Mol Biol Rep (2014) 41:779–785 DOI 10.1007/s11033-013-2917-4
Effects of vascular endothelial growth factor B on proliferation and migration in EA.Hy926 cells Guang-hong Zhang • Rui Qin • Shui-hua Zhang He Zhu
•
Received: 12 August 2012 / Accepted: 18 December 2013 / Published online: 30 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Vascular endothelial growth factor B (VEGF-B) was reported to be angiogenic, and it was considered as a neuroprotective agent in mouse retinal ganglion cells following optic nerve crush. Thus, it was necessary to investigate whether VEGF-B contributes to the process of retinal and choroidal neovascularization. We aimed to investigate the effects of VEGF-B on proliferation and migration in EA.Hy926 cells. The proliferation of cells was analyzed by cell counting kit 8 assay, and the migration of cells was evaluated by a modified Boyden chamber assay. The levels of phospho-ERK1/2 (P-ERK1/2), ERK1/2, phospho-p38 and p38 were detected by western blotting. The results showed that VEGF-B induced proliferation and migration of EA.Hy926 cells (P \ 0.01 and P \ 0.05, respectively), and ERK1/2 and p38 phosphorylation were significantly activated. Our study suggested that VEGF-B
Guang-hong Zhang and Rui Qin contributed equally to the article. G. Zhang Department of Ophthalmology, The No.474 Hospital of Chinese People’s Liberation Army, Urumchi 830011, China e-mail: [email protected] R. Qin Department of Ophthalmology, The People’s Hospital of Bozhou, Bozhou 236804, China e-mail: [email protected] S. Zhang (&) Department of Ophthalmology, The Second Clinical College, Harbin Medical University, Harbin 150086, China e-mail: [email protected] H. Zhu Department of Anesthesiology, The Second Clinical College, Harbin Medical University, Harbin 150086, China e-mail: [email protected]
was an angiogenesis factor in vitro and that ERK1/2 and p38-related signaling pathways were involved in these VEGF-B activities. Keywords VEGF-B Angiogenesis Proliferation Migration Phosphorylation
Introduction The VEGF family of growth factors presently comprises the following five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGF-E and placenta growth factor (PlGF) [1–5]. Three members of the VEGF receptor family have been identified: VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 [5]. Each of these distinct tyrosine kinase receptors binds only certain VEGF family members. Ligands of VEGFR-1 include VEGF-A, VEGF-B, and PlGF; ligands of VEGFR-2 include VEGF-A, VEGF-C, and VEGF-D. Finally, the ligands of VEGFR-3 are VEGF-C and VEGF-D; PlGF and VEGF-B uniquely bind VEGFR-1 [6–9]. VEGFs and their receptors have emerged as central regulators of the angiogenic process. VEGF-A, a primary regulator of angiogenesis and vasculogenesis [10], is a hypoxia-inducible endothelial cell (EC) mitogen [11]. These mitogens stimulate EC migration, vessel permeability, and promote EC proliferation and the survival of newly formed vessels [12]. However, the involvement of VEGF-B in angiogenesis is obscure [13–16], though VEGF-B is widely expressed in heart, skeletal muscle and vascular cells [17, 18]. VEGF-A exerts its effect through VEGFR-1 and VEGFR-2, and it may induce vessel growth by activating several pro-angiogenic pathways. VEGF-A-induced vessel growth may also imply activation of the serine/threonine kinase ERK1/2 and p38-related endothelial pathways [19].
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Interestingly, VEGF-induced endothelial cell proliferation requires the phosphorylation of ERK1/2, and migration requires the phosphorylation of p38. These results suggest that ERK1/2 and p38 are downstream effectors of VEGF-A signaling. In addition, VEGF-B forms heterodimers with VEGF-A, a property likely to alter its receptor specificity and biological effects. Nevertheless, the biological functions of VEGF-B remain obscure at present. Contrasting results were obtained by analyzing the angiogenic effects of VEGFB in normal and pathologic conditions, with some studies showing that VEGF-B was angiogenic [1, 20–24] and others reporting that it was not [25–30]. One of the reasons would be that they are under different conditions [15]. Moreover, because of its high sequence homology and similar receptor binding patterns with VEGF-A, we naturally predicted that VEGF-B might be an angiogenic factor. Moreover, the finding that anti-PlGF antibody inhibited pathological angiogenesis [30] demonstrated that VEGFR1 also plays a role. Therefore, we hypothesized that VEGF-B may affect the angiogenic process. To test this hypothesis, we analyzed the angiogenic effect of VEGF-B using two in vitro methods. Finally, we attempted to investigate the molecular events associated with VEGF-B-mediated effects by evaluating changes in phospho-ERK and phospho-p38 protein levels. A recent report [31] indicated that VEGF-B promoted retinal and choroidal neovascularization, and bloodretinal barrier (BRB) breakdown. These results suggest that VEGF-B may contribute to the complications of vasoproliferative ocular disorders and that it could be a therapeutic target for treatment of angiogenic ocular disorders and disorders leading to macular edema. Moreover, another study [32] demonstrated that VEGF-B was involved in the retinal recovery process and played an important neuroprotective role in RGCs following optic nerve crush [33]. Neuroprotection by VEGF-B led to retinal and choroidal neovascularization side effects. Therefore, it was necessary to investigate whether VEGF-B was a regulator of the angiogenesis process. In this study, the EA.Hy926 cell line was used to evaluate the cellular responses toward VEGF-B. Our results demonstrated that VEGF-B stimulated proliferation and migration in EA.Hy926 cells and that ERK1/2 and p38mediated signaling pathways were involved in these VEGF-B activities.
