Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice.

Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

Tropism of the Novel AAVBR1 Capsid Following Subretinal Delivery.

A serious limitation of current adeno-associated viral (AAV) capsids employed for subretinal delivery is achieving adequate lateral spread beyond the injection site, required for the efficient delivery of gene therapy to the outer retina and/or RPE. AAVBR1 is a unique AAV with exceptional tropism for CNS microvasculature following systemic delivery. Here, we used in vivo and ex vivo analysis to show that subretinal delivery of AAVBR1.GFP in mice achieves superior tropism to RPE and outer retina than either AAV2.GFP or AAV8.GFP, two of the most common capsids used for subretinal delivery. At a low (5 × 108 vg) subretinal dose, the AAVBR1.GFP signal was visible by 48 h and significantly surpassed peak fluorescence of other AAVs in retina and RPE. The co-injection of AAVBR1.GFP with the AAVBR1-specific heptapeptide, NRGTEWD, significantly blocked the AAVBR1.GFP signal, but had no effect on AAV2.GFP fluorescence, confirming that AAVBR1's enhanced tropism for RPE and outer retina derives from this 7AA modification within the capsid-binding motif. Enhanced dispersal and consequent transduction suggest that AAVBR1 can be employed at a lower dosage than the standard AAV2 capsid to achieve equivalent expression for gene therapy, warranting further evaluation of its utility as a therapeutic vehicle for subretinal delivery.

Detection of microvascular retinal changes in type I diabetic mice with optical coherence tomography angiography.

Optical coherence tomography (OCT) angiography is a dye-free and non-invasive angiography which allows visualization of retinal and choroid vascular flow, enabling observation of highly permeable and three dimensional vasculature. Although OCT angiography is providing new insights in human retinal and choroidal diseases, a few studies have been reported in experimental mice. In this study, to determine the potential of OCT angiography in experimental mice, we sought to examine whether OCT angiography can detect vascular change in type I diabetic mice. To conduct age dependent analysis, 2 and 6 month old male type 1 diabetic Ins2Akita/+ and age matched C57BL/6J mice were used. OCT angiography was performed by Heidelberg Spectralis OCT Angiography Module with 30° lens + mouse adapter lens. We acquired the OCT angiography image from the peripheral nasal position. For analysis of OCT angiography images, OCT angiography positive area were used for vascular density. We analyzed vascular density from the retinal surface (inner limiting membrane) to 120 µm depth with 4 µm steps in order to correlate vascular density vs depth (N = 4 per group). Vascular density of both mouse strains demonstrated three different peaks. By comparing with the OCT image, the first peak (superficial), second peak (intermediate) and third peak (deep) were located in nerve fiber layer/ganglion cell layer, inner plexiform layer/inner nuclear layer and outer plexiform layer/outer nuclear layer, respectively. We calculated vascular density of these peaks separately. In C57BL/6J mice, the vascular density in all three layers do not show significant difference between 2- and 6-month-old. On the other hand, 6-month-old Ins2Akita/+ mice showed a significant decrease of the vascular density in all three layers compared to 2-month-old Ins2Akita/+ mice. Also, the vascular density of 6-month-old Ins2Akita/+ mice in the deep layer showed a significant decrease compared to 2- and 6-month-old C57BL/6J mice. Thus, OCT angiography successfully detects retinal vascular difference between type I diabetic mice and control mice, and age-dependent vasculature change in type I diabetic mice. The diabetic mice demonstrated reduced vascular density due to reduced density of flowing deep vessels. Importantly, we observed this difference without retinal blood leakage, hemorrhage or neovascularization. Our analysis (vascular density vs retinal depth) suggests that OCT angiography is useful for in vivo detection of retinal vasculature alteration in experimental mice.

Targeted Intraceptor Nanoparticle for Neovascular Macular Degeneration: Preclinical Dose Optimization and Toxicology Assessment.

Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.

Subretinal AAV2.COMP-Ang1 suppresses choroidal neovascularization and vascular endothelial growth factor in a murine model of age-related macular degeneration.

To assess whether Tie2-mediated vascular stabilization ameliorates neovascular age-related macular degeneration (AMD), we investigated the impact of adeno-associated virus-mediated gene therapy with cartilage oligomeric matrix protein angiopoietin-1 (AAV2.COMP-Ang1) on choroidal neovascularization (CNV), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF) in a mouse model of the disease. We treated mice with subretinal injections of AAV2.COMP-Ang1 or control (AAV2.AcGFP, AAV2.LacZ, and phosphate-buffered saline). Subretinal AAV2 localization and plasmid protein expression was verified in the retinal pigment epithelium (RPE)/choroid of mice treated with all AAV2 constructs. Laser-assisted simulation of neovascular AMD was performed and followed by quantification of HIF, VEGF, and CNV in each experimental group. We found that AAV2.COMP-Ang1 was associated with a significant reduction in VEGF levels (29-33%, p < 0.01) and CNV volume (60-70%, p < 0.01), without a concomitant decrease in HIF1-α, compared to all controls. We concluded that a) AAV2 is a viable vector for delivering COMP-Ang1 to subretinal tissues, b) subretinal COMP-Ang1 holds promise as a prospective treatment for neovascular AMD, and c) although VEGF suppression in the RPE/choroid may be one mechanism by which AAV2.COMP-Ang1 reduces CNV, this therapeutic effect may be hypoxia-independent. Taken together, these findings suggest that AAV2.COMP-Ang1 has potential to serve as an alternative or complementary option to anti-VEGF agents for the long-term amelioration of neovascular AMD.

AAV2 delivery of Flt23k intraceptors inhibits murine choroidal neovascularization.

Long-term inhibition of extracellular vascular endothelial growth factor (VEGF) in the treatment of age-related macular degeneration (AMD) may induce retinal neuronal toxicity and risk other side effects. We developed a novel strategy which inhibits retinal pigment epithelium (RPE)-derived VEGF, sparing other highly sensitive retinal tissues. Flt23k, an intraceptor inhibitor of VEGF, was able to inhibit VEGF in vitro. Adeno-associated virus type 2 (AAV2)-mediated expression of Flt23k was maintained for up to 6 months postsubretinal injection in mice. Flt23k was able to effectively inhibit laser-induced murine choroidal neovascularization (CNV). VEGF levels in the RPE/choroid complex decreased significantly in AAV2.Flt23k treated eyes. Neither retinal structure detected by Heidelberg Spectralis nor function measured by electroretinography (ERG) was adversely affected by treatment with AAV2.Flt23k. Hence AAV2.Flt23k can effectively maintain long-term expression and inhibit laser-induced CNV in mice through downregulation of VEGF while maintaining a sound retinal safety profile. These findings suggest a promising novel approach for the treatment of CNV.

Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity.

Corneal transparency is a prerequisite for optimal vision and in turn relies on an absence of blood and lymphatic vessels, which is remarkable given the cornea's proximity to vascularized tissues. Membrane-bound vascular endothelial growth factor receptor 3 (VEGFR-3), with its cognate ligand vascular endothelial growth factor C (VEGF-C), is a major mediator of lymphangiogenesis. Here, we demonstrate that the cornea expresses a novel truncated isoform of this molecule, soluble VEGFR-3 (sVEGFR-3), which is critical for corneal alymphaticity, by sequestering VEGF-C. sVEGFR-3 binds and sequesters VEGF-C, thereby blocking signaling through VEGFR-3 and suppressing lymphangiogenesis induced by VEGF-C. sVEGFR-3 knockdown leads to lymphangiogenesis and hemangiogenesis in the mouse cornea, while overexpression of sVEGFR-3 inhibits lymphangiogenesis and hemangiogenesis in a murine suture injury model. Pax6(+/-) mice spontaneously develop corneal and lymphatic vessels and are deficient in sVEGFR-3. sVEGFR-3 suppresses hemangiogenesis by blocking VEGF-C-induced phosphorylation of VEGFR-2. Overexpression of sVEGFR-3 leads to a 5-fold increase in corneal transplant survival in mouse models. sVEGFR-3 holds promise as a molecule to control and regress lymphatic-vessel-based dysfunction. Therefore, sVEGFR-3 has the potential to protect the injured cornea from opacification secondary to infection, inflammation, or transplant rejection.

In vivo ZW800-microbead imaging of retinal and choroidal vascular leakage in mice.

The eye is an attractive organ for non-invasive discovery and monitoring of disease progression. Traditionally, fluorescein angiography (FA) and indocyanine green angiography (ICGA) have been used for dynamic evaluation of the retina and its vasculature. However, both fluorescein and indocyanine green (ICG) possess considerable disadvantages. FA is limited to assessing superficial retinal blood flow and often results in an unclear view due to fluorescein leakage. This obscures important pathologies such as neovascularization, ischemia and inflammation. ICG, a near-infrared fluorophore (NIRF), has nonspecific binding, high uptake and retention in tissues, as well as detrimental effects on the hepatobiliary tract. Here, we present a potential contrast agent for imaging ocular vascular permeability with ZW800, a heptamethine indocyanine NIRF, conjugated to polystyrene latex beads (ZW800m). ZW800 is an excellent alternative for near-infrared imaging, as it has excellent contrast, superior clearance, and is amendable to conjugation. ZW800m conjugation is an easy, attractive method of in vivo imaging and real-time tracking of ocular vascular pathologies. ZW800m is readily imaged via commercially available laser ophthalmoscope (SLO, HRA OCT, Spectralis) to assess vascular permeability in the mouse retina and choroid. In Type 1 diabetic Ins2Akita mice, ZW800m was observed in mouse retina but not in wild-type mice. After laser-induced choroidal neovascularization (CNV), ZW800m was observed in mouse choroid but not in control. In both CNV and diabetic mice, ZW800 imaging showed increased hyperfluorescence on ICG modality (ICGA) not seen on FA. Presence of ZW800m in respective tissues was confirmed ex vivo with flatmounts visualized with EVOS 800 nm light cube. ZW800 imaging may be easily employed in the research laboratory.

Dual suppression of hemangiogenesis and lymphangiogenesis by splice-shifting morpholinos targeting vascular endothelial growth factor receptor 2 (KDR).

The KDR gene, which participates in angiogenesis and lymphangiogenesis, produces two functionally distinct protein products, membrane-bound KDR (mbKDR) and its isoform, soluble KDR (sKDR). Since sKDR does not have a tyrosine kinase domain and does not dimerize, it is principally an antagonist of lymphangiogenesis by sequestering VEGF-C. Alternative polyadenylation of exon 30 or intron 13 leads to the production of mbKDR or sKDR, respectively, yet the regulatory mechanisms are unknown. Here we show that an antisense morpholino oligomer directed against the exon 13-intron 13 junction increases sKDR (suppressing lymphangiogenesis) and decreases mbKDR (inhibiting hemangiogenesis). The latent polyadenylation site in intron 13 of KDR is activated by blocking the upstream 5' splicing site with an antisense morpholino oligomer. Intravitreal morpholino injection suppressed laser choroidal neovascularization while increasing sKDR. In the mouse cornea, subconjunctival injection of the morpholino-inhibited corneal angiogenesis and lymphangiogenesis, and suppressed graft rejection after transplantation. Thus, this morpholino can be used for concurrent suppression of hemangiogenesis and lymphangiogenesis. This study offers new insight into the mechanisms and potential therapeutic modulation of alternative polyadenylation.

COMP-Ang1: Therapeutic potential of an engineered Angiopoietin-1 variant.

The Angiopoietin-1/2 system is an opportune target for therapeutic intervention in a wide range of vascular pathologies, particularly through its association with endothelium. The complex multi-domain structure of native human Angiopoietin-1 has hindered its widespread applicability as a therapeutic agent, prompting the search for alternative approaches to mimicking the Ang1:Tie2 signalling axis; a system with highly complex patterns of regulation involving multiple structurally similar molecules. An engineered variant, Cartilage Oligomeric Matrix Protein - Angiopoietin-1 (COMP-Ang1), has been demonstrated to overcome the limitations of the native molecule and activate the Tie2 pathway with several fold greater potency than Ang1, both in vitro and in vivo. The therapeutic efficacy of COMP-Ang1, at both the vascular and systemic levels, is evident from multiple studies. Beneficial impacts on skeletal muscle regeneration, wound healing and angiogenesis have been reported alongside renoprotective, anti-hypertensive and anti-inflammatory effects. COMP-Ang1 has also demonstrated synergy with other compounds to heighten bone repair, has been leveraged for potential use as a co-therapeutic for enhanced targeted cancer treatment, and has received considerable attention as an anti-leakage agent for microvascular diseases like diabetic retinopathy. This review examines the vascular Angiopoietin:Tie2 signalling mechanism, evaluates the potential therapeutic merits of engineered COMP-Ang1 in both vascular and systemic contexts, and addresses the inherent translational challenges in moving this potential therapeutic from bench-to-bedside.

Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy.

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

Systemic AAV10.COMP-Ang1 rescues renal glomeruli and pancreatic islets in type 2 diabetic mice.

INTRODUCTION: Diabetic hyperglycemia causes progressive and generalized damage to the microvasculature. In renal glomeruli, this results in the loss of podocytes with consequent loss of constitutive angiopoietin-1 (Ang1) signaling, which is required for stability of the glomerular endothelium. Repeated tail vein injection of adenovirus expressing COMP-Ang1 (a stable bioengineered form of Ang1) was previously reported to improve diabetic glomerular damage despite the liver and lungs being primary targets of adenoviral infection. We thus hypothesized that localizing delivery of sustained COMP-Ang1 to the kidney could increase its therapeutic efficacy and safety for the treatment of diabetes. RESEARCH DESIGN AND METHODS: Using AAVrh10 adeno-associated viral capsid with enhanced kidney tropism, we treated 10-week-old uninephrectomized db/db mice (a model of type 2 diabetes) with a single dose of AAVrh10.COMP-Ang1 delivered via the intracarotid artery, compared with untreated diabetic db/db control and non-diabetic db/m mice. RESULTS: Surprisingly, both glomerular and pancreatic capillaries expressed COMP-Ang1, compensating for diabetes-induced loss of tissue Ang1. Importantly, treatment with AAVrh10.COMP-Ang1 yielded a significant reduction of glycemia (blood glucose, 241±193 mg/dL vs 576±31 mg/dL; glycosylated hemoglobin, 7.2±1.5% vs 11.3±1.3%) and slowed the progression of albuminuria and glomerulosclerosis in db/db mice by 70% and 61%, respectively, compared with untreated diabetic db/db mice. Furthermore, COMP-Ang1 ameliorated diabetes-induced increases of NF-kBp65, nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase-2 (Nox2), p47phox and productions of myeloperoxidase, the inflammatory markers in both renal and pancreatic tissues, and improved beta-cell density in pancreatic islets. CONCLUSIONS: These results highlight the potential of localized Ang1 therapy for treatment of diabetic visceropathies and provide a mechanistic explanation for reported improvements in glucose control via Ang1/Tie2 signaling in the pancreas.

Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole.

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNA's from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5-10 microm, a phospholipase A(2) coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5-10 min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope.

Detection of mRNA in single living cells using AFM nanoprobes.

Examining messenger RNA (mRNA) expression is useful for the determination of cell and tissue conditions. Many methods of determining mRNA expression require total RNA extraction or cell fixation. These processes cause difficulties in examining mRNA expression in single living cells without causing cell death. We think that analysis of specific mRNA expression in single living cells will become important in cell biology. In this chapter, we present a method to examine mRNA expression of single living cells without killing the cells. The single-cell nanoprobe (SCN) method uses atomic force microscopy (AFM) to extract mRNA. We also present examples of beta-actin mRNA detection and multiple mRNA detection from single living cells.

Antiangiogenesis Effect of Albendazole on the Cornea.

Purpose: To investigate the anti-(lymph)angiogenic and anti-inflammatory effects of albendazole and to study whether these effects are additive with bevacizumab therapy in a murine corneal suture model. Methods: Corneal neovascularization (NV) and lymphangiogenesis (LY) were compared in a corneal suture model after administration of a subconjunctival injection of albendazole, bevacizumab, dexamethasone, or phosphate-buffered saline (PBS). Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines (tumor necrosis factor [TNF]-alpha and interleukin-6), vascular endothelial growth factor (VEGF)-A, VEGF-C, vascular endothelial growth factor receptor (VEGFR)-2, and VEGFR-3. To evaluate the additive effect of albendazole, corneal NV and LY were also analyzed in a combined group of albendazole and bevacizumab therapy and the additive effect was compared with that in the group of double dose of bevacizumab. Results: The albendazole group showed less NV and less LY compared with the PBS control group (P < 0.01). When albendazole was combined with bevacizumab therapy, a significant decrease in NV and LY was seen compared with bevacizumab treatment alone, and with albendazole alone (all P values <0.05). The combination group showed better antilymphangiogenesis effect than the group of double dose bevacizumab. The albendazole-treated group showed reduced expression of VEGF-A, VEGF-C, TNF-alpha, and VEGFR-2 compared with corneas from the PBS group (P value <0.05 in all respective comparisons). Conclusion: Albendazole significantly decreased NV and LY in the cornea. This beneficial effect is additively enhanced when combined with bevacizumab treatment.

Intravitreal AAV2.COMP-Ang1 Attenuates Deep Capillary Plexus Expansion in the Aged Diabetic Mouse Retina.

Purpose: We determine whether intravitreal angiopoietin-1 combined with the short coiled-coil domain of cartilage oligomeric matrix protein by adeno-associated viral serotype 2 (AAV2.COMP-Ang1) delivery following the onset of vascular damage could rescue or repair damaged vascular beds and attenuate neuronal atrophy and dysfunction in the retinas of aged diabetic mice. Methods: AAV2.COMP-Ang1 was bilaterally injected into the vitreous of 6-month-old male Ins2Akita mice. Age-matched controls consisted of uninjected C57BL/6J and Ins2Akita males, and of Ins2Akita males injected with PBS or AAV2.REPORTER (AcGFP or LacZ). Retinal thickness and visual acuity were measured in vivo at baseline and at the 10.5-month endpoint. Ex vivo vascular parameters were measured from retinal flat mounts, and Western blot was used to detect protein expression. Results: All three Ins2Akita control groups showed significantly increased deep vascular density at 10.5 months compared to uninjected C57BL/6J retinas (as measured by vessel area, length, lacunarity, and number of junctions). In contrast, deep microvascular density of Ins2Akita retinas treated with AAV2.COMP-Ang1 was more similar to uninjected C57BL/6J retinas for all parameters. However, no significant improvement in retinal thinning or diabetic retinopathy-associated visual loss was found in treated diabetic retinas. Conclusions: Deep retinal microvasculature of diabetic Ins2Akita eyes shows late stage changes consistent with disorganized vascular proliferation. We show that intravitreally injected AAV2.COMP-Ang1 blocks this increase in deep microvascularity, even when administered subsequent to development of the first detectable vascular defects. However, improving vascular normalization did not attenuate neuroretinal degeneration or loss of visual acuity. Therefore, additional interventions are required to address neurodegenerative changes that are already underway.

The Effect of 0.5% Timolol Maleate on Corneal(Lymph)Angiogenesis in a Murine Suture Model.

PURPOSE: To investigate the anti(lymph)angiogenic and anti-inflammatory effects of 0.5% timolol maleate in a murine corneal suture model. METHODS: Corneal neovascularization and lymphangiogenesis were compared in groups of mice that underwent corneal suture and were subsequently administered a subconjunctival injection of 0.5% timolol maleate, dexamethasone, or phosphate-buffered saline (PBS). Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines [TNF-alpha and interleukin (IL)-6], vascular endothelial growth factor (VEGF)-A, VEGF-C, vascular endothelial growth factor receptor (VEGFR)-2, and VEGFR-3. RESULTS: When corneas from the timolol-treated group were compared to the PBS-treated group, we observed decreases in angiogenesis, lymphangiogenesis, and inflammatory infiltration in the timolol-treated group (P value <0.05 in all respective comparisons). Corneas from the timolol-treated group showed reduced expression of VEGF-A, VEGF-C, TNF-alpha, IL-6, VEGFR-2, and VEGFR-3 compared to corneas from the PBS group (P value <0.05 in all respective comparisons). CONCLUSION: Blocking adrenergic signaling in the cornea with 0.5% timolol maleate decreased corneal neovascularization and lymphangiogenesis.

RNA activating-double stranded RNA targeting flt-1 promoter inhibits endothelial cell proliferation through soluble FLT-1 upregulation.

Short-activating RNA (saRNA), which targets gene promoters, has been shown to increase the target gene expression. In this study, we describe the use of an saRNA (Flt a-1) to target the flt-1 promoter, leading to upregulation of the soluble isoform of Flt-1 and inhibition of angiogenesis. We demonstrate that Flt a-1 increased sFlt-1 mRNA and protein levels, while reducing VEGF expression. This was associated with suppression of human umbilical vascular endothelial cell (HUVEC) proliferation and cell cycle arrest at the G0/G1 phase. HUVEC migration and tube formation were also suppressed by Flt a-1. An siRNA targeting Flt-1 blocked the effects of Flt a-1. Flt a-1 effects were not mediated via argonaute proteins. However, trichostatin A and 5'-deoxy-5'-(methylthio) adenosine inhibited Flt a-1 effects, indicating that histone acetylation and methylation are mechanistically involved in RNA activation of Flt-1. In conclusion, RNA activation of sFlt-1 can be used to inhibit angiogenesis.

mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization.

BACKGROUND: The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells. RESULTS: In order to evaluate the SCN method, we compared the SCN method with in situ hybridization (ISH). First, we examined spatial beta-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for beta-actin mRNA. In the SCN method, quantity of beta-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of beta-actin mRNA. We showed that intensity of ISH is higher; quantity of beta-actin mRNA detected by the SCN method increased more. CONCLUSION: In this study, we compare the SCN method with the ISH. We examined beta-actin mRNA expression in single cells using both methods. We picked up beta-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.

Effect of itraconazole on the cornea in a murine suture model and penetrating keratoplasty model.

AIM: To investigate the anti-(lymph)angiogenic and/or anti-inflammatory effect of itraconazole in a corneal suture model and penetrating keratoplasty (PK) model. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among itraconazole, amphotericin B, dexamethasone, phosphate buffered saline (PBS) and surgery-only groups following subconjunctival injection in mice that underwent PK and corneal suture. Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines (TNF-alpha, IL-6) and vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGFR-2, and VEGFR-3. RESULTS: In the suture model, the itraconazole group showed less angiogenesis, less lymphangiogenesis, and less inflammatory infiltration than the PBS group (all P<0.05). The itraconazole group showed reduced expression of VEGF-A, VEGFR-2, TNF-alpha, IL-6 than the PBS group (all P<0.05). In PK model, the two-month graft survival rate was 28.57% in itraconazole group, 62.50% in dexamethasone group, 12.50% in PBS group, 0 in amphotericin B group and 0 in surgery-only group. Graft survival in the itraconazole group was higher than that in the amphotericin, PBS and surgery-only group (P=0.057, 0.096, 0.012, respectively). The itraconazole group showed less total angiogenesis and lymphangiogenesis than PBS group (all P<0.05). CONCLUSION: Itraconazole decrease neovascularization, lymphangiogenesis, and inflammation in both a corneal suture model and PK model. Itraconazole has anti-(lymph)-angiogenic and anti-inflammatory effects in addition to its intrinsic antifungal effect and is therefore an alternative treatment option in cases where steroids cannot be used.