ObjectiveTo observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress. MethodsThe third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay. ResultsStable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05). ConclusionsCR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.
Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes, and it is the main cause of vision loss in diabetic patients. Angiopoietin (Ang), a superfamily of secreted proteins, is a vascular growth factor that regulates the stability of vascular environment, participates in angiogenesis and repair, and lipid metabolism. It plays an important role in the development of DR and has become a new target for the treatment of diabetic retinopathy. With the in-depth study of Ang and the research and development of various drugs for Ang, it is expected to bring new ideas and strategies for the treatment of DR in the future.
The mineralocorticoid receptor (MR) belongs to the nuclear receptor superfamily and is expressed in the retina and choroid. MR antagonist (MRA) has a long history of application in non-ophthalmic clinical practice. Various cellular and animal models indicated that inappropriate activation of MR participated in pathological angiogenesis, oxidative stress, inflammation, disturbance of ion/water homeostasis and neurodegenerative changes, while the application of MRA can reduce or reverse these pathological processes. After using MRA in central serous chorioretinopathy (CSC) patients, improved visual function, less subretinal fluid and reduced sub-foveal choroidal thickness were observed. Single nucleotide polymorphisms in MR and plasma aldosterone levels were significantly different between chronic CSC patients and CSC patients with spontaneous remission. Novel formulation for sustained-release MRA and the mechanisms involving inflammation may become the new focus of MR study. This review summarizes the research status of MR and MRA in order to provide a reference for future basic research and clinical treatment.
Purpose To identify the expression of alternatively spliced mRNA isoforms of the NMDA-R1 in the visual cortex of strabismic cats. Methods Two pai rs of normal and strabismic cats were used．The amblyopic cats had been made monocularly esotropic (by tenotomy) at the age of weeks，resulting in behavioral am blyopia．Animals were sacrificed about 6 months by intraperitoneal administration of Nembutal．Cryostat sections of fresh，frozen central visual cortex of the ats were cut to 20 micron thickness．A series of digoxygenin-labelled oligonucle otide probes basing on the human gene sequence were used for ISH．Control probes included sense oligonucleotides and short segment probes which were adjacent to ，but did not，span the splice junctions．A computer-assisted systematic morphometric ounting procedure was used to enumerate hybridising cells． Results The number of positive cells expressing NMDA-R1 mRNA in t he strabismic amblyopic cats was decreased，notably in layer IV of visual cortex (P＜0．0001)．The pattern of isoform expression varied between normal and strabismic amblyopic cats with decreased numbers of 1-a，1- b and 1-1 isoforms and apparently increased expression of 1-3 P ＜0．0001)，whereas no significant difference was found for the 1-2 and 1-4 isoforms (P＞0．05)． Conclusion Transcriptional inhibition of NMDA-R1 mRNA and of specifie isoforms may underlie the change in receptor expression．Alternatively，preferentialloss of neurones bearing particular NMDA-R1 isoforms and compensation with a proportional increase in cells expressing other isoforms may occurr during the critical period of visual plasticity． (Chin J Ocul Fundus Dis，2000，16：71-138)
Objective To identify the expression of nerve growth factor (NGF) and its high affinity receptor (tyrosine kinase receptor A, TrkA) during bone induction by recombined human bone morphogenetic protein 2 (rhBMP-2) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) and to discuss the role of NGF on the bone induction of the BMP. Methods Thirty-six ICR mice were divided into the experimental groupand the control group at random. rhBMP-2 /collagen sponge and collagen sponge were implanted into the right thigh muscle pouches of the mice in the experimental group and the control group, respectively. The tissues in the implanted site of the two groups were removed on the 7th, 14th and 21st day after the implantation. Histological, immunohistochemical and RT-PCR analyses were performed to detect osteoinductive effects of rhBMP-2 and the expression of NGF and TrkA. Results Gross observation showed that a solid lump was found in theright thigh in the experimental group on the 7th day and became harder on the 14th and 21st day, which was not found in the control group. rhBMP-2/collagen sponge displayed a potent ability to induce bone formation, while immunostaining for NGF and TrkA was observed in the course of osteoinduction by rhBMP-2. On the 7th day in the experimental group, NGF positive immunostaining reached the peak in thestage of chondrogenesis and there were a large number of cells expressing NGF, including fibroblasts, chondroblasts, chondrocytes, and osteoblasts; then, therewas a decrease in the number of the positivecells and in the intensity of immunostaining on the 14th and 21st day. Staining of TrkA wassimilar to that of NGF. The expression level of the mRNA of NGF during the course of bone induction peaked 7 days after the implantation and then decreased. Conclusion rhBMP-2/collagen is a kind of satisfactory osteoinductive material, and many different kinds of cells induced by rhBMP-2 can express NGF and TrkA, which suggests that NGF may play an important role in the osteogenesis initiated by exogenous BMP through direct and indirect pathways.
