Picroside II attenuates hyperhomocysteinemia-induced endothelial injury by reducing inflammation, oxidative stress and cell apoptosis.

Picroside II (P-II), one of the main active components of scrophularia extract, which have anti-oxidative, anti-inflammatory effects, but its effect on hyperhomocysteinemia (HHcy) induced endothelial injury remains to be determined. Here, we test whether P-II protects HHcy-induced endothelial dysfunction against oxidative stress, inflammation and cell apoptosis. In vitro study using HUVECs, and in hyperhomocysteinemia mouse models, we found that HHcy decreased endothelial SIRT1 expression and increased LOX-1 expression, subsequently causing reactive oxygen species generation, up-regulation of NADPH oxidase activity and NF-κB activation, thereby promoting pro-inflammatory response and cell apoptosis. Blockade of Sirt1 with Ex527 or siRNASIRT1 increased LOX-1 expression, whereas overexpression of SIRT1 decreased LOX-1 expression markedly. P-II treatment significantly increased SIRT1 expression and reduced LOX-1 expression, and protected against endothelial cells from Hcy-induced oxidative injury, inflammation and apoptosis. However, blockade of SIRT1 or overexpression of LOX-1 attenuated the therapeutic effects of P-II. In conclusion, our results suggest that P-II prevents the Hcy induced endothelial damage probably through regulating the SIRT1/LOX-1 signaling pathway.

Prognostic Utility of Soluble TREM-1 in Predicting Mortality and Cardiovascular Events in Patients With Acute Myocardial Infarction.

BACKGROUND: Triggering receptor expressed on myeloid cells-1 (TREM-1) is thought to be critical for inflammatory signal amplification and involved in the development of atherosclerosis. TREM-1 is significantly increased in patients with myocardial infarction. The aim of this study was to investigate the association between soluble TREM-1 (sTREM-1) and mortality and cardiovascular events in patients with acute myocardial infarction. METHODS AND RESULTS: We included 838 consecutive patients with acute myocardial infarction from October 7, 2012 to December 5, 2014. Blood samples were collected from patients with acute myocardial infarction immediately after diagnosis. During follow-up, 88 patients died, and 180 patients reached the combined end points of major adverse cardiovascular event (MACE). Patients with high sTREM-1 (higher than the median) had increased risk of all-cause mortality and MACE compared with those with low sTREM-1 (log-rank test, P<0.001). After adjustment for confounding risk factors by Cox regression analysis, high sTREM-1 remained an independent predictor of all-cause mortality (hazard ratio, 1.978; 95% confidence interval, 1.462-2.675; P<0.001) and MACE (hazard ratio, 2.413; 95% confidence interval, 2.022-2.879; P<0.001). After the addition of sTREM-1 to the reference model, the C-statistic for all-cause mortality increased from 0.86 to 0.89, and the difference was 0.023 (95% confidence interval, 0.0009-0.0477), and the C-statistic for MACE increased from 0.71 to 0.80, and the difference was 0.087 (95% confidence interval, 0.053-0.122). sTREM-1 levels were consistently positively associated with risks of all-cause mortality and MACE in various subpopulations, and there was no significant interaction among prespecified subgroups. CONCLUSIONS: sTREM-1 was significantly associated with all-cause mortality and MACE, independent of established conventional risk factors in patients with acute myocardial infarction.

Simvastatin suppresses vascular inflammation and atherosclerosis in ApoE(-/-) mice by downregulating the HMGB1-RAGE axis.

