Presence of autoantibodies against ß1-adrenoceptor aggravates the kidney injury in rats.

Since the autoantibodies against the second extracellular loop of ß(1)-adrenoceptor (ß(1)-AABs) have been found in the sera of patients with idiopathic dilated cardiomyopathy (IDCM), the involvement of autoimmune mechanisms in the pathogenesis of many cardiovascular diseases has extensively been investigated. Our previous study found that urinary occult blood and protein excretion were frequently found in the rats with positive ß(1)-AABs, but the mechanisms are unclear. Therefore, we infused the ß(1)-AABs into the vein periodically in an attempt to investigate whether ß(1)-AABs could induce morphological and functional changes in the kidneys of adult and aged rats and explore the possible mechanisms. The synthetic peptide according to the sequences of the second extracellular loop of ß(1)-adrenoceptor (ß(1)-AR-ECII) was used to immunize the adult rats to acquire enough ß(1)-AABs for use. Neonatal rat ventricular myocytes (NRVMs) culture was used to observe the biological effects of ß(1)-AABs on the beating rate. The purified ß(1)-AABs were transfused into the vein of rats. The sera level of blood urea nitrogen (BUN), creatinine (CR), uric acid (UA), urinary specific gravity, protein excretion, occult blood and urinary glucose were detected at the different time points by biochemistry and urine analyzers. HE and Masson's trichrome staining were used to detect the changes in kidney structure of passively immunized rats. Enhanced green fluorescent protein (EGFP) and ß(1)-AR-EGFP plasmids were transfected into the human embryonic kidney 293 (HEK293) cells in order to observe the changes in cell injury with the treatment of ß(1)-AABs. It was found that the sera level of BUN, CR and UA increased gradually and the ratio of BUN to CR decreased progressively with the administration of ß(1)-AABs. The increasing of proteinuria, urinary occult blood and urinary glucose was detected by urine analyzer in ß(1)-AABs group. By HE and Masson's coloration, lots of mononuclear cell infiltration and collagen fibers deposition could be observed at the 24th week of immunization. After the treatment of ß(1)-AABs, the caspase-3 activity increased significantly in the HEK293 cells transfected with ß(1)-AR-EGFP plasmids, while no significant changes were observed for lactate dehydrogenase (LDH) activity. The results indicate that long-term presence of ß(1)-AABs can induce the morphological and functional damage of the kidneys in adult and aged rats.

[The up-regulation of hypoxia inducible factor-1α by hypoxic postconditioning reduces hypoxia/reoxygenation-induced injury in heart-derived H9c2 cells].

The aim of the present study was to explore the effects of hypoxic postconditioning (PostC) on heart-derived H9c2 cells injury induced by hypoxia/reoxygenation (H/R) and the expression of hypoxia inducible factor-1α (HIF-1α), and to analyze the relationship between them. Cultured H9c2 cardiac muscle cells were subjected to 3-hour hypoxia and 2-hour reoxygenation to simulate ischemia and reperfusion, or underwent 3 cycles of 5-min reoxygenation and 5-min hypoxia preceding the long reoxygenation to simulate ischemic postconditioning. Cell viability, lactate dehydrogenase (LDH) activity, and caspase-3 activity were detected respectively to investigate the cell injury induced by H/R. The level of HIF-1α mRNA was measured by real-time PCR. Western blot was used to determine HIF-1α protein level. The results showed that postconditioning significantly increased H9c2 cell viability, reduced the activity of LDH and caspase-3. Simultaneously, postconditioning up-regulated the HIF-1α protein level. Moreover, after DMOG, an inhibitor of proline hydroxylase (PHD) which targeted to HIF-1α degradation, was used to stabilize HIF-1α protein level, the reduction of H9c2 cells injury was comparable to that by postconditioning. There was a significant linear positive relationship between HIF-1α protein level and cell viability (r = 0.743, P < 0.01). After HIF-1α gene was silenced by siRNA, the cardio-protective effects of postconditioning was significantly weakened. These data suggest that up-regulation of HIF-1α plays an important role in the cardio-protection of postconditioning.

