© 1997 American Heart Association, Inc.
Articles |
High-Density Lipoprotein-Binding Protein (HBP)/Vigilin Is Expressed in Human Atherosclerotic Lesions and Colocalizes With Apolipoprotein E
Correspondence to Kevin D. O'Brien, MD, Division of Cardiology, Box 356422, University of Washington, 1959 NE Pacific St, Seattle, WA 98195-6422. E-mail: cardiac{at}u.washington.edu
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Abstract Accumulation of cholesteryl esters within cells of the arterial intima is a hallmark of atherosclerosis. A small number of proteins have been shown in vitro to be upregulated by cellular cholesterol loading, including apolipoprotein E (apoE) and the recently cloned HDL-binding protein (HBP), but only apoE has been shown to be upregulated in cholesterol-loaded cells in atherosclerosis. To determine whether HBP (also called vigilin) might be expressed in human atherosclerosis, immunohistochemistry and in situ hybridization were performed on coronary arteries of 18 patients. HBP/vigilin was detected on all endothelial cells. HBP/vigilin mRNA and protein also were detected on a subset of macrophages and occasionally on smooth muscle cells (SMC) in atherosclerotic plaques but were not detected on these cell types in nondiseased coronary intima. The majority of HBP/vigilin-expressing macrophages were foam cells, but HBP/vigilin expression also was detected rarely in nonfoam cell macrophages. Foam cell macrophage HBP/vigilin expression was present in 100% of atherosclerotic quadrants, and nonfoam cell macrophage HBP/vigilin expression was present in 6% of atherosclerotic quadrants. HBP/vigilin-expressing human plaque cells also expressed apoE. However, HBP/vigilin was detected in cardiac myocyte foam cells of an apoE-deficient mouse, demonstrating that HBP/vigilin expression can occur independently of apoE. These results suggest that in vivo HBP/vigilin expression is upregulated by intracellular cholesterol loading but also that other factors present in atherosclerotic plaques may upregulate HBP/vigilin. Although the exact function of HBP/vigilin is unknown, its expression in plaque macrophages suggests a role for this molecule in atherogenesis.
Key Words: HDL-binding protein • vigilin • atherosclerosis • macrophage • apolipoprotein E
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Introduction |
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A hallmark of atherosclerosis is the accumulation of cholesteryl esters within cells of the arterial intima. Most of these intracellular esters are formed when macrophages and SMC ingest lipoproteins by endocytotic or phagocytotic processes, degrade the lipoproteins in lysosomes, and re-esterify the liberated free cholesterol in the cytoplasm.1 A fraction of this ingested cholesterol likely is excreted from cells in response to cell surface interactions of high-density lipoproteins (HDL).2–4
Although cellular processes for delivery of cholesterol have been well characterized, the molecular properties of pathways involved in handling and excretion of excess cellular cholesterol are poorly understood. Only a small number of cellular proteins have been shown to undergo positive regulation in response to cholesterol loading. Of those in macrophages, apolipoprotein E (apoE) has been studied the most extensively.5 Synthesis and secretion of apoE increase when cultured macrophages are overloaded with cholesterol.6–9 The secreted apoE interacts with the cell surface of macrophages to stimulate efflux of cholesterol.10,11 ApoE is expressed in human atherosclerotic lesions and is localized to foam cell macrophages.12–15 Thus, apoE may act in an autocrine fashion to mobilize excess cholesterol from macrophages of atherosclerotic lesions.
