Thrombomodulin: a multifunctional receptor modulating the endothelial quiescence.

Thrombomodulin (TM) is a type 1 receptor best known for its function as an anticoagulant cofactor for thrombin activation of protein C on the surface of vascular endothelial cells. In addition to its anticoagulant cofactor function, TM also regulates fibrinolysis, complement, and inflammatory pathways. TM is a multidomain receptor protein with a lectin-like domain at its N-terminus that has been shown to exhibit direct anti-inflammatory functions. This domain is followed by 6 epidermal growth factor-like domains that support the interaction of TM with thrombin. The interaction inhibits the procoagulant function of thrombin and enables the protease to regulate the anticoagulant and fibrinolytic pathways by activating protein C and thrombin-activatable fibrinolysis inhibitor. TM has a Thr/Ser-rich region immediately above the membrane surface that harbors chondroitin sulfate glycosaminoglycans, and this region is followed by a single-spanning transmembrane and a C-terminal cytoplasmic domain. The structure and physiological function of the extracellular domains of TM have been extensively studied, and numerous excellent review articles have been published. However, the physiological function of the cytoplasmic domain of TM has remained poorly understood. Recent data from our laboratory suggest that intracellular signaling by the cytoplasmic domain of TM plays key roles in maintaining quiescence by modulating phosphatase and tensin homolog signaling in endothelial cells. This article briefly reviews the structure and function of extracellular domains of TM and focuses on the mechanism and possible physiological importance of the cytoplasmic domain of TM in modulating phosphatase and tensin homolog signaling in endothelial cells.

Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin.

BACKGROUND: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. METHODS: We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. RESULTS: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a ß-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both ß-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by ß-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. CONCLUSIONS: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.

Thrombomodulin Regulates PTEN/AKT Signaling Axis in Endothelial Cells.

BACKGROUND: We recently demonstrated that deletion of thrombomodulin gene from endothelial cells results in upregulation of proinflammatory phenotype. In this study, we investigated the molecular basis for the altered phenotype in thrombomodulin-deficient (TM-/-) cells. METHODS: Different constructs containing deletions or mutations in the cytoplasmic domain of thrombomodulin were prepared and introduced to TM-/- cells. The phenotype of cells expressing different derivatives of thrombomodulin and tissue samples of thrombomodulin-knockout mice were analyzed for expression of distinct regulatory genes in established signaling assays. RESULTS: The phosphatase and tensin homolog were phosphorylated and its recruitment to the plasma membrane was impaired in TM-/- cells, leading to hyperactivation of AKT (protein kinase B) and phosphorylation-dependent nuclear exclusion of the transcription factor, forkhead box O1. The proliferative/migratory properties of TM-/- cells were enhanced, and cells exhibited hypersensitivity to stimulation by angiopoietin 1 and vascular endothelial growth factor. Reexpression of wild-type thrombomodulin in TM-/- cells normalized the cellular phenotype; however, thrombomodulin lacking its cytoplasmic domain failed to restore the normal phenotype in TM-/- cells. Increased basal permeability and loss of VE-cadherin were restored to normal levels by reexpression of wild-type thrombomodulin but not by a thrombomodulin construct lacking its cytoplasmic domain. A thrombomodulin cytoplasmic domain deletion mutant containing 3-membrane-proximal Arg-Lys-Lys residues restored the barrier-permeability function of TM-/- cells. Enhanced phosphatase and tensin homolog phosphorylation and activation of AKT and mTORC1 (mammalian target of rapamycin complex 1) were also observed in the liver of thrombomodulin-KO mice. CONCLUSIONS: These results suggest that the cytoplasmic domain of thrombomodulin interacts with the actin cytoskeleton and plays a crucial role in regulation of phosphatase and tensin homolog/AKT signaling in endothelial cells.

Activated protein C inhibits mesothelial-to-mesenchymal transition in experimental peritoneal fibrosis.

