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J Physiol (2003), 547.1, pp. 21-33
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.031534
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ABSTRACT |
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Early hypercholesterolaemia-induced vascular disease is characterized by an attenuated capacity for endothelial production of the antiatherogenic molecule nitric oxide (NO), which is generated by endothelial NO synthase (eNOS). In recent studies we have determined the impact of lipoproteins on eNOS subcellular localization and action, thereby providing a causal link between cholesterol status and initial abnormalities in endothelial function. We have demonstrated that eNOS is normally targeted to cholesterol-enriched caveolae where it resides in a signalling module. Oxidized low density lipoprotein (LDL; oxLDL) causes displacement of eNOS from caveolae by binding to endothelial cell CD36 receptors and by depleting caveolae cholesterol content, resulting in the disruption of eNOS activation. The adverse effects of oxLDL are fully prevented by high density lipoprotein (HDL) via binding to scavenger receptor BI (SR-BI), which is colocalized with eNOS in endothelial caveolae. This occurs through the maintenance of caveolae cholesterol content by cholesterol ester uptake from HDL. As importantly, HDL binding to SR-BI causes robust stimulation of eNOS activity in endothelial cells, and this process is further demonstrable in isolated endothelial cell caveolae. HDL also enhances endothelium- and NO-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, lipoproteins have potent effects on eNOS function in caveolae via actions on both membrane cholesterol homeostasis and the level of activation of the enzyme. These processes may be critically involved in the earliest phases of atherogenesis, which recent studies suggest may occur during fetal life.
(Received 23 August 2002; accepted after revision 18 November 2002; first published online 10 January 2003)
Corresponding author P. Shaul: Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9063, USA. Email: pshaul{at}mednet.swmed.edu
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INTRODUCTION |
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A key process in the early pathogenesis of hypercholesterolaemia-induced vascular disease and atherosclerosis is diminished bioavailability of the endothelium-derived signalling molecule nitric oxide (NO), which is generated by the endothelial isoform of NO synthase (eNOS). This review focuses on recent gains in our understanding of the impact of lipoproteins on eNOS subcellular localization and action, thereby providing a causal link between cholesterol status and initial abnormalities in endothelial function. First, we summarize investigations determining the role of NO in atherogenesis. The mechanisms underlying the normal localization of eNOS to plasma membrane caveolae, which are unique, cholesterol-enriched microdomains involved in the compartmentalization of signal transduction molecules, are then reviewed. The impact of lipoproteins on eNOS localization in caveolae is examined, and the existence of an eNOS signalling module in caveolae is considered. The capacity of lipoproteins to serve as agonists for eNOS is also presented. Finally, we summarize evidence from other investigators that cholesterol status during fetal life modifies later vascular structure and function in the offspring, thereby identifying the initial developmental phase during which lipoprotein modulation of eNOS action may be pathogenetically relevant. It is anticipated that issues of eNOS modulation by lipoproteins will continue to warrant strong consideration both in our efforts to understand the role of cholesterol in the initiation of atherogenesis, and our attempts to take preventative and therapeutic advantage of these mechanisms to modify the development of hypercholesterolaemia-induced vascular disease.
Nitric oxide and atherosclerosis
During the early stages of hypercholesterolaemia-induced vascular disease there is a dramatic decrease in bioavailable endothelium-derived NO, which is a potent vasodilator with multiple additional cardiovascular functions (Vane et al. 1990). The NO is generated by eNOS upon the conversion of L-arginine to L-citrulline, and eNOS activity is modulated by agonists of diverse G-protein-coupled cell surface receptors and by physical stimuli such as haemodynamic shear stress (Shaul, 2002). In the initial phase of hypercholesterolaemia there is exclusive impaired responsiveness to receptor-dependent stimuli such as acetylcholine (Ach), whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. As the disease progresses, there is nonspecific inhibition of NO bioavailability that is at least partly due to enhanced inactivation of NO by superoxide anions (Flavahan, 1992; Harrison, 1994; Cohen, 1995). These processes result in increased neutrophil adherence to the endothelium, thereby comprising key components of the pathogenesis of atherosclerosis (Lefer & Ma, 1993). It is also known that NO deficiency enhances smooth muscle cell proliferation and platelet aggregation and adhesion (Shaul, 2002). In vivo evidence of these mechanisms includes studies in rabbit models of hypercholesterolaemia in which the chronic inhibition of NO synthesis causes marked acceleration of the development of vascular dysfunction and intimal lesions (Cayatte et al. 1994; Naruse et al. 1994). In addition, mice lacking apolipoprotein E (apoE), which develop spontaneous atherosclerosis and display attenuated NO-mediated vasodilation, have more rapid progression of atherosclerosis when subjected to either long-term NOS antagonism or genetic eNOS deficiency (Yang et al. 1999; Kauser et al. 2000; Kuhlencordt et al. 2001). Thus, multiple lines of investigation indicate that NO is atheroprotective, and that NO deficiency is critically involved in the pathogenesis of hypercholesterolaemia-induced vascular disease.
