Tatsiana Suvorava

Endogenous Vascular Hydrogen Peroxide Regulates Arteriolar Tension In Vivo

Background— Although many studies suggested direct vasomotor effects of hydrogen peroxide (H2O2) in vitro, little is known about the vasomotor effects of H2O2 in vivo. Methods and Results— We have generated mice overexpressing human catalase driven by the Tie-2 promoter to specifically target this transgene to the vascular tissue. Vessels of these mice (cat++) expressed significantly higher levels of catalase mRNA, protein, and activity. The overexpression was selective for vascular tissue, as evidenced by immunohistochemistry in specimens of aorta, heart, lung, and kidney. Quantification of reactive oxygen species by fluorescence signals in cat++ versus catalase-negative (catn) mice showed a strong decrease in aortic endothelium and left ventricular myocardium but not in leukocytes. Awake male cat++ at 3 to 4 months of age had a significantly lower systolic blood pressure (sBP, 102.7±2.2 mm Hg, n=10) compared with their transgene-negative littermates (catn, 115.6±2.5 mm Hg, P=0.0211) and C57BL/6 mice (118.4±3.06 mm Hg, n=6). Treatment with the catalase inhibitor aminotriazole increased sBP of cat++ to 117.3±4.3 mm Hg (P=0.0345), while having no effect in catn (118.4±2.4 mm Hg, n=4, P>0.05). In contrast, treatment with the NO-synthase inhibitor nitro-l-arginine methyl ester (100 mg · kg BW−1 · d−1) increased sBP in cat++ and C57Bl/6 to a similar extent. Likewise, phosphorylation of vasodilator-stimulated phosphoprotein in skeletal muscle, left ventricular myocardium, and lung was identical in cat++ and catn. Endothelium- and NO-dependent aortic vasodilations were unchanged in cat++. Aortic KCl contractions were significantly lower in cat++ and exogenous H2O2 (10 &mgr;mol/L)–induced vasoconstriction. Conclusions— These data suggest that endogenous H2O2 may act as a vasoconstrictor in resistance vessels and contribute to the regulation of blood pressure.

Inhibition of Hyaluronan Synthesis Accelerates Murine Atherosclerosis Novel Insights Into the Role of Hyaluronan Synthesis

Background— Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. Methods and Results— Four-week-old apolipoprotein E–deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. Conclusions— The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis.Background— Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. Methods and Results— Four-week-old apolipoprotein E–deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. Conclusions— The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis. # Clinical Perspective {#article-title-39}

Reactive oxygen species as cardiovascular mediators: Lessons from endothelial-specific protein overexpression mouse models

The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.

Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo

Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low‐density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS++) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS++ mice with a several fold higher endothelial‐specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real‐time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5‐fold to 3.6‐fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS++ lung cytosols showed an increase of ecSOD protein level of 142 ± 10.5% as compared with transgene‐negative littermates (P < 0.05), which was abolished by Nω‐nitro‐L‐arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up‐regulation of vascular ecSOD may contribute to the reported antioxidative and anti‐atherosclerotic effects of PETN.

Left ventricular diastolic dysfunction in Nrf2 knock out mice is associated with cardiac hypertrophy, decreased expression of SERCA2a, and preserved endothelial function

Increased production of reactive oxygen species and failure of the antioxidant defense system are considered to play a central role in the pathogenesis of cardiovascular disease. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch controlling the expression of antioxidant and protective enzymes, and was proposed to participate in protection of vascular and cardiac function. This study was undertaken to analyze cardiac and vascular phenotype of mice lacking Nrf2. We found that Nrf2 knock out (Nrf2 KO) mice have a left ventricular (LV) diastolic dysfunction, characterized by prolonged E wave deceleration time, relaxation time and total diastolic time, increased E/A ratio and myocardial performance index, as assessed by echocardiography. LV dysfunction in Nrf2 KO mice was associated with cardiac hypertrophy, and a downregulation of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in the myocardium. Accordingly, cardiac relaxation was impaired, as demonstrated by decreased responses to β-adrenergic stimulation by isoproterenol ex vivo, and to the cardiac glycoside ouabain in vivo. Surprisingly, we found that vascular endothelial function and endothelial nitric oxide synthase (eNOS)-mediated vascular responses were fully preserved, blood pressure was decreased, and eNOS was upregulated in the aorta and the heart of Nrf2 KO mice. Taken together, these results show that LV dysfunction in Nrf2 KO mice is mainly associated with cardiac hypertrophy and downregulation of SERCA2a, and is independent from changes in coronary vascular function or systemic hemodynamics, which are preserved by a compensatory upregulation of eNOS. These data provide new insights into how Nrf2 expression/function impacts the cardiovascular system.

