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. 2022 Dec 16:2022:7497816.
doi: 10.1155/2022/7497816. eCollection 2022.

Short-Term In Vitro ROS Detection and Oxidative Stress Regulators in Epiretinal Membranes and Vitreous from Idiopathic Vitreoretinal Diseases

Bijorn Omar Balzamino  1 , Lucia Dinice  1 , Andrea Cacciamani  2 , Agnese Re  3 , Fabio Scarinci  2 , Luca Bruno  1 , Pamela Cosimi  2 , Alessandra Micera  1
Affiliations

Short-Term In Vitro ROS Detection and Oxidative Stress Regulators in Epiretinal Membranes and Vitreous from Idiopathic Vitreoretinal Diseases

Bijorn Omar Balzamino et al. Biomed Res Int. .

Abstract

Background: A plethora of inflammatory, angiogenic, and tissue remodeling factors has been reported in idiopathic epiretinal membranes (ERMs). Herein we focused on the expression of a few mediators (oxidative, inflammatory, and angiogenic/vascular factors) by means of short-term vitreal cell cultures and biomolecular analysis.

Methods: Thirty-nine (39) ERMs and vitreal samples were collected at the time of vitreoretinal surgery and biomolecular analyses were performed in clear vitreous, vitreal cell pellets, and ERMs. ROS products and iNOS were investigated in adherent vitreal cells and/or ERMs, and iNOS, VEGF, Ang-2, IFNγ, IL18, and IL22 were quantified in vitreous (ELISA/Ella, IF/WB); transcripts specific for iNOS, p65NFkB, KEAP1, NRF2, and NOX1/NOX4 were detected in ERMs (PCR). Biomolecular changes were analyzed and correlated with disease severity.

Results: The higher ROS production was observed in vitreal cells at stage 4, and iNOS was found in ERMs and increased in the vitreous as early as at stage 3. Both iNOS and NOX4 were upregulated at all stages, while p65NFkB was increased at stage 3. iNOS and NOX1 were positively and inversely related with p65NFkB. While NOX4 transcripts were always upregulated, NRF2 was upregulated at stage 3 and inverted at stage 4. No significant changes occurred in the release of angiogenic (VEGF, Ang-2) and proinflammatory (IL18, IL22 and IFNγ) mediators between all stages investigated.

Conclusions: ROS production was strictly associated with iNOS and NOX4 overexpression and increased depending on ERM stadiation. The higher iNOS expression occurred as early as stage 3, with respect to p65NFkB and NRF2. These last mediators might have potential prognostic values in ERMs as representative of an underneath retinal damage.

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Conflict of interest statement

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest, or nonfinancial interest in the subject matter or materials discussed in this manuscript.

Figures

Figure 1
Figure 1
Representative infrared (left) and related spectral domain-optical coherence tomography (OCT, right) images showing the epiretinal membrane (ERM) layered over retinal tissues. Note the ERM traction causing foveal distortion and alterations of the inner retinal structures in all stages investigated, according to the Govetto classification systems. (a) Stage 2, ERM with flattened foveal contour; (b) stage 3, ERM with the presence of ectopic inner foveal layer, and (c) stage 4, ERM with disorganization of inner and outer retinal layers and macular disruption. Green line indicates the examined region; scale bar = 200 μm.
Figure 2
Figure 2
Intracellular ROS immunoreactivity in short-term cultured vitreal cells depending on ERM severity. Adherent cells were evaluated for the ability to produce ROS products by using the Cellular ROS Assay Kit. Not-pooled samples were used for H2DCFDA assay. Fluorescent intensities were monitored and acquired. (a) Representative confocal images of intracellular ROS in vitreous adhering cells at different stages of disease progression. (b) Bar plots showing the increased ROS expression depending on ERM severity. IntDen, Integrated Density. ANOVA analysis followed by the Tukey-Kramer post hoc highlighted the significant effects indicated in the graphs, ∗p ≤ 0.05.
Figure 3
Figure 3
iNOS protein changes as function of disease severity. Epifluorescent and Western Blot analyses followed by densitometric analysis (below: ImageJ, IntDen). (a) ERMs. Representative epifluorescent images and IntDen histogram displaying the immunoreactivity of iNOS protein depending on ERM severity. Merged (green/blue) panels of iNOS (green) over a DAPI counterstaining (blue nuclei). Scale bar = 50 μm (b) vitreous. Vitreal iNOS immunoblotting and related iNOS band quantitation (120 kDa expected size). Immunoblots were destained and reprobed against β-actin (40 kDa expected band). Values are mean ± SD from 3 independent experiments carried out in triplicate.
Figure 4
Figure 4
Inflammatory and oxidative stress regulators' transcripts in ERMs. Relative expression ratio (fold-changes; mean ± SD; log2-scale) in ERMs at stage 3 and stage 4, with respect to stage 2 (Rest-ANOVA Tukey-Kramer's coupled analysis). (a) Histogram showing a significant upregulation for iNOS (stage 4), NOX1 (stage 3 and stage 4), and p65NFkB (stage 3) transcripts, with respect to stage 2 (∗p < 0.05). (b) Significant changes in the transcripts' expression were observed in NRF2 (upregulation at stage 3 and deregulation and stage 4) and NOX4 (upregulation at stage 3 and stage 4), as calculated with respect to stage 2 (∗p < 0.05).
Figure 5
Figure 5
Correlation between p65NFkB, KEAP1, and NRF2 upon disease severity. Plots showing the correlation between the inflammatory transcription factor (p65NFĸB) and the epigenetic genes KEAP1 (a) and NRF2 (b) and between NRF2 and KEAP1 (c). rho and p values are shown in the panels (Pearson's rho test analysis).
Figure 6
Figure 6
VEGF, Ang-2, IFNγ, IL18, and IL22 expression in vitreal samples. Untouched (cleared) vitreal samples were diluted and analyzed for VEGFA (a), Ang-2 (b), IFNγ (c), IL18 (d), and IL22 (e) protein expression and compared for ERM staging. As shown by scatter plots, no significant changes were detected in vitreal fluids depending on ERM severity/activation (pg/mg total protein).
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