ObjectiveTo observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).MethodsA three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.ResultsThe LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).ConclusionPSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.
ObjectiveTo measure and analyze the oxygen saturation and retinal blood vessel diameter in the eyes of patients with convalescence Vogt-Koyanagi-Harada (VKH) syndrome.
MethodsIn this cross-sectional study, 28 eyes of 14 patients with convalescence VKH syndrome (VKH group) and 20 eyes of 10 healthy subjects (control group) were enrolled. The oxygen saturation and retinal blood vessel diameter were detected by spectrophotometric oximetry unit. Retinal images were collected using filters with wavelengths of 570 nm and 600 nm in the darkroom by the same technologist and then the fused image was obtained. The oxygen saturation of retinal vessels was marked in different colors. The measurement was repeated 2-3 times for each patient, then take an average. A top-quality image in each eye was selected to detect the oxygen saturation and diameter of retinal vessel which located in 1.5-3.0 disc diameter from the optic disc. Image analysis and data acquisition were completed by another technologist.
ResultsRetinal venous oxygen saturation was (54.34±8.05)% in the VKH group and (60.07±7.91)% in the control group. The former was lower than the latter, the difference was significant (t=2.443, P=0.017). The mean diameter of retinal arteries was (102.8±18.1) μm in the VKH group and (112.9±19.8) μm in the control group. The former was smaller than the latter, the difference was significant (t=2.406, P=0.018). There was no significant difference of the mean diameter of retinal veins, oxygen saturation of retinal arteries and the arterial-venous difference between two groups (t=-0.330, 0.804, -0.631; P=0.743, 0.403, 0.536).
ConclusionsRetinal venous oxygen saturation and the mean diameter of retinal arteries are significantly decreased in patients with VKH syndrome. There is no significant difference of diameter of retinal veins, oxygen saturation of retinal arteries and the arterial-venous difference between VKH syndrome patients and healthy subjects.
ObjectiveTo observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.MethodshRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.ResultsDifferent concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant (t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant (t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1285-3p, hsa-miR-30d-3p, hsa-miR-3170, hsa-miR-7976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions.ConclusionFA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.
ObjectiveTo evaluate the protective effect of estrogen on survival of retinal ganglion cells (RGCs) after transient retinal ischemia-reperfusion (RIR) in rats.MethodsRIR was induced in 60 ovariectomized adult rats (OVX) by increasing intraocular pressure via an intracameral catheter. All of the rats were divided into two groups randomly: in experimental group, the rats underwent a subcutaneous injection with 17β-estrodiol（100 μg/kg） 2 hours before retinal ischemia; and in the control group, saline water was injected correspondingly. The number of RGCs and the thickness of the inner retinal layers were mesured by HE staining method before and 12, 24, 48, and 72 hours after reperfusion. TdT-mediated biotin-dUTP nick end labelling (TUNEL) staining technique was used to examine the apoptosis of RGCs.ResultsTwenty-four and 48 hours after reperfusion, the number of apoptotic cells in experimental group was obvious lower than that in the control group(Plt;0.05), and the number of RGCs in experimental group was higher than that in the control group(Plt;0.05).ConclusionEstrogen can protect retinal neurons from transient RIR in ovariectomized rats.(Chin J Ocul Fundus Dis, 2005,21:177-179)
ObjectiveTo investigate the relationship between retinal vessel diameters and cerebral infarction of carotid artery stenosis patients.
MethodsEighty-seven patients (174 eyes) with carotid stenosis were included in this study. There were 49 males and 38 females, with an average age of (65.25±7.85) years. Thirty-four patients were suffered from cerebral infarction (cerebral infarction group), and the other 53 patients had no cerebral infarction (control group). There was no significant difference in age (t=1.916), male rate (χ2=0.142) and carotid stenosis extent (χ2=0.785) between the two groups (P=0.059, 0.706, 0.675). All patients underwent color fundus photography after mydriasis. Retinal vascular caliber measurements were performed using IVAN software. The main parameters were central retinal artery diameter (central retinal artery equivalent, CRAE), the diameter of the central retinal vein (central retinal vein equivalent, CRVE) and the retinal arteriole to venular ratio (AVR). The relationship between retinal vessel diameter and cerebral vascular disease were analyzed with logistic regression analysis.
ResultsIn cerebral infarction group, CRVE, CRAE and AVR ratios were (132.90±20.67) μm, (243.47±43.92) μm and 0.56±0.10, while the control group was (145.26±21.59) μm, (224.99±32.35) μm and 0.68±0.13 respectively. There were significant differences between the two groups (t=-2.648, 2.257, -4.631; P < 0.05). After correction for risk factors, such as age, smoking history, CRAE reduction and CRVE increases were significantly correlated with cerebral infarction.
ConclusionCRAE reduction and CRVE increases are risk factors of cerebral infarction in patients with carotid stenosis, and it is useful in the prediction.
ObjectiveTo observe the stoichiometry of vascular endothelial growth factor receptor 2 (VEGFR2) on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.MethodsRhesus monkey retinal vascular endothelial cells (RF/6A) were divided into blank control group (normal culture) and plasmid transfection group [transfected with VEGFR2-green fluorescent protein (GFP) recombinant plasmid]. The expression of GFP in the plasmid transfected group was observed by confocal microscope, and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot. The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging. The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.ResultsGFP green fluorescence was observed in the transfected cells 12 hours after transfection. qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group (t=11.240, 12.330; P＜0.001, 0.001). Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group (t=8.346, P＜0.01). The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal, in which monomer and dimer were 86.0% and 14.0% respectively. By counting the steps of GFP fluorescence bleaching, the proportions of receptor monomer, dimer, trimer, and tetramer were 81.4%, 12.9%, 5.5%, and 0.3% respectively.ConclusionIn the absence of ligands, VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane, and monomers are dominant.
