Swenja Kröller-Schön

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.

Molecular Mechanisms of the Crosstalk Between Mitochondria and NADPH Oxidase Through Reactive Oxygen Species—Studies in White Blood Cells and in Animal Models

AIMS Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. RESULTS NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD(+/-)) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD(+/-) mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. INNOVATION These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. CONCLUSIONS Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension.

Glutathione Peroxidase-1 Deficiency Potentiates Dysregulatory Modifications of Endothelial Nitric Oxide Synthase and Vascular Dysfunction in Aging

Recently, we demonstrated that gene ablation of mitochondrial manganese superoxide dismutase and aldehyde dehydrogenase-2 markedly contributed to age-related vascular dysfunction and mitochondrial oxidative stress. The present study has sought to investigate the extent of vascular dysfunction and oxidant formation in glutathione peroxidase-1–deficient (GPx-1−/−) mice during the aging process with special emphasis on dysregulation (uncoupling) of the endothelial NO synthase. GPx-1−/− mice on a C57 black 6 (C57BL/6) background at 2, 6, and 12 months of age were used. Vascular function was significantly impaired in 12-month-old GPx-1−/− -mice as compared with age-matched controls. Oxidant formation, detected by 3-nitrotyrosine staining and dihydroethidine-based fluorescence microtopography, was increased in the aged GPx-1−/− mice. Aging per se caused a substantial protein kinase C– and protein tyrosine kinase–dependent phosphorylation as well as S-glutathionylation of endothelial NO synthase associated with uncoupling, a phenomenon that was more pronounced in aged GPx-1−/− mice. GPx-1 ablation increased adhesion of leukocytes to cultured endothelial cells and CD68 and F4/80 staining in cardiac tissue. Aged GPx-1−/− mice displayed increased oxidant formation as compared with their wild-type littermates, triggering redox-signaling pathways associated with endothelial NO synthase dysfunction and uncoupling. Thus, our data demonstrate that aging leads to decreased NO bioavailability because of endothelial NO synthase dysfunction and uncoupling of the enzyme leading to endothelial dysfunction, vascular remodeling, and promotion of adhesion and infiltration of leukocytes into cardiovascular tissue, all of which was more prominent in aged GPx-1−/− 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.

The Sodium-Glucose Co-Transporter 2 Inhibitor Empagliflozin Improves Diabetes-Induced Vascular Dysfunction in the Streptozotocin Diabetes Rat Model by Interfering with Oxidative Stress and Glucotoxicity

Objective In diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress. Methods Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE). Results Treatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy. Conclusions Empagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.

Crosstalk of mitochondria with NADPH oxidase via reactive oxygen and nitrogen species signalling and its role for vascular function

Cardiovascular diseases are associated with and/or caused by oxidative stress. This concept has been proven by using the approach of genetic deletion of reactive species producing (pro‐oxidant) enzymes as well as by the overexpression of reactive species detoxifying (antioxidant) enzymes leading to a marked reduction of reactive oxygen and nitrogen species (RONS) and in parallel to an amelioration of the severity of diseases. Likewise, the development and progression of cardiovascular diseases is aggravated by overexpression of RONS producing enzymes as well as deletion of antioxidant RONS detoxifying enzymes. Thus, the consequences of the interaction (redox crosstalk) of superoxide/hydrogen peroxide produced by mitochondria with other ROS producing enzymes such as NADPH oxidases (Nox) are of outstanding importance and will be discussed including the consequences for endothelial nitric oxide synthase (eNOS) uncoupling as well as the redox regulation of the vascular function/tone in general (soluble guanylyl cyclase, endothelin‐1, prostanoid synthesis). Pathways and potential mechanisms leading to this crosstalk will be analysed in detail and highlighted by selected examples from the current literature including hypoxia, angiotensin II‐induced hypertension, nitrate tolerance, aging and others. The general concept of redox‐based activation of RONS sources via “kindling radicals” and enzyme‐specific “redox switches” will be discussed providing evidence that mitochondria represent key players and amplifiers of the burden of oxidative stress.

