bims-mideyd 2025-07-13 papers

Eur J Med Res. 2025 Jul 07. 30(1): 583

Identifying the important involvement of cuproptosis in the pathophysiology of age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a neurodegenerative disease associated with severe visual impairment in the elders. Despite recent studies and advances, the pathophysiology underlying the development of AMD is still not fully understood, and current therapies remain limited. Programmed cell death (PCD) has been implicated in neurodegenerative diseases. Therefore, the aim of this study is to investigate and identify key types of PCD and PCD-related genes involved in the pathogenesis of age-related macular degeneration (AMD).
METHODS: This study employs transcriptomic analyses and animal experiments. For bulk tissue transcriptomic analysis, the enrichment scores of PCD forms in AMD samples were calculated using the single-sample gene set enrichment analysis (ssGSEA) algorithm. Single-cell transcriptomic analysis was conducted to examine the expression of PCD-related genes across different cell types. A mouse model was used to evaluate the therapeutic effects of a copper chelator on AMD.
RESULTS: Enrichment analysis indicated that cuproptosis was the most enriched type of PCD process in both AMD-affected macular and retinal pigment epithelium (RPE) samples. In addition, cuproptosis was associated with the progression of AMD. Single-cell transcriptomic analysis revealed that cuproptosis was highly activated in varies retinal cells from AMD samples when compared to normal ones. Pathway enrichment analysis showed that cuproptosis was associated with angiogenesis, inflammation, and cellular senescence in AMD. Furthermore, the copper chelator demonstrated a protective effect on retinal function in the AMD mouse model.
CONCLUSIONS: Our findings identified an important role of cuproptosis in the pathophysiology of AMD and suggested the potential of cuproptosis as a therapeutic target for AMD.

Keywords: Age-related macular degeneration; Copper; Cuproptosis; Programmed cell death; Single-cell RNA sequencing

DOI: https://doi.org/10.1186/s40001-025-02818-7

Cells. 2025 Jul 01. pii: 1007. [Epub ahead of print]14(13):

Porcine Single-Eye Retinal Pigment Epithelium Cell Culture for Barrier and Polarity Studies.

Philipp Dörschmann, Sina von der Weppen, Emi Koyama, Johann Roider, Alexa Klettner.

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein.

Keywords: 3R principle; Transwell; age-related macular degeneration; barrier; best practice protocol; polarity; primary cell culture; retinal pigment epithelium; single-eye culture; tight junctions

DOI: https://doi.org/10.3390/cells14131007

Invest Ophthalmol Vis Sci. 2025 Jul 01. 66(9): 20

DJ-1 Promotes Diabetic Corneal Epithelial Wound Healing by Attenuating Hyperglycemia-Induced Oxidative Stress Through Inhibiting PTEN.

Haoyu Li, Hanhan Peng, Benteng Ma, Xinyue Sun, Liwei Zhang, Baihua Chen.

Purpose: Diabetic keratopathy (DK) is characterized by delayed corneal epithelial wound healing and impaired nerve regeneration, primarily due to mitochondrial oxidative stress. DJ-1 plays a key role in redox regulation. This study explores the effects of DJ-1 downregulation on DK and its mechanisms.
Methods: Type 1 diabetes was induced in male C57BL/6J mice, and DJ-1 was overexpressed. Corneal oxidative stress and activity were assessed using DHE, Ki67, and TUNEL staining. Epithelial repair and nerve regeneration were evaluated by epithelial wound healing and nerve staining. Human corneal epithelial cells (HCE-T) and primary human corneal epithelial cells were exposed to high-glucose conditions, while DJ-1 and phosphatase and tensin homolog (PTEN) expression were modulated in HCE-T cells. Mitochondrial alterations were assessed by transmission electron microscopy, mitochondrial membrane potential staining, and mitoSOX staining. DJ-1, PTEN, and antioxidant protein levels were measured by immunofluorescence and Western blotting.
Results: In diabetic mice and high glucose-treated cells, DJ-1 and antioxidant enzyme levels were significantly reduced, while PTEN expression increased, accompanied by mitochondrial structural and functional impairments. DJ-1 overexpression alleviated oxidative stress and apoptosis, enhanced cell proliferation, and promoted epithelial wound healing and nerve regeneration. In HCE-T cells, DJ-1 downregulated PTEN, upregulated antioxidant proteins, and restored mitochondrial function, reducing reactive oxygen species accumulation and activity loss caused by high glucose. PTEN activation under high glucose diminished DJ-1's protective effects. DJ-1 also directly interacted with PTEN, indicating a regulatory mechanism.
Conclusions: DJ-1 deficiency disrupts mitochondrial function, upregulates PTEN, and suppresses antioxidant protein expression, exacerbating corneal oxidative stress. These findings provide insights into molecular mechanisms underlying DK.