BACKGROUND: In addition to its anticoagulant function in downregulating thrombin generation, activated protein C (APC) evokes pleiotropic cytoprotective signaling activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) in endothelial cells. OBJECTIVES: To investigate the protective effect of APC in a chlorhexidine gluconate (CG)-induced peritoneal fibrosis model. METHODS: Peritoneal fibrosis was induced in wild-type as well as EPCR- and PAR1-deficient mice via daily injection of CG (0.2 mL of 0.1% CG in 15% ethanol and 85% saline) for 21 days with or without concomitant injection of recombinant human APC derivatives (50 µg/kg of bodyweight). The expression of proinflammatory cytokines and profibrotic markers as well as collagen deposition were analyzed using established methods. RESULTS: CG significantly upregulated the expression of transforming growth factor-ß1 in peritoneal tissues, which culminated in the deposition of excessive extracellular matrix proteins, thickening of the peritoneal membrane, and mesothelial-to-mesenchymal transition in damaged tissues. APC potently inhibited CG-induced peritoneal fibrosis and downregulated the expression of proinflammatory cytokines, collagen deposition, Smad3 phosphorylation, and markers of mesothelial-to-mesenchymal transition (α-smooth muscle actin, vimentin, and N-cadherin). APC also inhibited transforming growth factor-ß1-mediated upregulation of α-smooth muscle actin, Smad3, and fibronectin in human primary mesothelial cells. Employing signaling-selective and anticoagulant-selective variants of APC and mutant mice deficient for either EPCR or PAR1, we demonstrated that the EPCR-dependent signaling function of APC through PAR1 activation was primarily responsible for its antifibrotic activity in the CG-induced peritoneal fibrosis model. CONCLUSION: APC and signaling-selective variants of APC may have therapeutic potential for preventing or treating pathologies associated with peritoneal fibrosis.

Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability.

Thrombin-induced endothelial permeability is associated with various pathological conditions. Apoptosis signal-regulating kinase-1 (ASK1), one of the upstream MAP3K, has been reported to be an important regulator of endothelial stress and apoptosis. Despite this, its role in endothelial permeability is unknown. The aim of this study was to determine the role of ASK1 in thrombin-induced endothelial permeability. To do so, a live cell monitoring system and transwell assay were used to evaluate in vitro endothelial permeability, while a Miles assay was used for in vivo permeability. Immunofluorescence and western blotting were used to visualize integrity of the junctions and phosphorylation of various proteins, respectively. We observed that in vivo thrombin-induced vascular permeability was attenuated in Ask1-/- mice. Pretreatment of human primary endothelial cells (ECs) with GS-4997 (ASK1 inhibitor) and deficiency of ASK1 in primary mouse lung ECs significantly attenuated the thrombin-induced endothelial permeability. Furthermore, in the presence of GS-4997, the following were also significantly reduced: thrombin-induced para-cellular gap formation, VE-cadherin proteolysis, and dislocation of VE-cadherin, JAM-A, and ZO1 from the junctions. Inhibition of ASK1 restored peripheral location of F-actin, similar to that induced by sphingosine-1-phosphate. These results suggest a unique role for ASK1 in regulating thrombin-induced endothelial permeability.

Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation.

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.

Extracellular Histones Bind Vascular Glycosaminoglycans and Inhibit the Anti-Inflammatory Function of Antithrombin.

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.

Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells.

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-TMf/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.

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

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.

Anticoagulant and signaling functions of antithrombin.

Antithrombin (AT) is a major plasma glycoprotein of the serpin superfamily that 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 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.

Hyperinsulinemia promotes endothelial inflammation via increased expression and release of Angiopoietin-2.

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.

PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells.

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.

Gly197Arg mutation in protein C causes recurrent thrombosis in a heterozygous carrier.

BACKGROUND: Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis. OBJECTIVE: The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation. METHODS: We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems. RESULTS: The activation of protein C-G197R by thrombin was improved ~10-fold; however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive because it showed no activity in an aPTT assay. Anti-inflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket. CONCLUSION: The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis.

Plasmodium falciparum histidine rich protein HRPII inhibits the anti-inflammatory function of antithrombin.

