Epigenetic Mechanisms Underlying The Biological Changes Of Vascular Endothelial Cells Induced By Serum From Patients With Diabetic Wounds

BackgroundDiabetes mellitus is a chronic disease worldwide,and its prevalence is on a significant increase.Among these diseases,diabetic wound is among the most common chronic complications in clinical practice.The area most commonly involved is the skin of the foot,which is characterized by a high disability rate,high mortality rate,and high economic burden.With the rising prevalence of diabetes,the problem of diabetic wound is becoming increasingly severe,which seriously affects the physical and mental health of patients and the quality of survival and brings enormous economic pressure to families and society.Therefore,elucidating the pathological molecular mechanism of diabetic wound refractory to healing and finding practical and feasible targets for intervention have become hot and harrowing research areas in diabetes-related fields.The vascular endothelium is the basic structure of the innermost layer of the blood vessel and serves as a mechanical barrier between blood and vascular smooth muscle and tissue.As a component of the vascular wall,vascular endothelial cells sense changes in hemodynamics and blood components,synthesize,metabolize and secrete a series of bioactive macromolecules to maintain vascular homeostasis and function,and play an essential role in physiological processes such as inflammatory response,coagulation,angiogenesis and wound healing.Impaired angiogenesis is one of the primary mechanisms underlying the difficulty in healing diabetic wounds.In a previous study,our group found that the distribution of vascular endothelial cells in diabetic wounds was disordered and failed to form vascular lumen effectively.The expression levels of inflammatory and pro-angiogenic factors were increased and decreased respectively in the wounds,indicating that endothelial cells in diabetic wounds were damaged and angiogenesis was impaired.In addition,we incubated skin tissues cultured in vitro using a human serum conditioned medium.We found that compared with skin tissues incubated with healthy control serum group,the number of blood vessels in tissues incubated with serum from patients with diabetic wounds was significantly reduced.The expression levels of inflammatory factors such as IL-1β and IL-6 were elevated,and the expression levels of pro-angiogenic factors VEGF were reduced,consistent with what we observed in diabetic wounds.Therefore,it was hypothesized that serum from patients with diabetic wounds was an important factor in causing vascular endothelial injury and dysfunction,so we further incubated umbilical vein endothelial cells with human serum conditioned medium and observed that serum from diabetic wound patients could induce senescence and cell cycle disorders in umbilical vein endothelial cells.The mechanism by which serum from patients with diabetic wounds induce endothelial cell injury and dysfunction needs to be answered.The answer to this question will provide new strategies to promote the prevention and treatment of chronic wound and other complications of diabetes.Post-translational modifications of histones are an essential part of epigenetics,mainly including methylation,acetylation,phosphorylation,adenylation,and ubiquitination of histones,which can affect gene transcriptional activity and consequently determine the state and fate of cells.In the nucleus of higher organisms,DNA strands and histones are wrapped around each other to form nucleosomes,the basic unit of chromatin in the nucleus,which needs to be opened to expose the DNA strands during gene transcription.Post-translational modifications of histones can significantly affect the opening of nucleosomes and thus the transcription of genes.Trimethylation of histone H3 at lysine 27(H3K27me3)is associated with transcriptional repression and heterochromatin,while acetylation at the same site(Acetylation of histone H3 at lysine 27,H3K27ac)is associated with transcriptional activation and euchromatin.These modifications can exert gene transcriptional regulation by affecting the structure of chromatin or by affecting the binding of other transcription factors to structural gene promoters.In diseases such as diabetes,neurodegeneration,and cancer,disruption of epigenetic modifications including histone H3K27me3 and H3K27 ac can cause abnormal expression of critical genes and play an essential role in developing these diseases.However,it is unclear whether histones H3K27me3 and H3K27 ac play a critical role in the biological changes of vascular endothelial cells induced by serum from patients with diabetic wounds,which key genes are affected,and whether there is an intrinsic link between genes associated with disordered H3K27me3 and H3K27 ac modifications and differential serum components.In summary,this study was conducted from a clinical point of view first to clarify the differentially expressed proteins in serum of patients with diabetic wounds and healthy controls,and then incubate umbilical vein endothelial cells with human serum conditioned medium to observe the effects of serum from patients with diabetic wounds on the apoptosis,migration and angiogenic ability of vascular endothelial cells,and to investigate the effects of serum from patients with diabetic wounds on the expression levels of H3K27me3 and H3K27 ac modifications in vascular endothelial cells,providing a specific theoretical basis and experimental foundation for the treatment of diabetic complications.Part 1 Serum proteomic analysis in patients with diabetic wounds Objective:To analyze differentially expressed proteins in serum from patients with diabetic wounds and healthy controls.Methods:Serum from patients with diabetic wounds was collected,and serum from healthy subjects was used as controls.Tandem mass tag(TMT)combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to screen differentially expressed proteins in serum.Combined with bioinformatics