Role of Leukocyte-Specific LDL Receptors on Plasma Lipoprotein Cholesterol and Atherosclerosis in Mice
Bone marrow–derived macrophages and lymphocytes express LDL receptors (LDL-R), which allow these cells to take up cholesterol-rich lipoproteins. Although these cells are ubiquitously distributed in the body, it is not known whether they influence plasma cholesterol. Macrophages and T lymphocytes also are found in atherosclerotic lesions, but it is not known whether their LDL-R expression plays a role in atherosclerosis. To address these questions, we subjected LDL-R −/− mice to total body irradiation to eliminate their endogenous bone marrow–derived cells and repopulated them with either LDL-R–expressing wild-type bone marrow (treated mice) or LDL-R −/− bone marrow (control mice). Thus, the only difference between the two groups of mice was the ability of the bone marrow–derived cells to express the LDL-R in the treated mice. Plasma cholesterol levels were similar in the two groups of mice at 8 and 16 weeks after transplantation. Chromatographic separation of the lipoproteins revealed similar lipoprotein cholesterol distributions. Although the extent of lesion area in the aortic valves of the high-fat-diet–fed mice was more severe than that in the chow-fed mice, lesions appeared similar between control and treated mice given either chow or high-fat diet. Abundant LDL-R expression was detected in the lesions of treated mice, whereas the lesions of control mice showed no LDL-R expression, indicating that donor-derived leukocytes had migrated into the lesions of the recipient mice. Thus, bone marrow transplantation can be used as a tool to replace the endogenous bone marrow–derived cells in the artery wall with those of the donor origin.
- Received December 28, 1995.
- Revision received June 19, 1996.
In humans, the LDL-R plays a crucial role in clearing atherogenic LDL particles from the circulation. The importance of this receptor is evidenced by the condition known as homozygous FH, which is characterized by lack of functional LDL-R.1 In these patients, plasma cholesterol levels typically reach 600 to 1200 mg/dL. Prominent atherosclerosis as well as cholesterol-rich cutaneous xanthomas are exhibited at an early age.1 An LDL-R −/− strain of mice was created by Ishibashi et al2 by homologous recombination in embryonic stem cells. These mice exhibit hypercholesterolemia with a twofold higher total plasma cholesterol than normal mice. This increase in plasma cholesterol is due to sevenfold to ninefold higher levels of IDL and LDL. HDL levels are slightly higher than those of wild-type mice.
The relatively moderate increase in serum cholesterol of the LDL-R −/− mice compared with that in human homozygous FH patients can be attributed to differences in lipoprotein metabolism between humans and mice. In humans, the liver synthesizes apo B100 that is incorporated into newly synthesized VLDL. VLDL is catabolized to IDL and eventually to LDL, which binds to the LDL-R via apo B100. Liver-derived mouse VLDL, however, contains ≈70% apo B48, a truncated version of apo B100 that lacks the LDL-R binding domain.3 Because of the large amount of apo B48 in mouse VLDL, it is cleared the same way as intestine-derived chylomicron remnants, which contain apo E that can be taken up by both chylomicron remnant receptors4 and LDL-R. For this reason, LDL does not accumulate to any significant degree in wild-type mice compared with normal humans. Thus, although LDL accumulates in the LDL-R −/− mice, this accumulation is not as severe as that found in human homozygous FH subjects.
