Promoting Angiogenesis via HGF Upregulation using Titanium Dioxide Nanoparticles

Titanium dioxide (TiO2) nanoparticles (NPs) have high biocompatibility, antimicrobial properties, and light-scattering abilities, making them a valuable tool across various industries. As such, TiO2 NPs have become strong points of interest in the modern biomedical field. Previous research has demonstrated that TiO2 NPs induce the activation of human umbilical vein endothelial cells (HUVECs) due to increased expression of adhesion and other molecules associated with inflammatory response [1]. Such NPs may be linked to promoting angiogenesis, the process by which new capillaries are formed from existing vascular structures in the human body. Hence, this study examines the effects of TiO2 NPs on HUVECs angiogenesis through flow cytometry and cell sorting based on TiO2 uptake, imaging time lapses of vascular development using EVOS microscopy, network quantification with Celleste Image Analysis software, scanning electron microscopy (SEM), and Reverse Transcription Polymerase Chain Reaction (RT-PCR).<br/><br/>HUVECs were cultured with and without 0.1mg/ml Rutile TiO2 particles for 24 hours. The cultures were then sorted using FACS, where cells were sorted by a low and high scattering angle window, and it was assumed that the high scattering fraction would increase with TiO2 uptake. The results showed that 91% of the treated cells contained TiO2 particles.<br/><br/>The high scattering fraction and the control untreated cells were then plated on a 10mg/ml matrigel substrate, and the progress of angiogenesis was recorded using an EVOS microscope with a CO2 onstage incubator. Images of triplicate cell cultures were captured every 20 minutes over 18 hours. The images were then analyzed using Celleste Image Analysis software. From the images, it was found that the culture with TiO2 nanoparticles nucleated a network, and the network was more robust and persisted longer than the control. SEM analysis of the networks indicated that TiO2 particles were present in the cells, which constituted the nodes, splines, and all aspects of the network.<br/><br/>RT-PCR was performed on the cultures, and the results of the treated cultures were compared to those of the control. The data showed that while the housekeeping gene, GAPDH, was significantly upregulated by the particles, no difference with the control group was observed for the other housekeeping gene, Actin Beta. Hence, only the latter was used for normalization.<br/><br/>The data demonstrated that TiO2 exposure upregulated the hepatocyte growth factor (HGF) expression by more than an order of magnitude. In contrast, Vascular endothelial growth factor A (VEGFA) expression, which was previously observed to be upregulated in tumors with HGF, did not occur. These results indicate that despite no evidence of TiO2 nanoparticle penetration into the nucleus, exposure to TiO2 can result in upregulation of multiple genes. Further research into the precise mechanism is underway to determine whether the findings can serve as a beneficial factor in promoting wound healing.<br/><br/>We acknowledge the Morin Charitable Trust for funding.<br/><br/>[1] Montiel-Dávalos, A., Ventura-Gallegos, J. L., Alfaro-Moreno, E., Soria-Castro, E., García-Latorre, E., Cabañas-Moreno, J. G., del Pilar Ramos-Godinez, M., & López-Marure, R. (2012). TiO2 nanoparticles induce dysfunction and activation of human endothelial cells. Chemical research in toxicology, 25(4), 920–930. https://doi.org/10.1021/tx200551u