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Cardiovascular Research 2002 53(1):253-262; doi:10.1016/S0008-6363(01)00432-1
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Copyright © 2001, European Society of Cardiology

Endothelial dysfunction of coronary resistance vessels in apoE–/– mice involves NO but not prostacyclin-dependent mechanisms

Axel Gödeckea,*, Martin Zieglera, Zhaoping Dinga and Jürgen Schradera,b

aInstitut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, 40001 Düsseldorf, Germany
bBiologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, 40001 Düsseldorf, Germany

* Corresponding author. Tel.: +49-211-811-2675, fax: +49-211-811-2672 axel.goedecke{at}uni-duesseldorf.de

Received 7 May 2001;
   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Experimental protocols
 4. Results
 5. Discussion
 References
 
Objective: We have analyzed the extent of endothelial dysfunction in cardiac resistance vessels of hyperlipidaemic apoE–/– mice and explored whether NO and/or prostacyclin dependent pathways are involved. Methods: Coronary resistance was measured in isolated perfused hearts from WT and apoE–/– mice. To discriminate between NO and PGI2-dependent flow responses, we made use of the finding that acetylcholine (ACh) predominantly activates the prostaglandin pathway whereas bradykinin (Bk) mainly acts via NO in murine cardiac resistance vessels. Results: Basal coronary flow as well as the ACh induced vasodilation (0.1–1 µM) were not different between WT and apoE–/– hearts (flow increase+100%). Similarly, vasodilation in response to the prostacyclin mimetic iloprost reached the same levels. In contrast, the Bk-stimulated [3.3 µM Bk] coronary flow was reduced from 31.6±4.2 in WT to 19.2±2.7 ml min–1 g–1 in apoE–/– hearts. NOS inhibition by ethylisothiourea (ETU, 10 µM) reduced basal as well as Bk-stimulated coronary flow in WT and apoE–/– hearts to the same extent. RT–PCR and Western analysis demonstrated that neither eNOS expression nor protein levels were reduced. Similarly, the flow response to the NO donor SNAP (0.3–33 µM) was not altered suggesting that soluble guanylyl cyclase was not affected. Intracoronary application of superoxide dismutase augmented the Bk-induced vasodilation of apoE–/– hearts almost back to WT levels (26.6±3.3 ml min–1 g–1). In line with this finding the NADPH induced O2– formation was enhanced in cardiac extracts from apoE–/– hearts. Conclusion: apoE–/– hearts develop a hemodynamically relevant endothelial dysfunction at the level of coronary resistance vessels most likely via inactivation of bioavailable NO by superoxide anions. The function of the prostacyclin system is not altered.

KEYWORDS Atherosclerosis; Coronary circulation; Endothelial function; Endothelial factors; Nitric oxide; Prostaglandins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Experimental protocols
 4. Results
 5. Discussion
 References
 
The endothelium-derived autacoids NO and prostacyclin (PGI2) represent not only potent vasodilators but also inhibit atherosclerotic processes. Using in vitro assays it was demonstrated that both agonists inhibit smooth muscle cell proliferation and migration in a cGMP (NO) and cAMP (PGI2)-dependent manner (for reviews see [1,2]). In vivo this property was used to inhibit neointima formation after balloon injury by overexpression of endothelial NO synthase [3] and PGI2 synthase [4] respectively in a rat restenosis model. Similarly, platelets are stabilized by NO and prostacyclin suggesting that both agonists are important in the control of platelet activation [5]. In addition, NO has been shown to attenuate the expression of monocyte chemoattractant protein-1 (MCP1) [6], reducing monocyte adhesion and transmigration through the vascular wall. P-selectin, involved in the first steps of leukocyte adhesion to the vascular wall and ICAM-1 are further targets of NO [7] leading to reduced leukocyte adhesion to the vascular wall.