The feasibility of using permanent gene transfer via bone marrow transplantation to correct severe hypercholesterolemia and the accompanying complications of atherosclerosis has been previously examined in mice. We and others have reported successful transfer of the apo E gene into apo E knockout mice by bone marrow transplantation.5 6 This gene transfer completely reversed the severe hypercholesterolemia of apo E-deficient mice and was accompanied by a clear decrease in the extent and severity of atherosclerosis.5 6 Therefore, we used this same method of permanent gene transfer to transfer the LDL-R gene into LDL-R knockout mice. If successful, this transplantation of wild-type bone marrow into LDL-R −/− mice should result in a rapid (within 4 weeks) decrease in plasma cholesterol. Among bone marrow–derived cells, macrophages7 8 9 10 11 and lymphocytes12 express the LDL-R under defined conditions. In these cells, as in all cells, LDL-R expression is strictly regulated by intracellular cholesterol levels13 along with HMG-CoA reductase, which is the rate-limiting enzyme for intracellular cholesterol synthesis. Binding of LDL to human peripheral blood lymphocytes has been demonstrated.14 LDL-R expression is induced severalfold in vitro by incubation of lymphocytes in lipoprotein-deficient medium.15 Furthermore, when endogenous cholesterol synthesis is blocked by adding mevinolin (an inhibitor of HMG-CoA reductase) to lipoprotein-deficient medium, mitogen-stimulated proliferation of T cells is markedly inhibited.16 The expression of LDL-R by monocyte/macrophages is dependent on both their state of differentiation and activation. Although mouse peritoneal macrophages take up relatively little unmodified LDL, peritoneal macrophages take up β-VLDL and chylomicron remnants, and this can be facilitated by the LDL-R.17 Unmodified LDL can cause moderate cholesteryl ester accumulation mediated by the LDL-R in human monocyte-derived macrophages, THP-1 cells,18 and murine macrophage cell lines.11 Interestingly, the most abundant bone marrow–derived leukocyte, the polymorphonuclear leukocyte, has not been studied, and it is not known whether these cells express the LDL-R.
Although polymorphonuclear leukocytes are not found in atherosclerotic lesions, both macrophages and T lymphocytes are a prominent cell type, particularly in early and developing lesions.19 One of the best-characterized mechanisms by which macrophages within the lesion become foam cells is by ingesting modified LDL through the scavenger receptor–mediated pathway.20 Although it is often implied that the macrophage LDL-R does not play a significant role in atherogenesis, this has never been confirmed in vivo. Involvement of LDL-R in atherosclerosis may be important because activated T lymphocytes, capable of expressing the LDL-R, are present in the lesions,21 and macrophages can accumulate cholesteryl ester by the LDL-R–mediated process.11 18 Lymphocytes and/or macrophages found in lesions may be metabolically altered in some way to exhibit dysfunctional LDL-R downregulation. Such a mechanism was proposed by Tabas et al11 to explain the foam-cell formation induced by incubating native LDL with murine macrophage J774 cells.
Because peripheral-blood leukocytes express LDL-R and these bone marrow–derived cells are ubiquitously distributed in the body, we questioned whether these LDL-R–expressing cells could influence plasma lipoprotein cholesterol. In addition, we wished to determine whether LDL-R expression within an atherosclerotic lesion played a role in lesion development. To address these questions, we subjected LDL-R −/− mice to total body irradiation to eliminate their endogenous bone marrow–derived progenitor cells. The bone marrow was then repopulated by intravenous injection of syngeneic bone marrow cells obtained from either wild-type mice possessing normal LDL-R–expressing cells or LDL-R −/− mice. The latter served as a control for the rigors of irradiation and bone marrow transplantation. The only difference between the two groups of LDL-R knockout mice after transplantation was the expression of the LDL-R by all bone marrow–derived cells in the mice injected with wild-type bone marrow.
LDL-R −/− mice backcrossed onto the C57BL/6J background were purchased from Jackson Laboratories. Wild-type C57BL/6J mice were obtained from the rodent breeding facility of the Scripps Research Institute. The mice were weaned at 4 weeks of age and were fed ad libitum either a standard chow diet (diet 5015, Harlan Teklad) or an atherogenic diet (henceforth referred to as high-fat diet) containing 15.8% (wt/wt) fat, 1.25% (wt/wt) cholesterol, and 0.5% (wt/wt) sodium cholate (diet 88051, Harlan Teklad). The mice were housed in autoclaved filter-top cages with autoclaved water and kept on a 12-hour light-dark cycle, and all procedures were in accordance with institutional guidelines. Blood was taken after an overnight fast (12 to 13 hours) by retro-orbital puncture under Metofane-induced anesthesia. Plasma was separated by centrifugation of the blood samples at 3000g for 30 minutes at 4°C.