eNOS localization in caveolae
Shortly after initial characterization in endothelial cells, eNOS was found to be primarily associated with the plasma membrane (Hecker et al. 1994). Since eNOS activity is acutely regulated by multiple extracellular stimuli and the NO produced is a labile, cytotoxic messenger molecule with primarily paracrine function (Moncada & Higgs, 1993; Nathan & Xie, 1994), the intracellular site of NO synthesis has a major influence on the biological activity of the molecule.
Caveolae are specialized, lipid-ordered plasma membrane microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules such as folate (Anderson et al. 1992). They are enriched in cholesterol, glycosphingolipids, sphingomyelin and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 °C. Once the identification of the caveola coat protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. The list of resident signalling molecules includes G-protein-coupled receptors such as the muscarinic acetylcholine receptor, G-proteins and molecules involved in the regulation of intracellular calcium homeostasis such as a plasma membrane calcium pump and protein kinase C (Shaul & Anderson, 1998). Knowledge that eNOS activity is acutely regulated by extracellular factors and that the protein is primarily associated with the plasma membrane prompted the initial examinations of eNOS in caveolae.
Using cultured endothelial cells, eNOS localization was first assessed in subcellular fractions including the caveolae and noncaveolae fractions of the plasma membrane (Shaul et al. 1996; Fig. 1A). Within the plasma membrane, caveolin was detected specifically in the caveolae fraction, and eNOS protein was also highly enriched in caveolae. The enrichment of eNOS protein in caveolae correlated with the distribution of NOS enzymatic activity (Fig. 1B), which was 7-fold greater in the plasma membrane than the cytosol. Within the plasma membrane, NOS activity was not detected in noncaveolae membranes, whereas it was 9- to 10-fold more prevalent in the caveolae fraction compared to whole plasma membrane. Over a range of experiments, 51 to 86 % of the total enzymatic activity in the postnuclear supernatant was recovered in plasma membrane, and 57 to 100 % of the activity in plasma membrane was recovered in caveolae, revealing that the majority of functional enzyme in quiescent endothelial cells is localized to caveolae. eNOS was also localized to caveolae by immunoelectron microscopy (Fig. 1C), indicating that the enrichment of eNOS protein and enzymatic activity in isolated endothelial caveolae membranes is not related to nonspecific association of the protein with the caveolae during purification; alternatively, it accurately reflects enzyme localization in the microdomain.
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Figure 1. eNOS is localized to endothelial cell caveolae A, immunoblot analysis for eNOS, caveolin-1 and calmodulin in subcellular fractions from endothelial cells. Samples of postnuclear supernatant (PNS), cytosol, plasma membrane (PM), noncaveolae membrane (NCM) and caveolae membrane (CM) were evaluated. B, NOS enzymatic activity in subcellular fractions from endothelial cells. [3H]-L-arginine conversion to [3H]-L-citrulline was measured in the presence of excess substrate, cofactors, calcium and calmodulin. Enzymatic activity was undetectable in NCM. Values are means ± S.E.M., n = 4-6, * P < 0.05 versus plasma membrane. C, localization of caveolin and eNOS in plasma membranes by immunoelectron microscopy. Immunogold labelling was performed using antibody to caveolin-1 in fibroblasts (panel 1) and endothelial cells (panel 2), and antibody to eNOS in fibroblasts (panel 3) and endothelial cells (panel 4). Caveolae (arrows) are evident in both cell types. Bar = 0.45 µm. Modified from Shaul et al. (1996). | ||
The basis for eNOS localization to caveolae was then explored. The process was faithfully reconstituted by transient transfection of wild-type eNOS cDNA into COS-7 cells which possess caveolae but do not express NOS constitutively. The distribution of wild-type eNOS in transfected COS-7 cells mimicked that in endothelial cells, with NOS activity in caveolae enriched 7- to 12-fold versus whole plasma membrane. The mechanisms underlying trafficking to the microdomain were then determined using the knowledge that eNOS is N-terminally myristoylated at the glycine residue in position 2 and palmitoylated at the cysteine residues at positions 15 and 26 (Lamas et al. 1992; Robinson & Michel, 1995). In distinct contrast to wild-type eNOS, myristoylation-deficient mutant eNOS, which is incapable of both myristoylation and palmitoylation, was not enriched in either whole plasma membranes or the caveolae fraction of plasma membranes. These findings indicated that acylation targets eNOS to caveolae. Experiments were then performed with a palmitoylation-deficient mutant of eNOS which is capable of myristoylation. The palmitoylation-deficient form of eNOS was partially enriched in plasma membrane versus cytosol, and it was partially enriched in caveolae versus