Oxidative stress-induced angiogenesis is mediated by miR-205-5p

miR-205-5p is known to be involved in VEGF-related angiogenesis and seems to reg-ulate associated cell signalling pathways, such as cell migration, proliferation and ap-optosis. Therefore, several studies have focused on the potential role of miR-205-5p as an anti-angiogenic factor. Vascular proliferation is observed in diabetic retinopathy and the ‘wet’ form of age-related macular degeneration. Today, the most common treatments against these eye-related diseases are anti-VEGF therapies. In addition, both AMD and DR are typically associated with oxidative stress; hence, the use of antioxidant agents is accepted as a co-adjuvant therapy for these patients. According to previous data, ARPE-19 cells release pro-angiogenic factors when exposed to oxi-dative insult, leading to angiogenesis. Matching these data, results reported here, indicate that miR-205-5p is modulated by oxidative stress and regulates VEGFA-angiogenesis. Hence, miR-205-5p is proposed as a candidate against eye-related pro-liferative diseasesUniversidad Católica de Valencia San Vicente Mártir research Fund and Centro de Investigación Traslacional San Alberto Magno, Universidad Católica de Valencia San Vicente Mártir, Grant/Award Number: 2018-128-001, 2019-128-001; Escuela de Doctorado Universidad Católica de Valencia San Vicente Mártir, Grant/Award Number: EDUCV-PRE-2015-006 and EDUCV-PRE-2016-005Medicin

miR302a and 122 are deregulated in small extracellular vesicles from ARPE-19 cells cultured with H2O2

Age related macular degeneration (AMD) is a common retina-related disease leading to blindness. Little is known on the origin of the disease, but it is well documented that oxidative stress generated in the retinal pigment epithelium and choroid neovascularization are closely involved. The study of circulating miRNAs is opening new possibilities in terms of diagnosis and therapeutics. miRNAs can travel associated to lipoproteins or inside small Extracellular Vesicles (sEVs). A number of reports indicate a signifcant deregulation of circulating miRNAs in AMD and experimental approaches, but it is unclear whether sEVs present a signifcant miRNA cargo. The present work studies miRNA expression changes in sEVs released from ARPE-19 cells under oxidative conditions (i.e. hydrogen peroxide, H2O2). H2O2 increased sEVs release from ARPE-19 cells. Moreover, 218 miRNAs could be detected in control and H2O2 induced-sEVs. Interestingly, only two of them (hsa-miR-302a and hsa-miR-122) were signifcantly under-expressed in H2O2-induced sEVs. Results herein suggest that the down regulation of miRNAs 302a and 122 might be related with previous studies showing sEVs-induced neovascularization after oxidative challenge in ARPE-19 cells.Te present project was supported by internal funds from Universidad Católica de Valencia San Vicente Mártir (2018-128-001), Centro de Investigación Traslacional San Alberto Magno de la UCV (2019-128-001), and Conselleria de Educación, Investigación, Cultura y Deporte; Generalitat Valenciana (PROMETEO/2016/094). Oltra M and Vidal-Gil L, PhD training program fellowship UCV (EDUCV-PRE-2016-005 and EDUCVPRE-2015-006 Personal Investigador en Formación UCV, respectively).Medicin

Ethanol-Induced Oxidative Stress Modifies Inflammation and Angiogenesis Biomarkers in Retinal Pigment Epithelial Cells (ARPE-19): Role of CYP2E1 and its Inhibition by Antioxidants

The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.LVG was recipient of a pre-doctoral fellowship (EDUCV-PRE-2015-006). Financial support by grant #94/2016 from the PROMETEO program from the Generalitat Valenciana, Valencia, Spain, to FJR

Role of retinal pigment epithelium-derived exosomes and autophagy in new blood vessel formation

