CircCAMTA1 facilitates atrial fibrosis by regulating the miR-214-3p/TGFBR1 axis in atrial fibrillation.

Circular RNAs (circRNAs) have been shown to be associated with cardiac fibrosis. Atrial fibrosis is an important pathophysiological event in the progression of atrial fibrillation (AF). Although a novel circRNA calmodulin binding transcription activator 1 (circCAMTA1) has been reported to be related with the development of AF, the detailed molecular mechanisms remain largely unknown. In this study, we found that circCAMTA1 was upregulated in atrial muscle tissues of AF patients and angiotensin-II (Ang-II)-treated human atrial fibroblasts (HAFs). Moreover, circCAMTA1 expression was positively correlated with the expression of collagen (I and III) and α-SMA in atrial muscle tissues of AF patients. In vitro experiments, knockdown of circCAMTA1 significantly suppressed Ang-II-induced HAFs proliferation and reduced the expression of atrial fibrosis-associated genes, but overexpression of circCAMTA1 exhibited opposite results. In vivo experiments, circCAMTA1 knockdown ameliorated Ang-II-induced atrial fibrosis by reducing AF incidence, AF duration, and collagen synthesis. Functionally, circCAMTA1 facilitated Ang-II-induced atrial fibrosis in vitro and in vivo via downregulating the inhibitory effect of miR-214-3p on transforming growth factor ß receptor 1 (TGFBR1) expression. In conclusions, circCAMTA1 knockdown alleviated atrial fibrosis through downregulating TGFBR1 expression intermediated by miR-214-3p in AF, suggesting circCAMTA1/miR-214-3p/TGFBR1 axis may be a novel therapeutic target for AF treatment in clinic.

Significantly improved detection performances of immunoassay for ractopamine in urine based on highly urea-tolerant rabbit monoclonal antibody.

Highly sensitive and accurate screening of ractopamine (RAC) residue in animal urine is greatly needed to ensure food security. The detection performance of immunoassay for RAC was always seriously harmed by the antibody inactivation derived from urea. Here, we first discovered one rabbit monoclonal antibody (RmAb) to RAC with a high affinity of 0.007 ng mL-1 and a surprising urea tolerance of 3 M urea, which is beneficial for developing robustly developed immunoassay in urine without sample pretreatment. The limits of detection of developed indirect competitive enzyme-linked immunosorbent assay based on RmAb1 for RAC were 0.0042-0.014 µg L-1 with the coefficient of variation below 11.7% in swine, sheep, and cow urine, significantly improved 10-100-fold in sensitivity. Moreover, the urea-tolerant mechanism of RmAb1 showed that more non-polar amino acids, more hydrogen bond donors on the surface, and preponderant Pi interaction of antibody-RAC all contributed to the stability of the RmAb1 in a high concentration of urea.

Effects of ticagrelor monotherapy vs. clopidogrel monotherapy on platelet reactivity: a randomized, crossover clinical study (SINGLE study).

Increasing clinical trials demonstrated that the discontinuation of aspirin while maintaining a P2Y12 inhibitor monotherapy could decrease the risk of bleeding without losing the antithrombotic effect. However, no data are available on the platelet reactivity of patients undergoing ticagrelor monotherapy vs. clopidogrel. Therefore, we performed this study to observe the efficacy of ticagrelor monotherapy vs. clopidogrel in Chinese patients with chronic coronary syndrome. This randomized, single-blinded, crossover trial enrolled 50 patients who were administered with ticagrelor (90 mg twice daily for 2 weeks) or clopidogrel (75 mg once daily for 2 weeks). Followed by a 2-week washout period, the two groups of patients underwent a crossover trial. Light transmission aggregometry (LTA) and thromboelastography (TEG) assays were used to test platelet reactivity. The platelet aggregation rate (PAgR) of ADP and AA was significantly lower with ticagrelor than clopidogrel (PAgR of ADP, 27.30% (7.30%-42.635%) vs. 35.55% (12.03%-69.25%), P = .0254; PAgR of AA, 77.80% (21.60%-86.43%) vs. 83.10% (67.13%-87.20%), P = .0400). There was no significant difference in PAgR of collagen and epinephrine between the two groups. The TEG assay showed that ADP and AA, which induced the inhibition of platelet aggregation, were significantly higher in the ticagrelor group than those in the clopidogrel group [ADP%, 69.00% (59.68%-88.95%) vs. 60.55% (35.98%-78.35%), P = .0020; AA%, 53.65% (22.75%-79.28%) vs. 15.15% (5.75%-70.25%), P = .0127]. High on-treatment platelet reactivity (HTPR) on ADP was 2.17% with ticagrelor and 19.57% with clopidogrel. HTPR on AA was 50.00% with ticagrelor and 69.57% with clopidogrel. Ticagrelor and clopidogrel caused the inhibition of ADP and AA-induced platelet aggregation. Moreover, ticagrelor monotherapy had a stronger inhibitory effect than clopidogrel monotherapy (except on collagen and epinephrine).

