Relation between the function and expression of VEGFR1 and the vasculogenic differentiation of dental pulp stem cells

Author(s)

Mariel Tavares de Oliveira Prado Bergamo

• Document type: Doctoral Thesis
• Press: Bauru.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Bauru (FOB/SDB)
• Defense date: 2020-03-13

Examining board members

Thais Marchini de Oliveira Valarelli; Fernando Borba de Araujo; Maria Aparecida de Andrade Moreira Machado; Jacques Eduardo Nor

• Advisor: Thais Marchini de Oliveira Valarelli

Abstract

This study aimed to evaluate the role of the Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) expression at the endothelial differentiation of Dental Pulp Stem Cell (DPSC) and Stem Cell from human deciduous tooth (SHED). Cells were sorted by High (positive/ VEGFR1HIGH) and Low (negative/VEGFR1LOW) levels of VEGFR1 expression through Flow Cytometry and cultured in alpha-MEM supplemented with 20% Fetal Bovine Serum (FBS) or Endothelial Growth Medium (EGM2-MV) with 50ng/mL rhVEGF (control or differentiation medium) and 0 or 25g/mL of Bevacizumab (Avastin or Bevacizumab). The following tests were performed in vitro to evaluate cell proliferation and endothelial differentiation of stem cells: SRB, Sprouting Assay, RT-PCR, Western Blot, and Immunofluorescence. To evaluate this process in vivo, scaffolds seeded with SHED expressing high and low levels of VEGFR1 were transplanted into the subcutaneous dorsum of immunodeficient mice and after 28 days the samples were retrieved for, HE staining, immunohistochemistry, and immunofluorescence. The new blood microvessels formation were counted through ImageJ software, the statistical analyses were performed using unpaired T Test or One-Way ANOVA followed by Tukey Test, and the threshold of statistical significance was set at p < 0.05. The results showed that the SHED VEGFR1LOW has more proliferation rate in 72h regardless of culture media. The differentiation of SHED/DPSC in endothelial-like cells in vitro was confirmed through the expression of endothelial cells markers and sprouting formation by those cells. SHED VEGFR1HIGH generated more quantity of sprouts in vitro and more quantity of microvessel in vivo, showing the important role of this receptor in vasculogenic differentiation of SHED and DPSC. (AU)

• FAPESP's process: 17/11396-3 - Protein gene expression by stem cells from pulp of deciduous teeth after the use of low intensity laser
• Grantee: Mariel Tavares de Oliveira Prado Bergamo
• Support Opportunities: Scholarships in Brazil - Doctorate