Irradiation and Bone Marrow Transplantation
Twelve 6-week-old male LDL-R knockout mice were subjected to 1000 rad total body irradiation to eliminate endogenous bone marrow stem cells and most of the bone marrow–derived cells. Bone marrow cells for repopulation were prepared from either wild-type C57BL/6J mice or LDL-R −/− mice as described previously.5 To control for the rigors of irradiation, 6 LDL-R −/− mice received bone marrow cells from LDL-R −/− mice (henceforth referred to as control mice). The other mice received LDL-R +/+ wild-type bone marrow cells (henceforth referred to as treated mice).
Plasma Cholesterol and Lipoprotein Analysis
Enzymatic measurements of plasma cholesterol and FPLC separation of lipoprotein particles were achieved as described earlier.5 FPLC separations were done with plasma samples pooled from either 6 mice per group (weeks 0 and 8, chow-fed animals) or 3 mice per group (week 16, chow- and high-fat-diet–fed animals). The cholesterol content of the FPLC fractions was measured enzymatically.
By the method of Chomczynski and Sacchi,22 total RNA was isolated from the liver, spleen, and peripheral blood mononuclear cells of chow-fed control and treated mice 20 weeks after bone marrow transplantation. Reverse transcription and PCR conditions were exactly as described for apo E transcripts5 except that a temperature of 58°C was used for primer annealing instead of 65°C. The primers used for LDL-R mRNA detection were designed to bind in the regions just upstream and downstream of the NEO cassette inserted into exon 4 to disrupt the LDL-R gene.2 The downstream primer (5′-ACACTGGAATTCATCAGGTC-3′) and upstream primer (5′-GCTGCAAATCATCCATATGC-3′) were synthesized by Operon. With this set of primers, wild-type LDL-R mRNA yielded a 275-bp product, whereas the mutant LDL-R mRNA yielded no product at all.
The presence of LDL-R antigen was assessed immunohistochemically with a rabbit anti-rat LDL-R polyclonal antibody (a generous gift of Dr Allen Cooper, Stanford University) that cross-reacts with mouse LDL-R. To detect LDL-R expression in the liver and spleen, the chow-fed control and treated mice were perfusion-fixed with 4% paraformaldehyde, and the tissues were dissected out and snap-frozen in OCT embedding compound in liquid nitrogen. Cryosections 6 μm thick were cut and dried overnight. The sections were fixed in acetone at −20°C for 2 minutes and immersed in PBS, pH 7.4, for 5 minutes to rehydrate the tissues. All subsequent incubations were done at room temperature in a humid chamber. The sections were incubated with normal goat serum (Vector Laboratories) diluted 1:50 in PBS with 0.1% BSA (PBS/BSA) for 30 minutes. The tissues were incubated with the anti–LDL-R antibody diluted 1:50 in PBS/BSA for 2 hours and washed for 30 minutes with three changes of PBS (3×10 minutes). The washed sections were incubated with biotinylated goat anti-rabbit IgG F(ab′)2 fragment (Vector Laboratories) diluted 1:200 in PBS/BSA for 1 hour and washed 3×10 minutes. The tissues were then incubated with FITC-conjugated streptavidin (PharMingen) diluted 1:200 in PBS/BSA for 30 minutes. The sections were washed 3×10 minutes in PBS and mounted with an aqueous mounting medium (Dako Corp). The slides were viewed under a fluorescence microscope equipped with appropriate emission and excitation filters to detect FITC staining.
LDL-R expression in the atherosclerotic lesions of the aortic valves was detected with the same strategy as above, with one exception. Instead of FITC-streptavidin, the sections were incubated with Vectastain ABC Elite solution (Vector Laboratories) and developed with aminoethylene carbazole (Vector Laboratories) according to the manufacturer's recommendations. The sections were counterstained with hematoxylin and mounted with a glycerol-based mounting medium (Dako Corp). This technique produced red positive staining and allowed the morphology of the lesions to show clearly.
Preparation and Examination of Aortic Valves for Atherosclerosis
To prepare the aortic valve sections, the mice were perfusion-fixed with formal-sucrose (4% paraformaldehyde and 5% sucrose in PBS, pH 7.4), and the heart was excised with a small portion of the ascending aorta remaining. The heart was cut horizontally parallel to the plane of the ascending aorta, and the basal half of the heart was immersed in PBS for 1 hour and fixed in formal-sucrose overnight. The heart was then embedded in OCT compound, snap-frozen in liquid nitrogen, and stored at −70°C until sectioning. Serial sections 10 μm thick were made through the entire aortic valve and the ascending aorta were collected on poly-l-lysine–coated slides. The extent of atherosclerosis was visually assessed by staining of the sections with oil red O (3 mg/mL oil red O in 60% acetone and 40% water), counterstaining with hematoxylin, and examination under a light microscope.
