Objective To detect expression of NF-κB in the inner retina and in vestigate the inhibitoryeffect of pyrrolidine dithiocarbamate on retinal neovascularization in rats. Methods The rat models with retinopathy were set up un der the hypoxia condition, and fluorescein fundus angiography (FFA) was used to observe the retinal neovascularization. The expressions of NF-κB in the inner retina in rats with and without neovascularization were detected by immunohisto chemical method. PDTC was intraperitoneally injected in rats with neovascularization to observe the expression of NF-κB in the inner retina and the effect on retinal neovascularization. Results Hypoxia induced NF-κB activation in the retinal glial cells and endothelial cells. But immuno-staining intensity for NF-κB and adhesion molecules were reduced by PDTC intraperitoneal injection. Retin al angiogenesis in rats were suppressed effectively (P＜0.05). Conclusions NF-κB activation correlates with retinal neovascularization closely. PDTC may inhibit the NF-κB activation and prove beneficial in the treatment of ischemic neovascularization. (Chin J Ocul Fundus Dis，2003，19：201-268)
ObjectiveTo observe the effects of angiostatin on the activity of extra-cellular signal-regulated protein kinase (ERK) of retinal microvascular endothelial cells of mice.MethodsAngiostatin was separated and purified by l-lysine sepharose 4B from human plasma. The primary retinal microvascular endothelial cells were divided into 4 groups: the control group, vascular endothelial growth factor (VEGF) 10 ng/ml group, angiostatin 130 μg/ml group, and VEGF (10 ng/ml) + angiostatin (130 μg/ml) group. The expression of ERK1 was assayed by Westernblotting method 1, 2, 5, 10, 15, and 30 minutes after the treatment of angiostatin.ResultsCompared with the control group, the expression of ERK-1 reduced 1 minute after treatment, reduced markedly after 10 minutes. After 30 minutes, no differences of the expression of ERK were seen between the control group and angiostatin group. The activation of ERK-1 of retinal microvascular endothelial cells occurred after stimulated by VEGF, and at the pitch at the peak after 5 minutes. The level of ERK in VEGF group increased 210% than that in the control (P＜0.05). After 30 minutes, no significant difference of the level of ERK between VEGF and the control group. And because of angiostatin, the expression of ERK-1 decreased 11.9%(1 minute)、17.9%(2 minutes)、38.7%(5 minutes)、49.3%(10 minutes) （P＜0.05）、27.9%(15 minutes)、1.12%(30 minutes) respectively.ConclusionsAngiostatin can effectively block the signal path through which VEGF transmits from outside of the cell to cellular nuclei. (Chin J Ocul Fundus Dis, 2005,21:170-173)
Neovascularization is a characteristic manifestation of a variety of retinal diseases. Vascular endothelial growth factor (VEGF) mainly regulates the proliferation and migration of endothelial cells. VEGF receptor 2 (VEGFR2) is the main receptor to mediate this effect. The activation of downstream signals requires the binding of VEGF and VEGFR2, followed by receptor dimerization and autophosphorylation. Blocking this process and inhibiting neovascularization is very attractive treatment ideas. Monoclonal antibodies and fusion protein drugs currently used in ophthalmology can bind free VEGF. In addition, there are also macromolecular antibodies binding VEGFR2 and small molecule tyrosine kinase inhibitors, which is expected to further expand into the field of ophthalmology. Although anti-VEGFR2 therapy is a revolutionary method to inhibit neovascularization, there are no sufficient clinical evidences at present. In-depth understanding of the application status and progress of anti-VEGFR2 in the treatment of retinal neovascular diseases has important clinical significance.
ObjectiveTo observe the inhibitory effect of lentivirus mediated small interference RNA (siRNA) targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) on retinal neovascularization (RNV) in mice. MethodsCREB1 siRNA construct was created, screened and packaged to produce CREB1 RNAi-lentivirus. One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into 4 groups including normal group, oxygen induced retinopathy (OIR) group, empty vector group and CREB1 therapy group with 35 mice in each group. Mice in the normal group were kept in normal room air, while in the other three groups retinal neovascularization was induced by hypoxia on postnatal day 7 (P7). The mice in the OIR group were not treated. The mice in the vector group received intravitreal injection of lentivirus-green fluorescent protein (lenti-GFP, 1 μl), and the CREB1 therapy group received CREB1 RNAi-lentivirus (1 μl) on P5.The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography. The areas of RNV and non-perfusion region were calculated. The expression of CREB1, phosphorylated-CREB1 (P-CREB1) and vascular endothelial growth factor (VEGF)-A levels, Akt and phosphoinositide 3-kinases (PI3K) in retinas were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. ResultsThe number of vascular cell nuclei breaking through the ILM of the OIR group and the empty vector group increased significantly compared with the normal group (P<0.05), while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group(P<0.05). The area of RNV and non-perfusion region of the OIR group and the empty vector group increased significantly compared with the normal group, while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group. The difference of area of RNV and non-perfusion region among 4 groups were significant (F=67.220, 110.090; P<0.05). The mRNA expression of CREB1 and protein expression of P-CREB1, the mRNA and protein expression of VEGF-A, Akt, PI3K in the retina were increased significantly in the OIR group and the empty vector group as compared with the normal group, while decreased significantly in the CREB1 therapy group as compared with the OIR group and the empty vector group. The difference of mRNA expression of CREB1, VEGF-A, Akt, PI3K in the retina among 4 groups were significant (F=6.087, 5.464, 6.191, 8.627; P<0.05). The protein expression of P-CREB1, VEGF-A, Akt, PI3K in the retina among 4 groups were significant (F=162.944, 13.861, 19.710, 22.827; P<0.05). ConclusionRNV in the mice is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1 down-regulation.