noncaveolae fractions of the plasma membrane, indicating a modest targeting to caveolae mediated by myristoylation alone. Repeated studies indicated that there is approximately a 10-fold enhancement in eNOS targeting to caveolae due to myristoylation alone, and the targeting is augmented another 10-fold by palmitoylation for a combined enhancement of 100-fold. Thus, both acylation processes are necessary for optimal targeting of eNOS to caveolae (Shaul et al. 1996). Importantly, the results of studies of caveolae obtained from rat lung agreed with those obtained in cultured endothelial cells (Garcia-Cardena et al. 1996), indicating that eNOS localization to caveolae occurs in intact endothelium. Further studies of the localization of the palmitoylation-deficient form of eNOS showed that the mutant protein is found principally in the Golgi apparatus, whereas the myristoylation-deficient mutant is ubiquitously distributed, and that normal dual acylation is necessary for optimal function of eNOS in a physiological context (Liu et al. 1996, 1997; Sowa et al. 1999).
Lipoproteins and eNOS localization in caveolae
In addition to the modifications of the eNOS protein described above, the specialized lipid environment within caveolae is critical to the targeting and regulation of the enzyme within the microdomain. Since membrane cholesterol is essential for normal caveolae function (Chang et al. 1992) and the initiating events in atherogenesis are characterized by diminished endothelial NO production in response to extracellular stimuli, the effects of oxLDL on eNOS subcellular localization and function were investigated (Blair et al. 1999). In cultured endothelial cells briefly exposed to control conditions including lipoprotein-deficient serum (LPDS), HDL or native LDL (nLDL), eNOS and caveolin both remained highly enriched in caveolae (Fig. 2A, panels 1 and 2). In contrast, oxLDL exposure induced eNOS and caveolin to move from caveolae and plasma membrane to an internal membrane fraction containing endoplasmic reticulum, Golgi apparatus, mitochondria and other intracellular organelles. However, PKC and GM1, two other resident proteins in caveolae, did not translocate from the caveolae fraction upon oxLDL exposure (Fig. 2A, panel 3). Comparable findings were obtained in cells treated with the protein synthesis inhibitor cycloheximide, and the removal of oxLDL and continued treatment with cycloheximide (recovery) enabled eNOS and caveolin to return to caveolae membranes (Fig. 2A, panels 1 and 2). Studies employing indirect immunofluorescence were confirmatory (Fig. 2B).
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Figure 2. Oxidized LDL displaces eNOS from endothelial cell caveolae due to depletion of caveolae cholesterol A, oxidized LDL (oxLDL) but not lipoprotein-deficient serum (LPDS), HDL or nLDL exposure (60 min) alters the subcellular distribution of eNOS and caveolin. Additional studies were performed in oxLDL-treated cells that were washed and incubated for an additional 120 min in LPDS only (Recovery). Samples of postnuclear supernatant (PNS), cytosol (CYTO), intracellular membranes (IM), plasma membrane (PM) and caveolae membrane (CM) were isolated, and immunoblot analysis was performed for eNOS (panel 1) and caveolin-1 (panel 2). Control experiments in oxLDL-treated cells included immunoblot analyses for the caveolae resident proteins protein kinase C and GM1, and for transferrin receptors which are found in coated pits (panel 3). B, oxLDL but not LPDS or nLDL induces eNOS and caveolin to colocalize in an internal membrane compartment. Cells were incubated for 60 min, fixed and processed for double label immunofluorescence. C, oxLDL depletes caveolae of cholesterol. For panels 1 and 2, endothelial cells were labelled with [3H]acetate for 18 h at 37 °C, washed and incubated with nLDL (panel 1) or oxLDL (panel 2) for 0-60 min at 37 °C. The medium was collected, and the nLDL or oxLDL was isolated by centrifugation. The cells were washed, processed to isolate caveolae and [3H]cholesterol was extracted and measured. For panels 3 and 4, unlabelled cells were incubated with oxLDL (panel 3) or HDL (panel 4) for 0-60 min at 37 °C. The cells were washed, caveolae were isolated, and the amount of total cholesterol associated with caveolae was determined. Values are means ± S.E.M., n = 8. Modified from Blair et al. (1999). | ||
Having demonstrated that oxLDL causes eNOS redistribution in endothelial cells, the effects of oxLDL on the activation of the enzyme by acetylcholine were evaluated. OxLDL attenuated eNOS stimulation at all concentrations of acetylcholine tested, yielding a shift in the dose-response curve to acetylcholine to the right by 100-fold. Potential effects of oxLDL on eNOS modification were assessed, and both the palmitoylation and myristoylation of the enzyme were unperturbed. In addition, eNOS phosphorylation was unaffected by oxLDL (Blair et al. 1999). Thus, oxLDL provokes dramatic subcellular redistribution of eNOS, and this process is not due to changes in the co- and post-translation modifications of the enzyme.