LDL-R Expression in Mice Receiving Bone Marrow Transplants
Repopulation of C57BL/6J mice undergoing total body irradiation and bone marrow transplantation has been observed within 5 weeks by FACS analysis of peripheral blood leukocytes.5 To detect by RT-PCR the expression of normal LDL-R mRNA in our wild-type bone marrow–transplanted LDL-R −/− mice, primers were designed to bind regions just upstream and downstream of the NEO gene insertion in exon 4, which was used to disrupt the LDL-R gene.2 This allowed detection of normal LDL-R mRNA, whereas no PCR product was obtained with the mutant mRNA. By use of RT-PCR, the expression of LDL-R mRNA was documented in the peripheral blood mononuclear cells, liver, and spleen of the wild-type bone marrow–transplanted mice (hereafter referred to as treated mice) 20 weeks after transplantation (Fig 1⇓). As expected, no LDL-R mRNA was detected in mice that received syngeneic bone marrow cells from an LDL-R −/− mouse (hereafter referred to as control mice). LDL-R antigen also was demonstrated in liver and spleen of the treated mice by immunohistochemical staining (Fig 2⇓). These organs of the LDL-R −/− bone marrow–transplanted control mice showed only background staining, whereas the treated mice displayed positive staining of a limited number of cells in both organs. This confirmed that the LDL-R gene was transferred successfully into the leukocytes of the treated mice and that these bone marrow–derived leukocytes expressed the LDL-R gene in liver and spleen.
Effect of LDL-R Expression on Plasma Lipoproteins
Total plasma cholesterol levels were considerably higher in all the LDL-R −/− mice fed a high-fat diet compared with those fed a chow diet (Fig 3⇓). However, the cholesterol levels were not different at any time between the bone marrow–transplanted control and treated groups fed either diet (Fig 3⇓). The cholesterol distribution of FPLC-separated lipoproteins from pooled plasma is shown in Fig 4⇓. The top panel displays the profile of a pooled C57BL/6J wild-type plasma, which was characterized by a preponderance of HDL cholesterol. Also shown in this panel is the chromatography of pooled plasma taken before bone marrow transplantation (week 0) from control and treated LDL-R −/− mice fed a chow diet. Compared with LDL-R +/+ wild-type mice, both chow-fed control and treated LDL-R −/− mice displayed greatly increased but similar amounts of VLDL and IDL/LDL cholesterol and somewhat higher levels of HDL cholesterol. The second and third panels of Fig 4⇓ show the profiles of pooled plasma from chow-fed LDL-R −/− control and treated mice at 8 and 16 weeks, respectively, after bone marrow transplantation. Unlike the week 0 FPLC separations, these later separations revealed a modest difference in the distribution of cholesterol among the IDL/LDL and HDL fractions between the two bone marrow–transplanted, chow-fed groups. The trend was observed as well with the pooled plasma samples taken 16 weeks after bone marrow transplantation (Fig 5⇓). Because only pooled plasma was analyzed, the statistical significance of this difference could not be determined. However, this pattern of distribution was not observed at week 0 (Fig 5⇓).
The lipoprotein profiles of high-fat-diet–fed mice 16 weeks after transplantation and 8 weeks after they were placed on a high-fat diet are displayed in the bottom panel of Fig 4⇑. The severe hypercholesterolemia in these mice was reflected by the higher range of cholesterol levels (relative fluorescence values). These high-fat-diet–fed mice had high levels of VLDL and IDL/LDL cholesterol and little HDL cholesterol. However, there were only slight differences in the cholesterol contents of the lipoprotein fractions between the two groups of high-fat-diet–fed mice when they were compared as shown in Fig 5⇑ (bottom panel). The small increase in plasma cholesterol seen in the pooled plasma of high-fat-diet–fed, treated mice was accounted for by an increase in their IDL/LDL cholesterol. Interestingly, although VLDL and IDL/LDL cholesterol levels in these animals were ≈15-fold and ≈5-fold higher, respectively, than the corresponding levels in the chow-fed control mice, their total HDL levels were only about half that of the chow-fed control mice.