AIM: High mobility group box protein 1 (HMGB1) and receptor for the advanced glycation end product (RAGE) play pivotal roles in vascular inflammation and atherosclerosis. The aim of this study was to determine whether the HMGB1-RAGE axis was involved in the actions of simvastatin on vascular inflammation and atherosclerosis in ApoE(-/-) mice. METHODS: Five-week old ApoE(-/-) mice and wild-type C57BL/6 mice were fed a Western diet. At 8 weeks of age, ApoE(-/-) mice were administered simvastatin (50 mg·kg(-1)·d(-1)) or vehicle by gavage, and the wild-type mice were treated with vehicle. The mice were sacrificed at 11 weeks of age, and the atherosclerotic lesions in aortic sinus were assessed with Oil Red O staining. Macrophage migration was determined with scanning EM and immunohistochemistry. Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. Western blots were used to quantify the protein expression of HMGB1, RAGE, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). RESULTS: Vehicle-treated ApoE(-/-) mice exhibited significant increases in aortic inflammation and atherosclerosis as well as enhanced expression of HMGB1, RAGE, VCAM-1, and MCP-1 in aortic tissues as compared to the wild-type mice. Furthermore, serum total cholesterol, triglyceride and LDL levels were markedly increased, while serum HDL level was decreased in vehicle-treated ApoE(-/-) mice. Administration with simvastatin in ApoE(-/-) mice markedly attenuated the vascular inflammation and atherosclerotic lesion area, and decreased the aortic expression of HMGB1, RAGE, VCAM-1, and MCP-1. However, simvastatin did not affect the abnormal levels of serum total cholesterol, triglyceride, LDL and HDL in ApoE(-/-) mice. Exposure of HUVECs to HMGB1 (100 ng/mL) markedly increased the expression of HMGB1, RAGE and VCAM-1, whereas pretreatment of the cells with simvastatin (10 µmol/L) blocked the HMGB1-caused changes. CONCLUSION: Simvastatin inhibits vascular inflammation and atherosclerosis in ApoE(-/-) mice, which may be mediated through downregulation of the HMGB1-RAGE axis.

[Role of triggering receptor expressed on myeloid cells-1 on coxsackievirus B3-induced inflammation and cardiomyocyte injury].

OBJECTIVE: To determine the expression of TREM-1 (triggering receptor expressed on myeloid cells-1) in macrophages after coxsackievirus B3 (CVB3) infection and the cardiomyocytes viability after culturing with supernatant of macrophages in the absence and presence of TREM-1 inhibitor LP-17 to explore if TREM-1 is involved in the pathogenesis of CVB3 infection induced inflammation and cardiomyocytes injury. METHODS: TREM-1 mRNA and TREM-1 and DAP-12 protein expression in macrophages were detected by Real-time PCR at 0, 1, 4, 8 and 12 h and by Western blot at 0, 16, 24 and 48 h post CVB3 infection. TNF-α secretion of macrophages was measure by ELISA, vitality and the apoptosis degree of cardiomyocytes was assessed by CCK8 and Annexin V-FITC after the cardiomyocytes were cultured with the supernatant of macrophages in normal control group, CVB3 infection group and LP-17 pretreated CVB3 infection group. RESULTS: TREM-1 mRNA expression was significantly upregulated at 4, 8, and 12 h (peaked at 8 h) and TREM-1 protein expression was significantly upregulated at 16 and 24 h and returned to baseline level at 48 h after CVB3 infection. The protein expression of DAP-12, a direct downstream signaling molecule of TREM-1, also significantly increased at 24 and 48 h post CVB3 infection (P < 0.01). Level of macrophages secreted TNF-α post CVB3 infection was significantly reduced in LP-17 pretreated cells (P < 0.01), LP-17 pretreatment also significantly improved viability and significantly reduced apoptosis of cardiomyocytes cultured with supernatant of CVB3 infected macrophages (P < 0.01). CONCLUSION: TREM-1 might be an important mediator post CVB3 infection and a major player on inducing excess macrophages-related inflammation and resulting in an indirect injury to cardiomyocytes.

