Portal de Periódicos da CAPES

Apolipoprotein E Inhibits Neointimal Hyperplasia after Arterial Injury in Mice

2000; Elsevier BV; Volume: 157; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)64823-7

1525-2191

Binghua Zhu, David G. Kuhel, David P. Witte, David Y. Hui,

Protease and Inhibitor Mechanisms

The potential cytostatic function of apolipoprotein (apo) E in vivo was explored by measuring neointimal hyperplasia in response to vascular injury in apoE-deficient and apoE-overexpressing transgenic mice. Results showed a significant increase in medial thickness, medial area, and neointimal formation after vascular injury in both apoE knockout and wild-type C57BL/6 mice. Immunochemical analysis with smooth muscle α-actin-specific antibodies revealed that the neointima contained proliferating smooth muscle cells. Neointimal area was 3.4-fold greater, and the intima/medial ratio as well as stenotic luminal area was more pronounced in apoE(−/−) mice than those observed in control mice (P < 0.05). The human apoE3 transgenic mice in FVB/N genetic background were then used to verify a direct effect of apoE in protection against neointimal hyperplasia in response to mechanically induced vascular injury. Results showed that neointimal area was reduced threefold to fourfold in mice overexpressing the human apoE3 transgene (P < 0.05). Importantly, suppression of neointimal formation in the apoE transgenic mice also abolished the luminal stenosis observed in their nontransgenic FVB/N counterparts. These results documented a direct role of apoE in modulating vascular response to injury, suggesting that increasing apoE level may be beneficial in protection against restenosis after vascular surgery. The potential cytostatic function of apolipoprotein (apo) E in vivo was explored by measuring neointimal hyperplasia in response to vascular injury in apoE-deficient and apoE-overexpressing transgenic mice. Results showed a significant increase in medial thickness, medial area, and neointimal formation after vascular injury in both apoE knockout and wild-type C57BL/6 mice. Immunochemical analysis with smooth muscle α-actin-specific antibodies revealed that the neointima contained proliferating smooth muscle cells. Neointimal area was 3.4-fold greater, and the intima/medial ratio as well as stenotic luminal area was more pronounced in apoE(−/−) mice than those observed in control mice (P < 0.05). The human apoE3 transgenic mice in FVB/N genetic background were then used to verify a direct effect of apoE in protection against neointimal hyperplasia in response to mechanically induced vascular injury. Results showed that neointimal area was reduced threefold to fourfold in mice overexpressing the human apoE3 transgene (P < 0.05). Importantly, suppression of neointimal formation in the apoE transgenic mice also abolished the luminal stenosis observed in their nontransgenic FVB/N counterparts. These results documented a direct role of apoE in modulating vascular response to injury, suggesting that increasing apoE level may be beneficial in protection against restenosis after vascular surgery. Despite the recent reduction in mortality from myocardial infarction and other forms of ischemic heart disease, atherosclerosis remains the major cause of death in approximately half the population in industrialized countries. Currently available treatment for vascular occlusive disease includes percutaneous transluminal coronary angioplasty, directional coronary atherectomy, and percutaneous delivery of balloon expandable stents.1Glagov S Intimal hyperplasia, vascular modeling, and the restenosis problem.Circulation. 1994; 89: 2888-2891Crossref PubMed Scopus (266) Google Scholar, 2Dussaillant GR Mintz GS Pichard AD Kent KM Satler LF Popma JJ Wong SC Leon MB Small stent size and intimal hyperplasia contribute to restenosis: a volumetric intravascular ultrasound analysis.J Am Coll Cardiol. 1995; 26: 720-724Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 3Kearney M Pieczek A Haley L Losordo DW Andres V Schainfeld R Rosenfield K Isner JM Histopathology of in-stent restenosis in patients with peripheral artery disease.Circulation. 