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Study of the mechanisms involved in the reduction of endothelin-1 signaling in the vascular bed of spontaneously diabetic mice

Grant number: 23/09528-0
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): November 01, 2023
Effective date (End): January 31, 2025
Field of knowledge:Biological Sciences - Pharmacology - Cardiorenal Pharmacology
Principal Investigator:Rita de Cassia Aleixo Tostes Passaglia
Grantee:Carina Amarante Pedersoli
Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:13/08216-2 - CRID - Center for Research in Inflammatory Diseases, AP.CEPID

Abstract

Diabetes Mellitus (DM) is one of the main risk factors for several cardiovascular diseases such as high blood pressure, atherosclerosis, stroke, coronary artery disease and heart failure. DM also promotes macro and microvascular complications, such as peripheral arterial disease, retinopathy, ischemia and limb amputation, which are largely responsible for reducing the life expectancy of patients with this metabolic disorder. The hyperglycemic state negatively modulates several regulatory factors of vascular endothelial cell activity, generating dysfunction and decreasing their ability to respond to humoral and mechanical stimuli. The changes produced by hyperglycemia in the release of dilating and constricting factors by endothelial cells compromise the fine adjustment of vascular tone, with a consequent increase in peripheral vascular resistance, contributing to damage to organs such as the kidneys, heart, eyes and brain. Diabetic patients and experimental models of DM show increased plasma concentrations of endothelin-1 (ET-1). In DM, there is also a reduction in the contractile response to increasing concentrations of ET-1, as observed in resistance arteries of spontaneously diabetic db/db mice, in mice with streptozotocin-induced diabetes, and in diabetic patients. Considering the recent advances in the study of the mechanisms involved in the control of signaling by receptors, such as the existence of canonical pathways (which involve activation of G proteins) and non-canonical pathways (activated, for example, by the recruitment of beta-arrestin and MAPKs) , the objective of this work is to investigate the mechanisms involved in the reduction of vascular responses to ET-1. We will test the hypothesis that the increase in the plasma concentration of ET-1 reduces the contractile response mediated by type A endothelin receptors (ETAR) by preferentially increasing its endosomal degradation. We will use mesenteric resistance arteries from db/db mice and respective control mice (db/+), as well as human kidney epithelial cells (HEK293T), which allow us to investigate intracellular signaling mechanisms, with heterologous expression of G protein-coupled receptors and luminescence assays such as BRET. Initially, the plasma concentration of ET-1 and transcription of genes associated with the ET-1 signaling pathway in mesenteric arteries will be quantified by ELISA and qRT-PCR, respectively. Arteries will be exposed to ET-1, and the contractile response and ETAR desensitization will be tested by vascular reactivity assays (resistance mesenteric arteries from db/db and db/+ mice), in the presence of chloroquine (to inhibit fusion of ET-1). endosomal vesicle with lysosomal vesicles and receptor degradation). HEK293T cells expressing human ETAR will be exposed to ET-1 and measurements of changes in intracellular calcium levels, in the presence of vehicle or modulators mentioned above will be performed. Finally, under these same experimental conditions, cells will be subjected to BRET assays to characterize endosomal trafficking and monitor receptor recycling after internalization.

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