Endogenous tissue factor pathway inhibitor in vascular smooth muscle cells inhibits arterial thrombosis.

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor-mediated coagulation. TFPI is expressed by endothelial and smooth muscle cells in the vasculature. Endothelium-derived TFPI has been reported to play a regulatory role in arterial thrombosis. However, the role of endogenous TFPI in vascular smooth muscle cells (VSMCs) in thrombosis and vascular disease development has yet to be elucidated. In this TFPIFlox mice crossbred with Sma-Cre mice were utilized to establish TFPI conditional knockout mice and to examine the effects of VSMC-directed TFPI deletion on development, hemostasis, and thrombosis. The mice with deleted TFPI in VSMCs (TFPISma) reproduced viable offspring. Plasma TFPI concentration was reduced 7.2% in the TFPISma mice compared with TFPIFlox littermate controls. Plasma TFPI concentration was also detected in the TFPITie2 (mice deleted TFPI in endothelial cells and cells of hematopoietic origin) mice. Plasma TFPI concentration of the TFPITie2 mice was 80.4% lower (P < 0.001) than that of the TFPIFlox mice. No difference in hemostatic measures (PT, APTT, and tail bleeding) was observed between TFPISma and TFPIFlox mice. However, TFPISma mice had increased ferric chloride-induced arterial thrombosis compared with TFPIFlox littermate controls. Taken together, these data indicated that endogenous TFPI from VSMCs inhibited ferric chloride-induced arterial thrombosis without causing hemostatic effects.

Conditional knockout of TFPI-1 in VSMCs of mice accelerates atherosclerosis by enhancing AMOT/YAP pathway.

BACKGROUND: Tissue factor pathway inhibitor-1 (TFPI-1) has multiple functions and its precise role and molecular mechanism during the development of atherosclerosis are not clear. OBJECTIVES: To determine the effect and molecular mechanism of TFPI-1 deficiency in vascular smooth muscle cells (VSMCs) in atherosclerosis in the apolipoprotein E knockout (ApoE-/-) mouse. METHODS AND RESULTS: A mouse model with a conditional knockout of TFPI-1 in VSMCs in an atherosclerosis-prone background (ApoE-/-) was generated. Mice were fed a high fat diet for 18weeks and were then euthanized. Arterial trees and aortas were stained with Sudan IV and were labeled via immunohistochemistry. Cell proliferation and migration of VSMCs in atherosclerotic plaques were assessed. More atherosclerotic lesions and higher levels of proliferation and migration of VSMCs were observed in TFPI-1fl/fl/Sma-Cre+ApoE-/-mice. An interaction between TFPI-1 and angiomotin (AMOT) was identified in human VSMCs by mass spectrometry, immunoprecipitation and co-localization analyses. Signal pathway changes were detected by Western blot analysis, and the expression levels of target genes were determined by real-time PCR. Decreased phosphorylation of AMOT and Yes-associated protein 1 (YAP) in TFPI-1fl/fl/Sma-Cre+ApoE-/- mice resulted in increased expression levels of snail family zinc finger 2 (SLUG) and connective tissue growth factor (CTGF), which are target genes of the Hippo signaling pathway that have been verified as atherosclerosis candidate genes. CONCLUSION: Deficiency in TFPI-1 in the VSMCs of ApoE-/- mice accelerated the development of atherosclerosis by promoting the proliferation and migration of VSMCs which may be caused by the decreased phosphorylation of AMOT and YAP. SIGNIFICANCE: TFPI-1 has been found to has an anticoagulant activity, induce cell apoptosis and prevent cell proliferation. For the first time, we constructed a line of conditional knockout mice in which the TPFI-1 gene is deleted in VSMCs. We found that TFPI-1 deficiency clearly promoted the development of atherosclerosis when these mice were crossed into an ApoE-/-background. One notable feature of atherosclerosis is the proliferation and migration of smooth muscle cells. Previous reports involved TFPI-1 do not completely explain the proliferation and migration of VSMCs because heterozygous TF deficient (TF±) mice bred in an ApoE-/- background did not show diminished atherosclerosis compared to TF+/+ mice bred in the same background. Our results first confirmed that TFPI-1 interacts with AMOT, which led to a decrease in the phosphorylation of YAP and further increased the genes expression of the proliferation and migration involved. Our results further confirmed that atherosclerosis was a localized disease.

