Precise treatment of acute antibody-mediated cardiac allograft rejection in rats using C4d-targeted microbubbles loaded with nitric oxide

BACKGROUND Antibody-mediated rejection (AMR) constitutes an important cause of cardiac allograft loss; however, all current therapeutic strategies represent systemic applications with unsatisfactory efficacy. Previously, we successfully non-invasively detected C4d, a specific marker for AMR diagnosis, in allografts using C4d-targeted microbubbles (MBC4d). In this study, we extended this approach by incorporating nitric oxide (NO), as high NO levels manifest immunosuppressive and anti-thrombotic effects. METHODS We designed novel MBC4d loaded with NO (NO-MBC4d). A rat model of AMR was established by pre-sensitization with skin transplantation. Contrast-enhanced ultrasound (CEUS) images were obtained and quantitatively analyzed following NO-MBC4d injection. Allograft survival and histologic features were analyzed to evaluate the therapeutic effect and underlying mechanism of NO-MBC4d toward AMR. RESULTS We successfully obtained CEUS images following NO-MBC4d injection and demonstrated that the ultrasound signal intensity of the myocardial area and clearance time of NO-MBC4d both increased with increased C4d grade, thereby realizing non-invasive diagnosis of AMR. Furthermore, allograft survival was significantly prolonged, and rejection was obviously attenuated following NO-MBC4d injection through significant suppression of thrombosis and reduction of inflammatory cell infiltrates. Overall, the therapeutic efficacy was significantly improved in the NO-MBC4d group compared with the control NO-MB group, demonstrating that precise treatment could significantly improve the therapeutic efficacy compared with that afforded by systemic applications. CONCLUSIONS This study presented a novel tool to provide simultaneous non-invasive diagnosis and precise treatment of AMR using NO-MBC4d CEUS imaging, which may be expected to provide a better option for recipients with AMR in clinic. Antibody-mediated rejection (AMR) constitutes an important cause of cardiac allograft loss; however, all current therapeutic strategies represent systemic applications with unsatisfactory efficacy. Previously, we successfully non-invasively detected C4d, a specific marker for AMR diagnosis, in allografts using C4d-targeted microbubbles (MBC4d). In this study, we extended this approach by incorporating nitric oxide (NO), as high NO levels manifest immunosuppressive and anti-thrombotic effects. We designed novel MBC4d loaded with NO (NO-MBC4d). A rat model of AMR was established by pre-sensitization with skin transplantation. Contrast-enhanced ultrasound (CEUS) images were obtained and quantitatively analyzed following NO-MBC4d injection. Allograft survival and histologic features were analyzed to evaluate the therapeutic effect and underlying mechanism of NO-MBC4d toward AMR. We successfully obtained CEUS images following NO-MBC4d injection and demonstrated that the ultrasound signal intensity of the myocardial area and clearance time of NO-MBC4d both increased with increased C4d grade, thereby realizing non-invasive diagnosis of AMR. Furthermore, allograft survival was significantly prolonged, and rejection was obviously attenuated following NO-MBC4d injection through significant suppression of thrombosis and reduction of inflammatory cell infiltrates. Overall, the therapeutic efficacy was significantly improved in the NO-MBC4d group compared with the control NO-MB group, demonstrating that precise treatment could significantly improve the therapeutic efficacy compared with that afforded by systemic applications. This study presented a novel tool to provide simultaneous non-invasive diagnosis and precise treatment of AMR using NO-MBC4d CEUS imaging, which may be expected to provide a better option for recipients with AMR in clinic.