Clinical and Genetic Analysis of Retinitis Pigmentosa with Primary Angle Closure Glaucoma in the Chinese Population.

PURPOSE: Retinitis pigmentosa (RP) constitutes a class of common inherited retinal dystrophies. Patients with RP and comorbid primary angle-closure glaucoma (PACG) have been described, but the relationship between the diseases remains unclear. This study investigated the clinical and genetic characteristics of Chinese patients with RP and comorbid PACG. METHODS: Of 1356 patients with RP, we analyzed the genetic features of 39 RP patients with PACG using next-generation sequencing and reviewed their clinical characteristics. RESULTS: In total, 18 patients with acute PACG and 21 patients with chronic PACG were included in this study; their age at examination was 50.54 ± 12.99 years (range, 25.0-71.0 years), and their age at PACG onset was 46.04 ± 14.50 years (range, 24.9-68.0 years). Additionally, the mean lens thickness (LT) was 4.49 ± 0.44 µm, and the mean axial length (AL) was 22.63 ± 1.17 mm. Notably, the prevalence of PACG in patients with RP was 2.88%; this was higher than the prevalence in the general population. This could be explained by nanophthalmos, thickened lentis, ectopia lentis, or zonular insufficiency. Furthermore, patients with a shorter AL, a greater LT, iridociliary cysts, or nanophthalmos exhibited earlier development of PACG. Overall, 30 disease-causing variants spanning 17 genes were identified in 56.41% of the patients, and PRPH2 was the most common mutation gene. CONCLUSIONS: Our findings revealed that there is a strong association between RP and PACG. Furthermore, intraocular pressure (IOP) should be measured in patients with RP to protect them from the aggravated damage of an elevated IOP.

Antidepressant effect of electroacupuncture on modulating the expression of c-Fos/AP-1 through the JNK signaling pathway.

The effectiveness and safety of electroacupuncture (EA) for depression have been identified by abundant clinical trials and experimental findings. The c-Jun-NH(2)-terminal kinase (JNK) signaling pathway is considered to be involved in the antidepressant mechanism of EA. However, the antidepressant effect of EA via modulating the expression of c-Fos/activator protein-1 (AP-1) under the condition of JNK inhibition remains unexplored. In this study, we investigated the antidepressant effect and possible mechanism of EA in regulating the expression of c-Fos/AP-1 under the condition of JNK inhibition by SP600125 in rats exposed to chronic unpredictable mild stress (CUMS). The depression-like behaviors were evaluated by the body weight, sucrose preference test (SPT), and open field test (OFT). The expression levels of c-Jun in the hypothalamus, c-Fos in the pituitary gland, and c-Fos and AP-1 in the serum of CUMS induced rat model of depression were detected by ELISA. The results indicated that treatment with EA and fluoxetine can reverse the CUMS-induced depression-like behaviors in rats and can up-regulate the expression levels of c-Jun in the hypothalamus, c-Fos in the pituitary gland, and c-Fos and AP-1 in the serum. Of note, the data demonstrated that SP600125, the inhibitor of JNK signaling pathway, can exert synergistic effect with EA in regulating CUMS-induced abnormal activation of the JNK signaling pathway. The antidepressant effect of EA might be mediated by modulating the expression of c-Fos/AP-1.

[Effect of moxibustion on IL-6/STAT3 signaling in frontal cortex of fatigue rats].

