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.

Effects of the Cdc2-like kinase-family and DNA topoisomerase I on the alternative splicing of eNOS in TNF-alpha-stimulated human endothelial cells.

Nitric oxide (NO) is synthesized by endothelial nitric oxide synthase (eNOS) and plays an important role in vascular homeostasis and cardiovascular diseases. It has recently been shown that increased expression of alternatively spliced eNOS isoforms eNOS 13A, B and C and heterodimerization with 'full-length' eNOS is associated with a decreased eNOS activity. The regulatory pathways enabling this phenomenon are completely unknown. This study examined the effect of Cdc2-like kinases and DNA topoisomerase I on eNOS splicing in TNF-alpha-induced human umbilical vein endothelial cells (HUVECs). We found that inhibition of DNA topoisomerase I, but not Cdc2-like kinases, prevents the TNF-alpha-induced increase in eNOS isoform expression and NO reduction in HUVEC. Moreover, we show that the inhibition of DNA topoisomerase I or the Cdc2-like kinases differently modulates the phosphorylation of the serine/arginine-rich proteins SRp75 and SRp55. Our results demonstrate, for the first time, that DNA topoisomerase I but not Cdc2-like kinases serves as an important regulator of the differential eNOS isoform expression in endothelial cells, thereby modulating the TNF-alpha-induced eNOS activity switch.

Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity.

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.

High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway.

BACKGROUND: Transient receptor potential canonical type 6 (TRPC6) channels mediating 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium entry have been identified on human platelets. In the present study we tested the hypothesis that hyperglycemia increases the expression of TRPC6 channels. METHODS AND RESULTS: Platelets from healthy control subjects and patients with type 2 diabetes mellitus were incubated with glucose and calcium influx was measured using the fluorescent dye technique. TRPC channel protein expression was investigated using immunofluorescence and fluorescence microscopy of single platelets. Administration of 25 mmol/L glucose significantly enhanced the OAG-induced calcium influx, which was attenuated by inhibitors of the phosphatidylinositol 3-kinase, wortmannin or LY294002. The glucose-enhanced and OAG-induced calcium influx was concentration- and time-dependent. Glucose significantly increased the TRPC6 protein expression in platelets to 131+/-12% (n=33; P<0.05), whereas the expression of TRPC1, TRPC3, TRPC4, or TRPC5 were unchanged. The glucose-induced TRPC6 expression was significantly attenuated in the presence of wortmannin or LY294002. Platelets from patients with type 2 diabetes mellitus showed increased TRPC6 expression compared to nondiabetic individuals (P<0.05). CONCLUSIONS: The study indicates that high glucose increases TRPC6 channel protein expression on the platelet surface which is mediated by a phosphatidylinositol 3-kinase-dependent pathway.