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Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism.

ABSTRACT: We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens. Herein we report that VEGF binds to the heparin-II domain of Fn and that the cell-binding and VEGF-binding domains of Fn, when physically linked, are necessary and sufficient to promote VEGF-induced endothelial cell proliferation, migration, and Erk activation. Using recombinant Fn domains, the C-terminal heparin-II domain of Fn (type III repeats 13 to 14) was identified as a key VEGF-binding site. Mutation of the heparin-binding residues on FnIII(13-14) abolished VEGF binding, and peptides corresponding to the heparin-binding sequences in FnIII(13-14) inhibited VEGF binding to Fn. Fn fragments containing both the alpha5beta1 integrin-binding domain (III 9 to 10) and the VEGF-binding domain (III 13 to 14) significantly enhanced VEGF-induced EC migration and proliferation and induced strong phosphorylation of the VEGF receptor and Erk. Neither the cell-binding or VEGF-binding fragment of Fn alone had comparable VEGF-promoting effects. These results suggest that the mechanism of VEGF/Fn synergism is mediated extracellularly by the formation of a novel VEGF/Fn complex requiring both the cell-binding and VEGF-binding domains linked in a single molecular unit. These data also highlight a new function for the Fn C-terminal heparin-binding domain that may have important implications for angiogenesis and tumor growth.

SUBMITTER: Wijelath ES

PROVIDER: S-EPMC3175430 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism.

Wijelath Errol S ES Rahman Salman S Namekata Mayumi M Murray Jacqueline J Nishimura Tomoaki T Mostafavi-Pour Zohreh Z Patel Yatin Y Suda Yasuo Y Humphries Martin J MJ Sobel Michael M

Circulation research 20060928 8

We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens. Herein we report that VEGF binds to the heparin-II domain of Fn and that the cell-binding and VEGF-binding domains of Fn, when physically linked, are necessary and sufficient to promote VEGF-induced ...[more]

PMID: 17008606

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Project description:Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress?LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress.Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness.First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF.Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins ?6?4 and ?1?1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2? expression.LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (?6?4-?1?1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2? expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling.N/A.The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available.These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders.This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Dataset Information

Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism.

Publications

Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism.

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