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Circulation. 2005;112:3644-3653
doi: 10.1161/CIRCULATIONAHA.105.587980
CLINICAL PERSPECTIVE
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(Circulation. 2005;112:3644-3653.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Decreased Neurotrophin TrkB Receptor Expression Reduces Lesion Size in the Apolipoprotein E–Null Mutant Mouse

Rosemary Kraemer, PhD; Peter James Baker, MS; K. Craig Kent, MD; Yuanfen Ye, MD; Jun Ji Han, BS; Rafael Tejada, BS; Michael Silane, MD; Rita Upmacis, PhD; Ruba Deeb, PhD; Yaoxin Chen, PhD; Daniel M. Levine, PhD; Barbara Hempstead, MD, PhD

From the Department of Pathology (R.K., P.J.B., Y.Y., J.J.H., R.U., R.D.), Division of Vascular Surgery (K.C.K.), and Division of Hematology (R.T., B.H.), Department of Medicine, Weill Medical College of Cornell University; The Rogosin Institute, Rockefeller University (Y.C., D.M.L.); and Beth Israel Medical Center (M.S.), New York, NY.

Correspondence to Dr Rosemary Kraemer, Weill Medical College of Cornell University, Department of Pathology, Room A631, 1300 York Ave, New York, NY 10021. E-mail rtkraeme{at}med.cornell.edu

Received November 30, 2004; de novo received May 11, 2005; revision received September 12, 2005; accepted September 19, 2005.


*    Abstract
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Background— Accumulation of macrophages and smooth muscle cells in the vascular wall is critical for the development of atherosclerotic lesions. Although much is known about the factors that regulate macrophage recruitment to the vascular wall, the ability of growth factors to regulate smooth muscle cell recruitment in lesion development in vivo is unclear. Our previous studies demonstrated that neurotrophins and their receptors, the Trk receptor tyrosine kinases, are potent chemotactic factors for smooth muscle cells, and the expression of brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB, is upregulated in human atherosclerotic lesions.

Methods and Results— TrkB+/– mice on a 129/B6 background were backcrossed to apolipoprotein E (ApoE)–null (ApoE–/–) mice on the C57Bl/6 background for 6 to 8 generations. Immunohistochemical analysis demonstrated BDNF immunoreactivity in areas of macrophage and smooth muscle cell infiltration, whereas TrkB immunoreactivity was predominant in areas of neointimal smooth muscle cells. Moreover, haplodeficient expression of TrkB in ApoE–/– mice was associated with a 30% to 40% reduction in lesion size compared with ApoE–/– mice with normal expression of TrkB and a 45% decrease in smooth muscle cell accumulation in the lesions. Finally, reconstitution with bone marrow from ApoE–/– mice with normal TrkB expression did not restore lesion development in TrKB+/–/ApoE–/– mice.

Conclusions— These results suggest that TrkB expression on smooth muscle cells contributes to lesion development in the cholesterol-fed ApoE–null mutant mouse. These data demonstrate, for the first time, a role for the neurotrophin TrkB receptor in atherosclerotic lesion development.


Key Words: arteriosclerosis • neurotrophins • smooth muscle cells • Trk receptors • vasculature


*    Introduction
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Atherosclerosis is a complex, chronic inflammatory process of the arterial wall that reflects aberrant recruitment and activation of cells in a developing intimal lesion. Among the initial events regulating this process is the activation of endothelial cells by inflammatory cytokines and oxidized lipoproteins, followed by the recruitment of blood-borne monocytes and the migration of vascular smooth muscle cells into the developing neointima. Further interactions of these 3 cell types to promote lipid accumulation and modulation of cell phenotypes by extracellular matrix lead ultimately to the advanced development of atherosclerotic plaque.

Clinical Perspective p 3653

Several classes of growth factors are known to regulate distinct aspects of this proinflammatory process. First, cytokines have been demonstrated to induce endothelial cell activation and expression of adhesion molecules to modulate monocyte and platelet adhesion.1–3 Second, the recruitment of circulating monocytes contributes significantly to the cellularity of early neointimal lesions.4 Thus, chemokines that regulate the transendothelial migration and activation of monocytes also play an important role in promoting lesion development. Selective chemoattractants and their receptors, such as monocyte chemoattractant protein-1 (MCP-1) and CC-chemokine receptor 2 (CCR2), promote lesion formation in murine models of atherogenesis, in which chemokine- or chemokine receptor–deficient animals exhibit impaired lesion development.5,6 In addition, vascular endothelial growth factor may also promote lesion development by increasing macrophage levels in bone marrow and peripheral blood.7

In contrast to the well-documented contribution of monocyte recruitment to the development of vascular lesions, little is known about the molecular mechanisms regulating smooth muscle chemotaxis and activation in atherogenesis. Recent studies document that increased platelet-derived growth factor (PDGF)Rß expression and activation in apolipoprotein E (ApoE) gene–targeted mice markedly accelerated smooth muscle cell recruitment and progression of lesion formation,8 whereas inhibition of PDGFRß activity reduced lesion formation and smooth muscle recruitment to the fibrous cap.9 Other studies, however, suggest that growth factors in addition to PDGF may play a role in maintaining smooth muscle cell accumulation in later lesion development.10

