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. 2023 Jul 25;9(1):44.
doi: 10.1186/s40942-023-00476-7.

The role of mitochondrial genes on nuclear gene expression in neovascular age related macular degeneration: analysis of nuclear VEGF gene expression after ranibizumab treatment in cytoplasmic hybrid retinal pigment epithelial cell lines correlated with clinical evolution

Rodrigo Donato Costa #  1   2 , Farid José Thomaz Neto #  1 , M Tarek Moustafa  1   3 , Shari R Atilano  1 , Marilyn Chwa  1 , Javier Cáceres-Del-Carpi  1   4 , Mohamed Hamid Mohamed  1   3 , M Cristina Kenney  1 , Baruch D Kuppermann  5
Affiliations

The role of mitochondrial genes on nuclear gene expression in neovascular age related macular degeneration: analysis of nuclear VEGF gene expression after ranibizumab treatment in cytoplasmic hybrid retinal pigment epithelial cell lines correlated with clinical evolution

Rodrigo Donato Costa et al. Int J Retina Vitreous. .

Abstract

Purpose: The present study tests the hypothesis that mitochondrial genes have retrograde signaling capacity that influences the expression of nuclear genes related to angiogenesis pathways. Cytoplasmic hybrid (cybrid) in vitro cell lines with patient specific mitochondria inserted into an immortalized retinal pigment epithelial cell line (ARPE-19) were used to test this hypothesis. This type of analysis can provide important information to identify the optimal regimen of anti-VEGF treatment, personalizing age-related macular degeneration (AMD) therapies.

Methods: Mitochondria deficient ARPE-19 cells (Rho0) were fused with AMD donor's platelets to create individual cybrid cell lines containing mitochondria from patients with phenotypic AMD disease and nuclear DNA from the immortalized RPE cell line. The cybrids were treated with Ranibizumab (Lucentis, Genentech, San Francisco, CA), at 4 different concentrations for 24 h, and subsequently the levels of reactive oxygen species (ROS), gene expression for VEGF-A, hypoxia-inducible factor 1-alpha (HIF1-a) and manganese superoxide dismutase (SOD2) were measured. The clinical evolution of the two AMD-donors were correlated with the molecular findings found in their 'personalized' cybrids.

Results: Cybrids from Patient-01 showed down-regulation of gene expression of VEGF-A and HIF-1a at both 1X and 4X Ranibizumab concentrations. Patient-01 AMD cybrid cultures had an increase in the ROS levels at 1X (P = 0.0317), no changes at 2X (P = 0.8350) and a decrease at 4X (P = 0.0015) and 10X (P = 0.0011) of Ranibizumab. Clinically, Patient-01 responded to anti-VEGF therapy but eventually developed geographic atrophy. Patient-02 cybrids demonstrated up-regulation of gene expression of VEGF-A and HIF-1a at Ranibizumab 1X and 4X concentrations. There was decreased ROS levels with Ranibizumab 1X (P = 0.1606), 2X (P = 0.0388), 4X (P = 0.0010) and 10X (P = < 0.0001). Clinically, Patient-02 presented with a neovascular lesion associated with a prominent production of intraretinal fluid in clinical follow-up requiring regular and repeated intravitreal injections of Ranibizumab with recurrent subretinal fluid.

Conclusions: Our cybrid model has the potential to help personalize the treatment regimen with anti-VEGF drugs in patients with neovascular AMD. Further investigation is needed to better understand the role that the mitochondria play in the cellular response to anti-VEGF drugs. Future studies that focus on this model have the potential to help personalize anti-VEGF treatment.

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Conflict of interest statement

Rodrigo Donato Costa: No competing interests Farid José Thomaz Neto: No competing interests M. Tarek Moustafa: No competing interests Shari R. Atilano: No competing interests Marilyn Chwa: No competing interests Javier Cáceres-del-Carpi: No competing interests Mohamed Hamid Mohamed: No competing interests M. Cristina Kenney: No competing interests Baruch D. Kuppermann: Clinical Research: Allegro Ophthalmics, Allergan, Genentech Inc, Ionis, IVERIC Bio, Novartis Pharmaceuticals, Regeneron Pharmaceuticals Inc, RegenXBio Consultant: Allegro Ophthalmics, Allergan, Aviceda Therapeutics, Clearside, EyeBio, Eyedaptic, Genentech Inc, Glaukos Corporation, InflammX Therapeutics, IVERIC Bio, jCyte, Novartis Pharmaceuticals, Regeneron Pharmaceuticals Inc, ReVana Therapeutics, Ripple Therapeutics, Theravance Biopharma Speaker Bureau: Allergan, Genentech.

Figures

Fig. 1
Fig. 1
Case report for patient-01 (CYBRID 14–141). Clinical history of blood donor of cybrid 14–141: a. Right Eye before treatment. b. Right Eye after 3 intravitreal injections of Ranibizumab. c. Right Eye after 4 intravitreal injections of Ranibizumab. d. Right Eye after 8 intravitreal injections of Ranibizumab. e. Right Eye after 8 intravitreal injections of Ranibizumab + 4 intravitreal injections of Aflibercept. No more injections in the last months, lesion remains dry in right eye
Fig. 2
Fig. 2
a. Left Eye before treatment. b. Left Eye after the 3rd intravitreal injection of Ranibizumab. c. Left Eye after the 4th intravitreal injection of Ranibizumab. d. Left Eye after the 12th intravitreal injection of Ranibizumab. e. Left Eye after the 18th intravitreal injection of Ranibizumab. Lesion remains producing fluid in the left eye
Fig. 3
Fig. 3
a–e Cybrid 14–141 ROS levels at 1X, 2X, 4X, 10X and untreated. Fold values of VEGF-A, HIF1-A, and SOD2 gene expression after treatment at 1X, 4X and untreated
Fig. 4
Fig. 4
a–e Cybrid 14–144 ROS levels at 1X, 2X, 4X, 10X and untreated. Fold values of VEGF-A, HIF1-A, and SOD2 gene expression after treatment at 1X, 4X and untreated
Fig. 5
Fig. 5
Correlation between SOD2 and Cellular Superoxide. SO, superoxides; GP, glutathione peroxidase; Catalas, catalase; Peroxynitrl, peroxynitrile; Oxyg, oxygen; NO, nitric oxide; O−2, singlet oxygen, superoxide
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