OBJECTIVE: To observe the effect of moxibustion on interleukin-6(IL-6)/signal transduction and transcriptional activator 3(STAT3) signaling pathway in the frontal cortex of fatigue rats, so as to reveal its mechanisms underlying alleviation of fatigue. METHODS: Twenty-one male SD rats were randomly divided into normal control, model, and moxibustion groups (n＝7 rats in each group). The fatigue model was established by forcing the rats to have an exhausted swim under load condition, once daily for 21 days. Moxibustion was applied to bilateral "Zusanli"(ST36) for about 15 min, once every other day for 21 days. The level of IL-6 in the frontal cortex was detected by ELISA, and the expression of Janus kinase 2 (JAK2), phosphorylated JAK2 (p-JAK2), signal transduction and transcriptional activator 3(STAT3) and phosphorylated STAT3 (p-STAT3) proteins in the frontal cortex was detected by Western blot. RESULTS: After modeling, the levels of IL-6 content and p-STAT3 protein expression and ratio of p-STAT3/STAT3 were significantly increased in the model group relevant to the normal control group (P<0.01). Follo-wing moxibustion, the duration of load swimming on the 21st day was significantly prolonged (P<0.01), content of IL-6 and levels of p-STAT3 protein expression and ratio of p-STAT3/STAT3 were significantly down-regulated in the moxibustion group compared with the model group (P<0.05). No significant differences were found between the model and control, and between moxibustion and model groups in the expression levels of JAK2, p-JAK2, STAT3 and p-JAK2/JAK2 (P>0.05). CONCLUSION: Moxibustion intervention can relieve fatigue in fatigue rats, which is associated with its function in inhibiting IL-6/STAT3 signaling pathway to reduce inflammatory injury.

Panel-based targeted exome sequencing reveals novel candidate susceptibility loci for age-related cataracts in Chinese Cohort.

BACKGROUND: Age-related cataracts (ARC) is the most common blinding eye disease worldwide, and its incidence tend to become younger. However, the relationship between genetic factors and mechanisms is not fully understood. The aim of the study was to further clarify the relationship between ARC and genetic mechanisms in East Asian populations and to elucidate the pathogenesis. METHODS: The study collected 191 sporadic cataracts and 208 healthy people from the eastern provinces of China, with an average age of about 60 years. All participants were subjected to a comprehensive ophthalmic clinical examination and peripheral blood samples were collected and their genomic DNA was extracted. Mutations were screened among 792 candidate genes to enhance understanding of the disease through targeted capture and high-throughput sequencing. RESULTS: We identified novel candidate susceptibility gene, which may serve as a potential susceptibility factor leading to an increase in the incidence of age-related cataracts. Three novel loci are associated with age-related cataracts significant significance: rs129882 in DBH (p = 5.27E-07, odds ratio = 3.9), rs1800280 in DMD (p = 2.85E-06, odds ratio = 1.4) and rs2871776 in ATP13A2 (p = 4.18E-05, odds ratio = 0.04). Gene-gene interaction analysis revealed that the most significant interactions between genes include the interaction between DBH and TUB (rs17847537 in TUB, rs129882 in DBH, p-value = 2.12E-14), and the interaction between DBH and DMD (rs1800280 in DMD, rs129882 in DBH, p-value = 2.12E-14). Pathway analysis shows that the most significant processes are concentrated in response to light stimulation (adjusted p-Value = 5.56E-03), response to radiation (adjusted P-Value = 5.56E-03), abiotic stimulus (adjusted p-Value = 5.56E-03). eQTL analysis shows that DBH rs129882 could regulate the expression of DBH mRNA in various tissues including retina. CONCLUSION: Our study indicates rs129882 and rs1800280 loci are associated with age-related cataracts, which enlarge the gene map of age-related cataracts.

Expanding the clinical and genetic spectrum of Heimler syndrome.

