The Mechanisms Of Free Fatty Acids Inhibiting The Endothelial Function And Angiogenesis

Background:Delayed wound healing,diabetic skin ulcer and increased premature cardiovascular complications events are the major complication of diabetes and metabolic syndrome that cause significant disability and mortality in these patients.Wound healing is a complex process of coagulation,inflammation,angiogenesis,tissue repair and remodeling,in which,impaired angiogenesis contributes a lot to the development of these vascular complications.The process involves proliferation,migration and functions of multiple cells including stem cells,endothelial cells,fibroblasts and keratinocytes.Abnormalities in these cells,particularly endothelial cells,have been implicated in the impaired wound healing in diabetes and metabolic syndrome.Metabolie stress produced by hyperglycemia and elevated free fatty acids has been shown to inhibit angiogenesis and wound healing.However,the underlying mechanisms remain 'poorly understood,the research of which,will provide new approach for the further study of pathophysiology mechanisms.Angiogenesis is a complex regulatory process,and many multiple signaling pathways and many functional protein molecules involve in.The Hippo pathway and its effector Yes-associated protein(YAP),widely expressed in various tissues and cells of the body,included in endothelial cell,are important in regulating cell proliferation and apoptosis,organ growth,tissue repair and regeneration,intercellular contact inhibition and participate in homeostatic regulation of the internal environment.Recent report has shown suggested its important role in the maintenance of endothelial function and integrity.The Hippo pathway contains mammalian Ste20-like kinases 1/2(MST1/2),Salvador(SAV)and large tumor suppressor(LATSI/2).YAP and transcriptional coactivator with PDZ-binding motif(TAZ)are the primary downstream effectors of the Hippo pathway.MST1/2 phosphorylates and activates LATSI/2,which in turn phosphorylates YAP at S127 or S381,leading to its cytoplasmic retention and degradation,and subsequent transcription suppression of its target genes.YAP promotes cell proliferation survival and angiogenesis.The Hippo pathway,by inactivating YAP,inhibits cell survival,proliferation and angiogenesis.The Hippo-pathway and YAP are key regulators in angiogenesis and wound healing.In addition to providing energy for the metabolism and survival,mitochondria also plays an important role in maintaining the endothelial stability and integrity,which extensively involves in the regulation of redox regulation in endothelial cell and referred an important part in maintaining the dynamic balance of endothelial cells.Due to a susceptible membrane,mitochondria is prone to depolarize when exposed to oxidative stress and result in the disintegration of its membrane,then the mitochondrial DNA(mtDNA)will break and leak into the cytoplasm,which could be identified by the DNA susceptors,the interrelated downstream pathway and molecules will be activated.As a regulatory molecules for innate immunity and inflammation,STING,could specifically recognise the mtDNA leaking into the cytoplasm and be activated,then STING wi1l recruits and combines with TBK1,the compound will promote the phosphorylation of IRF3 and trigger the following downstreams signal transduction and molecular regulation.STING was indicated to participate in the endothelial cell activation,and its excessive activation is also related to endothelial cell inflammation.A vasculopathy associated with STING was defined recently,while the role of STING in the mitochondrial damage and endothelial cell activation and the correlative mechanism remains anclear.The study of the mechanism,especially in the vascular disease,could bring a new field and therapeutic target for metabolic syndrome and diabetes.Objects:(1)To investigate the effects of palmitic acid on the function of human aortie endothelial cells and angiogenesis.(2)To elucidate the interaction between HIPPO and STING-IRF3 pathway under the influence of palmitic acid.(3)To confirm the role of mitochondrial damage and mtDNA in endothelial cells,and to verify the effects of mitochondrial damage induced by palmitic acid on STING-IRF3 signaling pathway and angiogenesis in endothelial cellsMethods:1.Cell culture and transfection.We use Endothelial cell culture medium(ECM)to culture the primary human aortic endothelial cells.Lipofectamine 2000 and siRNAs were used in the knocking down of genes according to the manufacturer' s instruction.Lipofectamine 3000 kit and plasmids were used in the overexpression assay following the instruction,cells were treated with conditioned mediums after transfection.2.Preparation for Palmitic acid.Palmitic acid(PA)was previously dissolved into 200mM by ethanol,and then dropped in and combined with 20%FFA-free BSA solution to make the PA storage solution at the concentrations of 1-5 nM,and stored at-20?,which will be diluted for 10 times by ECM at the concentrations of 0-0.5mM as the working solution and added to cells for different time points,the following experiment will then be carried on as protocol.3.Protein extraction and western blot experiment.Protein were extracted from the treated cells by the cell Lysis solution according to the manufacturer' s instruction,and storaged at-20? or-80,C.Samples will be aparted by the SDS-polyacrylamide gels and transferred to polyvinylidine fluoride membranes,the membranes with proteins will then be blocked and incubated with particular primary antibodies,the secondary antibodies with horse radish peroxidase and ECL luminal reagent helped to exposure the specific molecules.Image J were used in the analysis of results.4.Immunofluorescence experiment.Cells were transferred onto the 10mm coverslips and fixed with immunol staining fix solution after treatment,then treated with Trion X 100,blocked,and incubated with primary antibodies,fluorescently-labeled secondary antibodies and DAP I,observed and photographed by UltraVIEW(?)VOX confocal microscope.5.BrdU