Lipids DOI 10.1007/s11745-014-3888-5
ORIGINAL ARTICLE
15-Lipoxygenase-Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells Lucia Cutuli • Angela Pirillo • Patrizia Uboldi Hartmut Kuehn • Alberico L. Catapano
•
Received: 13 September 2013 / Accepted: 12 February 2014 Ó AOCS 2014
Abstract Caveolae are cholesterol and glycosphingolipidsenriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SRBI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenasemediated modification of HDL3 on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We
L. Cutuli P. Uboldi A. L. Catapano Department of Pharmacological and Biomolecular Sciences, Universita` degli Studi di Milano, Milan, Italy A. Pirillo (&) Center for the Study of Atherosclerosis, Bassini Hospital, Via M. Gorki 50, Cinisello Balsamo, Milan, Italy e-mail: [email protected] A. Pirillo A. L. Catapano IRCCS Multimedica, Milan, Italy H. Kuehn Institute of Biochemistry, University of Medicine Berlin-Charite´, Berlin, Germany
found that after modification, HDL3 failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL. Keywords High density lipoprotein Endothelial cells 15-Lipoxygenase Caveolae Nitric oxide Abbreviations 15LO 15-Lipoxygenase ABCG1 ATP binding cassette transporter G1 CAV-1 Caveolin-1 eNOS Endothelial nitric oxide synthase HDL High density lipoprotein HUVEC Human umbilical vein endothelial cell(s) LOX-1 Lectin-like oxidized low-density lipoprotein receptor-1 MAPK Mitogen-activated protein kinase NO Nitric oxide S1P3 Sphingosine-1-phosphate receptor 3 SR-BI Scavenger receptor type B class I
Introduction Endothelium, due to its localization between blood and the vessel wall, plays a central role in controlling vascular tone and homeostasis. Endothelial cells, in fact, produce several vasoactive factors in response to different stimuli, including vasoconstrictor and vasodilator substances [1]; among the latter, nitric oxide (NO) is the most crucial. The
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production of NO is regulated by the endothelial nitric oxide synthase (eNOS) activity, which in turn is regulated by a number of upstream factors [2]. Caveolae are specialized membrane microdomains enriched in cholesterol, sphingolipids and glycosphingolipids [3]; they are characterized by the presence of caveolin-1 as the main protein constituent [4], which in turn regulates the cholesterol content [5, 6]. Moreover, caveolae contain a number of other proteins, including receptors and molecules involved in signal transduction pathways [7, 8]. eNOS is localized in caveolae and interacts with caveolin-1 [9], being caveolin-1 the main negative regulator of eNOS activity, as the association of eNOS with caveolin-1 inhibits the enzyme activity [10]. Following extracellular stimuli, eNOS dissociates from caveolin-1, forms a complex with calcium/calmodulin or heat shock protein 90 and becomes activated, thus increasing the NO production [11]. eNOS is constitutively expressed in endothelial cells; however expression and activity of eNOS are finely modulated at transcriptional and post-translational levels by protein–protein interactions, Ca2? concentration, and subcellular distribution [12, 13]. eNOS activity is also regulated by phosphorylation [11], and the Akt-mediated phosphorylation of Ser1177 [14] positively regulates NO production [15]. Furthermore, caveolin negatively impacts eNOS activity through direct interaction with the enzyme [2]; finally, the cholesterol content in caveolae plays a critical role in eNOS function [16]. Plasma levels of high-density lipoprotein (HDL) cholesterol inversely correlate with the incidence of cardiovascular disease. The atheroprotective effects of HDL are mainly related to their role in reverse cholesterol transport (RCT), but HDL possesses, among others, also anti-oxidant, anti-inflammatory and anti-thrombotic activities [17]. HDL is a potent agonist of eNOS and promotes NO production by several mechanisms [18, 19]: it regulates eNOS abundance and its subcellular distribution; HDL and HDL-associated lysophospholipids, through the interaction with SR-BI (scavenger receptor class B type I) and S1P3 (sphingosine-1-phosphate receptor 3) respectively [19, 20], activate downstream kinases, resulting in eNOS activation. The activation of eNOS by HDL is, at least in part, Akt and MAPK-dependent [21]; however, Akt-independent mechanisms of eNOS activation by HDL exist [20]. Furthermore, SR-BI-mediated cholesterol flux in response to HDL is required for signal initiation by HDL [2]. The HDL modification induced by 15-lipoxygenase (15LO), an enzyme overexpressed in atherosclerotic lesions [22], impairs their athero-protective functions [23– 25]. In fact, 15LO-HDL3 are less effective in promoting cholesterol efflux from macrophages [23] and are less efficient in reducing the expression of TNF-a-induced
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adhesion molecules and LOX-1 in endothelial cells [24, 25]; furthermore, 15LO-HDL3 acquire pro-inflammatory properties, as they induce the expression of LOX-1 and adhesion molecules and chemoattractant factors in endothelial cells and increased the adhesion of monocytes to the endothelium [24, 25]. In the present work we aimed at studying the effect of 15LO-mediated HDL3 modification on the caveolae system. Specifically, we studied the effect of 15LO-HDL3 on the expression and activity of eNOS as well as on the expression of SR-BI, caveolin-1, and eNOS.
