Effect And Mechanism Of Tmod1 On Smooth Muscle Cell Proliferation And Vasoconstriction

According to the data of the China Cardiovascular Disease Health and Disease Report released by the National Cardiovascular Disease Center in2020,in 2019,cardiovascular diseases in rural areas and urban areas accounted for 46.7%and 44.26%of the causes of death,respectively,and the prevalence rate of cardiovascular diseases showed an increasing trend,and the number of people with cardiovascular diseases was estimated to reach330 million.Cardiovascular disease is a serious threat to human health.Vascular smooth muscle cells（VSMC）are located in the vascular media.Mature VSMC exhibits strong cell-specific functions,such as constricting blood vessels,regulating blood pressure and blood flow distribution.The change of cell proliferation and vasoconstriction ability are two important features in the phenotypic transformation of smooth muscle cells.When smooth muscle cells are affected by external factors or certain factors,the differentiation state is changed to the dedifferentiation state,and the proliferation ability is improved.The abnormal proliferation of vascular smooth muscle cells plays an important role in cardiovascular diseases,and the study of its molecular mechanism is of great clinical significance for understanding and preventing cardiovascular diseases.Vascular smooth muscle cells are located in the vascular media.Mature vascular smooth muscle cells have high cell specificity,mainly showing the functions of constricting blood vessels,regulating blood pressure and blood flow distribution.When smooth muscle cells are affected by the outside world or certain factors,they change from differentiated state to dedifferentiated state,and their proliferation ability is improved,which plays an important role in cardiovascular diseases.Changes in cell proliferation and vasoconstriction are two important features of phenotypic transformation,and the study of their molecular mechanisms is of great significance for understanding and preventing cardiovascular diseases.In this paper,the role and mechanism of Tmod1 in smooth muscle cell proliferation and vasoconstriction will be studied.The cytoskeleton serves as a bridge between the cell and extracellular signaling connections and consists of microfilaments,microtubules and intermediate fibers,which are composed of actin（Actin）.Tropomodulin1（Tmod1）is a cap protein of actin microfilaments（F-actin）,and changes in its expression in cells directly affect the assembly of actin microfilaments and thus the structure of the cytoskeleton.The objective of this study was to investigate the effects of tropomyosin regulatory protein 1（Tmod1）on vascular smooth muscle cell proliferation and vasoconstriction.Objective:To explore the effect of Tmod1 on vascular smooth muscle cell proliferation and vasoconstriction ability.Methods:1.In animal experiments1.1A mouse model of Tmod1 smooth muscle cell specific knockout was constructedTmod1 Flox homozygous mice were mated with SMMHC Cre tool mice,and the obtained mice were induced by Tamoxifen to obtain smooth muscle cell-specific knockout Tmod1 mice,and Tmod1flox/flox mice were used as control group.1.2 Detection of blood pressure in miceTmod1 smooth muscle cell-specific knockout mice and control mice,mouse tail blood pressure was measured for 7 days.In order to adapt to the environment,the first 3 days were not included in the statistics,and the rat tail blood pressure values of 4-7 days were used for statistics.1.3 Detection of vascular stiffness in miceThe pulse wave velocity（PWV）of the common carotid artery and thoracic aorta in vascular smooth muscle cell knockout Tmod1 mice and control mice were measured by ultrasonic photoacoustic imaging analyzer.2.Cell experiment2.1 Extraction of vascular smooth muscle cellsThe Smooth muscle cells in thoracic aorta of SD rats were extracted by enzyme digestion method,and the Smooth muscle 22 alpha（SM22α）,a specific marker of smooth muscle,was identified by immunofluorescence staining of the 4th to 7th generation cells.2.2 Adenovirus vector overexpression of Tmod1 in vascular smooth muscle cellsAdenoviral vectors were constructed for transfection,and the Ad-Tmod1 group was the overexpressed Tmod1 group and the Ad-Null group was the transfected control adenovirus group.Real-time quantitative PCR（RT-q PCR）and protein immunoblotting assay（Western blot）were performed to detect the transfection efficiency and the expression of proliferating nuclear antigen（PCNA）in both groups.2.3 RNA omics sequencing analysisThe cells of Ad-Tmod1 group and Ad-Null group were analyzed by RNA sequencing to explore the expression of differential genes,and the differential genes were verified.2.4 Cell cycle detectionThe cell cycle changes of smooth muscle cells in the overexpressed Tmod1 group and the control group were detected by flow cytometry.2.5 Western blot detection of changes of P53 signaling pathway-related proteins detected by overexpressed Tmod1.Results:1.Animal experiments1.1 Identification of a mouse model of smooth muscle cell specific knockout of Tmod1By gene identification,the Tmod1 F2R2 primer showed bands at the position of 461bp,and the photoadhesive results showed that the Tmod1gene was knocked out.1.2 Blood pressure test results of miceVascular smooth muscle cell-specific knockout Tmod1 mice and Tmod1^Flox/Floxcontrol mice,the blood pressure of vascular smooth muscle cell-specific knockout Tmod1 mice was statistically lower than that of control mice by rat tail blood pressure assay（P<0.01）.1.3 Results of vascular stiffness assay in miceSmooth muscle specific knockout Tmod1 mice and Tmod1f/f control mice,through the measurement of PWV,smooth muscle specific knockout Tmod1 mice PWV was lower than the control group,the difference was statistically significant（P<0.01）.2.Cell experiment2.1 Identification of vascular smooth muscle cellsAfter extracting SD rat vascular smooth muscle cells by enzyme elimination method,immunofluorescence staining was performed to identify SM22αin the cytoplasm of smooth muscle cells with green cytoplasm and blue nucleus,indicating that the extracted cells were smooth muscle cells.2.2 Detection of cell proliferation markers after adenovirus vector overexpression of Tmod1Compared with Ad-Null group,m RNA and protein levels of proliferating cell nuclear antigen（PCNA）in Ad-Tmod1 group were up-regulated,and the differences were statistically significant（P<0.05）.2.3 Cytoomics analysisThe prediction results of the database showed that overexpression of Tmod1 was associated with proliferation genes.2.4 Cell cycle detectionCompared with Ad-Null group,the proportion of cells entering S phase from G1 phase in Ad-Tmod1 group increased,and the differences were statistically significant（P<0.05）.2.5 Western Blot to detect P53 signaling pathway and some proteinsAfter overexpression of Tmod1,the expression of cell cycle checkpoint kinase 2（Chk2）decreased,the phosphorylation of P53 was inhibited,and the expression level of P21 decreased,with statistical significance（P<0.05）.Conclusions:Tmod1 may promote smooth muscle cell proliferation and regulate vasoconstriction by regulating cell cycle and P53 signaling pathway.