[The attenuation of myocardial susceptibility to ischemia/reperfusion injury by ischemic postconditioning in hypercholesteremia rats and the role of hypoxia inducible factor-1alpha].

OBJECTIVE: To explore whether ischemic postconditioning can attenuate the myocardial injury induced by ischemia/reperfusion (I/R) in hypercholesteremic rats and whether hypoxia inducible factor-1alpha (HIF-1alpha) play a role in the protection. METHODS: Adult male Wistar rats received a high fat diet for 8 weeks to prepare the hypercholesteremic models. Myocardial damage induced by ischemia/reperfusion was evaluated by infarct size, creatine kinase (CK) activity and myocardial apoptosis. HIF-1alpha mRNA level was detected by real time-RT-PCR and the protein level was detected by Western blot. RESULTS: Myocardial infarct size, CK activity, and caspase-3 activity induced by I/R were markedly increased in hypercholesteremic rats compared with those in normal rats. Ischemic postconditioning attenuated the myocardial injury in both normal rats and hypercholesteremic rats, and increased HIF-1alpha protein level. There was a significant linear inverse relationship between HIF-1alpha protein level and infarct size (r = -0.802, P <0.01). CONCLUSION: Hypercholesteremia enhanced the susceptibility of myocardia to ischemia/reperfusion injury. While ischemic postconditioning markedly attenuated the increase of myocardial susceptibility to I/R induced by hypercholesteremia. HIF-1alpha might be one of the mechanisms of protection by ischemic postconditioning.

Attenuation of myocardial injury by postconditioning: role of hypoxia inducible factor-1alpha.

Postconditioning (PostC) has regenerated interest as a mechanical intervention against myocardial ischemia/reperfusion injury, but its molecular mechanisms remain elusive. This study tested the hypothesis that hypoxia inducible factor-1alpha (HIF-1alpha) plays a role in PostC-induced cardioprotection. Male Wistar rats were subjected to 30 min ischemia followed by 3 h of reperfusion (Control). PostC with 3 cycles of 10 s reperfusion and 10 s re-occlusion was applied at the onset of reperfusion. Relative to the Sham group, HIF-1alpha protein level was increased by 2.9-fold in the Control group, but its level was enhanced by 5.8-fold with PostC (P < 0.01 vs. Control). However, HIF-1alpha protein level was further augmented by 2.0-fold and 3.3-fold, respectively, when the prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, 40 mg/kg, i.p.) was given at 24 h before ischemia in both Control and PostC groups. PostC reduced infarct size by 24% compared with the Control (27 +/- 4.2% vs. 36 +/- 5.2%, P < 0.01), consistent with significant lower levels of plasma creatine kinase activity, index of cardiomyocyte apoptosis and caspase-3 activity. Although pretreatment with DMOG significantly reduced infarct size relative to the Control, the infarct-sparing effect of PostC was remarkably enhanced when DMOG was given before PostC (18 +/- 2.0% vs. 27 +/- 4.2% in PostC alone, P < 0.05). There was a significant linear inverse relationship between HIF-1alpha protein level and infarct size (r = -0.799, P < 0.01) among all groups. Furthermore, along with up-regulated HIF-1alpha expression, the levels of iNOS mRNA and protein were significantly increased in the PostC alone and DMOG plus PostC groups. In conclusion, these data suggest that HIF-1alpha is involved in cardioprotection by PostC and pharmacological augmentation of HIF-1alpha expression that enhances the infarct-sparing effect of PostC; iNOS, the downstream gene of HIF-1alpha, may participate in signaling pathways in mediating PostC's protection.

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.

Autoantibody against cardiac beta1-adrenoceptor induces apoptosis in cultured neonatal rat cardiomyocytes.