Pathogenesis of coxsackievirus B3-induced myocarditis: role of macrophage migration inhibitory factor.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is an upstream regulator in immune and inflammatory responses. However, its role in viral myocarditis remains unknown. In this study, we investigated the role of the MIF in coxsackievirus B3 (CVB3)-induced myocarditis. METHODS: Mice were randomized into two groups receiving either Eagle's minimal essential medium (EMEM, control group) or virus solution (infected group). Subsets of mice in the infected group were sacrificed on days 3, 7, 14 and 28 after inoculation. Expression of MIF was detected using an enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction and immunohistochemistry. A neutralizing antibody (Ab) to MIF was injected intraperitoneally from day 0 to 7 after inoculation. Disease severity was estimated by histopathology of the heart and by the heart weight to body weight ratio, and the interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in the myocardium were measured by ELISA on day 14. RESULTS: The serum MIF concentration and expression levels of myocardial MIF mRNA and protein were significantly elevated in mice on days 7 and 14 post-infection. The survival rate was markedly higher and disease severity was obviously less in mice treated with anti-MIF Ab. Furthermore, MIF blockade significantly decreased the IL-1ß and TNF-α in the myocarditic heart. CONCLUSION: These results demonstrate that MIF is an important naturally occurring inflammatory cytokine in CVB3-induced myocarditis, and anti-MIF Ab may lessen the inflammatory response.

[Correlation of cardiac troponin T gene mutations to hypertrophic cardiomyopathy in Chinese patients].

OBJECTIVE: To study cardiac troponin T (TNNT2) gene mutations in Chinese patients with hypertrophic cardiomyopathy (HCM) and analyze the correlation between the genotype and phenotype. METHODS: Ninety-five unrelated Chinese patients with HCM and 120 control individuals were screened for TNNT2 gene mutations. Seven exons (8, 9, 10, 11, 14, 15, and 16) in the functional regions of TNNT2 gene were amplified using PCR and the products were sequenced. The patients with positive results underwent further family screening. RESULTS AND CONCLUSION: This study did not find any HCM-caused mutations in TNNT2 gene, a result different from the reported rates of TNNT2 gene mutation ranging from 10% to 20% in other nations, suggesting that TNNT2 gene is not a susceptible gene for HCM in Chinese population.

[Association between myocardial calpain activation and apoptosis in lipopolysaccharide-induced septic mouse model].

OBJECTIVE: in septic mice, myocardial calpain was activated and induced caspase-3 activation, the association between calpain activation and apoptosis was explored in this experiment. METHODS: in in vivo model, adult C57 mice were injected with lipopolysaccharide (LPS, 4 mg/kg, i.p.) to induce sepsis. Myocardial calpain and caspase-3 activities, protein levels of calpain-1, calpain-2, calpastatin, Bcl-2 and Bid were detected by Western blot analysis and myocardial apoptosis was detected by TUNEL, myocardiac function was evaluated by Langendorff system. In in vitro model, adult rat cardiomyocytes were incubated with LPS (1 microg/ml) or co-incubated with calpain inhibitor-III (10 micromol/L), calpain activity, caspase-3 activity, protein levels of Bcl-2 and Bid, and cardiomyocyte apoptosis were detected. RESULTS: in septic mice, myocardial calpain and caspase-3 activity were increased up to 2.7- and 1.8-folds, respectively. Both calpain inhibitor-III and PD150606 significantly attenuated the increase of caspase-3 activity. Myocardial protein levels of calpain-1, calpain-2, calpastatin, Bcl-2 and Bid were similar between control and septic mice, and no cleavage of both Bcl-2 and Bid was found in septic mice. Calpain inhibitor-III significantly improved myocardial function in septic mice. In in vitro model, calpain and caspase-3 activities were increased after 4 h LPS treatment, co-treatment with calpain inhibitor-III prevented caspase-3 activity increase, protein Bcl-2 and Bid were similar between normal cardiomyocytes and LPS-treated cardiomyocytes. Cardiomyocyte apoptosis was similar in in vivo and in vitro septic models. CONCLUSION: myocardial calpain activity is increased in LPS induced septic mice, subsequent caspase-3 activation may contribute to myocardial dysfunction in septic mice without aggravating myocardial apoptosis and Bcl-2 and Bid are not involved on calpain induced caspase-3 activation in our model.

