Enhancement of Angiogenic Potential of Endothelial Cells by Contact with Retinal Pigment Epithelial Cells in a Model Simulating Pathological Conditions

Dardik, Rima; Livnat, Tami; Nisgav, Yael; Weinberger, Dov

doi:10.1167/iovs.09-5095

Cited by 17 publications

(15 citation statements)

References 29 publications

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“…Similarly, polarised expression of membrane proteins such as Aquaporin-1, Bestrophin-1, and Na + /K + -ATPase, which play critical roles in exchanging metabolites, may also be observed (Ames et al, 1992;Juuti-Uusitalo et al, 2013;Maminishkis et al, 2006;Stamer et al, 2003). The interaction between RPE cells and the choroidal endothelial cells can also be modelled in culture conditions in order to gain a better understanding of the blood-retinal barrier (Dardik et al, 2010;Hamilton et al, 2007). As well as forming a barrier and interacting with cells of the choroid, RPE cells have an important role in supporting the photoreceptors in the retinoid cycle and phagocytosis of photoreceptor outer segments (POS) and these processes can also be studied in vitro (Ablonczy et al, 2011;Dowling, 1960;Finnemann et al, 1997;Young and Bok, 1969).…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Sorsby fundus dystrophy – A review of pathology and disease mechanisms

Christensen

Brown

Cree

et al. 2017

Experimental Eye Research

143

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Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 220,000 and an onset of disease around the 4th to 6th decade of life. Similar to age-related macular degeneration (AMD), ophthalmoscopy reveals accumulation of protein/lipid deposits under the retinal pigment epithelium (RPE), referred to as drusen, in the eyes of patients with SFD. SFD is caused by variants in the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which has been found in drusen-like deposits of SFD patients. TIMP3 is constitutively expressed by RPE cells and, in healthy eyes, resides in Bruch's membrane. Most SFD-associated TIMP3 variants involve the gain or loss of a cysteine residue. This suggests the protein aberrantly forms intermolecular disulphide bonds, resulting in the formation of TIMP3 dimers. It has been demonstrated that SFD-associated TIMP3 variants are more resistant to turnover, which is thought to be a result of dimerisation and thought to explain the accumulation of TIMP3 in drusen-like deposits at the level of Bruch's membrane. An important function of TIMP3 within the outer retina is to regulate the thickness of Bruch's membrane. TIMP3 performs this function by inhibiting the activity of matrix metalloproteinases (MMPs), which have the function of catalysing breakdown of the extracellular matrix. TIMP3 has an additional function to inhibit vascular endothelial growth factor (VEGF) signalling and thereby to inhibit angiogenesis. However, it is unclear whether SFD-associated TIMP3 variant proteins retain these functions. In this review, we discuss the current understanding of the potential mechanisms underlying development of SFD and summarise all known SFD-associated TIMP3 variants. Cell culture models provide an invaluable way to study disease and identify potential treatments. These allow a greater understanding of RPE physiology and pathophysiology, including the ability to study the blood-retinal barrier as well as other RPE functions such as phagocytosis of photoreceptor outer segments. This review describes some examples of such recent in vitro studies and how they might provide new insights into degenerative diseases like SFD. Thus far, most studies on SFD have been performed using ARPE-19 cells or other, less suitable, cell-types. Now, induced pluripotent stem cell (iPSC) technologies allow the possibility to non-invasively collect somatic cells, such as dermal fibroblast cells and reprogram those to produce iPSCs. Subsequent differentiation of iPSCs can generate patient-derived RPE cells that carry the same disease-associated variant as RPE cells in the eyes of the patient. Use of these patient-derived RPE cells in novel cell culture systems should increase our understanding of how SFD and similar macular dystrophies develop.

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Section: Accepted Manuscriptmentioning

confidence: 99%

Sorsby fundus dystrophy – A review of pathology and disease mechanisms

Christensen

Brown

Cree

et al. 2017

Experimental Eye Research

143

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“…A better characterization of the homeostatic RPE‐EC relationships is therefore particularly significant. Prior in vitro studies aimed at characterizing such interactions have focused on RPE‐dependent functions in pathological processes, mainly choroidal neovascularization (CNV) and epithelial–mesenchymal transition (EMT), using disease‐mimicking conditions such as hypoxia , direct cell contact , subconfluent or activated RPEs in high serum . While these models provided important insights on the cellular and molecular regulation of EC angiogenic status, the identification of the role of RPE‐EC heterotypic interactions on the formation and maintenance of the outer‐retinal complex requires the use of a more physiological model such as the one described in our study in which human RPE and EC were co‐cultured under quiescent conditions and their interdependent functions directly evaluated.…”

Section: Introductionmentioning

confidence: 99%

Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium

Spencer

Abend

McHugh

et al. 2017

J Cellular Molecular Medi

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The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long‐term co‐culture system, we determined the effect of retinal pigment epithelium‐endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle‐associated genes, accumulation of a Bruch's membrane‐like matrix and increase in retinal pigment epithelium phagocytic activity. Co‐culture conditions led to increased expression of anti‐angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube‐formation assays confirmed that co‐culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis.

