Genetic Deletion or Antibody Blockade of α1β1 Integrin Induces a Stable Plaque Phenotype in ApoE−/− Mice
Objective— Adhesive interactions between cells and the extracellular matrix play an important role in inflammatory diseases like atherosclerosis. We investigated the role of the collagen-binding integrin α1β1 in atherosclerosis.
Methods and Results— ApoE−/− mice were α1-deficient or received early or delayed anti-α1 antibody treatment. Deficiency in α1 integrin reduced the area of atherosclerotic plaques and altered plaque composition by reducing inflammation and increasing extracellular matrix. In advanced plaques, α1-deficient mice had a reduced macrophage and CD3+ cell content, collagen and smooth muscle cell content increased, lipid core sizes decreased, and cartilaginous metaplasia occurred. Anti-α1 antibody treatment reduced the macrophage content in initial plaques after early and delayed treatment, decreased the CD3+ cell content in advanced plaques after delayed treatment, and increased the collagen content in initial and advanced plaques after delayed treatment. Migration assays performed on α1-deficient macrophages on collagen I and IV substrata revealed that α1-deficient cells can migrate on collagen I, but not IV. Anti-α1 antibody treatment of ApoE−/− macrophages also inhibited migration of cells on collagen IV.
Conclusions— Our results suggest that α1β1 integrin is involved in atherosclerosis by mediating the migration of leukocytes to lesions by adhesion to collagen IV. Blocking this integrin reduces atherosclerosis and induces a stable plaque phenotype.
Inflammatory responses, as in atherosclerosis, involve adhesive interactions between cells and the extracellular matrix (ECM) that are necessary for cell attachment, extravasation and migration into tissues, proliferation, and differentiation. Major receptors for extracellular matrix ligands are integrins, which are transmembrane heterodimers consisting of an α and β subunit. The role of several integrins has previously been studied in atherosclerotic development. Integrin α4β1 has been shown to be expressed on smooth muscle cells (SMC) during pathological neointimal thickening and is involved in SMC differentiation.1 Furthermore, α4β1 integrin mediates adhesion of monocytes to the vascular endothelium, and anti-α4 antibody treatment decreases leukocyte entry in mice fed an atherogenic diet2 as well as attenuates neointimal growth following carotid injury in ApoE−/− mice.3 Agonists against αVβ3 integrin were also shown to reduce neointima formation after injury,4 and it has been suggested that this integrin regulates the maturation of macrophages into foam cells.5 Yet another model of endothelial injury has revealed that α2β1 integrin mediates the adhesion of platelets to collagen in the vessel wall, and α2β1-deficient mice exhibit delayed thrombus formation after injury to the carotid artery.6
The role of α1β1 integrin in atherosclerosis has not yet been investigated but has been shown to mediate inflammation in several other illnesses. Integrin α1β1 is a major collagen receptor that is highly expressed on activated monocytes7 and T-lymphocytes, including those found in atherosclerotic plaques.8 The role of α1β1 integrin has been studied in various models of inflammatory diseases including hypersensitivity and arthritis,9 kidney fibrosis,10 and colitis,11 in which it has been shown that a deficiency or blocking of this integrin using anti-α1 antibodies attenuates the inflammatory response as seen by a reduced leukocyte infiltrate. These studies suggest a role for α1β1 integrin in the migration of leukocytes to sites of inflammation.
Besides its expression on immune cells, α1β1 integrin is also expressed on mesenchymal cells, most notably SMCs and fibroblasts. Human aortic SMCs have been reported to express α1β1 integrin and is involved in SMC differentiation.12 It has been demonstrated that α1-deficient fibroblasts cannot adhere to or migrate on collagen IV13 and are deficient in collagen-dependent proliferation.14 A major function attributed to α1β1 integrin is the regulation of feedback inhibition of collagen synthesis. This was shown by enhanced collagen-dependent downregulation of collagen synthesis on receptor stimulation,15 and a later study reported an increase in collagen synthesis in the dermis of α1-deficient mice.16
Given that α1β1 integrin regulates collagen synthesis and mediates the migration of activated leukocytes during inflammation, we wanted to investigate its role in atherosclerosis. To examine the role of α1β1 integrin in plaque inflammation and development, we used a murine model of atherosclerosis in which α1 integrin was either knocked out or mice were treated with an α1-blocking antibody. Here we report that a deficiency or blocking of α1 integrin attenuates atherosclerosis and induces a stable plaque phenotype.