Mol Biol Rep (2014) 41:779–785
(Shanghai, China). The cell line is a hybridoma generated from human umbilical vein endothelial cells (HUVECs) and the epithelial lung tumor cell line A549. This cell line retains most of the features associated with HUVECs, including the expression of endothelial adhesion molecules and human factor VIII-related Ag [15]. EA.Hy926 cells were grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (Hyeclone, America). Antibodies and pharmacological reagents Recombinant Human VEGF-B167 was obtained from Peprotech (USA); Human VEGF R1/Flt-1 Antibody was purchased from R&D (USA). Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, 20G11) Rabbit mAb, p44/42 MAPK (Erk1/2) Antibody, Phospho-p38 MAPK (Thr180/ Tyr182, 12F8) Rabbit mAb, p38 MAPK Antibody and PD98059 (MEK1 Inhibitor) were obtained from Cell Signaling (USA); SB203580 was purchased from Sigma (USA). The Cell Counting Kit-8 (CCK-8) was acquired from Beyotime (China). Cell proliferation assay The EA.Hy926 cells (5 9 104/ml) were seeded into 96-well flat-bottom culture plates (Corning, America) with 100 ll culture medium (with 10 %FBS) and incubated overnight (medium with 0.1 %FBS) when they were at 80 % confluence. Subsequently, various concentrations (0, 0.1, 1.0, 10, 50 and 100 ng/ml) of VEGF-B (diluted in PBS with 0.1 %BSA) or vehicle were added to the wells (medium with 10 %FBS). For Flt-1 mAb and PD98059 groups, cells were pre-treated with Flt-1 Antibody (1 lg/ ml, the specificity and blocking activity of mAb) or PD98059 (25 lM, a specific inhibitor of MEK1) for 60 min prior to the addition of VEGF-B (1.0 ng/ml). The plates were then incubated at 37 °C and 5 % CO2 for 24 h. Following incubation, the cells were exposed to a solution of CCK-8 (100 ll/ml) for 4 h, and the optical density was measured spectrophotometrically. CCK-8 allowed for sensitive colorimetric assays to determine the number of viable cells in cell proliferation and cytotoxicity assays, and its detection sensitivity was higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1. Cell migration assay
Materials and methods Cell culture The EA.Hy926 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences
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To assess the cell migration ability of EA.Hy926 cells, a modified Boyden chamber assay was used. Cells (2 9 104 in 100 ll medium supplemented with 1 % v/v FBS) were added into each transwell filter chamber with 8 mm pore size (Corning, Lowell, MA, USA). Simultaneously, 600 ll
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of medium containing selected concentrations of VEGF-B (with 1 % v/v FBS) was added to the lower chambers. For the Flt-1 mAb and SB203580 groups, cells were pre-treated with Flt-1 mAb (1 lg/ml) or SB203580 (10 lM, a pyridinyl imidazole inhibitor of p38a and p38b MAPK isoforms) for 60 min. Cells were then allowed to migrate at 37 °C in 5 % CO2 for 6 h and were subsequently fixed by immersion of the filters in methanol at room temperature for 30 min. Filters were washed with deionized water and stained in 0.1 % crystal violet in a 20 % methanol solution for 30 min. Cells on the top surface of the filter membrane (non-migrated) were scraped with a cotton swab. Stained filters were photographed under the microscope (Olympus IX-71). Migrated cells were counted in five random fields (2009) and expressed as the average number of cells per field of view. Western blot analysis EA.Hy926 (1 9 106/ml) were seeded in 25 cm2 flasks and incubated for 24 h to allow cell attachment. Selected concentrations of VEGF-B were added to the flasks and incubated for 24 h. For the Flt-1 mAb, PD98059 and SB203580 groups, cells were pre-treated with PD98059 (25 lM), SB203580 (10 lM) or Flt-1 antibody (1 lg/ml) for 60 min prior to the addition of VEGF-B. Following treatment, cells were washed twice with phosphate buffer saline( PBS). The cells were then lysed with whole cell extraction buffer for 60 min on ice. The samples were heated at 95 °C for 10 min and then centrifuged at 12,0009g for 5 min at 4 °C. The supernatant proteins were resolved by electrophoresis on a 10 % SDS polyacrylamide gel and transferred to a 0.45 mm PVDF membrane (Immobilon, Millipore). The membrane was blocked with 5 % non-fat milk in Tris-buffered saline containing Tween-20 (20 mM Tris–HCl [pH 7.6], 150 mM NaCl, 0.1 % Tween20). The blots were incubated overnight with primary antibodies against ERK1/2, p-ERK1/2, p38, or p-p38. After incubation with the secondary horseradish peroxidaseconjugated antibodies (Beyotime, China) for 1 h, detection was performed using BeyoECL Plus (Beyotime, China). Statistial analysis Data were presented as mean ± SD of three independent experiments with four wells each (for proliferation assay) or five images (for migration assay). Differences among the treated and untreated control groups were determined by one-way ANOVA and multiple comparison (proliferation and migration assay). Relative phosphorylation was determined by scanning densitometry and was presented as the mean ± SD and differences between the groups were compared using the Student group t test.
