Study On The Molecular Mechanism Of Vascular Endothelial Cell Injury Induced By Hyperglycemia

Background:Diabetes mellitus is a major independent risk factor for the development of cardiovascular disease,and has been shown to accelerate the development of atherosclerosis.Despite its profound clinical importance,our understanding on diabetic vascular diseases is far from completion.Recent studies have shown that long non-coding RNAs(lncRNAs)play pivotal roles on the development of diabetic vasculopathies.However,its molecular mechanisms remain unclear.Methods:In our previous studies,we have found several differentially up-regulated novel human lncRNAs in response to high glucose stimulation in vascular endothelial cells.Among them,we identified HAVEN(Hyperglycemia-Activated Vascular Endothelial cell expressed Non-coding RNA)as a crucial candidate lncRNA.The expression of HAVEN and its adjacent genes was studied by constructing an in-vitro glucose-stimulated cell model.The plasmids of sphingosine-1-phosphate receptor 1(S1PR1),Kruppel-like factor 2(KLF2)were constructed.Small interfering RNA was used to knock down the expression of KLF2 and S1PR1 in human arterial endothelial cells,and to evaluate whether there was a direct or indirect effect between the three.Results:Previous studies showed that HAVEN is a new lncRNA expression induced by hyperglycemia.Its neighbor gene S1PR1 and S1PR1 ’s transcription factor KLF2 can participate in the regulation of HAVEN expression,and participate in the inflammatory response of vascular endothelial cells by regulating the expression of endothelial nitric oxide synthase(eNOS)and vascular cell adhesion molecule 1(VCAM-1).In order to study the specific molecular mechanism,we constructed an in vitro cell model and found that after 24 hours of high glucose stimulation of human aortic endothelial cells(HAEC),the expression of HAVEN was obviously up-regulated,while S1PR1 and KLF2 were downregulated.S1PR1 and KLF2 plasmids were constructed.In the hyperglycemic induced cell damage model,the overexpression of S1PR1 or KLF2 could reverse the expression of VCAM-1 and eNOS in HAEC respectively,and the overexpression of KLF2 could upregulate the expression of S1PR1.Small interfering RNA can be used to knock down the expression of KLF2 and S1PR1 in HAEC,and knocking down KLF2 can not only reduce S1PR1 expression but also downregulate HAVEN.However,short hairpin RNA(short hairpin RNA,shRNA)designed for HAVEN’s three loci can’t knock down HAVEN effectively.Conclusion:Based on the previous sequencing results,we found a new lncRNA(HAVEN),which mediates the inflammatory response of the arterial endothelial cells by interacting with the adjacent gene S1PR1 and its transcription factor KLF2,which provides a new molecular pathway to prevent the development of diabetic vascular diseases.Aim:DNA microarray is one of the most important methods in high-throughput study,compared with the growing number of microarray information,less data is well mined.By using methods of data mining,the transcriptional changes of aorta vessel under the short-time hyperglycemia condition can be directly showed off based on Matlab platform.Methods:We used a gene Fetch a gene expression data set GDS4016 from NCBI,which highlights transient hyperglycemia’s effect on transgenic diabetic aorta,and hybridizes on GPL1261.Data filter,differential gene expression,clustering analysis,gene ontology(GO)annotation,KEGG pathway analysis.Use Mathworks bioinformatics toolbox in Matlab,transfer the data set to a computer-readable structure.Filter out the background noise with the usage of pattern recognition.Get a subset of expression data with apparent changes under hyperglycemia condition.The data mining method includes K-means,principal component analysis(PCA),and self-organizing map(SOM).Based on the platform DAVID,GO annotation and KEGG enrichment analysis is performed.Results:The number of genes under filtering is 686,which can be divided into 9 clusters with two algorithms.These nine clusters indicate nine different expression patterns.One cluster among that has an apparent change immediate after stimuli,while the expression level doesn’t return to the original level long after the stimuli.This seems to explain the glycemic memory,referred as the prolonged harm produced by transient hyperglycemic stimulation.This finding enhances our understandings on vascular homeostasis and remodeling and provides novel avenues for early detection and effective prevention of the vascular diseases.