To study the role of α1 integrin in atherosclerosis, α1−/−//ApoE−/− mice were used. ApoE−/− mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, Me) and were backcrossed 10 times with α1−/− mice, originally on a Balb/c background13 to generate male and female α1−/−//Apo−/− (homozygous, n=13), α1+/−//ApoE−/− (heterozygous, n=8) and α1+/+//ApoE−/− (wild-type, WT, n=12) mice. Mice were fed normal chow for 27 weeks after which they were euthanized after an 8-hour fast. Approximately 0.5 to 1.0 mL of blood was drawn from the vena cava for lipoprotein analysis. To study effects of antibody blockade of α1 integrin, ApoE−/− mice on a C57BL/6 background were used, obtained from Iffa Credo S.A. (Charles River Co, Lyon, France) and fed normal chow. Mice were injected intraperitoneally twice a week with 200 μg9 of a murinized version of a hamster anti-rodent α1-blocking antibody (muHa31/8) or murine IgG isotype control antibody (MOPC21) (Biogen Idec, Cambridge, Mass). Mice received treatment for 12 weeks beginning at either 5 weeks of age when no atherosclerotic lesions are present in the aortic arch (early treatment: anti-α1 Ab n=13, control n=14) or 17 weeks of age when advanced lesions begin to appear in the aortic arch (delayed treatment: anti-α1 Ab n=11, control n=14). Animal experiments were approved by the institutional committee for the welfare of laboratory animals of the University of Maastricht.
To determine whether antibody infiltrated plaques, muHa31/8 monoclonal antibody (mAb) was labeled with Alexa488 using a protein labeling kit (Molecular Probes), and 200 μg were injected intraperitoneally into 53-week-old ApoE−/− mice 3 times in the period of one week. Control mice did not receive any antibody. After treatment, mice were euthanized and the arterial tree was briefly perfused with PBS. Aortic arches were snap frozen in Tissue Tek OCT compound for histological analysis, and carotid arteries and abdominal aortas were placed in PBS until analysis by two-photon laser scanning microscopy (TPLSM). Aortic arch cryosections were fixed in acetone, counterstained with hematoxylin and mounted with Prolong Anti-Fade (Molecular Probes), and viewed with a fluorescence microscope. For analysis by TPLSM, carotid arteries and abdominal aortas were placed in perfusion chambers and glass micro-pipettes were inserted into their ends so they could be infused with PBS and kept taut during imaging.
Lipid profiles were assessed as described previously.17
Histology and Morphometry
Histological and morphometric analyses were performed as described previously.17 To confirm the presence of cartilaginous metaplasia observed in plaques of α1-deficient mice, aortic arch sections were stained with alcian blue and toluidine blue.
Aortic arch sections were immunostained with Mac3 rat mAb (1:30; Pharmingen) to detect macrophages, CD3 rabbit polyclonal antibody (1:200; Dako) to detect T-lymphocytes, and αSMAFITC mAb (1:500; Sigma) as a marker for α-smooth muscle actin–positive vascular SMCs. To determine α1 integrin expression in macrophages in atherosclerotic plaques of aortic arches, double immunohistochemistry was performed using Alexa488-conjugated Ha31/8 anti-α1 mAb (Biogen Idec) and CD11b-PE (1:200; Pharmingen). To identify characteristics of cartilaginous metaplasia, sections were stained with antibodies against collagen II (goat polyclonal, 1:75; Santa Cruz Biotechnology, Inc), osteocalcin (OC) (rabbit polyclonal, 1:50; ANAWA Trading SA), osteonectin (ON) (rabbit polyclonal, 1:2000; Zymed Laboratories, Inc), osteopontin (OPN) (goat polyclonal, 1:25), bone morphogenetic protein 2 and 4 (BMP-2, mouse monoclonal, 1:20; Genetics Institute, Inc., BMP-4, goat polyclonal, 1:25; Santa Cruz), matrix GLA protein (MGP) (mouse monoclonal, 1:25), osteoprotegerin (OPG, 1:100) and osteoprotegerin ligand (OPGL, 1:75). To determine collagen IV expression in atherosclerotic plaques, mouse aortic arch and human carotid artery paraffin sections were stained with antibody against collagen IV (rabbit polyclonal anti-mouse 1:300; anti-human 1:50).
