CD40L controls obesity-associated vascular inflammation, oxidative stress, and endothelial dysfunction in high fat diet-treated and db/db mice.

Aims: CD40 ligand (CD40L) signaling controls vascular oxidative stress and related dysfunction in angiotensin-II-induced arterial hypertension by regulating vascular immune cell recruitment and platelet activation. Here we investigated the role of CD40L in experimental hyperlipidemia. Methods and results: Male wild type and CD40L-/- mice (C57BL/6 background) were subjected to high fat diet for sixteen weeks. Weight, cholesterol, HDL, and LDL levels, endothelial function (isometric tension recording), oxidative stress (NADPH oxidase expression, dihydroethidium fluorescence) and inflammatory parameters (inducible nitric oxide synthase, interleukin-6 expression) were assessed. CD40L expression, weight, leptin and lipids were increased, and endothelial dysfunction, oxidative stress and inflammation were more pronounced in wild type mice on a high fat diet, all of which was almost normalized by CD40L deficiency. Similar results were obtained in diabetic db/db mice with CD40/TRAF6 inhibitor (6877002) therapy. In a small human study higher serum sCD40L levels and an inflammatory phenotype were detected in the blood and Aorta ascendens of obese patients (body mass index > 35) that underwent by-pass surgery. Conclusion: CD40L controls obesity-associated vascular inflammation, oxidative stress and endothelial dysfunction in mice and potentially humans. Thus, CD40L represents a therapeutic target in lipid metabolic disorders which is a leading cause in cardiovascular disease.

Gliptins Suppress Inflammatory Macrophage Activation to Mitigate Inflammation, Fibrosis, Oxidative Stress, and Vascular Dysfunction in Models of Nonalcoholic Steatohepatitis and Liver Fibrosis.

AIMS: Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, panlobular inflammation, liver fibrosis, and increased cardiovascular mortality. Dipeptidyl peptidase-4 inhibitors (gliptins) are indirect glucagon-like peptide 1 agonists with antidiabetic and anti-inflammatory activity, used for the treatment of type 2 diabetes. Their potential and underlying mechanisms to treat metabolic liver inflammation and fibrosis as well as the associated vascular dysfunction remain to be explored. RESULTS: In the methionine/choline-deficient (MCD) diet and Mdr2-/- models of NASH and liver fibrosis, treatment with sitagliptin and linagliptin significantly decreased parameters of steatosis and inflammation, which was accompanied by suppression of hepatic transcript levels reflecting metabolic inflammation and fibrosis, including SREBP-1c, FAS, TNFα, iNOS, α-SMA, Col1α1, and MMP-12. Moreover, gliptins reduced the number of liver infiltrating CD11b+Ly6Chi proinflammatory monocytes/macrophages and liver-resident F4/80+ macrophages, with an increase of Ym1+ alternative macrophages and (anti-inflammatory) macrophage markers Arg1 and IL-10. This was paralleled by decreased hepatic and aortic reactive oxygen species (ROS) production and NOX-2 mRNA expression, a normalization of endothelial dysfunction, cardiac NADPH oxidase activity, mitochondrial ROS formation, and whole blood oxidative burst in the MCD model. Innovation and Conclusions: Gliptins via suppression of inflammation decrease steatosis, apoptosis, oxidative stress, and vascular dysfunction in murine models of NASH and liver fibrosis, with mild direct antifibrotic properties. They reduce the numbers of liver and vascular inflammatory monocytes/macrophages and induce their alternative polarization, with beneficial effect on NASH-associated hepatic and cardiovascular complications. Therefore, gliptins qualify as drugs for treatment of NASH and associated liver fibrosis and cardiovascular complications. Antioxid. Redox Signal. 28, 87-109.

The Endothelin Receptor Antagonist Macitentan Improves Isosorbide-5-Mononitrate (ISMN) and Isosorbide Dinitrate (ISDN) Induced Endothelial Dysfunction, Oxidative Stress, and Vascular Inflammation.

