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Cardiovascular Research 2001 50(3):566-576; doi:10.1016/S0008-6363(01)00251-6
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta

Benoît Darblade1,a, Dominique Caillauda,1, Marc Poirota, Marie-José Fouqueb, Jean-Claude Thiersc, Jacques Ramia,b, Francis Bayarda and Jean-François Arnala,b,*

aINSERM U397, CHU Rangueil, 31403 Toulouse, France
bLaboratoire de Physiologie, Faculté de médecine de Rangueil, 31062 Toulouse, France
cINSERM U466, CHU Rangueil, 31403 Toulouse, France

* Corresponding author. Present address: INSERM U397, CHU Rangueil, 31403 Toulouse Cedex, France. Tel.: +33-5-6132-2147; fax: +33-5-6132-2141 arnal{at}rangueil.inserm.fr

Received 22 September 2000; accepted 29 January 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production. Methods and Results: We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187 [GenBank] -induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their ‘grape-like’ clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-β-cyclodextrin (hp-β-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-β-CD (2%) or filipin (4 µg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 [GenBank] or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-β-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their ‘grape-like’ clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 [GenBank] remained unaltered. Conclusion: Fatty streak deposit is associated with a decrease in caveolae ‘transductosomes’ abundance which appears to represent a novel mechanism of endothelial dysfunction.

KEYWORDS Endothelial function; Atherosclerosis; Nitric oxide; Cholesterol


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelium has numerous functions, acting as a selective barrier between intravascular and interstitial compartments, and contributing to haemostasis through the control of vasomotion, platelet aggregation, coagulation and fibrinolysis. One of the major messengers of these endothelial functions is nitric oxide (NO) [1–3]. NO is a short-lived radical gas which acts in the smooth muscle cells and platelets by activating soluble guanylyl cyclase (sGC), to generate cyclic GMP (cGMP). NO promotes vasodilation, inhibits platelet aggregation, smooth muscle proliferation and monocyte adhesion to the endothelium. In the normal vessel wall, NO is mainly synthesized by the endothelium from the amino acid L-arginine by the endothelial NO synthase (eNOS or NOS III), one of three isoenzymes in the NO synthase family [4,5]. Blood flow and vasoactive agents (acetylcholine, bradykinin...) increase the free intracellular calcium and binding of the calcium/calmodulin complex to eNOS thereby promoting conversion of L-arginine to L-citrulline and NO [4]. Co-translational myristoylation and post-translational palmitoylation of eNOS target the enzyme to specialised plasmalemmal microdomains called caveolae [6,7]. These are non-coated microinvaginations (average size 50–100 nm) in the plasma membrane enriched with cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-linked proteins, and caveolin [8–10]. They ensure clathrin-independent endocytosis, transcytosis and potocytosis, and are involved in compartmentalization of signaling molecules. The caveolae contain seven-transmembrane domain receptors, G-coupled proteins, phospholipase C{gamma}, adenylyl cyclase, receptors coupled to tyrosine kinase activity and downstream molecules (Shc, Grb2, PI3Kinase, ras, MAP kinase), a Ca2+ pump, IP3-sensitive Ca2+ channel and protein kinase C.

Atherosclerosis, as well as other pathophysiological states, is linked to functional abnormalities of the endothelium. One major aspect of endothelial dysfunction is a reduction of the endothelium-dependent vasodilatation induced by acetycholine, or possible vasoconstriction due to the unopposed direct constrictive effect of acetylcholine on smooth muscle. Any decrease in NO bioavailability has major pathophysiological consequences, due to loss of the protective properties of NO against thrombosis and vasospasm. Numerous mechanisms of endothelial dysfunction have been proposed [11]. However, the role of caveolae in this process has not been investigated. Since caveolae are enriched in cholesterol (four- to eight-fold compared to the rest of the membrane [12]), two cholesterol-binding agents have been used to alter these cholesterol-sensitive microdomains: cyclodextrin [13,14] and filipin [15]. 2-Hydroxypropyl-β-cyclodextrin (hp-β-CD) is a membrane-impermeable molecule which depletes cellular cholesterol content through solubilization of the plasmalemmal cholesterol. It is a water-soluble cyclic oligosaccharide formed of seven glucopyranose units able to accept one molecule of cholesterol in its hydrophobic core [16]. The polyene antibiotic filipin interacts specifically with 3β-hydroxysterols in the membranes, modifying cholesterol interactions with other plasmalemmal components and consequently the organization and properties of the caveolae, and finally causes the caveolae components to disassemble [17].