Bone marrow–derived macrophages were isolated from the femur and tibia of α1−/−//Apo−/− and α1+/+//Apo−/− mice. Cells were cultured in standard RPMI containing l-glutamine, HEPES, 10% fetal calf serum, 100 IU/mL penicillin/streptomycin, and 15% L929 cell conditioned medium.
Cell migration was assayed using 24-well Transwell migration chambers (Costar) with a pore size of 8 μm. Membranes were coated with 110 μg/mL of type I collagen (isolated as previously described)18 or 50 μg/mL of type IV collagen (BD Biosciences) and incubated at 37°C for 1 hour until complete gel formation. Membranes were allowed to air dry, then 106 cells suspended in serum-free medium were added to each chamber. Cells isolated from WT mice were incubated with 100 μg/mL of α1-blocking antibody or control antibody for 30 minutes before addition to chambers. Complete medium including 100 ng/mL MCP-1 (R&D Systems, Inc) was added to lower chambers, and migration was carried out at 37°C for 4 and 12 hours. Nonmigrated cells were removed from membranes, and migrated cells within the membrane were fixed with methanol and stained with toluidine blue. Membranes were cut out of inserts and mounted onto slides in immersion oil. The number of migrated cells was counted on 5 randomly chosen microscopic fields of each membrane.
Fluorescence-activated cell sorting (FACS) analysis (FACS Calibur, Becton Dickinson) was performed on cells isolated from peripheral blood, spleen, and lymph nodes as well as peritoneal macrophages from antibody-treated mice (anti-α1 Ab n=6, control Ab n=6 of both early and delayed treatment groups). Cells were labeled with T lymphocyte–specific antibodies: CD3FITC, CD4Cy-Chrome, CD8PE and CD25PE, and antibodies to detect macrophages: Gr1FITC and Mac1PE (Pharmingen).
Statistical analyses were performed using a nonparametric Mann–Whitney U test. Data are expressed as mean±SEM, and differences were considered statistically significant at P<0.05.
Body weight did not differ in α1-deficient mice compared with controls and plasma total cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels did not differ between α1−/− and α1+/+ mice, but were elevated in α1+/− relative to α1+/+ mice (Table I, available online at http://atvb.ahajournals.org). Autopsy of organs revealed no abnormalities or pathologies.
Expression of α1 Integrin
Expression of α1 integrin was present on SMCs and slightly on T-lymphocytes (data not shown). Expression was also detected on macrophages in atherosclerotic plaques. Figure I (available online at http://atvb.ahajournals.org) shows CD11b+ macrophages in atherosclerotic plaques that stained positive for α1 integrin.
Extent of Atherosclerosis
Complete deletion of α1 integrin reduced total plaque area by 42% compared with WT mice (Figure 1). The decrease in plaque area was attributable to a 38% decrease in advanced atherosclerotic plaque area. Absence of one α1 allele was sufficient to significantly reduce the total plaque area as seen in heterozygous mice in which total plaque area was reduced by 37% and advanced plaque area was reduced by 16% compared with WT mice (P<0.05). Plaques representative of the three groups of mice are shown in Figure 1c, in which it can be seen that plaque area was significantly reduced in α1 homozygous and heterozygous mice compared with controls.
