Objective To observe the influence of prolyl hydroxylase 2 (PHD2) expression on endothelial barrier dysfunction induced by high glucose in human retinal vascular endothelial cells (HRECs). Methods The HRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 5 mmol/L glucose +25 mmol/L mannitol, 30 mmol/L glucose) as normal control group, mannitol control group and high glucose group, respectively. After the cells cultured for 24 and 48 hours, the protein levels of PHD2, hypoxia-inducible factor-1alpha; (HIF-1alpha;) and occludin was detected by Western blot; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by enzymelinked immuno sorbent assay (ELISA); the transcription levels of PHD2, HIF-1alpha;, VEGF and occludin were determined by the reversetranscription polymerase chain reaction (RT-PCR); the paracellular permeability between endotheliums was detected by 7times;104 molecular weight FITCdextran. Results Compared with normal control group, the protein level of PHD2 in mannitol control group and high glucose group firstly decreased and then increased, the protein level of HIF-1alpha; increased while that of occludin decreased; the secretion of VEGF increased in high glucose group but not in mannitol control group (PHD2:F=7.618, 8.627;P<0.05. HIF-1alpha;:chi;2=7.692, 7.652;P<0.05. occludin:F=23.23, 7.317;P<0.05. VEGF:F=10.768, 4.562; P<0.05). Compared with normal control group, the mRNA levels of PHD2, HIF-1alpha;, VEGF and occludin in mannitol control group and high glucose group increased (PHD2:F=5.69, 14.27;P<0.05. HIF-1alpha;:F=6.07, 10.47;P<0.05. VEGF:F=12.31, 9.14;P<0.05. occludin:F=8.77, 8.00;P<0.05). Compared with normal control group, the paracellular permeability of mannitol control group and high glucose group increased (chi;2=20.57,F=56.09;P<0.05). Conclusions High glucose induced altered expression of PHD2 which might play an important role in endothelial barrier dysfunction. The mechanism might be associated with HIF-1alpha; and VEGF.
Objective To observe the effects of dual targets intervention on the expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in diabetic rat retina. Methods Forty-eight Sprague -Dawley rats were randomly divided into control group (CON1 group) and diabetes mellitus group (DM group). The rats of DM group were induced with streptozotocin injection creating a diabetic model. Retinas were obtained at eight, 10, 12 weeks after DM induction from both groups. CTGF and VEGF mRNA levels were examined by realtime reverse transcriptionpolymerase chain reaction (RT-PCR). Based on the results of above experiments, 60 rats with same conditions were selected. Fifty rats were induced with streptozotocin injection creating a diabetic model, and 10 rats comprised the control group (CON2 group). Then the 50 diabetic rats were randomly divided into ranibizumab and CTGF shRNA dual targets intervention group, ranibizumab singletarget intervention group, CTGF shRNA singletarget intervention group and nonintervention group. Retinas were obtained at one week after intervention from all the groups. CTGF and VEGF mRNA levels were examined by RT-PCR. Results The levels of CTGF mRNA were significantly higher in DM group than that in CON1 group at the 8th weeks after DM induction, and this upregulation was maintained through the 12th week (t=-2.49, -2.67, -2.42;P<0.05). There was no difference on VEGF mRNA levels between DM group and CON1 group at the 8th weeks after DM induction(t=-0.443,P=0.669). VEGF mRNA levels of DM group started to be significantly elevated over those in the CON1 group at the 10th week, and remained to be higher at the 12th week (t=-2.35, -2.57;P<0.05). The VEGF mRNA of ranibizumab single-target intervention group was significantly lower than that in non-intervention group (t=-3.44,P=0.014), which was similar to CON2 group (t=-1.37,P>0.05); however, the CTGF mRNA level was significantly increased as compared to the nonintervention group (t=2.48,P<0.05). In the CTGF shRNA single-target intervention group, the levels of CTGF and VEGF mRNA were decreased as compared to the non-intervention group (t=0.23, -2.92;P<0.05). In the ranibizumab and CTGF shRNA dual targets intervention group, the levels of CTGF and VEGF mRNA were decreased as compared to the non-intervention group (t=-6.09, -5.11;P<0.001), which was similar to CON2 group (t=-1.16, 1.139; P>0.05). Conclusions Both CTGF and VEGF gene expression are up-regulated in early diabetic rat retina, and the level of CTGF increased earlier than VEGF. Ranibizumab combined with CTGF shRNA could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina.
