Skip Navigation

Cardiovascular Research 1997 34(3):590-596; doi:10.1016/S0008-6363(97)00061-8
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Lewis, T. V
Right arrow Articles by Chin-Dusting, J. P.F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lewis, T. V
Right arrow Articles by Chin-Dusting, J. P.F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Non-specific inhibition by human lipoproteins of endothelium dependent relaxation in rat aorta may be attributed to lipoprotein phospholipids

Tamara V Lewis, Anthony M Dart and Jaye P.F Chin-Dusting*

Alfred and Baker Medical Unit, Baker Medical Research Institute and Alfred Hospital, Commercial Road, Prahran, Vic. 3181, Australia

* Corresponding author. Tel.: +61 (3) 9276 3320; fax: +61 (3) 9276 3488.

Received 24 July 1996; accepted 8 January 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: In vitro incubation of low-density lipoprotein (LDL) is reported to attenuate endothelium dependent relaxation mediated by acetylcholine (ACh) while not affecting endothelium-independent relaxation. This study was designed to examine the effects of other lipid-carrying lipoproteins as well as to study their effects on responses mediated by endothelium dependent agonists other than ACh. Methods: The effects of human LDL, very-low-density lipoprotein (VLDL) and high density lipoprotein (HDL) on endothelium-dependent relaxation by ACh, histamine and the calcium ionophore, A23187 [GenBank] , and endothelium-independent relaxation by sodium nitroprusside (SNP) were investigated in rat isolated aortic rings. The effects of combined LDL and HDL incubation on responses mediated by ACh were also examined. Control experiments included experiments examining the effects of bovine serum albumin on responses mediated by ACh. Thiobarbituric-acid-reactive substances (TBARS) measured before and after organ bath incubation indicated little oxidation of the lipoproteins used.Results: Maximal responses to ACh were inhibited by LDL, VLDL and HDL (0.02 and 0.2 mg protein/ml), to histamine by LDL (0.2 mg protein/ml), VLDL (0.02 and 0.2 mg protein/ml) and HDL (0.02 and 0.2 mg protein/ml) and to A23187 [GenBank] by LDL (0.2 mg protein/ml), VLDL (0.2 mg protein/ml) and HDL (0.02 and 0.2 mg protein/ml). A small but significant correlation (r=0.54, P=0.01) was observed between the level of inhibition of the endothelium-dependent responses and lipoprotein phospholipid concentration in the organ bath but not between the level of inhibition and cholesterol (free and esterified) or triglyceride concentrations. Responses to SNP were unaffected by LDL, VLDL and HDL. Combined incubation of tissues with LDL (0.2 mg protein/ml) and HDL (0.2 mg protein/ml) significantly increased maximal responses to ACh (pre-lipoproteins 81.8±5.7 vs plus-LDL/HDL 100±0.0; P<0.05). Bovine serum albumin had no effect on the maximal responses to ACh. Conclusions: We conclude that inhibition by human lipoproteins of endothelium-dependent agonists occurs with LDL, HDL and VLDL and suggest that this may be due to the phospholipid content of each lipoprotein. However, combined incubation of HDL with LDL negates this effect and an increased maximal response to ACh is reported.

KEYWORDS Vascular reactivity; Nitric oxide; Human, lipoproteins; Phospholipids; Aorta; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The physical disruption of endothelial structure and function due to the formation of an atherosclerotic lesion results in, amongst other factors, a decreased vascular response to agents that stimulate the release of nitric oxide from the endothelium [1]. Endothelial dysfunction occurring before the formation of structural disruption [2]is less well-defined and has led to widespread interest in the role that lipoproteins may play. It is now clear that responses to endothelium-dependent vasodilators such as acetylcholine (ACh), but not direct smooth muscle dilators such as sodium nitroprusside (SNP), are impaired in forearm resistance vessels of hypercholesterolaemic patients, even though these vessels are not prone to atherosclerosis [3, 4]. It has been suggested that oxidation of low density lipoproteins (LDL) may play an important role. While in vitro incubation of rabbit aortic rings with non-oxidised LDL has been shown to have no effect on endothelium-dependent relaxation [5, 6], copper-oxidised LDL completely abolished relaxation responses mediated by ACh and reduced maximum relaxation to the calcium ionophore, A23187 [GenBank] , by approximately 50% [7].