DOI: https://doi.org/10.1167/iovs.66.9.20

Sci Rep. 2025 Jul 07. 15(1): 24304

Evaluation of retinal pigment epithelium changes in serous pigment epithelial detachment using synthesized multi-contrast polarization-sensitive optical coherence tomography.

Kosei Yanagida, Masahiro Miura, Hidetaka Noma, Toshihiro Mino, Shinnosuke Azuma, Thitiya Seesan, Shuichi Makita, Yoshiaki Yasuno.

Retinal pigment epithelium (RPE) melanin thickness maps, derived from multi-contrast images-including the degree of polarization uniformity (DOPU), optical coherence tomography (OCT) angiography, and the attenuation coefficient-are obtained using multi-contrast polarization-sensitive OCT (PS-OCT). These maps have demonstrated utility for three-dimensional assessment of changes in melanin within the RPE (RPE-melanin). While both OCT angiography and the attenuation coefficient can be derived from conventional OCT, measuring the DOPU requires PS-OCT, which is not available on standard commercial OCT systems. To overcome this limitation, we utilized a convolutional neural network to generate DOPU-like images from standard OCT images and used these to calculate a synthesized RPE-melanin thickness map. We evaluated 22 eyes from 20 patients with serous pigment epithelial detachment (PED) secondary to age-related macular degeneration. Both original and synthesized RPE-melanin thickness maps were calculated from multi-contrast PS-OCT datasets. Active RPE lesions were defined as areas with RPE-melanin thickness of ≥ 70 μm (originalRPE70 and synRPE70 for the original and synthesized maps, respectively). Both synthesized and original RPE-melanin thickness maps closely resembled near-infrared autofluorescence imaging. Furthermore, both the originalRPE70 area and synRPE70 area were significantly positively correlated with the PED volume. Synthesized RPE-melanin thickness maps may be useful for clinical quantification of RPE-melanin.

Keywords: Age-related macular degeneration; Convolutional neural network; Melanin; Pigment epithelial detachment; Polarization-sensitive optical coherence tomography; Retinal pigment epithelium

DOI: https://doi.org/10.1038/s41598-025-09302-6

Mediators Inflamm. 2025 ;2025 8586711

Evaluation of Gene Expression and the Regulatory Role of microRNAs Related to the Mitogen-Activated Protein Kinase Signaling Pathway in Human Retinal Pigment Epithelial Cells Treated With Lipopolysaccharide A and Tacrolimus.

Aleksandra Kiełbasińska, Katarzyna Krysik, Dominika Janiszewska-Bil, Martyna Machaj, Zuzanna Lelek, Mariola Szulik, Patrycja Mickiewicz, Anita Lyssek-Boroń, Beniamin Oskar Grabarek.

Background: The retinal pigment epithelium (RPE) is central to retinal health and immune regulation. In diseases, such as proliferative vitreoretinopathy (PVR), dysregulated RPE function, driven by aberrant signaling pathways like mitogen-activated protein kinase (MAPK), contributes to fibrotic membrane formation and retinal detachment. Tacrolimus, an immunosuppressive agent, has shown potential to modulate signaling beyond immune cells, but its effect on MAPK signaling in RPE cells remains unclear. This study aimed to investigate the impact of tacrolimus on MAPK pathway gene expression and microRNA (miRNA)-mediated regulation in human RPE (H-RPE) cells under inflammatory conditions induced by lipopolysaccharide (LPS). Methods: H-RPE cells were treated with LPS and tacrolimus, and cell viability was evaluated by 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Transcriptomic profiling of 300 MAPK-related genes and corresponding miRNAs was performed using Affymetrix microarrays. Key targets were validated via quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Gene interaction networks were analyzed with STRING. Results: LPS significantly suppressed MAPK pathway gene and protein expression, including transforming growth factor beta 1 (TGF-β-1), mitogen-activated protein kinase kinase 7 (MAP2K7), mitogen-activated protein kinase 3 (MAPK3), and dual specificity phosphatase 4 (DUSP4). Tacrolimus reversed these effects in a time-dependent manner, restoring expression levels and modulating regulatory miRNAs (e.g., miR-3196, miR-27a/b-3p, miR-190a-3p, miR-149-3p). STRING analysis revealed a highly connected protein network, with MAPK3, MAPK8, and TRAF6 acting as central nodes. Conclusion: Tacrolimus modulates MAPK signaling in H-RPE cells by reversing LPS-induced suppression and regulating specific miRNAs. These findings suggest a potential therapeutic role for tacrolimus in mitigating inflammatory and fibrotic responses associated with PVR.

Keywords: lipopolysaccharide A; mitogen-activated protein kinase signaling pathway; proliferative vitreoretinopathy; retinal pigment epithelium; tacrolimus

DOI: https://doi.org/10.1155/mi/8586711