Functional Evaluation of Intermediate Coronary Lesions with Integrated Computed Tomography Angiography and Invasive Angiography in Patients with Stable Coronary Artery Disease.

Background and objectives: The hemodynamic evaluation of coronary stenoses undergoes a transition from wire-based invasive measurements to image-based computational assessments. However, fractional flow reserve (FFR) values derived from coronary CT angiography (CCTA) and angiography-based quantitative flow ratio have certain limitations in accuracy and efficiency, preventing their widespread use in routine practice. Hence, we aimed to investigate the diagnostic performance of FFR derived from the integration of CCTA and invasive angiography (FFRCT-angio) with artificial intelligence assistance in patients with stable coronary artery disease (CAD). Methods: Forty stable CAD patients with 67 target vessels (50%-90% diameter stenosis) were included in this single-center retrospective study. All patients underwent CCTA followed by coronary angiography with FFR measurement within 30 days. Both CCTA and angiographic images were combined to generate a three-dimensional reconstruction of the coronary arteries using artificial intelligence. Subsequently, functional assessment was performed through a deep learning algorithm. FFR was used as the reference. Results: FFRCT-angio values were significantly correlated with FFR values (r = 0.81, P < 0.001, Spearman analysis). Per-vessel diagnostic accuracy of FFRCT-angio was 92.54%. Sensitivity and specificity in identifying ischemic lesions were 100% and 88.10%, respectively. Positive predictive value and negative predictive value were 83.33% and 100%, respectively. Moreover, the diagnostic performance of FFRCT-angio was satisfactory in different target vessels and different segment lesions. Conclusions: FFRCT-angio exhibits excellent diagnostic performance of identifying ischemic lesions in patients with stable CAD. Combining CCTA and angiographic imaging, FFRCT-angio may represent an effective and practical alternative to invasive FFR in selected patients.

Combined treatment with erythropoietin and granulocyte colony-stimulating factor enhances neovascularization and improves cardiac function after myocardial infarction.

BACKGROUND: Erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) are both potential novel therapeutics for use after myocardial infarction (MI). However, their underlying mechanisms remain unclear and the efficacy of monotherapy with EPO or G-CSF is also controversial. Therefore, we investigated the effects of combined treatment with EPO and G-CSF on neovascularization and cardiac function in post-infarction rats and explored the potential mechanisms. METHODS: Four groups of rats were used: control (saline injection after MI, i.h.), EPO (a single dose of 5 000 IU/kg after MI, i.h.), G-CSF (a dose of 50 µg× kg(-1)× d(-1) for 5 days after MI, i.h.), and both EPO and G-CSF (EPO+G-CSF, using the same regiment as above). Cardiac function was assessed by echocardiography before and 1 day, 7 days, 14 days and 21 days after MI. CD34(+)/Flk-1(+) cells in the peripheral blood were evaluated by flow cytometry before and 3 days, 5 days and 7 days after MI. The infarct area and angiogenesis in the peri-infarct area were analyzed. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and stromal-derived factor-1a (SDF-1α) in the peri-infarct area were detected by real-time quantitative RT-PCR and Western blotting. RESULTS: Compared with the control and monotherapy groups, the EPO+G-CSF group had significantly increased CD34(+)/Flk-1(+) endothelial progenitor cells (EPCs) in the peripheral blood (P < 0.05), up-regulated VEGF and SDF-1α levels in the peri-infarct region (P < 0.05), enhanced capillary density (P < 0.05), reduced infarct size (P < 0.05) and improved cardiac structure and function (P < 0.05). G-CSF alone did not dramatically increase EPCs in the peripheral blood, enhance capillary density in the peri-infarct area or reduce infarct size compared with the control group. CONCLUSIONS: Combined treatment with EPO and G-CSF increased EPCs mobilization, up-regulated VEGF and SDF-1α levels in the post-infarction microenvironment, subsequently enhanced neovascularization in the peri-infarct region and reduced infarct size. All factors contributed to its beneficial effects on cardiac function in post-infarction rats.

