Regulation of growth factor activity by heparin/HSPG

Regulation of growth factor activity by heparin/HSPG

1. The VEGF-induced incorporation of BrdU into HUVEC was examined by culturing the cells in the presence of 10 ng/mL VEGF₁₆₅ or VEGF₁₂₁ with various concentrations of heparin or modified heparins. 

The VEGF₁₆₅-induced proliferation was significantly enhanced dose-dependently only by heparin (heparin at 1 and 10 μg/mLstimulated proliferation 1.4- and 1.8-fold, respectively, compared to no added heparin) (Fig. 1A). None of modified heparins affected the proliferation of HUVEC. In contrast, the VEGF₁₂₁-dependent incorporation of BrdU was not enhanced by heparin or by any modified heparins at any concentrations tested (Fig. 1B). The results indicate that both 2-O- and 6-O-sulfate residues in heparin are necessary for the enhancement of VEGF₁₆₅-induced proliferation by heparin, but heparin is not involved in VEGF₁₂₁-dependent proliferation.

2. By Western blot analysis using a specific antibody for phospho-VEGFR-2, the tyrosine phosphorylation of VEGFR-2 was examined after the enhancement of VEGF₁₆₅ activity by heparin, since VEGF is known to stimulate the autophosphorylation of VEGFR-2.

Treatment with VEGF₁₆₅ alone for 5 min induced VEGFR-2 phosphorylation (Fig. 2A lanes 1 and 4). In the presence of heparin, the VEGF₁₆₅-induced phosphorylation of VEGFR-2 increased 1.7-fold (Figs. 2A lanes 4–6, 2B gray bars). Heparin alone did not substantially affect VEGFR-2 phosphorylation (Fig. 2A lanes 1–3). The VEGF₁₂₁-induced phosphorylation of VEGFR-2 did not increase in the presence of heparin (Figs. 2C and D), suggesting that the heparin-enhanced phosphorylation of VEGFR-2 occurred through the interaction with VEGF₁₆₅, but not with VEGF₁₂₁.

3. The involvement of cell surface HS in the VEGF₁₆₅-induced phosphorylation of VEGFR-2 was examined by the VEGF₁₆₅-induced receptor phosphorylation in HUVEC with and without the heparitinase mixture-treatment. Cells were treated with or without HSase mixture, washed (the digestion resulted in the removal of more than 95% of cell surface/ECM HS), and then stimulated with VEGF₁₆₅ in the absence or presence of heparin for 5 min at 37°C. VEGF₁₆₅-induced phosphorylation of VEGFR-2 was reduced to 75% by HSase mixture treatment (Figs. 3A lanes 1 and 2, 3B). In contrast, digestion with HSase mixture did not affect VEGF₁₂₁-induced phosphorylation of VEGFR-2 (Figs. 3C and D). The reduction of VEGF₁₆₅-induced phosphorylation by digestion with HSase mixture was rescued by the addition of heparin (Figs. 3A lanes 3–6, 3B). It is known that VEGFR-2 induces cell proliferation through activation of the classical extracellular regulated kinase (ERK) pathway, leading to gene transcription. As expected, phosphorylation of ERK1/2 increased 2.5-fold, 5 min after stimulation by VEGF₁₆₅ (Figs. 3E lanes 1 and 2, 3F); this phosphorylation was markedly decreased (to 25%) by HSase-mixture digestion, and restored by exogenous heparin (Figs. 3E lanes 3–5, 3F). The results suggest that cell-surface HS regulated VEGFR-2 activation mediated by heparin-binding VEGF.

    The experiments here demonstrated enhancement effects of heparin not only on the VEGF₁₆₅-induced proliferation of HUVEC but also on the VEGF-induced phosphorylation of VEGFR-2, which suggests that heparin facilitates to form a signaling complex with VEGF₁₆₅ and VEGFR-2 (see Fig. 4). The digestion of the cells with HSase mixture resulted in the reduction of the VEGF₁₆₅-induced phosphorylation of VEGFR-2 to 75% with significance (Fig. 3A), but the reduction was rescued by the addition of heparin (Figs.3A lanes 3–6, 3B), suggesting that the heparin-like domain of cell surface HS positively regulated VEGF₁₆₅ activity. Since the addition of heparin and digestion with HSase mixture did not affect VEGF₁₂₁-induced phosphorylation of VEGFR-2 (Fig. 1B & Figs. 3C and D, respectively), the cell surface HS was not essential for VEGFR-2 phosphorylation. However, mice expressing the VEGF₁₂₁ isoform exclusively via the Cre/loxP-mediated removal of exons 6 and 7, which encode the heparin-binding region, had impaired postnatal myocardial angiogenesis, and ultimately died of cardiac failur (Careliet, P., et al. 1999. Nat. Med., 5: 495–502). VEGF₁₂₁ alone was insufficient for normal angiogenesis, suggesting the important regulatory function of the interactions between the heparin-like domain of cell surface HS and the heparin binding domains of VEGF isoforms.

    Neither 2ODS heparin nor 6ODS heparin enhanced the VEGF₁₆₅-dependent proliferation (Fig.1A). Considering that heparin is known to interact with receptors for VE GF₁₆₅, VEGFR-2 and neuropilin (NRP)-1, 2ODS heparin and 6ODS heparin may lack the structure required for binding to those VEGF₁₆₅ receptors, and the heparin-like structural domain of HS may greatly contribute to the enhanced formation of a signaling complex between VEGF₁₆₅ and its receptors.