Materials and Methods Reagents Medium M199 was from Invitrogen. Fetal bovine serum (FBS), penicillin–streptomycin, and glutamine were from Sigma-Aldrich; PD10 columns and enhanced chemiluminescence (ECL) western blotting detection reagent were from Amersham Biosciences. Endothelial cell growth factor (ECGF) was from Boehringer Mannheim. Antibodies were purchased as follows: anti-SR-BI and anti-Cav-1 from Abcam; anti-eNOS, anti-phospho-eNOS, anti-Akt and anti-phospho-Akt from Cell Signaling; anti-b-actin and anti-mouse IgG peroxidase-conjugate from Sigma-Aldrich; anti-rabbit IgG peroxidase-conjugate from BioRad. Cell Culture HUVEC were isolated according to established procedures [26] and cultured in medium M199 supplemented with 20 % FBS, ECGF (20 lg/ml), heparin (15 U/ml), penicillin–streptomycin (1 %) and glutamine (1 %). Cells were used between the 3rd and 5th in vitro passage. Isolation and Modification of HDL3 HDL3 (d = 1.125–1.21 g/ml) were isolated by sequential ultracentrifugation at 4 °C from the plasma of healthy volunteers [27]. HDL3 were dialyzed using a Sephadex G25 column (PD10) with a PBS-EDTA 0.01 %, pH 7,4, then sterilized using a 0.22-lm filter (corning) and stored at 4 °C. The protein content was evaluated by the Lowry method [28], using bovine serum albumin as a standard. HDL3 were modified with 15-LO as described [23]. Caveolae Purification and Analysis HUVEC were incubated with 100 lg/ml HDL3 or 15LOHDL3 for 24 h, then washed twice with cold PBS and harvested by scraping. The samples were centrifuged at
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1,500 rpm for 5 min. The pellets were resuspended in 2 ml of Na2CO3 buffer (500 mM pH 11) and sonicated (3 9 30 s); 2 ml of the lysate were adjusted to 45 % sucrose by mixing with 2 ml of 90 % sucrose prepared in 25 mmol/l MES, pH 6.5, containing 150 mmol/l NaCl, and placed at the bottom of an ultracentrifuge tube. A 5–35 % discontinuous sucrose gradient was formed (in MES containing 100 mmol/l Na2CO3) and ultracentrifuged at 39,000 rpm in a SW41 rotor (Beckman) for 16 h. From the top, 1-ml fractions were removed sequentially and designated as fractions 1–12. Each fraction was subjected to western blotting analysis for Cav-1, SR-BI and eNOS expression. Western Blotting For protein analysis of fractions derived from sucrose gradient, equal volumes of each sample were mixed with loading buffer containing 2 % b-mercaptoethanol and 2 % SDS and proteins were separated on SDS-PAGE. For the analysis of cellular proteins, whole cell lysates were prepared in lysis buffer (2 % SDS, 62.5 mM TRIS, 50 mM DTT, 1 mM PMSF, 5 lg/ml aprotinin) and sonicated. Cellular proteins were separated on a 10 % SDS-PAGE, and then transferred onto a nitrocellulose membrane. Membranes were saturated with 5 % non-fat milk in PBS-T (PBS-0.1 % Tween) for 1 h at room temperature, followed by overnight incubation with the selected primary antibody at 4 °C and 1 h incubation with a 1:1,000 dilution of a goat anti-rabbit or anti-mouse IgG-HRP conjugate. Immunocomplexes were detected by ECL followed by autoradiography. The bands were quantified by a computer-assisted system for image analysis (ISF Image 1.52) and the expression of each antigen was corrected for b-actin content. Quantitative Real Time PCR (RT-PCR) Total RNA was extracted and reverse transcribed. Three ll of cDNA were amplified by real-time quantitative polymerase chain reaction (PCR) with 19 SYBR Green universal PCR mastermix (BioRad) [29]. The sequence of the primers used for RLP13A (housekeeping gene), SR-BI, eNOS, Cav-1 amplification are reported in Table 1. Each sample was analyzed in duplicate using the IQTM-Cycler (BioRad). For quantification, the target genes were normalized to the RLP13A content. NO Production Analysis by Flow Cytometry NO generation was determined by flow cytometry. HUVEC were preincubated for 1 h at 37 °C with the fluorescent
Table 1 Sequence of primers used in RT-PCR experiments Primer
Sequence
RLP Fw
50 -ATGCTGCCCCACAAAACC-30
RLP Rev
50 -TGCCGTCAACACCCTTGAGA-30
Cav-1 Fw
50 -TCCTTCCTCAGTTCCCTTAAAGC-30
Cav-1 Rev
50 -GCCCGTGGCTGGATGA-30
eNOS Fw
50 -CTCGTCCCTGTGGAAAGACAA-30
eNOS Rev
50 -TGACTTTGGCTAGCTGGTAACTGT-30
SR-BI Fw
50 -GACAAACTGGGAAGATTGAGCC-30
SR-BI Rev
50 -CCATGGCCCCGCTCTC-30
dye DAF-FM diacetate (4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate); cells were then washed with PBS and incubated with HDL3 or 15LO-HDL3 for 15 min at 37 °C. At the end of the incubation, cells were harvested by trypsinization and immediately subjected to flow cytometry analysis (FACScan, Becton–Dickinson). Lipoprotein–Cell Association Lipoproteins were labeled with the fluorescent dye DiO as previously described [25]. Endothelial cells were incubated at 37 °C with DiO-labeled lipoproteins (12.5 and 25 lg/ ml) for 1 h, then washed three times with cold PBS, detached by trypsinization, fixed in 1 % paraformaldehyde and immediately subjected to fluorescence flow cytometry using a FACScan (Becton–Dickinson). For each sample 10,000 events were analyzed; data were processed using the CellQuest program (Becton–Dickinson). Statistical Analysis Values are expressed as means ± SD. The statistical significance of the differences between groups was determined by the Student’s t test and values of P \ 0.05 were considered to be significant.
Results Effect of 15LO-Modified HDL3 on eNOS Phosphorylation and Activity The activation of eNOS by HDL3 is intimately related to the ability of this lipoprotein to induce the phosphorylation of eNOS at Ser1177 [20, 30]. Thus, we investigated the effect of HDL3 or 15LO-HDL3 on the phosphorylation of eNOS at this specific site. As expected, HDL3 significantly increased the phosphorylation of eNOS at Ser1177, while 15LO-HDL3 failed to induce such effect in HUVEC (Fig. 1a). eNOS
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*
a
*
p-eNOS/eNOS (fold induction)
2,0
Cont. HDL3 15LO-HDL3
1,5
p-eNOS eNOS
1,0
0,5
0,0 Control
b
15LO-HDL3
HDL3
**
*
3,0
NO production (fold induction)
Fig. 1 Effect of native and 15LO-modified HDL3 on eNOS phosphorylation (a) and NO production (b) and Akt phosphorylation (c) in endothelial cells. a, c HUVEC were incubated with HDL3 or 15LO-modified HDL3 (100 lg/ ml) for 100 ; eNOS and Akt phosphorylation were evaluated by western blotting. Results are given as means ± SD from 3 independent experiments. a *P \ 0.01; c *P \ 0.005, **P \ 0.0005. b HUVEC were pre-incubated with the fluorescent dye DAF-FM diacetate, then incubated with HDL3 or 15LO-HDL3 for 15 min. NO production was evaluated by flow cytometry. Results are given as means ± SD from 5 independent experiments. *P \ 0.01, **P \ 0.00005
2,5 2,0 1,5 1,0 0,5 0,0 Control
c
15LO-HDL3
HDL3
**
*
p-Akt/Akt (fold induction)
2,0
Cont. HDL3 15LO-HDL3 1,5
p-Akt Akt
1,0
0,5
0,0 Control
activation through phosphorylation at Ser1177 results in enzyme activation and nitric oxide generation [2]; accordingly to the ability to induce eNOS phosphorylation, HDL3 significantly increased the production of nitric oxide compared to control cells, while 15LO-HDL3 resulted appreciably less effective in generating this vasoactive compound compared to HDL3 (Fig. 1b). HDL-mediated eNOS stimulation involves Akt activation [21]; for this reason we evaluated the ability of native and 15LO-modified HDL3 to stimulate this kinase. We found that HDL3 significantly induced Akt phosphorylation, while 15LO-HDL3 were less effective (Fig. 1c).