[Effects of beta3-adrenergic receptor antagonist on myocardial UCP2 expression and energy metabolism in chronic heart failure rats].

OBJECTIVE: To observe the effects of beta3-adrenergic receptor(beta3-AR) antagonist on myocardial uncoupling protein 2 (UCP2) expression and energy metabolism in chronic heart failure rats. METHODS: Seven weight-matched normal adult rats (control group), 18 isoproterenol (ISO) induced heat failure (HR) rats (ISO group) and 21 ISO induced heart failure rats but received specific beta3-AR inhibitor SR59230A (ISO+ SR59230A group) for 6 weeks were included in this research. At the end of the study, echocardiography was performed, the ratio of left ventricular weight and body weight (LVW/BW) was calculated. The expression of beta3-AR ad UCP2 mRNA in myocardium were detected by reverse transcription-polymerase chain reaction (RT-PCR), the UCP2 protein in myocardium were detected by Western blot. The myocardial contents of creatine phosphate (PCr) and adenosine triphosphate (ATP) were measured by high performance liquid chromatography (HPLC). RESULTS: Compared with control group, the cardiac function was significantly reduced and myocardial beta3-AR mRNA significantly increased, UCP2 mRNA and protein were also significantly increased in ISO group, this change could be attenuated by the treatment with SR59230A, and the expression of myocardial UCP2 protein negatively correlated with the ratio of PCr/ATP. CONCLUSION: In the chronic stage of HF, the expression of UCP2 increases, which causes myocardial energy shortage, SR59230A improves myocardia energy efficiency and cardiac function by means of suppressing the expression of UCP2.

[Association between beta3-adrenergic receptor and oxidative stress in chronic heart failure rats].

OBJECTIVE: To investigate the association between beta(3)-adrenergic receptor (beta(3)-AR) and oxidative stress in isoproterenol (ISO)-induced chronic heart failure (HF) rats. METHODS: Seven weight-matched normal adult rats (control), 18 ISO induced heart failure rats and 21 ISO induced heart failure rats treated with specific beta(3)-AR inhibitor, SR59230A for 6 weeks were included in this study. Echocardiography was performed at the end of the study and the myocardial levels of total superoxide dismutase (T-SOD) and lipid peroxidation (LPO) were measured by colorimetry, myocardial expression of beta(3)-AR was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULT: Compared with control group, the cardiac function was significantly reduced and myocardial beta(3)-AR mRNA expression was significantly increased, LPO level was also significantly enhanced while T-SOD level was significantly reduced in ISO group and these changes could be significantly attenuated by treatment with SR59230A. CONCLUSION: Our results showed that myocardial upregulation of beta(3)-AR is associated with increased oxidative stress in this model and beta(3)-AR inhibitor may be a new therapeutic agent for heart failure treatment.

Adenovirus-mediated ICOSIg gene transfer alleviates cardiac remodeling in experimental autoimmune myocarditis.

To explore the therapeutic effects of adenovirus vector mediated transfer of the ICOSIg gene on immuno-inflammation-mediated cardiac remodeling in an experimental autoimmune myocarditis (EAM) model, pAdeno-ICOSIg was constructed and transfected into HEK 293 cells to produce the ICOSIg adenovirus. Ad-CMV-GFP was used as a control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. The immunized rats were divided into two groups. The inducible co-stimulatory molecule (ICOS) group received the adenovirus containing ICOSIg on day 14; the green fluorescent protein (GFP) group received the adenovirus containing GFP as the control adenovirus and 15 normal rats (Control group) consisted of the normal controls that were not immunized. On day 28, all rats were euthanized after echocardiography and histopathologically examined for cardiac fibrosis. Western blotting was performed to detect ICOS, ICOS ligand (ICOSL), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and real-time RT-PCR was performed to detect B7-1, B7-2 and interleukin (IL)-17 expression. ELISPOT was applied to detect Th1 and Th2 cytokine production. Collagen concentration and collagen cross-linking were determined as markers of cardiac fibrosis. It was found that blockade with ICOSIg exerted antifibrotic effects on cardiac remodeling in EAM. On day 28, cardiac function and inflammatory myocardial fibrosis improved significantly in the ICOS group compared to the GFP group. The expression of ICOS, the ICOSL, B7-1 and IL-17 was statistically significantly lower in the ICOS and Control groups compared to the GFP group. ICOSIg significantly augmented Th2 cytokine production and diminished Th1 and Th17 cytokine production. This blockade of the ICOS co-stimulatory pathway with ICOSIg alleviated autoimmune inflammation-mediated cardiac remodeling and improved cardiac function. Regulation of the Th1/Th2/Th17 balance may be one of the underlying mechanisms responsible for this effect.