BACKGROUND: Heimler syndrome (HS) is a rare hereditary systemic disorder, partial clinically overlapping with Usher syndrome. So far, our knowledge of HS is very limited, many cases are misdiagnosed or may not even be diagnosed at all. This study aimed to analyze the clinical and genetic characteristics of HS, and to evaluate potential phenotype-genotype correlations. RESULTS: Two HS cases caused by PEX1 mutations were identified, and a novel likely pathogenic mutation, PEX1 c.895_896insTATA, was found. The main ophthalmic finding of the two patients was consistent with retinitis pigmentosa accompanied by cystoid macular edema, but short axial length and hyperopia were also observed as two previously unreported ocular phenotypes. Analysis of the literature showed that of the 29 HS patients previously reported, 12 had PEX6 mutations, 10 had PEX1 mutations, two had PEX26 mutations, and the remaining patients were not genetically tested. Three novel genotype-phenotype correlations were revealed from analysis of these patients. First, most genotypes of every HS patient include at least one missense variant; second, at least one mutation in PEX1 or PEX6 gene affects the AAA-ATPase region in every HS patient with retinal dystrophy, suggesting AAA-ATPase region is a hypermutable region in patients with a retinal dystrophy; third, there are no significant differences between PEX1-, PEX6-, and PEX26-associated phenotypes. CONCLUSION: Next-generation sequencing is important for the diagnosis of HS. This study expands the clinical and genetic spectrum of HS, and provides additional insights into genotype-phenotype correlations, which is vital for accurate clinical practice, genetic counseling, and pathogenesis studies.

Calpastatin participates in the regulation of cell migration in BAP1-deficient uveal melanoma cells.

AIM: To detect how BRCA-associated protein 1 (BAP1) regulates cell migration in uveal melanoma (UM) cells. METHODS: Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, immunoprecipitations and surface plasmon resonance analyses were applied to identify BAP1 protein partners. Western blot and calpain activity assays were used to test the expression and function of calpastatin (CAST). RESULTS: CAST protein was confirmed as a new BAP1 protein partner, and loss of BAP1 reduced the expression and function of CAST in UM cells. The overexpression of CAST rescued the cell migration phenotype caused by BAP1 loss. CONCLUSION: BAP1 interacts with CAST in UM cells, and CAST and its subsequent calpain pathway may mediate BAP1-related cell migration regulation.

Targeting circular RNA-ZRANB1 for therapeutic intervention in retinal neurodegeneration.

Glaucoma is a neurodegenerative disease characterized by retinal ganglion cell (RGC) loss, optic disc excavation, and progressive visual field loss. Direct or indirect ameliorating retinal neurodegeneration is a promising therapeutic therapy for glaucoma. Circular RNAs (circRNAs) are a class of covalently closed circular RNA transcripts and have emerged as potential regulators in several neurodegenerative diseases. In this study, we show that cZRANB1 expression is significantly upregulated in retinal neurodegeneration induced by glaucoma. cZRANB1 knockdown decreases retinal reactive gliosis, glial cell activation, and facilitates RGC survival in vivo. cZRANB1 knockdown directly regulates Müller cell function and indirectly regulates RGC function in vitro. cZRANB1 acts as miRNA sponge to regulate Müller cell function through cZRANB1/miR-217/RUNX2 network. Intervention of cZRANB1 expression would become an effective strategy for treating retinal neurodegeneration.

Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus.

BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.

Granulomatous Lobular Mastitis Associated with Mycobacterium abscessus in South China: A Case Report and Review of the Literature.

Mycobacteria, which are known as rapidly growing bacteria, are pathogens that are responsible for cutaneous or subcutaneous infections that especially occur after injection, trauma, or surgery. In this report, we describe a species of Mycobacterium abscessus that was isolated from a breast abscess in a patient who was previously diagnosed with granulomatous lobular mastitis (GLM). This current case is the first ever presented case of GLM associated with M. abscessus documented in South China. The case presentation highlights the role of M. abscessus in GLM. The association of M. abscessus and GLM is discussed and a summary of breast infection due to Mycobacteria is given.

