Endothelial cell-specific overexpression of developmental endothelial locus-1 does not influence atherosclerosis development in ApoE-/- mice.
Endothelial cell-specific overexpression of developmental endothelial locus-1 does not influence atherosclerosis development in ApoE-/- mice.
复制标题
发育内皮基因座 1 的内皮细胞特异性过度表达不影响 ApoE-/- 小鼠的动脉粥样硬化发展。
DOI：
10.1160/th17-03-0160
复制
发表时间：
2017
影响因子：
6.7
通讯作者：
Chavakis,Triantafyllos
中科院分区：
文献类型：
--
作者：
Subramanian,Pallavi;Prucnal,Marta;Gercken,Bettina;Economopoulou,Matina;Hajishengallis,George;Chavakis,Triantafyllos
动脉粥样硬化的发展是由慢性炎症和炎性细胞和脂质在动脉壁上积聚导致动脉粥样硬化斑块(1-5)而促进的。白细胞亚群，如中性粒细胞、单核细胞和T细胞，在动脉粥样硬化形成的不同阶段渗入血管壁，并在血管病理中起关键作用(1-6)。鉴于白细胞募集对动脉粥样硬化发展的重要性，白细胞募集级联的内源性抑制物作为治疗干预的靶点可能特别令人感兴趣。发育内皮基因座-1(Del-1)是一种由内皮细胞衍生的52 kDa糖蛋白，由三个N端表皮生长因子(EGF)样重复序列和两个盘状蛋白样结构域(7-9)组成。一旦从内皮细胞释放，Del-1可以与内皮细胞表面和/或细胞外基质(9，10)物理结合。我们之前已经证明，DEL-1与β2整合素LFA-1和Mac-1结合，并干扰β2整合素依赖的白细胞募集(11，12)。在牙周炎的动物模型中，Del-1被证明可以抑制中性粒细胞聚集、IL-17依赖的炎症和炎症性骨丢失(14-16)。牙周炎是动脉粥样硬化发展的危险因素(13)。这些先前的研究表明，Del-1的抗炎功能可能代表了一种调节炎症以防止疾病发展的内源性动态平衡机制。动脉粥样硬化斑块进展到晚期的特征是细胞碎片的积累，主要来自于凋亡的富含脂质的巨噬细胞，导致形成无细胞的、促血栓形成的脂质核心(17，18)。氧化低密度脂蛋白(OxLDL)是动脉粥样硬化形成过程中的一个主要致病因素，它可以从这些未被吞噬清除的凋亡巨噬细胞中溢出。有趣的是，Del-1最近被证明与oxLDL结合并抑制oxLDL诱导的促炎基因的表达；此外，Del-1在小鼠体内的全球过度表达减缓了动脉粥样硬化的发展(19)。鉴于血管内皮细胞与动脉粥样硬化之间的密切联系，我们旨在研究内皮特异的Del-1过表达在动脉粥样硬化发生发展中的作用。为此，我们让内皮特异性过表达Del-1(Del-1-Tg)的小鼠参与实验(20)。选择这种方法的另一个理由是，与肺等小血管不同(11)，DEL-1在大动脉(如主动脉)中的表达严重减少(▶图1 A)。因此，通过在内皮细胞中过表达Del-1，我们的目标是赋予大动脉内皮细胞Del-1的抗黏附/抗炎特性。QRT-PCR(RNasy Micro Kit，Qiagen；iScrip cDNA合成试剂盒，Bio-Rad；SSOF-ast EvaGreen SuperMix，Bio-Rad)证实Del-1过表达。实际上，与Del-1-WT小鼠相比，Del-1-Tg小鼠主动脉中的Del-1mRNA增加了20倍(▶图1 A)。计算基于阈值周期(CT)方法(21)，并归一化为β-2微球蛋白RNA。此外，为了在蛋白质水平上证实Del-1的表达，我们对Del-1-TG和Del-1-WT小鼠的主动脉切片进行了Del-1的免疫荧光分析。我们发现大量的Del-1蛋白主要在Del-1-TG小鼠的内膜中表达，而在Del-1-WT小鼠中观察到的Del-1染色要少得多(见Suppl。图1，可在www..血栓形成-在线。Com)。此外，我们还研究了从野生…的血液和骨髓中获得的分选单核细胞在基因水平上的Del-1表达
Atherosclerosis development is promoted by chronic inflammation and the accumulation of inflammatory cells and lipids in the arterial wall leading to an atherosclerotic plaque (1–5). Leukocyte subsets such as neutrophils, monocytes and T cells infiltrate the vessel wall during various stages of atherogenesis and contribute critically to vessel pathology (1–6). Given the importance of leukocyte recruitment for atherosclerosis development, endogenous inhibitors of the leukocyte recruitment cascade could be of particular interest as targets of therapeutic intervention. Developmental endothelial locus-1 (Del-1) is an endothelial cell-derived 52-kDa glycoprotein, which consists of three N-terminal epidermal growth factor (EGF)-like repeats and two discoidin-like domains (7–9). Upon its release from endothelial cells, Del-1 can physically associate with the endothelial cell surface and/or with the extracellular matrix (9, 10). We have previously shown that Del-1 binds to the β2 integrins LFA-1 and Mac-1 and interferes with β2 integrin-dependent leukocyte recruitment (11, 12). In an animal model of periodontitis, a risk factor for atherosclerosis development (13), Del-1 was shown to inhibit neutrophil accumulation, IL-17-dependent inflammation and inflammatory bone loss (14–16). These previous studies suggest that the anti-inflammatory functions of Del-1 may represent an endogenous homeostatic mechanism that regulates inflammation to prevent disease development. Atherosclerotic plaque progression towards advanced stages is characterized by the accumulation of cell debris, derived mostly from apoptotic lipid-laden macrophages, resulting in formation of an acellular, pro-thrombotic, lipid core (17, 18). Oxidized low-density lipoprotein (oxLDL), which may be spilled from such apoptotic macrophages that are not cleared by phagocytosis, represents a major pathogenic trigger in the atherogenic process. Interestingly, Del-1 was recently shown to bind to oxLDL and suppress the oxLDL-induced pro-inflammatory gene expression; moreover, global Del-1 overexpression in mice attenuated atherosclerosis development (19). Due to the strong connection between endothelial cells and atherosclerosis, we aimed to study here the role of endothelialspecific Del-1 overexpression on atherosclerosis development. To this end, we engaged mice with endothelial-specific overexpression of Del-1 (Del-1-Tg)(20). An additional rationale for choosing this approach was that, in contrast to small vessels such as those of the lung (11), Del-1 expression in large arteries, like the aorta, is severely diminished (▶ Figure 1 A). Thus, by overexpressing Del-1 in the endothelium, we aimed to endow the endothelial cells of large arteries with the anti-adhesive/anti-inflammatory properties of Del-1. Del-1 overexpression was confirmed by qRT-PCR (RNeasy Micro kit, Qiagen; iScript cDNA synthesis kit, Bio-Rad; SsoF-ast EvaGreen Supermix, Bio-Rad). Indeed, Del-1 mRNA was increased 20-fold in the aorta of Del-1-Tg as compared to Del-1-WT mice (▶ Figure 1 A). Calculation was based on the threshold cycle (CT) method (21) and normalized to β-2 microglobulin RNA. Moreover, to confirm expression at the protein level, we performed immunofluorescence analysis for Del-1 in aorta sections of Del-1-Tg and Del-1-WT mice. We found substantial Del-1 protein expression predominantly in the intima of Del-1-Tg mice, whereas much less Del-1 staining was observed in the Del-1-WT mice (see Suppl. Figure 1, available online at www. thrombosis-online. com). Additionally, we have studied Del-1 expression at the mRNA level in sorted monocytes obtained from the blood and bone marrow of wild …