The Effect Of Neuropeptide Y On Atherosclerosis And Intimal Hyperplasia And Its Mechanism

Background and purposeCardiovascular disease is the leading cause of death,as the origin of many major cardiovascular diseases,further research are needed to understand the mechanism of atherosclerosis.Arteries predisposed to develop atherosclerosis are rich in innervation,and recent studies have found that some active components from nerves can directly affect atherosclerosis.NPY is a neurotransmitter containing 36-amino acid.In the peripheral nervous systems,it is expressed in sympathetic ganglia,and is stored and released together with norepinephrine during sympathetic nerve stimulation.Clinical studies have found that NPY gene polymorphism is significantly related to atherosclerosis,suggesting that NPY may play a role in atherosclerosis.However,conflicting findings have been shown in few existing animal studies,the role of NPY in atherosclerosis remains controversial,and its related mechanisms are still unclear.Therefore,in the first part of this study,we used NPY^-/-mice to study the effect of NPY deficiency on atherosclerosis,and performed proteomic analysis to explore the possible mechanisms of NPY in atherosclerosis.As the main effector cell in plaque,macrophage autophagy has multiple regulatory effects on plaque formation.In the second part of the study,we analysed the effects of NPY on macrophage autophagy in vitro to explored the cellular mechanism of NPY in atherosclerosis.Our group previously found an arcuate nucleus（Arc）-NPY-regulated neuronal circuit that controls sympathetic output and BAT thermogenesis.Therefore,in the third part we utilized Cre-loxp system to specifically regulate Arc NPY expression in transgenic mice,study the role of Arc NPY in atherosclerosis,and explore its central regulatory effect on atherosclerosis.In the fourth part,we further studied the effect of NPY deficiency on intimal hyperplasia（atherosclerotic complications）caused by arterial injury and its mechanism.MethodsPart I:For carotid atherosclerosis induction,NPY^-/-mice and wild-type control mice were given perivascular carotid collar placement combine a high-fat and high-cholesterol diet.Twelve weeks after collar placement and a high-fat and high-cholesterol diet feeding,mice were euthanized and dissected to obtain collared carotid artery.The following experiments were performed:1.Continuous frozen sections and HE staining of carotid artery near the proximal edge of the carotid collar were used to evaluate the formation of plaque;2.Proteomics was performed to detect protein expression at the collared segment of the carotid arteries in both groups of mice,in order to screen for differentially expressed proteins and conduct bioinformatics analysis;detection of m RNA expression levels was also performed for certain differentially expressed proteins;3.Immunohistochemical staining was conducted to determine the content of macrophages in the plaques;4.Real-time quantitative PCR was performed to detect inflammatory factors in diseased carotid arteries,and m RNA expression of autophagy markers;5.Western blotting was used to detect autophagy activation in diseased carotid arteries.Part II:NPY intervention was administered to the in vitro-cultured RAW264.7 murine macrophage cell line to detect changes in cellular autophagy.1.RAW264.7 cells were treated with rapamycin to activate autophagy and incubated with NPY,followed by detection of cellular autophagy markers by Western blotting and immunofluorescence staining;2.RAW264.7 cells in normal culture were treated with NPY for 6 h and Western blotting was performed to detect cellular autophagy levels;3.RAW264.7 cells were treated with graded doses of NPY for 24 h and cell viability was determined using the CCK8 method;4.Real-time quantitative PCR was performed to detect the m RNA expression of cellular autophagy-related molecules after NPY action on RAW264.7 cells.Part III:Adeno-associated virus carrying the Cre gene was first injected into the arcuate or paraventricular nucleus of transgenic mice via brain stereotactic injection,in order to induce the recovery of NPY expression in the arcuate nucleus of NPY^-/-mice and Y1 receptor knockout in the paraventricular nucleus of Y1R^flox/floxmice.Central nervous system（CNS）intervention was followed by the administration of a high-fat,high-cholesterol diet and perivascular carotid collar placement to induce carotid