Development of atherosclerosis is associated or even preceded by the development of endothelial dysfunction, characterized by a decreased vasodilation in response to acetylcholine (ACh) when measured in conduit vessels [8]. When endothelial dysfunction is severe, ACh, directly acting on smooth muscle cells, may even induce vasoconstriction. This effect has been mainly ascribed to a reduced level of bioavailable NO because in large conduit vessels NO is the main endothelium-dependent vasodilator released by ACh. Interestingly, endothelial dysfunction is not only found in large conduit vessels which develop atherosclerotic lesions but also seems to affect small resistance vessels usually not involved in atherosclerotic lesion formation [9]. This finding has led to the general view that endothelial dysfunction predisposes the vascular bed to the development of atherosclerosis at specific sites [10].

Mechanisms leading to the reduction of vascular NO levels involve downregulation of eNOS expression [11], inhibition of NOS activity [12] and inactivation of NO by an imbalance of NO and superoxide anions (O2–) resulting in formation of peroxynitrite (OONO–). In this context it is important to note that enhanced oxidative stress in the vascular wall may be one of the key mechanisms involved in development of endothelial dysfunction [13]. Besides NOS also prostacyclin synthase is subject to functional inactivation by e.g. oxidized LDL [14]. In addition it was shown that this enzyme is highly sensitive to low concentrations of peroxynitrite [15] representing a possible link between the NO inactivation by O2– and a decreased PGI2 release in dysfunctional endothelium.

Apolipoprotein E deficient mice (apoE–/–) are a well characterized model to study generation and progression of atherosclerotic lesions [16]. These mice exhibit massively elevated serum cholesterol levels and in contrast to WT mice develop spontaneous atherosclerotic lesions of conduit vessels when fed a normal low fat chow diet. Sites of lesion development in the vascular tree include the aortic arches and branch points of carotid and intercostal arteries [17]. Arterioles are usually not affected. Functional analysis using vessel segments from aorta or coronary artery revealed that similar to the human situation these conductance vessels develop a NO dependent endothelial dysfunction. Thus, apoE–/– mice can be considered a suitable model to study endothelial dysfunction [18,19].

While previous studies concentrated on large conduit vessels it is unknown whether endothelial dysfunction also exists on the level of cardiac resistance vessels of apoE–/– deficient mice. In addition, so far no study discriminated between the role of NO and prostacyclin in the pathogenesis of dysfunctional endothelium. Because analysis of conductance vessels runs short in judging the hemodynamic consequences of a functional alteration, the present study explored the relative contribution of the NO and prostaglandin pathways to endothelial dysfunction in coronary resistance vessels of apoE–/– hearts. In addition, the role of O2– release in the development of endothelial dysfunction was investigated.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Experimental protocols
 4. Results
 5. Discussion
 References
 
2.1 Mice
For this study apoE–/– mice backcrossed for 10 generations to C57BL6 strain were used [20]. Mice were obtained from Jackson laboratories and bred under conventional conditions at the Tierversuchsanlage Düsseldorf. Age matched C57BL6J mice were used as controls. Animals were fed a normal low fat chow diet and tap water ad libitum. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996)

2.2 Saline perfused hearts
Mice were injected i.p. with 250 U heparin and anesthetized with urethane (1.5 g kg–1; i.p.). Hearts were rapidly excised and transferred for preparation of the aortic trunk to warm, oxygenated Krebs–Henseleit buffer. The aorta was cannulated and hearts were perfused in a non-recirculating Langendorff mode at constant pressure (100 mm Hg, i.e., 140 cm H2O) with a modified Krebs–Henseleit buffer containing in mM 116 NaCl, 4.6 KCl, 1.1 MgSO4, 24.9 NaHCO3, 2.5 CaCl2, 1.2 KH2PO4, 10 glucose and 0.5 EDTA equilibrated with 95% O2 and 5% CO2. (pH 7.4, 37°C). Coronary flow was measured with a transit-time ultrasound flowmeter located above the aortic cannula (Transonics, Ithaca, NY). LVP was recorded by insertion of a buffer-filled balloon (prepared from thin PE foil) into the left ventricle which was connected to a Statham P23XL pressure transducer. The volume of the balloon was adjusted to induce an enddiastolic pressure of 5 mm Hg. Data were recorded using a MacLab data acquisition system. Contractile force data are given only for basal conditions. Contractility data under application of agonists are omitted, because mouse hearts are characterized by a pronounced Gregg effect. Therefore, application of vasodilators led to an increase in cardiac contractile force concomitant with the flow increase, vasoconstriction to a decrease in contractile force. No significant differences in left ventricular developed pressure (LVDP) were observed between WT and apoE–/– except for bradykinin application which induces a significantly lower vasodilation in apoE–/– hearts and consequently to a lower increase in LVDP.