Cyclodextrin [18], the oxidation of membrane cholesterol by cholesterol oxidase [12], and the exposure of endothelial cells to oxidized LDL (low density lipoprotein) [18] have all been shown to induce translocation of eNOS from plasmalemmal caveolae to the endoplasmic reticulum or Golgi apparatus in vitro. Colocalization of eNOS and numerous molecules known to be involved in the regulation of eNOS activity within the caveolae could contribute in optimizing its activation following binding of agonists (such as acetylcholine or bradykinin) to their receptors. In the present study, we evaluated the effect of hypercholesterolemia and fatty streak formation on endothelial function and on the endothelial caveolae, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
2-Hydroxypropyl-β-cyclodextrin (hp-β-CD, ref. C-0926), filipin (ref. F-9765), L-phenylephrine (ref. P-6126), acetylcholine (ref. A-2661), sodium nitroprusside (SNP, ref. S-0501), A23187 [GenBank] (ref. C-7522), bradykinin (ref. B-3259), and superoxide dismutase (SOD, ref. S-2515) were from Sigma. Fura-2 acetoxymethyl ester was purchased from Calbiochem, radiolabeled cholesterol from Isotopchim (ref. chol [4-14C], specific activity: 190 MBq/mmol). Filipin stocks were prepared in ethanol with final ethanol concentration of 0.01%.

2.2 Ex vivo experiments
2.2.1 Animals
New Zealand White male rabbits (2.5–3 kg body weight) were randomly assigned to regular (n=12) and hypercholesterolemic diets (1% cholesterol for 8 weeks) (n=9). Cholesterolemia was measured by the CHOD-PAP method (CHOL, Boehringer Mannheim) [19]. Experiments were performed in accordance with the recommendations of the French Accreditation of Laboratory Animal Care.

2.2.2 Isometric contraction–relaxation experiments in isolated rabbit aortic rings
Rabbits were sacrificed with an overdose of pentobarbital (50 mg/kg, i.v.). The middle part of the descending thoracic aorta was removed, cleaned of excess adventitial tissue, care being taken not to injure the endothelium during ring preparation. Then 3-mm segments of rabbit thoracic aorta (three segments per rabbit) were suspended in individual organ chambers filled with 20 ml Krebs buffer with the following millimolar composition: NaCl 118.3, KCl 4.69, CaCl2 1.25, MgSO4 1.17, K2HPO4 1.18, NaHCO3 25.0, and glucose 11.1, pH 7.4. The solution was aerated continuously with 95% O2–5% CO2 and maintained at 37°C. The rings were connected to force transducers and any variations in isometric force were recorded continuously. The resting tension was gradually increased to 5 g over a period of 40 min, and the ring segments exposed to 80 mM KCl until steady state was reached. When the vessels had recovered their resting tension, they were exposed to cumulative concentrations of an adrenergic {alpha}1 agonist, L-phenylephrine (10–8 to 3x10–5 M). A precontraction corresponding to 70% of the maximal phenylephrine contraction was selected for relaxation assessment. When the contraction plateau had been reached, the rings were exposed to cumulative acetylcholine concentrations (ACh, 10–9 to 3x10–5 M), sodium nitroprusside (SNP, 10–9 to 3x10–6 M), or calcium ionophore A23187 [GenBank] (10–9 to 3x10–6 M). These agents either elicited endothelium-dependent relaxation (ACh and A23187 [GenBank] ) or endothelium-independent relaxation (SNP). Basal NO production by aortic ring endothelium was evaluated in separate experiments from the contraction elicited with NG-nitro-L-arginine methyl ester (L-NAME, final concentration: 10–4 M) added to rings moderately preconstricted (3 g on average) with phenylephrine (10–8 M) [20]. Data were collected by Acknowledge (Biopac System) software and relaxation expressed as the percentage decrease in tension below the tension elicited by precontracting aortic rings with phenylephrine.

