[Kv1.3 potassium channel expression changes after CD4(+) and subsets CD28(null)/CD28(+)T cells activation in peripheral blood of patients with acute coronary syndrome].

OBJECTIVE: To study the Kv1.3 channel expression changes after CD4(+) and subsets CD28(null)/CD28(+)T cells activation in peripheral blood of patients with acute coronary syndrome (ACS). METHODS: CD4(+)T cell in 27 ACS patients and CD4(+)CD28(null)/CD4(+)CD28(+)T cells in 12 out of these 27 ACS patients were isolated from peripheral blood with magnetic cell sorting. The whole-cell Kv1.3 currents for three T cells were recorded with patch-clamp technique before and 72 hours after activation by purified anti-human CD3 Interferon gamma, tumor necrosis factor alpha (TNF-alpha), granzyme B mRNA expression were determined by reverse transcription-PCR before and 72 hours after activation by purified anti-human CD3 in the presence or absence of recombinant Margatoxin (rMgTX, 0.1, 1, 10 nmol/L), a specific Kv1.3 channel blocker. RESULTS: Peak Kv1.3 channel currents of CD4(+), CD4(+)CD28(null), CD4(+)CD28(+)T cells were significantly increased and the mean Kv1.3 channel numbers per cell of these cells were increased by about 90%, 60%, 80% (402 +/- 88 vs. 752 +/- 275, 553 +/- 328 vs. 874 +/- 400, 392 +/- 133 vs. 716 +/- 251, all P < 0.05) after activation compared to baseline values. Baseline CD4(+)CD28(null)T cell numbers were about 40% more than those of CD4(+)CD28(+)T cell (P < 0.05) and were similar after activation (P = 0.102). The mRNA expression of interferon gamma, TNF-alpha and granzyme B were dose-dependently down-regulated by rMgTX. CONCLUSIONS: Kv1.3 channels of peripheral CD4(+)T cell and CD28(null)/CD28(+)T cells from ACS patients significantly increased after activation and Kv1.3-specific channel blocker rMgTX could effectively abolish this effect suggesting a potential role of Kv1.3 channel blocker on plaque stabilization in ACS patients.

[Erythropoietin inhibits angiotensin II induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway].

OBJECTIVE: To explore the effect of erythropoietin (EPO) on angiotensin II (AngII) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. METHODS: Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by AngII in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. RESULTS: EPO (20 U/ml) significantly inhibited AngII induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P < 0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P < 0.05), increased the NO production (P < 0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P < 0.05). CONCLUSION: EPO attenuates AngII induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin-II in vitro.

OBJECTIVE: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin-II (Ang-II)-induced hypertrophy in cultured neonatal rat cardiomyocytes. MATERIAL AND METHODS: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang-II-induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor L-NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor-beta1 (TGF-beta1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. RESULTS: The results indicate that EPO significantly attenuates Ang-II-induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO-treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or L-NAME partially abolished the anti-hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down-regulated the protein expression of TGF-beta1. CONCLUSION: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K-Akt-eNOS-NO pathway and the down-regulation of TGF-beta1.

[Psychological stress status in patients with acute coronary syndrome and stable angina pectoris].

OBJECTIVE: To observe the psychological stress status in patients with acute coronary syndrome (ACS) and stable angina pectoris (SA). METHODS: The intensity of social psychological stress and the serum levels of IL-6, CRP and ICAM-1 were determined in patients with ACS (n = 67) and SA (n = 33). RESULTS: (1) The percentage of patients with psychological stress was significantly higher in ACS than that in SA group (78.8% vs. 21.2%, P < 0.01). (2) The serum levels of CRP [(14.82 +/- 5.07) g/L vs. (8.78 +/- 4.34) g/L], IL-6 [(101.7 +/- 22.2) ng/L vs. (71.1 +/- 23.5) ng/L] and sICAM-1 [(1.41 +/- 0.47) mg/L vs. (0.82 +/- 0.37) mg/L] were significantly higher in psychological stress group than those in non-psychological stress group (all P < 0.05). Serum CRP [(18.91 +/- 3.12) g/L vs. (6.20 +/- 2.46) g/L], IL-6 [(114.6 +/- 15.2) ng/L vs. (56.4 +/- 15.8) ng/L] and sICAM-1 [(1.67 +/- 0.39) mg/L vs. (0.63 +/- 0.28) mg/L] levels in ACS group were significantly higher than those in SA group (all P < 0.01). CONCLUSION: Higher psychological stress was associated with higher risk of ACS and increased serum inflammatory cytokines.

