Hyperhomocystein Induce Expresson Of New Protein Carom In EC And Its Function

Objective:Homocysteine(HHcy)is an independent risk factor for cardiovascular disease(CVD).We have previously demonstrated that homocysteine(Hcy)inhibits cell proliferation,migration,and repair of vascular endothelial cells(EC),but the molecular mechanisms of Hcy induced EC damage are not clear.In this study,we found a novel F-BAR protein Carom that mediates Hcy induced EC migration and angiogenesis inhibition.Methods:1 Treat the human aortic endothelial cells with 50 ?m DL-Hcy for 24 h,using RT-PCR and WB to dectect the expression of Carom in the endothelial cell.2 Exaim plasma Hcy levels in CBS KO mice by high flux liquid phase mass spectrometry,detect Carom expression in mice aorta and lung endothelial cell by Western blot.3 Treat endothelial cells with 50 ?m DL-Hcy for 24 h,examine Hcy,SAM,SAH level in EC.Treat HAEC with Actinnomyocin D and AZC,detect the expression of Carom by RT-PCR and WB.4 Tranfect HAEC with Adv-Carom or Sh-RNA Carom for 72 h,respectively extract total RNA to microarray screening to explore Carom related downstream genes in EC,and extract cell lysate to do cytokine array.5 endothelial cells(HAEC)were given Hcy,Sh-RNA Carom or Adv-Carom transfection,detect Carom and CXCLl0 m RNA levels by RT-PCR.6 endothelial cells(HAEC)first transfected Carom Sh-RNA for 48 h,and then give DL-Hcy 50 ?m treatment,do th cell scratch test,observe the effect of Hcy and Carom on the migration of endothelial cells.In addition,using a-cxcl10(5?g/ml),cell scratch test was carried out to observe the effect of CXCL10 on Carom mediating cell migration.7 Treat the HAEC with 50 ?m DL-Hcy for 24 h,detect the expression of Carom in different cell components(cytoplasm,cell membrane,nuclear and cytoskeletal).Synthesized Carom related NLS peptide,endothelial cells were treated with Hcy 50?m and NLS 40?m for 24 h,detect the effct of Carom NLS peptid on Carom's expression in chromatin binding cell nucleus(CB)by WB.At the same time,the effect of Carom NLS peptide on the expression of CXCLl0 was detected by RT-PCR.8 The inhibition of homocysteine and Carom on endothelial cell angiogenesis wasdetected by tube formation.9 Endothelial cells treated with Hcy or Carom related virus transfection,at the same time add two endocytosis inhibitor Mitmab(40 ?m)and Chroquine(50 ?m),detect the effect of homocysteine on the expression of VEGFR2 by flow cytometry.10 PIP array to detect interaction between Carom and Phosphatidylinositol,using membrane biotinlyation endocytosis assay to examin the endocytosis process of Carom and VEGFR2.Co IP combined with Mass Spectrium to find Carom potential binding partner proteinResults:1 We found that the expression of Carom mRNA in EC with Hcy treatment was higher than control group,the expression of Carom was 1.87(12h),2.72(24h)and 2.45(48h)P<0.05.Compared with the CT,the Carom protein increased to 2.2(P<0.05)by WB.2 Plasma Hcy levels of 12-16 weeks' Tg-h CBS Cbs-/-mice were 50-100 ?m,plasma Hcy levels of Tg-h CBS Cbs+/-and Tg-h CBS Cbs+/+ mice were less than 10 ?m.The expression of Carom in aorta and the lung endothelial cells of Tg-h CBS Cbs-/-was higher compared to CT mice(P<0.05).The expression of Carom in CD31+CD45-cell in lung sigel cell suspentsion is higer in CBS KO mice,P>0.05 3 Hcy intervention can effectively increase the level of SAH of endothelial cells(P<0.05),decreased the proportion of SAM/SAH from 2.2 to 0.5(P<0.05).Actinocyocin D can block Hcy induced Carom's exprssion,with increase of AZC concentration,the expression of Carom increased respectively 1.9 and 2.2(P<0.05).4 Compared with Adv-CT transfection group,Adv-Carom transfection can up-regulate 459 genes and down-regulate 460 genes(P<0.05,FC>1.2),Sh-RNA Carom can up-regulate 653 genes and down-regualte 537 gene(P<0.05,FC>1.2).We found CXCL10 as Carom downstream target gene among 9 Carom related genes(P<0.0.5,FC>2).5 With Hcy treatment for 12 h,24h,48 h,RT-PCR found that Hcy increased Carom and CXCL10 m RNA expression,and the highest expression was in 24 h to 22.1 FC(P<0.05).Adv-Caromd was transfected in EC,m RNA level of CXCL10 also increased to 27.5 FC(P<0.05).Sh-RNA Carom transfection can reverse Hcy-induced CXCL10 expression.6 Compared with control group,endothelial cell migration rate was 50%(P<0.05)with Hcy treatment,Hcy+sh-RNA Carom group was 82%(P<0.05),a-cxcl10(5 ?g/ml human)can reverse Adv-Carom induced cell migration inhibition from 60% to 77.4%(P<0.05).7 Except Carom mostly expressed in themembrane fraction(ME),Carom can also express in the cytoplasm(CE),soluble nuclear(NE),chromatin binding nucleus(CB)and cytoskeleton(PE).Homocysteine treatment can up-regulate Carom's expression in CB and PE respectively,2.2 FC and 1.75 FC compared to control group(P<0,05).The experiment found that Carom NLS peptide can inhibite Carom's expression in CB and block Adv-Carom induced CXCLl0 m RNA level from 27 to 1.57(P<0.05).8 The results showed that Sh-RNA Carom can revers Hcy induced tube formation inhibition from 35% to 80%(P<0.05)in EC,overexpression of Carom can inhibit endotheial cell tube formation down to 38%(P<0.05).9 Compared with Adv-CT group,expression of VEGFR2 was reduced to 28.6%(P<0.05),homocysteine can inhibit expression of VEGFR2 in endothelial cells to 41%(P<0.05),Mitmab can increased VEGFR2 expression compared to Hcy group from 39% to 58%(P<0.05),Chroquine has no effect on the expression of VEGFR2.It suggests that the effect of Carom on endothelial cell angiogenesis may be regulated by VEGFR2,which may be related to cell endocytosis.10 PIP array found that GST-Carom fusion protein likely bind to Phosphatidylinositol,especially bind to Ptdlns(3)P and Ptdlns(3,5)which is the component of endosome membrane.Cell biotinlyation endocytosis assay showed that Hcy can promote the endocytosis of Carom and VEGFR2 from membrane.11 Co IP showed that Carom may not bind to VEGFR2,CASK or MAGI1 under the Hcy stimulation.Over expression Carom using Co IP combind with mass specturim found that 13 poteintial Carom binding partner protein in in the Adv-Carom group.TRIM21 may be target partner which could participate in the Carom mediated endocytosis proess.Conclusion:1 Carom is highly expressed in Hcy treated endothelial cells,aorta and lung EC of HHcy mice,which may be related to the level of hypomethylation.2 Carom can regulate the expression of CXCLl0 and its mechanism may be related to Carom's expression in CB and the trafficking into nuleus via NLS;Hcy can inhibite EC migration via Carom,Carom may inhibite EC migration via CXCLl0.3 Carom can mediate the inhibition of EC angiogenesis via Carom,Hcy and Carom can deacrease the expression of VEGFR2 in endothelial cells and its mechanism may be related to endocytosis of VEGFR2 via Carom.Carom have 13 poteintial binding partner in endothelial cells.