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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2369-2375

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2369-2375.)
© 1997 American Heart Association, Inc.

Articles

Scavenger Receptors are Present on Rabbit Aortic Endothelial Cells In Vivo

Alan Daugherty; Joseph A. Cornicelli; Kathryn Welch; Sandra M. Sendobry; ; Debra L. Rateri

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


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
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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


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Methods
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 up arrowIntroduction
 *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.


*    Results
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 up arrowMethods
 *Results
down arrowDiscussion
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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 1ADown). 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 1BDown).



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  		Figure 1. Determination of antibody titres to scavenger receptor peptide–coated plates in sheep serum by ELISA. A, Immunoreactivity against the cytoplasmic tail–region peptide in the three sheep injected with this peptide ({blacktriangleup}), the three sheep injected with the collagen-like–region peptide ({blacksquare}), nonimmune serum ({bullet}), and after exposure to the secondary antibody alone ({blacktriangledown}). 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 2ADown) and collagen-like regions (Fig 2BDown) 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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  		Figure 2. Effects of preincubation of specific and irrelevant peptides on the immunoreactivity of antiserum by ELISA. A, Immunoreactivity against the scavenger receptor cytoplasmic tail for nonimmune serum (open bars). The remaining bars illustrate immunoreactivity of the antiserum raised against the cytoplasmic tail peptide (solid bars), after adsorption with the cytoplasmic tail peptide (horizontal-lined bars), or in the presence of rabbit LDL receptor peptide (cross-hatched bars). B, Similar data for immunoreactivity against the collagen-like region. Bars represent the mean of duplicate determinations.

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{approx}15 kD, whereas the collagen-like domain was {approx}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 3Down). 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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  		Figure 3. Western blot analysis of scavenger receptor antiserum against fragments of rabbit scavenger receptor protein expressed in bacteria. Antiserum against the cytoplasmic tail (A) and against the collagen-like region (B; both 1:300 dilution) was examined against whole-cell extracts of bacteria expressing the entire cytoplasmic domain (lane 1) and collagen-like domain (lane 2; both {approx}1 µg protein per lane). Reactivity was compared with that of nonimmune serum at the same dilution (lane 3). Reactivity against the expressed scavenger receptor fragments that included the peptide sequence used as antigen was also evaluated after preincubation of the antiserum with a solution of the same expressed fragment (lane 4).

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 {approx}220 and 150 kD exhibited specific reactivity (Fig 4ADown). 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 {approx}70 kD for both antisera, corresponding to the monomeric form (Fig 4BDown).



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  		Figure 4. Western blot analysis of scavenger receptor antiserum against liver membrane proteins. Liver membrane was isolated as described by Via et al 15 . Liver membrane protein (15 µg) was electrophoresed under nonreducing (A) and reducing (B) conditions and transferred to PVDF membranes. Reactivity was determined for antisera against the cytoplasmic tail (lane 1) and collagen-like region (lane 2). Reactivity of the nonimmune serum was also examined (lane 3) at the same dilution as the antisera (1:500). Under nonreducing conditions, major bands of immunoreactivity for serum from injected sheep corresponded to proteins of approximately 220 and 150 kD, consistent with the trimeric and dimeric forms of the scavenger receptor. Under reducing conditions, a single band of approximately 70 kD was detected for both antisera.

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 5ADown and 5BDown 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 5CDown 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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  		Figure 5. Immunoreactivity of scavenger receptor antiserum against sections of rabbit aorta. Tissue was perfusion fixed in situ, immersion fixed, and paraffin embedded as described in "Methods." Sections were incubated with the indicated antiserum at a dilution of 1:1000. Immunoreactivity was visualized with the red chromogen amino ethyl carbazole and counterstained with hematoxylin. Sections are representative of those incubated with antiserum against the collagen-like domain (A), antiserum against the cytoplasmic tail region (B), and nonimmune serum (C).

Immunocytochemical studies were performed with the preadsorbed serum (Fig 6Down). 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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  		Figure 6. Immunoreactivity of scavenger receptor antiserum against sections of rabbit aorta in the presence of specific or irrelevant peptides. Antiserum was incubated in the presence of the immunizing peptide or a peptide of the rabbit LDL receptor, as described in Fig 2AUp and 2BUp. A and B, Examples of reactivity of antiserum against the cytoplasmic tail peptide. C and D, Examples of reactivity of antiserum against the collagen-like peptide. Incubations shown in A and C were performed in the presence of specific scavenger receptor peptides, whereas sections in panels B and D were incubated in the presence of rabbit LDL receptor peptide. Adsorption with the specific peptide markedly attenuated immunostaining, while the irrelevant peptide was without effect.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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{gamma}-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.


*    Acknowledgments
 
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.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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