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Background: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism., Methods and Results: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice ( P <0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice ( P <0.01 and P <0.05), but not in TLR3 -/- mice, by reinforcing neutrophil recruitment ( P <0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion ( P <0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation ( P <0.05). Finally, eRNA triggered plasma clot formation in vitro ( P <0.01)., Conclusions: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening complication of a pulmonary embolism (PE) whose incidence and predictors are not precisely determined., Objective: To determine the frequency and predictors for CTEPH after a first unprovoked PE., Patients/methods: In a randomized trial comparing an additional 18-month warfarin versus placebo in patients after a first unprovoked PE initially treated with vitamin K antagonist for 6 months, we applied recommended CTEPH screening strategies through an 8-year follow-up to determine cumulative incidence of CTEPH. CTEPH predictors were estimated using Cox models. Pulmonary vascular obstruction (PVO) and systolic pulmonary arterial pressure (sPAP) at PE diagnosis and 6 months were studied by receiver operating curves analysis. All CTEPH cases and whether they were incident or prevalent were adjudicated., Results: During a median follow-up of 8.7 years, nine CTEPH cases were diagnosed among 371 patients, with a cumulative incidence of 2.8% (95% confidence interval [CI] 0.95-4.64), and of 1.31% (95% CI 0.01-2.60) after exclusion of five cases adjudicated as prevalent. At PE diagnosis, PVO > 45% and sPAP > 56 mmHg were associated with CTEPH with a hazard ratio (HR) of 33.00 (95% CI 1.64-667.00, p = .02) and 12.50 (95% CI 2.10-74.80, p < .01), respectively. Age > 65 years, lupus anticoagulant antibodies and non-O blood groups were also predictive of CTEPH. PVO > 14% and sPAP > 34 mmHg at 6 months were associated with CTEPH (HR 63.90 [95% CI 3.11-1310.00, p < .01]and HR 17.2 [95% CI 2.75-108, p < .01])., Conclusion: After a first unprovoked PE, CTEPH cumulative incidence was 2.8% during an 8-year follow-up. PVO and sPAP at PE diagnosis and at 6 months were the main predictors for CTEPH diagnosis., (© 2022 International Society on Thrombosis and Haemostasis.)

Reduced blood flow velocity in the vein triggers inflammation and is associated with the release into the extracellular space of alarmins or damage-associated molecular patterns (DAMPs). These molecules include extracellular nucleic acids, extracellular purinergic nucleotides (ATP, ADP), cytokines and extracellular HMGB1. They are recognized as a danger signal by immune cells, platelets and endothelial cells. Hence, endothelial cells are capable of sensing environmental cues through a wide variety of receptors expressed at the plasma membrane. The endothelium is then responding by expressing pro-coagulant proteins, including tissue factor, and inflammatory molecules such as cytokines and chemokines involved in the recruitment and activation of platelets and leukocytes. This ultimately leads to thrombosis, which is an active pro-inflammatory process, tightly regulated, that needs to be properly resolved to avoid further vascular damages. These mechanisms are often dysregulated, which promote fibrinolysis defects, activation of the immune system and irreversible vascular damages further contributing to thrombotic and inflammatory processes. The concept of thrombo-inflammation is now widely used to describe the complex interactions between the coagulation and inflammation in various cardiovascular diseases. In endothelial cells, activating signals converge to multiple intracellular pathways leading to phenotypical changes turning them into inflammatory-like cells. Accumulating evidence suggest that endothelial to mesenchymal transition (EndMT) may be a major mechanism of endothelial dysfunction induced during inflammation and thrombosis. EndMT is a biological process where endothelial cells lose their endothelial characteristics and acquire mesenchymal markers and functions. Endothelial dysfunction might play a central role in orchestrating and amplifying thrombo-inflammation thought induction of EndMT processes. Mechanisms regulating endothelial dysfunction have been only partially uncovered in the context of thrombotic diseases. In the present review, we focus on the importance of the endothelial phenotype and discuss how endothelial plasticity may regulate the interplay between thrombosis and inflammation. We discuss how the endothelial cells are sensing and responding to environmental cues and contribute to thrombo-inflammation with a particular focus on venous thromboembolism (VTE). A better understanding of the precise mechanisms involved and the specific role of endothelial cells is needed to characterize VTE incidence and address the risk of recurrent VTE and its sequelae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pilard, Ollivier, Gourdou-Latyszenok, Couturaud and Lemarié.)

