Krüppel-like Factor 4 In Endothelium Links Pro-inflammatory Cytokines Regulate The Expression Of Endothelial VWF And PAI-1 Gene

The vascular endothelium plays a critical role in vascular homeostasis, maintain blood fluidity through the production of factors that promote fibrinolysis, inhibit blood coagulation, and inhibit platelet activation. Both biomechanical and biochemical stimuli can affect endothelial gene expression, induce endothelial dysfunction, and confer a pro-adhesive and pro-thrombotic phenotype. The inflammatory cytokines tumor necrosis factor-α(TNF-α),IL-1βinduce the production of procoagulant factors, antifibrinolytic substances, while inhibiting the expression of natural anticoagulants. Pro-inflammatory cytokines can render the endothelium dysfunction, the endothelial surface loses its nonthrombogenic properties.The human KLF4 (Krüppel-like factors 4)gene localized to chromosome 9q31 and encoded a protein which consists of 470 amino acids. KLF4 might participate in the control of cell proliferation, cell differentiation, cell apoptosis, development of tumor, and acetylation or deacetylation of histone has been associated with the regulation of transcription of KLF4. Recently studies indicate that KLF4 is expressed in endothelial cells, inhibited the inflammatory cytokine-mediated induction of adhesion molecule expression, and may have an anti-inflammatory effect.The precise role of KLF4 in endothelial thrombotic function has not been elucidated. Therefore, our study wants to investigate the impossible role and related mechanisms of KLF4 in endothelium stimulated by inflammatory cytokines. The study raises the experimental evidence on the possible mechanisms that mediate the effects of these stimuli on endothelial function.ObjectiveTo investigate the effect of KLF4 under basal and IL-1β, TNF-αinflammatory cytokine on endothelial thrombotic function by stably transfecting recombinant adenoviral vector carrying antisense KLF4,and obseved differential effects of KLF4 on mRNA and protein levels of vWF,PAI-1 as endothelial key coagulant factors. At the same time, we design to demonstrate the mechanisms involved in it.MethodsFirstly, human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical vein and cultured in endothelial cell medium with 5% fetal bovine serum and 1% penicillin/streptomycin. Identified by microscopy and flow cytometry, HUVECs were treated with human IL-1β, human TNF-αat final concentrations of 2.5 ng/mL and 10 ng/mL, respectively, for 2 hours and 4 hours. HUVECs were subcultured using trypsin/EDTA solution. Total RNA was prepared from HUVECs using the Trizol RNA purification system. Meanwhile, HUVECs were suspended in ice-cold lysis buffer. To detected KLF4, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) mRNA expressions by Real Time-PCR, analyzed the changes of KLF4, PAI-1 protein by Western blot, observed the expression and location of KLF4, vWF by confocal laser microscopy, detected the influence of PAI-1 on migration abilities of endothelial cell by a standard wounding assay of HUVECs. Statistical analyses: all data were expressed as x±s. Differences between groups were assessed with variance analysis. P values less than 0.05 were considered to indicate a statistically significant difference.Secondly, Full-length human KLF4 cDNA was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pshuttle-CMV, with the resultant plasmid and the backbone plasmid AdEasy-l, the homologous recombination took place in the E.co1i BJ5183 and the recombinant adenoviral plasmid carrying the antisense KLF4 gene was constructed was generated. The adenoviruses (Ad-KLF4AS) were packaged and amplified in the HEK 293 ce1ls. Then the viral titer was checked by GFP. Recombinant adenoviral vector carrying antisense KLF4 infected the human HUVECs. HUVECs were infected with Ad-GFP or Ad-KLF4AS at 200 MOI for 48 h, and then harvested for total RNA, protein analysis. Study on the inhibitory action of Ad-KLF4AS to KLF4 mRNA and protein expression level in HUVEC.Finally, HUVECs, which has been transfected by recombinant adenoviral vector carrying KLF4 antisence gene at 200 MOI for 48 h, were treated with human IL-1βat final concentrations of 2.5ng/mL, for an additional 4 h. The mRNA expressions of KLF4, PAI-1 and vWF were determined by Real Time-PCR. The changes of protein of KLF4, PAI-1 were analyzed by Western blot. The expression and location of KLF4 and vWF were detected by confocal laser microscopy. The acetylation of KLF4 was performed by immunoprecipitation (IP). All data were expressed as x±s. Analysis of variance (ANOVA) was used to test for significant differences among multiple test groups. In all tests a p-value of 0.05 was considered significant.Results1. Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords following trypsin procedures, and cultured in endothelial cell medium with 5% fetal bovine serum and 1% penicillin/streptomycin. Identified by microscopy and flow cytometry, the common endothelial cell features such as"cobblestone"morphology, positive staining for HUVECs are characterized by flow cytometry with antibodies to factors VIII and CD31.2. Endothelial cells can express KLF4, and usually located on the cell nuclear. Endothelial KLF4 mRNA and protein levels are induced after treatment with TNF- and IL-1β. When time was added, KLF4 expression were increased gradually (P<0.05). Endothelial KLF4 is regulated by inflammatory cytokines. Pro-inflammatory treatment HUVECs led to different localization patterns of KLF4 with an increase in cell cytoplasm.3. Pro-inflammatory mediators significantly up-regulate endothelial PAI-1 and vWF expression.Compared with control group, the expression of PAI-1, vWF mRNA steadily increased since 2 hours to 4 hours after TNF- and IL-1βstimulation(p<0.05). The PAI-1 protein are induced after treatment with TNF- and IL-1βat higer level than in the absence of TNF- and IL-1β. The protein of vWF exhibited a similar result. Immunofluorescence confocal imaging suggested an increased expression of vWF at the protein level by pro-inflammatory stimuli.4. Antisense KLF4 mediated by adenovirus was constructed successfully, the sequence and correct site of adenovirus carring an antisense KLF4 were confirmed by PCR and sequencing assay. The strong green fluorescence was observed in HEK 293 cells by fluorescence microscopy.5. HUVECs were infected with Ad-GFP at a 200 MOI,more than 90% of the cells express GFP by fluorescence microscopy. This system was subsequently used to study the effect of KLF4 on endothelial coagulant function.6. Compared with Ad-GFP infection group and non-infection group, infection group with recombinant adenoviral vector carrying antisense KLF4 at a 200 MOI can cut down the expression of KLF4 gene in HUVEC(P<0.05). But, there were no significant difference in Ad-GFP infection group and non-infection group (p>0.05).7. KLF4 affects the level of endothelial cell vWF and PAI-1 expression. The vWF, PAI-1 mRNA and protein expression in endothelial cells were increased significantly after HUVECs were infected with Ad-KLF4AS as compared with HUVECs exposed to IL-1βand non-infected HUVECs(p<0.05).8. KLF4 in HUVEC is acetylated by pro-inflammatory cytokines.Conclusions1. KLF4 is expressed in vascular endothelial cells of the adult vasculature.2. Endogenous KLF4 expression is highly sensitive to pro-inflammatory stimuli such as TNF- and IL-1β. The remarkably stable transcriptional induction of KLF4 specifically by pro-inflammatory stimuli suggests a role for KLF4 as an intermediary transcriptional regulator of endothelial inflammatory specific gene expression.3. PAI-1 and vWF expression in endothelium are induced by pro-inflammatory stimuli.4. Down-regulated of Krüppel-like factor 4 leads to increase of tumor necrosis factor-αand IL-1βinduced endothelial cell vWF and PAI-1 expression. It implicate that KLF4 inhibit cytokine-mediated induction of the pro-coagulants vWF and PAI-1 expression.5. Acetylation is important for KLF4-mediated transcription in response of pro-inflammatory cytokines.6. The ability to modulate vWF and PAI-1 expression suggest that Krüppel-like factor 4 might play an important role in regulator of endothelial coagulant function in response to pro-inflammatory stimuli and may be an important target for acetylation effects.