There is ample evidence that it is the lipid component of LDL that diminishes vascular response to endothelium derived nitric oxide [8–10]and further that lysophosphatidylcholine, a substance produced when native LDL are oxidised [11], is responsible. It remains arguable, however, as to the effect of other lipid-carrying lipoproteins such as high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL). While unmodified HDL have been demonstrated to antagonise the vascular effects of oxidised LDL [10], oxidised HDL have also been shown to potently inhibit the effects of nitric oxide in their own right [9]. This non-specific inhibitory effect of lipoproteins appears to also extend to unmodified HDL and VLDL obtained from rabbit plasma [8]. It is not known whether this is also true of human lipoproteins.

The physiological significance of copper-oxidised lipoproteins is debatable. In the current paper we describe our studies on the effects of unmodified human lipoproteins, LDL, HDL and VLDL, on endothelium-dependent relaxations of the rat isolated aortic ring.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of lipoproteins
Approximately 150 ml of blood was taken from normal healthy volunteers. Individual lipoprotein fractions were obtained using discontinuous density gradient ultracentrifugation on a Beckman vertical rotor (70 Ti) with a Beckman ultracentrifuge; the VLDL fraction was obtained at 1.006 g/ml, LDL at 1.063 g/ml and HDL at 1.21 g/ml. To concentrate the fraction, VLDL was re-spun at 1.006 g/ml for a further 18–20 h. The isolated fractions were dialysed for a minimum of 18 h in saline containing EDTA 1 mM. All buffers contained EDTA 1 mM. Free and esterified cholesterol, phospholipids and triglycerides were measured using enzymatic colorimetric methods on a Cobas-Bio centriganalyser (Roche Diagnostics, Tegimenta, Switzerland). Apo A1 and apo B levels were measured using immunoturbidimetric methods on a Cobas-Bio centriganalyser. The protein content of each fraction was determined by a simplified protein assay method of Lowry [12]. All lipoprotein fractions were filtered through a 0.45 µm millex-HV filter unit (Millipore MA 01730; Bedford). Fractions were stored for a maximum of 10 days at 4°C.

The lipid peroxide content of the lipoprotein samples obtained was monitored fluorometrically as thiobarbituric-acid-reactive substances (TBARS) [13]; 300 ml of sample was vortexed with 600 ml of TBARS reagent (15% trichloroacetic acid, 0.375% thiobarbituric acid (TBA) and 0.25N HCl/100 ml). The samples were centrifuged at 10 000 r.p.m. for 10 min and the supernatant was measured for TBARS using a Cobas-Bio centriganalyser. This was repeated on samples which had been incubated in the organ bath experiments.

2.2 Isolation of rat thoracic aorta
Male Sprague-Dawley rats (250±50 g) were gassed with 80% CO2 and 20% O2 and then killed by exsanguination. The thoracic aorta was excised and placed in a Krebs modified solution (composition in mmol/L: NaCl 119, HCl 4.7, MgSO4·7H2O 1.17, NaHCO3 25, KH2PO4 1.18, CaCl2 2.5, glucose 11 and EDTA 0.03) kept ice-cold and then trimmed free of fat and connective tissue; 3-mm-wide rings were cut and mounted on 2 parallel stainless steel hooks in a water-jacketed, 5 ml organ bath containing Krebs solution maintained at 37°C and gassed with carbogen (95% O2, 5% CO2). Rings were set at a resting tension of 5 g, pre-determined in preliminary experiments to approximate an internal circumference equal to 0.9xL100, where L100 denotes the internal circumference at the level of passive stretch equivalent to a transmural pressure of 100 mmHg. The lower hook was attached to a plastic (PVC) support leg which was further attached to a micrometer for re-adjustment of initial tension. The upper hook was attached to a force transducer (FTO3 force transducer; Grass Scientific Instruments, Mitutoyo, Japan) for isometric recordings. Changes in isometric force were amplified (Quadbrige amplifier, Scientific Concepts Inc., Victoria, Australia) and recorded on a MacLab data acquisition system (MacLab 8E, Apple Computer Inc, Cupertina, CA) connected to an Apple Macintosh SE. Aortic ring preparations were allowed to equilibrate for 30 min at the initial stretch tension of 5 g. At the end of the equilibration period, rings were re-stretched to 5 g and allowed to equilibrate for a further 30 min.