A novel class of growth factors, the neurotrophins, exhibits chemotactic activities on vascular smooth muscle cells.11,12 The neurotrophins (nerve growth factor [NGF]; brain-derived neurotrophic factor [BDNF]; neurotrophin-3 [NT-3]; and neurotrophin-4 [NT-4]) have been most extensively studied as growth factors mediating neuronal survival and differentiation.13 These growth factors selectively activate distinct members of the Trk receptor tyrosine kinase family (NGF activating TrkA, BDNF and NT-4 activating TrkB, and NT-3 activating TrkC). Two neurotrophins, NGF and BDNF, are expressed in the vasculature by endothelial and vascular smooth muscle cells within the heart, skeletal muscle, and great vessels from late gestation through adulthood.11,14 Two distinct actions of neurotrophins in the vasculature have been identified. First, BDNF-induced activation of TrkB expressed on cardiac endothelial cells is required for the survival of these cells because BDNF gene–targeted neonatal mice exhibit vascular hemorrhage and lethality. Second, NGF and BDNF are potent chemotactic agents that induce the migration of smooth muscle cells bearing TrkA or TrkB receptors, respectively. Furthermore, NGF-induced activation of TrkA receptors expressed on vascular smooth muscle cells in vitro leads to the selective release and activation of matrix metalloproteinase (MMP)-9.15 These activities may be particularly relevant to the development of neointimal lesions because both BDNF and NGF, together with their cognate receptors TrkB and TrkA, are aberrantly upregulated in intimal lesions that develop in the rat balloon injury model and in human atherosclerotic lesions. These observations suggest that local secretion of neurotrophins in the developing neointima may serve to promote vascular smooth muscle cell accumulation or activation to promote atherogenesis.

The abundant expression of TrkB in the medial smooth muscle cells of large muscular arteries14 and in human atherosclerotic lesions11 suggests that it may play a role in atherosclerotic lesion development. To test whether TrkB-mediated activation by BDNF in smooth muscle cells contributes to the development and progression of atherosclerotic lesions, we generated gene-targeted mice that were haploinsufficient for TrkB and additionally ApoE–null. ApoE–null mutant mice maintained on a high-fat Western diet develop atherosclerotic lesions that have been well characterized and resemble human lesions in that they progress with age from the fatty streak stage to the intermediate stage of fibroproliferative lesions, characterized by fibrous and cholesterol clefts and macrophage and smooth muscle cell accumulation.16 Lesion development in ApoE–/– mice haplodeficient for TrkB expression were compared with TrkB wild-type littermates that were ApoE null to determine whether reduced expression and activation of TrkB/BDNF leads to alterations in neointimal lesion development. Because TrkB is prominently expressed by neointimal smooth muscle cells in human atherosclerotic lesions, the results of these studies will provide insight into the potential mechanisms regulating lesion development in human atherosclerosis and may lead to the development of novel therapeutics to limit the progression of the disease.


*    Methods
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Human Tissue Preparation
Human endarterectomy specimens were obtained as waste surgical material under institutional review board–approved protocols from patients undergoing surgical endarterectomy at the New York Presbyterian Hospital or Beth Israel Medical Center. Specimens were cryopreserved in 30% sucrose/OCT (1:1) within 1 hour of retrieval from the patient. Investigators were unaware of patient identifiers. Six specimens were analyzed and gave similar results.

Animals
All animal studies were approved by the Institutional Care and Use Committee. TrkB+/– mice on a 129/B6 background (Jackson Laboratories) were backcrossed to ApoE–/– mice on the C57Bl/6 background (Jackson Laboratories) for 6 to 8 generations. Animals that were to be evaluated for lesion development were maintained from 4 weeks of age on a diet composed of 21% (wt/wt) adjusted calories from fat and 0.15% (wt/wt) cholesterol (TD88137, Harland Tekland Laboratory) ad libitum. Genotyping was performed as recommended by Jackson Laboratory protocol to assess TrkB and ApoE alleles. TrkB+/– neonates were obtained at the predicted mendelian ratio. A detailed description of the methodology and data analysis is provided in an expanded Methods section, which can be found in the online-only Data Supplement.

Morphometry
The whole aorta and aortic sinus were evaluated for lesion development. Detailed descriptions are provided in the online-only Data Supplement.

Plasma Lipid Analysis
Mice were fasted 18 hours before euthanasia, and whole blood was collected at the time of euthanasia. Total serum cholesterol, triacylglycerol, and fatty acids were analyzed as previously described.17

Bone Marrow Transplantation Studies
Bone marrow transplantation was performed as previously described.18 A detailed description can be found in the online-only Data Supplement.

