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【摘要】
Objective- To determine the significance of fibroblast growth factor receptor (FGFR) expression for the development of atherosclerotic lesions in apoE-deficient (apoE -/- ) mice.
Methods and Results- ApoE -/- mice fed a high-fat diet were administered the FGFR tyrosine kinase inhibitor SU5402 (25 mg/kg/d sc), which inhibited neointima growth by 85%. We measured its effects on lesion size at the aortic sinus, macrophage and smooth muscle cell (SMC) accumulation, the expression of monocyte chemotactic and retention factors, as well as its effects on FGFR expression/phosphorylation. FGFR tyrosine kinase inhibition reduced phosphorylated FGFRs in lesions by 90%, associated with a 65% reduction in lesion size measured using Oil Red O. Macrophages and SMCs within lesions were reduced by 58% and 78%, respectively. Monocyte chemotactic protein-1 (MCP-1) expression was also reduced, as was the expression of hyaluronan synthase, cyclooxygenase-2, CD36, and endothelial monocyte-activating polypeptide-II. Although 3 FGFR types were expressed in lesions, the effects of SU5402 could be attributed largely to inhibition of FGFR-1 phosphorylation.
Conclusions- Atherosclerotic lesions in apoE -/- mice express multiple FGFRs and an active FGF:FGFR-1 signaling system that promotes atherosclerosis development via increased SMC proliferation, and by augmenting macrophage accumulation via increased expression of MCP-1 and factors promoting macrophage retention in lesions.
To assess the significance of fibroblast growth factors (FGFs) and their receptors (FGFRs) for atherosclerosis, we inhibited this system in apoE -/- mice. Such inhibition markedly reduced smooth muscle cell and macrophage accumulation, attenuated atherosclerotic lesion severity, and greatly reduced FGFR-1 phosphorylation. Thus, FGF:FGFR-1 signaling promotes the development of atherosclerotic lesions.
【关键词】 apoE / mice atherosclerosis fibroblast growth factor receptors proliferation macrophage retention
Introduction
Atherosclerosis remains an important cause of morbidity and mortality. It is characterized by aberrant smooth muscle cell and endothelial cell growth, extracellular matrix, lipid and macrophage accumulation which culminates in a neovascularized, smooth muscle, macrophage, and lipid rich atherosclerotic lesion. The mechanisms driving lesion progression are not fully defined, although there is evidence indicating a significant role for growth factors, including members of the fibroblast growth factor (FGF) family. FGF-1 and FGF-2 are pleiotropic growth factors that exert a broad spectrum of activities on cell types responsible for human atheromas and restenosis after angioplasty. FGF-1 and FGF-2 promote smooth muscle cell (SMC) proliferation, migration, and proteoglycan biosynthesis 1,2 and also stimulate endothelial cells to proliferate, migrate, and secrete matrix metalloproteinases. 3 FGF-1 and FGF-2 can be produced by macrophages, endothelial cells, and SMCs. 4-6 In atherosclerotic plaques, SMCs as well as macrophages express these growth factors. 7
Fibroblast growth factors exert their biological effects by interacting with and activating a family of 4 fibroblast growth factor receptors (FGFR-1 to -4) with protein tyrosine kinase activity. 8 Their ability to activate cells is in part dependent on the presence of tyrosine residues within a short noncatalytic insert between the split kinase domain; 2 phosphorylatable tyrosine residues are present in FGFR-1 and FGFR-2, 1 in FGFR-3, and none in FGFR-4. 8 FGF-1 is capable of activating all 4 FGFRs and their splice variants while other FGF family members exhibit more selective interactions. 9 Activation of FGFR-1, the most studied receptor on endothelial and SMCs, stimulates proliferation and increases motility of endothelial cells 10; it also stimulates proliferation of medial SMCs. 11 We have recently shown that neointimal SMCs in injured carotid arteries express multiple forms of FGFR-2 and FGFR-3, but only FGFR-2 contributes to their proliferation. 12 Multiple FGFRs are also expressed in human atherosclerotic lesions. Both FGFR-1 and FGFR-2 are expressed by intimal SMCs, foam cells, and plaque microvasculature whereas FGFR-3 and FGFR-4 exhibited more restricted patterns of distribution. 13
Because nothing is known about the significance of FGFR activation for the development and progression of atherosclerosis, in the current study we investigated in apoE -/- mice fed a high-fat diet the effects of chronic inhibition of FGFR tyrosine kinase activity using the specific inhibitor SU5402. 14 We demonstrate that development of atherosclerotic lesions in apoE -/- mice fed a high-fat diet is associated with the expression of multiple FGFR types. Inhibition of FGFR tyrosine kinase activity reduced lesion development with associated reductions in SMC and macrophage accumulation, expression of monocyte chemotactic protein-1, and monocyte retention factors. These effects could be attributed mostly to inhibition of activated FGFR-1.
