Primary Retinal Endothelial Cells Research Articles

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1.

Retinoblastoma (RB) is a prevalent type of eye cancer in youngsters. Prospero homeobox 1 (Prox1) is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic, hepatocyte, pancreatic, heart, lens, retinal, and cancer cells. The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance, as well as to explore the underlying Notch1 mechanism. Human RB cell lines (SO-RB50 and Y79) and a primary human retinal microvascular endothelial cell line (ACBRI-181) were used in this study. The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay. Drug-resistant cell lines (SO-RB50/vincristine) were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance. We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1. Finally, a xenograft model was constructed to assess the effect of Prox1 on RB in vivo. Prox1 was significantly downregulated in RB cells. Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine. Notch1 was involved in Prox1's regulatory effects. Notch1 was identified as a target gene of Prox1, which was found to be upregulated in RB cells and repressed by increased Prox1 expression. When pcDNA-Notch1 was transfected, the effect of Prox1 overexpression on RB was removed. Furthermore, by downregulating Notch1, Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo. These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1, implying that Prox1 could be a potential therapeutic target for RB.

An In Vitro Bilayer Model of Human Primary Retinal Pigment Epithelial and Choroid Endothelial Cells for Permeability Studies.

The blood-retinal barrier (BRB) present in the posterior chamber of the eye plays a major role in maintaining the proper function and integrity of the retina. Retinal pigment epithelium and choriocapillaris form the outer blood retinal barrier (oBRB), and breakdown of this barrier leads to vision-threatening diseases like macular edema, macular degeneration, and diabetic retinopathy. A simplified cell culture model of oBRB will be of great importance in elucidating the molecular mechanism of the disease progression. This chapter describes methods for primary cell isolation from donor eyes to culture human retinal pigment epithelial cells (hRPE) and choroidal endothelial cells (hCEC) and the protocol for construction of a simplified in vitro model of oBRB on fibronectin-coated Transwell inserts. Further, we explained the permeability study using FITC-dextran conjugated tracers for validating the bilayer model. The permeability experiments ensured that the system could easily be manipulated to recapitulate the pathological condition in vitro. Thus, it would be an optimal system for studying the disease mechanisms related to retinal and choroidal pathologies, for screening small molecules, and for performing drug permeability kinetics. Moreover, fundamental understanding of paracellular and transcellular trafficking of cargo in hRPE and hCEC could also be studied using this model.

Semaphorin 7a regulates inflammatory mediators and permeability in retinal endothelial cells

Research supports a key role for inflammation in damaging the retinal vasculature. Current work is designed to investigate regulation of key inflammatory pathways. In this study, we hypothesized that semaphorin 7a (Sema7a) was involved in the increased inflammatory mediators and permeability changes in retinal endothelial cells (REC) grown in high glucose. For these studies, we used diabetic mouse samples and REC to investigate our hypothesis. Primary retinal endothelial cells were grown in normal (5 mM) or high glucose (25 mM glucose) for measurements. In a subset of cells grown in high glucose, cells were transfected with Sema7a siRNA or scrambled siRNA. We measured levels of key inflammatory mediators and zonula occludens-1 (ZO-1) and occludin levels by Western blot. Data suggest that high glucose increased inflammatory mediators and reduced the tight junction proteins, which follows what is often observed in cells grown in high glucose. Sema7a siRNA significantly decreased inflammatory proteins and increased levels of ZO-1 and occludin. These data suggest that Sema7a mediates the actions of high glucose in REC. Use of Sema7a siRNA may offer a new avenue for treatment.

Loss of cystatin C regulates permeability and inflammatory pathways in retina.

