Published online before print March 3, 2005, doi: 10.1161/01.ATV.0000161420.55482.ef
© 2005 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Atherogenesis in Mice Does Not Require CD40 Ligand From Bone Marrow–Derived Cells
From Donald W. Reynolds Cardiovascular Clinical Research Center (U.B., A.Z., S.L., L.M., P.L., U.S.), Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; and Cardiology & Angiology (U.B.), Hannover Medical School, Germany. Current affiliation for Uwe Schönbeck: Boehringer Ingelheim Pharmaceutical Inc, Ridgefield, Conn.
Correspondence to Peter Libby, MD, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, NRB 741, Boston, MA 02115. E-mail plibby{at}rics.bwh.harvard.edu
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Abstract |
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Objective— Recent research suggests a central role for CD40 ligand (CD40L) in atherogenesis. However, the relevant cellular source of this proinflammatory cytokine remains unknown. To test the hypothesis that CD40L expressed on hematopoietic cell types (eg, macrophages, lymphocytes, platelets) is crucial to atherogenesis, we performed bone marrow reconstitution experiments using low-density receptor-deficient (ldlr–/–) and ldlr–/–/cd40l–/– compound-mutant mice.
Methods and Results— As expected, systemic lack of CD40L in hypercholesterolemic ldlr–/– mice significantly reduced the development of atherosclerotic lesions in the aortic arch, aortic root, and abdominal aorta compared with ldlr–/– mice. Furthermore, atheromata in ldlr–/–/cd40l–/– mice showed reduced accumulation of macrophages and lipids and increased content in smooth muscle cells and collagen compared with ldlr–/– mice. Surprisingly, reconstitution of irradiated ldlr–/– mice with ldlr–/–/cd40l–/– bone marrow did not affect the size or composition of atherosclerotic lesions in the root or arch of hypercholesterolemic ldlr–/– mice. Moreover, lipid deposition in the abdominal aorta diminished only marginally compared with mouse aortas reconstituted with ldlr–/– bone marrow.
Conclusions— These experiments demonstrate that CD40L modulates atherogenesis, at least in mice, primarily by its expression on nonhematopoietic cell types rather than monocytes, T lymphocytes, or platelets, a surprising finding with important pathophysiologic and therapeutic implications.
Although previous studies established CD40L as a mediator of atherogenesis, its relevant cellular source remains unknown. The present study demonstrates that CD40L modulates atherogenesis in mice, primarily by its expression on nonhematopoietic cell types in bone marrow chimeras. This surprising finding has important pathophysiologic and therapeutic implications.
Key Words: atherosclerosis • bone-marrow reconstitution • CD40 ligand • low-density lipoprotein receptor–deficient mice
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Introduction |
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The proinflammatory cytokine CD40L modulates experimental atherogenesis.1–3 Beyond its function in cell-mediated and humoral immunity,4 ligation of CD40 on vascular cells, eg, endothelial cells (ECs), smooth muscle cells (SMCs), and monocytes/macrophages (M
), induces the expression of a variety of mediators implicated in atherogenesis, including adhesion molecules, chemokines, cytokines, growth factors, matrix-degrading enzymes, coagulation factors, and others (eg, cyclooxygenase-2, caspase-1, serine proteinase inhibitor-9).1,5,6 In human as well as murine atherosclerotic lesions, the endothelium, SMC, M, and T cells express CD40L and its receptor CD40.7–9 Platelets also express functional CD40L.10 In vivo studies using hypercholesterolemic mice demonstrated the importance of CD40L for the development and progression of atherosclerotic lesions.11–14 In addition, CD40 signaling favors a plaque morphology associated in humans with elevated risk for rupture, a finding of potential clinical relevance.13,14 These findings suggested inhibition of CD40 signaling as a therapeutic target. Elevated plasma levels of the soluble form of CD40L correlate with future cardiovascular risk in healthy subjects or patients with acute coronary syndromes15–17 and with aspects of plaque composition.18 However, systemic inhibition of CD40 signaling would likely impair host defenses. Patients genetically deficient in functional CD40L have the hyper-IgM syndrome, have immunodeficiency, and usually die of severe infections in the first or second decade of life.19,20 Therefore, therapeutic targeting of CD40 signaling to mitigate atherosclerosis would require selective antagonism.