Materials and Methods
Animal Experiments
Male apoE -/- mice (n=40) on the C57BL/6 background were obtained from the Precinct Animal Facility, AMREP, Melbourne, Australia and were fed a high-fat diet containing 0.21% cholesterol and 21% fat from 14 weeks of age for 6 weeks. Mice also received by sc injection either vehicle (saline) or SU5402 (25 mg/kg/d) for the duration of the study. The dose of SU5402 was chosen on the basis of its ability to inhibit neointima development and macrophage accumulation 2 weeks after ligation of the carotid artery of apoE -/- mice (see Results). Body weights were measured every week to examine the consumption of food. All procedures and protocols were approved by the AMREP Animal Ethics Committee.
For expanded Materials and Methods used in this article, please see http://atvb.ahajournals.org.
Results
FGF and FGFR Expression
Multiple FGFs were expressed in atherosclerotic aortas from apoE -/- mice fed the high fat diet. These included FGF-1, FGF-2, FGF-11, and FGF-18, which were expressed in approximate equal abundance ( Figure 1 ). In addition, FGF-20 and FGF-21 were also expressed but their mRNAs were less abundant ( Figure 1 ). FGF-11 differs from the other FGFs in that it does not interact with known FGFRs; rather it interacts with the mitogen-activated protein kinase scaffolding protein, islet brain-2. 15 As the other FGFs can interact with FGFR-1, -2, and -3 9,16-18 to initiate biological effects, we also investigated such FGFR expression. FGFR-1 and FGFR-2 were similarly expressed whereas mRNA encoding FGFR-3 was less abundant ( Figure 1 ). The major FGFR-1 fragment encoding part of the extracellular domain was smaller in size ( 400 bp) than the predicted size for the 3 Ig-like-containing domain fragment (673 bp). Nucleotide sequencing of this fragment indicated that this FGFR-1 cDNA was devoid of the 267-bp exon encoding Ig-like domain 1. The 2 FGFR-2 fragments (400 to 500 bp) encoding part of the extracellular domain were also smaller than the predicted size of 772 bp ( Figure 1 ). Sequencing indicated that the larger of the 2 fragments (506 bp) was devoid of the 266-bp exon encoding Ig-like domain 1, whereas the smaller fragment (428 bp) was devoid of both the Ig-like domain 1 exon and the 78-bp exon encoding the acidic box in the extracellular domain of the receptor. The FGFR-3 fragment (447 bp) encoded all 3 Ig-like domains of the extracellular region ( Figure 1 ).
Figure 1. Expression of fibroblast growth factors and receptors in atherosclerotic aorta of apoE -/- mice. Top, mRNAs detected by RT-PCR encoding FGF-1, FGF-2, FGF-11, FGF-18, FGF-20, and FGF-21. Bottom, mRNAs detected by RT-PCR encoding FGFR-1 extracellular domain with a deleted Ig-like domain 1, FGFR-2 extracellular domain with splice variants in which Ig-like domain 1 has been deleted with/without deletion of the acidic box, and FGFR-3 extracellular domain. S denotes the X174-HaeIII size markers, with specific fragments shown in base pairs (bp).
