Zeaxanthin and α-tocopherol reduce the inhibitory effects of photodynamic stress on phagocytosis by ARPE-19 cells

Abstract

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, β5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/β-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Keywords: ARPE-19 cells; Antioxidants; Cholesterol hydroperoxides; Lipid peroxidation; Phagocytosis; Phagocytosis receptors MerTK and αvβ5 integrin; Photoreceptor outer segments; Zeaxanthin; α-Tocopherol.

Figure 1

Effect of antioxidants on cytotoxicity in ARPE-19 cells subjected to photodynamic stress mediated by merocyanine-540 (A) or rose Bengal (B). Control cultures (closed circles) or cultures enriched with zeaxanthin and α-tocopherol (open squares) were incubated with a range of concentrations of MC-540 or RB and exposed to green light followed by the MTT assay as an indicator of toxicity. Data are the percent of control cultures (irradiated without photosensitizers) expressed as means of triplicate cultures; error bars indicate SD.

Figure 2

Accumulation of lipid hydroperoxides 7α/β-OOH (open triangles) and 5α-OOH (black squares) in ARPE-19 cells undergoing photosensitized oxidation. Cells were incubated with a range of concentrations of MC-540 (A) or RB (B) and exposed to green light. After irradiation, lipids were extracted from 5 x10 ⁶ of cells and subjected to HPLC-EC (Hg) analysis. Values are means of triplicate cultures; error bars indicate SD.

Figure 3

The effect of antioxidants on the photosensitized generation of cholesterol hydroperoxide 5α-OOH (A, B) and 7α/β-OOH (C, D) in ARPE-19 cells without photosensitizers (controls) or in cells pre-loaded with (A,C) MC-540 or (B, D) RB. Cultures without antioxidants (black bars) or enriched with zeaxanthin and α-tocopherol (open bars) were incubated with a range of concentrations of MC-540 or RB and exposed to green light. After irradiation, lipids were extracted from 5 x10 ⁶ cells and subjected to HPLC-EC (Hg) analysis. Values are means of triplicate cultures; error bars indicate SD. Data were corrected for any variation in the amount of extracted lipids using cholesterol as an internal standard. Asterisks indicate significant differences between non-supplemented and antioxidant-treated cultures (t-test, P <0.05).

Figure 4

The effect of antioxidants on the specific phagocytic activity of ARPE-19 cells subjected to photoinduced oxidative stress with MC-540 (A) or RB (B). Cultures without antioxidants (black bars) or enriched with zeaxanthin and α-tocopherol (open bars) were incubated with a range of concentrations of MC-540 or RB and exposed to green light. POS-FITC were delivered to cultures and uptake was quantified by flow cytometry. Data were normalized to control cultures lacking photosensitizers. Values are means of six replicate cultures; error bars indicate SD. Asterisks indicate significant differences between non-supplemented and antioxidant-treated cultures (t-test, P <0.05).

Figure 5

Western blot analysis for (A) the αv integrin subunit, (B) the β5 integrin subunit, (C) MerTK, or (D) actin in antioxidant supplemented (open bars) or non-supplemented ARPE-19 cultures (black bars). Data are the mean band densities, given in arbitrary units (AU), from extracts of triplicate culture wells from each group in a representative experiment; error bars indicate SD (n= 4). Outcomes do not differ significantly for any protein (t-test analyses).

Figure 6

Western blot analysis for MerTK protein in ARPE-19 cultures without antioxidants or supplemented with zeaxanthin/α-tocopherol and subjected to photodynamic stress mediated by (A) 8 μM MC-540 or (B) 600 nM RB followed by light irradiation. Blots from triplicate cultures for each group are shown. Graphs show densitometric analysis of the bands for non-supplemented (black bars) or antioxidant supplemented cultures (open bars). Band densities in the photosensitizer-treated groups are expressed as a percent of their respective controls; error bars indicate SD. MerTK signals without versus with antioxidants differ significantly for both photosensitizers (t-test, P<0.05).

Figure 7

Western blot analysis for (A,B) αv integrin and (C,D) β5 integrin subunits in ARPE-19 cultures without antioxidants or supplemented with zeaxanthin/α-tocopherol and subjected to photodynamic stress mediated by (A,C) 8 μM MC-540 or (B,D) 600 nM RB followed by light irradiation. Blots from triplicate cultures for each group are shown. Graphs show densitometric analysis of the bands for non-supplemented (black bars) or antioxidant supplemented cultures (open bars) with the band densities in the photosensitizer-treated groups expressed as a percent of their respective controls. Error bars indicate SD. αv integrin and β5 integrin signals without versus with antioxidants differ significantly for both photosensitizers (t-test, P<0.05).

Figure 8

Western blot analysis for actin in ARPE-19 cultures without antioxidants or supplemented with zeaxanthin/α-tocopherol and subjected to photodynamic stress mediated by 8 μM MC-540 followed by green light irradiation. Blots from triplicate cultures for each group are shown. Graphs show densitometric analysis of the bands for non-supplemented (black bars) or antioxidant supplemented cultures (open bars). Band densities in the photosensitizer-treated groups are expressed as a percent of their respective controls; error bars indicate SD. Actin signals without versus with antioxidants do not differ significantly (t-test analysis, P<0.05).