Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes.

PURPOSE: To elucidate the potential role of angiopoietins and the Tie2 system in choroidal neovascularization. METHODS: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from five eyes with age-related macular degeneration, three eyes with idiopathic neovascular maculopathy, and two eyes had degenerative myopia and two eyes had angioid streaks. Light microscopic immunohistochemistry was performed to detect cytokines such as vascular endothelial growth factor (VEGF), Ang1, and Ang2 and cellular components such as retinal pigment epithelial (RPE) cells, macrophages, and endothelial cells. Immunofluorescent double staining using confocal microscopy was performed to identify the cell types that secrete specific cytokines. RESULTS: Ang1 and Ang2 were positive in all surgically excised CNVMs, regardless of the primary disease. Double staining revealed that many of the cytokeratin, CD68 and factor VIII positive cells also had Ang1 and Ang2 immunoreactivities. In contrast to Ang1, Ang2 immunoreactivity tends to be higher in the highly vascularized regions of many CNVMs, and the localization was very similar to that of VEGF staining. Almost all vascular structures had prominent immunoreactivity for Tie2, which was confirmed by double staining for Tie2 and factor VIII. Tie2 immunoreactivity was also observed in the RPE monolayer and in pigmented, polygonal, and fibroblast-like cells in the stroma. CONCLUSIONS: Present findings that Ang2 and VEGF are co-upregulated and that Tie2 is expressed in a variety of cell types in CNVMs further support a crucial role of the interaction between VEGF and Ang2 in pathologic angiogenesis of CNVM formation.     

Genetic polymorphisms of vascular endothelial growth factor and angiopoietin 2 in retinopathy of prematurity

Angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) contribute to development of retinopathy of prematurity (ROP). We aimed to test whether polymorphisms of these factors are associated with ROP. VEGF(-2578) and Ang2(-35) polymorphisms were analyzed with PCR-RFLP method in 200 preterm infants without ROP or with ROP stages 1-5. Our results suggest that there is no association between carrier state of VEGF(-2578) and Ang2(-35) and risk of ROP in preterm infants. The prevalence of VEGF(-2578) "A" allele was lower in preterm boys with severe ROP (stages 4-5) than in those without or with mild ROP (stages 1-3).     

Expression of hypoxia-inducible factor-1alpha and the protein products of its target genes in diabetic fibrovascular epiretinal membranes

AIMS: To investigate the expression of the hypoxia-inducible factor-1alpha (HIF-1alpha) and the protein products of its target genes vascular endothelial growth factor (VEGF), erythropoietin (Epo) and angiopoietins (Angs), and the antiangiogenic pigment epithelium-derived factor (PEDF) in proliferative diabetic retinopathy (PDR) epiretinal membranes. METHODS: Sixteen membranes were studied by immunohistochemical techniques. RESULTS: Vascular endothelial cells expressed HIF-1alpha, Ang-2 and VEGF in 15 (93.75%), 6 (37.5%) and 9 (56.25%) membranes, respectively. There was no immunoreactivity for Epo, Ang-1 and PEDF. There were significant correlations between the number of blood vessels expressing the panendothelial marker CD34 and the numbers of blood vessels expressing HIF-1alpha (r = 0.554; p = 0.026), Ang-2 (r = 0.830; p<0.001) and VEGF (r = 0.743; p = 0.001). The numbers of blood vessels expressing Ang-2 and VEGF in active membranes were higher than that in inactive membranes (p = 0.015 and 0.028, respectively). CONCLUSIONS: HIF-1alpha, Ang-2 and VEGF may play an important role in the pathogenesis of PDR. The findings suggest an adverse angiogenic milieu in PDR epiretinal membranes favouring aberrant neovascularisation and endothelial abnormalities.     

