Lee Chunsik, Dixelius Johan, Thulin Asa, Kawamura Harukiyo, Claesson-Welsh Lena, Olsson Anna-Karin
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
Exp Cell Res. 2006 Aug 1;312(13):2547-56. doi: 10.1016/j.yexcr.2006.04.022.
Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.
富含组氨酸的糖蛋白（HRGP）是一种丰富的肝素结合血浆蛋白。我们已经表明，从HRGP富含组氨酸/脯氨酸的中央结构域释放的一个片段在体外可阻断内皮细胞迁移，并在体内抑制小鼠纤维肉瘤的血管形成和生长。最近，发挥抗血管生成作用的最小活性HRGP结构域被缩小到一个由35个氨基酸组成的肽段，即HRGP330，它源自HRGP富含组氨酸/脯氨酸的结构域。通过使用一种代表400种不同信号转导分子的信号转导抗体阵列，我们现在表明，HRGP和合成肽HRGP330可特异性诱导内皮细胞中粘着斑激酶及其下游底物桩蛋白的酪氨酸磷酸化。用HRGP/HRGP330处理内皮细胞会导致肌动蛋白应力纤维的破坏，而用FAK抑制剂格尔德霉素处理细胞可逆转这一过程。此外，HRGP和HRGP330可抑制三维胶原基质中VEGF介导的内皮细胞管状形态发生。相比之下，VEGF诱导的增殖不受HRGP或HRGP330的影响，这表明细胞迁移在管形成过程中起核心作用。总之，我们的数据表明，HRGP以内皮细胞中的粘着斑为靶点，从而破坏细胞骨架组织以及内皮细胞组装成血管结构的能力。
