Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice

Abstract

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 alpha-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.

Figure 1.

Von Willebrand Factor immunohistochemical staining of control (a) and injured (b) carotid arteries. Paraffin sections were obtained from a C57BL/6 mouse 1 hour after mechanically induced injury of the left carotid artery. The sections were incubated with a polyclonal antibody against Von Willebrand Factor at a dilution of 1:100. Immunoreactivity was detected by incubation with 0.5% biotinylated anti-rabbit IgG, followed by incubation with avidin-peroxidase complex and visualized with 3,3′-diaminobenzidine. Scale bar, 50 μm.

Figure 2.

Representative 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.

Figure 3.

Response 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.

Figure 4.

Immunohistochemical staining of smooth muscle α-actin in control (a) and injured carotid arteries (b) of an apoE(−/−) mouse. Paraffin sections were obtained 14 days after mechanically induced injury of the mouse carotid artery. The sections were incubated with a monoclonal antibody against α-smooth muscle cell α-actin at a dilution of 1:3,000. Immunoreactivity was detected by incubation with 0.5% biotinylated anti-mouse IgG, followed by incubation with avidin-peroxidase complex and visualized with 3,3′-diaminobenzidine. Scale bar, 50 μm.

Figure 5.

Morphometric quantitation of control (open bars) and injured (filled bars) carotid arteries from C57BL/6 wild-type and apoE(−/−) mice. Carotid artery injury was performed using 3-0 sutures containing 0.45-mm epon beads. The animals were sacrificed after 14 days for tissue analysis. Medial thickness (A) was calculated as the average linear distance between the internal and external elastic lamina measured in four places at 90° apart. Medial area (B) was calculated as the area encircled by external elastic lamina minus the area encircled by the internal elastic lamina. Neointimal area (C) was determined by subtracting the luminal area from the area encircled by the internal elastic lamina. The intima-to-media ratio (D) was determined based on the data in B and C. Luminal stenosis (E) was calculated as the percentage of the area inside the internal elastic lamina occupied by the intimal area in the injured carotid arteries. The data represent the mean ± SEM from eight C57BL/6 and 12 apoE(−/−) mice. Scale bars with different letters indicate significant difference at P < 0.05.

Figure 6.

Immunohistochemical 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.

Figure 7.

Response of FVB/N wild-type and human apoE3 transgenic 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 FVB/N mouse (a–d) and apoE3 transgenic 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.

Figure 8.

Morphometric quantitation of control (open bars) and injured (filled bars) carotid arteries from FVB/N wild-type and human apoE3 transgenic mice. Carotid artery injury was performed using 3-0 sutures containing 0.45-mm epon beads. The animals were sacrificed after 14 days for tissue analysis. Medial thickness (A), medial area (B), neointimal area (C), the intima-to-media ratio (D), and luminal stenosis (E) were calculated as described in the legend to Figure 5 ▶ . The data represent the mean ± SEM from seven FVB/N and six apoE transgenic mice. Scale bars with different letters indicate significant difference at P < 0.05.

Figure 9.

Immunohistochemical staining of cyclin D1 in control (a, c, e, and g) and injured (b, d, f, and h) carotid arteries from C57BL/6 (a and b), apoE (−/−) (c and d), FVB/N (e and f), and apoE transgenic mice (g and h). Paraffin sections 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 cyclin D1 at a dilution of 1:20. Scale bar, 50 μm.