Effects and molecular mechanisms of AT1-AA in retinopathy of preeclampsia.

Preeclampsia not only seriously endangers maternal and fetal health during pregnancy but may incur many sequelae in postpartum women such as reduced visual acuity. Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) is closely associated with preeclampsia. The aim of the present study is to determine whether AT1-AA is associated with retinal impairment during the course of preeclampsia. A preeclampsia model was established by injecting AT1-AA into pregnant rats via the tail vein. Changes in the retinal histological structure were observed. Cell apoptosis and cytokines including reactive oxygen species (ROS), as well as apoptosis-related proteins such as Bcl-2, Bax, and caspase-3 were detected. In addition, flash electroretinograms obtained at different postpartum days were analyzed. Compared with the control group, the retinal structure became edematous and the cell density was reduced significantly in preeclampsia group. The cell apoptosis rate was increased significantly. In addition, the content of ROS, the levels of Bax and caspase-3 in the retina were increased, while the content of Bcl-2 was reduced significantly. Continuous observation of the electroretinograms showed loss of retinal ganglion cells postpartum. The present study demonstrated that AT1-AA induced retinal cell apoptosis by promoting ROS release and activating caspase, suggesting that the increased postpartum susceptibility of preeclamptic women to retinopathy is related to AT1-AA-induced cell apoptosis.

Role and mechanism of AT1-AA in the pathogenesis of HELLP syndrome.

HELLP syndrome remains a leading cause of maternal and neonatal mortality and morbidity worldwide, which symptoms include hemolysis, elevated liver enzymes and low platelet count. The objective of this study was to determine whether HELLP is associated with AT1-AA. The positive rate and titer of AT1-AA in plasma from pregnant women were determined, and the correlation of AT1-AA titer with the grade of HELLP was analyzed. A HELLP rat model established by intravenous injection of AT1-AA. Our experimental results show the AT1-AA titer and positive rate were significantly higher in HELLP group, and AT1-AA titer were positively correlated with the level of TNF-α and ET-1 in plasma and the grade of HELLP syndrome. The results of animal experiments showed that the typical features of HELLP in the pregnant rats after AT1-AA injection. The levels of TNF-α and ET-1 in plasma and liver tissue were significantly increased in AT1-AA-treated rats compared with control rats. The HELLP syndrome induced by AT1-AA was attenuated markedly after administration of losartan. These data support the hypothesis that one the potential pathway that AT1-AA induce damage to capillary endothelial cells and liver during pregnancy is through activation of TNF-α and ET-1.

Role of agonistic autoantibodies against type-1 angiotensin II receptor in the pathogenesis of retinopathy in preeclampsia.

To investigate the mechanism underlying AT1-AA-induced retinopathy in severe preeclampsia by measuring the positive rate and titer of AT1-AA in plasma from women with severe preeclampsia and normal pregnant women to see whether AT1-AA titer was correlated with the grade of retinopathy. A preeclampsia rat model was also established by intravenous injection of AT1-AA extracted from the plasma of patient suffering from severe preeclampsia. The results showed that the plasma titer and positive rate of AT1-AA were significantly higher in women with severe preeclampsia than normal pregnant women. The antibody titer in cases of severe preeclampsia was associated with the grade of retinopathy, and positively correlated with the level of TNF-α and VEGF. The animal experiment results showed that the modeled rats presented symptoms very similar to symptoms of human preeclampsia, including retinopathy. Ocular fundus examination showed retinal microvascular abnormalities, hemorrhaging and leakage in the severe preeclampsia. Morphological changes included edema, thickening of the INL and ONL, and pigment atrophy. TNF-α and VEGF levels were increased in the vitreous humor and retina of the model rats. Our studies results suggest that abnormal expression of AT1-AA could induce damage to retinal capillary endothelial cells and increase vascular permeability, resulting in retinopathy.

Mechanism of agonistic angiotensin II type I receptor autoantibody-amplified contractile response to Ang II in the isolated rat thoracic aorta.

Agonistic autoantibody to the angiotensin II type I receptor (AT1-AA) is highly associated with preeclampsia by increasing the sensitivity of Ang II during pregnancy in rats, thus leading to a preeclampsia-like syndrome. However, the mechanism underlying this phenomenon remains unclear. The purpose of this study was to observe AT1-AA amplification of Ang II-induced vasoconstriction in rat thoracic aortic rings. It was found that exposure to low concentrations of AT1-AA (0.4 nM) caused a contraction of <5% of the maximal response to 60 mM KCl. In addition, the Ang II-induced contractile response was amplified in the presence of a threshold contraction to AT1-AA, as manifested by a leftward shift of the midpoint of the concentration-response curve with no change in the maximal response. These results showed that preincubation with low AT1-AA could amplify the Ang II dose-response curve, and this amplification could be attenuated markedly by 0.1 µM heptapeptide AFHYESQ. In calcium-free Krebs solution, 10 µM of 2-aminoethoxydiphenyl borate (an IP3 receptor inhibitor) both blocked the AT1-AA base contraction and completely abolished the amplification. Both 5 µM of U-73122 (a phospholipase C inhibitor) and 10 µM of εV1-2 (an εPKC inhibitor) could partially inhibit the Ang II-induced contractile response. εV1-2, but not U-73122, could completely inhibit the amplification response of AT1-AA to Ang II. These results suggest that AT1-AA is able to cause amplification response to Ang II probably via the calcium-independent protein kinase C pathway, which may provide a new therapy strategy for preeclampsia.

