Increased AT(2)R protein expression but not increased apoptosis during cardioprotection induced by AT(1)R blockade

BACKGROUND: The angiotensin II type 2 receptor (AT2R) is considered to be antigrowth and to mediate apoptosis in several cell types. Whether AT2R upregulation, associated with angiotensin II type 1 receptor (AT1R) blockade and cardioprotection after ischemia-reperfusion (IR), might not result in increased cardiomyocyte (CM) apoptosis has not been documented. OBJECTIVES: To determine whether increased AT2R protein expression, during AT1R blockade after acute IR, is associated with no increase in CM apoptosis. MATERIALS AND METHODS: The recovery of left ventricular (LV) mechanical function after acute IR (30 min of ischemia, 40 min of reperfusion) was measured in isolated Langendorff rat hearts following pretreatment with the AT1R antagonist candesartan (CN) (CN 10 nmol/L) for 40 min before ischemia. The authors established with an initial dose-response curve using escalating concentrations of CN that 10 nmol/L abrogated vasoconstriction induced by angiotensin II (0.1 mol/L). AT1R and AT2R protein expression (Western immunoblot), CM apoptosis (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labelling assay and nuclear morphology) and apoptotic markers (Bax, Bcl-2, caspase-3, p53) were assessed in LV tissue. RESULTS: Compared with IR controls, CN improved peak systolic pressure, LV developed pressure and positive dp/dt, and increased AT2R (not AT1R) protein, but did not change the level of apoptosis or the expression of Bax, Bcl-2, caspase-3 or p53. CN also increased AT2R protein after ischemia alone but did not change CM apoptosis or expression of the markers. CONCLUSIONS: Increased AT2R protein expression during AT1R blockade after IR in the isolated Langendorff rat heart is associated with cardioprotection but no increase in CM apoptosis.     

Enhanced expression of angiotensin II type 2 receptor, inositol 1,4, 5-trisphosphate receptor, and protein kinase cepsilon during cardioprotection induced by angiotensin II type 2 receptor blockade

We hypothesized that the cardioprotective effect of angiotensin II type 2 receptor (AT(2)R) blockade with PD 123,319 (PD) on the recovery of left ventricular (LV) mechanical function after ischemia/reperfusion (IR) in the isolated working rat heart is associated with the enhanced expression of AT(2)R protein and mRNA as well as an increase in inositol 1,4,5-trisphosphate type 2 receptor (IP(3)R) and protein kinase Cepsilon (PKCepsilon) proteins. We assessed AT(2)R, angiotensin II type 1 receptor (AT(1)R), IP(3)R, and PKCepsilon protein expression (Western blots) and AT(2)R mRNA levels (Northern blots) in myocardium from isolated working rat hearts that were subjected to global ischemia (30 minutes) followed by reperfusion (30 minutes). Groups of adult rat hearts (n=6) were exposed to no IR, no IR+PD (0.3 micromol/L), IR, and IR+PD. Compared with no IR and no IR+PD, IR decreased (P<0.05) functional recovery and AT(2)R mRNA and protein, as well as AT(1)R mRNA (not protein) and IP(3)R and PKCepsilon proteins. Compared with IR, PD+IR improved LV functional recovery (P<0.05) and markedly increased AT(2)R mRNA and protein (P<0.001). However, PD did not change AT(1)R mRNA or protein. More importantly, PD+IR markedly increased IP(3)R and PKCepsilon proteins. The downregulation of AT(2)R mRNA and protein with IR and their upregulation with PD indicate that the effects of PD are AT(2)R specific. The overall results suggest that the cardioprotective effect of acute PD treatment on LV functional recovery after IR in the isolated working rat heart is specifically due to AT(2)R blockade and is associated with enhanced downstream IP(3)R and PKCepsilon signaling.     

AT1 receptor blockade prevents microvascular dysfunction induced by ischemia/reperfusion injury

Ischemia/reperfusion (I/R) in post-arterior post-capillary venules induces an acute inflammatory response, characterized by increased adherence and emigration of leukocytes and vascular permeability, all of which play important roles in cardiovascular disease. The aim of this study was to determine the roles of angiotensin II and AT1 receptor blockade in microvascular I/R injury in rats. Rats were anesthetized and intubated, then the peritoneum was opened and the mesentery was revealed. Small post-capillary venules were examined by in vivo fluorescence microscopy. The flow of erythrocytes and leukocytes was observed under the microscope and video recorded for later dynamic analyses. The superior mesenteric artery (SMA) was ligated with polyethylene tubing and released to induce I/R (20 min of ischemia/60 min of reperfusion). Subsequently, leukocyte adhesion, emigration and albumin leakage were compared with those of non-I/R controls. I/R injury was significantly suppressed by superfusing tissues with the AT1 receptor antagonist losartan (LO; 10 microM). The beneficial effects of LO were inhibited by topical application of either the bradykinin B2 receptor antagonist HOE140 (10 nM) or nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME 10 microM). The effects of LO were lost in the presence of AT2 receptor blocker PD 123319 (PD).In conclusion, LO suppressed and protected against I/R injuries. The possible interaction between AT1 and AT2 receptors was also suggested.     