After contraction with KCl and phenylephrine dose–response, rings were incubated in organ chambers in Krebs buffer with or without 2-hydroxypropyl-β-cyclodextrin (hp-β-CD), 2%, for 1 h, or with filipin or vehicle, 4 µg/ml, for 10 min. Contraction–relaxation experiments were then performed as described above.

2.2.3 Evaluation of fatty streak area
After organ chamber experiments, the fatty streak deposit on these aortic rings was quantified. Rings were fixed in paraformaldehyde 4% at 4°C for 24 h. They were then cut longitudinally and the fatty streak stained with lysochrome Sudan IV. The area of the streak infiltration (stained red) was evaluated by a macroscopic examination of the intima of each aortic ring. The surface of the intimal area infiltrated by fatty streak was expressed as a percentage of the total intimal area of the segment of aorta, as previously described [21]. A score of 0% was attributed to a segment showing no visible fatty streaks whereas a segment in which the intimal surface area was totally infiltrated was attributed a score of 100%.

2.3 In vitro experiments
2.3.1 Cell culture
Bovine aortic endothelial cells (BAEC) were obtained and grown as previously described [22] in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated newborn calf serum, fibroblast growth factor 2 (FGF2) (1 ng/ml every 2 days), gentamycin (0.1 mg/ml), amphotericin B (125 ng/ml), at 37°C in culture dishes (10 cm2) and in a humidified atmosphere containing 10% CO2. The cells used in this study were between the 9th and 13th passage. Several measures were taken to avoid artefacts of cell culture, due to proliferation, on the BAEC phenotype, as previously reported [23]. All passages were made using a splitting ratio of 1:6. Confluence was determined when >95% of the cells were in contact with adjacent cells. Under our culture conditions, cells invariably reached confluence 2 days after passage. All experiments were done in BAEC 5–6 days after confluence (100,000 cells/cm2). Cells were incubated with or without 2-hydroxypropyl-β-cyclodextrin 2%, 1 h in Krebs buffer supplemented with 20 mM of HEPES at 37°C and in a 10% CO2-containing humidified atmosphere.

2.3.2 Measurement of NO bioactivity using RFL-6 reporter cells
NO bioactivity was measured as the concentration of guanylyl cyclase stimulating activity produced in BAEC supernatant following the method described by Ishii et al. [24]. Briefly, BAEC were cultured in six-well plates (900,000 cells/well). The culture medium was then removed, and the BAEC washed twice with 2 ml of Locke's buffer (154.0 mM NaCl, 5.6 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5.6 mM glucose and 10.0 mM Hepes, pH 7.4) and equilibrated for 20 min in 1 ml of the same buffer at 37°C. Superoxide dismutase (SOD: 200 U/ml final concentration) was added to the incubation medium and the cells were stimulated for 2 min with either bradykinin (1 µM) or the calcium ionophore, A23187 [GenBank] (10 µM). Just-confluent RFL-6 cells, cultured in six-well plates (900,000 cells/well), were washed with Locke's buffer then equilibrated for 20 min in 1 ml of the same buffer containing 0.45 mM of 3-isobutyl-1-methylxanthine at 37°C. Then 5 min before transfer of the BAEC conditioned medium, SOD was added to the RFL-6 incubation medium. Conditioned medium (1 ml) was then transferred to the RFL-6 dishes for 3 min. Finally, RFL-6 cells cGMP production was stopped by adding 1 ml of ice-cold ethanol, just before the samples were stored frozen (–80°C). cGMP levels in RFL-6 cells were determined by radioimmunoassay (NEX-133, NEN) and expressed in pmol/106 cells. All measurements of a single experiment were made in the same radioimmunoassay series with an intraassay coefficient of variability of less than 10%.

