[Sensitization to laboratory animal allergens among students and researchers exposed to laboratory rodents in Hokkaido university].

BACKGROUND: Based on a case who developed anaphylaxis after mouse bite which occurred at Hokkaido University, we studied on allergic sensitization prevalence for laboratory animals among students and researchers who are exposed to laboratory rodents and rabbit, for the purpose of allergy prevention, particularly anaphylaxis. METHODS: We carried out the health check-up on laboratory animal allergy (LAA) by questionnaires and specific-IgE antibody test for 555 rodents and/or rabbit handlers from whom informed consent was obtained. RESULT: Prevalence of positive IgE antibody higher than class 1 to mice, rats, hamsters, guinea pigs, and/or rabbits in the examinees was 14.1% (62/441) , 17.9% (50/279) , 18.8% (6/32) , 17.4% (4/23) , and 11.3% (12/106) , respectively. Moreover, among users of mouse, those who had allergic symptoms during contact with animals resulted in significantly higher positive rate for anti-mouse IgE antibody test than the other (38.1% vs 8.8%, p<0.01) . CONCLUSION: Health check-up including measurement of specific-IgE antibody against laboratory animals is useful for understanding allergic sensitization.

Hepatocyte growth factor regulates E box-dependent plasminogen activator inhibitor type 1 gene expression in HepG2 liver cells.

OBJECTIVE: We sought to determine the etiologic mechanism of pleiotropic growth factor, hepatocyte growth factor (HGF), as a regulator of hepatic synthesis of plasminogen activator inhibitor (PAI)-1, the physiological inhibitor of fibrinolysis and a potential inducer of atherothrombosis. METHODS AND RESULTS: HGF increased PAI-1 mRNA expression and PAI-1 protein accumulation in the conditioned media of human liver-derived HepG2 cells, and increased hepatic PAI-1 mRNA expression in vivo in mice. HGF-inducible PAI-1 mRNA was attenuated by U0126, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, and genistein, an inhibitor of tyrosine kinase. HGF increased the human PAI-1 promoter (-829 to +36 bp) activity, and deletion and mutation analysis uncovered a functional E box (5'-CACATG-3') at positions -158 to -153 bp. Electrophoretic mobility shift assays demonstrated that this E box binds upstream stimulatory factors (USFs). HGF phosphorylated USFs through MAPK and tyrosine kinase pathways. Co-transfection of USF1 expression vector increased PAI-1 promoter activity. Sterol regulatory element-binding protein-1 attenuated HGF-inducible PAI-1 promoter activity. CONCLUSIONS: Because USFs are involved in the regulation of carbohydrates and lipid metabolism, HGF-mediated PAI-1 production may provide a novel link between atherothrombosis and metabolic derangements. Targeting HGF signaling pathway may modulate the thrombotic risk in high-risk patients.

Increased expression of plasminogen activator inhibitor-1 by mediators of the acute phase response: a potential progenitor of vasculopathy in hypertensives.

Hypertension is an important risk factor for coronary atherosclerosis, which is accelerated by inflammation and diminished fibrinolysis. We have previously shown that levels of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis, are increased with atherogenic metabolic derangement. Because the liver is one of the major sources of circulating PAI-1, we here examined the effects of two proinflammatory cytokines, interleukin (IL)-1beta, and IL-6, on PAI-1 production in a human hepatoma cell line, HepG2. IL-1beta (1 ng/ml) and IL-6 (1 ng/ml) increased the accumulation of PAI-1 in the conditioned media over 24 h (IL-1beta: 2.1 +/- 0.2 (mean +/- SD) fold over the control; IL-6:1.4 +/- 0.2 fold; Western blot, p < 0.05). The increase in PAI-1 protein accumulation correlated with the increased expression of PAI-1 mRNA (Northern blot). An HMG-CoA reductase inhibitor (mevastatin, 10 micromol/l) attenuated the PAI-1 production induced by IL-1beta and IL-6. The plasma PAI-1 activity level was higher in hypertensives than in normotensives (10.0 +/- 9.8 AU/ml vs. 6.2 +/- 4.5 AU/ml, p < 0.05). The plasma PAI-1 antigen level was also higher in hypertensives than in normotensives (30.9 +/- 22.4 ng/ml vs. 24.4 +/- 13.3 ng/ml, p < 0.05). Thus, 1) IL-1beta and IL-6 can increase PAI-1 production in hepatic cells and 2) mevastatin may exert anti-thrombotic effects by decreasing the PAI-1 protein production induced by these proinflammatory cytokines. These results provide further insights into how inflammation is involved in the atherothrombotic complications observed in hypertensives, which may be ameliorated by HMG-CoA reductase inhibitors.