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Results VEGF-B promotes proliferation of EA.Hy926 cells To specifically determine the role of VEGF-B on cell growth, proliferation of EA.Hy926 cells was examined utilizing the CCK-8 assay. The cells that were incubated with VEGF-B had a significant increase in cell viability after 24 h incubation when compared to cells incubated with media alone (P \ 0.01). However, there was no significant difference among groups containing different concentrations of VEGF-B (0.1,1.0,10, 50, 100 ng/ml) (Fig. 1a). To test whether the ERK1/2 pathway had a role in cell proliferation induced by VEGF-B, EA.Hy926 cells were pre-treated with the indicated concentration of Flt-1 mAb (1 lg/ml) or with PD98059 (25 lM) (Fig. 1b). The results indicated that cell proliferation was completely inhibited by Flt-1 mAb (P \ 0.01) and partially inhibited by PD98059 (P \ 0.05). VEGF-B promotes migration of EA.Hy926 cells EA.Hy926 cells were evaluated using a modified Boyden chamber assay. Many cells migrated from the upper chamber to the lower chamber through the membrane after a 6 h incubation when the lower chamber contained culture medium supplemented with selected concentrations of VEGF-B as a chemoattractant. As shown in Fig. 2, only VEGF-B (0.1 and 1.0 ng/ml) stimulated the migration of EA.Hy926 cells when compared to the media only control group (P \ 0.05). To test whether the p38 MAPK pathway plays a role in cell migration induced by VEGF-B, we pre-treated EA.Hy926 cells with Flt-1 mAb (1 lg/ml) and SB203580 (10 lM). Cell migration was comparably suppressed by Flt1 mAb and SB203580 (P \ 0.05) as shown in Fig. 2. Western blot VEGF-B induces phosphorylation of ERK1/2 To evaluate whether ERK1/2 phosphorylation was activated by VEGF-B, EA.Hy926 cells were treated with selected concentrations of VEGF-B. The concentration of VEGF-B 167 are presented in Fig. 3 and show an increase in ERK1/2 phosphorylation following addition of VEGF-B. Stimulation by VEGF-B (0.1, 1.0, 10, and 50 ng/ml) induced a significant increase in ERK1/2 phosphorylation following a 30 min incubation (P \ 0.01 or P \ 0.05). Pre-treatment of cells with Flt-1 mAb (1 lg/ml) or with PD98059 (25 lM) inhibited ERK1/2 phosphorylation by 36.6 % (P \ 0.01) and 25.3 % (P \ 0.05), respectively.
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Fig. 1 Effects of VEGF-B on proliferation of EA.Hy926 cells. a Cells were treated with increasing concentrations of VEGF-B for 24 h, and cell proliferation viability was determined by CCK-8 assay. b EA.Hy926 cells were left untreated or exposed to indicated concentrations of Flt-1 mAb or PD98059 for 60 min prior to the
addition of VEGF-B. Then the cultures were incubated with or without of VEGF-B (0.1 ng/ml) for 24 h and the viability of cells was determined by CCK-8 assay. **P \ 0.01, significantly different from the VEGF-B untreated group (a); *P \ 0.05, **P \ 0.01, significantly different from the VEGF-B treated alone group (b)
Fig. 2 Effects of VEGF-B on migration of EA.Hy926 cells in Boyden chambers. a Representative photomicrographs showing the stained cells on the lower side of membranes. The cells in the upper chambers were treated with media (control, 0 ng/ml) or 0.1, 1.0, 10, 50, 100 ng/ml. After 6 h incubation, those cells which had migrated to the lower chambers were stained and the numbers were counted. b Quantification of cell migration in EA.Hy926 cells were shown. c EA.Hy926 cells were left untreated or exposed to indicated
concentrations of Flt-1 mAb (1 lg/ml) or SB203580 (10 lM) for 60 min prior to the addition of VEGF-B. Then the cells in the upper chambers were treated with or without VEGF-B. After 6 h incubation, those cells which had migrated to the lower chambers were stained and the numbers were counted. d Quantification of cell migration in EA.Hy926 cells were shown. *P \ 0.05, significantly different from the VEGF-B untreated group (b); *P \ 0.05, significantly different from the VEGF-B treated alone group (d)
VEGF-B induces phosphorylation of p38
VEGF-B (0.1 and 1.0 ng/ml) increased p38 phosphorylation following a 30 min incubation (P \ 0.05). P38 phosphorylation was inhibited when EA.Hy926 cells were pre-treated with Flt-1 mAb or SB203580; inhibition was significant when compared to the VEGF-B treated group (P \ 0.05).