1997; 95: 1998-2002Crossref PubMed Scopus (273) Google Scholar, 4Hoffmann R Mintz GS Dussaillant GR Popma JJ Pichard AD Satler LF Kent KM Griffin J Leon MB Patterns and mechanisms of in-stent restenosis. A serial intravascular ultrasound study.Circulation. 1996; 94: 1247-1254Crossref PubMed Scopus (1240) Google Scholar, 5Ono K Imazu M Ueda H Hayashi Y Shimamoto F Yamakido M Differences in histopathologic findings in restenotic lesions after directional coronary atherectomy or balloon angioplasty.Coronary Artery Dis. 1998; 9: 5-12Crossref PubMed Scopus (5) Google Scholar However, despite a primary success rate of 90 to 95%, late restenosis of the artery occurs in 30 to 50% of patients within 3 to 6 months of the procedure.6McBride W Lange R Hillis L Restenosis after successful coronary angioplasty: pathophysiology and prevention.N Engl J Med. 1988; 318: 1734-1737Crossref PubMed Scopus (361) Google Scholar, 7Califf RM Fortin DF Frid DJ Harlan WR Ohman EM Bengtson JR Nelson CL Tcheng JE Mark DB Stack RS Restenosis after coronary angioplasty: an overview.J Am Coll Cardiol. 1991; 17: 2B-13BAbstract Full Text PDF PubMed Scopus (269) Google Scholar Accelerated coronary arteriosclerosis is also a prominent complication associated with allograft cardiac transplant.8Gravanis MB Roubin GS Histopathologic phenomena at the site of percutaneous transluminal coronary angioplasty: the problem of restenosis.Hum Pathol. 1989; 20: 477-485Abstract Full Text PDF PubMed Scopus (125) Google Scholar Accordingly, it would be worthwhile to gain additional understanding on the mechanism of restenosis and factors that determine individual differences in susceptibility to occlusive vasculopathy after these surgical operations. Accelerated coronary arteriosclerosis because of restenosis after surgical operations is different from progressive atherosclerosis in that lipid deposition and macrophage foam-cell appearance are late events. Pathological studies revealed that narrowing of the coronary vessels in both restenosis and allograft arteriosclerosis are related to intimal hyperplasia, with abnormal proliferation and migration of vascular smooth muscle cells from the tunica media to the intima.9Giraldo AA Esposo OM Meis JM Intimal hyperplasia as a cause of restenosis after percutaneous transluminal coronary angioplasty.Arch Pathol Lab Med. 1985; 109: 173-175PubMed Google Scholar, 10Forrester JS Fishbein M Helfant R Fagin J A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies.J Am Coll Cardiol. 1991; 17: 758-769Abstract Full Text PDF PubMed Scopus (481) Google Scholar, 11Mehra MR Ventura HO Stapleton DD Smart FW The prognostic significance of intimal proliferation in cardiac allograft vasculopathy: a paradigm shift.J Heart Lung Transplant. 1995; 14: S207-S211PubMed Google Scholar One hypothesis suggests that these accelerated forms of arteriosclerosis are because of immune-mediated endothelial injury, thus exposing the underlying vascular smooth muscle cells to mitogenic growth factors, which induces phenotypic conversion of the smooth muscle cells from the contractile-nonproliferating phenotype to the secretory proliferating phenotype.12Austin GE Ratliff MB Hollman J Tabei S Phillips DF Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty.J Am Coll Cardiol. 1985; 6: 369-375Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 13Jawien A Bowen-Pope DF Lindner V Schwartz SM Clowes AW Platelet-derived growth factor promotes smooth muscle cell migration and intimal thickening in a rat model of balloon angioplasty.J Clin Invest. 1992; 89: 507-511Crossref PubMed Scopus (594) Google Scholar, 14Jackson CL Raines E Ross R Reidy MA Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury.Arterioscler Thromb. 1993; 13: 1218-1226Crossref PubMed Scopus (229) Google Scholar, 15Davis SF Yeung AC Meredith IT Charbonneau F Ganz P Selwyn AP Anderson TJ Early endothelial dysfunction predicts the development of transplant coronary artery disease at 1 year posttransplant.Circulation. 