Another cellular protein shown to be regulated positively by cholesterol loading of macrophages is HDL-binding protein (HBP).16 HBP was cloned from a human cDNA expression library by using an antibody raised against a 110-kD cellular protein that binds HDL on ligand blots.16 The chicken homolog of HBP, called vigilin (based on the prevalence of valine-isoleucine-glycine repeats), was cloned independently from chondrocytes.17 HBP/vigilin is expressed primarily as a 150-kD membrane-bound protein localized in the cytoplasm of cells.16–18 This protein appears to undergo processing to form a 110-kD protein that binds HDL on ligand blots and that is localized, at least partially, to the plasma membrane.16
The specific function of HBP/vigilin has not been established. The protein is expressed in a wide variety of cell types and tissues,16–22 and its relative abundance is sensitive to the growth and differentiation states of cultured cells.16,18,19,21 Although it was cloned during an attempt to identify a cellular HDL receptor and although forms of HBP/vigilin appear to be associated with plasma membrane, this molecule lacks the classic signal peptide and the membrane-spanning domain that typify cell surface receptors. Thus, HBP/vigilin is unlikely to be an HDL receptor. Findings from several studies have suggested other possible functions for HBP/vigilin. The full-length protein comprises 14 imperfect tandem repeats of approximately 70 amino acids that contain KH domains, sequence motifs that are usually found in RNA-binding proteins.16,17,23 Some of these KH domains in HBP/vigilin have been shown to have weak nucleotide-binding activities,24 and HBP/vigilin has been found to be associated with cytoplasmic tRNA21 and mRNA.22 This protein also contains a nuclear localization sequence and has been detected in the nucleus of cells.25 These properties have led to the hypothesis that HBP/vigilin plays a role in modulating protein synthesis through its direct interaction with RNA.21,22,25
Several other lines of evidence suggest that HBP/vigilin may play a role in cellular sterol metabolism. First, a study in rats showed that the HBP/vigilin mRNA levels in the corpus luteum of the ovary increases with gonadotropin treatment,20 a finding suggesting that HBP/vigilin expression is associated with steriodogenesis. Second, estrogen and testosterone treatment of tissues alters HBP/vigilin expression.22 Third, cholesterol loading of cultured J774 macrophages increases HBP/vigilin mRNA and protein levels.16 This latter study raises the possibility that HBP/vigilin may be functionally important in the biology of foam cell macrophages in atherosclerotic lesions.
Therefore, this study was designed to examine the following questions: (1) Is HBP/vigilin present in human atherosclerotic lesions? (2) If so, in which cell types is it expressed? (3) Is HBP/vigilin expression associated with intracellular lipid accumulation? (4) How does the expression of HBP/vigilin compare to that of apoE, another molecule that is upregulated by cell cholesterol loading?
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Methods |
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Human Arterial Tissue
Human coronary artery segments were obtained from the excised native hearts of 18 cardiac transplant recipients and snap-frozen in OCT or fixed in 10% neutral buffered formalin (NBF) within 2 hours of organ removal. Six-micrometer-thick frozen sections were used for immunohistochemistry, histologic staining with oil red O (ORO, Sigma), and combined immunohistochemistry and ORO staining. NBF-fixed, paraffin-embedded tissue was used for in situ hybridization and for combined in situ hybridization and immunohistochemistry.
Antisera and Antibodies
Rabbit Anti-HBP/Vigilin IgG
A rabbit polyclonal antiserum was generated by immunization of
rabbits with a 21-amino-acid peptide (RGQVLREIAEEYGGVMVSFPRC)
corresponding to amino acid residues 819 to 840 of human
HBP/vigilin.16 To enhance immunization, the
peptide was synthesized with an additional cysteine residue and coupled
to rabbit serum albumin using a NHS-ester-maleimide
cross-linking agent (Pierce, Rockford, Illinois). A protein A column
was used to isolate the IgG fraction from this antiserum and the IgG
fraction was used (titer, 1:500) to detect HBP/vigilin. Specificity of
this IgG for HBP/vigilin was confirmed by ligand and Western blots of
HBP/vigilin-transfected BHK cells, which demonstrated the presence of a
major band at 150 kD and of a minor band at 110 kD, corresponding to
the predicted molecular weights of, respectively, the primary
HBP/vigilin gene product and its processed form.
Mouse Monoclonal Antibodies
To distinguish cell types in the arterial wall, the
following cell specific mouse monoclonal antibodies were used: (1)
HAM-56 (Dako; titer, 1:1000) or anti-CD68 (Dako; titer, 1:4000), which
are specific for human macrophages, and (2) anti-smooth-muscle
-actin (Boehringer Mannheim; titer, 1:250), which is, in
this context, specific for SMC. A mouse monoclonal antibody directed
against apoE (Monosan; titer, 1:75) was used to detect apoE. Before
immunohistochemistry, tissues were post-fixed in cold methanol (for
anti-HBP/vigilin IgG, HAM-56 or anti-CD68), 10% neutral buffered
formalin (for anti–-actin), or acetone (for anti-apoE). Slides were
counterstained with either hematoxylin or methyl green.