2.3.3 Evaluation of free intracellular calcium [Ca2+]i in cultured BAEC
BAEC were cultured on glass cover slips (5x10 mm) coated with fibronectin until confluence and [Ca2+]i mobilization was performed as previously described [25]. Briefly, after treatment, the cells were loaded with fura-2 acetoxymethyl ester (fura-2AM, 5 µM, dissolved in Krebs-HEPES buffer containing BSA 0.1% at 37°C in dark conditions for 45 min). After three washes, glass cover slips were placed in a quartz vat and then in a spectrophotometer (Spex Fluorilog). During recording, the cells were superfused with Krebs-HEPES buffer (8 ml/min at 37°C). The excitation wavelengths of free and Ca2+-bound fura-2AM were 380 and 340 nm, respectively, each with a 0.5-s period. The 520-nm-emitted light was collected by a photomultiplier tube and numerized. Cell autofluorescence (without fura-2AM) was subtracted from the total fluorescence detected in the presence of fura-2AM. Measurements were performed before and after the addition of bradykinin (1 µM). Results are expressed as the 340/380-nm ratio of fluorescence intensity.

2.3.4 High performance liquid chromatography (HPLC)
The BAEC were scraped and pelleted to measure cellular cholesterol content. Neutral cellular lipids were extracted according to a modified Folch method [26]. After adding radiolabeled cholesterol to measure the yield, the cell pellet was resuspended in a mix of methanol, chloroform and KCl 120 mM (4:8:3) and centrifuged at 230xg for 30 min. The organic layer was evaporated and the pellet, containing neutral lipids, was redissolved in 500 µl ethanol. The sample was prepurified on a Sep PakTM C18 (Millipore) and the flow through concentrated to 40 µl ethanol. Aliquots (10 µl) of this lipid extract were analyzed isocritically by reversed phase HPLC using a Beckman Gold system equipped with a Lichrosorb C18 5-µm column (25 cmx4 mm) fitted with a Lichrosorb C18 5-µm (0.5 cmx4 mm) guard cartridge, with methanol:water (96:4) at a flow rate of 0.7 ml/min. The effluent was monitored at 210 nm. Fractions were collected at 1-min intervals. The cholesterol peak had previously been determined by gas–liquid chromatography–mass spectroscopy (GLC–MS) (Kedjouar et al., in preparation). This process was also used to quantify the cholesterol solubilized by hp-β-CD in the BAEC supernatant after treatment.

2.4 Electron microscopy
Aortic samples, adjacent to those used for contraction–relaxation experiments, and BAEC were immediately fixed in 3% glutaraldehyde in PBS, postfixed in osmium tetroxide, dehydrated in graded ethanol series and embedded in Epon 812. Ultrathin sections were then cut (Reichert ultratome), placed on mesh copper grids, counterstained with uranyl acetate and lead citrate, and examined with a Hitachi 300 transmission electron microscope. Four independently prepared aortic samples from each group (A: control aortic endothelium, B: hp-β-CD treated aortic endothelium (2%, 1 h), C: aortic endothelium from hypercholesterolemic rabbit without fatty streak, D: aortic endothelium covering fatty streak from hypercholesterolemic rabbit) were used to photograph eight to nine randomly chosen fields of each sample. The abundance of caveolae was measured as previously described [27]. Only distinctly invaginated vesicles, found within 100 nm of the plasma membrane, were counted. The average number of caveolae per micrometre of sectioned plasma membrane is expressed as mean±S.E.M. (n=28–32 fields per group).

