College of Health and Life Sciences, Hamad Bin Khalifa University, Doha P.O. Box 34110, Qatar.
Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, UK.
Int J Mol Sci. 2023 Sep 22;24(19):14410. doi: 10.3390/ijms241914410.
The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.
微血管网络形成的损伤可能会延迟急性肢体缺血后血流的恢复。GSK 发布的激酶抑制剂文库的高通量筛选鉴定了一组 ROCK 抑制剂，这些抑制剂可促进内皮网络的形成。随后对 224 种蛋白激酶的激酶活性进行分析，结果表明两种基于吲唑的 ROCK 抑制剂对 ROCK1 和 ROCK2 同工型的选择性明显高于其他 ROCK 抑制剂。其中一种化学实体 GSK429286 被选中进行后续研究。我们发现 GSK429286 增强内皮管形成的效力比经典 ROCK 抑制剂法舒地尔高十倍。通过 RNAi 抑制 ROCK1 可模拟 GSK429286 化合物的血管生成表型。使用器官样血管生成共培养测定法，我们表明 GSK429286 可形成密集的血管网络，内皮管更厚。接下来，在诱导后肢缺血后，小鼠接受载体或 GSK429286（10mg/kg i.p.）治疗 7 天。如激光散斑对比成像评估所示，GSK429286 增强了缺血诱导后的血流恢复。在组织学水平上，我们发现与载体处理组相比，GSK429286 可显著增加缺血肌肉再生区新生微血管的大小。我们的研究结果表明，选择性 ROCK 抑制剂具有体外促血管生成特性和恢复肢体缺血血流的治疗潜力。
