bims-mideyd 2025-06-01 papers

Free Radic Biol Med. 2025 May 28. pii: S0891-5849(25)00722-1. [Epub ahead of print]

Hinokitiol Protects RPE Cells from Oxidative and Autophagic Dysfunction: Implications for AMD Therapy.

Ko-Chieh Huang, Yi-Fen Chiang, Kai-Lee Wang, Yun-Ju Huang, Tzong-Ming Shieh, Mohamed Ali, Shih-Min Hsia.

Age-related macular degeneration (AMD) is a leading cause of vision loss in the elderly, driven by dysfunction of retinal pigment epithelial (RPE) cells. Oxidative stress-induced reactive oxygen species (ROS) play a critical role in AMD progression, although the underlying mechanisms remain unclear. Autophagy is essential for maintaining retinal homeostasis by clearing damaged organelles and misfolded proteins through lysosomal degradation. However, excessive ROS can disrupt autophagy balance, leading to the excessive degradation of cell components and ultimately triggering autophagy dysfunction-induced cell death. Hinokitiol, a natural compound derived from the heartwood of Cupressaceae plants, possesses potent antioxidant properties. This study aimed to investigate its roles against oxidative damage in RPE cells exposed to H2O2-induced ROS generation. Cell viability was assessed using MTT and crystal violet staining. ROS were measured using H2DCFDA and MitoSOX probes, while catalase activity was evaluated as indicator of antioxidant capacity. DNA damage was assessed by γ-H2AX immunocytochemistry and comet assay. Mitochondrial membrane potential (MMP) was analyzed using JC-1, and autophagy markers were examined by Western blotting. Hinokitiol significantly enhanced RPE cell viability, reduced ROS by increasing catalase activity, preserved mitochondrial function, and mitigated DNA damage. Furthermore, it restored autolysosome fusion impaired by H2O2, thereby maintaining cellular homeostasis. These findings suggest that hinokitiol may be a promising therapeutic candidate for AMD treatment.

Keywords: AMD; RPE; hinokitiol; oxidative stress

DOI: https://doi.org/10.1016/j.freeradbiomed.2025.05.424

Antioxidants (Basel). 2025 May 10. pii: 575. [Epub ahead of print]14(5):

Aster-B Modulates Oxidative Stress Responses and Carotenoid Distribution in ARPE-19 Cells.

Vidya Gopakumar, Johannes von Lintig.

Lipid metabolism and oxidative stress are major contributors to ocular diseases, including drusen formation and photoreceptor damage. Aster-B, encoded by GRAMD1B, mediates the non-vesicular transport of cholesterol and carotenoids and is highly expressed in the human eye, though its specific ocular functions remain unknown. We investigated Aster-B's role in ARPE-19 cells, a model of the retinal pigment epithelium (RPE), using CRISPR/dCas9 to generate an Aster-B-expressing cell line. Aster-B expression significantly improved cell survival under oxidative stress induced by hydrogen peroxide (H2O2) and was associated with the activation of the p53 and TGFβ signaling pathways, indicating a role in modulating stress responses. To confirm its lipid transport activity, we treated cholesterol-depleted cells with carotenoids and tracked their localization. In Aster-B-expressing cells, carotenoids accumulated in mitochondria, while in control cells, they remained in other cellular compartments. Under oxidative stress, mitochondrial carotenoid levels declined in Aster-B-expressing cells but not in control cells. Interestingly, carotenoids enhanced survival in control cells exposed to H2O2 but had a detrimental effect in Aster-B-expressing cells, suggesting that carotenoid function is context and location dependent. These findings highlight Aster-B's role in coordinating lipid transport and stress responses in the RPE, with implications for oxidative stress-related eye diseases.

Keywords: Aster-B; CRISPR/dCas9; carotenoids; cholesterol; oxidative stress; retinal pigment epithelium

DOI: https://doi.org/10.3390/antiox14050575

Antioxidants (Basel). 2025 May 16. pii: 596. [Epub ahead of print]14(5):

Targeting Lysosomal Dysfunction and Oxidative Stress in Age-Related Macular Degeneration.

Ana S Falcão, Margarida L Pedro, Sandra Tenreiro, Miguel C Seabra.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the Western world, and it currently lacks effective therapy. It is believed that AMD initiates in the aged retinal pigment epithelium (RPE), which presents lysosomal dysfunction and oxidative stress (OxS) that ultimately leads to RPE damage and AMD progression. AMD is a complex pathology, so multitarget treatments are required to act on different pathways, presenting several challenges. In this review, we discuss the current knowledge on the pathogenesis of this disease, focusing mainly on lysosomal dysfunction and OxS. Because transcription factors regulate homeostasis, the transcription factor EB (TFEB), which controls lysosomal function and biogenesis, and the nuclear factor erythroid 2-related factor 2 (NRF2), which manages OxS, have been proposed as promising targets for disease intervention. Finally, we discuss the interplay of these pathways for a potential synergistic effect on AMD-targeted therapies, as they could change the course of today's available treatments for AMD.