2.5 Statistical analysis
The contraction and relaxation curves were compared using ANOVA with repeated measures. Values are presented as mean±S.E.M. Comparisons of means were performed by unpaired t-test or Mann–Whitney test. Values were considered significant when P was <0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of hypercholesterolemic diet on cholesterolemia, fatty streak formation, NO production and caveolae abundance
The cholesterolemia increased from 1.02±0.07 to 23.00±3.86 g/l in rabbits fed with 1%-cholesterol diet for 8 weeks. As the relaxant response to acetylcholine is conditioned by smooth muscle cell function, we first verified that hypercholesterolemia had not modified the reactivity to phenylephrine (not shown) and to sodium nitroprusside. The 100% relaxation of aortic rings from control and hypercholesterolemic rabbits was obtained with concentrations of 2.93±0.26 and 3.17±0.18 µM SNP, respectively (NS). We then assessed the relationships between the level of hypercholesterolemia, the level of fatty streak formation and the impairment of acetylcholine-stimulated relaxation in thoracic aorta. As shown in Fig. 1, large variations were apparent in the fatty streak infiltration of the middle part of the descending thoracic aorta from one hypercholesterolemic rabbit to another. The maximal response of the aortic rings to acetylcholine (3 µM) was not correlated with the level of hypercholesterolemia (P=0.44) but was inversely correlated with the percentage of intimal area infiltrated by the fatty streak (P<0.001, R2=0.82; Fig. 1A). When the fatty streak deposit infiltrated the entire intimal surface area of the aortic ring, relaxation was completely abrogated, whereas rings without fatty streak relaxed normally despite in vivo exposure to hypercholesterolemia. In contrast, the A23187 [GenBank] -maximal relaxant response (1 µM) was little altered (P=0.035, R2=0.18; Fig. 1B). The difference between the slopes of the regression lines obtained under acetylcholine or A23187 [GenBank] stimulation was highly significantly different (P<0.001).


Figure 1
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  		Fig. 1 Correlation between the percentage of the intimal area infiltrated by fatty streak of thoracic aortic rings obtained from control ({circ}) and hypercholesterolemic rabbits (bullet) and the maximal relaxation elicited by acetylcholine (ACh, 3x10–6 M) (A) or a calcium ionophore (A23187, 10–6 M) (B). Maximal relaxation values are expressed as the percentage of phenylephrine precontraction (at 70% of maximal contraction). The differences between the slopes of the regression lines of the correlations obtained in A (y=64.43–0.59x; P<0.001, R2=0.82) and in B (y=77–0.122x; P=0.035; R2=0.18) were highly significant (P<0.001).

 
The abundance of caveolae in the endothelium of rabbit aorta was estimated by transmission electron microscopy. As previously described [15,28], the caveolae appeared as non-coated plasmalemmal vesicles with a round lumen of up to 50–100 nm in diameter (Fig. 2). The caveolae in control aortic endothelium (Fig. 2A) and in endothelium from hypercholesterolemic rabbit aorta free of fatty streak deposit (Fig. 2C), were found next to the apical plasma membrane, either isolated or in clusters. The number of caveolae was less abundant in the endothelium covering the fatty streaks compared to control (Figs. 2D and Fig. 3) (P=0.02), and the ‘grape-like’ clustering, common in normal endothelium (Fig. 2A), was absent from the endothelium covering the fatty streaks (Fig. 2D).


Figure 2
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  		Fig. 2 Visualization of caveolae in endothelium using transmission electron microscopy. Representative sections of rabbit aortic endothelium and BAEC are shown. Following sampling and/or treatment, samples were thin sectioned and examined by electron microscopy as described in Methods. (A) Control aortic endothelium; (B) hp-β-CD-treated aortic endothelium (2%, 1 h); (C) aortic endothelium from hypercholesterolemic rabbit without fatty streak; (D) aortic endothelium covering fatty streak from hypercholesterolemic rabbit; (E) control BAEC; (F) hp-β-CD treated BAEC. Caveolae appear as vesicles 50–100 nm in diameter localized under the plasma membrane. Bar, 0.4 µm in each case.