Acetylcholine, bradykinin were from Sigma, iloprost from Schering AG, Berlin, superoxide dismutase from Serva, Heidelberg and SNAP from Biomol, Hamburg.


   3. Experimental protocols
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experimental protocols
4. Results
 5. Discussion
 References
 
3.1 Dose–response curves
After mounting to the perfusion column and insertion of the balloon hearts were allowed to equilibrate until a constant basal coronary flow was reached (approximately 30 min). Then the coronary flow response was analyzed first by examination of reactive hyperemia after 20 s of global no flow ischemia. Hearts were included in this study only when the peak flow of reactive hyperemia reached at least 200% of basal flow. Afterwards the endothelium-independent vasodilator adenosine was infused at a final concentration of 1 µM for 2 min leading to maximal vasodilation. After return to baseline experimental interventions were started. Dose response curves were generated by cumulative application of drugs from a side arm using infors infusor pumps (Braun Melsungen, Germany). The infusion volume did not exceed 1% of coronary flow and the in-flow volume was corrected for the flow increase to guarantee that in the plateau phase the desired agonist concentration was applied. Each agonist concentration was given for 4–5 min and a plateau of the flow response was recorded for 3–4 min. After reaching the maximal concentration infusion was stopped and after return to basal flow coronary function was controlled by another adenosine application (1 µM) and reactive hyperemia.

Data points for each concentration were calculated as mean flow during 1–2 min of the plateau phase except for the ACh induced vasoconstriction which represents the maximal vasoconstriction.

3.2 Effect of inhibitors/SOD on dose–response curves
When the effect of inhibitors (ETU, DF) or SOD was analyzed the general set up was the same as in Section 3.1. After the first adenosine application and return to basal flow hearts were treated with inhibitors or SOD. During the first 2–3 min a new stable basal flow level was reached. Agonist interventions (Bk, SNAP) were started 10 min after onset of inhibitor application and performed as described above under continuous inhibitor/SOD infusion.

Stock solutions of agonists and inhibitors were prepared in water (ETU, Bradykinin, SOD, Iloprost, ACh, adenosine). SNAP was dissolved in 5 µM potassium phosphate pH 5.9 which is known to prolong the half-life of this NO donor. Infusion of the acidic SNAP solution had no effect on the pH of Krebs–Henseleit perfusion buffer.

3.3 Biochemical techniques
Cardiac 6-keto-PGF1{alpha} release in coronary venous effluates was determined by ELISA (Amersham Pharmacia, Braunschweig, Germany). Fifty µl of effluates were measured in duplicate and quantified to known amounts of standard dissolved in Krebs–Henseleit buffer.

Cardiac eNOS protein levels were determined by Western analysis using monoclonal {alpha}-eNOS antibodies (Transduction Laboratories, KY, USA) as previously described [21].

Total RNA was isolated from whole hearts by guanidinium/phenol extraction [22]. Five µg of total RNA were reverse transcribed in a total of 50 µl using MMLV-reverse transcriptase (Life, Technologies, Eggenstein, Germany). RT-products were diluted 1:4 and 5 µl (i.e. corresponding to 125 ng) of the RT reaction and the dilutions were used for real time PCR quantitation (Perkin-Elmer 5700 sequence detection system) using myglobin specific primers for standardization — (1'94°C, 2' 55°C, 2' 72°C, 40 cycles). Primer sequences were eNOS: 5'-CTG GAC ATC ACT TCC CCG-3' and 5'-GAGCTGGCTCATCCACGT-3'; myoglobin: 5'-CAA TTA CCT GCT AAA GAT GGC C-3' and 5'-TAC TCC AGC AGT GCC TTG G-3'.

