Tanja Schönfelder

Angiotensin II–Induced Vascular Dysfunction Depends on Interferon-γ–Driven Immune Cell Recruitment and Mutual Activation of Monocytes and NK-Cells

Objective—Immune cells contribute to angiotensin II (ATII)–induced vascular dysfunction and inflammation. Interferon-&ggr; (IFN-&ggr;), an inflammatory cytokine exclusively produced by immune cells, seems to be involved in ATII-driven cardiovascular injury, but the actions and cellular source of IFN-&ggr; remain incompletely understood. Approach and Results—IFN-&ggr;−/− and Tbx21−/− mice were partially protected from ATII-induced (1 mg/kg per day of ATII, infused subcutaneously by miniosmotic pumps) vascular endothelial and smooth muscle dysfunction, whereas mice overexpressing IFN-&ggr; showed constitutive vascular dysfunction. Absence of T-box expressed in T cells (T-bet), the IFN-&ggr; transcription factor encoded by Tbx21, reduced vascular superoxide and peroxynitrite formation and attenuated expression of nicotinamide adenosine dinucleotide phosphate oxidase subunits as well as inducible NO synthase, monocyte chemoattractant protein 1, and interleukin-12 in aortas of ATII-infused mice. Compared with controls, IFN-&ggr;−/− and Tbx21−/− mice were characterized by reduced ATII-mediated vascular recruitment of both natural killer (NK)1.1+ NK-cells as the major producers of IFN-&ggr; and CD11b+Gr-1low interleukin-12 secreting monocytes. Selective depletion and adoptive transfer experiments identified NK-cells as essential contributors to vascular dysfunction and showed that T-bet+lysozyme M+ myelomonocytic cells were required for NK-cell recruitment into vascular tissue and local IFN-&ggr; production. Conclusions—We provide first evidence that NK-cells play an essential role in ATII-induced vascular dysfunction. In addition, we disclose the T-bet-IFN-&ggr; pathway and mutual monocyte–NK-cell activation as potential therapeutic targets in cardiovascular disease.

Inflammatory Monocytes Determine Endothelial Nitric-oxide Synthase Uncoupling and Nitro-oxidative Stress Induced by Angiotensin II

Background: Inflammatory monocytes are drivers of vascular injury and disease. Results: Depletion of lysozyme M-positive monocytes prevents eNOS uncoupling and iNOS-derived nitro-oxidative stress. Conclusion: Monocytes determine eNOS and iNOS function by directly modulating tetrahydrobiopterin bioavailability. Significance: Understanding the impact of inflammation on endothelial function in detail is essential to identify tailored therapeutic strategies. Endothelial nitric-oxide synthase (eNOS) uncoupling and increased inducible NOS (iNOS) activity amplify vascular oxidative stress. The role of inflammatory myelomonocytic cells as mediators of these processes and their impact on tetrahydrobiopterin availability and function have not yet been defined. Angiotensin II (ATII, 1 mg/kg/day for 7 days) increased Ly6Chigh and CD11b+/iNOShigh leukocytes and up-regulated levels of eNOS glutathionylation in aortas of C57BL/6 mice. Vascular iNOS-dependent NO formation was increased, whereas eNOS-dependent NO formation was decreased in aortas of ATII-infused mice as assessed by electron paramagnetic resonance (EPR) spectroscopy. Diphtheria toxin-mediated ablation of lysozyme M-positive (LysM+) monocytes in ATII-infused LysMiDTR transgenic mice prevented eNOS glutathionylation and eNOS-derived Nω-nitro-l-arginine methyl ester-sensitive superoxide formation in the endothelial layer. ATII increased vascular guanosine triphosphate cyclohydrolase I expression and biopterin synthesis in parallel, which was reduced in monocyte-depleted LysMiDTR mice. Vascular tetrahydrobiopterin was increased by ATII infusion but was even higher in monocyte-depleted ATII-infused mice, which was paralleled by a strong up-regulation of dihydrofolate reductase expression. EPR spectroscopy revealed that both vascular iNOS- and eNOS-dependent NO formation were normalized in ATII-infused mice following monocyte depletion. Additionally, deletion as well as pharmacologic inhibition of iNOS prevented ATII-induced endothelial dysfunction. In summary, ATII induces an inflammatory cell-dependent increase of iNOS, guanosine triphosphate cyclohydrolase I, tetrahydrobiopterin, NO formation, and nitro-oxidative stress as well as eNOS uncoupling in the vessel wall, which can be prevented by ablation of LysM+ monocytes.

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.

Heme oxygenase-1 suppresses a pro-inflammatory phenotype in monocytes and determines endothelial function and arterial hypertension in mice and humans

AIMS Heme oxygenase-1 (HO-1) confers protection to the vasculature and suppresses inflammatory properties of monocytes and macrophages. It is unclear how HO-1 determines the extent of vascular dysfunction in mice and humans. METHODS AND RESULTS Decreased HO-1 activity and expression was paralleled by increased aortic expression and activity of the nicotinamide dinucleotide phosphate oxidase Nox2 in HO-1 deficient Hmox1⁻/⁻ and Hmox1(⁺/⁻) compared with Hmox1⁺/⁺ mice. When subjected to angiotensin II-infusion, streptozotocin-induced diabetes mellitus and aging, HO-1 deficient mice showed increased vascular dysfunction inversely correlated with HO activity. In a primary prevention population-based cohort, we assessed length polymorphisms of the HMOX1 promoter region and established a bipolar frequency pattern of allele length (long vs. short repeats) in 4937 individuals. Monocytic HMOX1 mRNA expression was positively correlated with flow-mediated dilation and inversely with CD14 mRNA expression indicating pro-inflammatory monocytes in 733 hypertensive individuals of this cohort. Hmox1⁻/⁻ mice showed drastically increased expression of the chemokine receptor CCR2 in monocytes and the aorta. Angiotensin II-infused Hmox1⁻/⁻ mice had amplified endothelial inflammation in vivo, significantly increased aortic infiltration of pro-inflammatory CD11b⁺ Ly6C(hi) monocytes and Ly6G⁺ neutrophils and were marked by Ly6C(hi) monocytosis in the circulation and an increased blood pressure response. Finally, individuals with unfavourable HMOX1 gene promoter length had increased prevalence of arterial hypertension and reduced cumulative survival after a median follow-up of 7.23 years. CONCLUSIONS Heme oxygenase-1 is a regulator of vascular function in hypertension via determining the phenotype of inflammatory circulating and infiltrating monocytes with possible implications for all-cause mortality.