Chronic therapy with isosorbide-5-mononitrate causes endothelial dysfunction, oxidative stress, and a marked increase in vascular endothelin-1 expression

AIMS Isosorbide-5-mononitrate (ISMN) is one of the most frequently used compounds in the treatment of coronary artery disease predominantly in the USA. However, ISMN was reported to induce endothelial dysfunction, which was corrected by vitamin C pointing to a crucial role of reactive oxygen species (ROS) in causing this phenomenon. We sought to elucidate the mechanism how ISMN causes endothelial dysfunction and oxidative stress in vascular tissue. METHODS AND RESULTS Male Wistar rats (n= 69 in total) were treated with ISMN (75 mg/kg/day) or placebo for 7 days. Endothelin (ET) expression was determined by immunohistochemistry in aortic sections. Isosorbide-5-mononitrate infusion caused significant endothelial dysfunction but no tolerance to ISMN itself, whereas ROS formation and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase activity in the aorta, heart, and whole blood were increased. Isosorbide-5-mononitrate up-regulated the expression of NADPH subunits and caused uncoupling of the endothelial nitric oxide synthase (eNOS) likely due to a down-regulation of the tetrahydrobiopterin-synthesizing enzyme GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by bosentan in vivo/ex vivo treatment and suppressed by apocynin. In addition, a strong increase in the expression of ET within the endothelial cell layer and the adventitia was observed. CONCLUSION Chronic treatment with ISMN causes endothelial dysfunction and oxidative stress, predominantly by an ET-dependent activation of the vascular and phagocytic NADPH oxidase activity and NOS uncoupling. These findings may explain at least in part results from a retrospective analysis indicating increased mortality in post-infarct patients in response to long-term treatment with mononitrates.

Peroxisome Proliferator-activated Receptor γ, Coactivator 1α Deletion Induces Angiotensin II–Associated Vascular Dysfunction by Increasing Mitochondrial Oxidative Stress and Vascular Inflammation

Objective—Peroxisome proliferator-activated receptor &ggr;, coactivator 1&agr; (PGC-1&agr;) is an important mediator of mitochondrial biogenesis and function. Because dysfunctional mitochondria might be involved in the pathogenesis of vascular disease, the current study was designed to investigate the effects of in vivo PGC-1&agr; deficiency during chronic angiotensin II (ATII) treatment. Approach and Results—Although ATII infusion at subpressor doses (0.1 mg/kg per day for 7 days) did not cause vascular dysfunction in wild-type mice, it led to impaired endothelial-dependent and endothelial-independent relaxation in PGC-1&agr; knockout mice. In parallel, oxidative stress was increased in aortic rings from ATII-treated PGC-1&agr; knockout mice, whereas no change in nitric oxide production was observed. By using the mitochondrial-specific superoxide dye MitoSox and complex I inhibitor rotenone, we identified the mitochondrial respiratory chain as the major PGC-1&agr;–dependent reactive oxygen species source in vivo, accompanied by increased vascular inflammation and cell senescence. In vivo treatment with the mitochondria-targeted antioxidant Mito-TEMPO partially corrected endothelial dysfunction and prevented vascular inflammation in ATII-treated PGC-1&agr; mice, suggesting a causative role of mitochondrial reactive oxygen species in this setting. Conclusions—PGC-1&agr; deletion induces vascular dysfunction and inflammation during chronic ATII infusion by increasing mitochondrial reactive oxygen species production.

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.

Hyperglycemia and oxidative stress in cultured endothelial cells - a comparison of primary endothelial cells with an immortalized endothelial cell line

Diabetes mellitus is a major risk factor for the development of cardiovascular disease and oxidative stress plays an important role in this process. Therefore, we investigated the effects of hyperglycemia on the formation of reactive oxygen species (ROS) and nitric oxide/cGMP signaling in two different endothelial cell cultures. Human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells showed increased oxidative stress and impaired NO-cGMP signaling in response to hyperglycemia. The major difference between the two different cell types was the dramatic decrease in viability in HUVEC whereas EA.hy cells showed rather increased growth under hyperglycemic conditions. Starvation led to an additional substantial decrease in viability and increased superoxide formation in HUVEC. Both endothelial cell types, HUVEC and EA.hy 926, may be used as models for vascular hyperglycemia. However, high growth medium should be used to avoid starvation-induced oxidative stress and cell death.