The next series of studies determined whether modifications in the caveolae lipid environment underlie the effects of oxLDL on eNOS distribution. Endothelial cells were labelled with [3H]acetate, exposed to nLDL or oxLDL, the medium was collected and caveolae were isolated, and the amounts of sterol in the extracellular medium and caveolae were determined (Fig. 2C). nLDL did not alter the amount of radiolabelled sterol in caveolae, and negligible amounts accumulated extracellularly (Fig. 2C, panel 1). However, radiolabelled sterol was readily transferred from caveolae to oxLDL in the media, such that the caveolae were essentially devoid of labelled sterol by 30 min (Fig. 2C, panel 2). In parallel, the total cholesterol content of the caveolae fraction was depleted by oxLDL, whereas HDL chosen as a control had no effect (Fig. 2C, panels 3 and 4). Thus, by acting as an acceptor of cholesterol, oxLDL causes marked depletion of caveolae cholesterol. To determine if this process mediates eNOS displacement, the effect of the cholesterol-accepting agent cyclodextrin was determined. Similar to oxLDL, cyclodextrin caused redistribution of eNOS and caveolin from caveolae to the intracellular membrane fraction. Thus, oxLDL causes a depletion of caveolae cholesterol, and the alteration in the lipid environment results in eNOS redistribution and an attenuated capacity to activate the enzyme (Blair et al. 1999). Later experiments employing antibody blockade revealed that oxLDL-induced inhibition of eNOS localization and activation is mediated by the class B receptor CD36 (Uittenbogaard et al. 2000). Recent further studies by the Smart laboratory have shown that apoE-/- mice on a high fat diet do not have a normal acute fall in blood pressure with Ach infusion. In addition, caveolae isolated in vivo from apoE-/- vessels do not contain eNOS and are depleted of cholesterol. However, the Ach response and eNOS localization to caveolae are conserved in apoE/CD36 double knockout mice. Thus, the findings in cell culture have been effectively extrapolated to intact vasculature (Kincer et al. 2002). These cumulative observations indicate that oxLDL causes depletion of caveolae cholesterol in endothelial cells via CD36, leading to eNOS redistribution and an attenuated capacity to activate the enzyme. This process may play a critical role in the early pathogenesis of hypercholesterolaemia-induced vascular disease and atherosclerosis.
Since there is a strong negative correlation between HDL levels and the risk for atherosclerosis (Grundy, 1986; Tall, 1990) and HDL mediates cholesterol trafficking (Krieger, 1998; Fidge, 1999), the possibility was then tested that HDL modifies the effects of oxLDL on eNOS localization and activation in caveolae (Uittenbogaard et al. 2000). The addition of HDL to medium containing oxLDL prevented eNOS and caveolin displacement from caveolae (Fig. 3A), and it additionally restored Ach-induced stimulation of the enzyme (Fig. 3B). To determine the mechanisms underlying the effects of HDL on eNOS targeting, further studies of caveolae cholesterol homeostasis were performed (Fig. 3C). Whereas oxLDL alone caused a fall in caveolae sterol content (Fig. 3C, panel 1), cotreatment with HDL prevented the decline (Fig. 3C, panel 3). Further studies showed that the ability of HDL to maintain the concentration of caveolae-associated cholesterol in the face of oxLDL is not related to the inhibition of cholesterol transport from caveolae by oxLDL (Fig. 3C, panel 4); instead, it is due to the provision of cholesterol esters by HDL (Fig. 3C, panel 5). Thus, in the presence of oxLDL, HDL preserves the unique lipid environment within caveolae, thereby maintaining the normal subcellular localization and function of eNOS and possibly explaining at least a portion of the antiatherogenic properties of HDL.