Objective To investigate the insulin receptor expression and binding characteristics of H22 rat hepatic cancer cell membrane with 125Iinsulin and the possibility of using insulin as a carrier for the receptor mediated targeted therapy. MethodsInsulin was radiolabelled using ChT method, isolated and purified by polyacrylamide gel electrophoresis, the labelled 125Iinsulin was measured by specific activity selfreplacement and over dose receptor combination experiment. The rat H22 hepatocarcinoma cells, normal rat hepatic cells were made, continuing the value Kd and Bmax of 125Iinsulin binding with insulin receptor on rat H22 hepatocarcinoma and normal hepatic cells membrane were evaluated in vitro, the competed binding curve was pictured. The binding Kd and Bmax value of 125Iinsulin receptor were evaluated with t test and receptor binding curve was tested with Scatchard method. ResultsThe Bmax value of rat H22 hepatocarcinoma cell receptor ［(5.6±1.1) pmol/106 cell］ was higher than that of normal hepatic cell ［(3.2±0.8) pmol/106 cell］ significantly (P＜0.05). The Kd value of H22 cell ［high and lowaffinity vs (1.8±0.6) nmol/L and (32.0±10.7) nmol/L］ and normal hepatic cell ［high and low affinity vs (2.1±0.9) nmol/L and (37.0±12.3) nmol/L］ were not significant respectively. In this experiment, it was specific when 125Iinsulin combined with receptor on H22 hepatocarcinoma cell and rat normal hepatic cell membrane. Conclusion This experiment showed that the receptor expresses much more significantly on H22 hepatocarcinoma cell membrane than that on normal rat hepatic cell membrane, 125Iinsulin combining with receptor on H22 hepatocarcinoma cell and rat hepatic cell membrane is highly specific. We may use insulin as an anticancer carrier to mediate insulin combined with receptor on hepatocarcinoma cell membrane in the target treatment of hepatic cancer.
ObjectiveTo investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. MethodsA lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. ResultsImmunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P < 0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P < 0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P < 0.05).The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45;P < 0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P < 0.05). ConclusionsThe intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.
Objective To investigate the role of adenosine A2A receptor plays in retinal pathological neovascularization in mice. Methods A total of 202 mice were divided into room-air group (n=66) and oxygen induced retinopathy (OIR) group (n=136). Among room-air group, there were 18 A2A knock-out (KO) mice (KO subgroup) and 24 C57BL/6 mice as wide type (wide type subgroup). OIR group were divided into OIR control subgroup (n=48), A2A-OIR subgroup (n=24) and Caffeine-OIR subgroup (n=64). The retinal neovascularization of OIR group was induced by oxygen. The pathological neovascularization was determined by retinal sections. Fluorescent quantitative polymerase chain reaction (PCR) was used to measure the mRNA expression of A2A and vascular endothelial growth factor (VEGF). 0.1, 0.3, 1.0 g/L Caffeine was dissolve in drinking water of lactating females in Caffeine-OIR subgroup, non-perfusion areas of retina in mice at the age of 0 - 17, 0 - 7, 7- 17, 7-12, and 12- 17 days were analyzed in different dosage and when the dosage as 1.0 g/L. Results Compared with OIR control subgroup, the retinal non-perfusion areas and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in A2A- OIR subgroup were reduced significantly (t=7.694, 7.747;P＜0.001). Compared with wide type subgroup, the level of A2A and VEGF mRNA in OIR control subgroup increased significantly (t=4.036, 2.230;P＜0.05). Compared with OIR control subgroup, the level of VEGF mRNA in A2A- OIR subgroup decreased significantly (t=3.122,P＜0.01). Compared with OIR control subgroup, the retinal non-perfusion areas in mice at the dosage of 0.1 and 1.0 g/L (t=2.397, 4.533) and at the age of 0 -17, 0 -7 days when the dosage as 1.0 g/L (t=4.070, 2.399) were reduced significantly (P＜0.05). Conclusions The expression of adenosine A2A receptor increases in oxygen-induced retinal pathological neovascularization. Adenosine A2A receptor may regulate the expression of VEGF. A2A receptor inactivation can inhibit oxygen-induced retinal pathological neovascularization.
