Objective: Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MB cAbVcam1-5 ) and microbubble with a control nanobody (MB VHH2E7 ) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MB cAbVcam1-5 and MB VHH2E7 . Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×10 5 per microbubble. Compared with MB VHH2E7 , MB cAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm 2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MB cAbVcam1-5 in early ( P =0.0003 versus MB VHH2E7 ) and late atherosclerosis ( P =0.007 versus MB VHH2E7 ); in wild-type mice, there were no differences between MB cAbVcam1-5 and MB VHH2E7 . CEUMI in human endarterectomy specimens showed a 100% increase in signal for MB cAbVcam1-5 versus MB VHH2E7 (20.6±27.7 versus 9.6±14.7, P =0.0156). Conclusions: CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.

中文翻译：

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纳米抗体对动脉粥样硬化的超声分子成像

客观的：VCAM（血管细胞粘附分子）-1 内皮表达的对比增强超声分子成像 (CEUMI) 可以改善动脉粥样硬化的风险分层。为临床前研究开发的微泡造影剂不适合临床转化。我们的目的是使用临床可翻译的单变量域免疫球蛋白（纳米抗体）配体来表征和验证微泡造影剂。 方法和结果：具有靶向 VCAM-1 的纳米抗体的微泡 (MB抗体Vcam1-5）和带有对照纳米体的微泡（MBVHH2E7）在体外制备和表征。使用活化的鼠内皮细胞研究了连续和脉动流下 VCAM-1 的附着效率。动脉粥样硬化双敲除小鼠和野生型小鼠注射MB后进行体内主动脉CEUMI抗体Vcam1-5和MBVHH2E7。人动脉内膜切除术标本的离体 CEUMI 是在闭环循环模型中进行的。纳米抗体配体的表面密度为3.5×105每个微泡。与MB相比VHH2E7,MB抗体Vcam1-5随着剪切应力增加 1-8 达因/厘米，在连续流动下显示出附着力增加2而在脉动流下，附着发生在较高的剪切应力下。双敲除小鼠中的 CEUMI 显示 MB 信号增强抗体Vcam1-5早在（磷=0.0003 与 MBVHH2E7）和晚期动脉粥样硬化（磷=0.007 与 MBVHH2E7）；在野生型小鼠中，MB 之间没有差异抗体Vcam1-5和MBVHH2E7。人动脉内膜切除术标本中的 CEUMI 显示 MB 信号增加 100%抗体Vcam1-5与MB相比VHH2E7（20.6±27.7 与 9.6±14.7，磷=0.0156）。 结论：使用带有 VCAM-1 靶向纳米抗体的临床可翻译微泡，CEUMI 表达 VCAM-1 在动脉粥样硬化小鼠模型和人体组织中是可行的。