Keywords: NRF2; TFEB/mTORC1 axis; age-related macular degeneration; lysosomal dysfunction; oxidative stress; retinal pigment epithelium

DOI: https://doi.org/10.3390/antiox14050596

Exp Eye Res. 2025 May 22. pii: S0014-4835(25)00216-7. [Epub ahead of print]257 110445

CRB1 mutations cause structural and molecular defects in patient-derived retinal pigment epithelium cells.

Yini Wang, Yuqin Liang, Yalan Zhou, Zekai Cui, Jianing Gu, Siqi Xiong, Jiansu Chen.

Mutations in the CRB1 gene can cause retinitis pigmentosa (RP), Leber congenital amaurosis, and other retinopathies, with retinal pigment epithelium (RPE) being a primary affected cell type. However, the effects of CRB1 variants on RPE cells remain poorly defined. Here, for the first time, we report an in vitro model of patient-specific RPE cells carrying the CRB1 mutations (c.2249G > A and c.2809G > A) to study CRB1-associated RP disease. The patient-derived RPE cells exhibited irregular cell morphology, sparse apical microvilli, abnormal tight junctions, and reduced expression of RPE markers. We also observed that impaired barrier function and phagocytosis lead to increased apical-to-basal movement of fluorescent molecules in disease RPE cells. Notably, transcriptomic analysis revealed decreased expression of cell junction-related genes. In addition, aggregated RPE cells on polydimethylsiloxane (PDMS) microwells significantly enhanced RPE phenotype and cell survival, which was associated with anti-epithelial-mesenchymal transition, anti-aging, and anti-apoptosis. In this study, our results reveal that CRB1-mutated RPE cells generated using RP patient-derived iPSCs could recapitulate the genotype-phenotype features of the disease and provide insights into the pathogenesis of CRB1-associated RPE cells. In addition, our study developed a cell aggregation culture method based on PDMS microwell platforms for the production of highly active and mature iPSC-derived RPE cells.

Keywords: CRB1 variant; Disease modeling; Retinal pigment epithelium; Retinitis pigmentosa

DOI: https://doi.org/10.1016/j.exer.2025.110445

Biomedicines. 2025 May 10. pii: 1167. [Epub ahead of print]13(5):

Cannabidiol-Loaded Retinal Organoid-Derived Extracellular Vesicles Protect Oxidatively Stressed ARPE-19 Cells.

Peggy Arthur, Sangeetha Kandoi, Anil Kalvala, Breana Boirie, Aakash Nathani, Mounika Aare, Santanu Bhattacharya, Tanmay Kulkarni, Li Sun, Deepak A Lamba, Yan Li, Mandip Singh.

Background/Objectives: Age-related macular degeneration (AMD) is the third leading cause of irreversible blindness in elderly individuals aged over 50 years old. Oxidative stress plays a crucial role in the etiopathogenesis of multifactorial AMD disease. The phospholipid bilayer EVs derived from the culture-conditioned medium of human induced pluripotent stem cell (hiPSC) differentiated retinal organoids aid in cell-to-cell communication, signaling, and extracellular matrix remodeling. The goal of the current study is to establish and evaluate the encapsulation of a hydrophobic compound, cannabidiol (CBD), into retinal organoid-derived extracellular vesicles (EVs) for potential therapeutic use in AMD. Methods: hiPSC-derived retinal organoid EVs were encapsulated with CBD via sonication (CBD-EVs), and structural features were elucidated using atomic force microscopy, nanoparticle tracking analysis, and small/microRNA (miRNA) sequencing. ARPE-19 cells and oxidative-stressed (H2O2) ARPE-19 cells treated with CBD-EVs were assessed for cytotoxicity, apoptosis (MTT assay), reactive oxygen species (DCFDA), and antioxidant proteins (immunohistochemistry and Western blot). Results: Distinct miRNA cargo were identified in early and late retinal organoid-derived EVs, implicating their roles in retinal development, differentiation, and functionality. The therapeutic effects of CBD-loaded EVs on oxidative-stressed ARPE-19 cells showed greater viability, decreased ROS production, downregulated expression of inflammation- and apoptosis-related proteins, and upregulated expression of antioxidants by Western blot and immunocytochemistry. Conclusions: miRNAs are both prognostic and predictive biomarkers and can be a target for developing therapy since they regulate RPE physiology and diseases. Our findings indicate that CBD-EVs could potentially alleviate the course of AMD by activating the targeted proteins linked to the adenosine monophosphate kinase (AMPK) pathway. Implicating the use of CBD-EVs represents a novel frontline to promote long-term abstinence from drugs and pharmacotherapy development in treating AMD.

Keywords: AMPK; ARPE-19 cells; cannabinoid; extracellular vesicles; oxidative stress; retinal organoids; small RNA profiling

DOI: https://doi.org/10.3390/biomedicines13051167