Background: Epidemiologic studies indicate that early life arsenic exposures are linked to an increased risk of cardiovascular diseases. Different oxidation and methylation states of arsenic exist in the environment and are formed in vivo via the action of arsenic ( + 3 oxidation state) methyltransferase (As3MT). Methylated arsenicals are pro-atherogenic postnatally, but pre- and perinatal effects are unclear. This is particularly important because methylated arsenicals are known to cross the placenta., Objectives: We tested the effects of early life exposure to inorganic and methylated arsenicals on atherosclerotic plaque formation and its composition in apolipoprotein E knock-out ( apoE - / - ) mice and evaluated whether apoE - / - mice lacking As3MT expression were susceptible to this effect., Methods: We exposed apoE - / - or apoE - / - / As 3 MT - / - mice to 200  ppb inorganic or methylated arsenic in the drinking water from conception to weaning and assessed atherosclerotic plaques in the offspring at 18 wk of age. Mixed regression models were used to estimate the mean difference in each outcome relative to controls, adjusting for sex and including a random effects term to account for within-litter clustering., Results: Early life exposure to inorganic arsenic, and more profoundly methylated arsenicals, resulted in significantly larger plaques in the aortic arch and sinus in both sexes. Lipid levels in these plaques were higher without a substantial difference in macrophage numbers. Smooth muscle cell content was not altered, but collagen content was lower. Importantly, there were sex-specific differences in these observations, where males had higher lipids and lower collagen in the plaque, but females did not. In mice lacking As3MT, arsenic did not alter the plaque size, although the size was highly variable. In addition, control apoE - / - / As 3 MT - / - mice had significantly larger plaque size compared with control apoE - / - ., Conclusion: This study shows that early life exposure to inorganic and methylated arsenicals is pro-atherogenic with sex-specific differences in plaque composition and a potential role for As3MT in mice. https://doi.org/10.1289/EHP8171.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells., Competing Interests: The authors have declared that no competing interests exist.

Morbidity and mortality from venous thromboembolism (VTE), which refers to deep vein thrombosis and pulmonary embolism, have a substantial effect on the global burden of disease. The field of venous thrombosis research has been dramatically changed over the past 10 years with the improvement of animal models that shed some light on the interaction between inflammation and thrombosis. Important recent advances provided evidence of the implication of the innate immune system in venous thrombosis. In this review, we highlighted the cytokines and chemokines that regulate mechanisms of thrombus formation and resolution. Cytokines are pleiotropic, redundant, and multifunctional endogenous mediators orchestrating the inflammatory responses leading to thrombus formation or resolution. The use of experimental models has revealed the pro-thrombotic activity of some cytokines including interferon-γ, interleukin (IL)-6, chemokine ligand 2, IL-17A, IL-9, IL-1β, and transforming growth factor-β. Other cytokines such as IL-10, tumor necrosis factor-α, and IL-8 appear to promote thrombus resolution in late phase of venous thromboembolism. The purpose of this review is to bring together the current knowledge regarding the cytokines and chemokines that have been involved in thrombosis formation and resolution. We postulate that an imbalance between pro-thrombotic and anti-thrombotic cytokines/chemokines may be involved in the pathophysiology of VTE. However, in-depth basic and clinical research in venous thrombosis is still require to fully understand the precise mechanism of action of these cytokines., (© 2020 International Society on Thrombosis and Haemostasis.)

Essentials Neutrophil extracellular traps (NETs) are generated during deep vein thrombosis (DVT). The role of interferon γ (IFNγ) and natural killer (NK) cells in NET formation was studied. IFNγ promote venous thrombosis through NET formation. NK cell depletion reduces DVT. SUMMARY: Background Neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs), but the mechanism triggering their formation remains unclear. In vitro data show that interferon (IFN)-γ induces the formation of NETs. Objectives To determine whether IFN-γ and the transcription factor T-box expressed on T cells (Tbet) promote venous thrombosis through neutrophil activation. Methods Venous thrombosis was induced by flow restriction in the inferior vena cava in IFN-γ -/- , Tbet -/- or wild-type (WT) mice. After 48 h, thrombus size was measured by the use of high-frequency ultrasound. NET formation was determined by immunofluorescence. Results and Conclusions Thrombus formation was reduced in Tbet -/- and IFN-γ -/- mice, suggesting that Tbet/IFN-γ-expressing cells are required for venous thrombosis. The number of NETs formed during thrombosis was significantly lower in Tbet -/- and IFN-γ -/- mice. NET formation was also decreased in WT mice treated with an IFN-γ-blocking antibody. Injection of recombinant IFN-γ into IFN-γ -/- mice rescued the phenotype. Natural killer (NK) cells were specifically depleted prior to venous thrombosis induction. NK cell depletion results in decreased NET formation and smaller thrombi, suggesting that NK cells are required for thrombus development. In depleted mice, adoptive transfer of WT NK cells induced a similar thrombosis burden as in WT mice. In contrast, adoptive transfer of IFN-γ -/- NK cells resulted in thrombi similar in size to those in depleted mice. In vitro, we showed that WT neutrophils released fewer NETs when they were cocultured with IFN-γ -/- NK cells. This study demonstrates that NK cell-dependent IFN-γ production is crucial for thrombus development by promoting the formation of NETs by neutrophils., (© 2018 International Society on Thrombosis and Haemostasis.)