Curculigoside attenuates human umbilical vein endothelial cell injury induced by H2O2.

AIM OF THE STUDY: Vessel endothelium injury caused by reactive oxygen species (ROS) including H(2)O(2) plays a critical role in the pathogenesis of cardiovascular disorders. Therefore, agents or antioxidants that can inhibit production of ROS has highly clinical values in cardiovascular therapy. Curculigoside is the major bioactive compounds present in Curculigo orchioides, and possess potent antioxidant properties against oxidative stress insults through undefined mechanism(s). The present study was designed to test the hypothesis that curculigoside can inhibit H(2)O(2)-induced injury in human umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with curculigoside in the presence/absence of hydrogen peroxide (H(2)O(2)). The protective effects of curculigoside OP-D against H(2)O(2) were evaluated. RESULTS: HUVECs incubated with 400 µM H(2)O(2) had significantly decreased the viability of endothelial cells, which was accompanied with apparent cells apoptosis, the activation of caspase-3 and the upregulation of p53 mRNA expression. In addition, H(2)O(2) treatment induced a marked increase of MDA, LDH content and in intracellular ROS, decreased the content of nitric oxide (NO) and GSH-Px activities in endothelial cells. However, pretreatment with 0.5.5,10 µM curculigoside resulted in a significant recovery from H(2)O(2)-induced cell apoptosis. Also, it decreased other H(2)O(2)-induced damages in a concentration-dependent manner. Furthermore, pretreatment with curculigoside decreased the activity of caspase-3 and p53 mRNA expression, which was known to play a key role in H(2)O(2)-induced cell apoptosis. CONCLUSION: The present study shows that curculigoside can protect endothelial cells against oxidative injury induced by H(2)O(2), suggesting that this compound may constitute a promising intervention against cardiovascular disorders.

[Link between cardiac myosin binding protein-C gene mutation of Pro1208fs and Gly507 Arg and hypertrophic cardiomyopathy in Chinese patients].

OBJECTIVE: To detect gene mutations associated with hypertrophic cardiomyopathy (HCM) in Chinese patients and possible correlations between genotype and phenotype. METHODS: Twenty-one unrelated patients with hypertrophic cardiomyopathy were studied. The clinical data including symptoms, physical examination, echocardiography and electrocardiography were collected. The full ecoding exons of cardiac myosin-binding protein C gene (cMYBPC3) were amplified with PCR and the products were sequenced. RESULTS: Two mutations were identified in probands from two families. One mutation was frame shift mutation Pro1208fs in the exon 32 of the cMYBPC3 gene. Pro1208fs mutation was identified in a 59 years old female patient with familial hypertrophic cardiomyopathy. Symptom onset was late and a favorable clinical course was evidenced in this patient. Another mutation was missence mutation Gly507Arg in the exon 17 of the MYBPC3 gene identified in a 24 years old male patient. Diffuse thickness of left ventricular wall, impaired diastolic function and enlarged left atria were evidenced in echocardiography. No mutation was identified in the 80 control healthy individuals. CONCLUSION: cMYBPC3 might be the disease-causing genes in Chinese patients with hypertrophic cardiomyopathy.

[Association between expression of chromogranin A and myocardial fibrosis in patients with dilated cardiomyopathy].