analysis,we explored the main functions,involved signaling pathways,and protein interaction networks of differentially expressed proteins;The ELISA protein quantification technique was used to validate the differential proteins;Multiplex microsphere flow immunofluorescence luminescence assay was applied to determine the expression levels of 12 cytokines in serum.Results:One hundred seventy-three differential proteins were identified between serum from patients with diabetic wounds and healthy controls,including 100 upregulated proteins and 73 down-regulated proteins.Bioinformatics analysis showed that the differential proteins were mainly involved in extracellular matrix(ECM)-receptor interactions and complement and coagulation cascades.With ELISA validation,LRG1,CD5 L,CRP,IGHA1,and LBP were upregulated in serum from patients with diabetic wounds with statistically significant differences(p<0.05).Serum cytokine analysis revealed IL-6,IL-8,and IL-12p70 expression levels were upregulated,and IFN-α expression levels were downregulated in serum from patients with diabetic wounds compared to controls,with statistically significant differences(p<0.05).Conclusion:Differentially expressed proteins in serum from patients with diabetic wounds activate the complement and coagulation cascades and immune response via ECMreceptor interaction,which were closely associated with prolonged low-level systemic inflammation and vascular endothelial dysfunction in patients with diabetic woundsPart 2 Effect of serum from patients with diabetic wounds on the biological changes of vascular endothelial cells Objective:To study the effect of serum from patients with diabetic wounds on the apoptosis,migration,and angiogenic capacity of vascular endothelial cells.Methods:The primary culture system for human umbilical vein endothelial cells(HUVECs)was established,and the cells were identified using CD31 immunofluorescence staining.The 15% human serum conditioned medium was prepared by adding 15% serum from patients with diabetic wounds or healthy controls.HUVECs were randomly divided into diabetic wound serum and healthy control serum groups and incubated with 15% human serum conditioned medium for48 or 72 hours.The effect of serum from diabetic wound patients on the apoptosis of HUVECs was detected by flow cytometry;the effect of serum from patients with diabetic wounds on the migration ability of HUVECs was detected by transwell migration assay;the effect of serum from patients with diabetic wounds on the angiogenic ability of HUVECs was observed by tube formation assay;Meanwhile,q RT-PCR was used to detect the effects of serum from patients with diabetic wounds on the m RNA expression levels of apoptosis,angiogenesis,cycle disturbance and senescence related genes(EGFR,Bcl-2,VEGF,P21,P53)in HUVECs.Results:HUVECs were successfully isolated and cultured from the umbilical vein by enzymatic elimination.Cell membrane were diffusely and strongly positive for CD31 expression by immunofluorescence staining,demonstrating the isolated cells were endothelial cells.Compared with the healthy control serum group,the apoptosis rate of HUVECs in the diabetic wound serum group was significantly higher,the migration ability and the angiogenic ability of HUVECs was significantly inhibited,with statistically significant differences(p<0.05).Meanwhile,q RT-PCR results showed that the expression levels of EGFR,Bcl-2,and VEGF were significantly decreased,and the expression levels of P21 and P53 were significantly increased in HUVECs from diabetic wound serum group compared with those from healthy control serum group.The differences were statistically significant(p<0.05).Conclusion:Serum from patients with diabetic wounds can induce abnormal expression of critical genes,which disrupt vascular endothelial cells homeostasis and induce endothelial cell dysfunction.Part 3 The role and molecular mechanisms of histone H3K27 modifications underlying the biological changes of vascular endothelial cells induced by serum from patients with diabetic wounds Objective:To explore the molecular mechanisms of serum from patients with diabetic wounds-induced biological change of vascular endothelial cells from the perspective of histone H3K27me3 and H3K27 ac modifications.Methods:HUVECs were incubated with human serum conditioned medium for 6 hours.Western Blot detected the overall expression levels of H3K27me3 and H3K27 ac,and Chromatin Immunoprecipitation Sequencing(ChIP-Seq)was used to detect the enrichment and distribution of H3K27me3 and H3K27 ac in the whole genome region.ChIP-q PCR was performed to verify the enrichment level of H3K27me3 in the promoter region of the EGFR.Results:Compared with healthy control serum group,the overall expression level of H3K27me3 was reduced and H3K27 ac was increased in HUVECs from diabetic wound serum group,indicating that these two histone modifications were disordered in HUVECs incubated with serum from patients with diabetic wounds.ChIP-seq analysis showed that these two histone modifications were mainly distributed in the TSS region of the genome,suggesting that they are mainly involved in gene transcriptional regulation.We found that H3K27me3 was significantly enriched in the promoter regions of EGFR and TNXB and significantly less enriched in the promoter regions of TLR5 and RTEN;H3K27ac was significantly enriched in the genome functional regions of P21,VDAC1,ADAMTS1,and EFEMP1,and significantly less enriched PLXDC2.ChIP-q PCR results showed that H3K27me3 was significantly enriched in the promoter region of EGFR in HUVECs from diabetic wound serum group compared with those from healthy control serum group,and the difference was statistically significant(p<0.05).Conclusion:Serum from patients with diabetic wounds induces disorder of H3K27me3 and H3K27 ac modifications in vascular endothelial cells,leading to transcription abnormalities of key genes,which play an important role in the biological changes of vascular endothelial cells such as apoptosis,migration and angiogenesis.