Angiopoietin/Tie2 signalling and its role in retinal and choroidal vascular diseases: a review of preclinical data

The angopoietin/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Ang/Tie) pathway is an emerging key regulator in vascular development and maintenance. Its relevance to clinicians and basic scientists as a potential therapeutic target in retinal and choroidal vascular diseases is highlighted by recent preclinical and clinical evidence. The Ang/Tie pathway plays an important role in the regulation of vascular stability, in angiogenesis under physiological and pathological conditions, as well as in inflammation. Under physiological conditions, angiopoietin-1 (Ang-1) binds to and phosphorylates the Tie2 receptor, leading to downstream signalling that promotes cell survival and vascular stability. Angiopoietin-2 (Ang-2) is upregulated under pathological conditions and acts as a context-dependent agonist/antagonist of the Ang-1/Tie2 axis, causing vascular destabilisation and sensitising blood vessels to the effects of vascular endothelial growth factor-A (VEGF-A). Ang-2 and VEGF-A synergistically drive vascular leakage, neovascularisation and inflammation, key components of retinal vascular diseases. Preclinical evidence suggests that modulating the Ang/Tie pathway restores vascular stabilisation and reduces inflammation. This review discusses how targeting the Ang/Tie pathway or applying Ang-2/VEGF-A combination therapy may be a valuable therapeutic strategy for restoring vascular stability and reducing inflammation in the treatment of retinal and choroidal vascular diseases.Subject terms: Mechanisms of disease, Medical research     

Phosphorylation of alphaB-crystallin in epiretinal membrane of human proliferative diabetic retinopathy

AIM: To examine phosphorylation of alphaB-crystallin (p-?BC), a vascular endothelial growth factor (VEGF) chaperone, and immunohistochemically investigate relationship between p-?BC, VEGF and phosphorylated p38-mitogen-activated protein kinase (p-p38 MAPK) in the epiretinal membrane of human proliferative diabetic retinopathy (PDR). METHODS: Eleven epiretinal membranes of PDR surgically excised were included in this study. Two normal retinas were also collected from enucleation tissues due to choroidal melanoma. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with anti-p-?BC, VEGF, CD31, and p-p38 MAPK antibodies. RESULTS: Immunoreactivity for p-?BC was observed in all of the epiretinal membranes examined, where phosphorylation on serine (Ser) 59 showed strongest immunoreactivity in over 70% of the membranes. The immunolocalization of p-?BC was detected in the CD31-positive endothelial cells, and co-localized with VEGF and p-p38 MAPK in PDR membranes. Immunoreactivity for p-?BC, however, was undetectable in endothelial cells of the normal retinas, where p-p38 MAPK immunoreactivity was less marked than PDR membranes. CONCLUSION: Phosphorylation of ?BC, in particular, phosphorylation on Ser59 by p-p38 MAPK may play a potential role as a molecular chaperon for VEGF in the pathogenesis of epiretinal membranes in PDR.     

Expression of ephrinB2 and its receptors on fibroproliferative membranes in ocular angiogenic diseases

PURPOSE: To determine whether ephrinB2 plays a role in ocular angiogenesis, we investigated the expression of ephrinB2 and EphB receptors on retinal fibroproliferative membranes. DESIGN: Experimental study of the expression of ephrinB2 and EphB receptors within fibroproliferative membranes in patients with ocular angiogenic diseases collected during vitrectomy. METHODS: Fibroproliferative membranes were obtained at the time of vitrectomy from 20 patients with proliferative diabetic retinopathy (PDR) and from 40 patients who had stage 5 retinopathy of prematurity. Specimens were investigated with immunohistochemistry using polyclonal antibodies directed against ephrinB2 and the EphB2, EphB3, and EphB4 receptors. Immunoreactivity for von Willebrand factor (factor VIII) and alpha-smooth muscle actin (alpha-SMA) was also determined to confirm the identity of the target vascular endothelial cells. RESULTS: Positive staining for ephrinB2 was observed on fibroproliferative membranes that were obtained from patients with PDR (65.0%) and retinopathy of prematurity (25.0%). Specifically, ephrinB2 was found to be present on endothelial cells, as confirmed by its colocalization with factor VIII and alpha-SMA staining. EphB2 and EphB3 expression was observed on fibroproliferative membranes that were harvested from patients with PDR (EphB2, 90.0%; EphB3, 70.0%) and retinopathy of prematurity (EphB2, 35.0%; EphB3, 45.0%). However, EphB4 expression was not observed in any of the membranes derived from patients with PDR or retinopathy of prematurity. The rate of ephrinB2 expression in patients with PDR was significantly higher than that seen in patients with retinopathy of prematurity, which probably reflected differences in the vascular density of their fibroproliferative membranes. CONCLUSION: These data suggest that the ephrinB2-EphB2/B3 system may play an important role in ocular angiogenesis.     

