Objective To investigate if lactic acid can promote the expression of vascular endothelial growth factor (VEGF) in the rat retinal explants.Methods The retinas of two-week neonatal SD rats were placed onto the culture plate inserts and incubated with Dulbeccoprime;s modified Eagleprime;s medium （DMEM） plus 2% fetal bovine serum (FBS) containing 10,20,30 mmol/L of lactic acid, respectively. Each group had 24 retinas. At 24 hours after incubation, the retinas were sectioned for light microscopy and the expression of VEGF was measured by real time PCR and Western blot. Results The cultured retinas maintained intact construction, and no cytolysis and apoptosis were observed under light microscope. RT-PCR showed the levels of VEGF mRNA were 0.74plusmn;0.06 for 10 mmol/L lactic acid group, 0.99plusmn;0.12 for 20 mmol/L group, and 1.45plusmn;0.17 for 30 mmol/L group respectively. VEGF expression was 0.34plusmn;0.15 for 10 mmol/L, 0.54plusmn;0.16 for 20 mmol/L, and 0.93plusmn;0.23 for 30 mmol/L group respectively by Western blot. Both PCR and Western blot showed 30 mmol/L of lactic acid significantly increased the levels of VEGF mRNA and VEGF expression. Conclusion The induction of retinal VEGF by lactic acid is concentration-dependent.
Proliferative vitreoretinopathy (PVR) is a common complication and major cause of blindness of ocular trauma. Many cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), participate in the process of the pathogenesis of traumatic PVR. VEGF competitively inhibits binding of PDGF to its receptor (PDGFRα), enables indirect activation of PDGFRα by non-PDGF ligands, resulting in reduced p53 expression, cell proliferation and migration, which is a key point in the pathogenesis of traumatic PVR.
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 expression of the pigment epithelium derived growth factor (PEDF) in retina and the effects of PEDF for vascular endothelial growth factor (VEGF) and retinal microvascular after recombinant adeno-associated virus mediated pigment epithelium derived factor（rAAV-mediated-PEDF）transferred retina of diabetic rats. Methods Male Wister rats were induced diabetes with intraperitoneal injection of streptozocin, then divided into 1 month group (DM1), 3month group(DM3) and 6 month group randomly. In diabetic rats, right eyes were injected rAAV2CMVhPEDF into vitreum as treat group,left eyes were injected into rAAV2-CMV-GFP (1times;1011 v.g/ml) as self-control group. In normal rats, right eyes were punctured but not injected (CONf), left eyes let it be without any disposal. The levels of VEGFmRNA and hPEDFmRNA in retina were evaluated using RT-PCR in different period. The protein levels of VEGF, PEDF within the retina were determined using western blot. The change of retinal capillary were observed through retinal vascular flattening. Result The expression of hPEDFmRNA in retina was enhanced persistence after rAAV2-CMV-hPEDF injected, achieved climax until 6 months. The levels of PEDF were also incerased consecutive, the differences were statistically significant in treatment group compared with own control group （Plt;0.01）. Levels of mRNA and protein of VEGF at different time-points among therapy group were not statistically significant, all obviously higher than normal （Plt;0.05），but all lower obviously than respectively own control group at the same timepoint （Plt;0.01）. The morphology of retinal capillary was not different significant with normal rats in 1 month diabetic rats. Morphology changes of therapy groups were less than those of respective own control group in DM3 and DM6. Conclusion Intravitreous injection rAAV2-CMV-hPEDF can increase expression of mRNA and protein of PEDF，alleviate lesion of retinal microvascular in early period of diabetic rats and supress expression of VEGF in retina of diabetic rats.The regulation occur on mRNA level. （Chin J Ocul Fundus Dis，2008，24：259-264）
ObjectiveTo analyze the concentrations of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in aqueous humor of patients with proliferative diabetic retinopathy (PDR) before and after intravitreal injection of ranibizumab. MethodsTwenty-five eyes of 20 PDR patients were collected as the PDR group. Twenty-five eyes of 21 senile cataract patients were collected as the control group. There were no statistical significance in gender (χ2=0.223), age (Z=-1.555) and intraocular pressure (Z=-0.225) between the two groups (P > 0.05). Samples of aqueous humor (0.1 ml) were collected just before and 7 days after the injection of ranibizumab in PDR group. Samples of aqueous (0.1 ml) humor were collected just before cataract surgery in control group. The concentrations of VEGF and PEDF in the aqueous humor were measured by enzyme-linked immunosorbent assay. ResultsThe VEGF and PEDF concentration in the aqueous humor were reduced significantly after intravitreal injection of ranibizumab in PDR group (Z=-4.072, -4.319; P < 0.05). The concentrations of VEGF and PEDF in the aqueous humor before intravitreal injection of ranibizumab in PDR group were significantly higher than the control group (Z=-5.228, 4.706; P < 0.05). The VEGF concentration in the aqueous humor after intravitreal injection of ranibizumab in PDR group were similar to control group (Z=-1.557, P > 0.05). However, the concentration of PEDF in the aqueous humor after intravitreal injection of ranibizumab in PDR group still higher than control group (Z=-2.475, P < 0.05). The ratio of VEGF/PEDF before and after intravitreal injection of ranibizumab was statistically different (Z=-2.058, P < 0.05), but was the same between PDR group and control group (Z=-0.456, -0.844; P > 0.05). The aqueous humor concentrations of VEGF and PEDF were not significantly correlated with each other, neither in PDR group (r=-0.195, -0.174; P > 0.05) nor in control group (r=-0.286, P > 0.05). ConclusionsAqueous humor concentrations of VEGF and PEDF are significantly elevated in eyes with PDR. Intravitreal injection of ranibizumab significantly decreased the VEGF and PEDF in the aqueous humor after 7 days.
