© 1997 American Heart Association, Inc.
Articles |
Scavenger Receptors are Present on Rabbit Aortic Endothelial Cells In Vivo
From the Cardiovascular Division, Department of Medicine (A.D., S.M.S., D.L.R.), and the Department of Biochemistry and Molecular Biophysics (A.D.), Washington University School of Medicine, St Louis, Mo; and the Department of Vascular and Cardiac Diseases, Parke-Davis, Ann Arbor, Mich (J.A.C., K.W.).
Correspondence to Alan Daugherty, Division of Cardiovascular Medicine, University of Kentucky, L-543 KY Clinic, Lexington, KY 40536. E-mail adaug0{at}pop.uky.edu
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Abstract |
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Abstract Endothelial cells metabolize modified LDL, but attempts to detect scavenger receptors in this cell type in vitro have been unsuccessful. To determine whether scavenger receptors are present on endothelial cells in vivo, species-specific reagents were developed to detect rabbit scavenger receptor protein. Antiserum against the rabbit scavenger receptor was generated with the use of synthetic peptides of two distinct regions: residues 3 to 21 in the cytoplasmic tail and residues 282 to 304 in the collagen-like region. Reactivity of antiserum against the synthetic peptides was confirmed with an enzyme-linked immunosorbent assay. Positive reactivity was also observed against fragments of scavenger receptor protein expressed in bacteria. Antiserum to both regions reacted with liver membrane proteins of sizes consistent with the scavenger receptor, as confirmed by Western blotting under reduced and nonreduced conditions. Immunocytochemical examination of rabbit aortic tissue by use of antiserum to both regions of scavenger receptor protein produced striking and identical patterns of staining of aortic endothelium. Immunostaining was abolished for both antisera by preadsorption with the specific peptide region used as immunogen. In contrast, incubation of scavenger receptor antiserum with a peptide of a region of the rabbit LDL receptor failed to influence immunoreactivity against endothelium. These data demonstrate the presence of scavenger receptors in rabbit endothelium in vivo, which may have fundamental implications for lipoprotein metabolism by the arterial wall.
Key Words: scavenger receptors • endothelium • rabbits • immunocytochemistry
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Introduction |
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The development of atherosclerotic lesions is characterized by the unregulated intracellular deposition of lipids. Macrophages are the most common cell type to accumulate excessive lipid in lesions, although smooth muscle cells are also well-known foam cell progenitors.1 2 3 While it has not been widely reported, lipid droplets have also been observed in the endothelial cells that cover developing atherosclerotic lesions.4 Excessive intracellular lipid deposition is assumed to be derived from unregulated receptor-mediated entry of modified lipoproteins.5 6 Scavenger receptors represent one potentially important mechanism for the cellular recognition of modified lipoproteins. Consistent with their possible involvement in atherogenesis, both scavenger receptor mRNA and protein have been detected in atherosclerotic lesions.7 8 9 10 11 12 13
Scavenger receptors were originally defined as mediators of the metabolism of acetylated LDL (AcLDL) by cultured macrophages.14 Scavenger receptor protein was identified in phorbol ester–stimulated cultured murine macrophages15 and isolated from bovine liver.16 The gene was subsequently cloned from cells from a number of species, including cows,17 18 humans,19 mice,20 and rabbits.20 21 While scavenger receptors are commonly considered to be restricted to macrophages, AcLDL is also metabolized by smooth muscle cells and fibroblasts. Both scavenger receptor protein and mRNA have been detected in these cell types.22 23 The ability of endothelium to metabolize modified forms of LDL both in vitro and in vivo has also been documented.24 25 26 27 Furthermore, the accumulation of fluorescent dye–labeled AcLDL28 has been used to identify and isolate cultured aortic endothelium.29 30 Thus, there is abundant evidence that modified forms of LDL are catabolized by endothelial cells, but scavenger receptor mRNA has not been detected in this cell type in culture.21 However, this may be due to the limited sensitivity of the detection system rather than the absence of scavenger receptor mRNA.