1996; 93: 457-462Crossref PubMed Scopus (224) Google Scholar However, more recent data revealed that differences in abnormal vascular remodeling, associated with inefficient compensatory enlargement of the arterial wall, is the contributory factor toward restenosis.16Kakuta T Currier JW Hauderschild CC Ryan TJ Faxon DP Differences in compensatory enlargement, not intimal formation, account for restenosis after angioplasty in the atherosclerotic rabbit model.Circulation. 1994; 89: 2809-2815Crossref PubMed Scopus (286) Google Scholar, 17Post MJ Borst C Kuntz RE The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty.Circulation. 1994; 89: 2816-2821Crossref PubMed Scopus (368) Google Scholar Although coronary stents have been used successfully to reduce vascular wall remodeling with a decrease in the rate of restenosis,18Savage MP Douglas JS Fischman DL Pepine CJ King SB Werner JA Bailey SR Overlie PA Fenton SH Brinker JA Leon MB Goldberg S Stent placement compared with balloon angioplasty for obstructed coronary bypass grafts. Saphenous vein de novo trial investigators.N Engl J Med. 1997; 337: 740-747Crossref PubMed Scopus (514) Google Scholar, 19Serruys PW van Hout B Bonnier H Legrand V Garcia E Macaya C Sousa E van der Giessen W Colombo A Seabra-Gomes R Kiemeneij F Ruygrok P Ormiston J Emanuelsson H Fajadet J Haude M Klugmann S Morel MA Randomised comparison of implantation of heparin-coated stents with balloon angioplasty in selected patients with coronary artery disease.Lancet. 1998; 352: 673-681Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar restenosis because of smooth muscle cell hyperplasia occurs after stenting in 20 to 30% of patients.20Santoian EC King III, SB Intravascular stents, intimal proliferation and restenosis.J Am Coll Cardiol. 1992; 19: 877-879Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 21Bai H Masuda J Sawa Y Nakano S Shirakura R Shimazaki Y Ogata J Matsuda H Neointima formation after vascular stent implantation. Spatial and chronological distribution of smooth muscle cell proliferation and phenotypic modulation.Arterioscler Thromb. 1994; 14: 1846-1853Crossref PubMed Scopus (86) Google Scholar The key factors that are important for regulating smooth-muscle cell proliferation and in determining the severity of neointimal hyperplasia have not been completely elucidated. Recent studies revealed that apolipoprotein (apo) E4 homozygosity is associated with increased risk of restenosis after percutaneous transluminal coronary angioplasty in human patients.22van Bockxmeer FM Mamotte CDS Gibbons FR Taylor RR Apolipoprotein E4 homozygosity—a determinant of restenosis after coronary angioplasty.Atherosclerosis. 1994; 110: 195-202Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 23van Bockxmeer FM Mamotte CDS Gibbons FA Burke V Taylor RR Angiotensin-converting enzyme and apolipoprotein E genotypes and restenosis after coronary angioplasty.Circulation. 1995; 92: 2066-2071Crossref PubMed Scopus (67) Google Scholar The relationship between the ε4/4 genotype and restenosis seemed to be independent of serum cholesterol and apo(a) levels.23van Bockxmeer FM Mamotte CDS Gibbons FA Burke V Taylor RR Angiotensin-converting enzyme and apolipoprotein E genotypes and restenosis after coronary angioplasty.Circulation. 1995; 92: 2066-2071Crossref PubMed Scopus (67) Google Scholar These results suggested a lipid transport-independent role of apoE in protection against vascular disease. Our previous studies showed that apoE inhibits oxidized low-density lipoprotein- and platelet-derived growth factor-induced smooth-muscle cell migration and proliferation in vitro.24Ishigami M Swertfeger DK Granholm NA Hui DY Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase.J Biol Chem. 1998; 273: 20156-20161Crossref PubMed Scopus (132) Google Scholar Thus, apoE may have direct cell regulatory functions in the vessel wall. ApoE-deficient mice have been used previously to assess neointimal formation after various treatments.25De Geest B Zhao Z Collen D Holvoet P Effects of adenovirus-mediated human apoA-I gene transfer on neointima formation after endothelial denudation in ApoE-deficient mice.Circulation. 