Atherosclerotic Plaque Composition
To further characterize atherosclerosis, the composition of lesions was analyzed. Histological analysis of advanced plaques demonstrated that α1-deficiency resulted not only in a decreased size, but also a dramatic alteration in plaque composition. Lesions from α1-deficient mice were more fibrotic and less lipid-rich than those of WT mice, whereas plaques of α1 heterozygous mice were of an intermediate phenotype (Figure 2). Analysis of initial lesions in the aortic arch revealed that the relative number of CD3+ cells, macrophages, collagen, and αSMA content did not differ in initial atherosclerotic plaques. Similarly, initial lesions in heterozygous mice were not altered compared with those in WT mice.
Differences in plaque composition were more pronounced in advanced lesions of homozygous and heterozygous mice. Inflammatory cell content significantly decreased in homozygous mice, reflected by a decrease in the percentage of macrophages and CD3+ cells. Furthermore, lipid cores of plaques were smaller whereas extracellular matrix as measured by collagen and αSMA content increased. Atherosclerotic plaques of heterozygous mice showed an intermediate plaque phenotype with a significant decrease in CD3+ cells and a trend toward a decrease in macrophage content (P=0.07) and collagen content (P=0.06).
The presence of cartilaginous metaplasia observed in advanced plaques of α1-deficient mice was confirmed with immunohistochemical staining against collagen II, which is the major matrix protein of cartilage tissue, as well as with alcian blue and toluidine blue, which stain chondroid tissue (Figure II, available online at http://atvb.ahajournals.org). To further identify characteristics of cartilaginous metaplasia, we performed immunohistochemistry using bone markers. The regulators of bone formation OC, ON, OPN, BMP-2, and BMP-4 were present in areas of cartilaginous metaplasia (Figure III, available online at http://atvb.ahajournals.org). MGP, another protein involved in bone formation, was only present in plaque macrophages. Two regulators of osteoclastogenesis, OPG and OPGL, did not show expression.
Average body weight of mice did not differ in the anti-α1 antibody–treated group compared with controls in either of the early or delayed treatment groups. There were no differences in plasma total cholesterol, triglycerides, or LDL, however HDL was slightly elevated in anti-α1 antibody–treated mice compared with controls in the early treatment group (Table I). Autopsy of organs revealed no abnormalities or pathologies.
To ascertain possible systemic effects of antibody treatment on atherosclerotic lesion development, FACS analysis was performed on cells isolated from peripheral blood, spleen, and lymph nodes and on peritoneal macrophages of anti-α1 antibody–treated and control mice. Analysis revealed no differences in the amount of Mac1+ macrophages between the two groups in either blood or peritoneum. Similarly, there were no differences in the amount of CD4+ (helper), CD8+ (cytotoxic) T-lymphocytes, nor in the activation status of T-lymphocytes (reflected by CD4+/CD8+ ratio and CD4+CD25+ cells) between the 2 groups in any of the tissues. There was, however, an increase in the amount of CD3+ T-lymphocytes (total T-lymphocytes) in peripheral blood of anti-α1 antibody–treated mice compared with controls in both the early and delayed treatment groups (Figure IV, available online at http://atvb.ahajournals.org).
To ascertain whether anti-α1 antibody infiltrated atherosclerotic plaques, we used TPLSM and fluorescence microscopy. Atherosclerotic plaques of mice injected with Alexa488-labeled anti-α1 antibody displayed more fluorescence in plaques of aortic arches compared with control mice, which were not injected with antibody. Similarly, using TPLSM, which combines 3-D resolution and large penetration depth,19 showed a positive signal for antibody labeled with the green fluorescent label Alexa488 in atherosclerotic plaques in carotid arteries (Figure V, available online at http://atvb.ahajournals.org).
Extent of Atherosclerosis
There were no significant differences in the individual plaque area between anti-α1 antibody–treated and control mice in either of the early (initial: anti-α1 mAb 14 596±1455 versus control 15 761±1955, P=0.06; advanced: anti-α1 mAb 49 593±8051 versus control 91 519±14468, P>0.05) or delayed treatment groups (initial: anti-α1 mAb 45 184±9635 versus control 31 076±5729, P>0.05; advanced: anti-α1 mAb 141 300±19 809 versus control 120 498±10 671, P>0.05).