超速起搏预适应的延迟保护与热休克蛋白70的表达

目的观察在超速心室起搏（ventricular overdrive pacing,VOP）预适应延迟保护阶段热休克蛋白70（HSP70）的表达水平。方法新西兰兔24只,随机分为3组,单纯结扎组,起搏组,起搏+放线菌素D组。制作超速起搏预适应和缺血/再灌注的动物模型,检测CK和CK-MB的变化,动态描记再灌注时心电图,免疫组织化染色检测HSP70抗原。结果缺血后起搏组心肌酶的水平在再灌注时均低于单纯结扎组和起搏+放线菌素D组（P<0.01）;单纯结扎组中在再灌注过程中共有5只发生心律失常,起搏+放线菌素组也有4只,而起搏组无心律失常发生。起搏组和单纯结扎组之间有显著性差别（P<0.05）。起搏组HSP70的阳性表达的程度明显高于其他两组。结论超速起搏预适应可以模拟缺血预适应,其延迟保护作用可能与HSP70表达增加密切相关。     

超速起搏预适应的延迟保护与热休克蛋白27的表达

目的：观察超速心室起搏（VOP）预适应延迟保护阶段热休克蛋白27（HSP27）的表达水平。方法：新西兰兔24只,随机分为3组,缺血再灌注（I/R）空白对照组,起搏组,起搏＋放线菌素D组。制作I/R和超速起搏预适应的动物模型,检测肌酸激酶（CK）和肌酸激酶-同工酶（CK-MB）水平的变化,动态描记再灌注时心电图,以免疫组织化染色法检测HSP27抗原表达水平。结果：I/R后起搏组心肌酶的水平均明显低于I/R空白对照组和起搏＋放线菌素D组（P〈0.01）;I/R空白对照组在再灌注过程中共有5只（62.5%）发生心律失常,起搏＋放线菌素D组有4只（50.0%）,而起搏组无心律失常发生。起搏组心律失常发生率明显低于I/R空白对照组和起搏＋放线菌素D组（P〈0.05）。起搏组HSP27阳性表达＋＋者（37.5%）明显高于I/R空白对照组和起搏＋放线菌素D组（0,12.5%,P〈0.05）。结论：心室超速起搏预适应有心脏保护作用,其机制可能与热休克蛋白27表达增加密切相关。     

Angiotensin AT2 receptor contributes to cardiovascular remodelling of aged rats during chronic AT1 receptor blockade

Ang II acting at AT(1)Rs has well documented effects on cardiovascular structure such as the promotion of cardiovascular hypertrophy and fibrosis, effects which are believed to be opposed by AT(2)R stimulation. AT(1) and AT(2)R expression are up regulated in senescent hearts, and other components of the local renin-angiotensin system are also dramatically increased in the ageing heart. Therefore, the aim of this study was to determine the role of the AT(2)R in aged rats by determining their potential contribution to the chronic antihypertensive and cardiovascular effects of AT(1)R blockade. Radiotelemetry probes were implanted into senescent (20 months) male Wistar-Kyoto (WKY) rats, and baseline recordings of mean arterial pressure (MAP) were made for 1 week. Candesartan cilexetil (2 mg/kg per day) was given in drinking water, while an additional group simultaneously received the AT(2)R antagonist, PD123319 (10 mg/kg per day) via osmotic mini-pump. At the end of the 4 weeks treatment period, animals were perfusion-fixed to enable histological analysis of cardiovascular structure. MAP was decreased by candesartan cilexetil, however, this effect was not further influenced by PD123319. Cardiac hypertrophy and fibrosis, and aortic hypertrophy were all significantly reduced by candesartan cilexetil. Most interestingly, these structural changes were reversed by concomitant PD123319 administration, despite the lack of AT(2)R-mediated effects on MAP. These results suggest that the AT(2)R does not exert a significant influence on chronic blood pressure regulation in senescent rats. However, PD123319 did reverse AT(1)R-mediated regression of cardiovascular hypertrophy and fibrosis, highlighting the important role of the AT(2)R on cardiovascular structure in the ageing heart and vasculature.     

