(H-307) Evaluation of regeneration and immune response of functionalized cell-free arterial grafts in a large animal model

Introduction:: cardiovascular disease (CVD) is a leading cause of death in the United States, and surgical intervention is often necessary in severe cases. Autologous grafts, harvested from the patient's own body, have traditionally been used but are limited by the availability of suitable grafts. Tissue-engineered vessels (TEVs) have been developed as an alternative but require extensive cell culture time and autologous cell sources. We previously developed acellular(A)TEVs made from small intestinal submucosa (SIS) tubes functionalized with heparin and a novel protein named H2R5, which attracted circulating blood monocytes (MCs) to the graft lumen where they formed endothelial (EC) and smooth muscle cell (SMC) layers in the small animal model [1]. In this study, we implanted these A-TEVs in sheep to investigate their regenerative capacity and immune response in a large animal model.


Materials and Methods:: SIS tubes, with a diameter of 4.5mm and length of 4-5 cm, were functionalized with heparin and H2R5, following established protocols [1,2]. These tubes were used to construct arterialized tissue-engineered vessels (ATEVs) which were surgically implanted in the carotid arteries of six 1-year-old sheep and monitored for a period of 3 months. Patency and blood flow of the A-TEVs were assessed using doppler ultrasound, while explanted A-TEVs were evaluated for the presence of cells, extracellular matrix (ECM) deposition and their mechanical properties. The SMC function in response to vasoactive agonists was assessed by subjecting the explanted A-TEVs to a vasoreactivity test as described before [2].


Results, Conclusions, and Discussions:: Our previous study in mice has demonstrated that functionalized-H2R5 ATEVs can successfully recruit circulating MCs to the graft lumen by simulating the interaction between MCs and the endothelium. To provide direct evidence of MC/Macrophage(Mφ) recruitment, we conducted lineage tracing analysis using a novel CX3CR1-confetti mouse model(Fig.1). The results revealed the presence of fluorescently labeled MCs co-expressing vascular markers in the lumen of the ATEV, indicating trans-differentiation of circulating MCs into ECs and SMCs in an arterial environment[1]. To investigate the immunological response and efficacy of H2R5 ATEVs in a large animal model, we implanted them in sheep and observed patency and cellularization over a 3-month period as demonstrated in Fig.2. Immunostaining of the ATEV’s lumen revealed the presence of ECs, as indicated by eNOS expression, and the ATEV’s wall was infiltrated by SMCs, as indicated by α-SMA expression(Fig.3). A continuous endothelium co-expressing both EC and MC/Mφ-specific markers was observed on the ATEV lumen, along with multiple cell layers beneath the endothelial layer co-expressing both SMC and MC/Mφ-specific markers(Fig.4). These results were consistent with previous findings from recruited cells to the H2R5 ATEV in CX3CR1 confetti mice models and provide direct evidence of the direct contribution of circulating MCs to the endothelialization and vascular wall formation of ATEVs implanted in large animal models. Furthermore, the ATEVs exhibited biological function to vasoagonists and maintained their mechanical properties through ECM deposition and degradation of SIS overtime.

Currently, we apply spatial transcriptomics to gain a comprehensive understanding of the spatial distribution of distinct cell types within the ATEV explants as a function of time and location within the vessel, including the luminal and medial layers, based on the corresponding gene expression profiles. By elucidating the interactions between these cells, we can further comprehend the underlying mechanisms that contribute to successful ATEV regeneration and long-term patency.

Our data highlighted the potential of H2R5-functionalized ATEVs as tissue replacement grafts in a large pre-clinical animal model by modulating the immune response to promote vascular regeneration. The ATEVs offer off-the-shelf options for on-demand grafts with anti-occlusive properties and self-endothelializing capabilities that may have significant impact on regenerative medicine.


Acknowledgements (Optional): :

References (Optional): : 1- Nasiri, Bita, Tai Yi, Yulun Wu, Randall J. Smith Jr, Ashis Kumar Podder, Christopher K. Breuer, and Stelios T. Andreadis. "Monocyte Recruitment for Vascular Tissue Regeneration." Advanced Healthcare Materials 11, no. 22 (2022): 2200890.

2- Koobatian, Maxwell T., Sindhu Row, Randall J. Smith Jr, Carmon Koenigsknecht, Stelios T. Andreadis, and Daniel D. Swartz. "Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model." Biomaterials 76 (2016): 344-358.