ObjectiveTo study the inhibitory effects of pigment epithelium derived factor (PEDF) on oxygen-induced retinal neovascularization in mice, and to investigate the possible involvement of interleukin-1β (IL-1β) in the neovascular-inhibitory function of PEDF. Methods A total of 140 postnatal day (P)7 C57BL/6 mice were randomly divided into normal control group, oxygen-induced retinopathy (OIR) model group, PEDF treatment group and PBS treatment control group. All mice except normal control group with their mothers were exposed to (75±2)% oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model. Mice in normal control group were kept in room air only. At P12 and P14, respectively, mice in PEDF treatment group received intravitreous injections of 1 μl PEDF (2 μg/μl), while PBS treatment control group received the same volume of PBS (10 mmol/L, pH7.4).All mice were euthanized at P17 and eyes were isolated. The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy. Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR). ResultsChanges of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina, the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group (t=15.02, P < 0.01), and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group (t=5.96, P < 0.01). Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer (OPL) to ganglion cell layer (GCL) of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group. The specific expression levels of IL-1β protein in retinas of OIR mice by Western-blot analysis were higher than those of normal control group(t=3.35, P < 0.05), While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.764, P > 0.05). Similarly, expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group(t=4.43, P < 0.01). After treated with PEDF, expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.15, P > 0.05). ConclusionsPEDF can inhibit oxygen-induced retinal neovascularization. The mechanism may be related to that PEDF can downregulate the expression of IL-1β in retina.
Objective To investigate the method and effect of krypton laser photocoagulation for neovascularization in retinal vein occlusion . Methods Tweenty eight eyes of 27 patients with retinal vein occlusion with neovascularization were photocoagulated by krypton green and red laser.The fundus changes were observed by fundus fluorescein angiography after photocoagulation. Results The neov ascularization disappeared completely in 20 eyes and became smaller in 6 eyes,re mained no change in 2 eyes,and the visual acuity improved in 17 eyes (60.7%) after 6 monthes to 2.5 years of follow-up. Conclusion Krypton laser photocoagulation is obviously effective on regression of n eovascularization and prevenion of vitreous hemorrhage in retinal vein occlusion . (Chin J Ocul Fundus Dis, 2001,17:12-14)
Objective To investigate the role of ephrin A genes in the development of oxygen induced retinalneovascularization (OIR) in mice.Methods The OIR model was established by oxygen induction in new born C57BL/6J mice.Reversed transcript polymerase chain reaction (RT-PCR) was used to measure the expression levels of ephrin A1-A5 in retinas of mice in experimental and normal control group.Results All of the ephrin A family genes expressed in normal retinas. Ephrin A1 mRNA was significantly higher in OIR group(t=3.19,P=0.019); ephrin A2 mRNA was higher in the 15-day-old OIR retinas(t=3.71,P=0.033）; ephrin A3-A5 mRNA decreased or disappeared in 12 and 13-day-old RNV mice, and increased in 15-day-old OIR mice. Conclusion Ephrin A genes are involved in the development of retina and OIR.
