Maafi Foued, Li Baoqiang, Gebhard Catherine, Brodeur Mathieu R, Nachar Walid, Villeneuve Louis, Lesage Frédéric, Rhainds David, Rhéaume Eric, Tardif Jean-Claude
Montreal Heart Institute and Université de Montréal, Quebec, Canada.
Montreal Heart Institute and Université de Montréal, Quebec, Canada; Institute of Biomedical Engineering, École Polytechnique de Montréal, Montreal, Quebec, Canada.
Atherosclerosis. 2017 Mar;258:8-19. doi: 10.1016/j.atherosclerosis.2017.01.026. Epub 2017 Jan 21.
The potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities.
Fluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05).
We detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (-54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr atherosclerotic mice (ATX) as compared to wild-type mice. In vivo observations were confirmed by ex vivo aorta imaging quantification where a 10-fold increase in fluorescent signal in ATX mice (p ≤ 0.05 vs. control mice) was observed.
The use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.
高密度脂蛋白（HDL）对动脉粥样硬化的潜在益处归因于其主要蛋白质成分载脂蛋白A-I（apoA-I）。血管壁中的大多数apoA-I似乎处于脂质缺乏形式。然而，后者会被定位在动脉粥样硬化斑块中的蛋白酶降解，这反过来已被证明会对其抗动脉粥样硬化功能产生负面影响。在此，我们报告了一种可生物激活的近红外全长apoA-I-Cy5.5荧光探针的研发及其在体内的应用，用于评估apoA-I降解性蛋白水解活性。
通过apoA-I蛋白上的Cy5.5荧光团分子饱和实现荧光猝灭。apoA-I的裂解导致近红外荧光增强。包括丝氨酸蛋白酶、半胱氨酸蛋白酶和金属蛋白酶在内的多种蛋白酶对apoA-I探针进行体外蛋白水解，导致荧光增加高达11倍（n = 5，p≤0.05）。
我们使用原位酶谱法在动脉粥样硬化小鼠主动脉切片中检测到探针的激活，并表明广谱蛋白酶抑制剂可保护探针不被降解，从而导致荧光降低（-54%，每组n = 6，p≤0.0001）。在体内，与野生型小鼠相比，注射的探针在人apoB转基因Ldlr动脉粥样硬化小鼠（ATX）的主动脉中显示出更强的荧光发射。通过离体主动脉成像定量证实了体内观察结果，其中在ATX小鼠中观察到荧光信号增加了10倍（与对照小鼠相比，p≤0.05）。
在不同应用中使用该探针可能有助于评估动脉粥样硬化的新分子机制，并可能通过增强apoA-I功能来改善当前基于HDL的治疗方法。
