Susanne Karbach

Lysozyme M–Positive Monocytes Mediate Angiotensin II–Induced Arterial Hypertension and Vascular Dysfunction

Background— Angiotensin II (ATII), a potent vasoconstrictor, causes hypertension, promotes infiltration of myelomonocytic cells into the vessel wall, and stimulates both vascular and inflammatory cell NADPH oxidases. The predominant source of reactive oxygen species, eg, vascular (endothelial, smooth muscle, adventitial) versus phagocytic NADPH oxidase, and the role of myelomonocytic cells in mediating arterial hypertension have not been defined yet. Methods and Results— Angiotensin II (1 mg · kg−1 · d−1 for 7 days) increased the number of both CD11b+Gr-1lowF4/80+ macrophages and CD11b+Gr-1highF4/80− neutrophils in mouse aorta (verified by flow cytometry). Selective ablation of lysozyme M-positive (LysM+) myelomonocytic cells by low-dose diphtheria toxin in mice with inducible expression of the diphtheria toxin receptor (LysMiDTR mice) reduced the number of monocytes in the circulation and limited ATII-induced infiltration of these cells into the vascular wall, whereas the number of neutrophils was not reduced. Depletion of LysM+ cells attenuated ATII-induced blood pressure increase (measured by radiotelemetry) and vascular endothelial and smooth muscle dysfunction (assessed by aortic ring relaxation studies) and reduced vascular superoxide formation (measured by chemiluminescence, cytochrome c assay, and oxidative fluorescence microtopography) and the expression of NADPH oxidase subunits gp91phox and p67phox (assessed by Western blot and mRNA reverse-transcription polymerase chain reaction). Adoptive transfer of wild-type CD11b+Gr-1+ monocytes into depleted LysMiDTR mice reestablished ATII-induced vascular dysfunction, oxidative stress, and arterial hypertension, whereas transfer of CD11b+Gr-1+ neutrophils or monocytes from gp91phox or ATII receptor type 1 knockout mice did not. Conclusions— Infiltrating monocytes with a proinflammatory phenotype and macrophages rather than neutrophils appear to be essential for ATII-induced vascular dysfunction and arterial hypertension.

Glucose-independent improvement of vascular dysfunction in experimental sepsis by dipeptidyl-peptidase 4 inhibition

AIMS Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel class of drugs for the treatment of hyperglycaemia. Preliminary evidence suggests that their antioxidant and anti-inflammatory effects may have beneficial effects on the cardiovascular complications of diabetes. In the present study, we investigate in an experimental sepsis model whether linagliptin exerts pleiotropic vascular effects independent of its glucose-lowering properties. METHODS AND RESULTS Linagliptin (83 mg/kg chow for 7 days) was administered in a rat model of lipopolysaccharide (LPS) (10 mg/kg, single i.p. dose/24 h)-induced sepsis. Vascular relaxation, reactive oxygen species (ROS) formation, expression of NADPH oxidase subunits and proinflammatory markers, and white blood cell infiltration in the vasculature were determined. Oxidative burst and adhesion of isolated human neutrophils to endothelial cells were measured in the presence of different DPP-4 inhibitors, and their direct vasodilatory effects (isometric tension in isolated aortic rings) were compared. In vivo linagliptin treatment ameliorated LPS-induced endothelial dysfunction and was associated with reduced formation of vascular, cardiac, and blood ROS, aortic expression of inflammatory genes and NADPH oxidase subunits in addition to reduced aortic infiltration with inflammatory cells. Linagliptin was the most potent inhibitor of oxidative burst in isolated activated human neutrophils and it suppressed their adhesion to activated endothelial cells. Of the inhibitors tested, linagliptin and alogliptin had the most pronounced direct vasodilatory potency. CONCLUSION Linagliptin demonstrated pleiotropic vasodilatory, antioxidant, and anti-inflammatory properties independent of its glucose-lowering properties. These pleiotropic properties are generally not shared by other DPP-4 inhibitors and might translate into cardiovascular benefits in diabetic patients.