In our study, species-specific antisera were generated against synthetic peptides of two distinct regions of the predicted rabbit scavenger receptor peptide sequence.20 21 In preliminary studies using these antisera to define scavenger receptors in atherosclerotic lesions, marked immunoreactivity was observed against endothelium. A formal study was initiated to substantiate the presence of scavenger receptor protein in endothelial cells on normal aortic intima in vivo since the presence of scavenger receptors on endothelial cells could profoundly influence the atherogenic process.
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Methods |
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Preparation of Liver Membrane Proteins and Aortic Tissue Sections
New Zealand White rabbits of both sexes, on normal diet, and weighing 2 to 3 kg were supplied by Shady Grove Rabbitry (Pacific, Mo). All procedures on animals were approved by the Washington University Animal Studies Committee. Rabbits were killed by administration of sodium pentobarbital (150 mg/kg) and exsanguinated via the abdominal aorta.
Liver tissue was excised and immediately flash-frozen in liquid nitrogen, then pulverized by use of a stainless steel pestle and mortar at the temperature of liquid nitrogen. Liver cell membranes were prepared by making a fresh liver homogenate in a suspension buffer (50 mmol/L Tris, pH 8.0, 160 mmol/L NaCl, 2 mmol/L EDTA, 23 µg · mL-1 PMSF, 1000 U · mL-1 aprotinin). Membrane proteins were then isolated and solubilized with an octylglucoside-containing buffer (40 mmol/L), as described previously.15 Total protein was quantified by the method described by Lowry et al.31 Protein solutions were stored at -20°C.
For immunocytochemical analyses, aortas were perfusion fixed in vivo with 4% wt/vol paraformaldehyde in PBS (0.05 mol/L phosphate, 0.154 mol/L NaCl, pH 7.5). Thoracic aortas were removed, sliced into rings, embedded in paraffin, and 2-µm sections placed on MicroProbe slides.
Generation of Antisera to Scavenger Receptor Peptides
Antisera were generated against peptides derived from two
distinct regions of the scavenger receptor protein corresponding to the
cytoplasmic tail (residues 3 to 21; QWDSFTDQEDTDSCSESV) and the
collagen-like domain (residues 281 to 304; KGDRGPTGESGPPGVPGPVGPPGL).
An additional cysteine residue was incorporated at the amino terminal
of the collagen-region peptide to facilitate coupling to the carrier
protein. Peptides were coupled to the keyhole limpet hemocyanin carrier
with the Imject Activated Immunogen Conjugation kit from
Pierce. This method uses a stable maleimide-activated carrier
protein that is capable of reacting with a sulfhydryl-containing
peptide. The coupled peptides were injected subcutaneously into Suffolk
sheep (Grantshire Farms, Brighton, Mich) at a concentration of 330
mg · mL-1 of Freund's complete
adjuvant. Booster injections were made with the same amount of peptide
in Freund's incomplete adjuvant on weeks 4, 16, and 28. Blood samples
were drawn at weeks 16 and 28 for determination of titer.
Reactivity of Antisera by Enzyme-Linked Immunosorbent
Assay
The enzyme-linked immunosorbent assay procedure was based on the
method described by Vector Laboratories. All incubations were performed
at room temperature, and plates were washed extensively between
additions. Briefly, 96-well microtiter plates (Nunc) were coated with
relevant or irrelevant peptide (0.5 µg per well) or PBS and left for
at least 4 hours. Plates were subsequently incubated with BSA (3%
wt/vol) for 2 hours to block nonspecific binding. Dilutions of sheep
serum ranging from 1:100 to 1:1 000 000 were added and incubated
overnight. Biotinylated anti-sheep IgG and conjugated avidin-biotin
complex were incubated for 1 hour each. The chromogen 2,2'-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid)-diammonium (Sigma Chemical
Company) was used to visualize reactivity. Further color development
was inhibited by the addition of sodium azide.