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.

NOX2 amplifies acetaldehyde-mediated cardiomyocyte mitochondrial dysfunction in alcoholic cardiomyopathy

Alcoholic cardiomyopathy (ACM) resulting from excess alcohol consumption is an important cause of heart failure (HF). Although it is assumed that the cardiotoxicity of the ethanol (EtOH)-metabolite acetaldehyde (ACA) is central for its development and progression, the exact mechanisms remain obscure. Murine cardiomyocytes (CMs) exposed to ACA or EtOH showed increased superoxide (O2•−) levels and decreased mitochondrial polarization, both being normalized by NADPH oxidase (NOX) inhibition. C57BL/6 mice and mice deficient for the ACA-degrading enzyme mitochondrial aldehyde dehydrogenase (ALDH-2−/−) were fed a 2% EtOH diet for 5 weeks creating an ACA-overload. 2% EtOH-fed ALDH-2−/− mice exhibited a decreased cardiac function, increased heart-to-body and lung-to-body weight ratios, increased cardiac levels of the lipid peroxidation product malondialdehyde (MDA) as well as increased NOX activity and NOX2/glycoprotein 91phox (NOX2/gp91phox) subunit expression compared to 2% EtOH-fed C57BL/6 mice. Echocardiography revealed that ALDH-2−/−/gp91phox−/− mice were protected from ACA-overload-induced HF after 5 weeks of 2% EtOH-diet, demonstrating that NOX2-derived O2•− contributes to the development of ACM. Translated to human pathophysiology, we found increased gp91phox expression in endomyocardial biopsies of ACM patients. In conclusion, ACM is promoted by ACA-driven mitochondrial dysfunction and can be improved by ablation of NOX2/gp91phox. NOX2/gp91phox therefore might be a potential pharmacological target to treat ACM.

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.

Lack of T-bet reduces monocytic interleukin-12 formation and accelerates thrombus resolution in deep vein thrombosis

The role of leukocytes in deep vein thrombosis (DVT) resolution is incompletely understood. We determined how depletion of lysozyme positive (LysM+) cells and a switched-off type 1 immune response influences thrombus resolution. DVT was induced in 12-week-old male mice by inferior vena cava (IVC) stenosis. Toxin mediated depletion of myeloid cells improved thrombus resolution in mice with Cre-inducible expression of the diphtheria toxin receptor in LysM+ cells. This correlated with decreased CD45+ cells, a population shift of Gr-1+ to Gr-1− CD11b+ myelomonocytic cells (flow cytometry) and an increase in CC-chemokine ligand 2, interleukin-4 and interleukin-10 mRNA expressions. Tbx21−/− mice (lacking transcription factor T-bet and marked by an attenuated type 1 immune response) with DVT had faster thrombus resolution, a reduction of pro-inflammatory Ly6Chi monocytes in thrombi and decreased interleukin-12p40 mRNA expression than control mice resulting in increased vascular endothelial growth factor mRNA expression and improved neovascularization of thrombotic veins. Transfer of Tbx21−/− bone marrow into irradiated Tbx21+/+ recipients lead to accelerated thrombus resolution with lower T-bet-dependent interleukin-12p40 mRNA levels following IVC-stenosis. We conclude that inhibition of Tbet+ interleukin-12 forming myelomonocytic cells accelerated thrombus resolution. Modulating the inflammatory immune response might be an approach to improve therapy of DVT.

Pulmonary Arterial Hypertension and Endothelial Dysfunction Is Linked to NADPH Oxidase-Derived Superoxide Formation in Venous Thrombosis and Pulmonary Embolism in Mice

Pulmonary embolism (PE) results from deep vein thrombosis (DVT) and can lead to chronic thromboembolic pulmonary hypertension (CTEPH) involving vascular dysfunction. Mechanisms are incompletely understood, in part due to lack of mouse models. We induced PE in C57BL/6 mice by intravenous injection of thrombin (166 U/kg BW), confirmed by a sudden bradycardia, bradypnea, and an increase in pulmonary artery (PA) pressure observed by high-frequency ultrasound. While symptoms resolved rapidly after single thrombin application, repeated PEs resulted in sustained PA-pressure increase, increased PA superoxide formation assessed by oxidative fluorescent microtopography, increased PA gp91phox expression, and endothelial dysfunction assessed by isometric tension studies of isolated PA segments after 24 hours. DVT was modeled in C57BL/6 mice by ligation of the inferior vena cava (IVC). Importantly, small pulmonary emboli could be detected along with a mild phenotype of PA endothelial dysfunction and oxidative stress in the absence of PA-pressure elevation. mRNA expression of plasminogen activator inhibitor-1 was increased in PAs of mice with recurrent PE after repetitive thrombin injections and to a lesser extent in DVT mice. In summary, our data suggest that PA endothelial dysfunction, induced by gp91phox-derived ROS, is an early event upon repetitive PE. This phenomenon might help to elucidate the mechanisms of PA dysfunction in the pathogenesis of CTEPH.