Postoperative lower limb ischemic necrosis following xenogeneic heart transplantation from gene-edited pigs to rhesus macaques_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

WANG Xianzhi ¹ , REN Zhipeng ¹ , DAI Ziqiang ¹ , ZHOU Rong ² , ZHANG Gen ¹ , YAN Jie ³ , GUAN Yulong ⁴ , PAN Guangyu ⁵ , HE Xingzhuang ⁶ , LIU Tingting ⁶ , LI Shangxuan ¹ , CUI Guanzheng ¹ , LAI Chenghong ⁷ ,  PAN Dengke ³ , LI Dianyuan ¹

1. Department of Cardiovascular Surgery, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Suzhou, 215000, Jiangsu, P. R. China;
2. Department of Ultrasound, First People's Hospital of Neijiang, Neijiang, 641000, Sichuan, P. R. China;
3. Chengdu Aoge Biotechnology Co, Ltd., Chengdu, 610094, P. R. China;
4. Extracorporeal Circulation Center, National Cardiovascular Disease Center, Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100037, P. R. China;
5. Department of Cardiovascular Surgery, Peking University People's Hospital, Beijing, 100032, P. R. China;
6. Department of Pathology, First People's Hospital of Guangyuan, Guangyuan, 628000, Sichuan, P. R. China;
7. Medical Affairs Department, First People's Hospital of Neijiang, Neijiang, 641000, Sichuan, P. R. China;

Corresponding?author：

PAN Dengke, Email: drdianyuanli@163.com

Keywords：

Gene editing; Xenotransplantation from gene-edited pigs to rhesus macaques; heart transplantation; lower extremity ischemia; vascular thrombosis; pathology; imnunological rejection

DOI：

10.7507/1007-4848.202512033

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Objective To investigate the causes and management strategies for lower limb ischemic necrosis following xenogeneic heterotopic heart transplantation from a multigene-edited pig to a rhesus monkey. Methods A xenogeneic heterotopic heart transplantation was performed on December 16, 2023, at the Institute of Experimental Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, using a quintuple-gene-edited pig as the donor and a rhesus monkey as the recipient. On postoperative day (POD) 9, the recipient monkey underwent left lower limb amputation due to ischemic necrosis. Blood samples were collected at various time points after transplantation for analysis of hematologic parameters, liver and renal function, myocardial enzymes, and coagulation profiles. Ultrasound and computed tomography (CT) were used to evaluate anastomotic patency and cardiac structure. Immunological assays, including complement-dependent cytotoxicity (CDC) and IgG/IgM antibody detection, combined with clinical observations, were employed to assess rejection type and therapeutic response. Results The recipient monkey survived for 46 days after transplantation. Echocardiography demonstrated preserved biventricular systolic function in the recipient’s native heart, with left ventricular ejection fraction (LVEF) consistently exceeding 50%. In the donor pig heart, left ventricular endocardial thickening was noted on POD 9, followed by right ventricular endocardial thickening on POD 24, while LVEF remained around 35%. No hyperacute or acute rejection was detected immunologically. CDC positivity ranged between 3.4% and 5.1%, with IgG/IgM antibody binding trends consistent with CDC results. Following amputation, the recipient exhibited elevated inflammatory markers, coagulopathy, and reactive thrombocytosis, which later normalized. Immunohistochemical staining of the necrotic limb revealed arterial and venous thrombosis; however, no T-cell or B-cell infiltration was observed in vascular structures, thrombi, nerves, muscles, fascia, or skin tissues, with CD3 and CD20 staining both negative. Conclusion Limb ischemia after xenogeneic heart transplantation may be associated with lower extremity vascular thrombosis triggered by local trauma in the context of transplantation-induced inflammatory activation and coagulation dysfunction. While no clear lymphocyte-mediated rejection was observed, further studies are needed to explore the potential role of non-lymphocyte-mediated immune mechanisms.

