Inter-patient heterogeneity in the hepatic ischemia-reperfusion injury transcriptome: implications for research and diagnostics [MUG data]

Background & Aims: Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles. We have investigated this effect to obtain insight into molecular ischemia responses during surgical procedures and characterize pre-analytical effects impacting on molecular analyses. Methods: 143 non-malignant liver samples were obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts, treated either with the Pringle maneuver or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-qPCR. Results: Transcriptional profiles of both cohorts displayed 179 genes that were mutually deregulated confirming elevated cytokine signaling, and NFkB as the dominant pathways in ischemia response. Contrary to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFkB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were clearly present in the non-tumor tissue. Conclusions: We identified transcripts mutually deregulated during ischemia and reperfusion injury in both cohorts that can be used to monitor ischemia during liver surgery and highlight the importance of pre-analytical quality control. The marked inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a comprehensive and pre-analytically highly standardized in vivo transcriptome profile of histologically normal liver and identified 230 genes with substantial pre-analytical robustness (<2 % covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications. Conclusions: We identified transcripts mutually deregulated during ischemia and reperfusion injury in both cohorts that can be used to monitor ischemia during liver surgery and highlight the importance of pre-analytical quality control. The marked inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a comprehensive and pre-analytically highly standardized in vivo transcriptome profile of histologically normal liver and identified 230 genes with substantial pre-analytical robustness (<2 % covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications. Ischemia related changes of RNA profiles obtained from frozen liver tissue samples collected before, during and after routine surgery and compared between the different time points. Medical University of Graz, 83 samples of a total of 21 patients at 4 different time points per patient.

背景与目的：手术操作或缺血再灌注损伤（ischemia-reperfusion injury, IRI）诱导的细胞应答可显著改变转录组图谱。本研究旨在探究该效应，以深入解析外科手术过程中的分子缺血应答，并表征影响分子分析的分析前效应。
方法：从两个独立队列的30名患者的手术中不同缺血时间点获取143份非恶性肝脏样本，受试者分别接受普林格尔手法（Pringle maneuver）或全血管阻断（total vascular exclusion）处理。采用Affymetrix基因芯片（Affymetrix microarrays）分析转录组图谱，并通过逆转录实时定量聚合酶链式反应（RT-qPCR）验证选定mRNA的表达。
结果：两个队列的转录谱均存在179个共同失调基因，证实细胞因子信号通路激活，核因子κB（NFkB）是缺血应答中的核心通路。与缺血过程不同，再灌注可诱导涉及肿瘤坏死因子（TNF）、NFkB及丝裂原活化蛋白激酶（MAPK）通路的促凋亡与促炎症级联反应。与正常肝脏中表达上调的情况相反，脂肪变性组织中FOS与JUN的表达下调。令人意外的是，非肿瘤组织中可清晰检测到潜在原发性及继发性癌症的分子特征。
结论：我们在两个队列中均鉴定出缺血与再灌注损伤过程中共同失调的转录本，这些转录本可用于监测肝脏手术中的缺血状态，并凸显了分析前质量控制的重要性。显著的患者间差异可能反映了个体应激应答的差异及基础疾病状态的影响。此外，我们提供了一套全面且分析前高度标准化的组织学正常肝脏的体内转录组图谱，并鉴定出230个具有优异分析前稳定性的基因（两个队列间协变性<2%），这些基因可作为参考基因，尤其适用于未来的诊断应用。
本研究同时分析了常规手术前后、术中采集的冷冻肝脏组织样本的RNA谱缺血相关变化，并对不同时间点的样本进行了比较。该数据集来自格拉茨医科大学，包含21名患者共83份样本，每名患者采集4个不同时间点的样本。