The activities and distributions of succinate dehydrogenase(SDH),malic dehydrogenase(MDH),lactic dehydrogenase(LDH),acid phosphatase(ACP) and alkaline phosphatase(AKP) in retinal vessels were studied and observed with enzymatic histochemical techniques. The retinal vessels showed a b LDH activity, moderate SDH and MDH activity. The dehydrogenase activity described above was evenly and equally distributed in the microvasculature between arterioles and venules, and was the best in arteries. AKP showed predominant activity in the endothelial cells of capillaries and arterioles which were stained bly. No activity for ACP observed in the retinal vessels. The observations above indicate that the retinal vessels are metabolically active and have a great capacity for glycolysis.
（Chin J Ocul Fundus Dis,1992,8:6-9）
ObjectiveTo explore the effect of estrogen on the permeability of retinal blood vessel by ovariectomy.MethodsTwenty-two healthy rats were divided into experimental and control group randomly. Estrogen level of rats decreased due to ovariectomy in the experimental group while stabilized by sham-ovariectomy in the control group. The results were confirmed by vaginal epithelium smearing. Retinal vein occlusion was established by photodynamic method, and leakage of Evan's blue in retina was determined by spectrophotometer.ResultsMature value of vaginal epithelium decreased significantly in ovariectomy rats（t=21.008，P=0.000） while not significantly in sham-ovariectomy ones （t=0.319，P=0.756）; the mean leakage of Evans blue was (25.503 0±4.378 47) ng/mg in experimental group, and (17.830 0±4.265 69) ng/mg in the control group, and the difference between the two groups is significant（t=3.969 36，P=0.001）.ConclusionOvariectomy is an useful method to study the effect of estrogen on ocular diseases, and when estrogen level decreases, the permeability of retinal blood vessel increases.(Chin J Ocul Fundus Dis, 2005,21:174-176)
Objective To observe the changes of tortuosity and bifurcation angle of retinal arteries and veins in each quadrant of the posterior pole in eyes with high myopia.Methods The tortuosity and bifurcation angle of retinal vessels in each quadrant of the posterior pole in 32 patients (52 eyes) with high myopia and 22 healthy people (30 eyes) were observed and compared. The outcomes were analyzed by multivariate analysis of variance. Results The tortuosity of macular vessels and the artery from optic disc in eyes with high myopia was (1.29plusmn;1.10)times;10-4 and (5.39plusmn;1.93)times;10-5 respectively, and in the normal eyes was (4.15plusmn;2.38) times;10-4 and (9.75plusmn;4.99)times;10-5 respectively; there was significant difference between the two groups （t=1.99, 2.00;Plt;0.05）. The bifurcation angle of superior nasal and inferior nasal retinal arteries in eyes with high myopia was（66.17plusmn;14.04）deg; and (61.20plusmn;11.02) deg; respectively, and in the normal eyes was （77.66plusmn;14.12）deg; and (85.86plusmn;16.45) deg; respectively; there was significant difference between the two groups （F=0.77, 0.83; Plt;0.05）. The bifurcation angle of superior temporal and inferior temporal retinal veins in eyes with high myopia was（92.39plusmn;20.36）deg; and (83.56plusmn;23.50) deg; respectively, and in the normal eyes was （79.45plusmn;15.94）deg; and (70.59plusmn;17.27) deg;; there was significant difference between the two groups （F=2.34, 1.83; Plt;0.05）.Conclusions The vessel tortuosity of retinal arteries and the vessels extending from the optic disc to macula is smaller in eyes with high myopia, while the venous tortuosity has no change. The bifurcation angle of retinal arteries in the superior nasal and inferior nasal field was smaller in eyes with high myopia, while the venous tortuosity has no change. The bifurcation angle of retinal veins in the superior temporal and inferior temporal field was larger in eyes with high myopia.
Objective
It has been shown that pigment epitheliumderived factor (PEDF) is an effective anti-apoptosis agent on several kinds of cells of the central nervous system.This study aimed to evaluate the effect of PEDF on pressure induced retinal ischemia in a rat model.
Methods
Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 45 minutes via an intracameral catheter.Ten microlit ers (0.1 mu;g/mu;l) PEDF was injected into the vitreous of 4 eyes of each group im mediately after reperfusion and 4 additional eyes received only normal saline as vehicle controls.The animals were euthanized at 2 or 7 days after reperfusion.T he effect of PEDF on retinal degeneration was assessed by measuring the thicknes s of the inner retinal layers (MTIRL) and counting the retinal ganglion cells (R GC) on plastic embedded retinal sections.
Results
The MTIRL and the RGC counting in eyes treated with intravitreal PEDF were significantly higher than those in vehicle controls (118.1plusmn;5.0) mu;m vs(94.9plusmn;3.0) mu;m (Plt;0.05);(6.0plusmn;1.0) cells/100 mu;m vs (4.5 plusmn;0.5) cells/100 mu;m (Plt;0.05) 7 days after reperfusion,respectively．
Conclusion
Intravitreal administration of PEDF can ameliorate an ischemiareperfusion retinal injury and may be useful to prevent neuronal degeneration in the inner retina.
(Chin J Ocul Fundus Dis, 2001,17:138-140)
