Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

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Abstract

Background

Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).

Results

We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.

Conclusions

RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

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Most cited references 39

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Mechanisms of age-related macular degeneration.

Jayakrishna Ambati, Benjamin J Fowler (2012)

Age-related macular degeneration (AMD), a progressive condition that is untreatable in up to 90% of patients, is a leading cause of blindness in the elderly worldwide. The two forms of AMD, wet and dry, are classified based on the presence or absence of blood vessels that have disruptively invaded the retina, respectively. A detailed understanding of the molecular mechanisms underlying wet AMD has led to several robust FDA-approved therapies. In contrast, there are no approved treatments for dry AMD. In this review, we provide insight into the critical effector pathways mediating each form of the disease. A recurring theme that spans most aspects of AMD pathogenesis is defective immune modulation in the classically immune-privileged ocular haven. Interestingly, the latest advances in AMD research also highlight common molecular disease pathways with other neurodegenerative disorders. Finally, the therapeutic potential of intervening at known mechanistic steps of AMD pathogenesis is discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

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Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.

Arvydas Maminishkis, Shan Chen, Stephen Jalickee … (2006)

Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.

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Physiology of vitreous surgery.

Einar Stefánsson (2009)

Vitreous surgery has various physiological and clinical consequences, both beneficial and harmful. Vitrectomy reduces the risk of retinal neovascularization, while increasing the risk of iris neovascularization, reduces macular edema and stimulates cataract formation. These clinical consequences may be understood with the help of classical laws of physics and physiology. The laws of Fick, Stokes-Einstein and Hagen-Poiseuille state that molecular transport by diffusion or convection is inversely related to the viscosity of the medium. When the vitreous gel is replaced with less viscous saline, the transport of all molecules, including oxygen and cytokines, is facilitated. Oxygen transport to ischemic retinal areas is improved, as is clearance of VEGF and other cytokines from these areas, thus reducing edema and neovascularization. At the same time, oxygen is transported faster down a concentration gradient from the anterior to the posterior segment, while VEGF moves in the opposite direction, making the anterior segment less oxygenated and with more VEGF, stimulating iris neovascularization. Silicone oil is the exception that proves the rule: it is more viscous than vitreous humour, re-establishes the transport barrier to oxygen and VEGF, and reduces the risk for iris neovascularization in the vitrectomized-lentectomized eye. Modern vitreous surgery involves a variety of treatment options in addition to vitrectomy itself, such as photocoagulation, anti-VEGF drugs, intravitreal steroids and release of vitreoretinal traction. A full understanding of these treatment modalities allows sensible combination of treatment options. Retinal photocoagulation has repeatedly been shown to improve retinal oxygenation, as does vitrectomy. Oxygen naturally reduces VEGF production and improves retinal hemodynamics. The VEGF-lowering effect of photocoagulation and vitrectomy can be augmented with anti-VEGF drugs and the permeability effect of VEGF reduced with corticosteroids. Starling's law explains vasogenic edema, which is controlled by osmotic and hydrostatic gradients between vessel and tissue. It explains the effect of VEGF-induced vascular permeability changes on plasma protein leakage and the osmotic gradient between vessel and tissue. At the same time, it takes into account hemodynamic changes that affect the hydrostatic gradient. This includes the influence of arterial blood pressure, and the effect oxygen (laser treatment) has in constricting retinal arterioles, increasing their resistance, and thus reducing the hydrostatic pressure in the microcirculation. Reduced capillary hydrostatic pressure and increased osmotic gradient reduce water fluxes from vessel to tissue and reduce edema. Finally, Newton's third law explains that vitreoretinal traction decreases hydrostatic tissue pressure in the retina, increases the pressure gradient between vessel and tissue, and stimulates water fluxes from vessel into tissue, leading to edema.

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Author and article information

Contributors

Masayuki Takeyama: mtakeyam@aichi-med-u.ac.jp

Masahiko Yoneda: yoneda@nrs.aichi-pu.ac.jp

Masahiko Gosho: mgosho@md.tsukuba.ac.jp

Masayoshi Iwaki: iwaki@aichi-med-u.ac.jp

Masahiro Zako: zako@aichi-med-u.ac.jp

Journal

Journal ID (nlm-ta): Biol Res

Journal ID (iso-abbrev): Biol. Res

Title: Biological Research

Publisher: BioMed Central (London )

ISSN (Print): 0716-9760

ISSN (Electronic): 0717-6287

Publication date (Electronic): 30 July 2015

Publication date PMC-release: 30 July 2015

Publication date Collection: 2015

Volume: 48

Issue: 1

Electronic Location Identifier: 42

Affiliations

[ ]Department of Ophthalmology, Aichi Medical University, Nagakute, 480-1195 Aichi Japan

[ ]Department of Biochemistry and Molecular Biology, School of Nursing and Health, Aichi Prefectural University, Nagoya, 463-8502 Aichi Japan

[ ]Department of Clinical Trial and Clinical Epidemiology, Faculty of Medicine, University of Tsukuba, Tsukuba, 305-8575 Ibaraki Japan

Article

Publisher ID: 34

DOI: 10.1186/s40659-015-0034-7

PMC ID: 4518530

SO-VID: 65f15eed-4cc8-41f5-992a-7d5533f44dcd

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 27 December 2014

Date accepted : 21 July 2015

Custom metadata

Keywords: age-related macular degeneration (amd),hypothermia,pigment epithelium–derived factor (pedf),retinal pigment epithelium (rpe),vascular endothelial growth factor (vegf),vitrectomy

Data availability:

Keywords: age-related macular degeneration (amd), hypothermia, pigment epithelium–derived factor (pedf), retinal pigment epithelium (rpe), vascular endothelial growth factor (vegf), vitrectomy

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

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Abstract

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Most cited references 39

Mechanisms of age-related macular degeneration.

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.

Physiology of vitreous surgery.

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