Periadventitial delivery of anti-EGF receptor antibody inhibits neointimal macrophage accumulation after angioplasty in a hypercholesterolaemic rabbit

Abstract

Monocyte recruitment and their differentiation into macrophages are both early events in native and accelerated atherosclerosis that follows angioplasty. We have investigated the putative functional role of the epidermal growth factor receptor (EGFR) present on rabbit monocytes/macrophages. The impact of periadventitial delivery of an EGFR-specific, blocking monoclonal antibody (ICR62, which inhibits EGF-binding to its receptor) was investigated in a rabbit model of accelerated atherosclerosis induced by a combination of carotid injury and 4 weeks of a 2% cholesterol-diet. Two weeks after the initiation of the diet, a balloon-catheter angioplasty of the left common carotid artery was performed and a collar placed around the injured carotid artery immediately, for the delivery of ICR62 antibody, isotype-matched antibody or saline control. Monocyte/macrophage accumulation, cell proliferation and neointimal thickening were determined 2 weeks after the delivery of the antibodies. The function of the EGFR on rabbit monocytes was also investigated in vitro, using chemotaxis assays. Treatment with ICR62 was associated with a significant reduction in macrophage accumulation and neointimal thickening and a 76% reduction in neointimal area of the vessel wall compared with controls. In vitro ICR62 inhibited macrophage and smooth muscle cell migration towards EGFR ligands including EGF and HB-EGF. These findings suggest that EGFR ligation may be important in the development of early atherosclerotic lesions following balloon-catheter angioplasty, and periadventitial delivery may provide a feasible approach for administration of the inhibitors of EGFR-binding such as ICR62.

Figure 1

Neointima formation in representative H & E stained cross sections of balloon-catheter injured and uninjured carotid arteries of cholesterol-fed rabbits 2 weeks post delivery of antibody or saline. The uninjured contralateral carotid artery show a normal morphology (a, b), whereas injured saline-treated (c, d) and ICR62 antibody treated (e, f) showed various degree of neointimal formation. The vessel lumen of saline-treated (c) was very much smaller than the ICR62-treated artery (e). Arrows indicate IEL. L, lumen; M, media.

Figure 2

Periadventitial delivery of ICR62 inhibits neointimal formation in injured carotid arteries of hypercholesterolaemic rabbits. VSMCs identified by immunostaining with anti-α-actin (top panel ai–iii). Arrows indicate IEL. L, lumen; M, media. Quantification of absolute intima/media ratios (b) of the ICR62 (n= 8), isotype matched control antibody (n= 3) and saline-treated (n= 7) were as described in Methods. ICR62 antibody significantly reduced intimal thickening compared with the saline or isotype controls: ***P< 0.0001. Bars represent mean ± SEM.

Figure 3

Macrophage accumulation and their colocalization with EGFR in injured carotid arteries. The total macrophage content of the saline-treated (n= 6), IgG2b Isotype control (n= 3) and ICR62 antibody (n= 7) injured arteries was determined by immunostaining with RAM 11 (top panel a). In the saline-injured carotid artery (ai) numerous tissue macrophages and foam cells are apparent within the neointima and adjacent media, where as in the ICR62 fewer numbers are noticed and the neointima thickness is less (aiii). In IgG2b isotype-treated carotid artery (aii) the number of RAM 11 +ve cells appeared to be reduced compared to the saline carotid (ai). (aiv) a negative control (isotype matched control antibody for RAM 11). Arrows indicate IEL. Macrophages numbers are expressed as neointimal RAM 11 positive cells in injured carotid artery sections from six rabbits per group, except for IgG2b isotype controls (n= 3) (b). Macrophage density is expressed as neointimal RAM 11 positive cells per mm² (total neointimal area) (c). ICR62 antibody significantly reduced neointimal macrophage content compared with the saline and IgG2b isotype matched control. **P< 0.002; ***P< 0.0001.

Figure 4

Macrophage proliferative activity as assessed by double immunostaining of rabbit carotid artery sections and adherent macrophages in vitro. Panel a shows a representative colocalization of PCNA-positive stained nuclei (purple staining) with RAM 11 positive cells (brown cytoplasmic staining). Arrowheads in the carotid neointimal lesion indicate proliferating macrophages (ai & aii; double-positive) and the open-arrows points to non-proliferating single-positive cells (only cytoplasmic staining without nuclear involvement) at a higher magnification (aii). (aiii) a negative control for RAM 11 and PCNA antibodies. Arrows indicate IEL. M, Media. (b) Quantification of neointimal macrophage proliferation expressed as percentage means of PCNA(+)-RAM 11 +ve cells (double-positive)/total number of RAM 11 +ve cells (single positive) in sections from the saline- (n= 4), IgG2b isotype (n= 3) and ICR62 antibody (n= 6) treated carotid arteries. (c) Inhibition of EGF-induced PCNA expression by ICR62 antibody in adherent macrophages in vitro. Bars represent the mean ± SEM of four independent experiments performed in duplicates. Significance of difference compared to the saline or isotype-matched antibody; *P< 0.05; **P< 0.005.

Figure 5

ICR62 antibody inhibits HB-EGF and EGF-induced (a) smooth muscle cells and (b) monocytes migration. A Chemotaxis assay was used to determine whether increasing concentrations of ICR62 (10, 50 and 200 nM) or the isotype control antibody (200 nM) inhibited HB-EGF and EGF-induced migration of peripheral blood monocytes and aortic SMCs. (c) Comparison of peripheral blood monocyte and monocyte-derived macrophage chemotactic response to EGF. The chemotactic response is expressed as mean fold induction over medium control ± SEM of three independent experiments performed in triplicate, except for the isotype control in monocyte migration, where n= 2. The chemotactic response of the cells induced by HB-EGF, EGF, MCP-1 and PDGF-BB was significantly higher as compared to the medium control; ***P< 0.0001. Significance of differences in the inhibition of HB-EGF and EGF-induced cell migration by ICR62 antibody compared to the relevant controls: ***P< 0.0001; **P< 0.001. Significance of difference of the chemotactic response of monocyte-derived macrophages compared to monocytes; ***P< 0.0005.