To clarify whether apoptosis is involved in the injury processes induced by autoantibody against cardiac beta1-adrenoceptor, we investigated the biological and apoptotic effects of antibodies on cultured neonatal rat cardiomyocytes. Wistar rats were immunized with peptides corresponding to the second extracellular loop of the beta1-adrenoceptor to induce the production of anti-beta1-adrenoceptor antibodies in the sera. Immunoglobulin (Ig) G in the sera was detected using synthetic antigen enzyme-linked immunosorbent assay and purified using the diethylaminoethyl cellulose ion exchange technique. Apoptosis of cardiomyocytes was evaluated using agarose gel electrophoresis and flow cytometry. Our results showed that the positive serum IgG greatly increased the beating rates of cardiomyocytes and showed an agonist-like activity. Furthermore, positive serum IgG induced cardiomyocyte apoptosis after treatment with beta1-adrenoceptor overstimulation for 48 h. The effects of monoclonal antibody against beta1-adrenoceptor were also found to be similar to those of positive serum IgG. It was suggested that the autoantibody could induce cardiomyocyte apoptosis by excessive stimulation of beta1-adrenoceptor.

Detection of serum autoantibodies against AT1A-receptor during the development of the four types of hypertensive rat models.

Using two-kidney one-clip renal hypertensive (2K1C group), stress-induced hypertensive (neural group), DOCA-salt treated hypertensive (DOCA group) and spontaneously hypertensive rats (SHR group), to investigate the change in AT(1A)-receptor autoantibodies (AT(1A)-AAs) during the development of the four types of hypertension. The biological activities of AT(1A)-AAs were examined. It was shown that the frequency of occurrence and titres of AT(1A)-AAs increased significantly during the development of hypertension. In the four hypertensive groups studied, the occurrence of AT(1A)-AAs was most prominent in SHR, 2K1C and neural groups. The biological effects of AT(1A)-AAs were shown to increase the beating frequency of cultured neonatal myocardial and vascular contractile tension. It is suggested that autoimmune mechanisms are involved the pathogenesis of different types of hypertension and the AT(1A)-AAs may be one of the mechanisms leading to cardiac hypertrophy.

Comparative studies of vasodilating effects of angiotensin-(1-7) on the different vessels.

The purpose of this study was to compare the vasodilating effects of angiotensin-(1-7) [Ang-(1-7)] on the different vessels and to clarify its mechanisms by using relaxing responses of preconstricted vascular rings. The results showed: (1) Ang-(1-7) dose-dependently induced vasorelaxation in all the vessels studied. However, there is apparent heterogeneity in the responsiveness of vessels from different origin. (2) The Ang-(1-7)-induced vasorelaxation was endothelium dependent and largely mediated by NO system. (3) The vasodilator action of Ang-(1-7) was not mediated by AT1 or AT2 receptor subtypes. It is suggested that the Ang-(1-7)-induced vasorelaxation is endothelium dependent by some other unclarified angiotensin receptor subtypes and is largely mediated by NO system.

Changes in autoantibodies against beta1-adrenoceptor and M2-muscarinic receptor during development of renovascular hypertension in rats.

In an experimental rat's renovascular hypertension model, we studied the genesis of anti-cardiac beta1-adrenoceptor and M2-muscarinic receptor autoantibodies in relation to the changes in immunological function during the development of renal hypertension. The biological activities of these autoantibodies were also examined. It was shown that after two weeks of operation both the frequency of occurrence and the titre of autoantibodies to cardiac beta1-adrenoceptor and M2-muscarinic receptor were significantly increased as compared with the control of pre-treatment. The increased autoantibodies lasted for several weeks and then automatically decreased gradually to the pre-clipping level at 10 weeks. Meanwhile the ratio of CD4+/CD8+ was also undergone an initial increase followed by gradual recovery and correlated well with the changes in antibody titre. The biological effects of these autoantibodies displayed an "gonistic-like" activities on the beating frequency of cultured neonatal cardiomyocyte. It is suggested that autoimmune mechanisms are involved in the pathogenesis of renal hypertension and the cardiac receptor autoantibodies might be one of the mechanisms leading to cardiac dysfunction.

