*2.4. VEGF Secretion*

The e ffect of the three LH sulfated fucans on the secretion of VEGF was determined in the primary, porcine RPE cells and the human RPE cell line ARPE-19. ARPE-19 secreted nearly 4 times less VEGF compared to the primary RPE cells (ARPE-19: 66 pg/h and RPE: 243 pg/h). The VEGF content secreted into the supernatant of the cells was collected for ARPE-19 over 24 h and for RPE over 4 h because of the higher VEGF outcome. At this time point a medium exchange with added sulfated fucans was performed. The VEGF content [pg/mL] was normalized to the cell viability (after 72 h sulfated fucan treatment), giving the ratio of VEGF/cell viability (in arbitrary units [arb. unit]). The cell viability of both cell types with all sulfated fucans and all test concentrations was essentially una ffected (data not shown).

The secreted VEGF was lowered by all tested sulfated fucans in ARPE-19 (Figure 8). Fuc3 reduced VEGF to 0.84 ± 0.05 [arb. unit], *p* < 0.001, and Fuc2 lowered it to 0.66 ± 0.01 [arb. unit], *p* < 0.001, at 100 μg/mL. The e ffect of Fuc2 and Fuc3 on VEGF was concentration dependent. This did not apply for Fuc1, the sulfated fucan with the highest molecular weight, which reduced VEGF to 0.48 ± 0.04 [arb. unit], *p* < 0.01, at 50 μg/mL and had the strongest effect of all sulfated fucans at this concentration.

**Figure 8.** Vascular endothelial growth factor (VEGF) secretion of ARPE19 cells after incubation with Fuc1 (**a**), Fuc2 (**b**), and Fuc3 (**c**). VEGF content was analyzed with ELISA and normalized to cell viability. All LH extracts reduced VEGF content with 50 μg/mL Fuc1 as the most efficient. Significance was evaluated with ANOVA; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to the control (*n* = 4).

Due to the high amount of VEGF production in RPE cells, weaker effects could be expected, but again VEGF was reduced by Fuc2 and Fuc1 (Figure 9). Fuc3, the smallest LH sulfated fucan, seemed to increased VEGF secretion at 10 μg/mL (1.21 ± 0.04 [arb. unit]); however, this change was not significant. Fuc2 reduced VEGF at 50 μg/mL to 0.64 ± 0.12 and at 100 μg/mL to 0.63 ± 0.10 [arb. unit], *p* < 0.01, and Fuc1 reduced VEGF at 50 μg/mL to 0.30 ± 0.09 [arb. unit], *p* < 0.001. Once more, Fuc1 had the highest efficiency and, similar to what was seen in ARPE-19, again showed the strongest effect at 50 μg/mL.

**Figure 9.** VEGF secretion of RPE cells after incubation with Fuc1 (**a**), Fuc2 (**b**), and Fuc3 (**c**). VEGF content was analyzed via ELISA and normalized to the cell viability. Fuc2 and Fuc1 reduced the VEGF content. Significance was evaluated via ANOVA; \* *p* < 0.05, \*\**p* < 0.01, compared to the control (*n* = 3).