Autophagy and exosome secretion play important roles in a variety of physiological and disease states, including the development of age‐related macular degeneration. Previous studies have demonstrated that these cellular mechanisms share common pathways of activation. Low oxidative damage in ARPE‐19 cells, alters both autophagy and exosome biogenesis. Moreover, oxidative stress modifies the protein and genetic cargo of exosomes, possibly affecting the fate of surrounding cells. In order to understand the connection between these two mechanisms and their impact on angiogenesis, stressed ARPE‐19 cells were treated with a siRNA‐targeting Atg7, a key protein for the formation of autophagosomes. Subsequently, we observed the formation of multivesicular bodies and the release of exosomes. Released exosomes contained VEGFR2 as part of their cargo. This receptor for VEGF—which is critical for the development of new blood vessels—was higher in exosome populations released from stressed ARPE‐19. While stressed exosomes enhanced tube formation, exosomes became ineffective after silencing VEGFR2 in ARPE‐19 cells and were, consequently, unable to influence angiogenesis. Moreover, vessel sprouting in the presence of stressed exosomes seems to follow a VEGF‐independent pathway. We propose that abnormal vessel growth correlates with VEGFR2‐expressing exosomes release from stressed ARPE‐19 cells, and is directly linked to autophagy

Mechanisms of neurodegeneration in a preclinical autosomal dominant retinitis pigmentosa knock-in model with a RhoD190N mutation

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.We greatly appreciate the assistance of the members of the Bernard & Shirlee Brown Glaucoma laboratory, especially to Chun-Wei Hsu for technical support. SHT is a Burroughs-Wellcome Program in Biomedical Sciences Fellow, and is also supported by the Charles E. Culpeper-Partnership for Cures 07-CS3, Crowley Research Fund, Schneeweiss Stem Cell Fund, New York State N09G-302, Foundation Fighting Blindness [TA-NMT-0116-0692- COLU] (Owings Mills, MD), TS080017 from US Department of Defense, NIH Grants [P30EY019007, R01EY018213, R01EY024698, R01EY026682, R21AG050437], Research to Prevent Blindness (New York, NY), and Joel Hoffmann Scholarship. CSL is the Homer McK. Rees Scholar. JSP is a BEST2016 awardee (BEST/ 2016/030, Conselleria de Educación, Investigación, Cultura y Deporte; Generalitat Valenciana) and his research is supported by a Prometeo Grant (PROMETEO/2016/094; Conselleria de Educación, Investigación, Cultura y Deporte; Generalitat Valenciana) and by internal funds from Universidad Católica de Valencia San Vicente Mártir (2018-128-001). VBM is supported by NIH Grants K08EY020530, R01EY016822, The Doris Duke Charitable Foundation Grant #2013103, and Research to Prevent Blindness (New York, NY); GV is supported by NIH Grants [F30EYE027986 and T32GM007337].Author manuscriptMedicin

Animal-Assisted Therapy in the Residential Treatment of Dual Pathology

Animal-assisted therapy (AAT) is a complementary intervention of therapy that has shown positive results in the treatment of various pathologies. This study assesses the viability of the implementation and the effectiveness of an AAT program in patients diagnosed with substance abuse disorder and associated mental disorders (dual pathology). For the study, a dynamic prospective cohort was used, consisting of 43 patients in residential treatment. The program consisted of 10 sessions with a duration of about 60 min, where data was collected in the 3rd, 6th and 10th sessions. The Life Skills Profile questionnaire (LSP) and the Barratt Impulsiveness Scale (BIS-11) were used for subsequent evaluation. Patients who participated in the program showed an improvement in daily skills, which favoured a better quality of life and decreased impulsiveness, enabling them to regain self-control. These results suggest that the dog can be a multi-sensory stimulus that captures attention, and improves motivation, cooperation and patient involvement in therapy. It was concluded that AAT can serve as an adjunctive therapy in the rehabilitation processes of people diagnosed with dual pathology

