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.

Retinoblastoma vulnerability to combined de novo and salvage pyrimidine ribonucleotide synthesis pharmacologic blockage.

Retinoblastoma is an eye cancer that commonly affects young children. Despite significant advances, current treatments cause side effects even when administered locally, and patients may still have to undergo enucleation. This is particularly disheartening in cases of bilateral retinoblastoma. Hence, there is an urgent need for novel therapeutic strategies. Inhibitors of the enzyme dihydroorotate dehydrogenase (DHODH), which is involved in the de novo pyrimidine ribonucleotide synthesis pathway, have proven to be effective in preclinical trials against several cancers including pediatric cancers. Here we tested whether blocking pyrimidine ribonucleotide synthesis promotes retinoblastoma cell death. Cultured retinoblastoma cell lines were treated with small molecule inhibitors of DHODH alone or in combination with inhibitors of nucleoside uptake to also block the salvage pathway for pyrimidine ribonucleotide formation. On their own, DHODH inhibitors had a moderate killing effect. However, the combination with nucleoside uptake inhibitors greatly enhanced the effect of DHODH inhibition. In addition, we observed that pyrimidine ribonucleotide synthesis blockage can cause cell death in a p53 mutant retinoblastoma cell line derived from a patient with metastasis. Explaining these results, the analysis of a published patient cohort revealed that loss of chr16q22.2 (containing the DHODH gene) is amongst the most frequent alterations in retinoblastoma and that these tumors often show gains in chromosome regions expressing pyrimidine ribonucleotide salvage factors. Furthermore, these genome alterations associate with malignancy. These results indicate that targeting pyrimidine ribonucleotide synthesis may be an effective therapeutic strategy to consider as a treatment for retinoblastoma.

Prognostic implications of tenascin C in peripheral blood and primary tumours at the time of uveal melanoma diagnosis.

OBJECTIVE: To examine the prognostic implication of tenascin C (TNC) in posterior uveal melanoma (UM). DESIGN: Retrospective cohort study. PARTICIPANTS: A total of 162 patients diagnosed with posterior UM. METHODS: A peripheral blood sample was obtained from 82 patients at the time of UM diagnosis between 1996 and 1999. Samples were kept frozen at -80°C until the concentration of TNC was measured in 2021. Primary tumour TNC RNA sequencing data were collected from another 80 patients (The Cancer Genome Atlas cohort). Patients were separated based on median TNC values. Cumulative incidences of metastatic death (UM mortality) from competing risks data were calculated as well as Cox regression hazard ratios. RESULTS: Patients with high and low TNC levels had tumours of similar size and American Joint Committee on Cancer stage at Bonferroni-corrected significance levels. The exception was a significantly smaller tumour diameter in patients with high serum TNC levels (p = 0.003). In competing risks analysis, patients with high serum TNC levels (≥7 ng/mL) had a higher UM mortality rate (44% vs 17% at 20 years; p = 0.008). Similarly, patients with higher primary tumour TNC RNA levels (≥1 transcripts per million) had higher UM mortality (83% vs 27% at 5 years; p = 0.003). In multivariate Cox regressions, TNC levels in peripheral blood and primary tumours were predictors of metastatic death independent of American Joint Committee on Cancer stage. CONCLUSIONS: TNC is a prognostic biomarker in UM. At the time of primary tumour diagnosis, it is measured in higher levels in both peripheral blood and tumour tissue from patients who will eventually suffer from metastatic death.

Obesity paradox in uveal melanoma: high body mass index is associated with low metastatic risk.

BACKGROUND: Metabolic factors and obesity may influence the development and progression of cancer. In this study, we examine their association with the risk of developing metastases of uveal melanoma. METHODS: Data on metabolic factors, medications, serum leptin levels, tumour leptin receptor RNA expression and clinical outcomes were examined in three cohorts. HRs for metastasis and cumulative incidences of melanoma-related mortality were calculated, and the levels of tumour leptin receptor expression were compared with prognostic factors including BAP1 mutation, and tumour cell morphology. RESULTS: Of 581 patients in the main cohort, 116 (20%) were obese and 7 (1 %) had metastatic disease at presentation. In univariate Cox regressions, tumour diameter, diabetes type II and use of insulin were associated with metastases, but patients with obesity had a lower risk. The beneficial prognostic implication of obesity was retained in multivariate regressions. In competing risk analyses, the incidence of melanoma-related mortality was significantly lower for patients with obesity. Serum leptin levels≥median were associated with a reduced risk for metastasis, independent of patient sex and cancer stage in a separate cohort (n=80). Similarly, in a third cohort (n=80), tumours with BAP1 mutation and epithelioid cells had higher leptin receptor RNA expression levels, which have a negative correlation with serum leptin levels. CONCLUSION: Obesity and elevated serum leptin levels are associated with a lower risk for developing metastases and dying from uveal melanoma.