1997; 96: 4349-4356Crossref PubMed Scopus (90) Google Scholar However, the direct role of apoE in dictating the severity of the neointima in vivo remains unclear. The current study used apoE-deficient mice as well as mice with transgenic overexpression of human apoE3 to explore the importance of apoE level in dictating neointimal hyperplasia after mechanically induced injury of the vessel wall. Male apoE-null mice back-crossed to a C57BL/6 genetic background were obtained from The Jackson Laboratory (Bar Harbor, ME). The human apoE-transgenic mice were generously provided by Dr. John Taylor (Gladstone Institute, San Francisco, CA). Detailed characterization of these apoE transgenic mice was reported previously by de Silva et al.26de Silva HV Lauer SJ Mahley RW Weisgraber KH Taylor JM Apolipoproteins E and C-III have opposing roles in the clearance of lipoprotein remnants in transgenic mice.Biochem Soc Trans. 1993; 21: 483-487PubMed Google Scholar These apoE transgenic mice were originally produced in the ICR strain background and were back-crossed with FVB/N mice in our institutional facility for seven generations to >99% genetic homogeneity in FVB/N background before experiments. The wild-type C57BL/6 and FVB/N mice were obtained initially from The Jackson Laboratory and were maintained as breeding colonies in our institution. These animals were used as controls for the apoE(−/−) and apoE transgenic mice in all experiments. The animals were maintained on a 12-hour light/12-hour dark cycle and were fed a normal mouse chow diet (Harlan Teklad, Madison, WI). Food and water were available ad libitum. The animals were used for experimentation when they reached 6 to 8 weeks of age, weighing ∼25 to 30 g. All animal experimentation protocols were performed under the guidelines of animal welfare by the University of Cincinnati, in accordance with National Institutes of Health guidelines. Human apoE level in the transgenic mice was measured by enzyme-linked immunosorbent assay. A 96-well microtiter plate was incubated overnight with 100 μl of a 2 μg/ml solution of mouse anti-human apoE monoclonal antibody 1D7 (Ottawa Heart Institute Research Corporation, Ottawa, Ontario, Canada). Nonspecific sites were then blocked with phosphate-buffered saline (PBS) containing 5 mg/ml Tween-20 and 5 mg/ml bovine serum albumin. One hundred μl of mouse serum was added to each well and the incubation was continued for 2 hours, followed by a 2-hour incubation with a 1:500 dilution of rabbit anti-human apoE polyclonal antibody (DAKO, Carpinteria, CA). The plates were washed and then incubated for an additional 2 hours with a 1:5,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG obtained from Sigma Chemical Co. (St. Louis, MO). Immunoreactivity was determined by addition of ALP 10 substrate (Sigma Chemical Co.) and measuring absorbance at 405 nm. Purified human apoE isolated according to Rall et al27Rall SC Weisgraber KH Mahley RW Isolation and characterization of apolipoprotein E.Methods Enzymol. 1986; 128: 273-287Crossref PubMed Scopus (94) Google Scholar was used as the standard. Mechanically induced endothelial denudation was performed by modification of the method originally described by Lindner et al.28Lindner V Fingerle J Reidy MA Mouse model of arterial injury.Circ Res. 1993; 73: 792-796Crossref PubMed Scopus (313) Google Scholar In this modification,29Zhou M Sutliff RL Paul RJ Lorenz JN Hoying JB Haudenschild CC Yin M Coffin JD Kong L Kranias EG Luo W Boivin GP Duffy JJ Pawlowski SA Doetschman T Fibroblast growth factor 2 control of vascular tone.Nat Med. 1998; 4: 201-207Crossref PubMed Scopus (303) Google Scholar an epon resin probe made by forming an epon bead slightly larger than the diameter of the carotid artery (0.45 mm) on a 3-0 nylon suture instead of a guide wire was used for the arterial injury. The animals were anesthetized by intraperitoneal injection with a solution composed of ketamine (80 mg/kg body weight; Fort Dodge Laboratories, Inc., Fort Dodge, IA) and xylazine (16 mg/kg; The Butler Co., Columbus, OH) diluted in 0.9. NaCl. The mice were immobilized and the fur covering the neck from sternum to chin were removed with lotion hair remover (Nair. Carter-Wallace, Inc., New York, NY). Surgery was performed using a dissection microscope (Leica GZ6; Leica, Buffalo, NY). The entire length of left carotid artery was exposed and the artery was ligated immediately proximal from the point of bifurcation with a 7-0 silk suture (Ethicon, Inc., Somerville, NJ). Another 7-0 suture was placed around the common carotid artery immediately distal from the branch point of the external carotid. A transverse arteriotomy was made between the 7-0 sutures and the resin probe was inserted and advanced toward the aorta arch and withdrawn five times. The probe was removed and the proximal 7-0 suture was ligated. Once restoration of blood flow through the carotid branch points was confirmed, the incision was closed with a 5-0 sterile surgical gut (Ethicon, Inc.). All these procedures were performed within 20 minutes. Animals were allowed to recover in a 37°C heat box. The identical surgical procedure was applied to each animal to assure reproducibility of the results. Fourteen days after inducing arterial injury, animals were anesthetized and perfused with 0.9% NaCl by placement of a 22-gauge butterfly angiocatheter in the left ventricle. The mice were subsequently perfusion-fixed in situ by infusion with 10. buffered formalin (pH 7.0) for 20 minutes at a constant pressure of 100 mmHg. The entire neck was dissected from each mouse and fixed in 10. buffered formalin for an additional 48 hours. The whole neck was decalcified for 48 hours before embedding in paraffin. Identical whole-neck cross-sections of 5 μm were made from the distal side of the neck beginning at the point of the distally ligated 7-0 suture. The whole-neck sections were used to evaluate both the injured and the uninjured control vessels on the same section. For each mouse, four levels of serial sections at 500-μm intervals were made, and the data collected were averaged which allowed for the measurement to represent lesion formation along the entire length of the artery. Parallel sections were subjected to routine hematoxylin and eosin (H&E) staining as well as to Verhoeff Van-Gieson staining of elastic lamina. Four unstained sections from each level were used for immunohistochemistry. Morphometric analyses were performed on elastin-stained tissue. For each animal, four whole-neck cross-sections with both injured left and uninjured-control right carotid arteries were measured. Images were digitized and captured using a Sony video camera (Sony, New York, NY) connected to a personal computer. Measurements were performed at a magnification of ×200 using a Scion Image analysis computer program (Scion, Frederick, MD). For each artery, luminal area, area inside the internal elastic lamina, and the area encircled by external elastic lamina were measured. Medial area was calculated as area encircled by external elastic lamina-area inside the internal elastic lamina and intimal area was calculated as area inside the internal elastic lamina-luminal area. To calculate the medial thickness for each vessel cross-section, the linear distance between internal elastic lamina and external elastic lamina was measured independently in four places, each at 90° apart and averaged. From these measurements, the ratio of intimal area and medial area, and the percent of luminal stenosis (100 × intimal area/area inside the internal elastic lamina) were calculated. For all staining, sections were deparaffinized with xylene by incubating for 10 minutes three times and then dehydrated with a series of graded ethanol from 70 to 100% for 10 minutes each. Slides were then washed in distilled water for 5 minutes, and endogenous peroxidase activities were blocked by incubating for 30 minutes with 0.5. hydrogen peroxide in PBS containing Triton X-100 (Sigma Chemical Co.). Slides were then washed three times in the same solution without H2O2 for 15 minutes each. Nonspecific binding sites were blocked by incubation for 30 minutes with 1.5% serum in PBS containing Triton X-100. For the identification of smooth muscle cells and endothelial cells, sections were incubated overnight at 4°C with anti-smooth muscle α-actin (Clone 1A4; Sigma Chemical Co.) at 1:3,000 dilution or anti-Von Willebrand Factor (DAKO, Carpinteria, CA) at 