To determine if p38 MAPK is activated by VEGF-B, EA.Hy926 cells were treated with concentrations of VEGF-B that induced cell migration. The concentration of VEGF-B are shown in Fig. 4. Only stimulation by
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Fig. 3 ERK1/2 phosphorylation was activated by VEGF-B. EA.Hy926 cells were treated to selected concentrations of VEGF-B for 30 min and tyrosine phosphorylation of P-ERK and ERK were analysed by Western blot analysis. a Images of immunoblots illustrate relative levels of P-ERK tyrosine phosphorylation. b Relative phosphorylation was determined by scanning densitometry. c EA.Hy926 cells were left untreated or exposed to indicated concentrations of Flt1 mAb or PD98059 for 60 min prior to the addition of VEGF-B. Then
the cultures were incubated with or without indicated VEGF-B (0.1 ng/ml) for 30 min and tyrosine phosphorylation of P-ERK and ERK were analysed by Western blot analysis. Images of immunoblots illustrate relative levels of P-ERK tyrosine phosphorylation. d Relative phosphorylation was determined by scanning densitometry. *P \ 0.05 versus VEGF-B untreated group (b) or VEGF-B treated alone group (d)
Fig. 4 P-p38 MAP Kinase tyrosine phosphorylation was activated by VEGF-B. a EA.Hy926 cells were treated to indicated concentrations of VEGF-B for 30 min and tyrosine phosphorylation of P-p38 and p38 were analysed by Western blot analysis. Images of immunoblots illustrate relative levels of P-p38 tyrosine phosphorylation. b Relative phosphorylation was determined by scanning densitometry. *P \ 0.05 versus VEGF-B untreated group. c EA.Hy926 cells were left untreated or exposed to indicated concentrations of Flt-1 mAb or
SB203580 for 60 min prior to the addition of VEGF-B. Then the cultures were incubated with or without indicated VEGF (0.1 ng/ml) for 30 min and tyrosine phosphorylation of P-p38 and p38 were analysed by Western blot analysis. Images of immunoblots illustrate relative levels of p38MAPK tyrosine phosphorylation. d Relative phosphorylation was determined by scanning densitometry. *P \ 0.05 versus VEGF-B treated alone group
Discussion
The pro-angiogenic effect of VEGF-B was likely mediated by VEGF-R1 signaling, which is associated with activation of phospho-ERK1/2 and -p38 signaling pathways.
The main results of this study were that VEGF-B promoted proliferation and migration of EA.Hy926 cells.
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We demonstrated VEGF-B-mediated proliferation of EA.Hy926 cells by using the CCK-8 assay, and there was no significant difference in all selected concentrations. Surprisingly, the level of phosphorylated ERK1/2 protein was increased in a dose-dependent manner under the same concentrations of VEGF-B. Another signal transduction pathway may be involved in mediating VEGF-B effects. According to some studies, VEGF-B and its receptor may activate new vessel growth via several complementary mechanisms such as the AKT-mediated pathway [1]. The blockade of Flt-1-mediated signals completely suppressed the proliferation of EA.Hy926 cells, and the addition of an ERK1/2 inhibitor partially decreased cell proliferation. The results further confirmed that VEGF-R1mediated signaling might play a significant role in the angiogenic process; however, activation of ERK1/2 phosphorylation may not play a major role in proliferation. VEGF-B stimulated the migration of EA.Hy926 cells in a dose-dependent manner, consistent with the change in the level of phosphorylated p38. Our results indicated that VEGF-B-mediated signals modulated migration via the activation of p38 MAP kinase. Blocking Flt-1 and inhibiting p38 comparably decreased the migration of EA.Hy926 cells, indicating that activation of p38 MAP kinase might be the major signal transduction pathway responsible for the VEGF-B-mediated effects. VEGF-B also bound to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins [34]. Previous reports have suggested that NP-1 enhances the mitogenic effect of VEGF-R1 following VEGF-A165 stimulation [35], and others [2] have indicated that NP-1 plays a role in mediating the vascular survival effects induced by VEGF-B. Binding of VEGF-B to NP-1 may also mediate some of the biological responses to VEGF-B [36]. Thus, future studies should evaluate the angiogenic effects of NP-1. Angiogenesis requires a cascade of events, including endothelial cell proliferation, survival, migration, extracellular matrix remodeling, and maturation to form capillary tubes. Moreover, many studies [37, 38] have demonstrated the heterogeneity of endothelial cells; therefore, experiments conducted on one type of endothelial cell may be misleading. In our study, we evaluated the proliferation and migration of EA.Hy926 cells in vitro. Thus, future studies should be conducted on at least two types of ECs, such as HUVECs, human (dermal) microvascular endothelial cell (HMEC-1) and human retinal endothelial cell (HREC). Nevertheless, it will be necessary to include in vitro proliferation, migration and tube formation and further in vivo assays such as the murine ischemic hindlimb model. In conclusion, we have demonstrated that VEGF-B promoted the proliferation and migration of EA.Hy926 cells. Additionally, the activation of ERK1/2 phosphorylation was in part associated with proliferation, and activation of p38
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MAP kinase played a major role in the signal transduction pathway mediated by VEGF-B. The results here suggest that VEGF-B effects may also depend on its concentration. Acknowledgments The authors declare that they have no conflicts of interest to disclose. Funding Shui-hua Zhang acknowledges that the project was supported by the Scientific Research Starting Foundation for Returned Overseas Chinese Scholars, Ministry of Education, China (Grant No. 1001). The research was also supported by the Scientific Research Starting Foundation for Returned Overseas Chinese Scholars, Ministry of Education, Heilongjiang (Grant No. LC2009C24).
