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

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

Feasibility of laser-targeted photoocclusion of the choriocapillary layer in rats

Purpose. A new method, laser-targeted photoocclusion, was developed to occlude choroidal neovascularization while minimizing damage to the overlying retina. The ability to occlude normal choriocapillary layer in rats was evaluated as a first test of die feasibility of treating choroidal neovascularization with this method. Method. A photosensitive agent, aluminum phdialocyanine tetrasulfonate, encapsulated in heat-sensitive liposomes, was administered intravenously along with carboxyfluorescein liposomes. A low-power argon laser (retinal power density of 5.7 W/cm 2 ) locally released a photosensitizer bolus, monitored by the simultaneous release of carboxyfluorescein. A diode laser (operating at 675 nm with a retinal power density of 0.27 W/cm 2 ) activated the photosensitizer with its release. Results. Vessels in the choriocapillary layer were occluded at day 3 after laser treatment and remained unchanged during die 30-day follow-up. Larger choroidal vessels and retinal capillaries remained perfused. Control experiments excluded possible effects of heat or activation of free photosensitizer. Pilot histologic studies showed no damage to the retinal pigment epithelium. Conclusions. Laser-targeted photoocclusion caused selective occlusion of normal choriocapillaries while sparing overlying retinal pigment epithelium and retinal vessels. The method has potential as a treatment of choroidal neovascularization diat may minimize iatrogenic loss of vision. Invest Ophthalmol Vis Sci. 1997;38:2702-2710 Age-related macular degeneration (ARMD) is one of the leading causes of severe loss of vision in people more than 50 years old. &quot; 3 Choroidal neovascularization (CNV), which occurs in ARMD, is often treated by laser photocoagulation. However, the thermal damage and the scarring of large macular areas can caus

Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy and temozolomide

<p>Abstract</p> <p>Background</p> <p>Expression of neuronal elements has been identified in various glial tumors, and glioblastomas (GBMs) with neuronal differentiation patterns have reportedly been associated with longer survival. However, the neuronal class III β-tubulin has been linked to increasing malignancy in astrocytomas. Thus, the significance of neuronal markers in gliomas is not established.</p> <p>Methods</p> <p>The expressions of class III β-tubulin, neurofilament protein (NFP), microtubule-associated protein 2 (MAP2) and neuron-specific enolase (NSE) were investigated in five GBM cell lines and two GBM biopsies with immunocytochemistry and Western blot. Moreover, the expression levels were quantified by real-time qPCR under different culture conditions. Following NSE siRNA treatment we used Electric cell-substrate impedance sensing (ECIS) to monitor cell growth and migration and MTS assays to study viability after irradiation and temozolomide treatment. Finally, we quantitated NSE expression in a series of human glioma biopsies with immunohistochemistry using a morphometry software, and collected survival data for the corresponding patients. The biopsies were then grouped according to expression in two halves which were compared by survival analysis.</p> <p>Results</p> <p>Immunocytochemistry and Western blotting showed that all markers except NFP were expressed both in GBM cell lines and biopsies. Notably, qPCR demonstrated that NSE was upregulated in cellular stress conditions, such as serum-starvation and hypoxia, while we found no uniform pattern for the other markers. NSE knockdown reduced the migration of glioma cells, sensitized them to hypoxia, radio- and chemotherapy. Furthermore, we found that GBM patients in the group with the highest NSE expression lived significantly shorter than patients in the low-expression group.</p> <p>Conclusions</p> <p>Neuronal markers are aberrantly expressed in human GBMs, and NSE is consistently upregulated in different cellular stress conditions. Knockdown of NSE reduces the migration of GBM cells and sensitizes them to hypoxia, radiotherapy and chemotherapy. In addition, GBM patients with high NSE expression had significantly shorter survival than patients with low NSE expression. Collectively, these data suggest a role for NSE in the adaption to cellular stress, such as during treatment.</p

Potent Inhibition of Cicatricial Contraction in Proliferative Vitreoretinal Diseases by Statins

OBJECTIVE—Despite tremendous progress in vitreoretinal surgery, certain postsurgical complications limit the success in the treatment of proliferative vitreoretinal diseases (PVDs), such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). One of the most significant complications is the cicatricial contraction of proliferative membranes, resulting in tractional retinal detachment and severe vision loss. Novel pharmaceutical approaches are thus urgently needed for the management of these vision-threatening diseases. In the current study, we investigated the inhibitory effects of statins on the progression of PVDs

Genome-wide identification of hypoxia-inducible factor binding sites and target genes by a probabilistic model integrating transcription-profiling data and in silico binding site prediction

The transcriptional response driven by Hypoxia-inducible factor (HIF) is central to the adaptation to oxygen restriction. Hence, the complete identification of HIF targets is essential for understanding the cellular responses to hypoxia. Herein we describe a computational strategy based on the combination of phylogenetic footprinting and transcription profiling meta-analysis for the identification of HIF-target genes. Comparison of the resulting candidates with published HIF1a genome-wide chromatin immunoprecipitation indicates a high sensitivity (78%) and specificity (97.8%). To validate our strategy, we performed HIF1a chromatin immunoprecipitation on a set of putative targets. Our results confirm the robustness of the computational strategy in predicting HIF-binding sites and reveal several novel HIF targets, including RE1-silencing transcription factor co-repressor (RCOR2). In addition, mapping of described polymorphisms to the predicted HIF-binding sites identified several single-nucleotide polymorphisms (SNPs) that could alter HIF binding. As a proof of principle, we demonstrate that SNP rs17004038, mapping to a functional hypoxia response element in the macrophage migration inhibitory factor (MIF) locus, prevents induction of this gene by hypoxia. Altogether, our results show that the proposed strategy is a powerful tool for the identification of HIF direct targets that expands our knowledge of the cellular adaptation to hypoxia and provides cues on the inter-individual variation in this response

Molecular application of aptamers in the diagnosis and treatment of cancer and communicable diseases

Cancer and infectious diseases such as Ebola, HIV, tuberculosis, Zika, hepatitis, measles and human schistosomiasis are serious global health hazards. The increasing annual morbidities and mortalities of these diseases have been blamed on drug resistance and the inefficacy of available diagnostic tools, particularly those which are immunologically-based. Antibody-based tools rely solely on antibody production for diagnosis and for this reason they are the major cause of diagnostic delays. Unfortunately, the control of these diseases depends on early detection and administration of effective treatment therefore any diagnostic delay is a huge challenge to curbing these diseases. Hence, there is a need for alternative diagnostic tools, discovery and development of novel therapeutic agents. Studies have demonstrated that aptamers could potentially offer one of the best solutions to these problems. Aptamers are short sequences of either DNA or RNA molecules, which are identified in vitro through a SELEX process. They are sensitive and bind specifically to target molecules. Their promising features suggest they may serve as better diagnostic agents and can be used as drug carriers for therapeutic purposes. In this article, we review the applications of aptamers in the theranostics of cancer and some infectious diseases