Induction of Tissue Factor Expression in Endothelial Cells by Basic Fibroblast Growth Factor and its Modulation by Fenofibric acid.

BACKGROUND: Tissue factor (TF), expressed in endothelial cells (ECs) and enriched in human atherosclerotic lesions, acts as a critical initiator of blood coagulation in acute coronary syndrome. Basic fibroblast growth factor (bFGF) induces the proliferation and migration of ECs and plays a role in angiogenesis and restoration of endothelial integrity. As TF is implicated in angiogenesis, we studied the effect of bFGF on TF gene and protein expression. Methods: Human umbilical vein ECs (HUVECs) were exposed to bFGF. TF mRNA was assessed by Northern blot and TF protein was assessed by Western blot. TF promoter activity was assessed by transient transfection assay and transcription factor was identified by electro mobility shift assay. RESULTS: bFGF increased TF mRNA and protein expression in HUVECs. Increased TF mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. Transient transfection assays of the human TF promoter-luciferase construct (-786/+121 bp) demonstrated that bFGF induced transcription was dependent on the elements within the -197 to -176 bp relative to the transcription start site of the human TF gene. This region contains NF-kappaB like binding site. Electro mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of NF-kappaB transcription factor to TF promoter. Nucleotide substitution to disrupt NF-kappaB like site reduced bFGF stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator activated receptor-alpha, reduced basal and bFGF stimulated TF expression. CONCLUSIONS: These results indicate that bFGF may increase TF production in ECs through activation of transcription at NF-kappaB binding site, and control coagulation in vessel walls. Fibrate can inhibit TF expression and therefore reduce the thrombogenecity of human atherosclerotic lesions.

Intracellular signal transduction modulating expression of plasminogen activator inhibitor-1 in adipocytes.

The concentrations in blood of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis and proteolysis, are elevated in obese and insulin-resistant subjects, predispose them to the risk of thrombosis, and may accelerate atherogenesis. Adipose tissue is a prominent source. Accordingly, intracellular signaling pathways that may influence PAI-1 expression in adipocytes have been the focus of considerable study. Rho, a small GTP binding and GTPase protein, when activated in turn activates its target, Rho-associated coiled-coil forming protein, to yield an active kinase, Rho-kinase, an effector in the Rho pathway. Rho-kinase exerts calcium-sensitizing effects in vascular smooth muscle cells and inhibitory effects on transforming growth factor-beta (TGF-beta) expression in chicken embryonic heart cells. Because TGF-beta is a powerful agonist of PAI-1 expression, we characterized the effects of inhibition of Rho-kinase in 3T3-L1 adipocytes. PAI-1 mRNA was determined by Northern blotting, and PAI-1 protein was determined by Western blotting. The Rho-kinase inhibitor, Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide], increased PAI-1 expression markedly. Although genistein, a flavonoid tyrosine kinase, attenuated the increase of PAI-1 induced by Y-27632, other non-flavonoid tyrosine kinase inhibitors did not. However, another flavonoid, daidzein, which lacks tyrosine kinase activity, decreased basal PAI-1 expression and attenuated the induction of PAI-1 expression by Y-27632. Thus, the Rho/Rho-kinase system inhibits PAI-1 expression by a flavonoid-sensitive mechanism in adipocytes. Therefore, flavonoids may be useful in decreasing elevated PAI-1 expression in adipose tissue and its consequent pathophysiologic sequelae.

Induction of plasminogen activator inhibitor-1 in endothelial cells by basic fibroblast growth factor and its modulation by fibric acid.

Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis and proteolysis. Basic fibroblast growth factor (bFGF) stimulates angiogenesis, which requires regional proteolysis. Because modulation of vasculopathy requires tight control of proteolysis, effects of bFGF on PAI-1 expression in endothelial cells (ECs) were characterized. bFGF increased PAI-1 mRNA and accumulation of PAI-1 protein in conditioned media in human umbilical vein ECs. The bFGF-mediated increase in PAI-1 mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. The rate of decrease in PAI-1 mRNA after actinomycin D treatment was not affected by bFGF. Transient transfection assays of the human PAI-1 promoter-luciferase construct demonstrated that bFGF-induced PAI-1 transcription was dependent on the elements within the -313 to -260 bp relative to the transcription start site. This region contains an E26 transformation specific 1 (Ets-1)-like site. Electrophoretic mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of the Ets-1-like transcription factor to the PAI-1 promoter. Nucleotide substitution to disrupt the Ets-1-like site reduced bFGF-stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator-activated receptor-alpha, inhibited basal and bFGF-stimulated PAI-1 expression. By inducing PAI-1 expression from ECs, bFGF may control proteolysis and fibrinolysis in vessel walls.