[Show abstract][Hide abstract] ABSTRACT: Background
Netrin-1 has been reported to promote retinal neovascularization in oxygen-induced retinopathy (OIR). However, netrin-1 receptors, which may mediate netrin-1 action during retinal neovascularization, have not been characterized. In this study, we investigated netrin-1 receptor subtype expression and associated changes in the retinas of mice with OIR.
C57BL/6J mice were exposed to 75±2% oxygen for 5 days and then returned to normal air to induce retinal neovascularization. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to examine the expression of netrin-1 receptor subtypes in the mouse retinas. Double staining of netrin-1 receptor subtypes and isolectin B4 was used to determine the location of the netrin-1 receptor subtypes in the retinas. Inhibition of retinal neovascularization was achieved by UNC5B shRNA plasmid intravitreal injection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections.
RT-PCR results showed that, except for UNC5A, netrin-1 receptor subtypes UNC5B, UNC5C, UNC5D, DCC, neogenin, and A2b were all expressed in the retinas of OIR mice 17 days after birth. Western blots showed that only UNC5B expression was significantly increased on that day, and immunofluorescence results showed that only UNC5B and neogenin were expressed in retinal vessels. Treatment of OIR mice with the UNC5B shRNA plasmid dramatically reduced neovascular tufts and neovascular outgrowth into the inner limiting membrane.
UNC5B may promote retinal neovascularization in OIR mice.
[Show abstract][Hide abstract] ABSTRACT: To study two methods for culturing and purifying Sprague-Dawley (SD) rat retinal Müller cells and determine which one is better.
The passage culture method of Müller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method. After culturing retinal cells for one month through these two methods, fluorescence-activated cell sorter (FACS), RT-PCR, and immunohistochemistry technology were performed to examine the enrichment and purity of Müller glial cells, and carried out two-sample approximate t test using SSPS 13.0 to further compare the Müller cell positive rate in both methods.
The statistical results showed that the purity of Müller cells was 83.2%±5.16% in group A, and the purity was 98.5%±1.08% in group B. The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant (t=-9.178, P<0.005). The results clearly exhibited a difference between the purity of Müller cells cultured by the complete pancreatic enzyme digestion method (group A) and the repeated incomplete pancreatic enzyme digestion method (group B).
Compared with the complete pancreatic enzyme digestion method, this novel method was more efficient and a higher purity of Müller cells could be obtained using this approach.
International Journal of Ophthalmology 01/2013; 6(6):778-84. DOI:10.3980/j.issn.2222-3959.2013.06.07 · 0.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glaucoma is a chronic, neurodegenerative disease that often leads to blindness. A common treatment is to reduce intraocular pressure (IOP), but this approach does not halt visual loss caused by the death of retinal ganglion cells (RGCs). Therefore, there is an important need for therapies that protect against RGCs degeneration. The present study in a rat glaucoma model aimed to determine whether retinal stem cells (RSCs) transplantation plus vaccination with a glatiramer acetate copolymer-1 (COP-1) could confer neuroprotection. Rats were immunized with COP-1 on the same day as IOP induction by argon laser photocoagulation of the episcleral veins and limbal plexus. RSCs were cultured and transplanted intravitreally 1week after laser treatment. The expression of brain-derived neurotrophic factor (BDNF) and insulin-like growth factor I (IGF-I) were detected by immunohistochemical staining, RT-PCR, and Western blotting. RGCs survival was assessed by TUNEL staining and RGCs counting . We found that the expression of BDNF and IGF-I in the RSCs/COP-1 group was significantly higher than in other groups (P<0.05). In addition, the number of the apoptotic RGCs in the RSCs/COP-1 group was notably lower than in other groups (P<0.05), and number of RGCs in the RSCs/COP-1 group was higher than in other groups(P<0.05). We conclude, therefore, that the combined effects between RSCs transplantation and COP-1 immunization protects RGCs from apoptosis in our rat model of glaucoma. The increase in levels of secreted BDNF and IGF-I may be one of the mechanisms underlying the Neuro-protection of RGCs.