See page 1088
Development of such selective therapy would require knowledge of the relevant cellular source(s) of CD40L. Therefore, the present study tested whether CD40L expressed by hematopoietic cells (such as, monocytes/M, lymphocytes, and platelets) or by nonhematopoietic cells (including cells of the vascular wall, eg, ECs, SMCs) promotes the development of atherosclerotic lesions by bone marrow reconstitution of hypercholesterolemic ldlr–/– mice with bone marrow from ldlr–/–/cd40l–/– compound-mutant or ldlr–/– mice.
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Materials and Methods |
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Treatment of Mice
Low-density lipoprotein receptor (LDLR)-deficient and CD40L-deficient mice (C57Bl/6) were obtained from Jackson Laboratories (Bar Harbor, Me). Crossbreeding ldlr–/– mice with cd40l–/– mice generated ldlr–/–/cd40l–/– compound-mutant mice. The genotype of each mouse was verified by polymerase chain reaction (PCR) using genomic DNA (tail tip digest) and the following primer: LDLR, 5'-ACC CCA AGA CGT GCT CCC AGG ATG A-3' (sense), 5'-CGC AGT GCT CCT CAT CTG ACT TGT-3' (antisense); CD40L, 5'-CCC AAG TGT ATG AGC ATG TGT GT-3' (sense), 3'-GTT CCT CCA CCT AGT CAT trichloroacetic acid TC-3' (antisense); and neomycin-cassette, 5'-GCC CTG AAT GAA CTG CAG GAC G-3' (sense), 5'-CAC GGG TAG CCA ACG CTA TGT C-3' (antisense). In the first study group, 8- to 10-week-old ldlr–/– mice and ldlr–/–/cd40l–/–compound-mutant mice (each group, n=8) consumed a high-cholesterol diet (product D12108; Research Diets, New Brunswick, NJ; 1.25% cholesterol, 0% cholate) for 16 weeks. In the second study group, ldlr–/– mice (n=16) were lethally irradiated (2x600 rad, 3 hours apart) at the age of 8 to 10 weeks and were randomly assigned to receive bone marrow derived from ldlr–/– or ldlr–/–/cd40l–/– donor mice 6 to 8 weeks of age (both groups, n=8). Consumption of the high-cholesterol diet for 16 weeks began 8 weeks after successful bone marrow reconstitution. Subsequently, the mice were euthanized, and the hearts and aortas were removed and analyzed as described. The aortic roots and arches were frozen in OCT (OCT compound; Tissue-Tek, Torrance, Calif) and the thoracic and abdominal aortas were fixed in 10% buffered formalin, as described previously.11,13 In this protocol, successful reconstitution of bone marrow-derived cells was verified by transplanting bone marrow-derived cells from CD45.1-positive/CD45.2-negative mice into ldlr–/– mice (CD45.2-positive/CD45.1-negative). Overall reconstitution of peripheral blood cells was >95% (Figure 1a), and cell type-specific reconstitution was >98% for CD11b-positive cells (monocytic marker) and CD19-positive cells (B-cell marker), as well as >95% for CD3-positive cells (T-cell marker) (Figure 1b). Furthermore, successful reconstitution of the mononuclear cell-enriched splenic tissue with CD40L-deficient bone marrow-derived cells was verified by PCR, using the primers described, and genomic DNA was obtained from spleens harvested from the respective mice at the end of the experiment. In contrast to the recipients receiving CD40L wild-type bone marrow-derived cells, which yielded a strong signal for the CD40L wild-type allele, no PCR product was detected for the CD40L wild-type allele in recipients receiving CD40L-deficient bone marrow-derived cells (Figure 1c).
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All mice were housed under specific pathogen-free conditions and all procedures were approved by the Institutional Animal Care and Use Committee at the Harvard Medical School.
