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Background/aims Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. Methods We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. Results Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. Conclusion We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.

Antithrombin (AT) is a major plasma glycoprotein of the serpin superfamily which regulates the proteolytic activity of the procoagulant proteases of both intrinsic and extrinsic pathways. Two important structural features that participate in the regulatory function of AT include a mobile reactive center loop (RCL) that binds to active-site of coagulation proteases, trapping them in the form of inactive covalent complexes and a basic D-helix that binds to therapeutic heparins and heparan sulfate proteoglycans (HSPGs) on vascular endothelial cells. The binding of D-helix of AT by therapeutic heparins promotes the reactivity of the serpin with coagulation proteases by several orders of magnitude by both a conformational activation of the serpin and a template (bridging) mechanism. In addition to its essential anticoagulant function, AT elicits a potent anti-inflammatory signaling response when it binds to distinct vascular endothelial cell HSPGs, thereby inducing prostacyclin synthesis. Syndecans-4 has been found as a specific membrane-bound HSPG receptor on endothelial cells that relays the signaling effect of AT to the relevant second messenger molecules in the signal transduction pathways inside the cell. However, following cleavage by coagulation proteases and/or by spontaneous conversion to a latent form, AT loses both its anti-inflammatory activity and high affinity interaction with heparin and HSPGs. Interestingly, these low-affinity heparin conformers of AT elicit potent proapoptotic and antiangiogenic activities by also binding to specific HSPGs by unknown mechanisms. This review article will summarize current knowledge about mechanisms through which different conformers of AT exert their serine protease inhibitory and intracellular signaling functions in these biological pathways.

Background and aims Angiopoietin-2 (ANG-2) mediates endothelial inflammation to initiate atherosclerosis and angiogenesis. Here we determined the serum levels of ANG-2 in hyperinsulinemic subjects and whether insulin increases its expression and release. Methods Healthy male subjects were recruited from the D-CLIP and CURES studies and, based on their fasting insulin levels, were classified as normoinsulinemic (n = 228) and hyperinsulinemic (n = 32). Serum proteins were estimated by ELISA. Endothelial inflammation was scored as the number of THP-1 monocytes adhered to HUVEC monolayer. Gene expression was determined with promoter reporter assays and semi-quantitative RT-PCR. Western blotting was used to assess changes in protein expression and activation. Immunofluorescence imaging and ChIP assay were used for nuclear localization and promoter binding studies, respectively. Results ANG-2 and sTIE2 levels were higher in hyperinsulinemic subjects. Hyperinsulinemic serum elicited endothelial inflammation, which was abrogated by an ANG-2 blocker antibody. Insulin (100 nM) increased mRNA and protein expression of ANG-2, and its release from HUVECs. It induced activation of p38 MAPK and an increase in protein levels and nuclear localization of cFOS. Binding of cFOS to the −640 to −494 promoter region mediated insulin dependent ANG-2 transcription. p38 MAPK inhibitor (25 μM) blocked insulin-induced nuclear localization of cFOS, expression of ANG-2 and ICAM-1, and release of ANG-2 into the culture medium. Spent medium collected from insulin treated cells enhanced endothelial inflammation, which was lost upon ANG-2 knockdown as well as upon p38 MAPK inhibition. Conclusions ANG-2 levels are high in hyperinsulinemic subjects and insulin induces expression and release of ANG-2 from HUVECs through p38 MAPK-cFOS pathway to elicit endothelial inflammation.

Objective: Native and latent conformers of AT (antithrombin) induce anti-inflammatory and proapoptotic signaling activities, respectively, in vascular endothelial cells by unknown mechanisms. Synd-4 (syndecan-4) has been identified as a receptor that is involved in transmitting signaling activities of AT in endothelial cells. Approach and Results: In this study, we used flow cytometry, signaling assays, immunoblotting and confocal immunofluorescence microscopy to investigate the mechanism of the paradoxical signaling activities of high-affinity heparin (native) and low-affinity heparin (latent) conformers of AT in endothelial cells. We discovered that native AT binds to glycosaminoglycans on vascular endothelial cells via its heparin-binding D-helix to induce anti-inflammatory signaling responses by recruiting PKC (protein kinase C)-δ to the plasma membrane and promoting phosphorylation of the Synd-4 cytoplasmic domain at Ser179. By contrast, the binding of latent AT to endothelial cells to a site(s), which is not competed by the native AT, induces a proapoptotic effect by localizing PKC-δ to the perinuclear/nuclear compartment in endothelial cells. Overexpression of a dominant-negative form of PKC-δ resulted in inhibition of anti-inflammatory and proapoptotic signaling activities of both native and latent AT. Conclusions: These results indicate that the native and latent conformers of AT may exert their distinct intracellular signaling effects through differentially modulating the subcellular localization of PKC-δ in endothelial cells.

