Julian Widder

Endothelial Nitric Oxide Synthase Uncoupling Impairs Endothelial Progenitor Cell Mobilization and Function in Diabetes

Uncoupling of the endothelial nitric oxide synthase (eNOS) resulting in superoxide anion (O2−) formation instead of nitric oxide (NO) causes diabetic endothelial dysfunction. eNOS regulates mobilization and function of endothelial progenitor cells (EPCs), key regulators of vascular repair. We postulate a role of eNOS uncoupling for reduced number and function of EPC in diabetes. EPC levels in diabetic patients were significantly reduced compared with those of control subjects. EPCs from diabetic patients produced excessive O2− and showed impaired migratory capacity compared with nondiabetic control subjects. NOS inhibition with NG-nitro-l-arginine attenuated O2− production and normalized functional capacity of EPCs from diabetic patients. Glucose-mediated EPC dysfunction was protein kinase C dependent, associated with reduced intracellular BH4 (tetrahydrobiopterin) concentrations, and reversible after exogenous BH4 treatment. Activation of NADPH oxidases played an additional but minor role in glucose-mediated EPC dysfunction. In rats with streptozotocin-induced diabetes, circulating EPCs were reduced to 39 ± 5% of controls and associated with uncoupled eNOS in bone marrow. Our results identify uncoupling of eNOS in diabetic bone marrow, glucose-treated EPCs, and EPCs from diabetic patients resulting in eNOS-mediated O2− production. Subsequent reduction of EPC levels and impairment of EPC function likely contributes to the pathogenesis of vascular disease in diabetes.

Endothelial mechanotransduction, nitric oxide and vascular inflammation.

Numerous aspects of vascular homeostasis are modulated by nitric oxide and reactive oxygen species (ROS). The production of these is dramatically influenced by mechanical forces imposed on the endothelium and vascular smooth muscle. In this review, we will discuss the effects of mechanical forces on the expression of the endothelial cell nitric oxide synthase, production of ROS and modulation of endothelial cell glutathione. We will also review data that exercise training in vivo has a similar effect as laminar shear on endothelial function and discuss the clinical relevance of these basic findings.

Uncoupled Cardiac Nitric Oxide Synthase Mediates Diastolic Dysfunction

Background— Heart failure with preserved ejection fraction is 1 consequence of hypertension and is caused by impaired cardiac diastolic relaxation. Nitric oxide (NO) is a known modulator of cardiac relaxation. Hypertension can lead to a reduction in vascular NO, in part because NO synthase (NOS) becomes uncoupled when oxidative depletion of its cofactor tetrahydrobiopterin (BH4) occurs. Similar events may occur in the heart that lead to uncoupled NOS and diastolic dysfunction. Methods and Results— In a hypertensive mouse model, diastolic dysfunction was accompanied by cardiac oxidation, a reduction in cardiac BH4, and uncoupled NOS. Compared with sham-operated animals, male mice with unilateral nephrectomy, with subcutaneous implantation of a controlled-release deoxycorticosterone acetate pellet, and given 1% saline to drink were mildly hypertensive and had diastolic dysfunction in the absence of systolic dysfunction or cardiac hypertrophy. The hypertensive mouse hearts showed increased oxidized biopterins, NOS-dependent superoxide production, reduced NO production, and dephosphorylated phospholamban. Feeding hypertensive mice BH4 (5 mg/d), but not treating with hydralazine or tetrahydroneopterin, improved cardiac BH4 stores, phosphorylated phospholamban levels, and diastolic dysfunction. Isolated cardiomyocyte experiments revealed impaired relaxation that was normalized with short-term BH4 treatment. Targeted cardiac overexpression of angiotensin-converting enzyme also resulted in cardiac oxidation, NOS uncoupling, and diastolic dysfunction in the absence of hypertension. Conclusions— Cardiac oxidation, independently of vascular changes, can lead to uncoupled cardiac NOS and diastolic dysfunction. BH4 may represent a possible treatment for diastolic dysfunction.