Immunohistochemistry
Single-label immunohistochemistry (IHC) was performed as
described previously.26–28 Briefly, tissues were
blocked with 3% H2O2
(Sigma Corp, St Louis, Missouri), washed with phosphate-buffered saline
(PBS), incubated for 60 minutes with the primary IgG or monoclonal
antibody, and then washed again with PBS. A biotin-labeled anti-mouse
secondary antibody then was applied for 30 minutes, followed by an
avidin-biotin-peroxidase conjugate (ABC Elite, Vector Laboratories,
Burlingame, Calif) for 30 minutes. Standard peroxidase enzyme
substrate, 3,3'-diaminobenzidine (Sigma), was added to yield a brown
reaction product or 3,3'-diaminobenzidine with
NiCl2 was added to yield a black reaction
product.
Oil-Red O Staining
Intracellular lipid accumulation was determined using ORO, which
stains neutral lipids such as esterified cholesterol and
triglycerides. Frozen sections were brought to room
temperature and covered with filtered, working ORO solution for 4
minutes. Slides then were rinsed four times in 60% isopropanol, washed
in H2O, and counterstained with hematoxylin.
Combined ORO Staining and Immunohistochemistry
Staining with ORO was combined with immunohistochemical staining
with either anti-CD68 or anti--actin to identify, respectively,
macrophage or smooth muscle cell foam cells. Frozen sections
were brought to room temperature and covered with filtered, working ORO
solution for 4 minutes. Slides were rinsed four times in 60%
isopropanol, then washed in 1X PBS. Immunohistochemistry then was
performed as described previously27 using mouse
monoclonal antibody anti-CD68 (titer, 1:4000) or mouse monoclonal
antibody anti -actin (titer, 1:250). Slides were counterstained with
hematoxylin, and a coverslip was applied.
Riboprobe Preparation
To confirm the expression of HBP/vigilin by cells of the artery
wall, in situ hybridization was performed on NBF-fixed,
paraffin-embedded tissue with 35S-labeled
antisense riboprobes to detect HBP/vigilin and apoE mRNA. The antisense
HBP/vigilin riboprobe was transcribed from a 1545-bp fragment of the
human HBP/vigilin cDNA, corresponding to bases 2810 to 4354. Antisense
riboprobes were transcribed from the cDNA using reagents from Promega,
except 35S-UTP 1000 to 3000 Ci/mmol, which was
obtained from New England Nuclear (Boston, Massachusetts). The sense
(control) HBP/vigilin riboprobe was transcribed from a fragment
containing the first 848 bp of the human HBP/vigilin cDNA.
The HBP/vigilin antisense or sense riboprobe transcription reaction mixtures contained 1 µg of DNA; 250 µCi of 35S-UTP; 500 µmol/L each of rATP, rCTP, and rGTP; 40 U RNasin (Promega); 10 mm of dithiothreitol; 40 mm of Tris; and either 10 U of SP6 polymerase (for antisense riboprobe transcription) or 10 U of T7 polymerase (for sense riboprobe transcription). ApoE antisense riboprobe was transcribed from a 1-kb human apoE cDNA using SP6 polymerase as described previously.15 After 75 minutes of incubation at 37°C, the cDNA was digested by adding 1 U of RQ1 DNAse (Promega), and incubation was continued for an additional 15 minutes at 37°C. Free nucleotides were separated by using a Sephadex G-50 column, and the riboprobes were used within 24 hours of synthesis.
In Situ Hybridization
Formalin-fixed, paraffin-embedded, 6-µm arterial
sections were washed with 0.5X standard saline citrate (SSC) (1X
SSC=150 mm NaCl, 15 mm Na citrate, pH 7.0) and digested
with proteinase K (1 mg/mL) (Sigma) in RNAse A (Promega) buffer.