Expression and Distribution of Mesencephalic Astrocyte-Derived Neurotrophic Factor in the Retina and Optic Nerve.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), otherwise named Arginine-Rich, Mutated in Early-stage Tumors (ARMET), is a secretory endoplasmic reticulum stress (ERS) protein that is widely expressed in mammalian tissues. To date, little is known about the distribution and expression of MANF in the retina and optic nerve (ON). Therefore, we studied the expression and distribution of MANF in the ON and retina by real-time PCR, immunofluorescence staining and western blotting. Results from rat and mouse were highly consistent in the retina. MANF was detected in both tissues in rat, wherein it was principally localized to the ganglion cell layer (GCL), followed by the inner nuclear layer (INL). The MANF protein levels in the rat retina were 3.33-fold higher than in the rat ON. Additionally, MANF was robustly expressed by retinal ganglion cells (RGCs) in the human retina. In human ON, MANF was partially co-localized with glial fibrillary acidic protein (GFAP), suggesting that it was not restricted to astrocytes. In vitro studies confirmed that MANF could be robustly expressed in RGCs and was found principally within the cytoplasm. Hypoxia can stimulate up-regulation by of MANF expression over time, suggesting that MANF may play a vital role in the functional regulation of RGCs both in health and disease. We believe that the present study improves our understanding of the distribution and expression of MANF in the retina and ON and could help in further analysis of its interact and correlate with the relevant ophthalmic diseases.

[Roles of retinal Müller cells in health and glaucoma].

In the vertebrate retina, Müller cells are principal glial cells which stretch across the whole thickness of the retina and contact with the somata and processes of all retinal neurons, thus forming an anatomical and functional link between glial cells and retinal neurons. Numerous studies have shown that Müller cells express various neurotransmitter receptors, transporters, ion channels and enzymes that are relative to cellular activities. In addition, the cells also release factors, such as D-serine and glutamate etc., to regulate the neuron excitability. Therefore, retinal Müller cells may play more curious roles in addition to supporting the retinal neurons. The information exchange and interaction between Müller cells and neurons may regulate and maintain retinal neuronal functions. In the glaucomatous retina, Müller cells are reactivated (gliosis). Reactivated Müller cells undergo a variety of changes in cellular physiology, biochemistry and morphological features. Meanwhile, the reactivated Müller cells may produce and release cytotoxic factors, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), reactive oxygen species (ROS) and prostaglandin E2 (PGE2), thus involving in the induction of retinal ganglion cell apoptosis and death. Here, we reviewed the physiological properties of retinal Müller cells, and the functional changes of Müller cells in the glaucomatous retina.

RNAi screening identifies GSK3ß as a regulator of DRP1 and the neuroprotection of lithium chloride against elevated pressure involved in downregulation of DRP1.

Elevated intraocular pressure (IOP) is considered as the major risk factor for the loss of retinal ganglion cells (RGCs) and their axons in glaucoma. Emerging evidence suggests elevated IOP can induce Drp1 upregulation and mitochondrial fission, which is involved in cell death. However, the underlying mechanism for these effects remains unknown. The present study used RNAi screening to investigate the effects of 24 kinases associated with mitochondrial activities on DRP1 expression under hydrostatic pressure. We identified, for the first time, that glycogen synthase kinase 3 beta (GSK3ß) knockdown suppressed the upregulation of DRP1 induced by elevated pressure. Use of the pharmacological inhibitor of GSK3ß inhibitor, lithium chloride (LiCl), confirmed this result. Furthermore, we demonstrated that one of the mechanisms of lithium chloride neuroprotection might be via inhibition of mitochondrial fission through downregulation of Drp1.

Physico-chemical and antioxidant properties of four mango (Mangifera indica L.) cultivars in China.

Four principal mango cultivars (Tainong No.1, Irwin, JinHwang and Keitt) grown in southern China were selected, and their physico-chemical and antioxidant properties were characterized and compared. Of all the four cultivars, Tainong No.1 had highest content of total phenols, ρ-coumaric acid, sinapic acid, quercetin, titratable acidity, citric acid, malic acid, fructose, higher antioxidant activities (DPPH, FRAP) and L(*), lower pH, PPO activity and individual weight. Keitt mangoes showed significantly (p<0.05) higher contents of ß-carotene, ρ-hydroxybenzoic acid, sucrose, total sugar, total soluble solid, catechin, succinic acid and higher PPO activity. JinHwang mangoes exhibited significantly (p<0.05) higher individual weight and PPO activity, but had lower content of total phenols, ß-carotene and lower antioxidant activity. Principal component analysis (PCA) allowed the four mango cultivars to be differentiated clearly based on all these physico-chemical and antioxidant properties determined in the study.