Attenuation of Angiotensin II–Induced Vascular Dysfunction and Hypertension by Overexpression of Thioredoxin 2

Reactive oxygen species increase in the cardiovascular system during hypertension and in response to angiotensin II. Because mitochondria contribute to reactive oxygen species generation, we sought to investigate the role of thioredoxin 2, a mitochondria-specific antioxidant enzyme. Mice were created with overexpression of human thioredoxin 2 (TghTrx2 mice) and backcrossed to C57BL/6J mice for ≥6 generations. Twelve-week-old male TghTrx2 or littermate wild-type mice were made hypertensive by infusion of angiotensin II (400 ng/kg per minute) for 14 days using osmotic minipumps. Systolic arterial blood pressure was not different between TghTrx2 and wild-type animals under baseline conditions (101±1 respective 102±1 mm Hg). The angiotensin II–induced hypertension in wild-type mice (145±2 mm Hg) was significantly attenuated in TghTrx2 mice (124±1 mm Hg; P<0.001). Aortic endothelium-dependent relaxation was significantly reduced in wild-type mice after angiotensin II infusion but nearly unchanged in transgenic mice. Elevated vascular superoxide and hydrogen peroxide levels, as well as expression of NADPH oxidase subunits in response to angiotensin II infusion, were significantly attenuated in TghTrx2 mice. Mitochondrial superoxide anion levels were augmented after angiotensin II infusion in wild-type mice, and this was blunted in TghTrx2 mice. Angiotensin II infusion significantly increased myocardial superoxide formation, heart weight, and cardiomyocyte size in wild-type but not in TghTrx2 mice. These data indicate a major role for mitochondrial thioredoxin 2 in the development of cardiovascular alterations and hypertension during chronic angiotensin II infusion. Thioredoxin 2 may represent an important therapeutic target for the prevention and treatment of hypertension and oxidative stress.

Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress

An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the &agr; prime (&agr;′) subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of endothelial NO synthase, increased endothelial cell superoxide production, and prevented the increase in NO production caused by shear. Thus, shear stress not only increases endothelial NO synthase levels but also stimulates production of H4B by markedly enhancing GTPCH-1 activity via casein kinase 2–dependent phosphorylation on serine 81. These findings illustrate a new function of casein kinase 2 in the endothelium and provide insight into regulation of GTPCH-1 activity.

Improvement in Left Ventricular Remodeling by the Endothelial Nitric Oxide Synthase Enhancer AVE9488 After Experimental Myocardial Infarction

Background— Reduced endothelial nitric oxide (NO) bioavailability contributes to the progression of heart failure. In this study, we investigated whether the transcription enhancer of endothelial NO synthase (eNOS) AVE9488 improves cardiac remodeling and heart failure after experimental myocardial infarction (MI). Methods and Results— Starting 7 days after coronary artery ligation, rats with MI were treated with placebo or AVE9488 (25 ppm) as a dietary supplement for 9 weeks. AVE9488 therapy versus placebo substantially improved left ventricular (LV) function, reduced LV filling pressure, and prevented the rightward shift of the pressure-volume curve. AVE9488 also attenuated the extent of pulmonary edema, reduced LV fibrosis and myocyte cross-sectional area, and prevented the increases in LV gene expression of atrial natriuretic factor, brain natriuretic peptide, and endothelin-1. eNOS protein levels and calcium-dependent NOS activity were decreased in the surviving LV myocardium from placebo MI rats and normalized by AVE9488. The beneficial effects of AVE9488 on LV dysfunction and remodeling after MI were abrogated in eNOS-deficient mice. Aortic eNOS protein expression and endothelium-dependent NO-mediated vasorelaxation were significantly enhanced by AVE9488 treatment after infarction, whereas increased vascular superoxide anion formation was reduced. Moreover, AVE9488 prevented the marked depression of circulating endothelial progenitor cell levels in rats with heart failure after MI. Conclusions— Long-term treatment with the eNOS enhancer AVE9488 improved LV remodeling and contractile dysfunction after MI. Molecular alterations, circulating endothelial progenitor cell levels, and endothelial vasomotor dysfunction were improved by AVE9488. Pharmacological interventions designed to increase eNOS-derived NO constitute a promising therapeutic approach for the amelioration of postinfarction ventricular remodeling and heart failure.