FGFR Inhibition and Intima Growth
To determine the significance of FGF/FGFR expression for development of atherosclerotic lesions we initially performed a dose finding study with the FGFR tyrosine kinase inhibitor SU5402 by examining its effects on intima growth in apoE -/- mice after ligating the carotid artery. Remodelling of this artery after cessation of blood flow has been shown to be dependent on FGF-2. 19 SU5402 markedly reduced intima formation in apoE -/- mice 2 weeks after cessation of carotid artery blood flow. The 2 doses examined (25 and 50 mg/kg/d sc) exhibited similar reductions in intima size, which averaged 85% (supplemental Figure I, available online at http://atvb.ahajournals.org). Influx of macrophages into the intima was also markedly reduced, by 71% (supplemental Figure I). Consequently, the lower dose of SU5402 was chosen to determine the effects of FGFR tyrosine kinase inhibition on development of atherosclerosis.
FGFR Tyrosine Kinase Inhibition and Development of Atherosclerosis
Treatment of mice with SU5402 while on a high-fat diet did not affect body weight, their general well-being, or plasma cholesterol levels, which averaged 3.31±0.15 mmol/L in SU5402-treated mice and 2.91±0.18 mmol/L in vehicle-treated mice ( P 0.05). Analysis of Oil Red O-stained histological cross sections of atherosclerotic lesions that developed at the level of the aortic sinus indicated a 65% reduction in lesion size in the SU5402-treated mice ( P <0.001; Figure 2 ). Macrophage accumulation was also reduced in the SU5402-treated mice, by 58% ( P <0.01; Figure 2 ). The number of -SM actin positive cells that accumulated within the lesions was also reduced, by 78% in the SU5402-treated mice ( P <0.05; Figure 3 ). The number of PCNA-positive cells expressed as % of total cells was reduced by 68% ( P <0.01; Figure 3 ). In contrast, CD31-positive endothelial cells within the lesions were unaffected ( Figure 3 ).
Figure 2. Photomicrographs of aortic sinus from vehicle- (left) and SU5402-treated (right) apoE -/- mice fed a high-fat diet. Top, Sections stained with Oil Red O and mean area of staining (bar graph) in the 2 groups of mice. Bottom, Immunohistochemistry using anti-CD68 (macrophage) antibody and mean area of staining (bar graph) in the 2 groups of mice. vehicle-treated; SU5402-treated, * P <0.05 from vehicle. Size bars represent 100 µm.
Figure 3. Immunohistochemistry of aortic sinus from vehicle- (left) and SU5402-treated (right) apoE -/- mice fed a high-fat diet. Cross-sections were stained with anti- -SM actin antibody to detect smooth muscle cells (top), anti-proliferating nuclear antigen (PCNA) antibody to detect cell proliferation (middle), and anti-CD31 antibody to detect endothelial cells (bottom). Bar graphs represent differences between the 2 groups; vehicle-treated; SU5402-treated, * P <0.05 from vehicle. Size bars represent 100 µm.
FGFR Tyrosine Kinase Inhibition and FGFR/Phospho-FGFR Expression
Atherosclerotic lesions expressed all 3 FGFRs, FGFR-1, -2, and -3, with a somewhat different spatial distribution. In lesions of vehicle-treated mice FGFR-1 and FGFR-2 were mostly localized to the inner regions of the plaque whereas FGFR-3 was more associated with the luminal regions ( Figure 4 ). SU5402 treatment ( Figure 4 ) markedly reduced their expression, FGFR-1 and FGFR-2 by 70% ( P <0.05), and FGFR-3 by 60% ( P <0.05). Phosphorylated FGFR was also expressed in lesions of vehicle-treated apoE -/- mice, and appeared to colocalize more with FGFR-1 and FGFR-2 than with FGFR-3, indicating activated FGFRs in the lesions. We next performed dual immunohistochemistry to precisely colocalize phospho-FGFR with the 3 FGFR types. Phospho-FGFR colocalized predominantly with FGFR-1 (supplemental Figure IIA). No colocalization was apparent between phospho-FGFR and FGFR-2, whereas with FGFR-3 colocalization was low ( 5%; supplemental Figure IIB and IIC, respectively). Thus, FGFR-1 is the major phosphorylated FGFR type in developing atherosclerotic lesions. SU5402 treatment markedly reduced (by 90%) phosphorylated FGFR within the lesions, confirming the inhibitory effect of SU5402 on FGFR tyrosine kinases ( Figure 4 ).