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.
The introduction of anti-vascular endothelial growth factor (VEGF) therapy represents a landmark in the management of wet age-related macular degeneration (AMD). However, as a new therapy, several problems such as durability of the therapeutic effects, medication side effects, and medication selection have emerged. We should make appoint of improving the therapeutic effect and safety by realizing the limitation of the therapy, monitoring the clinical potential adverse reactions of anti-VEGF agents, and recommending individualized treatment.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
Objective To observe the change of serum associated factors concentrations in the patients with idiopathic choroidal neovascularization (CNV).Methods The clinical data of 21 patients (21 eyes) with idiopathic CNV (CNV group) and 20 normal individuals (control group) were retrospective analyzed. Serous concentrations of vascular endothelial growth factor (VEGF), tumor necrosis factor alpha;(TNFalpha;), interleukin 1-beta; (IL1-beta;), IgG, IgA, IgM, IgE, CH50, C3, C4 and Creactive protein (CRP) were assayed by enzymelinked immunosorbent assay (ELISA) and immunonephelometry. Results The level of VEGF in CNV group was significantly higher than that in control group(t=2.340，P=0.025). The level of IgE in CNV group was significantly lower than that in control group(Z=-2.765，P=0.006). The other factors were not significantly different between the two groups（Pgt;0.05）.Conclusion VEGF and IgE may play an important role in the formation of idiopathic CNV.
ObjectiveTo study the changes and correlation of cytokines in aqueous humor before and after intravitreal injection of conbercept (IVC) treatment in patients with proliferative diabetic retinopathy (PDR).MethodsA prospective clinical study. From March to December 2019, 36 patients (42 eyes) of PDR patients treated with IVC combined with pars plana vitrectomy (PPV) (the observation group) and 27 patients (31 eyes) underwent cataract surgery in the same period (control group) in Department of Ophthalmology of the First Affiliated Hospital of Guangzhou University of Chinese Medicine were included in this study. Before PPV 5-7 days, IVC treatment was performed, and the aqueous humor were extracted during IVC and second-stage PPV in the observation group. The aqueous humor was extracted during cataract surgery in the control group. Luminex assay was used to detect VEGF-A, placental growth factor (PLGF), platelet-derived growth factor-AA (PDGF-AA), platelet-derived growth factor-BB (PDGF-BB), angiopoietin-like protein 4 (ANGPTL4), IL-6, IL-8, IL-1β, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) cytokine expression. For normally distributed data, the independent sample t test was used for comparison between two independent samples; for non-normally distributed data, the Wilcoxon rank sum test was used for comparison between two independent samples. The correlation analysis used Spearman rank correlation test.ResultsBefore IVC treatment, the concentrations of VEGF-A, PLGF, PDGF-AA, ANGPTL4, IL-6, IL-8, MCP-1 and ICAM-1 in the aqueous humor of PDR patients were significantly higher than those in the control group (P＜0.05). After IVC treatment, the concentration of VEGF-A in the aqueous humor was significantly lower than that before treatment, and the concentrations of ANGPTL4 and IL-8 were significantly higher than those before treatment (P＜0.05). There were no significant differences in the concentrations of PLGF, PDGF-AA, PDGF-BB, IL-6, IL-1β, MCP-1, ICAM-1 and TNF-α before and after IVC treatment (P＞0.05). Before IVC treatment, the concentration of VEGF-A was positively correlated with PLGF, PDGF-AA, PDGF-BB, ANGPTL4, IL-6, IL-8, MCP-1 and TNF-α (P＜0.05).ConclusionsIVC treatment can reduce the concentration of VEGF-A and increase the concentrations of ANGPTL4 and IL-8 in aqueous humor in PDR patients before PPV.