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“…However, contacting co-culture did not show a difference in response to hypoxia treatment from that of non-contacting co-cultures. Recently, Dardix and coworkers [41] also investigated the effects of co-culture (with retina pigmented epithelial cells - RPE) and hypoxia on endothelial cell expression of VEGF. These researchers reported that contact co-cultures upregulated both VEGF mRNA and protein expression to the same degree as hypoxia alone.…”

Section: Discussionmentioning

confidence: 99%

Co-culture of Retinal and Endothelial Cells Results in the Modulation of Genes Critical to Retinal Neovascularization

et al. 2011

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BackgroundNeovascularization (angiogenesis) is a multistep process, controlled by opposing regulatory factors, which plays a crucial role in several ocular diseases. It often results in vitreous hemorrhage, retinal detachment, neovascularization glaucoma and subsequent vision loss. Hypoxia is considered to be one of the key factors to trigger angiogenesis by inducing angiogenic factors (like VEGF) and their receptors mediated by hypoxia inducible factor-1 (HIF-1α) a critical transcriptional factor. Another factor, nuclear factor kappa B (NFκB) also regulates many of the genes required for neovascularization, and can also be activated by hypoxia. The aim of this study was to elucidate the mechanism of interaction between HRPC and HUVEC that modulates a neovascularization response.MethodsHuman retinal progenitor cells (HRPC) and human umbilical vein endothelial cells (HUVEC) were cultured/co-cultured under normoxia (control) (20% O2) or hypoxia (1% O2) condition for 24 hr. Controls were monolayer cultures of each cell type maintained alone. We examined the secretion of VEGF by ELISA and influence of conditioned media on blood vessel growth (capillary-like structures) via an angiogenesis assay. Total RNA and protein were extracted from the HRPC and HUVEC (cultured and co-cultured) and analyzed for the expression of VEGF, VEGFR-2, NFκB and HIF-1α by RT-PCR and Western blotting. The cellular localization of NFκB and HIF-1α were studied by immunofluorescence and Western blotting.ResultsWe found that hypoxia increased exogenous VEGF expression 4-fold in HRPC with a further 2-fold increase when cultured with HUVEC. Additionally, we found that hypoxia induced the expression of the VEGF receptor (VEGFR-2) for HRPC co-cultured with HUVEC. Hypoxia treatment significantly enhanced (8- to 10-fold higher than normoxia controls) VEGF secretion into media whether cells were cultured alone or in a co-culture. Also, hypoxia was found to result in a 3- and 2-fold increase in NFκB and HIF-1α mRNA expression by HRPC and a 4- and 6-fold increase in NFκB and HIF-1α protein by co-cultures, whether non-contacting or contacting.Treatment of HRPC cells with hypoxic HUVEC-CM activated and promoted the translocation of NFκB and HIF-1α to the nuclear compartment. This finding was subsequently confirmed by finding that hypoxic HUVEC-CM resulted in higher expression of NFκB and HIF-1α in the nuclear fraction of HRPC and corresponding decrease in cytoplasmic NFκB and HIF-1α. Lastly, hypoxic conditioned media induced a greater formation of capillary-like structures (angiogenic response) compared to control conditioned media. This effect was attenuated by exogenous anti-human VEGF antibody, suggesting that VEGF was the primary factor in the hypoxic conditioned media responsible for the angiogenic response.ConclusionsThese findings suggest that intercellular communications between HRPC and HUVEC lead to the modulation of expression of transcription factors associated with the production of pro-angiogenic factors under hypoxic conditions, which are n...

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Enhancement of Angiogenic Potential of Endothelial Cells by Contact with Retinal Pigment Epithelial Cells in a Model Simulating Pathological Conditions

Cited by 17 publications

References 29 publications

Sorsby fundus dystrophy – A review of pathology and disease mechanisms

Sorsby fundus dystrophy – A review of pathology and disease mechanisms

Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium

Co-culture of Retinal and Endothelial Cells Results in the Modulation of Genes Critical to Retinal Neovascularization

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