OBJECTIVE: Organic nitrates such as isosorbide-5-mononitrate (ISMN) and isosorbide dinitrate (ISDN) are used for the treatment of patients with chronic symptomatic stable coronary artery disease and chronic congestive heart failure. Limiting side effects of these nitrovasodilators include nitrate tolerance and/or endothelial dysfunction mediated by oxidative stress. Here, we tested the therapeutic effects of the dual endothelin (ET) receptor antagonist macitentan in ISMN- and ISDN-treated animals. METHODS AND RESULTS: Organic nitrates (ISMN, ISDN, and nitroglycerin (GTN)) augmented the oxidative burst and interleukin-6 release in cultured macrophages, whereas macitentan decreased the oxidative burst in isolated human leukocytes. Male C57BL/6j mice were treated with ISMN (75 mg/kg/d) or ISDN (25 mg/kg/d) via s.c. infusion for 7 days and some mice in addition with 30 mg/kg/d of macitentan (gavage, once daily). ISMN and ISDN in vivo therapy caused endothelial dysfunction but no nitrate (or cross-)tolerance to the organic nitrates, respectively. ISMN/ISDN increased blood nitrosative stress, vascular/cardiac oxidative stress via NOX-2 (fluorescence and chemiluminescence methods), ET1 expression, ET receptor signaling, and markers of inflammation (protein and mRNA level). ET receptor signaling blockade by macitentan normalized endothelial function, vascular/cardiac oxidative stress, and inflammatory phenotype in both nitrate therapy groups. CONCLUSION: ISMN/ISDN treatment caused activation of the NOX-2/ET receptor signaling axis leading to increased vascular oxidative stress and inflammation as well as endothelial dysfunction. Our study demonstrates for the first time that blockade of ET receptor signaling by the dual endothelin receptor blocker macitentan improves adverse side effects of the organic nitrates ISMN and ISDN.

Role of Protein Kinase C and Nox2-Derived Reactive Oxygen Species Formation in the Activation and Maturation of Dendritic Cells by Phorbol Ester and Lipopolysaccharide.

Aims. Activation/maturation of dendritic cells (DCs) plays a central role in adaptive immune responses by antigen processing and (cross-) activation of T cells. There is ongoing discussion on the role of reactive oxygen species (ROS) in these processes and with the present study we investigated this enigmatic pathway. Methods and Results. DCs were cultured from precursors in the bone marrow of mice (BM-DCs) and analyzed for ROS formation, maturation, and T cell stimulatory capacity upon stimulation with phorbol ester (PDBu) and lipopolysaccharide (LPS). LPS stimulation of BM-DCs caused maturation with moderate intracellular ROS formation, whereas PDBu treatment resulted in maturation with significant ROS formation. The NADPH oxidase inhibitors apocynin/VAS2870 and genetic gp91phox deletion both decreased the ROS signal in PDBu-stimulated BM-DCs without affecting maturation and T cell stimulatory capacity of BM-DCs. In contrast, the protein kinase C inhibitors chelerythrine/Gö6983 decreased PDBu-stimulated ROS formation in BM-DCs as well as maturation. Conclusion. Obviously Nox2-dependent ROS formation in BM-DCs is not always required for their maturation or T cell stimulatory potential. PDBu/LPS-triggered BM-DC maturation rather relies on phosphorylation cascades. Our results question the role of oxidative stress as an essential "danger signal" for BM-DC activation, although we cannot exclude contribution by other ROS sources.

Gliptin and GLP-1 analog treatment improves survival and vascular inflammation/dysfunction in animals with lipopolysaccharide-induced endotoxemia.

