The retinal pigment epithelium ( RPE ), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species ( ROS ). As the RPE , because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell‐to‐cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors ( VEGFR ) were analysed by both flow cytometry and Western blot. RT ‐ PCR and qPCR were conducted to assess mRNA content of VEGFR s in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA . Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.

Background and Purpose: Large-scale observational studies of acute ischemic stroke (AIS) promise to reveal mechanisms underlying cerebral ischemia. However, meaningful quantitative phenotypes attainable in large patient populations are needed. We characterize a dynamic metric of AIS instability, defined by change in National Institutes of Health Stroke Scale score (NIHSS) from baseline to 24 hours baseline to 24 hours (NIHSS _baseline – NIHSS _24hours = ΔNIHSS _6-24h ), to examine its relevance to AIS mechanisms and long-term outcomes. Methods: Patients with NIHSS prospectively recorded within 6 hours after onset and then 24 hours later were enrolled in the GENISIS study (Genetics of Early Neurological Instability After Ischemic Stroke). Stepwise linear regression determined variables that independently influenced ΔNIHSS _{6 –24h} . In a subcohort of tPA (alteplase)-treated patients with large vessel occlusion, the influence of early sustained recanalization and hemorrhagic transformation on ΔNIHSS _6–24h was examined. Finally, the association of ΔNIHSS _{6 –24h} with 90-day favorable outcomes (modified Rankin Scale score 0–2) was assessed. Independent analysis was performed using data from the 2 NINDS-tPA stroke trials (National Institute of Neurological Disorders and Stroke rt-PA). Results: For 2555 patients with AIS, median baseline NIHSS was 9 (interquartile range, 4–16), and median ΔNIHSS _{6 –24h} was 2 (interquartile range, 0–5). In a multivariable model, baseline NIHSS, tPA-treatment, age, glucose, site, and systolic blood pressure independently predicted ΔNIHSS _{6 –24h} (R ² =0.15). In the large vessel occlusion subcohort, early sustained recanalization and hemorrhagic transformation increased the explained variance (R ² =0.27), but much of the variance remained unexplained. ΔNIHSS _{6 –24h} had a significant and independent association with 90-day favorable outcome. For the subjects in the 2 NINDS-tPA trials, ΔNIHSS _{3 –24h} was similarly associated with 90-day outcomes. Conclusions: The dynamic phenotype, ΔNIHSS _6–24h , captures both explained and unexplained mechanisms involved in AIS and is significantly and independently associated with long-term outcomes. Thus, ΔNIHSS _{6 –24h} promises to be an easily obtainable and meaningful quantitative phenotype for large-scale genomic studies of AIS.

Rationale:: Ischemic stroke is among the leading causes of adult disability. Part of the variability in functional outcome after stroke has been attributed to genetic factors but no locus has been consistently associated with stroke outcome. Objective:: Our aim was to identify genetic loci influencing the recovery process using accurate phenotyping to produce the largest GWAS (genome-wide association study) in ischemic stroke recovery to date. Methods and Results:: A 12-cohort, 2-phase (discovery-replication and joint) meta-analysis of GWAS included anterior-territory and previously independent ischemic stroke cases. Functional outcome was recorded using 3-month modified Rankin Scale. Analyses were adjusted for confounders such as discharge National Institutes of Health Stroke Scale. A gene-based burden test was performed. The discovery phase (n=1225) was followed by open (n=2482) and stringent joint-analyses (n=1791). Those cohorts with modified Rankin Scale recorded at time points other than 3-month or incomplete data on previous functional status were excluded in the stringent analyses. Novel variants in PATJ (Pals1-associated tight junction) gene were associated with worse functional outcome at 3-month after stroke. The top variant was rs76221407 (G allele, β=0.40, P =1.70×10 ⁻⁹ ). Conclusions:: Our results identify a set of common variants in PATJ gene associated with 3-month functional outcome at genome-wide significance level. Future studies should examine the role of PATJ in stroke recovery and consider stringent phenotyping to enrich the information captured to unveil additional stroke outcome loci.

Background and Purpose—: Clopidogrel is one of the most used antiplatelet drugs in patients with cardiovascular disease. However, 16% to 50% of patients have a high on-clopidogrel platelet reactivity and an increased risk of ischemic events. The pathogenesis of high on-treatment platelet reactivity in patients with stroke is only partially explained by genetic variations. This study aims to find differentially methylated sites across the genome associated with vascular recurrence in ischemic stroke patients treated with clopidogrel. Methods—: From a cohort of 1900 patients with ischemic stroke, we selected 42 patients treated with clopidogrel, including 21 with a recurrent vascular event and 21 without vascular recurrence during the first year of follow-up. Over 480 000 DNA methylation sites were analyzed across the genome. Differentially methylated CpG sites were identified by nonparametric testing using R. Replication analysis was performed in a new cohort of 191 subjects and results were correlated with platelet reactivity in a subset of 90 subjects using light transmission aggregometry. Results—: A total of 73 differentially methylated CpG sites ( P <1×10 ⁻⁰⁵ ) were identified; 3 of them were selected for further replication: cg03548645 ( P =1.42×10 ⁻⁰⁵ , TRAF3 ), cg09533145 ( P =7.81×10 ⁻⁰⁶ , ADAMTS2 ), and cg15107336 ( P =1.89×10 ⁻⁰⁵ , XRCC1 ). The cg03548645 CpG remained significant in the replication study ( P =0.034), a deep analysis of this region revealed another methylation site associated with vascular recurrence, P =0.037. Lower cg03548645 ( TRAF3 ) DNA methylation levels were correlated with an increased platelet aggregation (ρ=−0.29, P =0.0075). Conclusions—: This study suggests for the first time that epigenetics may significantly contribute to the variability of clopidogrel response and recurrence of ischemic events in patients with stroke.

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.