Ouabain, angiotensin II and cyclooxygenase-2: cellular interactions on the endothelial pathophysiology control

• Grant number: 22/04201-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: December 01, 2022
• Status: Discontinued
• Field of knowledge: Biological Sciences - Physiology - Physiology of Organs and Systems
• Principal Investigator: Luciana Venturini Rossoni
• Grantee: Henrique Charlanti Reis Assunção
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Ouabain (Oua) is a glycoside cardenolide, found in the plant kingdom and mammals. In addition to inhibiting the activity of Na+-K+-ATPase (NKA), this interaction is capable of regulating protein kinases through its signalosome activity, causing cellular effects such as hypertrophy and generation of reactive oxygen species (ROS). Elevated levels of this glycoside are associated with several cardiovascular pathologies. The chronic administration of Oua supports a hypertensive action, alongside mechanical and physical changes in vessels, which are highly bed-specific. It is observed in this model that vascular actions of Oua interact or depend on angiotensin II (Ang II) or cyclooxygenase (COX)-2, or both. These interactions have also been demonstrated in other tissues. In addition, the intracellular signaling of Oua and Ang II have some similarities, such as cSrc, EGFR and ROS. Studies of Oua in the endothelial cells are still scarce and the knowledge of its interactions with Ang II and COX-2 in vessels may provide a better understanding of its action. Therefore, the present project aims to evaluate Oua signaling in endothelial cells, and possible interactions with the Ang II and COX-2 pathways. To do so, an Oua concentration-response curve will be performed in human endothelial cells, at various times, evaluating cSrc phosphorylation. From there, the participation of Ang II and COX-2 will be evaluated in detail, through the inhibition of the Ang II-converting enzyme and COX-2, and the AT1R antagonism. in parallel studies, the small interfering RNA technique will be used to silence AT1R, COX-2 and EGFR. A signalosome pathway antagonist will also be used. The aspects evaluated will be gene and protein expression of the pathways involved, through the qRT-PCR and Western Blot techniques; synthesis of NO and ROS, using fluorescent probes; synthesis of prostanoids and Ang II, using ELISA kits; and cell proliferation.