[Effect of beta3-adrenoceptor antagonist on the cardiac function and expression of endothelial nitric oxide synthase in a rat model of heart failure].

OBJECTIVE: To investigate the effect of beta(3)-adrenoceptor (beta(3)-AR) antagonist (SR59230A) on the cardiac function and left ventricular remodeling in a rat model of heart failure induced by isoproterenol (ISO), and to probe into its mechanism. METHODS: Eight rats were randomly selected to serve as controls from 85 male adult Wistar rats. After a heart failure model was reproduced, twenty remain rats were randomly divided into ISO group (n = 10) and SR59230A group (SR group, n = 10). ISO group received intraperitoneal injection of 1 ml saline twice a day; SR group received intraperitoneal injection of 85 nmol SR59230A in 1 ml saline twice a day; control group received no treatment. The parameters determined included echocardiogram, the expression of nitric oxide synthase (eNOS) of left ventricle by the technique of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, cyclic guanosine monophosphate (cGMP) level by enzyme linked immunosorbent assay (ELISA), the ratio of left ventricular weight and body weight (LVW/BW), and the ratio of lung weight and body weight (PW/BW). RESULTS: Compared with control group, the left ventricular end systolic pressure (LVESP), the maximum and minimum first derivative of left ventricular pressure (+/-dp/dtmax) were significantly decreased (all P<0.01), while heart rate (HR) and left ventricular end-diastolic pressure (LVEDP) were significantly increased (both P<0.01) in ISO group. Compared with ISO group, LVESP, +/-dp/dtmax were markedly higher (P<0.05 or P<0.01) respectively, whereas HR and LVEDP were markedly lower (P<0.05 and P<0.01) in SR group, and there was no difference in HR between SR group and control group (P>0.05), while LVEDP was higher in RS group than control group (P<0.01). The levels of eNOS mRNA, protein and cGMP were significantly lower in SR group compared with ISO group (all P<0.01). In addition, when compared with ISO group, LVW/BW ratio and PW/BW ratio in SR group were also decreased (both P<0.05). CONCLUSION: beta(3)-AR antagonist SR59230A can block the beta(3)-AR-NOS-cGMP pathway and improve cardiac function in heart failure in rat when if is administered for a long term. SR59230A can also improve left ventricular remodeling in a certain degree.

[Effects of adenovirus-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocarditis in Lewis rats].

OBJECTIVE: To explore the effects of adenovirus vector-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocarditis (EAM) in Lewis rats. METHODS: Expression vector containing ICOSIg (p-Adeno-ICOSIg) was constructed by fusion of human ICOS and IgGFc segment. Adenovirus vector was digested by PacI enzyme and transfected into HEK 293 cells. Adenovirus expressing ICOSIg was produced. EGFP was constructed into adenovirus vector and used as control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. All immunized Lewis rats were divided into 4 groups. Group A (n = 15) and B (n = 15) received adenovirus containing ICOSIg on day 0 and day 14 respectively to study the effects of costimulatory molecules gene therapy on T cell activation and inflammation; group C (n = 10) and group D (n = 10) received adenovirus containing EGFP on day 0 and day 14 respectively as controls. Group E (n = 10) was normal controls that did not receive immunization. On day 28, all rats were killed after echocardiography examination. Histopathological examination was performed to observe myocardial inflammation. Protein levels of ICOS, ICOSL, B7-1 and B7-2 were detected by Western blot. INF-gamma, IL-2 and IL-4 mRNA were determined by realtime RT-PCR. RESULTS: On day 28, cardiac function was significantly improved and myocardial inflammation significantly attenuated in group B compared to group A, C and D (all P < 0.05). B7-1 expression at protein level was significantly lower in group B than that of group C (P < 0.05). ICOS and ICOSL expressions at protein level were significantly decreased in both group A and B compared with group C and D (P < 0.05). IFN-gamma mRNA level significantly decreased and IL-4 mRNA significantly increased in group A and B compared to group C and D (P < 0.05). CONCLUSIONS: Blockade of costimulatory pathway with gene therapy of ICOSIg alleviated autoimmune inflammatory damage and improved cardiac function in Lewis rats with EAM. Down-regulated costimulatory molecules in the myocardium and reduced inflammatory cytokine secretion might be responsible for the beneficial effects of ICOSIg in this model.