1:100 dilution, respectively. The slides were washed three times for 15 minutes each with PBS containing Triton X-100 and then incubated for 1 hour at 23°C with 0.5% biotinylated anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA) for anti-smooth muscle cell α-actin or 0.5. biotinylated anti-rabbit IgG (Vector Laboratories, Inc.) for Von Willebrand Factor in the same solution containing 1.5% normal serum. Slides were then washed as described above, and then incubated with the avidin-peroxidase complex reagent (Peroxidase Vectastain Elite ABC kit, Vector Laboratories, Inc.) for 1 hour at 23°C. The reaction was visualized with 3,3′-diaminobenzidine. Identification of proliferating cells in the S phase of the growth cycle was accomplished by injecting the mice with three doses of 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg; Sigma Chemical Co. intraperitoneally at 24, 8, and 1 hour before their sacrifice, followed by immunohistochemical analysis with mouse monoclonal anti-BrdU (Clone BU33, diluted 1:300; Sigma Chemical Co.). Cells in the G1 growth phase were identified by immunohistochemical staining with the mouse monoclonal anti-cyclin D1 antibody, DCS-6 (Oncogene Research Products, Boston, MA) at a dilution of 1:20. The sections were pretreated by incubation with 4 mol/L HCl for 30 minutes at 37°C and neutralized in 0.2 mol/L borate buffer, pH 9.0. After a 15-minute washing with PBS containing Triton X-100, the sections were further incubated with 0.1% trypsin for 30 minutes at 37°C, followed by blocking endogenous peroxidase and nonspecific binding sites as described above. The reaction was visualized using the Vectastain Elite ABC kit as described above. All values were expressed as mean ± SEM. When only two groups (injured arteries and contralateral control arteries) were compared, differences were assessed by a paired Student's t-test. Multiple comparisons were first tested by analysis of variance. When the analysis of variance demonstrated significant differences, individual mean differences were analyzed by the Student-Newman-Keuls test. Statistical software SigmaStat (Version 2.0, Jandel Co., San Rafael, CA) was used in statistical analysis. For all statistical analyses, P < 0.05 was considered significant. This study used an epon resin probe on a 3-0 nylon suture to induce arterial injury in mice. Immunohistochemical staining with Von Willebrand Factor antibodies demonstrated that the resin probe could introduce consistent denudation of the endothelium (Figure 1). Importantly, endothelial denudation by resin probe did not result in damage of the elastic lamina (Figure 2, Figure 3). Thus, this procedure is ideally suitable to evaluate neointimal hyperplasia as a consequence of endothelial denudation with minimal trauma to the underlying medial smooth muscle cells. Animals with damaged elastic lamina after mechanically induced injury were excluded from subsequent characterization.Figure 2Representative photomicrographs of a whole-neck section with H&E staining. Mechanically induced endothelial denudation was performed on the left carotid artery of an apoE-null mouse using a 3-0 nylon suture with a 0.45-mm epon bead. The mouse was sacrificed after 14 days, perfusion-fixed with 10% buffered formalin, and the whole neck was decalcified and embedded in paraffin. Four levels of serial sections at 500-μm intervals were made. a: Whole neck section with both the injured left carotid artery and the uninjured right carotid artery. Scale bar, 100 μm. b and c: Magnified versions of the uninjured and injured arteries, respectively. Scale bars, 50 μm. The arrows indicate the external elastic laminae and the arrowheads indicate the internal elastic laminae in each section.