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Figure 3. HDL prevents oxLDL-induced inhibition of eNOS localization and activation in caveolae A, endothelial cells were treated for 60 min with nLDL, HDL, or oxLDL, or with oxLDL followed by HDL for an additional 15 min. Samples of postnuclear supernatant (PNS), cytosol (CYTO), intracellular membranes (IM), plasma membrane (PM) and caveolae membrane (CM) were isolated, and immunoblot analysis was performed for eNOS and caveolin-1. B, after treating endothelial cells as described above, NOS activity was evaluated in intact cells by measuring [3H]L-arginine conversion to [3H]L-citrulline over 15 min in the absence of exogenous stimulation (Basal) or in the presence of acetylcholine (Ach, 10-6 M). Values are means ± S.E.M., n = 4, * P < 0.05 versus basal. C, HDL maintains the sterol content of caveolae. Endothelial cells were incubated with oxLDL (panel 1) or HDL (panel 2) for 0-60 min at 37 °C. For panel 3, cells were pretreated with oxLDL and then HDL was added for an additional 15 min. The cells were fractionated to isolate caveolae and the mass of cholesterol associated with caveolae was determined. In panel 4, cells were radiolabelled with [14C]acetate for 18 h, and HDL and oxLDL were added simultaneously for 0-60 min. The cells were processed to measure the amount of [14C]cholesterol associated with caveolae and the mass of cholesterol associated with caveolae. In panel 5, cellular cholesterol pools were radiolabelled as described for panel 4, and HDL was labelled with [3H]cholesterol ester. [3H]HDL and oxLDL were incubated with cells as described for panel 4, and the amount of [14C]cholesterol and [3H]cholesterol associated with caveolae was determined. Values are means ± S.E.M., n = 3. Modified from Uittenbogaard et al. (2000). | ||
Studies of the principal HDL receptor, scavenger receptor BI, were done to determine if the receptor is expressed in endothelial cells, and SR-BI protein was indeed detected and found to be highly enriched in endothelial cell caveolae. Antibody blockade experiments were performed to determine if HDL binding to SR-BI underlies the reversal of the impact of oxLDL on eNOS localization and function. Endothelial cells were treated with oxLDL or oxLDL plus HDL in the presence of nonspecific IgG or SR-BI IgG, and eNOS and caveolin-1 distribution was evaluated (Fig. 4A). In the presence of nonspecific IgG, eNOS and caveolin-1, localization to caveolae was restored by HDL. However, SR-BI blocking antibody prevented the restoration of eNOS and caveolin-1 localization by HDL. In parallel, SR-BI blocking antibody prevented HDL-mediated restoration of eNOS stimulation by acetylcholine (Fig. 4B) (Uittenbogaard et al. 2000). These cumulative observations indicate that SR-BI mediates the capacity of HDL to preserve eNOS localization and function in the presence of oxLDL.
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Figure 4. SR-BI mediates the effects of HDL on eNOS localization and activation in caveolae A, endothelial cells were treated for 1 h with oxLDL, or oxLDL and HDL in the presence of nonspecific IgG or SR-BI IgG. Samples of postnuclear supernatant (PNS), cytosol (CYTO), intracellular membranes (IM), plasma membrane (PM) and caveolae membrane (CM) were isolated, and immunoblot analysis was performed for eNOS and caveolin-1. Representative data from four independent experiments are shown. B, after treating endothelial cells as described above, NOS activity was evaluated in intact cells by measuring [3H]-L-arginine conversion to [3H]-L-citrulline over 15 min in the absence of exogenous stimulation (Basal) or in the presence of acetylcholine (Ach, 10-6 M). Values are means ± S.E.M., n = 4, * P < 0.05 versus basal. Reprinted with permission (Uittenbogaard et al. 2000). | ||
eNOS signalling module in caveolae
Over the past several years, there has been an accumulation of observations indicating that many of the signal transduction molecules modulating eNOS activity are colocalized with the enzyme in caveolae (Shaul & Anderson, 1998). Such colocalization has suggested the existence of a functional signalling module which compartmentalizes the multiple events regulating the level of NO production. However, direct evidence of such a module was unavailable until recent studies were performed to further understand how eNOS is stimulated by oestradiol via nongenomic actions of the oestrogen receptor (ER), which was previously known to serve solely as a nuclear transcription factor (Chen et al. 1999). These mechanisms were pursued because oestradiol is an important agonist for endothelial eNOS in both physiological and pathophysiological circumstances, affording atheroprotection to premenopausal women compared to men and to postmenopausal women receiving certain forms of oestrogen replacement, and also modifying responses to vascular injury (Mendelsohn & Karas, 1999).