Objective lt;brgt;To investigate the morphological features of choroidal neovascularization (CNV) in central exudative chorioretinopathy (CEC) using optical coherence tomography（OCT）. lt;brgt; lt;brgt;Methods lt;brgt;OCT and fundus fluorescein angiography (FFA) were performed in 41 cases (43 eyes) of CEC,and the course of CEC disease was from 1 week to 10 months. Twenty-seven of 43 eyes were also examined by indocyanine green angiography (ICGA). lt;brgt; lt;brgt;Results lt;brgt;OCT images revealed 5 kinds of morphological features of CEC: well-defined CNV（41.86 %）,poorly-defined CNV(30.23 %),hemorrhagic pigment epithelium detachment （PED）(16.28 %), CNV companied with serous (6.98 %) or hemorrhagic neurosensory retina detachment (4.65 %). CNV mainly showed well-defined and poorly-defined CNV (72.09 %).In those eyes that could clear define the CNV boundary,there were 12 eyes on FFA examination and 20 eyes on ICGA examination which defined the boundary from retinal horizontal plane, while there were 23 eyes on OCT examination which defined the boundary from retinal vertical section. Classic CNV on FFA consistently presented with well-defined boundaries on OCT, whereas non-classic CNV had a variable cross-sectional appearance. lt;brgt; lt;brgt;Conclusions lt;brgt;The OCT morphological features of CNV in CEC is mainly well-defined CNV and poorly-defined CNV; OCT examination can precisely observe the retinal and choriocapillaries pathological anatomy of CEC from retinal vertical section, in making the CEC diagnosis as an important complementary examination of FFA and ICGA which observe the focus from retinal horizontal plane. lt;brgt; lt;brgt;(Chin J Ocul Fundus Dis, 2002, 18: 121-124)
Objective To invesitgate the influence of recombinant adenovirus vector of human pigment epithelium-derived factor（AV-hPEDF）on retinal new vessels mediated by recombinant adenovirus vector. Methods Twenty 7-days-old Sprague-Dawley (SD) rat were divided into two groups randomly after the establishment of retinal neovascularization model. At postnatal 14 day, they were accepted intravitreal injection with blankadenovirus-vector (AV-Blank group) and adenovirus-vector PEDF(AV-PEDF group) respectively. The retinal vascular endothelial cells were counted, the PEDF mRNA and protein expression in retina and vitreous were determined by reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistochemistry. Results After injection with medicine, the number of RNV was decreased obviously in AV-PEDF group（t＝42.009，Plt;0.001）;the protein expression of retinal PEDF was increased obviously in AV-PEDF group（t=36.638，Plt;0.001）; the PEDF mRNA expression in vitreous was also increased obviously in AV-PEDF group （t=9.128，Plt;0.001）. Conclusion Recombinant Adenovirus vector mediated PEDF can raise the PEDF expression in the retinal and vitreous neovascularized tissues in rat, which suggested that the expression of PEDF may be related to inhibition and reduction of RNV.
Objective To investigate the therapeutic efficacy of transpupillary thermal therapy (TTT) for age-related macular degeneration (AMD) accompanied with subfoveal choroidal neovascularization (CNV). Methods Fifty-one eyes of 47 patients whose illness had been diagnosed as AMD by fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) were treated with diode 810 laser. There are 42 eyes of 39 patients had occult CNV and 9 eyes of 8 patients had classic CNV, and the average visual acuity in their fist diagnosis was 0.12. According to the focus size, the diameters of beam spot varied from 0.8, 1.2, 2.0, and 3.0 mm; and the power was 120, 160, 260 and 360mW correspondingly, with the duration of 60 seconds. The follow-up examination was performed once a month after the treatment, and repetitious treatment would be taken once to thrice if necessary. The follow-up period was 3~33 months with the mean of 10 months. Visual acuity, haemorrhage in ocular fundus, absorption of exudation, and the closure of CNV were examined in the follow-up examination. Results No immediate decrement of visual acuity or any other discomforts were found in all of the treated eyes soon after the treatment. The average visual acuity of 51 eyes was 0.16 in the last diagnosis, which remained no change in 68.62%; increased in 23.53% and decreased in 7.84% compared with that in the first diagnosis. The results of FFA and ICCG demonstrated that at the 3rd months after the treatment, the closure rate was 42.86% in occult CNV and 22.22% in classic CNV; and at the 6th month, the closure rate was 73.81% in occult CNV and 66.67% in classic CNV. The results of ophthalmoscopy showed that at the 3rd month after the treatment, partial or complete absorption of hemorrhage and/or exudates with various thickness of organized scarring tissue was found in 42 eyes with occult CNV; decrement of hemorrhage and exudates was observed in 7 out of 9 eyes with classic CNV; and new hemorrhage occurred in 1 eye. At the 6th month, in 27 eyes with occult CNV, new hemorrhage occurred in 3 including 2 eyes with occult CNV, new hemorrhage occurred in 3 including 2 eyes with faster absorption and remaining unchanged for 12 months; in 5 eyes with classic C NV, new hemorrhage occurred in 2, which was absorbed after treated again and remained stable in the 16-month followed-up. In 19 eyes with occult CNV which had been followed up for more than 6 months, hemorrhage disappeared in 5 and new hemorrhage occurred in 5. In the followed-up over 6 months, new hemorrhage occurred in 8 eyes with the recurrent rate of 15.6%. Conclusion TTT is effective for AMD with either classic or occult CNV. In the long-term followed-up, CNV recurs in 15.6% of the treated eyes which may be improved after the further treatment. (Chin J Ocul Fundus Dis，2004，20：280-284)