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HDL3
15LO-HDL3
Effect of 15LO-Modified HDL3 on Gene Expression of SR-BI, Caveolin-1 and eNOS To analyze the effect of 15LO-modified HDL3 on the expression at transcriptional level of proteins present in the caveolae membranes, HUVEC were incubated overnight with 100 lg/ml HDL3 or 15LO-HDL3. HDL3 significantly increased SR-BI mRNA levels, while in the presence of 15LO-HDL3 the expression of SR-BI was significantly lower compared to native HDL3, although higher compared to control cells (Fig. 2a). Caveolin-1 mRNA was significantly and similarly reduced in cell incubated with HDL3
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a
**
SR-BI/RLP (fold induction)
2,5 2,0
#
1,5 1,0 0,5 0,0
Control
Cav-1/RLP (fold induction)
b
HDL3
1,0
15LO-HDL3
* **
0,8 0,6 0,4 0,2 0,0
Control
eNOS/RLP (fold induction)
c
1,0
HDL3
*
15LO-HDL3
*
0,8 0,6 0,4 0,2
Incubation of HUVEC with HDL3 significantly increased SR-BI protein expression; on the contrary, the incubation of endothelial cells with 15LO-HDL3 resulted in a decreased expression of SR-BI protein compared to either control or HDL3-treated cells (-43 and -60 %, respectively) (Fig. 3a). HDL3 did not alter the levels of caveolin1 compared to controls, while 15LO-HDL3 increased it by about 30 % compared to either control or HDL3-treated cells (Fig. 3b). No relevant changes were observed in the expression of eNOS protein in cells treated with HDL3 or 15LO-HDL3 compared to controls (Fig. 3c). Effect of HDL3 and 15LO-HDL3 on Caveolin-1, SR-BI and eNOS Distribution Next, we examined the effect of HDL3 or 15LO-HDL3 on caveolin-1, SR-BI and eNOS distribution in caveolae. We found that most caveolin-1 was localized in caveolae fractions (4, 5) in cells treated with HDL3 or 15LO-HDL3, without appreciable differences in the caveolin-1 content (Fig. 4a); however, a significant increase of caveolin-1 content was found in fractions other than caveolae in 15LO-HDL3 treated cells (fold induction 1.68; P \ 0.0005). As expected, both SR-BI and eNOS proteins were localized in the caveolae fraction 4; however, in 15LO-HDL3-treated cells we found a lower amount of SRBI protein associated with caveolae fractions compared to HDL3-treated cells (-23.8 %), even when adjusted for the caveolin-1 content (-31.2 %) (Fig. 4b), without any change in the amount of eNOS protein associated with caveolae (Fig. 4c). Lipoprotein–Endothelial Cell Association
0,0
Control
HDL3
15LO-HDL3
Fig. 2 Effect of native and 15LO-modified HDL3 on SR-BI, Cav-1 and eNOS mRNA expression in endothelial cells. HUVEC were incubated with HDL3 or 15LO-modified HDL3 (100 lg/ml) for 18 h; total mRNA was isolated and the expression of the indicated genes was evaluated by real time PCR. RLP13A was used as an internal control. Results are given as means ± SD from 5 (a) or 4 (b, c) independent experiments. *P \ 0.05 and **P \ 0.005 vs control; # P \ 0.05 vs HDL3
or 15LO-HDL3 compared to controls (Fig. 2b); similar results were obtained for eNOS mRNA, with no difference between treatment with HDL3 or 15LO-HDL3 (Fig. 2c). Effect of 15LO-Modified HDL3 on Protein Expression of SR-BI, Caveolin-1 and eNOS Then we evaluated the effect of HDL3 or 15LO-HDL3 on the expression of SR-BI, eNOS and Cav-1 at protein level.
We previously showed that 15LO-mediated modification significantly reduces the ability of HDL3 to associate to macrophages and that this was due to a reduced ability to interact with SR-BI [23]. We found that 15LO-HDL3 associated less efficiently also to endothelial cells compared to native HDL3 (Fig. 5).
Discussion In this study we have shown that 15-lipoxygenase-mediated modification significantly reduces the endothelial protective effect of HDL3. In fact, after modification, HDL3 induces changes in the expression/activity of proteins involved in eNOS activity, resulting in a reduced production of nitric oxide. The main mechanism by which HDL exert their antiatherogenic role is through the promotion of cholesterol efflux from macrophages and smooth muscle cells in the
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a
**
* Cont.
SR-BI/ß-actin (fold induction)
1,5
HDL3
15LO-HDL 3
1,2
SR-BI
0,9
ß-actin 0,6 0,3 0,0
Control
b
Cav-1/ß-actin (fold induction)
15LO-HDL3
HDL3
* 1,5
Cont. HDL3 15LO-HDL 3
1,2
Cav-1
0,9
ß-actin
0,6 0,3 0,0
c
1,2
eNOS/ß-actin (fold induction)
Control
0,9
HDL3
15LO-HDL3
Cont.
HDL3
15LO-HDL 3
eNOS
0,6
ß-actin
0,3
0,0
Control
HDL3
15LO-HDL3
Fig. 3 Effect of native and 15LO-modified HDL3 on SR-BI, Cav-1 and eNOS protein expression in endothelial cells. HUVEC were incubated with HDL3 or 15LO-modified HDL3 (100 lg/ml) for 24 h.
SR-BI, Cav-1 and eNOS protein expression were evaluated by western blotting. Results are given as means ± SD from 6 independent experiments. *P \ 0.005, **P \ 0.0005
arterial wall [31]; however, HDL possess several other athero-protective properties that include anti-inflammatory, anti-oxidant and endothelial-protective functions [32]. Caveolae are specialized microdomains that compartmentalize signal transduction molecules involved in the regulation of endothelial functions; among them, a key role is played by the process of nitric oxide generation, that involves several players, including eNOS, the enzyme responsible for the production of NO, caveolin-1, the major coat protein of caveolae, SR-BI, the main HDL receptors, all of which are localized within the caveolae domain, and
HDL [2]. HDL induce NO production by several different mechanisms. ApoA-I and HDL induce eNOS activation and NO production following the interaction with SR-BI [19, 30]. The interaction of HDL with SR-BI activates multiple kinases, including Akt, resulting in the phosphorylation of eNOS at Ser1177 and subsequent activation of the enzyme [21]. In addition, other HDL-associated components can stimulate eNOS-mediated NO generation [20, 33]; also, ABCG1-mediated cholesterol efflux to HDL from endothelial cells reduces the interaction between caveolin-1 and eNOS, resulting in eNOS activation [34].
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Lipids Fig. 4 Effect of native and 15LO-modified HDL3 on caveolin-1, SR-BI and eNOS distribution in caveolae. HUVEC were incubated with 100 lg/ml HDL3 or 15LOHDL3 for 24 h, then cells were subjected to sucrose gradient fractionation for caveolae isolation. Cav-1, SR-BI and eNOS distribution and expression were evaluated by western blotting. Quantification of the amount of protein in each fraction was obtained by densitometry analysis. Experiments were performed in triplicate; representative images are shown
Sucrose gradient (5-45%)
Fraction n. 1
2 3
4
5
6
7
8
9
10 11 12 HDL3
a
Caveolin-1 15LO-HDL3 Fraction n.