Cystatin C has been linked to inflammation in other diseases, such as epilepsy and Alzheimer's disease. These studies were designed to investigate whether Cystatin C regulates retinal inflammation and permeability. To address this question, we used Cystatin C knockout mice in a retinal ischemia/reperfusion model to determine whether Cystatin C regulated retinal damage, as well as inflammatory mediators and retinal permeability. To support the mouse work, we also used primary retinal endothelial cells cultured in normal and high glucose. Ischemia/reperfusion in Cystatin C knockout mice caused increased formation of degenerate capillaries. Loss of Cystatin C increased fluorescein leakage in the retina, which was accompanied by reduced levels of zonula occludin 1 (ZO-1) and occludin proteins. When REC were grown in high glucose, recombinant Cystatin C decreased retinal permeability, while Cystatin C siRNA increased dextran flux compared to high glucose alone. Recombinant Cystatin C decreased levels of interleukin-1-beta (IL-1β) and high mobility group box 1 (HMGB1) levels. In conclusion, loss of Cystatin C increased vascular damage in response to ischemia/reperfusion. Cystatin C regulated permeability and inflammatory mediators in the retina in response to stressors. Cystatin C offers a new target for retinal disease therapeutic development.

Human retinal endothelial cells express functional interleukin-6 receptor

BackgroundInterleukin (IL)-6 is an inflammatory cytokine present in the eye during non-infectious uveitis, where it contributes to the progression of inflammation. There are two major IL-6 signaling pathways: classic signaling and trans-signaling. Classic signaling requires cellular expression of the IL-6 receptor (IL-6R), which exists in membrane-bound (mIL-6R) and soluble (sIL-6R) forms. Prevailing dogma is that vascular endothelial cells do not produce IL-6R, relying on trans-signaling during inflammation. However, the literature is inconsistent, including with respect to human retinal endothelial cells.FindingsWe examined IL-6R transcript and protein expression in multiple primary human retinal endothelial cell isolates, and assessed the effect of IL-6 on the transcellular electrical resistance of monolayers. Using reverse transcription-polymerase chain reaction, IL-6R, mIL-6R and sIL-6R transcripts were amplified in 6 primary human retinal endothelial isolates. Flow cytometry on 5 primary human retinal endothelial cell isolates under non-permeabilizing conditions and following permeabilization demonstrated intracellular stores of IL-6R and the presence of mIL-6R. When measured in real-time, transcellular electrical resistance of an expanded human retinal endothelial cell isolate, also shown to express IL-6R, decreased significantly on treatment with recombinant IL-6 in comparison to non-treated cells across 5 independent experiments.ConclusionsOur findings indicate that human retinal endothelial cells produce IL-6R transcript and functional IL-6R protein. The potential for classic signaling in human retinal endothelial cells has implications for the development of therapeutics targeted against IL-6-mediated pathology in non-infectious uveitis.

Selective Transcription Factor Blockade Reduces Human Retinal Endothelial Cell Expression of Intercellular Adhesion Molecule-1 and Leukocyte Binding

The interaction between leukocytes and cytokine-activated retinal endothelium is an initiating step in non-infectious uveitis involving the posterior eye, mediated by cell adhesion molecules. However, because cell adhesion molecules are required for immune surveillance, therapeutic interventions would ideally be employed indirectly. Using 28 primary human retinal endothelial cell isolates, this study sought to identify transcription factor targets for reducing levels of the key retinal endothelial cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, and limiting leukocyte binding to the retinal endothelium. Five candidate transcription factors-C2CD4B, EGR3, FOSB, IRF1, and JUNB-were identified by differential expression analysis of a transcriptome generated from IL-1β- or TNF-α-stimulated human retinal endothelial cells, interpreted in the context of the published literature. Further filtering involved molecular studies: of the five candidates, C2CD4B and IRF1 consistently demonstrated extended induction in IL-1β- or TNF-α-activated retinal endothelial cells and demonstrated a significant decrease in both ICAM-1 transcript and ICAM-1 membrane-bound protein expression by cytokine-activated retinal endothelial cells following treatment with small interfering RNA. RNA interference of C2CD4B or IRF1 significantly reduced leukocyte binding in a majority of human retinal endothelial cell isolates stimulated by IL-1β or TNF-α. Our observations suggest that the transcription factors C2CD4B and IRF1 may be potential drug targets for limiting leukocyte-retinal endothelial cell interactions in non-infectious uveitis involving the posterior eye.

Association of Placental Growth Factor and Angiopoietin in Human Retinal Endothelial Cell-Pericyte co-Cultures and iPSC-Derived Vascular Organoids

PurposePlacental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids.MethodsIn this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1.ResultsInhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network.ConclusionCombined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.