Electrical stimulation ameliorates light-induced photoreceptor degeneration in vitro via suppressing the proinflammatory effect of microglia and enhancing the neurotrophic potential of Müller cells.

Many types of electrical stimulation (ES) devices have been shown to promote the survival of degenerated neural cells, such as dopaminergic neurons in the medial forebrain bundle-transected rats, ischemic-injured cortical neurons and inner-and outer-nuclear-layer cells in degenerated retina. Using a rat photic injury model, our lab previously proved the neuroprotective effect of transcorneal electrical stimulation (TCES) on apoptotic photoreceptor cells. To delineate the mechanisms that might underlie this process, the effects of ES on light-damaged photoreceptor degeneration-induced microglia and Müller cell activation were investigated in the present in vitro study. Our data showed that ES (3 ms, 20 Hz, 300-1600 µA) increased survival among light-reared cone-derived cells (661W) cultured alongside microglia or Müller cells analyzed by LDH and TUNEL assays. The degree of rescue was found to depend on the different intensities of the ES current. The immunocytochemistry revealed that ES significantly decreased the numbers of activated microglia cells with ameboid shapes and increased the numbers of reactive gliotic Müller cells with larger soma when they were co-cultured with light-damaged 661W cells. Real-time RT-PCR and Western blotting indicated that ES which was applied to different co-cultures and 661W cell-conditioned media (661WCM)-treated glia cultures had a prominent inhibitive effect on the secretion of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in microglia and a positive regulative effect on the production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in Müller cells. The death rate of light-exposed 661W cells cultured with microglia was decreased significantly by the addition of neutralizing antibodies against IL-1ß and TNF-α. On the other hand, the death rate of light-exposed 661W cells cultured with Müller cells was prominently increased when the co-culture was incubated in the presence of neutralizing antibody against BDNF while anti-CNTF neutralizing antibody did not induce additional exacerbation of the cell death among those 661W cells. These findings indicate the feasibility of using ES to create a nurturing environment for light-damaged photoreceptor cells. This environment is characterized by diminished microglial activation and fortified Müller cells reactive gliosis, which may have great potential in ameliorating photoreceptor damage. In this way, ES was here determined to be a novel, potent therapeutic option for delaying the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa (RP).

Excessive milk intake as a risk factor, probably associated with oxidative stress, in experimental naphthalene-initiated cataract in rats.

AIM: To determine whether a diet containing excessive amounts of milk aggravates naphthalene-initiated cataracts in a common animal model of age-related human cataract. METHODS: Ninety Sprague-Dawley rats were fed a natural diet supplemented with either water (group A), normal amounts of milk (group B), excessive amounts of milk (group C), naphthalene plus water (group D), naphthalene plus normal amounts of milk (group E), naphthalene plus excessive amounts of milk (group F). Cataract development was monitored weekly using a slit lamp and lens gray value analysis. Concentrations of reactive oxygen species (ROS), reduced glutathione (GSH) and malondialdehyde (MDA) in rat lenses were measured to determine the role of oxidative stress in cataract induction. RESULTS: By week 4, the cortical gray value was significantly higher in group F than that in group D, and the cortical gray value was significantly higher in group D than in group A. However, by week 8, no significant differences were observed among groups C, F, B, E and A. ROS concentrations in lenses of rats of groups C and F were slightly higher than in those of groups B, E and A, but ROS concentrations in group F were significantly higher than in the other groups receiving naphthalene (i.e. groups D and E). GSH concentrations in group F were significantly lower than in the other groups. MDA concentrations in group F were significantly higher than in the other groups receiving naphthalene, indicating increased lipid peroxidation induced by naphthalene plus excessive intake of milk. CONCLUSIONS: Our results provide quantitative evidence that excessive intake of milk aggravates naphthalene-initiated cataracts, which is probably due to oxidative damage caused by increased ROS.