Objective To observe the effect of ginsenoside Rg3 on the proliferation, migration, and tube formation of human retinal capillary endothelial cell (HRCEC) cultured in normal and hypoxia condition. Methods HRCEC was cultured in normal condition and treated with 0.0 mmol/L (group A), 0.1 mmol/L (group B) and 0.5 mmol/L (group C) ginsenoside Rg3. HRCEC was also cultured in hypoxia condition and treated with 0.0 mmol/L (group D), 0.1 mmol/L (group E) and 0.5 mmol/L (group F) ginsenoside Rg3. The effects of ginsenoside Rg3 on HRCEC proliferation were measured by methylthiazoletrazolium assay in 24, 48 and 72 hours after culture. In 24 hours after culture, the effect of cell migration was evaluated by transwell chamber; the effect of tube formation was evaluated by Matrigel; the expression of vascular endothelial growth factor (VEGF) protein and mRNA were detected by Western blot and real-time quantitative reverse transcription-polymerase chain reaction. Results Ginsenoside Rg3 could inhibit proliferation of HRCEC, depending on the concentration (F=30.331 and 33.402 in normal and hypoxia condition, respectively; P<0.05) and time (F=85.462 and 136.045 in normal and hypoxia condition, respectively; P<0.05). The number of cell migration was 103.33plusmn;3.54, 92..25plusmn;3.68, 78.64plusmn;4.66 in group A, B and C, the difference among three groups was statistically significant (F=28.801, P<0.05). The number of cell migration was 125.76plusmn;3.11, 90.27plusmn;3.55, 77.81plusmn;5.01 in group D, E and F, the difference among three groups was statistically significant (F=117.594, P<0.05). The number of tube formed in Matrigel was 24.3plusmn;2.2, 15.7plusmn;1.7, 10.1plusmn;2.3 in group A, B and C, the difference among three groups was statistically significant (F=35.364, P<0.05). The number of tube formed in Matrigel was 26.2plusmn;1.9, 15.1plusmn;2.6, 8.6plusmn;1.9 in group D, E and F, the difference among three groups was statistically significant (F=50.989, P<0.05). The expression of VEGF mRNA was 1.00plusmn;0.06, 0.79plusmn;0.06, 0.68plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=31.303, P<0.05). The expression of VEGF mRNA was 3.88plusmn;0.12, 2.83plusmn;0.09, 1.15plusmn;0.05 in group D, E and F, the difference among three groups was statistically significant (F=682.668, P<0.05). The expression of VEGF protein was 0.62plusmn;0.03, 0.41plusmn;0.02, 0.32plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=125.471, P<0.05). The expression of VEGF protein was 0.91plusmn;0.03, 0.82plusmn;0.03, 0.71plusmn;0.02 in group D, E and F, the difference among three groups was statistically significant (F=41.045, P<0.05). Conclusion Ginsenoside Rg3 can inhibit the proliferation, migration, and tube formation of HRCEC through the inhibition of VEGF expression.
