Published online before print September 25, 2003, doi: 10.1161/01.ATV.0000097282.22923.EF
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© 2003 American Heart Association, Inc.
Thrombosis |
2ß1 Integrin and Development of Atherosclerosis in a Mouse Model
Assessment of Risk
From the Departments of Pathology & Immunology (D.G.G., S.A.S., M.M.Z.), Medicine (T.C., C.F.S.), and Cell Biology and Physiology (C.F.S.), Washington University School of Medicine, St Louis, Mo.
Correspondence to Mary M. Zutter, Department of Pathology, Vanderbilt University School of Medicine, C-3321A, 1161 21st Ave S, Nashville, TN 37232-2561. E-mail mary.zutter{at}vanderbilt.edu
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Abstract |
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Objectives— The
2ß1 integrin serves as a collagen or collagen/laminin receptor on many cell types, including endothelial cells and platelets. Many studies indicate that the 2ß1 integrin is a critical mediator of platelet adhesion to collagen. Epidemiologic studies suggest a direct correlation between the genetically determined platelet surface density of the 2ß1 integrin and the risk of thrombotic diseases, such as myocardial infarction and stroke, in the young, which are well-established complications of atherosclerosis. We have now used the 2ß1 integrin–deficient mouse to evaluate the contributions of the 2ß1 integrin to the development of atherosclerosis.
Methods and Results— We generated wild-type (2+/+) or 2ß1 integrin–deficient (2-/-) mice that were also deficient in the apolipoprotein E (ApoE) gene (ApoE-/-) and compared atherosclerotic lesion development in 2+/+ ApoE-/- and 2-/- ApoE-/- mice that were fed a high-fat, cholesterol-containing diet for 6 or 15 weeks. Total lesional area did not differ significantly between the 2-null animals and the wild-type animals at either 6 or 15 weeks.
Conclusions— Our results suggest that risk for arterial thrombotic disease associated with high-level 2ß1 integrin expression is not attributable to enhanced development of atherosclerosis per se but may rather be a consequence of thrombotic complications at the plaques.
Key Words: 2ß1 • integrin • collagen • atherosclerosis • thrombosis
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Introduction |
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The
2ß1 integrin serves as a collagen or collagen/laminin receptor on many cell types, including endothelial cells and platelets.1–3 By virtue of its expression and function on both platelets and endothelial cells, the 2ß1 integrin is well positioned to play a significant role in vascular pathobiology. The integrin has been extensively studied as a collagen receptor on platelets. Several studies have suggested that the 2ß1 integrin is a critical mediator of platelet adhesion to collagen. Recent epidemiologic studies suggest that a direct correlation exists between the genetically determined platelet surface density of the 2ß1 integrin and the risk of thrombotic and vascular diseases, such as myocardial infarction, diabetic retinopathy, and stroke, in the young.4–6
We recently developed a genetically engineered mouse in which expression of the 2ß1 integrin was completely eliminated. The 2ß1 integrin–deficient mice are viable, fertile, and develop normally. This animal model provides a unique and valuable tool to evaluate the role of the 2ß1 integrin in platelet biology and vascular disease. We previously reported that 2ß1 integrin–deficient platelets fail to adhere to type I collagen substrates under both static and flow conditions and that platelet aggregation is diminished.7 More recently, we have demonstrated that the 2ß1 integrin–deficient mice have defects in thrombus formation after acute vascular injury in vivo (Blood, in press, 2003).
Platelet thrombus formation after injury is an essential component of normal hemostasis but also contributes to the pathology of myocardial infarction and stroke. The development and progression of atherosclerosis is complex. Two mouse models have provided essential insight into the relative contributions of inflammation, glucose metabolism, blood pressure, and the coagulation and fibrinolytic systems in atherogenesis. The mouse models of atherosclerosis use genetic deletion of either the apolipoprotein E (APOE) gene or the LDL receptor (LDLR) gene.8,9
As noted above, the role of the 2ß1 integrin has been identified as a risk factor for myocardial infarction and stroke, both of which are complications of atherosclerotic disease. However, the epidemiologic studies have not identified the level of 2ß1 integrin expression as a risk factor for atherosclerosis per se. Given the complex, multigenic determinants of atherosclerosis, the studies may have lacked sufficient sensitivity to make such a determination. The recent availability of suitable inbred mouse models has allowed us to address the role of the 2ß1 integrin in the development of atherosclerotic lesions. In this report, we describe the results of our studies in mice lacking both the 2ß1 integrin and the ApoE protein.
