27 - Investigating the Angiogenic Effects of Titanium Dioxide Nanoparticles on Human Umbilical Vein Endothelial Cell Networking via HGF, VEGF, and VEGFR2 Upregulation

Poster Board Number: 27
There are separate poster presentation times for odd and even posters.
Odd poster #s – first hour
Even poster #s – second hour

Co-authors:

Grace Wang - Detroit Country Day School; Michael Lotwin - Rambam Mesivta; Jacqueline Han - Great Neck South High School; Annika Joshi - Johns Creek High School; Chunuo Chu - Shenzhen Middle School; Sahana Dhama - The Wheatley School; Rachel Na - Washington University in St. Louis; Shi Fu - Stony Brook University; Miriam Rafailovich - Stony Brook University

Abstract Body : Titanium dioxide nanoparticles (TiO2 NPs) are versatile due to their biocompatibility, antimicrobial properties, and light-scattering capabilities, making them highly sought-after in multiple industries, particularly in biomedicine. Previous research demonstrated that they induce the activation of human umbilical vein endothelial cells (HUVECs) due to increased expression of adhesion and other inflammation-associated molecules. This study aims to investigate the impact of TiO2 NPs on HUVECs angiogenesis using flow cytometry and cell sorting based on TiO2 uptake, time-lapse imaging of vascular development with EVOS fluorescence microscope, network analysis with Celleste Image Analysis software, scanning electron microscopy (SEM) to evaluate the presence of TiO2 NPs within treated HUVECs and reverse transcription polymerase chain reaction (RT-PCR) to determine the relevance of genetically-upregulated growth factors within the treated HUVECs.



HUVECs were cultured with and without 0.1 and 0.2 mg/mL rutile TiO2 particles for 24 hours. Treated and untreated HUVECs were plated on a 10 mg/mL Matrigel substrate, and the progress of angiogenesis was recorded using an EVOS fluorescence microscopy with a CO2 onstage incubator. Images of triplicate cell culture per group were captured every 20 minutes over 24 hours, and analyzed using Celleste Image Analysis. The DISC method was then used to measure the mechanical field applied on the substrates by HUVEC network formation. RT-PCR was performed on the cells cultured with TiO2 NPs (0.1 and 0.2 mg/mL) for an initial 24-hour period and an additional 24-hour period after washing off the excess NPs with PBS. Fluorescence-activated Cell Sorting (FACS) was performed on 0.1 mg/mL treated HUVECs to determine the cell uptake percentage of the TiO2 NPs.



FACS analysis showed that the uptake of TiO2 NPs occurred in 91% of the treated HUVECs. Transmission electron microscopy (TEM) showed these NPs to be sequestered in vacuoles, agglomerating near the mitochondria without penetrating the nucleus. RT-PCR confirmed these particles’ impact on genetic expression within the treated HUVECs, showing significant upregulation of VEGF, VEGFR2, and HGF, growth factors that are heavily involved in angiogenesis. When plated on Matrigel, treated HUVECs produced a more robust network than the untreated HUVECs. SEM demonstrated that cells containing TiO2 participated in all aspects, from splines to junction points. DISC analysis indicated maximum stress occurring at the center of the network unit cells, with cells containing TiO2 exerting less force on the network. Future studies will focus on identifying the mechanisms responsible for genetic upregulation without penetration into the nucleus.