■垂体腺苷酸环化酶激活多肽(PACAP)作为抗动脉粥样硬化神经肽,在细胞保护中起重要作用,以及炎症过程,和心血管调节。因此,本研究的目的是研究PACAP及其受体VPAC1对不同巨噬细胞(MΦ)亚型炎症过程和脂质稳态的调节作用.
■为了研究体内标准食物(SC)或富含胆固醇的饮食(CED)下PACAP缺乏在动脉粥样硬化发病机理中的作用,将PACAP-/-小鼠与ApoE-/-杂交以产生PACAP-/-/ApoE-/-小鼠。通过定量免疫组织化学分析主动脉弓的管腔狭窄和动脉粥样硬化斑块中的不同MΦ亚型。分离来自30周龄ApoE-/-和PACAP-/-/ApoE-/-小鼠的未分化骨髓来源细胞(BMDC),分为BMDM1-和BMDM2-MΦ,并与氧化低密度脂蛋白(oxLDL)孵育。此外,PMA分化的人THP-1MΦ进一步分化为M1-/M2-MΦ,随后用VPAC1激动剂[(Ala11,22,28)VIP]处理,拮抗剂(PG97-269),和/或oxLDL。通过oxLDL-DyLight™488和Bodipy™493/503分析oxLDL的摄取/积累。通过qRT-PCR分析mRNA表达,蛋白质水平通过蛋白质印迹,和通过ELISA的细胞因子释放。
■体内,SC30周后,PACAP-/-/ApoE-/-小鼠与ApoE-/-小鼠相比,管腔狭窄增加。在CED下PACAP-/-/ApoE-/-小鼠的动脉粥样硬化斑块中,与ApoE-/-小鼠相比,VPAC1,CD86和CD163的免疫反应面积增加。体外,与ApoE-/-BMDM相比,PACAP-/-/ApoE-/-BMDM中的VPAC1蛋白水平升高,导致BMDM1-MΦ中TNF-αmRNA表达增加,而BMDM2-MΦ中TNF-α释放减少。关于脂质稳态,PACAP缺乏减少了BMDM1-/M2-MΦ中的脂滴面积,并伴随着增加的脂肪分化相关蛋白水平。在THP-1M1-/M2-MΦ中,VPAC1拮抗剂增加了oxLDL的摄取,而VPAC1激动剂降低了oxLDL诱导的细胞内甘油三酯含量。
■我们的数据表明,通过VPAC1信号传导的PACAP在动脉粥样硬化斑块的炎症过程和不同MΦ亚型的脂质稳态中起着重要的调节作用,从而影响泡沫细胞的形成。因此,VPAC1激动剂或PACAP可能代表一类新的抗动脉粥样硬化治疗剂。
UNASSIGNED: Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as an anti-atherogenic neuropeptide and plays an important role in cytoprotective, as well as inflammatory processes, and cardiovascular regulation. Therefore, the aim of this study is to investigate the regulatory effects of PACAP and its receptor VPAC1 in relation to inflammatory processes and lipid homeostasis in different macrophage (MΦ) subtypes.
UNASSIGNED: To investigate the role of PACAP deficiency in the pathogenesis of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Lumen stenosis in the aortic arch and different MΦ-subtypes were analyzed in atherosclerotic plaques by quantitative immunohistochemistry. Undifferentiated bone marrow-derived cells (BMDC) from 30-weeks-old ApoE-/- and PACAP-/-/ApoE-/- mice were isolated, differentiated into BMDM1- and BMDM2-MΦ, and incubated with oxidized low-density lipoprotein (
oxLDL). In addition, PMA-differentiated human THP-1 MΦ were further differentiated into M1-/M2-MΦ and subsequently treated with PACAP38, the VPAC1 agonist [(Ala11,22,28)VIP], the antagonist (PG 97-269), and/or
oxLDL. Uptake/accumulation of
oxLDL was analyzed by
oxLDL-DyLight™488 and Bodipy™ 493/503. The mRNA expression was analyzed by qRT-PCR, protein levels by Western blot, and cytokine release by ELISA.
UNASSIGNED: In vivo, after 30 weeks of SC, PACAP-/-/ApoE-/- mice showed increased lumen stenosis compared with ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice under CED, immunoreactive areas of VPAC1, CD86, and CD163 were increased compared with ApoE-/- mice. In vitro, VPAC1 protein levels were increased in PACAP-/-/ApoE-/- BMDM compared with ApoE-/- BMDM, resulting in increased TNF-α mRNA expression in BMDM1-MΦ and decreased TNF-α release in BMDM2-MΦ. Concerning lipid homeostasis, PACAP deficiency decreased the area of lipid droplets in BMDM1-/M2-MΦ with concomitant increasing adipose differentiation-related protein level. In THP-1 M1-/M2-MΦ, the VPAC1 antagonist increased the uptake of
oxLDL, whereas the VPAC1 agonist decreased the
oxLDL-induced intracellular triglyceride content.
UNASSIGNED: Our data suggest that PACAP via VPAC1 signaling plays an important regulatory role in inflammatory processes in atherosclerotic plaques and in lipid homeostasis in different MΦ-subtypes, thereby affecting foam cell formation. Therefore, VPAC1 agonists or PACAP may represent a new class of anti-atherogenic therapeutics.