Bacterial Expression of Fragments of Rabbit Scavenger Receptor
Protein
Cytoplasmic (bases 1 to 150) and collagen-like (bases 331 to
993) regions of the rabbit scavenger receptor type I cDNA were
engineered with 5' Sal I and 3' Not I restriction
sites by polymerase chain reaction. Primer sets used to engineer the
restriction sites for the cytoplasmic region were
5'-CGCGGGGTCGACATGGCGCAGTGGGACAGC and
5'-CGCGGGGCGGCCGCTTTGAAGGATTTCAGCTT; those for the collagen-like region
were 5'-CGCGGGGTCGACCGAGAAGTTGTTATGGAAC and
5'-CGCGGGGCGGCCGCTATCCTGTCCTCCCAGTC. Resultant polymerase chain
reaction products were cloned into a pET23b system vector and
transformed into DE3 cells per manufacturer's instructions (Novagen).
Expressed fragments of rabbit scavenger protein were isolated according
to the manufacturer's recommendation.
Western Blot Analyses
Purified liver membrane proteins from normal rabbits and
fragments of scavenger receptor expressed in bacteria were subjected to
SDS–polyacrylamide gel electrophoresis under both reducing and
nonreducing conditions. Prestained markers were used for determination
of molecular weight (Bio-Rad Laboratories). Resolved proteins were
transferred to PVDF Plus membranes (Millipore Corp). Membranes were
blocked with powdered milk (5% wt/vol) in PBS for 1 hour at room
temperature. Membranes were incubated for 20 minutes at room
temperature in either sheep antiserum or nonimmune sheep serum diluted
in PBS plus milk (0.5% wt/vol). Horseradish peroxidase–conjugated
secondary antibody, also diluted in powdered milk buffer, was incubated
with the membranes for 15 minutes at room temperature. Membranes were
washed extensively with PBS containing Tween (0.05% vol/vol)
between successive incubations. Immunoreactive proteins were visualized
by chemiluminescent emission detection (ECL, Amersham Life Science)
with exposure onto autoradiography film.
Immunocytochemistry
Immunocytochemical analysis was performed as described
previously with a Fisher MicroProbe system.32 Tissue
sections were dewaxed after mild heating and incubation with
limolene/xylene (3:1), incubated with hydrogen peroxide to abolish
endogenous peroxidase activity, and extensively washed.
Sections were incubated with serum or antiserum at the indicated
dilutions for 15 minutes at 40°C. Immunoreactivity was detected with
biotinylated anti-sheep IgG and a biotin-avidin-peroxidase complex and
visualized after incubation with the red chromogen 3-amino-9-ethyl
carbazole (Biomeda). Control experiments were performed in which
chromogen alone was used to determine complete quenching of
endogenous peroxidase. Results with nonimmune sheep serum
(as with antisera, obtained from Grantshire Farms) were compared with
those with anti-scavenger receptor peptide–injected sheep serum to
determine the specificity of the primary antibody. Exclusion of primary
antibody allowed determination of nonspecific reactivity of the
secondary antibody.
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Results |
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Determination of Specificity of Immunoreactivity of the Two Antisera Against Rabbit Scavenger Receptor Protein
To allow characterization of scavenger receptor protein, antisera to two independent regions of the protein (the cytoplasmic tail region and the collagen-like region) were developed with use of synthetic peptides coupled to keyhole limpet hemocyanin and were injected into sheep. The specific sequences are described in the "Methods" section.
As a primary screen to determine antibody titer, sheep serum was tested
for reactivity against the immunizing peptide in an ELISA format. Serum
from three sheep injected with the cytoplasmic tail region peptide
reacted with this immunogen. Conversely, no reactivity was observed
against the cytoplasmic tail peptide in serum from sheep injected with
the collagen-like peptide (Fig 1A
).
Nonimmune serum also failed to react against the peptide, as did the
second antibody alone. Similar data were obtained from sheep injected
with the collagen-like domain when their serum was screened against the
immunizing peptide (Fig 1B).
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), the three sheep
injected with the collagen-like–region peptide (
), nonimmune
serum (
), and after exposure to the secondary antibody alone
(
). B, Immunoreactivity against the collagen-like–region
peptide.