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Effects of vascular endothelial growth factor B on proliferation and migration in EA.Hy926 cells Guang-hong Zhang • Rui Qin • Shui-hua Zhang He Zhu
•
Received: 12 August 2012 / Accepted: 18 December 2013 / Published online: 30 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Vascular endothelial growth factor B (VEGF-B) was reported to be angiogenic, and it was considered as a neuroprotective agent in mouse retinal ganglion cells following optic nerve crush. Thus, it was necessary to investigate whether VEGF-B contributes to the process of retinal and choroidal neovascularization. We aimed to investigate the effects of VEGF-B on proliferation and migration in EA.Hy926 cells. The proliferation of cells was analyzed by cell counting kit 8 assay, and the migration of cells was evaluated by a modified Boyden chamber assay. The levels of phospho-ERK1/2 (P-ERK1/2), ERK1/2, phospho-p38 and p38 were detected by western blotting. The results showed that VEGF-B induced proliferation and migration of EA.Hy926 cells (P \ 0.01 and P \ 0.05, respectively), and ERK1/2 and p38 phosphorylation were significantly activated. Our study suggested that VEGF-B
Guang-hong Zhang and Rui Qin contributed equally to the article. G. Zhang Department of Ophthalmology, The No.474 Hospital of Chinese People’s Liberation Army, Urumchi 830011, China e-mail: [email protected] R. Qin Department of Ophthalmology, The People’s Hospital of Bozhou, Bozhou 236804, China e-mail: [email protected] S. Zhang (&) Department of Ophthalmology, The Second Clinical College, Harbin Medical University, Harbin 150086, China e-mail: [email protected] H. Zhu Department of Anesthesiology, The Second Clinical College, Harbin Medical University, Harbin 150086, China e-mail: [email protected]
was an angiogenesis factor in vitro and that ERK1/2 and p38-related signaling pathways were involved in these VEGF-B activities. Keywords VEGF-B Angiogenesis Proliferation Migration Phosphorylation
Introduction The VEGF family of growth factors presently comprises the following five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGF-E and placenta growth factor (PlGF) [1–5]. Three members of the VEGF receptor family have been identified: VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 [5]. Each of these distinct tyrosine kinase receptors binds only certain VEGF family members. Ligands of VEGFR-1 include VEGF-A, VEGF-B, and PlGF; ligands of VEGFR-2 include VEGF-A, VEGF-C, and VEGF-D. Finally, the ligands of VEGFR-3 are VEGF-C and VEGF-D; PlGF and VEGF-B uniquely bind VEGFR-1 [6–9]. VEGFs and their receptors have emerged as central regulators of the angiogenic process. VEGF-A, a primary regulator of angiogenesis and vasculogenesis [10], is a hypoxia-inducible endothelial cell (EC) mitogen [11]. These mitogens stimulate EC migration, vessel permeability, and promote EC proliferation and the survival of newly formed vessels [12]. However, the involvement of VEGF-B in angiogenesis is obscure [13–16], though VEGF-B is widely expressed in heart, skeletal muscle and vascular cells [17, 18]. VEGF-A exerts its effect through VEGFR-1 and VEGFR-2, and it may induce vessel growth by activating several pro-angiogenic pathways. VEGF-A-induced vessel growth may also imply activation of the serine/threonine kinase ERK1/2 and p38-related endothelial pathways [19].
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Interestingly, VEGF-induced endothelial cell proliferation requires the phosphorylation of ERK1/2, and migration requires the phosphorylation of p38. These results suggest that ERK1/2 and p38 are downstream effectors of VEGF-A signaling. In addition, VEGF-B forms heterodimers with VEGF-A, a property likely to alter its receptor specificity and biological effects. Nevertheless, the biological functions of VEGF-B remain obscure at present. Contrasting results were obtained by analyzing the angiogenic effects of VEGFB in normal and pathologic conditions, with some studies showing that VEGF-B was angiogenic [1, 20–24] and others reporting that it was not [25–30]. One of the reasons would be that they are under different conditions [15]. Moreover, because of its high sequence homology and similar receptor binding patterns with VEGF-A, we naturally predicted that VEGF-B might be an angiogenic factor. Moreover, the finding that anti-PlGF antibody inhibited pathological angiogenesis [30] demonstrated that VEGFR1 also plays a role. Therefore, we hypothesized that VEGF-B may affect the angiogenic process. To test this hypothesis, we analyzed the angiogenic effect of VEGF-B using two in vitro methods. Finally, we attempted to investigate the molecular events associated with VEGF-B-mediated effects by evaluating changes in phospho-ERK and phospho-p38 protein levels. A recent report [31] indicated that VEGF-B promoted retinal and choroidal neovascularization, and bloodretinal barrier (BRB) breakdown. These results suggest that VEGF-B may contribute to the complications of vasoproliferative ocular disorders and that it could be a therapeutic target for treatment of angiogenic ocular disorders and disorders leading to macular edema. Moreover, another study [32] demonstrated that VEGF-B was involved in the retinal recovery process and played an important neuroprotective role in RGCs following optic nerve crush [33]. Neuroprotection by VEGF-B led to retinal and choroidal neovascularization side effects. Therefore, it was necessary to investigate whether VEGF-B was a regulator of the angiogenesis process. In this study, the EA.Hy926 cell line was used to evaluate the cellular responses toward VEGF-B. Our results demonstrated that VEGF-B stimulated proliferation and migration in EA.Hy926 cells and that ERK1/2 and p38mediated signaling pathways were involved in these VEGF-B activities.
