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. Overall design: 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.