Cyclin G2 Promotes The Formation Of Smooth Muscle Cells Derived Foam Cells In Atherosclerosis By Upregulating LOX-1

Objective:Atherosclerosis（AS）is a chronic progressive arterial vascular obstructive disease.It is the common pathophysiological basis of ischemic heart disease,stroke and peripheral vascular disease.The incidence rate of AS is increasing year by year and showing a younger trend,and it seriously threatens human health.According to the Global Burden of Cardiovascular Diseases and Risk Factors 1990-2019,the prevalence and mortality of cardiovascular diseases worldwide have increased significantly since 1990,with China having the highest mortality rate from cardiovascular diseases.The formation of foam cells and its massive accumulation under the intima to form lipid plaques are important pathological events in the development of AS.Recent studies have found that40-70%of foam cells are derived from vascular smooth muscle cells.Vascular smooth muscle cells pass through scavenger receptors on the cell membrane to excess endocytosis of lipids to form foam cells.LOX-1 is the main scavenger receptor.Cyclin G2 is a non-specific cyclin which is encoded by the CCNG2 gene that promotes the differentiation and maturation of preadipocytes and suppresses osteoporosis.PP2A is an important serine threonine phosphatase in cells and is involved in regulating various signaling pathways.Studies have shown that cyclin G2 can bind PP2A to affect its phosphatase activity,NF-κB is a transcription regulator of LOX-1,and its phosphorylation activates translocation into the nucleus and up-regulates the transcription of LOX-1.Based on the above research,we speculated that cyclin G2 may affect the activity of NF-κB by binding to PP2A and regulate the transcription of LOX-1 to affect foam cell formation and the development of atherosclerosis.In this study,we used Ccng2 knockout mice which is constructed in the previous period to observe the relationship between cyclin G2 and atherosclerosis in vivo,and further study the effects and molecular mechanisms of cyclin G2 on the formation of smooth muscle-derived foam cells were observed in vitro and in vivo and provided a new target for atherosclerosis-related diseases.Methods:1.Effect of Ccng2^-/-on atherosclerosis in mice:construction of Apoe^-/-Ccng2^-/-mice,identification of the mouse genotypes,the experimental mice were divided into two groups:Apoe^-/-mice and Apoe^-/-Ccng2^-/-mice,10 mice in each group.Both groups were selected from male mice.Two groups of mice were given a high-fat diet（Western diet）at8 weeks of age.After 8 weeks,we measured the weight of the two groups of mice,collected blood and aortic vessel of the mice,measured the lipid indicators of the mice,observed the aortic atherosclerotic plaques by HE staining and oil red O staining and used immunohistochemistry to detect the expression ofα-SMA and CD68 in the aortic roots of the mice.2.Cyclin G2 affected NF-κB activity,regulates LOX-1 expression,and promotes the formation of smooth muscle-derived foam cells:In vivo experiments we detected the expression of p-NF-κB and LOX-1 in the aortic roots of two groups of mice by immunohistochemistry;In vitro experiments,we applied MOVAS,a mouse aortic root smooth muscle cell line,to give cells 80μg/ml ox LDL to induce foam cell formation and identify by oil red O staining;Through liposome transfection,the cells were divided into overexpression group（FLAG-Ccng2）and its control group（FLAG-Vector）,and the total cholesterol content in the two groups was detected;Oil red O staining was used to observe the formation of foam cells;Observation the uptake of Dil-ox LDL on vascular smooth muscle cells by immunofluorescence;q RT-PCR and Western blot were used to detect the expression of cyclin G2 and scavenger receptor LOX-1 at the m RNA and protein levels in two groups of cells;immunofluorescence and Western blot were used to detect the expression of nuclear p-NF-κB;3.Cyclin G2 increases the expression of LOX-1 in vascular smooth muscle cells by activating the NF-κB signaling pathway through PP2A:administration of PP2A agonists to interfere with cells in the overexpression group and its control group;detection of total intracellular cholesterol content;oil red O staining and Dil-ox LDL Uptake experiments were performed to observe foam cell formation and lipid uptake;q RT-PCR,Western blot,and immunofluorescence were used to observe the expression of related factors.Results:1.The degree of atherosclerosis in Ccng2^-/-mice was reduced:Apoe^-/-Ccng2^-/-mice were less weight compared with Apoe^-/-mice,total cholesterol and LDL-c were relatively lower,triglycerides were not changed.Oil red O staining of aortic sections and aorta in mice showed that the lipid plaques of aortic arteries and the foam cells of aortic root were reduced in Apoe^-/-Ccng2^-/-mice.HE staining showed that the plaque area of aortal root of Apoe^-/-Ccng2^-/-mice was smaller than that of Apoe^-/-mice.Further immunohistochemistry was used to detect the expression of smooth cell markerα-SMA and macrophage marker CD68.The results showed that there was no significant difference in the expression of CD68 between the two groups and the expression ofα-SMA was higher than that of CD68.The expression ofα-SMA in Apoe^-/-Ccng2^-/-mice was higher than that in the control group.Ccng2 knockoutmainly reduced smooth muscle derived foam cells.2.Cyclin G2 affects the activity of NF-κB and regulates the expression of LOX-1 and promotes the formation of smooth muscle derived foam cells.The expression of p-NF-κB and LOX-1 in the aortic root of mice was detected by immunohistochemistry.The results showed that the positive expression of Apoe^-/-Ccng2^-/-mice was reduced compared with that of the control group.Further,the mechanism of cyclin G2 affecting the mechanism of Apoe^-/-Ccng2^-/-was further studied in vitro.Foam cells were successfully modeled by ox LDL at 80μg/ml.FLAG-Ccng2 group had more lipid droplets compared with FLAG-Vector group by Oil red O staining,it was observed by immunofluorescence that more Dil-ox LDL were internalizated in FLAG-Ccng2 group.q RT-PCR and Western blot test results showed that the m RNA and protein expression of scavenger receptor LOX-1 of smooth muscle-derived foam cells were increased in the FLAG-Ccng2 group,The expression of p-NF-κB protein in the nucleus of the FLAG-Ccng2 group was increased.In addition,immunofluorescence showed that p-NF-κB of nucleus increased in FLAG-Ccng2 group.3.Cyclin G2 increased the expression of LOX-1 in vascular smooth muscle cells through activation of p-NF-κB signaling pathway through PP2A.PP2A activity decreased in FLAG-Ccng2 group smooth muscle foam cells compared with control group,and PP2A activity agonist DT-061 increased the activity of PP2A in both groups,and decreased the uptake of lipids in the cells of FLAG-Ccng2 group.There was no significant difference between control group with DT-061.Conclusion:The deletion of Ccng2 reduces the degree of atherosclerosis in mice.Cyclin G2 promotes the formation of vascular smooth source foam cells in vitro;Cyclin G2increases the phosphorylation level of NF-κB by reducing the phosphatase effect of PP2A and promotes its entry into the nucleus to play a role in up-regulating LOX-1 expression.