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Methods |
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Mice
Mice with complete deletion of the
2ß1 integrin have been previously described.7 To generate mice lacking expression of both 2ß1 and ApoE, 2-null female mice were crossed with ApoE-null males on a C57BL/6 background (Jackson Laboratories, Bar Harbor, Maine), and the resulting double-heterozygous offspring were mated. Progeny with a 2-/+ ApoE-/- genotype were crossed to produce littermates with the desired 2+/+ ApoE-/- and 2-/- ApoE-/- genotypes. These animals were maintained on a standard rodent chow diet until 8 weeks of age and were then started on a Western diet containing 0.15% cholesterol and providing 42% of calories from fat (Harlan Teklad). Mice were allowed to eat ad libitum for an additional 6 or 15 weeks. All experiments were conducted according to Washington University School of Medicine Division of Comparative Medicine’s guidelines for animal care.
Quantitation of Atherosclerosis
Animals were fasted for 4 hours and then euthanized by carbon dioxide asphyxiation in accordance with institutional guidelines. Blood was immediately collected by cardiac puncture, and the serum was stored at -20°C for later biochemical analysis. In addition, sera from genotypically identical mice were pooled and stored at 4°C for up to 24 hours before lipoprotein analysis. Animals were perfused with 30 mL of cold 4% paraformaldehyde in PBS. The heart was immediately frozen in Tissue-Tek OCT Compound (Sakura Finetek), and the aorta was dissected in its entirety from the adventitia beginning at its origin in the heart to the iliac bifurcation. Aortas were fixed overnight in 4% paraformaldehyde and then transferred to PBS and stored at 4°C until morphometric analysis could be performed.
The aorta was incised longitudinally, and a second longitudinal incision was made at the arch to allow the aorta to be pinned flat. Pinned aortas were photographed with a digital camera, and the pictures were subjected to image analysis using imaging software (Scion Image, version 4.0.2). Atherosclerotic lesions were clearly visible as white, opaque areas within the intima. Lesions were reported as percent intimal involvement for the arch (extending from the origin to a point just distal to the left subclavian artery), thoracic aorta (ending at the final intercostal artery), and abdominal aorta (ending at the iliac bifurcation). Total intimal involvement was determined similarly.
Serum Biochemical and Lipoprotein Analysis
For each animal, concentrations of serum total cholesterol, triglyceride, and glucose were determined enzymatically with kits purchased from Sigma-Aldrich Corp. Additionally, lipoproteins from serum pooled from the 4 to 8 mice euthanized on any single day and stored at 4°C for no longer than 24 hours were fractionated by FPLC containing a single Superose column.10 Fractions (0.5 mL) were collected and assayed for cholesterol as described above. The areas under the lipoprotein peaks were quantitated and expressed as a percentage of the total area, and this value was used to calculate the concentrations of VLDL and IDL/LDL cholesterol.
Immunohistochemistry
Ten-micron sections from the aortic sinus or skin of wild-type and 2ß1 integrin–deficient mice were cut on a cryostat, collected on glass slides, and immediately fixed with acetone. Wild-type skin served as a positive control for the 2ß1 integrin. Sections were air dried and washed 3 times for 5 minutes with PBS and then incubated for 10 minutes with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. Blocking was performed for 30 minutes using nonimmune serum before incubation with the primary antibody for 1.5 hours. Immunostaining was performed using Vectastain kits with peroxidase-conjugated secondary antibodies (Vector Labs), as described by the manufacturer. Primary antibodies used were MOMA-2 (Serotec), a rat monoclonal antibody with specificity for mouse macrophages, and a previously described polyclonal rabbit antibody with specificity for the cytoplasmic domain of the 2 integrin subunit.11 Peroxidase was visualized with 3,3'-diaminobenzidine (Sigma-Aldrich Corp), and tissue sections were counterstained with hematoxylin.
Statistical Analysis
Data are reported as the mean±SEM and were analyzed by the Mann-Whitney test. Significance was defined as P<0.05.
