma agonists may exert their cardioprotective effects by eliminating those risk factors that increase ischaemic injury remains unknown. METHODS AND RESULTS Male New Zealand rabbits were fed with a normal or a high-cholesterol diet for 8 weeks, treated with vehicle or rosiglitazone (RSG, 3 mg/kg/day for the last 5 weeks) and subjected to myocardial ischaemia/reperfusion (1 h/4 h). MPO expression, activity, and distribution, cardiac caspase-3 activity, and myocardial infarct size were determined. Diet-induced hypercholesterolaemia caused a significant increase in neutrophil MPO expression/activity (7.2-/5.4-fold). Hypercholesterolaemia also tripled MPO activity in ischaemic/reperfused hearts when compared with rabbits fed with a normal diet. Surprisingly, MPO immunostaining was not only observed in perivascular and extracellular spaces in ischaemic/reperfused hearts, but also in cardiomyocytes. This intracardiomyocyte MPO staining was further intensified by hypercholesterolaemia. There is a strong positive correlation between cardiac MPO activity and caspase-3 activity, and treatment with an MPO inhibitor significantly reduced post-ischaemic caspase-3 activation. Treatment with RSG markedly inhibited hypercholesterolaemia-induced leucocyte MPO overexpression and activation, reduced MPO activity in ischaemic/reperfused hearts, decreased caspase-3 activity, and reduced myocardial infarct size (P < 0.01). CONCLUSION Our results demonstrated that hypercholesterolaemia and MPO overexpression are causally related and that PPARgamma agonists may have great therapeutic value in ischaemic heart disease patients with multiple complications such as hypercholesterolaemia and diabetes.

Early anti-apoptosis treatment reduces myocardial infarct size after a prolonged reperfusion

Objective: Significant myocardial apoptosis occurs in ischemia/reperfused hearts. However, the contribution of apoptosis to the development of myocardial injury remains controversial. The present study attempted to obtain evidence that inhibition of apoptosis at early reperfusion can reduce myocardial infarction after prolonged reperfusion. Methods: Adult male rats were subjected to 30 min ischemia and 4 (apoptosis assay) or 24 h (myocardial infarction determination) of reperfusion and treated with vehicle, SB 239063, insulin or insulin plus wortmannin. Results: Treatment with SB 239063 or insulin markedly decreased myocardial apoptosis (10.6 ± 1.5% and 7.9 ± 0.9% respectively, P < 0.01 vs. vehicle) and significantly reduced infarct size (43 ± 3.6% and 35 ± 2.9%, respectively, P < 0.01 vs. vehicle). Most interestingly, inhibition of insulin signaling with wortmannin to block insulin signaling not only blocked insulins anti-apoptotic effect, but also abolished its infarct reduction property. Conclusion: These data indicate that apoptosis contributes to the development of myocardial infarction, and inhibition of apoptosis at early reperfusion reduces the myocardial infarction.

Long-term active immunization with a synthetic peptide corresponding to the second extracellular loop of β1-adrenoceptor induces both morphological and functional cardiomyopathic changes in rats

BACKGROUND β1-adrenoceptors (β1-ARs) are the predominant receptors in regulating heart functions. β1-ARs contain 7-transmembrane domains (7TM), 3 extracellular loops and 3 intracellular loops. Among these loops, the second extracellular loop of β1-AR (β1-AR-ECII) plays an important role in the pathogenesis of heart failure. METHODS (1) Select the sera-negative rats for antibodies against β1-AR-ECII. (2) Detect the level of antibodies against β1-AR-ECII in the process of active immunization with artificial synthetic peptides according to the sequence of human β1-AR-ECII. (3) Observe its long-term role on cardiac structure and function in vivo by immunizing rats. (4) Detect the changes of T lymphocytes subsets in the process of active immunization. RESULTS The peptides induced the production of specific autoantibody against β1-AR-ECII. Furthermore, immunization with β1-AR-ECII peptide induced both morphological and functional alterations in the hearts of rats, which potentially results in heart failure. In addition, induction of the autoantibodies against β1-AR-ECII increased the CD4+/CD8+ ratio in the peripheral blood of rats. DISCUSSION In this study, we determined the effect of anti-β1-AR-ECII autoantibody on morphological and functional cardiomyopathic alterations by immunization of rats with a synthetic peptide corresponding to the β1-AR-ECII for 18 months. These results provide further evidence that autoantibody against β1-AR-ECII is involved in the pathogenesis of heart failure. But the underlying mechanisms need further study.