Distinctive gene transduction efficiencies of commonly used viral vectors in the retina.

The transduction efficiency and cell tropism of viral vectors rAAV2/1, rAAV2, Ad5, Ad5/F35, and Lentivirus were evaluated in retina. All viral vectors achieved efficient transduction in living rat retina. However, each vector showed distinctive efficiency in vitro especially for rAAV2/1, which displayed poor transduction in cultured retinal cells. Distinctive cell-specific GFP expression was observed in vivo and in vitro for the same viral vector. The cell-specific tropism was not strictly correlated with the correspondent distribution of viral receptors in retina. These results provided important insights into the selection of appropriate vectors when specific retinal diseases are considered for gene therapy.

[Transduction efficacy of recombinant type 1 and type 2 adeno-associated virus in the retinal cells].

OBJECTIVE: To investigate the transduction and gene expression of the recombinant adeno-associated viruses (rAAV) of the serotypes 1 and 2 in the retinal cells. METHODS: rAAV vectors of type 1 and type 2 encoding EGFP were infected into the cultured retinal pigmentary epithelium (RPE) cells of the line CRL-2302 and primarily cultured retinal neural cells from normal SD rats, and primarily cultured RPE cells from an adult cornea donor. The cultured RPE cells transduced by rAAV2-EGFP or rAAV2/1-EGFP were harvested at the 7 th and 14 th day after infection to be detected by fluorescence-activated cell sorter. The onset of EGFP gene expression and EGFP positive rate were detected by flow cytometry and fluorescence microscopy. Then, rAAV2/1-EGFP and rAAV2-EGFP were injected into the subretinal spaces of 32 SD rats to investigate the onset of EGFP fluorescence and its distribution in the fundus in vivo via fluorescence stereoscope. HE staining and immunohistochemistry were used to observe the infected cell type and immune response in the retina. RESULTS: The percentage of EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2-EGFP 7 and 14 days after transduction were 13.50% +/- 1.70% and 15.60% +/- 0.82%, and 2.75 +/- 0.12 and 3.80 +/- 0.72 respectively; and the EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2/1-EGFP were 1.09% +/- 0.5% and 1.98% +/- 0.45%, and 1.12 +/- 0.09 and 1.75 +/- 0.2 respectively. The EGFP fluorescence area in the retina were (5389 +/- 211) microm(2), (9832 +/- 364) microm(2), (14 454 +/- 446) microm(2), (20 528 +/- 648) microm(2), and (20 264 +/- 683) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after transduction by rAAV2-EGFP in vivo; In the rat retina transduced by rAAV2/1-EGFP, the EGFP fluorescence areas were (9666 +/- 348) microm(2), (12 160 +/- 439) microm(2), (19 794 +/- 621) microm(2), (26 172 +/- 923) microm(2), and (26 022 +/- 965) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after infection. CONCLUSION: rAAV2 efficiently transduces retinal cells both in vitro and in vivo. rAAV2/1 is a more effective gene-transferring vector to be used in retinal cells in vivo than rAAV2.

[Tumor targeting gene transfer mediated by electroporation for growth suppression of retinoblastoma in vivo].

