CD93 in macrophages: A novel target for atherosclerotic plaque imaging?

Abstract

Noninvasive imaging atherosclerotic (AS) plaque is of great importance for early diagnosis. Recently, CD93 in MΦ was linked to atherosclerosis development. Herein, we have investigated whether CD93 in MΦ is a potential novel target for atherosclerotic plaque imaging. CD93^hi and CD93^lo MΦ were prepared with or without LPS stimulation, before biological activity was evaluated. A rat AS model was produced with left carotid artery clamped. Whole-body/ex vivo phosphor autoradiography of the artery and biodistribution were investigated after incorporation of ³ H-2-DG into CD93^hi and CD93^lo MΦ or after ¹²⁵ I-α-CD93 (¹²⁵ I-anti-CD93mAb) injection. The plaque tissue was subjected to CD93/CD68 immunofluorescence/immunohistochemistry staining. CD93^hi and CD93^lo MΦ cells were successfully prepared without significant effect on bioactivity after incorporative labelled with ³ H-2-DG. The AS model was successfully established. Biodistribution studies showed that adoptive transfer of ³ H-2-DG-CD93^hi MΦ or ¹²⁵ I- α-CD93 injection resulted in accumulation of radioactivity within the atherosclerotic plaque in the clamped left carotid artery. T/NT (target/non-target, left/right carotid artery) ratio was higher in the ³ H-2-DG-CD93^hi MΦ adoptive transfer group than in the ³ H-2-DG-CD93^lo MΦ group (p < .05). Plaque radioactivity in the ¹²⁵ I-α-CD93 injection group was significantly higher than in the ¹²⁵ I-IgG control group (p < .01). The higher radioactivity accumulated in the clamped left carotid artery was confirmed by phosphor autoradiography. More importantly, CD93/CD68 double-positive MΦ accumulated at the atherosclerotic plaque in ³ H-2-DG-CD93^hi MΦ adoptive transfer group, which correlated with plaque radioactivity (r = .99, p < .01). In summary, both adoptive-transferred ³ H-2-DG-labelled CD93^hi MΦ and ¹²⁵ I-α-CD93 injection specifically targeted CD93 in atherosclerotic plaque. CD93 is a potential target in atherosclerotic plaque imaging.

Keywords: CD93; atherosclerosis; macrophage; molecular imaging; radionuclide.

FIGURE 1

H&E and Oil red staining for rat artery and aorta. (A) Left/right carotid artery and aorta arch from the rat AS model, with representative H&E staining images at 100× and 400× magnification. The black arrow points to the atherosclerotic plaque in the carotid artery (left) and the aorta. (B) Oil red O staining of aorta in the AS model, with black arrow pointing to lipid droplets. (C) Immunofluorescence staining of CD93^hi and CD93^lo MΦ and representative images (400×), CD93 (red), CD68 (green) and nucleus (blue). Data were obtained from three independent experiments

FIGURE 2

Effect of LPS stimulation and ³H‐2‐DG labelling on CD93 expression and MΦ function. (A) CD93 mRNA expression after LPS stimulation and ³H‐2‐DG labelling was detected by RT‐PCR. (B) CD93 protein expression after LPS stimulation and ³H‐2‐DG labelling was detected by Western Blot. (C) Phagocytosis of Dil‐Ox‐LDL by MΦ (400×), with Dil‐Ox‐LDL (red) and nuclear (blue). NT: non treated by LPS, no ³H‐2‐DG labelling, LPS: LPS‐stimulated, ³H‐2‐DG: labelled with ³H‐2‐DG. Data were analysed by Student's t‐test (n = 3), **p < .01

FIGURE 3

Effect of LPS stimulation and ³H‐2‐DG labelling on CD93 expression and MΦ function. (A) Morphology (400×) after LPS stimulation and ³H‐2‐DG labelling. (B) Apoptosis of MΦ detected by flow cytometry. (C) Cell cycle of MΦ detected by flow cytometry. G1: pre‐DNA synthesis stage, NT: non treated by LPS, no ³H‐2‐DG labelling, LPS: LPS‐stimulated, ³H‐2‐DG: labelled with ³H‐2‐DG. Data were obtained from three independent experiments

FIGURE 4

Evaluation of radiolabelled tracers. (A) Representative saturation binding curve and Scatchard plots with increasing ¹²⁵I‐α‐CD93 binding to CD93^hi MΦ; (B) the same for CD93^lo MΦ. (C) Competition binding curve between ¹²⁵I‐α‐CD93 and increasing unlabelled α‐CD93, n = 5

FIGURE 5

Tritium‐phosphor autoradiography and biodistribution of ex vivo aorta arch 72 h after ³H‐2‐DG‐labelled CD93^hi and CD93^lo MΦ adoptive transfer. (A) Representative ex vivo tritium‐phosphor autoradiography images of aortic arch in AS model after 72 h; (B/C) radioactivity biodistribution (%ID/g) of representative tissues in the ³H‐2‐DG‐labelled CD93^hi and CD93^lo MΦ adoptive transfer group after 72 h, with L (left carotid artery), R (right carotid artery) and A (aortic arch). n = 5, *** p < .001

FIGURE 6

Whole‐body and ex vivo artery phosphor autoradiography and biodistribution. (A) Representative images of ¹²⁵I‐α‐CD93 and ¹²⁵I‐IgG in AS model whole‐body phosphor autoradiography after 24 and 48 h, with black arrow pointing to the left carotid artery. (B) In vivo radioactivity ratio (left carotid artery/right carotid artery) between ¹²⁵I‐α‐CD93 and ¹²⁵I‐IgG; representative images of ex vivo (C) left/right carotid artery and (D) aorta artery after 48 h. (E) Biodistribution of ¹²⁵I‐α‐CD93 and ¹²⁵I‐IgG after 48 h. (F) Biodistribution of ¹²⁵I‐α‐CD93 and ¹²⁵I‐IgG in left/right carotid artery and aorta after 48 h. L (left carotid artery), R (right carotid artery) and A (aorta). Data were obtained from three independent experiments (n = 5), * p < .05, ** p < .01, *** p < .001

FIGURE 7

Immunofluorescence and immunohistochemistry imaging of diseased vessels. (A) Immunofluorescence staining of nucleus with DAPI (blue), CD68 (green) and CD93 (red). (B) Immunohistochemistry staining (400×) of CD93 and CD68, with positive staining shown as brown colour. (C) Correlation between IOD/Area of CD93 in immunohistochemistry staining and ¹²⁵I‐α‐CD93 (DLU/mm², digital light units) in ex vivo artery phosphor autoradiography. (D) Correlation between IOD/Area of CD68 in immunohistochemistry staining and ³H‐2‐DG‐CD93^hi MΦ in ex vivo aorta tritum‐phosphor autoradiography. L (left carotid artery), R (right carotid artery) and A (aorta). n = 3