Related Research On Phenotypic Heterogeneity Of Vascular Smooth Muscle Cells In Atherosclerosis And Its Molecular Regulatory Mechanism

Part 1: Revealing the master regulators of cell clusters of smooth muscle cell lineage in atherosclerosis in mice through single cell RNA sequencingBackground and objective: Atherosclerosis is the main pathological basis of cardiovascular disease,leading to a heavy burden on the family and society.Though the current clinical prevention and treatment strategies can effectively delay the progression of the disease and improve the prognosis,there are still residual risks to be resolved.The recent development of single-cell technology has made it possible for researchers to unbiasedly explore the heterogeneity of cells in atherosclerotic lesions at the single-cell level;gene regulatory network inference algorithms developed based on single-cell data,especially SCENIC,allows us to comprehensively characterize the key regulators of each cell population.Methods: The atherosclerotic model was firstly constructed using Apo E-/-mice and the aortic root to the abdominal aorta was dissected and processed to perform single-cell RNA sequencing using 10 X Genomics technology.To obtain more powerful insights,we expanded the data by retrieving 6 public datasets that met the screening criteria,resulting in more than 100,000 cells included in this analysis.For all cells,the Seurat package was used to cluster all cells and construct cell atlas in the atherosclerotic plaques,and the py SCENIC package was used to identify the master regulators of interested cell clusters derived from the smooth muscle cell lineage.Results: After dimensionality reduction and clustering of all cells,classic marker genes published in the literatures were used to assign biological cell identity to each cluster.Firstly,the cell clusters in normal arterial tissues were annotated,and the following main cell types were obtained: smooth muscle cells,fibroblasts,endothelial cells,macrophages,T cells,and a small number of endothelial cells and neuronal cells.Compared with normal arterial tissue,there were two main characteristics in the cellular composition of atherosclerotic plaques: 1.The proportion of immune cells including macrophages and T cells was increased.2.There existed a substantial cell population that appear only in atherosclerotic lesions;in addition,this cell population were derived from smooth muscle cells as indicated by cell lineage information;that is to say,the disease-associated cells are phenotypically modulated smooth muscle cells.We then divide these cells into three subclusters(namely m SMC1,m SMC2,and m SMC3)according to the similarity of the transcription profiles with classical smooth muscle cells.As the disease progresses,the proportion of m SMC3 gradually increases.Functional analysis showed that m SMC1 tends to have highly proliferative and synthetic properties,while the markers genes of m SMC3 were mainly enriched in multiple calcification-related pathways.Then we selected more than 80,000 cells to perform gene regulatory network inference.Results showed that the highly m SMC1-specific regulons were mainly associated with promoting phenotypic modulation and regulating cell proliferation,such as Nfe2l1,Glis3,Foxq1,E2f3,Stat5 a,Sreb1,etc.Among them,Foxq1 has been proven to directly target and regulate the differentiation genes of VSMCs.The top active regulons of m SMC3 were mainly related to calcification,such as Runx2,Sox9 and Msx1.These regulatory inferences are consistent with the results of functional analysis based on transcriptional profiles.Conclusion: In atherosclerotic lesions,phenotypically modulated smooth muscle cells possess a high functional heterogeneity.Gene regulatory network inference indicates a hierarchical organization of regulatory modules that work together in fine-tuning cellular states.The analysis here provides a valuable resource that can provide guidance for subsequent biological experiments.Part 2: The effect and its molecular mechanism of Twist1 on the phenotypic transformation of vascular smooth muscle cellsBackground and objective: This part of the research aims to evaluate the effect of TWIST1 on the phenotype of vascular smooth muscle and explore the mechanism of its effect on vascular smooth muscle.Methods: Firstly,atherosclerosis factors PDGF-BB and ox-LDL were used to stimulate VSMCs to determine the expression of Twist1 and VSMCs-specific marker genes,and meanwhile a public single cell dataset were used to characterize the expression pattern of Twist1 in atherosclerotic plaques.The sh Twist1-gene knockdown model was constructed using adenovirus was used as a vector to evaluate the effect of Twist1 on vascular smooth muscle cells.Finally,bulk RNA sequencing was performed on PDGF-BBstimulated Twist1-knockdown or non-knockdown VSMCs,so as to explore the underlying molecular mechanisms of Twist1 on the phenotypic modulation of VSMCs.Single-cell data analysis showed that in atherosclerotic tissues,the proportion of Twist1 positive cells was significantly increased;Results of cell experiments displayed that Twist1 expression was significantly increased both at the transcriptome level and protein level under the induction of the PDGF-BB.After knocking down the expression of Twist1 with Twist1-sh RNA,the synthesis and proliferation ability of VSMC cells decreased,and apoptosis potential increased.On the contrary,with downregulated expression of Twist1,the expression of the VSMC contraction markers ACTA2 and MYH11 increased.The above evidence supports that Twist1 expression is positively correlated with VSMC phenotypic changes.RNA sequencing results showed that Twist1 is associated with a variety of biological processes during phenotypic changes of VSMCs,and down-regulation of its expression can lead to up-regulation of the expression of genes related to inflammation and calcification related biological process.Conclusion: The expression level of Twist1 is closely related to the VSMC phenotype,which can promote VSMCs synthesis and proliferation potential and also inhibit VSMCs apoptosis;Furthermore,Twist1-induced VSMCs phenotype possess athero-protective properties.Thus,Twist1 can be a new promising target to promote a modulated VSMCS phenotype which can reduce the risk of CVDs.