Titanate Nanomaterials Affect KLF Signaling Pathway In Human Umbilical Vein Endothelial Cells

Titanate nanomaterials(TiNMs)have been proposed as relatively biocompatible nanomaterials.There is currently lacking systemic studies which investigated the toxicity of Titanate nanofibers(TiNFs)and titanate nanotubes(TiNTs)to endothelium.Our results showed that TiNTs were less effectively internalized into HUVECs compared with TiO₂NPs,but none of the NMs induced cytotoxicity or activation of endoplasmic reticulum(ER)stress biomarkers.In addition,intracellular reactive oxygen species(ROS)was only modestly induced by TiNTs and TiO₂NPs.However,both types of NMs significantly promoted the protein levels of vascular cell adhesion molecule-1(VCAM-1).TiNTs also promoted the release of soluble(s VCAM-1).TiNTs decreased the production of NO,associated with a decrease of protein levels of endothelial NO synthase(eNOS).The transcription factors of eNOS,including Krüppel-like factor 2(KLF2)and KLF4,were more effectively down-regulated by TiNTs compared with TiO₂NPs.In conclusion,our results indicated that TiNTs,albeit not cytotoxic,might impair NO signaling pathway in human endothelial cells leading to the activation of endothelial cells.Next,we developed endothelial monolayer model by using cell culture inserts,and systemically investigated the toxicity of TiNFs and TiNTs by RNA-seq,with a focus on Krüppel-like factor(KLF)-mediated effects,since KLF are transcription factors(TF)involved in the regulation of vascular biology.The expression of many KLF was altered,and Western blot further confirmed that NMs down-regulated KLF2proteins.Ingenuity pathway analysis(IPA)revealed that NMs altered the expression of KLF2-targed genes,typically the genes involved in inflammatory responses.KLF2-related Gene Ontology(GO)terms and Kyoto Encyclopedia of Gene and Genomes(KEGG)pathways were also altered,and it should be noticed that NMs altered GO terms and KEGG pathways related with endothelial NO synthase(eNOS).This study further verified that NMs decreased intracellular NO and eNOS proteins.All the observed effects were more obvious for TiNFs compared with TiNTs.Combined,this study showed that TiNFs or TiNTs were non-cytotoxic to endothelial monolayer model,but TiNFs and more modestly TiNTs decreased KLF2 leading to decreased eNOS proteins and NO productionWe further analyzed the influence of TiNFs on KLF6 pathway in an endothelial monolayer model.Ingenuity Pathway Analysis(IPA)revealed that TiNFs altered the expression of KLF6-related genes.Meanwhile,TiNFs also significantly altered KLF6-related GO terms,including cytokine-mediated signaling pathways,TF functions and membrane-bound organelles.Real-time RT-PCR confirmed that TiNTs increased KLF6 and activating transcription factor 3(ATF3),a TF involved in endoplasmic reticulum(ER)stress.In addition,TiNFs also increased the protein levels of soluble monocyte chemotactic protein 1(s MCP-1),a KLF6-related inflammatory cytokine.Knock-down of KLF6 by si RNA decreased the expression of ATF3.Combined,there data suggested that TiNFs were not cytotoxic but affected KLF6 expression and KLF6-regulated downstream events in endothelial monolayer model..Our data may provide novel understanding about the toxicity of TiNFs as well as other Ti-based NMs to endothelium.