Microchamber Chemotaxis Assay
BDNF-induced migration of vascular smooth muscle cells was assessed with the use of the microchamber chemotaxis assay, as previously described.12 A detailed description is provided in the online-only Data Supplement.

Immunohistochemistry and Double Immunofluorescence
Immunohistochemical and double immunofluorescence analysis was performed as previously described.11,14 Detailed descriptions are provided in the online-only Data Supplement. Collagen staining was performed with a Masson trichrome kit from Poly Scientific R&D Corp.

Statistical Analysis
Statistical differences in lesion size and smooth muscle cell, macrophage, and collagen content in the whole aorta and the aortic root of ApoE–/– mice with normal and haplodeficient expression of TrkB were determined by the Mann-Whitney test. Statistical significance was determined at a value of P≤0.05.


*    Results
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Expression of TrkB and BDNF in Atherosclerotic Lesions of ApoE–/– Mice
We previously demonstrated that TrkB and its ligand, BDNF, were expressed in the neointimal lesions that develop after balloon injury of the rat thoracic aorta and in human atherosclerotic lesions.11 To determine whether TrkB and BDNF were similarly upregulated in the vascular atherosclerotic lesions that develop in ApoE-null mutant mice, immunohistochemical analysis was performed with the use of frozen sections of aortic root lesions from ApoE–/– mice maintained on a high-fat diet for 9 or 12 weeks. BDNF immunoreactivity was present in both media and plaque of murine atherosclerotic lesions (Figure 1B) and was found to be both cell and matrix associated. Double immunofluorescence staining of BDNF and either smooth muscle cell {alpha}-actin or a murine macrophage marker (the antigen recognized by the MOMA-2 antibody) (Figure 2), demonstrated that BDNF was expressed in areas adjacent to cells expressing either smooth muscle {alpha}-actin or the macrophage marker, the MOMA antigen (arrows, Figure 2). However, there was very little overlap between BDNF immunoreactivity and either cell marker, suggesting that in murine atherosclerotic lesions, BDNF is predominantly matrix associated. The relatively widespread expression of BDNF in lesions is consistent with BDNF secretion and immobilization in extracellular matrix. TrkB immunoreactivity was noted in medial smooth muscle cells (Figure 1A). In the murine plaque, TrkB immunoreactivity was prominent in the areas of the lesion that exhibited smooth muscle cell {alpha}-actin immunoreactivity, as assessed in serial sections (Figure 1C). Double immunofluorescence studies demonstrated that the majority of TrkB-expressing cells coexpress the smooth muscle–specific marker, with little colocalization with the MOMA-2 antigen (Figure 3). Quantitative analysis of 27 high-power fields in 6 ApoE–/– mice demonstrated that 75%±5% of the TrkB+ cells colocalized with smooth muscle cell {alpha}-actin, whereas only 17%±4% of the TrkB+ cells colocalized with the MOMA-2 antigen. Moreover, 71%±8% of neointimal smooth muscle cells but only 11%±3% of macrophages expressed TrkB. These results suggest that neointimal smooth muscle cells are the predominant cell type expressing TrkB.



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  		Figure 1. Immunohistochemistry for detection of TrkB and BDNF in atherosclerotic lesions from aortic sinus of ApoE–/– mice maintained on high-fat diet for 12 weeks. Sections A through D were incubated with the indicated primary antibodies. Immunoreactivity was detected by incubating the sections with the appropriate biotinylated secondary antibody, followed by ABC-horse radish peroxidase and VIP, colorimetric indicator solution. a, Control section incubated with rabbit IgG ({alpha}-TrkB antibody); b, control section incubated with chicken IgY ({alpha}-BDNF antibody); c, control section incubated with BT-murine IgG ({alpha}-smooth muscle cell [SMC] {alpha}-actin); d, control section incubated with rat IgG (MOMA-2 antibody). Sections were counterstained with hematoxylin. Bar=100 µm. L indicates lumen; P, plaque; M, media. For TrkB, lesions from 9 mice were assessed, covering a distance of 50–100 µm. For BDNF, lesions from 8 mice were assessed, covering a distance of 50–100 µm.



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  		Figure 2. Double immunofluorescence detection of BDNF with either smooth muscle cell (SMC) {alpha}-actin or MOMA-2 antigen in aortic root lesions from ApoE–/– mice maintained on high-fat diet for 12 weeks. Sections were incubated with the indicated primary antibodies. Insets are control sections incubated with chicken IgY (anti-BDNF antibody), biotinylated (BT)-murine IgG (BT-anti–smooth muscle cell {alpha}-actin), or FITC-conjugated rat IgG (MOMA-2 antibody). BDNF immunoreactivity was detected by incubating the sections with a biotinylated-anti-chick IgG followed by rhodamine-conjugated avidin. SMC {alpha}-actin immunoreactivity was detected using a FITC-conjugated avidin. L indicates lumen; P, plaque; M, media. Arrowheads indicate colocalization of BDNF and smooth muscle cell {alpha}-actin immunoreactivity; arrows, colocalization of BDNF and MOMA-2 immunoreactivity. BDNF colocalization with smooth muscle cell {alpha}-actin or MOMA-2 antigen assessed in lesions from 3 mice, covering distance of 50–100 µm.



