Copper-Catalyzed Aza-Benzyl Transfer Michael Addition via C-C Bond Cleavage.

A non-noble Cu-catalyzed transfer aza-benzyl Michael addition via the C-C bond cleavage of aza-benzyl alcohols has been disclosed. The unstrained C(sp3)-C(sp3) bond of an alcohol was selectively cleaved. This aza-benzyl transfer strategy provides a selective and environmentally benign approach for the C-alkylation of α,ß-unsaturated carbonyl compounds that employs readily available alcohols as carbon nucleophiles and is characterized by a wide range of substrates and good to excellent yields.

Silver-Catalyzed 1,3-Aza-Benzyl Migration of Allyl Alcohol.

Carbon migration of alkenyl alcohols has been recognized as an increasingly viable methodology in organic synthesis. Herein, we disclose a silver-catalyzed 1,3-aza-benzyl migration of allyl alcohols by utilizing chelation-assisted selective cleavage of an unstrained C(sp3)-C(sp3) bond. This approach provides an available, efficient, high atom-economic, and environmentally benign procedure, leading to alkylation products with broad substrate scopes and excellent yields. The migration proceeds via a one-pot, two-step process involving a free-state alkyl metal species.

Morphological Characteristics Predict Postoperative Outcomes After Vitrectomy in Myopic Traction Maculopathy Patients.

BACKGROUND AND OBJECTIVES: To provide the surgical indication for patients with myopic traction maculopathy (MTM) by investigating the postoperative outcomes after vitrectomy among different types of morphological characteristic groups. PATIENTS AND METHODS: This was a retrospective cohort study that included patients (37 eyes) diagnosed with MTM at a single institution. All 37 eyes from 37 patients with MTMs were classified into three groups: foveal retinoschisis (FS), lamellar macular hole (LMH), and foveal retinal detachment (FRD). The ratios of anatomic recovery, central retinal thickness (CRT), and best-corrected visual acuity (BCVA) were statistically analyzed among the three groups preoperatively and at 1, 3, 6, and 12 months after vitrectomy. RESULTS: Anatomical recovery could be found in all patients of the FS group at 6 months postoperatively and in the LMH group at 12 months postoperatively. Only 83.33% patients in the FRD group showed anatomic recovery until 12 months. The time taken for CRT to reduce to 200 µm was gradually increased between the FS, LMH, and FRD groups. Postoperative BCVA was better in the FS group than the LMH and FRD groups (P < .05), but the LMH and FDR groups had no difference (P ≥ .05) at any point. The visual acuity was significantly improved in the FS group (P < .01) and FRD group (P = .018), but not in the LMH group (P = .196) at 12 months postoperatively. CONCLUSIONS: The FS group achieved anatomical recovery in the shortest time and had the best postoperative BCVA. FRD patients could get visual gain but need too much time for the anatomical recovery. LMH patients experienced anatomic success with surgery, but not in BCVA. Early surgery might be considered for eyes at FS prior to the occurrence of LMH or FRD. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:574-582.].

Diabetic retinopathy: Focus on NADPH oxidase and its potential as therapeutic target.

Diabetic retinopathy is a common complication of diabetes that affects the retina due to a sustained high blood sugar level. Recent studies have demonstrated that high glucose-driven oxidative stress plays an important role in the microvascular complications of retina in diabetes. Oxidative stress occurs due to the excess of reactive oxygen species, which causes oxidative damage to retina, leading to the leak of tiny blood vessels, or acts as signaling molecules to trigger neovascularization, resulting in new fragile vessels. NADPH oxidase (NOX) is a key enzymatic source of reactive oxygen species in the retina, and it is involved in the early as well as the advanced stage of diabetic retinopathy. To date, at least 7 NOX isoforms, including NOX1 to NOX5, dual oxidase1 and dual oxidase 2, have been identified. It has been shown that NOX isoforms exert different roles in the pathogenesis of diabetic retinopathy. Intervention of NOX by its inhibitors or modulators shows beneficial effect on improving the retinal functions in the models of diabetic retinopathy in vivo or in vitro. Thereby, NOX might be a potential target for the therapy of diabetic retinopathy. The present review focuses on the role of NOX, particularly the NOX isoforms, in promoting the development of diabetic retinopathy. In addition, NOX isoforms as potential targets for therapy of diabetic retinopathy are also discussed.

Reliability and validity of the Chinese version of the new fear of hypoglycemia scale: FH-15.

OBJECTIVE: This study aimed to evaluate the reliability and validity of the Chinese version of the Fear of Hypoglycemia scale with 15 items (FH-15). METHODS: After obtaining the original author's authorization, the English version of the FH-15 scale was translated, back translated, and culturally debugged to obtain the Chinese version of FH-15. A convenient sampling method was used to extract patients with type 2 diabetes from four tertiary hospitals in Tianjin. A total of 408 patients with type 2 diabetes were investigated in the hospital to test the reliability and validity of Chinese version FH-15 scale. RESULTS: The content validity index of the scale was 0.92, and the content validity index of each item was 0.8-1.0. The exploratory factor analysis extracted three common factors (fear, avoidance, and interference), which contained 15 items, and the cumulative variance contribution rate was 71.245%. The confirmatory factor analysis results showed that the model fit was better at 1.981 χ 2/df, GFI = 0.901, CGI = 0.962, TLI = 0.952, and RMSEA = 0.070. The cut-off value for the total hypoglycemia fear scale was 30.5. The Cronbach's α coefficient of the three dimensions of the scale was 0.918, the Cronbach's α coefficient of each dimension is 0.876-0.916, the test-retest reliability was 0.903, and the test-retest reliability of each factor was 0.733-0.930. CONCLUSION: The Chinese version of the FH-15 scale can be considered reliable and valid. The item expression is concise, clear, and easy to understand. It is suitable for clinical practice as an initial screening tool to identify and evaluate the severity of fear of hypoglycemia in patients with type 2 diabetes.