Exposure to AT1 receptor autoantibodies during pregnancy increases susceptibility of the maternal heart to postpartum ischemia-reperfusion injury in rats.

Epidemiological studies have demonstrated that women with a history of preeclampsia have a two-fold increased risk of developing cardiovascular diseases in later life. It is not known whether or not this risk is associated with angiotensin II receptor type 1 autoantibody (AT1-AA), an agonist acting via activation of AT1 receptor (AT1R), which is believed to be involved in the pathogenesis of preeclampsia. The objective of the present study was to confirm the hypothesis that AT1-AA exposure during pregnancy may change the maternal cardiac structure and increase the susceptibility of the postpartum heart to ischemia/reperfusion injury (IRI). In the present study, we first established a preeclampsia rat model by intravenous injection of AT1-AA extracted from the plasma of rats immunized with AT1R, observed the susceptibility of the postpartum maternal heart to IRI at 16 weeks postpartum using the Langendorff preparation, and examined the cardiac structure using light and transmission electron microscopy. The modeled animals presented with symptoms very similar to the clinical symptoms of human preeclampsia during pregnancy, including hypertension and proteinuria. The left ventricular weight (LVW) and left ventricular mass index (LVMI) in AT1-AA treatment group were significantly increased as compared with those of the control group (p < 0.01), although there was no significant difference in final weight between the two groups. AT1-AA acting on AT1R not only induced myocardial cell hypertrophy, mitochondrial swelling, cristae disorganization and collagen accumulation in the interstitium but affected the left ventricular (LV) function and delayed recovery from IRI. In contrast, co-treatment with AT1-AA + losartan completely blocked AT1-AA-induced changes in cardiac structure and function. These data indicate that the presence of AT1-AA during pregnancy was strongly associated with the markers of LV geometry changes and remodeling, and increased the cardiac susceptibility to IRI in later life of postpartum maternal rats.

Maternal treatment with agonistic autoantibodies against type-1 angiotensin II receptor in late pregnancy increases apoptosis of myocardial cells and myocardial susceptibility to ischemia-reperfusion injury in offspring rats.

Epidemiological studies have demonstrated that offspring born to mothers preeclampsia (PE) are at increased risk for developing cardiovascular diseases after birth, but the underlying mechanism is unknown. Angiotensin II receptor type 1 autoantibody (AT1-AA), an agonist acting via activation of the AT1 receptor, is believed to be involved in the pathogenesis of both PE and fetal growth restriction. The aim of the present study was to confirm the hypothesis that prenatal AT1-AA exposure increases the heart susceptibility to ischemia/reperfusion injury (IRI) in the offspring in an AT1-AA-induced animal model of PE, and determine whether or not the increase of maternal AT1-AA level is a factor contributing to sustained abnormalities of the heart structure during infancy. The hearts of 45-day-old offspring rats were studied using Langendorff preparation to determine the susceptibility of the heart to IRI. The results showed that the body weight of the maternal rats was not significantly different between the study and control groups, but the body weight of their offspring in AT1-AA group was decreased slightly at day 21 of gestational age, and at day 3 after birth. Although the heart weight index was not significantly affected at all ages examined, AT1-AA significantly increased the size of myocardial cells of the left ventricle (LV) at the age of 45 days. AT1-AA gained access to fetal circulation via the placenta and induced apoptosis of fetal myocardial cells. AT1-AA also significantly delayed recovery from IRI and affected the LV function of 45-day-old offspring. This was associated with a significant increase in IRI-induced LV myocardial infarct size. These results suggest that AT1-AA induced abnormal apoptosis of fetal myocardial cells during the fetal period and increased the cardiac susceptibility to IRI in adult offspring.

Antibodies against AT1 receptors are associated with vascular endothelial and smooth muscle function impairment: protective effects of hydroxysafflor yellow A.