Fluorescence-Activated Cell Sorter Analysis
50 µL of mouse blood and 2 µL of Fc block (ebioscience) were diluted 1:1 in fluorescence-activated cell sorter (FACS) buffer (FACS buffer 2% bovine serum albumin, 0.1% sodium azide in phosphate-buffered saline) and incubated for 30 minutes at room temperature. Subsequently, the fluorescent-labeled antibodies were added for another 30 minutes at room temperature. Finally, 1 mL of FACS lysis buffer (Becton Dickinson) was added for 10 minutes and samples were washed with FACS buffer and analyzed by FACS. Antibodies for CD45.1-PE, CD45.2-PE, CD45.2-fluorescein isothiocyanate (FITC), CD11b-FITC, CD19-PECy, CD3-APC, as well as corresponding fluorescently labeled isotype controls were purchased from ebioscience.
Lipoprotein Measurement
Blood samples were collected by retro-orbital venous plexus puncture before assignment to study groups and also at the end of the experiment. Serum total cholesterol and triglyceride concentrations were assayed by enzymatic methods using Sigma Diagnostics (kits 401 and 343; Sigma-Aldrich, St. Louis, Mo, respectively), adapted for microtiter plate assay.
Immunohistochemistry
Serial cryostat sections (6 µm) of mouse aortic arches were fixed in acetone (–20°C, 5 minutes), air-dried, and stained by the avidin-biotin-peroxidase method, as previously described.13,21 After limiting endogenous peroxidase activity with 0.3% H2O2 and nonspecific binding of primary antibody with 5% species-appropriate normal serum (Vector Laboratories, Burlingame, Calif), sections were incubated with primary antibodies diluted in phosphate-buffered saline supplemented with 5% species-appropriate normal serum for 90 minutes at room temperature. Incubation with secondary antibodies for 45 minutes was followed by avidin-biotin complex (ABC; Vector Laboratories) for 30 minutes. The reaction was visualized with 3-amino-9-ethylcarbazole (ready-to-use AEC; DAKO Corp, Carpinteria, Calif), followed by counterstaining with Gill’s hematoxylin solution (Sigma-Aldrich). Controls for specificity used staining with the respective nonimmune IgG subclass (Pharmingen, Dako). Antibodies used were: rat anti-mouse Mac-3 (1:1000; Pharmingen, San Diego, Calif) and monoclonal anti-human smooth muscle 
-actin (1:100; Dako).
Picrosirius Red Staining for Type I Collagen
Formalin-fixed frozen sections were incubated for 4 hours in a freshly prepared 0.1% solution of picrosirius red F3BA (Polysciences Inc, Warrington, Pa) in saturated aqueous picric acid. After rinsing twice in 0.01 N HCl and distilled water, sections were briefly dehydrated in 70% ethanol and mounted in Permount (Vector Laboratories). Picrosirius red staining was analyzed by polarization microscopy.
Oil Red O Staining for Lipids
Deposition of lipids in en face preparations of abdominal aortas (fixed with 10% formalin) was determined by oil red O staining. Subsequently, the aortas were opened longitudinally to the aortic bifurcation, pinned on the surface of black wax with 0.2-mm steel pins, stained with oil red O solution (2.5 hours, room temperature), and washed 4 times in 85% propylene glycol solution. Formalin-fixed frozen sections of the aortic arch were stained after dehydration with propylene glycol with oil red O solution (25 minutes, 60°C).
Tissue Analysis
To quantify the extent and composition of the aortic lesions, longitudinal sections of the aortic arch as well as cross-sections of the aortic root were analyzed microscopically in all mice, as described previously.11,13 In the aortic arch, a 2-mm proximal segment of the inner curvature, starting at a perpendicular dropped from the left side of the left subclavian artery origin (Figure 2a), was analyzed for the total wall area. Within the aortic root, lesion areas were analyzed in cross-sections obtained at the level of all 3 leaflets of the aortic valve, immediately proximal to the right coronary artery ostium. The total aortic wall area, lesion area in the aortic root, and the percentage of area stained for M, lipids, SMCs, or collagen were determined via computer-assisted image quantification (ImagePro Plus Software; Media Cybernetics, Silver Spring, Md).
