Axel Gödecke

HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3

HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.

Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.

Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice

For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS-/-). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS-/- mice. Aortic endothelial cells derived from eNOS-/- mice displayed only background levels of NOx formation compared with wild-type (WT) cells (88 versus 1990 pmol NOx x h-1/mg protein-1). eNOS-/- mice were hypertensive (mean arterial pressure, 135 +/- 15 versus 107 +/- 8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with Nomega-nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, -35%) in WT hearts was followed by sustained vasodilation (+190%). L-NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS-/- hearts, the initial vasoconstriction was augmented (-70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (-49% decrease of basal flow). This effect was even more pronounced in eNOS-/- hearts (-71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOS-/- mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh-induced vasodilation in both WT and eNOS-/- mice.

Targeted Disruption of cd73/Ecto-5′-Nucleotidase Alters Thromboregulation and Augments Vascular Inflammatory Response

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5′-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (−14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73−/− mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73−/− mice (−35%). In vivo platelet cAMP levels were 0.96±0.46 in WT versus 0.68±0.27 pmol/106 cells in cd73−/− mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 &mgr;mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia–reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73−/− compared with WT littermates (WT 98% versus cd73−/− 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo–perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73−/− mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Increased nitrovasodilator sensitivity in endothelial nitric oxide synthase knockout mice: role of soluble guanylyl cyclase.

Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS(-/-)) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS(-/-) mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N(omega)-Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS(-/-) but increased the sensitivity in WT mice so it was no longer different than that of eNOS(-/-). Basal cGMP levels in aortic rings were significantly lower in eNOS(-/-) mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOS(-/-) mice than in WT mice. The maximal SNP-induced (10 micromol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS(-/-) mice than in WT mice. Incubation of aortic segments from WT mice with N(omega)-nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 micromol/L). The aortic expressions of the sGC alpha1 and beta1 subunits in WT and eNOS(-/-) mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS(-/-) mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents.

Role of myoglobin in the antioxidant defense of the heart

Although the primary function of myoglobin (Mb) has been considered to be cellular O2 storage and supply, recent studies have shown that Mb in addition can act as NO oxidase. Here we report that Mb also significantly contributes to the attenuation of oxidative stress in cardiac muscle. In support of this hypothesis, we found that in isolated perfused hearts of Mb‐deficient (myo‒/‒) mice oxidative challenge by intracoronary infused H2O2 (1‐300 µM) or superoxide formed by 2,3‐dimethoxy‐1,4‐naphtoquinone (0.1‐30 µM), respectively, depressed cardiac contractility to a greater extent than in wild‐type (WT) hearts, e.g., up to [H2O2] = 10 µM there was a significant left ventricular developed pressure (LVDP) decrease in myo‒/‒ hearts only (90.4±4.2 vs. 98.1±0.7% of control, n=6, P<0.05). Likewise in an ischemia/reperfusion protocol, myo‒/‒ hearts showed a delayed recovery of postischemic function as compared with WT controls (e.g., LVDP was 35.6±7.5 vs. 22.4±5.3 mmHg, respectively, after 10 min of reperfusion, P<0.05, n=8), which correlated well with an enhanced release of reactive oxygen species in myo‒/‒ hearts as measured by online lucigenin‐enhanced chemiluminescence [e.g. 465±87 relative light units (RLU) in myo‒/‒ vs. 287±73 RLU in WT after 2.5 min of reperfusion, P<0.05, n=8]. 31P NMR spectroscopy revealed concomitantly a more pronounced phosphocreatine overshoot during reperfusion in the knockout but only minute alterations in ATP and pHi. Our data show that lack of Mb leads to increased vulnerability of cardiac function to oxidative challenge either pharmacologically induced or endogenously generated. We propose that Mb is a key element influencing redox pathways in cardiac muscle to functionally and metabolically protect the heart from oxidative damage.

Enhanced Blood Pressure Variability in eNOS Knockout Mice

It has been shown previously that endogenous nitric oxide can buffer arterial blood pressure variability in dogs and rats. In these former studies, all isoforms of the nitric oxide synthase were blocked pharmacologically and an increased blood pressure variability was observed. Thus the question as to which isoform of the nitric oxide synthase is responsible for the blood pressure buffering effect of endogenous nitric oxide remains unraveled. In the present study, we therefore compared blood pressure variability in knockout mice that lack specifically the gene for endothelial nitric oxide synthase with their respective wild-type controls. One day after carotid artery cannulation, blood pressure was recorded in these conscious mice. During resting conditions, blood pressure variability was markedly enhanced in knockout mice compared with wild-type mice (10.5+/-1.5 mm Hg2 vs 6.0+/-0.8 mm Hg2, P<0.05). Power spectral analysis revealed that this increase in blood pressure variability is manifested at low frequencies that range from 0.05 to 0.40 s-1 (Hz) (5.1+/-1.0 mm Hg2 vs 2.5+/-0.5 mm Hg2, P<0.05). On the basis of these results, we conclude that the blood pressure buffering effect of endogenous nitric oxide is mediated by the endothelial isoform of the nitric oxide synthase. In addition, endothelial nitric oxide is most effective in buffering blood pressure oscillations at frequencies that range from 0.05 to 0.40 s-1 (Hz) in conscious mice.