The subcellular site of interaction between ER and eNOS was initially determined in experiments employing isolated endothelial cell plasma membranes (Chambliss et al. 2000). Oestradiol caused an increase in eNOS activity in the isolated membranes in the absence of added calcium, calmodulin or eNOS cofactors, the activation was blocked by the ER antagonist ICI 182 780 and ER antibody, and immunoidentification experiments detected ER protein in the plasma membrane fraction. Plasma membranes from COS-7 cells expressing eNOS and ER also displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ER plus a myristoylation-deficient mutant eNOS were insensitive. Further fractionation of endothelial cell plasma membranes revealed ER protein in caveolae, and oestradiol caused stimulation of eNOS in isolated caveolae that was ER-dependent; in contrast, noncaveolae membranes were insensitive. Responses to oestradiol and the more classical eNOS agonists acetylcholine and bradykinin were also compared in isolated caveolae, and equivalent, robust eNOS activation was observed with all three agents (Fig. 5). In addition, calcium chelation prevented oestradiol-stimulated eNOS activity in both isolated whole plasma membranes and caveolae. These cumulative findings reveal that a subpopulation of ER and multiple other receptors for eNOS agonists are localized to endothelial cell caveolae where they are coupled to the enzyme in a functional signalling module which regulates the local calcium environment (Chambliss et al. 2000). Mechanisms occurring within this signalling module are probably critical to the atheroprotective properties of oestradiol (Mendelsohn & Karas, 1999).
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Figure 5. eNOS is localized to a functional signalling module in endothelial cell caveolae [3H]-L-arginine conversion to [3H]-L-citrulline was measured in isolated noncaveolae and caveolae fractions of endothelial cell plasma membranes, in the absence (basal, B) or presence of 10-8 M oestradiol (E2), 10-6 M acetylcholine (Ach), or 10-6 M bradykinin (BK). Values are means ± S.E.M., n = 4-6, * P < 0.05 vs. basal. Modified from Chambliss et al. (2000). | ||
Lipoprotein-induced activation of eNOS
More recent work has determined whether the effects of various lipoproteins on antiatherogenesis are further explained by caveolae-related mechanisms other than those involving changes in membrane cholesterol content. Because of its potent antiatherogenic properties, studies of the effects of HDL on eNOS function in the absence of oxLDL were undertaken (Yuhanna et al. 2001). They demonstrated that HDL causes rapid and dramatic stimulation of the enzyme in cultured endothelial cells (Fig. 6A). HDL stimulation of eNOS was concentration-dependent (Fig. 6B), it was detected within 3 min of exposure to HDL, the maximal effect was seen by 6 min, and stimulation persisted for at least 60 min. The HDL fraction of human serum caused eNOS activation whereas the non-HDL fraction did not, and whole serum yielded an effect comparable to HDL but lipoprotein-deficient serum did not (Fig. 6C). In contrast to HDL, nLDL did not stimulate eNOS, and simultaneous exposure to excess nLDL prevented the HDL response (Fig. 6D,E). Importantly, eNOS was not activated by purified forms of apoA-I or apoA-II (Fig. 6F). Heterologous expression experiments in chinese hamster ovary cells further revealed that SR-BI mediates the effects of HDL on the enzyme, and SR-BI mRNA was detected in cultured endothelial cells by RT-PCR. Endothelial cell subfractionation experiments revealed that HDL activation of eNOS is demonstrable in isolated caveolae but not in noncaveolae membranes, and that SR-BI and eNOS are colocalized in caveolae (Yuhanna et al. 2001). Thus, HDL is a potent agonist for eNOS, and SR-BI is another important component of the eNOS signalling module in caveolae.