Dipeptidyl peptidase (DPP)-4 inhibitors are used to treat hyperglycemia by increasing the incretin glucagon-like peptide-1 (GLP-1). Previous studies showed anti-inflammatory and antiatherosclerotic effects of DPP-4 inhibitors. Here, we compared the effects of linagliptin versus sitagliptin and liraglutide on survival and vascular function in animal models of endotoxic shock by prophylactic therapy and treatment after lipopolysaccharide (LPS) injection. Gliptins were administered either orally or subcutaneously: linagliptin (5 mg/kg/day), sitagliptin (50 mg/kg/day) or liraglutide (200 µg/kg/day). Endotoxic shock was induced by LPS injection (mice 17.5-20 mg/kg i.p., rats 10 mg/kg/day). Linagliptin and liraglutide treatment or DPP-4 knockout improved the survival of endotoxemic mice, while sitagliptin was ineffective. Linagliptin, liraglutide and sitagliptin ameliorated LPS-induced hypotension and vascular dysfunction in endotoxemic rats, suppressed inflammatory parameters such as whole blood nitrosyl-iron hemoglobin (leukocyte-inducible nitric oxide synthase activity) or aortic mRNA expression of markers of inflammation as well as whole blood and aortic reactive oxygen species formation. Hemostasis (tail bleeding time, activated partial thromboplastin time) was impaired in endotoxemic rats and recovered under cotreatment with linagliptin and liraglutide. Finally, the beneficial effects of linagliptin on vascular function and inflammatory parameters in endotoxemic mice were impaired in AMP-activated kinase (alpha1) knockout mice. The improved survival of endotoxemic animals and other data shown here may warrant further clinical evaluation of these drugs in patients with septic shock beyond the potential improvement of inflammatory complications in diabetic individuals with special emphasis on the role of AMP-activated kinase (alpha1) in the DPP-4/GLP-1 cascade.

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.

In vitro and in vivo characterization of a new organic nitrate hybrid drug covalently bound to pioglitazone.

BACKGROUND/AIMS: Organic nitrates represent a group of nitrovasodilators that are clinically used for the treatment of ischemic heart disease. The new compound CLC-3000 is an aminoethyl nitrate (AEN) derivative of pioglitazone, a thiazolidinedione antidiabetic agent combining the peroxisome proliferator-activated receptor γ agonist activity of pioglitazone with the NO-donating activity of the nitrate moiety. METHODS: In vitro and in vivo characterization was performed by isometric tension recording, platelet function, bleeding time and detection of oxidative stress. RESULTS: In vitro, CLC-3000 displayed more potent vasodilation than pioglitazone alone or classical nitrates. In vitro, some effects on oxidative stress parameters were observed. Authentic AEN or the AEN-containing linker CLC-1275 displayed antiaggregatory effects. In vivo treatment with CLC-3000 for 7 days did neither induce endothelial dysfunction nor nitrate tolerance nor oxidative stress. Acute or chronic administration of AEN increased the tail vein bleeding time in mice. CONCLUSION: In summary, the results of these studies demonstrate that CLC-3000 contains a vasodilative and antithrombotic activity that is not evident with pioglitazone alone, and that 7 days of exposure in vivo showed no typical signs of nitrate tolerance, endothelial dysfunction or other safety concerns in Wistar rats.

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.

CD40L contributes to angiotensin II-induced pro-thrombotic state, vascular inflammation, oxidative stress and endothelial dysfunction.

