Interaction of cowpea mosaic virus nanoparticles with surface vimentin and inflammatory cells in atherosclerotic lesions

Abstract

Aims: Detection of atherosclerosis has generally been limited to the late stages of development, after cardiovascular symptoms present or a clinical event occurs. One possibility for early detection is the use of functionalized nanoparticles. The aim of this study was the early imaging of atherosclerosis using nanoparticles with a natural affinity for inflammatory cells in the lesion.

Materials & methods: We investigated uptake of cowpea mosaic virus by macrophages and foam cells in vitro and correlated this with vimentin expression. We also examined the ability of cowpea mosaic virus to interact with atherosclerotic lesions in a murine model of atherosclerosis.

Results & conclusion: We found that uptake of cowpea mosaic virus is increased in areas of atherosclerotic lesion. This correlated with increased surface vimentin in the lesion compared with nonlesion vasculature. In conclusion, cowpea mosaic virus and its vimentin-binding region holds potential for use as a targeting ligand for early atherosclerotic lesions, and as a probe for detecting upregulation of surface vimentin during inflammation.

Figure 1. Characterization of labeled cowpea mosaic virus

(A) Space-filling model of the cowpea mosaic virus capsid structure assembled in Chimera, using coordinates obtained from [103]. The large capsid subunit is in green, small capsid subunit is in blue and surface-accessible lysines are highlighted in pink. (B) Fast protein liquid chromatography chromatogram of Alexa Fluor® 647-labeled cowpea mosaic virus particles. The three traces reflect the absorbance in mAU at three wavelengths (from top to bottom: 260, 280 and 650 nm, respectively).

Figure 2. Development and progression of atherosclerotic lesions

(A) Design schematic for development and study of atherosclerotic lesion in vivo. (B-D) Shows three orientations of 3D confocal stacks. CPMV-A555 particles (red) interact with endothelial cells (CD31, green), but do not progress into deeper smooth muscles layers, best seen in (D), where the lumen side is marked with a white asterisk (4′,6-diamidino-2-phenylindole, blue). (E) Confocal image (40×) of endothelial staining (CD31, red) of aorta atherosclerotic lesion (white in small panel) from a low-density lipoprotein-receptor knockout mouse on western diet for 20 weeks. (F-H) Brightfield image (40×) of picrosirius red staining of collagen in control aorta from C57Bl/6J mice (F) and in aortic atherosclerotic lesions from low-density lipoprotein-receptor knockout mice on diet for (G) 12 and (H) 20 weeks. CPMV: Cowpea mosaic virus.

Figure 3. Vimentin, macrophages and cowpea mosaic virus uptake in atherosclerotic lesion

(A) Vimentin (red) on the surface of endothelial cells in nonlesion tissue from low-density lipoprotein receptor knockout (LDLR^−/−) mice (40×). (B) Vimentin (red) on the surface of endothelial cells in atherosclerotic lesion tissue from LDLR^−/− mice (40×, white in small panel). (C) Lipid staining (Oil Red O, red) and CPMV (green) in atherosclerotic lesions from LDLR^−/− mouse on western diet for 20 weeks (40×). (D) Macrophage staining (MOMA-2, red) and CPMV (green) in atherosclerotic lesions from LDLR^−/− mouse on western diet for 20 weeks (40×). CPMV: Cowpea mosaic virus; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 4. Quantification of vimentin and cowpea mosaic virus in lesion versus nonlesion tissue

Analysis of vimentin and CPMV in tissue sections using ImageJ. (A) Mean fluorescence of vimentin staining in the endothelium for C57Bl/6J control mice on normal chow, and lesion and nonlesion areas of LDLR^−/− mice on a high-fat diet for 12 weeks. Mean fluorescence of vimentin was statistically significant between control C57Bl6/J tissue and LDLR^−/− lesion (p < 0.01) and not statistically significant between LDLR^−/− nonlesion and lesion tissue. (B) Mean fluorescence of CPMV in endothelium for control C57BL6/J mice on normal chow and lesion and nonlesion areas of LDLR^−/− mice on high-fat diet for 20 weeks (p < 0.0001, between LDLR^−/− lesion and both LDLR^−/− nonlesion and control). (C) Percent of endothelial area positive for CPMV particle fluorescence in control and atherosclerotic mouse aorta after 12 and 20 weeks on a high-fat diet using the color threshold plugin for ImageJ. LDLR^−/− lesion tissue compared with both nonlesion LDLR^−/− tissue and control tissue was statistically significant (p < 0.001). (D) Percent total lesion area positive for CPMV particle fluorescence using color threshold plugin for ImageJ. CPMV: Cowpea mosaic virus; LDLR^−/−: Low-density lipoprotein receptor knockout; MFI: Median fluorescence intensity.

Figure 5. Reduced cowpea mosaic virus interaction with differentiated macrophages in vitro

(A) Confocal image of lipid staining of untreated RAW macrophages with ORO (red). (B) Confocal image of lipid staining of oxLDL-treated RAW macrophages with ORO (red). (C) Confocal image of CPMV uptake by untreated RAW macrophages (green). (D) Confocal image of CPMV uptake by oxLDL-treated RAW macrophages (green). (E) Confocal image of lipid staining of untreated TSPM with ORO (red). (F) Confocal image of lipid staining of oxLDL treated TSPM with ORO (red). (G) Confocal image of CPMV uptake by untreated TSPM (green). (H) Confocal image of CPMV uptake by oxLDL-treated TSPM (green). All confocal images were obtained at 60× and the nucleus has been stained with 4′,6-diamidino-2-phenylindole (blue). (I) FACS analysis of CPMV uptake in RAW macrophages with and without oxLDL treatment. (J) FACS analysis of number of RAW macrophages stained positive for surface vimentin with oxLDL treatment for 24 h (p = 0.22, not significant) and 72 h (p = 0.09, not significant). CPMV: Cowpea mosaic virus; ORO: Oil Red O; oxLDL: Oxidized low-density lipoprotein; TSPM: Thioglycolate-stimulated mouse peritoneal macrophages.