AT2 receptor-mediated relaxation is preserved after long-term AT1 receptor blockade

Angiotensin II type 2 receptor (AT2R) stimulation may cause vasodilation per se and may contribute to the antihypertensive effect produced by Angiotensin II type 1 receptor (AT1R) antagonists, given that AT1R blockade increases endogenous levels of Ang II, suggesting a physiological role for the unblocked AT2R. Thus, we first directly assessed whether or not there is desensitization to AT2R-mediated vasorelaxation because this is an important consideration, given the raised Ang II levels and the marked desensitization that is known to occur after AT1R stimulation. Second, we examined if AT2R-mediated vasorelaxation is preserved after long-term treatment with the AT1R antagonist candesartan cilexetil. Consecutive concentration-response curves to AT2R stimulation, with either Ang II (with AT1R blockade) or the selective agonist CGP42112, were studied in rat isolated mesenteric resistance arteries mounted in an arteriograph. AT2R stimulation with Ang II induced a concentration-dependent relaxation without desensitization. Similarly, CGP42112 evoked highly reproducible relaxation, which, like Ang II, was abolished by the AT2R antagonist PD123319. By contrast, AT1R-mediated contraction exhibited marked desensitization. In rats treated with candesartan cilexetil (2 mg/kg per day for 2 weeks), AT1R-mediated contraction was abolished, whereas AT2R-mediated relaxation evoked by either Ang II or CGP42112 was highly reproducible, PD123319-sensitive, and of a magnitude similar to that observed in na?ve animals. Therefore, this study has provided unequivocal evidence for the reproducible nature of AT2R-mediated vasorelaxation during short-term and long-term AT1R blockade. Such preservation of AT2R function is a prerequisite for the consideration of physiological role(s) of AT2R during AT1R blockade.     

Denervation stimulates apoptosis but not Id2 expression in hindlimb muscles of aged rats

Inhibitors of differentiation (Id) proteins are repressors of myogenic regulatory factors and have been implicated in apoptosis and muscle atrophy during aging. Indeed, we have previously found that Id levels are elevated in muscles from old rodents, possibly as a consequence of loss of alpha-motoneurons during senescence. To determine if Id2 proteins increase after denervation and if this is accompanied by increased apoptosis in aged as compared with adult animals, the gastrocnemius and soleus muscles were denervated in 1 limb of Fischer 344 x Brown Norway rats aged 9 months (adult, n = 12) and 33 months (aged, n = 9), while the contralateral limb served as the intra-animal control. After 14 days, the muscles in each limb were removed. The levels of Id1, Id2, and Id3 mRNA and protein were significantly greater in muscles of old as compared with young adult rats. Denervation, however, did not significantly increase Id1, Id2, and Id3 mRNA in soleus or gastrocnemius muscles from either young or old rats. Also Id2 protein levels were similar in denervated and control muscles from young adult and old rats. In young adult rats only, denervation induced an increase in Id1 and Id3 protein levels in both the soleus (Id1 113%; Id3 900%) and gastrocnemius (Id1 86%; Id3 80%). Denervation induced a significant increase in caspase 8 in both soleus and gastrocnemius muscles from young (101% and 147%, respectively) and old rats (167% and 190%, respectively). Bax protein levels, as estimated by western blots, increased by 726% and 1087% after denervation in the soleus and by 368% and 49% in the gastrocnemius muscles of young and old rats, respectively. The data suggest that the denervation-induced muscle loss was at least partly due to apoptosis as indicated by elevated caspase 8 and Bax levels in denervated muscles. While Id2 may have a role in aging-induced sarcopenia, Id2 does not appear to directly regulate apoptosis during denervation. The elevated Id expression in muscles from aged animals is therefore not a direct consequence of loss of alpha-motoneurons during senescence.     

AT1-receptor blockade with irbesartan improves peripheral but not coronary endothelial dysfunction in patients with stable coronary artery disease

Activation of the renin-angiotensin-aldosterone system plays an important role in the pathogenesis of endothelial dysfunction and atherosclerosis. Studies evaluating the effect of AT1-receptor blockers on endothelial dysfunction in patients with coronary artery disease (CAD) revealed mixed results. Studies addressing the effects of AT1-receptor blockers on the coronary and peripheral function in the same study population, are still lacking. We therefore aimed to test the effects of long-term therapy with the AT1-receptor blocker irbesartan (IRB) on both, the coronary and peripheral endothelial function in patients with CAD. Seventy-two patients with CAD were randomly assigned to double-blinded treatment for 6 months with IRB 300 mg per day or placebo, respectively. Coronary and peripheral endothelial function were measured by intracoronary infusion of acetylcholine (final intracoronary concentration 10(-7.3) to 10(-5.6)M) and by determining flow-dependent dilation (FMD) of the brachial artery, respectively. IRB significantly improved FMD, while no change of coronary endothelial function was observed. Interestingly, plasma levels of N(G),N(G)-dimethyl-arginine, and the isoprostane excretion rate were not modified. IRB treatment improves peripheral but not coronary endothelial dysfunction in patients with CAD. Since reduced FMD of the brachial artery has been shown to be associated with a high-cardiovascular event rate, improvement of FMD by IRB may lead to better prognosis of patients with CAD.     