Background: Arsenic is metabolized through a series of oxidative methylation reactions by arsenic (3) methyltransferase (As3MT) to yield methylated intermediates. Although arsenic exposure is known to increase the risk of atherosclerosis, the contribution of arsenic methylation and As3MT remains undefined., Objectives: Our objective was to define whether methylated arsenic intermediates were proatherogenic and whether arsenic biotransformation by As3MT was required for arsenic-enhanced atherosclerosis., Methods: We utilized the apoE −/− mouse model to compare atherosclerotic plaque size and composition after inorganic arsenic, methylated arsenical, or arsenobetaine exposure in drinking water. We also generated apoE −/− /As3mt −/− double knockout mice to test whether As3MT-mediated biotransformation was required for the proatherogenic effects of inorganic arsenite. Furthermore, As3MT expression and function were assessed in in vitro cultures of plaque-resident cells. Finally, bone marrow transplantation studies were performed to define the contribution of As3MT-mediated methylation in different cell types to the development of atherosclerosis after inorganic arsenic exposure., Results: We found that methylated arsenicals, but not arsenobetaine, are proatherogenic and that As3MT is required for arsenic to induce reactive oxygen species and promote atherosclerosis. Importantly, As3MT was expressed and functional in multiple plaque-resident cell types, and transplant studies indicated that As3MT is required in extrahepatic tissues to promote atherosclerosis., Conclusion: Taken together, our findings indicate that As3MT acts to promote cardiovascular toxicity of arsenic and suggest that human AS3MT SNPs that correlate with enzyme function could predict those most at risk to develop atherosclerosis among the millions that are exposed to arsenic. https://doi.org/10.1289/EHP806.

Objective: Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis., Approach and Results: Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl 3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6 -/- mice contain less inflammatory (CCR2 hi CX 3 CR1 lo ) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2 hi CX 3 CR1 lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase)., Conclusions: This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2 hi CX 3 CR1 lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis., (© 2017 American Heart Association, Inc.)

Venous thromboembolism is a common complication of cancer. Based on recent evidence that (1) growth arrest-specific 6 (Gas6) regulates the expression of tissue factor during venous thrombosis, and (2) cancer promotes a procoagulant milieu, we hypothesize that Gas6 may be involved in cancer-induced coagulopathy. Venous thrombi were induced in both wild-type (WT) and Gas6-deficient ((-/-)) mice with cancer. WT mice with cancer developed larger thrombi than their healthy counterparts; these larger thrombi induced by cancer were not seen in Gas6(-/-) mice. Whole genome microarray analysis of differential gene expression in WT and Gas6(-/-) endothelial cells exposed to M27 murine lung carcinoma cells reveal that Gas6 increases prostaglandin E synthase (Ptges) expression in endothelial cells. This was confirmed using real-time polymerase chain reaction and immunofluorescence staining. Culture of WT endothelial cells with M27 increases the secretion of prostaglandin E2 (PGE2), the enzymatic product of Ptges, in WT but not in Gas6(-/-) endothelial cells. In WT endothelial cells, Ptges expression was regulated through extracellular signal-regulated kinase 1/2 phosphorylation (ERK1/2). In vitro, PGE2 activates platelets after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced venous thrombosis., (© 2016 by The American Society of Hematology.)

Background: Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase-Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE., Objectives: To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression., Methods: Thrombin was used to stimulate endothelial cells (ECs). Wild-type (WT) and Gas6(-/-) ECs were transfected with small interfering RNA targeting Axl or FoxO-1, a luciferase-coupled plasmid containing the FoxO-1 consensus sequence, and a phosphorylation-resistant FoxO-1 mutant, or treated with an Akt inhibitor. VCAM-1 mRNA expression was measured by real time-qPCR. VCAM-1 protein expression and FoxO-1 and Akt phosphorylation were assessed by western blot analysis. FoxO-1 localization was assessed by immunofluorescence. Adhesion of bone marrow mononuclear cells (BM-MCs) on ECs was assessed by fluorescence., Results and Conclusions: Thrombin induces both VCAM-1 expression and FoxO-1 phosphorylation and nuclear exclusion in WT ECs only. Silencing of FoxO-1 enhances VCAM-1 expression in both WT and Gas6(-/-) ECs. Inhibition of Akt or FoxO-1 phosphorylation prevents VCAM-1 expression in WT ECs. These data show that Gas6 induces FoxO-1 phosphorylation, leading to derepression of VCAM-1 expression. BM-MC-EC adhesion is increased by thrombin in WT ECs. BM-MC-EC adhesion is further increased when FoxO-1 is silenced, but decreased when FoxO-1 phosphorylation is inhibited. These results demonstrate that the Gas6-FoxO-1 signaling axis plays an important role in VCAM-1 expression in the context of VTE by promoting BM-MC-EC adhesion., (© 2015 International Society on Thrombosis and Haemostasis.)

