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

Pernomian, L.; Waigi, E. W.; Nguyen, V.; Mohammed, A.; Costa, T. J.; Fontes, M. T.; Kubinak, J. L.; Aitken, A.; Biancardi, V. C.; Sinclair, D. A.; McCarthy, C. G.; Wang, Y.; Tan, W.; Wenceslau, C. F.

doi:10.1101/2024.05.20.595057

bioRxiv · 10.1101/2024.05.20.595057

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

Pernomian, L.·Waigi, E. W.·Nguyen, V.·Mohammed, A.·Costa, T. J.·Fontes, M. T.·Kubinak, J. L.·Aitken, A.·Biancardi, V. C.·Sinclair, D. A.·McCarthy, C. G.·Wang, Y.·Tan, W.·Wenceslau, C. F.

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Abstract

BackgroundSmall artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire additional mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. MethodsUsing a partial reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Primary ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Cellular migration, senescence, and apoptosis were evaluated. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, cardiac function, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. ResultsOSK overexpression induced partial EC reprogramming in vitro, and these cells showed endothelial progenitor cell (EPC)-like features with lower migratory capability. OSK treatment of hypertensive BPH/2J mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of EndMT and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of both BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis only in male BPN/3J mice. OSK-treated human ECs from hypertensive patients 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 human ECs from hypertensive patients. ConclusionOverall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

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Pernomian, L., Waigi, E. W., Nguyen, V., Mohammed, A., Costa, T. J., Fontes, M. T., Kubinak, J. L., Aitken, A., Biancardi, V. C., Sinclair, D. A., McCarthy, C. G., Wang, Y., Tan, W., Wenceslau, C. F.. 2024-05-21. A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice. https://doi.org/10.1101/2024.05.20.595057

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