[Erythropoietin suppresses the expressions of TGF-beta1 and collagen in rat cardiac fibroblasts induced by angiotensin II].

OBJECTIVE: Recent studies have shown cardiac protection effects of erythropoietin (EPO). The present experiment was designed to investigate the effects of EPO on TGF-beta1, nitric oxide synthase (NOS), collagen contents induced by angiotensin II (Ang II) in rat cardiac fibroblasts (CFs) and explore the roles of PI3-K/Akt signaling pathway on related effects. METHODS: Neonatal rat CFs was isolated by collagenase and trypsinase digestion methods. PBS, EPO, Ang II in the absence or presence of LY294002, an inhibitor of PI3-K, or L-NAME, an inhibitor of NOS, were added to CFs and cultured for 24 hours. The concentration of collagen I and collagen III in culture medium were quantitated by ELISA. The levels of nitric oxide (NO) and the activities of NOS as well as NOS isoforms were measured by chemical enzymic method. Western blot was applied to detecting the protein expressions of Akt, p-Akt, eNOS, iNOS, and TGF-beta1. RESULTS: The concentrations of collagen I and collagen III in CFs culture medium were significantly increased while the level of NO was significantly decreased by Ang II and these changes were significantly suppressed by EPO in a dose dependent manner. The effects of EPO on eNOS and NO could be blocked by LY294002. L-NAME could block EPO's effect on NO but not on the eNOS expression. The suppression effects on expressions of TGF-beta1 and collagen by Ang II in CFs were blocked by both LY294002 and L-NAME. CONCLUSION: EPO suppresses the upregulated expressions of TGF-beta1 and increased production of collagen induced by Ang II through activating the PI3-K/Akt signaling pathway in neonatal rat CFs.

[Effects of adrenomedullin on angiotensin II-induced collagen synthesis in vascular adventitial fibroblasts].

OBJECTIVE: To explore the effects of adrenomedullin (ADM) on Angiotensin II (AngII)-induced collagen synthesis in cultured rat vascular adventitial fibroblasts. METHODS: Rat vascular adventitial fibroblasts were cultured in vitro. ADM produced and secreted from adventitia in the presence of AngII was detected by radioimmunoassay, type I, III collagen contents in adventitia fibroblasts were measured by ELISA and the expressions of TGFbeta1 and MMP-2 were determined by RT-PCR and Western blotting. RESULTS: AngII significantly induced ADM secretion in adventitial fibroblasts in a dose-dependent manner. These effects could be reduced by 45%, 3% and 46%, through pre-treatment with Losartan, PD123319 or both, respectively for 30 min in culture medium. The AngII-induced type I, III collagen secretion in adventitial fibroblasts was significantly reduced by AMD in a dose-dependent manner, (P < 0.01) while ADM agonist ADM(22 - 52) significantly potentiated the effect of AngII; ADM also reduced AngII-induced expression of TGFbeta1 at mRNA and protein levels in a dose-dependent manner. AngII reduced the expression of MMP-2 at mRNA and protein levels in adventitial fibroblasts and these effects could be reversed by AMD (10(-8) mol/L). CONCLUSION: AngII stimulated ADM secretion in adventitia fibroblasts, ADM in turn can inhibit AngII-induced type I, III collagen synthesis in adventitial fibroblasts probably by downregulating the TGFbeta1 expression and upregulating MMP-2 expression. ADM therefore served as an antifibrotic factor in vascular remodeling process.

[The effects of calmodulin kinase II inhibitor on ventricular arrhythmias in rabbits with cardiac hypertrophy].

