Sven Jäckel

Gut Microbiota Promote Angiotensin II–Induced Arterial Hypertension and Vascular Dysfunction

Background The gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown. Methods and Results Unchallenged germ‐free mice (GF) had a dampened systemic T helper cell type 1 skewing compared to conventionally raised (CONV‐R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T‐box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV‐R mice, angiotensin II (AngII; 1 mg/kg per day for 7 days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein 1 (MCP‐1), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic‐acid receptor‐related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)‐17 synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G+ neutrophils and Ly6C+ monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to AngII. Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII. Conclusion Gut microbiota facilitate AngII‐induced vascular dysfunction and hypertension, at least in part, by supporting an MCP‐1/IL‐17 driven vascular immune cell infiltration and inflammation.

Cofactor Independent Human Antiphospholipid Antibodies Induce Venous Thrombosis in Mice

Essentials Cofactor‐independent antiphospholipid antibodies (CI‐aPL) are generally considered non‐pathogenic. We analyzed the effects of human monoclonal CI‐aPL in a mouse model of venous thrombosis. As shown in vitro, CI‐aPL induce a procoagulant state in vivo by activation of endosomal NADPH‐oxidase. Contrary to common belief, CI‐aPL induce venous thrombosis in vivo.

Platelet-localized FXI promotes a vascular coagulation-inflammatory circuit in arterial hypertension

Blockade of an inflammatory, thrombin-activated feedback loop on platelets controls high blood pressure. Spotlight on factor XI Hypertension, cardiovascular disease, and vascular inflammation are inextricably linked, often co-occurring. Kossmann et al. have now discovered a regulatory pathway linking these pathologies that could be inhibited to allow the control of treatment-resistant high blood pressure. In rats and mice with hypertension, the authors found that vascular disease is driven by an overactive thrombin-driven factor XI feedback loop on platelets. Inhibition of this feedback loop with an antisense molecule against factor XI reduced both the vascular pathology and hypertension. The authors show that this factor XI–dependent feedback loop also operates in patients with uncontrolled hypertension, raising the possibility that factor XI inhibition may prove a useful addition to our armamentarium for treating high blood pressure. Multicellular interactions of platelets, leukocytes, and the blood vessel wall support coagulation and precipitate arterial and venous thrombosis. High levels of angiotensin II cause arterial hypertension by a complex vascular inflammatory pathway that requires leukocyte recruitment and reactive oxygen species production and is followed by vascular dysfunction. We delineate a previously undescribed, proinflammatory coagulation-vascular circuit that is a major regulator of vascular tone, blood pressure, and endothelial function. In mice with angiotensin II–induced hypertension, tissue factor was up-regulated, as was thrombin-dependent endothelial cell vascular cellular adhesion molecule 1 expression and integrin αMβ2– and platelet-dependent leukocyte adhesion to arterial vessels. The resulting vascular inflammation and dysfunction was mediated by activation of thrombin-driven factor XI (FXI) feedback, independent of factor XII. The FXI receptor glycoprotein Ibα on platelets was required for this thrombin feedback activation in angiotensin II–infused mice. Inhibition of FXI synthesis with an antisense oligonucleotide was sufficient to prevent thrombin propagation on platelets, vascular leukocyte infiltration, angiotensin II–induced endothelial dysfunction, and arterial hypertension in mice and rats. Antisense oligonucleotide against FXI also reduced the increased blood pressure and attenuated vascular and kidney dysfunction in rats with established arterial hypertension. Further, platelet-localized thrombin generation was amplified in an FXI-dependent manner in patients with uncontrolled arterial hypertension, suggesting that platelet-localized thrombin generation may serve as an inflammatory marker of high blood pressure. Our results outline a coagulation-inflammation circuit that promotes vascular dysfunction, and highlight the possible utility of FXI-targeted anticoagulants in treating hypertension, beyond their application as antithrombotic agents in cardiovascular disease.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.

Gut Microbial Colonization Orchestrates TLR2 Expression, Signaling and Epithelial Proliferation in the Small Intestinal Mucosa

The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs). Here, we report that colonization of germ-free (GF) Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R) showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

Deep vein thrombus formation induced by flow reduction in mice is determined by venous side branches

BACKGROUND Interaction between vascular wall abnormalities, inflammatory leukocytes, platelets, coagulation factors and hemorheology in the pathogenesis of deep vein thrombosis (DVT) is incompletely understood, requiring well defined animal models of human disease. METHODS AND RESULTS We subjected male C57BL/6 mice to ligation of the inferior vena cava (IVC) as a flow reduction model to induce DVT. Thrombus size and weight were analyzed macroscopically and sonographically by B-mode, pulse wave (pw) Doppler and power Doppler imaging (PDI) using high frequency ultrasound. Thrombus size varied substantially between individual procedures and mice, irrespective of the flow reduction achieved by the ligature. Interestingly, PDI accurately predicted thrombus size in a very robust fashion (r2 = 0.9734, p < 0.0001). Distance of the insertion of side branches from the ligature significantly determines thrombus weight (r2 = 0.5597, p < 0.0001) and length (r2 = 0.5441, p < 0.0001) in the IVC, regardless of the flow measured by pw-Doppler with distances <1.5 mm drastically impairing thrombus formation. Occlusion of side branches prior to ligation of IVC did not increase thrombus size, probably due to patent side branches inaccessible to surgery. CONCLUSION Venous side branches influence thrombus size in experimental DVT and might therefore prevent thrombus formation. This renders vessel anatomy and hemorheology important determinants in mouse models of DVT, which should be controlled for.