After several 0.5X SSC washes, 50 µL of prehybridization buffer (0.3
mol/L NaCl, 20 mmol/L Tris pH 8.0, 5
mmol/L ethylenediaminetetraacetic acid, 1X Denhardt's solution,
1X dextran sulfate, 10 mmol/L dithiothreitol) was applied
for 2 hours at 50°C. For hybridizations,
35S-labeled anti-sense riboprobe (300 000 cpm in
50 µL of prehybridization buffer) was added, and hybridizations were
allowed to proceed overnight at 50°C. After hybridization, sections
were washed with 0.5X SSC, treated with RNAse A (20 µg/mL) for
30 minutes, and washed twice in 2X SSC, followed by three
high-stringency washes in 0.1X SSC/Tween 20 (Sigma) at 37°C, followed
by several 2X SSC washes. After air drying, the tissue was dipped in
NTB2 nuclear emulsion (Kodak) and incubated in the dark for 10 to 35
days. After development, the sections were counterstained with
hematoxylin.
Combined Immunohistochemistry and in Situ Hybridization
Immunohistochemistry with HAM-56 or anti-smooth-muscle -actin
was performed as described above, with the following exceptions: (1)
RNasin (Promega) was added at a concentration of 180 U/mL to the
primary and secondary antibodies as well as the avidin-biotin complex
to inhibit endogenous RNases, (2) antibodies were used at
twofold to fourfold higher concentrations, and (3) PBS was treated with
diethyl pyrocarbonate (Sigma) to denature endogenous
RNases. In situ hybridization with the apoE riboprobe then was
performed as reported previously15 according to
the above protocol.
HBP/Vigilin Expression in apoE (apoE-/-)
Mice
Because HBP/vigilin protein has been demonstrated in murine
macrophages16 and by Western blot
analyses of murine tissues (Oram JF, unpublished observations,
1996), immunohistochemistry also was performed on 6-µm-thick frozen
sections obtained at the level of the aortic sinuses from a 17.5-week
old apoE-/- mouse that had been fed a
high-fat, high-cholesterol diet containing 15% fat, 1.25%
cholesterol, and 0.5% cholate for 8
weeks.29 This mouse had markedly elevated plasma
lipids with total cholesterol of 6844 mg/dL and
triglycerides of 343 mg/dL.
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Results |
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HBP/Vigilin Presence in All Endothelial Cells
Immunohistochemical staining of nonatherosclerotic segments with rabbit polyclonal HBP/vigilin antiserum localized HBP/vigilin to endothelial cells, both at the arterial lumen (Fig 1
) and in
endothelium of the adventitial vasa vasorum. However,
HBP/vigilin was not detected in nonendothelial cells of
morphologically normal regions of the intima. Further, HBP/vigilin was
not detected either in medial SMCor in nonlipid-laden resident
macrophages of the adventitia.
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B HBP/Vigilin Expression by Lipid-Laden
Nonendothelial Cells of Atherosclerotic
Plaques
In contrast, HBP/vigilin (Fig 2a)
frequently was detected in nonendothelial cells of
atherosclerotic arteries, particularly those with lipid inclusions as
detected by ORO staining (Fig 2b). The vast majority of these
lipid-laden nonendothelial cells were
macrophages, as determined by immunohistochemistry of adjacent
sections (Fig 2c).
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Further confirmation of the foam cell types that expressed HBP/vigilin
was obtained by comparison of slides stained with HBP/vigilin to
adjacent sections double-stained with ORO plus the macrophage
marker, anti-CD68, or with ORO and the smooth muscle cell marker,
anti--actin (Fig 3). This technique
allowed the unambiguous identification of either macrophage or
smooth-muscle-cell foam cells. The vast majority of
HBP/vigilin-positive foam cells (Fig 3b) were
macrophage-derived (Fig 3a), while HBP/vigilin-positive SMC
foam cells were detected only rarely (Fig 3c).
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Infrequent HBP/Vigilin Expression by Nonfoam Cell
Macrophages
A second pattern of HBP/vigilin expression, specifically
HBP/vigilin expression by ORO-negative (ie, nonfoam cell)
nonendothelial cells, also was seen (Figs 4a, 4b). Immunohistochemical staining of
adjacent sections with cell-specific antibodies indicated that these
HBP/vigilin-positive nonfoam cells were macrophages.