Measurements of O2– formation by cardiac protein extracts were performed according to Rajagopalan et al. [23]. In brief, blood free perfused hearts were homogenized for 2 min in 50 mM potassium phosphate buffer, pH 7.5 (10 ml buffer/g wet weight) using an Ultraturrax homogenizer. Crude extracts were centrifuged for 5 min at 3000 rpm in a microfuge (Kendro instruments). Five µl of protein extract were incubated in a total volume of 100 µl potassium phosphate buffer containing lucigenin (5 µM final concentration) in a Berthold biolumat LB9700T at 37°C. O2– formation was stimulated with NADPH (50 µM final concentration) and chemoluminescence (CL) was recorded for three min. Then, either diphenylene iodonium (50 µM), SOD (100 U), ETU (20 µM) or rotenone (20 µM) was added to the assay mixture and CL was recorded for another three min. Basal CL was not different from background CL (w/o) protein. Activity measured as counts per min (cpm) was normalized to protein concentration (cpm mg protein–1).

3.4 Statistics
Data derived from repeated measures were analyzed by two-way-ANOVA followed by Bonferroni post-hoc test using Prism 3.0 software (GraphPad). Otherwise data were compared with Student's t-test. Differences were considered to be significant at P<0.05.


   4. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experimental protocols
 4. Results
5. Discussion
 References
 
The analysis of endothelial function of cardiac resistance vessels was performed in isolated hearts of 6–8 month old apoE–/– mice and compared with age matched WT (C57BL6) controls. At this age total serum cholesterol levels were increased ten-fold when compared with WT mice (WT: 77±26 mg dl–1; apoE–/–: 486±110 mg dl–1). This was almost exclusively due to an increase in the pro-atherogenic LDL cholesterol fraction (WT: 4.6±4.1 mg dl–1, apoE–/–: 362±95 mg dl–1). Levels of HDL-bound cholesterol were not altered (WT: 72±20 mg dl–1; apoE–/–: 93±10 mg dl–1).

4.1 NO and prostacyclin-dependent coronary vasodilation in the mouse heart
Bradykinin (1 µM) enhanced coronary flow three-fold (+214%) (Table 1) in the murine heart. NOS inhibition (100 µM L-NMMA) decreased basal coronary flow by 40% and the Bk-induced vasodilation amounted only to 25% of the vasodilation under control conditions. Cyclooxygenase inhibition by diclofenac (3 µM) did not alter the Bk-induced vasodilation excluding a role for prostaglandins in the Bk-mediated flow response.


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  		Table 1 Coronary vasodilation in murine hearts in response to bradykinin

 
In an earlier report we have shown that ACh induces a biphasic flow response in the murine heart: A short initial and transient vasoconstriction is followed by a pronounced vasodilation which was predominantly mediated by the activation the cyclooxygenase pathway [21]. Only a minor component was mediated by NO. Thus, Bk is a suitable agonist to analyze the integrity of the NO system while ACh allows to investigate the role of prostaglandins.

4.2 Basal parameters of apoE–/– hearts
In a first set of experiments control parameters of cardiac function were measured. Basal coronary flow was not different between WT and apoE–/– deficient hearts (WT: 11.2±1.9 ml min–1 g–1, apoE–/–: 11.1±2.22 ml min–1 g–1). The endothelium-independent vasodilator adenosine (1 µM) elevated coronary flow in WT hearts to 38.0±3.53 ml min–1 g–1 (n=13). A similar increase was found in apoE–/– hearts (39.2±5.6 ml min–1 g–1, n=9, n.s.). Also, no differences in basal left ventricular developed pressure (LVDP) (WT:81.4±23.0 mm Hg; apoE–/–: 78.7±18.2 mm Hg) were detected. The normalized heart weights (heart weight/body weight) were not different (WT: 5.1±0.7 mg g–1; n=73, apoE–/–: 5.4±0.7 mg g–1, n=33, n.s.).