BACKGROUND: It has been reported that histidine-rich protein II (HRPII), secreted by the malaria parasite, Plasmodium falciparum (Pf), inhibits the heparin-dependent anticoagulant activity of antithrombin (AT) in vitro and in plasma-based assay systems. OBJECTIVE: The objective of this study was to test the hypothesis that HRPII may also interact with the AT-binding vascular glycosaminoglycans (GAGs), thereby inhibiting the anti-inflammatory signaling function of the serpin. METHODS: We expressed HRPII in bacteria, purified it to homogeneity and studied its effect on endothelial cell signaling in the absence and presence of AT employing established signaling assays. RESULTS: We demonstrate that a low concentration of HRPII potently disrupts the barrier permeability function of endothelial cells. Moreover, HRPII competitively inhibits the protective effect of AT by a concentration-dependent manner. Similarly, AT inhibits the pro-inflammatory activity of HRPII by a concentration-dependent manner. The siRNA knockdown of 3-O-sulfotransferase 1 (3-OST-1), the enzyme responsible for the essential 3-O-sulfation of the AT-binding GAGs, downregulates the pro-inflammatory function of HRPII in endothelial cells, supporting the hypothesis that HRPII competitively inhibits the interaction of AT with 3-OS containing vascular GAGs. Histidine-rich protein II elicits its barrier-disruptive effect by the Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and AT counteracts this effect. We further demonstrate that inorganic polyphosphates bind HRPII with a high affinity to amplify the pro-inflammatory signaling function of HRPII in both cellular and in vivo permeability models. CONCLUSION: We postulate that Pf-derived HRPII and polyphosphate can contribute to the pathogenesis of malaria infection by downregulating the AT-dependent anti-inflammatory and anticoagulant pathways.

The Cardioprotective Signaling Activity of Activated Protein C in Heart Failure and Ischemic Heart Diseases.

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.

Thrombomodulin Regulation of Mitogen-Activated Protein Kinases.

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.

Activated protein C inhibits lipopolysaccharide-mediated acetylation and secretion of high-mobility group box 1 in endothelial cells.

Essentials APC elicits cytoprotective responses in endothelial cells via EPCR-dependent cleavage of PAR1. APC inhibits LPS-mediated translocation and extracellular secretion of HMGB1 in endothelial cells. Signaling activity of APC inhibits LPS-mediated acetylation of HMGB1 by epigenetic mechanisms. APC inhibits LPS-mediated HMGB1 expression in CD31-positive endothelial cells in cremaster muscle. SUMMARY: Background Activated protein C (APC) inhibits high-mobility group box 1 (HMGB1) signaling and its lipopolysaccharide (LPS)-mediated release by endothelial protein C receptor (EPCR)-dependent activation of protease-activated receptor 1 (PAR1) in endothelial cells. Post-translational acetylation is known to modulate the subcellular localization of HMGB1, and its hyperacetylated form is translocated to the cytoplasm of innate immune cells before being secreted into the extracellular space. Objective To determine whether APC inhibits LPS-mediated HMGB1 secretion from endothelial cells by modulating its acetylation status. Methods The subcellular localization of HMGB1 in LPS-treated endothelial cells was monitored in the absence and presence of APC by western blot analysis of fractionated cell lysates and confocal immunofluorescence microscopy. Results Both western blot and immunofluorescence data indicated that APC effectively inhibits LPS-mediated translocation of HMGB1 from the nucleus to the cytoplasm by EPCR-dependent and PAR1-dependent mechanisms. When EPCR was ligated by the Gla-domain of protein C/APC, thrombin also inhibited LPS-mediated HMGB1 translocation. Further studies revealed that APC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm by inhibiting LPS-mediated hyperacetylation of HMGB1 by (de)acetylating enzymes. Furthermore, the translocated HMGB1 was found to be associated with lysosome-associated membrane protein 1 in LPS-treated endothelial cells. The in vivo relevance of these findings was investigated in the mouse cremaster muscle, and this demonstrated that both wild-type APC and a signaling-selective mutant of APC inhibit LPS-mediated HMGB1 expression and translocation in CD31-positive endothelial cells. Conclusion These results suggest that APC inhibits LPS-mediated cytoplasmic translocation and secretion of HMGB1 in endothelial cells by epigenetic mechanisms.

Tie1 controls angiopoietin function in vascular remodeling and inflammation.

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 ß1 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.

Astaxanthin inhibits JAK/STAT-3 signaling to abrogate cell proliferation, invasion and angiogenesis in a hamster model of oral cancer.

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.