Plasmodium falciparum-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) not only to disrupt the barrier-permeability function of endothelial cells but also to inhibit the antiinflammatory signaling function of AT. Here we investigated the mechanisms of the proinflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration, and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation, and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of P falciparum-derived HRPII in both cellular and animal models.

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM(−/−)) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM(−/−) cells that resulted in enhanced adhesion of leukocytes to TM(−/−) cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM(−/−) cells. In a flow chamber assay, isolated platelets adhered to TM(−/−) cells, forming characteristic VWF–platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TM(f/f) mice. Reexpression of the TM construct in TM(−/−) cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Protein C belongs to vitamin K-dependent family of serine protease zymogens in plasma which upon activation by thrombin in complex with thrombomodulin (TM) functions as an anticoagulant to degrade factors Va and VIIIa (FVa and FVIIIa) by limited proteolysis [1–3]. Activated protein C (APC) has a multi-domain structure composed of an N-terminal non-catalytic light chain linked by a disulfide bond to the C-terminal catalytic heavy chain with a trypsin-like specificity [4,5]. APC functions as an anticoagulant by proteolytic cleavage of at least two peptide bonds on heavy chains of factors FVa and FVIIIa bound to negatively charged membrane surfaces in extrinsic and intrinsic pathways of the clotting cascade [6–9]. The APC cleavage of cofactors culminates in disassembly of procoagulant activation complexes and inhibition of thrombin generation. The anticoagulant function of APC in degradation of FVa and FVIIIa requires the cofactor function of protein S, bound to the same negatively charged membrane surfaces in the presence of calcium [10,11]. In addition to its key anticoagulant role, APC also exhibits potent cytoprotective and antiinflammatory activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 [12–14]. Thus, protein C deficiency is associated with dysregulation of both coagulation and inflammatory pathways. Protein C deficiency has an autosomal dominant pattern of inheritance with heterozygous deficiency increasing the risk of venous thromboembolism and homozygous deficiency causing purpura fulminans, which is fatal if not treated by protein C replacement therapy [15,16]. Complete deficiency of protein C is not compatible with life in knockout mice [17]. Protein C deficiency is divided into type-I and type-II. Type-I deficiency is characterized by equally low antigen (PC:Ag) and activity (PC:A) levels, whereas type-II is defined by only a lower activity level [18]. Type-I deficiency is characterized by specific ELISA assays and type-II deficiency is measured by analysis of either amidolytic activity of APC after activating plasma protein C by the snake venom Protac or by anticoagulant activity in plasma-based assays using aPTT reagents [19]. If both amidolytic and anticoagulant activities are decreased, the deficiency is called type-IIa and if only anticoagulant activity is reduced the deficiency is classified as type-IIb [18,19]. Most type-IIa deficiencies occur on the catalytic doamin since mutations on the non-catalytic light-chain rarely influence APC amidolytic activity, whereas mutations on both heavy and light chains can adversely affect the APC anticoagulant activity. In this study, we identified novel and not previously reported protein C-deficient patients who experience venous thrombosis. Initial analysis of subjects’ plasma PC:Ag levels by a commercial ELISA and PC:A levels by both chromogenic and clotting assays showed these values are ~50% of normal, suggesting patients have type-I deficiency. However, results from a second commercial ELISA indicated PC:Ag levels of 84% and 102% for patients. Genetic analysis indicated subjects carry a heterozygous mutation (c.715G>A) in PROC, leading to Gly197 to Arg substitution (p.Gly197Arg) on the catalytic domain (G43R in chymotrypsin numbering) [20]. We expressed this protein C variant and discovered the variant is activated by thrombin with ~10-fold improved rate, however, TM has no cofactor activity in promoting its activation as tested in purified and plasma-based assay systems. Moreover, the APC mutant exhibited several hundred-fold impaired activity in both amidolytic and plasma-based coagulation assays. Structural-based analysis by molecular modeling indicated an Arg side-chain in APC can cause major distortions of the local structure, and adversely affect the folding of catalytic pocket and/or the calcium-binding-loop of the mutant. In support of this hypothesis, we discovered the initial commercial ELISA used to determine the PC:Ag level in subjects’ plasma, is incapable of detecting this mutant, explaining the 50% antigen level in the plasma. Thus the first commercial ELISA mischaracterized this type-IIa protein C deficiency as type-I.

