Establishment Of Vascular Smooth Muscle Cell-Specific Scap Transgenic Mice Model And Phenotype Identification

Objective:Atherosclerosis (atherosclerosis, AS) has become one of the major cardiovascular disease and threats to human health. Dyslipidemia, particularly abnormally high plasma cholesterol levels, has been widely recognized as an important risk factor for atherosclerosis. Sterol regulatory element-binding protein cleavage-activating protein, also known as SREBP cleavage-activating protein (SCAP) is a protein that in humans is encoded by the SCAP gene. SCAP can cleave and activate SREBPs to promote the expression of target gene LDLr and HMGCoAR, which were involved in regulation of lipid homeostasis (synthesis/uptake of cholesterol, fatty acids, triglycerides and phospholipids). This study developed vascular smooth muscle specific SCAP transgenic mice, and investigate the effect of overexpression of vascular SCAP on vascular plaque formation and cellular cholesterol metabolism.Methods:The expression eukaryotic expressive vector (pGL3-SM22-SCAP) was constructing with a smooth muscle specific promoter SM22 and SREBP cleavage-activating protein (SCAP) mutant (D443N). Eight vascular smooth muscle specific SCAP transgenic mice (FOUNDER:numbered 1-8) were generated by microinjecting the vector into 1.5 days follicles and the genotype was identified by PCR. Founder mice were bred with non-transgenic C57 mouse, whose offspring were conducted primary screening by PCR to identify the genotype. The positive progeny were bred with non-transgenic C57 mouse for five generations to purify the genetic background. Primary screening positive progeny of the five groups (1,2,5,6,7, number 3/4/8 FOUNDER had been dead) were conducted Southern Blot to eliminate false positives. The most significant SCAP expression change occurred in the progeny of group 2, which was chosen for the next further study. Immunohistochemistry was conducted to identify the expression of SCAP, SREBP2, LDLr and HMGCoAR in the mice aortic root. The expression of SCAP regulation related gene and protein (SREBP2, LDLr and HMGCoAR) of the mice aortic root were detected by RT-PCR.12 of SCAPTg/+(positive) and 12 SCAP+/+(negative) femle mice about 6-8 weeks from the progeny of group 2 were both given high cholesterol diet for 48 weeks. Intracellular lipid accumulation of the aortic root was measured using Oil-Red O.Results:Eight FOUNDER transgenic mice was established by prokaryotic microinjection of the SCAP(D443N) eukaryotic expressive vector (pGL3-SM22-SCAP). Immunohistochemistry indicated the expression of SCAP, SREBP2, LDLr and HMGCoAR in SCAPTg/+ transgenic mice was more significant. RT-PCR also confirmed that the expression of SREBPs, LDLr and HMGCoAR increased significantly compared with the control group. Founder mice were bred with non-transgenic C57 mouse at least 5generation. The line of transgenic mice who expresses SCAP the most was chosen for further study. After high cholesterol diet for 48 weeks, intracellular lipid accumulation of the SCAPTg/+ transgenic mice was more obvious and statistically significant comparing with SCAP+/+ transgenic mice.Conlusions:Overexpressing of vascular SCAP upregulates the expression of SREBPs, LDLr and HMGCoAR. In the condition of high cholesterol diet, blood vessel overexpressed SCAP siginificantly, which thereby promot the accumulation of lipid.