CD40 ligand (CD40L) is involved in the vascular infiltration of immune cells and pathogenesis of atherosclerosis. Additionally, T cell CD40L release causes platelet, dendritic cell and monocyte activation in thrombosis. However, the role of CD40L in angiotensin II (ATII)-driven vascular dysfunction and hypertension remains incompletely understood. We tested the hypothesis that CD40L contributes to ATII-driven vascular inflammation by promoting platelet-leukocyte activation, vascular infiltration of immune cells and by amplifying oxidative stress. C57BL/6 and CD40L-/- mice were infused with ATII (1 mg/kg/day for 7 days) using osmotic minipumps. Vascular function was recorded by isometric tension studies, and reactive oxygen species (ROS) were monitored in blood and heart by optical methods. Western blot, immunohistochemistry, FACS analysis and real-time RT-PCR were used to analyze immune cell distribution, pro-inflammatory cytokines, NAPDH oxidase subunits, T cell transcription factors and other genes of interest. ATII-treated CD40L-/- mice showed improved endothelial function, suppression of blood platelet-monocyte interaction (FACS), platelet thrombin generation (calibrated automated thrombography) and coagulation (bleeding time), as well as decreased oxidative stress in the aorta, heart and blood compared to wild-type mice. Moreover, ATII-treated CD40L-/- mice displayed decreased levels of TH1 cytokines released by splenic CD4⁺ T cells (ELISA) and lower expression levels of NOX-2, T-bet and P-selectin as well as diminished immune cell infiltration in aortic tissue compared to controls. Our results demonstrate that many ATII-induced effects on vascular dysfunction, such as vascular inflammation, oxidative stress and a pro-thrombotic state, are mediated at least in part via CD40L.

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 γ, coactivator 1α (PGC-1α) 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-1α 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-1α knockout mice. In parallel, oxidative stress was increased in aortic rings from ATII-treated PGC-1α 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-1α-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-1α mice, suggesting a causative role of mitochondrial reactive oxygen species in this setting. CONCLUSIONS: PGC-1α deletion induces vascular dysfunction and inflammation during chronic ATII infusion by increasing mitochondrial reactive oxygen species production.

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-γ (IFN-γ), 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-γ remain incompletely understood. APPROACH AND RESULTS: IFN-γ(-/-) 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-γ showed constitutive vascular dysfunction. Absence of T-box expressed in T cells (T-bet), the IFN-γ 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-γ(-/-) 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-γ and CD11b(+)Gr-1(low) 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-γ 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-γ pathway and mutual monocyte-NK-cell activation as potential therapeutic targets in cardiovascular disease.

Effects of telmisartan or amlodipine monotherapy versus telmisartan/amlodipine combination therapy on vascular dysfunction and oxidative stress in diabetic rats.

Our previous studies identified potent antioxidant effects and improvement of vascular function by telmisartan therapy in experimental diabetes and nitrate tolerance. The present study compared the beneficial effects of single telmisartan or amlodipine versus telmisartan/amlodipine combination therapy (T+A) in streptozotocin (STZ)-induced type 1 diabetic rats. Male Wistar rats were injected once with STZ (60 mg/kg, i.v.) and 1 week later the drugs (telmisartan, amlodipine, or T+A) were administrated orally by a special diet (2.5-5 mg kg(-1) day(-1)) for another 7 weeks. We only observed a marginal beneficial on-top effect of T+A therapy over the single drug regimen that was most evident in the improvement of endothelial function (acetylcholine response) and less pronounced in the reduction of whole blood, vascular and cardiac oxidative stress (blood leukocyte oxidative burst, aortic dihydroethidine and 3-nitrotyrosine staining, as well as cardiac NADPH oxidase activity and uncoupling of endothelial nitric oxide synthase) in diabetic rats. These effects on oxidative stress parameters were paralleled by those on the expression pattern of NADPH oxidase and nitric oxide synthase isoforms. In addition, development of mild hypotension in the T+A-treated rats was observed. Reasons for this moderate synergistic effect of T+A therapy may be related to the potent beneficial effects of telmisartan alone and the fact that amlodipine and telmisartan share similar pathways to improve endothelial function. Moreover, hypotension in the T+A-treated rats could partially antagonize the beneficial additive effects by counter-regulatory mechanisms (e.g., activation of the renin-angiotensin-aldosterone system).

Characterization of new organic nitrate hybrid drugs covalently bound to valsartan and cilostazol.

