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.

[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.

[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.

[Effect of astragaloside on myocardial fibrosis in chronic myocarditis].

OBJECTIVE: To investigate the effect of astragaloside (Astr), one of the active components of the Chinese medical herb Astragulus membranaceus, on cardiac fibrosis in chronic myocarditis and its relevant mechanisms. METHODS: Eighty mice were randomized into 3 groups, the control group (n=20), the model group (n=30) and the Astr group (n=30). Mice in the model group and the Astr group were monthly intraperitoneally inoculated with CVB3, but to the control group equal amount of culture fluid was given instead. Mice in the control and the model group were fed with drinking water while those in the Astr group with drinking water containing Astr-sodium carboxymethycellulose at a concentration of 300 mg/L. All the survived mice were sacrificed 3 months later. Heart tissue of mice was stained by picrosirius red for calculating collagen volume fraction (CVF) with an automatic image analysis system. Expressions of transforming growth factor beta1 (TGF-beta1), platelet derived growth factor (PDGF), matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1), MMP-13 and MMP-14 in heart tissue were detected by Western blot analysis. RESULTS: As compared with the model group, in the Astr group, the mortality and CVF were significantly lower (53.3% vs. 23.3%, chi2 = 4.23, P < 0.05), and (17.4 +/- 1.2% vs. 8.6 +/- 0.9%, chi2 = 5.38, P < 0.05), respectively. As compared with the control group, Western blot analysis showed that expression of TGF-beta1 was decreased, MMP-1 and TIMP-1 were down-regulated, while expressions of MMP-13 and MMP-14 were up-regulated after Astr treatment. CONCLUSION: Astr could lower the mortality and alleviate the myocardial fibrosis of mice with chronic myocarditis. Its antifibrotic effect might be realized by way of inhibiting TGF-beta1 expression and up-regulating the expressions of MMP-13 and MMP-14 in the heart tissues.

[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.

Hepatitis B virus infection and coronary atherosclerosis: results from a population with relatively high prevalence of hepatitis B virus.

AIM: To investigate the possible association between hepatitis B virus (HBV) infection and angiographically proven coronary artery disease (CAD) in a population with relatively high prevalence of HBV. METHODS: Sera from 434 patients who underwent coronary angiography were tested for HBV antigens (HBsAg, HBeAg) and antibodies (Anti-HBs, Anti-HBc and Anti-HBe) by ELISA. RESULTS: Seventy-seven percent (224/291) of the patients with CAD and 73.4% (105/143) of the patients without angiographic evidence of atherosclerosis were seropositive for HBV (P > 0.05). However, C-reactive protein (CRP) levels were significantly higher in patients with CAD (P = 0.008), while lower in HBV seropositive population (P = 0.043 and P = 0.021 after adjustment for conventional risk factors). CONCLUSION: Our results suggested HBV infection negatively correlates with CRP levels, but seems not to be associated with coronary atherosclerosis.

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.

[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.

Cloning, Sequencing and Expression of the atpE Gene of Broad Bean Chloroplast.

The ninth KpnI fragment (about 4.0 kb) of broad bean chloroplast DNA contains the entire chloroplast atpE gene and atpB gene. This fragment was inserted into the pUC18 polylinker region, yielding plasmid pUK1. Southern hybridization using atpE gene from maize chloroplast as probe revealed that atpE gene encoding epsilon subunit of broad bean chloroplast was localized in 0.9 kb ClaI fragment of pUK1. The whole sequence of atpE gene of broad bean chloroplast was determined by using primer F and primer R of the polylinker region of pBluescript KS (+) DNA. The entire atpE gene of broad bean chloroplast was inserted into the polylinker region of vector pJLA505 to form recombinant plasmids pJLA505-atpE. The expression plasmid was transformed into E. coli DH5alpha cells which were then induced at 42 degrees. By analysis with SDS-PAGE, the product of interest accounted for more than 38% of total E. coli cell proteins. Identification of the expressed product by Western blot analysis demonstrated that it can react specifically with anti-epsilon serum of maize chloroplast CF(1). The expressed product aggregated to form insoluble inclusion bodies and was purified. The purified product had the same function as that of the native epsilon subunit.