Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity

Abstract

Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A165 and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A165 binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A165 showed that heparin can also modulate the conformation and secondary structure of the protein.

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

“…We also examined lung ECs derived from mice lacking Ndst2, the other major NDST isozyme expressed in ECs. Ndst1 f/f Ndst2 -/-ECs express normal HS, whereas Ndst1 -/-Ndst2 -/-(Ndst1/2 -/-) ECs express nonsulfated HS (8,22). Ndst1 f/f Ndst2 -/-ECs, but not Ndst1/2 -/-cells, responded strongly to SLIT3-induced cell migration (Supplemental Figure 4C).…”

Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

“…The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Georgia. Mouse lung endothelial cell (MLEC) lines were derived from 8-weekold Ext1f/f mice as described previously (12,25). Briefly, the lungs were minced, digested at 37°C for 1 h in Delbecco's modified Eagles medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and various enzymes including collagenase Type II (200 U/ml, Invitrogen, 1 The abbreviations used are: BCA, bicinchoninic acid; DTT, dithiothreitol; DMEM, Dulbecco's Modified Eagle Medium; Ext, Exostosin; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FDR, false discovery rate; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; Glc, glucuronic acid; GlcNAc, N-acetylglucosamine; GO, gene ontology; HGF, hepatocyte growth factor; HS, heparan sulfate; IGF-1, insulin-like growth factor 1; IGF-1R, insulin growth factor-1 receptor; IGFBP, insulin-like growth factor binding protein; LC, liquid chromatography; MLEC, mouse lung endothelia cells; Ndst, N-deacetylase/N-sulfotransferase; PDGF, platelet-derived growth factor; PE-cy5, R-Phycoerythrin-Cyanine dye 5; RTK, receptor tyrosine kinase; p-RTK, Phospho-receptor tyrosine kinase; SCX, strong cation exchange chromatography; SDF-1, stromal cellderived factor 1; TFA, trifluoroacetic acid; SILAC, stable isotope labeling by amino acids; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.…”

“…Flow Cytometry Analysis (25,28)-Confluent MLECs in culture were detached with PBS containing 2 mM EDTA and 1% bovine serum albumin (BSA) (PBS-EB) and stained on ice for 30 min with FITC-conjugated-anti-CD31 (1:500, Invitrogen), PE-conjugated anti-VEGFR2 (1:500, BD Biosciences, San Jose, CA), anti-HS antibody 10E4 (1:50, Seigagaku, Tokyo, Japan), or their corresponding isotype control antibodies. After washing, the cells were analyzed by flow cytometry (Cell Lab Quanta TM SC, Beckman Coulter).…”

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

“…Immortalized mouse lung endothelial cells (22,23) were seeded at 1.5 ϫ 10 4 per well in the upper chamber and became confluent after 3-4 days in culture. The integrity of the endothelium-covered trans-wells was controlled after each migration by May-Grunwald-Giemsa staining and followed by visualization by microscopy.…”