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.

Exploring the Potential of Wild Olive (Acebuche) Oil as a Pharm-Food to Prevent Ocular Hypertension and Fibrotic Events in the Retina of Hypertensive Mice.

SCOPE: Our laboratory has previously described the antioxidant and anti-inflammatory potential of a wild olive (acebuche, ACE) oil against hypertension-associated vascular retinopathies. The current study aims to analyze the antifibrotic effect of ACE oil on the retina of hypertensive mice. METHODS AND RESULTS: Mice are rendered hypertensive by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) and simultaneously subjected to dietary supplementation with ACE oil or a reference extra virgin olive oil (EVOO). Intraocular pressure (IOP) is measured by rebound tonometry, and retinal vasculature/layers are analyzed by fundus fluorescein angiography and optical coherence tomography. Different fibrosis-related parameters are analyzed in the retina and choroid of normotensive and hypertensive mice with or without oil supplementation. Besides preventing the alterations found in hypertensive animals, including increased IOP, reduced fluorescein signal, and altered retinal layer thickness, the ACE oil-enriched diet improves collagen metabolism by regulating the expression of major fibrotic process modulators (matrix metalloproteinases, tissue inhibitors of metalloproteinases, connective tissue growth factor, and transforming growth factor beta family). CONCLUSION: Regular consumption of EVOO and ACE oil (with better outcomes in the latter) might help reduce abnormally high IOP values in the context of hypertension-related retinal damage, with significant reduction in the surrounding fibrotic process.

Anatomy of the complete mouse eye vasculature explored by light-sheet fluorescence microscopy exposes subvascular-specific remodeling in development and pathology.

Eye development and function rely on precise establishment, regression and maintenance of its many sub-vasculatures. These crucial vascular properties have been extensively investigated in eye development and disease utilizing genetic and experimental mouse models. However, due to technical limitations, individual studies have often restricted their focus to one specific sub-vasculature. Here, we apply a workflow that allows for visualization of complete vasculatures of mouse eyes of various developmental stages. Through tissue depigmentation, immunostaining, clearing and light-sheet fluorescence microscopy (LSFM) entire vasculatures of the retina, vitreous (hyaloids) and uvea were simultaneously imaged at high resolution. In silico dissection provided detailed information on their 3D architecture and interconnections. By this method we describe successive remodeling of the postnatal iris vasculature, involving sprouting and pruning, following its disconnection from the embryonic feeding hyaloid vasculature. In addition, we demonstrate examples of conventional and LSFM-mediated analysis of choroidal neovascularization after laser-induced wounding, showing added value of the presented workflow in analysis of modelled eye disease. These advancements in visualization and analysis of the respective eye vasculatures in development and complex eye disease open for novel observations of their functional interplay at a whole-organ level.

Efficient targeting of HIF-1α mediated by YC-1 and PX-12 encapsulated niosomes: potential application in colon cancer therapy.

A number of molecular biofactors have been documented in pathogenesis and poor prognosis of colorectal cancer (CRC). Among them, the Hypoxia-Inducible Factor (HIF-1a) is frequently reported to become over-expressed, and its targeting could restrict and control a variety of essential hallmarks of CRC. Niosomes are innovative drug delivery vehicles with the encapsulating capacity for co-loading both hydrophilic and hydrophobic drugs at the same time. Also, they can enhance the local accumulation while minimizing the dose and side effects of drugs. YC-1 and PX-12 are two inhibitors of HIF-1a. The purpose of this work was to synthesize dual-loaded YC-1 and PX-12 niosomes to efficiently target HIF-1α in CRC, HT-29 cells. The niosomes were prepared by the thin-film hydration method, then the niosomal formulation of YC-1 and PX-12 (NIO/PX-YC) was developed and optimized by the central composition method (CCD) using the Box-Behnken design in terms of size, polydispersity index (PDI), entrapment efficiency (EE). Also, they are characterized by DLS, FESEM, and TEM microscopy, as well as FTIR spectroscopy. Additionally, entrapment efficiency, in vitro drug release kinetics, and stability were assessed. Cytotoxicity, apoptosis, and cell cycle studies were performed after the treatment of HT-29 cells with NIO/PX-YC. The expression of HIF-1αat both mRNA and protein levels were studied after NIO/PX-YC treatment. The prepared NIO/PX-YC showed a mean particle size of 185 nm with a zeta potential of about-7.10 mv and a spherical morphology. Also, PX-12 and YC-1 represented the entrapment efficiency of about %78 and %91, respectively, with a sustainable and controllable release. The greater effect of NIO/PX-YC than the free state of PX-YC on the cell survival rate, cell apoptosis, and HIF-1α gene/protein expression were detected (p < 0.05). In conclusion, dual loading of niosomes with YC-1 and PX-12 enhanced the effect of drugs on HIF-1α inhibition, thus boosting their anticancer effects.

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.