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ldlr–/–) or ldlr–/–/cd40l–/– mice (black bar, ldlr–/–/cd40l–/–En face analysis of lipid depositions in the pinned thoracic and abdominal aorta used measurement of the percentage of surface area (15 mm from the iliac bifurcation to the aortic section of the aorta) stained by oil red O (Figure 3a) by using computer-assisted analysis (ImagePro Plus Software).
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Statistical Analysis
Morphometric calculations of the tissue sections were performed independently by 2 blinded observers. Statistical analysis used the Statistical Package for Social Sciences. Data were presented as mean±SEM. Comparison of the respective study groups used the Student t test. A value of P<0.05 was considered significant.
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Results |
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The Development of Atherosclerotic Lesions Does Not Require Expression of CD40L by Bone Marrow–Derived Cells
To examine whether systemic deficiency of CD40L inhibits atherogenesis and confers characteristics associated with stability in human atheromata in hypercholesterolemic ldlr–/– mice (as found previously in mice treated with
-CD40L antibody),13,14 ldlr–/– mice and ldlr–/–/cd40l–/– compound-mutant mice consumed an atherogenic diet (1.25% cholesterol, 0% cholate) for 16 weeks and were analyzed for the extent of atherosclerosis in the aortic arch and the abdominal aorta and plaque morphology. Total serum cholesterol and triglyceride concentrations did not differ significantly between ldlr–/– mice and ldlr–/–/cd40l–/– compound-mutant mice on the atherogenic diet (total cholesterol 1372±64 mg/dL and 1581±172 mg/dL, total triglycerides 68±6 mg/dL and 53±4 mg/dL, respectively). Compared with ldlr–/– mice, systemic deficiency of CD40L yielded significantly reduced atherosclerotic lesion size in the aortic arch (0.41±0.04 versus 0.27±0.03 mm2 total wall area, respectively; P<0.01; Figure 2b) and lipid deposition in the abdominal aorta (17.2±2.7 versus 1.6±0.6% lipid-positive area; P<0.001; Figure 3b). Furthermore, systemic CD40L deficiency affected the composition of atherosclerotic lesions (Figure 4), as reported previously after -CD40L antibody treatment in hypercholesterolemic mice or apolipoprotein E–/–/cd40l–/– compound-mutant mice.13,14
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To test the hypothesis that CD40L expressed on hematopoietic cells (eg, monocytes/M, lymphocytes, and platelets) promotes the development of atherosclerotic lesions, we reconstituted lethally irradiated ldlr–/– mice with bone marrow obtained from ldlr–/– mice or ldlr–/–/cd40l–/– compound-mutant mice. Eight weeks after engraftment, these mice consumed an atherogenic diet for 16 weeks. Again, total cholesterol and triglyceride serum concentrations in ldlr–/– mice on the atherogenic diet receiving bone marrow-derived cells obtained from ldlr–/– mice versus ldlr–/–/cd40l–/– compound-mutant mice did not differ (total cholesterol, 1532±84 mg/dL versus 1654±54 mg/dL; total triglycerides, 89±4 mg/dL versus 99±6 mg/dL, respectively). Surprisingly, CD40L deficiency of bone marrow-derived cells in ldlr–/– mice did not affect the development of atherosclerotic lesions in the aortic arch compared with ldlr–/– mice receiving ldlr–/–/cd40l+/+ bone marrow (0.27±0.02 versus 0.28±0.01 mm2 total wall area; Figure 2c). In accord with previous studies,22 bone marrow transplantation after 
-irradiation significantly reduced lesion size in the aortic arch compared with nonirradiated mice that underwent transplantation in the present study (