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Figure 6. HDL activates eNOS in endothelial cells A, [3H]L-arginine conversion to [3H]L-citrulline was measured under basal conditions or in the presence of HDL or A23187. B, the dose-response to HDL was also assessed. C, eNOS activation was evaluated under basal conditions or in the presence of the HDL fraction or the non-HDL fraction of human serum, or whole serum, or lipoprotein-deficient serum (LPDS) added at equal volumes. D, the responses to HDL and LDL were compared. E, eNOS activation by HDL was assessed in the absence or presence of excess LDL. F, eNOS activation was determined in the presence of HDL or recombinant apoA-I or lipid-free apoA-II purified from human plasma. Values are means ± S.E.M., n = 3-6, * P < 0.05 vs. basal. Reprinted with permission from Yuhanna et al. (2001). | ||
The effect of HDL on endothelial NO production has also been evaluated in a physiological system by assessing the impact of the lipoprotein on endothelium-dependent vascular relaxation (Yuhanna et al. 2001). HDL caused direct relaxation of phenylephrine-precontracted rings of thoracic aortae from wild-type CD-1 mice, the response was blocked by pretreatment with the NOS antagonist nitro-L-arginine methyl ester (L-NAME), and relaxation was not observed in endothelium-denuded rings (Fig. 7A,B). The ability of HDL to alter acetylcholine (Ach)-mediated vasorelaxation was also examined. Relaxation with intermediate concentrations of Ach was greater with coexposure to HDL, whereas responses with higher levels of Ach were not (Fig. 7C). The role of SR-BI in endothelial NO production was also determined by assessing both direct responses to HDL and Ach-stimulated relaxation in the absence or presence of HDL using aortae from homozygous null SR-BI knockout mice (Rigotti et al. 1997). Aortae from wild-type 129/C57BL/6 mice (SR-BI+/+) exhibited direct relaxation to HDL, mimicking the observations in the CD-1-derived vascular rings. In contrast, HDL did not cause discernable relaxation in aortae from homozygous null mutants (SR-BI-/-) (Fig. 7D). In addition, relaxation to Ach was increased 2.8-fold by HDL in aortae from SR-BI+/+ mice, whereas HDL did not modify the Ach response in aortae from SR-BI-/- mice (Fig. 7E). Thus, HDL enhances endothelial NO production in intact arteries, and this process is mediated by SR-BI. This process may be critical to the atheroprotective features of HDL.
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Figure 7. HDL enhances endothelial NO production in aortae from wild-type mice, but not in aortae from homozygous SR-BI null mutant mice A, following precontraction of rings of thoracic aortae from wild-type CD-1 mice with phenylephrine (arrow), direct relaxation responses to control buffer (CON), HDL at 10 µg ml-1 (open arrowhead) or 25 µg ml-1 (filled arrowhead), HDL with prior L-NAME treatment, or HDL with endothelium-denuded (- ENDO) rings were evaluated. B, cumulative findings (means ± S.E.M., for maximal relaxation to 25 µg ml-1 HDL in n = 10, 10, 3 and 5 studies, respectively, which were performed as in A. C, HDL augments the relaxation response to acetylcholine (Ach) in aortae from wild-type CD-1 mice. Studies were performed in control rings (open circles) and rings exposed to HDL (filled circles). D, the direct effects of control buffer (CON) or HDL were tested on phenylephrine-contracted rings of thoracic aortae from wild-type 129/C57BL/6 mice (SR-BI+/+) or homozygous null mutant mice (SR-BI-/-) mice. E, rings of thoracic aortae from SR-BI+/+ or SR-BI-/- mice were precontracted with phenylephrine, and the response to Ach was determined before and after exposure to HDL. In C, D and E, values are means ± S.E.M., and a minimum of 4 rings from 4 different mice were studied in each group. * P < 0.05 vs. no HDL. Reprinted with permission from Yuhanna et al. (2001). | ||
Cholesterol status during fetal life and the initiation of adult vascular disease
Now knowing that lipoproteins have potent, rapid impact on endothelial cell function via the modulation of eNOS, one should consider the developmental period during which these processes may be pathogenetically relevant. Animal models have revealed that cholesterol status during fetal life has marked impact on later vascular function and disease. In studies of maternal diet and blood pressure in rat progeny by Langley-Evans (1996), high fat intake during pregnancy led to marked increases in systolic blood pressure in the mature offspring. Further direct assessments of vascular function in rat offspring following maternal diets high in saturated fat in the Poston laboratory showed enhanced constrictor responses and attenuated endothelium-dependent relaxation, whereas direct smooth muscle responses to a NO donor were unaltered (Koukkou et al. 1998). Investigations in humans have included assessments of fatty streak formation in human fetal and childhood aortae in offspring of normocholesterolaemic mothers, hypercholesterolaemic mothers, and mothers who were hypercholesterolaemic only during pregnancy. In fetuses younger than 6 months, fetal plasma cholesterol levels were strongly correlated with maternal levels, whereas no relationship was evident later in gestation. Fatty streak formation was found to be greater in fetal aortas from mothers with transient or long-term hypercholesterolaemia compared with fetuses of normocholesterolaemic mothers (Napoli et al. 1997). The later evolution of these lesions was assessed in studies of aortae from normocholesterolaemic children, ages 1 to 13 years. Fatty streak lesions increased linearly with age, and lesion progression was markedly greater in the offspring of hypercholesterolaemic mothers (Napoli et al. 1999). Thus, maternal hypercholesterolaemia during pregnancy is associated with greater fatty streak development in the human fetus and more rapid progression of atherosclerosis under normocholesterolaemic conditions during childhood. The robust correlation between maternal and fetal cholesterol concentrations during early gestation (Napoli et al. 1997) suggests that this is due to the impact of elevated fetal cholesterol levels on the fetal vascular wall.