Objective To observe the effect of tetramethypyrazine (TMP) on the expression of hypoxia-related factors in human umbilical vein endothelial cells (HUVECs). Methods The second to fifth passage cultured HUVECs were divided into five groups: control group, CoCl2induced hypoxic group and 50, 100, 200 mu;mol/L TMP treatment groups. HUVECs in control group were not treated. HUVECs inCoCl2induced hypoxic group were treated with 150 mu;mol/LCoCl2for four hours. HUVECs in 50, 100, 200 mu;mol/L TMP treated groups were pretreated with 150 mu;mol/LCoCl2 for four hours, followed by treatment with 50, 100, 200 mu;mol/L TMP for eight hours. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of prolyl hydroxylase 2 (PHD2), hypoxia-induced factor-1alpha;(HIF-1alpha;) and vascular endothelial growth factor (VEGF). Protein levels of PHD2, HIF-1alpha;, and VEGF were detected using Western blot. Results Compared with the control group, theCoCl2 induced hypoxic group showed decreased mRNA and protein levels of PHD2 (t=3.734, 3.122;P<0.05), while those of HIF-1alpha; and VEGF increased (HIF-1alpha; mRNA:t=4.589,P<0.05; HIF-1alpha; protein:t=3.778,P<0.05. VEGF mRNA:t=3.926,P<0.05; VEGF protein:t=3.257,P<0.05). Compared with theCoCl2 induced hypoxic group, 50, 100, 200 mu;mol/L TMP treated groups showed increased mRNA and protein levels of PHD2 (PHD2 mRNA: t=3.286, 3.617, 3.886;P<0.05. PHD2 protein: t=2.813, 3.026, 3.078; P<0.05); while those of VEGF decreased (VEGF mRNA: 50 mu;mol/L TMP: t=1.696,P>0.05; 100 mu;mol/L TMP:t=2.974,P<0.05; 200 mu;mol/L TMP: t=3.492,P<0.05; VEGF protein: 50 mu;mol/L TMP: t=1.986,P>0.05; 100 mu;mol/L TMP: t=2.976,P<0.05; 200 mu;mol/L TMP:t=3.136,P<0.05); although changes in HIF-1alpha;mRNA levels were not statistically significant (t=1.025, 0.726, -1.386;P>0.05), showed a decrease in HIF-1alpha;protein levels (50 mu;mol/L TMP: t=2.056,P>0.05; 100 mu;mol/L TMP:t=3.058,P<0.05; 200 mu;mol/L TMP:t=3.828,P<0.05). ConclusionIn HUVECs, TMP can upregulate the mRNA and protein expression of PHD2, while down regulating HIF-1alpha; protein expression and VEGF mRNA and protein expression under acute hypoxic conditions.
Objective To observe the application and effectiveness of bevacizumab intravitreal injection as adjunctive treatment for laser coagulation to treat retinopathy of prematurity (ROP). MethodsFrom March 2008 to October 2010, 17 infants (31 eyes) with ROP received bevacizumab intravitreal injection and were analyzed. Ten infants were male (18 eyes) and 7 were female (13 eyes).Their gestational age was from 24.7 to 31.0 weeks, with a mean of (28.2±1.9) weeks. Their birth weight was from 750 to 1600 grams, with a mean of (1150±264) grams. The indications for treatment included poor papillary dilation and refractive media opacity precluding complete laser coagulation and that ROP could not be controlled after complete laser coagulation treatment. The duration of followup was 1.4 to 40.8 months, with a mean of (20.8±13.2) months. It was noted whether the diseases were completely controlled or not, unfavourable structural outcome occurred or not and if there were complications regarding treatment. Results During the follow-up of all 31 eyes, ROP of 27 eyes (87.0%) was controlled by bevacizumab intravitreal injection as adjunctive treatment for laser coagulation. Increasing neovascularization and traction retinal detachment occurred in 2 eyes (6.5%). These 2 eyes underwent vitreoretinal surgery. The posterior retinal structure returned to normal in 1 eye and posterior vitreoretinal traction occurred in 1 eye. After bevacizumab intravitreal injection ROP continued progressing and traction retina detachment occurred in 2 eyes. The overall health of this infant remained good during and after operation. No systemic adverse drug reactions were found. No endophthalmitis occurred. No ocular complications such as corneal burn, cataract, and anterior segmental ischemia were found. Conclusions During the follow-up,the effectiveness of bevacizumab intravitreal injection as adjunctive treatment for laser coagulation to treat ROP was positive. No complications regarding the treatment were found.