 

Figure 3
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  		Fig. 3 Evaluation of caveolae abundance in endothelium using transmission electron microscopy. Caveolae number per micrometre of sectioned plasma membrane is expressed as mean±S.E.M. *P<0.05 versus control. (A) Control aortic endothelium; (B) hp-β-CD-treated aortic endothelium (2%, 1 h); (C) aortic endothelium from hypercholesterolemic rabbit without fatty streak; (D) aortic endothelium covering fatty streak from hypercholesterolemic rabbit.

 
3.2 Effect of hp-β-CD and filipin on NO production in isolated aortic rings
The effect of hp-β-CD (2%, 1 h) was then assessed on aortic ring contraction–relaxation in rabbits fed a regular diet. Contraction elicited by phenylephrine was similar in the cyclodextrin-treated and control (untreated) rings (Table 1). Hp-β-CD markedly increased the EC50 of acetylcholine and reduced acetylcholine-induced maximal relaxation (Fig. 4A and Table 1). Neither the endothelium-dependent relaxant response to the calcium ionophore A23187 [GenBank] nor the endothelium-independent relaxant response to the NO donor SNP were altered (Fig. 4B,C and Table 1). Coincubation with hp-β-CD (2%, 1 h) and cholesterol (80 µg/ml) completely abolished the decrease in acetylcholine-elicited relaxation, showing that hp-β-CD acted through membrane depletion of cholesterol and not through a direct effect of the molecule (data not shown). When the relaxant response to acetylcholine was analyzed over a period of 1 h, the acetylcholine-induced relaxation (EC50 and maximal relaxation) of hp-β-CD-treated aortic rings tended to normalize (Fig. 4D, Table 1). We also used filipin to modify plasmalemmal cholesterol. Again, acetylcholine-induced relaxation was decreased, whereas the contractile response to phenylephrine and the relaxant response to A23187 [GenBank] and SNP were not altered by filipin (4 µg/ml for 10 min) (Fig. 4A–C and Table 2). The alterations in acetylcholine-induced relaxation in filipin-treated aortic rings were sustained over time (Fig. 4D, Table 2).


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  		Table 1 Maximal contraction (g) to phenylephrine and maximal relaxations (% of precontraction value) of control and hp-β-CD-treated (2%, 1 h) thoracic aortic ringsa

 

Figure 4
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  		Fig. 4 Effect of hp-β-CD and filipin on the relaxation of rabbit thoracic aortic rings. {circ}: hp-β-CD-control group; bullet: hp-β-CD-treated group (2% for 1 h); {bigtriangleup}: filipin-control group; and {blacktriangleup}: filipin-treated group (4 µg/ml for 10 min). Aortic rings were exposed immediately after either treatment to cumulative doses of acetylcholine (A), calcium ionophore A23187 (B), or sodium nitroprusside (SNP) (C). Cumulative doses of acetylcholine were applied with a delay of 1 h after treatment (D). Values are presented as percentage of relaxation relative to phenylephrine precontraction (at 70% of maximal contraction). Values are expressed as mean±S.E.M. *P<0.05 versus control.

 

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  		Table 2 Maximal contraction (g) to phenylephrine and maximal relaxations (% of precontraction value) of control and filipin-treated (4 µg/ml, 10 min) thoracic aortic ringsa

 
Basal NO production of aortic rings was evaluated from the L-NAME-induced contraction. After addition of L-NAME, the contraction elicited by phenylephrine increased from 8.11±0.40 to 11.10±0.56 g in control rings and from 8.09±0.41 to 11.23±0.89 g in hp-β-CD treated rings. Thus cholesterol depletion did not alter basal eNOS activity. Similarly, the exposure of aortic rings to diclofenac (2 µM), a cyclooxygenase inhibitor, did not alter endothelium-dependent relaxation to acetylcholine in control or in hp-β-CD treated rings (not shown), suggesting that the cyclooxygenase-relaxant pathway was not involved in this process. Finally, examination of the endothelium of hp-β-CD-treated aortic rings by electron microscopy showed that, in comparison to the control, hp-β-CD decreased the number of caveolae and disassembled their ‘grape-like’ clustering (compare Fig. 2A and B).