Figure 4. Photomicrographs of the distribution of FGFRs in aortic sinus from vehicle- (left) and SU5402-treated (right) apoE -/- mice fed a high-fat diet. Serial sections were stained with anti-FGFR1, anti-FGFR2, anti-FGFR3, and anti-phospho-FGFR antibody and the mean area of staining within lesions determined. Sections were counter stained with hematoxylin. Bar graphs represent area stained as % of lesion area; vehicle-treated; SU5402-treated, * P <0.05 from vehicle. Size bars represent 100 µm.
Chemokine and Adhesion Molecule Expression
To determine the mechanism by which FGFR tyrosine kinase inhibition reduced macrophage accumulation in the lesions we initially investigated the effects of SU5402 treatment on the expression of monocyte chemotactic factors, MCP-1, and connective tissue growth factor (CTGF). Both MCP-1 and CTGF were highly expressed in lesions of vehicle-treated mice ( Figure 5 ). SU5402 treatment markedly reduced MCP-1 expression, by 80% ( P <0.05); the small reduction ( 20%) in CTGF expression was not statistically significant ( P 0.05). SU5402 treatment also tended to induce a reduction in vascular cell adhesion molecule (VCAM)-1 expression, although this was not statistically significant ( Figure 5, P 0.05). To determine whether phosphorylated FGFR-1 was associated with MCP-1 and VCAM-1 expression we carried out dual immunohistochemistry to colocalize the FGFR types with this chemokine and adhesion molecule. MCP-1 colocalized with FGFR-1, but not FGFR-2 or FGFR-3 (supplemental Figure IIIA, IIIB, and IIIC, respectively). FGFR-1 also colocalized with VCAM-1, although VCAM-1 expression was also apparent in areas not expressing FGFR-1; neither FGFR-2 nor FGFR-3 colocalized significantly with VCAM-1 (supplemental Figure IIID, IIIE, and IIIF, respectively). Thus, the effects of SU5402 on MCP-1 expression, and to a lesser degree on VCAM-1 expression, appear to be mediated by inhibition of FGFR-1 phosphorylation.
Figure 5. Photomicrographs of the distribution of cytokines and VCAM-1 in aortic sinus from vehicle- (left) and SU5402-treated (right) apoE -/- mice fed a high-fat diet. Cross-sections were stained for MCP-1, CTGF, and VCAM-1. Bar graphs represent area stained as % of lesion area, vehicle-treated; SU5402-treated, * P <0.05 from vehicle. Size bars represent 100 µm.
Factors Affecting Monocyte Retention
Because adhesive interactions between monocytes and SMCs can contribute to subendothelial monocyte-macrophage retention in atherosclerosis, 20,21 we initially examined whether inhibiting FGFR signaling would affect expression of hyaluronan synthase. Hyaluronan synthase can be induced by fibroblast growth factor 22 and when overexpressed in SMCs increases monocyte binding 21,23; also overexpression of hyaluronan, the product of hyaluronan synthase (HAS), promotes atherosclerosis. 23 Real-time polymerase chain reaction (PCR) analysis of lesions from SU5402 and vehicle-treated mice (supplemental Figure IV) indicated large reductions in the expression of mRNAs encoding HAS-1 ( 68%) and HAS-2 ( 85%) after inhibition of FGFR signaling. Similarly, the level of the oxLDL receptor CD36 mRNA, the expression of which is known to increase when monocytes interact with SMCs, 24 was reduced by 90%. Strikingly, cyclooxygenase-2 (COX-2), the expression of which also enhances SMC-monocyte interactions 20 and which can be induced by FGF-2 in SMCs, 24 was almost completely abolished by treatment with SU5402. Finally, endothelial monocyte-activating polypeptide-II (EMAP-II), which can also promote binding of monocytes to SMCs, 25 was also reduced in expression after inhibition of FGFR signaling, although to a lesser extent ( 45%) than the other retention factors.