Objective To observe the effect of intravitreal injection of bevacizumab (Avastin, IVB) on the expression of integrin-linked kinase (ILK) in fibrovascular membranes and the number of vascular endothelial cells (VECs) in proliferative diabetic retinopathy (PDR). Methods Twenty-four fibrovascular membrane samples were collected during pars plana vitrectomy in 24 patients with PDR. 12 PDR patients had received a single 1.25 mg IVB 7 days preoperatively (bevacizumab group), the other 12 patients (non-bevacizumab group) had not received IVB. For each of 24 fibrovascular membranes specimen, the number of VECs in the membranes were counted after staining with hematoxylin-eosin and von willebrand factor. Expressions of ILK in the fibrovascular membranes were detected through immunohistochemistry analysis. Results Immunohistochemistry revealed that ILK was highly expressed in all of 24 fibrovascular membranes of PDR.The average optical density of ILK expression level in bevacizumab and non-bevacizumab group were (127.78plusmn;15.08) and (129.03plusmn;16.26) respectively, the difference was not statistically significant (t=0.330,P=0.745).The number of VECs in fibrovascular membranes in bevacizumab and non-bevacizumab group were 21.50plusmn;3.94 and 41.33plusmn;7.44 respectively, the difference was statistically significant (t=3.872,P=0.003). Conclusions ILK was expressed in fibrovascular membranes of PDR. IVB can decrease the number of VECs during the process of PDR, but it can not affect the expression of ILK protein.
Objective To observe the expression of matrix metalloproteinase(MMP-2, MMP-9 and vascular endothelial growth factor (VEGF) in retinoblastoma (RB) and its relationship with the differentiation and optic nerve infiltration of RB.Methods Forty paraffin specimens of pathological confirmed RB were studied. They were divided into differentiated group (15 cases) and undifferentiated group (25 cases) , optic nerve infiltration group(13 cases) and without optic nerve infiltration group(27 cases). The expression of MMP-2, MMP-9 and VEGF were detected by immunohistochemistry, their relationships with the differentiation and optic nerve infiltration were also analyzed.Results The positive rate of MMP-2, MMP-9 and VEGF expression in 40 RB cases were 52.5%,57.5% and 72.5% respectively.The expression of MMP-2, MMP-9 and VEGF in the undifferentiated group were significantly higher than those in the differentiated group (chi;2=9.037, 9.253, 8.095; P<0.05). The expression of MMP-2, MMP-9 and VEGF in RB with optic nerve infiltration group were significantly higher than those in RB without optic nerve infiltration group (chi;2=11.045,10.243, 8.956;P<0.05). The expression of MMP-2,MMP-9 had a positive correlation with the expression of VEGF in RB (r=0.126,0.314;P<0.05). Conclusions MMP-2, MMP-9 and VEGF expressed in RB tumor tissues. The expression of MMP-2, MMP-9 has a positive correlation with the expression of VEGF. The levels of MMP-2, MMP-9 and VEGF expression are related to optic nerve infiltration of RB cells.
Objective To observe the effects of immunologic cytokines or anti-angiogenesis gene transfer mediated by electroporation for choroidal melanoma cells.Methods The human embryo kidney cells and malignant choroidal melanoma cells were transfected with plasmids pNGVL-mIL2, pNGVL-mIL12, pCI-sFLK-1, pCR3.1-antiVEGF121,pCI-ExTek. Then the expression of mIL2, mIL12, sFLK-1, VEGF and ExTek were detected by enzymelinked immunosorbentassay (ELISA) and Western blot. Nude mice models of malignant choroidal melanoma were established and they were divided into four groups randomly. Each group was treated with 30 mu;l of 0.9% NaCl, 30 mu;g pNGVL, 30 mu;g antiVEGF121+sFLK-1+ExTek and 30 mu;g mIL2+mIL12 respectively by electroporation. Seven,14, 21, 28, 35 and 42 days after treatment, the tumor volumes were measured to calculate the tumor inhibition rate. Results ELISA and Western blot showed that mIL2,mIL12,sFLK-1 and ExTek were expressed after electroporation,VEGF expression was decreased remarkably. After treatment,the tumors of mIL2+mIL12 group were greatly inhibited with a tumor inhibition rate of 97.33%,while the tumors of antiVEGF121+sFLK-1+ExTek and pNGVL group were partially inhibited with tumor inhibition rates of 53.33% and 36.33% respectively.Conclusions Immunologic cytokines transfer mediated by electroporation can inhibit the growth of melanoma,but anti-angiogenesis only have a mild effects.
