The Role Of Smooth Muscle Cell P2Y₁₂ Receptor During Artherogenesis

Part I The relationship between P2Y₁₂ receptor and the progression of atherosclerosisPurpose P2Y₁₂ is a well-recognized receptor expressed on platelets,and the target of thienopyridine-type anti-platelet drugs.However,recent evidence suggests that P2Y₁₂ expressed in vessel wall plays a role in atherogenesis,but the mechanisms remain elusive.P2Y₁₂ was also found in human carotid plaque and mainly distributed in vascular smooth muscle cells?VSMCs?,which suggests that VSMCs P2Y₁₂ may be associated with the progression of atherosclerosis.In the present study,we further examined the expression of P2Y₁₂ receptor by smooth muscle cells during atherogenesis and explored the potential mechanisms underling oxidized low density lipoprotein?OxLDL?-mediated P2Y₁₂ expression.Method Apolipoprotein E null?ApoE-/-?mice were fed with high-fat diet containing 1.25%cholesterol and 20%fat 4 or 12 weeks,after which aortas were harvested.Whole aorta or aortic root was then performed with oil red O staining or hematoxylin-eosin?H&E?for assessing atherosclerotic burden.Immunofluorescent staining was performed on aortic root sections to analyze the content of plaque.Mouse aortic smooth muscle cells from 6-week-old C57BL/6 mice were obtained by explants culture.Western blots and quantitative real-time polymerase chain reaction?PCR?was conducted to study the expression of P2Y₁₂ in VSMCs.Results Immunofluroscent staining showed that the number of P2Y₁₂-positive VSMCs as well as the percentage of P2Y₁₂-positive VSMCs in total VSMCs of plaque increased in a time-dependent manner and plaque area is positively related to the number of P2Y₁₂-positive VSMCs in plaque?y = 0.0119x + 0.095,R2 = 0.6318,P<0.001?.In cultured mice aortic smooth muscle cells,both Western blots and quantitative real-time.reverse transcription-PCR?qRT-PCR?analysis showed that OxLDL,which is an important pro-atherogenic factor stimulated P2Y₁₂ expression in VSMCs.OxLDL stimulated NF-?B activation in VSMCs and NF-?B inhibitor BAY 11-7082?10 ?M?abolished OxLDL?50?g/ml?-induced P2Y₁₂ up-regulation.Conclusion Smooth muscle cell P2Y₁₂ receptor was highly associated with the progression of atherosclerosis.Part II P2Y₁₂ receptor mediates smooth muscle cell migration and proliferation during atherogenesisPurpose Smooth muscle cells migrate from medium to intimal and play a role in the progression of atherosclerosis.In the present study,we examined whether P2Y₁₂ receptor could mediate smooth muscle cells migration or proliferation and explored the underlying mechanisms of it.Method To study the function of receptor,P2Y₁₂ receptor agonist adenosine diphosphate?ADP?and antagonist 2-methylthioadenosine 5 '-monophosphate?2-MeSAMP?were applied o study the function of receptor.Cell migration was determined by transwell system and scratch assay.Filamentous actin?F-actin?was stained with green fluorescent phalloidin.Protein expression was determined by western blot.Cyclic adenosine monophosphate?cAMP?or protein kinase A?PKA?activity was detected by enzyme linked immunosorbent assay?Elisa?.Mouse aortic smooth muscle cell treated with ADP or ADP(3s or?and?2-MeSAMP for 6 or 72 h and then we performed Ki67?a molecular marker of cell proliferation?immunofluorescent staining to evaluate cell proliferation.Results ADP?10 p,M?stimulation induced significant smooth muscle cell migration and F-actin disassembly,while 2-MeSAMP abolished ADP-induced cell migration and F-actin disassembly.What's more,ADP induced cofilin dephosphorylation was also abolished by 2-MeSAMP.When cofilin was mutated to constitutively inactive phosphorylated form,ADP can not induce significant cell migration as well as F-actin disassembly.As compared to untreated control cells,direct activation of P2Y₁₂ with ADPps?10 uM?for 72 h promoted vascular smooth muscle cell proliferation.Whereas when ADPPs was added in the presence of 2-MeSAMP,the increased effect of cell proliferation was abolished.Conclusion P2Y₁₂ receptor could mediate smooth muscle cell migration and proliferation and may thereby playing a role in the progression of atherosclerosis.Part III P2Y₁₂ receptor inhibitor clopidogrel has an anti-atherosclerotic effectPurpose To exam whether P2Y₁₂ receptor inhibitor clopidogrel has an anti-atherosclerotic effect and examined if clopidogrel could influence VSMCs migration and proliferation during atherogenesis.Method Apolipoprotein E null mice were fed with high-fat diet containing 1.25%cholesterol and 20%fat for 4 or 12 weeks accompanied by either daily clopidogrel?Plavix,20 mg/kg?or saline?control vehicle?by means of intragastric administration,after which aortas were harvested.Whole aortic or aortic root was then performed with oil red O staining or hematoxylin-eosin for for assessing atherosclerotic burden.Immunofluorescent staining was performed on aortic root sections to analyze the content of plaque.Results Oil Red o staining and hematoxylin-eosin staining both showed that four weeks of clopidogrel treatment did not influence lesion size at either whole aorta or aortic arch or aortic root as compared to vehicle.However,twelve weeks of clopidogrel treatment significantly reduced lesion area both at whole aorta?12.53 ± 1.22%vs control 20.19 ±1.79%,P<0.05?and aortic arch?23.07 ± 3.10%vs control 33.05± 2.18%,P<0.05?.Clopidogrel administration significantly increased cAMP level as well as PKA activity and cofilin phosphorylation as compared to vehicle administration.Clopidogrel adminstration also decreased the number of P2Y₁₂-positive smooth muscle cells in plaque.In addition,compared to vehicle,clopidogrel administration decreased Ki67-positive smooth muscle cells in plaque.Conclusion Clopidogrel may inhibit atherosclerosis via inhibit smooth muscle migration and proliferation.