2
3
4
5
6
7 HDL3
SR-BI
b
15LO-HDL3 Fraction n. 3
4
5
6 HDL3
eNOS
c
15LO-HDL3
Lipoprotein-cell association
HDL3 HDL3
15LO-HDL3 15LO-HDL3
*
100 90 80 70 60 50 40 30 20 10 0
*
0
12,5
25
µg/ml Fig. 5 Native and 15LO-modified HDL3 association to endothelial cells. HUVEC were incubated with DiO-labeled lipoproteins (12.5 and 25 lg/ml) for 1 h at 37 °C. Cell-associated fluorescence was evaluated by flow cytometry. Data are means ± SD of 3 independent experiments performed in duplicate. *P \ 0.0005
Oxidative modification, however, impairs the atheroprotective functions of HDL [35, 36]. We have previously shown that oxidative modification mediated by 15-lipoxygenase, an enzyme overexpressed in atherosclerotic lesions [22, 37], severely altered the properties of HDL, as it resulted in a reduced efficiency to promote cholesterol efflux [23] and in a decreased anti-inflammatory activity [24, 25], and also conferred pro-inflammatory characteristics, as 15LO-modified HDL3 induced the expression of
adhesion molecules [24] and LOX-1 [25] in endothelial cells. In the present work we found that 15LO-modified HDL3 affected the expression and/or the activity of proteins involved in the nitric oxide generation pathway in endothelial cells, including SR-BI, caveolin-1 and eNOS. Caveolin-1 and caveolae are present in all the cells of the arterial wall, but the role of caveolin-1 in atherosclerosis is strictly dependent on the cell type. In fact, while macrophage and smooth muscle cell caveolin-1 seems to play an inhibitory role in the development of atherosclerosis, endothelial caveolin-1 may act as a pro-atherogenic protein [38]. In agreement with this, genetic ablation of caveolin-1 markedly reduced the extent of atherosclerosis in apoE knockout mice [39], whereas endothelial-specific overexpression of caveolin-1 enhanced the progression of mouse atherosclerosis by increasing the infiltration of LDL particles within the arterial wall, by inducing the expression of leukocyte adhesion molecules and by decreasing the production of nitric oxide [40]. In our experimental conditions, we found that, although similar effects at mRNA level, native HDL3 did not alter caveolin-1 protein expression, while cells incubated in the presence of 15LO-modified HDL3 increased total caveolin-1 protein content. Nevertheless, this change did not translate in an enrichment of caveolin-1 in caveolae, suggesting that 15LO-HDL3 effects on eNOS activation are not related to the observed increase of caveolin-1 protein. It has been reported that HDL increased eNOS protein abundance in endothelial cells by increasing the protein
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half-life, without any significant effect on eNOS mRNA levels [41]; however, other studies did not find any effect of HDL on eNOS and caveolin-1 abundance [34]. In our experiments we did not detect any significant change in eNOS protein in HUVEC incubated with native or 15LOmodified HDL3, despite a significant decrease of eNOS mRNA with both treatments. In agreement with this finding, no differences were observed in eNOS content in caveolae of cells incubated with HDL3 or 15LO-HDL3. However, a significant different ability of lipoproteins to induce eNOS activity was observed. In fact native HDL3 increase the phosphorylation of eNOS at Ser1177, which translates in an increased production of nitric oxide. After 15LO-mediated modification, HDL3 exhibited a reduced ability to activate eNOS and to induce NO generation. In addition, HDL3 significantly induced the phosphorylation of Akt, the kinase that directly activates eNOS by phosphorylation at Ser1177 [14]; 15LO-mediated modification of HDL3 significantly reduced Akt phosphorylation; this result may partly explain the reduced eNOS phosphorylation observed in cells treated with 15LO-HDL3. Several observations have been reported suggesting a reduced ability of HDL isolated from subjects with atherosclerosisrelated disease to induce nitric oxide production. In fact, while HDL from healthy subjects stimulated endothelial NO production, HDL from patients with stable coronary artery disease or acute coronary syndrome (ACS) exhibited an impaired ability to induce NO generation [33, 42]. This effect was mainly due to a reduced capacity to stimulate Akt phosphorylation and subsequent eNOS phosphorylation at Ser1177 [33]. Similarly, HDL isolated from diabetic women failed to stimulate NO production without affecting the levels of eNOS, compared to HDL from healthy subjects [43], and HDL from obese adolescents exhibited a reduced ability to induce eNOS-Ser1177 phosphorylation compared to HDL from adolescents with normal body mass index [44]. Altogether these findings suggest that under pathological conditions HDL may undergo structural and functional modifications that significantly impair their endothelial-protective activities. In addition to being central in reverse cholesterol transport in the liver, SR-BI is involved in several other processes in different cell types [45]. In endothelial cells, SR-BI is localized in caveolae; by binding to SR-BI, HDL stimulates eNOS via activation of Akt kinase which in turn induces eNOS phosphorylation and NO generation [19, 21]; SR-BI is also involved in HDL-induced endothelial cell migration and repair [46]. In HUVEC, native HDL3 upregulated SR-BI mRNA and protein expression, whereas 15LO-HDL3 significantly reduced the expression of this receptor, thus contributing to the reduction of SR-BI-mediated processes. This in agreement with the finding that, while HDL from healthy subjects significantly increased SR-BI expression in
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endothelial cells, HDL from diabetics downregulate cell surface SR-BI expression, an effect similar to that obtained with in vitro modified HDL (glycated or copper-oxidized HDL) [47]; the downregulation of SR-BI was supposed to be a primary mechanism for the reduced efficiency of diabetic HDL in inducing endothelial cell migration and proliferation [47]. Here, we suggest that SR-BI downregulation may be one of the mechanisms accounting for the reduced ability of 15LO-HDL3 to induce eNOS activation. In summary, the results obtained in this study confirm that 15LO-mediated modification induces functional changes of HDL3 properties. Thus, besides reduced ability to promote cholesterol efflux and to protect endothelial cells from pro-inflammatory stimuli, here we have shown that HDL3 modified with 15LO fail to induce eNOS activation and, as consequence, NO generation. The reduced efficiency of 15LO-HDL3 to stimulate nitric oxide production is not due to changes in eNOS or caveolin-1 amount in the caveolae system, but seems to result from the modulation of a number of mechanisms by 15LO-modified HDL3. As first, HDL3 modification impairs its immediate ability to activate Akt and eNOS mainly due to a reduced interaction with SR-BI. Modified HDL3 also reduced the long-term expression of SR-BI, thus resulting in a further reduction of the interaction lipoprotein/receptor. The role of other mechanisms that may be involved in the observed effects (i.e. the effect of sphingosine-1-phosphate or other bioactive lipids) remains to be addressed. Acknowledgments This work was supported by SISA (Societa` Italiana per lo Studio dell’Aterosclerosi, Lombardia section). Conflict of interest interest.
