Sulochana Konerirajapuram N, Fan Huapeng, Jois Seetharama, Subramanian Vivekanandan, Sun Feng, Kini R Manjunatha, Ge Ruowen
Department of Biological Sciences, National University of Singapore, Singapore.
J Biol Chem. 2005 Jul 29;280(30):27935-48. doi: 10.1074/jbc.M414320200. Epub 2005 May 27.
Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.
过度血管生成与许多人类疾病相关，抑制血管生成是药物研发的一个重要领域。关于核心蛋白聚糖作为细胞外可溶性因子时是否能发挥血管生成抑制剂的作用，一直存在相互矛盾的报道。在本研究中，我们证明不仅纯化的核心蛋白聚糖，而且核心蛋白聚糖核心蛋白的26个氨基酸的富含亮氨酸重复序列5（LRR5）通过抑制血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子诱导的血管生成，发挥血管生成抑制剂的作用。肽LRR5通过多种机制抑制血管生成，包括抑制VEGF刺激的内皮细胞（EC）迁移、在基质胶上形成管腔、细胞与纤连蛋白的附着，以及诱导EC凋亡，而对其增殖没有显著影响。我们进一步证明LRR5的不同亚区域抑制血管生成的不同方面，其中间区域（LRR5M，12个氨基酸）抑制内皮细胞管腔形成的效力比LRR5高1000倍。虽然C末端区域（LRR5C）强烈抑制VEGF刺激的内皮细胞迁移，但N末端区域（LRR5N）在抑制内皮细胞与纤连蛋白的附着方面与LRR5活性相当。虽然LRR5M和LRR5N都通过半胱天冬酶依赖性途径剂量依赖性地诱导EC凋亡，与LRR5相似，但LRR5C没有这种功能。我们进一步表明，LRR5和LRR5M对管腔形成的抑制与其抑制VEGF诱导的粘着斑激酶磷酸化以及EC中粘着斑和肌动蛋白应力纤维组装的能力有关，而与其干扰内皮细胞与基质附着的能力无关。圆二色性研究表明，LRR5在溶液中经历3(10)螺旋和β-折叠结构之间的相互转换，这一特性可能对其抗血管生成活性很重要。因此，肽LRR5及其衍生物是新型血管生成抑制剂，可作为进一步开发抗血管生成药物的原型。