OBJECTIVE: To observe the possible correlation between expression of chromogranin A (CGA) and myocardial fibrosis and investigate the potential role of CGA in the development of myocardial fibrosis in patients with dilated cardiomyopathy (DCM). METHODS: Surgical myocardial specimen from 10 DCM patients underwent successful orthotopic cardiac transplantation, and 3 normal myocardial specimen from brain-dead organ donors were obtained. CGA-mRNA, COLI-mRNA, COLIII-mRNA and ADAMTS-1-mRNA were analyzed by real-time PCR. The location and expression of CGA were assessed by immunohistochemistry(INH)with anti-CGA antibody. The collagen specific picrosirius red staining was applied on transversal myocardial slides and the collagen volume fraction (CVF) was calculated. The correlation between CGA and CVF was analyzed. RESULTS: Cytoplasmic expression of CGA assessed by INH showed large amount of strong positive granules densely arranged in the epicardial and endocardial myocardiocytes in DCM specimen while there was only few sparse granules in the normal myocardium (P < 0.05). CVF was significantly higher in DCM myocardial specimen than that in normal specimen (P < 0.001). CGA-mRNA was significantly correlated with COLI-mRNA (r = 0.729), COLIII-mRNA (r = 0.95) and ADAMTS-1-mRNA (r = 0.665, all P < 0.05). Moreover, collagen deposition location was almost identical with the strong positive expression location of CGA. CONCLUSION: We demonstrated for the first time that the deposition of CGA was related with the myocardial fibrosis in DCM heart, therefore, CGA might play an important role by influencing myocardial remodeling and fibrosis in DCM patients.

[Association between myocardial ADAMTS-1 expression and myocardial fibrosis in a murine model of viral myocarditis].

OBJECTIVE: To investigate the association between myocardial ADAMTS-1 expression and myocardial fibrosis in coxsackievirus B(3) (CVB(3))-induced acute and chronic murine myocarditis model. METHODS: Balb/c mice were infected with CVB(3) (single injection or monthly injection for 3 months) to establish acute or chronic myocarditis model. Normal controls received equal-volume Eagles minimal essential medium (EMEM) without CVB(3). Hearts were examined at 7 days or 3 months post CVB(3) infection. Heart slides were stained with collagen specific picrosirius red staining and the collagen volume fraction (CVF) was calculated with image analysis software. The expressions of ADAMTS-1 were determined by RT-PCR and immunohistochemistry. RESULTS: Compared with controls, the CVF levels and myocardial expressions of ADAMTS-1 were significant increased in two myocarditis groups, especially in mice with chronic myocarditis (P < 0.01). The increased expression of ADAMTS-1 was located in endochylema as visualized by immunohistochemistry. Myocardial ADAMTS-1 mRNA was positively correlated with CVF in both myocarditis groups (r(7 days) = 0.65, P < 0.05; r(3 months) = 0.73, P < 0.01). CONCLUSIONS: ADAMTS-1 increased in proportion with collagen accumulation in acute and chronic myocarditis, which might play an important role in the development of myocardial fibrosis by modulating the collagen metabolism.

Assessment of left ventricular systolic synchronicity by real-time three-dimensional echocardiography in patients with dilated cardiomyopathy.

BACKGROUND: Recent advances in real-time three-dimensional echocardiography (RT3DE) offer the potential to assess the left ventricular (LV) dyssynchrony simultaneously by analyzing the 17 segments time-volume curves. The purpose of this study was to test the feasibility and accuracy of RT3DE for quantitative evaluation of left ventricular systolic synchronicity. METHODS: Twenty-four patients with dilated cardiomyopathy (DCM) and twenty-five healthy volunteers were enrolled in this study. Full volume RT3DE was performed by using Philips IE33 with X3-1 probe. The global and 17-segmental time-volume curves were obtained by the on-line Qlab software (version 4.2). The time to minimal systolic volume in each segment (T(msv)) was taken to derive the following indexes of systolic asynchrony: T(msv) 16-SD, T(msv) 16-Dif, T(msv) 12-SD, T(msv) 12-Dif, T(msv) 6-SD and T(msv) 6-Dif, which meant the standard deviation or the maximal difference of T(msv) among the 16, 12 and 6 segments of the left ventricle respectively. The software also provided with each of the above parameters as a percentage of the cardiac cycle. RESULTS: T(msv) 16-SD, T(msv) 12-SD and T(msv) 6-SD were all significantly larger in the DCM group than those of the control group [T(msv) 16-SD: (52.9 +/- 40.6) ms vs (8.8 +/- 6.2) ms; T(msv) 12-SD: (29.5 +/- 30.8) ms vs (6.9 +/- 4.0) ms; T(msv) 6-SD: (28.9 +/- 34.6) ms vs (7.0 +/- 4.7) ms, all P < or = 0.001]. T(msv) 16-Dif, T(msv) 12-Dif and T(msv) 6-Dif were also significantly larger in the DCM group. There were close negative relations between the LVEF determined by RT3DE and each of the indexes of systolic asynchrony, among which the indexes of T(msv)-16-SD% and T(msv)-16-Dif% correlated most closely (r = -0.703 and r = -0.701, respectively). The DCM patients had significantly larger EDV and ESV, with significantly reduced LVEF compared with the healthy subjects. CONCLUSION: RT3DE provides a simple, useful and unique approach to assess the systolic synchronicity of all the left ventricular segments simultaneously.