Effects of laser photocoagulation on serum angiopoietin-1, angiopoietin-2, angiopoietin-1/angiopoietin-2 ratio, and soluble angiopoietin receptor Tie-2 levels in type 2 diabetic patients with proliferative diabetic retinopathy

AIM: To determine the effects of laser photocoagulation on serum levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), soluble angiopoietin receptor Tie-2 (Tie-2), Ang-1/Ang-2 ratio and vascular endothelial growth factor (VEGF) in patients with type 2 diabetes mellitus (T2DM) and proliferative diabetic retinopathy (PDR). We also explored the role of the Ang/Tie system in PDR.METHODS: 160 patients with T2DM, including 50 patients with non-diabetic retinopathy (NDR), 58 patients with non-proliferative diabetic retinopathy (NPDR), and 52 patients with PDR were enrolled in this study. Serum Ang-1, Ang-2, Tie-2 receptor and VEGF levels were measured using enzyme-linked immunosorbent assays for all patients and were repeated in 26 patients who underwent laser photocoagulation two months after the procedure.RESULTS: The median levels of Ang-2 and VEGF in serum were significantly higher in the NPDR group (4.23 ng/mL and 303.2 pg/mL, respectively) compared to the NDR group (2.67 ng/mL and 159.8 pg/mL, respectively, P<0.01), with the highest level in the PDR group (6.26 ng/mL and 531.2 pg/mL, respectively, P<0.01). The median level of Ang-1 was significantly higher in the NPDR group (10.77 ng/mL) compared to the NDR group (9.31 ng/mL) and the PDR groups (9.54 ng/mL) (P<0.05), while no difference was observed between the PDR and NDR groups. Ang-1/Ang-2 ratio of PDR group was lowest in three groups (1.49 vs 2.69 and 2.90, both P<0.01). The median level of Tie-2 was not significantly different among three groups (P>0.05). Ang-2 was positively correlated with VEGF and Tie-2 in the PDR and NPDR groups (both P<0.05). Among the 26 patients who underwent laser photocoagulation, serum Ang-2 and VEGF levels significantly decreased (both P<0.05), whereas serum Ang-1 level and Ang-1/Ang-2 ratio were weakly increased (P>0.05). The median levels of Ang-2 and VEGF in serum were highest in PDR group, however, Ang-1/Ang-2 ratio of PDR group was lowest in three groups.CONCLUSION: Laser photocoagulation can reduce serum Ang-2 and VEGF levels. The Ang/Tie system and VEGF play an important role in the development and progression of T2DM patients with PDR.     

Ang/Tie信号轴在新生血管性年龄相关性黄斑变性发病及治疗中的作用

血管生成素(Ang)/酪氨酸激酶受体Tie信号轴（Ang/Tie信号轴）在调节血管稳定性、血管生成以及炎症等方面发挥着重要作用。Ang1支持静止和成熟的血管表型,而Ang2常与异常的血管结构形成、血管渗漏和炎症有关。新生血管性年龄相关性黄斑变性(nAMD)是一种能导致患者出现严重视力损害的疾病,其病理特征是脉络膜新生血管的形成及炎症反应。Ang/Tie信号轴是治疗nAMD的新靶点。靶向Ang/Tie信号轴的治疗方法可促进血管稳定,抑制渗漏及炎症,与抗血管生成药物联合治疗时可产生协同作用。本文对Ang/Tie信号轴在nAMD发病及治疗中的作用进行综述,以期为nAMD的治疗提供新思路。     

Expression of growth factors in the conjunctiva from patients with active trachoma

