Abstract Body: Introduction
Chronic mental stress accelerates atherosclerosis by amplifying macrophage-driven inflammation within the vasculature. Pro-inflammatory M1 macrophages play a central role in plaque instability and rupture; however, real-time in vivo assessment of pro-inflammatory macrophage activity within atherosclerotic plaques, particularly in the context of chronic stress, remains challenging. Given that M1 macrophages exhibit high surface expression of CD44 receptors with strong binding affinity for hyaluronic acid (HA), we hypothesized that an HA-targeted cellular trafficking imaging strategy could enable in vivo assessment of the dynamic biological behavior of pro-inflammatory macrophages within murine atheroma under chronic stress conditions.

Methods and Results
An M1 macrophage-targeted NIRF imaging agent was newly synthesized by conjugating HA with cyanine-7 (Cy7). In vitro studies using RAW264.7 macrophages polarized toward the M1 (LPS/IFN-γ) or M2 (IL-4) phenotype demonstrated selective uptake of HA-Cy7 by M1 macrophages, yielding a 7.2-fold higher fluorescence signal compared with M2 macrophages (p < 0.01), as quantified by custom fluorescence microscopy (Figure). For in vivo studies, ApoE^-/- mice were subjected to a 4-week cyclic chronic variable stress (CVS) protocol. Forty-eight hours after intravenous administration of HA-Cy7 (equivalent to Cy7 0.5 mg/kg), selective accumulation of HA-Cy7 within plaque macrophages was prominently visualized using a novel heartbeat- and respiration-synchronized multispectral intravital imaging as compared to controls (p<0.01) (Figure). In vivo NIRF imaging revealed significantly enhanced M1-associated signals within carotid atheromata of stressed mice, accompanied by increased rolling and adherent cellular trafficking, providing real-time evidence of stress-augmented pro-inflammatory macrophage activity (p < 0.01). These findings were further corroborated by quantitative ex vivo fluorescence imaging and immunohistochemical analysis (p < 0.01).

Conclusions
This study provides robust in vivo evidence linking chronic psychological stress to heightened pro-inflammatory macrophage activity within atherosclerotic plaques. CD44-targeted intravital macrophage imaging represents a promising strategy for elucidating the mechanistic interplay between stress-associated plaque inflammation and high-risk vascular events, and could offer new opportunities for imaging-guided assessment of plaque vulnerability.