Methylglyoxal increases cardiomyocyte ischemia-reperfusion injury via glycative inhibition of thioredoxin activity

Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality, but the specific molecular basis linking DM with increased vulnerability to cardiovascular injury remains incompletely understood. Methylglyoxal (MG), a precursor to advanced glycation end products (AGEs), is increased in diabetic patient plasma, but its role in diabetic cardiovascular complications is unclear. Thioredoxin (Trx), a cytoprotective molecule with antiapoptotic function, has been demonstrated to be vulnerable to glycative inhibition, but whether Trx is glycatively inhibited by MG, thus contributing to increased cardiac injury, has never been investigated. Cultured H9c2 cardiomyocytes were treated with MG (200 muM) for 6 days. The following were determined pre- and post-simulated ischemia-reperfusion (SI-R; 8 h of hypoxia followed by 3 h of reoxygenation): cardiomyocyte death/apoptosis, Trx expression and activity, AGE formation, Trx-apoptosis-regulating kinase-1 (Trx-ASK1) complex formation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity. Compared with vehicle, MG significantly increased SI-R-induced cardiomyocyte LDH release and apoptosis (P < 0.01). Prior to SI-R, Trx activity was reduced in MG-treated cells, but Trx expression was increased moderately. Moreover, Trx-ASK1 complex formation was reduced, and both p38 MAPK activity and phosphorylation were increased. To investigate the effects of MG on Trx directly, recombinant human Trx (hTrx) was incubated with MG in vitro. Compared with vehicle, MG incubation markedly increased CML formation (a glycation footprint) and inhibited Trx activity. Finally, glycation inhibitor aminoguanidine administration during MG treatment of cultured cells reduced AGE formation, increased Trx activity, restored Trx-ASK1 interaction, and reduced p38 MAPK phosphorylation and activity, caspase-3 activation, and LDH release (P < 0.01). We demonstrated for the first time that methylglyoxal sensitized cultured cardiomyocytes to SI-R injury by posttranslational modification of Trx via glycation. Therapeutic interventions scavenging AGE precursors may attenuate ischemic-reperfusion injury in hyperglycemic state diseases such as diabetes.

Inhibition of iNOS protects endothelial-dependent vasodilation in aged rats

AbstractAim:To examine whether iNOS contributes to endothelial dysfunction in aged rats.Methods:Male Sprague Dawley rats were divided into three groups: young rats, aged rats treated with vehicle and aged rats treated with N-[3-(Aminomethyl) benzyl] acetamidine (1400W, 1 mg/kg, ip). Vasorelaxation was measured in isolated thoracic aorta. iNOS expression of thoracic aortic arteries was detected using immunohistochemistry and Western blot. Nitrotyrosine (a marker for peroxynitrite formation) content and expression in thoracic aortic tissue were determined using enzyme linked immunosorbent assay and immunohistochemistry.Results:Maximal relaxation induced by acetylcholine (10-9 to 10-5 mol/L) in the aged rats treated with vehicle was significantly decreased (70%±15%, P<0.01), as compared with the young rats (95%±8%). However, the maximal relaxation induced by acidified NaNO2 (an endothelium-independent vasodilator) had no significant difference between the two groups. Moreover, iNOS and nitrotyrosine expression increased in the vessels of the aged rats. In the aged rats treated with 1400W (a highly selective iNOS inhibitor) nitrotyrosine expression was reduced and acetylcholine-induced vasorelaxation was markedly improved (maximal relaxation was increased to 87%±8%, P<0.05), but the acidified NaNO2-induced vasorelaxation had no significant change.Conclusion:Our study demonstrated that inhibition of iNOS by 1400W increased endothelium-dependent vasodilation in aged rats. The mechanism was related with attenuation of peroxynitrite formation.

Antiapoptotic mechanisms of benidipine in the ischemic/reperfused heart

Considerable evidence indicates that calcium plays a critical role in apoptosis. We have previously shown that benidipine, a vasodilatory calcium channel blocker, attenuates postischemia myocardial apoptosis. The present study was designed to determine the mechanisms by which benidipine exerts its antiapoptotic effect. Adult male rats were subjected to 30 min of ischemia followed by 3 h of reperfusion. Rats were randomized to receive either vehicle or benidipine (10 μg kg−1, i.v.) 10 min before reperfusion. Compared with rats receiving vehicle, those rats treated with benidipine had reduced postischemic myocardial apoptosis as evidenced by decreased TUNEL‐positive staining (8.4±1.2 vs 15.3±1.3%, P<0.01) and caspase‐3 activity (1.94±0.25 vs 3.43±0.29, P<0.01). Benidipine treatment significantly reduced mitochondrial cytochrome c release and caspase‐9 activation, but had no effect on caspase‐8 activation, suggesting that benidipine exerts its antiapoptotic effect by inhibiting the mitochondrial‐mediated, but not death receptor‐mediated, apoptotic pathway. Benidipine treatment not only increased the maximal activity of ERK1/2 at 10 min after reperfusion, but also prolonged the duration of ERK1/2 activation. Benidipine treatment had no significant effect on other apoptotic regulating molecules, such as p38 MAPK. Taken together, our present study demonstrated for the first time the differential regulation of a calcium channel blocker. Benidipine tilted the balance between ERK1/2 and p38 MAPK toward an antiapoptotic state, decreased mitochondrial cytochrome c release, reduced caspase‐9 activation, and attenuated subsequent caspase‐3 activation and postischemic myocardial apoptosis.