Deficiency of MAPKAPK-2 reduces ROS formation and improved endothelium- independent vasorelaxation in hypercholesterolemic mice.
Kumaravelu Jagavelu1, Carola Doerries1, Ulf Landmesser1, Matthias Gaestel2, Helmut Drexler1, Bernhard Schieffer1, Udo Bavendiek1
1Department of Cardiology & Angiology, Hannover Medical School, Germany (K. J., C.D., U.L., H. D., B. S., U. B.)
2 Department of Biochemistry, Hannover Medical School, Germany (M. G.)
Address of Correspondence:
Udo Bavendiek, MD
Department of Cardiology & Angiology Hannover Medical School
Carl-Neuberg-Str. 01 30625 Hannover, Germany Tel: (0)511-532-9597 Fax: (0)511-532-3357
Email: Bavendiek.Udo@mh-hannover.de
Journal Subject Heads: endothelial dysfunction, ROS formation, hypercholesterolemic mice Total word count: (3606)
Abstract
Atherosclerosis is a multifactorial chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. Endothelial dysfunction and reactive oxygen species (ROS) are important mediators of atherogenesis. We investigated the role of the mitogen -activated protein kinase -activated protein kinase-2 (MK2) in endothelial dysfunction and ROS production in hypercholesterolemic ldlr-/-- deficient mice. Deficiency of MK2 leads to reduced blood pressure in ldlr-/--deficient mice before and after feeding a high cholesterol diet. MK2 deficiency did not affect endothelium-dependent vasorelaxation induced by acetylcholine, but improved endothelium-independent vasorelaxation induced by nitroglycerine and reduced total ROS formation as well as NAD(P)H oxidase dependent ROS formation in aortas isolated from hypercholesterololemic ldlr-/- deficient mice. These data suggest that MK2 may be critically involved in atherogenesis by fostering ROS formation. In addition MK2 seems to impair endothelial-independent vasorelaxation, probably by desensitizing the vascular response to NO.
Keywords: Atherosclerosis, MK2, Endothelial dysfunction, hypercholesterolemic mice, ROS
Introduction
Atherosclerosis is a chronic inflammatory disease characterised by the recruitment of inflammatory cells to the inflamed vessel wall and deposition of lipids1-3. The integrity and function of endothelial cells are fundamental for normal homeostasis of the vessel wall. In health, the endothelium has an anti-inflammatory phenotype that maintains the uninterrupted circulation of leukocytes and platelets, whereas endothelial dysfunction and injury results in the increased adhesion and recruitment of leukocyte and platelets to the vascular wall, initial stages of atherosclerosis. Also, clinical and experimental studies have shown that a marked reduction in endothelial-dependent vasodilation is a characteristic of an early stage of atherosclerosis4, 5. Therefore, endothelial dysfunction seems to be a key factor in the initiation and progression of atherosclerosis6. Endothelial dysfunction is mainly caused by the reduced bioavailability of Nitric oxide (NO), a potent vasodilator formed in endothelial cells from L-arginine by eNOS. NO released towards the vascular lumen is a potent inhibitor of platelet aggregation and adhesion to the vascular wall7, 8. It also inhibits leukocyte adhesion to the vessel wall by interfering with the leukocyte adhesion molecules CD11 and CD189, 10. As leukocyte adherence is an early event in the development of atherosclerosis NO may protect against the onset of atherogenesis. A reduction in the bioavailability of NO has been implicated in various diseases and also in atherosclerosis and hypertension. Studies on hypercholesterolemic primates have demonstrated a loss of endothelium-dependent vasodilatation in response to acetylcholine and bradykinin4 because of a reduced availability of NO and an increased formation of reactive oxygen species (ROS)11. Reactive oxygen species (ROS) are mainly derived from the vascular NAD(P)H oxidase system. ROS are thought to promote atherosclerosis through multiple mechanisms, including enhanced oxidation of lipoproteins12, activation of proinflammatory mediators13, reduction in the bioavailability of NO and proliferation of smooth-muscle cell growth14 which are all known as key events in atherogenesis.
The mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAP-2, MK2) is a direct physiological substrate of MAPK p38α/β. MK2 regulates the expression of several proinflammatory genes by regulating the mRNA-stability through targeting AU-rich elements15. In smooth muscle cells angiotensin II induced ROS-dependent p38-activation through the angiotensin type1 (AT1) receptor resulting in activation of MAPKAP kinase-2 and phosphorylation of HSP2716.In synovial neutrophils of rheumatoid arthritis (RA) patients ROS
generation is dependent on p47phox (a subunit of NAD(P)H oxidase). The activation of this subunit occurs via phosphorylation of p47phox-Ser345. The activation of NAD(P)H oxidase is probably dependent on p38-MAPK as pharmacological inhibition of p38-MAPK decreased TNF-α induced phosphorylation of p47phox-Ser345 and subsequently reduced ROS formation17. In addition, long term treatment with a selective p38 MAPK inhibitor suppresses both NAD(P)H oxidase expression and ROS generation and protects against Ang II-induced target-organ damage in rats 18. Furthermore, inhibition of p38 MAP kinase in rats with chronic heart failure reduced MK-2 phosphorylation, preserved acetylcholine-induced vasorelaxation and reduced vascular superoxide formation 19. In this regard, Ju et.al (2003) has shown that hypertension induced endothelial dysfunction depends on p38-MAPK activation.
Therefore, in the present study we investigated the functional role of MK2 in endothelial dysfunction and ROS formation in hypercholesterolemic mice deficient for MK2 compared to ldlr-/- deficient control mice.
Materials and Methods
Materials
All chemicals were purchased from Sigma-Aldrich unless otherwise stated.
Mouse model:
All procedures followed were in accordance with approved guidelines set by the Institutional Animal Care and Use Committee at Hannover Medical School and the Bezirksregierung Niedersachsen. To understand the pathophysiological alteration of atherosclerosis and to assess the potential role of MK2 in atherosclerosis, low-density-lipoprotein-receptor-deficient mice (ldlr-/- C57Bl/6, Jackson Laboratories) and MK2-deficient mice (mk2-/- C57Bl/6, backcrossed
>10 generations) 21were crossbred to generate ldlr-/-/mk2-/-compound mutant mice. The genotype of each mouse was confirmed by PCR after isolating genomic DNA (tail tip digest) using the following primers: LDLR: 5’- ACC CCA AGA CGT GCT CCC AGG ATG A –3’ (sense), 5’ – CGC AGT GCT CCT CAT CTG ACT TGT – 3’ (antisense); MK2: 5’ – CGT GGG GGT GGGGTG ACA TGC TGG TTG AC –3’ (sense), 3’ – GGT GTC ACC TTG ACA TCC CGG TGA G–3’ (antisense); Neomycin-Cassette: 5’ – TGC TCG CTC GAT GCG ATG TTT CGC – 3’(sense). 12 week old male ldlr-/-and ldlr-/-/mk2-/- compound-mutant mice (each group n=8-14)
consumed a high cholesterol diet (product #D12108, Research Diets; 1.25% Cholesterol without added cholate) for 7 weeks. Subsequently, mice were sacrificed and the aortas were removed and analyzed as described below. All mice were housed under specific pathogen-free conditions.
Blood Pressure Measurement:
Systolic blood pressure and heart rate were recorded by tail cuff measurement with the aid of a computerized system (BP2000 Blood Pressure Analysis System, Visitech System, Apex, NC, USA) in conscious animals. Animals were pretrained for blood pressure measurements 3 consecutive days prior to the real measurements. On each day blood pressure determination was measured at the same time, 30 measurements were taken and averaged for each individual animal every day. Blood pressure reading was taken before starting the high cholesterol diet and after high cholesterol diet and mice were pretrained in both cases.
