Atherogenesis Research Group, Department of Cardiology, University of Freiburg, Germany.
Circ Res. 2011 Nov 11;109(11):1269-79. doi: 10.1161/CIRCRESAHA.111.247684. Epub 2011 Oct 13.
CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor.
Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo.
CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo.
We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.
CD40L 在动脉粥样硬化等慢性炎症性疾病中起着重要作用。然而，由于 CD40L 通过与 CD40 受体和血小板整合素 GPIIb/IIIa 的相互作用，强有力地调节免疫功能和止血作用，其全球抑制作用会损害宿主防御，并在临床试验中产生血栓栓塞并发症。我们最近报道 CD40L 通过独立于 CD40 的途径介导动脉粥样硬化形成，并提出 Mac-1 是替代受体。
本研究旨在从分子水平上阐明 CD40L-Mac-1 相互作用，并检测其通过小肽选择性抑制来调节体内炎症和动脉粥样硬化形成的作用。
CD40L 在固相结合测定中浓度依赖性地与 Mac-1 I 结构域结合，表面等离子体共振分析显示高亲和力相互作用。我们确定 Mac-1 上的 EQLKKSKTL 基序（α1 螺旋和β-sheet B 之间的暴露环）是 CD40L 的结合位点。模拟该序列的线性肽 M7 特异性抑制 CD40L 和 Mac-1 的相互作用。为体内应用优化的环化版本 cM7，减少了体内的腹膜炎症和炎症细胞募集。最后，用腹腔注射 cM7 治疗的 LDLr(-/-) 小鼠形成更小、炎症反应较轻的动脉粥样硬化病变，具有稳定性特征。然而，cM7 不干扰体外 CD40L-CD40 结合和体内 CD40L-GPIIb/IIIa 介导的血栓形成。
我们提出了新的发现，即 CD40L 结合到 Mac-1 上的 EQLKKSKTL 基序，介导白细胞募集和动脉粥样硬化形成。CD40L-Mac-1 结合的特异性抑制可能是动脉粥样硬化和其他炎症性疾病的一种有吸引力的抗炎治疗策略，可能避免 CD40L 全球抑制的不必要的免疫和血栓形成作用。
