Effect of anti-ApoA-I antibody-coating of stents on neointima formation in a rabbit balloon-injury model

Abstract

Background and aims: Since high-density lipoprotein (HDL) has pro-endothelial and anti-thrombotic effects, a HDL recruiting stent may prevent restenosis. In the present study we address the functional characteristics of an apolipoprotein A-I (ApoA-I) antibody coating in vitro. Subsequently, we tested its biological performance applied on stents in vivo in rabbits.

Materials and methods: The impact of anti ApoA-I- versus apoB-antibody coated stainless steel discs were evaluated in vitro for endothelial cell adhesion, thrombin generation and platelet adhesion. In vivo, response to injury in the iliac artery of New Zealand white rabbits was used as read out comparing apoA-I-coated versus bare metal stents.

Results: ApoA-I antibody coated metal discs showed increased endothelial cell adhesion and proliferation and decreased thrombin generation and platelet adhesion, compared to control discs. In vivo, no difference was observed between ApoA-I and BMS stents in lumen stenosis (23.3±13.8% versus 23.3±11.3%, p=0.77) or intima surface area (0.81±0.62 mm2 vs 0.84±0.55 mm2, p=0.85). Immunohistochemistry also revealed no differences in cell proliferation, fibrin deposition, inflammation and endothelialization.

Conclusion: ApoA-I antibody coating has potent pro-endothelial and anti-thrombotic effects in vitro, but failed to enhance stent performance in a balloon injury rabbit model in vivo.

Fig 1. HMEC-1 cell growth on coated and uncoated disks.

HMEC-1 cell growth on ApoA-I-coated discs in 100%, 10% and 1% density, incubated in HDL after 1, 2 and 4 days, compared to similarly incubated uncoated discs (control). Error bars indicate Standard Deviation (SD) of mean (n = 3). (* = P<0.05). HMEC-1 cell adhesion (B) on disks—HMEC-1 cell adhesion on ApoA-I-, ApoB- or Isotype-coated discs with 100% and 10% antibody density, incubated for 4 days. Error bars indicate Standard Deviation (SD) of mean (n = 4). (* = P<0.05)

Fig 2. Thrombin generation on coated discs.

Thrombin generation for discs coated with either ApoA-I-, ApoB-, Isotype- or no antibody (empty discs), compared to thrombin generation in absence of a disc (negative control). (n = 4).

Fig 3

A. Platelet adhesion on coated disks. Platelet adhesion: SEM analysis of adhered platelets on surfaces coated with ApoA-I-, ApoB- or isotype antibody after corresponding pre-treatment. The number of adhered platelets are given as the mean ± SD (n = 3). (* = P<0.05). B. Platelet morphology. The morphology of the adhered platelets was divided into 4 different classes representing various degrees of activation, ranging from “round” (weak), “dendritic” (intermediate), “spread/dendritic” (strong) to “spread” (very strong). The data are presented as the percentage of platelets exhibiting the indicated morphology. Data error bars are the standard deviation of the mean (n = 3).

Fig 4. Morphometric analysis results.

Lumen stenosis (A1 and A2), intima surface (B1 and B2) and IM-ratio (C1 and C2) of anti ApoA-I coated stent and BMS stent 28 days after implantation. Bars indicate mean value per section with error bars indicating the standard deviation (SD). Three left panels (A1, B1 and C1) show results of five stent regions together. No significant differences were observed between the two stent types. Three right panels (A2, B2 and C2) show results of individual stent regions in the two stent types. No significant differences were observed between the corresponding regions in the two stent types.

Fig 5. Immunohistochemical analysis.

Results of immunohistochemical analysis (top) with corresponding representative examples of the immunohistochemical staining (20x objective). KI-67 (first panels), Fibrin (second panel), RAM11 (third panel) and VWF (fourth panel) are shown. Bars indicate mean score or count with error bars indicating the standard deviation (SD). Proliferation (KI-67), fibrin deposition, macrophage infiltration (RAM11) and endothelialization (VWF) were not significantly different between the two stent types.

Fig 6. Scanning electron microscopy overview.

SEM images of a 28-day BMS (upper half) and anti- ApoA-I coated stent (lower half) implanted in the rabbit common iliac artery. High magnification details of stent strut coverage are shown of three randomly chosen spots, next to the low magnification overview in the centre. Complete endothelial lining of the lumen is shown in all high-magnification details, with similar endothelial cell aspect.