Ample evidence has shown that autoantibodies against AT1 receptors (AT1-AA) are closely associated with human cardiovascular disease. The aim of this study was to investigate mechanisms underlying AT1-AA-induced vascular structural and functional impairments in the formation of hypertension, and explore ways for preventive treatment. We used synthetic peptide corresponding to the sequence of the second extracellular loop of the AT1 receptor (165-191) to immunize rats and establish an active immunization model. Part of the model received preventive therapy by losartan (20 mg/kg/day) and hyroxysafflor yellow A (HSYA) (10 mg/kg/day). The result show that systolic blood pressure (SBP) and heart rate (HR) of immunized rats was significantly higher, and closely correlated with the plasma AT1-Ab titer. The systolic response of thoracic aortic was increased, but diastolic effects were attenuated markedly. Histological observation showed that the thoracic aortic endothelium of the immunized rats became thinner or ruptured, inflammatory cell infiltration, medial smooth muscle cell proliferation and migration, the vascular wall became thicker. There was no significant difference in serum antibody titer between losartan and HSYA groups and the immunized group. The vascular structure and function were reversed, and plasma biochemical parameters were also improved significantly in the two treatment groups. These results suggest that AT1-Ab could induce injury to vascular endothelial cells, and proliferation of smooth muscle cells. These changes were involved in the formation of hypertension. Treatment with AT1 receptor antagonists and anti oxidative therapy could block the pathogenic effect of AT1-Ab on vascular endothelial and smooth muscle cells.

Possible arrhythmiogenic mechanism produced by ibuprofen.

AIM: The aim of the present study was to investigate the electrophysiological effect of ibuprofen on the cardiac action potentials (AP) and electrocardiograms (ECG), and to identify its arrhythmiogenic mechanism. METHODS: The intracellular microelectrode recording technique was employed to record the fast- and slowresponse AP in guinea pig papillary muscles. The cardiac responses of ibuprofen were monitored by ECG, both in in vivo and in vitro studies. RESULTS: The ECG recording revealed that ibuprofen could induce arrhythmias, both in vitro and in vivo. Fatal ventricular fibrillations are readily produced in in vitro experiments by ibuprofen. Our results show that ibuprofen could dose dependently shorten the duration of AP and the effective refractory period (ERP), and it could also decrease the maximum depolarization velocity of phase 0 (V(max)) in both the fast- and slow-response AP. The duration of the QRS complex wave (QRS duration) in ECG was prolonged. Although the heart rate was depressed by ibuprofen, the corrected QT interval duration (QTc) decreased. CONCLUSION: Ibuprofen could inhibit cardiac Na+ and Ca2+ channels as it slows V(max) in both fast- and slowresponse AP. Furthermore, ibuprofen shortens the ERP and decreases the excitation propagation within the heart, which might provide a substrate for an arrhythmiogenic re-entry circuit. Taken together, we conclude that ibuprofen, when used improperly, may impose a potential hazard in inducing cardiac arrhythmias in patients with existing heart diseases.

Voltage and ionic regulation of human serotonin transporter in Xenopus oocytes.

1. The serotoninergic system is known to be involved in the control of multiple behavioural and physiological functions. The serotonin (5-hydroxtryptamine; 5-HT) transporter (SERT), which controls the synaptic 5-HT concentration through re-uptake of this neurotransmitter into presynaptic terminals, has been a primary therapeutic target for various psychiatric and peripheral disorders. The aim of the present study was to identify the regulatory mechanism(s) of the human SERT (hSERT) in heterologously expressed oocytes. 2. The hSERT cRNA was transcribed in vitro and injected into Xenopus oocytes. The 5-HT-induced transporter currents were measured by voltage clamp. The effects of extracellular sodium or chloride were studied by replacement perfusion with tetramethylammonium-chloride (96 mmol/L) or sodium acetate (96 mmol/L). In addition, to alter the internal calcium concentration, CaCl2 (50 micromol/L) and inositol triphosphate (IP3; 50 micromol/L), with or without EGTA (2.5 mmol/L), were injected into oocytes. The specificity of 5-HT-sensitive currents was determined by the use of the SERT antagonist desipramine and niflumic acid to block background chloride currents. 3. The hSERT-expressing oocytes displayed voltage-dependent, 5-HT-induced currents that increased at negative potentials. Replacing extracellular sodium or chloride significantly decreased the hSERT currents by 89 and 45%, respectively (P < 0.05, n = 7 each). Injection of IP3 or CaCl2 increased the hSERT currents by approximately 65% (P < 0.05; n = 10 each) and the effect of IP3 was abolished by preinjection of EGTA. 4. These results demonstrate that hSERT activity is not only voltage dependent, but is also affected by intracellular calcium and extracellular sodium and chloride.

Reduced sinoatrial cAMP content plays a role in postnatal heart rate slowing in the rabbit.