Angiotensin-(1–7) Modulates Renal Vascular Resistance Through Inhibition of p38 Mitogen-Activated Protein Kinase in Apolipoprotein E–Deficient Mice

Apolipoprotein E–deficient (apoE(−/−)) mice fed on Western diet are characterized by increased vascular resistance and atherosclerosis. Previously, we have shown that chronic angiotensin (Ang)-(1–7) treatment ameliorates endothelial dysfunction in apoE(−/−) mice. However, the mechanism of Ang-(1–7) on vasoconstrictor response to Ang II is unknown. To examine Ang-(1–7) function, we used apoE(−/−) and wild-type mice fed on Western diet that were treated via osmotic minipumps either with Ang-(1–7) (82 &mgr;g/kg per hour) or saline for 6 weeks. We show that Ang II–induced renal pressor response was significantly increased in apoE(−/−) compared with wild-type mice. This apoE(−/−)-specific response is attributed to reactive oxygen species–mediated p38 mitogen–activated protein kinase activation and subsequent phosphorylation of myosin light chain (MLC20), causing renal vasoconstriction. Here, we provide evidence that chronic Ang-(1–7) treatment attenuated the renal pressor response to Ang II in apoE(−/−) mice to wild-type levels. Ang-(1–7) treatment significantly decreased renal inducible nicotinamide adenine dinucleotide phosphate subunit p47phox levels and, thus, reactive oxygen species production that in turn causes decreased p38 mitogen-activated protein kinase activity. The latter has been confirmed by administration of a specific p38 mitogen-activated protein kinase inhibitor SB203580 (5 &mgr;mol/L), causing a reduced renal pressor response to Ang II in apoE(−/−) but not in apoE(−/−) mice treated with Ang-(1–7). Moreover, Ang-(1–7) treatment had no effect in Mas(−/−)/apoE(−/−) double-knockout mice confirming the specificity of Ang-(1–7) action through the Mas-receptor. In summary, Ang-(1–7) modulates vascular function via Mas-receptor activation that attenuates pressor response to Ang II in apoE(−/−) mice by reducing reactive oxygen species–mediated p38 mitogen-activated protein kinase activity.

Hydrogen peroxide inhibits exercise-induced increase of circulating stem cells with endothelial progenitor capacity

Abstract The number of circulating stem cells with endothelial progenitor capacity (EPCs) inversely correlates with the number of cardiovascular risk factors. In this study we sought to investigate the effects of vascular H2O2 on circulating EPC levels. In C57BL/6 mice 3 weeks of freely moving or forced physical activity or voluntary exercise failed to increase circulating EPCs defined as double positive for Flk-1 and CD34, CD133 or Sca-1. Likewise, neither insertion of additional genes encoding for catalase (cat++) or eNOS nor eNOS knock-out changed EPCs in resting mice. In striking contrast, inhibition of catalase by aminotriazole strongly reduced circulating EPCs in sedentary cat++ and their transgen-negative littermates (catn), while forced or voluntary exercise training of cat++ mice significantly increased the number of circulating EPCs. The latter effect was completely inhibitable by aminotriazole. These data suggest that endogenous vascular H2O2 likely contributes to the impairment of important stem cell-induced vascular repair mechanisms in cardiovascular disease.

Regulation of vascular guanylyl cyclase by endothelial nitric oxide-dependent posttranslational modification

In isolated cells, soluble guanylyl cyclase (sGC) activity is regulated by exogenous nitric oxide (NO) via downregulation of expression and posttranslational S-nitrosylation. The aim of this study was to investigate whether such regulatory mechanism impact on endothelium-dependent vasodilation in a newly developed mouse strain carrying an endothelial-specific overexpression of eNOS (eNOS++). When compared with transgene negative controls (eNOSn), eNOS++-mice showed a 3.3-fold higher endothelial-specific aortic eNOS expression, increased vascular cGMP and VASP phosphorylation, a L-nitroarginine (L-NA)-inhibitable decrease in systolic blood pressure, but normal levels of peroxynitrite and nitrotyrosine formation, endothelium-dependent aortic vasodilation and vasodilation to NO donors. Western blot analysis for sGC showed similar protein levels of sGC-α1 and sGC-β1 subunits in eNOSn and eNOS++. In striking contrast, the activity of isolated sGC was strongly decreased in lungs of eNOS++. Semiquantitative evaluation of sGC-β1-S-nitrosylation demonstrated that this loss of sGC activity is associated with increased nitrosylation of the enzyme in eNOS++, a difference that disappeared after L-NA-treatment. Our data suggest the existence of a physiologic NO-dependent posttranslational regulation of vascular sGC in mammals involving S-nitrosylation as a key mechanism. Because this mechanism can compensate for reduction in vascular NO bioavailability, it may mask the development of endothelial dysfunction.

Catalase activity prevents exercise-induced up-regulation of vasoprotective proteins in venous tissue.

Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up‐regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up‐regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9‐ and 10‐fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up‐regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise‐induced up‐regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation.

Deletion of Hyaluronan Synthase 3 Inhibits Neointimal Hyperplasia in Mice

Objective— Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. Approach and Results— Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. Conclusions— HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB–mediated migration, and in turn enhances neointimal hyperplasia in vivo.