Endothelial cell-anchored tissue factor pathway inhibitor regulates tumor metastasis to the lung in mice.

Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of the tissue factor (TF)-initiated coagulation pathway. Both circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis. However, the significance of endothelial cell-anchored TFPI in cancer biology remains largely unexplored. We generated mice with full-length disruption of TFPI (including TFPIα and TFPIß isoforms) in endothelial cells, using a Cre-LoxP system and gene inactivation (GI) strategy. Experimental pulmonary tumor metastasis models were used with TFPI-deficient mice to evaluate the role of endothelial cell-anchored TFPI in cancer progression. Finally, lung microvascular permeability and microenvironment were investigated. TFPI-deficient mice were viable and fertile, and showed decreased plasma TFPI levels and lung TFPI levels as compared with their control littermates. TFPI deficiency in endothelial cells promoted pulmonary tumor metastasis with an increased vascular permeability and altered lung microenvironment. Our observations suggest that endothelial cell-anchored TFPI controls lung tumor metastasis, and does so largely through the inhibition of local TF-induced thrombin generation and the regulation of the lung microenvironment in mice.

Antioxidant and prooxidant effects of lanthanum ions on Vicia faba L. seedlings under cadmium stress, suggesting ecological risk.

The present study combined chemical analyses and biological measurements to investigate biphasic effects of La on Cd stress in leaves of Vicia faba seedlings, which were hydroponically cultivated for 15 d in the combination of 6 µM CdCl(2) and 2 to 480 µM La(NO(3))(3), respectively. The results showed that contents of Cd first elevated above and then declined below the 6 µM single Cd treatment when 2 to 30 µM extraneous La were combined. Contents of mineral nutrients altered differentially and became imbalanced. No distinct band was observed in catalase (CAT), guaiacol peroxidase (GPX), or ascorbate peroxidase (APX) patterns, but in superoxide dismutase (SOD) isozymes by the supplementation with 8 to 480 µM of extraneous La. Superoxide dismutase and APX activities changed as a U-shaped curve; however, CAT and GPX changed as an inverted U-shaped curve along with increasing La. Moreover, heat shock protein 70 (HSP 70) production was reduced below the single treatment of Cd at 2 to 8 µM of extraneous La and enhanced thereafter. Thus, La at lower concentrations promoted antioxidation against Cd stress; La at higher concentrations turned to prooxidant effects, implicating potential ecological risk. Heat shock protein 70, combined with the antioxidant enzymes, constitutes an integrative defense system, which can be used to estimate the degree of antioxidation or prooxidation of extraneous La to Cd-induced oxidative stress in the seedlings.

Comparative impact of cadmium on two phenanthrene-degrading bacteria isolated from cadmium and phenanthrene co-contaminated soil in China.

Alcaligenes faecalis strain J08 and Brevundimonas sp. strain X08 were isolated from soils co-contaminated by cadmium (Cd) and polycyclic aromatic hydrocarbons (PAHs) in Northeast China. The two strains of bacteria were identified by phenotypic tests and 16S rDNA. Different Cd treatments (0.01 mM, 0.1mM, 0.5mM) showed no significant influence (p>0.05) on the biodegradation of phenanthrene by A. faecalis strain J08. Brevundimonas sp. strain X08 also presented no significant differences in the biodegradation of phenanthrene in Cd treatments (0.01 mM, 0.1mM). The growth of Brevundimonas sp. strain X08 was prohibited significantly (p<0.05) by Cd in the concentration of 0.5mM, but the biodegradation of phenanthrene in this group was not impaired. The specific biodegradation rate of Brevundimonas sp. strain X08 in the 0.5mM Cd group was significantly (p<0.05) higher than rates in other Cd treatments (0mM, 0.01 mM, 0.1mM).