4.3 Role of prostaglandins
In the next set of experiments we compared the coronary flow response to ACh in WT and apoE–/– hearts. As shown in Fig. 1A (VC) ACh-dose-dependently increased the extent of the initial vasoconstriction in WT hearts reducing coronary flow from basal 13.6±2.8 ml min–1 g–1 to 47% (1 µM ACh). During vasodilation following the initial vasoconstriction coronary flow reached 200% of basal flow (Fig. 1A, VD). In apoE–/– hearts basal coronary flow was not different from WT hearts. Similarly, both the ACh-induced vasoconstriction as well as maximal vasodilation reached the same levels as in WT hearts.


Figure 1
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  		Fig. 1 Role of prostaglandins in apoE–/– hearts. (A) Extent of the transient ACh induced vasoconstriction (VC, triangles) and vasodilation (VD, circles) in WT (black, n=7) and apoE–/– (gray, n=7) hearts. (B) Iloprost-induced vasodilation in WT and apoE–/– deficient hearts (n=6 each). WT: black circles, apoE–/– gray circles. Data represent means±S.D. n.s.=not significant.

 
Because ACh is known also to activate M3 receptors on smooth muscle cells resulting in vasoconstriction we used the prostacyclin mimetic Iloprost to stimulate IP3 receptors directly. As shown in Fig. 1B iloprost (0.1–10 nM) dose-dependently elevated coronary flow in WT hearts from basal values by 270%. In apoE–/– hearts a similar dose–response was obtained. In addition to the functional analysis we examined prostacyclin synthase activity by measuring the cardiac release of the stable prostacyclin metabolite 6-keto-PGF1{alpha}. As shown in Table 2, ACh (330 nM) caused an almost 20-fold PGI2 release in WT hearts. Similar results were obtained in apoE–/– hearts.


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  		Table 2 Prostacyclin release by ACh and Bk in WT and apoE–/– hearts

 
4.4 Role of NO
Intracoronary application of Bk (Fig. 2A) dose-dependently increased coronary flow in WT hearts (maximal response: 300% of basal). In apoE–/– hearts starting from the same basal flow the dose response curve was flattened and maximal vasodilation reached 200% of basal flow. To address the question whether the Bk-mediated NO release was attenuated, hearts were perfused with the NO synthase inhibitor ethylisothiourea (ETU) [24]. As shown in Fig. 2A ETU decreased basal coronary flow in WT hearts by 40%. The flow response to Bk was also attenuated. The maximal flow increase amounted only to 25% of the amplitude without NOS inhibition. In apoE–/– hearts the Bk-dose–response curve under NOS inhibition was not different from WT hearts.


Figure 2
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  		Fig. 2 Role of NO in apoE–/– hearts. (A) Dose–response curve for intracoronary Bk application in WT (black circles/triangles) and apoE–/– (gray circles/triangles) hearts. Circles: Bk-application, Triangles Bk+ETU. Data represent means±S.D. of the number of n=7 experiments experiments each. ** P<0.01 WT versus apoE–/– (two-way ANOVA followed by Bonferroni post-hoc test) n.s.=not significant. (B) SNAP induced dose–response curve in WT and apo E–/– hearts. WT: Black circles, apoE–/–: Gray circles. Data represent means±S.D. of the number of n=6 experiments for each group. n.s.=not significant.

 
To analyze whether changes might be observed at the level of the soluble guanylyl cyclase (sGC), the NO donor S-nitroso-penicillamine (SNAP) was used. As shown in Fig. 2B SNAP induced a dose dependent increase in coronary flow to maximal 370% of basal flow. The coronary flow response of apoE–/– hearts was not different from WT hearts.

We further analyzed whether the reduced function of the NO system was due to changes of eNOS expression. RT–PCR analysis of total cardiac RNA isolated from WT and apoE–/– hearts using myoglobin as internal standard revealed no differences in eNOS expression levels between WT and apoE–/– hearts (Fig. 3A). Likewise by Western analysis no differences in cardiac eNOS protein levels were found (Fig. 3B).