To further characterize the specificity of these antisera,
immunoadsorption experiments were performed. The ability of specific
and nonspecific peptides to adsorb antiserum to the cytoplasmic (Fig 2A) and collagen-like regions (Fig 2B)
was determined by ELISA. Immunoreactivity of antiserum was effectively
reduced by prior incubation with the specific peptide. As expected,
prior incubation of antiserum with a peptide of part of the rabbit LDL
receptor had little effect on immunoreactivity to scavenger
receptor.
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Further validation of the immunoreactivity of the two antisera was
provided by Western blot analysis of fragments of scavenger
receptor expressed in bacteria. The expressed scavenger receptor
fragments of the cytoplasmic tail were
15 kD, whereas the
collagen-like domain was 29 kD (this includes the T7 and His Tag
regions). For both scavenger receptor regions, antiserum reacted
against the protein fragments that included the peptide sequence used
to generated them, but not against the other region (Fig 3). Nonimmune serum failed to react at
equivalent dilutions. Furthermore, the immunoreactivity against the
specific expressed protein on the membrane was ablated by preincubation
of the antiserum with a solution of the expressed protein.
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To characterize the reactivity of these antisera against native
scavenger receptor protein, Western blot analysis was performed
against liver membrane preparations under reduced and nonreduced
conditions. This analysis demonstrated that the same size
proteins were immunoreactive to both scavenger receptor antisera. Under
nonreducing conditions, two bands of 220 and 150 kD exhibited
specific reactivity (Fig 4A). The
molecular weights of these two proteins are consistent with the
trimeric and dimeric forms of the scavenger receptor. Under
reducing conditions, immunoreactivity was demonstrated against a single
band of 70 kD for both antisera, corresponding to the monomeric form
(Fig 4B).
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As further evidence of specific reactivity against rabbit scavenger receptors, immunostaining was performed on rabbit liver sections to determine whether antiserum recognition was similar to that described for scavenger receptor activity. In agreement with results of these ligand-based studies,25 26 27 immunoreactivity of rabbit liver sections was predominantly against hepatic endothelium (data not shown).
Immunocytochemical Detection of Scavenger Receptors in
Endothelium
Having established the specificity of the antisera to the two
regions of the scavenger receptor protein, immunocytochemical
analysis was performed on rabbit aortic sections that had been
perfusion fixed and paraffin embedded. Immunoreactivity was visualized
with the use of Vector ABC kits and the red chromogen amino ethyl
carbazole. Fig 5A and 5B shows reactivity
of antiserum at a dilution of 1:1000: panel A is immunoreactivity
against the collagen-like region and B that against the cytoplasmic
tail region. There was intense immunoreactivity of the
endothelial monolayer to antisera against both regions
of the scavenger receptor. Fig 5C shows the low background of
reactivity of nonimmune serum at the same dilution used in the other
reactions. Counterstaining with hematoxylin demonstrated the presence
of endothelial nuclei close to the intimal aspect of
the internal elastic lamina.
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Immunocytochemical studies were performed with the preadsorbed serum
(Fig 6). Immunostaining
of the cytoplasmic tail region is shown in panels A and B, and that of
the collagen-like region in panels C and D. Panels A and C demonstrate
the marked attenuation of immunostaining when antiserum
was previously incubated with the specific peptide used to generate the
antibody. In contrast, there was little demonstrable effect after
preincubation with an irrelevant peptide of the rabbit LDL receptor
(panels B and D).
