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

doi:10.1016/j.healun.2020.02.002

The Journal of Heart and Lung Transplantation

Volume 39, Issue 5, May 2020, Pages 481-490

https://doi.org/10.1016/j.healun.2020.02.002 Get rights and content

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 (MB_C4d). 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 MB_C4d loaded with NO (NO-MB_C4d). 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-MB_C4d injection. Allograft survival and histologic features were analyzed to evaluate the therapeutic effect and underlying mechanism of NO-MB_C4d toward AMR.

RESULTS

We successfully obtained CEUS images following NO-MB_C4d injection and demonstrated that the ultrasound signal intensity of the myocardial area and clearance time of NO-MB_C4d 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-MB_C4d injection through significant suppression of thrombosis and reduction of inflammatory cell infiltrates. Overall, the therapeutic efficacy was significantly improved in the NO-MB_C4d 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-MB_C4d CEUS imaging, which may be expected to provide a better option for recipients with AMR in clinic.

Section snippets

Preparation of MBs loaded with NO

All phospholipids including 18 mg 1,2-dipalmitoyl-rac-glycero-3-phosphocholine, 3.5 mg 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], 1 mg 1,2-dipalmitoyl-sn-glycero-3-phosphate, and 0.72 µg biotin-DSPE-PEG₂₀₀₀ were dissolved in 4 ml of chloroform. Chloroform was then extracted via evaporation until a thin film was observed. Next, 4 ml of phosphate buffered saline containing 0.72 mg streptavidin (Sigma, St. Louis, MO, S4762) was used to hydrate the film,

Characterization of the MBs

To determine the proper proportion of NO in MBs, we investigated the stability and contrast ability in vitro for MBs with different NO proportions including 0, 25%, 50%, 75%, and 100%. Compared with MBs without NO, the clearance time and intensity signals of MBs at different time points including 0, 10, 20, 30, 40, and 50 minutes were decreased with increasing NO proportion (Figure 1). These results showed that the stability and contrast ability of MBs decreased following NO addition and

Discussion

In this study, using CEUS imaging based on NO-MB_C4d, we developed a novel approach for simultaneous non-invasive diagnosis and precise treatment of AMR, which is expected to be applicable for the clinical management of cardiac transplant recipients with AMR.

This study provides a more precise, local, effective, and safe means to treat AMR. Using the rat model of AMR, which was established and analyzed in detail in our previous study,¹⁰ we demonstrated here that NO-MB_C4d injection significantly

Disclosure statement

The authors have no conflicts of interest to disclose.

This study was supported by the National Natural Science Foundation of People's Republic of China (No. 81800663, 81970649, 81770753); Natural Science Foundation of Guangdong Province (2019A1515011942), National Key R&D Program of People's Republic of China (2018YFA0108804); the Leading Scientific, Technical, and Innovation Talents of Guangdong special support program (No. 2015TX01R112); the Science and Technology Project of Guangdong

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¹: These authors have contributed equally to this work.

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ORIGINAL PRE-CLINICAL SCIENCEPrecise treatment of acute antibody-mediated cardiac allograft rejection in rats using C4d-targeted microbubbles loaded with nitric oxide

BACKGROUND

METHODS

RESULTS

CONCLUSIONS

Section snippets

Preparation of MBs loaded with NO

Characterization of the MBs

Discussion

Disclosure statement

J Heart Lung Transplant

Am J Transplant

J Heart Lung Transplant

Kidney Int

EBioMedicine

Trends Immunol

Trends Mol Med

Am J Transplant

Am J Transplant

Am J Transplant

Am J Transplant

Am J Transplant

Am J Transplant

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