Increased vascular expression of iNOS at day but not at night in asthmatic subjects with increased nocturnal airway obstruction.

Nitric oxide production by endothelial cells may have important consequences for the development of airway inflammation as well as for airway obstruction. The present study investigated whether the expression of vascular inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in human bronchi differs between asthmatic and healthy subjects, and whether it shows a circadian rhythm, especially in subjects with increased nocturnal airway obstruction. Bronchial biopsy samples were taken at 16:00 and 04:00 h from 13 healthy and 25 asthmatic subjects, 18-45 yrs. Biopsy samples were snap-frozen and double-immunostained for iNOS and eNOS in combination with a common vascular antigen (CD31). The degree of immunopositivity was expressed as a percentage of CD31-positive vessels encountered in complete biopsy sections. Asthmatic subjects showed greater iNOS expression than healthy controls: 23+/-15 versus 7+/-17% (mean+/-SD) at 16:00 h (p<0.001) and 19+/-15 versus 8+/-11% at 04:00 h (p<0.05). Asthmatic subjects with a fall in forced expiratory volume in one second of >10% of the predicted value between 16:00 and 04:00 h showed greater iNOS expression at 16:00 than at 04:00 h: 32+/-16 versus 20+/-13% (p<0.05). eNOS expression did not differ between healthy controls and asthmatic patients, nor did it differ between 16:00 and 04:00 h. It is suggested that asthmatic subjects with increased nocturnal airway obstruction demonstrate increased activation of inducible nitric oxide synthase during the day. The resulting nitric oxide production might protect against airway obstruction during the day. However, at night, nitric oxide production is probably insufficient to counterbalance the bronchoconstricting forces.     

Increased expression of uncoupling protein 2 in HepG2 cells attenuates oxidative damage and apoptosis.

INTRODUCTION: Oxidative damage plays a major part in the pathogenesis of liver disease. Uncoupling proteins (UCPs) may be able to limit the generation of reactive oxygen species (ROS) and be cytoprotective. METHODS: We investigated the effect of up-regulation of UCP2 in a hepatoblastoma cell line exposed to menadione or hypoxia/re-oxygenation. RESULTS: Lipid and protein oxidation was increased in HepG2 cells exposed to ROS but this increase was significantly lower in cells over-expressing UCP2 under identical conditions. LDH release increased 2.5-fold in response to hypoxia/re-oxygenation in control HepG2 cells with no significant increase in UCP2 transfected cells. Hypoxia/re-oxygenation resulted in a reduction in liver-specific protein secretion that was attenuated in transfected cells and UCP2 over-expression also resulted in a 66% reduction in apoptosis compared with non-transfected controls. CONCLUSIONS: These data suggest that UCP2 can limit oxidative damage in HepG2 cells in response to oxidative stress resulting in improved cell function and resistance to apoptosis.     

Cough induced by enalapril but not by captopril

We report a 68 yr old woman with hypertension who developed a dry cough on enalapril but not on captopril therapy. Pulmonary function tests, methacholine inhalation challenges, total blood eosinophil counts, and changes in plasma concentrations of prostaglandin E2 and thromboxane B2 did not explain the difference in the adverse reaction between these two angiotensin converting enzyme inhibitors.     

Dysregulated bcl-2 expression inhibits apoptosis but not differentiation of retinoic acid-induced HL-60 granulocytes

The bcl-2-proto-oncogene appears to contribute to the development of certain malignancies by inhibiting programmed cell death (apoptosis). Mature granulocytes show a markedly limited life span and rapidly undergo apoptosis. To further define the relationship between apoptosis and granulocyte differentiation, we used retroviral vector-mediated gene transduction to introduce the normal bcl-2 gene into the HL-60 myeloid leukemia cell line and determined the response of these bcl-2- transduced HL-60 cells to the induction of granulocyte differentiation by retinoic acid (RA). Although the bcl-2-transduced HL-60 cells showed the same differentiative response to RA as did the parental HL-60 cells, the life span of the RA-induced, bcl-2-transduced HL-60 granulocytes was markedly prolonged compared with that of the RA- induced parental HL-60 granulocytes. DNA fragmentation studies indicate that this prolonged life span resulted from diminished apoptosis in the bcl-2-transduced cells. These studies indicate that bcl-2 is involved in regulating apoptosis in maturing granulocytes. Because bcl-2 over- expression did not interfere with RA-induced granulocyte differentiation, it appears that granulocyte differentiation and apoptosis are under distinct and separate regulatory controls.