Figure 3
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  		Fig. 3 eNOS expression and protein levels. (A) Result of real time RT–PCR analysis. Expression levels are given as arbitrary units normalized to myoglobin expression. Bars represent means±S.D. of each 6 WT and apoE–/– hearts analyzed. (B) Western blot analysis of eNOS expression in cardiac protein extracts. One hundred and fifty µg of total cardiac protein were loaded onto a 7.5% SDS–PAGE, transferred to nitrocellulose and incubated with {alpha}-eNOS antibodies (Transduction Laboratories). Arrow indicates the eNOS specific band, numbers indicate molecular weight standards (kDa) of the BenchMarkTM prestained protein Ladder (Life Technologies).

 
4.5 Role of superoxide anions
To study whether elevated superoxide anions might have caused a reduction in the level of bioavailable NO in cardiac resistance vessels of apoE-deficient mouse hearts, the effect of superoxide dismutase (SOD, 5 U/ml) on the Bk-induced flow–response of WT and apoE–/– hearts was investigated. As shown in Fig. 4 neither basal nor the Bk-induced coronary flow in WT hearts was affected by SOD. Similarly, SOD did not alter basal flow in apoE–/– hearts. However, SOD significantly augmented the Bk-induced vasodilation (flow increase+140% versus 70% without SOD). As shown in Fig. 4B SOD did neither alter the SNAP induced vasodilation in WT nor in apoE–/– hearts.


Figure 4
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  		Fig. 4 Effect of SOD on coronary flow in WT and apoE–/– hearts. (A) Effect on Bk induced vasodilation. (B) Effect on SNAP induced vasodilation. WT: black circles/triangles; apoE–/–: gray circles/triangles. Circles: Bk/SNAP only, triangles Bk/SNAP+SOD. Data represent means±S.D. of n=6 experiments in each group. * P<0.05, ** P<0.01 two-way ANOVA followed by Bonferroni post-hoc test.

 
To directly compare the extent of cardiac O2– formation by WT and apoE–/– hearts, protein extracts were analyzed using the lucigenin assay. Under basal conditions no O2– release could be measured. However, NADPH stimulated substantial lucigenin enhanced chemoluminescence which reached higher levels in extracts from apoE–/– hearts (P=0.08, n=7). The chemoluminescence could be completely inhibited by diphenylene iodonium (50 µM) and significantly attenuated by SOD (100 U/ml) (Fig. 5). Inhibition of NOS by ETU (20 µM) or mitochondrial flavoproteins by rotenone (20 µM) did not affect the lucigenin enhanced chemoluminescence (data not shown).


Figure 5
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  		Fig. 5 Superoxide formation by cardiac protein extracts. Bars represent mean values±S.D. of n=7 experiments for NADPH-stimulated lucigenin enhanced chemoluminescence and with inhibition by diphenylene iodonium (DPI) or superoxide dismutase (SOD) ** P<0.01 (Student's t-test).

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experimental protocols
 4. Results
 5. Discussion
References
 
The present paper demonstrates that hearts from hypercholesterolemic apoE-deficient mice are characterized by endothelial dysfunction at the level of resistance vessels which are known not to develop atherosclerotic plaques. This endothelial dysfunction is due to an impaired function of the NO system and is not associated with alterations of the prostaglandin-mediated vasodilation. The reduced NO formation was not the result of a reduced eNOS expression but rather due to inactivation of bioavailable NO by superoxide anions. This study, therefore, complements data obtained in segments of conduit vessels of hyperlipidaemic mice (apoE–/–, LDLR–/–) [18,19].

In order to evaluate the relative contribution of the NO and prostacyclin systems to endothelial dysfunction we made use of the fact that Bk and ACh preferentially stimulate the release of either NO or PGI2. As shown in an earlier report ACh elevates coronary flow predominantly via prostaglandins [21]. In the present study we demonstrate that the Bk-induced vasodilation was insensitive to COX inhibition and does not result in augmented PGI2 release. The Bk-induced vasodilation could be almost completely blocked by NOS inhibition, while there is a minor NOS–COX inhibitor resistant response, which is most likely mediated by EDHF (unpublished results). Earlier reports on vascular function in mice [18,25,26] demonstrated that in the aorta NO is the main vasodilator released by ACh and that this is also the case in epicardial coronary arteries [19]. Thus, along the vascular tree ACh switches from NO to PGI2 as the preferred endothelial vasodilator. A similar change of function is observed for Bk, which did not induce vasodilation in isolated aortic ring preparations of mice, most likely due to lacking B2 receptors [27] but dilates coronary resistance vessels via NO and an EDHF like factor.