Atherosclerotic Plaque Composition
Differences in plaque composition between anti-α1 antibody–treated and control mice were less striking than in α1-deficient mice. Significant differences observed were a decreased macrophage content in initial atherosclerotic plaques of anti-α1 antibody–treated mice compared with controls in both the early and delayed treatment groups (Figure 3a), a decreased CD3+ cell content in advanced plaques of anti-α1 antibody–treated mice of the delayed treatment group compared with controls (Figure 3b), and an increased collagen content in initial and advanced plaques of anti-α1 antibody–treated mice compared with controls of the delayed treatment group (Figure 3c). There were no differences in αSMA content in any of the groups (early treatment, initial: anti-α1 Ab 3.0±0.6 versus control 1.8±0.4, P>0.05; early treatment, advanced: anti-α1 Ab 1.9±1.1 versus control 3.7±0.5, P>0.05; delayed treatment, initial: anti-α1 Ab 0.7±0.5 versus control 2.8±1.1, P>0.05; delayed treatment, advanced: anti-α1 Ab 3.4±0.6 versus control 4.5±0.4, P>0.05).
Cell Migration Assay
Because we observed a reduced macrophage content in both α1-deficient and anti-α1 antibody–treated mice, we wanted to test the ability of α1-deficient bone marrow–derived macrophages to migrate on collagen substrata. Cells were allowed to migrate on collagen I and IV monolayers as it has previously been reported that α1 integrin binds these types of collagen.13 After 4 and 12 hours of incubation, significantly less α1−/− cells had migrated on collagen IV compared with WT cells (Figure 4a). There was no significant difference in the number of α1-deficient cells migrating on collagen I compared with WT cells after 4 or 12 hours of migration (Figure 4b). Treatment of WT cells with α1-blocking antibody inhibited migration on collagen IV compared with control antibody after 4 and 12 hours of migration (Figure 4c).
Collagen IV Expression
Because α1-deficient macrophages as well as macrophages treated with α1-blocking antibody were inhibited from migrating on collagen type IV, the expression of collagen IV was determined in atherosclerotic plaques. Collagen IV is strongly expressed in the region of endothelial cells and slightly in areas surrounding cells in both mouse and human atherosclerotic plaques (Figure VI, available online at http://atvb.ahajournals.org).
To investigate the role of α1β1 integrin in atherosclerosis we have used α1 integrin, ApoE double knockout mice as well as administered an α1-blocking antibody to normal ApoE knockout mice in an early and delayed treatment setting. Deficiency in α1 integrin did not prevent the initiation of lesion formation, but did reduce the size of advanced atherosclerotic plaques and induced a more stable plaque phenotype as characterized by decreased inflammation and increased extracellular matrix content. In both the early and delayed anti-α1 antibody treatment groups, macrophage content was decreased in initial plaques whereas collagen content increased in advanced plaques. Furthermore, the CD3+ cell content was decreased in advanced plaques after delayed treatment.
