Single-Chain VEGF/Cy5.5 Targeting VEGF Receptors to Indicate Atherosclerotic Plaque Instability

Abstract

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). 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. 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. 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.