Organ Chamber Experiments
After excision of the descending aorta, the vessel was placed in ice cold buffer I (mmol/L, NaCl 118.0, CaCl2 2.5, KCL 4.73, MgCl2 1.2, KH2PO4 1.2, NaHCO3 25.0, C8H17N2NaO4S 1.355, D(+)glucose 5.5) 22. The aorta was cut into 3 mm rings, after the removal of surrounding fat, the rings were placed in the organ baths filled with the buffer I (37° C continuously aerated with 95%
O2 and 5% CO2). After inserting the force transducers into the rings while maintaining the endothelial integrity, the rings were stretched gradually using a passive tension of 1 g, which was maintained throughout the experiment. Rings were precontracted with KCl (80 mmol/L) and again precontracted to 50% of the maximal tension (KCL) with phenylepherine. To assess endothelium-dependent and -independent relaxation,acetylcholine (ACH 10-9 to 10-5 µmol/L) or nitroglycerine, (NTG 10-9 to 10-5 µmol/L) was cumulatively added to the organ chambers respectively. The isometric tension was recorded using Biopac systems and analysis using Acqknowledge 3.7.2 software.
Measurement of Vascular Reactive oxygen species (ROS) and NAD(P)H-Oxidase dependent ROS formation by Electron Paramagnetic Resonance Spectroscopy/ESR Detection of Superoxide Radicals.
After feeding the high cholesterol diet as mentioned above mice were sacrificed by CO2
inhalation and the aorta from the thoracic region was dissected. Care was taken to excise off the
fat and extra tissue without destroying the endothelium and aortas were cut into 4 mm rings from ldlr-/-and ldlr-/-/mk2-/-(each group n = 10-12 mice) The dissected rings were placed in ice cold buffer 1containingdeferoxamine (25 µmol/litre, metal chelator). The rings were placed in 50 µl capillary tube (Corning). ESR spectra were recorded as mentioned previously 23 by using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Noxygen GmBH) to measure the total superoxide anions. To measure the NAD(P)H oxidase dependent superoxide anion production, NADH was used as a substrate and NAD(P)H dependent ROS formation was measured using CMH using a MiniScope ESR spectrometer (Magnettech, Berlin, Germany). The ESR setting was the following: field center: 3356.53G; field sweep width: 50G; microwave frequency: 9.82GHz; microwave power: 32mW (5dB); magnetic field modulation frequency: 100 kHz; modulation amplitude: 2.5G. The intensity of ESR spectra was quantified after subtraction of the ESR signal of samples and without the sample. Data from each spectrum were quantified as sum of the total 3-peak intensities and two rings were analysed for each mouse and averaged.
Statistical analysis
All values are given as mean ± SEM. Results were analyzed using Student's t-test or analysis of variance followed by Bonferroni/Tukey for multiple comparisons using Instat software (Graph pad). A value of P<0.05 was considered statistically significant.
Reduced Systolic Blood Pressure in hypercholesterolemic ldlr-/- and ldlr-/-/mk2-/-mice.
Systolic blood pressure was monitored by tail cuff plethysmography in ldlr-/- and ldlr-/- /mk2-/- mice before and after feeding a high cholesterol diet for 7 weeks. Ldlr-/-/mk2-/- mice (n=20) showed a significantly reduced blood pressure before the start of the high cholesterol diet (ldlr-/- vs ldlr-/- /mk2-/-: 127±2.8 mmHg vs. 110±3.4 mmHg n=20, P<0,002) and after feeding the high cholesterol diet compared to age-matched ldlr-/- mice. (ldlr-/- vs ldlr-/- /mk2-/-: 116±2.6 mmHg vs. 106±2.2 mmHg, n=20, P<0,007) (Fig. 1).
MK2 deficiency improved endothelial independent vasorelaxation, but not endothelium-dependent vasorelaxation in hypercholesterolemic mice.
To examine the role of MK2 in vascular function of hypercholesterolemic mice, we fed ldlr-/- /mk2-/- mice and ldlr-/- control mice with a high cholesterol diet for 7 weeks and the endotheliumdependant and independent vasorelaxation was studied. Endothelium -dependent vasorelaxation of aortic rings was not different between ldlr-/- mice and ldlr-/- /mk2
-/- mice elicited by acetylcholine concentrations ranging from 10-9 to 10-4.5 mol/L. (n=14 in both groups) (Fig.2a). In contrast, endothelium-independent vasorelaxation in response to nitroglycerine was substantially improved at a concentration range of (10-8 to 10-5 mol/L, n=
14, P<0, 01) in ldlr-/- /mk2-/- compared to ldlr-/- mice.(Fig. 2b).