Chronic blocking of beta 3-adrenoceptor ameliorates cardiac function in rat model of heart failure.

BACKGROUND: Stimulation of the heart beta 3-adrenoceptor (AR) may result in a negative inotropic effect. Being up-regulated, beta 3-AR plays a more important role in the regulation of cardiac function during heart failure. However, the effect of chronic blocking of beta 3-AR on heart failure has not been fully elucidated. In this study, we used a selective beta 3-AR antagonist SR59230A to treat a well defined heart failure rat model chronically, then evaluated its effect on cardiac function and investigated the mechanism. METHODS: Male Wistar rats were chosen randomly as controls (n = 8). Isoproterenol induced heart failure rats were randomly divided into ISO group (n = 10) and SR group (n = 10). The ISO group received intraperitoneal injection of 1 ml saline twice a day; the SR group received intraperitoneal injection of SR59230A 85 nmol in 1 ml saline twice a day; and the control group received no treatment. The treatment was started 24 hours after the last isoproterenol injection and continued for 7 weeks. Then we measured the following indexes: the ratio of heart weight to body weight (HW/BW) and the ratio of left ventricular weight to body weight (LVW/BW), collagen volume fraction (CVF), left ventricular end diastolic dimension (LVEDd), left ventricular end systolic dimension (LVESd), ejection fraction (EF), fractional shortening (FS) and the ratio of E wave to A wave (E/A), the mRNA and protein expression of beta 3-AR and eNOS, and cGMP level in the heart. RESULTS: The ratios HW/BW and LVW/BW were significantly increased in the ISO group compared with the control group (P < 0.01), but they were limited in the SR group (P < 0.05 compared with the ISO group). CVF increased in the ISO group and the SR group (P < 0.01), but it was significantly attenuated in the SR group (P < 0.01). LVEDd, LVESd and E/A ratio were significantly increased in the ISO group compared with the control group (P < 0.01), while EF and FS were significantly decreased (P < 0.01). Compared with the ISO group, the SR group showed that LVEDd, LVESd and E/A ratio were significantly decreased (P < 0.01), whereas EF and FS were significantly increased (P < 0.01). beta(3)-AR and eNOS mRNA and protein in the ISO group were significantly increased when compared with the control group (P < 0.01). These increases were all attenuated in the SR group compared with the ISO group (P < 0.01). The level of cGMP in myocardial tissue was significantly increased in the ISO group compared with the control group (P < 0.01), whereas SR59230A treatment normalized this increment (P < 0.01). CONCLUSIONS: Chronic blocking of beta 3-AR could ameliorate cardiac function in heart failure rats and its mechanism involves inhibition of the negative inotropic effect and attenuation of cardiac remodeling.

[IL-10 gene modification on immature dendritic cells induces antigen-specific tolerance in experimental autoimmune myocarditis].

OBJECTIVE: To investigate whether IL-10 gene modification on immature dendritic cells (iDC) could induce autoimmune tolerance in rat experimental autoimmune myocarditis (EAM). METHODS: EAM was induced by cardiac myosin immunization on day 0 and day 7 in rats. A total of 2 x 10(6) mature DC (mDC), iDC, pcDNA3 transfected iDC, pcDNA3-IL-10 transfected iDC or PBS were injected intravenously at 5th immunization day. Three weeks later, echocardiography and HE staining were performed to observe the cardiac function and myocardial inflammation. Th1/Th2 cytokines were detected by ELISA and MHC-II molecules, costimulatory molecules were identified by flow cytometry. In vitro T lymphocyte proliferation assay and adoptive transfer of DCs were performed to determine the antigen specific tolerance induced by IL-10 gene modification on iDCs. RESULTS: EAM rats treated with pcDNA3-IL-10 transfected iDC showed improved cardiac function and reduced inflammatory cells infiltration into myocardium. Moreover, lower Th1 and higher Th2-type response was induced, MHC-II and costimulatory molecules down-regulated and antigen specific immunological responses towards cardiac myosin inhibited in pcDNA3-IL-10-iDC treated EAM rats. CONCLUSION: Treatment with IL-10 gene modified iDCs could ameliorates EAM by inducing Th2 polarization and down-regulation of MHC-II molecules and costimulatory molecule expressions.

Association of HLA class II DRB1, DPA1 and DPB1 polymorphism with genetic susceptibility to idiopathic dilated cardiomyopathy in Chinese Han nationality.