Infection with Plasmodium species affects nearly 200 million annually with ~600,000 fatalities [1]. The clinical manifestation of the infection ranges from severe anemia, respiratory distress and cerebral malaria [2–4]. Infection with Plasmodium falciparum (Pf) is responsible for most of the fatalities associated with cerebral malaria and the survivors are often afflicted with long-term neurological problems and cognitive impairment [5]. The primary cause of death in the Pf-mediated cerebral malaria has been attributed to brain swelling, inflammation and perivascular edema resulting from the blood-brain barrier breakdown [3]. Upon entering circulation, Pf invades its host red blood cells and expresses hundreds of proteins as an essential process for maintaining viability and virulence [1]. One of these proteins, Pf Erythrocyte Membrane Protein 1 (PfEMP1) has been shown to bind endothelial protein C receptor (EPCR), thereby inhibiting the protein C anticoagulant and antiinflammatory pathways [6,7]. Another Pf secretory protein that modulates immune responses and coagulation is a histidine rich protein called HRPII [1,4,8]. HRPII is synthesized by Pf during asexual growth cycle in infected red blood cells and released upon their rupture [1]. The physiological role of HRPII is not known, though it has been hypothesized that it may be involved in detoxifying free heme released after hemoglobin degradation and in binding host glycosaminoglycans (GAGs) [1]. A recent study evaluating the effect of HRPII on blood coagulation discovered that HRPII specifically binds to some anticoagulant GAGs including heparan sulfate and dermatan sulfate [9]. Thus, HRPII was shown to effectively inhibit the cofactor function of high molecular weight heparins in inhibition of coagulation proteases, including factor Xa and thrombin, by antithrombin (AT) in in vitro assays [9]. Given its ability to bind anticoagulant heparins, it has been hypothesized that HRPII, released by the Pf-infected erythrocytes, can bind to GAGs in the microvasculature to prevent their interaction with AT, thereby contributing to coagulopathy associated with parasite infection [9,10]. Moreover, it was demonstrated that HRPII through activation of the inflammasome decreases the integrity of tight junctions and increases endothelial permeability, supporting the hypothesis that HRPII is a virulence factor that may contribute to cerebral malaria by disrupting the blood-brain barrier [8]. Recent results have indicated that AT elicits potent antiinflammatory signaling responses in vascular endothelial cells in response to cytokines and other proinflammatory stimuli [11–14]. The antiinflammatory activity of AT is mediated through the basic residues of the D-helix of the serpin binding to 3-OS containing vascular GAGs [15]. It has been demonstrated that the interaction of AT with vascular GAGs initiates antiinflammatory responses by inducing prostacyclin production that culminates in the inhibition of NF-κB activation, down-regulation of the expression vascular cell adhesion molecules and inhibition of the barrier-disruptive effects of proinflammatory cytokines [11–14,16]. In light of the findings that the Pf-derived HRPII can bind heparan sulfates to inhibit their cofactor activity in the AT-dependent inhibition of coagulation proteases together with the observation that HRPII may function as a virulence factor to contribute to cerebral malaria by compromising endothelial barrier integrity within the central nervous system, we hypothesized that HPRII counteracts the GAG-dependent antiinflammatory function of AT. To address this question, we expressed HRPII and evaluated its vascular activity in both cellular and animal models. The results demonstrate that a low concentration of HRPII is highly barrier-disruptive in vascular endothelial cells. In agreement with our hypothesis, HRPII effectively competes with AT for binding vascular GAGs. Moreover, we discovered that polyphosphates (polyP) bind HRPII with a high affinity to promote the inflammatory function of HRPII. This finding may have pathophysiological significance since it is known that Pf stores large amounts of short- and long-chain polyP’s in different blood stages of the infection [17].

Activated protein C (APC) is a vitamin-K dependent plasma serine protease, which functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. APC also modulates cellular homeostasis by exhibiting potent cytoprotective and anti-inflammatory signaling activities. The beneficial cytoprotective effects of APC have been extensively studied and confirmed in a number of preclinical disease and injury models including sepsis, type-1 diabetes and various ischemia/reperfusion diseases. It is now well-known that APC modulates downstream cell signaling networks and transcriptome profiles when it binds to the endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) on various cell types. However, despite much progress, details of the downstream signaling mechanism of APC and its crosstalk with other signaling networks are far from being fully understood. In this review, we focus on the cardioprotective properties of APC in ischemic heart disease and heart failure with a special emphasis on recent discoveries related to the modulatory effect of APC on AMP-activated protein kinase (AMPK), PI3K/AKT, and mTORC1 signaling pathways. The cytoprotective properties of APC might provide a novel strategy for future therapies in cardiac diseases.

