Endothelial glycocalyx shedding as a trigger for phenotypic change in endothelial cells and evaluation of the restoration of this structure through cellular reprogramming and rejuvenation

• Grant number: 25/00126-1
• Support Opportunities: Scholarships abroad - Research
• Start date: August 01, 2025
• End date: July 31, 2026
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Carlos Henrique Miranda
• Grantee: Carlos Henrique Miranda
• Host Investigator: Camilla Ferreira Wenceslau
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: University of South Carolina, United States

Abstract

Introduction: Endothelial dysfunction plays a central pathophysiological role in the onset of several cardiovascular diseases, such as arterial hypertension, atherosclerosis, pulmonary hypertension, vascular graft degeneration, etc. Until recently, endothelial dysfunction was primarily considered an imbalance between the production of vasodilators and vasoconstrictor agents. However, increasing evidence shows an association between endothelial dysfunction and the presence of endothelial-to-mesenchymal transition (EndMTs). This is a process of cellular transdifferentiation in which the endothelial cell loses its identity and acquires a mesenchymal phenotype. However, the triggers for this phenotypic change still need to be fully understood.The endothelial glycocalyx (EG) is constituted of proteoglycans (syndecan, glypican, CD44) attached to the luminal surface of endothelial cells interconnected with numerous glycosaminoglycan (GAGs), such as heparan sulfate and hyaluronic acid. These membrane proteoglycans are intimately connected to the endothelial cell cytoskeleton. The EG shedding has been described in many cardiovascular diseases, such as arterial hypertension. The EG has several functions, highlighting its role in transducing physiological signals from the vascular content to the endothelial cell. We hypothesize that the EG plays a vasculoprotective role, inhibiting the formation of EndMTs and that situations that cause EG shedding would lead to the formation of these EndMTs, which would play a determining role in the endothelial dysfunction onset. Furthermore, to show that partial reprogramming of endothelial cells via overexpression of OSK transcription factors could lead to GE reconstitution. Methods: The experimental study will be divided into two stages. In the first stage, endothelial cells originating from the macrocirculation (aorta) and microcirculation (coronary) will be cultured in nine different conditions in a static situation and under laminar flow. The stimulus for the formation of EndMTs will be performed through TGF beta. The nine different conditions will be: PBS (control), TGF beta alone, TGF beta + heparinase III (degrades heparan sulfate from GE), TGF beta + hyaluronidase (degrades hyaluronic acid from GE), TGF beta + metalloproteinase MT1-MMP (degrades syndecan-1 from GE) and in different combinations of GE degrading agents: TGF beta + heparinase III + hyaluronidase; TGF beta + heparinase III + MT1-MMP; TGF beta + hyaluronidase + MT1-MMP and finally, TGF beta + heparinase + hyaluronidase + MT1-MMP. The occurrence of EndMTs will be evaluated by confocal microscopy with immunofluorescence, Western blotting for endothelial/mesenchymal cell proteins, cell migration, and genetic sequencing. In the second stage, the culture of aortic endothelial cells originating from hypertensive patients will be performed under two conditions. In the first condition, they will be infected with Sendai virus (SeV) carrying the polycistronic Klf4-Oct3/4-Sox2 and Klf4, referred to as OSK-SeV. In a second condition, these endothelial cells will be infected only with SeV. The reconstitution of the GE will be evaluated by confocal microscopy with immunofluorescence for heparan sulfate, hyaluronic acid and syndencan-1 and Western blotting for these same proteins.