BACKGROUND AND PURPOSE: Organic nitrates represent a group of nitrovasodilators that are clinically used for the treatment of ischemic heart disease. With the present studies we synthesized and characterized new organic nitrate hybrid molecules. Compounds CLC-1265 (valsartan mononitrate) and CLC-1280 (valsartan dinitrate) are derivatives of the angiotensin receptor blocker valsartan, with CLC-1265 containing a single organic nitrate linker and CLC-1280 also containing a second, different linker. Compounds CLC-2000 (cilostazol mononitrate) and CLC-2100 (cilostazol dinitrate) are nitrate derivatives of the phosphodiesterase III inhibitor cilostazol. All compounds are designed as hybrid molecules, potentially combining the NO-donating properties of organic nitrates with the AT1-blocking activity of valsartan or the phosphodiesterase-III-inhibiting effect of cilostazol. EXPERIMENTAL APPROACH: The properties of new drugs were assessed by isometric tension recording, inhibition of platelet aggregation and formation of mitochondrial reactive oxygen and nitrogen species. KEY RESULTS: In this report, all new nitrate compounds are shown, in vitro, to induce vasodilation in the range of other, classical organic nitrates, without inducing oxidative stress or classical nitrate tolerance. In addition, the new hybrid nitrate molecules displayed superior antiaggregatory properties over classical mono- and dinitrates. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that organic nitrates can be successfully linked to existing therapeutic molecules to create a new class of molecular entities with a potential dual mechanism of action via combining the established pharmacological properties of valsartan or cilostazol with the vasodilating properties of organic nitrates. Future experimental studies have to demonstrate whether the combined action of these compounds translates to superior therapeutic effects.

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.

α1AMP-activated protein kinase mediates vascular protective effects of exercise.

OBJECTIVE: We investigated whether AMP-activated protein kinase (AMPK) may be involved in the signaling processes leading to exercise-mediated vascular protection. METHODS AND RESULTS: The effects of voluntary exercise on AMPK activity, endothelial NO synthase expression and phosphorylation, vascular reactive oxygen species formation, and cell senescence were tested in α1AMPK knockout and corresponding wild-type mice. Exercise significantly improved endothelial function, and increased plasma nitrite production in wild-type mice, associated with an activation of aortic AMPK assessed by its phosphorylation at threonine 172. In addition, regular physical activity resulted in an upregulation of endothelial NO synthase protein, serine 1177 endothelial NO synthase phosphorylation, and an increase of circulating Tie-2(+)Sca-1(+)Flk-1(+) myeloid progenitor cells. All these changes were absent after α1AMPK deletion. In addition, exercise increased the expression of important regulators of the antioxidative defense including heme oxygenase-1 and peroxisome proliferator-activated receptor γ coactivator 1α, decreased aortic reactive oxygen species levels, and prevented endothelial cell senescence in an α1AMPK-dependent manner. CONCLUSIONS: Intact α1AMPK signaling is required for the signaling events leading to the manifestation of vascular protective effects during exercise. Pharmacological AMPK activation might be a novel approach in the near future to simulate the beneficial vascular effects of physical activity.

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.

Vascular dysfunction in experimental diabetes is improved by pentaerithrityl tetranitrate but not isosorbide-5-mononitrate therapy.