atherosclerosis in mice.At 12 weeks after carotid collar placement,collared segments of the carotid arteries were removed for the following experiments:1.The effect of CNS intervention on carotid atherosclerosis in mice was evaluated using serial frozen sections and HE staining;2.Real-time quantitative PCR was performed to detect the m RNA expression of inflammatory factors in diseased carotid arteries.Part IV:Intimal hyperplasia of the carotid artery was induced via focal injury with ferric chloride in NPY^-/-mice and wild-type control mice.At 3 weeks after injury,the injured segment of the carotid artery and the contralateral uninjured carotid artery were sampled for the following experiments:1.Serial frozen sections and HE staining were performed to evaluate intimal hyperplasia after carotid artery injury;2.Immunohistochemical staining was performed to determine the NPY expression,macrophage content and smooth muscle cell content of the hyperplastic intima;3.Real-time quantitative PCR was performed to detect the m RNA expression of inflammatory mediators in injured segments of the carotid artery.ResultsPart I:1.Compared with WT control mice,NPY^-/-mice had a smaller carotid artery plaque area[（1.725±0.264）×10⁴μm²in WT mice versus（0.891±0.086）×10⁴μm² in NPY^-/-mice,p<0.05]and a lower plaque/media ratio(0.701±0.122 in WT mice versus 0.355±0.038 in NPY^-/-mice,p<0.05).2.Based on the proteomic analysis of the collared carotid artery segments of WT and NPY^-/-mice,1641 proteins were identified,including 71 differentially expressed proteins.3.NPY deficiency led to a decrease in the content of macrophages in the plaque.4.NPY deficiency inhibited the m RNA expressions of the inflammatory factor IL-6and the adhesion molecule ICAM-1,VCAM-1in the diseased carotid arteries of mice.5.Compared with WT mice,the expression of autophagy marker LC3Ⅱin the carotid artery of NPY^-/-mice was up-regulated.Part II:1.Rapamycin upregulated the expression of LC3Ⅱin RAW264.7 cells,and 10^-6 M NPY can significantly inhibited the expression of LC3Ⅱin RAW264.7 cells induced by rapamycin.2.After 6 hours of NPY incubation,the basal state macrophages LC3Ⅱdid not change.3.NPY downregulated the expression of Atg5 m RNA,an autophagy-related molecule in macrophages.Part III:1.Compared with the control group NPY^-/-mice without CNS intervention,the NPY^-/-mice whose arcuate nucleus NPY expression restored had a smaller carotid artery plaque area and a lower plaque/media ratio.2.The expression of IL-1β,ICAM-1 and VCAM-1 m RNA in mice with restored NPY expression decreased,which is the inflammatory mediator of the diseased carotid artery.3.Compared with Y1R^flox/flox control mice without CNS injection,Y1R^flox/floxmice with Y1 receptor knockout in the paraventricular nucleus increased the carotid plaque area[（2.103±0.179）×10⁴μm²in Y1R^flox/flox mice versus（4.315±0.528）×10⁴μm²in Y1R^flox/flox+PVN cre mice,p<0.05]and had a higher plaque/media ratio(0.830±0.062 in Y1R^flox/flox mice versus 1.647±0.173 in Y1R^flox/flox+PVN cre mice,p<0.05).Part IV:1.Compared with wild-type mice,the area of neointima in NPY^-/-mice is significantly smaller[（2.562±0.241）×10⁴μm²in WT mice versus（1.145±0.156）×10⁴μm²in NPY^-/-mice,p<0.05]after ferric chloride injury,and the ratio of intima/media is significantly reduced(1.142±0.091 in WT mice versus 0.559±0.067 in NPY^-/-mice,p<0.05).2.NPY-positive areas were observed in the hyperplastic intima,media and perivascular tissues of the left injured carotid artery in WT mice.3.Lower macrophage content was observed in the hyperplastic intima of injured carotid arteries in NPY^-/-mice.4.Compared with wild-type mice,NPY^-/-mice have reduced carotid inflammatory mediators（IL-6,TGF-β1）and adhesion molecule（ICAM-1）m RNA expression levels after injury.Conclusions1.NPY deficiency can inhibits the formation of carotid atherosclerotic plaques in mice.This inhibition is due to the down-regulation of inflammation and the up-regulation of autophagy in the diseased blood vessels.2.NPY can inhibit the autophagy of macrophages induced by rapamycin,but does not affect the autophagy of macrophages under the basal state.3.CNS intervention in the arcuate nucleus NPY can inhibit carotid atherosclerosis in mice,and this inhibition may also be mediated by the Y1 receptor in the paraventricular nucleus.4.NPY deficiency inhibits intimal hyperplasia after arterial injury by suppressing the local inflammatory response.