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Results |
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2ß1 Integrin, Body Weight, and Serum Lipoprotein LevelsBecause hypercholesterolemia and hyperlipidemia are well-defined risk factors that contribute to atherosclerotic development in humans and increased development of atherosclerosis in mice, we generated mice that were deficient in the ApoE gene and either expressed high, wild-type levels of the
2ß1 integrin (2+/+ ApoE-/-) or also lacked the integrin (2-/- ApoE-/-). To examine the influence of the loss of 2ß1 integrin expression on the development of atherosclerosis, 2+/+ ApoE-/- and 2-/- ApoE-/- mice were fed a high-fat, cholesterol-containing atherogenic diet for either 6 or 15 weeks. Weight at either 6 or 15 weeks was not affected by the expression or lack thereof of the 2ß1 integrin (Table 1).
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After either 6 or 15 weeks on the high-cholesterol diet, mice were fasted for 4 hours before analysis of plasma lipoproteins. The levels of serum cholesterol, triglycerides, and glucose were all higher in both 2+/+ ApoE-/- and 2-/- ApoE-/- mice after 15 weeks on the high-fat diet than after 6 weeks on the diet, but with the exception of glucose, these time-dependent differences did not reach significance. Neither total cholesterol, triglyceride, nor glucose concentrations were significantly different between the 2+/+ ApoE-/- and 2-/- ApoE-/- mice at either time point. Size-exclusion chromatography of lipoproteins obtained from pooled serum specimens revealed no differences in the proportions of lipoproteins or the concentrations of VLDL or IDL/LDL cholesterol between the 2+/+ ApoE-/- and 2-/- ApoE-/- mice (Table 2). Thus, expression of the 2ß1 integrin did not seem to influence lipid metabolism.
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2ß1 Integrin and Atherosclerotic Lesion Formation
Because other genes that do not influence lipid metabolism but that are involved in inflammation or endothelial damage have been shown to influence the initiation or progression of atherosclerosis, we next addressed the role of the 2ß1 integrin in the development of atherosclerotic lesions. After 6 or 15 weeks on a high-cholesterol diet, 2+/+ ApoE-/- and 2-/- ApoE-/- mice were euthanized, their aortas were photographed, and the images were subjected to image analysis using Scion Image software. Atherosclerotic lesions were clearly visible as white, opaque areas within the intima (Figures 1A and 1B). Lesions were reported as the percentage of the intimal surface affected by lesions in the aortic arch (extending from the origin to a point just distal to the left subclavian artery), thoracic aorta (ending at the final intercostal artery), and abdominal aorta (ending at the iliac bifurcation). Total intimal involvement was also ascertained. Figure 2A and Table 3 show the percentage of the aorta involved by atheroma in animals fed a high-fat Western diet for 6 weeks. The mean total atherosclerotic lesion area in 2-/- ApoE-/- mice (n=16) was 0.63±0.11% (mean±SEM; range, 0% to 1.35%). In 2+/+ ApoE-/- mice (n=19), the total lesional area was 1.06±0.19% (mean±SEM; range, 0.03% to 2.63%). Although the total lesional area was not significantly lower in 2-null animals than wild-type animals (P=0.17), there did seem to be a trend toward a decrease in the total lesional area in the 2-/- ApoE-/- animals. This same trend was also apparent but not statistically significant in the lesions of the aortic arch and thoracic aorta. These data suggested that the lack of 2 integrin expression may be protective against the development of atheroma and prompted us to maintain animals on the high-fat diet for a longer period of time.
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When 2-/- ApoE-/- mice (n=16) were fed a high-fat diet for 15 weeks, the mean total lesion area was 5.00±0.87% (mean±SEM; range, 1.77 to 16.21%). In comparison, 2+/+ ApoE-/- littermates (n=19) developed a total lesional area of 4.48±0.59% (mean±SEM; range, 1.49% to 9.95%) (Figure 2B and Table 3). There was no significant difference in the total involved area (P=0.56). Similarly, there were no significant differences in lesion development in any of the aortic segments. The trends apparent at 6 weeks were not present at 15 weeks.