    Here we propose an action mechanism of VEGF₁₆₅ activity enhancement by heparin considering the reported roles of NRP-1, as shown in Fig. 4. In the absence of heparin, both VEGF₁₆₅ binding to VEGFR-2 and VEGF₁₆₅-dependent phosphorylation of VEGFR-2 are regulated by cell surface HSPG via binding to the heparin-like domain. In the presence of heparin, VEGF₁₆₅, VEGFR-2 and NRP-1 form a complex through the intermediary molecule of heparin. In consequence, VEGF₁₆₅-induced signaling processes, such as the phosphorylation of VEGFR-2, are increased by heparin. This mechanism is also suggested by the following results reported by others: Heparin enhances VEGF₁₆₅ binding to VEGFR, the affinity of VEGF₁₆₅ for NRP-1 is also enhanced by the addition of heparin, NRP-1 in turn enhances VEGF₁₆₅ binding to VEGFR-2 and its bioactivity, heparin induced the binding of NRP-1 to VEGF₁₆₅, and in consequence, heparin may regulate VEGF₁₆₅ activity selectively via NRP-1. We should also consider another mechanism of VEGF₁₆₅ activity enhancement by heparin: VEGF₁₆₅ stability was increased by complexing with heparin.

Figure & Legends

Fig. 1. Effect of heparin and modified heparins on VEGF-induced proliferation of HUVEC

HUVEC were seeded on a 96-well plate. The medium contained 10 ng/mL VEGF₁₆₅ (A) or VEGF₁₂₁ (B) in the absence (open bars), or presence of 1 (striped bars) or 10 μg/mL (closed bars) heparin or modified heparins. After 24 h of incubation, BrdU was added and the cells reincubated for an additional 24 h. Each bar represents the relative mitogenic activity (ratio of incorporation to that in VEGF alone). Data are the averages of triplicate measurements with standard deviation. Asterisks indicate significant differences from VEGF alone as determined by t test (p<0.05). The same results were obtained from two independent experiments. (Redrawn, with permission, from Ashikari-Hada S., et al. 2005. J. Biol. Chem. 280: 31508–15)

This figure was originally published in J Biol Chem. Ashikari-Hada S. et al. "Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins" 2005, 280(36):31508–15. © the American Society for Biochemistry and Molecular Biology.

Fig. 2. Increase of VEGF165-induced phosphorylation of VEGFR-2 by the addition of heparin

HUVEC were stimulated with 10 ng/mL VEGF₁₆₅ (A) or VEGF₁₂₁ (C) in the absence (open bars) or presence of heparin (gray bars) or PIO heparin (striped bars) for 5 min at 37°C. Samples were subjected to gel electrophoresis, with the subsequent transfer of membranes. Phospho-VEGFR-2 was stained with its specific antibody. VEGF₁₆₅-dependent (B) and VEGF₁₂₁-dependent (D) VEGFR-2 phosphorylation were measured using Image Gauge. Each bar represents the mean value of four (B) and three (D) independent experiments. Asterisks indicate significant differences from VEGF alone as determined by t test (p<0.05). (Redrawn, with permission, from Ashikari-Hada S. et al. 2005)

This figure was originally published in J Biol Chem. Ashikari-Hada S. et al. "Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins" 2005, 280(36):31508–15. © the American Society for Biochemistry and Molecular Biology.

Fig. 3. Restoration of VEGF165-induced phosphorylation in heparinase-treated HUVEC by heparin

HUVEC were digested with or without HSase mixture, and then stimulated with 10 ng/mL VEGF₁₆₅ (A and E) or VEGF₁₂₁ (C) in the presence or absence of heparin for 5 min at 37°C. Samples were subjected to gel electrophoresis, and then toWestern blotting using anti-phospho-VEGFR-2 antibody (A and C) or anti-phospho-ERK1/2 antibody (E). VEGFR-2 phosphorylation (B and D) and ERK1/2 phosphorylation (F) were quantified, and each bar (B and F) indicates the relative phosphorylation to that of VEGF alone and the average of three independent experiments and standard deviation. Asterisks indicate significant differences from VEGF alone as determined by t test (p<0.05). (Redrawn, with permission, from Ashikari-Hada S. et al. 2005)

This figure was originally published in J Biol Chem. Ashikari-Hada S. et al. "Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins" 2005, 280(36):31508–15. © the American Society for Biochemistry and Molecular Biology.

Fig. 4. Postulated model of the signaling by the interactions of VEGF165, VEGFR-2 and heparin

The VEGF₁₆₅-dependent phosphorylation of VEGFR-2 was increased by interaction with HS proteoglycans on the cell surface. Added heparin also regulates VEGF₁₆₅ activity by participating in the formation of a complex of VEGF, VEGFR-2 and NRP-1. Further, it is also involved in VEGF₁₆₅ stability. (Redrawn, with permission, from Ashikari-Hada S. et al. 2005)

This figure was originally published in J Biol Chem. Ashikari-Hada S. et al. "Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins" 2005, 280(36):31508–15. © the American Society for Biochemistry and Molecular Biology.