UNLABELLED Essentials Mechanism of thrombin-induced inflammation is not fully understood. Thrombin induced monocyte adhesion and barrier loss require Angiopoietin-2 (Ang-2). Ang-2 mediates vessel leakage and monocyte adhesion through SHP-2/p38MAPK pathway. Calcium dependent SHP2/p38MAPK activation regulates Ang-2 expression through a feedback loop. SUMMARY Background Thrombin imparts an inflammatory phenotype to the endothelium by promoting increased monocyte adhesion and vascular permeability. However, the molecular players that govern these events are incompletely understood. Objective The aim of this study was to determine whether Angiopoietin-2 (Ang-2) has a role, if any, in regulating inflammatory signals initiated by thrombin. Methods Assessment of vascular leakage by Miles assay was performed by intra-dermal injection on the foot paw. Surface levels of intercellular adhesion molecule-1 (ICAM-1) were determined by flow cytometry. Overexpression, knockdown and phosphorylation of proteins were determined by Western blotting. Results In time-course experiments, thrombin-stimulated Ang-2 up-regulation, peaked prior to the expression of adhesion molecule ICAM-1 in human umbilical vein-derived endothelial cells (HUVECs). Knockdown of Ang-2 blocked both thrombin-induced monocyte adhesion and ICAM-1 expression. In addition, Ang-2(-/-) mice displayed defective vascular leakage when treated with thrombin. Introducing Ang-2 protein in Ang-2(-/-) mice failed to recover a wild-type phenotype. Mechanistically, Ang-2 appears to regulate the thrombin-activated calcium spike that is required for tyrosine phosphatase SHP2 and p38 MAPK activation. Further, down-regulation of SHP2 attenuated both thrombin-induced Ang-2 expression and monocyte adhesion. Down-regulation of the adaptor protein Gab1, a co-activator of SHP2, as well as overexpression of the Gab1 mutant incapable of interacting with SHP2 (YFGab1), inhibited thrombin-mediated effects, including downstream activation of p38 MAPK, which in turn was required for Ang-2 expression. Conclusions The data establish an essential role of the Gab1/SHP2/p38MAPK signaling pathway and Ang-2 in regulating thrombin-induced monocyte adhesion and vascular leakage.

The multifaceted role of mitogen-activated protein kinases (MAPKs) in modulating signal transduction pathways in inflammatory conditions such as infection, cardiovascular disease, and cancer has been well established. Recently, coagulation factors have also emerged as key players in regulating intracellular signaling pathways during inflammation. Among coagulation factors, thrombomodulin, as a high affinity receptor for thrombin on vascular endothelial cells, has been discovered to be a potent anti-inflammatory and anti-tumorigenic signaling molecule. The protective signaling function of thrombomodulin is separate from its well-recognized role in the clotting cascade, which is to function as an anti-coagulant receptor in order to switch the specificity of thrombin from a procoagulant to an anti-coagulant protease. The underlying protective signaling mechanism of thrombomodulin remains largely unknown, though a few published reports link the receptor to the regulation of MAPKs under different (patho)physiological conditions. The goal of this review is to summarize what is known about the regulatory relationship between thrombomodulin and MAPKs.

The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.

Background and aims: Studies suggest that Gentiana lutea (GL), and its component isovitexin, may exhibit anti-atherosclerotic properties. In this study we sought to investigate the protective mechanism of GL aqueous root extract and isovitexin on endothelial inflammation, smooth muscle cell migation, and on the onset and progression of atherosclerosis in streptozotocin (STZ)-induced diabetic rats. Methods and results: Our results show that both GL extract and isovitexin, block leukocyte adhesion and generation of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and rat aortic smooth muscle cells (RASMCs), following TNF-alpha and platelet derived growth factor-BB (PDGF-BB) challenges respectively. Both the extract and isovitexin blocked TNF-alpha induced expression of ICAM-1 and VCAM-1 in HUVECs. PDGF-BB induced migration of RASMCs and phospholipase C-gamma activation, were also abrogated by GL extract and isovitexin. Fura-2 based ratiometric measurements demonstrated that, both the extact, and isovitexin, inhibit PDGF-BB mediated intracellular calcium rise in RASMCs. Supplementation of regular diet with 2% GL root powder for STZ rats, reduced total cholesterol in blood. Oil Red O staining demonstrated decreased lipid accumulation in aortic wall of diabetic animals upon treatment with GL. Medial thickness and deposition of collagen in the aortic segment of diabetic rats were also reduced upon supplementation. Immunohistochemistry demonstrated reduced expression of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and vascular endothelial cadherin (VE-cadherin) in aortic segments of diabetic rats following GL treatment. Conclusions: Thus, our results support that GL root extract/powder and isovitexin exhibit anti-atherosclerotic activities. (C) 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Identifying agents that inhibit STAT-3, a cytosolic transcription factor involved in the activation of various genes implicated in tumour progression is a promising strategy for cancer chemoprevention. In the present study, we investigated the effect of dietary astaxanthin on JAK-2/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by examining the mRNA and protein expression of JAK/STAT-3 and its target genes. Quantitative RT-PCR, immunoblotting and immunohistochemical analyses revealed that astaxanthin supplementation inhibits key events in JAK/STAT signaling especially STAT-3 phosphorylation and subsequent nuclear translocation of STAT- 3. Furthermore, astaxanthin downregulated the expression of STAT-3 target genes involved in cell proliferation, invasion and angiogenesis, and reduced microvascular density, thereby preventing tumour progression. Molecular docking analysis confirmed inhibitory effects of astaxanthin on STAT signaling and angiogenesis. Cell culture experiments with the endothelial cell line ECV304 substantiated the role of astaxanthin in suppressing angiogenesis. Taken together, our data provide substantial evidence that dietary astaxanthin prevents the development and progression of HBP carcinomas through the inhibition of JAK-2/STAT-3 signaling and its downstream events. Thus, astaxanthin that functions as a potent inhibitor of tumour development and progression by targeting JAK/STAT signaling may be an ideal candidate for cancer chemoprevention.