The use of laboratory animals was approved by the Animal Ethics Committee, Baker Medical Research Institute which adheres to National Health and Medical Research Council of Australia guidelines.

2.3 Organ bath experimental protocol
All rings were contracted with a submaximal dose of noradrenaline (NA; 0.1–1 mM). When a steady level of active force was obtained, cumulative concentration–relaxation responses to the endothelium-dependent relaxant agonists, ACh (1 nM–30 µM) and histamine (1 nM–30 µM), the calcium ionophore, A23187 [GenBank] (1 pM–3 nM) and the endothelium-independent vasodilator, SNP (1 nM–30 µM), were obtained. Only one relaxing agonist was tested on any one aortic ring from any one rat.

Any one of the 3 lipoprotein fractions, VLDL, LDL or HDL, was then added to the organ bath to give a final concentration of either 0.02 or 0.2 mg protein/ml. Lipoproteins obtained from only one individual were tested at any one time. Tissues were left to incubate in the lipoprotein-rich solution for 60 min. In the continued presence of the lipoprotein, cumulative concentration–relaxation curves were repeated with the same agonist as used prior to addition of lipoprotein. At the end of this second curve, rings were thoroughly washed (replacement of fresh Krebs' solution 3–4 times organ bath volume) every 10–15 min for 60 min. Cumulative concentration–relaxation curves were then repeated for the second time. In addition to these single lipoprotein experiments, the combined effects of HDL (0.2 mg protein/ml) and LDL (0.2 mg protein/ml) were also examined. As before, concentration–relaxation response curves to ACh on noradrenaline pre-constricted rat aortic rings were obtained before and in the continued presence, following a 60 min incubation, of LDL and HDL.

Two series of control experiments were performed. The first were time-control experiments, where relaxation responses to ACh were obtained and repeated at the same time points as the experiments described above, but in the absence of any lipoprotein. The second studied the specificity of the effects of the lipoproteins. Thus, relaxation–response curves to ACh were performed initially and then repeated after a 60 min incubation with bovine serum albumin (0.2 mg protein/ml).

2.4 Data analysis
Individual concentration response curves for each agonist were fitted to a logistic equation of the form:


Formula

where E is the response, M is the maximum response, A is the concentration eliciting E, K is the concentration eliciting 50% of the maximum response (i.e., EC50) and P is the slope parameter [14]. The location of the EC50 value provided a measure of drug potency.

Data are presented as mean±standard deviation (s.d.) unless otherwise stated. Results were analysed by analysis of variance (ANOVA) followed by Student's unpaired t-test using Sigmastat Statistical Software (Jandel Scientific, San Rafael, CA) which inherently analyses data for normality prior to performing parametric analysis. Correlations between % inhibition of maximal relaxation of endothelium-dependent agonists by the lipoproteins and calculated organ bath concentrations of cholesterol (free, esterified and total), triglycerides and phospholipids were performed using simple regression analysis (Sigmastat, Jandel Scientific, San Rafael, CA). Percent inhibition of maximal relaxation by lipoproteins is defined as the difference in the maximal response obtained to the endothelium-dependent agonist used (ACh, histamine, A23187 [GenBank] ) in the presence, compared with that obtained in the absence, of each lipoprotein (LDL, VLDL, HDL). Calculated organ bath concentrations of each lipoprotein component (free and esterified cholesterol, triglyceride and phospholipid) were grouped into deciles of concentrations and matched against the relevant mean % inhibition. Statistical significance was set at P<0.05.

