Vascular Smooth Muscle Cell-specific Deletion Of SCAP Results In Mouse Embryonic Lethality

Objective: Sterol Regulatory Element Binding Proteins(SREBPs)Cleavage Activating Protein(SCAP),also known as cholesterol sensor,can regulate the expression of the low density lipoprotein receptor(LDLr)and Hydroxy Methylglutaryl Coenzyme A Reductase(HMGCoAR),thus regulating cholesterol synthesis and uptake,thereby maintaining a stable homeostasis of intracellular cholesterol.Recent studies on SCAP mainly focus on its relevance to lipid metabolic diseases and its role in maintaining intracellular cholesterol homeostasis.In recent years,its role of lipid metabolism in cell growth,organ development and embryonic lethality has been paid more and more attention.In this study,by constructing vascular smooth muscle cell(VSMC)-specific SCAP knockout mouse to investigate the effects of SCAP gene deletion on mouse embryos,and to preliminarily discuss its underlying molecular mechanism.Methods: SCAPflox/flox(stock number: 004162)and SM22-Cre(stock Number: 004746)mice were purchased from Jackson Laboratories,USA.Female SCAPflox/flox mice were mated with male SM22-Cre+/-transgenic mice to obtain VSMC-specific SCAP knockout mice.The mouse genomic DNA was extracted and the mouse genotype was identified by PCR amplification.The obtained SM22-Cre/SCAPflox/+transgenic mice were mated between male and female.At embryonic E12.5d,E14.5d,E16.5d,E18.5d,Embryos or mice(amniotic or livers)were examined for genotype identification and the proportion of mice in each genotype was calculated.The morphology of the mouse embryos in homozygotes group(experimental group)and wild type group(control group)were observed and recorded by microscope respectively.The effect of VSMC-specific SCAP gene knockout on the expression of various genes in mice was detected by Genome-Wide Transcriptome Analysis in wild and homozygous E14.5d hearts to preliminarily explore the possible molecular mechanism of SCAP gene deletion effect on embryonic development.Results:1.We successfully constructed VSMC-specific SCAP gene knockout mice(Hereafter it will be abbreviated as SCAP knockout mice),and compared with wild type,the deletion efficiency of SCAP in heterozygous vascular smooth muscle cells is about 50%..However,by mouse genotype identification it was interesting to note that homozygous mice(SM22-Cre/SCAPflox/flox)were not found in all offspring mice,The newborn mice were all heterozygous(SM22-Cre/SCAPflox/+)or other wild-type mice,which suggested that homozygous SCAP gene knockout led to embryonic lethality.2.By the genotype identification of mouse embryos we further found that the ratio of wild type,heterozygous and homozygous was 1: 2: 1 before E14.5d,but from E16.5d later,the proportion of homozygous significantly reduced,and the ratio was reduced to 0 at E18.5d.While dissecting E12.5d,E13.5d,E14.5d,E15.5d embryos,the E14.5d embryos were found to have obvious morphological abnormalities,and the E15.5d embryos were atrophic and necrotic,and there was slight bleeding in the fetus,indicating that the deletion of SCAP mouse embryos began to appear obvious abnormalities from E14.5d,and gradually lead to embryonic lethality.3.RT-PCR results showed that compared with wild type,the deletion efficiency of SCAP in homozygous E14.5d hearts is about 80%.Genome-Wide transcriptome analysis shown as below: in the E14.5d hearts of the VSMC-specific SCAP gene knockout homozygous mice,compared with the wild group,the expression level of SCAP/SREBP2 signaling pathway and glucose metabolism in the experimental group was significantly lower than that in the control group.Lack of lipid and sugar metabolism,resulting in lack of energy metabolism and inadequate supply;PPAR signal pathway-related gene expression levels decreased significantly,leading to placental dysfunction;platelet activity and coagulation dysfunction,leading to embryo bleeding or prolonged clotting time;innate immune system hyperthyroidism,increased inflammation,bacterial virus signal activation,may lead to intrauterine infection,down-regulation of gene expression associated with embryo or cardiac development may lead to heart developmental disorders.All of those can lead to embryonic dysplasia,which can finally lead to embryonic lethality.Conlusions : VSMC-specific SCAP gene knockout can cause homozygous embryonic development disorders leading to embryonic lethality,the mechanism may be related to the lack of SCAP affecting lipid and cholesterol metabolism,PPAR signal pathway,platelet activity and coagulation,inflammatory response,innate immunity,embryo or cardiac development and other comprehensive factors,which may finally cause embryonic lethality.