Vascular endothelial growth factor-B gene transfer exacerbates retinal and choroidal neovascularization and vasopermeability without promoting inflammation

The role of vascular endothelial growth factor (VEGF)-B in the eye is poorly understood. The present study was conducted to evaluate the effect of overexpression of VEGF-B via adeno-associated virus (AAV) gene transfer on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB).Three recombinant AAV vectors were prepared, expressing the 167 (AAV-VEGF-B167) or 186 amino acid isoform (AAV-VEGF-B186) of VEGF-B or the green fluorescent protein (GFP) reporter gene (AAV-GFP). Approximately 1 x 10\u2079 viral genome copies of AAV-VEGF-B167, AAV-VEGF-B186, or AAV-GFP were intraocularly injected. The efficacy of the gene transfer was assessed by directly observing GFP, by immunohistochemistry, or by real-time PCR. A leukostasis assay using fluorescein isothiocyanate-conjugated Concanavalin A was used to evaluate inflammation. The BRB was assessed using a quantitative assay with \ub3H-mannitol as a tracer. Retinal neovascularization (NV) was assessed at postnatal day 17 in oxygen-induced ischemic retinopathy after intravitreal injection of AAV-VEGF-B in left eyes and AAV-GFP in right eyes at postnatal day 7. Two weeks after injection of AAV vectors, choroidal NV was generated by laser photocoagulation and assessed 2 weeks later.GFP expression was clearly demonstrated, primarily in the retinal pigment epithelium (RPE) and outer retina, 1-6 weeks after delivery. mRNA expression levels of VEGF-B167 and VEGF-B186 were 5.8 and 12 fold higher in the AAV-VEGF-B167- and AAV-VEGF-B186-treated groups, respectively. There was no evidence of an inflammatory response or vessel abnormality following injection of the vectors in normal mice; however, VEGF-B increased retinal and choroidal neovascularization. AAV-VEGF-B186, but not AAV-VEGF-B167, enhanced retinal vascular permeability.VEGF-B overexpression promoted pathological retinal and choroidal NV and BRB breakdown without causing inflammation, which is associated with the progression of diabetic retinopathy and age-related macular degeneration, showing that these complications are not dependent on inflammation. VEGF-B targeting could benefit antiangiogenic therapy

En masse nascent transcription analysis to elucidate regulatory transcription factors

Despite exhaustively informing about steady-state mRNA abundance, DNA microarrays have been used with limited success to identify regulatory transcription factors (TFs). The main limitation of this approach is that altered mRNA stability also strongly governs the patterns of expressed genes. Here, we used nuclear run-on assays and microarrays to systematically interrogate changes in nascent transcription in cells treated with the topoisomerase inhibitor camptothecin (CPT). Analysis of the promoters of coordinately transcribed genes after CPT treatment suggested the involvement of TFs c-Myb and Rfx1. The predicted CPT-dependent associations were subsequently confirmed by chromatin immunoprecipitation assays. Importantly, after RNAi-mediated knockdown of each TF, the CPT-elicited induction of c-Myb- and/or Rfx1-regulated mRNAs was diminished and the overall cellular response was impaired. The strategies described here permit the successful identification of the TFs responsible for implementing adaptive gene expression programs in response to cellular stimulation

GESS: a database of global evaluation of SARS-CoV-2/hCoV-19 sequences

The COVID-19 outbreak has become a global emergency since December 2019. Analysis of SARS-CoV-2 sequences can uncover single nucleotide variants (SNVs) and corresponding evolution patterns. The Global Evaluation of SARS-CoV-2/hCoV-19 Sequences (GESS, https://wan-bioinfo.shinyapps.io/GESS/) is a resource to provide comprehensive analysis results based on tens of thousands of high-coverage and high-quality SARS-CoV-2 complete genomes. The database allows user to browse, search and download SNVs at any individual or multiple SARS-CoV-2 genomic positions, or within a chosen genomic region or protein, or in certain country/area of interest. GESS reveals geographical distributions of SNVs around the world and across the states of USA, while exhibiting time-dependent patterns for SNV occurrences which reflect development of SARS-CoV-2 genomes. For each month, the top 100 SNVs that were firstly identified world-widely can be retrieved. GESS also explores SNVs occurring simultaneously with specific SNVs of user's interests. Furthermore, the database can be of great help to calibrate mutation rates and identify conserved genome regions. Taken together, GESS is a powerful resource and tool to monitor SARS-CoV-2 migration and evolution according to featured genomic variations. It provides potential directive information for prevalence prediction, related public health policy making, and vaccine designs.National Institutes of Health [P30CA082709 to J.W.]; Walther Cancer Foundation. Funding for open access charge: National Institutes of Health

A collagen IV-derived peptide disrupts α5β1 integrin and potentiates Ang2/Tie2 signaling