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  		Figure 3. Double immunofluorescence detection of TrkB with either smooth muscle cell (SMC) {alpha}-actin or MOMA-2 antigen in aortic root lesions from ApoE–/– mice maintained on high-fat diet for 9 weeks. Sections were incubated with the indicated primary antibodies. Insets are control sections incubated with rabbit IgG (anti-TrkB antibody), BT murine IgG (BT-anti–smooth muscle cell {alpha}-actin), or FITC-conjugated rat IgG (MOMA-2 antibody). Trk B immunoreactivity was detected by incubating the sections with a biotinylated-anti-rabbit IgG followed by rhodamine-conjugated avidin. SMC {alpha}-actin immunoreactivity was detected using FITC-conjugated avidin. L indicates lumen; P, plaque; M, media. Arrowheads indicate colocalization of TrkB and smooth muscle cell {alpha}-actin immunoreactivity. TrkB colocalization with smooth muscle cell {alpha}-actin assessed in 9 mice, covering distance of 50–100 µm.

Expression of TrkB in Human Atherosclerotic Lesions
Our prior studies indicated that TrkB was expressed in human atherosclerotic lesions, although the cell types expressing this receptor were not identified. In addition, BDNF mRNA has been detected in human atherosclerotic lesions; however, localization of BDNF protein has not been reported. To determine whether a similar pattern of expression of TrkB and BDNF was observed in human atherosclerotic lesions compared with murine atherosclerotic lesions, immunohistochemistry was performed on advanced atherosclerotic lesions in human endarterectomy specimens. Immunohistochemical analysis of human atherosclerotic lesions demonstrated significant TrkB and BDNF immunoreactivity in both the media and plaque (Figure 4). Similar to what was observed in murine atherosclerotic lesions, TrkB immunoreactivity was detectable in areas of lesions containing cells that by morphological and immunological criteria were characteristic of smooth muscle cells (Figure 4A, 4C, 4D, 4E). Moreover, double immunofluorescence detection of TrkB and either smooth muscle cell {alpha}-actin or the human macrophage marker, the Ham 56 antigen, demonstrated that TrkB colocalized to areas expressing smooth muscle cell {alpha}-actin but not the Ham 56 antigen (Figure 5A to 5F). In contrast to observations in murine atherosclerotic lesions, neointimal smooth muscle cells in human atherosclerotic lesions did not uniformly express TrkB. This may be due to the advanced state of the atherosclerotic plaque resected from the endarterectomy, as well as variability in human atherosclerotic lesions. Although BDNF immunoreactivity was present adjacent to cells that had characteristics of either smooth muscle cells or macrophages, most of the BDNF immunoreactivity was not cell associated (Figure 4K to 4M). Moreover, colocalization studies failed to demonstrate significant colocalization of BDNF with smooth muscle cell {alpha}-actin or the Ham 56 antigen, indicating that BDNF was predominately associated with the extracellular matrix (Figure 5G to 5L). Thus, TrkB and BDNF are expressed in a largely similar pattern in murine and human atherosclerotic lesions. The predominant expression of TrkB by smooth muscle cells suggests that these are the primary cells activated by BDNF in atherosclerotic lesions.



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  		Figure 4. Immunohistochemistry for detection of TrkB (A, C, D, G, H) and BDNF (K to M) in human atherosclerotic lesions. A, Low-power image of section incubated with anti-Trk B antibody. B, Control section incubated with rabbit IgG (anti-TrkB antibody). C, High-magnification image of lower area marked in A. D, Higher magnification of area marked in C. E, Area shown in D from serial section incubated with anti–smooth muscle cell {alpha}-actin. F, Area shown in E from serial section incubated with anti-human macrophage antibody Ham-56. G, Higher magnification of upper area marked in A. H, Higher magnification of area marked in G. I, Area shown in H from serial section stained with Ham-56 antibody. J, Area shown in I from serial section incubated with anti–smooth muscle cell {alpha}-actin antibody. K, Low-power image of section incubated with anti-BDNF antibody. L, High-power image of BDNF plus cells from area of lesion that in serial section stained positive for smooth muscle cell {alpha}-actin. M, High-power image of BDNF plus cells from area of lesion in serial section that stained positive for Ham-56 antigen. N–O, Control sections incubated with chick IgY (anti-BDNF antibody). Sections counterstained with hematoxylin. For A, B, K, and N, bar=100 µm; for C and G, bar=25 µm; for D, E, F, H, I, and J, bar=10 µm. L indicates lumen; P, plaque; M, media. For TrkB, similar results observed in 6 specimens, with 3–9 sections/specimen. For BDNF, similar results were observed in 5 specimens, with 2–7 sections/specimen.