[Inhibitory effect of CVB3-VP1 siRNA on CVB3 replication].

AIM: To inhibit the expression of CVB3 VP1 protein and the replication of CVB3 with synthesized siRNAs. METHODS: According to the sequence and secondary structure of CVB3 VP1 protein, four pieces of siRNAs were designed following the requirement from Journal of Nature Cell Biology were synthesized in Shanghai GeneChem Company. Then they were transfected into HeLa cells by liposome (Lipofectamine 2000), but the non-transfected cells and non-specific siRNAs were taken as control. 48 hours later, the patho-morphous changes were observed, virus titer changes were examined by TCID50, CVB3-VP1 protein expression were detected by immunofluorescence with FITC dyeing, and CVB3-RNA level was tested by semi-quantitative RT-PCR. RESULTS: Two pieces of the four specific synthesized siRNAs (VP1-1 and VP1-2) were found to have obvious inhibitory effect on CVB3 replication and VP1 protein expression were reduced greatly. Besides, the changes of pathological cells were obviously mitigated. CONCLUSION: Specific siRNAs can effectively inhibit the expression of CVB3 VP1 protein and the replication of CVB3 in HeLa cells.

[The expression and significance of myocardial cathepsin L in dilated cardiomyopathy].

OBJECTIVE: To clarify the expression of myocardial cathepsin L and its significance in the pathogenesis of dilated cardiomyopathy (DCM). METHODS: Myocardial tissue specimens derived from 20 patients undergoing orthotopic cardiac transplantation and 5 normal myocardium samples collected at autopsy from sudden death as controls were studied. The expression of cathepsin L was detected with immunohistochemistry, real time PCR and Western Blotting, respectively. At the same time, the relationship between the Cathepsin L mRNA expressional levels and the myocardial function (ejection fraction, EF) was investigated. RESULTS: The expression levels of cathepsin L mRNA and protein in DCM group were markedly elevated compared with those in control group and the expression levels of cathepsin L mRNA had significantly negative correlation with the myocardial function (ejection fraction, EF). CONCLUSION: Cathepsin L is markedly elevated in DCM myocardial tissue and it may participate in the pathogenesis of DCM.

[Upregulating the expression of angiogenesis-related genes by transplanting autologous mononuclear bone marrow cells into myocardial infarction scar and the periphery].

