SCAP Knockout In SM22α-Cre Mice Induces Defective Angiogenesis In The Placental Labyrinth

Objective:The placenta is a highly specialized and unique organ connecting the fetus to the mother.During pregnancy,it is responsible for the exchange of nutrients and gases between the mother and the embryo in mice.Among the multilayer structures of placenta,the labyrinth is the most important layer,which contains the fetal capillaries and the maternal blood sinuses,and it is the area where nutrients,gases,and waste products are exchanged.Inadequacies in placental development and function may cause pregnancy complications,such as fetal intrauterine growth restriction,preeclampsia and so on,while the development of fetal vascular network in the labyrinth of the placenta greatly affects the function of the placenta.Although the importance of the placenta has been recognized,the molecular mechanisms underlying its development remain unclear.Sterol regulatory element-binding protein（SREBP）cleavage-activator protein（SCAP）is considered to be a cholesterol sensor that regulates lipid metabolism in cells,tissues and organs.Studies have shown that the knockout of SCAP in multiple organs can lead to developmental disorders,while the role of SCAP in the placenta has not been reported.Recently,we intended to generate smooth muscle 22 alpha（SM22α）-Cre recombinase（Cre）-specific SCAP KO mice(SM22α-Cre⁺SCAP^flox/flox mice,hereafter referred to as SCAP KO mice)to study the function of SCAP in vascular smooth muscle cells（VSMCs）during certain pathological processes.However,we unexpectedly found that SCAP KO in SM22α-Cre mice is lethal.The SCAP KO embryos and placentas showed phenotypic differences compared with the wildtype.The objective of this study was to investigate the effect of knockout of SCAP in SM22α⁺cells on placental labyrinth,and to investigate the primary mechanism.Methods:Mice with conditional lox P-flanked SCAP(SCAP^loxp/loxp)alleles and SM22α-Cre mice were used.To gain SM22α-Cre-specific SCAP KO embryos and placentas,the SCAP^loxp/loxp mice were crossed with the SM22α-Cre mice to generate SM22α-Cre⁺SCAP^flox/+mice,which were then crossed with each other.SM22αis expressed in smooth muscle cells（SMCs）and pericytes,while there are no VSMCs within the placental labyrinth.To examine the expression patterns of SCAP in pericytes in the placental labyrinth and to confirm the lack of SCAP expression in pericytes in the KO placental labyrinth,we performed Immunohistochemical（IHC）and Immunofluorescence（IF）analyses.After that,the differences of embryo morphology,placental and yolk sac blood vessels between the knockout group and the control group were observed by stereomicroscope;to further clarify fetal vascular phenotypes in the placental labyrinth,hematoxylin and eosin（H&E）staining,IHC and IF were performed after sectioning.To explore the specific reasons and mechanism for the defects of the placental labyrinth,we analyzed and compared the transcript profile of E12.5 WT and KO placentas using RNA-seq;Ki67 IF analysis and proliferating cell nuclear antigen（PCNA）IHC analysis were used to detect cell proliferation;western blotting was used to detect the expression of vascular endothelial growth factor（VEGF）-A and glial cells missing 1（GCM1）in placenta.Finally,the expression of multiple genes regarding pericyte/endothelial interactions was detected by real-time quantitative PCR（RT-q PCR）.Results:1.The IHC results showed that SCAP is highly expressed in the WT and KO placental labyrinth,including in trophoblasts and endothelial cells.We then further cultured primary pericytes for IF,and found that there was no SCAP expression in pericytes from SCAP KO placental tissues.2.SCAP KO embryos exhibited morphological abnormalities（i.e.,they were curled up）;in these KO embryos,vascular branching in the yolk sac were noticeably reduced compared to in WT embryos;the vascular branching on the surface of KO placentas was disorganized and barely visible,compared to the WT placentas.3.The thickness of SCAP KO placental labyrinth layer was basically the same as in WT mice,but the fetal blood vessels in the labyrinth were fewer and narrower.In addition,in the placental labyrinth of knockout group,the number of nucleated erythrocytes and pericytes was decreased,and the distance of substance exchange was increased.The changes of these indexes indicated lower efficiency of gas transport and the exchange of other substances.4.RNA-seq results showed that multiple GO terms were associated with our phenotypes and suggested abnormal cell proliferation.SCAP KO placentas showed an obvious reduction in the proportions of Ki67⁺cells and PCNA⁺cells,suggesting that missing SCAP in pericytes decreases the cell proliferation in the labyrinth,while there was an aberrant increase in the number of trophoblasts.Western blotting results showed that,although there was no difference in GCM1 expression,VEGFA expression in placenta of knockout group was significantly down-regulated.5.KEGG pathway analysis showed changes in multiple angiogenesis-related pathways,and RT-q PCR results showed down-regulation of multiple pericyte/endothelial interaction genes,including transforming growth factor（TGF）-β1,TGF-β2,angiopoietin-2（Ang2）,and TGF-βreceptor 2（TGF-βR2）.Conclusions:SCAP KO in SM22-positive cells leads to the absence of SCAP in pericytes in placental labyrinth.The loss of SCAP further affects multiple genes regarding pericyte/endothelial interactions and multiple pathways related to angiogenesis,resulting in reduced microvascular density and diameter in placental labyrinth.In addition,there was a decrease in the number of nucleated erythrocytes and an increase in the number of trophoblasts.These results suggested that the efficiency of nutrients and gases exchange between the embryo and the mother was reduced,which may be one of the causes of fetal death.