Antibody treatment against α1 integrin was less effective in attenuating atherosclerosis compared with complete genetic deletion; however, the consequences of antibody intervention do not necessarily correlate with the phenotype of corresponding null animals. Knockout animals are deficient in a particular protein from birth, whereas antibody-treated mice receive treatment against the already existing protein beginning at a later time point for a limited period of time. Furthermore, in the present study we euthanized α1-deficient mice after 26 weeks of age, whereas in the antibody intervention study mice were euthanized after 17 or 29 weeks of age in the early and delayed treatment groups, respectively. Previous studies have also demonstrated differences between effects of using knockout mice and antibody intervention. For instance, complete genetic deletion of CD154 (CD40 ligand) in ApoE−/− mice resulted in smaller advanced plaques,20 whereas with anti-CD40 ligand antibody treatment, advanced plaque area was not reduced compared with controls.21
Despite differences in the effects of α1-deficiency and anti-α1 antibody treatment, plaque composition was altered in both experiments to a more stable phenotype in two important respects, by reducing the macrophage content and increasing the collagen content. Indeed, the two major functions of α1β1 integrin are the regulation of collagen synthesis and the mediation of migration of activated leukocytes into inflamed tissues.7,16 During the course of inflammation, leukocytes migrate through the subendothelial basement membrane,7 which is rich in collagen type IV.22 Central to the migration of cells into inflammatory sites are adhesive interactions between cells and extracellular matrix proteins that are widely mediated by the integrin family of adhesion molecules.23 α1β1 is a major collagen-binding integrin with a preference for collagen IV and is expressed on activated leukocytes. It has been demonstrated in vitro that lipopolysaccharide (LPS)-activated monocytes highly express α1β1 integrin.24 Activated T-lymphocytes also express α1β1 and it has been shown in vivo to be involved in the migration and retention of leukocytes in tissues.25
T-lymphocytes found in atherosclerotic plaques express α1β1 integrin,8 and we have demonstrated here that macrophages in normal mouse atherosclerotic plaques also express this integrin. However, the role of α1β1 integrin in lesion development has not yet been determined. Our study revealed a reduced inflammatory cell content particularly in lesions of α1-deficient, but also in anti-α1 antibody–treated mice, suggesting a role for α1β1 integrin in the accumulation of leukocytes in atherosclerotic plaques. To shed light on the mechanism by which this occurs we performed migration assays, which demonstrated that α1-deficient and anti-α1 antibody–treated macrophages are inhibited from migrating on collagen IV. The subendothelial basement membrane of vessel walls consists largely of collagen IV, through which leukocytes must migrate to enter the intima during atherogenesis.26 In the present study, we have also shown by immunohistochemical staining that collagen IV is expressed in the region of endothelial cells in both human and mouse atherosclerotic plaques. It appears, therefore, that α1β1 integrin is necessary for the infiltration of leukocytes during atherogenesis. Results of the migration assays also suggest that antibody treatment against α1β1 integrin was successful in blocking its function in vivo.
Furthermore, there was an increase in the level of CD3+ T-lymphocytes in the peripheral blood of anti-α1 antibody–treated mice as determined by FACS analysis as well as a significantly reduced CD3+ cell content in advanced plaques after delayed anti-α1 antibody treatment. These findings are consistent with a role of α1β1 in the migration and/or retention of activated T-lymphocytes in collagen-rich tissues.27 By preventing migration and localization of α1β1-positive T-lymphocytes in tissues such as the vessel wall, antibody treatment against α1β1 results in an increased circulating T-lymphocyte population.
Our findings are consistent with previous studies on α1β1 integrin in various models of inflammatory diseases. Monocyte accumulation and activation was found to be reduced in mouse models of colitis in which α1 integrin was deleted or blocked by antibody treatment.11 A decrease in macrophage accumulation was also observed in a model of kidney fibrosis in which α1 integrin was deleted.10 A deficiency in α1β1 integrin as well as anti-α1 antibody treatment was reported to be protective against experimental murine arthritis, delayed-type hypersensitivity, and contact hypersensitivity as shown by a reduced leukocyte infiltrate.9 In addition, in vitro α1-blocking antibody treatment attenuated the proliferation of T-lymphocytes isolated from draining lymph nodes of arthritic rats28 and inhibited T-lymphocytes cultured from peripheral blood of arthritic patients from migrating on collagen IV.29
Besides mediating activated leukocyte migration to sites of inflammation, α1β1 integrin regulates collagen synthesis by negative feedback inhibition. This was demonstrated in α1-deficient mice in which steady state collagen synthesis was observed in normal and wounded dermis.16 Furthermore, receptor stimulation has been shown to increase the downregulation of collagen synthesis.15 In our study we found an increased collagen content in atherosclerotic plaques of both α1-deficient and anti-α1 antibody–treated mice. This was particularly evident in advanced plaques, which is to be expected given that initial plaques consist of little or no collagen. An increased collagen content in atherosclerotic lesions contributes to their stability as does a thick fibrous cap, which we also observed in α1-deficient mice. Atherosclerotic lesions consist primarily of collagen types I and III and although α1β1 prefers to bind collagen IV, it is also a receptor for collagen I. It has been reported that α1β1 integrin downregulates collagen I mRNA levels in cells suspended in collagen gels,30 and collagen I synthesis was increased in the dermis of α1-deficient mice because of elevated collagen I mRNA levels.16 This suggests that negative feedback inhibition of collagen synthesis mediated by α1β1 integrin occurs in atherosclerosis.