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Prevalence of HBP/Vigilin Expression by Foam Cells and Nonfoam
Cells
The relative prevalences of foam cell–associated HBP/vigilin
expression and of nonfoam cell–associated HBP/vigilin expression were
determined semiquantitatively. Each arterial segment was
divided into four quadrants, and each quadrant then was scored for the
presence or absence of HBP/vigilin-expressing foam cells and for the
presence or absence of HBP/vigilin-expressing nonfoam cells. One
hundred percent of plaque quadrants contained HBP/vigilin-positive foam
cells. In contrast, only 6% of plaque quadrants contained
HBP/vigilin-positive nonfoam cells. Both patterns of HBP/vigilin
expression were specific to atherosclerosis, as neither
pattern of HBP/vigilin was found in any of the nonatherosclerotic
control quadrants examined (Fig 5).
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Association of HBP/Vigilin With apoE
Cell-associated apoE has been detected in atherosclerotic plaques,
particularly in foam cell macrophages, a finding suggesting
that intracellular lipid accumulation might upregulate the expression
of that protein,13–15,30 as has been suggested
for HBP/vigilin. Immunohistochemistry demonstrated that apoE protein
was present on both foam cell macrophages (Fig 2d
) and
nonfoam cell macrophages (Fig 4c) that expressed HBP/vigilin
(comparison with Figs 2a, 2b, and 2c and Figs 4a and 4b) in
atherosclerotic sections. Cell-associated HBP/vigilin and apoE were
present in all plaque quadrants with a highly statistically
significant correlation between HBP/vigilin-positive foam cells and
apoE-positive foam cells (
2 value: 33.601,
P < .001, Table
1). ApoE was not detected in SMC within
atherosclerotic plaques. Extracellular apoE was present in all
atherosclerotic quadrants and was detected in 22 of 37
nonatherosclerotic control quadrants.
Detection of HBP/Vigilin mRNA in Plaque Macrophages by in
Situ Hybridization
To confirm local production of HBP/vigilin
production of HBP/vigilin in atherosclerotic plaques, in situ
hybridization was performed with antisense and sense (ie, control)
HBP/vigilin riboprobes. Concordant with the immunohistochemical
location of HBP/vigilin protein, strong in situ hybridization signal
was detected with the antisense HBP/vigilin riboprobe (Figs 6c, 6d) in cells identified by
immunohistochemistry on adjacent sections as
macrophage-derived foam cells (Figs 6a, 6b), thus
confirming that macrophage-derived foam cells synthesize
HBP/vigilin in atherosclerotic plaques. Specificity of the in situ
hybridizations was confirmed by the absence of specific signal on
sections to which the sense HBP/vigilin riboprobe had been hybridized
(Figs 6e, 6f).
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Comparison of HBP/Vigilin and apoE mRNA Expression in Plaques by in
Situ Hybridization
To confirm the expression of HBP/vigilin by plaque cells, in situ
hybridization was performed to detect HBP/vigilin mRNA using a
35S-labeled antisense riboprobe on formalin-fixed
sections (Fig 7b). On neighboring
sections, combined in situ hybridization for apoE mRNA (Figs 7a, 7c)
and immunohistochemistry for either macrophages (Fig 7a) or
SMCs (Fig 7c) was performed. HBP/vigilin mRNA was detected in foam
cells (Fig 7b), which also expressed apoE mRNA (Figs 7a, 7c) and were
identified by immunohistochemistry as macrophages (Fig 7a)
rather than SMCs (Fig 7c). The presence of HBP/vigilin mRNA in apoE
mRNA–expressing macrophages is consistent with the
hypothesis that similar factors may upregulate the expression of both
genes in these cells.