Although the aetiology of idiopathic dilated cardiomyopathy (IDC) remains unclear, many immunological abnormalities involving changes in cell-mediated and humoral immunity may be associated with cardiac impairment in IDC. Autoimmune mechanisms are likely to participate in the pathogenesis of at least a subgroup of IDC and components of the major histocompatibility complex may serve as markers for the propensity to develop immune-mediated myocardial damage. Human leukocyte antigen (HLA) class II genes, which are highly polymorphic, play an important role in the activating of immune responses and thus control the predisposition for or protection from IDC. This study explores the possible contribution of HLA-DRB and DP polymorphisms to IDC susceptibility. DNA genotyping for HLA-DRB1, DPA1 and DPB1 was performed using polymerase chain reaction-sequencing based typing (PCR-SBT) method in 198 IDC patients and 136 random selected healthy Han ethnic individuals living in Northern China. IDC patients were, sub-grouped into asymptomatics (subgroup A), with arrhythmia (subgroup B) and with overt congestive heart failure (subgroup C) according to the clinical manifestations and electrocardiogram or echocardiographic characteristics. ADP/ATP autoantibody was detected in IDC group by immunoblot analysis. The results revealed that HLA-DR15, -DPB*0601 frequencies were significantly elevated in IDC group compared with normal control. The DPB1*0601 allele in homozygous form or in combination with allele DPB1*2301 or *3901, was found present more often in IDC patients. The predominance of HLA-DR4 allele was observed in subgroup B after stratification. However, the frequency of DPB1*0101 allele increased in the control than in the IDC group. The frequency of HLA-DPB1*0601 allele was significantly higher in IDC patients with positive autoantibody against ADP/ATP carrier of myocardial mitochondria in contrast to those with negative autoantibody. We conclude that HLA-DR4, -DR15, -DPB1*0601 alleles confers susceptibility to, while DPB1*0101 allele confers protection from IDC among individuals of northern Chinese Han nationality. The glutamate at position 69 in the second exon of DPB1*0601, as a key residue for special conformation of HLA-DP, may confer predisposition to IDC. HLA-DR and -DP alleles polymorphisms may serve as genetic markers for IDC and be involved in the regulation of the immune specific response to auto or exterior anti-myocardium antibodies.

Antigen-specific tolerance induced by IL-10 gene modified immature dendritic cells in experimental autoimmune myocarditis in rats.

BACKGROUND: Experimental autoimmune myocarditis (EAM) in rats is a T-cell-mediated disorder. The initiation and maintenance of autoimmune responses in EAM depend on the maturation state of dendritic cells. IL-10 is a pleiotrophic immunomodulatory cytokine that functions at different levels of the immune response, so it has emerged as a promising therapeutic factor for the treatment of autoimmune/inflammatory diseases. This study was designed to test the hypothesis that IL-10 gene modified bone marrow-derived immature dendritic cells (iDCs) ameliorate EAM and to explore the underlying mechanisms. METHODS: EAM was induced using the methods of cardiac myosin immunization on day 0 and day 7. Immature and mature bone marrow-derived dendritic cells (BMDCs) were generated without or with the stimulation by lipopolysaccharide (LPS) and the phenotype was analyzed by flow cytometry. Some of the iDCs were transfected by pcDNA3-IL-10 plasmid. 2 x 10(6)/per rat mature DC (mDC), immature DC (iDC), pcDNA3 transfected iDC, pcDNA3-IL-10 transfected iDC or phosphate buffered saline (PBS) were injected intravenously for treatment 5 days after the first immunization. On day 21, HE staining was performed to detect the myocardial inflammation and T lymphocyte proliferation assay was used to determine the effects of IL-10 gene transfected iDC on autoreactive T cell proliferation. Expression of IkappaB, the inhibitor of NF-kappaB pathway, was determined by Western blot. RESULTS: BMDCs generated in a medium supplemented with granulocyte-macrophage-colony-stimulating factor (GM-CSF) were relatively immature, as determined by flow cytometry. However, stimulation with LPS induced these cells to become mature (m) DCs with higher levels of surface major histocompatibility complex (MHC)-II and costimulatory molecules. Intravenous administration of iDCs, especially pcDNA3-IL-10 transfected iDC, ameliorated the histopathological severity of the myosin induced-EAM, and the effect was lost after the DCs underwent maturation induced by in vitro exposure to LPS. IL-10 gene modified iDC inhibited the antigen specific T cell responses towards cardiac myosin. IkappaB protein was up-regulated significantly in the IL-10 gene modified iDC group. CONCLUSIONS: IL-10 gene modified iDC induced antigen-specific tolerance in EAM. The underlying mechanisms may be related to costimulatory molecules down-regulation and NF-kappaB pathway inhibition.