3.3 Effect of hp-β-CD on cultured BAEC
The stimulated NO production in the BAEC supernatant was assessed as the amount of cGMP generated after transfer of their conditioned media on to guanylyl cyclase rich reporter cells (RFL-6). Bradykinin (1 µM) and A23187 [GenBank] (10 µM) stimulations of BAEC increased the cGMP content of RFL-6 supernatant from undetectable levels to 2.33±0.27 and 3.11±0.25 pmol/106 cells, respectively. Hp-β-CD treatment of BAEC induced a significant decrease in bradykinin-induced cGMP production to 1.13±0.13 pmol/106 cells (P<0.01), whereas cGMP production in response to A23187 [GenBank] remained unaltered (3.02±0.13 pmol/106; P=0.79).

Studies with the fluorescent Ca2+-binding probe fura-2AM showed that bradykinin induced [Ca2+]i mobilization in BAEC. In the control BAEC, bradykinin elicited a transient two-phase increase in free intracellular calcium (Fig. 5A) consisting of a transient peak of Ca2+ followed by a prolonged plateau of lesser intensity. In hp-β-CD-treated BAEC, both peak and plateau were attenuated in response to bradykinin (Fig. 5B).


Figure 5
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  		Fig. 5 Bradykinin-elicited intracellular Ca2+ ([Ca2+]i) increase in control (A) and hp-β-CD-treated (2%, 1 h) (B) BAEC. Fura 2AM-loaded post-confluent BAEC were superfused with Krebs-HEPES buffer, stimulated with bradykinin (BK, 1 µM) and changes in [Ca2+]i monitored. The data presented are representative of three independent experiments.

 
Hp-β-CD decreased BAEC cholesterol content from 10.77±0.61 to 5.58±0.50 µg/106 cells (P<0.01). The cholesterol counterpart removed from the cellular plasmalemmal membrane was recovered in the culture supernatant (not shown). Again, cholesterol depletion was associated with a decreased number and disorganization of the caveolae (Fig. 2E,F).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
An initial set of experiments had shown that the intimal fatty streak deposit was associated with a loss of endothelium-dependent vasodilatation in response to acetylcholine, whereas the endothelium-dependent vasodilatation in response to A23187 [GenBank] and the endothelium-independent vasodilatation in response to SNP were both preserved. Similar alterations in acetylcholine-induced endothelium-derived NO release, i.e. impairment of acetylcholine-induced relaxation with normal A23187 [GenBank] - and SNP-elicited maximal relaxations, had previously been reported in human atherosclerosis and in cholesterol-fed rabbit [11,29]. In the present study, we observed that the maximal relaxation to acetylcholine and the level of fatty streak deposit were inversely correlated, in agreement with a previous report [30]. Hypercholesterolemia is a major cause of atherosclerosis and endothelial dysfunction. However, in the present study, some aortic segments of hypercholesterolemic rabbits showed a normal response to acetylcholine when they were free of fatty streak infiltration. Endothelial dysfunction appeared to be proportional to the presence of underlying fatty streak. Similar observations were made in genetically altered hyperlipidemic mice [31]. This demonstrates that hypercholesterolemia alone, at least in these models, is not sufficient to impair the acetylcholine-induced relaxation. We can thus conclude that the dysfunctional endothelium covering fatty streaks is still able to release NO, but carries an abnormality in signal transduction located upstream of eNOS

Interestingly, a similar pattern of endothelial dysfunction has been previously promoted experimentally by cytokines generated in the atherosclerotic lesions. Exposure of rabbit carotids to IL-1β, or of porcine coronary arteries to a combination of TNF{alpha}, IFN{gamma} and lipopolysaccharides induced an alteration in receptor-mediated endothelium-dependent relaxation, whereas relaxation to A23187 [GenBank] or to SNP remained normal [32]. Oxidized LDL and lysophosphatidylcholine also induce a decrease in serotonin or thrombin-induced relaxation, without any change in relaxation to A23187 [GenBank] or SNP [33–36] and alter the mobilization of free intracellular Ca2+ and of inositol triphosphate in cultured endothelial cells [37–39]. Finally, Blair et al. using cultured porcine artery endothelial cells recently demonstrated that oxidized LDL depletes the cholesterol content of caveolae which then delocalizes eNOS from the plasmalemmal caveolae [18]. These data fit with our observations and suggest that alteration in the plasmalemmal cholesterol could alter the caveolae and lead to endothelial dysfunction.

