Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic ex vivo model.

Rubeosis Iridis (RI) is characterized by an increase in neovascularization and inflammation factors in the iris. During angiogenesis, the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a pivotal role in extracellular matrix remodeling, where uPAR regulates endothelial cell migration and proliferation through assembly with transmembrane receptors. Here, in the context of hypoxia-induced angiogenesis, the uPA/uPAR system blockage was investigated by using UPARANT in a novel ex vivo human iris organotypic angiogenesis assay. The effects of uPA/uPAR system antagonism in the humanized model of ocular pathologic angiogenesis were analyzed by sprouting angiogenesis and protein assays (western, dot blots, and co-immunoprecipitation) and correlated to vascular endothelial growth factor (VEGF) inhibition. Phosphoprotein and co-immunoprecipitation assay illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low-density lipoprotein receptor-related protein-1 (LRP-1), resulting in inhibition of ß-catenin-mediated angiogenesis in this model. The effects of uPA/uPAR system inhibition were focal to endothelial cells ex vivo. Comparison between human iris endothelial cells and human retinal endothelial revealed an endothelial-specific mechanism of ß-catenin-mediated angiogenesis inhibited by uPA/uPAR system blockage and not by VEGF inhibition. Collectively, these findings broaden the understanding of the effects of the uPA/uPAR system antagonism in the context of angiogenesis, revealing non-canonical ß-catenin downstream effects mediated by LRP-1/uPAR interaction.

Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells.

BACKGROUND AIMS: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. METHODS: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. RESULTS: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. CONCLUSIONS: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

Photoreceptor laminin drives differentiation of human pluripotent stem cells to photoreceptor progenitors that partially restore retina function.

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32 days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2 weeks post transplantation as measured by full-field electroretinogram. At 4 weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20 weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy.

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.

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

BACKGROUND: Echinomycin (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. METHODS: Effects 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. RESULTS: aRPE 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. CONCLUSIONS: Our 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.

Intussusceptive Vascular Remodeling Precedes Pathological Neovascularization.

Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.

INJECTION FREQUENCY OF AFLIBERCEPT VERSUS RANIBIZUMAB IN A TREAT-AND-EXTEND REGIMEN FOR CENTRAL RETINAL VEIN OCCLUSION: A Randomized Clinical Trial.

PURPOSE: To prospectively investigate the injection frequency of aflibercept and ranibizumab in the treatment of macular edema in central retinal vein occlusion. METHODS: Patients with treatment-naive central retinal vein occlusion and macular edema were randomized to receive intravitreal injections with aflibercept (n = 22) or ranibizumab (n = 23) in a treat-and-extend regimen with a follow-up time of 18 months. After 3 loading doses, the treatment intervals were extended by 2 weeks to a maximum of 12 weeks. Intervals were shortened by 2 weeks if macular edema recurred. RESULTS: The number of injections was significantly lower in the aflibercept group with a mean of 10.9 injections (95% confidence interval, 9.6-12.3) compared with 14.4 in the ranibizumab group (95% confidence interval 12.7-16.1) at study completion (P = 0.0017). The mean treatment interval was significantly longer in the aflibercept group compared with the ranibizumab group 10.0 (95% confidence interval, 8.7-11.3) and 6.6 (95% confidence interval, 5.2-8.0) weeks, respectively (P < 0.001). No significant difference between the groups regarding visual acuity or central retinal thickness was observed. CONCLUSION: Patients with macular edema secondary to central retinal vein occlusion required significantly fewer intravitreal injections of aflibercept compared with ranibizumab when treated with a treat-and-extend regimen. This may reduce the treatment burden and, to some extent, the need for close monitoring of patients.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY OF THE FOVEAL MICROVASCULATURE IN GEOGRAPHIC ATROPHY.

PURPOSE: To examine the retinal and choroidal foveal and parafoveal vasculature in patients with bilateral geographic atrophy (GA) secondary to age-related macular degeneration using optical coherence tomography angiography (OCTA). METHODS: Fourteen eyes from 7 patients with and without fovea-sparing bilateral GA at St. Erik Eye Hospital. All patients were examined by optical coherence tomography angiography, en face OCT and fundus autofluorescence (FAF). Segmented optical coherence tomography angiography flow scans were obtained from the superficial retinal vascular layer (SL) and the choriocapillaris (CC) and correlated with areas of retinal pigment epithelial (RPE) loss on fundus autofluorescence. The foveal avascular zone (FAZ) was measured on superficial retinal vascular layer scans and compared to the GA area of each patient. RESULTS: No significant correlation (r = -0.17, P = 0.58) was found between superficial retinal vascular layer foveal avascular zone (0.49 mm ± 0.23 mm) and GA area (7.36 mm ± 4.36 mm). Absent or severely impaired CC flow was observed inside all GA lesions and to varied extent outside the GA margins including areas of fovea sparing. A high level of symmetry was observed in CC flow between fellow eyes. CONCLUSION: In this cross-sectional study, no relation was found between superficial retinal vascular layer foveal avascular zone and GA area. CC flow inside the GA was severely impaired, whereas CC flow outside the GA correlated poorly with both RPE integrity and visual acuity. Fellow eye symmetry suggests that CC monitoring may be a relevant clinical end point in interventional GA studies.

