WNT5A/Ror2 Pathway Contributes To The Regulation Of Cholesterol Homeostasis And Inflammation Response

Objective: To investigate the effect of WNT5 A on cholesterol homeostasis and inflammation response in VSMCs,and elucidate the mechanism of WNT5 A regulation of cholesterol homeostasis and inflammation response in VSMCs.Methods: Collecting the blood of patients with atherosclerosis and volunteers,then determining the content of WNT5 A in blood by Elisa.Establishing atherosclerosis model with apoE knockout mice,and we determine the expression of WNT5 A in the detection of blood vessels by Western blot.Building a stable overexpressing WNT5 A VSMCs cell line(VSMC-WNT5A),VSMC-WNT5 A cells treat with siWNT5 A and siRor2,respectively.Lipid loaded cells model were induced using ox-LDL treatmenting after 48h;employing oil red O,Di I and enzyme fluorescence detected the intracellular cholesterol levels,respectively.In addition,the level of mRNA of inflammation factors was detected by qRT-PCR,and the level of protein of WNT5 A,Ror2 and ABCA1 was detected by Western blot,finally NF-?B nuclear translocation wasdetected by immunofluorescence.CO-IP was used to verify the binding of WNT5 A to Ror2 receptor.LPS treated VSMC and VSMC-WNT5 A cells as positive control reagents,and ?-CD treated VSMC-WNT5 A cells as cholesterol depleting reagents.Lipid loaded cells model were induced using ox-LDL treatmenting after 48h;employing oil red O,Di I and enzyme fluorescence detected the intracellular cholesterol levels,respectively.In addition,the level of mRNA of inflammation factors was detected by qRT-PCR,and the level of protein of WNT5 A,Ror2 and ABCA1 was detected by Western blot,finally NF-?B nuclear translocation was detected by immunofluorescence.Results:1.WNT5 A was highly expressed in the blood vessels of AS patients and in the aortic arteries of AS animal models.2.Overexpression of WNT5 A promoted the accumulation of cholesterol in VSMCs.3.Transient knockdown of WNT5 A inhibited the accumulation of cholesterol in VSMCs.4.Overexpression of WNT5 A inhibited the expression of ABCA1,and transient knockdown of WNT5 A promoted the expression of ABCA1.5.Transient knockdown of ABCA1 could reverse the effect of transient knockdown of WNT5 A on the inhibition of VSMCs cholesterolaccumulation.6.WNT5 A promoted the expression of Ror2 and was able to specifically bind to Ror2.7.Transient knockdown of Ror2 reversed the effect overexpression WNT5 A on the accumulation of VSMCs cholesterol.8.WNT5 A promoted inflammatory response of VSMCs and induced the nuclear translocation of NF-?B.9.Transient knockdown of WNT5 A inhibited the inflammation response of VSMCs and the nuclear translocation of NF-?B.10.Transient knockdown of Ror2 reversed the effect of overexpression WNT5 A on the inflammation response of VSMCs and the nuclear translocation of NF-?B.11.LPS promoted the expression of WNT5 A and Ror2,and cholesterol accumulation and inflammation response in VSMCs.12.?-CD inhibited the inflammation response induced by WNT5 A,but promoted the expression of WNT5 A and Ror2 in VSMCs.Conclusions:1.WNT5 A promotes the accumulation of cholesterol in VSMCs,which may be mediated by inhibiting of ABCA1 expression2.WNT5 A promotes the inflammation response in VSMCs,which may be mediated by promoting NF-?B nuclear translocation3.Ror2 participates in the process which WNT5 A regulation ofVSMCs cholesterol accumulation and inflammation response4.WNT5A/Ror2 signaling may be one of the hubs that mediate VSMCs cholesterol accumulation and inflammatory response.