Inotropic response to β-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts

1 The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti‐adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS–/–) mice. 2 eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT‐PCR analysis. 3 In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dtmax) of eNOS–/– hearts were not significantly different from those of WT hearts (LVP: 97 ± 11 mmHg WT vs. 111 ± 11 mmHg eNOS–/–; dP/dtmax: 3700 ± 712 mmHg s−1 WT vs. 4493 ± 320 mmHg s−1 eNOS–/–). 4 The dobutamine (10‐300 nm)‐induced increase in LVP was enhanced in eNOS–/– hearts. In contrast, L‐type Ca2+ currents (ICa,L) in isolated cardiomyocytes of WT and eNOS–/– hearts showed no differences after β‐adrenergic stimulation. Dibutyryl‐cGMP (50 μm) reduced basal ICa,L in WT cells to 72 ± 12 % while eNOS–/–ICa,L was insensitive to the drug. The pre‐stimulated ICa,L (30 nm isoproterenol) was attenuated by dibutyryl‐cGMP in WT and eNOS–/– cells to the same extent. 5 The Ca2+ (1.5‐4.5 mm)‐induced increase in inotropy was not different between the two experimental groups and β‐adrenergic receptor density was increased by 50 % in eNOS–/– hearts. 6 The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti‐adrenergic effect of both compounds was identical in WT and eNOS–/– hearts. Measurement of ICa,L in isolated cardiac myocytes yielded similar results. 7 These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to β‐adrenergic stimulation and with elevated β‐adrenergic receptor density, (2) the unaltered response of ICa,L in eNOS–/– cardiac myocytes to β‐adrenergic stimulation suggests that endothelium‐derived NO is important in mediating the whole‐organ effects and (3) eNOS is unimportant for the anti‐adrenergic effect of ACh and adenosine.

Acute Inhibition of Myoglobin Impairs Contractility and Energy State of iNOS-Overexpressing Hearts

Abstract— Elevated cardiac levels of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) have been implicated in the development of heart failure. The surprisingly benign phenotype of recently generated mice with cardiac-specific iNOS overexpression (TGiNOS) provided the rationale to investigate whether NO scavenging by oxymyoglobin (MbO2) yielding nitrate and metmyoglobin (metMb) is involved in preservation of myocardial function in TGiNOS mice. 1H nuclear magnetic resonance (NMR) spectroscopy was used to monitor changes of cardiac myoglobin (Mb) metabolism in isolated hearts of wild-type (WT) and TGiNOS mice. NO formation by iNOS resulted in a significant decrease of the MbO2 signal and a concomitantly emerging metMb signal in spectra of TGiNOS hearts only (&Dgr;MbO2: −46.3±38.4 &mgr;mol/kg, &Dgr;metMb: +41.4±17.6 &mgr;mol/kg, n=6; P <0.05) leaving contractility and energetics unaffected. Inhibition of the Mb-mediated NO degradation by carbon monoxide (20%) led to a deterioration of myocardial contractility in TGiNOS hearts (left ventricular developed pressure: 78.2±8.2% versus 96.7±4.6% of baseline, n=6; P <0.005), which was associated with a profound pertubation of cardiac energy state as assessed by 31P NMR spectroscopy (eg, phosphocreatine: 13.3±1.3 mmol/L (TGiNOS) versus 15.9±0.7 mmol/L (WT), n=6; P <0.005). These alterations could be fully antagonized by the NOS inhibitor S-ethylisothiourea. Our findings demonstrate that myoglobin serves as an important cytoplasmic buffer of iNOS-derived NO, which determines the functional consequences of iNOS overexpression.

Nitric oxide differentially regulates proliferation and mobilization of endothelial progenitor cells but not of hematopoietic stem cells

To investigate the role of nitric oxide in controlling endothelial progenitor (EPC) and hematopoietic stem cell (HSC) mobilization, wild-type mice, L-NAME treated WT and eNOS-/- mice received either PBS or G-CSF for 5 days. Under unstimulated conditions bone marrow of either L-NAME treated WT and eNOS-/- mice, representing acute and chronic NO-deficiency, showed higher CD34(+)Flk-I+ EPC numbers compared to their WT littermates. Furthermore, CD34(+)Flk-I+ progenitors under NO-deficient conditions showed a higher cell turn over since the proliferation and apoptosis activity under in vivo as well as in vitro conditions were enhanced. In line with this finding bone marrow derived EPC differentiation towards endothelial cells was modulated in an NO-dependent manner. Administration of G-CSF resulted in an increase of EPC within the bone marrow of WT animals with a consecutive release of these cells into the peripheral circulation. Under NO-deficient conditions G-CSF failed to increase EPC numbers. In contrast, the HSC population c-kit(+)Lin- was not influenced by nitric oxide. Thus, NO differentially supports the mobilization of the endothelial committed progenitor subpopulation in bone marrow but does not have an effect on HSC in vivo.