Real-World Weekly Efficacy Analysis of Faricimab in Patients with Age-Related Macular Degeneration.

Objectives: This study entailed a weekly analysis of real-world data (RWD) on the safety and efficacy of intravitreal (IVT) faricimab in neovascular age-related macular degeneration (nAMD). Methods: A retrospective, single-centre clinical trial was conducted at the Department of Ophthalmology, University Hospital Zurich, University of Zurich, Switzerland, approved by the Cantonal Ethics Committee of Zurich, Switzerland. Patients with nAMD were included. Data from patient charts and imaging were analysed. The safety and efficacy of the first faricimab injection were evaluated weekly until 4 weeks after injection. Results: Sixty-three eyes with a complete 4-week follow-up were enrolled. Six eyes were treatment-naïve; fifty-seven eyes were switched to faricimab from another treatment. Neither group showed signs of retinal vasculitis during the 4 weeks after injection. Central subfield thickness (CST) and volume (CSV) showed a statistically significant decrease compared to the baseline in the switched group (CST: p = 0.00383; CSV: p = 0.00702) after 4 weeks. The corrected visual acuity returned to the baseline level in both groups. The macular neovascularization area decreased in both groups, but this was not statistically significant. A complete resolution of sub- and intraretinal fluid after 4 weeks was found in 40% (switched) and 75% (naïve) of the treated patients. Conclusions: The weekly follow-ups reflect the structure-function relationship beginning with a fast functional improvement within two weeks after injection followed by a return to near-baseline levels after week 3. The first faricimab injection in our cohort showed a high safety profile and a statistically significant reduction in macular oedema in switched nAMD patients.

Macular hole Delphi consensus statement (MHOST).

PURPOSE: To derive a Delphi method-based consensus for the surgical management of Full Thickness Macular Hole (FTMH) and Lamellar Macular Hole (LMH). METHODS: 37 expert VR surgeons from 21 mainly European countries participated in Delphi method-based questionnaire for diagnosis and treatment of FTMHs and LMHs. RESULTS: A total of 36 items were rated in round 1 by 37 participants, of which 10 items achieved consensus: intraoperative verification of PVD; clinical superiority of OCT-based FTMH classification; practical ineffectiveness of ocriplasmin; circular 360° ILM peeling for small macular holes; use of regular surgical technique for the size of the hole in concomitant retinal detachment; performing complete vitrectomy; SF6 gas as preferred tamponade; cataract surgery if crystalline lens is mildly/moderately opaque; removal of both ILM and LHEP in LMH surgery. In round 2, 18 items with moderate consensus (45-70% agreement) in round 1 were rated by 35 participants. Final consensus was reached in 35% of questions related to both diagnosis and surgical procedures. CONCLUSIONS: This Delphi study provides valuable information about the consensus/disagreement on different scenarios encountered during FTMH and LMH management as a guide tosurgical decision-making. High rate of disagreement and/or variable approaches still exist for treating such relatively common conditions.

Bevacizumab for macular edema in central retinal vein occlusion: a prospective, randomized, double-masked clinical study.

PURPOSE: To evaluate the efficacy of intraocular injections with bevacizumab in patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO). DESIGN: Prospective, randomized, sham injection-controlled, double-masked clinical trial. PARTICIPANTS: Sixty patients with ME secondary to CRVO. METHODS: At baseline, patients were randomized 1:1 to receive intraocular injections of bevacizumab or sham injections every 6 weeks for 6 months. MAIN OUTCOME MEASURES: The primary outcome measure was the proportion of patients gaining at least 15 letters at 6 months. Secondary outcome measures included mean change from baseline best-corrected visual acuity (BCVA), foveal thickness, and neovascular glaucoma. RESULTS: At the end of follow-up, 18 of 30 patients (60.0%) in the study group had gained ≥15 letters compared with 6 of 30 patients (20.0%) in the control group (P=0.003). The BCVA improved by 14.1 letters at 24 weeks compared with a decrease of 2.0 letters in the control group (P < 0.003). The mean decrease in central retinal thickness (CRT) was significantly greater in the study group (426 µm) than in the control group (102 µm) at all time points up to week 24 (P < 0.001). No residual edema, defined as CRT <300 µm at 24 weeks, was found in 26 of 30 patients (86.7%) in the treatment group compared with 6 of 30 patients (20%) in the control group (P < 0.001). In the sham group, 5 of 30 patients (16.7%) had developed iris rubeosis at week 24. No patients in the study group had rubeosis at week 24 (P=0.052). There were no events of endophthalmitis, retinal tear, or retinal detachment during the 24-week treatment period. No serious non-ocular adverse events were reported. CONCLUSIONS: Intraocular injections of bevacizumab given every 6 weeks for 6 months improve visual acuity (VA) and reduce ME significantly compared with sham. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Benefit from bevacizumab for macular edema in central retinal vein occlusion: twelve-month results of a prospective, randomized study.