OBJECTIVE: Diabetes is associated with vascular oxidative stress, activation of NADPH oxidase, and uncoupling of nitric oxide (NO) synthase (endothelial NO synthase [eNOS]). Pentaerithrityl tetranitrate (PETN) is an organic nitrate with potent antioxidant properties via induction of heme oxygenase-1 (HO-1). We tested whether treatment with PETN improves vascular dysfunction in the setting of experimental diabetes. RESEARCH DESIGN AND METHODS: After induction of hyperglycemia by streptozotocin (STZ) injection (60 mg/kg i.v.), PETN (15 mg/kg/day p.o.) or isosorbide-5-mononitrate (ISMN; 75 mg/kg/day p.o.) was fed to Wistar rats for 7 weeks. Oxidative stress was assessed by optical methods and oxidative protein modifications, vascular function was determined by isometric tension recordings, protein expression was measured by Western blotting, RNA expression was assessed by quantitative RT-PCR, and HO-1 promoter activity in stable transfected cells was determined by luciferase assays. RESULTS: PETN, but not ISMN, improved endothelial dysfunction. NADPH oxidase and serum xanthine oxidase activities were significantly reduced by PETN but not by ISMN. Both organic nitrates had minor effects on the expression of NADPH oxidase subunits, eNOS and dihydrofolate reductase (Western blotting). PETN, but not ISMN, normalized the expression of GTP cyclohydrolase-1, extracellular superoxide dismutase, and S-glutathionylation of eNOS, thereby preventing eNOS uncoupling. The expression of the antioxidant enzyme, HO-1, was increased by STZ treatment and further upregulated by PETN, but not ISMN, via activation of the transcription factor NRF2. CONCLUSIONS: In contrast to ISMN, the organic nitrate, PETN, improves endothelial dysfunction in diabetes by preventing eNOS uncoupling and NADPH oxidase activation, thereby reducing oxidative stress. Thus, PETN therapy may be suited to treat patients with cardiovascular complications of diabetes.

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-1(low)F4/80(+) macrophages and CD11b(+)Gr-1(high)F4/80(-) 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 (LysM(iDTR) 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 gp91(phox) and p67(phox) (assessed by Western blot and mRNA reverse-transcription polymerase chain reaction). Adoptive transfer of wild-type CD11b(+)Gr-1(+) monocytes into depleted LysM(iDTR) mice reestablished ATII-induced vascular dysfunction, oxidative stress, and arterial hypertension, whereas transfer of CD11b(+)Gr-1(+) neutrophils or monocytes from gp91(phox) 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.

Nitroglycerin-induced endothelial dysfunction and tolerance involve adverse phosphorylation and S-Glutathionylation of endothelial nitric oxide synthase: beneficial effects of therapy with the AT1 receptor blocker telmisartan.

OBJECTIVE: Continuous administration of nitroglycerin (GTN) causes tolerance and endothelial dysfunction by inducing reactive oxygen species (ROS) production from various enzymatic sources, such as mitochondria, NADPH oxidase, and an uncoupled endothelial nitric oxide synthase (eNOS). In the present study, we tested the effects of type 1 angiotensin (AT(1))-receptor blockade with telmisartan on GTN-induced endothelial dysfunction in particular on eNOS phosphorylation and S-glutathionylation sites and the eNOS cofactor synthesizing enzyme GTP-cyclohydrolase I. METHODS AND RESULTS: Wistar rats were treated with telmisartan (2.7 or 8 mg/kg per day PO for 10 days) and with GTN (50 mg/kg per day SC for 3 days). Aortic eNOS phosphorylation and S-glutathionylation were assessed using antibodies against phospho-Thr495 and Ser1177 or protein-bound glutathione, which regulate eNOS activity and eNOS-dependent superoxide production (uncoupling). Expression of mitochondrial aldehyde dehydrogenase was determined by Western blotting. Formation of aortic and cardiac ROS was assessed by fluorescence, chemiluminescence, and 3-nitrotyrosine/malondialdehyde-positive protein content. Telmisartan prevented endothelial dysfunction and partially improved nitrate tolerance. Vascular, cardiac, mitochondrial, and white blood cell ROS formation were significantly increased by GTN treatment and inhibited by telmisartan. GTN-induced decrease in Ser1177, increase in Thr495 phosphorylation or S-glutathionylation of eNOS, and decrease in mitochondrial aldehyde dehydrogenase expression were normalized by telmisartan. CONCLUSIONS: These data identify modification of eNOS phosphorylation as an important component of GTN-induced endothelial dysfunction. Via its pleiotropic "antioxidant" properties, telmisartan prevents, at least in part, GTN-induced oxidative stress, nitrate tolerance, and endothelial dysfunction.