proliferation assay.BrdU was added into the medium as 10ug/ml at 6h before the end of the treatment,the following steps were similar with the immunofluorescence experiment,after treated with Triton X 100,2mol/L HCL was added to fix the chromatin,and 0.lmol/L PH8.3 Sodium Borate was used as neutralization,the following blocking,incubating with primary antibody,fluorescently-labeled secondary antibody and DAPI was similar with the immunofluorescence experiment.For flow cytometry experiment,cells were harvested by 0.125%trypsin after treated,centrifuged at 1000rpm,10min,the following steps were carried on as above.6.Migration assay.Cells were cultured in the 6-well plates to the 100%confluence and then scratched with 200ul pipette tips,observed and photographed at different time points,the gap area was measured by Image J.We use a formula as:(the initial gap area-time point gap area)/the initial gap area,to make the comparation.7.Tube formation assay.Matrigel was previously added into 96-well plate and concreted at 37?.Cells were harvested by trypsin and resuspended by ECM after treatment,and then transfer into the wells containing matrigel at 8000-12000 cells pre well.The total tube length were recorded at 4h and 12h and calculated by Image J,8.Real-time quantitative RT-PCR.Trizol was used to extract the total RNA and reverse transcription kit was used to get the cDNA according to the manufacturer' s protocol.The mRNAs were detected and measured by Real-time PCR.9.Mitochondrial membrane potential detection.JC-1 was used in this experiment,the whole process were according to the manufacturer' s protocol.10.Chromatin immunoprecipitation experiment(ChIP).The EZ-ChIPTM Kit was used in this experiment,and carried on as the manufacturer' s protocol.11.Statistical analysis.Data were shown as means ± SD,The independent sample t test was used to compare between groups,one-way ANOVA test was used among multiple groups.P<0.05 is statistically significant.Results:1.Palmitic acid inhibits endothelial proliferation,migration and angiogenesis.(1)BrdU staining and flow cytometry analysis of BrdU proliferation assay showed that PA treatment induced a dose-dependent inhibition of endothelial cell proliferation.(2)Scratch assay showed that PA treatment induced a dose-dependent inhibition of endothelial cell migration.(3)In vitro Matrigel tube formation assay showed that PA reduced endothelial cell tube formation as a dose-dependent way.2.Palmitic acid upregulates MST1 and inhibits YAP.(1)Western blot analysis showed that PA induced MST1 expression and phosphorylation,and YAP phosphorylation.(2)iununostaining showed that PA prevented YAP nuclear translocation.(3)BrdU proliferation assay showed that overexpression of YAP WT or YAP S127A increased endothelial cell proliferation.(4)In vitro tube formation assay showed that overexpression of YAP WT or YAP S127A enhanced endothelial tube formation.3.MST1 is involved in palmitic acid-induced inhibition of endothelial proliferation and angiogenesis.(5)MST1 is involved in palmitic acid-induced inhibition of endothelial proliferation and angiogenesis.(6)Immunostaining showed that knocking down MST1 prevented the PA-induced YAP cytoplasm retention.(7)BrdU staining and flow cytometry analysis of BrdU proliferation assay showed that silencing MST1 prevented PA-induced inhibition of endothelial proliferation.(8)In vitro tube formation assay showed that MST1 siRNA treatment partially reversed PA-induced impairment of endothelial tube formation.4.PA induces mitochondrial DNA(mtDNA)release to cytosol and activates the cytosolic DNA sensor cGAS-STING-IRF3 pathway.(1)JC-1)staining and quantification showed that PA treatment induced mitochondrial depolarization.(2)Double staining of mitochondria(mito tracker)and double strand DNA(dsDNA)showed that PA triggered dsDNA release to cytosol.(3)PCR analysis of mtDNA in cytosolic fraction showed that PA treatment increased cytosolic mtDNA.(4)Western blot analysis showed that PA activated cGAS,STING,IRF3 and MST1 by time sequence.(5)Western blot analysis showed that mitochondrial damage induced by CCCP treatment lead to the upregulation of cGAS,STING,IRF3.(6)Immunostaining showed that CCCP treatment induced STING perinuclear translocation and IRF3 nuclear translocation.5.The cGAS-STING-IRF3 pathway mediates PA-induced MST1 upregulation and YAP inhibition.(7)Western blot analysis showed that knocking down cGAS or STING or IRF3 prevented PA-induced activation MSTI expression and YAP phosphorylation.(8)immunostaining showed that knocking down STING or IRF3 prevented PA-induced YAP cytoplasm retention.6.IRF3 directly binds to MST1 promoter and mediates PA-induced induction of MST1 expression.(1)RT-PCR analysis showed that PA treatment increased MSTI mRNA,which can be prevented by knocking down IRF3 or STING.(2)Putative IRF3 binding sites upstream of the s' UTR of MST1 gene are shown.(3)Chromatin immunoprecipitation assay showed that IRF3 bound to MST1 gene promoter.The binding was increased in PA or CCCP treated cells.7.The cGAS-STING-IRF3 signaling is involved in PA-induced inhibition of endothelial angiogenesis.(1)BrdU staining and flow cytometry analysis of BrdU proliferation assay showed that silencing cGAS or STING or IRF3 prevented PA-induced inhibition of endothelial proliferation.(2)Tube formation assay showed that knocking down cGAS or STING or IRF3 reversed PA-induced inhibition of endothelial angiogenesis.Conclusions:(1)Palmitic acids could inhibit endothelial cells function,induce its apoptosis and inhibit the angiogenesis.(2)Palmitic acids could affect the function and express of HIPPO pathway.(3)HIPPO pathway is involved in palmitic acid-induced inhibition of endothelial proliferation and angiogenesis.(4)Palmitic acids could cause mitochondrial damage and activate the cGAS-STING-IRF3 pathway.(5)The cGAS-STING-IRF3 pathway is involved in the regulation of HIPPO pathway induced by PA(6)The cGAS-STING-IRF3 pathway is involved in the palmitic acid-induced inhibition of endothelial proliferation and angiogenesis.