Gene expression modulation in human dermal substitute exposed to reversible electroporation (ECT or GET types)

In this project we aimed at deciphering modulation in genes expression induced by external pulsed electric fields applied in reversible electroporation-based treatments. Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel extracellular matrix, which was the point of interest in our to be published study. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute, a tissue model rich in endogeneous extracellular matrix such as collagens, was used to demonstrate the effects of microsecond and millisecond pulsed electric fields applied respectively in electrochemotherapy (ECT) treatment and gene electrotransfer (GET) strategy. Our analyses, focused on matrisome genes and extracellular matrix remodeling, underpin that pulsed electric fields, a technology already approved for clinical use combined with anti-cancer agents, are particularly promising to provide local and effective treatment of abnormal extracellular matrix. Part of this dataset was used to describe how pulsed electric field on its own (with no addition of external drugs) induce extracellular matrix (ECM) remodeling at human dermal scale, by focusing at genes related to matrisome subset. In this manuscript to be published, electrochemotherapy (ECT) parameters were named SP for "short pulses" and gene electrotransfer (GET) parameters were named LP for "long pulses". W demonstrated that these both types of electric parameters inducing reversible electroporation of the cells whitin the dermal tissue substitute induced 1) a rapid modulation (4h after electrostimulation) of mRNA’s genes composing the matrisome, particularly a down-regulation of pro-collagens and ECM maturation’s enzymes such as transglutaminase TG2 and LOX-like; 2) a transient decrease in pro-collagens production and hydroxyproline tissue content within a week after electrostimulation; 3) a long-lasting ROS-dependent over-activation of MMPs for at least 48h and 4) a down-regulation at both mRNA and protein level of pro-fibrotic TGF-β. 3 distinct conditions were produced and analyzed, each with 4 independant biological replicates. The first condition is the control condition (CTL) used as reference. The second one is a treatment using electric parameters applied in electrochemotherapy treatment (ECT) consisting in 8 pulses lasting 100µs, at 1000V/cm at a 1 Hz frequecy. The third condition is a treatment using electric parameters applied in gene electrotransfer (GET) consisting in 10 pulses lasting 5ms, at 600V/cm at a 1 Hz frequency. Please note that for conditions consisting in pulsed electric field application no additional drug was added, only the electric parameters. Grant number: ANR-17-CE19-0013-01 Grantee: Laure Gibot Funding source: Agence Nationale de la Recherche

本研究旨在解析可逆电穿孔类治疗中施加的外部脉冲电场所诱导的基因表达调控模式。
得益于其局部施加且效应短暂的特性，物理刺激已成为重塑细胞外基质（extracellular matrix, ECM）的极具吸引力的手段，这也是本待发表研究的核心关注点。
我们评估了经典用于药物递送的脉冲电场技术在组织层面诱导胶原重塑的潜力。
本研究采用一种构建精细的体外组织工程化人体皮肤替代物——该模型富含胶原等内源性细胞外基质——来分别验证应用于电化学治疗（electrochemotherapy, ECT）与基因电转染（gene electrotransfer, GET）方案的微秒级与毫秒级脉冲电场的效应。
我们针对基质组（matrisome）基因与细胞外基质重塑展开分析，结果证实：脉冲电场作为一项已获批联合抗癌药物用于临床的技术，在异常细胞外基质的局部高效治疗方面极具应用前景。
本数据集的部分数据用于阐明：仅施加脉冲电场（未添加外源性药物）时，如何通过靶向基质组相关基因，在人体皮肤组织层面诱导细胞外基质重塑。
在本待发表的手稿中，电化学治疗（ECT）参数被命名为“短脉冲（short pulses, SP）”，基因电转染（GET）参数则被命名为“长脉冲（long pulses, LP）”。
我们证实，这两类可诱导皮肤组织替代物内细胞发生可逆电穿孔的电参数，可引发如下效应：1） 电刺激后4小时内，构成基质组的基因mRNA表达发生快速调控，尤其表现为前胶原以及细胞外基质成熟相关酶（如转谷氨酰胺酶TG2与赖氨酰氧化酶样蛋白）的下调；2） 电刺激后一周内，前胶原的合成与组织羟脯氨酸含量出现一过性降低；3） 基质金属蛋白酶（matrix metalloproteinases, MMPs）出现依赖活性氧（reactive oxygen species, ROS）的持续过度激活，时长至少达48小时；4） 促纤维化转化生长因子-β（transforming growth factor-β, TGF-β）在mRNA与蛋白水平均出现下调。
本研究设置了3组独立实验条件并开展分析，每组均包含4次独立生物学重复。
第一组为对照条件（CTL），作为实验参照；第二组采用电化学治疗（ECT）参数施加处理：8个脉冲，时长100微秒，电场强度1000V/cm，频率1Hz；第三组采用基因电转染（GET）参数施加处理：10个脉冲，时长5毫秒，电场强度600V/cm，频率1Hz。
请注意：所有仅施加脉冲电场的实验条件均未添加外源性药物，仅施加对应电参数。
资助编号：ANR-17-CE19-0013-01
受资助人：Laure Gibot
资助方：法国国家研究署（Agence Nationale de la Recherche, ANR）