The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5 β1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV–derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5 β1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5 β1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5 β1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5 β1 with AXT107 provides a potentially more effective approach to treat these diseases.Fil: Mirando, Adam C.. University Johns Hopkins; Estados UnidosFil: Shen, Jikui. University Johns Hopkins; Estados UnidosFil: Silva, Raquel Lima E.. University Johns Hopkins; Estados UnidosFil: Chu, Zenny. University Johns Hopkins; Estados UnidosFil: Sass, Nicholas C.. University Johns Hopkins; Estados UnidosFil: Lorenc, Valeria Erika. University Johns Hopkins; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Green, Jordan J.. University Johns Hopkins; Estados Unidos. AsclepiX Therapeutics; Estados UnidosFil: Campochiaro, Peter A.. University Johns Hopkins; Estados UnidosFil: Popel, Aleksander S.. University Johns Hopkins; Estados UnidosFil: Pandey, Niranjan B.. University Johns Hopkins; Estados Unidos. AsclepiX Therapeutics; Estados Unido

Genetic Spectrum and Distinct Evolution Patterns of SARS-CoV-2

Four signature groups of frequently occurred single-nucleotide variants (SNVs) were identified in over twenty-eight thousand high-quality and high-coverage SARS-CoV-2 complete genome sequences, representing different viral strains. Some SNVs predominated but were mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguishable evolution patterns over time. A few hundred patients were detected with multiple viral strain-representing mutations simultaneously, which may stand for possible co-infection or potential homogenous recombination of SARS-CoV-2 in environment or within the viral host. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tended to switch between bat RaTG13 coronavirus sequence and Wuhan-Hu-1 genome, indicating the higher genetic instability or tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.This work was partially supported by the National Institutes of Health (Grant Number: P30CA082709) and Walther Cancer Foundation (Grant Number: 4301-80519/0187.01). Funding for open access charge: National Institutes of Health

Updated SARS-CoV-2 Single Nucleotide Variants and Mortality Association

Since its outbreak in December 2019, COVID-19 has caused 100,5844,555 cases and 2,167,313 deaths as of Jan 27, 2021. Comparing our previous study of SARS-CoV-2 single nucleotide variants (SNVs) before June 2020, we found out that the SNV clustering had changed considerably since June 2020. Apart from that the group SNVs represented by two non-synonymous mutations A23403G (S: D614G) and C14408T (ORF1ab: P4715L) became dominant and carried by over 95% genomes, a few emerging groups of SNVs were recognized with sharply increased monthly occurrence ratios up to 70% in November 2020. Further investigation revealed that several SNVs were strongly associated with the mortality, but they presented distinct distribution in specific countries, e.g., Brazil, USA, Saudi Arabia, India, and Italy. SNVs including G25088T, T25A, G29861T and G29864A were adopted in a regularized logistic regression model to predict the mortality status in Brazil with the AUC of 0.84. Protein structure analysis showed that the emerging subgroups of non-synonymous SNVs and those mortality-related ones in Brazil were located on protein surface area. The clashes in protein structure introduced by these mutations might in turn affect virus pathogenesis through conformation changes, leading to the difference in transmission and virulence. Particularly, we found that SNVs tended to occur in intrinsic disordered regions (IDRs) of Spike (S) and ORF1ab, suggesting a critical role of SNVs in protein IDRs to determine protein folding and immune evasion

Blockade of VEGFR1 and 2 Suppresses Pathological Angiogenesis and Vascular Leakage in the Eye

VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents

Cbx2, a PcG Family Gene, Plays a Regulatory Role in Medaka Gonadal Development

Chromobox homolog 2 (CBX2), a key member of the polycomb group (PcG) family, is essential for gonadal development in mammals. A functional deficiency or genetic mutation in cbx2 can lead to sex reversal in mice and humans. However, little is known about the function of cbx2 in gonadal development in fish. In this study, the cbx2 gene was identified in medaka, which is a model species for the study of gonadal development in fish. Transcription of cbx2 was abundant in the gonads, with testicular levels relatively higher than ovarian levels. In situ hybridization (ISH) revealed that cbx2 mRNA was predominately localized in spermatogonia and spermatocytes, and was also observed in oocytes at stages I, II, and III. Furthermore, cbx2 and vasa (a marker gene) were co-localized in germ cells by fluorescent in situ hybridization (FISH). After cbx2 knockdown in the gonads by RNA interference (RNAi), the sex-related genes, including sox9 and foxl2, were influenced. These results suggest that cbx2 not only plays a positive role in spermatogenesis and oogenesis but is also involved in gonadal differentiation through regulating the expression levels of sex-related genes in fish

Blockade of VEGFR1 and 2 suppressed retinal NV in OIR.

<p>Neovascular tufts were labeled with griffonia simplicifolia lectin-594. Representative retinal NV for controls (a) and the combination treatment of MF1 and DC101 (b) were demonstrated. (c) The quantification of retinal NV.</p