MK2-deficiency reduced ROS formation in hypercholesterolemic ldlr-/-mice.
To elucidate the underlying mechanism of the observed impact of MK2 deficiency in the endothelium-dependent and -independent vasorelaxation, we investigated if deficiency of MK2 affects the formation of ROS in the aortas of hypercholesterolemic ldlr-/- by electron spin resonance (ESR). Total ROS formation in ldlr-/-/mk2-/- mice was significantly reduced (1.17 pmol O2.- / min, n=12, P<0,009) compared to ldlr-/- mice (2.55 pmol O2.- / min) (Fig. 3a).
Furthermore, to determine if the NADPH dependent ROS formation is affected by MK2-deficiency we also analysed the NAD(P)H oxidase/activity in these hypercholesterolemic mice. We found a significantly reduced NAD(P)H oxidase dependent ROS formation in ldlr-/-
/mk2-/- mice (0.977 pmol O2.- / min, n= 12, P<0,01) compared to ldlr-/- mice (1.817 pmol O2
.-/ min) (Fig. 3b).
Discussion
Hypercholesterolemia and modified lipoproteins (oxLDL) are known to cause endothelial dysfunction and ROS formation and therefore seems to be key factors in the initiation of atherosclerosis6. Earlier studies by Widder et.al (2004) showed that pharmacological inhibition of p38-MAPkinase with SB239063 reduced MAPKAPK-2 phosphorylation, preserved acetylcholine-induced vasorelaxation and reduced vascular superoxide formation in CHF rats, revealing that the p38-MAPK downstream target MK2 may increase endothelial dysfunction. The unimpaired endothelium-dependent vasodilator response in MK2-deficient hypercholesterolemic mice (this study) could be due to the presence of high concentrations of proatherogenic lipoproteins, known to decrease the expression of endothelial nitric oxide synthase (eNOS) in endothelial cells and thereby impaired formation of NO and endothelial-dependent vasorelaxation24, masking a potentially increased endothelium-dependent vasorelaxation caused by MK2- deficiency in hypercholesterolemic ldlr-/- mice. The increased endothelial-independent relaxation caused by MK2-deficiency in hypercholesterolemic ldlr -/-mice may be due to an increased sensitivity of the sGC-cGMP signalling cascade to NO in smooth muscle cells. This might result in the observed increased endothelium-independent vasorelaxation caused by the potentially reduced NO formation in hypercholesterolemic mk2-/- mice, similarly as demonstrated in the eNOS-/- mice25. In line, overexpressing of bovine eNOS was followed by a reduced NO-elicited vasorelaxation26, 27.
Furthermore, contraction and relaxation of smooth muscle cells (SMC) are dependent on cytoskeletal changes. The increased endothelium independent vasorelaxation in MK2 deficient mice could also be due to the cytoskeletal changes observed in endothelial cells due to a casual link between MK2 activation, HSP27 phosphorylation and actin redistribution28 or may be due to the improper actin reorganization mediated by MK2 phosphorylation/activation of LIMK1 as shown in endothelial cells29. Cytoskeletal changes are also mediated via the cGMP signalling cascade. Cyclic GMP-dependent protein kinase I (PKGI) interacts with formin homology (FH) domain-containing protein (FHOD1)30. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics and cell migration. Recently it has been shown that p38 MAPK also interact with FHOD131, which may result in increased vasorelaxation.
Previous studies18,19 show that the endothelial-dependent vasorelaxation response is dependent on ROS production. To further elucidate the unimpaired endothelial-dependent
vasorelaxation in MK2-defecient hypercholesterolemic mice, we hypothesised that this could be due to reduced level of ROS formation. As expected hyperchosterolemic mice lacking MK2 showed less aortic ROS level. The reduced ROS formation could be attributed to a reduced NAD(P)H oxidase/activity. Previous studies have shown that the ROS production is regulated through NAD(P)H oxidase, constituting a major contributor for ROS production, and is also shown to be increased in early stages of atherosclerosis through renin-angiotensin system (RAS) activation 32. We hypothesised that in MK2-defecient mice NAD(P)H oxidase-dependent ROS formation may be less. As expected MK2 deficient mice showed less production of NADH-dependent ROS formation. It is shown that in bovine leukocytes p38MAP kinase regulates the activation of the NAD(P)H-oxidase and that pharmacological inhibition of p38MAPK reduces phosphorylation of p47phox leading to reduced production of ROS 33. The phosphorylation of p47phox could be directly mediated by MK2, a direct substrate of p38, thereby regulating the production of ROS. A recent study by Dang et.al., 2006 showed that TNFα primes p38-dependent ROS production through phosphorylation of Ser345 of the p47phox subunit of NAD(P)H oxidase. However, further studies are essential to prove that the MK2 is actually regulating NAD(P)H oxidase /activity.