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(Shanghai, China). The cell line is a hybridoma generated from human umbilical vein endothelial cells (HUVECs) and the epithelial lung tumor cell line A549. This cell line retains most of the features associated with HUVECs, including the expression of endothelial adhesion molecules and human factor VIII-related Ag [15]. EA.Hy926 cells were grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (Hyeclone, America). Antibodies and pharmacological reagents Recombinant Human VEGF-B167 was obtained from Peprotech (USA); Human VEGF R1/Flt-1 Antibody was purchased from R&D (USA). Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, 20G11) Rabbit mAb, p44/42 MAPK (Erk1/2) Antibody, Phospho-p38 MAPK (Thr180/ Tyr182, 12F8) Rabbit mAb, p38 MAPK Antibody and PD98059 (MEK1 Inhibitor) were obtained from Cell Signaling (USA); SB203580 was purchased from Sigma (USA). The Cell Counting Kit-8 (CCK-8) was acquired from Beyotime (China). Cell proliferation assay The EA.Hy926 cells (5 9 104/ml) were seeded into 96-well flat-bottom culture plates (Corning, America) with 100 ll culture medium (with 10 %FBS) and incubated overnight (medium with 0.1 %FBS) when they were at 80 % confluence. Subsequently, various concentrations (0, 0.1, 1.0, 10, 50 and 100 ng/ml) of VEGF-B (diluted in PBS with 0.1 %BSA) or vehicle were added to the wells (medium with 10 %FBS). For Flt-1 mAb and PD98059 groups, cells were pre-treated with Flt-1 Antibody (1 lg/ ml, the specificity and blocking activity of mAb) or PD98059 (25 lM, a specific inhibitor of MEK1) for 60 min prior to the addition of VEGF-B (1.0 ng/ml). The plates were then incubated at 37 °C and 5 % CO2 for 24 h. Following incubation, the cells were exposed to a solution of CCK-8 (100 ll/ml) for 4 h, and the optical density was measured spectrophotometrically. CCK-8 allowed for sensitive colorimetric assays to determine the number of viable cells in cell proliferation and cytotoxicity assays, and its detection sensitivity was higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1. Cell migration assay
Materials and methods Cell culture The EA.Hy926 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences
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To assess the cell migration ability of EA.Hy926 cells, a modified Boyden chamber assay was used. Cells (2 9 104 in 100 ll medium supplemented with 1 % v/v FBS) were added into each transwell filter chamber with 8 mm pore size (Corning, Lowell, MA, USA). Simultaneously, 600 ll
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of medium containing selected concentrations of VEGF-B (with 1 % v/v FBS) was added to the lower chambers. For the Flt-1 mAb and SB203580 groups, cells were pre-treated with Flt-1 mAb (1 lg/ml) or SB203580 (10 lM, a pyridinyl imidazole inhibitor of p38a and p38b MAPK isoforms) for 60 min. Cells were then allowed to migrate at 37 °C in 5 % CO2 for 6 h and were subsequently fixed by immersion of the filters in methanol at room temperature for 30 min. Filters were washed with deionized water and stained in 0.1 % crystal violet in a 20 % methanol solution for 30 min. Cells on the top surface of the filter membrane (non-migrated) were scraped with a cotton swab. Stained filters were photographed under the microscope (Olympus IX-71). Migrated cells were counted in five random fields (2009) and expressed as the average number of cells per field of view. Western blot analysis EA.Hy926 (1 9 106/ml) were seeded in 25 cm2 flasks and incubated for 24 h to allow cell attachment. Selected concentrations of VEGF-B were added to the flasks and incubated for 24 h. For the Flt-1 mAb, PD98059 and SB203580 groups, cells were pre-treated with PD98059 (25 lM), SB203580 (10 lM) or Flt-1 antibody (1 lg/ml) for 60 min prior to the addition of VEGF-B. Following treatment, cells were washed twice with phosphate buffer saline( PBS). The cells were then lysed with whole cell extraction buffer for 60 min on ice. The samples were heated at 95 °C for 10 min and then centrifuged at 12,0009g for 5 min at 4 °C. The supernatant proteins were resolved by electrophoresis on a 10 % SDS polyacrylamide gel and transferred to a 0.45 mm PVDF membrane (Immobilon, Millipore). The membrane was blocked with 5 % non-fat milk in Tris-buffered saline containing Tween-20 (20 mM Tris–HCl [pH 7.6], 150 mM NaCl, 0.1 % Tween20). The blots were incubated overnight with primary antibodies against ERK1/2, p-ERK1/2, p38, or p-p38. After incubation with the secondary horseradish peroxidaseconjugated antibodies (Beyotime, China) for 1 h, detection was performed using BeyoECL Plus (Beyotime, China). Statistial analysis Data were presented as mean ± SD of three independent experiments with four wells each (for proliferation assay) or five images (for migration assay). Differences among the treated and untreated control groups were determined by one-way ANOVA and multiple comparison (proliferation and migration assay). Relative phosphorylation was determined by scanning densitometry and was presented as the mean ± SD and differences between the groups were compared using the Student group t test.
