Angiogenesis refers to the formation of new blood vessels from preexisting vasculature, and is essential in a series of physiologic processes such as embryonic development, wound healing and female reproductive cycle. Excessive or inefficiency angiogenesis may lead to pathologic response and diseases such as rheumatoid arthritis, diabetic retinopathy as well as tumor growth and metastasis. Rho kinase is the major effector of GTPase Rho and plays an important role in regulation of rearrangement of cytoskeleton underlying several biological processes such as cell proliferation and migration as well as muscle contraction. The over activation of ROCK was found to associate with some cardiovascular diseases and thus the inhibition of ROCK became a potential therapy target of these diseases. Fasudil and Y-26732 are some examples of ROCK inhibitors which had been successfully developed into clinical stages. Computational virtual screening coupled with high-throughput assays are viable approaches to the discovery of ROCK inhibitor candidates. In present study, ten ROCK inhibitor candidates had been identified and further determined for their inhibitory effects on proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as blood vessel formation of zebrafish. Three of the compounds, namely analogs 2, 4 and 7, were found to exert significant anti-angiogenesis effect on intersegmental vessel (ISV) formation of zebrafish. Since angiogenesis involves multiple biological processes such as proliferation, migration, invasion and differentiation of endothelial cell, we further investigated the differential effects of analogs 2, 4 and 7 on proliferation and migration assays. The results revealed that analog 7 exerted higher potency than analogs 2 and 4 on inhibiting HUVECs proliferation. By contrary, only analog 2 but not analog 4 and 7 suppressed HUVECs migration. These results suggest that different mechanisms of actions may be involved in their underlying anti-angiogenesis activities. In short, the present study, for the first time, identified three novel ROCK inhibitors and demonstrated their anti-angiogenic effects on HUVEC and zebrafish. These evidences supporting that these three compounds are potential therapeutic agents for vascular dysfunction with excessive angiogenesis.

血管新生是在已有的血管組織網絡中形成新血管的過程。血管新生涉及胚胎 發育，創傷癒合和女性生理週期等多個生理過程，但過多或不足的血管新生會導 致一些病理變化甚至發展為疾病，例如類風濕關節炎，糖尿病視網膜病變，腫瘤 的形成和轉移。 Rho 激酶是 Rho 激活后主要的效應器，能够調節細胞骨架的重構，與細胞遷 移、黏附、肌細胞收縮等多項生理功能相關。有研究報導在一些心血管疾病中發 現了 Rho 激酶的高度激活，提示了 Rho 激酶的抑制是此類疾病可能的治療途徑。 Y-26732 和法舒地爾是臨床前研究和臨床應用最常用 Rho 激酶抑製劑。計算機擬 合和酶反應檢測在 Rho 激酶抑製劑的篩選中應用廣泛，一些有潛力的 Rho 激酶 抑製劑也相继被发现。 本課題研究篩選了十種化合物的對 Rho 激酶抑製活性，并研究了這十種化合 物對人臍靜脈內皮細胞增值、遷移和對斑馬魚節間血管新生的影響。其中三種化 合物 2, 4 和 7 表現出較強的 Rho 激酶抑製劑活性且具有抑制斑馬魚節間血管的 形成的活性，但是這三種化合物對人臍靜脈內皮細胞的增值和遷移的影響不同。 化合物 7 表現出顯著的抗增殖作用而化合物 2 表現出顯著的抗遷移作用，這提示 了他們可能潛在的不同的作用機理。 綜上所述，此次研究首次鑒定了化合物 2, 4 和 7 的 Rho 激酶抑製劑活性和在 斑馬魚和人臍靜脈內皮細胞的抗血管新生活性，對與以後利用其作為治療血管功 能紊亂類疾病中的血管新生過剩癥狀的提供了依據。 關鍵字： Rho 激酶抑製劑；抗血管新生；斑馬魚；人臍靜脈內皮細胞；細胞遷 移；細胞增殖