OBJECTIVE: To explore the effects and mechanisms of electroporation on the therapy of transplanted retinoblastoma tumor (RB) by transferring the expressive plasmids of sFlk-1 and ExTek. METHODS: The effects and mechanisms of sFlk-1 and ExTek on RB is validated both in vitro and in vivo. In vitro, pCI-sFlk-1 was transfected into HEK293 (human embryonic kidney 293) cells with DMRIE-C Reagent, and its supernatant was collected 24 h later to mix with RPMI-1640 medium as 1:1, 2:1 and 3:1. RB cells were cultured in the mixed medium and the cell growth curve were analyzed 6 days later. The suspension of 1 x 10(7) RB cells were inoculated in 40 nude mice subcutaneously. The nude mice with RB were divided into 2 groups. The experimental group was treated with transfer of pCI-sFlk-1 (15 microg) and pCI-ExTek (15 microg) by electroporation once a week, while the control group was treated with physiological saline solution. After 3 weeks' therapy, the animals were observed for one more week. During this period the tumors were measured to draw the growth curve of the tumors and to calculate the anti-tumor rate. The vessel endothelium were tagged with smooth muscle actin (SMA) by immunohistochemical methods to inspect the micro vessel density (MVD) of the tumors. Moreover, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1 of the tumors was confirmed by immunohistochemical staining. RESULTS: sFlk-1 exhibited no inhibiting effects on the growth of RB cells in vitro, while sFlk-1 combined with ExTek restrained the growth of the transplanted tumors obviously in vivo, and the anti-growth rate is 80.04%. The MVD of the experimental group is 26.69 +/- 2.95, which is evidently lower than that of the control group (44.51 +/- 6.11). The difference between these 2 groups was statistically significant (P < 0.05). As to the expression of VEGF, there was no significant difference between these 2 groups, while the expression of Flk-1 of the experimental group was much stronger than that of the control group. CONCLUSION: Transfer the expressive plasmids of sFlk-1 and ExTek by electroporation in vivo restrained the growth of the transplanted RB tumor, and the effect is due to the inhibition of angiogenesis process.

[Analysis of angiogenesis associated factors of retinoblastoma cell line and tumor tissues].

OBJECTIVE: To investigate the factors regulating angiogenesis of retinoblastoma. METHODS: Cultured retinoblastoma cells from HXO-Rb-44 cell lines were inoculated subcutaneously into the leg of nude-mice. Experimental retinoblastoma was studied by immunohistochemistry staining for the expression of angiogenesis factors (VEGF-A, VEGF-C, VEGF-D and bFGF), vascular endothelial cell membrane receptor tyrosine kinases (Flt-1, Flt-4, Flk-1, Tie-2 and FGFR), transcription regulated factor, Hif-1, matrix metalloproteinase (MMP-2, MMP-7 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1 and TIMP-2). RESULTS: There were plenty of capillaries and small vessels in the retinoblastoma. Some retinoblastoma cells expressed Hif-1 and most cells expressed VEGF at a high level, especially VEGF-D. Flt-1, Flt-4 and FGFR were found in vascular endothelial cells in the retinoblastoma. Three matrix metalloproteinases were positive and two tissue inhibitors of metalloproteinases were negative in the retinoblastoma. CONCLUSIONS: Down-regulated TIMP and up-regulated MMP and VEGF signal transduction play an important role in the occurrence of angiogenesis in the retinoblastoma. This could be considered as the target of retinoblastoma treatment.

[In vitro transdifferentiation of adult human retinal pigment epithelium cells to neuro-like cells induced by blocking VEGF expression].

OBJECTIVE: To investigate the effects of blocking VEGF expression on the retinal pigment epithelium (RPE) in vitro. METHODS: RPE was transfected with plasmid encoding anti-sense of VEGF by lipofectin. RPE clones with stable expression of anti-sense of VEGF were screened by neomycin (G418). The changes of RPE and the level of VEGF secreted by RPE were observed by immunofluorescence immunohistochemical staining, Elisa and inverted-microscope. RESULTS: Transdifferentiation of cultured adult human retinal pigment epithelium cells to neurons was induced by blocking VEGF expression. The transdifferentiation was a direct phenotypic change from RPE cell to neurons without cell differentiation. The transdifferentiated neurons showed up-regulated expression of VEGF receptor 2 (Flk-1) and expressed several markers of retinal ganglion cell, including Thy1.1 and neurofilament. CONCLUSION: Blocking of VEGF expression and up-regulation of Flk-1 can induce transdifferetiation of adult human retinal pigment epithelium cells into ganglion cells.