Effect of Leukocyte LDL-R Expression on Atherosclerosis
Representative oil red O–stained frozen sections of the aortic valves from the four experimental groups of mice are displayed in Fig 6⇓. Although all sections were examined, these sections were taken from comparable regions of the aortic valve for direct visual comparison of the extent of lesion formation among the experimental groups. The early fatty-streak lesions of the chow-fed animals (Fig 6A and 6B⇓⇓) are seen as intensely red-stained regions near the valve stems, as identified by arrows. Compared with the high-fat-diet–fed mice, the atherosclerosis was considerably less pronounced in the chow-fed mice and covered only small areas of the aortic valve. The lesions of the high-fat-diet–fed mice were inordinately prominent and covered most of the opening in the valve (Fig 6C and 6D⇓⇓). However, the extent of lesions appeared similar between the control and treated groups fed either the chow or the high-fat diet.
Aortic valve cryosections from the high-fat-diet–fed, LDL-R −/− mice were analyzed immunohistochemically for the presence of LDL-R antigen in the lesions using a polyclonal antibody against rat LDL-R that cross-reacts with mouse LDL-R (Fig 7⇓). Panels A and B display sections from a high-fat-diet–fed, LDL-R −/− control mouse, which, as expected, expressed no LDL-R antigen. Aortic valve sections from a high-fat-diet–fed, LDL-R −/− treated mouse showing intense staining of the LDL-R antigen are shown in panels C and D. Because bone marrow–derived cells were the sole source of LDL-R in the treated mice, the intense staining reflects the expression of LDL-R by the leukocytes that had migrated into the lesion. Furthermore, all bone marrow–derived cells, including lymphocytes and macrophages, appeared to be expressing the LDL-R, because no specific localization of the staining pattern to macrophage- versus lymphocyte-predominant regions was evident.
Although hepatic LDL-R play a pivotal role in plasma LDL clearance, their specific role in either cholesterol metabolism or atherogenesis expressed by bone marrow–derived cells is unknown. Bone marrow–derived cells give rise to monocytes, which become resident macrophages in connective tissue (histiocytes), liver (Kupffer cells), lungs (alveolar macrophages), peritoneal cavity (serosal macrophages), bone (osteoclasts), brain (microglia), and kidney (intraglomerular mesengial macrophages). Bone marrow cells also give rise to all lymphoid and erythroid tissues, including lymph nodes, thymus, and spleen. Bone marrow–derived cells synthesize LDL-R and are found ubiquitously throughout the body. Therefore, these cells may have an impact on lipoprotein clearance from plasma by their ability to take up and degrade apo B– and apo E–containing lipoproteins through the action of the LDL-R. Furthermore, LDL-R expressed by monocytes and T lymphocytes may affect lesion development after their migration into the microenvironment of the vascular intima. To address these questions, we performed bone marrow transplantations on irradiated LDL-R −/− mice with either wild-type bone marrow cells that expressed LDL-R or, to serve as controls, syngeneic LDL-R −/− bone marrow cells. Although all mice underwent total body irradiation and bone marrow transplantation, control mice lacked the LDL-R, whereas in treated mice the LDL-R was expressed exclusively by all bone marrow–derived cells. Subsequently, the mice were fed a standard chow diet for 20 weeks or a chow diet for 8 weeks and then a high-fat diet for an additional 12 weeks. At the end of 20 weeks, RT-PCR and immunohistochemical analyses were used to confirm the expression of LDL-R mRNA and antigen, respectively, in the treated mice.