2.5 Drugs and chemicals
Norepinephrine bitartrate salt (Sigma, USA), ACh chloride (Sigma, USA), (2-[4-imidazolyl]ethylamine) dihydrochloride (Sigma, USA) and sodium nitroferricyanide (Sigma USA) were all dissolved and diluted in Krebs' solution. Calcium ionophore, A23187 [GenBank] (Sigma, USA), was made up in ethanol as a 2 mM stock and diluted in Krebs' solution. Boehringer Mannheim kit numbers for analysis of lipoprotein composition were as follows: free cholesterol 310 328, total cholesterol 144 2 350, triglycerides 450 032, phospholipids 691 844, apo A1 1 378 686 and apo B 1 378 694.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Lipoprotein content
The mean (±standard error of the mean) percentage composition (free and esterified cholesterol, triglycerides, phospholipids and protein) of LDL, VLDL and HDL are shown in Fig. 1. As expected, the higher the density of the lipoproteins, the higher the proportion of protein, and the lower the triglyceride concentrations. The levels of malonaldehyde (MDA) reflected the minimally oxidised state of the lipoproteins (Fig. 1). These levels were not significantly altered by incubation in the organ baths (data not shown).


Figure 1
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Mean (±s.e.m.) percentage composition (free and esterified cholesterol, triglycerides, phospholipids and protein) of LDL, VLDL and HDL.

 
3.2 Organ bath experiments
3.2.1 Control experiments: time controls and bovine serum albumin
Time control experiments where concentration–response curves were obtained to ACh at the same time periods (i.e., before and after 1 h), but without any lipoprotein intervention, revealed little effect of time on maximal responses to ACh (mean±s.d.: Curve 1, Time 0, 89.0±11.0% vs Curve 2, Time 60 min, 75.6±27.1%; P>0.05).

Mean maximum relaxation responses to ACh were not affected after incubation with albumin at 0.2 mg protein/ml (mean±s.d.: pre-albumin 68.5±8.1% vs plus-albumin 80.8±14.0%, P<0.05). Albumin had no effect on negative log EC50 values of ACh (mean±s.d. pre-albumin 6.77±0.37 vs plus-albumin 6.29±0.26).

3.2.2 LDL
Fig. 2a represents the mean concentration–response curve to ACh obtained in the absence and presence of LDL (0.02 mg protein/ml). Table 1 details the mean maximal responses and potency of all the agonists used in the absence and presence of LDL. Maximal relaxation to ACh was significantly attenuated in the presence of LDL (0.02 mg protein/ml). The EC50 (negative log of the concentration required to produce 50% of the maximum response) of ACh shifted significantly to the right following incubation with LDL. Although maximal relaxation responses to histamine and A23187 [GenBank] were also blunted by LDL (0.02 mg protein/ml), this was not significant at P<0.05.


Figure 2
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Mean concentration–response curves to ACh obtained in the absence and presence of (a) LDL (0.02 mg protein/ml), (b) VLDL (0.02 mg protein/ml), and (c) HDL (0.02 mg protein/ml).

 

View this table:
[in this window]
[in a new window]

  		Table 1 Effect of LDL (0.02 mg protein/ml and 0.2 mg protein/ml) on mean % maximal relaxation response±standard deviation (s.d.) and mean potency (neg. log M EC50)±s.d. to acetylcholine, histamine, A23187 and sodium nitroprusside

 
Maximal relaxation responses and EC50 values to ACh were significantly attenuated in the presence of LDL 0.2 mg protein/ml (see Table 1). Maximal relaxation responses to histamine and A23187 [GenBank] were also significantly attenuated. There was no significant shift in the dose–response curves to these agonists. Neither maximal relaxation responses nor potency to SNP were significantly affected by LDL (0.02 or 0.2 mg protein/ml).