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Response of C57BL/6 wild-type and apoE(−/−) mice to vascular injury. Mechanically induced injury of left carotid arteries in mice were performed 14 days before their sacrifice. Representative photomicrographs of the uninjured (left) and injured (right. carotid arteries from a C57BL/6 mouse (a–d) and apoE(−/−) mouse (e–h). Paraffin sections of the carotid arteries were stained with Verhoeff Van-Gieson (a and b and e and f) and H&E (c and d and g and h). The arrows indicate the external elastic laminae and the arrowheads indicate the internal elastic laminae in each section. Scale bar, 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Examination of uninjured carotid arteries in normal C57BL/6 mice revealed three clearly delineated elastic lamina separating two layers of medial smooth muscle cells (Figure 3). Representative histology data comparing injured and uninjured carotid arteries from a control and two apoE-null mice are shown in Figure 3. Although little or no cells could be observed in the intima, within the innermost internal elastic lamina, of either apoE(+/+) or apoE(−/−) mice before injury (Figure 2, Figure 3), a significant number of cells was found to be accumulated in the intima 14 days after injury of the carotid arteries (Figure 2, Figure 3). Immunohistochemical analysis with smooth muscle cell-specific α-actin antibodies identified the majority of cells in injured and uninjured media, as well as in the neointima after arterial injury, as smooth muscle cells (Figure 4). Morphometric analysis of the complete set of data clearly demonstrated significant increases in medial thickness, medial area, and neointimal formation (defined as an increase in intimal area) after vascular injury in both C57BL/6 control and apoE(−/−) mice (Figure 5, A–C). Interestingly, the severity of neointimal formation in response to arterial injury was found to be 3.4-fold greater in apoE(−/−) mice in comparison with that in the C57BL/6 control mice (11,223.75 ± 2,715.4 μm2 versus 3,323.98 ± 2,231.65 μm2, P < 0.05) (Figure 5C). Analysis of BrdU-labeled cells revealed an eightfold and fivefold increase in the number of proliferating cells in the media and intima of apoE(−/−) mice compared to that in control animals (P < 0.05) (Figure 6a and Table 1). The greater neointimal formation in apoE(−/−) mice also resulted in the significant increase of intima/media ratio in apoE-null mice in comparison with that observed in the C57BL/6 control mice (0.11 ± 0.073 versus 0.359 ± 0.091, P < 0.05) (Figure 5D). In addition, luminal stenosis was found to be more severe in the apoE(−/−) mice after arterial injury (32.8 ± 8.01% versus 7.0 ± 3.58% in control mice, P < 0.05) (Figure 5E).Figure 6Immunohistochemical staining of BrdU in control (a, c, e, and g) and injured (b, d, f, and h ) carotid arteries from C57BL/6, apoE (−/−), FVB/N, and apoE transgenic mice. Paraffin section were obtained 14 days after mechanically induced injury of the mouse carotid artery. The sections were pretreated with 4 mol/L HCl and 0.1% trypsin for 30 minutes at 37°C, followed by incubation with a monoclonal antibody against BrdU at a dilution of 1:300. Scale bar, 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Analysis of Proliferating Cells in Injured Carotid Arteries of MiceBrdU-labeled cells/cross-sectionMicenMediaIntimaC57Bl/671.44 ± 0.586.78 ± 1.66ApoE(−/−)1012.56 ± 3.13*Significant difference from their respective control group at P < 0.05.36.44 ± 10.84*Significant difference from their respective control group at P < 0.05.FVB/N516.00 ± 4.0949.60 ± 9.94ApoE Transgenics93.00 ± 1.01*Significant difference from their respective control group at P < 0.05.15.00 ± 5.29*Significant difference from their respective control group at P < 0.05.Proliferating cells in the media and intima after mechanically induced arterial injury was determined by counting the number of BrdU-positive cells in each section. The data represent the mean ± SEM.* Significant difference from their respective control group at P < 0.05. Open table in a new tab Proliferating cells in the media and intima after mechanically induced arterial injury was determined by counting the number of BrdU-positive cells in each section. The data represent the mean ± SEM. Consideration was given to the fact that, in addition to difference in apoE level, the apoE(−/−) mice also had significantly high plasma lipid levels than the C57BL/6 control mice even under basal low-fat low-cholesterol diet.30Zhang SH Reddick RL Piedrahita JA Maeda N Spontaneous hyperch