[Show abstract][Hide abstract] ABSTRACT: This study investigated whether brain-derived neurotrophic factor (BDNF) regulates the L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Müller cells (RMCs) under normal and hypoxic conditions. Mouse RMCs were treated with recombinant human BDNF (50, 75, 100, 125, or 150 ng/ml) for 24 h or underwent hypoxia induced by CoCl(2) (125 µM; 6, 12, 24, 48, or 72 h). An additional group underwent combined treatment with BDNF (100 ng/ml; 24, 48, 72, or 96 h) and CoCl(2) (125 µM/ml; 72 h). GLAST and GS mRNA and protein expression, L-[3,4-3H]-glutamic acid uptake, and apoptosis were assessed. BDNF dose-dependently up-regulated GLAST and GS mRNA and protein and increased glutamate uptake. Similarly, in early-stage CoCl(2)-induced hypoxia, GLAST and GS were up-regulated and glutamate uptake increased, but these decreased over time. BDNF also up-regulated GLAST and GS and increased glutamate uptake when RMCs under CoCl(2) induced hypoxic condition. However, BDNF treatment 24 h before CoCl(2) had no effect on GLAST or GS expression. CoCl(2) alone or combined with BDNF did not induce apoptosis. Hypoxia rapidly increased GLAST and GS expressions. This effect was transient, perhaps due to compensatory mechanisms that reduce GLAST and GS by 72 h. BDNF can up-regulate GLAST and GS and increase glutamate uptake during hypoxia, and these functions may underlie its neuroprotective effects.
[Show abstract][Hide abstract] ABSTRACT: To study the inhibition effect of HIF-1α specific siRNA expression vector pSUPERH1-siHIF-1α on retinal neovascularization in a mouse model of retinopathy of prematurity (ROP).
The mouse model of ROP was prepared by the method Smith described. Forty-eight ROP mice were randomly divided into two groups: an experimental group that was intravitreously injected with pSUPERH1-siHIF-1α and a control group that was injected with pSUPER retro vector. The levels of HIF-1α and vascular endothelia growth factor (VEGF) in the retina were examined by Western blot. The retinal neovascularization was evaluated by angiography using FITC Dextran and quantitated histologically.
The levels of HIF-1α and VEGF in the retina in the experimental group were reduced 90% and 65% respectively compared with those in the control group. Meanwhile, the number of retinal neovascular endothelial nucleus outbreaking the inner limit membrane in the experimental group was significantly reduced compared with that in the control group.
The development of retinal neovascularization of ROP can be markedly inhibited by RNA interference targeting HIF-1α.
Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 08/2011; 13(8):680-3.
[Show abstract][Hide abstract] ABSTRACT: Caveolin-1 expression correlates with the permeability of endothelial barriers and angiogenesis. However, the role of caveolin-1 in retinal neovascularization remains unknown. We evaluated the effect of caveolin-1 on the blood-retina barrier and retinal neovascularization in a murine model of oxygen-induced retinopathy.
Starting at postnatal day 7, mice were exposed to 75 ± 5% oxygen for 5 days and then returned to room air conditions to induce retinal neovascularization. Effects on blood-retina barrier were evaluated by Western blot analysis of extravasated albumin in the retina. Retinal neovascularization morphology was studied by fluorescence angiography and was quantified by counts of the endothelial nuclei that protruded into the vitreous cavity. Reverse transcription-polymerase chain reaction and Western blot analysis was used to examine retinal expression levels of caveolin-1. siRNA against caveolin-1 was injected intravitreally in the oxygen-induced retinopathy models. Effects on caveolin-1 mRNA and protein, and retinal neovascularization were assessed as described above.
Caveolin-1 expression was found to increase during hypoxia and overexpression of caveolin-1 correlated with the appearance of extravascular albumin. Caveolin-1 siRNA reduced caveolin-1 mRNA and protein levels by 47.94% and 54.76%, respectively. Furthermore, caveolin-1 siRNA inhibition reduced retinal neovascularization by 51.3% and reduced albumin leakage by 56.32%.
Caveolin-1 may play an important role in induction of retinal neovascularization. SiRNA against caveolin-1 can inhibit experimental retinal hyperpermeability and neovascularization. Therefore, the inhibition of caveolin-1 may be a powerful and novel therapeutic tool for the treatment of ischaemia-induced retinal diseases.