Effect of pyridone agent on blood-retinal barrier in diabetic mice.

AIM: To evaluate the therapeutic effect of fluorofenidone on disrupted blood-retinal barrier in the diabetic mice and uncover its underlying mechanism. METHODS: db/db mice were randomly chosen for treatment with daily doses of fluorofenidone or placebo at 5-week-old, treatment continued until mice reach 24-week-old. Then, expression of transcriptiona factor insulin gene enhancer binding protein-1 (Islet-1) and vascular endothelial growth factor (VEGF) in murine retinas were evaluated. Retinal vascular permeability was assessed by examining the level of albumin in db/db murine retinas. Furthermore, the retinal vessel tight junction was estimated by checking the level of occludin in the murine retinal tissues. RESULTS: After occurrence of diabetic retinopthy in db/db mice, expressions of transcritpional factor Islet-1 was found to be upregulated in db/db murine retinas compared with non-diabetic controls. Similar to expression pattern of Islet-1, VEGF were also demonstrated to be increased in retinas of db/db mice, which was accompanied by increased retinal vascular leakage and decreased tight junction protein level. Systemetic administration of fluorofenidone repaired broken retinal vascular tight junction by restoring occludin expression in db/db retinal tissue. Consequently, retinal vascular premeability were indicated to be reduced by examining the transudative albumin level in diabetic retinal tissues. Both Islet-1 and VEGF expression were inhibited in the retinas of db/db mice after treatment with fluorofenidone. CONCLUSION: Fluorofenidone significantly protectes retinal tight junction and reduces retinal vascular leakage. The phenomenon can be partially attributed to reducing overexpression of Islet-1 and VEGF in diabetic retinal tissues.

Treatment of myopic foveoschisis via macular buckling and vitrectomy.

The aim of the present study was to evaluate the efficacy and safety of the treatment of myopic foveoschisis patients using the macular buckling with L-shaped titanium plate and silicon sponge combined with vitrectomy. The data of the patients who underwent macular buckling combined with vitrectomy was collected. The study recorded the following parameters: best corrected visual acuity (BCVA), axial length, intraocular pressure, central macular thickness, and the position of the titanium plate. Following the surgery, the BCVA of the included patients were improved, whereas the axial lengths were reduced followed by resolution of the foveoschisis compared with that noted prior to the operations. All patients had orbital CT examination and the results indicated that the titanium plates were appropriately placed and were not in contact with the optic nerve. Therefore, it is effective to treat myopic foveaschisis by macular buckling using the L-shaped titanium plate and silicon sponge in the presence of vitrectomy.

Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis.

OBJECTIVE: To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. METHODS: siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor-reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. RESULTS: The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. CONCLUSIONS: Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF.

Expression of netrin-1 receptors in retina of oxygen-induced retinopathy in mice.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: UNC5B may promote retinal neovascularization in OIR mice.

HIF-1α siRNA reduces retinal neovascularization in a mouse model of retinopathy of prematurity.

OBJECTIVE: To explore the effect of HIF-1α specific siRNA expression vector pSUPERH1-siHIF-1α on retinal neovascularization in a mouse model of retinopathy of prematurity (ROP). METHODS: Forty-eight newborn C57BL/6J mice were randomly divided into the control and experimental groups (n=24 apiece) to create the model of ROP following the methods described by Smith et al. Twelve days after birth, the experimental group received intravitreal injection with pSUPERH1-siHIF-1α; meanwhile, mice in the control group were injected with empty vectors. The expressions of HIF-1α and vascular endothelia growth factor (VEGF) in the retina were examined by Western blotting in both groups. The differences in the neovascular endothelial cell count were compared based on the FITC-Dextran fluorescence stretched preparation/sections. RESULTS: Compared with the control group, the expressions of HIF-1α and VEGF significantly decreased in the experimental group (P<0.01). Meanwhile, the number of retinal neovascular endothelial nuclei that had protruded the internal limiting membrane was significantly lower in the experimental group than in control group (P<0.01). CONCLUSIONS: RNA interference targeting HIF-1α can effectively inhibit the retinal neovascularization of ROP.

Comparison of two methods used to culture and purify rat retinal Müller cells.

AIM: To study two methods for culturing and purifying Sprague-Dawley (SD) rat retinal Müller cells and determine which one is better. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Neuro-protection of retinal stem cells transplantation combined with copolymer-1 immunization in a rat model of glaucoma.

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) was 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 the 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 protect 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.

Caveolin-1 expression regulates blood-retinal barrier permeability and retinal neovascularization in oxygen-induced retinopathy.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.

BDNF regulates GLAST and glutamine synthetase in mouse retinal Müller cells.

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.

[HIF-1α siRNA reduces retinal neovascularization in a mouse model of retinopathy of prematurity].

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: The development of retinal neovascularization of ROP can be markedly inhibited by RNA interference targeting HIF-1α.

Netrin-1 overexpression in oxygen-induced retinopathy correlates with breakdown of the blood-retina barrier and retinal neovascularization.

PURPOSES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The netrin-1 level increases in murine retina under hypoxia and may be key in inducing retinal neovascularization.

Suppression of retinal neovascularization by small-interference RNA targeting erythropoietin.

OBJECTIVE: To observe the effect of inhibition of retinal neovascularization by small-interference RNA (siRNA) targeting erythropoietin (EPO). METHOD: 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. RESULTS: 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. CONCLUSION: 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.

Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF-1alpha and VEGF.

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.

Inhibition of retinal neovascularization by siRNA targeting VEGF(165).

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Advancement of targeting therapy for retinal neovascularization].

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.