Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
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)
The retinal vessel changes are the primary and major features of retinal vascular diseases. The retinal vessel is part of systemic vessels with its own characteristics to sustain normal retinal function. These basic characteristics are important to the correct understanding and proper treatment of retinal vascular diseases. Always keep in mind that the retinal vessels is one part of the systemic vascular system, thus retinal vascular diseases may have systemic etiology, and systemic drug administration may have a profound effects to the whole body. However retinal vascular system also has its own structural and functional characteristics, thus retinal vascular diseases are also different from the systemic diseases. Finally the main function of retinal vascular network is to maintain the neuro-retinal function, thus we should balance the vision protection and treatments against abnormal retinal blood vessels. Over-treatments may damage the retinal vision.
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.
OBJECTIVE :To investigate ocular vessel flow velocity in normal eyes by color Doppler imaging(CDI). METHODS: Ninety people (180 normal eyes)had flow velocity measured by CDI in three vessels,ophthalmic artery (OA),central retinal artery(CRA) and posterior ciliary artery (PCA),and the relations between velocity and affecting factors were analysed. RESULT: The diastolic and systolic velocity of OA,CRA and PCA were (31.7plusmn;10.9)cm/s, (7.2plusmn;2.6)cm/,s, (10.2plusmn;3.4)cm/s, (2.8plusmn;1.2)cm/s and (11.3plusmn;3.6)cm/s, (3.2plusmn;1.4)cm/s respectively. The velocity of OA had possitive correl-ativity with RBC,HCT and negative correlativity with age,while it revealed no relationship with sex, laterality of right or left eye,and normal IOP. CDI of ocular vessels in normal eyes is a triangle with three peaks and double sunkens in its frenquency spectum, revealed highly resistant,and both the pulsatility and resistive indexes were relatively high. The width of each frequency band basically was the same,and there was no obvious frequency window. CONCLUSION: The ocular application of CDI might lay the foundation of a comparatively comprehensive knowledge of the ocular hemodynamics. （Chin J Ocul Fundus Dis,1997,13: 99-101）
Objective To investigate the effect of hypoxia on the exp ression and function of integrin receptor αvβ3 of bovine retinal vascular endotheliocytes. Methods Bovine retinal vascular endotheliocy tes in the culture dishes coated by vitronectin was put into the normal and hypoxemic condition, respectively. Enzyme linked immunosorbent assay and cell adhesion analysis were used to detect the expression and function of integrin receptor αvβ3 in bovine retinal vascular endotheliocytes, respectively. Results Under the condition of hypoxia, the expression of αvβ3 increased gradually, and reached the peak at the 48th hour. The expression of αvβ3 at the 60th and 72nd hour in hypoxia group was higher than that in the normal group. Bovine retinal vascular endotheliocytes absorbed more Vn of extra-cellular matrixes (ECM) after cultured under hypoxemic condition for 24 hours.Conclusion Hypoxia may up-regulate the expression of αvβ3, which promote the adsorbability of endotheliocytes.(Chin J Ocul Fundus Dis，2004，20：360-363)
Objective To investigate the spatial and temporal regulation effect of VEGF on human fetal retinal vascularization and angiogenesis. Methods The posterior segmental retinas from 54 human fetuses of the 9th week to the 40th week were studied by immunohistodhemistry standing for the expressions of VEGF and PCNA. Results 1. The distribution of VEGF espression was spiking and the peaks were during the 9th-13th and around the 26th week. 2. PCNA immunoreactivity was localized in spindle cells and vascular endothelial cells. The expression level was fluctuated during the developmental process. The peaks were during the 9th-13th and around the 21st week. In these periods, the spindle cells kept proliferating and differentiating, and remodelled subsequently to form the inner side retinal vessels. From the 26th or 34th week, the PCNA immununoreactivity is fully expressed in the vascular endothelial cells of the inner and outer margin of inner nuclear layer(INL) and kept to full terms. 3. Significant positive correlation were shown between the content of VEGF in the retina and that of PCNA in spindle cells and vascular endothelial cells(r=0.736,p＜0.01). Conclusion VEGF was positively involved in modulating human fetal retinal vascularization and angiogenesis. (Chin J Ocul Fundus Dis,1999,15:12-15)