OBJECTIVE: To investigate the effect of KN-93, a calmodulin kinase II inhibitor, on ventricular arrhythmias in rabbits with cardiac hypertrophy. METHODS: Female New Zealand white rabbits were randomly divided into four groups (n = 10 each): Sham; LVH; LVH + KN-92 and LVH + KN-93 group. LVH was induced by partially constricting the abdominal aorta. In Sham group, the abdominal aorta was exposed without constriction. Eight weeks later, the arterially perfused left ventricular wedge preparations were made and transmembrane action potentials (TAP) from epicardium and endocardium and transmural ECG were simultaneously recorded. Incidence of early after depolarization (EAD) and torsade de pointes (Tdp), QT interval, action potential duration (APD) and transmural depolarization dispersion (TDR) at different cycle lengths were observed under slow stimulation (2000 - 4000 ms), hypokalemic (2 mmol/L) and hypomagnesaemic (0.25 mmol/L) Tyrode's solution perfusion. RESULTS: Left ventricular hypertrophy was detected in LVH group by echocardiography and not affected by KN-92 and KN-93. Perfused with hypokalemic, hypomagnesaemic Tyrode's solution and under slow stimulation (2000 - 4000 ms), the incidences of EAD and Tdp in Sham group, LVH group, LVH + KN-92 group (0.5 micromol/L) and LVH + KN-93 group (0.5 micromol/L) were 0/10, 10/10, 9/10, 5/10 and 0/10, 5/10, 4/10, 1/10, respectively. With 1 micromol/L KN-92 and KN-93, the incidences of EAD and Tdp in LVH + KN-92 and LVH + KN-93 group were 9/10, 3/10 and 4/10, 1/10 respectively. The QT interval, APD and TDR were not affected by KN-93. CONCLUSION: The calmodulin kinase II inhibitor KN-93 can effectively suppress ventricular arrhythmias in rabbits with cardiac hypertrophy by decreasing EAD.

[Effects of RNA interference targeting angiotensin 1a receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C hypertension].

OBJECTIVE: To investigate the effects of RNA interference (RNAi) targeting angiotensin 1a (AT1a) receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C (2-kidney, 1-clip) hypertension. METHODS: Two kinds of RNAi plasmids, pAT1a-shRNA1 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 928 - 946 and pAT1a-shRNA2 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 978 - 996, and a blank plasmid pCon carrying a nonspecific shRNA-coding sequence were constructed. Thirty Sprague-Dawley rats underwent clipping of the left renal artery so as to establish two-kidney, one-clip (2K1C) hypertension models and then were randomly divided into 5 equal groups: pAT1a-shRNA1 group (injected with pAT1a-shRNA1 4 mg/kg only one time), pAT1a-shRNA2 group (injected with pAT1a-shRNA2 4 mg/kg only one time), pCon group (injected with pCon 4 mg/kg only one time), valsartan group (perfused into the stomach with valsartan, a AT1 receptor inhibitor 30 mg.kg(-1).d(-1), for 3 weeks), and control blank group (without any treatment). Three weeks later, the systolic pressure of the caudal artery was measured, catheterization through carotid artery was conducted to measure the systolic blood pressure (SBP) and diastolic blood pressure (DBP), and the left ventricular pressure curve was drawn. Then the rats were killed; the weight of the heart was measured, the ratio of left ventricle weight to body weight (LV/BW) was calculated, and pathological examination of the heart and thoracic aorta was performed. Western blotting was used to detect the protein expression of AT21 in the ventricle and aorta. Six age-matched healthy rats were used as normal controls. RESULTS: There was no significant difference in the caudal artery pressure among the 5 groups (all P > 0.05) before intervention. Three weeks later the caudal artery pressures of the blank control group and pCon group continued to significantly increase by about 25 mm Hg compared to the values before the intervention (both P < 0.001) and without significant difference between these 2 groups; however, the caudal artery pressures of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 15.1 mm Hg +/- 5.4 mm Hg, 16.4 mm Hg +/- 8.4 mm Hg, and 30.6 mm Hg +/- 18.2 mm Hg lower than those before the intervention respectively (all P < 0.01); and were also significantly lower than those of the blank groups (P < 0.01 or P < 0.05). There was no significant differences in the +/- dp/dt value and indicators of renal function among these groups. The carotid artery pressure of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 194 mm Hg +/- 5 mm Hg, 200 mm Hg +/- 5 mm Hg, and 164 mm Hg +/- 5 mm Hg, all significantly lower than those of the blank and pCon groups (234 mm Hg +/- 10 mm Hg and 232 mm Hg +/- 7 mm Hg respectively, all P < 0.01). The LV/BW of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 2.27 +/- 0.37, 2.31 +/- 0.26, and 2.26 +/- 0.39, all significantly lower than that of the blank and pCon groups (3.24 +/- 0.38 and 2.94 +/- 0.06, respectively, all P < 0.01), similar to that of the normal control group (P > 0.05). The myocardiocytes were significantly hypertrophic and the arterial tunica media was significantly thickened in the blank group and such changes were all improved to different degrees in the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups. The protein expression levels of AT1 receptor in the myocardium of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 53.3% and 47.8% respectively than that of the blank group, and the protein expression levels of AT1 receptor in the thoracic aorta of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 58.7% and 49.3% respectively than that of the blank group (all P < 0.01); however, there were no significant difference in the protein expression levels of AT1 receptor in the myocardium and thoracic aorta between the valsartan and blank groups (both P > 0.05). CONCLUSION: RNA interference targeting AT1a receptor inhibits the development of renovascular hypertension and the accompanying cardiac hypertrophy. The RNAi technology may become a new strategy of gene therapy for hypertension.

