Table_2_Preservation solution Custodiol containing human alpha-1-antitrypsin improves graft recovery after prolonged cold ischemic storage in a rat model of heart transplantation.docx

IntroductionThe shortage of available donor hearts and the risk of ischemia/reperfusion injury restrict heart transplantation (HTX). Alpha-1-antitrypsin (AAT), a well-characterized inhibitor of neutrophil serine protease, is used in augmentation therapy to treat emphysema due to severe AAT deficiency. Evidence demonstrates its additional anti-inflammatory and tissue-protective effects. We hypothesized that adding human AAT in a preservation solution reduces graft dysfunction in a rat model of HTX following extended cold ischemic storage.
MethodsThe hearts from isogenic Lewis donor rats were explanted, stored for either 1h or 5h in cold Custodiol supplemented with either vehicle (1h ischemia, n=7 or 5h ischemia, n=7 groups) or 1 mg/ml AAT (1h ischemia+AAT, n=7 or 5h ischemia+AAT, n=9 groups) before heterotopic HTX. Left-ventricular (LV) graft function was evaluated in vivo 1.5h after HTX. Immunohistochemical detection of myeloperoxydase (MPO) was performed in myocardial tissue and expression of 88 gene quantified with PCR was analyzed both statistical and with machine-learning methods.
ResultsAfter HTX, LV systolic function (dP/dtmax 1h ischemia+AAT 4197 ± 256 vs 1h ischemia 3123 ± 110; 5h ischemia+AAT 2858 ± 154 vs 5h ischemia 1843 ± 104mmHg/s, p<0.05) and diastolic function (dP/dtmin 5h ischemia+AAT 1516 ± 68 vs 5h ischemia 1095 ± 67mmHg/s, p<0.05) at an intraventricular volume of 90µl were improved in the AAT groups compared with the corresponding vehicle groups. In addition, the rate pressure product (1h ischemia+AAT 53 ± 4 vs 1h ischemia 26 ± 1; 5h ischemia+AAT 37 ± 3 vs 5h ischemia 21 ± 1mmHg*beats/min at an intraventricular volume of 90µl; p<0.05) was increased in the AAT groups compared with the corresponding vehicle groups. Moreover, the 5h ischemia+AAT hearts exhibited a significant reduction in MPO-positive cell infiltration in comparison to the 5h ischemia group. Our computational analysis shows that ischemia+AAT network displays higher homogeneity, more positive and fewer negative gene correlations than the ischemia+placebo network.
DiscussionWe provided experimental evidence that AAT protects cardiac grafts from prolonged cold ischemia during HTX in rats.

引言
可获取的供体心脏短缺与缺血再灌注损伤（ischemia/reperfusion injury）的风险，制约了心脏移植（Heart Transplantation, HTX）的临床应用。α1-抗胰蛋白酶（Alpha-1-antitrypsin, AAT）是一种特性明确的中性粒细胞丝氨酸蛋白酶（neutrophil serine protease）抑制剂，目前通过增强疗法用于治疗重度AAT缺乏症引发的肺气肿。现有研究证实其还具备额外的抗炎与组织保护功效。本研究提出假设：在器官保存液中添加人源AAT，可减轻大鼠HTX模型在长时间冷缺血储存后的移植物功能障碍。

方法
将同基因Lewis系供体大鼠的心脏离体取出后，置于低温Custodiol保存液中储存1小时或5小时；保存液分别添加载体溶剂（分别对应1小时缺血组，n=7；5小时缺血组，n=7）或1mg/ml的AAT（分别对应1小时缺血+AAT组，n=7；5小时缺血+AAT组，n=9），随后开展异位HTX。HTX术后1.5小时，体内评估左心室（Left-ventricular, LV）移植物功能。对心肌组织进行髓过氧化物酶（myeloperoxydase, MPO）的免疫组织化学检测，通过聚合酶链式反应（PCR）定量88个基因的表达水平，并分别采用统计学方法与机器学习方法对实验数据进行分析。

结果
HTX术后，在心室内容积为90µl时，与相应载体溶剂组相比，AAT组的LV收缩功能[1小时缺血+AAT组：4197±256，1小时缺血组：3123±110 mmHg/s；5小时缺血+AAT组：2858±154，5小时缺血组：1843±104 mmHg/s；P<0.05]与舒张功能[5小时缺血+AAT组：1516±68，5小时缺血组：1095±67 mmHg/s；P<0.05]均得到显著改善。此外，在心室内容积为90µl时，与相应载体溶剂组相比，AAT组的速率压力乘积[1小时缺血+AAT组：53±4，1小时缺血组：26±1；5小时缺血+AAT组：37±3，5小时缺血组：21±1 mmHg·次/分钟；P<0.05]显著升高。进一步分析显示，相较于5小时缺血组，5小时缺血+AAT组的心肌组织中MPO阳性细胞浸润量显著减少。本研究的计算分析表明，与缺血+安慰剂组网络相比，缺血+AAT组网络具有更高的同质性、更多的正相关基因关联与更少的负相关基因关联。

讨论
本研究提供了实验证据，证实于大鼠HTX模型中，AAT可保护心脏移植物免受长时间冷缺血的损伤。