IL-10 gene modified dendritic cells induced antigen-specific tolerance in experimental autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) in rats is a T-cell-mediated disorder, and the involvement of Th1/Th2 unbalance has been demonstrated. The induction of antigen-specific tolerance is critical for the treatment of EAM and maintenance of immune tolerance. IL-10 is a pleiotrophic immunomodulatory cytokine that functions at different levels of the immune response, so it has emerged as a promising therapeutic factor for the treatment of autoimmune/inflammatory diseases. This study was designed to explore the effects of IL-10 gene modified bone-marrow-derived immature dendritic cells (iDCs) on the in vitro and in vivo immune response to cardiac myosin in EAM. EAM was induced using the classic methods of cardiac myosin immunization on day 0 and day 7. 2 x 10(6)/per rat mature DC (mDC), immature DC (iDC), pcDNA3 transfected iDC, pcDNA3-IL-10 transfected iDC or PBS were injected intravenously for treatment 5 days after the first immunization. On day 21, transthoracic echocardiogram and HE staining were performed to detect the cardiac function and myocardial inflammation. Th1/Th2 cytokines were detected by ELISA and MHC-II molecules, costimulatory molecules were identified by flow cytometry. In vitro T lymphocyte proliferation assay and adoptive transfer of DCs were performed to determine the antigen-specific tolerance induced by IL-10 gene modified iDCs. IL-10 gene modified iDC-treated EAM rats showed improved cardiac function and reduced infiltration of inflammatory cell into myocardium. Serum cytokines data indicated lower Th1 while higher Th2-type responses were induced in the pcDNA3-IL-10-iDC-treated group, suggesting a Th2 polarization. Moreover, IL-10 gene modified iDCs down-regulated MHC-II and costimulatory molecules on the surface of splenocytes and inhibited the antigen-specific immunological responses towards cardiac myosin. Adoptive transfer of IL-10 producing DCs prevented EAM induction. IL-10 gene modified iDCs ameliorates EAM histopathologically and functionally. The underlying mechanisms may be related to the IL-10 induced Th2 polarization and down-regulation of MHC-II molecules and costimulatory molecules expression.

[The research on associating the single nucleotide polymorphism of CTLA-4 gene promoter region with idiopathic dilated cardiomyopathy].

OBJECTIVE: To investigate the expression of cytotoxic T lymphocyte associated antigen-4 (CTLA-4) in patients with idiopathic dilated cardiomyopathy (IDC) and to explore genetic susceptibility to IDC caused possibly by single nucleotide polymorphism (SNP) of CTLA-4 gene promoter. METHODS: PCR-restriction fragment length polymorphism techniques were used to analyze the SNPs of CTLA-4 gene at position -1772, -1661 and -318 in the promoter region. Serum sCTLA-4, IFN-gamma and IL-4 were tested by ELISA. RESULTS: sCTLA-4 levels of IDC patients were associated with the haplotype and genotype. Patients with -1772 TC genotype or -1772 TC -1661 AA, -1772 TC -1661 AG haplotypes had higher sCTLA-4 levels than patients with other haplotypes did. The frequency of -1772 TC genotype was significantly high in patients with low ejection factor(EF) values. Whereas the frequencies of -1661 G allele and -1661 GG genotype were lower in IDC patients. Levels of IL-4 were increased in IDC group. CONCLUSION: Patients with IDC have an aberrant expression of the CTLA-4 products, and the -1772 C/T and -1661 A/G polymorphisms. The two SNPs may function as genetic markers for disease susceptibility.

[Effects of NG-nitro-L-arginine methyl ester on hemodynamics and beta-adrenoreceptors mRNA in rats with heart failure after beta3-adrenergic receptors agonist injection].