Release of Retinal Extracellular Vesicles in a Model of Retinitis Pigmentosa

Extracellular vesicles (EVs) are membranous structures released by cells, including those of the retinal pigment epithelium (RPE) and photoreceptors. The cargo of EVs includes genetic material and proteins, making these vesicles essential in cell communication. Among the genetic materials, we find a large number of microRNAs (miRNAs), small chains of noncoding RNA. In the case of EVs from the retina, changes have also been observed in the number and cargo of EVs.Our group confirmed that damaged RPE cells in vitro release a greater number of EVs with a higher pro-angiogenic factor (VEGFR-1 and VEGFR-2) than control non-damaged cells, thus increasing neovascularization in endothelial cell cultures. This indicates that something similar could happen in patients suffering from some types of retinal degeneration that occur with angiogenesis, such as wet AMD or RD.Here, we investigated the role of EVs in photoreceptor degeneration, and we report for the first time on CD9 and CD81, closely related tetraspanins, in wild-type and rd1 retinae. Our study demonstrates the involvement of EVs in the process of inherited photoreceptor degeneration in a PDE6 mutationMedicin

Poly ADP ribosylation and extracellular vesicle activity in rod photoreceptor degeneration

Abstract Retinitis Pigmentosa is a group of inherited neurodegenerative diseases that result in selective cell death of photoreceptors. In the developed world, RP is regarded as the main cause of blindness among the working age population. The precise mechanisms eventually leading to cell death remain unknown and to date no adequate treatment for RP is available. Poly ADP ribose polymerase (PARP) over activity is involved in photoreceptor degeneration and pharmacological inhibition or genetic knock-down PARP1 activity protect photoreceptors in mice models, the mechanism of neuroprotection is not clear yet. Our result indicated that olaparib, a PARP1 inhibitor, significantly rescued photoreceptor cells in rd10 retina. Extracellular vesicles (EVs) were previously recognized as a mechanism for discharging useless cellular components. Growing evidence has elucidated their roles in cell–cell communication by carrying nucleic acids, proteins and lipids that can, in turn, regulate behavior of the target cells. Recent research suggested that EVs extensively participate in progression of diverse blinding diseases, such as age-related macular (AMD) degeneration. Our study demonstrates the involvement of EVs activity in the process of photoreceptor degeneration in a PDE6 mutation. PARP inhibition protects photoreceptors via regulation of the EVs activity in rod photoreceptor degeneration in a PDE6b mutation

PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP

Role of Exosomal miR-205-5p Cargo in Angiogenesis and Cell Migration

Exosomes or small extracellular vesicles (sEVs) represent a pivotal component in intercellular communication, carrying a diverse array of biomolecules. Several factors can affect sEVs release dynamics, as occurs in hyperglycemia or inflammation. In fact, sEVs release has been associated with the promotion of physio-pathological processes. Among the sEVs cargo, microRNAs play an essential role in cell-to-cell regulation. More concretely, miR-205-5p is related to angiogenesis and cell proliferation. The aim of this study is to understand the specific role of sEVs containing miR-205-5p under high glucose conditions. ARPE-19 cells were cultured with high glucose (HG) for 5 days. sEVs were isolated and characterized. sEVs from ARPE-19 were used for angiogenesis and cell proliferation. HG increased sEVs release but downregulated miR-205-5p cargo expression compared to the control. sEVs from HG-treated ARPE-19 cells promoted tube formation and migration processes. In contrast, miR-205-5p overexpression (by mimic transfection) decreased angiogenesis and cell migration. Our results demonstrate how ARPE-19 cells respond to HG challenge by increasing sEVs with weak miR-205-5p cargo. The absence of this miRNA in sEVs is enough to promote angiogenesis. In contrast, restoring sEVs-miR-205-5p levels decreased it. These findings open new possibilities in sEVs-based therapies containing miR-205-5p against angiogenesis.This research was funded by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación Española, grant number PID2020-117875GB-10; Instituto de Salud Carlos III grant number PI21/00083; and the European Union research fund HORIZON MSCA 2021-DN-01-01_RETORNA 101073316.Medicin