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.

Objective: Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial cells, vascular smooth muscle cells, and leukocytes. It regulates cell survival, migration, and inflammatory response, but its role in atherogenesis is unknown., Approach and Results: To investigate the role of FHL2 in atherosclerosis, FHL2-deficient mice were crossed with ApoE-deficient mice, to generate ApoE/FHL2-/- mice. After high-fat diet, ApoE/FHL2-/- mice had significantly smaller atherosclerotic plaques than ApoE-/- mice in the aortic sinus, the brachiocephalic artery, and the aorta. This was associated with enhanced collagen and smooth muscle cell contents and a 2-fold reduction in macrophage content within the plaques of ApoE/FHL-2-/- versus ApoE-/- mice. This could be explained, in part, by the reduction in aortic ICAM-1 (intracellular adhesion molecule) mRNA and VCAM-1 (vascular cell adhesion molecule) protein expression in the plaque. Aortic gene expression of the chemokines CX3CL1 and CCL5 was increased in ApoE/FHL2-/- versus ApoE-/- mice. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of proinflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- versus ApoE-/- mice. Furthermore, mRNA levels of CX3CR1 were 2-fold higher in monocytes from ApoE/FHL2-/- versus ApoE-/- mice. Finally, we investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. After being irradiated, ApoE-/- or ApoE/FHL2-/- mice were transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After high-fat diet, both chimeric groups developed smaller plaques than ApoE-/- transplanted with ApoE-/- bone marrow., Conclusions: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting proinflammatory chemokine production, adhesion molecule expression, and proinflammatory monocyte recruitment., (© 2015 American Heart Association, Inc.)

Rationale: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair., Objective: We investigated the role of FHL2 in the regulation of EOCs., Methods and Results: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell-derived factor-1-induced EOC migration through upregulation of αv/β3, αv/β5, and β2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2(-/-) mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2(-/-) versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2(-/-) versus WT EOCs., Conclusions: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.

Background: Gas6 has been shown to interact with Axl in endothelial cells and to induce several signaling pathways involved in cell survival and proliferation. However, the interaction of Gas6/Axl with lipid raft/caveolin-1 in endothelial cells and its role in thrombosis are unknown., Objectives: We tested whether Axl and/or caveolin-1 is involved in Gas6-induced Akt, ERK1/2, and c-Src activation leading to altered tissue factor expression in endothelial cells., Methods: Gas6-treated endothelial cells were transfected with small interfering RNA (siRNA) for Axl, caveolin-1, c-Src, and Akt or treated with pharmacological inhibitors of c-Src and ERK1/2. Sucrose gradient centrifugation and confocal microscopy were used to study lipid raft/caveolin-1-enriched fractions. Akt, ERK1/2, p38, and c-Src activation was analyzed by Western blot analysis. Tissue factor expression was assessed by real-time quantitative polymerase chain reaction and immunofluorescence., Results and Conclusion: Gas6 induced Axl and c-Src localization into lipid raft/caveolin-1-enriched fractions. Gas6 increased the phosphorylation of Akt, ERK1/2, and c-Src but not p38. Using siRNA, we demonstrated that Axl is required for Akt, ERK1/2, and c-Src activation after Gas6 stimulation. siRNA for caveolin-1 blocked Gas6-induced phosphorylation of Akt, ERK1/2, and c-Src. c-Src downregulation inhibited Gas6-induced Akt but not ERK1/2 phosphorylation. Finally, Gas6 increased tissue factor mRNA and protein expression in endothelial cells. Tissue factor expression was blocked by siRNA for Axl, caveolin-1, or Akt as well as c-Src inhibition. These data demonstrate that the signaling pathway Gas6/Axl/caveolin-1/c-Src/Akt is required for tissue factor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature., (© 2013 International Society on Thrombosis and Haemostasis.)