Using the approach outlined above we demonstrate a reduced response of coronary resistance vessels of apoE–/– mice to Bk stimulation which could be ascribed unequivocally to a reduced function of the NO system. Although conduit vessels of apoE–/– mice display also attenuated NO release [18,19] our results imply that only the endothelial dysfunction of resistance vessels is responsible for the observed hemodynamic alterations because:

1. The coronary flow response to ACh, acting via NO in conduit vessels, was not reduced in apoE–/– hearts.
2. Adenosine and reactive hyperemia elicited the same peak flow in WT and apoE–/– deficient hearts ruling out the possibility that plaque formation in conduit arteries significantly obstructed coronary in-flow.

The conclusion that the NO system was impaired in the microvasculature of apoE–/– hearts was based on the observation that NOS inhibition resulted in reduction of the Bk induced vasodilation to the same levels in WT and apoE–/– deficient hearts. Therefore, endothelial dysfunction did not affect the EDHF-like part of the Bk-dependent vasodilation. Surprisingly, basal NO release appeared not to be impaired in apoE–/– hearts because basal coronary flow did not differ from WT hearts and was reduced by NOS inhibition to the same extent. Thus, the dysfunction of the NO system concerned only the agonist-stimulated NO release. In line with the finding of an unaltered basal NO function we found that endothelial dysfunction was not the result of a transcriptional or translational downregulation of eNOS since both, RT–PCR as well as Western blot analysis revealed no differences in eNOS expression and protein levels in apoE–/– deficient hearts. Although these analyses do not allow to specifically measure eNOS expression in cardiac resistance vessels our results suggest that eNOS expression is not the cause for endothelial dysfunction in apoE–/– hearts. In the murine heart only 20% of cardiac eNOS expression is found in cardiac myocytes [28] and, therefore, the expression analysis reflects primarily eNOS levels in the endothelial compartment. Together with the finding of Lamping et al. [19] it can be concluded that endothelial dysfunction seems to generally affect the micro- and macrovascular endothelium. Therefore, a constant eNOS expression level highly suggests that changes in eNOS expression might not be relevant for the development of endothelial dysfunction in apoE–/– hearts.

An alternative mechanism leading to a dysfunctional NO system may involve enhanced levels of reactive oxygen species (ROS) in the vascular wall, which would lead to scavenging and thereby reduction of bioavailable NO [13]. In our experiments, superoxide dismutase (SOD) substantially improved the NO mediated flow response to bradykinin in apoE–/– deficient hearts suggesting that elevated O2– formation represents the main mechanism leading to endothelial dysfunction in apoE–/– hearts. SOD had no effect on basal coronary flow demonstrating that basal function of the NOS system was not compromised in apoE–/– hearts. In line with this finding we found an enhanced NADPH-stimulated superoxide formation in cardiac protein extracts from apoE–/– hearts. Whereas the Bk-stimulated NO release could be augmented by SOD in apoE–/– hearts, vasodilation induced by the NO donor SNAP was not altered by SOD. Taken together these data suggest that basal O2– formation does not affect NO availability under control conditions but substantially reduces NO levels under inducing conditions.

NADH/NADPH oxidase, xanthine oxidase, a dysfunctional NO synthase, or mitochondrial flavoproteins might be responsible for vascular ROS generation. By pharmacological means we could rule out that NO synthase or mitochondrial flavoproteins contributed to the elevated O2– formation. Thus, NADH/NADPH oxidase and/or xanthine oxidase activity seem to be augmented in apoE-/deficient hearts. According to more recent reports NADPH oxidase a multi-subunit complex is considered to be of major importance in the development of endothelial dysfunction and endothelial cells themselves are an important source for reactive oxygen species [29]. Also in vascular smooth muscle cells subunits of NADPH oxidase and enzymatic activity were detected. The subunit composition, however, appears to be different from that of endothelial cells [30]. NADPH oxidase activity can be activated by a variety of agonists including angiotensin II, thrombin and TNF-{alpha}. Most interestingly, Greene et al. recently demonstrated that Bk, at least in vascular smooth muscle cells, can rapidly induce ROS formation by activation of NADPH oxidase [31]. Thus, a similar mechanism might explain why the bradykinin-stimulated but not the basal NO release or the SNAP effects were altered in apoE–/– hearts.