Cholesterol depletion by hp-β-CD and cholesterol sequestration by filipin were also found to induce endothelial dysfunction. Moreover, cholesterol depletion of endothelial cells led to disorganization and an apparent decrease in the number of caveolae. The effects of hp-β-CD and filipin were first studied on the production of endothelial EDRF-NO of rabbit aortic rings. Both treatments decreased acetylcholine-mediated NO production but completely preserved (a) the receptor-independent endothelial NO production (A23187 [GenBank] -elicited), (b) the basal release of NO, and (c) response to the NO-donor SNP. The integrity of receptor-independent relaxation in response to A23187 [GenBank] suggests that the availability of eNOS substrate and cofactors remained unaltered despite membrane cholesterol disturbance. As similar results were obtained in hp-β-CD treated BAEC, the biochemical mechanisms involved could be investigated. Loss of muscarinic receptors in secondary cultures did not allow stimulation with acetylcholine, whereas the bradykinin receptor B2 was well preserved [40,41]. Hp-β-CD treatment led to a two-fold decrease in NO generation in bradykinin-stimulated cells, and at the same time, attenuated the bradykinin-elicited increase in free intracellular Ca2+. Both the transient peak (due to intracellular Ca2+ mobilization), and the following plateau (due to extracellular Ca2+ entry), induced by bradykinin [42] were attenuated in hp-β-CD-treated cells. In contrast, the receptor-independent NO bioactivity stimulated by A23187 [GenBank] was not modified by hp-β-CD. Other mechanisms involving seven-transmembrane domains receptors, G proteins and coupling with transmembrane receptor and phospholipase C, might also contribute to this alteration of the transmembrane signal transduction pathway [43,44]. Alternatively, as also observed by Blair et al. [18], the delocalization of eNOS from the caveolae induced by cholesterol depletion alters Ca2+ dependent receptor-mediated eNOS activation whereas eNOS remains sensitive to A23187 [GenBank] whatever its subcellular location. The finding that delocalization of eNOS is reversible with a return to the caveolae within 2 h [18], is also consistent with the progressive normalization of acetylcholine reactivity (i.e. maximal relaxation and EC50) of hp-β-CD-treated aortic rings.

In conclusion, we show that in the hypercholesterolemic rabbit, endothelial dysfunction is associated with a decrease in the amount of caveolae in the endothelium covering fatty streaks. Hypercholesterolemia alone, in the absence of fatty streak deposit, is not sufficient to elicit endothelial dysfunction. We used hp-β-CD and filipin to demonstrate that disturbance of the caveolae, brought about by modification of the plasmalemmal cholesterol, induces a selective impairment of receptor-mediated NO production, whereas receptor-independent eNOS activation remains unaltered. As the number of caveolae was apparently decreased under all these conditions, our data strongly suggest a link between fatty streak deposit, decreased endothelial caveolae abundance and endothelial dysfunction. The precise mechanisms leading to this decrease in caveolae abundance and to alteration of receptor-mediated eNOS activation will require further studies.

Time for primary review 25 days.


   Acknowledgements
 
This work was supported by INSERM, the Fondation pour la Recherche Médicale, and the Fondation de France. We thank Dr M.-T. Pieraggi for electron microscopy, and Drs J.-L. Bascands and J. Schranstra for evaluation of the free intracellular calcium experiments, and Dr. J.B. Michel for fruitful discussions.


   Notes
 
1 These authors contributed equally to this paper. Back


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

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