Most fatalities from trauma occur due to severe blood loss. There is a need for improved hemostatic biomaterials that can address this problem. The aim of this study was to determine the in vivo efficacy of nanofibrous microspheres (NFM) loaded with hemostatic peptides, specifically ideal amphipathic peptides (IAP) that have been demonstrated to possess both procoagulant and antifibrinolyic activities. We demonstrate that IAP can be coupled to NFM (IAP-NFM) to form matrices that exhibit substantial hemostatic activity. IAP-NFM matrices were compared to a commercial zeolitic hemostatic biomaterial (QuikClot) and have superior efficacy in reducing bleeding in vivo. In both a murine tail transection and a murine hepatic injury model, bleeding times were significantly reduced (P<0.05) with the use of IAP-NFM as compared with equal masses of either QuikClot or NFM alone, or no treatment. Importantly, histological examination revealed no tissue injury when IAP-NFM or NFM were applied to hepatic lacerations. In contrast, QuikClot caused widespread hepatocyte degeneration and necrosis, with higher average injury zone thickness as determined by semiquantitative analysis. In summary, NFM was able to maintain the pro-coagulant properties of IAP in our preclinical model, caused no observable tissue damage at the site of application and had better performance than QuikClot controls., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (-/-) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5 μM ADP and 10 μM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6(-/-) mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6(-/-) endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.

Gas6 (growth arrest-specific 6) belongs structurally to the family of plasma vitamin K-dependent proteins. Gas6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition. Interestingly, despite the presence of a γ-carboxyglutamic acid domain in its structure, no role in the coagulation cascade has been identified for gas6. Gas6 has been shown to be involved in vascular homeostasis and more precisely is involved in proliferation, apoptosis, efferocytosis, leukocyte migration, and sequestration and platelet aggregation. It is also involved in the activation of different cell types, from platelets to endothelial and vascular smooth muscle cells. Thus, it has been shown to play a role in several pathophysiological processes such as atherosclerosis, cancer, and thrombosis. Interestingly, studies using gas6 null mice highlighted that gas6 may represent a novel potential target for anticoagulant therapy, because these animals are protected from lethal venous thromboembolism without excessive bleeding. However, the mechanism in thrombus occurrence remains to be further explored. In the present review, we will focus on the role of gas6 in innate immunity, atherosclerosis, thrombosis, and cancer-related events.

Background: Venous thrombosis (VT) is an important cause of morbidity and mortality in clinical medicine. Animal models studying venous thrombosis are scarce and, in most cases, very crude and rely on sacrificing the animals to excise formed thrombi. Developing an in vivo murine model of venous thrombosis can be a powerful tool for studying venous thrombosis., Objectives: We sought to use a high-frequency ultrasound system (HFUS) to dynamically and non-invasively monitor thrombus formation in the inferior vena cava (IVC) of mice., Methods: We developed a murine model of venous thrombosis using, for detection, the Vevo 770(®), a micro-imaging HFUS. Two different thrombosis models were used to generate thrombi in the IVC of C57Bl/6NCr mice: (i) ligation and (ii) application of ferric chloride (FeCl(3)). We then assessed venous thrombosis by HFUS., Results: In both models, measurements of the clot pathologically correlated favorably with measurements acquired with HFUS. Thrombus develops less than an hour after ligation or FeCl(3) -induced injury of the IVC and the size of the clot increases over time for up to 24 h. Importantly, we demonstrate that HFUS can be used to monitor the effect of an anticoagulant such as dalteparin until complete resolution of the thrombus., Conclusions: These data show that HFUS assesses venous thrombosis in mice reliably and non-invasively. Developing a murine model of thrombosis using more accurate, and clinically more relevant, techniques such as ultrasonography, is a step towards a better understanding of the pathophysiology of venous thromboembolism., (© 2011 International Society on Thrombosis and Haemostasis.)

Proprotein convertase (PC) 5/6 belongs to a family of secretory proteases involved in proprotein proteolysis. Several studies suggest a role for PC5/6 in cardiovascular disease. Because lethality at birth of mice lacking PC5/6 precluded elucidation of its function in the adult, we generated mice in which the gene of PC5/6 (pcsk5) is specifically inactivated in endothelial cells (ecKO), which are viable and do not exhibit overt abnormalities. In order to uncover the function of PC5/6 in the cardiovascular system, the effect of ecKO was studied in aging mice. In 16 to 18-month-old ecKO mice, the left ventricle (LV) mass, media cross-sectional area of aorta and coronary arteries, and media-to-lumen ratio of mesenteric arteries were decreased. The LV presented decreased diastolic function, and mesenteric arteries showed decreased stiffness. Collagen was decreased in the LV myocardial interstitium and perivascularly in coronary arteries and aorta. Cardiovascular hypotrophy likely develops with aging, since no significant changes were observed in 2-month-old ecKO mice. Fibroblasts, as a source of collagen in myocardium and vasculature, may play a role in the decrease in collagen deposition. Fibroblasts co-cultured with ecKO endothelial cells showed decreased collagen production, decreased insulin-like growth factor (IGF)-1/Akt/mTOR signaling, and enhanced autophagic activation. PC5/6 inactivation in endothelial cells results in cardiovascular hypotrophy associated with decreased collagen deposition, decreased LV diastolic function, and vascular stiffness, suggesting a trophic role of endothelial PC5/6 in the cardiovascular system, likely mediated by IGF-1/Akt/mTOR signaling and control of autophagy.

Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction in part as a result of enhanced oxidative stress. Function and survival of endothelial progenitor cells (EPCs, defined as sca1(+) c-kit(+) flk-1(+) bone marrow-derived cells), which significantly contribute to neovascularization and endothelial regeneration, depend on controlled production of reactive oxygen species (ROS). Mice heterozygous for the gene deletion of methylenetetrahydrofolate reductase (Mthfr(+/-)) have a 1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy significantly reduced the number of circulating EPCs as well as their differentiation. Mthfr deficiency was also associated with increased ROS production and reduced nitric oxide (NO) generation in Mthfr(+/-) EPCs. Treatment of EPCs with sepiapterin, a precursor of tetrahydrobiopterin (BH(4)), a cofactor of endothelial nitric oxide synthase (eNOS), significantly reduced ROS and improved NO production. mRNA and protein expression of eNOS and the relative amount of eNOS dimer compared with monomer were decreased by Mthfr deficiency. Impaired differentiation of EPCs induced by Mthfr deficiency correlated with increased senescence, decreased telomere length, and reduced expression of SIRT1. Addition of sepiapterin maintained cell senescence and SIRT1 expression at levels comparable to the wild type. Taken together, these results demonstrate that Mthfr deficiency impairs EPC formation and increases EPC senescence by eNOS uncoupling and downregulation of SIRT1.

Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.

Angiotensin II (Ang II) is considered the major final mediator of the renin-angiotensin system. The actions of Ang II have been implicated in many cardiovascular conditions, such as hypertension, atherosclerosis, coronary heart disease, restenosis, and heart failure. Ang II can act through two different receptors: Ang II type 1 (AT(1)) receptor and Ang II type 2 (AT(2)) receptor. The AT(1) receptor is ubiquitously expressed in the cardiovascular system and mediates most of the physiological and pathophysiological actions of Ang II. The AT(2) receptor is highly expressed in the developing foetus, but its expression is very low in the cardiovascular system of the normal adult. Expression of the AT(2) receptor can be modulated by pathological states associated with tissue remodelling or inflammation such as hypertension, atherosclerosis, and myocardial infarction. The precise role of the AT(2) receptor remains under debate. However, it appears that the AT(2) receptor plays a vasodilatory role, and may be enhanced as a countervailing mechanism in cardiac hypertrophy, and in presence of vascular injury in hypertension and atherosclerosis. Signalling pathways induced by the stimulation of the AT(2) receptor are poorly understood, but three main mechanisms have been described: (a) activation of protein phosphatases causing protein dephosphorylation; (b) activation of bradykinin/nitric oxide/cyclic guanosine 3',5'-monophosphate pathway; and (c) stimulation of phospholipase A(2) and release of arachidonic acid. Vasodilatory effects of the AT(2) receptor, probably the only well-established role of the AT(2) receptor, have been attributed to the second of these mechanisms. The participation of the AT(2) receptor in cardiovascular remodelling and inflammation is more controversial. In vitro, AT(2) receptor stimulation clearly inhibits cardiac and vascular smooth muscle growth and proliferation, and stimulates apoptosis. In vivo, the situation is less clear, and depending on the studies, the AT(2) receptor appears to be required for cardiac hypertrophic growth or contrariwise, the AT(2) receptor has demonstrated no effects on cardiac hypertrophy. Similar controversial findings have been reported in atherosclerosis. Here we discuss the role of the AT(2) receptor on cardiovascular structure and disease, and the signalling pathways induced by its activation.