[Selective knockdown of Angiotensin II receptor subtype 1a in rat vascular smooth muscle cells by RNA interference].

OBJECTIVE: To selectively knockdown the expression of Angiotensin II receptor subtype 1a (AT1aR) in rat vascular smooth muscle cells (VSMCs) by RNA interference and the sequential effects on cellular viability and proliferation. METHODS: The primary cultured rat aortic VSMCs were transfected by plasmids pAT1a-shRNA1 and pAT1a-shRNA2, each carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence, or by a control plasmid pGenesil-Control (pCon) carrying a nonspecific shRNA-coding sequence. The mRNA and protein expressions of AT1a, AT2 were analyzed by semi-quantified RT-PCR and Western blot, respectively and normalized to the internal control gene beta-actin. Cellular viability and proliferation were determined with methylthiazoletetrazolium (MTT) assay. RESULTS: AT1a mRNA and protein were reduced by 82% and 69% by pAT1a-shRNA1, 77% and 56% by pAT1a-shRNA2, respectively while no change was found in pCon treated VSMCs. AT2 receptor level in VSMCs remains unchanged after various treatments. The A(490nm) values obtained by MTT measurements were similar among groups in the absence of Ang II but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 treated VSMCs in the presence of Ang II. CONCLUSION: RNA interference can selectively knockdown AT1a expression in cultured VSMCs and attenuate the Ang II induced cell proliferation. Future studies are warranted to explore the potential role of RNA interference on AT1 function and as a new gene therapy tool for cardiovascular diseases.

[Effects of endocannabinoids on cardiovascular system].

Endocannabinoids and its corresponding receptors exist in myocardium, vascular smooth muscle cells, endothelial cells, nerve cells within blood vessel walls and some of circulating cells in blood. Endocannabinoids have different roles such as modulating blood pressure and vascular dilation in cardiovascular system in different models and organs of animal or human. It might mediate cardiac protection and modulating circulation in shock and myocardial infarction. Also, endocannabinoids might play a key role in cardiac preconditioning. The study of endocannabinoids in cardiovascular system was just start. We introduced the progress in the source and distribution of endocannabinoids in cardiovascular system, and the action and mechanisms of endocannabinoids in blood vessels and hearts in the present review.

Endogenous nitric oxide mediates lipoteichoic acid induced preconditioning on reoxygenation injury of cultured human coronary artery endothelial cells.