To avoid the potential impact of genetic differences between patients, Napoli and Palinski employed a rabbit model and showed that a hypercholesterolaemic diet during pregnancy enhances fatty streak formation in the offspring, both at birth and into adulthood (Napoli et al. 2000; Palinski et al. 2001). More recently, they have also demonstrated that the offspring of LDL receptor-deficient hyypercholesterolaemic mice have greater aortic lesion formation (Napoli et al. 2002). When these cumulative observations in humans, rabbits and mice are combined with those demonstrating a strong influence of maternal sterol metabolism on fetal sterol metabolism with diet manipulation in hamsters (McConihay et al. 2001), it appears that alterations in maternal or fetal cholesterol homeostasis early in pregnancy have considerable long-term impact on vascular structure and function. Thus, the earliest phases of atherogenesis during which lipoprotein modulation of eNOS function is potentially of mechanistic importance may be during fetal life.
Summary
Attenuated NO production by eNOS is a critical feature of early hypercholesterolaemia-induced vascular disease. In studies in cell culture and in mature animal models determining causal linkage between cholesterol status and alterations in endothelial function, we have shown that eNOS is normally targeted to cholesterol-enriched caveolae where it resides in a signalling module, and that oxLDL disrupts eNOS localization and action in caveolae by depleting caveolae cholesterol content. In contrast, HDL fully prevents these adverse effects of oxLDL, and HDL also causes robust activation of eNOS. Thus, we now know that lipoproteins have potent effects on eNOS function in caveolae via actions on membrane cholesterol homeostasis and enzyme activation, thereby altering endothelial NO production. Work by others suggests these processes would be pathogenetically relevant during the earliest phases of atherogenesis, which may occur in fetal life. One of the many current challenges is to determine if eNOS modulation within caveolae is indeed operative in the fetal vasculature, and whether it is under the direct influence of changes in cholesterol status. As importantly, we must determine if these specific mechanisms modify the cardiovascular phenotype of the offspring. Further work is now warranted that is focused on the initiating events occurring during early development. It is only through diligent commitment to investigation of the onset of the disease that the next major advancement will be made in applying the robust biological effects of lipoproteins and NO to the prevention of atherosclerosis and its sequelae later in life.
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Acknowledgements
The author would like to thank his multiple colleagues and collaborators, past and present, who have contributed to the data summarized in this review. These include Richard G. W. Anderson, Alison Blair, Ken L. Chambliss, Patricia A. Conrad, Blair E. Cox, Zohre German, Lisa D. Hahner, Helen H. Hobbs, Richard E. Karas, Pingsheng Liu, Ping Lu, Yves L. Marcel, Michael E. Mendelsohn, Thomas Michel, Chieko Mineo, Sherri Osborne-Lawrence, Margaret C. Pace, Lisa J. Robinson, Todd S. Sherman, Eric J. Smart, Annette Uittenbogaard, Yunshu Ying, Ivan S. Yuhanna and Yan Zhu. The author also expresses gratitude to Ms Marilyn Dixon for preparing this manuscript. This work was supported by National Institutes of Health grants HL58888, HL53546 and HD30276, the Lowe Foundation and the Crystal Charity Ball.
This report was presented at The Journal of Physiology Symposium on Fetal Programming: from gene to functional systems, Los Angeles, USA, 20 March 2002. It was commissioned by the Editorial Board and reflects the views of the author.
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