PURPOSE: To evaluate the efficacy of intraocular injections with bevacizumab over 12 months in patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO). DESIGN: A prospective study including a randomized 6-month, sham injection-controlled, double-masked clinical trial followed by a 6-month open-label extension. PARTICIPANTS: Sixty patients with ME secondary to CRVO. METHODS: At baseline, patients were randomized 1:1 to receive intraocular injections of bevacizumab or sham injections every 6 weeks for 6 months. From month 6, all patients received intraocular injections of bevacizumab every 6 weeks for 6 months. MAIN OUTCOME MEASURES: The primary outcome measure was the proportion of patients gaining at least 15 letters at 12 months. Secondary outcome measures included mean change from baseline best-corrected visual acuity (BCVA), change in foveal thickness, and development of neovascular glaucoma. RESULTS: At the end of follow-up, 18 of 30 patients (60.0%) in the bevacizumab/bevacizumab (bz/bz) group had gained ≥ 15 letters compared with 10 of 30 patients (33.3%) in the sham/bevacizumab (sh/bz) group (P < 0.05). The BCVA improved by 16.0 letters at 12 months in the bz/bz group compared with 4.6 letters in the sh/bz group (P < 0.05). In an unplanned retrospective analysis, patients aged >70 years had a significantly worse outcome when receiving delayed treatment, losing 1.4 letters (95% confidence interval [CI], -9.7 to 8.4) in the sh/bz group compared with a gain of 20.1 letters (95% CI, 13.9-26.3) in the bz/bz group in patients aged <70 years (P < 0.003). The mean decrease in central retinal thickness (CRT) was 435 µm in the bz/bz group compared with 404 µm in the sh/bz group (P = not significant). No patients developed iris rubeosis during the 6-month open-label extension period. There were no events of endophthalmitis, retinal tear, or retinal detachment during the 12-month treatment period. No serious nonocular adverse events were reported. CONCLUSIONS: Intraocular injections of bevacizumab given every 6 weeks for 12 months improve visual acuity (VA) and reduce ME significantly. Patients receiving delayed treatment have a limited visual improvement. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Hormetic Heat Shock Enhances Autophagy through HSF1 in Retinal Pigment Epithelium Cells.

To maintain homeostasis, cells have evolved stress-response pathways to cope with exogenous and endogenous stress factors. Diverse stresses at high doses may be detrimental, albeit low doses of stress, known as hormesis, can be beneficial. Upon exposure to stress, such as temperature rise, the conventional heat shock response (HSR) regulated by the heat shock transcription factor 1 (HSF1) facilitates refolding of misfolded proteins with the help of heat shock proteins (HSPs). However, the role and molecular mechanisms underlying the beneficial effects of HSR with other clearance processes, such as autophagy, remain poorly understood. In this study, human ARPE-19 cells, an in vitro model of retinal pigment epithelium, were treated with hormetic heat shock (HHS) and the autophagy expression profile was examined using quantitative PCR (qPCR), immunoblotting, immunoprecipitation, and immunofluorescence. We demonstrate that HHS enhances the expression of fundamental autophagy-associated genes in ARPE-19 cells through the activation of HSF1. HHS transiently increases the level of SQSTM1 and LC3B-II and activates autophagy. These findings reveal a role for autophagic HSF1-regulated functions and demonstrate the contribution of autophagy to hormesis in the HSR by improving proteostasis.

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

Autophagy Involvement in the Postnatal Development of the Rat Retina.

During retinal development, a physiologic hypoxia stimulates endothelial cell proliferation. The hypoxic milieu warrants retina vascularization and promotes the activation of several mechanisms aimed to ensure homeostasis and energy balance of both endothelial and retinal cells. Autophagy is an evolutionarily conserved catabolic system that contributes to cellular adaptation to a variety of environmental changes and stresses. In association with the physiologic hypoxia, autophagy plays a crucial role during development. Autophagy expression profile was evaluated in the developing retina from birth to post-natal day 18 of rat pups, using qPCR, western blotting and immunostaining methodologies. The rat post-partum developing retina displayed increased active autophagy during the first postnatal days, correlating to the hypoxic phase. In latter stages of development, rat retinal autophagy decreases, reaching a normalization between post-natal days 14-18, when the retina is fully vascularized and mature. Collectively, the present study elaborates on the link between hypoxia and autophagy, and contributes to further elucidate the role of autophagy during retinal development.