3.2.3 VLDL
Mean concentration–response curves to ACh in the absence and presence of VLDL 0.02 mg protein/ml are shown in Fig. 2b. Table 2 details the mean maximal responses and potency of all the agonists used in the absence and presence of VLDL. Maximal relaxation mediated by ACh and histamine, but not by A23187 [GenBank] , was significantly attenuated in the presence VLDL at 0.02 mg protein/ml. Similarly, EC50 values to ACh and histamine, but not to A23187 [GenBank] were significantly decreased in the presence of VLDL 0.02 mg protein/ml. On the other hand, maximal relaxation mediated by the 3 endothelium-dependent relaxing agonists used (ACh, histamine and A23187 [GenBank] ) were all significantly attenuated by VLDL 0.2 mg protein/ml. The EC50 value for histamine was significantly attenuated following incubation with VLDL 0.2 mg protein/ml. EC50 values to A23187 [GenBank] were unaltered. Mean EC50 values for ACh could not be calculated from fitted curves because of the very dampened response to ACh following VLDL incubation.


View this table:
[in this window]
[in a new window]

  		Table 2 Effect of VLDL (0.02 mg protein/ml and 0.2 mg protein/ml) on mean % maximal relaxation response±standard deviation (s.d.) and mean potency (neg. log M EC50)±s.d. to acetylcholine, histamine, A23187 and sodium nitroprusside

 
Neither maximal relaxations nor EC50 values to SNP were affected by VLDL (0.02 or 0.2 mg protein/ml).

3.2.4 HDL
Mean concentration–response curves to ACh in the absence and presence of HDL 0.02 mg protein/ml are shown in Fig. 2c. Maximal relaxation responses to ACh, histamine and A23187 [GenBank] were all significantly attenuated in the presence of HDL at 0.02 mg protein/ml and at 0.2 mg protein/ml (Table 3), but there was no significant change in the potency of any of these agonists (Table 3).


View this table:
[in this window]
[in a new window]

  		Table 3 Effect of HDL (0.02 and 0.2 mg protein/ml) on mean % maximal relaxation response±standard deviation (s.d.) and mean potency (neg. log M EC50)±s.d. to acetylcholine, histamine, A23187 and sodium nitroprusside

 
Responses to SNP were unaltered by HDL (Table 3).

3.2.5 Combined LDL and HDL
Mean maximum relaxation responses±s.d. mediated by ACh were significantly higher following incubation of LDL combined with HDL (pre-lipoproteins 81.8±5.7 vs plus-LDL/HDL 100±0.0, P<0.05). There was no difference in the negative log EC50 values for ACh after lipoprotein incubation (pre-lipoproteins 6.69±0.15 vs plus-LDL/HDL, 6.62±0.61).

3.3 Correlations of lipoprotein composition and % maximal inhibition of responses
To investigate whether the inhibition of endothelium-dependent dilatation could be attributed to the composition of the lipoproteins used, correlation analyses were performed comparing the % maximal level of inhibition of all the endothelium-dependent agonists used against the calculated organ bath concentration of each lipoprotein component—cholesterol (total, free and esterified), triglyceride, phospholipid; see data, Section 2.4)—regardless of the density at which each fraction was obtained. The calculated organ bath concentration of phospholipids, but not of cholesterol (free, esterified and total) or triglycerides, correlated positively with the maximal level of inhibition of relaxation obtained to the endothelium-dependent agonists (r=0.54, P=0.01).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The major new finding from the current study is that the inhibitory effect reported of human low-density lipoproteins (LDL) on endothelium-dependent relaxation appears not to be specific to this lipoprotein but is also true of lipoproteins of high density (HDL) and very low density (VLDL). We attribute this non-specific action of lipoproteins to the phospholipid composition of each fraction and suggest that it is this component that is responsible for the inhibitory actions of the lipoproteins on endothelium derived relaxation.