Hypertension secondary to nitric oxide depletion produces oxidative imbalance and inflammatory/fibrotic outcomes in the cornea of C57BL/6 mice

Arterial hypertension (AH) leads to oxidative and inflammatory imbalance that contribute to fibrosis development in many target organs. Here, we aimed to highlight the harmful effects of severe AH in the cornea. Our experimental model was established by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) to C57BL/6 mice, which were monitored weekly for arterial blood pressure and intraocular pressure (IOP). Morphological studies of ocular tissues were accompanied by analyses of reactive oxygen species generation, and localization/expression of NAPDH oxidase isoforms (NOX1, NOX2, NOX4) and inflammatory biomarkers (PPARα, PPARγ, IL-1β, IL-6, IL-10, TNF-α, and COX-2). Masson’s trichrome and Sirius Red staining were used to explore the fibrotic status of the cornea. The expression of collagen isoforms (COL1α1, COL1α2, COL3α1, COL4α1, COL4α2) and relevant metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were also quantified to evaluate the participation of collagen metabolism in AH-related corneal damage. Hypertensive animals showed an increase in IOP values, and a thinner cornea compared with normotensive controls. Moreover, AH increased NADPH oxidase activity and reactive oxygen species generation in the cornea, which was accompanied by transcriptional upregulation of NOX isoforms and inflammatory biomarkers, while reducing PPAR expression. L-NAME-treated animals also developed corneal fibrosis with overexpression of collagen isoforms and reduction of factors responsible for collagen degradation. This is the first study reporting structural changes in the cornea and elevated IOP in L-NAME-treated mice. Overexpression of the NADPH oxidase system and collagen deposition might play a substantial role in the pathogenic mechanisms contributing to ocular disturbances in a context of severe hypertension. (AU)

Hypertension secondary to nitric oxide depletion produces oxidative imbalance and inflammatory/fibrotic outcomes in the cornea of C57BL/6 mice.

Arterial hypertension (AH) leads to oxidative and inflammatory imbalance that contribute to fibrosis development in many target organs. Here, we aimed to highlight the harmful effects of severe AH in the cornea. Our experimental model was established by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) to C57BL/6 mice, which were monitored weekly for arterial blood pressure and intraocular pressure (IOP). Morphological studies of ocular tissues were accompanied by analyses of reactive oxygen species generation, and localization/expression of NAPDH oxidase isoforms (NOX1, NOX2, NOX4) and inflammatory biomarkers (PPARα, PPARγ, IL-1ß, IL-6, IL-10, TNF-α, and COX-2). Masson's trichrome and Sirius Red staining were used to explore the fibrotic status of the cornea. The expression of collagen isoforms (COL1α1, COL1α2, COL3α1, COL4α1, COL4α2) and relevant metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were also quantified to evaluate the participation of collagen metabolism in AH-related corneal damage. Hypertensive animals showed an increase in IOP values, and a thinner cornea compared with normotensive controls. Moreover, AH increased NADPH oxidase activity and reactive oxygen species generation in the cornea, which was accompanied by transcriptional upregulation of NOX isoforms and inflammatory biomarkers, while reducing PPAR expression. L-NAME-treated animals also developed corneal fibrosis with overexpression of collagen isoforms and reduction of factors responsible for collagen degradation. This is the first study reporting structural changes in the cornea and elevated IOP in L-NAME-treated mice. Overexpression of the NADPH oxidase system and collagen deposition might play a substantial role in the pathogenic mechanisms contributing to ocular disturbances in a context of severe hypertension.

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.

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.

Anti-Inflammatory Action of Dietary Wild Olive (Acebuche) Oil in the Retina of Hypertensive Mice.

Inflammation plays a crucial role in the course of eye diseases, including many vascular retinopathies. Although olive oil is known to have beneficial effects against inflammatory processes, there is no information available on the anti-inflammatory potential of the wild olive tree (namely, acebuche (ACE) for the primitive Spanish lineages). Here we investigate the anti-inflammatory effects of ACE oil in the retina of a mouse model of arterial hypertension, which was experimentally induced by administration of L-NAME (NG-nitro-L-arginine-methyl-ester). The animals were fed supplements of ACE oil or extra virgin olive oil (EVOO, for comparative purposes). Retinal function was assessed by electroretinography (ERG), and different inflammation-related parameters were measured in the retina and choroid. Besides significant prevention of retinal dysfunction shown in ERG recordings, ACE oil-enriched diet upregulated the expression of the anti-inflammatory markers PPARγ, PPARα and IL-10, while reducing that of major proinflammatory biomarkers, IL-1ß, IL-6, TNF-α and COX-2. This is the first report to highlight the anti-inflammatory properties of an ACE oil-enriched diet against hypertension-related retinal damage. Noteworthy, dietary supplementation with ACE oil yielded better results compared to a reference EVOO.

The monoamine stabilizer OSU6162 has anxiolytic-like properties and reduces voluntary alcohol intake in a genetic rat model of depression.