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Discussion |
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Several investigators have demonstrated that modification of LDL by acetylation or maleyation promotes metabolism of the particles by endothelial cells both in vivo and in vitro.24 25 26 27 28 29 30 Such metabolic characteristics had been attributed to recognition by scavenger receptors. However, a number of other membrane proteins have been identified that may act as receptors for modified lipoproteins, including CD36,33 34 Fc
-RII-B2,35 macrosialin,36 37 a protein
structurally related to the c-type lectin family,38 and
other unidentified proteins.39 40 Thus,
metabolism of modified lipoproteins cannot be ascribed to a
specific receptor solely on the basis of recognition of a modified
lipoprotein. Our findings demonstrate that scavenger receptor protein is present in the endothelial monolayer of aortas of normolipidemic rabbits. The presence of the protein in endothelium in vivo was characterized by antisera that were developed against two distinct regions of the scavenger receptor by use of synthetic peptides as immunogens. The specificity of the antisera was defined initially by reactivity against the immunizing peptides. This immunoreactivity was further verified by Western blotting against fragments of scavenger receptors expressed in bacteria, and against the native protein under reducing and nonreducing conditions. The immunocytochemical staining on rabbit aortic tissue was identical for the antisera to the two regions of the scavenger receptor. Furthermore, this immunoreactivity was ablated by preadsorption with the immunizing peptide, but not by that with an irrelevant peptide of the rabbit LDL receptor. Thus, these results are consistent with presence of scavenger receptor on endothelial cells in vivo.
To our knowledge, only one study has directly compared the metabolism of modified LDL by cultured endothelial cells to the presence of scavenger receptors as identified by detection of mRNA.21 In contrast to our results, Bickell and Freeman21 were unable to detect the presence of scavenger receptor mRNA in cultured endothelial cells, possibly because of their use of Northern blotting, which is a relatively insensitive technique. The other major confounding factor in comparing these results is that we performed our experiments in endothelial cells in vivo, not in culture, as did Bickell and Freeman. It is generally accepted that some level of dedifferentiation occurs during culturing of cells that may generate a phenotype not necessarily representative of the cell in vivo. Examples of proteins that are expressed in lesser amounts or are absent from cultured endothelial cells include Ia antigen,41 von Willebrand factor,42 and the brain glucose transporter GLUT1.43 44 It is therefore not surprising that expression of scavenger receptors may differ when cultured endothelial cells are compared with those in vivo. Indeed, we have been unable to demonstrate the presence of scavenger receptor protein or mRNA in cultured rabbit endothelial cells (data not shown).
The function of the endothelium in the atherogenic process is a focus of considerable attention.45 It is now realized that the initiation of lesions does not require endothelial denudation.1 2 Instead, it is assumed that functional abnormalities in endothelium provide a substrate for atherosclerotic lesion formation. LDL-derived lipid enters the subendothelial space via a transcytotic process46 that does not involve LDL receptors47 and is not affected by the disease process.48 Aortic endothelial cells metabolize native LDL with high avidity49 by a mechanism that is influenced by the extent of endothelial confluence.50 Although endothelial cells are able to metabolize native LDL, one of the earliest events in atherogenesis is the deposition of extracellular lipid in the subendothelial space.51 52 53 This lipid deposition may be related to lipoprotein aggregation by physical and enzymatic events54 55 56 and retention of these modified lipoproteins by proteoglycans,57 58 the most prominent component in the subendothelial space of a normal artery.59 Subendothelial retention of modified lipoproteins has recently been proposed as the primary event in the development of atherosclerotic lesions.60 Indeed, aggregated lipoproteins present in an environment rich in proteoglycans may promote interactions with scavenger receptors.61 62 63 Therefore, the presence of scavenger receptors in endothelium could provide a mechanism for removal of modified lipoproteins from the subendothelial space and could account for the intracellular lipid inclusions that have been observed in this cell type in areas overlying atherosclerotic lesions.3
In conclusion, we have demonstrated that scavenger receptor protein is present on endothelial cells of normolipidemic rabbits in vivo. Further studies will be needed to determine the relative abundance of the type I and II forms of scavenger receptors in this cell type.17 18 The functional significance of scavenger receptors in the endothelium has not been defined but could be evaluated with genetic manipulations whereby scavenger receptor activity is modified in an endothelial cell–specific manner with a preproendothelin-1 promoter.64 Our current research addresses this issue, as well as the mechanisms of regulation in this cell.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health (HL-55487). Alan Daugherty is an Established Investigator of the American Heart Association. We thank Dr Kodama (University of Tokyo) for supplying the rabbit scavenger receptor cDNA, Marthelia Ellison for preparation of tissue sections, Beth Engeszer for editorial assistance, and Kelly Hall for secretarial assistance.
Received February 19, 1997; accepted May 12, 1997.
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