A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice

Small artery remodeling and endothelial dysfunction are hallmarks of hypertension. Evidence supports a likely causal association between cardiovascular diseases and endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. We hypothesized that arteries from hypertensive mice present high levels of EndMT and that specific EC reprogramming can decrease blood pressure values and restore vascular function in resistance arteries in hypertensive mice. Here, we demonstrated OSK overexpression induced partial EC reprogramming in vitro, and these cells had lower migratory capability. Using spatial whole transcriptome atlas, we showed that OSK treatment of hypertensive BPH/2J mice attenuated EndMT and elastin breaks, and by other assays, we showed that OSK treatment reduced blood pressure and resistance arteries hypercontractility. OSK-treated hypertensive HAoECs showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in HAoECs from hypertensive patients. Overall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage. All animal procedures and protocols used were approved by the Animal Care and Use Committee at the University of South Carolina School of Medicine Columbia. Lentiviral vector production and utilization in cells and mice had the School of Medicine Columbia University of South Carolina ethics committee approval (IACUC #2596-101690-041122, and IBC protocol #300322). Experiments were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Animal Research Reporting of in Vivo Experiments (ARRIVE) guidelines. Male and female BPN/3J (RRID:IMSR_JAX:003004) and BPH/2J (RRID:IMSR_JAX:003005) mouse strains were obtained from The Jackson Laboratory and maintained as an inbred colony at the Animal Facility (School of Medicine Columbia, University of South Carolina). In addition, all male and female mice were used at 40-44 weeks of age for the intravenous (i.v.) treatment with the LVCO (control) or LV-OSK. One intravenous (i.v.) injection of lentiviral vectors (100 µL) carrying control plasmid (LVCO) or containing the transcription factors Oct-3/4, Sox-2 and Klf-4 (LV-OSK) was performed in male and female BPN/3J and BPH/2J mice through the tail vein. The i.v. injection consisted of 5 µL LVCO or LV-OSK (stock solution at 2.81x108 TU/mL) combined with 95 µL sterile saline. All mice were maintained on a 12-hour light/dark cycle with water and standard chow diet ad libitum. Digital spatial profiling (DSP) was performed on formalin-fixed paraffin-embedded (FFPE) thoracic aortas isolated from the i.v. LV-OSK-treated (40-44-week-old) mice, and the Mouse Whole Transcriptome Atlas (RNA v1.0) was applied to the samples (Nanostring, Seattle, WA, USA) in this study.

小动脉重构与内皮功能障碍是高血压的标志性病理特征。已有证据表明，心血管疾病与内皮细胞向间充质细胞转化（endothelial-to-mesenchymal transition, EndMT）之间存在潜在的因果关联；该过程是一种细胞转分化事件，在此过程中内皮细胞（endothelial cells, ECs）会部分丧失自身表型并获得间充质细胞特性。内皮细胞重编程（endothelial cell reprogramming, EC reprogramming）是再生医学中用于预防心血管疾病引发有害损伤的创新策略。本研究提出假说：高血压小鼠的动脉组织中EndMT水平较高，而特异性内皮细胞重编程可降低高血压小鼠的血压水平，并恢复其阻力动脉的血管功能。本研究证实，体外实验中OSK过表达可诱导部分内皮细胞重编程，且此类细胞的迁移能力有所降低。借助空间全转录组图谱技术，本研究发现对高血压BPH/2J小鼠进行OSK处理可减轻其EndMT程度与弹性蛋白断裂情况；通过其他实验检测，我们还证实OSK处理可降低小鼠血压，并改善其阻力动脉的高收缩性。经OSK处理的高血压人主动脉内皮细胞（human aortic endothelial cells, HAoECs）表现出更高的内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）激活水平与一氧化氮（nitric oxide, NO）生成量，同时活性氧（reactive oxygen species, ROS）生成量较低。单细胞RNA测序分析显示，OSK可缓解高血压患者HAoECs的内皮细胞衰老与EndMT过程，恢复其正常表型。综上，上述实验数据表明，OSK处理与内皮细胞重编程可降低血压，并逆转高血压诱导的血管损伤。本研究中所有动物实验操作与方案均获得南卡罗来纳大学哥伦比亚分校医学院动物护理与使用委员会的批准。慢病毒载体（lentiviral vector）的制备以及在细胞与小鼠中的应用均获得南卡罗来纳大学哥伦比亚分校医学院伦理委员会批准（IACUC编号：2596-101690-041122，生物安全委员会协议编号：300322）。本实验严格遵循美国国立卫生研究院（National Institutes of Health, NIH）《实验动物护理与使用指南》以及《动物研究体内实验报告指南》（Animal Research Reporting of in Vivo Experiments, ARRIVE）开展。本研究使用的雌雄BPN/3J（RRID:IMSR_JAX:003004）与BPH/2J（RRID:IMSR_JAX:003005）小鼠品系均购自杰克逊实验室（The Jackson Laboratory），并在南卡罗来纳大学哥伦比亚分校医学院动物设施中作为近交系种群饲养。此外，本研究使用的雌雄小鼠均为40~44周龄，通过尾静脉给予LVCO（对照）或LV-OSK处理。通过尾静脉向雌雄BPN/3J与BPH/2J小鼠单次静脉注射100μL慢病毒载体：携带对照质粒的LVCO，或携带转录因子Oct-3/4、Sox-2与Klf-4的LV-OSK。本次静脉注射制剂为5μL LVCO或LV-OSK（原液浓度为2.81×10^8 TU/mL）与95μL无菌生理盐水的混合液。所有小鼠均饲养于12小时光暗循环环境中，自由饮水并进食标准饲料。本研究对经LV-OSK静脉处理的40~44周龄小鼠的福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）胸主动脉组织进行数字空间图谱分析（Digital spatial profiling, DSP），并使用小鼠全转录组图谱（Mouse Whole Transcriptome Atlas, RNA v1.0）对样本进行检测（Nanostring公司，美国华盛顿州西雅图）。