Anti-inflammatory properties of antiangiogenic fucoidan in retinal pigment epithelium cells

**Background:** Age-related macular degeneration (AMD) is a leading cause of blindness, with inflammation, oxidative stress, and angiogenesis as key pathogenic drivers. Current treatments only target late exudative AMD via anti-VEGF therapy. Fucoidans, sulfated polysaccharides from brown algae, have shown anti-angiogenic and anti-oxidative properties. This study investigates the anti-inflammatory effects of fucoidan from Fucus vesiculosus (FV) in primary porcine retinal pigment epithelium (RPE) cells, a well-established model for adult human RPE.

**Methods:** Primary porcine RPE cells were isolated from freshly slaughtered pig eyes and cultured for two weeks. Inflammation was induced using 1 μg/mL lipopolysaccharide (LPS, TLR-4 agonist), 10 μg/mL polyinosinic:polycytidylic acid (Poly I:C, TLR-3 agonist), 50 ng/mL tumor necrosis factor alpha (TNF-α), or 10 ng/mL Pam2CSK4 (Pam, TLR-2 agonist). Cells were treated with 50 μg/mL FV fucoidan (Sigma-Aldrich, lot SLBT5471) for up to 28 days. Cell viability was assessed by MTT assay. Barrier function was measured by transepithelial electrical resistance (TEER) at days 0, 1, 3, 7, and 28. Secretion of IL-6 and IL-8 was quantified by ELISA at days 1, 3, 7, and 28. Gene expression of IL6, CXCL8, MERTK, and PIK3CA was analyzed by qPCR after 3 days (normalized to GAPDH). Protein expression of CD59 (protectin) and RPE65 was evaluated by Western blot after 1 and 3 days (normalized to β-actin). Phagocytosis of fluorescent latex beads opsonized with photoreceptor outer segments was measured after 1, 3, and 7 days. Statistical tests included one-sample t-test, ANOVA with student's t-test, or Kruskal-Wallis with Mann-Whitney U test, as appropriate; significance set at p ≤ 0.05.

**Key Results:** FV fucoidan did not reduce cell viability under any condition; after 1 day, it significantly increased MTT signal compared to untreated control (p < 0.05). TEER decreased over 28 days in all groups, but only co-stimulation with FV fucoidan and Poly I:C showed no significant reduction compared to day 0 (p > 0.05), indicating a protective effect on barrier integrity. For cytokine secretion, all inflammatory stimuli induced IL-6 and IL-8. FV fucoidan significantly reduced IL-6 secretion under Poly I:C stimulation at days 3, 7, and 28 (p < 0.05 to p < 0.01), and at day 28 also reduced TNF-α-induced IL-6 (p < 0.05). IL-8 secretion was significantly reduced by FV fucoidan only under Poly I:C at day 28 (p < 0.01). In qPCR, FV fucoidan alone significantly reduced IL6 expression (RQ 0.259, p = 0.008) and CXCL8 expression (RQ 0.032, p = 0.033) compared to Poly I:C control. For CD59 protein, Poly I:C (0.60 ± 0.19 a.u., p = 0.0352) and TNF-α (0.64 ± 0.04 a.u., p = 0.0005) significantly reduced expression at day 1; at day 3, TNF-α (0.77 ± 0.18 a.u., p = 0.0348) and LPS (0.76 ± 0.19 a.u., p = 0.0401) reduced CD59. In all cases, co-treatment with FV fucoidan resulted in loss of statistical significance, suggesting protection. RPE65 expression was significantly reduced by LPS (0.57 ± 0.22 a.u., p = 0.0186) and TNF-α (0.39 ± 0.43 a.u., p = 0.0463) at day 3; this reduction was not significant when cells were co-treated with FV fucoidan. Phagocytosis was reduced by inflammatory stimuli at various time points (e.g., after 7 days, LPS, TNF-α, and combinations with FV fucoidan reduced beads/cell to 2–6 vs. control 8 ± 3, p < 0.05), but FV fucoidan did not ameliorate this reduction. Gene expression of MERTK and PIK3CA showed no relevant changes (all RQ < 2-fold; only Pam increased PIK3CA to RQ 1.598, p = 0.049).

**Clinical Implications:** This study demonstrates that FV fucoidan possesses anti-inflammatory properties in RPE cells, specifically targeting TLR-3-mediated pathways (Poly I:C), while also modestly protecting CD59 and RPE65 expression under LPS and TNF-α stimulation. The compound did not affect cell viability or phagocytosis. Given that fucoidan also exhibits anti-angiogenic and anti-oxidative activities, it may simultaneously address three major AMD pathogenic pathways. The specificity for TLR-3 is particularly relevant, as TLR-3 activation is implicated in AMD-related chronic inflammation and RPE dysfunction. These in vitro findings support further investigation of fucoidan in in vivo AMD models and potentially as a preventive or early-stage therapeutic for AMD. However, the lack of effect on phagocytosis and the stimulus-dependent nature of the anti-inflammatory action highlight the need for additional mechanistic studies.