OBJECTIVE: To detect the regulation of angiogenic genes involved in the processes of collateral development. METHODS: Myocardial infarction (MI) scar was induced by cryoinjury in New Zealand rabbits. Four weeks after MI, 24 hours before cell transplantation, bone marrow was aspirated from the right thigh bone and mononuclear bone marrow cells (BMCs) were isolated by Ficoll density gradient centrifugation. Then the mononuclear BMCs (n = 8) or IMDM culture medium (n = 8) were transplanted into infarction scar and the periphery. Four weeks after mononuclear BMCs transplantation, DNA microarray analysis was performed to detect the regulation of angiogenesis-related genes in infarction scar and the periphery. And the differences of angiogenic genes expression were compared among several important growth factors by Western blot. RESULTS: DNA microarray analysis showed the detail regulation of genes involved in the angiogenic processes. There were 15 genes upregulated over 3 times in the infarction scar. In addition, we also found more genes are involved in the process of angiogenesis in its periphery than in the infarction scar (40 genes vs. 15 genes). Western bolt analysis further demonstrated that mononuclear BMCs transplantation was capable of increasing the levels of VEGF, FGF and Angiopoietin-I expression in the infarction scar and its periphery, compared with the control group, P < 0.05. CONCLUSION: These findings indicate that the natural angiogenic processes leading to collateral development are extremely complex, since many kinds of bone marrow-derived growth factors involved in the processes after mononuclear BMCs transplantation into infarction sites.

Microarray analysis of extracellular matrix genes expression in myocardium of mouse with Coxsackie virus B3 myocarditis.

BACKGROUND: Extracellular matrix (ECM) orchestrates cell behaviour including growth, death, apoptosis, adhesion, migration, and invasion by activating several signalling pathways. Certain components of ECM, such as integrins, may act as receptors or co-receptors of enterovirus. ECM-activated gene expressions in myocardium of viral heart disease including myocarditis and partial cardiomyopathy remain elusive. This study was to investigate the expression of ECM-activated genes in myocardium of mouse with viral myocarditis. METHODS: BALB/c mice were infected with Coxsackie virus B3 (CVB3) to establish an animal model of myocarditis. Uninfected mice were also prepared and served as controls. Specific mRNA expression pattern in myocarditic mouse heart was analysed by an in-house cDNA microarray containing 8,192 genes. Overexpressed ECM genes were selected and subsequently confirmed by Northern blot analysis. RESULTS: Nine ECM genes were isolated, from the array of 8,192 genes, as overexpressed genes in hearts of myocarditic mice in comparison with controls. Subsequent Northern blot analysis confirmed that four of the nine genes were highly expressed. Expression of these four genes, Fin15, ILk, Lamr1 and ADAMTS-1, has not been reported previously to be induced by Coxsackie virus. CONCLUSION: CVB3-induced myocarditis is associated with gene expression profiles of certain ECM components.

[A novel DNA vaccine containing endosome-fusogenic peptide prevents CVB3-induced myocarditis in mice].

OBJECTIVE: To enhance the effect of chitosan-pcDNA3-VP1 DNA vaccine to prevent onset of coxsackievirus B3 (CVB3)-induced myocarditis. METHODS: HA-pLys16 "carrier peptide" containing "endosome fusogenic peptide" and poly-Lysine was synthesized and complexed with DNA and chitosan sequentially, resulting chitosan-pcDNA3-VP1-HApLys16 vaccine. It was administrated to BALB/c mice with a total dose of 200 micro g pcDNA3-VP1. Mice were infected with 3 (LD(50) CVB3 3 weeks later to induce myocarditis. Loss of body weight, body/heart weight ratio, outlook and HE staining of heart tissues was observed and compared as an indicative of severity of myocarditis. RESULTS: chitosan-pcDNA3-VP1-HApLys16 vaccine intranasal immunization enhanced secretion of mucosal sIgA. After 3 x LD(50) CVB3 infection, pcDNA3 immunized mice groups lost their 5.75% weight following CVB3 infection, while chitosan-pcDNA3-VP1 and chitosan-pcDNA3-VP1-HApLys16 immunized mice increased their body weight by 1.75% and 5.44%. Body/heart weight ratio of chitosan-pcDNA3-VP1-HAplys16 group reached 193.77 suggesting lightened myocarditis. No myonecrosis or infiltrating immune cells existed in heart of chitosan-pcDNA3-VP1-HAplys16 immunized mice, and 1/6 of incidence and very light myocarditis were seen in chitosan-pcDNA3-VP1 immunized mice. While in pcDNA3 immunized mice 6/6 incidence and extremely severe myocarditis were observed. Heart from pcDNA3-immunized mice was bigger than normal ones and had a lot of white stripes, which were not observed in that of chitosan-pcDNA3-VP1 and chitosan-pcDNA3-VP1-HApLys16 immunized mice. CONCLUSION: Novel chitosan-pcDNA3-VP1-HApLys16 vaccine containing "endosome fusogenic peptide" could more effectively prevent CVB3-induced myocarditis than chitosan-pcDNA3-VP1 vaccine.