ObjectiveTo evaluate the association between the Single Nucleotide Polymorphism (SNP) BsmI (rs1544410) in the vitamin D receptor gene and the susceptibility of coronary artery disease. MethodsDatabases including PubMed, Web of Science, CNKI, WanFang Data, VIP and CBM were searched from inception to May, 2016 to collect case-control studies about SNP BsmI (rs1544410) in the vitamin D receptor gene and the susceptibility of coronary artery disease. Two reviewers independently screened literature, extracted data, and assessed the risk of bias of included studies. Then Meta-analysis was performed by using RevMan 5.3. ResultsA total of seven studies were included, which involved 2182 patients and 5925 controls. The results of meta-analyses showed that the B allele and BB genotype in rs1544410 was associated with the risk of coronary artery disease (B vs. b:OR=1.36, 95%CI 1.03 to 1.79, P=0.03; BB vs. bb:OR=1.70, 95%CI 1.06 to 2.72, P=0.03; BB+Bb vs. bb:OR=1.52, 95%CI 1.00 to 2.30, P=0.05). Subgroup analysis by age showed that rs1544410 was associated with the risk of coronary artery disease in the age <65(B vs. b:OR=1.65, 95%CI 1.00 to 2.73, P=0.05; BB vs. Bb+ bb:OR=1.79, 95%CI 1.08 to 2.97, P=0.02; BB vs. bb:OR=2.64, 95%CI 1.12 to 6.25, P=0.03). Subgroup analysis by ethnicity showed that rs1544410 was associated with the risk of coronary artery disease in Caucasians (B vs. b:OR=1.47, 95%CI 1.10 to 1.97, P=0.01; BB+Bb vs. bb:OR=1.71, 95%CI 1.09 to 2.68, P=0.02; BB vs. Bb+bb:OR=1.39, 95%CI 1.01 to 1.92, P=0.05; BB vs. bb:OR=1.80, 95%CI 1.10 to 2.95, P=0.03). Subgroup analysis by genotyping methods showed that rs1544410 was associated with the risk of coronary artery disease in the TaqMan (B vs. b:OR=2.18, 95%CI 1.06 to 4.45, P=0.03; BB+Bb vs. bb:OR=3.32, 95%CI 1.06 to 10.40, P=0.04; BB vs. bb:OR=3.31, 95%CI 1.06 to 10.30, P=0.04). Subgroup analysis by diagnostic criteria for cases showed that rs1544410 was associated with the risk of coronary artery disease in the ECG (B vs. b:OR=1.15, 95%CI 1.02 to 1.29, P=0.02; BB+Bb vs. bb:OR=1.22, 95%CI 1.02 to 1.45, P=0.03; BB vs. bb:OR=1.31, 95%CI 1.03 to1.67, P=0.03). ConclusionBsmI (rs1544410) B allele may have a significant association with the high risk of coronary artery disease especially the Caucasians and the ones with age <65.
Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR), and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2). Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study. The PDR patients were divided into non-injection group (30 patients, 32 eyes) and injection group (27 patients, 28 eyes). The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery. The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy. Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls. The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression of Notch1, Dll4 and VEGFR2. In the meantime, the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted. Results The immunohistochemical staining revealed that there were positive expression of Notch1, Dll4 and VEGFR2 in all PDR membranes, regardless of the injection of the ranibizumab. The levels of Notch1, Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45, 6.01, 4.08;P=0.030, 0.008, 0.023). In injection group, the number of endothelial cells in the membranes reduced (17.17±2.48) compared with that of the non-injection group (41.50±5.57). There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05). RT-PCR showed that the differences of the mRNA expression of Notch1, Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50, 12.50, 12.02;P＜0.05).The expression of Notch1, Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR group, the expression of Notch1, Dll4 and VEGFR2 of non-injection group was higher than that of injection group. Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83), VEGFR2 and Notch1 (r=0.81), Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05). Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group, and it is positively correlated with the expression of the VEGFR2. Notch1 and Dll4 play a regulatory rule in the neovascularization in PDR, the acting way may be correlated with VEGFR2.