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  		Figure 5. Double immunofluorescence detection of TrkB (A–F) or BDNF (G–L) in human atherosclerotic lesions with either smooth muscle cell (SMC) {alpha}-actin (B, C, H, I) or Ham-56 antigen (E, F, K, L). Sections were incubated with the indicated primary antibodies. Control sections incubated with rabbit IgG and mouse IgG (a–f) or chick IgY and mouse IgG (g–l). Trk B and BDNF immunoreactivity were detected by incubation with biotinylated secodary antibodies followed by incubation with rhodamine-conjugated avidin. SMC {alpha}-actin and Ham 56 immunoreactivity were detected using a FITC-conjugated anti-mouse IgG. L indicates lumen; P, plaque. Arrows in A, B, and C indicate colocalization of TrkB with smooth muscle cell {alpha}-actin immunoreactivity. TrkB colocalization with smooth muscle cell {alpha}-actin observed in 4 specimens over 6–10 serial sections ({approx}200 µm).

Decreased Lesion Formation in ApoE–/– Mice With Reduced Expression of the TrkB Receptor System
The coexpression of TrkB and its ligand, BDNF, in the atherosclerotic lesions of the ApoE-null mutant mouse, together with prior studies demonstrating that neurotrophins induce vascular smooth muscle cell migration and MMP-9 expression in vitro, suggests that this ligand/receptor system could be exerting an autocrine or paracrine action to promote vascular smooth muscle cell recruitment and lesion development in this model of atherogenesis. To test this hypothesis, we explored the effect of TrkB haploinsufficiency on lesion development in ApoE–/– mice. TrkB+/– mice (129/B6 background) were generated that were additionally ApoE–/– (C57BL/6 background) by backcrossing TrkB+/– (129/B6) with ApoE–/– (C57BL/6). Evaluation of animals with the TrkB–/– genotype was not possible because these animals die perinatally.19 TrkB+/–/ApoE–/– mice exhibited reduced expression of TrkB in medial smooth muscle cells, as assessed by immunoprecipitation/Western blot analysis (Figure I, online-only Data Supplement). Haploinsufficiency of TrkB did not affect total serum cholesterol, triglycerides, or fatty acid after 6, 9, 12, or 16 weeks on the high-fat Western diet (Table I, online-only Data Supplement). Evaluation of total surface area of the whole aorta and aortic root lesion size after 6 and 9 weeks on the high-fat diet indicated that there was no significant difference in lesion development in TrkB+/–/ApoE–/– mice compared with TrkB+/+/ApoE–/– mice (Figures 6 and 7 Down). However, in mice maintained on a high-fat diet for 12 and 16 weeks, reduced TrkB expression was associated with a 30% to 40% decrease in lesion development (Figures 6 and 7Down). After 12 weeks on the high-fat diet, the percentage of aortic surface occupied by lesion was decreased by 38% in TrkB+/–/ApoE–/– compared with wild-type mice (Figure 6), and a similar reduction in lesion area was observed in the aortic root of TrkB+/–/ApoE–/– mice (Figure 7). Evaluation of mice maintained on a Western diet for 16 weeks confirmed that the reduction in lesion size was maintained at 25% in both the whole aorta (Figure 6) and the aortic sinus (Figure 7). These results demonstrate that reduced TrkB expression is associated with a decrease in atherosclerotic lesion development in ApoE–/– mice.



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  		Figure 6. Morphometric analysis of lesion size in whole aorta of TrkB+/+/ApoE–/– and TrkB+/–/ApoE–/– mice maintained on high-fat diet for 9 and 12 weeks. Lesion size presented as percentage of surface area of total aortic surface occupied with lesion. Nine weeks, TrkB+/+/ApoE–/–, n=13; TrkB+/–/ApoE–/–, n=6; 12 weeks, TrkB+/+/ApoE–/–, n=17; TrkB+/–/ApoE–/–, n=17.



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  		Figure 7. Morphometric analysis of lesion size in aortic root of TrkB+/+/ApoE–/– and TrkB+/–/ApoE–/– mice maintained on high-fat diet for 6 to 16 weeks. A to D, Representative hematoxylin and eosin–stained sections of aortic root lesions. A, C, Lesions from TrkB+/+/ApoE–/– mice. B, D, Lesions from TrkB+/–/ApoE–/– mice. A, B, Mice maintained on high-fat diet for 12 weeks. C, D, Mice maintained on high-fat diet for 16 weeks. E, Quantitative analysis of lesion size in aortic root. Six weeks, TrkB+/+/ApoE–/–, n=12; TrkB+/–/ApoE–/–, n=4; 9 weeks, TrkB+/+/ApoE–/–, n=12; TrkB+/–/ApoE–/–, n=6; 12 weeks, TrkB+/+/ApoE–/–, n=16; TrkB+/–/ApoE–/–, n=11; 16 weeks, TrkB+/+/ApoE–/–, n=10; TrkB+/–/ApoE–/–, n=6.

