Korean J Ophthalmol.  2004 Dec;18(2):121-131. 10.3341/kjo.2004.18.2.121.

Effect of Basic Fibroblast Growth Factor-Saporin (bFGF-SAP) Conjugate on Bovine Choriocapillary Endothelial Cells

• Kim YH
• Kim HS
• Yoo JS

• KMID: 754377
• DOI: http://doi.org/10.3341/kjo.2004.18.2.121

Abstract

We evaluated the effect of a basic fibroblast growth factor (bFGF) and saporin conjugate (bFGF-SAP) on proliferation, migration and tubule formation in bovine choriocapillary endothelial cells (BCECs). Cell proliferation and MTS assays were done with 0.01, 0.1, 1, 10, and 100 nM bFGF-SAP, and an equimolar concentration of bFGF and saporin. TUNEL assay was performed to confirm apoptosis. Cells were treated with 1, 10, and 100 nM bFGF-SAP and migration assay and tubule formation assay were done. Results were evaluated with image analysis. All experiments were performed in triplicate and repeated three times. Viable cells (ID50 = 0.62) and cell proliferation by MTS assay (ID50 = 0.75 nM) were inhibited. Saporin caused cytotoxicity and inhibition of proliferation at high concentration. DNA fragmentation was identified by TUNEL assay. Migration and tubule formation were also inhibited. All mechanisms responsible for neovascularization were inhibited, and this could be applied in the management of subretinal choroidal neovascularization (SRN).

Keyword

basic FGF; saporin; choriocapillary endothelial cell; immunotoxin; choroidal neovascularization

MeSH Terms

Animals
Apoptosis/drug effects
Cattle
Cell Count
Cell Movement/drug effects
Cell Proliferation/drug effects
Cells, Cultured
Comparative Study
Cytotoxins/*pharmacology
Endothelium, Vascular/*drug effects
Fibroblast Growth Factor 2/*pharmacology
In Situ Nick-End Labeling
Neovascularization, Physiologic/drug effects
Plant Proteins/*pharmacology
Recombinant Fusion Proteins/*pharmacology

Figure

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  Fig. 1. Characterization of bovine choriocapillary endothelial cells. Bovine choriocapillary endothelial cells showed dark brown colored positive immuno-staining for von Willebrand factor. (magnification, ×10).

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  Fig. 2. Effect of bFGF-SAP, bFGF and saporin on bovine choriocapillary endothelial cells growth. A: bFGF-SAP showed a dose-dependent cytotoxicity to choriocapillary endothelial cells. Analysis by nonlinear regression using Sigmaplot V7.0 revealed a typical reverse sigmoid standard curve. ID50 was 0.62 nM. B: bFGF induced the proliferation of bovine choriocapillary endothelial cells dose-dependently. Saporin had no significant effect at lower concentrations, but showed cytotoxicity at a high concentration of 50 nM. Results are given as a mean S.D. ∗P < 0.01 compared with untreated control.

• 

  Fig. 3. Effect of bFGF-SAP, bFGF and saporin on bovine choriocapillary endothelial cells proliferation. A: bFGF-SAP showed dose-dependent inhibition of proliferation. Analysis by nonlinear regression using Sigmaplot V7.0 revealed a typical reverse sigmoid standard curve. ID50 was 0.75 nM. B: bFGF induced proliferation dose-dependently. Saporin showed inhibition of proliferation at a high concentration of 50 nM. Results are given as a mean S.D. ∗P < 0.01 compared with untreated control.

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  Fig. 4. Effect of serum on bFGF-SAP-induced inhibition of bovine choriocapillary endothelial cells. In comparison to the 10% FBS group, cell proliferation was decreased in the serum-free group at a given concentration of bFGF-SAP, but still showed a dose-dependent inhibition. The effect of serum was significant. Results are given as a mean S.D. ∗P < 0.01 compared between serum-free and 10% FBS at a given concentration of bFGF-SAP.

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  Fig. 5. bFGF-SAP-induced apoptosis of bovine choriocapillary endothelial cells. A: Control (bFGF-SAP-free). B: bFGF-SAP (10 nM). Cells positive for the TUNEL apoptosis marker were seen as green fluorescein labeled nuclei. (magnification, ×20).

• 

  Fig. 6. Migration assay of bovine choriocapillary endothelial cells. Migration area represents cellular migration indirectly. The control group showed a migration area of 85.48%. bFGF-SAP showed a migration area of 73.66 ± 4.09, 61.68 ± 5.07 and 56.21 ± 4.79% at the concentration of 1, 10 and 100 nM, respectively. Inhibition rate was dose-dependently increased, showing 13.8, 27.7 and 34.3% inhibition at 1, 10 and 100 nM of bFGF-SAP, respectively. Results are given as a mean S.D. ∗P < 0.01 compared with untreated control.

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  Fig. 7. Phase contrast photomicrograph of bovine choriocapillary endothelial cell migration. Migration area was dose-dependently decreased by bFGF-SAP. A: Initial control (bFGF-SAP-free) at day 0. B: Control after 72 hours culture. C: bFGF-SAP (1 nM). D: bFGF-SAP (10 nM). E: bFGF-SAP (100 nM). (magnification, ×10).

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  Fig. 8. Tubule formation assay of bovine choriocapillary endothelial cells. Tubule density represents tubule formation indirectly. The control group showed a tubule area of 27.75 ± 3.37%. bFGF-SAP showed a tubule density of 24.46 ± 2.18, 10.38 ± 0.84 and 5.15 ± 0.56% at the concentration of 1, 10 and 100 nM, respectively. Inhibition rate was dose-dependently increased, showing 11.84, 62.58 and 81.42% inhibition at 1, 10 and 100 nM of bFGF-SAP, respectively. Results are given as a mean S.D. ∗P < 0.01 compared with untreated control.

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  Fig. 9. Phase contrast photomicrograph of tubule formation. bFGF-SAP shows a dose-dependent inhibition of tubule formation. A: Control (bFGF-SAP-free). B: bFGF-SAP (1 nM). C: bFGF-SAP (10 nM). D: bFGF-SAP (100 nM). (magnification, ×15).

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