Conversion of biliverdin to bilirubin by biliverdin reductase contributes to endothelial cell protection by heme oxygenase-1—evidence for direct and indirect antioxidant actions of bilirubin

Heme oxygenase-1 (HO-1) is highly protective in various pathophysiological states such as cardiovascular and neurodegenerative diseases. HO-1-derived bilirubin is an efficient scavenger of reactive oxygen and nitrogen species (RONS). It remains to determine whether conversion of biliverdin to bilirubin is an essential step for HO-1-conferred protection of endothelial cells. RONS scavenging activities of biliverdin versus bilirubin were assessed by different RONS generating systems and detection techniques. We also silenced the biliverdin reductase (BVR) or HO-1 gene in cultured primary human endothelial cells (HUVECs) and measured the effect on RONS formation upon stimulation with lipopolysaccharide (LPS). In addition, effects of bilirubin and biliverdin on expression of GTP-cyclohydrolase were assessed in an endothelial cell line (EA.hy 926). HO-1- and BVR-silenced cells have increased levels of oxidative stress and bilirubin but not biliverdin increased expression of the protective protein GTP-cyclohydrolase. Moreover, protection by hemin-induced HO-1 expression or biliverdin-triggered bilirubin formation was impaired upon silencing of the HO-1 or BVR gene, respectively. Since bilirubin significantly scavenged RONS but chronic treatment was even more protective our observations support direct and indirect antioxidant properties of BVR and bilirubin and an important role for BVR and bilirubin in HO-1 conferred protection of endothelial cells.

An Alternative Pathway of Imiquimod-Induced Psoriasis-Like Skin Inflammation in the Absence of Interleukin-17 Receptor A Signaling

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RA(del) mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RA(del) mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs.

Interleukin 17 Drives Vascular Inflammation, Endothelial Dysfunction, and Arterial Hypertension in Psoriasis-Like Skin Disease

Objective— Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease. Approach and Results— Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17Aind/+ mice), associated with increased reactive oxygen species formation and circulating CD11b+ inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17Aind/+ mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidase+CD11b+GR1+F4/80− cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-&agr; and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17Aind/+ mice. Conclusions— Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidase+CD11b+GR1+F4/80− inflammatory cells. Depletion of the GR-1+ immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17Aind/+ mice.

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.

Pentaerythritol Tetranitrate Improves Angiotensin II–Induced Vascular Dysfunction via Induction of Heme Oxygenase-1

The organic nitrate pentaerythritol tetranitrate is devoid of nitrate tolerance, which has been attributed to the induction of the antioxidant enzyme heme oxygenase (HO)-1. With the present study, we tested whether chronic treatment with pentaerythritol tetranitrate can improve angiotensin II–induced vascular oxidative stress and dysfunction. In contrast to isosorbide-5 mononitrate (75 mg/kg per day for 7 days), treatment with pentaerythritol tetranitrate (15 mg/kg per day for 7 days) improved the impaired endothelial and smooth muscle function and normalized vascular and cardiac reactive oxygen species production (mitochondria, NADPH oxidase activity, and uncoupled endothelial NO synthase), as assessed by dihydroethidine staining, lucigenin-enhanced chemiluminescence, and quantification of dihydroethidine oxidation products in angiotensin II (1 mg/kg per day for 7 days)–treated rats. The antioxidant features of pentaerythritol tetranitrate were recapitulated in spontaneously hypertensive rats. In addition to an increase in HO-1 protein expression, pentaerythritol tetranitrate but not isosorbide-5 mononitrate normalized vascular reactive oxygen species formation and augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase I and dihydrofolate reductase in angiotensin II–treated rats, thereby preventing endothelial NO synthase uncoupling. Haploinsufficiency of HO-1 completely abolished the beneficial effects of pentaerythritol tetranitrate in angiotensin II–treated mice, whereas HO-1 induction by hemin (25 mg/kg) mimicked the effect of pentaerythritol tetranitrate. Improvement of vascular function in this particular model of arterial hypertension by pentaerythritol tetranitrate largely depends on the induction of the antioxidant enzyme HO-1 and identifies pentaerythritol tetranitrate, in contrast to isosorbide-5 mononitrate, as an organic nitrate able to improve rather than to worsen endothelial function.

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.

Chronic skin inflammation leads to bone loss by IL-17–mediated inhibition of Wnt signaling in osteoblasts

Skin inflammation inhibits bone formation through IL-17A. IL-17 in skin and bones Patients with psoriasis develop red, itchy, scaly patches on the skin in part because of the proinflammatory cytokine interleukin (IL)-17A. Now, Uluçkan et al. report that IL-17A may also lead to bone loss in affected individuals. They found in two different mouse models that in contrast to other types of inflammatory bone loss where osteoclast activation leads to bone degradation, IL-17A prevented new bone formation. Blocking IL-17A restored bone formation in vivo. If these data hold true in humans, targeting IL-17A in psoriasis may have the added benefit of blocking psoriasis-related bone loss. Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A–mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A–mediated skin inflammation, K14-IL17Aind and JunBΔep, strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients.