Buspirone Enhances Cell Survival and Preserves Structural Integrity during Oxidative Injury to the Retinal Pigment Epithelium.

Chronic oxidative stress impairs the normal functioning of the retinal pigment epithelium (RPE), leading to atrophy of this cell layer in cases of advance age-related macular degeneration (AMD). The purpose of our study was to determine if buspirone, a partial serotonin 1A (5-HT1A) receptor agonist, protected against oxidative stress-induced changes in the RPE. We exposed differentiated human ARPE-19 cells to paraquat to induce oxidative damage in culture, and utilized a mouse model with sodium iodate (NaIO3)-induced oxidative injury to evaluate the effect of buspirone. To investigate buspirone's effect on protective gene expression, we performed RT-PCR. Cellular toxicities and junctional abnormalities due to paraquat induction in ARPE-19 cells and buspirone's impact were assessed via WST-1 assays and ZO-1 immunostaining. We used spectral-domain optical coherence tomography (SD-OCT) and ZO-1 immunostaining of RPE/choroid for structural analysis. WST-1 assays showed dose-dependent protection of viability in buspirone-treated ARPE-19 cells in culture and preservation of RPE junctional integrity under oxidative stress conditions. In the NaIO3 model, daily intraperitoneal injection (i.p.) of buspirone (30 mg/kg) for 12 days improved the survival of photoreceptors compared to those of vehicle-treated eyes. ZO-1-stained RPE flat-mounts revealed the structural preservation of RPE from oxidative damage in buspirone-treated mice, as well as in buspirone-induced Nqo1, Cat, Sqstm1, Gstm1, and Sod2 genes in the RPE/choroid compared to untreated eyes. Since oxidative stress is implicated in the pathogenesis AMD, repurposing buspirone, which is currently approved for the treatment of anxiety, might be useful in treating or preventing dry AMD.

Pattern Classification Using Quantized Neural Networks for FPGA-Based Low-Power IoT Devices.

With the recent growth of the Internet of Things (IoT) and the demand for faster computation, quantized neural networks (QNNs) or QNN-enabled IoT can offer better performance than conventional convolution neural networks (CNNs). With the aim of reducing memory access costs and increasing the computation efficiency, QNN-enabled devices are expected to transform numerous industrial applications with lower processing latency and power consumption. Another form of QNN is the binarized neural network (BNN), which has 2 bits of quantized levels. In this paper, CNN-, QNN-, and BNN-based pattern recognition techniques are implemented and analyzed on an FPGA. The FPGA hardware acts as an IoT device due to connectivity with the cloud, and QNN and BNN are considered to offer better performance in terms of low power and low resource use on hardware platforms. The CNN and QNN implementation and their comparative analysis are analyzed based on their accuracy, weight bit error, RoC curve, and execution speed. The paper also discusses various approaches that can be deployed for optimizing various CNN and QNN models with additionally available tools. The work is performed on the Xilinx Zynq 7020 series Pynq Z2 board, which serves as our FPGA-based low-power IoT device. The MNIST and CIFAR-10 databases are considered for simulation and experimentation. The work shows that the accuracy is 95.5% and 79.22% for the MNIST and CIFAR-10 databases, respectively, for full precision (32-bit), and the execution time is 5.8 ms and 18 ms for the MNIST and CIFAR-10 databases, respectively, for full precision (32-bit).

Impact of age and sex on hyperoxia-induced cardiovascular pathophysiology.

Hyperoxia is characterized by pronounced inflammatory responses, pulmonary cell apoptosis, and adverse cardiac remodeling due to an excess supply of oxygen. Hyperoxic episodes are frequent in mechanically ventilated patients and are associated with in-hospital mortality. This study extends the analysis of prior published research by our group as it investigates the influence of age in male and female rodents exposed to hyperoxic conditions. Age is an independent cardiovascular risk factor, often compounded by variables like obesity, diabetes, and a decline in sex hormones and their receptors. This study simulates clinical hyperoxia by subjecting rodents to > 90 % of oxygen for 72 h and compares the changes in cardiac structural and functional parameters with those exposed to normal air. While in both sexes conduction abnormalities with ageing were discernible, aged females owing to their inherent higher baseline QTc, were at a higher risk of developing arrhythmias as compared to age-matched males. Quantitative real-time RT-PCR and western blot analysis reflected altered expression of cardiac potassium channels, resulting in conduction abnormalities in aged female rodents. Unaffected by age and sex, hyperoxia-treated mice had altered body composition, as evidenced by a considerable reduction in body weight. Interestingly, compensatory hypertrophy observed as a protective mechanism in young males was absent in aged males, whereas protection of hearts from hyperoxia-induced cardiac hypertrophy was absent in aged female mice, both of which may be at least in part due to a reduction in sex steroid receptors and the systemic steroid levels. Finally, statistical analysis revealed that hyperoxia had the greatest impact on most of the cardiac parameters, followed by age and then sex. This data established an imperative finding that can change the provision of care for aged individuals admitted to ICU by elucidating the impact of intrinsic aging on hyperoxia-induced cardiac remodeling.

A Novel 65 nm Active-Inductor-Based VCO with Improved Q-Factor for 24 GHz Automotive Radar Applications.

The inductor was primarily developed on a low-voltage CMOS tunable active inductor (CTAI) for radar applications. Technically, the factors to be considered for VCO design are power consumption, low silicon area, high frequency with reasonable phase noise, an immense quality (Q) factor, and a large frequency tuning range (FTR). We used CMOS tunable active inductor (TAI) topology relying on cascode methodology for 24 GHz frequency operation. The newly configured TAI adopts the additive capacitor (Cad) with the cascode approach, and in the subthreshold region, one of the transistors functions as the TAI. The study, simulations, and measurements were performed using 65nm CMOS technology. The assembled circuit yields a spectrum from 21.79 to 29.92 GHz output frequency that enables sustainable platforms for K-band and Ka-band operations. The proposed design of TAI demonstrates a maximum Q-factor of 6825, and desirable phase noise variations of -112.43 and -133.27 dBc/Hz at 1 and 10 MHz offset frequencies for the VCO, respectively. Further, it includes enhanced power consumption that varies from 12.61 to 23.12 mW and a noise figure (NF) of 3.28 dB for a 24 GHz radar application under a low supply voltage of 0.9 V.

Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function.

The systemic transport of dietary vitamin A/all-trans retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant rbpr2 zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body Rbpr2 knockout mouse (Rbpr2-/-) model. These mice were viable on both vitamin A-sufficient and -deficient diets. Rbpr2-/- mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when Rbpr2-/- mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

A 65 nm Duplex Transconductance Path Up-Conversion Mixer for 24 GHz Automotive Short-Range Radar Sensor Applications.

A 24 GHz highly-linear upconversion mixer, based on a duplex transconductance path (DTP), is proposed for automotive short-range radar sensor applications using the 65-nm CMOS process. A mixer with an enhanced transconductance stage consisting of a DTP is presented to improve linearity. The main transconductance path (MTP) of the DTP includes a common source (CS) amplifier, while the secondary transconductance path (STP) of the DTP is implemented as an improved cross-quad transconductor (ICQT). Two inductors with a bypass capacitor are connected at the common nodes of the transconductance stage and switching stage of the mixer, which acts as a resonator and helps to improve the gain and isolation of the designed mixer. According to the measured results, at 24 GHz the proposed mixer shows that the linearity of output 1-dB compression point (OP1dB) is 3.9 dBm. And the input 1-dB compression point (IP1dB) is 0.9 dBm. Moreover, a maximum conversion gain (CG) of 2.49 dB and a noise figure (NF) of 3.9 dB is achieved in the designed mixer. When the supply voltage is 1.2 V, the power dissipation of the mixer is 3.24 mW. The mixer chip occupies an area of 0.42 mm2.

Design and Analysis of a Novel 24 GHz Up-Conversion Mixer with Improved Derivative Super-Position Linearizer Technique for 5G Applications.

A 24 GHz high linear, high-gain up-conversion mixer is realized for fifth-generation (5G) applications in the 65 nm CMOS process. The mixer's linearity is increased by applying an Improved Derivative Super-Position (I-DS) technique cascaded between the mixer's transconductance and switching stage. The high gain and stability of amplifiers in the transconductance stage of the mixer are achieved using novel tunable capacitive cross-coupled common source (TCC-CS) transistors. Using the I-DS, the third-order non-linear coefficient of current is closed to zero, enhancing the linearity. Additionally, a TCC-CS, which is realized by varactors, neutralizes the gate-to-drain parasitic capacitance (Cgd) of transistors in the transconductance stage of the mixer and contributes to the improvement of the gain and stability of the mixer. The measured 1 dB compression point OP1dB of the designed mixer is 4.1 dBm and IP1dB is 0.67 dBm at 24 GHz. The conversion gain of 4.1 dB at 24 GHz and 3.2 ± 0.9 dB, from 20 to 30 GHz is achieved in the designed mixer. Furthermore, a noise figure of 3.8 dB is noted at 24 GHz. The power consumption of the mixer is 4.9 mW at 1.2 V, while the chip area of the designed mixer is 0.4 mm2.

Role of Nuclear Factor of Activated T Cells (NFAT) Pathway in Regulating Autophagy and Inflammation in Retinal Pigment Epithelial Cells.

Nuclear factor of activated T cells (NFAT) family of transcription factors are substrates of calcineurin and play an important role in integrating Ca2+ signaling with a variety of cellular functions. Of the five NFAT proteins (NFAT1-5), NFAT1-4 are subject to dephosphorylation and activation by calcineurin, a Ca2+-calmodulin-dependent phosphatase. Increased levels of intracellular Ca2+ activates calcineurin, which in turn dephosphorylates and promotes nuclear translocation of NFAT. We investigated the functions of NFAT proteins in the retinal pigment epithelial cells (RPE). Our results show that NFAT-mediated luciferase activity was induced upon treatment with the bacterial endotoxin, lipopolysaccharide (LPS) and treatment with the NFAT peptide inhibitor, MAGPHPVIVITGPHEE (VIVIT) decreased LPS-induced NFAT luciferase activity. LPS-induced activation of NFAT-regulated cytokines (IL-6 and IL-8) is inhibited by treatment of cells with VIVIT. We also investigated the effects of NFAT signaling on the autophagy pathway. Our results show that inhibition of NFAT with VIVIT in cells deprived of nutrients resulted in cytosolic retention of transcription Factor EB (TFEB), decreased expression of TFEB-regulated coordinated Lysosomal Expression and Regulation CLEAR network genes and decreased starvation-induced autophagy flux in the RPE cells. In summary, these studies suggest that the NFAT pathway plays an important role in the regulation of autophagy and inflammation in the RPE.

Loss of Motor Protein MYO1C Causes Rhodopsin Mislocalization and Results in Impaired Visual Function.

Unconventional myosins, linked to deafness, are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice, specifically causes rhodopsin mislocalization, leading to impaired visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with MYO1C. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is important for rhodopsin localization to the photoreceptor OS, and for normal visual function.

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function.

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.

Erythropoietin Gene Therapy Delays Retinal Degeneration Resulting from Oxidative Stress in the Retinal Pigment Epithelium.

Erythropoietin (EPO) plays an important role in erythropoiesis by its action in blocking apoptosis of progenitor cells and protects both photoreceptors and retinal ganglion cells from induced or inherited degeneration. A modified form of EPO, EPO-R76E has attenuated erythropoietic activity but is effective in inhibiting apoptosis, oxidative stress, and inflammation in several models of retinal degeneration. In this study, we used recombinant Adeno Associated Virus (AAV) to provide long-term sustained delivery of EPO-R76E and demonstrated its effects in a mouse model of dry-AMD in which retinal degeneration is induced by oxidative stress in the retinal pigment epithelial (RPE) cells. Experimental vector AAV-EPO-R76E and control vector AAV-GFP were packaged into serotype-1 (AAV1) to enable RPE selective expression. RPE oxidative stress-mediated retinal degeneration was induced by exon specific deletion of the protective enzyme MnSOD (encoded by Sod2) by cre/lox mechanism. Experimental mice received subretinal injection of AAV-EPO-R76E in the right eye and AAV-GFP in the left eye. Western blotting of RPE/choroid protein samples from AAV-EPO-R76E injected eyes showed RPE specific EPO expression. Retinal function was monitored by electroretinography (ERG). EPO-R76E over-expression in RPE delayed the retinal degeneration as measured by light microscopy in RPE specific Sod2 knockout mice. Delivery of EPO-R76E vector can be used as a tool to prevent retinal degeneration induced by RPE oxidative stress, which is implicated as a potential cause of Age-Related Macular Degeneration.

Carbon Dot Nanoparticles: Exploring the Potential Use for Gene Delivery in Ophthalmic Diseases.

Ocular gene therapy offers significant potential for preventing retinal dystrophy in patients with inherited retinal dystrophies (IRD). Adeno-associated virus (AAV) based gene transfer is the most common and successful gene delivery approach to the eye. These days, many studies are using non-viral nanoparticles (NPs) as an alternative therapeutic option because of their unique properties and biocompatibility. Here, we discuss the potential of carbon dots (CDs), a new type of nanocarrier for gene delivery to the retinal cells. The unique physicochemical properties of CDs (such as optical, electronic, and catalytic) make them suitable for biosensing, imaging, drug, and gene delivery applications. Efficient gene delivery to the retinal cells using CDs depends on various factors, such as photoluminescence, quantum yield, biocompatibility, size, and shape. In this review, we focused on different approaches used to synthesize CDs, classify CDs, various pathways for the intake of gene-loaded carbon nanoparticles inside the cell, and multiple studies that worked on transferring nucleic acid in the eye using CDs.

Emerging Nano-Formulations and Nanomedicines Applications for Ocular Drug Delivery.

Ocular diseases can deteriorate vision to the point of blindness and thus can have a major impact on the daily life of an individual. Conventional therapies are unable to provide absolute therapy for all ocular diseases due to the several limitations during drug delivery across the blood-retinal barrier, making it a major clinical challenge. With recent developments, the vast number of publications undergird the need for nanotechnology-based drug delivery systems in treating ocular diseases. The tool of nanotechnology provides several essential advantages, including sustained drug release and specific tissue targeting. Additionally, comprehensive in vitro and in vivo studies have suggested a better uptake of nanoparticles across ocular barriers. Nanoparticles can overcome the blood-retinal barrier and consequently increase ocular penetration and improve the bioavailability of the drug. In this review, we aim to summarize the development of organic and inorganic nanoparticles for ophthalmic applications. We highlight the potential nanoformulations in clinical trials as well as the products that have become a commercial reality.

Carbon Dots Fabrication: Ocular Imaging and Therapeutic Potential.

Vision loss is a major complication in common ocular infections and diseases such as bacterial keratitis, age-related macular degeneration (AMD) and diabetic retinopathy (DR). The prevalence of such ophthalmic diseases represents an urgent need to develop safe, effective, and long-term treatments. Current therapies are riddled with drawbacks and limitations which calls for the exploration of alternative drug delivery mechanisms. Toxicity of the inorganic metals and metal oxides used for drug delivery raise safety concerns that are alleviated with the alternate use of, a natural and organic polymer which is both biocompatible and environmentally friendly. Carbon dots (CDs) represent a great potential in novel biomedical applications due to their tunable fluorescence, biocompatibility, and ability to be conjugated with diverse therapeutic materials. There is a growing interest on the exploitation of these properties for drug delivery with enhanced bio-imaging. However, there are limited reports of CD applications for ophthalmic indications. In this review, we focus on the CD potential and the development of translational therapies for ophthalmic diseases. The current review presents better understanding of fabrication of CDs and how it may be useful in delivering anti-bacterial agents, anti-VEGF molecules as well as imaging for ophthalmic applications.

Advances in Translational Nanotechnology: Challenges and Opportunities.

The burgeoning field of nanotechnology aims to create and deploy nanoscale structures, devices, and systems with novel, size-dependent properties and functions. The nanotechnology revolution has sparked radically new technologies and strategies across all scientific disciplines, with nanotechnology now applied to virtually every area of research and development in the US and globally. NanoFlorida was founded to create a forum for scientific exchange, promote networking among nanoscientists, encourage collaborative research efforts across institutions, forge strong industry-academia partnerships in nanoscience, and showcase the contributions of students and trainees in nanotechnology fields. The 2019 NanoFlorida International Conference expanded this vision to emphasize national and international participation, with a focus on advances made in translating nanotechnology. This review highlights notable research in the areas of engineering especially in optics, photonics and plasmonics and electronics; biomedical devices, nano-biotechnology, nanotherapeutics including both experimental nanotherapies and nanovaccines; nano-diagnostics and -theranostics; nano-enabled drug discovery platforms; tissue engineering, bioprinting, and environmental nanotechnology, as well as challenges and directions for future research.

CCL20-CCR6 axis modulated traumatic brain injury-induced visual pathologies.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability in the USA and the world; it constitutes 30% of injury-related deaths (Taylor et al., MMWR Surveill Summ 66:1-16, 2017). Contact sports athletes often experience repetitive TBI (rTBI), which exerts a cumulative effect later in life. Visual impairment is a common after-effect of TBI. Previously, we have shown that C-C chemokine 20 (CCL20) plays a critical role in neurodegeneration and inflammation following TBI (Das et al., J Neuroinflammation 8:148, 2011). C-C chemokine receptor 6 (CCR6) is the only receptor that CCL20 interacts with. The objective of the present study was to investigate the role of CCL20-CCR6 axis in mediating rTBI-induced visual dysfunction (TVD). METHODS: Wild type (WT) or CCR6 knock out (CCR6-/-) mice were subjected to closed head rTBI. Pioglitazone (PG) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist which downregulates CCL20 production. Subsets of WT mice were treated with PG following final rTBI. A subset of mice was also treated with anti-CCL20 antibody to neutralize the CCL20 produced after rTBI. Histopathological assessments were performed to show cerebral pathologies, retinal pathologies, and inflammatory changes induced by rTBI. RESULTS: rTBI induced cerebral neurodegeneration, retinal degeneration, microgliosis, astrogliosis, and CCL20 expression. CCR6-/- mice showed reduced retinal degeneration, microgliosis, and inflammation. Treatment with CCL20 neutralization antibody or PG showed reduced CCL20 expression along with reduced retinal degeneration and inflammation. rTBI-induced GFAP-positive glial activation in the optic nerve was not affected by knocking out CCR6. CONCLUSION: The present data indicate that rTBI-induced retinal pathology is mediated at least in part by CCL20 in a CCR6-dependent manner.

Daily zeaxanthin supplementation prevents atrophy of the retinal pigment epithelium (RPE) in a mouse model of mitochondrial oxidative stress.

Oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). The dry form of AMD (geographic atrophy) is characterized by loss of RPE, photoreceptors, and macular pigments. The cumulative effects of oxidative stress impact mitochondrial function in RPE. In Sod2flox/floxVMD2-cre mice, the RPE specific deletion of Sod2, the gene for mitochondrial manganese superoxide dismutase (MnSOD), leads to elevated oxidative stress in retina and RPE, and causes changes in the RPE and underlying Bruch's membrane that share some features of AMD. This study tested the hypothesis that zeaxanthin supplementation would reduce oxidative stress and preserve RPE structure and function in these mice. Zeaxanthin in retina/RPE/choroid and liver was quantified by LC/MS, retinal function and structure were evaluated by electroretinogram (ERG) and spectral domain optical coherence tomography (SD-OCT), and antioxidant gene expression was measured by RT-PCR. After one month of supplementation, zeaxanthin levels were 5-fold higher in the retina/RPE/choroid and 12-fold higher in liver than in unsupplemented control mice. After four months of supplementation, amplitudes of the ERG a-wave (function of rod photoreceptors) and b-wave (function of the inner retina) were not different in supplemented and control mice. In contrast, the c-wave amplitude (a measure of RPE function) was 28% higher in supplemented mice than in control mice. Higher RPE/choroid expression of antioxidant genes (Cat, Gstm1, Hmox1, Nqo1) and scaffolding protein Sqstm1 were found in supplemented mice than in unsupplemented controls. Reduced nitrotyrosine content in the RPE/choroid was demonstrated by ELISA. Preliminary assessment of retinal ultrastructure indicated that supplementation supported better preservation of RPE structure with more compact basal infoldings and intact mitochondria. We conclude that daily zeaxanthin supplementation protected RPE cells from mitochondrial oxidative stress associated with deficiency in the MnSOD and thereby improved RPE function early in the disease course.

