Expression Of Keratin 9 In Arterial Endothelial Cells And Its Effect On The Occurrence And Development Of Atherosclerosis

Objectives:Atherosclerosis（AS）is a complex chronic inflammatory disease,the pathogenesis of which has yet to be fully explored.A large number of studies have shown that vascular endothelial cell dysfunction is closely related to its occurrence.Keratin is the main component of the cytoskeleton intermediate filament,and its main function is to maintain the integrity of epithelial tissue.Currently,the role of keratin goes far beyond its traditional function as a stabilizing cytoskeletal element.In the context of injury and disease,the expression levels、cellular localization and post-transcriptional modifications of keratins are involved in many pathological processes,such as atherosclerosis、diabetes,suggesting that they may be potential therapeutic targets.Keratin 9 belongs to type I intermediate filament keratin,and is mainly expressed in differentiated palm and toe epidermis.In the previous experimental work,our research group found that the expression level of keratin 9 was increased in human carotid atherosclerotic tissue.By consulting relevant databases and literature,it was also found that keratin 9 was expressed in endothelial cell lines and its expression level was increased in atherosclerosis.This suggests that keratin 9,as a cytokeratin,may be involved in the occurrence and development of atherosclerosis.Therefore,this experiment aims to study the expression of keratin 9 in atherosclerotic vascular endothelial cells and its effect and mechanism on the occurrence and development of atherosclerosis.Methods:This study combined with 14 human autopsy specimens and cultured human aortic endothelial cells（HAEC）in vitro.Through bioinformatics,multiple databases combined with morphological analysis、HE staining、immunohistochemical staining（IHC）、cellular immunofluorescence（IF）、real-time fluorescence quantitative PCR（q RT-PCR）,Western Blot（WB）、CCK8、Hoechst staining、cell scratch test and other experimental methods to study the expression of keratin9 in atherosclerosis and how keratin 9participates in the occurrence and development of atherosclerosis.By using lentivirus interference technique,KRT9 was stably transfo-rmed into endothelial cells by lentivirus,and overexpression-KRT9（OE-KRT9）and knockdown KRT9（SH-KRT9）endothelial cell lines were established.Combined with the in vitro atherosclerosis model induced by exogenous H₂O₂,to further study whether Keratin 9 is involved in the regulation of biological behavior of endothelial cells and how to participate in the occurrence and development of atherosclerosis.Results:1.Keratin 9 was found to be expressed in human carotid atherosclerotic plaques by searching the proteomic database.In addition,by consulting the GEO database,it was found that the expression level of Keratin9 in the atherosclerotic model group was higher than that in the normal group,and the expression of apoptosis、inflammation and Endothelial–mesenchymal transition（EndMT）related indexes changed in different groups.2.In human autopsy aorta tissues,WB and q RT-PCR experiments showed that Keratin 9 expression level was significantly increased in atherosclerotic tissues.The IHC results showed that Keratin 9 expression level in endothelial cells of atherosclerotic tissue was higher than that in normal aorta group.3.In vitro experiments,WB results showed that compared with the negative control Ig G group,Keratin 9 was significantly expressed in HAEC.Meanwhile,cell immunofluorescence results showed that Keratin 9 was expressed in HAEC and co-localized with endothelial cell marker CD31.In vitro atherosclerosis model induced by exogenous addition of H₂O₂:CCK8 test results showed that the survival rate of HAEC was about 50%when treated with 600μmol/L H₂O₂ for 8 hours.Therefore,600μmol/L H₂O₂ treatment for 8 hours was selected as the condition for constructing an in vitro atherosclerosis model in this experiment.q RT-PCR detection showed that the expression level of KRT9 m RNA was significantly higher than that of the control group when treated with 600μmol/L H₂O₂ for 8 hours.4.Western Blot assay was used to detect the expression of Proliferating Cell Nuclear Antigen（PCNA）protein:After OE-KRT9,the expression of PCNA protein decreased;after SH-KRT9,the expression of PCNA protein increased;Cell CCK8 assay showed that the proliferation activity of HAEC decreased after overexpression of KRT9,and the viability of HAEC was further decreased by overexpression of KRT9 after adding H₂O₂,and showed the opposite trend after knocking down KRT9.Cell scratch assay was used to detect the effect of Keratin 9 on the migration ability of HAE.Compared with the control group and OE-NC group,the HAEC migration rate was lower after overexpression of KRT9,and the HAEC migration rate was increased after knocking down KRT9 compared with the control group and SH-NC group.5.Western Blot assay was used to detect apoptosis-related markers,and the results showed that:compared with normal aortic tissue,The expression of pro-apoptotic indicators Bax and Caspase-3 was increased and the expression of anti-apoptotic indicator Bcl-2 was decreased in human atherosclerotic tissue.After transfection of KRT9 with lentivirus in endothelial cells,the expression of Bax and Caspase-3 increased and the expression of Bcl-2 decreased in the overexpression of KRT9 group;The trend was reversed after KRT9 was knocked down.In addition,the results of Hoechst experiment showed that compared with the OE-NC group,the dense brilliant blue fluorescence intensity of the overexpressed KRT9 group increased,while the dense brilliant blue fluorescence intensity of the knock-down KRT9 group had no significant change compared with its corresponding NC group.6.Compared with the normal control group,in human atherosclerotic tissue,The expression of pro-inflammatory cytokines IL-6 and TNF-alpha increased,while the expression of anti-inflammatory cytokines IL-10 decreased.the expression of endothelial cell marker CD31 and VE-Cadherin protein decreased,and the expression of mesenchymal marker Vimentin and FN1 protein increased.The expression of these indicators in normal tissues was the opposite.The results of in vitro experiments showed that after induction with exogenous H₂O₂,compared with the control group,the expression of pro-inflammatory cytokines IL-6 and TNF-alpha increased,and the expression of anti-inflammatory cytokine IL-10 decreased;Compared with OE-NC,overexpression of KRT9 further promoted the H₂O₂induced release of pro-inflammatory factor IL-6,TNF-ɑprotein expression level was increased and anti-inflammatory cytokine IL-10 protein expression level was decreased in HAEC.Endothelial cell marker CD31,VE-Cadherin protein expression decreased,mesenchymal marker Vimentin and FN1 expression increased;The opposite was observed when KRT9was knocked down.Conclusion:1.The expression level of Keratin 9 was increased in human AS tissues and in AS vascular endothelial cells.2.Keratin 9 was expressed in cultured HAEC and increased in endothelial cells induced by H₂O₂.3.Overexpression of KRT9 inhibits the proliferation and migration of endothelial cells,promotes endothelial cell apoptosis,and affects the repair of damaged endothelium,thereby further promoting the occurrence and development of AS.4.Overexpression of KRT9 promotes H₂O₂induced HAEC release of inflammatory cytokines and endothelial-mesenchymal transition（EndMT）,the trend is opposite after knocking down KRT9.It is suggested that KRT9 may promote the occurrence and development of AS by promoting endothelial cell inflammation and EndMT.