1. Decreasing heart rate during development is known to be the result of parasympathetic nervous system maturation that depresses the pacemaker current (If) by acetylcholine (ACh). However, a direct effect of ACh on If has been ruled out and the involvement of other secondary messengers, such as cAMP, was verified in previous studies. Therefore, we hypothesized that reduced basal cAMP production in sinoatrial (SA) nodal cells may contribute to the slowing of heart rate after birth. 2. The electrocardiogram and heart rate variability (HRV) were documented and measured in vivo and in vitro (in isolated perfused Langendorff preparations) for rabbits aged 2, 4, 6, 8 and 12 weeks. Sinoatrial node action potential (AP) recording and perforated patch-clamp analyses were used to investigate the spontaneous depolarization rate and pacemaker If currents. Concentrations of cAMP in SA nodal tissues were determined by radioimmunoassay. Relative expression of adenylate cyclases (ADCY1, 5) and phosphodiesterases (PDE1A, 4A and 8A) were quantified by real-time reverse transcription-polymerase chain reaction. 3. Significantly reduced heart rate, but unchanged HRV, was observed in perfused hearts in the older age groups, accompanied with a slowed phase 4 spontaneous depolarization rate (90.5 +/- 4.7 vs 49.6 +/- 2.6 mV/s for 2 week vs 4 week hearts, respectively; n = 5; P < 0.05), a negative shift of the If threshold potential (-45.5 +/- 3.0 vs -51.1 +/- 6.0 mV for 2 week vs 4 week hearts, respectively; n = 9; P < 0.05) and decreasing basal levels of SA nodal cAMP (0.31 +/- 0.05 vs 0.025 +/- 0.002 micromol/L for 2 week vs 4 week hearts, respectively; n = 6; P < 0.05). Gene expression levels of PDE1A, 4A and 8A were increased in the 12 week group compared with the 2 week group 1.5-, 2- and 1.8-fold, respectively (P < 0.05), with little change in ADCY1 and 5. 4. These data suggest that, in addition to autonomic innervation, slowing of heart rate during postnatal maturation can be attributed to a negative shift of the If activation caused by diminished baseline cAMP content in SA nodal cells.

Electrophysiological effect of fluoxetine on Xenopus oocytes heterologously expressing human serotonin transporter.

AIM: To investigate the electrophysiological effect of fluoxetine on serotonin transporter. METHODS: A heterologous expression system was used to introduce human serotonin transporter (hSERT) into Xenopus oocytes. A 2-electrode voltage clamp technique was used to study the pharmacological properties of fluoxetine. RESULTS: hSERT-expressing oocytes were perfused with 10 micromol/L serotonin (5-HT) to induce hSERT-current. The 5-HT-induced hSERT currents were dose-dependently reversed by fluoxetine. The RC50 (concentration that achieved a 50% reversal) was approximately 3.12 micromol/L. Fluoxetine took more time to combine with hSERT than 5-HT did, and it was also slow to dissociate from hSERT. This long-lasting effect of fluoxetine affected normal 5-HT transport. Fluoxetine significantly prolonged the time constant for 5-HT-induced hSERT current. These results might be used to explain the long-lasting anti-anxiety effect of fluoxetine in clinical practice, because it increases the concentration of 5-HT in the synaptic cleft by its enduring suppression of the function of 5-HT transporters. CONCLUSION: Fluoxetine inhibits 5-HT reuptake by competing with 5-HT and changing the normal dynamics of hSERT.

Effect of mexiletine on long QT syndrome model.

AIM: To make a LQT3 model (one form of the long QT syndromes) and to investigate the effect of mexiletine on LQT3. METHODS: Sea anemone toxin (ATX II) was used to produce the LQT3 model. The Effect of mexiletine on LQT3 was performed on single Na channel, action potential, and electrocardiography in guinea pigs. RESULTS: With the binding of ATX II to the Na+ channels, the probability of being in the open state and the open time constant of single Na+ channel with long opening mode increased significantly. Action potential duration APD50, APD90, and the maximal upstroke velocity of phase 0 were increased by 25.8 %, 26.1 %, and 12 %, respectively. The QT interval and QTc, a rectified QT interval, increased by 12.8 % and 16.9 %. On the contrary, after application of mexiletine, the open probability of single Na+ channel was reduced greatly. In the presence of ATX II (40 nmol/L), mexiletine (1, 5, 15, 45, 70 micromol/L) shortened the APD50 by 0.5 %, 6.7 %, 14.4 %, 19.4 %, and 18.8 %, respectively, and decreased the APD90 and Vmax accordingly. In the experiments with ECG, mexiletine reversed the ATX II-produced prolongation effects on QTc in a dose-dependent manner. CONCLUSION: Mexiletine may be an effective drug in the treatment of LQT3.