Objective To investigate the influence of vascular endothelial growth factor (VEGF) antagonist bevacizumab on the growth of human choroidal melanoma (CM) OCM-1 cell xenografts in nude mice, and to explore the probable mechanism.Methods OCM-1 cells were subcutaneously implanted on 18 nude mice to establish ectopic model of human CM. The nude mice with the tumor of 5 mm in diameter were randomly divided into three groups: untreated group (group A), normal saline (NS) group (group B), drug treated group (group C). Bevacizumab was intraperitoneally injected for 14 consecutive days in group C, and the same volume of NS was used at a same way in group B. The volume and weight of implanted tumor as well as inhibitory rates of drug on tumor were calculated, ki67 and survivin proteins were measured with immunohistochemistry, and the mRNA expression of VEGF and survivin were assessed by RT-PCR.Results The volume and weight of tumor was (598.86plusmn;321.81) mm3, (0.66plusmn;0.15) g; (1 715.15plusmn;278.16) mm3, (1.54plusmn;0.39) g and (1 750.23plusmn;206.36) mm3, (1.54plusmn;0.31) g in groups C, A and B, respectively. There were significant differences between group C and A (F=34.53, P=0.00) and group C and group B (F=8.69, P=0.01). The inhibitory rate of these three groups were 57.14%, 5.31%, 6.25%, respectively, and the proliferation index (PI) of ki67 in these three groups were (51.85plusmn;1.32)%, (46.30plusmn;1.39)%, (27.90plusmn;0.90)%, respectively, there were significant differences in ki67 PI between C group and A or B group (H=15.17, P=0.00). The expression of survivin mRNA was (0.49plusmn;0.02), (0.82plusmn;0.05) and (0.61plusmn;0.05) in groupss C, A and B, respectively, there were significant differences between C group and A or B group (F=15.17, P<0.05) . The expression of VEGF mRNA was (0.32plusmn;0.08), (0.73plusmn;0.07), (0.80plusmn;0.04) in groups C, A and B, significant difference was found between group C and A or B group (F=12.05,P<0.05). Conclusion Bevacizumab can inhibit the growth of human CM in nude mice probably by inhibiting the activity of VEGF and downregulating survivin expression of the tumor as well as inhibiting the growth of the tumor.
Objective To observe the microvessel density(MVD)and expression of vascular endothelial growth factor (VEGF) in retinoblastoma(RB)before and after comprehensive treatment and explore its clinical correlations with tumor infiltration and recurrence. Methods Sixty-one paraffin specimens of RB were divided into enucleation without comprehensive treatment group (untreated group,52 cases), planned enucleation before comprehensive treatment group (planned enucleation group,six cases) and tumor recurrence after comprehensive treatment group(recurrence group,three cases). There were optic nerve invasion in 19 cases,no optic nerve infiltration in 33 cases. The MVD and VEGF expressions of 61 paraffin specimens were detected by streptavidin biotin-peroxidase (SP) immunohistochemistry with monoclonal antibody of CD34 and VEGF. Real time PCR was performed for the VEGF expression of planned enucleation group and recurrence group.Results In the untreated group,the MVD and VEGF expression of optic nerve infiltration cases were significantly higher than those of cases without optic nerve infiltration(t=-2.4685, P=0.017; chi;2=8.416 6,P=0.032 8).Tumor microvessel regression, decreased MVD, occlusion and hyaline changes of blood vessels were observed in the planned enucleation group in the course of systemic chemotherapy. Many neovascularized capillaries and increased MVD were observed in tumor tissues of the recurrence group. The VEGF expression of planned enucleation group was lower than that in the recurrence group.Conclusions There was no significant difference on VEGF expression in RB between with and without comprehensive treatment. The increasing MVD and VEGF expression of cases without comprehensive treatment were related to the optic nerve infiltration. And the increasing MVD may also play an important role in RB recurrence after comprehensive treatment.