Peroxisome proliferator activated‐receptor agonism and left ventricular remodeling in mice with chronic myocardial infarction

Peroxisome proliferator activated receptor γ (PPARγ) has been implicated in several cellular pathways assumed to beneficially affect heart failure progression. In contrast, population‐based studies demonstrate an increased incidence of heart failure in patients treated with PPARγ agonists. Therefore, we examined the effect of pioglitazone, a PPARγ agonist, on chronic left ventricular remodeling after experimental myocardial infarction (MI) in mice. Mice were treated with placebo or pioglitazone (20 mg kg−1 by gavage) from week 1 to week 6 after ligation of the left anterior descending artery. Serial transthoracic echocardiography was performed at weeks 1, 3, and 6. Over 6 weeks, there was no difference in mortality (placebo 12%, pioglitazone 10%). Echocardiography showed significant left ventricular dilatation in animals with MI (week 6, end‐systolic area, placebo sham 9.6±1.3 vs placebo MI 14.4±2.5 mm2). However, there was no difference between the placebo and pioglitazone groups (week 6, end‐systolic area, pioglitazone MI 14.8±2.9 mm2, P=NS vs placebo). Moreover, there were no changes in metabolic parameters, inflammation, and collagen deposition. Endothelial function in the aorta was not changed by PPARγ activation. In conclusion, PPARγ activation did not adversely affect left ventricular remodeling and survival in mice with chronic MI. However, we were also not able to identify a protective effect of pioglitazone.

Improvement of Endothelial Dysfunction by Selective Estrogen Receptor-α Stimulation in Ovariectomized SHR

Abstract—Both known estrogen receptors, ER&agr; and ER&bgr;, are expressed in blood vessels. To gain further insight into the role of ER&agr; in a functional setting, we investigated the effect of the novel highly selective ER&agr; agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17&bgr;-estradiol (E2, 2 &mgr;g/kg body wt per day), the selective ER&agr; agonist Cpd1471 (30 &mgr;g/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (Rmax, 53%±3% versus sham, 79%±2%; P <0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (Rmax, 70%±2%; resp, 73%±2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor N&ohgr;-nitro-l-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53±2% versus 77%±5%; P <0.01) and was normalized by both E2 (70%±2%) and Cpd1471 (70%±3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ER&agr; agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ER&agr; activation mediates positive vascular effects.

Role of the Multidrug Resistance Protein-1 in Hypertension and Vascular Dysfunction Caused by Angiotensin II

Objective—Human endothelial cells use the multidrug resistance protein-1 (MRP1) to export glutathione disulfide (GSSG). This can promotes thiol loss during states of increased glutathione oxidation. We investigated how MRP1 modulates blood pressure and vascular function during angiotensin II-induced hypertension. Methods and Results—Angiotensin II–induced hypertension altered vascular glutathione flux by increasing GSSG export and decreasing vascular levels of glutathione in wild-type (FVB) but not in MRP1−/− mice. Aortic endothelium-dependent vasodilatation was reduced in FVB after angiotensin II infusion, but unchanged in MRP1−/− mice. Aortic superoxide (O2·−) production and expression of several NADPH oxidase subunits were increased by angiotensin II in FVB. These effects were markedly blunted in MRP1−/− vessels. The increase in O2·− production in FVB vessels caused by angiotensin II was largely inhibited by L-NAME, suggesting eNOS uncoupling. Accordingly, aortic tetrahydrobiopterin and levels of NO were decreased by angiotensin II in FVB but were unchanged in MRP1−/−. Finally, the hypertension caused by angiotensin II was markedly blunted in MRP1−/− mice (137±4 versus 158±6 mm Hg). Conclusion—MRP1 plays a crucial role in the genesis of multiple vascular abnormalities that accompany hypertension and its presence is essential for the hypertensive response to angiotensin II.

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.