Immune cells have been implicated in the pathogenesis of hypertension. We hypothesized that under the influence of chromosome (chr)2, T lymphocytes contribute to vascular inflammation in genetic salt-sensitive hypertension. Normotensive (Brown Norway), hypertensive (Dahl salt-sensitive), and consomic rats (SSBN2; in which chr2 has been transferred from Brown Norway to Dahl rats) were studied. Systolic blood pressure, measured by tail cuff, and aortic preproendothelin mRNA, measured by quantitative RT-PCR, were elevated in Dahl rats compared with Brown Norway rats and were reduced in SSBN2 rats compared with Dahl rats (P < 0.01). Compared with Brown Norway rats, Dahl rats exhibited increased inflammatory markers and mediators such as nuclear translocation of the aortic p65 subunit of NF-kappaB as well as VCAM-1, ICAM-1, chemokine (C-C motif) receptor 5, and CD4 mRNA, all of which were reduced in SSBN2 rats. Aortic CD8 mRNA was equally increased in Dahl and SSBN2 rats relative to Brown Norway rats. CD4(+) T cell infiltration in the aorta of SSBN2 rats was reduced compared with Dahl rats, whereas the aortic protein expression of Foxp3b and immunosuppressors transforming growth factor (TGF)-beta(1) and IL-10, the three markers associated with the regulatory T cell lineage, were enhanced in SSBN2 rats. Activation in vitro of T cells demonstrated that CD4(+)CD25(+) and CD8(+)CD25(+) cells (Tregs) produce IL-10 in SSBN2 rats. Thus, increased vascular inflammatory responses and hypertension in a genetic salt-sensitive hypertensive rodent model are reduced by transfer of chr2 from a normotensive strain, and this is associated with enhanced levels of immunosuppressive mediators.

Rationale: Aldosterone has been shown to induce vascular damage, endothelial dysfunction, and myocardial fibrosis, which depend in part on activation of angiotensin II (Ang II)-mediated pathways. However, mechanisms underlying crosstalk between Ang II type 1 receptor (AT(1)R) and mineralocorticoid receptor (MR) are mostly unknown., Objectives: We tested whether the lack of Ang II type 1a receptor (AT(1a)R) or Ang II type 1b receptor (AT(1b)R) would decrease cellular effects induced by aldosterone., Methods and Results: We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT(1a)R, AT(1b)R, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-kappaB phosphorylation after a 20-minute stimulation. Small interference RNA for AT(1a)R downregulated phosphorylation of ERK1/2, JNK, and NF-kappaB after aldosterone stimulation compared to controls. Downregulation of AT(1b)R or MR only abolished the activation of NF-kappaB. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT(1a)R knockout mice., Conclusion: We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT(1a)R and AT(1b)R. Our data suggest that aldosterone augments AT(1)R-dependent activation of ERK1/2, JNK, and NF-kappaB in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT(1)R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

Angiotensin II (Ang II) is considered the main final mediator of the renin-angiotensin-aldosterone system (RAAS). The actions of Ang II have been implicated in many cardiovascular conditions, such as hypertension, atherosclerosis, coronary heart disease, restenosis after injury, and heart failure. The Ang II type 1 receptor (AT(1)R), a G-protein-coupled receptor, mediates most of the physiological and pathophysiological actions of Ang II. This receptor is predominantly expressed in cardiovascular cells, such as vascular smooth muscle cells where it activates various signaling cascades leading to vascular remodeling and inflammation. Besides Ang II, aldosterone has emerged as an important component and mediator of the effects of the RAAS. Aldosterone-induced genomic effects mediated through binding to the mineralocorticoid receptor (MR), a member of the steroid hormone receptor superfamily, which functions as a ligand-dependent transcription factor, are characterized by a delay of minutes to hours corresponding to a long series of subcellular events that include gene activation and protein synthesis. Besides its well-known genomic actions, there is evidence of aldosterone-mediated rapid effects which lead to the activation of ion channels and other signaling pathways. Some of the effects of aldosterone occur through similar pathways as Ang II-induced signaling events. Indeed, recent studies suggest complex interactions between Ang II and aldosterone: it has become evident that aldosterone may influence the signaling or trafficking of the AT(1)R. Thus, growing evidence demonstrates the existence of cross-talk between Ang II and aldosterone which could potentially modulate Ang II signal transduction. These interactions between Ang II and aldosterone activate specific signaling pathways, sometimes in ways distinct from those that they induce on their own, one which may lead to pathogenic effects on target organs. Here we focus on recent findings and concepts that suggest the existence of novel signaling mechanisms whereby the cross-talk between Ang II and aldosterone plays a role in cardiovascular disease. We also discuss the importance of investigating Ang II/aldosterone cross-talk as a mean of developing new therapeutic strategies to combat cardiovascular disease.