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Results VEGF-B promotes proliferation of EA.Hy926 cells To specifically determine the role of VEGF-B on cell growth, proliferation of EA.Hy926 cells was examined utilizing the CCK-8 assay. The cells that were incubated with VEGF-B had a significant increase in cell viability after 24 h incubation when compared to cells incubated with media alone (P \ 0.01). However, there was no significant difference among groups containing different concentrations of VEGF-B (0.1,1.0,10, 50, 100 ng/ml) (Fig. 1a). To test whether the ERK1/2 pathway had a role in cell proliferation induced by VEGF-B, EA.Hy926 cells were pre-treated with the indicated concentration of Flt-1 mAb (1 lg/ml) or with PD98059 (25 lM) (Fig. 1b). The results indicated that cell proliferation was completely inhibited by Flt-1 mAb (P \ 0.01) and partially inhibited by PD98059 (P \ 0.05). VEGF-B promotes migration of EA.Hy926 cells EA.Hy926 cells were evaluated using a modified Boyden chamber assay. Many cells migrated from the upper chamber to the lower chamber through the membrane after a 6 h incubation when the lower chamber contained culture medium supplemented with selected concentrations of VEGF-B as a chemoattractant. As shown in Fig. 2, only VEGF-B (0.1 and 1.0 ng/ml) stimulated the migration of EA.Hy926 cells when compared to the media only control group (P \ 0.05). To test whether the p38 MAPK pathway plays a role in cell migration induced by VEGF-B, we pre-treated EA.Hy926 cells with Flt-1 mAb (1 lg/ml) and SB203580 (10 lM). Cell migration was comparably suppressed by Flt1 mAb and SB203580 (P \ 0.05) as shown in Fig. 2. Western blot VEGF-B induces phosphorylation of ERK1/2 To evaluate whether ERK1/2 phosphorylation was activated by VEGF-B, EA.Hy926 cells were treated with selected concentrations of VEGF-B. The concentration of VEGF-B 167 are presented in Fig. 3 and show an increase in ERK1/2 phosphorylation following addition of VEGF-B. Stimulation by VEGF-B (0.1, 1.0, 10, and 50 ng/ml) induced a significant increase in ERK1/2 phosphorylation following a 30 min incubation (P \ 0.01 or P \ 0.05). Pre-treatment of cells with Flt-1 mAb (1 lg/ml) or with PD98059 (25 lM) inhibited ERK1/2 phosphorylation by 36.6 % (P \ 0.01) and 25.3 % (P \ 0.05), respectively.
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Fig. 1 Effects of VEGF-B on proliferation of EA.Hy926 cells. a Cells were treated with increasing concentrations of VEGF-B for 24 h, and cell proliferation viability was determined by CCK-8 assay. b EA.Hy926 cells were left untreated or exposed to indicated concentrations of Flt-1 mAb or PD98059 for 60 min prior to the
addition of VEGF-B. Then the cultures were incubated with or without of VEGF-B (0.1 ng/ml) for 24 h and the viability of cells was determined by CCK-8 assay. **P \ 0.01, significantly different from the VEGF-B untreated group (a); *P \ 0.05, **P \ 0.01, significantly different from the VEGF-B treated alone group (b)
Fig. 2 Effects of VEGF-B on migration of EA.Hy926 cells in Boyden chambers. a Representative photomicrographs showing the stained cells on the lower side of membranes. The cells in the upper chambers were treated with media (control, 0 ng/ml) or 0.1, 1.0, 10, 50, 100 ng/ml. After 6 h incubation, those cells which had migrated to the lower chambers were stained and the numbers were counted. b Quantification of cell migration in EA.Hy926 cells were shown. c EA.Hy926 cells were left untreated or exposed to indicated
concentrations of Flt-1 mAb (1 lg/ml) or SB203580 (10 lM) for 60 min prior to the addition of VEGF-B. Then the cells in the upper chambers were treated with or without VEGF-B. After 6 h incubation, those cells which had migrated to the lower chambers were stained and the numbers were counted. d Quantification of cell migration in EA.Hy926 cells were shown. *P \ 0.05, significantly different from the VEGF-B untreated group (b); *P \ 0.05, significantly different from the VEGF-B treated alone group (d)
VEGF-B induces phosphorylation of p38
VEGF-B (0.1 and 1.0 ng/ml) increased p38 phosphorylation following a 30 min incubation (P \ 0.05). P38 phosphorylation was inhibited when EA.Hy926 cells were pre-treated with Flt-1 mAb or SB203580; inhibition was significant when compared to the VEGF-B treated group (P \ 0.05).