ObjectiveTo explore the effects of transthyretin (TTR) on biological behavior of retinal microvascular epithelial cell (RMVEC). MethodsRMVEC was cultured in medium with 0 μmol/L and 4 μmol/L TTR. The proliferation, migration and healing abilities (0, 24, 48 hours) of RMVEC with different concentrations of TTR were measured by methyl thiazol tetrazolium (MTT) assay, transwell assay and scarification test. ResultsMTT assay shows that RMVEC with the concentrations of 4 μmol/L TTR [absorbance (A) value=0.17±0.02] glows faster than with the concentrations of 0 μmol/L TTR (A value=0.40±0.03), the difference was statistically significant (t=15.47, P=0.000 1). The transwell assay shows RMVEC with the concentration of 4 μmol/L TTR [(140±7) cells] migrants faster than RMVEC with the concentration of 0 μmol/L TTR [(227±14) cells], the difference was statistically significant (t=5.44, P=0.000 6). The scarification test shows that the RMVEC with the concentration of 4 μmol/L TTR [(134.4±45.4) μm] heals faster than the RMVEC with the concentration of 0 μmol/L TTR [(330.0±23.1) μm], the difference was statistically significant (t=8.25, P<0.01). The cells in 48 hours and 4 μmol/L group were healed completely, but not healed in 0 μmol/L group. ConclusionTTR can promote the proliferation, migration and healing abilities of RMVEC.
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.
ObjectiveTo investigate the protection and the corresponding molecular mechanisms of polypyramidine tract binding protein-associated splicing factor (PSF) overexpression on human retinal microvascular endothelial cells (hRMECs) induced by advanced glycation end-products (AGEs).MethodsThe hRMECs were divided into the normal group, the vector group, PSF group, zinc protoporphyrin (ZnPP) group and PSF+ZnPP group for experiment. Cells in the normal group were cultured in a DMEM medium containing 10% fetal calf serum, penicillin/streptomycin, and placed in a closed constant temperature incubator at 37 °C, 95% air, and 5% CO2. Cells in the vector group were infected with empty lentivirus. The cells in the PSF group were infected with overexpressing PSF lentivirus. Cells in the ZnPP group were treated with ZnPP (10 mol/L) for 2 h. The PSF+ZnPP group cells were infected with overexpressing PSF lentivirus, and then pretreated with ZnPP (10 mol/L) for 2 h. With the last four groups of cells stimulated with AGEs, HE, Hoechst33258 staining and flow cytometry were used to observe the protective effect of high expression of PSF on cell damage and the antagonistic effect of ZnPP on PSF. Western blot was used to detect the protein expression of heme oxygenase-1 (HO-1), phosphorylated (p) extracellular regulatory protein kinase (ERK), and Nrf2 in the cells. U0126, a specific antagonist of ERK pathway, was introduced, and Western blot verified the reversal effect of U0126 on the expression of HO-1 induced by PSF protein.ResultsHE staining and Hoechst33258 staining showed that the number of nuclei of damaged cells of PSF group were significantly increased compared with control group, while decreased compared with PSF+ZnPP group (F=27.5, 38.7; P＜0.05). The results of flow cytometry showed that the ROS produced by cells in the PSF group was significantly increased compared to the normal group, and significantly decreased compared to the PSF+ZnPP group, the difference was statistically significant (F=126.4, P＜0.05). Western blot results showed that HO-1 expression of PSF group was significantly increased compared with control and the vector group (F=70.1, P＜0.05). AGEs inducement of 30, 60, 120 and 240 min could significantly improve pERK expression compared with 15 min (F=474.0, P＜0.05). The expression of HO-1 and Nrf2 proteins in the PSF+/U0126－ group was significantly more than those in the PSF－/U0126－ group, the expression of HO-1 and Nrf2 proteins in the PSF+/U0126+ group was significantly lower than that in the PSF+/U0126－ group, and the differences were statistically significant (F=30.2, 489.4; P＜0.05).ConclusionOver expression of PSF can promote the HO-1 expression by activating ERK pathway and promoting the Nrf2 to the nucleus, thus protect hRMECs against AGEs-induced oxidative damage.