Characterization of Lesions in TrkB+/–/ApoE–/– Mice
To assess potential mechanisms underlying the reduction in lesion development in the TrkB+/–/ApoE–/– mice, we examined smooth muscle cell and macrophage accumulation and collagen content in the lesions that developed in ApoE-null mutant mice after 12 weeks on a high-fat diet. In lesions of TrkB+/+/ApoE–/– mice, smooth muscle cell {alpha}-actin and macrophage immunoreactivity was present in 18% and 30% of the lesion area, respectively (Figure 8A). Reduced TrkB expression inversely affected smooth muscle cell and macrophage accumulation in the lesions of ApoE–/– mice, in which the area of smooth muscle cell immunoreactivity was significantly decreased, representing only 10% of the total area of the lesion in the aortic sinus (Figure 8A), a 45% reduction compared with TrkB+/+ mice. In contrast, macrophage immunoreactivity was increased from 30% to 49% of the total lesion area. Collagen content in the lesions of TrkB+/–/ApoE–/– mice was also significantly decreased compared with TrkB+/+ mice (Figure 8B), decreasing from 49% to 32% of the total lesion area. This decrease is most likely caused by the decrease in smooth muscle cell accumulation in the lesions of TrkB haplodeficient mice. No change in the expression of MMP-9 was observed in the lesions that developed in TrkB+/–/ApoE–/– mice versus TrkB+/+/ApoE–/– mice (results not shown). These results indicate that in lesions of TrkB+/–/ApoE–/–, there is a decrease in smooth muscle cell accumulation and collagen, resulting in an overall decrease in lesion development.



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  		Figure 8. A, Quantitative analysis of smooth muscle cell (SMC) and macrophage content in lesions of aortic sinus from TrkB+/+/ApoE–/– mice (n=5) and TrkB+/–/ApoE–/– mice (n=6). Data presented as percent lesion area demonstrating immunoreactivity for either smooth muscle cell {alpha}-actin or MOMA-2 antigen and quantified in 10–15 sections/mouse, covering a distance of 200–300 µm. B, Collagen content of lesions from TrkB+/+/ApoE–/– mice (n=11) and TrkB+/–/ApoE–/– mice (n=5). Data presented as percent area of lesion demonstrating collagen staining, as assessed by Masson trichrome staining, and assessed in 10–15 sections/mouse, covering distance of 250–300 µm. C, Quantitative analysis of BDNF-induced migration of smooth muscle cells transfected with a cDNA expressing Trk B. C indicates control; P, PDGF-BB (10 ng/mL). This represents 1 of 3 replicate experiments. D, Morphometric analysis of lesion development in aortic root of TrkB+/+/ApoE–/– and TrkB+/–/ApoE–/– mice after bone marrow transplantation. TrkB+/+/ApoE–/– mice receiving bone marrow from TrkB+/+/ApoE–/– mice, n=4; TrkB+/+/ApoE–/– mice receiving bone marrow from TrkB+/–/ApoE–/– mice, n=8; TrkB+/–/ApoE–/– mice receiving bone marrow from TrkB+/+/ApoE–/– mice, n=5; TrkB+/–/ApoE–/– mice receiving bone marrow from TrkB+/–/ApoE–/– mice, n=5.

Previous studies from our laboratory demonstrated that neurotrophins are potent chemotactic agents for smooth muscle cells. Thus, the decrease in smooth muscle cell accumulation in TrkB+/–/ApoE–/– mice may result from decreased migration of medial smooth muscle cells into the developing lesion. To determine whether alterations in TrkB expression by vascular smooth muscle cells affect BDNF-induced cell migration, vascular smooth muscle cell lines expressing low levels of TrkB were transfected with a plasmid encoding TrkB or plasmid alone. Cells transfected with plasmid alone migrated to the known smooth muscle cell chemotactic factor PDGF-BB but did not migrate in response to BDNF (Figure 8C). In contrast, cells transfected with the plasmid encoding TrkB migrated in response to BDNF in a dose-dependent manner (Figure 8C). These results indicate that BDNF-induced migration of smooth muscle cells is dependent on TrkB expression.