To determine if p38 MAPK is activated by VEGF-B, EA.Hy926 cells were treated with concentrations of VEGF-B that induced cell migration. The concentration of VEGF-B are shown in Fig. 4. Only stimulation by
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Fig. 3 ERK1/2 phosphorylation was activated by VEGF-B. EA.Hy926 cells were treated to selected concentrations of VEGF-B for 30 min and tyrosine phosphorylation of P-ERK and ERK were analysed by Western blot analysis. a Images of immunoblots illustrate relative levels of P-ERK tyrosine phosphorylation. b Relative phosphorylation was determined by scanning densitometry. c EA.Hy926 cells were left untreated or exposed to indicated concentrations of Flt1 mAb or PD98059 for 60 min prior to the addition of VEGF-B. Then
the cultures were incubated with or without indicated VEGF-B (0.1 ng/ml) for 30 min and tyrosine phosphorylation of P-ERK and ERK were analysed by Western blot analysis. Images of immunoblots illustrate relative levels of P-ERK tyrosine phosphorylation. d Relative phosphorylation was determined by scanning densitometry. *P \ 0.05 versus VEGF-B untreated group (b) or VEGF-B treated alone group (d)
Fig. 4 P-p38 MAP Kinase tyrosine phosphorylation was activated by VEGF-B. a EA.Hy926 cells were treated to indicated concentrations of VEGF-B for 30 min and tyrosine phosphorylation of P-p38 and p38 were analysed by Western blot analysis. Images of immunoblots illustrate relative levels of P-p38 tyrosine phosphorylation. b Relative phosphorylation was determined by scanning densitometry. *P \ 0.05 versus VEGF-B untreated group. c EA.Hy926 cells were left untreated or exposed to indicated concentrations of Flt-1 mAb or
SB203580 for 60 min prior to the addition of VEGF-B. Then the cultures were incubated with or without indicated VEGF (0.1 ng/ml) for 30 min and tyrosine phosphorylation of P-p38 and p38 were analysed by Western blot analysis. Images of immunoblots illustrate relative levels of p38MAPK tyrosine phosphorylation. d Relative phosphorylation was determined by scanning densitometry. *P \ 0.05 versus VEGF-B treated alone group
Discussion
The pro-angiogenic effect of VEGF-B was likely mediated by VEGF-R1 signaling, which is associated with activation of phospho-ERK1/2 and -p38 signaling pathways.
The main results of this study were that VEGF-B promoted proliferation and migration of EA.Hy926 cells.
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We demonstrated VEGF-B-mediated proliferation of EA.Hy926 cells by using the CCK-8 assay, and there was no significant difference in all selected concentrations. Surprisingly, the level of phosphorylated ERK1/2 protein was increased in a dose-dependent manner under the same concentrations of VEGF-B. Another signal transduction pathway may be involved in mediating VEGF-B effects. According to some studies, VEGF-B and its receptor may activate new vessel growth via several complementary mechanisms such as the AKT-mediated pathway [1]. The blockade of Flt-1-mediated signals completely suppressed the proliferation of EA.Hy926 cells, and the addition of an ERK1/2 inhibitor partially decreased cell proliferation. The results further confirmed that VEGF-R1mediated signaling might play a significant role in the angiogenic process; however, activation of ERK1/2 phosphorylation may not play a major role in proliferation. VEGF-B stimulated the migration of EA.Hy926 cells in a dose-dependent manner, consistent with the change in the level of phosphorylated p38. Our results indicated that VEGF-B-mediated signals modulated migration via the activation of p38 MAP kinase. Blocking Flt-1 and inhibiting p38 comparably decreased the migration of EA.Hy926 cells, indicating that activation of p38 MAP kinase might be the major signal transduction pathway responsible for the VEGF-B-mediated effects. VEGF-B also bound to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins [34]. Previous reports have suggested that NP-1 enhances the mitogenic effect of VEGF-R1 following VEGF-A165 stimulation [35], and others [2] have indicated that NP-1 plays a role in mediating the vascular survival effects induced by VEGF-B. Binding of VEGF-B to NP-1 may also mediate some of the biological responses to VEGF-B [36]. Thus, future studies should evaluate the angiogenic effects of NP-1. Angiogenesis requires a cascade of events, including endothelial cell proliferation, survival, migration, extracellular matrix remodeling, and maturation to form capillary tubes. Moreover, many studies [37, 38] have demonstrated the heterogeneity of endothelial cells; therefore, experiments conducted on one type of endothelial cell may be misleading. In our study, we evaluated the proliferation and migration of EA.Hy926 cells in vitro. Thus, future studies should be conducted on at least two types of ECs, such as HUVECs, human (dermal) microvascular endothelial cell (HMEC-1) and human retinal endothelial cell (HREC). Nevertheless, it will be necessary to include in vitro proliferation, migration and tube formation and further in vivo assays such as the murine ischemic hindlimb model. In conclusion, we have demonstrated that VEGF-B promoted the proliferation and migration of EA.Hy926 cells. Additionally, the activation of ERK1/2 phosphorylation was in part associated with proliferation, and activation of p38
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MAP kinase played a major role in the signal transduction pathway mediated by VEGF-B. The results here suggest that VEGF-B effects may also depend on its concentration. Acknowledgments The authors declare that they have no conflicts of interest to disclose. Funding Shui-hua Zhang acknowledges that the project was supported by the Scientific Research Starting Foundation for Returned Overseas Chinese Scholars, Ministry of Education, China (Grant No. 1001). The research was also supported by the Scientific Research Starting Foundation for Returned Overseas Chinese Scholars, Ministry of Education, Heilongjiang (Grant No. LC2009C24).
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