OBJECTIVE: To evaluate the effects of different doses of N(G)-nitro-L-arginine methyl ester (L-NAME) on hemodynamics, cyclic guanosine monophosphate (cGMP) production and the level of beta-adrenergic receptors (beta-ARs) mRNA in a heart failure rat model after BRL-37344 (beta(3)-ARs agonist) injection. Meanwhile, to investigate the influence of beta(3)-ARs and L-NAME on signal transduction in failing heart. METHODS: The rats were randomly divided into six groups, control group (group I), Iso (isoproterenol) group (group II), Iso + BRL group (group III), Iso + BRL + low dose of L-NAME group (5 mg/kg, group IV), Iso + BRL + moderate dose of L-NAME group (50 mg/kg, group V), Iso + BRL + high dose of L-NAME group (100 mg/kg, group VI). The hemodynamics [left ventricular end systolic pressure (LVESP), +/- dp/dt, left ventricular end diastolic pressure (LVEDP)], cardiac cGMP and the levels of beta(1)-, beta(2)-, and beta(3)-ARs mRNA were measured. RESULTS: (1) LVESP, +/- dp/dt values in group II were significantly lower, and LVEDP was significantly higher than that in group I (except -dp/dt P < 0.05, the rest were P < 0.01). Comparing with group II, group III had lower -dp/dt value and LVESP, higher LVEDP (P < 0.05). The level of +dp/dt had a trend to be lower but lacked statistical significance between two groups. The value of +/- dp/dt got higher and LVEDP got lower along with higher dose of L-NAME, but a large dose of L-NAME had more deteriorated cardiac functions. (2) The cardiac cGMP in group I, II and III had a higher tendency (P < 0.01). The tendency of cardiac cGMP in group IV, V and VI was inversed with the dose of L-NAME. After a large dose of L-NAME was applied, cGMP returned to the same level as Group I. (3) Among groups I, II and III, the level of beta(1)-AR mRNA was the highest in group I and the lowest in group III (P < 0.01). The levels of beta(2)-AR mRNA were also tended to be lower among three groups but with no significance. While the level of beta(3)-AR mRNA was the highest in group III. The levels of beta-AR mRNA were all the same in group VI, V and VI. CONCLUSIONS: The negative inotropic effect of beta(3)-ARs stimulation was mediated by activation of the NOS pathway. L-NAME blocked beta(3)-ARs agonist negative chronotropic effect on failing heart partly and improved hemodynamics, but a large dose of L-NAME had more deteriorated cardiac functions.

[Effect of beta3-adrenoreceptors agonist on beta3-adrenoreceptors expression and myocyte apoptosis in a rat model of heart failure].

OBJECTIVE: To evaluate the effects of beta(3)-adrenoreceptor (AR) agonist (BRL-37344) on the expression of beta(3)-AR in a isoproterenol (ISO)-induced heart failure (HF) rat model and to investigate the influence on the levels of beta(3)-AR in failing heart. METHODS: The rats were randomly divided into four groups: I group (control group, n=10); II group (normal with BRL group, n=10); III group (HF group, n=30); IV group (HF with BRL group, n=35).II and IV groups received BRL 0.4 nmol.kg-1.min-1 through caudal vein for 10 minutes twice a week. I and III groups received saline at the same time. The measure included hemodynamics, the expression of beta3-AR in left ventricular myocytes by the techniques of immunohistochemistry, beta3-AR proteins by western blot, expression levels of beta3-AR mRNA in myocardium by reverse transcription- polymerase chain reaction (RT-PCR) and the levels of apoptotic cells with a terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL) kit. RESULTS: (1)Hemodynamic: The tendency of left ventricular end systolic pressure (PES), maximal rates of rise of ventricular pressure(dp/dtmax), maximal rates of decline of ventricular pressure (dp/dtmin) in II, III and IV groups were lower (P<0.01), time constant of left ventricular relaxation (Tc) and left ventricular end diastolic pressure (PED) were higher than those of the I group (all P<0.01). There was no difference between I and II group except PED (P<0.05). Compared with III group, PES, dp/dtmax, dp/dtmin in IV group were dramatically decline (P<0.05): Tc, PED were markedly increased (Tc P<0.05, PED P<0.01). (2)The levels of beta3-AR mRNA and beta3-AR proteins were higher in III and IV groups when compared with I and II groups. There was no difference between I and II group. IV group's levels were higher than III group's. (3)The apoptotic rates in III group and IV group were significantly higher than those in I and II group (all P<0.01). When compared with III group's apoptotic cell rate, IV group's was higher (P<0.01). CONCLUSION: The levels of beta(3)-AR mRNA and proteins show an increase in failing heart compared with nonfailing heart. The effect of beta(3)-AR agonist aggravate markedly cardiac function and stimulate cardiac myocytes apoptosis in failing heart. If the levels of beta(3)-AR were too high, they might contribute to the loss of cardiac function and be the foundation of the functional degradation of HF.