Inhibition of coagulation factor Xa improves myocardial function during CVB3-induced myocarditis.

INTRODUCTION: Myocarditis is induced by coxsackievirus B3 (CVB3). Myocardial inflammation is tied to the activation of coagulation. Coagulation factor (F) Xa, a central player in coagulation, activates matrix metalloproteinases (MMP), which modulate the remodeling. AIMS: In this study, we investigated the effects of pharmacological FXa inhibition on myocardial function, inflammation, and remodeling during a CVB3-induced myocarditis. METHODS AND RESULTS: Immune cells and matrix proteins were detected by immunohistochemistry. The expression of cytokines was measured by real-time PCR and the activity of MMP-2 by zymography. Left ventricular function was analyzed using microconductance pressure catheter. Treatment with the FXa inhibitor fondaparinux led to an improved left ventricular function in CVB3-induced mice compared to saline-treated controls (dPdtmax: fondaparinux 4632 ± 499.6 vs. saline 3131 ± 374.0 [mmHg/s], P = 0.0503; SV: fondaparinux 33.19 ± 4.893 vs. saline 19.32 ± 2.236 [µL], P < 0.118; CO: fondaparinux 15124 ± 2183 vs. saline 8088 ± 1035 [µL/min], P < 0.05). Therapy with fondaparinux reduced the activity of MMP-2 (fondaparinux 1.208 ± 0.1247 vs. saline 1.565 ± 0.05476, P < 0.05). The collagen type I/III ratio as well as the expression of TIMP-1 was comparable in both infection groups postinfectionem (p.i.), despite an increased infiltration of macrophages into the hearts of mice treated with fondaparinux 8 days p.i. (CD68+: fondaparinux 494.2 ± 64.73 vs. saline 306.9 ± 43.73 [cells/mm(2) ], P < 0.05). Anti-inflammatory CD206-positive M2-type macrophages were increased in the infected hearts after fondaparinux treatment (CD206+: fondaparinux 182.1 ± 18.18 vs. saline 111.6 ± 21.07 [cells/mm(2) ], P < 0.05), whereas CD80-positive M1-type macrophages were comparable in both groups. CONCLUSION: In conclusion, selective inhibition of FXa improves the left ventricular function during CVB3-induced myocarditis and seems to be associated with an improved myocardial remodeling.

PAR-1 contributes to the innate immune response during viral infection.

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3-induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1(-/-) mice expressed reduced levels of IFN-ß and CXCL10 during the early phase of infection compared with Par1(+/+) mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-ß and CXCL10 expression. Par1(-/-) mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1(+/+) mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-ß expression and contributes to the innate immune response during single-stranded RNA viral infection.

Overexpression of alternatively spliced tissue factor induces the pro-angiogenic properties of murine cardiomyocytic HL-1 cells.

BACKGROUND: Tissue factor (TF) is expressed in 2 isoforms: membrane-bound "full length" (fl)TF and soluble alternatively spliced (as)TF. flTF is the major thrombogenic form of TF. Although the function of asTF is poorly understood, it was suggested that asTF contributes to tumor-associated growth and angiogenesis. In the heart of a developing embryo, asTF is expressed much later compared to flTF, but in adult heart, asTF exhibits a distribution pattern similar to that of flTF. Thus, it is possible that asTF may play a role in heart development via pro-angiogenic signaling. The purpose of the present study was to examine the effects of murine asTF overexpression in murine cardiomyocyte-like HL-1 cells on their pro-angiogenic potential, the chemotaxis of monocytic cells, and the expression of fibroblast growth factor-2 (FGF2), cysteine-rich 61 (Cyr61), and vascular endothelial growth factor (VEGF). METHODS AND RESULTS: Expression of FGF2, Cyr61 and VEGF was assessed on reverse transcription-polymerase chain reaction and western blot. Cell migration, proliferation, and endothelial tube formation assays were carried out. It was found that overexpression of murine asTF in HL-1 cells increases their proliferation and pro-angiogenic properties. The supernatant of murine asTF-overexpressing HL-1 cells induces the chemotaxis of monocytic cells. CONCLUSIONS: Overexpression of murine asTF in murine cardiomyocytic cells increases their proliferation, monocyte migration, and pro-angiogenic properties -possibly- mediated by the induction of the pro-migratory and pro-angiogenic factors FGF2, Cyr61 and VEGF. Thus, we propose that murine asTF may serve as a migration- and angiogenesis-promoting factor.

Role of the phosphatidylinositol 3-kinase/protein kinase B pathway in regulating alternative splicing of tissue factor mRNA in human endothelial cells.

BACKGROUND: Tissue factor (TF) is the primary initiator of blood coagulation. In response to tumor necrosis factor (TNF)-alpha human umbilical vein endothelial cells (HUVECs) express 2 TF isoforms: a soluble alternatively spliced isoform (asHTF) and membrane-bound "full length" (fl)TF. How the differential TF isoform expression is regulated is still unknown. This study compared the impact of PI3K/Akt pathway inhibition on alternative splicing of TF in HUVECs, to the influence of transcriptional regulation by inhibiting nuclear factor kappa B (NFkappaB). METHODS AND RESULTS: The mRNA expression of TF isoforms was assessed by real-time PCR, the thrombogenic activity was measured by a chromogenic TF activity assay and the phosphorylation state of serine/arginine-rich (SR) proteins was analyzed by western blotting. Transfection of HUVECs was done 72 h before the inhibition experiments were performed. PI3K/Akt pathway inhibition reduced the mRNA expression of asHTF but not flTF. Inhibition of NFkappaB reduced the expression of both isoforms. Moreover, the PI3K/Akt pathway inhibition, but not that of NFkappaB, modified the phosphorylation of the SR proteins SRp75, SRp55 and SF2/ASF. Additionally, overexpression of SF2/ASF and SRp75 influenced the differential TF-isoform expression in HUVECs. CONCLUSIONS: The PI3K/Akt pathway modulates alternative splicing of TF in HUVECs, distinct from transcriptional regulation.