*2.4. VEGF Secretion of ARPE-19*

We tested the influence of the ten di fferent fucoidans on the VEGF secretion of the human RPE cell line ARPE-19. The optimal parameters for VEGF determination after fucoidan treatment were determined in a previous study [11]. In brief, cells were incubated for three days with the fucoidans and media exchange was done 24 h before taking of the supernatant for a subsequent ELISA analysis. VEGF in % was set in relation to cell viability in % both compared to untreated control (in arbitrary units [arb. unit]. The cell viability of both cell types was essentially una ffected by treatment with any sulfated fucans and at any tested concentrations (data not shown).

LD\_SiAT2 lowered secreted VEGF at 10, 50 and 100 μg/mL to 0.92 ± 0.08 [arb. unit (arbitrary unit)] (*p* < 0.05), 0.88 ± 0.04 [arb. unit] (*p* < 0.01) and 0.81 ± 0.07 [arb. unit] (*p* < 0.001), respectively (Figure 5). SL\_F2 and SL\_F3 reduced VEGF significantly at all tested concentration, in contrast to the first fraction SL\_F1 and the unfractionated SL\_SiAT2 with the highest e ffect at 100 μg/mL (SL\_F2 with 0.40 ± 0.07 [arb. unit], *p* < 0.001 and SL\_F3 with 0.37 ± 0.04 [arb. unit], *p* < 0.001). The FE extracts did not show any significant VEGF reducing e ffects, which could be also due to the high standard deviation and heterogeneous results, with the only exception of FE\_SiAT2, which lowered VEGF significant at 10 μg/mL to 0.80 ± 0.21 [arb. unit], *p* < 0.05 and at 100 μg/mL to 0.72 ± 0.10 [arb. unit], *p* < 0.001.

**Figure 5.** Secreted VEGF (vascular endothelial growth factor) of ARPE-19 after three days of incubation with 1, 10, 50 and 100 μg/mL LD fucoidan (**a**), SL fucoidans (**b**) and FE fucoidans (**c**). VEGF amount was determined with ELISA and normalized to cell survival, making a quotient of VEGF and cell viability. 10–100 μg/mL LD\_SiAT2 and 1–100 μg/mL SL\_F2 and SL\_F3 decreased VEGF significantly. Significant values were analyzed with ANOVA, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to the control (*n* ≥ 4, number of independent experiments).
