High-Throughput Screening Identifies Idarubicin as a Preferential Inhibitor of Smooth Muscle versus Endothelial Cell Proliferation

Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection). We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization.

内膜增生（intimal hyperplasia）是复发性闭塞性血管病，即再狭窄（restenosis）的致病原因。当前用于治疗再狭窄的药物虽可有效抑制平滑肌细胞（smooth muscle cell, SMC）增殖，但同时也会对具有保护作用的管腔内皮细胞（endothelial cell, EC）层的生长造成抑制，进而引发血栓形成（thrombosis）。为筛选出可选择性抑制平滑肌细胞而非内皮细胞增殖的化合物，我们开发了一种基于人体细胞的高通量筛选（high-throughput screening, HTS）体系，并利用该体系对多种化合物集合（美国国立卫生研究院临床化合物库，NIH Clinical Collection）完成了筛选。我们采用人主动脉平滑肌细胞与内皮细胞，在96孔板中构建了自动化、高精度的增殖检测方法。借助该高通量筛选体系，我们对包含447种药物的美国国立卫生研究院临床化合物库进行了筛选。最终我们鉴定出11种可使平滑肌细胞增殖抑制率超过50%的化合物，其中伊达比星（idarubicin）展现出独特的优势：可优先抑制平滑肌细胞增殖，而非内皮细胞。浓度反应分析显示，这种差异化增殖抑制效应在约10 nM至5 µM的浓度区间内最为显著。在大鼠颈动脉损伤模型中开展的伊达比星体内实验结果表明，给药14天后，内膜增生程度降低了80%，管腔尺寸增加了45%，且对再内皮化（re-endothelialization）过程无显著影响。综上，我们成功建立了人体血管细胞增殖的高通量筛选检测方法，并在体外与体内实验中均确认伊达比星可选择性抑制平滑肌细胞而非内皮细胞的增殖。对更大规模、更具多样性的化合物库进行筛选，有望开发出既能抑制内膜增生，又不影响再内皮化的下一代治疗药物。