Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization.

AIM: To investigate whether ultrasound-targeted cationic microbubbles (CMBs) destruction could deliver endostatin-green fluorescent protein (GFP) plasmids efficiently to the human retinal endothelial cells (HRECs) and inhibit retinal neovascularization in mice. METHODS: CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy. Experiments assessing HRECs migration and vascular formation were performed to evaluate gene therapy's efficiency in vitro. A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl, Bcl-2, vascular endothelial growth factor (VEGF) and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction (qPCR). The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5, 14, and 28d after treatment. RESULTS: The gene expression of endostatin was the highest in the group of the CMBs. Besides, the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro. In vivo, retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups. CONCLUSION: The research illustrates ultrasound-targeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

Clinical relevance of the glucocorticoid receptor gene polymorphisms in glucocorticoid-induced ocular hypertension and primary open angle glaucoma.

AIM: To avoid the side effects of ocular hypertension of glucocorticoid (GC) usage in eye, we must identify susceptible individuals, which exists in about one-third of all population. Further, the majority of all primary open angle glaucoma (POAG) patients show this phenotype. Glucocorticoid receptor (GR) regulates C responsiveness in trabecular meshwork (TM) cells. In this study, single nucleotide polymorphism (SNP) genotyping was used to determine whether there are differences in the BclI (rs41423247) and N363S (rs6195) polymorphisms of the GR gene in healthy and POAG patients, and glucocorticoid-induced ocular hypertension (GIOH) populations. METHODS: Three hundred and twenty-seven unrelated Chinese adults, including 111 normal controls, 117 GIOH subjects and 99 POAG patients, were recruited. DNA samples were prepared and the BclI and N363S polymorphisms were screened using real-time polymerase chain reaction (RT-PCR)-restriction fragment length polymorphism (RFLP) analysis. Frequencies of the BclI and N363S polymorphisms were determined and compared using Fisher's exact test and the Chi-squared test. RESULTS: Only the BclI polymorphism was identified in the Chinese Han population. The frequency of the G allele was 21.6 % in normal controls, 18.3% in GIOH patients, and 13.64% in the POAG patients. There was no significant difference in polymorphism or allele frequency in the 3 groups. Furthermore, no N363S polymorphism was found in the study subjects. CONCLUSION: The BclI polymorphisms in GR gene had no association with GIOH and POAG patients, and N363S polymorphism might not exist in the Chinese Han population. Therefore, the BclI polymorphism might not be responsible for the development of GC-induced ocular hypertension or POAG.

Quantitation of 47 human tear proteins using high resolution multiple reaction monitoring (HR-MRM) based-mass spectrometry.

Tear proteins are intimately related to the pathophysiology of the ocular surface. Many recent studies have demonstrated that the tear is an accessible fluid for studying eye diseases and biomarker discovery. This study describes a high resolution multiple reaction monitoring (HR-MRM) approach for developing assays for quantification of biologically important tear proteins. Human tear samples were collected from 1000 subjects with no eye complaints (411 male, 589 female, average age: 55.5±14.5years) after obtaining informed consent. Tear samples were collected using Schirmer's strips and pooled into a single global control sample. Quantification of proteins was carried out by selecting "signature" peptides derived by trypsin digestion. A 1-h nanoLC-MS/MS run was used to quantify the tear proteins in HR-MRM mode. Good reproducibility of signal intensity (using peak areas) was demonstrated for all 47 HR-MRM assays with an average coefficient of variation (CV%) of 4.82% (range: 1.52-10.30%). All assays showed consistent retention time with a CV of less than 0.80% (average: 0.57%). HR-MRM absolute quantitation of eight tear proteins was demonstrated using stable isotope-labeled peptides. BIOLOGICAL SIGNIFICANCE: In this study, we demonstrated for the first time the technique to quantify 47 human tear proteins in HR-MRM mode using approximately 1µl of human tear sample. These multiplexed HR-MRM-based assays show great promise of further development for biomarker validation in human tear samples. Both discovery-based and targeted quantitative proteomics can be achieved in a single quadrupole time-of-flight mass spectrometer platform (TripleTOF 5600 system).

Association between polymorphisms in lysyl oxidase-like 1 and susceptibility to pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