Objective: The aim of this study was to determine subclinical inflammation in umbilical vein derived endothelial cells (HUVECs) obtained from Asian Indian subjects with gestational diabetes (GDM) and to determine levels of angiogenic factors and arginase activity in their cord blood. Methods: This case-control study included 38 control and 30 GDM subjects. Subjects were confirmed as GDM based on 75g oral glucose tolerance test (OGTT) conducted in the second trimester of pregnancy. Angiogenic markers and arginase activity were measured in cord blood by ELISA and colorimetric methods respectively. Endothelial inflammation was assessed through adhesion of PKH26-labelled leukocytes onto HUVEC monolayer obtained from the study groups. Gene and surface expression of adhesion molecules were confirmed via reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry respectively. Results: The study revealed increased adhesion of leukocytes to HUVECs isolated from GDM subjects compared to controls. HUVECs of babies born to GDM mothers had increased surface and mRNA expression of E-selectin. sTie2 levels were significantly higher in the cord blood for GDM subjects (3869 � 370 ng/L) compared to controls (3045 � 296 ng/L). Furthermore, arginase activity was higher in cord blood of GDM mothers as opposed to the control group (7.75 � 2.4 ?moles of urea/ml/hour vs 2.88 �0.49 ?moles of urea/ml/hour; p-value= 0.019). Spearman's correlation analysis revealed positive correlation of cord blood arginase activity with glucose intolerance (?=0.596, p=0.004) and post load glucose values (?=0.472, p=0.031) of mothers observed during the second trimester of pregnancy. Conclusions: HUVECs derived from Asian Indian GDM mothers, exhibit signs of sub-clinical endothelial inflammation along with increased levels of sTie2 and arginase activity in their cord blood serum. � 2013 Giri et al.

Objective-Insulin promotes adhesion of leukocytes to the endothelium through increased expression of surface adhesion molecules. We determined whether src-homology domain-2-containing protein tyrosine phosphatase 2 (SHP2), a downstream effecter of insulin signaling, is involved in insulin-induced endothelial inflammation. Methods and Results-In human umbilical vein-derived endothelial cells, treatment with insulin (100 nmol/L) increased Tyr phosphorylation, activity, and subsequently expression of SHP2. Increase in SHP2 accompanied a parallel decrease in the availability of the anti-inflammatory molecule, NO. This consequently enhanced the expression of cell adhesion molecules. Decrease in NO index was caused by endothelial NO synthase (eNOS) uncoupling and increased arginase activity. Among the 2 isoforms, insulin treatment induced the expression of arginase II. Inactivation of endogenous SHP2 via NSC87877 [8-hydroxy-7-(6-sulfonapthalen-2-yl)-diazenyl-quinoline-5-sulfonic acid] and its knockdown by small interfering RNA decreased arginase activity by blocking arginase II expression, however, it failed to restore eNOS coupling. Inactivation of SHP2 also abrogated insulin-mediated leukocyte adhesion by blocking the expression of adhesion molecules. Finally, downregulation of endogenous arginase II blocked insulin-mediated endothelial inflammation. Conclusion-SHP2 mediates chronic insulin-induced endothelial inflammation by limiting the production of NO in an eNOS-independent and arginase-II-dependent manner. � 2012 American Heart Association, Inc.