Expression and functional characterization of two natural heparin-binding site variants of antithrombin.

Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions. SUMMARY: Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-κB or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.

Inorganic polyphosphate promotes cyclin D1 synthesis through activation of mTOR/Wnt/ß-catenin signaling in endothelial cells.

Essentials Polyphosphate (polyP) activates mTOR but its role in Wnt/ß-catenin signaling is not known. PolyP-mediated cyclin D1 expression (ß-catenin target gene) was monitored in endothelial cells. PolyP and boiled platelet-releasates induced the expression of cyclin D1 by similar mechanisms. PolyP establishes crosstalk between mTOR and Wnt/ß-catenin signaling in endothelial cells. SUMMARY: Background Inorganic polyphosphate (polyP) elicits intracellular signaling responses in endothelial cells through activation of mTOR complexes 1 and 2. Glycogen synthase kinase 3 (GSK-3) is known to be a negative regulator of mTOR and Wnt/ß-catenin signaling pathways. Objective The objective of this study was to investigate the effect of polyP on the expression, degradation and subcellular localization of the Wnt/ß-catenin target gene, cyclin D1, in endothelial cells. Methods Regulation of cyclin D1 expression, phosphorylation and subcellular localization by polyP or platelet releasates was monitored in the absence and presence of pharmacological inhibitors and/or siRNA for specific molecules of the upstream mTOR/Wnt/ß-catenin signaling network by established methods. Results Both synthetic polyP and boiled-platelet releasates induced the phosphorylation-dependent inactivation of GSK-3, thereby increasing the expression and nuclear localization, but inhibiting the degradation of cyclin D1. Inhibitors of mTORC1 (PI3K, AKT, PLC, PKC), rapamycin and siRNA for raptor (mTORC1-specific component) and ß-catenin, all inhibited polyP-mediated regulation of cyclin D1 expression, phosphorylation and subcellular localization in endothelial cells. The signaling effect of polyP was effectively inhibited by the recombinant extracellular domain of the receptor for advanced glycation end products (RAGE) and/or by the RAGE siRNA. Specific pharmacological inhibitors and siRNA knockdown of ERK1/2 and NF-κB pathways indicated that polyP-mediated cyclin D1 expression and nuclear localization are IKKɑ and ERK1/2 dependent, whereas its inhibitory effect on phosphorylation-dependent degradation of cyclin D1 is IKKß-dependent. Conclusion We conclude that a RAGE-dependent polyP-mediated crosstalk between mTOR and the GSK-3/Wnt/ß-catenin signaling network can modulate important physiological processes in endothelial cells.

Inorganic polyphosphate elicits pro-inflammatory responses through activation of the mammalian target of rapamycin complexes 1 and 2 in vascular endothelial cells.

BACKGROUND: Inorganic polyphosphate (polyP) elicits pro-inflammatory signaling responses in endothelial cells through interaction with two receptors, RAGE and P2Y1 . It is known that polyP activates mTOR signaling in breast cancer cells. OBJECTIVES: The objective of this study is to understand the mechanism of the polyP-mediated signaling pathway in endothelial cells and to determine whether polyP exerts its pro-inflammatory effect through activation of mTOR. METHODS: mTOR activation by polyP or platelet releasates in cellular and animal models was monitored in the absence and presence of pharmacological inhibitors and/or siRNA knockdown of specific signaling molecules. RESULTS: PolyP effectively induced phosphorylation of mTOR complex 1 (mTORC1) substrate, p70S6K, in endothelial cells by an AKT-dependent but ERK-independent mechanism. The siRNA knockdown of both RAGE and P2Y1 or specific inhibitors of the PI3K/PLC/PKC/Ca(2+) signaling axis inhibited polyP-mediated p70S6K phosphorylation. Moreover, either rapamycin or siRNA knockdown of raptor (mTORC1-specific component) abrogated polyP-mediated phosphorylation of p70S6K. By contrast, the siRNA knockdown of rictor (mTOR complex 2-specific component) but not raptor eliminated the barrier-disruptive effect of polyP. Specific NF-κB inhibitors abrogated polyP-mediated phosphorylation of p70S6K and rapamycin suppressed polyP-induced activation of NF-κB. Finally, specific inhibitors of the mTOR signaling network eliminated polyP-mediated vascular leakage and leukocyte recruitment in animal models. CONCLUSIONS: PolyP, through interaction with RAGE and P2Y1 , activates both the mTORC1 and mTORC2 signaling network. Both the pro-inflammatory and mTOR signaling functions of polyP are linked.

Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin.

Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-κB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders.

Trends in weights, heights, BMI and comparison of their differences in urban and rural areas for Iranian children and adolescents 2-18-year-old between 1990-1991 and 1999.

BACKGROUND: Secular trends in height and weight are interesting because in middle- and low-income countries they are a marker for changes in population health. The present study aims to evaluate the secular trend in height and weight and body mass index (BMI) of Iranian children and adolescents aged 2-18 years old between 1990-1991 and 1999 and compare the magnitude of urban-rural differences during this period for the first time in an Asian country. METHODS: Data from two national health surveys in 1990-1991 and 1999, of 22,349 and 25,196 weight and height measures of Iranian children and adolescents were used to study the trend and compare its difference in urban and rural children. Logarithmic transformation of weight, height and BMI was modelled as a polynomial in age for urban and rural boys and girls in each survey separately. The trend in urban and rural growth indexes (weight, height and BMI) and also the comparisons of urban-rural differences between two national surveys were tested in logarithmic scale using a weighted form of Z statistic for comparison of two means adjusted for age groups. RESULTS: Urban and rural boys and girls became taller and heavier (P≤ 0.02) with no change of BMI (P > 0.05) during the period. There was not any significant difference between the magnitudes of urban-rural difference between two surveys (P≥ 0.61). CONCLUSION: Although generally positive weight and height trend was observed among urban and rural residents, the magnitude of their differences was not changed.