[Infection of Coxsackievirus group B type 3 regulates the expression profile of chemokines in myocardial tissue/cells].

OBJECTIVE: To investigate the role of Coxsackievirus group B type 3 (CVB3) infection on the expression profile of chemokines (ChKs) in myocardial tissue/cells. METHODS: CVB3 was inoculated into male BALB/c intraperitoneally and primary neonatal myocardial cells of BALB/c to establish CVB3 infection models in vivo and in vitro, where the expression profile of ChKs was detected at different time points post-infection as well as under different loading of CVB3 qualitatively and quantitatively by RT-PCR. RESULTS: The expression of MIP-2 and IP-10 was induced post-infection, while SDF-1, MCP-1, MCP-2, MCP-3, MCP-5, MDC, FKN and Ltn were constitutively expressed in myocardial tissue. The expression of MCP-1, MCP-2, MCP-3, MCP-5, MDC and Ltn increased 1.8, 1.9, 3.7, 1.7, 1.3 and 1.2 folds post-infection higher than that of uninfected control (P < 0.01). There was not significant difference in the expression of SDF-1 and FKN between infected myocardial tissue and uninfected myocardial tissue (P > 0.05). The expression of Eot was not detected in infected and uninfected myocardial tissue. Every chemokine had different expression at different infection time points. For example, the expression of MIP-2 at the 4th day was 1.1 and 1.5 times than that of the 7th and 14th day (P < 0.01). IP-10 showed similar expression between the 4th and 7th days (P > 0.05), which is 2.47 and 2.54 times compared to that of the 9th day (P < 0.01). And the expression of MCP-1 at the 14th day post-infection was 1.3, 1.2 and 1.0 times comparing to that of the 4th, 7th, 9th day post-infection, which showed statistical meaning. The expression of MCP-2 at the 4th was 1.4, 1.5 and 2.2 times comparing to that of the 7th, 9th, 14th day, and moreover, the expression was lower than that of the basal expression. The expression of MCP-1 and MCP-3 was up-regulated significantly, which occurred at different time points post-infection in vitro. While the expression of MIP-2 and MCP-5 was down-regulated in vitro. The expression patterns of MCP-2, MCP-3, MCP-5 and MDC were consistent with CVB3 loading. But that of the others (FKN, SDF-1, et al) were inconsistent with CVB3 loading. There was a correlation between change patterns of MCP-3 and CVB3 loading post-infection (r = 0.881, P < 0.05) within 14 days after infection. The varied expression trend of MCP-1 and MCP-3 was similar to the titer of anti-CVB3 antibody (r = 0.913, P = 0.031), while the expression of MCP-2, MCP-5 and MDC shows contrary change. There was not a significant correlation between change patterns of other ChKs (IP-10, SDF-1, et al) and the titer of anti-CVB3 antibody. A positive correlation between anti-CVB3 antibody and MCP-1 (r = 0.976, P < 0.05) was showed. CONCLUSION: The expression level and kind of ChKs in vivo and in vitro was varied significantly in clusters after CVB3 infection. The ChKs changed in clusters consisted of the expression profiles of ChKs. There were complexity and unbalance in the change of the expression of ChKs in every expression profile. It suggested that CVB3 infection could regulate the expression of ChKs in myocardial tissue/cells likely in different ways.