Cell-specific gene therapy driven by an optimized hypoxia-regulated vector reduces choroidal neovascularization.

Aberrant growth of blood vessels in the choroid layer of the eye, termed choroidal neovascularization (CNV), is the pathological hallmark of exudative age-related macular degeneration (AMD), causing irreversible blindness among the elderly. Co-localization of proangiogenic factors and hypoxia inducible factors (HIF) in neovascular membranes from AMD eyes suggests the role of hypoxia in pathogenesis of CNV. In order to utilize hypoxic conditions in RPE for therapeutic purposes, we developed an optimized hypoxia regulated, RPE cell-specific gene therapy to inhibit choroidal neovascularization. An adeno-associated virus (AAV2) vector comprising a RPE-specific promoter and HIF-1 response elements (HRE) was designed to regulate production of human endostatin (a powerful angiostatic protein) in RPE. The vector was tested in a mouse model of laser-induced CNV using subretinal delivery. Spectral domain optical coherence tomography (SD-OCT) images from live mice and confocal images from lectin stained RPE flat mount sections demonstrated reduction in CNV areas by 80% compared to untreated eyes. Quantitative real-time polymerase chain reaction (qPCR) confirmed exogenous endostatin mRNA expression from the regulated vector that was significantly elevated 3, 7, and 14 days following laser treatment, but its expression was completely shut off after 45 days. Thus, RPE-specific, hypoxia-regulated delivery of anti-angiogenic proteins could be a valuable therapeutic approach to treat neovascular AMD at the time and in the ocular space where it arises. KEY POINTS: An optimized gene therapy vector targeting hypoxia and tissue-specific expression has been designed. The inhibitory role of gene therapy vector was tested in a mouse model of laser-induced CNV. An 80% reduction in choroidal neovascularization was achieved by the optimized vector. The expression of endostatin was limited to retinal pigment epithelium and regulated by hypoxia.

Analyzing cytokines as biomarkers to evaluate severity of glaucoma.

AIM: To analyze cytokines as biomarkers for evaluation of severity of glaucoma. METHODS: This was a prospective case-control study including 29 eyes with glaucoma. Besides, 28 eyes with senile cataract were used as control. Patients were classified into four groups: acute angle closure glaucoma (AACG), chronic angle closure glaucoma (CACG), primary open angle glaucoma (POAG) and senile cataract. Undiluted vitreous samples were collected, then vitreous concentrations of 9 types of cytokines were determined by cytometric bead assay system: γ-interferon (IFNg), interleukin (IL)-10, IL-2, IL-4, IL-5, interferon-γ-inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF). We also recorded the intraocular pressure (IOP) of patients in each group and Pearson correlated analysis was performed to analysis the correlation between each type of cytokine with IOP. RESULTS: Vitreous levels of IL-2, IL-5, MCP-1, TNF-α and IP-10 were significantly higher (P<0.05) in AACG group. Patients with AACG, CACG and POAG have higher IOP than senile cataract, but we didn't find any significant correlation between IOP with any type of the cytokines. CONCLUSION: Inflammation and immune reaction have a strong link with the pathology of glaucoma especially AACG. Some cytokines may act as biomarkers to evaluate the severity of glaucoma. Anti-inflammatory treatments and controlling of IOP are necessary for the therapy of glaucoma.