Although LDL modified extensively by negative charge is readily taken up by macrophage scavenger receptors,23 native LDL also can be taken up by the LDL-R in extrahepatic tissues, such as macrophages, and this can, in some instances, result in cholesteryl ester accumulation. Human peripheral blood monocytes,8 macrophages,18 and murine and human macrophage cell lines10 11 can take up native LDL. The uptake of this LDL is facilitated by the LDL-R and is inhibited by treatment of the cells with an acyl coenzyme A:cholesterol acyl transferase inhibitor.24 Thus, it is feasible that the bone marrow–derived cells in our chow-fed, treated mice participated in the removal of LDL from plasma. Furthermore, T lymphocytes in the chow-fed, treated mice probably utilized a portion of the LDL for their cholesterol requirement by synthesizing the LDL-R. T lymphocytes obtain exogenous cholesterol by binding and catabolizing LDL via the LDL-R.15 However, when exogenous lipoproteins are limited, T lymphocytes are capable of de novo synthesis of the cholesterol they require for proliferation.15 16 Thus, in the case of the LDL-R −/−, chow-fed, control mice, the T cells probably synthesized the cholesterol they needed de novo. Nevertheless, our studies demonstrated that extrahepatic LDL-R expression had no influence on total plasma cholesterol levels. This was probably because only cholesterol that is delivered to hepatocytes can be converted to bile acids for excretion from the body.
No difference in the lipoprotein cholesterol distribution was observed in the high-fat-diet–fed mice either. However, whether LDL-R is expressed and, if so, the role that it may play in lesions of the vessel wall were addressed by examination of the large prominent lesions of the high-fat-diet–fed animals. In the high-fat-diet–fed, treated mice, we detected abundant expression of LDL-R in the region of the lesions in which macrophages and T cells are present. As expected, the layer of smooth muscle cells forming the fibrous cap did not express the LDL-R, because only bone marrow–derived cells would express the LDL-R. Also as expected, expression was not detected in the lesions of the control mice. The extent of LDL-R expression in the lesions of our high-fat-diet–fed, treated mice was surprising in light of findings that LDL-R expression in cultured macrophages9 13 25 and T lymphocytes15 are downregulated by added LDL. Moreover, LDL diffuses freely into and out of the artery wall, and the concentration of LDL can be twice as high in the intima as in the plasma.26 Thus, we expected that LDL-R expression would be minimal within the lesion. However, LDL-R staining was observed in the LDL-R −/− treated mice, suggesting that LDL-R antigen was present in the bone marrow–derived cells within the lesions of these animals. Other investigators have reported that LDL-R expression in macrophage cell lines such as J774 is resistant to LDL-mediated downregulation and that foam cell formation is possible by metabolically altered macrophages interacting with native LDL.11 Therefore, our observation may mean that the lesion macrophages were altered in some way by the lesion microenvironment so as to express the LDL-R even in the presence of large amounts of lipid and cholesterol within the lesion. However, because no direct comparisons could be made between the staining intensity of upregulated versus downregulated LDL-R expression in intimal macrophages and T cells, no conclusion could be made regarding the degree of LDL-R expression observed. Further studies are necessary to understand the structural properties, functional integrity, and regulation of the LDL-R in the lipid-rich environment of the atheromatous lesion.
To the best of our knowledge, this is the first attempt to assess the role of bone marrow–derived LDL-R expression within the microenvironment of the atheromatous lesion. The fact that both high-fat-diet–fed control and treated mice developed extensive atherosclerosis in the aortic valves despite the clear absence and presence of LDL-R expression in the control and treated mice, respectively, indicates that LDL-R expression by the lesion leukocytes may not play a major role in lesion development. Despite previous reports asserting that macrophage uptake of β-VLDL, chylomicron remnants, and unmodified LDL mediated by the LDL-R can lead to foam cell formation, these mechanisms do not appear to contribute to any significant degree to lesion formation in vivo, based on the findings in this report. The expression of scavenger receptors, well known for their involvement in cholesterol accumulation by the macrophages in the lesion by taking up modified LDL,20 was not studied in the present report but probably played a prominent role in the lesions of both control and treated mice.
In the studies reported here, the LDL-R gene was permanently transferred into LDL-R −/− mice by bone marrow transplantation. The apo E gene was also permanently transferred into apo E–deficient mice by bone marrow transplantation.5 6 However, the effectiveness of macrophage-derived apo E in facilitating hepatic clearance of cholesterol in apo E–deficient mice was not duplicated with bone marrow–derived cell expression of the LDL-R in LDL-R −/− mice. Presumably, the importance of macrophage-derived apo E for hepatic clearance of cholesterol resides in the fact that apo E is secreted by Kupffer cells in the liver in amounts sufficient to facilitate hepatocyte clearance, whereas macrophage expression of the LDL-R in the liver of LDL-R −/− mice played no role in the delivery of cholesterol to hepatocytes.