Discussion
The present study has assessed whether FGFR-dependent pathways play a role in the development of atherosclerosis in apoE -/- mice. Development of atherosclerosis was associated with expression of multiple FGFRs and FGFs. Furthermore, the tyrosine kinase inhibitor of FGFRs, SU5402, was able to attenuate both intima growth and atherosclerosis. In atherosclerotic lesions this was associated with reduced accumulation of SMCs and macrophages, reductions in cell proliferation, the expression of the leukocyte chemokine MCP-1, as well as reductions in the mRNAs encoding proteins that facilitate monocyte retention in lesions. Activated FGFR-1 appears to be the major FGFR type implicated in these effects. These findings indicate for the first time that activated FGFR-dependent pathways contribute to the development of atherosclerosis.
Previous immunohistochemical studies suggest that multiple FGFRs may participate in the development of human atherosclerosis. 7,13 FGFR-1, -2, and -3 are expressed by intimal SMCs in early human lesions as well as in simple and advanced plaques. FGFR expression in lesions is also associated with intimal macrophage foam cells and microvessels, with FGFR-1 and FGFR-2 being most highly expressed. Our findings in atherosclerotic lesions of apoE -/- mice confirm the expression of multiple FGFRs in developing atherosclerotic lesions. These receptors can potentially be activated by FGFs expressed in the lesions, FGF-1, FGF-2, and FGF-18. FGF-1 and FGF-2 are known to interact with high affinity with FGFR-1 and FGFR-2, 9 whereas FGFR-3 can be activated by FGF-1 9 and FGF-18 26; FGF-11 (fibroblast homologous factor-1; FHF-1), which is also expressed within lesions, does not activate FGFRs. 15 To determine which splice variants of FGFRs are expressed in lesions we subjected FGFR RT-PCR products encoding the extracellular domains to nucleotide sequencing. The extracellular ligand-binding domains of FGFRs are composed of 3 immunoglobulin (Ig)-like domains and contain an acidic box of 8 amino acids between the first and second domain. In atherosclerotic lesions of apoE -/- mice, 3 Ig-like domain containing FGFR-1 and FGFR-2 were low in abundance. Rather, the major splice variants were 2 Ig-like containing FGFR-1 and FGFR-2. Deletion of the first Ig-like domain of these receptors does not affect their affinity for FGFs. Rather, deletion of Ig-like domain I of FGFR-1 has been associated with differential signaling attributable to less pronounced translocation of FGF-1/FGFR-1 to a perinuclear location and potentially a greater mitogenic response. 27 Deletion of Ig-like domain I in FGFR-2 also has important effects on posttranslational modifications and cell signaling. Two Ig-like domain containing FGFR-2 is extensively modified by glycosaminoglycan close to the N-terminal region of the acid box, an effect abrogated by the presence of Ig-like domain I. 28 The resultant high affinity receptor exhibits enhanced receptor autophosphorylation, substrate phosphorylation, and sustained mitogen activated protein kinase activity, resulting in sustained responses to FGF stimulation. Activation of this receptor accounts for much of the SMC proliferation within the neointima of injured arteries. 12 Despite 3 FGFR types being expressed in developing lesions, our colocalization studies indicate that FGFR-1 is the major activated FGFR type. Precisely how the other receptor types contribute to other stages of lesion progression remains to be determined.