–35%, 0.27±0.02 versus 0.41±0.04, P<0.01; Figure 2b and 2c), although the reduction observed in the present study was less than previously demonstrated by Schiller et al.22 However, CD40L may contribute mainly to the later stages of plaque formation. Therefore, the transplantation-induced reduction of lesion size in the aortic arch might dampen the effect of CD40L on atherosclerosis. Because hypercholesterolemic ldlr–/– mice have more extended atherosclerotic lesions earlier in the aortic root than in the aortic arch,23 we additionally analyzed whether lack of CD40L on bone marrow-derived cells reduced the size of more advanced aortic root lesions in these mice. As expected, ldlr–/–/cd40l–/– compound-mutant mice had significantly fewer atherosclerotic lesions in the aortic root compared with ldlr–/– mice (–43.6%, 0.39±0.06 versus 0.68±0.05 mm2 lesion area, P<0.01; Figure 5b), similar to the reduction observed in the aortic arch (–35.1%, P<0.01; Figure 2b). However, in agreement with the data obtained in the aortic arch, ldlr–/– mice receiving bone marrow cells from ldlr–/– mice or ldlr–/–/cd40l–/– compound-mutant mice showed no differences in more advanced atherosclerotic lesion size (0.80±0.05 versus 0.78±0.06 mm2 lesion area; Figure 5c) or composition (data not shown) in the aortic root. Importantly, bone marrow transplantation after -irradiation did not reduce and even increased the lesion size in the aortic root compared with nonirradiated mice (0.80±0.05 versus 0.68±0.05 mm2; Figure 5b and 5c). In addition, CD40L deficiency of bone marrow-derived cells in ldlr–/– mice compared with those receiving ldlr–/–/cd40l+/+ bone marrow diminished lipid deposition in the abdominal aorta (13.4±2.8 versus 26.1±3.2% lipid-positive area, P<0.01; Figure 3c). This reduction was markedly (5.5-fold) less compared with mice systemically deficient for CD40L (P<0.01; Figure 3b and 3c), although irradiated mice showed significantly more extensive lipid deposition in the abdominal aorta compared with nonirradiated mice (26.1±3.2% versus 17.2±2.7% lipid deposition area, P<0.05; Figure 3b and 3c). These data suggest that expression of CD40L on hematopoietic cells (eg, monocytes/M, lymphocytes, and platelets) contributes little to the formation of atherosclerotic lesions in mice.
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Deficiency of CD40L on Bone Marrow–Derived Cells Does Not Alter Plaque Morphology
Because deficiency of CD40L on bone marrow-derived cells did not affect atherosclerotic lesion size in the aortic arch and root, we analyzed whether CD40L deficiency of bone marrow-derived cells might favor plaques with characteristics of stability based on human studies. Therefore, we determined the relative content of M (Mac-3), lipid deposition (oil red O), SMCs (-SMC-actin), and collagen (picrosirius red) in lesions of the aortic arch (Figure 4) and root (data not shown). Analysis of hypercholesterolemic mice lacking CD40L systemically reaffirmed a profound influence of CD40L on plaque morphology. Atheromata of ldlr–/–/cd40l–/– compound-mutant mice had features of enhanced plaque stability compared with those of ldlr–/– mice, namely reduced accumulation of M (–45.2%, 6.3±1.1% versus 11.5±0.9% Mac-3–positive area, P<0.001; Figure 4B) and lipids (–63.1%, 2.7±0.4% versus 7.3±1.1% lipid-positive area, P<0.001; Figure 4D), as well as an increased content in SMCs (+65.0%, 28.9±4.4% versus 19.0±2.2% -actin–positive area, P<0.05; Figure 4F) and collagen (+41.5%, 14.8±3.9% versus 8.8±2.4% picrosirius red-positive area, P<0.05; Figure 4H).