The present knowledge on the association of single nucleotide polymorphisms (SNPs) of lysyl oxidase-like 1 (LOXL1) with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG) is controversial and inconclusive. This meta-analysis sought to derive a more precise estimation of the effects of LOXL1 SNP loci (rs1048661, rs3825942, and rs2165241) on PEXS/PEXG. Literature searches were conducted on the PubMed, EMBASE, ISI Web of Science, and Cochrane Library databases through October 2013. Twelve studies describing 1810 cases and 1790 controls met the inclusion criteria. The strengths of the associations found through the meta-analysis were assessed with pooled odds ratios and their 95% confidence intervals (CI). A meta-regression analysis was also used to examine the influence of the study and population characteristics. The results indicated that rs1048661 TT carriers had 92.1% and 40.4% less risk of developing PEXS/PEXG than did the controls in the Caucasian and Asian populations, respectively. Carriers of rs3825942 AA or rs2165241 CC also had significantly less PEXS/PEXG susceptibility than did the non-carriers. Meta-regression showed that in Caucasians, the male proportion (slope: 0.272; 95% CI: 0.167-0.376; P = 0.0001) and mean age (slope: 0.796; 95% CI: 0.375-1.217; P = 0.0002) of the PEXS/PEXG subjects correlated positively with the effect of rs3825942 on PEXS/PEXG susceptibility. The meta-analysis suggested that LOXL1 rs1048661 TT, rs3825942 AA, and rs2165241 CC were associated with a reduced risk of developing PEXS/PEXG.

Experimental research of RB94 gene transfection into retinoblastoma cells using ultrasound-targeted microbubble destruction.

The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma.

Prevalence of eye diseases and causes of visual impairment in school-aged children in Western China.

BACKGROUND: The present study investigated the prevalence of refractive error, visual impairment, and eye diseases in school-aged children in western China. METHODS: The survey was done in a representative county (Yongchuan District, Chongqing Municipality) of western China. Cluster random sampling was used to select children aged 6 to 15 years. We conducted door-to-door surveys and eye examinations including optometry, stereoscopic vision test, eye position and eye movement, slit lamp examination of the anterior segment, retinoscopy, and fundus examination after cycloplegia with 1% cyclopentolate. RESULTS: Among 3469 children, data were available for 3079 (88.76%). The prevalences of eye diseases were, in descending order, refractive error (20.69%; 637/3079), conjunctivitis (11.76%; 362/3079), amblyopia (1.88%; 58/3079), color vision defect (0.52%; 16/3079), keratitis (0.36%; 11/3079), strabismus (0.29%; 9/3079), cataract (0.23%; 7/3079), pathologic myopia (0.19%; 6/3079), and ocular trauma (0.13%; 4/3079). The prevalence of corneal leucoma, corneal staphyloma, optic neuropathy, macular degeneration, and myelinated nerve fibers was 0.03% (1/3079) for each. The prevalence of visual impairment was 7.70% (237/3079), and the major causes of visual impairment were uncorrected refractive error (86.08%; 204/237), amblyopia (9.70%; 23/237), pathologic myopia (1.27%; 3/237), congenital cataract (0.42%; 1/237), and others (2.11%; 5/237). CONCLUSIONS: Among school-aged children in a less developed area of western China, refractive error was the most prevalent eye disorder, and uncorrected refractive error was the main cause of visual impairment.

Elevation of plasma membrane permeability upon laser irradiation of extracellular microbubbles.

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high controllability of laser energy and direction. In this report, we describe a combination laser-microbubble system that enables membrane-impermeable molecules to penetrate cell membranes. The main theories we apply are optical breakdown and photoacoustic generation, which are induced by laser irradiation. Firstly, different types of laser light (Ar-green, Novus Varia poly-wavelength and Nd:YAG laser) were adopted to blast liposome microbubble contrast medium; subsequently, the Nd:YAG laser (1064 nm, 4 ns), which could successfully blast microbubbles, and ultrasound were used in combination to irradiate a mixture of liposome microbubbles and retinoblastoma (Rb) cells. After irradiation, membrane permeability was evaluated by flow cytometric assay using propidium iodide (PI) and fluorescein diacetate (FDA). The proportion of permeabilized resealed cells was affected by changes in the light energy. All of the Nd:YAG laser, Nd:YAG combination laser-microbubble and combination ultrasound-microbubble systems were able to permeabilize the Rb cells. These results suggest that this combination laser-microbubble system is a new means of delivering exogenous materials into living cells.

Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization.

BACKGROUND: Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV. METHODS: Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDF gene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography. RESULTS: In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively. CONCLUSIONS: Ultrasound-microbubble technique could increase PEDF gene transfer into rats' retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.

Knockdown of damage-specific DNA binding protein 1 (DDB1) enhances the HBx-siRNA-mediated inhibition of HBV replication.

Recent studies have demonstrated that the effect of inhibition of HBV replication can be achieved by RNA interference (RNAi) at both the cellular and organismal levels. However, HBV replication cannot be completely inhibited by this method. To completely inhibit HBV replication, new strategies for improving the inhibition efficacy of HBV-specific siRNAs are needed. In this study, we demonstrated that knockdown of damage-specific DNA binding protein 1(DDB1), a protein involved in nucleotide-excision repair and HBV replication, significantly enhanced the HBx-siRNA-mediated inhibition of HBV replication. Although knockdown of DDB1 may be toxic to normal liver cells, our results indeed suggest a new direction to enhance the efficacy of HBV-siRNA-mediated inhibition of HBV replication.