Feasibility of ex vivo fluorescence imaging of angiogenesis in (non-) culprit human carotid atherosclerotic plaques using bevacizumab-800CW

Abstract

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques (n = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis (p < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo.

Figure 1

Ex vivo fluorescent signal analyses of a culprit and non-culprit plaque. (a) Odyssey (800 nm) scans of a culprit (top row) and non-culprit plaque (bottom row) tissue sections, and the corresponding VEGF-A stainings of the sections (VEGF-A is stained blue). The sections were taken at approximately 1–2 mm depth of the whole tissue specimens. Fluorescent intensity measurements are highest at sites of strong VEGF-A staining, as seen in the culprit plaque. On the contrary, the non-culprit plaque displays low fluorescent intensity and has no visible VEGF-A expression. (b) Fluorescence microscopy of a culprit plaque demonstrates the near-infrared signal of the tracer (red; 800 nm channel; left column) overlaps the cell cytoplasm, indicating the intracellular localization of bevacizumab-800CW (H&E staining; right column). Cell nuclei are displayed by Hoechst staining (blue; DAPI channel; left column). The asterisk indicates the same place in the corresponding specimens.

Figure 2

Fluorescent pattern intensity of tissue sections and VEGF-A staining intensity. (A) Based on whole tissue fragments, a higher mean fluorescence intensity of all culprit plaques (36,425; range 22,478–55,530) was observed in comparison to non-culprit plaques (8855; range 3515–16,586) after incubation with bevacizumab-800CW. The median MFI of the culprit plaques was higher than the median MFI of the non-culprit plaques (42,129 vs. 6450). (B) VEGF-A staining intensities (H-score) of both culprit and non-culprit plaques displayed by a boxplot. The average H-score was the highest in the culprit plaques. (C) Representative example of a tissue section taken at approximately 1–2 mm depth of a culprit plaque hot spot displaying the overlap of the pattern of fluorescence and VEGF-A immunohistochemistry (blue staining).

Figure 3

Representative examples of histologic parameters of hot spots of a culprit, the asymptomatic and a non-culprit plaque. The stainings were performed on subsequent slides, depicting a ROI in the specimens. (A–C) H&E stain, demonstrating extensive intraplaque hemorrhage in the culprit plaque (the yellow asterisks indicate areas with red blood cells, orange spots); (D–F) VEGF-A stain, revealing elevated VEGF-A expression quantified by the H-score in both the culprit and asymptomatic plaque as opposed to the non-culprit plaque; (G–I) CD34 stain indicating the association between VEGF-A and microvessel density; (J–L) CD68 stain, demonstrating an influx of macrophages around the micro vessels in the culprit and asymptomatic plaque; (M–O) Von Kossa stain, revealing no differences between the culprit, asymptomatic and non-culprit plaques; (P–R) SMA stain, showing elevated SMA expression in the non-culprit plaque.