Mechanism Studies For Homeostatic Regulation Of Human Cardiovascular Cells By ApoE

The ApoE gene,which is located on chromosome 19,is a well-established longevity gene.ApoE is considered as an apolipoprotein,playing a critical role in lipid and lipoprotein metabolism.There are three major human alleles,ApoE2,ApoE3,and ApoE4,encoding three different ApoE isoforms.Among them,ApoE3 is the most prevalent,while ApoE4 is associated with the development of age-related diseases,such as cardiovascular disease,Alzheimer’s disease,and Parkinson’s syndrome.Unlike humans,wild-type mice do not develop spontaneous atherosclerosis during aging,whereas,Apoe knockout mice develop atherosclerosis spontaneously with a shortened lifespan,showing more severe cardiac fibrosis under high-fat diet.Therefore,Apoe knockout mice are considered as a classic model for cardiovascular disease research.Based on this model,mounting studies have shown that ApoE is involved in the development of cardiovascular diseases by regulating lipid metabolism,inflammatory signaling,mitochondrial function,endoplasmic reticulum stress,and other pathways.These rodent-based studies are urgently needed to be extended and validated in human systems.However,although ApoE has been intensively studied in the human neuronal cells,relatively little study has been done in human cardiovascular cells and related stem cells.Currently,cardiovascular disease has become the predominant cause of mortality.Functional alterations in the heart and blood vessels caused by aging are considered as critical factors in the development of cardiovascular disease.However,the mechanism underneath of functional decline in cardiovascular systems is far from being clear,which has become one of the hot topics for biological studies.The artery wall consists of three layers,including the intima(innermost layer consisting of vascular endothelial cells(VEC)),tunica media(middle layer consisting of vascular smooth muscle cells(VSMC)),and tunica adventitia(outermost layer consisting of connected tissue cells,fibroblasts,and mesenchymal progenitor cells(MPC)).In this thesis,we devoted to explore the mechanisms for homeostatic regulation of different cardiovascular cell types by ApoE.We generated ApoE protein-deficient human embryonic stem cells(hESC)and their derived cardiovascular cells,including hMPC,hVEC,hVSMC,and cardiomyocytes(CM),based on CRISPR/Cas9 gene editing technology and in vitro directed differentiation method.Subsequent analysis displayed that the function of ApoE was cell-type specific.Deletion of ApoE did not affect the pluripotency of human embryonic stem cells.However,it retarded MPC senescence,and promoted apoptosis in VEC,VSMC,and CM.We noticed that ApoE protein levels were elevated in senescent hMPC and human fibroblasts.Through a series of hMPC aging models,including replicative senescence,pathological senescence,physiological senescence,and stress-induced senescence models,we found that ApoE proteins bind with nuclear membrane associated protein Emerin,Lamin B receptor(LBR)and heterochromatin-associated KRAB-associated protein-1(KAP1)to form a complex that destroys nuclear membrane proteins and heterochromatin proteins through the autophagy-lysosome pathway.The decreased nuclear membrane proteins and heterochromatin proteins destabilize the heterochromatin,which results in the abnormal expression of silent repetitive sequences and ultimately contributes to accelerated cellular senescence.In contrast,the reduction or elimination of ApoE proteins delayed cellular senescence including human fibroblasts.These results demonstrated that ApoE functions as an epigenetic regulator and therefore mediates cellular senescence in hMPC and human fibroblasts.In contrast,deletion of ApoE protein promotes apoptosis in VEC,VSMC,and CM,accompanied by an increase in the secretory phenotype associated with cellular senescence.With the integrative analysis of transcriptome data generated from different cell types(ESC,VEC,VSMC,MPC,CM),we found that APOE deficiency specifically induced the expression of the long non-coding RNA MEG3 in VEC,VSMC and CM.ChIP-qPCR experiments further demonstrated that ApoE protein binds to the promoter region of the MEG3 gene and possibly serves as a transcriptional repressor to inhibit the expression of the MEG3 gene.In summary,with human stem cell-directed differentiation and gene editing technologies,we have elucidated the functions of ApoE in different human cardiovascular cell types,and revealed novel roles of ApoE proteins as epigenetic or transcriptional regulators.This provides a theoretical basis for subsequent studies to uncover the role and regulatory mechanisms of ApoE in cardiovascular aging and its related diseases.