Our findings with human lipoproteins are entirely consistent with findings using lipoproteins of rabbit origin [8]. In the previous work, the inhibition observed of ACh- and A23187 [GenBank] -mediated dilatation was demonstrated to be lipoprotein phospholipid, but not cholesterol, concentration-dependent. It would also appear from both studies that lipoproteins are capable of potently inhibiting endothelium-dependent dilatation without first having to undergo substantial oxidation as previously suggested. Kugiyama et al. [5]found native LDL to have no effect on endothelium-dependent relaxation; however, the concentrations used were half that in the current study and it has been clearly demonstrated that impairment of endothelium-dependent relaxation is concentration-dependent [6]. Organ bath concentrations of LDL used in the current study equate with those observed at physiological plasma levels, those of VLDL are similar to those observed with pathological levels of VLDL and those of HDL somewhat underestimates physiological levels.

We are confident that the non-specific effect of lipoproteins on endothelium-dependent relaxation is not merely a kinetic phenomenon (e.g., binding of the compounds to the protein) since tissue incubation with bovine serum albumin had no effect on endothelium-dependent relaxation mediated by ACh. Nor is it due to time-dependent changes in endothelial function as no change in relaxation responses over time was observed.

The inhibitory effect of LDL on endothelial-dependent relaxation has previously been advocated to be contributory to the atherogenic and possibly vaso-spasmogenic actions of this lipoprotein. Following from our data, the corollary would be that both VLDL and HDL are potentially harmful since both these lipoproteins potently inhibit endothelial-dependent relaxation. The latter finding is especially startling since a positive correlation between HDL concentration and ACh-induced relaxation has previously been reported in vivo [15]. HDL is also recognised for its anti-atherogenic properties including reverse cholesterol transport in vivo [16]and protection of LDL oxidation in vitro [17]. To further explore the role of HDL under the current in vitro setting, we studied the combined effect of HDL and LDL. When placed in the organ bath together, endothelium-dependent relaxation to ACh was not impaired, but in fact increased. One explanation may be that the lipoproteins undergo some form of minimal oxidation in the organ bath, which was not detected by the TBARS assay used in the current study, but which is necessary for their inhibitory effect on endothelium-dependent agonists. Co-incubation of HDL with LDL may inhibit this oxidation as has previously been shown in vitro [17].

The in vivo effect of VLDL is not known. Since VLDL is the main carrier for triglycerides, our data suggest that pathological levels of VLDL observed in hypertriglyceridaemia would be inhibitory of endothelium-dependent dilatation.

The exact mechanism by which LDL exerts its inhibitory actions on endothelium-dependent relaxation remain unclear. Since the nitric oxide precursor, L-arginine, reverses the endothelium impairment seen in hypercholesterolaemic rabbits [18]and humans [19], it may be that hypercholesterolaemia depletes L-arginine stores [20]or that LDL blocks binding of nitric oxide synthase enzyme (NOS) to L-arginine or that there is increased metabolism of nitric oxide. Other evidence has implicated free radicals sequestering or inactivating NO after it has been released from the endothelial cell [9, 21, 22]. Yet other studies suggest that LDL specifically inactivates some part of the G-protein receptor-coupled pathway of bradykinin and ACh [23]. As the disease progresses, however, this selective receptor inactivation becomes less apparent [24]. Our data using LDL are consistent with the hypothesis that at lower concentrations the effect of LDL appears to be selective, only inhibiting responses to ACh and that this selectivity becomes less apparent at the higher concentration of 0.2 mg protein/ml where inhibition is seen to responses mediated not only by ACh but also by histamine which stimulates synthesis of nitric oxide through phospholipase C linked to the Gq protein [25], and the receptor-independent agonist, A23187 [GenBank] , which opens calcium channels directly. Our data hence support the premise by Flavahan [24]that the mechanism of LDL action is concentration-dependent such that at lower concentrations there is selective inhibition of the Gi pathway whereas at higher concentrations the inhibition becomes more global affecting further along the nitric oxide pathway. It is interesting that VLDL appears slightly more potent, inhibiting both ACh and histamine at the concentration of 0.02 mg protein/ml and that HDL is even more potent, inhibiting all 3 endothelium-dependent agonists used at this concentration. This may be entirely dependent on phospholipid content; the design of the current study, however, precludes us from confirming this hypothesis.

