Single-chain VEGF/Cy5.5 targeting vegf receptors to indicate atherosclerotic plaque instability.
OBJECTIVE
Unstable plaques may cause clinical events. Plaque destabilization results from the synergy between intraplaque angiogenesis and inflammation. Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are considered to be involved in these processes. We investigated the efficacy of the anti-VEGFR mimic single-chain VEGF (scVEGF) to map intra-plaque VEGFR expression and atherosclerotic plaque instability using near-infrared fluorescence (NIRF).


METHODS
Human carotid plaques were retrieved from 15 symptomatic and five asymptomatic patients. NIRF plaque imaging was performed pre-/post-incubation with scVEGF/Cy5.5. Biopsies taken from regions with high (hot spot) and low (cold spot) NIRF signals were examined for VEGF-A, VEGFR-1 and VEGFR-2 mRNA expression levels using real-time RT-PCR analysis. Immunohistochemistry for CD31 (endothelium), CD68 (macrophages) and αSMA (smooth muscle cells) was performed to evaluate plaque composition.


RESULTS
NIRF imaging of 20 plaques revealed a heterogeneous distribution of scVEGF/Cy5.5 binding. After incubation NIRF activity increased from 3.9×10(-5) ± 5.2×10(-6) to 3.0×10(-4) ± 2.2×10(-5) and 5.8×10(-5) ± 1.9×10(-5) to 3.1×10(-4) ± 1.9×10(-5) photons/s/cm(2)/sr/illumination intensity on the intraluminal and extraluminal side, respectively (both p < 0.001). Real-time RT-PCR analysis showed a ~1.2- and ~16.4-fold increased mRNA expression of VEGFR-1 and VEGFR-2, respectively, in hot spots (vs. cold spots). Immunohistochemistry exhibited higher intraplaque capillary density in hot spots (vs. cold spots) (17.2 ± 3.7 vs. 5.4 ± 2.2 capillary/mm(2); p = 0.037). Hot spots contained significantly reduced numbers of α-SMA-positive cells (vs. cold spots) (2.2 ± 0.7 % vs. 6.9 ± 1.5 %; p = 0.038). Finally, a ~2-fold increase of CD68(+) infiltrating macrophages within hot spots (vs. cold spots) was observed (not significant, p = 0.17). Significant higher capillary density in hot spots (vs. cold spots) was observed in plaques from symptomatic patients but not in plaques from asymptomatic patients.


CONCLUSIONS
Our data support that scVEGF/Cy5.5 is a suitable indicator for plaque instability and a promising diagnostic tool for risk assessment in cardiovascular diseases.
Division of Vascular Surgery, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Ming Kai
MK
Lam
Sali
S
Al-Ansari
Gooitzen M
GM
van Dam
René A
RA
Tio
Jan-Cees
JC
Breek
Riemer H J A
RH
Slart
Jan-Luuk
JL
Hillebrands
Clark J
CJ
Zeebregts
D000368:Aged; D002232:Carbocyanines; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D010641:Phenotype; D058226:Plaque, Atherosclerotic / Q000175:diagnosis*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D019265:Spectroscopy, Near-Infrared; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2