The authors have no financial conflict of
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26. Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756 27. Havel RJ, Eder HA, Bragdon JH (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345–1353 28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275 29. Norata GD, Tibolla G, Seccomandi PM, Poletti A, Catapano AL (2006) Dihydrotestosterone decreases tumor necrosis factor-alpha and lipopolysaccharide-induced inflammatory response in human endothelial cells. J Clin Endocrinol Metab 91:546–554 30. Drew BG, Fidge NH, Gallon-Beaumier G, Kemp BE, Kingwell BA (2004) High-density lipoprotein and apolipoprotein AI increase endothelial NO synthase activity by protein association and multisite phosphorylation. Proc Natl Acad Sci USA 101:6999–7004 31. Rosenson RS, Brewer HB Jr, Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang XC, Phillips MC, Rader DJ, Remaley AT, Rothblat GH, Tall AR, Yvan-Charvet L (2012) Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport. Circulation 125:1905–1919 32. Tall AR (2008) Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins. J Intern Med 263:256–273 33. Besler C, Heinrich K, Rohrer L, Doerries C, Riwanto M, Shih DM, Chroni A, Yonekawa K, Stein S, Schaefer N, Mueller M, Akhmedov A, Daniil G, Manes C, Templin C, Wyss C, Maier W, Tanner FC, Matter CM, Corti R, Furlong C, Lusis AJ, von Eckardstein A, Fogelman AM, Luscher TF, Landmesser U (2011) Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease. J Clin Invest 121:2693–2708 34. Terasaka N, Westerterp M, Koetsveld J, Fernandez-Hernando C, Yvan-Charvet L, Wang N, Sessa WC, Tall AR (2010) ATPbinding cassette transporter G1 and high-density lipoprotein promote endothelial NO synthesis through a decrease in the interaction of caveolin-1 and endothelial NO synthase. Arterioscler Thromb Vasc Biol 30:2219–2225 35. Norata GD, Pirillo A, Catapano AL (2006) Modified HDL: biological and physiopathological consequences. Nutr Metab Cardiovasc Dis 16:371–386 36. Feng H, Li XA (2009) Dysfunctional high-density lipoprotein. Curr Opin Endocrinol Diabetes Obes 16:156–162 37. Yla-Herttuala S (1992) Gene expression in atherosclerotic lesions. Herz 17:270–276 38. Frank PG (2010) Endothelial caveolae and caveolin-1 as key regulators of atherosclerosis. Am J Pathol 177:544–546 39. Frank PG, Lee H, Park DS, Tandon NN, Scherer PE, Lisanti MP (2004) Genetic ablation of caveolin-1 confers protection against atherosclerosis. Arterioscler Thromb Vasc Biol 24:98–105 40. Fernandez-Hernando C, Yu J, Davalos A, Prendergast J, Sessa WC (2010) Endothelial-specific overexpression of caveolin-1 accelerates atherosclerosis in apolipoprotein E-deficient mice. Am J Pathol 177:998–1003 41. Ramet ME, Ramet M, Lu Q, Nickerson M, Savolainen MJ, Malzone A, Karas RH (2003) High-density lipoprotein increases the abundance of eNOS protein in human vascular endothelial cells by increasing its half-life. J Am Coll Cardiol 41:2288–2297 42. Gomaraschi M, Ossoli A, Favari E, Adorni MP, Sinagra G, Cattin L, Veglia F, Bernini F, Franceschini G, Calabresi L (2013) Inflammation impairs eNOS activation by HDL in patients with acute coronary syndrome. Cardiovasc Res 100:36–43
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ORIGINAL ARTICLE
15-Lipoxygenase-Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells Lucia Cutuli • Angela Pirillo • Patrizia Uboldi Hartmut Kuehn • Alberico L. Catapano
•
Received: 13 September 2013 / Accepted: 12 February 2014 Ó AOCS 2014
Abstract Caveolae are cholesterol and glycosphingolipidsenriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SRBI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenasemediated modification of HDL3 on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We
L. Cutuli P. Uboldi A. L. Catapano Department of Pharmacological and Biomolecular Sciences, Universita` degli Studi di Milano, Milan, Italy A. Pirillo (&) Center for the Study of Atherosclerosis, Bassini Hospital, Via M. Gorki 50, Cinisello Balsamo, Milan, Italy e-mail: [email protected] A. Pirillo A. L. Catapano IRCCS Multimedica, Milan, Italy H. Kuehn Institute of Biochemistry, University of Medicine Berlin-Charite´, Berlin, Germany
found that after modification, HDL3 failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL. Keywords High density lipoprotein Endothelial cells 15-Lipoxygenase Caveolae Nitric oxide Abbreviations 15LO 15-Lipoxygenase ABCG1 ATP binding cassette transporter G1 CAV-1 Caveolin-1 eNOS Endothelial nitric oxide synthase HDL High density lipoprotein HUVEC Human umbilical vein endothelial cell(s) LOX-1 Lectin-like oxidized low-density lipoprotein receptor-1 MAPK Mitogen-activated protein kinase NO Nitric oxide S1P3 Sphingosine-1-phosphate receptor 3 SR-BI Scavenger receptor type B class I
Introduction Endothelium, due to its localization between blood and the vessel wall, plays a central role in controlling vascular tone and homeostasis. Endothelial cells, in fact, produce several vasoactive factors in response to different stimuli, including vasoconstrictor and vasodilator substances [1]; among the latter, nitric oxide (NO) is the most crucial. The
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production of NO is regulated by the endothelial nitric oxide synthase (eNOS) activity, which in turn is regulated by a number of upstream factors [2]. Caveolae are specialized membrane microdomains enriched in cholesterol, sphingolipids and glycosphingolipids [3]; they are characterized by the presence of caveolin-1 as the main protein constituent [4], which in turn regulates the cholesterol content [5, 6]. Moreover, caveolae contain a number of other proteins, including receptors and molecules involved in signal transduction pathways [7, 8]. eNOS is localized in caveolae and interacts with caveolin-1 [9], being caveolin-1 the main negative regulator of eNOS activity, as the association of eNOS with caveolin-1 inhibits the enzyme activity [10]. Following extracellular stimuli, eNOS dissociates from caveolin-1, forms a complex with calcium/calmodulin or heat shock protein 90 and becomes activated, thus increasing the NO production [11]. eNOS is constitutively expressed in endothelial cells; however expression and activity of eNOS are finely modulated at transcriptional and post-translational levels by protein–protein interactions, Ca2? concentration, and subcellular distribution [12, 13]. eNOS activity is also regulated by phosphorylation [11], and the Akt-mediated phosphorylation of Ser1177 [14] positively regulates NO production [15]. Furthermore, caveolin negatively impacts eNOS activity through direct interaction with the enzyme [2]; finally, the cholesterol content in caveolae plays a critical role in eNOS function [16]. Plasma levels of high-density lipoprotein (HDL) cholesterol inversely correlate with the incidence of cardiovascular disease. The atheroprotective effects of HDL are mainly related to their role in reverse cholesterol transport (RCT), but HDL possesses, among others, also anti-oxidant, anti-inflammatory and anti-thrombotic activities [17]. HDL is a potent agonist of eNOS and promotes NO production by several mechanisms [18, 19]: it regulates eNOS abundance and its subcellular distribution; HDL and HDL-associated lysophospholipids, through the interaction with SR-BI (scavenger receptor class B type I) and S1P3 (sphingosine-1-phosphate receptor 3) respectively [19, 20], activate downstream kinases, resulting in eNOS activation. The activation of eNOS by HDL is, at least in part, Akt and MAPK-dependent [21]; however, Akt-independent mechanisms of eNOS activation by HDL exist [20]. Furthermore, SR-BI-mediated cholesterol flux in response to HDL is required for signal initiation by HDL [2]. The HDL modification induced by 15-lipoxygenase (15LO), an enzyme overexpressed in atherosclerotic lesions [22], impairs their athero-protective functions [23– 25]. In fact, 15LO-HDL3 are less effective in promoting cholesterol efflux from macrophages [23] and are less efficient in reducing the expression of TNF-a-induced
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adhesion molecules and LOX-1 in endothelial cells [24, 25]; furthermore, 15LO-HDL3 acquire pro-inflammatory properties, as they induce the expression of LOX-1 and adhesion molecules and chemoattractant factors in endothelial cells and increased the adhesion of monocytes to the endothelium [24, 25]. In the present work we aimed at studying the effect of 15LO-mediated HDL3 modification on the caveolae system. Specifically, we studied the effect of 15LO-HDL3 on the expression and activity of eNOS as well as on the expression of SR-BI, caveolin-1, and eNOS.