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Immunohistochemical Detection of HBP/Vigilin in Cardiac Myocyte
Foam Cells in an ApoE-/- Mouse
To further investigate the relationships of HBP/vigilin expression
to both intracellular lipid accumulation and apoE expression,
immunohistochemistry was performed by using the rabbit anti-HBP/vigilin
IgG or nonimmune (control) rabbit IgG at a titer of 1:500 on frozen
sections from the level of the cardiac aortic sinuses in an
apoE-/- mouse (Fig 8). This mouse, which had extraordinarily
high plasma cholesterol owing to a combination of the
genetic apoE deficiency and a high-fat diet, had cardiac myocyte foam
cells, which also contained immunohistochemically detectable
HBP/vigilin (Fig 8a). Specificity of immunohistochemical staining for
HBP/vigilin was confirmed by demonstration of the absence of
immunohistochemical staining with the nonimmune rabbit IgG (Fig 8b).
The presence of HBP/vigilin in cardiac myocyte foam cells of
apoE-/- mice further strengthens the
association of HBP/vigilin expression with excessive cell
cholesterol accumulation. In addition, the presence of
HBP/vigilin in apoE-/- mice demonstrates that
HBP/vigilin expression does not require expression of apoE.
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HBP/vigilin is a ubiquitous cellular protein that is upregulated by treatment of cells with cholesterol and steroid hormones.16–25 In this study, immunohistochemistry and in situ hybridization for HBP/vigilin were used to characterize the expression of HBP/vigilin in normal and atherosclerotic human coronary arteries. In both nondiseased and diseased specimens, HBP/vigilin was localized to endothelial cells. However, nonendothelial HBP/vigilin expression was found only in cells with significant intimal lipid accumulation, as demonstrated by ORO staining. HBP/vigilin was associated primarily with foam cell macrophages, although a small subset of nonfoam cell macrophages also expressed HBP/vigilin. Rare SMC foam cells also showed positive HBP/vigilin staining. Overall, 100% of plaque quadrants were found to contain HBP/vigilin-positive foam cells, while only 6% of plaque quadrants contained HBP/vigilin-positive nonfoam cells. The expression of HBP/vigilin by nonendothelial cells of atherosclerotic lesions but not by nonendothelial cells of normal intima suggests that this protein is important in atherosclerosis. Additionally, the observation that nonendothelial cell HBP/vigilin expression is restricted primarily to foam cells suggests a prominent role for intracellular lipid accumulation in the regulation of HBP/vigilin expression. This observation is consistent with previous in vitro studies demonstrating that HBP/vigilin mRNA, and protein levels are increased with cholesterol loading in SMCs and in the J774 mouse macrophage cell line.16
ApoE, a lipid transport protein that is hypothesized to be important in atherosclerosis, is synthesized and secreted by several peripheral tissues,5 including atherosclerotic plaque macrophages.12,15 ApoE expression is upregulated by increases in cellular cholesterol content6–9 as well as by macrophage-activating growth factors and cytokines.31–33 Interestingly, 100% of atherosclerotic plaque quadrants exhibited HBP/vigilin and apoE colocalization in foam cell macrophages. A much smaller proportion of atherosclerotic plaque quadrants also had HBP/vigilin and apoE colocalization in nonfoam cell macrophages. The demonstration of HBP/vigilin and apoE coexpression by foam cell macrophages is consistent with in vitro observations that cell cholesterol loading upregulates expression of both proteins.
The observation that a few nonfoam cell macrophages expressed
both proteins suggests that these proteins also may be coordinately
regulated by other factors specific to plaques, such as
cytokines or growth factors. Though several in vitro studies
have elucidated how various plaque-associated cytokines, such
as TGF-ß1,33
IFN-
,31 and
macrophage-colony-stimulating factor,32
affect cellular apoE expression, the effect of these cytokines
on HBP/vigilin expression is, as yet, unknown. Alternatively, these
nonfoam cell macrophages may represent cells that have
recently unloaded most of their sterol but have not yet downregulated
their expression of apoE and HBP/vigilin.