In summary, this study demonstrated that expression of LDL-R in bone marrow–derived cells by transplantation of wild-type bone marrow into the LDL-R −/− mice had no effect on total plasma cholesterol. Although LDL-R expression was clearly absent in the lesions of the control mice and present in the lesions of the treated mice, this did not seem to affect lesion formation. This study indicates that bone marrow transplantation can be used to replace the bone marrow–derived leukocytes in the artery wall with cells from a donor origin, which would allow studies of the role of specific donor leukocytes in lesion development.
Selected Abbreviations and Acronyms
|FPLC||=||fast protein liquid chromatography|
|LDL-R −/−||=||LDL-R knockout|
|RT-PCR||=||reverse-transcription polymerase chain reaction|
This work was supported by National Institutes of Health (NIH) grant HL-35297 to Dr Curtiss. Dr Spangenberg is supported by a fellowship from the California Affiliate of the American Heart Association and Dr Boisvert by NIH training grant AI-07244. The authors thank Dr Allen Cooper for generously providing us with the anti–LDL-R antibody and Anna Meyers for administrative assistance. This is manuscript 9716-IMM from The Scripps Research Institute.
Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. New York, NY: McGraw-Hill Inc; 1995:1981-2030.
Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hyper-cholesterolemia in LDL receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993;92:883-893.
Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest. 1994;93:1885-1893.
Higuchi K, Kitagawa K, Kogishi K, Takeda T. Developmental and age-related changes in apoprotein B mRNA editing in mice. J Lipid Res. 1992;33:1753-1764.
Boisvert WA, Spangenberg J, Curtiss LK. Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation. J Clin Invest. 1995;96:1118-1124.
Linton ME, Atkinson JB, Sergio F. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science. 1995;267:1034-1037.
Fogelman AM, Schecter I, Seager J, Hokom M, Child JS, Edwards PA. Malondialdehyde alterations of low density lipoproteins leads to cholesterol ester accumulation in human monocyte-macrophages. Proc Natl Acad Sci U S A. 1980;77:5466-5470.
Traber MG, Kayden HJ. Low density lipoprotein receptor activity in human monocyte-derived macrophages and its relation to atheromatous lesions. Proc Natl Acad Sci U S A. 1980;77:5466-5470.
Fogelman AM, Haberland ME, Seager J, Hokom M, Edwards PA. Factors regulating the activities of the low density lipoprotein receptor and the scavenger receptor on human monocyte-macrophages. J Lipid Res. 1981;22:1131-1141.
Tabas I, Weiland DA, Tall AR. Unmodified low density lipoprotein causes cholesteryl ester accumulation in J774 macrophages. Proc Natl Acad Sci U S A. 1985;82:416-420.
Curtiss LK, Edgington TS. Identification of a lymphocyte surface receptor for low density lipoprotein inhibitor, an immunoregulatory species of normal human serum low density lipoprotein. J Clin Invest. 1978;61:1298-1308.
Cuthbert JA, Russell DW, Lipsky PE. Regulation of low density lipoprotein receptor gene expression in human lymphocytes. J Biol Chem. 1989;264:1298-1304.
Cuthbert JA, Lipsky PE. Provision of cholesterol to lymphocytes by high density and low density lipoproteins: requirement for low density lipoprotein receptors. J Biol Chem. 1987;262:7808-7818.
Ellsworth JL, Kraemer FB, Cooper AD. Transport of β-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway. J Biol Chem. 1987;262:2316-2325.
Banka CL, Black AS, Dyer CA, Curtiss LK. THP-1 cells form foam cells in response to coculture with lipoproteins but not platelets. J Lipid Res. 1991;32:35-43.
Tabas I, Weiland DA, Tall AR. Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced accumulation. J Biol Chem. 1986;261:3147-3155.
Schechter I, Fogelman AM, Haberland ME, Seager J, Hokom M, Edwards PA. The metabolism of native and malondialdehyde-altered low density lipoproteins by human monocyte-macrophages. J Lipid Res. 1981;22:63-71.