EA4245 Transplantation, Immunologie et Inflammation, Loire Valley Cardiovascular Collaboration and FHU SUPORT, Tours University, Tours, France; Department of Cardiac Surgery, Tours University Hospital, Tours, France.
EA4245 Transplantation, Immunologie et Inflammation, Loire Valley Cardiovascular Collaboration and FHU SUPORT, Tours University, Tours, France.
J Thorac Cardiovasc Surg. 2019 Sep;158(3):780-790.e1. doi: 10.1016/j.jtcvs.2018.12.014. Epub 2018 Dec 21.
Myocardial ischemia reperfusion is a major cause of cell injury during cardiac transplantation and is responsible for increased graft rejection. Several in vitro studies demonstrated the protective effect of P2Y11-like purinoreceptor stimulation in the context of myocardial ischemia/reperfusion. In this study, we hypothesized a possible cardioprotective role of P2Y11R stimulation against ischemia/reperfusion lesions and validated its clinical effect in vivo in a heart transplantation model.
We subjected H9c2 rat cardiomyocyte-derived cell line to 5 hours of hypoxia and 1 hour of reoxygenation. P2Y11R selective agonist NF546 and antagonist NF340 were added at the onset of reoxygenation. Cell injuries were assessed by microculture tetrazolium reduction and intracellular adenosine triphosphate level. Clinical effect of P2Y11R stimulation was further investigated in vivo. Hearts from BALB/c mice were transplanted intra-abdominally into allogenic C57BL/6 mice (n = 104). Recipient mice were injected with P2Y11R agonist. Mice in the sham group were injected with saline solution. In the control group, hearts from C57BL/6 were transplanted into syngeneic C57BL/6 mice. Rejection lesions were investigated using histology and immunohistochemistry at days 3, 5, and 7 after transplantation. We measured caspase activities to quantify apoptosis. Production of proinflammatory and anti-inflammatory cytokines was investigated.
P2Y11R stimulation at the onset of reoxygenation significantly reduced in vitro hypoxia/reoxygenation injuries. This protection was suppressed with P2Y11R antagonist. In vivo, cardiac allograft survival was significantly prolonged after P2Y11R stimulation. Rejection lesions, classified according to the International Society of Heart Lung Transplantation guidelines and quantified using the mean number of inflammatory cells per field, were significantly reduced in the treated group. At day 5 after transplantation, P2Y11R agonist pretreated allografts also demonstrated less apoptotic lesions.
Our data suggest a novel cardioprotective role of P2Y11R at the onset of reoxygenation/reperfusion against reperfusion injuries. Pharmacologic conditioning using P2Y11 agonist may be beneficial after cardiac transplantation in improving myocardial ischemia/reperfusion outcomes and decreasing graft rejection lesions.
心肌缺血再灌注是心脏移植过程中细胞损伤的主要原因，并导致移植物排斥增加。几项体外研究表明，在心肌缺血/再灌注的情况下，P2Y11 样嘌呤能受体刺激具有保护作用。在这项研究中，我们假设 P2Y11R 刺激可能对缺血/再灌注损伤具有保护作用，并在心脏移植模型中体内验证了其临床效果。
我们将 H9c2 大鼠心肌细胞衍生细胞系置于 5 小时缺氧和 1 小时复氧中。在复氧开始时加入 P2Y11R 选择性激动剂 NF546 和拮抗剂 NF340。通过微量培养四唑减少和细胞内三磷酸腺苷水平评估细胞损伤。进一步在体内研究 P2Y11R 刺激的临床效果。将来自 BALB/c 小鼠的心脏通过腹部移植到同种异体 C57BL/6 小鼠（n=104）中。受体小鼠注射 P2Y11R 激动剂。假手术组的小鼠注射生理盐水。在对照组中，将来自 C57BL/6 的心脏移植到同基因 C57BL/6 小鼠中。在移植后 3、5 和 7 天，使用组织学和免疫组织化学研究排斥病变。我们测量了半胱天冬酶活性以量化细胞凋亡。研究了促炎和抗炎细胞因子的产生。
复氧开始时 P2Y11R 刺激可显著减轻体外缺氧/再氧合损伤。这种保护作用被 P2Y11R 拮抗剂抑制。在体内，P2Y11R 刺激后心脏同种异体移植物的存活时间明显延长。根据国际心肺移植协会指南分类的排斥病变，并通过每个视野的炎症细胞数进行量化，在治疗组中明显减少。在移植后第 5 天，用 P2Y11R 激动剂预处理的同种异体移植物也显示出较少的凋亡病变。
我们的数据表明，在再灌注/再灌注开始时，P2Y11R 具有新的心脏保护作用，可防止再灌注损伤。心脏移植后使用 P2Y11 激动剂进行药物处理可能有利于改善心肌缺血/再灌注的结果并减少移植物排斥病变。