Materials and Methods Reagents Medium M199 was from Invitrogen. Fetal bovine serum (FBS), penicillin–streptomycin, and glutamine were from Sigma-Aldrich; PD10 columns and enhanced chemiluminescence (ECL) western blotting detection reagent were from Amersham Biosciences. Endothelial cell growth factor (ECGF) was from Boehringer Mannheim. Antibodies were purchased as follows: anti-SR-BI and anti-Cav-1 from Abcam; anti-eNOS, anti-phospho-eNOS, anti-Akt and anti-phospho-Akt from Cell Signaling; anti-b-actin and anti-mouse IgG peroxidase-conjugate from Sigma-Aldrich; anti-rabbit IgG peroxidase-conjugate from BioRad. Cell Culture HUVEC were isolated according to established procedures [26] and cultured in medium M199 supplemented with 20 % FBS, ECGF (20 lg/ml), heparin (15 U/ml), penicillin–streptomycin (1 %) and glutamine (1 %). Cells were used between the 3rd and 5th in vitro passage. Isolation and Modification of HDL3 HDL3 (d = 1.125–1.21 g/ml) were isolated by sequential ultracentrifugation at 4 °C from the plasma of healthy volunteers [27]. HDL3 were dialyzed using a Sephadex G25 column (PD10) with a PBS-EDTA 0.01 %, pH 7,4, then sterilized using a 0.22-lm filter (corning) and stored at 4 °C. The protein content was evaluated by the Lowry method [28], using bovine serum albumin as a standard. HDL3 were modified with 15-LO as described [23]. Caveolae Purification and Analysis HUVEC were incubated with 100 lg/ml HDL3 or 15LOHDL3 for 24 h, then washed twice with cold PBS and harvested by scraping. The samples were centrifuged at
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1,500 rpm for 5 min. The pellets were resuspended in 2 ml of Na2CO3 buffer (500 mM pH 11) and sonicated (3 9 30 s); 2 ml of the lysate were adjusted to 45 % sucrose by mixing with 2 ml of 90 % sucrose prepared in 25 mmol/l MES, pH 6.5, containing 150 mmol/l NaCl, and placed at the bottom of an ultracentrifuge tube. A 5–35 % discontinuous sucrose gradient was formed (in MES containing 100 mmol/l Na2CO3) and ultracentrifuged at 39,000 rpm in a SW41 rotor (Beckman) for 16 h. From the top, 1-ml fractions were removed sequentially and designated as fractions 1–12. Each fraction was subjected to western blotting analysis for Cav-1, SR-BI and eNOS expression. Western Blotting For protein analysis of fractions derived from sucrose gradient, equal volumes of each sample were mixed with loading buffer containing 2 % b-mercaptoethanol and 2 % SDS and proteins were separated on SDS-PAGE. For the analysis of cellular proteins, whole cell lysates were prepared in lysis buffer (2 % SDS, 62.5 mM TRIS, 50 mM DTT, 1 mM PMSF, 5 lg/ml aprotinin) and sonicated. Cellular proteins were separated on a 10 % SDS-PAGE, and then transferred onto a nitrocellulose membrane. Membranes were saturated with 5 % non-fat milk in PBS-T (PBS-0.1 % Tween) for 1 h at room temperature, followed by overnight incubation with the selected primary antibody at 4 °C and 1 h incubation with a 1:1,000 dilution of a goat anti-rabbit or anti-mouse IgG-HRP conjugate. Immunocomplexes were detected by ECL followed by autoradiography. The bands were quantified by a computer-assisted system for image analysis (ISF Image 1.52) and the expression of each antigen was corrected for b-actin content. Quantitative Real Time PCR (RT-PCR) Total RNA was extracted and reverse transcribed. Three ll of cDNA were amplified by real-time quantitative polymerase chain reaction (PCR) with 19 SYBR Green universal PCR mastermix (BioRad) [29]. The sequence of the primers used for RLP13A (housekeeping gene), SR-BI, eNOS, Cav-1 amplification are reported in Table 1. Each sample was analyzed in duplicate using the IQTM-Cycler (BioRad). For quantification, the target genes were normalized to the RLP13A content. NO Production Analysis by Flow Cytometry NO generation was determined by flow cytometry. HUVEC were preincubated for 1 h at 37 °C with the fluorescent
Table 1 Sequence of primers used in RT-PCR experiments Primer
Sequence
RLP Fw
50 -ATGCTGCCCCACAAAACC-30
RLP Rev
50 -TGCCGTCAACACCCTTGAGA-30
Cav-1 Fw
50 -TCCTTCCTCAGTTCCCTTAAAGC-30
Cav-1 Rev
50 -GCCCGTGGCTGGATGA-30
eNOS Fw
50 -CTCGTCCCTGTGGAAAGACAA-30
eNOS Rev
50 -TGACTTTGGCTAGCTGGTAACTGT-30
SR-BI Fw
50 -GACAAACTGGGAAGATTGAGCC-30
SR-BI Rev
50 -CCATGGCCCCGCTCTC-30
dye DAF-FM diacetate (4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate); cells were then washed with PBS and incubated with HDL3 or 15LO-HDL3 for 15 min at 37 °C. At the end of the incubation, cells were harvested by trypsinization and immediately subjected to flow cytometry analysis (FACScan, Becton–Dickinson). Lipoprotein–Cell Association Lipoproteins were labeled with the fluorescent dye DiO as previously described [25]. Endothelial cells were incubated at 37 °C with DiO-labeled lipoproteins (12.5 and 25 lg/ ml) for 1 h, then washed three times with cold PBS, detached by trypsinization, fixed in 1 % paraformaldehyde and immediately subjected to fluorescence flow cytometry using a FACScan (Becton–Dickinson). For each sample 10,000 events were analyzed; data were processed using the CellQuest program (Becton–Dickinson). Statistical Analysis Values are expressed as means ± SD. The statistical significance of the differences between groups was determined by the Student’s t test and values of P \ 0.05 were considered to be significant.
Results Effect of 15LO-Modified HDL3 on eNOS Phosphorylation and Activity The activation of eNOS by HDL3 is intimately related to the ability of this lipoprotein to induce the phosphorylation of eNOS at Ser1177 [20, 30]. Thus, we investigated the effect of HDL3 or 15LO-HDL3 on the phosphorylation of eNOS at this specific site. As expected, HDL3 significantly increased the phosphorylation of eNOS at Ser1177, while 15LO-HDL3 failed to induce such effect in HUVEC (Fig. 1a). eNOS
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*
a
*
p-eNOS/eNOS (fold induction)
2,0
Cont. HDL3 15LO-HDL3
1,5
p-eNOS eNOS
1,0
0,5
0,0 Control
b
15LO-HDL3
HDL3
**
*
3,0
NO production (fold induction)
Fig. 1 Effect of native and 15LO-modified HDL3 on eNOS phosphorylation (a) and NO production (b) and Akt phosphorylation (c) in endothelial cells. a, c HUVEC were incubated with HDL3 or 15LO-modified HDL3 (100 lg/ ml) for 100 ; eNOS and Akt phosphorylation were evaluated by western blotting. Results are given as means ± SD from 3 independent experiments. a *P \ 0.01; c *P \ 0.005, **P \ 0.0005. b HUVEC were pre-incubated with the fluorescent dye DAF-FM diacetate, then incubated with HDL3 or 15LO-HDL3 for 15 min. NO production was evaluated by flow cytometry. Results are given as means ± SD from 5 independent experiments. *P \ 0.01, **P \ 0.00005
2,5 2,0 1,5 1,0 0,5 0,0 Control
c
15LO-HDL3
HDL3
**
*
p-Akt/Akt (fold induction)
2,0
Cont. HDL3 15LO-HDL3 1,5
p-Akt Akt
1,0
0,5
0,0 Control
activation through phosphorylation at Ser1177 results in enzyme activation and nitric oxide generation [2]; accordingly to the ability to induce eNOS phosphorylation, HDL3 significantly increased the production of nitric oxide compared to control cells, while 15LO-HDL3 resulted appreciably less effective in generating this vasoactive compound compared to HDL3 (Fig. 1b). HDL-mediated eNOS stimulation involves Akt activation [21]; for this reason we evaluated the ability of native and 15LO-modified HDL3 to stimulate this kinase. We found that HDL3 significantly induced Akt phosphorylation, while 15LO-HDL3 were less effective (Fig. 1c).