Furthermore, we have previously shown (Jagavelu et al., 2007 in revision) that mice deficient for MK2 have significantly less atherosclerosis. This could be potentially due to the observed reduced ROS production in these mice with subsequently reduced lipid oxidation. Other studies have shown that ROS are known to be important for monocyte migration34 and oxidation of lipids35. Thereby the initiation and progression of the disease may be attenuated or delayed. In addition, ROS are known to quench NO with the formation of peroxynitrite 36. MK2 deficient mice showed reduced ROS formation thereby potentially an increased bioavailability of NO, eventually recovering the potentially reduced NO caused by increased proatherogenic lipoproteins compared to the hypercholesterolemic ldlr-/- mice. This could be one reason for the observed unimpaired endothelium-dependent vasodilation.
Taken together, these data indicate a potential role for MK2 in atherogenesis by regulating the vascular production of ROS. In addition, MK2 seems to impair the endothelial independent vasorelaxation, probably by desensitizing the vascular response to NO. Therefore, MK2 may provide a feasible target for a therapeutical regime to treat patients suffering from atherosclerotic cardiovascular disease.
Figure Legends
Fig. 1: MK2-deficiency reduced systolic blood pressure in hypercholesterolemic ldlr-/- mice.
Systolic blood pressure was measured using tail cuff plethysmography before and after feeding a high cholesterol diet for 7 weeks (Pre and Post HCD n=20, ldlr-/- vs ldlr-/- /mk2-/- * p<0.05).
Fig. 2: MK2-deficiency improved endothelial-independent vasorelaxation but did not affect endothelium-dependent vasorelaxation in hypercholesterolemic ldlr-/- mice.
Vasorelaxation was determined in aortas isolated from ldlr-/- and ldlr-/-/mk2-/- in response to acetylcholine (Ach) and nitroglycerine (NTG). Vascular reactivity was determined as changes in isometric tension of aortic rings submaximally precontracted with phenylepherine.
Endothelium-dependent relaxation of the aortas from ldlr-/- (squares) and ldlr-/- /mk2-/- (triangles) induced by cumulative additions of ACh (10-8 to 10-4.5 mol/L, A) and endothelium-independent relaxation by NTG (10-8.5 to 10-5 mol/L, B) were demonstrated. (ldlr-/- vs ldlr-/- /mk2-/- , n= 14, *P < 0.05).
Fig. 3: MK2-deficiency reduced ROS formation in hypercholesterolemic ldlr-/-/mk2-/-mice.
Vascular superoxide production was measured by electron spin resonance (ESR). Total ROS formation (A) and NAD(P)H oxidase dependent ROS formation (B) was determined (ldlr-/- vs.
ldlr-/- /mk2-/-, n=12, *P < 0.05).
Fig. 1
Systolicbloodpressure(mm Hg)
0 20 40 60 80 100 120 140
Pre HCD Post HCD
* *
Systolic blood pressure measurement
ldlr -/-ldlr-/-/mk2
-/-Fig. 1
Systolicbloodpressure(mm Hg)
0 20 40 60 80 100 120 140
Pre HCD Post HCD
* *
Systolic blood pressure measurement
ldlr -/-ldlr-/-/mk2
-/-Fig. 2 A
-/-ROS formation
A B
NADPH oxid ROS foFi g. 3
ROS formation
A B
NADPH oxid ROS fo ldlr-/-ldlr-/-/mk2-/- ldlr-/-/mk2-/-ldlr-/-Fi g. 3
asedependent rmation
ldlr-/-/mk2-/-ldlr-/-References
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