P2Y12/LRP1 Signaling Pathway Regulates The Progression Of Atherosclerosis And Related Biomarkers Research

Objective:Atherosclerosis（AS）,the formation of fibrofatty lesions in the artery wall,is the pathological basis of most cardiovascular diseases.The previous research of our group found a new target for the diagnosis and treatment of atherosclerosis:P2Y purinoceptor 12（P2Y12）on vascular smooth muscle cells（VSMCs）,and preliminarily confirmed the antiatherosclerotic effect of clopidogrel,a P2Y12 receptor inhibitor,through clinical research.The high expression of P2Y12 receptor in atherosclerotic plaques is the pathological basis for the benefit of P2Y12 receptor inhibitor in the treatment of atherosclerotic plaques,but it is difficult to directly detect the level of P2Y12 receptor in plaques.Therefore,we hope to find serum biomarkers reflecting the level of P2Y12 receptor in atherosclerotic plaques in order to guide clinical medication.Approach:In this study,we included patients in the atorvastatin+clopidogrel group（20 mg ATV+75 mg CDL,po.qd）in the Clopidogrel-enhanced statin-based atherosclerosis regression study（CESAR）study.The maximum carotid plaque thickness（MCPT）was measured by ultrasound before and 6 months after treatment.ELISA was used to detect the content of circulating various proteins that may be related to the expression of P2Y12receptor in plaques.The content of circulating soluble low density lipoprotein receptor associated protein 1（sLRP1）was detected by ELISA and LRP1⁺α-smooth muscle actin⁺（α-SMA⁺）,P2Y12⁺and P2Y12⁺LRP1⁺cells was detected by immunofluorescence assay in plaques from apo E^-/-mice.The protein expression was detected by western blot and cellular immunofluorescence.The m RNA level was detected by real-time quantitative PCR.ELISA was used to detect the content of 3’-5’-cyclic adenosine monophosphate（c AMP）and the activity of protein kinase A（PKA）.Result:We found that patients in clopidogrel regression group had higher levels of sLRP1compared with those in clopidogrel non-regression group.Moreover,clopidogrel was more effective in patients with higher levels of sLRP1 compared with those with lower levels of sLRP1 at reducing the maximum carotid plaque thickness.Furthermore,there were positive correlation between the level of circulating sLRP1 and the number of LRP1⁺α-SMA⁺,P2Y12⁺,or P2Y12⁺LRP1⁺cells in plaques from apo E^-/-mice fed a high-fat diet.In addition,in vitro experiments showed that 5′-α,β-methylene adenosine diphosphate（ADPβs）,agonist of P2Y12 receptor,could promote the expression of LRP1 by inhibiting c AMP/PKA/sterol regulatory element binding transcription factor 2（SREBP2）signaling pathway in VSMCs.Furthermore,activation of the P2Y12 receptor could also promote the cleavage of LRP1 by upregulating the expression of ADAM metallopeptidase domain 12（ADAM12）and membranetype1 matrix metalloproteinase（MT1-MMP）through c AMP/PKA/SREBP2 in VSMCs,so the content of sLRP1 increased in circulation.Conversely,knockout or inhibition of the P2Y12 receptor had the opposite results.In addition,P2Y12 receptor inhibitor CDL could decreased the number of lesional LRP1⁺α-SMA⁺cells and the levels of circulating sLRP1 by activating c AMP/PKA/SREBP2 in apo E^-/-mice fed a high-fat diet.Conclusions:Our study shows that sLRP1 may be a biomarker to reflect the P2Y12 receptor level in atherosclerotic plaques,which provides a basis for clinical screening of patients suitable for the treatment of atherosclerosis with P2Y12 receptor inhibitors.