Nuclear expression of BAP-1 in transvitreal incisional biopsies and subsequent enucleation of eyes with posterior choroidal melanoma.

BACKGROUND: As a majority of patients with choroidal melanoma do not undergo enucleation, tumour tissue for prognostic testing has to be obtained with alternate methods. Transvitreal incisional biopsies enable histological examination as well as immunohistochemical staining of BRCA1-associated protein-1 (BAP-1). METHODS: Fifty-nine patients diagnosed with choroidal melanoma in transvitreal biopsies between years 2003 and 2019 were included. Twenty-one of these patients subsequently underwent enucleation. The level of nuclear expression of BAP-1 in transvitreal biopsies and enucleations was evaluated and the concordance calculated. Metastasis-free survival and HR for metastasis were analysed. RESULTS: The mean tumour thickness and diameter at biopsy was 3.8 mm (SD 2.1) and 9.3 mm (SD 4.8), respectively. For biopsies, 37 of 59 tumours (63%) were classified as having high nuclear BAP-1 expression, and 22 (37%) as low. For enucleations, 13 of 21 tumours (62%) were classified as having high nuclear BAP-1 expression, and 8 (38%) as low. Eighty-six per cent of biopsies had an identical BAP-1 classification as the subsequent enucleation, yielding a Cohen's kappa coefficient of 0.70. Patients with low nuclear BAP-1 expression in transvitreal biopsies had a significantly shorter metastasis-free survival (p=0.001), with a size-adjusted Cox regression HR for metastasis of 13.0 (95% CI 3.1 to 54.4, p=0.0004). CONCLUSION: Loss of nuclear BAP-1 expression occurred in a large proportion of the small tumours included in this study. BAP-1 immunoreactivity in transvitreal incisional biopsies of choroidal melanoma is substantially concordant with immunoreactivity in enucleated specimens and identifies patients with poor metastasis-free survival.

Vascular permeability in retinopathy is regulated by VEGFR2 Y949 signaling to VE-cadherin.

Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy. Whereas therapies targeting vascular endothelial growth factor A (VEGFA) can suppress leakage, side-effects include vascular rarefaction and geographic atrophy. By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 (VEGFR2)-induced vascular permeability, we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding. Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy, similarly sized lesions leak less in mutant mice, separating regulation of permeability from angiogenesis. Strikingly, vascular endothelial (VE)-cadherin phosphorylation at the Y685, but not Y658, residue is reduced when VEGFR2 pY949 signaling is impaired. These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage. Therapeutically, targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact.

The number of people with impaired vision and blindness is increasing in Western society due to the aging population and the increased prevalence of diabetes. This has led to eye diseases, such as age-related macular degeneration and diabetic retinopathy becoming more common. In both these eye diseases, new blood vessels grow in the retina ­ the light-sensitive part of the eye ­ to bring oxygen and nutrients to the tissue. However, these new blood vessels are leaky and allow molecules to leave the bloodstream and enter the retinal tissue. This causes the retina to swell and impair a person's vision. The leaky blood supply also reduces the amount of oxygen that gets to the tissue, resulting in further damage to the retina. When tissues experience low levels of oxygen, cells start making a protein called vascular endothelial growth factor (or VEGF for short). Whilst this protein is important for helping form new blood vessels, it also makes these vessels leaky. Current treatments for age-related macular degeneration and diabetic retinopathy decrease swelling in the eye by blocking the action of VEGF. However, these treatments also cause existing blood vessels and nerve cells to die, leading to irreversible damage. Now, Smith et al. have set out to find whether the effects of VEGF can be blocked without causing further damage to existing cells. To investigate this possibility, the eyes and retinas of mice were treated with a laser or exposed to changing oxygen levels to create injuries that resembled human age-related macular degeneration and diabetic retinopathy. Each of the tested mice had specific mutations in proteins known to interact with VEGF. Fluorescent particles were injected into the bloodstream of the mice to assess how these different mutations affected blood vessel leakage: if fluorescent particles could no longer be detected outside the blood vessels, this suggested that the mutation had stopped the vessels from leaking. Further experiments showed these specific mutations affected leakage and did not prevent new blood vessels from forming. In the future it will be important to see if drugs, rather than mutations, can also decrease the leakiness of blood vessels in the retina. Such chemical compounds could then be tested in mouse experiments. If successful, these drugs might be used to treat patients with age-related macular degeneration and diabetic retinopathy.

Preclinical safety studies of human embryonic stem cell-derived retinal pigment epithelial cells for the treatment of age-related macular degeneration.

As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology.

Publisher Correction: Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells.

In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.