Peptidic Targeting of Phosphatidylserine for the MRI Detection of Apoptosis in Atherosclerotic Plaques
Peptidic Targeting of Phosphatidylserine for the MRI Detection of Apoptosis in Atherosclerotic Plaques
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DOI：
10.1021/mp900106m
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发表时间：
2009-11-01
影响因子：
4.9
通讯作者：
Muller, Robert N.
中科院分区：
文献类型：
--
作者：
Burtea, Carmen;Laurent, Sophie;Muller, Robert N.
动脉粥样硬化的分子和细胞成像在21世纪初获得了更多的关注，目的是成像斑块病变的体内生物学特性。细胞凋亡似乎是一个有吸引力的目标，以诊断易损动脉粥样硬化斑块血栓形成事件。本研究的目的是通过噬菌体展示筛选凋亡肽结合物，最终目的是通过磁共振成像（MRI）检测动脉粥样硬化斑块中的凋亡细胞。因此，通过噬菌体展示鉴定的磷脂酰丝氨酸特异性肽被用于设计MRI造影剂（CA），该造影剂被评估为凋亡细胞的潜在体内报告细胞。筛选了具有固定化磷脂酰丝氨酸作用的线性6聚随机肽库。分离噬菌体DNA并测序，用酶联免疫吸附法评价多肽对磷脂酰丝氨酸的亲和力。磷脂酰丝氨酸特异性肽及其混乱的同源物被连接到一个连接体上并偶联到dtpa -异硫氰酸酯上。产物经透析和柱层析纯化，并与氯化钆络合。在用凋亡细胞和小鼠肝凋亡模型对其进行评估后，利用磷脂酰丝氨酸靶向CA对ApoE（-/-）转基因小鼠的动脉粥样硬化病变进行成像。用磷脂酰丝氨酸和活性caspase-3免疫染色法检测肝脏和主动脉细胞凋亡。噬菌体基因组测序显示了9种不同的肽。它们与相关蛋白的氨基酸序列的比对揭示了与Ca2+通道的频繁同源性，使人联想到膜联蛋白的功能。与参与细胞凋亡的分子对齐提供了肽选择和效用之间的直接关联。4.7 T时进行的体内MRI研究证明，使用低分子量顺磁剂可以通过MRI诊断凋亡相关病理。新的CA在动脉粥样硬化性疾病和其他细胞凋亡相关病理（如癌症、缺血、慢性炎症、自身免疫性疾病、移植排斥反应、神经退行性疾病和糖尿病）的诊断和治疗监测方面具有真正的潜力。噬菌体展示衍生肽也可以通过模仿磷脂酰丝氨酸内源性配体膜联蛋白V的作用，发挥潜在的抗凝血作用。
Molecular and cellular imaging of atherosclerosis has garnered more interest at the beginning of the 21st century, with aims to image in vivo biological properties of plaque lesions. Apoptosis seems an attractive target for the diagnosis of vulnerable atherosclerotic plaques prone to a thrombotic event. The aim of the present work was to screen for apoptosis peptide binders by phage display with the final purpose to detect apoptotic cells in atherosclerotic plaques by magnetic resonance imaging (MRI). A phosphatidylserine-specific peptide identified by phage display was thus used to design an MRI contrast agent (CA), which was evaluated as a potential in vivo reporter of apoptotic cells. A library of linear 6-mer random peptides was screened in vitro against immobilized phosphatidylserine. Phage DNA was isolated and sequenced, and the affinity of peptides for phosphatidylserine was evaluated by enzyme-linked immunosorbent assay. The phosphatidylserine-specific peptide and its scrambled homologue were attached to a linker and conjugated to DTPA-isothiocyanate. The products were purified by dialysis and by column chromatography and complexed with gadolinium chloride. After their evaluation using apoptotic cells and a mouse model of liver apoptosis, the phosphatidylserine-targeted CA was used to image atherosclerotic lesions on ApoE(-/-) transgenic mice. Apoptotic cells were detected on liver and aorta specimens by the immunostaining of phosphatidylserine and of active caspase-3. Sequencing of the phage genome highlighted nine different peptides. Their alignment with amino acid sequences of relevant proteins revealed a frequent homology with Ca2+ channels, reminiscent of the function of annexins. Alignment with molecules involved in apoptosis provides a direct correlation between peptide selection and utility. The in vivo MRI studies performed at 4.7 T provide proof of concept that apoptosis-related pathologies could be diagnosed by MRI with a low molecular weight paramagnetic agent. The new CA could have real potential in the diagnosis and therapy monitoring of atherosclerotic disease and of other apoptosis-associated pathologies, such as cancer, ischemia, chronic inflammation, autoimmune disorders, transplant rejection, neurodegenerative disorders, and diabetes mellitus. The phage display-derived peptide could also play a potential therapeutic role through anticoagulant activity by mimicking the role of annexin V, the endogenous ligand of phosphatidylserine.