Funding Acknowledgements Type of funding sources: None. Introduction Despite major attempts aiming to revascularize an ischemic heart, prognosis of cardiac ischemia remains poor. A major obstacle for the induction of new blood vessels, referred as therapeutic angiogenesis, is inefficient gene transfer in cardiac endothelial cells (ECs). This poor permissiveness to genetic modification is a limiting step in studies of pathological conditions with inadequate, but also with increased vascularization. To study the function of a specific gene and its potential implications in angiogenesis, the most straightforward approach would be to either overexpress or silence it in ECs. We and others have tested multiple protocols for either transfection and transduction of primary ECs in vitro and in vivo, with no success. Vectors based on Adeno-associated virus (AAV) and Lentivirus (LV) stand as ideal tools for gene delivery, however they do not work in cardiac ECs. Purpose To enhance the permissiveness of EC, we optimized and performed high-throughput screening on adult cardiac ECs using a library of FDA approved drugs, to search for compounds increasing transduction by AAV6 and LV, with the final aim to identify the major determinants inhibiting genetic modification of ECs. Methods Cardiac ECs were isolated from 2 months old C57BL/6 mice and plated on 384-well plates. After 48 hours, the FDA drug library (> 1500 compounds) was added together with AAV6-dsRed and LV-GFP. Cells were fixed with 4% PFA 72 hours post-transduction, and subsequently stained for the EC marker ERG. The levels of AAV6-dsRed, LV-GFP and ERG expression were assessed by automated, high-content microscopy. Results After two rounds of screening at different drug concentrations, we obtained a list of candidate compounds able to increase adult cardiac EC transduction by either AAV6 or LV over two folds. Among these, our best hit increasing AAV6-mediated transduction was Vatalanib, a tyrosine kinase inhibitor acting on VEGFRs, PDGFR, and c-Kit. Valatanib not only increased permissiveness to AAV6, but it also changed the morphology of the cells, which became elongated and similar to mesenchymal cells. This suggested ongoing endothelial mesenchymal transition (EndMT), which was verified by decreased CD31 and increased α-SMA expression. Interestingly, AAV mostly transduced cells with the lowest CD31 expression. Conclusions Our high-throughput screening identified Vatalanib as the most potent drug in increasing permissiveness of cardiac ECs to AAV transduction. Because the drug induced loss of CD31 and gain in α-SMA, we hypothesized that the reason for increased permissiveness was EndMT. This hypothesis is consistent with the known mechanism of action of Valatanib, which inhibits VEGFR2 and thus interferes with VEGF signalling, that in turn is essential to maintain EC identity.

中文翻译：

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转导过渡：如何使心脏内皮细胞更容易接受 AAV

资金确认 资金来源类型：无。引言 尽管进行了旨在对缺血性心脏进行血运重建的重大尝试，但心脏缺血的预后仍然很差。诱导新血管（称为治疗性血管生成）的主要障碍是心脏内皮细胞 (EC) 中的低效基因转移。这种对基因改造的不良许可是研究不充分但血管化增加的病理状况的一个限制步骤。为了研究特定基因的功能及其在血管生成中的潜在影响，最直接的方法是在 ECs 中过表达或沉默它。我们和其他人已经在体外和体内测试了多种原代 EC 转染和转导方案，但均未成功。基于腺相关病毒 (AAV) 和慢病毒 (LV) 的载体是基因传递的理想工具，但它们在心脏 ECs 中不起作用。目的 为了提高 EC 的允许性，我们使用 FDA 批准的药物库对成人心脏 EC 进行优化和高通量筛选，以寻找增加 AAV6 和 LV 转导的化合物，最终目的是确定抑制遗传的主要决定因素EC的修改。方法 从 2 个月大的 C57BL/6 小鼠中分离出心脏 ECs，并将其铺在 384 孔板上。48小时后，将FDA药物库(＞1500种化合物)与AAV6-dsRed和LV-GFP一起添加。细胞在转导后 72 小时用 4% PFA 固定，随后对 EC 标记 ERG 进行染色。通过自动评估 AAV6-dsRed、LV-GFP 和 ERG 表达的水平，高内涵显微镜。结果在不同药物浓度的两轮筛选后，我们获得了一系列候选化合物，这些化合物能够通过 AAV6 或 LV 将成人心脏 EC 转导增加两倍以上。其中，增加 AAV6 介导的转导的最佳打击是 Vatalanib，一种作用于 VEGFR、PDGFR 和 c-Kit 的酪氨酸激酶抑制剂。Valatanib 不仅增加了对 AAV6 的允许性，而且还改变了细胞的形态，细胞变得拉长并类似于间充质细胞。这表明正在进行的内皮间质转化 (EndMT)，这通过 CD31 减少和 α-SMA 表达增加得到证实。有趣的是，AAV 主要转导具有最低 CD31 表达的细胞。结论 我们的高通量筛选确定 Vatalanib 是增加心脏 ECs 对 AAV 转导的许可的最有效药物。因为药物导致 CD31 的损失和 α-SMA 的增加，我们假设增加许可的原因是 EndMT。这一假设与 Valatanib 的已知作用机制一致，后者抑制 VEGFR2 并因此干扰 VEGF 信号传导，而这反过来又是维持 EC 特性所必需的。