[Show abstract][Hide abstract] ABSTRACT: Recent research has shown netrin-1 to promote neovascularization. We evaluate the expression of netrin-1 during retinal neovascularization in a murine model of oxygen-induced retinopathy.
C57BL/6J mice were exposed to 75 ± 5% oxygen for 5 days and returned to room air to induce retinal neovascularization. Retinal neovascularization was observed by fluorescence angiography and was quantified by counting the endothelial nuclei protruding into the vitreous cavity after hematoxylin-eosin staining. RT-PCR and Western blot analyses were used to determine retinal netrin-1 mRNA and protein levels at postnatal days (PN) 13, 15 and 17.
In fluorescence angiograms, irregular neovascularization and fluorescein leakage were observed surrounding the unperfused areas in the hypoxic group. The hypoxic group had, on average, 50.70 ± 4.56 neovascular nuclei protruding into the vitreous body, while similar nuclei were absent in the control group. Compared to the normoxic group, there were significant increases in both retinal netrin-1 mRNA and protein levels in the hypoxic group at PN13, PN15 and PN17.
The netrin-1 level increases in murine retina under hypoxia and may be key in inducing retinal neovascularization.
[Show abstract][Hide abstract] ABSTRACT: To observe the effect of inhibition of retinal neovascularization by small-interference RNA (siRNA) targeting erythropoietin (EPO).
Three siRNAs against EPO were designed and synthesized. Then they were transfected to NIH/3T3 cells by liposomes. RT-PCR and Western blot were used to evaluate the efficacy of siRNA in attenuating EPO expression in NIH/3T3 cells. One-week-old C57BL/6J mice were exposed to 75 +/- 2% oxygen for 5 days, then they were returned to room air to induce retinal neovascularization. The siRNA type shown as most powerful in reducing EPO expression in vitro was intravitreally injected in the treatment group. Retinal neovascularization was evaluated by angiography with injection of fluorescein-dextran and quantification of neovascular proliferative retinopathy after 5 days in room air. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of siRNA could affect the expression of EPO in murine retinas.
Among the 3 designed siRNAs (named siEPO1-3), siEPO2 is the most efficient in inhibiting EPO expression. In this murine model of oxygen-induced retinopathy, retinal neovascularization in the eyes with siEPO2 injection was significantly reduced compared with that of the contralateral control eyes. Similarly, histological analysis indicates that the number of neovascular nuclei protruding into the vitreous cavity was decreased compared to the control eyes. Furthermore, the expression of EPO in the retinas injected with siEPO2 was dramatically decreased.
siRNA against EPO could inhibit experimental retinal neovascularization by reducing EPO expression in the retinas of mice. It may provide a powerful and novel therapeutic tool for ischemia-induced retinal diseases.
[Show abstract][Hide abstract] ABSTRACT: Retinal neovascularization (NV) occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in blindness. Vascular endothelial growth factor (VEGF) is an essential growth factor for angiogenesis, and is particularly regulated by hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions. Therefore, HIF-1alpha and VEGF could provide targets for therapeutic intervention on retinal NV. In this study, we investigated the inhibitory effects of small interfering RNA (siRNA) targeting HIF-1alpha and VEGF on the expression of HIF-1alpha and VEGF in human umbilical vein endothelial cells (HUVEC) in vitro and on retinal NV in vivo. siRNA-expressing plasmids targeting human HIF-1alpha (HIF-1alpha siRNA) and human VEGF(165) (VEGF siRNA) were constructed. They were transfected and co-transfected to HUVEC and C57BL/6J mice of ischemic retinopathy model. HIF-1alpha siRNA and VEGF siRNA specifically downregulated HIF-1alpha and VEGF at both mRNA and protein levels in vitro and in vivo. Neovascular tufts and neovascular nuclei were decreased in gene therapy group compared to control hypoxia group. Co-transfection of HIF-1alpha siRNA and VEGF siRNA resulted in maximal effects on VEGF suppression in vitro and in vivo. It also manifested the maximal inhibitory effect on retinal NV. These results indicate that the application of HIF-1alpha siRNA and VEGF siRNA technology holds great potential as a novel therapeutic for retinal NV.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the inhibitory effect of small interfering RNA (siRNA) on the expressions of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in retinal neovascularization in the mouse.