The function of HBP/vigilin is unknown. Most studies suggest that HBP
may be a complex protein with multiple functions. This protein contains
14 repeated KH domains that are usually found in nucleic acid–binding
proteins, including the FMR protein involved in fragile X mental
retardation and the -poly (C)-binding protein associated with the
-globulin mRNA ribonucleic protein complex.23
The KH domains in HBP/vigilin have been shown to interact directly with
polynucleotides.24 HBP/vigilin
has also been found to be associated with cytoplasmic t-RNA-protein
complexes isolated from human liver cells21 and
from Xenopus liver bound to vitellogenin
mRNA.22 HBP/vigilin has also been reported to
contain a functional nuclear localization sequence and to be
present in the nucleus.25 On the basis of
these findings, it has been postulated that HBP/vigilin has several
functions related to RNA metabolism, including protecting
tRNA,21 stabilizing mRNA,22
and transporting RNA between the nucleus and
cytoplasm.25 Despite these RNA-binding properties
and the lack of a classic membrane-spanning domain, HBP/vigilin is
tightly associated with cellular
membranes,16,18,25 partially localizes to the
plasma membrane when overexpressed in cells,16
and was identified in a screen for adipocyte surface proteins (Scherer
PE, Bichel PE, Kotler M, Lodish HF, unpublished observations, 1997).
Moreover, overexpression of HBP/vigilin increases HDL binding to
cultured cells,16 and antibodies for HBP/vigilin
can inhibit cell surface binding of HDL.34 These
results raise the possibility that HBP/vigilin may play some role in
recognition of extracellular molecules, particularly HDL.
Expression of HBP/vigilin appears to be regulated tightly and varies considerably between tissues and cell types.16–22 It has been suggested that expression of HBP/vigilin correlates with the degree of protein production among various tissues.21 This hypothesis, however, does not explain why cholesterol loading of cultured J774 macrophages would cause a greater than sixfold increase in HBP/vigilin mRNA levels16 under conditions in which a net increase in total protein synthesis would not be expected. Also, the degree of expression of HBP/vigilin among cells of atherosclerotic lesions appears to reflect variations in intracellular lipid content. Interestingly, hearts of apoE-/- mice also contained lipid-laden myocytes that express higher levels of HBP/vigilin than do surrounding cells. In addition to cholesterol, estrogen and gonadotropin have been shown to induce HBP/vigilin mRNA in Xenopus liver and rat ovaries.20,22 One possible explanation for these findings is that HBP/vigilin plays a role in coupling sterol metabolism to protein synthesis.
In summary, this study demonstrates the following novel findings regarding HBP/vigilin expression: (1) HBP/vigilin expression is constitutive in endothelium, (2) nonendothelial HBP/vigilin expression is specific for atherosclerosis, (3) HBP/vigilin expression is associated with lipid accumulation in macrophages but also is detected in a minor subset of macrophages without lipid accumulation, (4) HBP/vigilin expression in macrophages is associated with apoE expression in human atherosclerotic plaques, and (5) HBP/vigilin is expressed in lipid-laden cardiac myocytes of apoE-/- mice. Also, we demonstrate the utility of double labeling with ORO and cell-specific antibodies for identifying foam cell types within atherosclerotic lesions. The findings that HBP/vigilin is expressed in human atherosclerotic lesions, that it is coexpressed with apoE, and that is expressed in murine cardiac foam cells suggest a role for HBP/vigilin in cellular cholesterol metabolism. Further studies are required to elucidate the precise mechanisms by which it participates in these processes.
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Acknowledgments |
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This study was supported in part by Grants-in-Aid 94-WA-518R and 96-WA-304 (KDO) from the American Heart Association, Washington Affiliate, Seattle, WA, and by grants DK02456 (KDO), HL02788 (KDO), HL18645 (JFO), HL50367 (RCL, JFO), and HL47151 (CEA) from the National Institutes of Health, Bethesda, MD. The authors gratefully acknowledge Lisa Anne Billings for assistance in manuscript preparation and Winnie Chiu, Marina Ferguson, Susan Rozell and Randy Small for expert technical support.
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Footnotes |
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Division of Cardiology (D.S.C, K.D.O), Division of Metabolism, Endocrinology and Nutrition (J.F.O., R.C.L.), Department of Medicine; Department of Nutritional Science (R.C.L.); Department of Pathology (C.E.A.) University of Washington, Seattle, Wash.
Presented in part to the Western Section, American Federation for Clinical Research, Carmel, CA, February 11, 1995, and to the American Federation for Clinical Research, San Diego, CA, May 6, 1995.
Received May 23, 1996; accepted August 19, 1997.
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