Bone Marrow Transplantation
The present data indicate that smooth muscle cells are the predominant Trk-expressing cell in both human and murine atherosclerotic lesions. Previous studies, however, have demonstrated that TrkB is expressed by hematopoietic cell types in bone marrow20 and that circulating bone marrow leukocytes contribute to lesion development in ApoE–/– mice.4,21,22 Thus, to distinguish between the effects of TrkB in vessel-derived cells from its potential effects on hematopoietic elements, we performed bone marrow transplantation studies using ApoE–/– mice that were wild-type or heterozygous for TrkB expression, maintained on a high-fat diet for 12 weeks and reconstituted with bone marrow from animals that were TrkB+/+/ApoE–/– or TrkB+/–/ApoE–/–. Successful engraftment of marrow occurred in {approx}50% of animals (54% survival at 4 weeks after irradiation). Chimerism was confirmed by polymerase chain reaction analysis of elicited peritoneal macrophages in a cohort of TrkB+/+ mice transplanted with bone marrow from TrkB+/– mice (Figure II, online-only Data Supplement). These results suggest that mice that had undergone bone marrow transplantation after lethal irradiation had undergone successful engraftment. No significant difference in serum cholesterol, triglycerides, or phospholipids was observed between the 4 experimental groups (Table II, online-only Data Supplement). Lesion development in the aortic root of TrkB+/+/ApoE–/– mice receiving bone marrow cells from TrkB+/+/ApoE–/– mice and TrkB+/–/ApoE–/– animals receiving TrkB+/–/ApoE–/– bone marrow was similar to that observed in the previous experiment (compare Figure 7 with Figure 8C), in which a reduction in lesion size of 30% to 35% was observed in the aortic root in animals haplosufficient for TrkB. However, bone marrow from TrkB+/–/ApoE–/– mice transplanted into TrkB+/+/ApoE–/– mice did not significantly reduce lesion formation, nor did reconstitution of TrkB+/+/ApoE–/– marrow into TrkB+/–/ApoE–/– mice significantly enhance lesion formation. These results suggest that the predominant effect of TrkB haploinsufficiency on lesion development in ApoE–/– mice is the result of its local effects on neointimal smooth muscle cell accumulation and activity and not through an effect on the recruitment and activity of bone marrow–derived cells in the atherosclerotic lesion.


*    Discussion
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 up arrowMethods
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 *Discussion
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The migration and activation of medial smooth muscle cells into the neointimal space are integral processes in the development of atherosclerotic lesions. Smooth muscle cells are present in the early stages of lesion development, the fatty streak stage, and are essential for the later formation of the fibrous cap and to maintain plaque stability. During atherosclerotic lesion development, smooth muscle cells promote the inflammatory response as they express monocyte chemotactic factors and vascular cell adhesion molecules,23,24 further promoting macrophage infiltration into the vascular wall. Finally, smooth muscle cells are a major source of extracellular matrix molecules, the synthesis of which contributes to increased lesion development, as well as plaque stability. Most recently, the role of extracellular matrix in sequestering local growth factors, as well as the ability of proteinases to facilitate growth factor release, has been uncovered as a mechanism that modulates neointimal cell recruitment and activation.25,26 Moreover, extracellular matrix molecules may be important for lipoprotein retention in the vascular wall.27

Gene deletion studied in both ApoE knockout mice and LDL-R knockout mice have identified the chemokines and their receptors as playing a prominent role in the recruitment of monocyte/macrophages to the vessel wall and subsequent lesion development (reviewed by Lucas and Greaves,28 Boisvert,29 and Bursill et al30). In contrast, little is known about specific growth factors regulating smooth muscle cell activity as it relates to lesion development. Studies have demonstrated that a variety of growth factors and cytokines, including transforming growth factor-ß and interferon-{gamma}, can regulate lesion development and smooth muscle cell accumulation in the neointimal plaques that develop in murine models of atherogenesis.31–33 However, transforming growth factor-ß and interferon-{gamma} have a broad array of activities on other lesional cells, including macrophages, T and B cells, and smooth muscle cells; therefore, their activity is not smooth muscle cell specific.

The present study provides the first in vivo evidence that a member of the Trk family of receptor tyrosine kinases contributes to lesion development in a murine model of atherogenesis. First, both TrkB and its ligand, BDNF, are expressed in the lesions that develop in ApoE–null mutant mice maintained on a high-fat diet. Second, haploinsufficiency for TrkB was associated with a decrease in lesion development in the advanced stages of atherogenesis after 12 and 16 weeks on the high-fat diet, with little effect in the early stages of lesion development. The decrease in lesion size was accompanied by a decrease in smooth muscle cell accumulation and collagen content in the lesion. The preferential expression of TrkB in neointimal smooth muscle cells may explain the lack of effect on lesion size in early atherogenesis in the haploinsufficient TrkB animals. Although smooth muscle cells are present in early lesion development, their contribution is primarily a later event in atherogenesis, perhaps as a mechanism to maintain the inflammatory response and support the progression of the disease. Alternatively, other growth factors, such as PDGF-BB, may mediate smooth muscle cell activity and may compensate for decreased TrkB receptor activation in early lesion development.9,10

Studies by our laboratory and others demonstrated that BDNF induces angiogenesis through activation of TrkB on vascular endothelial cells.34 This activity, coupled with the BDNF-induced recruitment of hematopoietic progenitors, promotes neoangiogenesis in ischemic tissue.35 Several studies have indicated that neovascularization is observed both in human atherosclerotic lesions and in ApoE–null mutant mice maintained on a high-fat diet for >32 weeks.36 Moreover, inhibitors of angiogenesis limit advanced lesion development in the cholesterol-fed ApoE–/– mouse.37,38 In the present study, the mice were maintained on the high-fat diet for only 16 weeks, a time when neovascularization is not observed. Thus, the potential proangiogenic actions of BDNF in lesion development are important questions that are best addressed in future studies.