A serum protein signature at the time of Uveal Melanoma diagnosis predicts long-term patient survival.

PURPOSE: To develop a prognostic test based on a single blood sample obtained at the time of uveal melanoma diagnosis. METHODS: 83 patients diagnosed with posterior uveal melanoma between 1996 and 2000 were included. Peripheral serum samples were obtained at diagnosis and kept at -80 °C until this analysis. Protein profiling of 84 cancer-related proteins was used to screen for potential biomarkers and a prognostic test that stratifies patients into metastatic risk categories was developed (serUM-Px) in a training cohort and then tested in a validation cohort. RESULTS: Low serum leptin levels and high osteopontin levels were found to identify patients with poor prognosis and were therefore selected for inclusion in the final test. In the validation cohort, patient sex and American Joint Committee on Cancer stages were similarly distributed between the low, intermediate, and high metastatic risk categories. With increasing metastatic risk category, patients had shorter metastasis-free- and overall survival, as well as greater cumulative incidence of uveal melanoma-related mortality in competing risk analysis (P = 0.007, 0.018 and 0.029, respectively). In multivariate Cox regression, serUM-Px was an independent predictor of metastasis with tumor size and patient sex as covariates (hazard ratio 3.2, 95% CI 1.5-6.9). CONCLUSIONS: A prognostic test based on a single peripheral venous blood sample at the time of uveal melanoma diagnosis stratifies patients into low, intermediate, and high metastatic risk categories. Prospective validation will facilitate its clinical utility.

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.

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.

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.

MicroRNAs and the HIF/VEGF axis in ocular neovascular diseases.

Ocular neovascular diseases, such as proliferative diabetic retinopathy, retinopathy of prematurity and neovascular age-related macular degeneration, are the leading causes of visual impairment worldwide. The hypoxia-inducible factors and vascular endothelial growth factors are key molecular promoters of ocular neovascularization. Moreover, the role of microRNAs as regulators of angiogenesis has been expanding, particularly hypoxia-associated microRNA; hypoxamiRs. This review provides a summary of hypoxamiRs that directly and specifically target HIF1A and VEGF mRNAs, thus critically involved in the regulation of ocular neovascular pathologies. The discussed microRNAs highlight putative diagnostic markers and therapeutic agents in choroidal and retinal angiogenic diseases, including proliferative diabetic retinopathy, retinopathy of prematurity and neovascular age-related macular degeneration.

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.

When Is a Control Not a Control? Reactive Microglia Occur Throughout the Control Contralateral Pathway of Retinal Ganglion Cell Projections in Experimental Glaucoma.

Purpose: Animal models show retinal ganglion cell (RGC) injuries that replicate features of glaucoma and the contralateral eye is commonly used as an internal control. There is significant crossover of RGC axons from the ipsilateral to the contralateral side at the level of the optic chiasm, which may confound findings when damage is restricted to one eye. The effect of unilateral glaucoma on neuroinflammatory damage to the contralateral pathway of RGC projections has largely been unexplored. Methods: Ocular hypertensive glaucoma was induced unilaterally or bilaterally in the rat and RGC neurodegenerative events were assessed. Neuroinflammation was quantified in the retina, optic nerve head, optic nerve, lateral geniculate nucleus, and superior colliculus by high-resolution imaging, and in the retina by flow cytometry and protein arrays. Results: After ocular hypertensive stress, peripheral monocytes enter the retina and microglia become reactive. This effect is more marked in animals with bilateral ocular hypertensive glaucoma. In rats where glaucoma was induced unilaterally, there was significant microglia activation in the contralateral (control) eye. Microglial activation extended into the optic nerve and terminal visual thalami, where it was similar across hemispheres in unilateral ocular hypertension. Conclusions: These data suggest that caution is warranted when using the contralateral eye as a control and in comparing visual thalami in unilateral models of glaucoma. Translational Relevance: The use of a contralateral eye as a control may confound the discovery of human-relevant mechanism and treatments in animal models. We also identify neuroinflammatory protein responses that warrant further investigation as potential disease-modifiable targets.