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15LO-HDL3
Effect of 15LO-Modified HDL3 on Gene Expression of SR-BI, Caveolin-1 and eNOS To analyze the effect of 15LO-modified HDL3 on the expression at transcriptional level of proteins present in the caveolae membranes, HUVEC were incubated overnight with 100 lg/ml HDL3 or 15LO-HDL3. HDL3 significantly increased SR-BI mRNA levels, while in the presence of 15LO-HDL3 the expression of SR-BI was significantly lower compared to native HDL3, although higher compared to control cells (Fig. 2a). Caveolin-1 mRNA was significantly and similarly reduced in cell incubated with HDL3
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a
**
SR-BI/RLP (fold induction)
2,5 2,0
#
1,5 1,0 0,5 0,0
Control
Cav-1/RLP (fold induction)
b
HDL3
1,0
15LO-HDL3
* **
0,8 0,6 0,4 0,2 0,0
Control
eNOS/RLP (fold induction)
c
1,0
HDL3
*
15LO-HDL3
*
0,8 0,6 0,4 0,2
Incubation of HUVEC with HDL3 significantly increased SR-BI protein expression; on the contrary, the incubation of endothelial cells with 15LO-HDL3 resulted in a decreased expression of SR-BI protein compared to either control or HDL3-treated cells (-43 and -60 %, respectively) (Fig. 3a). HDL3 did not alter the levels of caveolin1 compared to controls, while 15LO-HDL3 increased it by about 30 % compared to either control or HDL3-treated cells (Fig. 3b). No relevant changes were observed in the expression of eNOS protein in cells treated with HDL3 or 15LO-HDL3 compared to controls (Fig. 3c). Effect of HDL3 and 15LO-HDL3 on Caveolin-1, SR-BI and eNOS Distribution Next, we examined the effect of HDL3 or 15LO-HDL3 on caveolin-1, SR-BI and eNOS distribution in caveolae. We found that most caveolin-1 was localized in caveolae fractions (4, 5) in cells treated with HDL3 or 15LO-HDL3, without appreciable differences in the caveolin-1 content (Fig. 4a); however, a significant increase of caveolin-1 content was found in fractions other than caveolae in 15LO-HDL3 treated cells (fold induction 1.68; P \ 0.0005). As expected, both SR-BI and eNOS proteins were localized in the caveolae fraction 4; however, in 15LO-HDL3-treated cells we found a lower amount of SRBI protein associated with caveolae fractions compared to HDL3-treated cells (-23.8 %), even when adjusted for the caveolin-1 content (-31.2 %) (Fig. 4b), without any change in the amount of eNOS protein associated with caveolae (Fig. 4c). Lipoprotein–Endothelial Cell Association
0,0
Control
HDL3
15LO-HDL3
Fig. 2 Effect of native and 15LO-modified HDL3 on SR-BI, Cav-1 and eNOS mRNA expression in endothelial cells. HUVEC were incubated with HDL3 or 15LO-modified HDL3 (100 lg/ml) for 18 h; total mRNA was isolated and the expression of the indicated genes was evaluated by real time PCR. RLP13A was used as an internal control. Results are given as means ± SD from 5 (a) or 4 (b, c) independent experiments. *P \ 0.05 and **P \ 0.005 vs control; # P \ 0.05 vs HDL3
or 15LO-HDL3 compared to controls (Fig. 2b); similar results were obtained for eNOS mRNA, with no difference between treatment with HDL3 or 15LO-HDL3 (Fig. 2c). Effect of 15LO-Modified HDL3 on Protein Expression of SR-BI, Caveolin-1 and eNOS Then we evaluated the effect of HDL3 or 15LO-HDL3 on the expression of SR-BI, eNOS and Cav-1 at protein level.
We previously showed that 15LO-mediated modification significantly reduces the ability of HDL3 to associate to macrophages and that this was due to a reduced ability to interact with SR-BI [23]. We found that 15LO-HDL3 associated less efficiently also to endothelial cells compared to native HDL3 (Fig. 5).
Discussion In this study we have shown that 15-lipoxygenase-mediated modification significantly reduces the endothelial protective effect of HDL3. In fact, after modification, HDL3 induces changes in the expression/activity of proteins involved in eNOS activity, resulting in a reduced production of nitric oxide. The main mechanism by which HDL exert their antiatherogenic role is through the promotion of cholesterol efflux from macrophages and smooth muscle cells in the
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a
**
* Cont.
SR-BI/ß-actin (fold induction)
1,5
HDL3
15LO-HDL 3
1,2
SR-BI
0,9
ß-actin 0,6 0,3 0,0
Control
b
Cav-1/ß-actin (fold induction)
15LO-HDL3
HDL3
* 1,5
Cont. HDL3 15LO-HDL 3
1,2
Cav-1
0,9
ß-actin
0,6 0,3 0,0
c
1,2
eNOS/ß-actin (fold induction)
Control
0,9
HDL3
15LO-HDL3
Cont.
HDL3
15LO-HDL 3
eNOS
0,6
ß-actin
0,3
0,0
Control
HDL3
15LO-HDL3
Fig. 3 Effect of native and 15LO-modified HDL3 on SR-BI, Cav-1 and eNOS protein expression in endothelial cells. HUVEC were incubated with HDL3 or 15LO-modified HDL3 (100 lg/ml) for 24 h.
SR-BI, Cav-1 and eNOS protein expression were evaluated by western blotting. Results are given as means ± SD from 6 independent experiments. *P \ 0.005, **P \ 0.0005
arterial wall [31]; however, HDL possess several other athero-protective properties that include anti-inflammatory, anti-oxidant and endothelial-protective functions [32]. Caveolae are specialized microdomains that compartmentalize signal transduction molecules involved in the regulation of endothelial functions; among them, a key role is played by the process of nitric oxide generation, that involves several players, including eNOS, the enzyme responsible for the production of NO, caveolin-1, the major coat protein of caveolae, SR-BI, the main HDL receptors, all of which are localized within the caveolae domain, and
HDL [2]. HDL induce NO production by several different mechanisms. ApoA-I and HDL induce eNOS activation and NO production following the interaction with SR-BI [19, 30]. The interaction of HDL with SR-BI activates multiple kinases, including Akt, resulting in the phosphorylation of eNOS at Ser1177 and subsequent activation of the enzyme [21]. In addition, other HDL-associated components can stimulate eNOS-mediated NO generation [20, 33]; also, ABCG1-mediated cholesterol efflux to HDL from endothelial cells reduces the interaction between caveolin-1 and eNOS, resulting in eNOS activation [34].