Mechanical factors regulate both blood vessel growth and the development and progression of vascular disease. Acting on apoptotic and inflammatory signaling, the transcription factor nuclear factor kappaB (NF-kappaB) is a likely mediator of these processes. Nevertheless, pressure-dependent NF-kappaB activation pathways remain mostly unknown. Here we report that high intraluminal pressure induces reactive oxygen species (ROS) in arteries and that inhibition of NADPH oxidase prevents both the generation of ROS and the activation of NF-kappaB associated with high pressure. We also identify the epidermal growth factor receptor (EGFR) as a ROS-dependent signaling intermediate. In arteries from EGFR mutant mice (waved-2), pressure fails to activate NF-kappaB. Moreover, using vessels from EGFR ligand-deficient mice, we show that transforming growth factor (TGF)-alpha, but neither heparin-binding EGF-like growth factor nor epiregulin, transduces NF-kappaB activation by high pressure. Preventing the release of the active form of TGF-alpha also abolishes NF-kappaB induction by strain. The role of TGF-alpha signaling in vascular remodeling is substantiated in vivo; angiotensin II-induced activation of NF-kappaB and associated cell proliferation and wall thickening are much reduced in TGF-alpha-mutant mice compared with wild-type, despite equivalent hypertension in both groups. Conversely, apoptotic cells are detected only in vessels from hypertensive TGF-alpha-mutant mice, outlining the role of NF-kappaB in cell survival. Finally, the NF-kappaB activation pathway contrasts with that of extracellular signal-regulated kinase 1/2, which is activated by stretch through the EGFR but does not implicate TGF-alpha. Hence, our data identify TGF-alpha as a potential specific target to modulate mechanosensitive NF-kappaB activation and associated vascular remodeling.

Background: High blood pressure causes a change in vascular wall structure involving altered extracellular matrix composition, but how this process occurs is not fully understood., Methods and Results: Using mouse carotid arteries maintained in organ culture for 3 days, we detected increased gelatin zymographic activity of matrix metalloproteinase (MMP)-2 (168+/-13%, P<0.05) in vessels kept at low intraluminal pressure (10 mm Hg) compared with vessels at 80 mm Hg (100%), whereas in vessels maintained at high pressure (150 mm Hg), both MMP-2 and MMP-9 activity was induced (182+/-32%, P<0.05, and 194+/-21%, P<0.01, respectively). MMPs were detected in endothelial and smooth muscle cells by immunohistochemistry and in situ gelatin zymography. In vessels at 150 mm Hg, MMP activation was associated with a shift in the pressure-diameter curve toward greater distensibility (P<0.01) compared with vessels at 80 mm Hg. However, distensibility was not altered in vessels at 10 mm Hg, in which only activated MMP-2 was detected. The role of MMPs in high pressure-induced vessel distensibility was confirmed by use of the MMP inhibitor FN-439, which prevented the shift in the pressure-diameter relationship. Furthermore, in carotid arteries from MMP-9-deficient mice, the pressure-dependent increase in MMP-2 and in situ gelatinolytic activity were maintained, but the upward shift in the pressure-diameter curve was abolished., Conclusions: MMP-9 seems to play a key role in the early stages of hypertensive vascular remodeling.

The effects of mechanical factors on nuclear factor (NF)-kappaB activation and its potential functional roles have been very little explored in the intact vessel. Thus, we chose to study the regulation of NF-kappaB by intraluminal pressure using an organ culture model of mouse carotid arteries maintained at 80 or 150 mm Hg during 24 hours. Gel shift analysis revealed an increase in the DNA-binding capacity of NF-kappaB in vessels at high pressure compared with vessels at normal pressure (304+/-49%; P<0.001). This coincided with reduced levels of the endogenous NF-kappaB inhibitor IkappaBalpha in arteries at 150 mm Hg (52+/-7%; P<0.001), as detected by Western blot. To study the functional role of the pressure-induced activation of NF-kappaB, we evaluated the rate of apoptosis (TUNEL method) in carotid arteries cultured with or without an inhibitor peptide blocking nuclear translocation of NF-kappaB. No apoptosis was detected in control arteries either at 80 or 150 mm Hg. However, in the presence of the NF-kappaB inhibitor peptide, we observed apoptosis in vessels at 80 mm Hg (5+/-1%; P<0.001 versus untreated controls), which was markedly increased in vessels at 150 mm Hg (14+/-2%; P<0.001). These results were corroborated by immunohistochemical analysis showing positive staining for cleaved caspase 3 in vessels at 80 mm Hg treated with the NF-kappaB inhibitor peptide, which was additionally enhanced in treated vessels at 150 mm Hg. Our findings demonstrate that high intraluminal pressure activates NF-kappaB in arteries. Moreover, the activation of NF-kappaB seems to play a key role in preventing apoptosis in vascular cells, especially when vessels are exposed to high intraluminal pressure.