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1. Niu D, Wei HJ, Lin L, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science, 2017, 357(6357): 1303-1307.
2. Anand RP, Layer JV, Heja D, et al. Design and testing of a humanized porcine donor for xenotransplantation. Nature, 2023, 622(7982): 393-401.
3. Moazami N, Stern JM, Khalil K, et al. Pig-to-human heart xenotransplantation in two recently deceased human recipients. Nat Med, 2023, 29(8): 1989-1997.
4. 張根, 王歡, 管玉龍, 等. 5基因編輯豬-恒河猴異種心臟移植實驗報道. 中華胸心血管外科雜志, 2024, 40(6): 379-384.Zhang G, Wang H, Guan YL, et al. Report on xenogeneic heart transplantation from 5-gene-edited pig to rhesus macaque. Chin J Thorac Cardiovasc Surg, 2024, 40(6): 379-384.
5. 周容, 張亞萍, 廖宇, 等. 超聲在基因編輯豬-猴異種并聯式心臟移植術中的應用價值. 中華醫學超聲雜志(電子版), 2024, 21(6): 617-623.Zhou R, Zhang YP, Liao Y, et al. The application value of ultrasound in xenogeneic parallel heart transplantation from gene-edited pig to monkey. Chin J Med Ultrasound (Electron Ed), 2024, 21(6): 617-623.
6. Zidan A, El-Sherbini AH, Noureldin A, et al. Characterizing coagulation responses in humans and nonhuman primates following kidney xenotransplantation-a narrative review. Am J Hematol, 2024, 100(2): 285-295.
7. Mohiuddin MM, Singh AK, Scobie L, et al. Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report. Lancet, 2023, 402(10399): 397-410.
8. Eisenson D, Hisadome Y, Santillan M, et al. Consistent survival in consecutive cases of life-supporting porcine kidney xenotransplantation using 10GE source pigs. Nat Commun, 2024, 15(1): 3361.
9. Chen X, Ghanizada M, Mallajosyula V, et al. Differential roles of human CD4(+) and CD8(+) regulatory T cells in controlling self-reactive immune responses. Nat Immunol, 2025, 26(2): 230-239.
10. Gierej B, Górnicka B, Wasiutyński A. Role of C3d and C4d complement fragments in the diagnostics of acute allograft rejection after transplantations. Ann Transplant, 2009, 14(4): 61-70.
11. Loupy A, Goutaudier V, Giarraputo A, et al. Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study. Lancet, 2023, 402(10408): 1158-1169.
12. Estrada CC, Cardona S, Guo Y, et al. Endothelial-specific loss of Krüppel-Like factor 4 triggers complement-mediated endothelial injury. Kidney Int, 2022, 102(1): 58-77.
13. Eisenson DL, Hisadome Y, Yamada K, et al. Progress in xenotransplantation: immunologic barriers, advances in gene editing, and successful tolerance induction strategies in pig-to-primate transplantation. Front Immunol, 2022, 13: 899657.
14. Peterson L, Yacoub MH, Ayares D, et al. Physiological basis for xenotransplantation from genetically modified pigs to humans. Physiol Rev, 2024, 104(3): 1409-1459.
15. Kim H, Hawthorne WJ, Kang HJ, et al. Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno-immune response. Xenotransplantation, 2015, 22(4): 260-272.
16. Kubagawa H, Honjo K, Ohkura N, et al. Functional roles of the IgM Fc receptor in the immune system. Front Immunol, 2019, 10: 945.
17. Stark K, Kilani B, Stockhausen S, et al. Antibodies and complement are key drivers of thrombosis. Immunity, 2024, 57(9): 2140-2156. e10.
18. Parienti JJ, Mongardon N, Mégarbane B, et al. Intravascular complications of central venous catheterization by insertion site. N Engl J Med, 2015, 373(13): 1220-1229.
19. Baskin JL, Pui CH, Reiss U, et al. Management of occlusion and thrombosis associated with long-term indwelling central venous catheters. Lancet, 2009, 374(9684): 159-169.
20. Koupenova M, Clancy L, Corkrey HA, et al. Circulating platelets as mediators of immunity, inflammation, and thrombosis. Circ Res, 2018, 122(2): 337-351.
21. Jian H, Cui X, Dou Y, et al. Adoption of plasma D-dimer testing, color Doppler ultrasound, and enoxaparin sodium in predicting and preventing lower extremity deep vein thrombosis in high-risk parturients undergoing cesarean section. Medicine (Baltimore), 2025, 104(50): e45827.
22. Luderer V, Jung F, Brandenstein M, et al. First assessment of flow phenomena of acute and chronic thrombosis in the jugular veins using new ultrasound vector-flow imaging. Clin Hemorheol Microcirc, 2024, 86(1-2): 133-142.
23. Talabieke S, Yang X, Yang J, et al. Arachidonic acid synergizes with aspirin preventing myocardial ischaemia-reperfusion injury and mitigates bleeding risk. Cardiovasc Res, 2025, 121(5): 775-787.
24. Poddighe D, Maulenkul T, Zhubanova G, et al. Natural killer T (NKT) cells in autoimmune hepatitis: current evidence from basic and clinical research. Cells, 2023, 12(24): 2854.
25. Liu L, Zhang H, Shi Y, et al. Prostaglandin E1 improves cerebral microcirculation through activation of endothelial NOS and GRPCH1. J Mol Neurosci, 2020, 70(12): 2041-2048.
26. Videm V, Sharma A, Dahl T. Complement and diverse enrichment of antibodies in intraluminal thrombi from abdominal aortic aneurysms may contribute to increased inflammation. Sci Rep, 2025, 16(1): 220.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Latest ArticlesPostoperative lower limb ischemic necrosis following xenogeneic heart transplantation from gene-edited pigs to rhesus macaques

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