Short Pulse of Clinical Concentration of Bevacizumab Modulates Human Retinal Pigment Epithelial Functionality

Subramani, Murali; Murugeswari, Ponnalagu; Dhamodaran, Kamesh; Chevour, Priyanka; Gunasekaran, Sindhu; Kumar, Rajesh S.; Jayadev, Chaitra; Shetty, Rohit; Begum, Naheeda

doi:10.1167/iovs.15-18330

Cited by 9 publications

(31 citation statements)

References 54 publications

(65 reference statements)

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“…Human PRPE Cell Culture PRPE cells were isolated and cultured within 24 hours of enucleation from 10 noninfectious human cadaver eyes obtained from Shankar Anand Eye Bank, Narayana Nethralaya, Bangalore, India. 12 The age of the donor eyes range from 2 to 45 years (mean ± SD, 32 ± 15.8). Cells were cultured with 2% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) in an incubator with 21% O 2 for normoxia (HERAcell 240i; Heraeus, Germany) and 40% O 2 for hyperoxia (SMA-80DS/165; ASTEC, Japan) with 5% CO 2 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed on cultured PRPE cells using specific primers, as described previously 12,13 (See Supplementary Table S1).…”

Section: Reverse Transcriptase Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

“…The VEGF levels in cell supernatants from different experimental conditions (normoxia, hyperoxia, normoxia + VIT-D, and hyperoxia + VIT-D) were estimated using the human VEGF DuoSet ELISA (R&D Systems, Minneapolis, MN, USA), as described previously. 12,13 Immunofluorescence Staining PRPE cells were cultured on cover slips (12 mm; Blue Star, India) in a 12-well plate with a cell density of 1 × 10 4 cells/well in normoxia and hyperoxia with or without VIT-D for 5 days. Immunofluorescence staining was carried out with specific antibodies (See Supplementary Table S2), as previously described.…”

Section: Secreted Vegf Level Estimationmentioning

confidence: 99%

“…Immunofluorescence staining was carried out with specific antibodies (See Supplementary Table S2), as previously described. 12,13 For VEGF and DLL4, fluorescent images were captured using the ProgRes Capture Pro version 2.5 software on an Olympus BX41 fluorescent microscope (Olympus, Tokyo, Japan). For other proteins, fluorescent images were captured using the Olympus BX41 Qimaging software.…”

Section: Secreted Vegf Level Estimationmentioning

confidence: 99%

“…Transmembrane potential assay was performed on PRPE cells in different experimental conditions (normoxia, hyperoxia, normoxia + VIT-D, and hyperoxia + VIT-D), as previously described. 12,13…”

Section: Transmembrane Potential Assaymentioning

confidence: 99%

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Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults

Murugeswari

Firoz

Subramani

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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Citation: Murugeswari P, Firoz A, Murali S, et al. Vitamin-D3 (α-1, 25(OH) 2D3) protects retinal pigment epithelium from hyperoxic insults. Invest Ophthalmol Vis Sci. 2020;61(2):4. https://doi.org/10.1167/iovs.61.2.4 PURPOSE.Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. METHODS.Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O 2 ) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. RESULTS.Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. CONCLUSIONS. VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult.These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.Keywords: retinal pigment epithelium, vitamin D, hyperoxia, vascular endothelial growth factor (VEGF), tubulogenesis O xidative stress is the result of an imbalance between the synthesis of reactive oxygen species (ROS) and the levels of antioxidants in the cells. It plays a major role in the pathophysiology of various ocular diseases, such as agerelated cataract, macular degeneration, glaucoma, diabetic retinopathy, and retinitis pigmentosa, by affecting cellular and vascular physiological aspects. 1,2 Antioxidants, such as enzymatic antioxidants, vitamins, minerals, carotenoids, and flavonoids, are the primary scavengers of ROS reducing levels of oxidative stress. 3 Cholecalciferol (vitamin-D3 (VIT-D)) has diverse functions, including modulation of inflammation, angiogenesis, oxidative stress, and fibrosis. 4,5 Recent studies have demonstrated an association between VIT-D and retinal pathophysiological conditions, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinopathy of prematurity (ROP). 6−8 These studies implicated deficiency of VIT-D to higher risk for early/late AMD, whereas a supplementation leads to delay or prevention in the progression of AMD. 6,9 Although VIT-D receptors as well as enzymes for VIT-D metabolism are present in the retina, choroid, and retinal pigment epithelium (RPE) cells, their functions are still not well understood. 6 VIT-D has been studied as a potential inhibitor of angiogenesis in a mouse model of oxygen-induced ischemic retinopathy (OIR). 10 A correlation of VIT-D and vascular endothelial growth factor (VEGF) has been ob...

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Section: Methodsmentioning

confidence: 99%

Section: Reverse Transcriptase Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

Section: Secreted Vegf Level Estimationmentioning

confidence: 99%

Section: Secreted Vegf Level Estimationmentioning

confidence: 99%

Section: Transmembrane Potential Assaymentioning

confidence: 99%

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Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults

Murugeswari

Firoz

Subramani

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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Retinal pigment epithelium-secretome: A diabetic retinopathy perspective

et al. 2017

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Protective Role of Decellularized Human Amniotic Membrane from Oxidative Stress-Induced Damage on Retinal Pigment Epithelial Cells

Krishna

Dhamodaran

Subramani

et al. 2018

ACS Biomater. Sci. Eng.

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Oxidative stress is an important cause for several retinal aging diseases. Cell therapy using a decellularized human amniotic membrane (dHAM) as a tissue scaffold for retinal pigment epithelial cells has a potential therapeutic role under such pathological conditions. This is attributed by the anti-inflammatory, antimicrobial, low-immunogenicity aspects of dHAM, apart from harboring a drug reservoir potential. The underlying mechanisms for maintaining the physiological properties of transplanted cells and their survival in a diseased milieu using dHAM has remained unexplored/unanswered. Hence, we investigated the potential role of dHAM in preserving the cellular functions of retinal pigment epithelium in an oxidative stress environment. Adult human retinal pigment epithelial (ARPE-19) cells were cultured on dHAM or tissue culture dishes under hyperoxia. Gene expression, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), and scanning electron microscopy (SEM) were performed to assess the levels of reactive oxygen species, proliferation, apoptosis, epithelial-mesenchymal transition, phagocytosis, and secretion of vascular endothelial factors. These results indicate reduced epithelial-mesenchymal transition, generation of reactive oxygen species (p ≤ 0.0001), and apoptosis (p ≤ 0.05) in cells cultured on dHAM, compared to those on tissue culture dishes under oxidative stress conditions. Concomitantly, the secretion of the vascular endothelial growth factor was significantly reduced (p ≤ 0.01) on dHAM. Phagocytic activity was significantly higher (p ≤ 0.001) in cells cultured on dHAM and were comparable to those cells cultured on tissue culture dishes. SEM images showed a clustered growth pattern on dHAM compared to an elongated morphology when cultured on tissue culture dishes under oxidative stress conditions. These findings demonstrate the utility of dHAM as a scaffold for growing retinal epithelial cells and to maintain their physiological properties in an oxidative stress condition with a potential to develop regenerative medicine strategies to treat degenerative eye diseases.

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Short Pulse of Clinical Concentration of Bevacizumab Modulates Human Retinal Pigment Epithelial Functionality

Cited by 9 publications

References 54 publications

Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults

Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults

Retinal pigment epithelium-secretome: A diabetic retinopathy perspective

Protective Role of Decellularized Human Amniotic Membrane from Oxidative Stress-Induced Damage on Retinal Pigment Epithelial Cells

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