A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis.

We have discovered a novel small-molecule TAFIa inhibitor, BX 528, which is potent, highly selective against other carboxypeptidases and safe. The present study was to determine if BX 528 can enhance exogenous and endogenous thrombolysis in four different animal models. In the first three models, a thrombus was induced by FeCl (2) (dogs) or laser (rats) injury of the femoral artery, or formed ex vivo and implanted in the jugular vein in rabbits. A low dose of exogenous t-PA was given to induce a low-level thrombolysis on an established thrombus. Co-treatment with BX 528 further enhanced the thrombolytic effects induced by the exogenous t-PA and, thus, reduced thrombosis in all three animal models. In a second rat model, fibrin deposition in the lungs was induced by batroxobin, which was spontaneously resolved in 30 minutes due to the activation of endogenous fibrinolysis. Pre-treatment with lipopolysaccharide (LPS) attenuated this spontaneous fibrinolysis. Co-treatment with 10 mg/kg BX 528 prevented the LPS-induced attenuation of endogenous fibrinolysis. Thus, these studies demonstrated that inhibition of TAFIa by BX 528, our newly discovered small-molecule TAFIa inhibitor, enhanced both the exogenous (induced by a low dose of t-PA) and endogenous (LPS-induced resistance) thrombolysis without increasing the bleeding risk in four different animal models of thrombosis in different species (rat, dog and rabbit) employing different thrombogenic stimuli (FeCl (2) , laser, ex vivo and batroxobin) to induce thrombus formation in different tissues (artery, vein and lung microcirculation).

Inhibition of rho-kinase by hydroxyfasudil prevents vasopressin-induced myocardial ischemia in Donryu rats by attenuating coronary vasoconstriction.

BACKGROUND: Inhibition of rho-kinase has been shown to attenuate vasopressin (AVP)-induced myocardial ischemia measured as S-wave depression in Donryu rats. This has been attributed to a direct inhibitory effect on AVP-induced coronary vasoconstriction. However, since AVP also increased mean arterial blood pressure (MAP) which was attenuated by the rho-kinase inhibitors used, the prevention of myocardial ischemia could have been due to effects on afterload. RESULTS: The purpose of this study was to determine if rho-kinase inhibition prevents S-wave depression independent of the effects on blood pressure. In anesthetized Donryu rats (200-340 g), infusion of AVP (0.1 IU/kg) resulted in a sustained increase in MAP (DeltaMAP=46+/-7 mm Hg) and a transient S-wave depression (-90+/-20 microV). Infusion of phenylephrine titrated to achieve a comparable pressor response (DeltaMAP=52+/-2 mm Hg) resulted in a significantly smaller S-wave depression (-30+/-20 microV). Pretreatment with the rho-kinase inhibitor, hydroxyfasudil (3 mg/kg), decreased MAP by -28+/-2 mm Hg and significantly attenuated AVP-induced S-wave depression (-10+/-10 microV) compared to AVP. When rats were pretreated with phenylephrine titrated to maintain MAP, hydroxyfasudil still significantly attenuated AVP-induced S-wave depression (-14+/-12 microV). Hydralazine (1 mg/kg), which lowered MAP by -36+/-5 mm Hg, had no significant effect on AVP-induced S-wave depression (-105+/-32 microV). CONCLUSION: These data indicate that inhibition of rho-kinase with hydroxyfasudil attenuates AVP-induced myocardial ischemia independent of changes in MAP and are consistent with an inhibitory effect on coronary vasoconstriction.

Rev-erbalpha upregulates NF-kappaB-responsive genes in vascular smooth muscle cells.

Rev-erbalpha and retinoic acid receptor-related orphan receptor-alpha (RORalpha) are orphan nuclear receptors but their effects on transcription are opposed. Here, we show that Rev-erbalpha was expressed predominantly in vascular smooth muscle cells (VSMCs) rather than endothelial cells. Overexpression of Rev-erbalpha upregulated the expression of interleukin-6 and cyclooxygenase-2, and increased transactivation by NF-kappaB and translocation of p65 to the nucleus in A7r5 VSMCs. Furthermore, the expression of Rev-erbalpha was upregulated by RORalpha1 but that upregulation was attenuated by Rev-erbalpha itself in A7r5 VSMCs. These results suggest a regulatory link between Rev-erbalpha and the NF-kappaB pathway.

RORalpha1 and RORalpha4 suppress TNF-alpha-induced VCAM-1 and ICAM-1 expression in human endothelial cells.

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) is a nuclear orphan receptor. Adenovirus-mediated overexpression of RORalpha1 and RORalpha4 suppressed tumor necrosis factor-alpha (TNF-alpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells. Overexpression of RORalpha1 and RORalpha4 also suppressed TNF-alpha-stimulated translocation of p50 and p65 to the nucleus. In contrast, dominant-negative deletion mutants of RORalpha1 and RORalpha4 failed to suppress the induction of VCAM-1 and ICAM-1 and translocations of p50 and p65. These results suggest that RORalpha1 and RORalpha4 regulate the inflammatory responses via inhibition of the nuclear factor-kappaB signaling pathway in endothelial cells.