A Comprehensive Profiling of Cellular Sphingolipids in Mammalian Endothelial and Microglial Cells Cultured in Normal and High-Glucose Conditions.

Sphingolipids (SPLs) play a diverse role in maintaining cellular homeostasis. Dysregulated SPL metabolism is associated with pathological changes in stressed and diseased cells. This study investigates differences in SPL metabolism between cultured human primary retinal endothelial (HREC) and murine microglial cells (BV2) in normal conditions (normal glucose, NG, 5 mM) and under high-glucose (HG, 25 mM)-induced stress by sphingolipidomics, immunohistochemistry, biochemical, and molecular assays. Measurable differences were observed in SPL profiles between HREC and BV2 cells. High-glucose treatment caused a >2.5-fold increase in the levels of Lactosyl-ceramide (LacCer) in HREC, but in BV2 cells, it induced Hexosyl-Ceramides (HexCer) by threefold and a significant increase in Sphingosine-1-phosphate (S1P) compared to NG. Altered SPL profiles coincided with changes in transcript levels of inflammatory and vascular permeability mediators in HREC and inflammatory mediators in BV2 cells. Differences in SPL profiles and differential responses to HG stress between endothelial and microglial cells suggest that SPL metabolism and signaling differ in mammalian cell types and, therefore, their pathological association with those cell types.

Topical Nanoemulsion of a Runt-related Transcription Factor 1 Inhibitor for the Treatment of Pathologic Ocular Angiogenesis.

Purpose:To test the efficacy of runt-related transcription factor 1 (RUNX1) inhibition with topical nanoemulsion containing Ro5-3335 (eNano-Ro5) in experimental ocular neovascularization.Design:Preclinical experimental study.Participants:In vitro primary culture human retinal endothelial cell (HREC) culture. C57BL/6J 6- to 10-week-old male and female mice.Methods:We evaluated the effect of eNano-Ro5 in cell proliferation, cell toxicity, and migration of HRECs. We used an alkali burn model of corneal neovascularization and a laser-induced model of choroidal neovascularization to test in vivo efficacy of eNano-Ro5 in pathologic angiogenesis in mice. We used mass spectrometry to measure penetration of Ro5-3335 released from the nanoemulsion in ocular tissues.Main Outcome Measures:Neovascular area.Results:RUNX1 inhibition reduced cell proliferation and migration in vitro. Mass spectrometry analysis revealed detectable levels of the active RUNX1 small-molecule inhibitor Ro5-3335 in the anterior and posterior segment of the mice eyes. Topical treatment with eNano-Ro5 significantly reduced corneal neovascularization and improved corneal wound healing after alkali burn. Choroidal neovascularization lesion size and leakage were significantly reduced after treatment with topical eNano-Ro5.Conclusions:Topical treatment with eNano-Ro5 is an effective and viable platform to deliver a small-molecule RUNX1 inhibitor. This route of administration offers advantages that could improve the management and outcomes of these sight-threatening conditions. Topical noninvasive delivery of RUNX1 inhibitor could be beneficial for many patients with pathologic ocular neovascularization.

Prohibitin 1 Regulates Inflammatory Mediators and Reactive Oxygen Species in Retinal Endothelial Cells.

Diabetic retinopathy is associated with increased inflammatory mediator levels. In these studies, we focused on prohibitin 1. We performed western blotting for retinal lysates from diabetic mice and Epac1 floxed and cdh5Cre-Epac1 mice. We also grew primary retinal endothelial cells (REC) in normal (5 mM) and high (25 mM) glucose, and treated some cells with an Epac 1 agonist or prohibitin 1 siRNA. Western blotting was done to confirm knockdown of prohibitin 1 and Epac 1 agonism. We measured the tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1β), phosphorylated prohibitin 1, phosphorylated nuclear factor kappa beta (NFkB), high mobility group box 1 (HMGB1) and reactive oxygen species (ROS) levels in REC after transfection with prohibitin 1 siRNA. Results showed that high glucose increased the inflammatory mediators, as well as HMGB1 and ROS. The levels of ROS, HMGB1, and inflammatory pathways were all reduced after cells were transfected with prohibitin 1 siRNA. Epac1 reduced prohibitin 1 phosphorylation. In conclusion, decreased prohibitin 1 significantly reduced the inflammatory mediator and ROS levels in REC. Epac1 regulates the prohibitin 1 levels in REC.

Drp1 knockdown represses apoptosis of rat retinal endothelial cells by inhibiting mitophagy

Diabetic retinopathy (DR) is the leading clinical cause of blindness in diabetic patients. Mitophagy participates in the pathogenesis of DR. Dynamin related protein 1 (Drp1) is associated with mitophagy. Here, we investigated whether Drp1 can regulate mitophagy to affect the progression of DR. We constructed DR rat model by administration of streptozocin. Primary rat retinal endothelial cells (RECs) were treated with high glucose (HG) as a DR cell model. Drp1 was highly expressed in the retinal tissues of DR rats and HG-treated RECs. Drp1 knockdown inhibited HG-mediated increase of reactive oxygen species (ROS) levels and apoptosis in RECs. Moreover, Drp1 silencing inhibited the expression of autophagy-related proteins LC3-II/LC3–1 and Beclin-1 and reduced LC3 puncta in HG-treated RECs. The expression of mitochondrial marker Tom20 was reduced and the levels of mitophagy were increased in the HG-treated RECs, which was rescued by Drp1 silencing. Drp1 knockdown repressed LC3-II expression in HG-treated RECs, indicating that autophagy flux was inhibited. Rapamycin (autophagy activator) enhanced ROS levels and apoptosis in HG-treated RECs by activating autophagy, which was rescued by Drp1 knockdown. In conclusion, these data demonstrated that Drp1 knockdown repressed apoptosis of rat retinal endothelial cells by inhibiting mitophagy. Thus, this work suggests that targeted regulation of Drp1 may become a treatment for DR.

Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1

BackgroundEchinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. MethodsEffects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. ResultsaRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. ConclusionsOur data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.

Rank Order of Small Molecule Induced Hypoxiamimesis to Prevent Retinopathy of Prematurity.

Here we rank order small molecule inhibitors of hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) using severity of oxygen induced retinopathy (OIR) as an outcome measure. Dose response analyses in cell cultures of hepatoma (Hep3B), retinal Müller cells (MIO-M1) and primary retinal endothelial cells were conducted to evaluate potency by comparing dose to HIF-1,2 protein levels by western blotting. In vivo dose response was determined using the luciferase-transgene HIF reporter (luc-ODD). Each compound was placed in rank order by their ability to reduce neovascularization and capillary drop out in the OIR mouse model. An Epas1 KO confined to retinal Müller cells was used to determine whether successful protection by HIF stabilization requires HIF-2. Two candidate small molecules can prevent OIR by stabilizing HIF-1 to prevent oxygen induced growth attenuation and vascular obliteration. Müller cell HIF-2, the mediator of pathologic retinal angiogenesis, is not required for protection. The lack of dependence on Müller cell HIF-2 predicts that inhibition of HIF PHD will not drive pathological angiogenesis.

All-trans-Retinaldehyde Contributes to Retinal Vascular Permeability in Ischemia Reperfusion.

PurposeExtracellular accumulation of all-trans-retinaldehyde (atRAL), a highly reactive visual cycle intermediate, is toxic to cells of the outer retina and contributes to retinal and macular degenerations. However, the contribution of atRAL to retinal capillary function has not been studied. We hypothesized that atRAL released from the outer retina can contribute to retinal vascular permeability. We, therefore, tested the contribution of atRAL to retinal ischemia-reperfusion (IR)-induced vascular permeability.MethodsIR was induced in mice by transient increase in intraocular pressure followed by natural reperfusion. The visual cycle was ablated in the Lrat−/− mice, reduced by dark adaptation or the use of the RPE65 inhibitor and atRAL scavenger emixustat. Accumulation of FITC-BSA was used to assess vascular permeability and DNA fragmentation quantified cell death after IR. Primary bovine retinal endothelial cell (BREC) culture was used to measure the direct effects of atRAL on endothelial permeability and cell death.ResultsInhibition of the visual cycle by Lrat−/−, dark adaptation, or with emixustat, all reduced approximately half of IR induced vascular permeability at 48 hours. An increase in BREC permeability with atRAL coincided with lactate dehydrogenase (LDH) release, a measure of cell death. Both permeability and toxicity were blocked by emixustat.ConclusionsOuter retinal pathology may contribute to vascular permeability by release of atRAL, which can act directly on vascular endothelial cells to alter barrier properties and induce cell death. These studies may have implications for a variety of blinding eye diseases that include outer retinal damage and retinal vascular permeability.

