Contribution of angiotensin II-induced Netosis in the vascular dysfunction and Hypertension induced by ethanol consumption

• Grant number: 24/13353-3
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2025
• End date: April 30, 2028
• Field of knowledge: Biological Sciences - Pharmacology - Cardiorenal Pharmacology
• Principal Investigator: Carlos Renato Tirapelli
• Grantee: Alessandra Oliveira Silva
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Arterial hypertension resulting from ethanol consumption affects a significant portion of the world population. This condition is associated with high morbidity and mortality due to its complications. Ethanol consumption activates the renin-angiotensin-aldosterone system (RAAS) and blockade of the angiotensin II AT1 receptor (AT1R) prevents arterial hypertension and vascular dysfunction in response to ethanol. Vascular dysfunction promoted by ethanol occurs through multiple mechanisms and involves an increase in thromboxane (TX)A2 and intracellular Ca2+ concentration, activation of the enzyme NADPH oxidase with a consequent increase in the production of reactive oxygen species (ROS), and a reduction in nitric oxide (NO) bioavailability. Furthermore, ethanol favors the infiltration of neutrophils into the vasculature, and this response is concomitant with an increase in pro-inflammatory cytokines. Thus, it is possible that neutrophils contribute to vascular damage and arterial hypertension induced by ethanol consumption. Angiotensin II, via AT1R, induces neutrophil activation during arterial hypertension, a response that culminates in the vascular release of neutrophil extracellular traps (NETs) through a cell death process called NETosis. Although it functions as a defense mechanism, NETosis can cause tissue damage. Recent data show that in the vasculature, NETosis maintains and amplifies an inflammatory condition that is characterized by the activation of NFºB, an increase in pro-inflammatory cytokines (TNF-±, IL-6 and IL-1²), TGF-², endothelin- 1, TXA2 and increased ROS generation. Thus, hypercontractility and reduced vascular relaxation are common consequences in different conditions in which NETosis induces vascular damage. NETosis is a mechanism by which ethanol promotes its deleterious effects on the liver. However, the role of NETosis in the cardiovascular changes induced by ethanol remains elusive. Here, we aim to fill a gap in the understanding of the mechanism that leads to the hypertensive state induced by ethanol, focusing on NETosis, which, in the last decade, has been established as an important mechanism and therapeutic target in different conditions that lead to vascular dysfunction. The objective is to investigate the contribution of NETosis, induced by angiotensin II, in vascular dysfunction and arterial hypertension induced by ethanol consumption. Two hypotheses were put forward: 1) Ethanol consumption will stimulate the RAAS, increasing the production of angiotensin II, which in turn will stimulate (via AT1R) the infiltration of neutrophils into the vasculature through increased production of chemokines (CXCL1 and CXCL2) and the expression of adhesion molecules (P-selectin, VCAM-1, ICAM-1). Angiotensin II will activate AT1R in neutrophils, triggering an intracellular that will lead to NETosis; 2) NETs released into the vessel will promote vascular dysfunction through mechanisms that involve increased generation of pro-contractile (ROS, TXA2 and endothelin-1) and pro-inflammatory mediators (TNF-±, IL-6 and IL-1²). NETosis will cause vascular dysfunction and propagate a local inflammatory response that will contribute to vascular dysfunction and hypertension. With this purpose, male C57Bl6 mice will be treated with an ethanol solution for 12 weeks. The role of AT1R in vascular infiltration of neutrophils and induction of NETosis will be evaluated using losartan (a selective AT1R antagonist). The involvement of NETosis will be evaluated using PAD4-deficient mice (PAD4 is essencial for NETosis). Functional (blood pressure, vascular reactivity) and molecular (flow cytometry, ELISA, immunofluorescence, Western immunoblotting) assessments will be carried out using blood and the mesenteric arterial bed.