Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro-and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-a/b activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-c and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.Therapeutic application of long, double-stranded (ds)RNAmediated RNAi and sequence-specific gene silencing through RNAi by short synthetic RNA duplexes is challenging because mammalian cells do not uptake 'naked' siRNA (whether chemically modified or not) without cell-permeating entities [1][2][3][4] . To minimize systemic exposure, initial clinical trials of siRNA were launched using intraocular injection in patients with CNV. CNV, wherein the retina is invaded by choroidal vessels beneath the retinal pigmented epithelium (RPE), is a late stage of age-related macular degeneration that afflicts 30-50 million people globally. The preclinical bases for trials of naked VEGFA siRNA (Bevasiranib) or VEGFR1 siRNA (AGN211745/ siRNA-027) were single reports in mice 5,6 that such siRNAs suppressed laser-injury-induced CNV, a model predictive of efficacy in humans 7,8 . These findings were interpreted as anomalous examples of local delivery surmounting the impediment to intracellular entry 9-11 . Instead, we show in two animal models that suppression of neovascularization is a generic property of siRNAs independent of sequence, target and internalization.Sequence-independent angiogenesis suppression by siRNA Numerous synthetic non-targeted 21-nucleotide duplex siRNAs from multiple vendors, when injected into the vitreous humour of wild-type mice, uniformly and dose-dependently suppressed CNV (Fig. 1a, b and Supplementary Fig. 1). siRNAs targeting jellyfish green fluorescent ...

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularization (CNV). We report that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in CNV endothelial cells in humans with AMD, and that, despite the expression of its ligands eotaxin-1, -2, and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation as it occurred in mice lacking eosinophils or mast cells and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor-A (VEGF-A) neutralization, which is currently in clinical use, and, unlike VEGF-A blockade, not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m), compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK) and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell.

Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. The findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte.

FKN is expressed in various ocular tissues and cells. Inflammatory mediator modulation of ocular FKN expression suggests that this adhesive chemokine may play important roles in regulating leukocyte efflux in inflammatory eye diseases, such as anterior uveitis and retinochoroiditis.

Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of tachyzoites to the retina.

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