In conclusion, we have demonstrated that the in vitro inhibitory effect of LDL on endothelium (nitric oxide)-dependent dilatation is not specific to this lipoprotein but is also true of HDL and VLDL. We further conclude that this non-specific action may be attributed to the phospholipid concentration of each fraction.

Time for primary review 26 days.


   Acknowledgements
 
The authors would like to thank Bridget Sherrard for her assistance with lipoprotein analysis. Tamara Lewis is a recipient of an Alfred Hospital Postgraduate Research Scholarship.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Zeiher A. Modulation of coronary vasomotor tone in humans. Progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation (1991) 83:391–401.[Abstract/Free Full Text]
  2. Cohen R.A., Zitnay K.M., Haudenschild C.C., Cunningham L.D. Loss of selective endothelial cell vasoactive functions caused by hypercholesterolemia in pig coronary arteries. Circ Res (1988) 63:903–910.[Abstract/Free Full Text]
  3. Chowiencyzk P.J., Watts G.F., Cockcroft J.R., Ritter J.M. Impaired endothelium-dependent vasodilation of forearm resistance vessels in hypercholesterolaemia. Lancet (1992) 340:1430–1432.[CrossRef][Web of Science][Medline]
  4. Chin J.P.F., Dart A.M. Therapeutic restoration of endothelial function in hypercholesterolaemic subjects: effect of fish oils. Clin Exp Pharmacol Physiol (1994) 21:749–755.[Web of Science][Medline]
  5. Kugiyama K., Kerns S.A., Morrisett J.D., Roberts R., Henry P.D. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature (1990) 344:160–162.[CrossRef][Medline]
  6. Plane F., Bruckdorfer K.R., Kerr P., Steuer A., Jacobs M. Oxidative modification of low-density lipoproteins and the inhibition of relaxations mediated by endothelium-derived nitric oxide in rabbit aorta. Br J Pharmacol (1992) 105:216–222.[Web of Science][Medline]
  7. Plane F., Kerr P., Bruckdorfer K.R., Jacobs M. Inhibition of endothelium-dependent relaxation by oxidized LDLs. Biochem Soc Trans (1990) 18:1177–1178.[Web of Science][Medline]
  8. Takahashi I.M., Yui Y., Yasomoto H., Morishita H., Hattori R., Kawai C. Lipoproteins are inhibitors of endothelium-dependent relaxation of rabbit aorta. Am J Physiol (1990) 258:H1–H8.[Web of Science][Medline]
  9. Chin J.H., Azhar S., Hoffman B. Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. J Clin Invest (1992) 89:10–18.[Web of Science][Medline]
  10. Schmid K., Klatt P., Graier W.F., Kostner G.M., Kukovetz W.R. High-density lipoprotein antagonizes the inhibitory effects of oxidized low-density lipoprotein and lysolecithin on soluble guanylylate cyclase. Biochem Biophys Res Commun (1992) 182:302–308.[CrossRef][Web of Science][Medline]
  11. Yokohoma M., Hirata K., Miyake R., Akita H., Ishikawa Y., Fukuzaki H. Lysophosphatidylcholine: Essential role in the inhibition of endothelium-dependent vasorelaxation by oxidised low density lipoprotein. Biochem Biophys Res Commun (1990) 168:301–308.[CrossRef][Web of Science][Medline]
  12. Peterson G.L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem (1977) 83:346–356.[CrossRef][Web of Science][Medline]
  13. Abbey M., Nestel P.J., Baghurst P.A. Antioxidant vitamins and low-density-lipoprotein oxidation. Am J Clin Nutr (1992) 58:525–532.[Web of Science]
  14. Nakashima A., Angus J.A., Johnston C.I. Comparison of angiotensin converting enzyme inhibitors captopril and MK421-diacid in guinea-pig atria. Eur J Pharmacol (1982) 81:487–492.[CrossRef][Web of Science][Medline]