The scavenging of superoxide anions by NO may, therefore, represent an important protective mechanism limiting the extent of atherosclerotic lesions. This view is supported by recent studies of Knowles et al. [32] who demonstrated that apoE/eNOS double knockout mice exhibit a much more severe atherosclerosis with larger plaques and more advanced stages than age-matched apoE–/– mice which included also small vessels of the heart.

Earlier work in eNOS knockout mice has demonstrated that the activity of soluble guanylyl cyclase (sGC) is elevated under conditions of low vascular NO release [25,26,33] and, vice versa, is reduced in transgenic mice with vascular overexpression of eNOS [34]. Further it was shown that the response to nitrovasodilators is preserved in atherosclerotic vessels up to the advanced stage and is even elevated under conditions of endothelium removal occurring in late stage atherosclerosis (for review see Shimokawa [8]). In apoE–/– hearts characterized by a general dysfunction of the NOS system, the SNAP induced dose–response curves did not differ significantly between WT and apoE–/– hearts. Thus, we found no evidence of sGC sensitization. The effect of other endothelium independent vasodilators such as adenosine and iloprost was also not altered in apoE–/– hearts. In addition, the vasoconstrictory response to ACh reflecting most likely direct activation of M3-receptors on smooth muscle cells was not different from WT mice making it unlikely that an altered sensitivity of vascular smooth muscle cells might have contributed to the reduced vasodilation in apoE–/– hearts.

A large number of reports support the view that also vasodilatory prostaglandins and especially PGI2, exhibit vasoprotective functions similar to NO [2]. It was shown, that prostacyclin release by atherosclerotic vessels may be reduced [35] possibly by direct inhibition of PGI2 synthase (PGIS) by oxidized LDL [14]. In addition, Zou et al. [15] demonstrated that PGI2 synthase (PGIS) is a highly sensitive target of peroxynitrite the reaction product of NO and O2–. PGIS inactivation by tyrosine nitration with IC50 values as low as 50 nM in vitro or 100 nM peroxynitrite in isolated bovine coronary arteries has been demonstrated. In view of these findings, the surprising result of the present report is that alterations of prostacyclin release are not involved in the pathogenesis of endothelial dysfunction in apoE–/– hearts. This the more since Pratico et al. [36] demonstrated a systemic increase of PGI2 release by apoE–/– mice. In the heart, however, we found comparable coronary responses in apoE–/– and WT hearts to ACh and also similar levels of ACh induced PGI2-release. In addition, there were no changes on the level of IP3 receptors because the flow response to the prostacyclin mimetic iloprost was not altered in apoE–/– hearts. Thus, in contrast to the NO system we found the prostacyclin synthase activity not compromized in apoE–/– hearts.

In hypercholesterolemic guinea pig hearts a reduced function of the NOS system characterized by a reduced flow response to Bk and a lower cGMP release was reported. This was associated with a reduction of eNOS expression and protein levels. PGI2 release was even elevated, but obviously this was not sufficient to compensate for the eNOS defect [37]. In humans, macro- and micro-vascular coronary endothelial dysfunction as characterized by a reduced ACh-dependent vasodilation can be reduced by L-arginine demonstrating the involvement of NO. However, the underlying cause for attenuated NO bioavailability may involve multiple mechanisms, such as oxidant stress, eNOS expression levels, endogenous NOS inhibitor (ADMA) concentrations or limiting L-arginine [38].

Time for primary review 23 days


   Acknowledgements
 
This work was supported by grant GO875/1-1 by the Deutsche Forschungsgemeinschaft. The authors wish to thank S. Küsters and B. Patzer for excellent technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Experimental protocols
 4. Results
 5. Discussion
 References
 

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