Previous studies have demonstrated that TrkB is expressed by hematopoietic cell types present in bone marrow, including eosinophils, mastocytes, and stromal macrophages.20 In the present study TrkB was expressed by both medial and neointimal smooth muscle cells. Thus, although this would suggest that TrkB activation of vascular smooth muscle cells is primarily responsible for its effect on lesion development, the expression of TrkB by bone marrow cells that have the potential to be recruited to developing atherosclerotic lesions could indicate an overall systemic effect of TrkB on lesion development. To differentiate between systemic and vascular effects of TrkB haploinsufficiency on lesion development, we performed bone marrow transplantation studies. This technique has been well established to distinguish between the effect of proteins expressed by both smooth muscle cells and hematopoietic cells in the development of atherosclerotic lesions in murine models of atherogenesis.4,21,22 Indeed, prominent effects of growth factors or cytokines, including MCP-1,21,22 tumor necrosis factor,39 and interferon-{gamma},40 on hematopoietic elements have been dissected with the use of this technique. Of note, the contribution of monocyte/macrophage recruitment appears early, within 3 to 4 weeks after the start of administration of a high-fat, cholesterol-enriched diet, in lesion development in the ApoE system, with maximal effects noted after 5 to 10 weeks on the high-fat Western diet.16 At about this time, smooth muscle cells begin to accumulate in the neointimal lesion, with maximal recruitment occurring at {approx}10 weeks of hypercholesterolemia, when early fibrous cap formation can be observed.16 These data suggest that the activity of smooth muscle cell–specific growth factors and cytokines occurs later in lesion development. Reconstitution of lethally irradiated TrkB+/–/ApoE–/– mice with bone marrow from TrkB+/+/ApoE–/– did not significantly reverse the reduction in lesion size observed in TrkB+/–/ApoE–/– mice reconstituted with bone marrow cells from TrkB+/–/ApoE–/– mice. These data, coupled with the later effect of reduced TrkB expression on plaque formation at 12 to 16 weeks, indicate that the effect of TrkB haploinsufficiency on lesion development is primarily caused by decreased TrkB signaling in the local vascular wall rather than by its reduced activity on bone marrow–derived hematopoietic cells.

Thus, previous studies describing the expression of Trk receptors and their ligands in neointimal lesions and their effects on smooth muscle cell activity in vitro suggested that this growth factor/receptor system may contribute to vascular lesion development. The present in vivo data support this hypothesis. The modest but significant effect on lesion development in the ApoE model of murine atherogenesis that is observed is caused by the limitation of the animal model. Full TrkB knockout mice show perinatal lethality, limiting our analysis to ApoE knockout mice that were haplodeficient for TrkB expression. However, neurotrophins are exquisitely regulated, with haploinsufficient neurotrophin and Trk animals yielding identifiable, intermediate phenotypes.41,42 Moreover, studies in ApoE–/– mice haplodeficient for the chemokine receptor CCR2 demonstrated reduced lesion formation after 5 weeks on a high-fat diet, an effect that was lost by 9 weeks.5 In the present study, the effect of TrkB haploinsufficiency was observed later in lesion development and was maintained for up to 16 weeks. Finally, the decrease in lesion development that is observed in TrkB+/–/ApoE–/– mice is similar to results in other studies that have assessed the role of different genes in lesion development in the ApoE–/– mice, in which lesion development in a double-knockout mouse was able to be analyzed.5,23,43 Thus, these data represent the first in vivo evidence that Trk receptor activation is an important contributor to atherogenesis.


*    Acknowledgments
 
Disclosures

None.

This work was supported by National Institutes of Health Public Service grant PO1 HL46403.


*    References
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CLINICAL PERSPECTIVE

The development of atheromas is the principal cause of ischemic heart disease, the major cause of death and morbidity in the Western world. Atheromas are complex vascular lesions composed of macrophages, smooth muscle cells, cholesterol, and extracellular matrix. Despite the success of lipid-lowering agents, cardiovascular events are still common, suggesting that other strategies are needed to optimally limit atherogenesis. Although the cytokines that regulate macrophage accumulation in the vessel wall have been well studied, little is known about the molecular mechanisms that recruit smooth muscle cells to the developing lesion. This study identifies a novel growth factor/receptor system of the neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor, the TrkB receptor tyrosine kinase, as critical mediators of smooth muscle cell accumulation and lesion development in atherogenesis. TrkB is preferentially expressed by neointimal smooth muscle cells in human atherosclerotic lesions and in the apolipoprotein E (ApoE)–null mutant mouse, a murine model of atherogenesis. Moreover, reduced expression (haplodeficiency) of TrkB in the ApoE-null mutant mouse decreases lesion size, smooth muscle cell accumulation, and collagen content in the lesion. This study provides the first in vivo evidence that Trk receptors contribute to the development of atherosclerotic lesions and identifies neurotrophins and TrkB as potential therapeutic targets to modulate smooth muscle cell function and atherogenesis.


*    Footnotes
 
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/112/23/3644/DC1.





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