Abstract Body (Do not enter title and authors here): Introduction. Plaque erosion (PE) has a distinct pathogenesis, clinical presentation, and prognosis than other acute coronary syndrome (ACS) causes such as plaque rupture (PR). At present the ability to distinguish between PE and PR using in vivo imaging is limited. A critical feature of PE is the enrichment of hyaluronic acid (HA) at the luminal interface, which is poorly present in the other types of plaque. To better resolve mechanisms of ACS in vivo, we designed and investigated a new hyaluronan-targeted near-infrared fluorescence (NIRF) molecular imaging agent and tested the agent in vitro, in mouse PE in vivo, and in resected human carotid specimens. NIRF has emerged as a translational intravascular imaging modality that allows visualization of plaque pathobiology in vivo.
Methods. . The NIRF agent was generated via microwave-assisted solid phase peptide synthesis and was conjugated to the NIR fluorophore CyAl5 (ex/em 643/661 nm), to yield the hyaluronan-targeted NIRF agent (HAT-CyAl5). In vitro binding of HAT-CyAl5 to HA was assessed by fluorescence reflectance imaging (FRI). HAT-CyAl5 or a scramble control peptide-CyAl5 (scrambled-CyAl5) were intravenously injected in an established electrical injury-based mouse model of carotid artery PE; intravital microscopy (IVM, n=7) was performed in mice injected with HAT-CyAl5 (n=4) or scrambled-CyAl5 (n=3). Next, freshly resected human carotid specimens were incubated with HAT-CyAl5 (n=4) or PBS (n=4). Histological analyses then assessed specific binding of HAT-CyAl5 in murine PE and human atheroma.
Results. The blood half-life of HAT-CyAl5 was 5.7 minutes (95% CI, 3.4-9.8min). In vitro HA binding analyses showed a significant higher target-to-background ratio (TBR) in the HAT-CyAl5 group (P < 0.03 vs. Scrambled-CyAl5). In carotid PE undergoing IVM, HAT-CyAl5 double the TBR in vivo (4.7 ±1.36 vs. 2.38 ± 0.17 Scrambled-CyAl5, P< 0.05). In human carotid plaques, HAT-CyAl5 markedly increased plaque NIRF signal (TBR _{post-incubation - pre-incubation} 4.95 ±3.36 vs. 0.00 ± 0.15 PBS, P< 0.03). Histology of human plaques showed that HAT-CyAl5 localized well with HA immunohistochemical expression.
Conclusion. HAT-CyAl5 is a new NIRF probe that avidly and specifically binds HA in murine PE in vivo and in human plaques. HAT-CyAl5 coupled with intravascular NIRF imaging has the potential to specifically diagnose PE and provide new insights into the pathogenesis of PE.