  15. Kuhn F.E., Mohler E.R., Satler L.F., Reagan K., Lu D.Y., Rackley C.E. Effects of high-density lipoprotein on ACh-induced coronary vasoreactivity. Am J Cardiol (1991) 68:1425–1430.[CrossRef][Web of Science][Medline]
  16. Miyazaki A., Rahim A.T.A., Morino O.Y., Horiuchi S. HDL mediates selective reduction in cholesterol esters from macrophages foam cells. Biochim Biophys Acta (1992) 1126:73–80.[Medline]
  17. Parthasarathy S., Barnett J., Fong L.G. High-density lipoprotein inhibits the oxidative modification of low density lipoprotein. Biochim Biophys Acta (1990) 1044:275–283.[Medline]
  18. Cooke J.P., Andon N.A., Girerd X.J., Hirsch A.T., Creager M.A. Arginine restores cholinergic relaxation of hypercholesterolaemic rabbit thoracic aorta. Circulation (1991) 83:1057–1062.[Abstract/Free Full Text]
  19. Creager M.A., Gallagher S.J., Girerd X.J., Coleman S.M., Dzau V.J., Cooke J.P. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolaemic humans. J Clin Invest (1992) 90:1248–1253.[Web of Science][Medline]
  20. Jeserich M., Munzel T., Just H., Drexler H. Reduced plasma L-arginine in hypercholesterolaemia. Lancet. (1992) 339:561.[Web of Science][Medline]
  21. Galle J., Mulsch A., Busse R., Bassenge E. Effects of native and oxidized LDLs on formation and inactivation of endothelium-derived relaxing factor. Arterioscler Thromb (1991) 11:198–203.[Abstract/Free Full Text]
  22. White C.R., Brock T.A., Chang L.Y., et al. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci USA (1994) 91(Feb):1044–1048.[Abstract/Free Full Text]
  23. Liao J.K. Inhibition of Gi proteins by LDL attenuates bradykinin-stimulated release of endothelial-derived nitric oxide. J Biol Chem (1994) 269:12987–12992.[Abstract/Free Full Text]
  24. Flavahan N.A. Atherosclerosis or lipoprotein induced endothelial dysfunction: potential mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation (1992) 85(5):1927–1938.[Free Full Text]
  25. Yuan Y., Granger H.J., Zawieja D.C., Defily D.V., Chilian W.M. Histamine increases venular permeability via a phospholipase C–NO synthase guanylate cyclase cascade. Am J Physiol (1993) 264:H1734–H1739.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
F. H. de Man, A. W. E. Weverling-Rijnsburger, A. van der Laarse, A. H. M. Smelt, J. W. Jukema, and G. J. Blauw
Not Acute but Chronic Hypertriglyceridemia Is Associated With Impaired Endothelium-Dependent Vasodilation : Reversal After Lipid-Lowering Therapy by Atorvastatin
Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 744 - 750.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. M Dart and J. P.F Chin-Dusting
Lipids and the endothelium
Cardiovasc Res, August 1, 1999; 43(2): 308 - 322.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
T. V. Lewis, A. M. Dart, and J. P. F. Chin-Dusting
Endothelium-dependent relaxation by acetylcholine is impaired in hypertriglyceridemic humans with normal levels of plasma LDL cholesterol
J. Am. Coll. Cardiol., March 1, 1999; 33(3): 805 - 812.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Y. Numaguchi, M. Harada, H. Osanai, K. Hayashi, Y. Toki, K. Okumura, T. Ito, and T. Hayakawa
Altered gene expression of prostacyclin synthase and prostacyclin receptor in the thoracic aorta of spontaneously hypertensive rats
Cardiovasc Res, March 1, 1999; 41(3): 682 - 688.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Lewis, T. V
Right arrow Articles by Chin-Dusting, J. P.F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lewis, T. V
Right arrow Articles by Chin-Dusting, J. P.F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?