Several lines of evidence indicate that FGF-mediated activation of FGFRs may be important in both inflammatory cytokine and growth factor-stimulated atherogenesis. Interleukin (IL)-1ß is considered to play a significant role in vascular inflammation and atherosclerosis, in part by stimulating the expression of MCP-1 29 and augmenting intimal SMC proliferation. 30 This latter effect of IL-1ß is mediated by the cytokine-enhancing FGF-2 release in arteries. 30 Inhibiting FGFR tyrosine kinase activity in the apoE -/- mice is associated with reduced proliferation and the expression of MCP-1. Similarly, platelet-derived growth factor-BB (PDGF-BB) has been implicated in the development of atherosclerosis, by stimulating the expression of MCP-1 31 and proliferation of SMCs. 32 This ability of PDGF-BB to induce SMC proliferation is also dependent on FGF-2 release and FGFR-1 activation. 33 Consistent with these observations, we found colocalization of FGFR-1 with MCP-1 in the lesions. Hypercholesterolemia-induced atherosclerosis has also been associated with activation of angiotensin type 1 (AT1) receptors. 34 Activation of this receptor also upregulates FGF-1 and FGFR-1 on SMCs. 35
Phosphorylation of FGFRs is critical for FGF-induced mitogenesis and most likely accounts for much of the accumulation of SMCs within the intima of developing atherosclerotic lesions. SU5402 was highly effective in inhibiting FGFR phosphorylation and reducing SMC numbers within developing lesions. Intimal SMCs within atherosclerotic lesions are essential for lesion development and exhibit a proinflammatory phenotype. 36 They express P-selectin and chemokines GRO- and fractalkine, important for monocyte and lymphocyte arrest on SMCs; they are also an important source of MCP-1. Such expression by intimal SMCs is associated with enhanced NF- B activity, 36 which can be stimulated by FGF-2. 37 Inhibition of FGFR tyrosine kinase activity not only reduces MCP-1 expression but may also reduce the arrest of migrating macrophages, by reducing their interaction with intimal SMCs. Inhibition of FGFR signaling was associated with large reductions in the expression of hyaluronan synthases-1 and -2 which are responsible for hyaluronan synthesis. Hyaluronan expression is increased in atherosclerotic lesions and promotes recruitment of macrophages. 38 Ligation of hyaluronan to its main receptor CD44 is required for activation of inflammatory cells and for phenotypic dedifferentiation of SMCs to the synthetic phenotype. 39 VCAM-1, a marker of SMC dedifferentiation, 39 tended to also be reduced in lesions of the SU5402-treated apoE -/- mice. Our findings also indicate that FGFRs can promote atherosclerosis by inducing COX-2 expression; COX-2 is highly expressed in human atherosclerotic lesions. 40
It is also possible that FGFs regulate lesion development by modulating endothelial cell function and responses. Activation of FGFR-1 has been associated with enhanced endothelial cell survival, augmented proliferation, and angiogenesis. 10 Because neovascularization of lesions affects macrophage accumulation, 41 we also assessed the effects of FGFR tyrosine kinase inhibition on endothelial cells within lesions. Inhibition of FGFR phosphorylation did not affect CD31-positive endothelial cells within the lesions, indicating that activated FGFRs are not required for lesion vascularization during development of atherosclerosis. Similarly, inhibition of FGFR tyrosine kinase activity did not affect endothelial cell coverage of the lesions, suggesting that activated FGFRs are not required for endothelial cell survival during development of atherosclerosis. An issue not addressed in the current study is whether FGFR-1 is activated via autocrine or paracrine mechanisms. This can be addressed using laser capture microdissection of phospho-FGFR-stained frozen sections for mRNA analysis of FGFs. Unfortunately, the anti-phospho-FGFR antibody used in this study was not able to detect phospho-FGFR-positive cells within the time-frame required for adequate mRNA isolation from laser capture microdissected cells. 42 Development of higher affinity phospho-FGFR antibodies that bind rapidly to phospho-FGFRs will be required to address this issue.
In conclusion, our data extend earlier findings on FGFR expression in human atherosclerotic lesions and demonstrate that although multiple FGFRs are expressed in atherosclerotic lesions, FGFR-1 is the major activated FGFR type that contributes to lesion development, modulating cell proliferation and SMC numbers, the expression of MCP-1, and macrophage accumulation.
Acknowledgments
Source of Funding
This study was supported by a Program Grant from the National Health and Medical Research Council of Australia.
Disclosure(s)
None.
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作者单位：Baker Heart Research Institute, Melbourne, Victoria, Australia.