2ß1 Integrin and Foamy Macrophage Accumulation
The infiltrative macrophage plays a major role in development of the incipient fatty streak early in the process of atherosclerosis. In fact, deficiency of P- or E-selectin or intercellular adhesion molecule (ICAM)-1, which significantly diminishes the development of atherosclerotic lesions in the ApoE-/- mouse model, is believed to exert its protective effect by diminishing macrophage infiltration into the fatty streak.12,13 We therefore assessed the presence of macrophages as identified using anti–MOMA-2, a rat monoclonal antibody with specificity against mouse macrophages. The percentage of macrophages and the expression of the MOMA-2 antigen were similar between the fatty streaks of 2-null animals and wild-type animals (Figures 3A and 3B). We also assessed the expression of the 2ß1 integrin by the macrophages in 2+/+ ApoE-/- animals. The macrophages did not express the 2ß1 integrin, although the integrin was expressed at high levels in many other sites, including the stratified squamous epithelium of the epidermis (Figure 3C and data not shown). As expected, macrophages in the lesions of 2-/- ApoE-/- also failed to express the integrin (Figure 3D). It is noteworthy that monocytes and macrophages in several sites and organs, including the peritoneal cavity, spleen, lymph node, and bone marrow, did not express the 2ß1 integrin when evaluated with several antibodies by flow cytometric analysis. These data suggest that macrophages do not require the 2ß1 integrin for infiltration into the fatty streaks of early atherosclerotic lesions.
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Discussion |
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Platelet aggregate formation is essential for normal hemostasis after vascular injury and is also responsible for the acute pathology of myocardial infarction and stroke. Thrombosis and coagulation are complex multicomponent processes. Animal models have proven especially useful in dissecting the relative contributions of individual receptors and enzymes. Mouse models have contributed greatly to our understanding of the atherosclerotic process. The two most useful mouse models of atherosclerosis utilize inactivation of the APOE or LDLR genes.8,9 Animals deficient in either ApoE or the LDLR can, depending on diet and the appropriate time course, develop complex atherosclerotic lesions resembling those seen in humans. The APOE and LDLR mouse models have also made it possible to assess the contributions of other genes in the development and progression of atherosclerosis. Many factors, including inflammation, glucose metabolism, blood pressure, and the coagulation and fibrinolytic systems that affect atherosclerosis, have been studied in one or both of the models.14 We have now used the
2ß1 integrin–deficient mouse to evaluate whether alterations in 2ß1 integrin expression affect the development or progression of atherosclerosis in the APOE model.
2ß1 integrin–mediated adhesion of platelets to collagen results in platelet activation leading to thrombus formation.15 An important role for the 2ß1 integrin in vascular disease is suggested by epidemiologic data, demonstrating an association between the genetically determined 2ß1 integrin receptor density on platelets and pathologic thrombosis. Platelet surface expression of the 2ß1 integrin can vary substantially from individual to individual and is determined by several linked polymorphisms within the 2 integrin subunit gene.16,17 High-level 2ß1 integrin expression on platelets has been identified as an independent risk factor for myocardial infarction in younger individuals.18,19 Other studies also suggest that high-level 2ß1 integrin expression is a risk factor for the development of diabetic retinopathy in patients with type II diabetes mellitus and for stroke in the young.5,6 The intriguing cause-and-effect relationship between 2ß1 integrin levels and vascular disease suggested by the epidemiology studies requires experimental evaluation in vivo through the use of animal models to establish the existence of a mechanistic relationship.
Our results suggest that 2ß1 integrin expression is not required for the development of atherosclerosis per se in uninjured mice with a targeted deletion of the ApoE gene. Furthermore, the absence of the integrin does not influence the level of cholesterol or lipoproteins when animals lacking ApoE are fed a high-fat diet. These results suggest that the 2ß1 integrin is not involved in fat metabolism or in the formation of the early atherosclerotic lesions.
These data demonstrating the lack of a requirement for the 2ß1 integrin in the development of atherosclerosis complement our recent findings on the role of the integrin in acute thrombosis. We recently compared the responses of wild-type mice and 2ß1 integrin–deficient mice in two models of in vivo thrombosis.20 In a model of Rose-Bengal–induced photochemical injury of the carotid artery endothelium, the absence of the 2ß1 integrin significantly delayed by almost 2-fold the time to complete vessel occlusion. The results obtained with the carotid artery endothelial injury model in vivo are consistent with the previously established role of the 2ß1 integrin in platelet adhesion, aggregation, and thrombus formation under both static and flow conditions in vitro as well as the correlation between high-level 2ß1 integrin expression and an increased risk for thrombosis involving coronary and cerebral vessels.4–6 Based on epidemiologic data, we expected to observe a gene dosage effect in mice heterozygous for 2ß1 integrin deficiency. However, no gene dosage effect in the carotid injury model was observed.20 Only homozygous-deficient animals were protected. In contrast, we observed that the 2ß1 integrin was not required for the formation of thrombi and pulmonary emboli after intravascular injection of collagen. In addition, as now demonstrated, the integrin is not required for the initial development of atherosclerosis.