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Lipids Fig. 4 Effect of native and 15LO-modified HDL3 on caveolin-1, SR-BI and eNOS distribution in caveolae. HUVEC were incubated with 100 lg/ml HDL3 or 15LOHDL3 for 24 h, then cells were subjected to sucrose gradient fractionation for caveolae isolation. Cav-1, SR-BI and eNOS distribution and expression were evaluated by western blotting. Quantification of the amount of protein in each fraction was obtained by densitometry analysis. Experiments were performed in triplicate; representative images are shown
Sucrose gradient (5-45%)
Fraction n. 1
2 3
4
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6
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Lipoprotein-cell association
HDL3 HDL3
15LO-HDL3 15LO-HDL3
*
100 90 80 70 60 50 40 30 20 10 0
*
0
12,5
25
µg/ml Fig. 5 Native and 15LO-modified HDL3 association to endothelial cells. HUVEC were incubated with DiO-labeled lipoproteins (12.5 and 25 lg/ml) for 1 h at 37 °C. Cell-associated fluorescence was evaluated by flow cytometry. Data are means ± SD of 3 independent experiments performed in duplicate. *P \ 0.0005
Oxidative modification, however, impairs the atheroprotective functions of HDL [35, 36]. We have previously shown that oxidative modification mediated by 15-lipoxygenase, an enzyme overexpressed in atherosclerotic lesions [22, 37], severely altered the properties of HDL, as it resulted in a reduced efficiency to promote cholesterol efflux [23] and in a decreased anti-inflammatory activity [24, 25], and also conferred pro-inflammatory characteristics, as 15LO-modified HDL3 induced the expression of
adhesion molecules [24] and LOX-1 [25] in endothelial cells. In the present work we found that 15LO-modified HDL3 affected the expression and/or the activity of proteins involved in the nitric oxide generation pathway in endothelial cells, including SR-BI, caveolin-1 and eNOS. Caveolin-1 and caveolae are present in all the cells of the arterial wall, but the role of caveolin-1 in atherosclerosis is strictly dependent on the cell type. In fact, while macrophage and smooth muscle cell caveolin-1 seems to play an inhibitory role in the development of atherosclerosis, endothelial caveolin-1 may act as a pro-atherogenic protein [38]. In agreement with this, genetic ablation of caveolin-1 markedly reduced the extent of atherosclerosis in apoE knockout mice [39], whereas endothelial-specific overexpression of caveolin-1 enhanced the progression of mouse atherosclerosis by increasing the infiltration of LDL particles within the arterial wall, by inducing the expression of leukocyte adhesion molecules and by decreasing the production of nitric oxide [40]. In our experimental conditions, we found that, although similar effects at mRNA level, native HDL3 did not alter caveolin-1 protein expression, while cells incubated in the presence of 15LO-modified HDL3 increased total caveolin-1 protein content. Nevertheless, this change did not translate in an enrichment of caveolin-1 in caveolae, suggesting that 15LO-HDL3 effects on eNOS activation are not related to the observed increase of caveolin-1 protein. It has been reported that HDL increased eNOS protein abundance in endothelial cells by increasing the protein
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half-life, without any significant effect on eNOS mRNA levels [41]; however, other studies did not find any effect of HDL on eNOS and caveolin-1 abundance [34]. In our experiments we did not detect any significant change in eNOS protein in HUVEC incubated with native or 15LOmodified HDL3, despite a significant decrease of eNOS mRNA with both treatments. In agreement with this finding, no differences were observed in eNOS content in caveolae of cells incubated with HDL3 or 15LO-HDL3. However, a significant different ability of lipoproteins to induce eNOS activity was observed. In fact native HDL3 increase the phosphorylation of eNOS at Ser1177, which translates in an increased production of nitric oxide. After 15LO-mediated modification, HDL3 exhibited a reduced ability to activate eNOS and to induce NO generation. In addition, HDL3 significantly induced the phosphorylation of Akt, the kinase that directly activates eNOS by phosphorylation at Ser1177 [14]; 15LO-mediated modification of HDL3 significantly reduced Akt phosphorylation; this result may partly explain the reduced eNOS phosphorylation observed in cells treated with 15LO-HDL3. Several observations have been reported suggesting a reduced ability of HDL isolated from subjects with atherosclerosisrelated disease to induce nitric oxide production. In fact, while HDL from healthy subjects stimulated endothelial NO production, HDL from patients with stable coronary artery disease or acute coronary syndrome (ACS) exhibited an impaired ability to induce NO generation [33, 42]. This effect was mainly due to a reduced capacity to stimulate Akt phosphorylation and subsequent eNOS phosphorylation at Ser1177 [33]. Similarly, HDL isolated from diabetic women failed to stimulate NO production without affecting the levels of eNOS, compared to HDL from healthy subjects [43], and HDL from obese adolescents exhibited a reduced ability to induce eNOS-Ser1177 phosphorylation compared to HDL from adolescents with normal body mass index [44]. Altogether these findings suggest that under pathological conditions HDL may undergo structural and functional modifications that significantly impair their endothelial-protective activities. In addition to being central in reverse cholesterol transport in the liver, SR-BI is involved in several other processes in different cell types [45]. In endothelial cells, SR-BI is localized in caveolae; by binding to SR-BI, HDL stimulates eNOS via activation of Akt kinase which in turn induces eNOS phosphorylation and NO generation [19, 21]; SR-BI is also involved in HDL-induced endothelial cell migration and repair [46]. In HUVEC, native HDL3 upregulated SR-BI mRNA and protein expression, whereas 15LO-HDL3 significantly reduced the expression of this receptor, thus contributing to the reduction of SR-BI-mediated processes. This in agreement with the finding that, while HDL from healthy subjects significantly increased SR-BI expression in
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endothelial cells, HDL from diabetics downregulate cell surface SR-BI expression, an effect similar to that obtained with in vitro modified HDL (glycated or copper-oxidized HDL) [47]; the downregulation of SR-BI was supposed to be a primary mechanism for the reduced efficiency of diabetic HDL in inducing endothelial cell migration and proliferation [47]. Here, we suggest that SR-BI downregulation may be one of the mechanisms accounting for the reduced ability of 15LO-HDL3 to induce eNOS activation. In summary, the results obtained in this study confirm that 15LO-mediated modification induces functional changes of HDL3 properties. Thus, besides reduced ability to promote cholesterol efflux and to protect endothelial cells from pro-inflammatory stimuli, here we have shown that HDL3 modified with 15LO fail to induce eNOS activation and, as consequence, NO generation. The reduced efficiency of 15LO-HDL3 to stimulate nitric oxide production is not due to changes in eNOS or caveolin-1 amount in the caveolae system, but seems to result from the modulation of a number of mechanisms by 15LO-modified HDL3. As first, HDL3 modification impairs its immediate ability to activate Akt and eNOS mainly due to a reduced interaction with SR-BI. Modified HDL3 also reduced the long-term expression of SR-BI, thus resulting in a further reduction of the interaction lipoprotein/receptor. The role of other mechanisms that may be involved in the observed effects (i.e. the effect of sphingosine-1-phosphate or other bioactive lipids) remains to be addressed. Acknowledgments This work was supported by SISA (Societa` Italiana per lo Studio dell’Aterosclerosi, Lombardia section). Conflict of interest interest.
The authors have no financial conflict of
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