Innate Effector-Memory T-Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution

RATIONALE Immune cells play an important role during the generation and resolution of thrombosis. T cells are powerful regulators of immune and nonimmune cell function, however, their role in sterile inflammation in venous thrombosis has not been systematically examined. OBJECTIVE This study investigated the recruitment, activation, and inflammatory activity of T cells in deep vein thrombosis and its consequences for venous thrombus resolution. METHODS AND RESULTS CD4+ and CD8+ T cells infiltrate the thrombus and vein wall rapidly on deep vein thrombosis induction and remain in the tissue throughout the thrombus resolution. In the vein wall, recruited T cells largely consist of effector-memory T (TEM) cells. Using T-cell receptor transgenic reporter mice, we demonstrate that deep vein thrombosis-recruited TEM receive an immediate antigen-independent activation and produce IFN-γ (interferon) in situ. Mapping inflammatory conditions in the thrombotic vein, we identify a set of deep vein thrombosis upregulated cytokines and chemokines that synergize to induce antigen-independent IFN-γ production in CD4+ and CD8+ TEM cells. Reducing the number of TEM cells through a depletion recovery procedure, we show that intravenous TEM activation determines neutrophil and monocyte recruitment and delays thrombus neovascularization and resolution. Examining T-cell recruitment in human venous stasis, we show that superficial varicose veins preferentially contain activated memory T cells. CONCLUSIONS TEM orchestrate the inflammatory response in venous thrombosis affecting thrombus resolution.

Gut microbiota regulate hepatic von Willebrand Factor synthesis and arterial thrombus formation via Toll-like receptor-2

The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in hepatic endothelial cells, as a critical factor that is regulated by gut microbiota and determines thrombus growth in Tlr2-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, Tlr2-/- , and heterozygous Vwf+/- mice that are all characterized by a modest reduction in plasma VWF levels. Defective platelet matrix interaction can be restored by exposure to WT plasma or to purified VWF depending on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in Tlr2-/- mice in vivo. These experiments delineate an unexpected pathway in which microbiota-triggered TLR2 signaling alters the synthesis of proadhesive VWF by the liver endothelium and favors platelet integrin-dependent thrombus growth.

Malondialdehyde Epitopes Are Sterile Mediators of Hepatic Inflammation in Hypercholesterolemic Mice

Diet‐related health issues such as nonalcoholic fatty liver disease and cardiovascular disorders are known to have a major inflammatory component. However, the exact pathways linking diet‐induced changes (e.g., hyperlipidemia) and the ensuing inflammation have remained elusive so far. We identified biological processes related to innate immunity and oxidative stress as prime response pathways in livers of low‐density lipoprotein receptor‐deficient mice on a Western‐type diet using RNA sequencing and in silico functional analyses of transcriptome data. The observed changes were independent of the presence of microbiota and thus indicative of a role for sterile triggers. We further show that malondialdehyde (MDA) epitopes, products of lipid peroxidation and markers for enhanced oxidative stress, are detectable in hepatic inflammation predominantly on dying cells and stimulate cytokine secretion as well as leukocyte recruitment in vitro and in vivo. MDA‐induced cytokine secretion in vitro was dependent on the presence of the scavenger receptors CD36 and MSR1. Moreover, in vivo neutralization of endogenously generated MDA epitopes by intravenous injection of a specific MDA antibody results in decreased hepatic inflammation in low‐density lipoprotein receptor‐deficient mice on a Western‐type diet. Conclusion: Accumulation of MDA epitopes plays a major role during diet‐induced hepatic inflammation and can be ameliorated by administration of an anti‐MDA antibody. (Hepatology 2017;65:1181‐1195)

Anti-Inflammatory and Anti-Thrombotic Effects of the Fungal Metabolite Galiellalactone in Apolipoprotein E-Deficient Mice

Patients suffering from chronic inflammatory diseases have an increased mortality risk resulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic events. Until now, it is not completely understood in which way an abnormal expression of pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a need for new drugs that on the one hand suppress the expression of pro-inflammatory mediators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-inflammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiellalactone lowered the inflammatory expression profile and improved blood clotting times, as well as platelet adhesion to the injured common carotid artery. The results indicate that administration of Galiellalactone is able to reduce the extent of inflammation and arterial platelet adhesion in this mouse model.