Our findings that the 2ß1 integrin does not contribute to atherosclerotic lesion development differs from results of studies addressing the role of some other adhesion molecules, such as P- and E-selectin and ICAM-1. Deletion of any of these adhesion receptors resulted in a statistically significant decrease (ranging from 1.3- to 2-fold) in the size of the atherosclerotic lesion area in ApoE-deficient mice on the same or similar genetic background as our mice.12,13,21 The adhesion receptors P-selectin, E-selectin, and ICAM-1 are expressed primarily on inflammatory cells, specifically on the monocytes that infiltrate and compose the early fatty streak. The marked decrease in atherosclerosis in mice deficient in these receptors is thought to be attributable to the inability of the monocytes and inflammatory cells to enter the lesions and perpetuate the atherosclerotic lesion. Our immunohistochemical studies indicated that the 2ß1 integrin is not expressed on the monocytes in fatty streaks of wild-type animals. Therefore, it seems logical that the lack of the integrin would not influence the inflammatory recruitment of monocytes into the lesion.
Manipulation of other genetic modifiers of atherosclerosis, including genes that alter lipid storage, glucose metabolism, insulin sensitivity, and blood pressure, altered the development of atherosclerotic lesion in the ApoE-deficient mouse model that was used in our study.22–25 In addition, localized reduction of atherosclerosis was reported in von Willebrand factor (vWf)-deficient mice (vWf-/-) when bred with mice lacking the LDLR.26 LDLR-/-vWf-/- mice developed 40% smaller fatty streaks that contained fewer monocytes than LDLR-/-vWf+/+ mice after both 8 and 22 weeks on a high-fat diet. The lesions were prominently located at the branch points of the renal and mesenteric arteries, sites where we observed almost no lesions at all. In contrast, atherosclerotic lesions were significantly increased in ß3 integrin–deficient mice bred to mice lacking either ApoE or the LDLR.27 Such animals were also more likely to die of pneumonitis when fed a high-fat diet but not a standard chow diet. These finding were attributed to CD36, CD40L, and CD40, proinflammatory and proatherogenic mediators that are known to associate with the ß3 integrin and whose expression was increased in ß3-null animals. Similar to our findings, alterations in genes related to coagulation, including fibrinogen, plasminogen, and plasminogen activator inhibitor-1 (PAI-1), did not effect the size of the atherosclerotic lesions in the ApoE-deficient mouse model.28 The lack of an effect on atherosclerosis by deletion of the 2ß1 integrin is similar to lack of an affect on atherosclerosis by changes in these other coagulation factors. In fact, although PAI-1 deficiency did not alter the development of atherosclerosis in the aorta, lack of PAI-1 did lead to a prolonged time to complete occlusive thrombosis after Rose-bengal–initiated photochemical injury to the carotid artery endothelium.29 The protection against thrombosis after neointimal injury of the carotid artery in PAI–1 deficient mice is similar to that observed in our recent studies in the 2ß1 integrin–deficient mouse.
In conclusion, epidemiologic studies suggest that expression of 2ß1 integrin is associated with an elevated risk of myocardial infarction, stroke, and diabetic retinopathy, all directly related to atherosclerotic disease. Based on the results reported here, we suggest that the 2ß1 integrin is not involved in the development of the early atherosclerotic plaque and is not involved in lipid metabolism. The 2ß1 integrin does seem to play an important role in acute vascular thrombosis after endothelial injury. These combined results suggest the hypothesis that the increased risk of disease associated with high 2ß1 integrin expression is a consequence of thrombotic complications at the site of atherosclerotic plaques attributable to plaque rupture.
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Acknowledgments |
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Acknowledgments
This study was supported by grants from the National Institutes of Health (CA070275, CA098027, HL58427, DK56341, DK20579, and HL63446).
Received August 22, 2003; accepted September 12, 2003.
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