Studies on the correlation among the fucosylation index, concentration of alpha-fetoprotein and des-gamma-carboxy prothrombin as prognostic indicators in hepatocellular carcinoma.

Our previous results showed that the fucosylation index (FI) was considered to be a useful prognostic factor in patients with hepatocellular carcinoma (HCC). On the other hand, serum concentrations of alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) were regarded as prognostic indicators. However, the relationship among FI, AFP, and DCP as prognostic factors remained unknown. The aim of this study was to elucidate the correlation among these three prognostic factors. One hundred and seventy-six patients with HCC from 1990 to 1998, who showed increment of serum AFP concentrations more than 30ng/ml before treatment, were examined in the present study. FI was determined in these patients by crossed immunoaffino-electrophoresis in the presence of Lens culinaris agglutinin. FI of AFP was defined as the percentage of the L. culinaris agglutinin (LCA)-reactive species in total AFP (same as L3 fraction). Serum concentrations of DCP were also measured. Enrolled patients with HCC underwent transcatheter arterial embolization, chemoembolization, percutaneous ethanol injection, and/or percutaneous microwave coagulation therapy. The current patients status was the one which was confirmed at the end of March 2001. Analysis by the Cox's proportional hazards model showed that FI, AFP, and DCP were significant prognostic factors. When the tentative demarcation levels of FI, AFP, and DCP were set at 18%, 200ng/ml, and 0.06arbitrary units (AU)/ml, respectively, the following results for the prognostication of patients with HCC were obtained. First, the survival rates in the groups with one out of the three optional markers over the demarcation level were significantly lower than the survival rates of other groups, whose optional one marker was equal to or less than the demarcation level, respectively. Next, the survival rates in the groups in which two out of three optional markers were over the demarcation levels were lower than the survival rates of other groups, whose optional two markers were equal to or less than the demarcation levels, with high significance. On the contrary, there was absence or attenuation of statistically significant differences in the survival rates between the groups in which two of the three optional markers showed no accordant results (high FI and low AFP versus low FI and high AFP, low FI and high DCP versus high FI and low DCP, high DCP and low AFP versus low DCP and high AFP). Finally, we compared the survival rates between the HCC groups, whose optional one marker was over the demarcation level and whose remainders were equal to or less than the demarcation levels and another HCC group whose optional one marker was equal to or less than the demarcation level and whose remainders were over the demarcation levels to reconfirm the weight of each prognostic factor. These comparisons together with Cox's analysis showed that the weight of each prognostic factor in the survival rates is consecutively ordered as DCP, FI, and AFP. The present study indicates that measurements of FI, AFP, and DCP from the sera before the initial treatment improve prognostic estimates and appraisal of the therapeutic outcome in patients with HCC.

The fucosylation index of serum alpha-fetoprotein as useful prognostic factor in patients with hepatocellular carcinoma in special reference to chronological changes.

Aim of this study was to establish fucosylation index (FI) of alpha-fetoprotein (AFP) before and after initial treatment as a useful prognostic factor in patients with hepatocellular carcinoma (HCC). One hundred ninety-seven patients with HCC from 1990 to 2000, in whom an increment of serum AFP concentrations more than 30 ng/ml was observed before treatment, were examined in the present study. Enrolled patients with HCC underwent transcatheter arterial embolization, chemoembolization, percutaneous ethanol injection and/or percutaneous microwave coagulation therapy. The current patients status was confirmed as of the end of March 2001. FI was determined by crossed immunoaffinoelectrophoresis in the presence of Lens culinaris agglutinin (LCA). FI of AFP was defined as the percentage of the LCA-reactive species in total AFP (same as L3 fraction). When the tentative discriminating line of FI was set at 18%, the mean survival rate in the HCC group, whose FI-1 (before treatment) was higher than 18% (high FI), was significantly lower than that in another HCC group, whose FI-1 was equal to or less than 18% (low FI) by the generalized Wilcoxon test and the log rank test (P<0.0001). There were statistical significant differences of survival rate when FI-2 (2 months after treatment) and FI-3 (at the time of HCC recurrence or 2 years after treatment in the case of no recurrence) were introduced in the same analysis. Additionally, statistical significant differences of survival rates were obtained between HCC groups with high and low FI-1 when the patient stage was limited to II, III, IVA or IVB. The HCC group, FI-1, FI-2 and FI-3 of which were persistently equal to or less than 18%, showed considerably better prognosis than the group, whose FI-1, FI-2 and FI-3 were persistently higher than 18%. The univariate analysis in the prognostic factor by the Cox's proportional hazards model showed that FI-1, FI-2 and FI-3 were independent prognostic factors. The present study indicates that measuring FI from the sera before and after the treatment serves as a new prognostic indicator and may improve prognostic estimates and appraisal of therapeutic outcome in patients with HCC.