HIF-1alpha siRNA recombinant plasmid was constructed. Liposome mediated the expressive plasmid of enhanced green fluorescent protein (EGFP) pEGFP-N1 complex was injected into the vitreous of C57BL/6J mice. The expression of GFP was observed in retinal flat-mounts one day after injection. Randomized controlled trial was performed. There were totally ninety (seven-day-old) C57BL/6J mice in which seventeen mice were chosen as normal group and seventy-three mice were divided into five groups randomly including control model group, vector group and gene therapy group (HIF-1alpha siRNA group, VEGF siRNA group and co-transfection group), in which retinal neovascularization was induced by hypoxia. Liposome with vector plasmid, HIF-1alpha siRNA and VEGF165 siRNA were injected into the vitreous in the vector group, HIF-1alpha siRNA group and VEGF siRNA group respectively one day before mice were moved out to room air from the cabin. Liposome with HIF-1alpha siRNA and VEGF165 siRNA was injected in the co-transfection group at the same time point. Fluorescent angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. HIF-1alpha and VEGF levels in retinas were measured by reverse transcriptase-polymerase chain reaction and Western blot. Significant differences between groups were evaluated by one-way analysis of variance, followed by a least-significant difference analysis.
The GFP expression in retinal cells was observed one day after injection of liposome mediated pEGFP-N1 complex. Neovascular tufts and fluorescein leakage were decreased in gene therapy group especially in co-transfection group compared to the control model group. The neovascular nuclei were decreased in gene therapy group [HIF-1alpha siRNA group (27.73 +/- 2.33), VEGF siRNA group (15.43 +/- 3.23), co-transfection group (8.70 +/- 2.88)] compared to the other three groups (F = 3016.537, P < 0.01). The expression of HIF-1alpha mRNA and protein in retinas were increased in control model group (1.08 +/- 0.06, 0.383 +/- 0.009) and vector group (1.09 +/- 0.05, 0.386 +/- 0.010) as compared with normal group (0.81 +/- 0.07, 0.035 +/- 0.003), while decreased 57.4% and 52.5% respectively in the HIF-1alpha siRNA group (0.46 +/- 0.06, 0.182 +/- 0.008) as compared with control model group (F = 139.804, 2686.001; P < 0.01). The expression of VEGF mRNA and protein in retinas were increased significantly in control model group (1.53 +/- 0.07, 0.340 +/- 0.004) and vector group (1.59 +/- 0.06, 0.337 +/- 0.009) as compared with normal group (0.27 +/- 0.08, 0.051 +/- 0.008), while decreased significantly in gene therapy group especially co-transfection group (decreased 85.6% and 80.9% respectively) as compared with control model group (F = 421.423, 2513.583; P < 0.01).
HIF-1alpha siRNA and VEGF165 siRNA can inhibit retinal neovascularization in the mouse effectively. Co-transfection of these two siRNAs shows the greatest inhibitory effect.
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology 11/2008; 44(10):921-8.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether vector-based HIF-1alpha -targeted shRNA expression system (pSUPER(siHIF-1alpha)) can inhibit HIF-1alpha and VEGF expression in vitro and suppress retinal neovascularization in the murine model of oxygen-induced retinopathy.
pSUPER(siHIF-1alpha) from which siRNA targeting HIF-1alpha could be generated was constructed and transfected to human umbilical vein endothelial cell lines (HUVECs). Then the expression levels of HIF-1alpha and VEGF in the cultured cells were measured by RT-PCR, immunoblot, and ELISA assays. Subsequently, pSUPER(siHIF-1alpha)was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated by angiography using fluorescein-labeled dextran and quantitated histologically. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of pSUPER(siHIF-1alpha)could affect the expression levels of HIF-1alpha and VEGF in murine retinas.
HIF-1alpha and hypoxia-induced vascular endothelial growth factor (VEGF) increase in cultured cells were greatly abolished by pSUPER(siHIF-1alpha). Meanwhile, retinal neovascularization in the eye with pSUPER(siHIF-1alpha)injection was significantly reduced compared with that of the contralateral control eye. Histological analysis indicates that neovascular nuclei protruding into the vitreous cavity were decreased by nearly 65%. Furthermore, HIF-1alpha and VEGF expression levels were down-regulated in the murine retinas treated with pSUPER(siHIF-1alpha).
