Angiotensin II in the modulation of apical human papilla cells in vitro

Author(s)

Laís Nicolay Pizzatto

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia (FO/SDO)
• Defense date: 2018-08-03

Examining board members

Carla Renata Sipert; Celso Luiz Caldeira; Marinilce Fagundes dos Santos; Marcia Carneiro Valera

• Advisor: Carla Renata Sipert; Carlos Ferreira dos Santos

Abstract

Angiotensin II (Ang II), the main effector peptide of the Renin Angiotensin System (SRA), has been reported as an important mediator of inflammatory processes. It is considered of extreme relevance to detect this system and understand the modulation of Ang II in the apical papilla cells (CPA) considering that this cellular population is the key in the success in pulpar revascularization. It is also worth noting that infection of the root canal system can alter the function of these cells. The aim of the present work was to elucidate the role of lipopolysaccharide (LPS) in the apical papilla cells, to detect the components of the Renin Angiotensin System (SRA) and to understand the modulation of Ang II in the CPA. To overcome the scientific challenge, cultures of papilla cells were established and characterized phenotypically by flow cytometry and functionally by Alizarin Red. When stimulated by LPS, cytotoxicity by MTT and cytokine and Osteoprotegerin (OPG) were analyzed by ELISA. Next, the detection of the gene expression of the components of the Renin Angiotensin System was performed by Reverse Transcription followed by quantitative Polymerase Chain Reaction (RT-qPCR) and the Ang II peptide by ELISA. The cells were stimulated with LPS, Ang II itself and an Angiotensin II receptor antagonist type 1 (AT1) and evaluated for cytotoxicity by MTT assay, alizarin red mineralized nodule formation capacity, and production of inflammatory cytokines and OPG by ELISA. The results demonstrated that CPA cultures expressed typical levels of CD146, CD24 and STR0-1 mesenchymal stem cell markers and formed mineralized nodules with the differentiation medium. The stimulus with LPS at 0.1; 1 and 10?g/mL in the period of 1, 3, 5, 7 and 14 days did not affect the viability of CPA. LPS-stimulated cells had higher levels of 1?g/mL chemokine MCP-1/CCL-2 at one and 24 hours and OPG expression decreased in the LPS presence at 0.1 and 10?g/mL. At the concentration of 1 ?g/mL, the gene expression of Angiotensinogen, Renin, Angiotensin Converting Enzyme (ECA) and AT1 was detected. There was greater expression of ACE in the experimental period of 1 hour with LPS and in 24 hours, only AT1 and ACE remained present. Being the protein production sovereign, Ang II was found in the cells by the ELISA. Functional assays demonstrated that Ang II increased cell proliferation at 24, 48 and 72 hours. In 24 hours, the presence of the effector peptide of SRA increased the production of the chemokine MCP-1/CCL-2 and the activator of the nuclear factor receptor-KB (RANKL). We conclude that LPS was able to functionally alter CPA, that the Renin Angiotensin System is present and Ang II has the capacity for pro-inflammatory, proliferative and bone resorption modulation in this cell population. (AU)

• FAPESP's process: 16/16473-3 - Study of the role of angiotensin II on in vitro differentiation of human apical papilla cells
• Grantee: Laís Nicolay Pizzatto
• Support Opportunities: Scholarships in Brazil - Master