Treatment of diabetic retinopathy through neuropeptide Y-mediated enhancement of neurovascular microenvironment.

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N‐methyl‐D‐aspartate (NMDA)‐induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)‐induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic‐induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA‐induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR.

Efficacy of echinomycin in hypoxia‐inducible factor‐mediated ocular angiogenesis

AbstractPurposeEchinomycin (EKN), an inhibitor of hypoxia‐inducible factor (HIF)‐1 DNA‐binding activity, has been implied as a possible therapeutic agent in ischemic diseases. We assessed EKN in hypoxia‐driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE), and in vivo using the laser‐induced mouse choroidal neovascularization (CNV) model.MethodsAdult retinal endothelial were kept at normoxia (20% O2) or exposed to hypoxia (1% O2), in the presence or absence of EKN. Epithelial wound‐healing was used to determine the dose of EKN. Effects of EKN on hypoxia‐mediated pathways were analyzed by western blot for HIF‐1a, qPCR of HIF‐target genes, and proteome array for soluble angiogenic factors. Inhibition of angiogenesis by EKN was determined in human primary adult retinal endothelial cells (aREC), exposed to EKN‐treated hypoxic conditioned aRPE medium by spheroid sprouting assay. 8‐week‐old C57BL/6J mice underwent laser‐induced CNV, as a model of HIF‐associated ocular neovascularization. Animals were treated with EKN and compared to a mouse VEGFR1‐Fc chimera protein (equivalent of aflibercept). CNV lesion area was determined by fluorescein angiography.ResultsAdult retinal endothelial treated with EKN demonstrated hypoxia‐dependent decreased cell proliferation in the wound‐healing assay. Lower HIF‐mediated transcripts were detected in hypoxic aRPE cells treated with EKN compared with non‐treated controls. These results were confirmed by proteome profiler for angiogenic factors, and subsequently reduced hREC sprouting. Laser‐induced mice treated with 1 mg of intravitreally injected EKN demonstrated a significant decrease in vascular lesion area when compared with 1 mg of VEGFR1 chimera or vehicle‐treated controls.ConclusionsOur data suggest EKN as a potent inhibitor of HIF‐mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age‐related macular degeneration.

Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir–Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration.In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. Statement of significanceArtificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.

Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury.

Oxidative stress can cause injury in retinal endothelial cells. Carboxymethyl cellulose modified with collagen peptide (CMCC) is of a distinct antioxidant capacity and potentially a good drug carrier. In this study, the protective effects of CMCC against H2O2‐induced injury of primary retinal endothelial cells were investigated. In vitro, we demonstrated that CMCC significantly promoted viability of H2O2‐treated cells, efficiently restrained cellular reactive oxygen species (ROS) production and cell apoptosis. Then, the CMCC was employed as both drug and anti‐inflammatory drug carrier for treatment of retinal ischaemia/reperfusion (I/R) in rats. Animals were treated with CMCC or interleukin‐10‐loaded CMCC (IL‐10@CMCC), respectively. In comparisons, the IL‐10@CMCC treatment exhibited superior therapeutic effects, including better restoration of retinal structural thickness and less retinal apoptosis. Also, chemiluminescence demonstrated that transplantation of IL‐10@CMCC markedly reduced the retinal oxidative stress level compared with CMCC alone and potently recovered the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that CMCC provides a promising platform to enhance the drug‐based therapy for I/R‐related retinal injury.

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells.

Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.