RNAi targeting HIF-1alpha could inhibit the retinal neovascularization by approximately 65% through down-regulating the expression of HIF-1alpha and VEGF in the murine retinas, which may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.
Current eye research 11/2008; 33(10):892-902. DOI:10.1080/02713680802416670 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinal angiogenesis could be attributed to complex interactions among multiple angiogenic stimulators. Under pathological condition, angiogenic molecules, such as metalloproteinase, erythropoietin, integrin, promote retinal neovascularization through different pathway. Therapy target to these molecules involving in retinal angiogenesis could suppress retinal neovascularization effectively. This article reviews recent progress in studies on the molecules participating in retinal angiogenesis and therapy targeting it.
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology 05/2008; 44(4):381-4.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether vector-based vascular endothelial growth factor 165 (VEGF)(165) targeted siRNA expression system (pSilencer(siVEGF)) could inhibit VEGF(165) expression in vitro and suppresses retinal neovascularization in the murine model of oxygen-induced retinopathy.
pSilencer(siVEGF), from which siRNA targeting VEGF(165) could be generated, was constructed and transfected to human umbilical vein endothelial cells. Then the level of VEGF isoforms in cultured cells was measured by RT-PCR and ELISA. Intravitreal injection of pSilencer(siVEGF) was performed in mice with ischemic retinopathy. Retinal neovascularization was evaluated by angiography using fluorescein-labeled dextran and quantitated histologically. The levels of VEGF(164), which is equivalent to human VEGF(165) in murine retinas were determined by RT-PCR and western immunoblotting.
Expression of VEGF(165) in cultured cells was greatly curtailed by pSilencer(siVEGF) under both normoxia and hypoxia conditions. However, the other isoforms, VEGF(189) and VEGF(121), were expressed to a similar degree regardless of whether pSilencer(siVEGF) was administered. Based on angiography and histological analysis, retinal neovascularization in the eyes treated with pSilencer(siVEGF) were significantly reduced compared to the control eyes. Furthermore, the VEGF(164) levels in the murine retinas were suppressed by pSilencer(siVEGF).
Retinal neovascularization in the murine model was significantly attenuated by pSilencer(siVEGF) through decreasing VEGF(164) levels in the retinas. pSilencer(siVEGF) seems to be a potential therapeutic tool for ischemic-induced retinal diseases.
[Show abstract][Hide abstract] ABSTRACT: To evaluate suppression efficiency of hypoxia inducible factor-1 alpha (HIF-1 alpha) specific siRNA derived from recombinant plasmid (pSUPER(H1-siHIF-1 alpha)) on both HIF-1 alpha mRNA and protein expression and concomitant downregulation of expression of downstream angiogenic factor vascular endothelial growth factor (VEGF).
Stable pSUPER(H1-siHIF-1 alpha) expression cell lines were constructed by transient transfection of pSUPER(H1-siHIF-1 alpha) eukaryotic expression vector, followed by puromycin selection. Stable expression pSUPER(H1-siHIF-1 alpha) cell line with highest HIF-1 alpha inhibition efficiency determined by reverse transcription-polymerase chain reaction (RT-PCR) was cultured under normoxia (20% O(2)) and hypoxia conditions (1% O(2)) together with control cells. RT-PCR, western blot and ELISA were used to measure inhibition ability of pSUPER(H1-siHIF-1 alpha) on HIF-1 alpha and VEGF expression.
Compared to the control cells, both mRNA and protein level of HIF-1 alpha and VEGF were dramatically decreased by pSUPER(H1-siHIF-1 alpha) under hypoxia conditions. Under sufficient oxygen supply situation, HIF-1 alpha mRNA level was downregulated by pSUPER(H1-siHIF-1 alpha), but pSUPER(H1-siHIF-1 alpha) did not cause suppression of VEGF expression.
pSUPER(H1-siHIF-1 alpha) could decrease HIF-1 alpha expression under both normoxia and hypoxia conditions. VEGF expression was downregulated under hypoxia conditions only. Consequently, pSUPER(H1-siHIF-1 alpha) might be a powerful tool for the inhibition of retinal neovascularization.
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology 12/2007; 43(11):1028-35.