AIM: To explore the effects of lipoteichoic acid (LTA) induced delayed preconditioning (PC) on hypoxia-reoxygenation (H/R) injury of cultured human coronary artery endothelial cells (HCAECs), and to investigate the potential role of endogenous nitric oxide (NO) participated in the protective mechanism. METHODS: HCAECs were incubated for 2 h in a hypoxic atmosphere and reoxygenated for 4 h in a normoxic atmosphere. The delayed PC was induced by pretreatment with LTA (30 or 300 microg x L(-1)) for 4 h before 24 h recovery. The extent of cellular injury after reoxygenation was assessed by the percentage of cellular injury with Trypan blue exclusion and by the amount of lactate dehydrogenase (LDH) in culture media. The NO level of the culture media was measured spectrophotometrically. Furthermore, HCAECs were exposed to 300 microg x L(-1) of LTA for 4 h, and to detect the expression of eNOS mRNA by RT-PCR method after cells were recovered from different points. RESULTS: LTA pretreatment significantly decreased the percentage of the killed cell and the concentration of LDH in media. Also, LTA pretreatment obviously raised the concentrations of NO in culture media. The protective effects of LTA were abrogated by pretreatment with N-monomethyl-L-arginine (L-NMMA). Moreover, the expression of eNOS mRNA was significantly upregulated after HCAECs exposure to LTA for 4 h following 2 h or 4 h recovery. CONCLUSION: LTA could induce the delayed protection against H/R induced endothelial injury and dysfunction of cultured HCAECs. NO produced by eNOS acts initially as a trigger and subsequently as a mediator of delayed PC.

[Development and applications of an auto-analyzing system for Model TJ-IV vector-cardiogram].

A new computer-assisted vector-cardiogram analyzing system Model TJ-IV developed based on Model TJ-III, has been using in the routine clinical work in order to evaluate its features and performances. The system employs a 586 computer with a CPU of 120 MHz, a special low-noise amplifier, a 12 bit A/D tranducer and the C language for programming. The examinations of 206 cases were performed and all the vector-cardiograms were analyzed by the computer system and by manipulative methods respectively. In comparison with the manipulative methods the system has a very high accuracy of picture-recognition. The accuracy for distinguishing the onsets and terminals of orthogonal ECG waves is 98% while that for distinguishing the peaks and troughs of the waves is 100%. These waves include P, Q, R, S, R' and S' waves. The new system is capable to provide the parameters of more than 591 items, including 46 newly-developed diagnostic parameters. The testing and analyzing of 12 parameters of orthogonal ECG and plane VCG have proved that the results of the aboved two methods have no difference. The new system has a very high accuracy of picture-recognition and index calculation with many technical problems existing in the old versions, solved--a great improvement of safety and anti-interference and an increase of the detecting & diagnostic speed.

Effects of transforming growth factor-beta1 and signal protein Smad3 on rat cardiomyocyte hypertrophy.

BACKGROUND: SMAD proteins have recently been identified as the first family of putative transforming growth factor-beta1 (TGF-beta1) signal transducers. This study was to investigate the effects of TGF-beta1 and signal protein Smad3 on rat cardiac hypertrophy. METHODS: The incorporation of [(3)H]-leucine was measured to determine the hypertrophy of cardiomyocyte incubated with different doses of TGF-beta1 in cultured neonatal cardiomyocytes. The model of rat cardiac hypertrophy was produced with constriction of the abdominal aorta. At different times after the operation, rats were killed, and their left ventricular mass index (LVMI) determined. The mRNA expression of TGF-beta1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR. The protein expression of Smad3 was assessed by Western blot. RESULTS: In cultured neonatal cardiomyocytes, TGF-beta1 significantly promoted incorporation of [(3)H]-leucine. With the concentration of 3 pg/L, it increased the expression of Smad3 in mRNA and protein levels after 15 minutes, and continued for up to 8 hours of cultured cardiomyocytes. The LVMI and the expression of TGF-beta1 (mRNA) and Smad3 (mRNA and protein) of hypertrophic left ventricle were increased by day 3 after the operation and continued to the 4th week. The peak expression of these was in the second week after operation. CONCLUSION: TGF-beta1 has positive effects on rat cardiomyocyte hypertrophy. Signal protein Smad3 could be related to the pathologic progression of rat cardiac hypertrophy.