Abstract：
BACKGROUND: Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis.
RESULTS: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs.
CONCLUSIONS: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.
RESULTS: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs.
CONCLUSIONS: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.
摘要：
背景：控制血管生成被广泛认为是一种治疗策略，但是可靠的控制方法仍在开发中。肌球蛋白轻链2（MLC2）的磷酸化，调节肌动蛋白-肌球蛋白相互作用，对于血管生成过程中血管内皮细胞（ECs）的行为至关重要。MLC2被MLC激酶(MLCK)磷酸化,并被含有催化亚基PP1的MLC磷酸酶(MLCP)去磷酸化。我们研究了MLC2在血管生成的药理学控制中的潜在作用。
结果：我们将转基因斑马鱼Tg（fli1a：Myr-mCherry）ncv1胚胎暴露于化学抑制剂，并观察到血管发育。互变异构霉素对PP1的抑制作用增加了节间血管（ISVs）的长度，而ML7对MLCK的抑制作用降低了它；这些作用不伴有结构发育异常。Y-27632的ROCK抑制也减少了血管长度。人脐静脉内皮细胞(HUVECs)的体外血管生成模型显示,而ML7和Y-27632降低了它。这些作用似乎受细胞形态的调节而不是细胞活力或运动性的影响。肌动蛋白与磷酸化MLC2（pMLC2）共定位在血管样细长形EC中丰富，但在非细长EC中较差。pMLC2与紧密排列的肌动蛋白相关，但不具有松散排列的肌动蛋白。此外，编码MLC2的MYL9基因的敲减会减少总MLC2和pMLC2蛋白并抑制HUVEC中的血管生成。
结论：本研究发现MLC2是血管生成的关键调节因子。MLC2磷酸化可能参与细胞形态发生和细胞伸长的调节。功能相反的抑制剂正或负控制血管生成，可能是通过调节EC形态。这些发现可以为血管生成提供独特的治疗靶标。
结果：我们将转基因斑马鱼Tg（fli1a：Myr-mCherry）ncv1胚胎暴露于化学抑制剂，并观察到血管发育。互变异构霉素对PP1的抑制作用增加了节间血管（ISVs）的长度，而ML7对MLCK的抑制作用降低了它；这些作用不伴有结构发育异常。Y-27632的ROCK抑制也减少了血管长度。人脐静脉内皮细胞(HUVECs)的体外血管生成模型显示,而ML7和Y-27632降低了它。这些作用似乎受细胞形态的调节而不是细胞活力或运动性的影响。肌动蛋白与磷酸化MLC2（pMLC2）共定位在血管样细长形EC中丰富，但在非细长EC中较差。pMLC2与紧密排列的肌动蛋白相关，但不具有松散排列的肌动蛋白。此外，编码MLC2的MYL9基因的敲减会减少总MLC2和pMLC2蛋白并抑制HUVEC中的血管生成。
结论：本研究发现MLC2是血管生成的关键调节因子。MLC2磷酸化可能参与细胞形态发生和细胞伸长的调节。功能相反的抑制剂正或负控制血管生成，可能是通过调节EC形态。这些发现可以为血管生成提供独特的治疗靶标。