In addition to an increased collagen content, we found an increase in SMC content as revealed by αSMA-positive cells in advanced atherosclerotic plaques of α1-deficient mice. SMCs are extracellular matrix–producing cells that are responsible for the collagens present in atherosclerotic plaques and are particularly involved in the formation of fibrous caps. The abundance of SMCs in atherosclerotic plaques is therefore important to plaque stability, however the role of α1β1 integrin in SMC proliferation and accumulation in atherosclerotic plaques is unclear.
Along with a change in plaque composition to a more stable plaque phenotype, cartilaginous metaplasia was observed in plaques of α1-deficient mice. Cartilaginous metaplasia may be a pathway by which calcification develops in atherosclerotic lesions,31 and increasing morphological and molecular evidence suggests that atherosclerotic calcification shares similarities with bone formation. Several proteins involved in osteogenesis have been identified in human atherosclerotic lesions such as OC, ON, OPN, BMP-2, BMP-4, and MGP and are associated with calcification.32 OC is an osteoblast-specific protein that can also be expressed by macrophages, whereas ON is a noncollagenous protein that accumulates in the ECM of bone tissue. BMP-2 and 4 are osteogenic factors that trigger osteoblast differentiation. OPG, a member of the tumor necrosis factor (TNF)-α receptor superfamily, is expressed on osteoblast-like cells and inhibits their differentiation.
In our study, we observed the expression of all of these bone markers to various degrees in areas of cartilaginous metaplasia, which suggests that the cartilaginous tissue found in these lesions may be a precursor to calcification. It is important to note that although cartilaginous metaplasia was more extensive in lesions of α1-deficient mice relative to controls, there was no gross difference in the expression pattern of cartilage and bone markers between the two groups. The role of α1β1 integrin in cartilaginous metaplasia is unknown; however, its occurrence in atherosclerotic plaques may be an indirect effect of α1-deficiency. It has been reported that two of the matrix components known to be important in bone formation, fibronectin and collagen I, are also important in promoting mineralization of vascular cells.33 Because collagen type I is abundantly present in atherosclerotic plaques and an increased collagen content was observed in plaques of α1-deficient mice, this may have led to the formation of cartilaginous metaplasia observed in these plaques.
These results indicate a role of α1β1 integrin in atherosclerosis and emphasizes the importance of integrin-mediated adhesive interactions in this inflammatory disease. Complete deletion of α1β1 integrin not only reduced plaque area, but also altered plaque composition by reducing inflammation and increasing the ECM content, which are crucial features of a stable atherosclerotic plaque. Antibody intervention against α1 integrin, although less effective in attenuating atherosclerosis compared with complete genetic deletion, modulated plaque characteristics in 2 very important respects by decreasing the leukocyte content and increasing collagen content. It appears that α1β1 integrin is involved in the inflammatory process of atherosclerosis by mediating the migration of leukocytes to lesions.
E.L. is a post-doctoral fellow of the Dr E. Dekker program of the Dutch Heart Foundation (2000T41). The authors thank Linda Beckers, Anique Janssen, Ine Middendorp, Mat Rousch, and Marjan Smook for excellent technical assistance.
K.S. and E.L. contributed equally to this work.
- Received January 14, 2005.
- Accepted June 2, 2005.
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