Alcohol use disorders (AUD) often co-occur with anxiety and depressive disorders, and anxiety often drives relapse during alcohol abstinence. Optimal AUD pharmacotherapies may thus need to target both excessive alcohol intake and elevated anxiety. (-)-OSU6162 (OSU) is a monoamine stabilizer that attenuates alcohol-mediated behaviors in both preclinical and clinical settings. However, OSU's effect on anxiety-like behavior following long-term drinking remains unknown. To this end, we utilized a genetic rat model that exhibits increased anxiety- and depression-like behaviors (Flinders Sensitive Line; FSL) and their controls (Flinders Resistant Line; FRL). Using the novelty suppressed feeding (NSF) test, we evaluated anxiety-like behaviors (1) at baseline, (2) following long-term voluntary drinking and after 24 h of alcohol deprivation, and (3) following OSU administration in the same animals. At baseline, FSL animals displayed significantly elevated anxiety-like characteristics compared to FRL. Compared to alcohol-naïve animals, long-term drinking significantly reduced anxiety-like behaviors in FSL, without any significant effects in FRL animals. Compared to vehicle, OSU administration significantly reduced anxiety-like behaviors in alcohol-naïve FSL and long-term drinking FRL animals. While there was no significant difference in alcohol intake between FSL and FRL, OSU attenuated alcohol intake in both strains. Conclusively, in addition to the compound's previously identified ability to suppress alcohol-mediated behaviors, OSU may also possess anxiolytic properties, warranting further clinical evaluation in both AUD and anxiety disorder settings.

NADPH oxidase-induced oxidative stress in the eyes of hypertensive rats.

Purpose: Increased reactive oxygen species (ROS) released by NADPH oxidase and inflammation are associated with arterial hypertension and eye diseases associated with high blood pressure, including glaucoma, retinopathies (e.g., age-related macular degeneration), and choroidopathies affecting ocular function; however, the mechanisms underlying these adverse outcomes remain undefined. The present study was designed to highlight the importance of oxidative stress in severe hypertension-related eye damage. Methods: Male Wistar rats (n = 7, unless otherwise specified for specific experiments) were administered an oral dose of 30 mg of Nω-nitro-L-arginine methyl ester (L-NAME) per kilogram of bodyweight and day for 3 weeks; chronic administration with L-NAME is a validated experimental approach resulting in severe hypertension secondary to nitric oxide (NO) depletion and subsequent vasoconstriction in the systemic circulation. Upon treatment completion, histomorphometric studies, NADPH oxidase activity, and ROS production were measured in eyecup homogenates and paraffin-embedded sections from control and L-NAME-treated animals. In addition, immunohistofluorescence, western blotting, and real-time PCR (RT-qPCR) analyses were performed in the eye and the retina to evaluate the expression of i) NADPH oxidase main isoforms (NOX1, NOX2, and NOX4) and subunits (p22phox and p47phox); ii) glial fibrillary acidic protein (GFAP), as a marker of microglial activation in the retina; iii) antioxidant enzymes; and iv) endothelial constitutive (eNOS) and inflammation inducible (iNOS) nitric oxide synthase isoforms, and nitrotyrosine as a versatile biomarker of oxidative stress. Results: Increased activity of NADPH oxidase and superoxide anion production, accompanied by transcriptional upregulation of this enzyme isoforms, was found in the retina and choroid of the hypertensive rats in comparison with the untreated controls. Histomorphometric analyses revealed a significant reduction in the thickness of the ganglion cell layer and the outer retinal layers in the hypertensive animals, which also showed a positive strong signal of GFAP in the retinal outer segment and plexiform layers. In addition, L-NAME-treated animals presented with upregulation of nitric oxide synthase (including inducible and endothelial isoforms) and abnormally elevated nitrotyrosine levels. Experiments on protein and mRNA expression of antioxidant enzymes revealed depletion of superoxide dismutase and glutathione peroxidase in the eyes of the hypertensive animals; however, glutathione reductase was significantly higher than in the normotensive controls. Conclusions: The present study demonstrated structural changes in the retinas of the L-NAME-treated hypertensive animals and strengthens the importance of NADPH oxidase as a major ROS-generating enzyme system in the oxidative and inflammatory processes surrounding hypertensive eye diseases. These observations might contribute to unveiling pathogenic mechanisms responsible for developing ocular disturbances in the context of severe hypertension.

Liver bioengineering: Recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.

Development of novel technologies provides the best tissue constructs engineering and maximizes their therapeutic effects in regenerative therapy, especially for liver dysfunctions. Among the currently investigated approaches of tissue engineering, scaffold-based and scaffold-free tissues are widely suggested for liver regeneration. Analogs of liver acellular extracellular matrix (ECM) are utilized in native scaffolds to increase the self-repair and healing ability of organs. Native ECM analog could improve liver repairing through providing the supportive framework for cells and signaling molecules, exerting normal biomechanical, biochemical, and physiological signal complexes. Recently, innovative cell sheet technology is introduced as an alternative for conventional tissue engineering with the advantage of fewer scaffold restrictions and cell culture on a Thermo-Responsive Polymer Surface. These sheets release the layered cells through a temperature-controlled procedure without enzymatic digestion, while preserving the cell-ECM contacts and adhesive molecules on cell-cell junctions. In addition, several novelties have been introduced into the cell sheet and decellularization technologies to aid cell growth, instruct differentiation/angiogenesis, and promote cell migration. In this review, recent trends, advancements, and issues linked to translation into clinical practice are dissected and compared regarding the decellularization and cell sheet technologies for liver tissue engineering.

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.