[Effect of losartan on produce of sera autoantibodies to angiotensin II-1 receptor in renovascular hypertension rats].

AIM AND METHODS: The effects of losartan (after operation 2 week to 10 week, 5 mg/kg d ig) on generation of AT1R-AA in sera were observed during development of hypertension in rats. The renovascular hypertension (RVH) model was established by two-kidney one-clip method, a synthetic peptide corresponding to amino acid sequence 165-191 of the second extracellular loop of the angiotensin II-1 receptor (AT1R) was used as antigen, SA-ELISA were used to examine sera AT1R autoantibody (AT1R-AA). RESULTS: The frequencies and titres of AT1R-AA after operation one week rats were significantly increased (P < 0.05). The treatment with losartan not only inhibited structural and functional changes, but also the frequencies and titres of AT1R-AA was significantly lower (P < 0.05) than RVH group. CONCLUSION: It is suggested that the losartan significantly inhibits generation of the AT1R-AA.

[Changes in autoantibody against cardiovascular AT1-receptor during development of renovascular hypertension in rats].

The aim of this study was to observe the change in angiotensin II receptor subtype 1 (AT(1)) autoantibody during the development of renovascular hypertension (RVH). The Goldblatt renovascular hypertension model was established by the two-kidney one-clip method, and a synthetic peptide corresponding to amino acid sequence 165-191 of the second extracellular loop of the AT(1)-receptor was used as the antigen. Sera AT(1)-receptor autoantibody was detected by SA-ELISA. It was shown that two weeks after operation both the frequency of occurrence and the titre of autoantibodies to AT(1)-receptor were significantly increased as compared with the pre-treatment control. The increase in autoantibodies lasted several weeks and then decreased gradually to the pre-clipping level at 12 weeks. It is suggested that autoimmune mechanisms are involved in the pathogenesis of renovascular hypertension and the AT(1) autoantibodies may be one of the mechanisms leading to cardiac hypertrophy.

Relationship of myocardial remodeling to the genesis of serum autoantibodies to cardiac beta(1)-adrenoceptors and muscarinic type 2 acetylcholine receptors in rats.

OBJECTIVES: We sought to investigate the mechanism responsible for the occurrence of anticardiac receptor autoantibodies. BACKGROUND: Increasing evidence suggests the involvement of autoimmune mechanisms in the pathogenesis of a number of cardiovascular diseases. Among them, the biologic, functional and pathogenic properties of anticardiac receptor antibodies have been extensively investigated. However, the mechanism responsible for the occurrence of anticardiac receptor autoantibodies remains poorly understood. METHODS: Two rat models (aortic banding [AB] and adriamycin [ADR] groups) were constructed. Determination of cardiac function and morphology and T-lymphocyte subtypes, enzyme-linked immunosorbent assay and cardiomyocyte cultures were performed. RESULTS: It was shown, in the AB and ADR groups, that the frequency and titer of autoantibodies to beta(1) and muscarinic type 2 receptors were increased when myocardial remodeling occurred, as evidenced by significant cardiac morphologic changes, deposition of collagen and obvious functional impairment. This suggests that cardiac remodeling itself, in two disparate models of heart failure and cardiomyopathy, was able to trigger the genesis of anticardiac receptor autoantibodies. These autoantibodies have biologic effects similar to those seen in human autoantibodies. They have also shown a characteristic self-growth, as well as a time-course decline, suggesting that a negative finding of anticardiac receptor autoantibodies in sera of patients with heart disease does not necessarily imply that there is no autoimmune reaction involved in the pathogenesis. CONCLUSIONS: Our results demonstrated that myocardial damage was able to trigger the occurrence of an autoimmune reaction, resulting in the genesis of anticardiac receptor autoantibodies with properties similar to those seen in patients with idiopathic dilated cardiomyopathy.