In contrast, lesions in ldlr–/– mice receiving bone marrow-derived cells from ldlr–/– mice or ldlr–/–/cd40l–/– compound-mutant mice showed no significant difference in the content of M (11.5±1.4% versus 11.8±2.5% Mac-3–positive area, respectively; Figure 4C), lipids (4.7±0.7% versus 5.7±0.8% oil red O–positive area; Figure 4E), SMCs (24.8±2.2% versus 21.7±2.8% -actin–positive area; Figure 4G), or collagen (5.1±1.4% versus 5.3±1.4% picrosirius red-positive area; Figure 4I). Therefore, CD40L deficiency of bone marrow-derived cells affected neither these aspects of atherogenesis nor composition of atherosclerotic lesions in the aortic root and arch in hypercholesterolemic ldlr–/– mice.
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Discussion |
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Considerable evidence supports a central role for the CD40/CD40L receptor-ligand dyad in the pathogenesis of atherosclerosis.1–3 Cells of the arterial wall, such as ECs and SMCs, as well as blood cells such as mononuclear phagocytes, T lymphocytes, and platelets, can express functional CD40 and CD40L.5,7,10,24,25 Notably, these cell types express increased levels of the ligand and receptor within human and experimental atheroma,7–9 and ligation of CD40 on these cell types in vitro induces a range of inflammatory processes pivotal to the pathogenesis of atherosclerosis.1,5,7,24,26,27 Additionally, in vivo studies in hypercholesterolemic mice support the relevance of CD40L in the pathogenesis of atherosclerosis. Furthermore, interruption of CD40/CD40L interactions either by anti-CD40L treatment in ldlr–/– mice or genetic deficiency in apolipoprotein E–/– mice markedly slows the development of atherosclerotic lesions and favors features of plaque morphology considered to reduce risk for rupture in humans.11–14 The latter finding implicated CD40L in the pathogenesis of the acute coronary syndromes. Further supporting its pathogenic role, recent reports show that CD40L can directly enhance thrombosis.28–30 However, because an intact immune defense requires CD40 signaling, systemic blockade of this receptor/ligand dyad may cause unwanted effects.19,20 The design of therapies that target CD40 signaling specifically in a cell-selective manner requires identification of the cell type(s) that promote(s) atherogenesis by expressing CD40L. In this regard, the present study made the surprising finding that CD40L modulates atherogenesis primarily by its expression on nonhematopoietic cell types, at least in hypercholesterolemic mice.
Control studies verified that the systemic lack of CD40L caused by deficiency of the functional gene in hypercholesterolemic ldlr–/– mice yielded markedly reduced lesion size and enhanced features associated in humans with plaque stability, as anticipated from previous studies administering -CD40L antibody to atherosclerosis-prone mice.11,13 Interestingly, however, absence of CD40L on bone marrow-derived cells, the originally recognized predominant cellular sources for the ligand, did not limit size or alter composition of atherosclerotic lesions within 2 distinct locations, the aortic arch and the root. Importantly, this finding did not depend on lesion stage because these anatomic locations represented less (aortic arch) and more (aortic root) advanced stages of atherogenesis.
These data suggest that the pro-atherogenic role of CD40L in mice does not require expression of the ligand on bone marrow-derived cell types, including mononuclear phagocytes, T lymphocytes, or platelets, but does require CD40L expression on cells of the vascular bed. Initially, the reports of the ability of cells of extramedullary origin (eg, ECs and SMCs) to express CD40L engendered controversy.7,8,31,32 However, the present study affirms the functional significance of CD40L on these nontraditional cellular sources in atherogenesis. These findings have considerable impact on our understanding of the inflammatory mechanisms in atherogenesis as well as therapeutic implications in the design of selective anti-inflammatory strategies in this disease.
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Acknowledgments |
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We thank R. Reynolds, E. Shvartz, and E. Simon-Morrissey for skillful technical assistance, G. Sukhova and N. Gerdes for technical advice, and K. Williams for editorial assistance (all from Brigham & Women’s Hospital).
This work was supported by grants from the Fondation Leducq, National Institutes of Health (HL66086) to U.S., and by grants from the Deutsche Forschungsgemeinschaft to U.B. (BA 1997/1-1) and AZ (ZI 743/1-1).
Received September 3, 2004; accepted February 24, 2005.
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