PURPOSE: The blinding complications of trachoma are associated with progressive conjunctival fibrosis due to excessive accumulation of extracellular matrix (ECM) components. We studied the processes involved in the regulation of fibrosis in trachoma by investigating the expression of the fibrogenic and angiogenic connective tissue growth factor (CTGF) and basic fibroblast growth factor (bFGF), the angiogenic vascular endothelial growth factor (VEGF), the angiogenesis-associated endothelial cell marker CD105 (endoglin), and the ECM protein tenascin in the conjunctiva. METHODS: Conjunctival biopsy specimens from six patients with active trachoma, and six control subjects were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against CTGF, bFGF, VEGF, CD105, and tenascin. RESULTS: In the normal conjunctiva, weak immunoreactivity for VEGF was observed in epithelial cells. There was no immunoreactivity for the other antibodies. In all trachoma specimens, immunoreactivity for CTGF and bFGF was localized in monocytes/macrophages, positive for the CD68 marker. Strong immunoreactivity for VEGF was observed in epithelial cells and on vascular endothelial cells. CD105 immunoreactivity was observed on vascular endothelial cells. Immunoreactivity for tenascin was noted in the upper substantia propria. CONCLUSIONS: These findings suggest that macrophages play an active role in conjunctival scarring, upregulated local production of CTGF, bFGF, and VEGF contributes to both fibrous tissue growth and angiogenesis, vascular endothelial cells are activated and are undergoing active angiogenesis, and deposition of tenascin reflect remodelling of the conjunctiva in trachomatous conjunctivitis.     

Expression of pigment epithelium derived factor and vascular endothelial growth factor in choroidal neovascular membranes and polypoidal choroidal vasculopathy

AIMS: To determine whether pigment epithelium derived factor (PEDF), a protein that inhibits angiogenesis, is expressed in human choroidal neovascular membranes (CNVMs) and in tissues from an eye with polypoidal choroidal vasculopathy (PCV). In addition, to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF), a known stimulator of angiogenesis, in these tissues. METHODS: CNVMs, associated with age related macular degeneration (AMD), angioid streaks, and PCV, were obtained during surgery. The expression of PEDF and VEGF in the excised subretinal fibrovascular membranes was determined by immunohistochemistry. RESULTS: PEDF and VEGF were strongly expressed in the vascular endothelial cells and retinal pigment epithelial (RPE) cells in the CNVMs where numerous new vessels were prominent (clinically active CNVMs). On the other hand, immunoreactivity for PEDF and VEGF was weak in the new vessels where fibrosis was prominent (clinically quiescent CNVMs). However, the RPE cells were still positive for PEDF and VEGF. The specimens from the eye with PCV also showed strong expression of PEDF and VEGF in the vascular endothelial cells and the RPE cells. CONCLUSION: Because PEDF is an inhibitor of ocular angiogenesis and an inhibitor of ocular cell proliferation, our results suggest that PEDF along with VEGF may modulate the formation of subfoveal fibrovascular membranes.     

The clinical role of vascular endothelial growth factor (VEGF) system in the pathogenesis of retinopathy of prematurity

BACKGROUND: Recent experimental studies suggest that vascular endothelial growth factor (VEGF) can play an important role in the development of retinopathy of prematurity (ROP). There are interesting observations of VEGF concentration in the serum, depending on its gene polymorphism which can have an impact on abnormal vessel development in the retina. AIM: Analysis of: (1) association of VEGF gene polymorphisms and the incidence of ROP, (2) correlation between serum concentration of VEGF and soluble VEGF receptor 1 (sVEGFR-1) during the 1st month of life and the risk of ROP, and (3) correlation between VEGF gene polymorphisms and VEGF serum concentrations. METHODS: A sample of 181 newborns with mean birthweight 1054 g (range: 500-1500 g) was prospectively evaluated. Molecular analysis of VEGF -460T>C and 405G>C polymorphisms were performed in the whole studied population, and concentrations of VEGF and sVEGFR-1 were measured by Elisa assay in the 2nd, 3rd and 4th weeks of life in the group of 128 children. The infants were divided into 3 groups: A) no ROP (n = 101), B) ROP not requiring treatment (n = 20), and C) ROP requiring laser or cryotherapy (n = 60). RESULTS: The frequency of VEGF 405G>C polymorphism was similar in all studied groups. The carriage of polymorphic allele -460 T was significantly overrepresented in ROP newborns who required treatment as compared to the no ROP group (54.2% vs 42.6%; OR: 1.63; 95% CI: 1.03-2.55). VEGF serum concentrations in the patients ascribed to different groups depending on the 405G>C or -460 T>C polymorphisms were similar. VEGF and sVEGFR-1 concentration on the 10th day of life did not differ significantly between the studied groups. Consecutive measurements showed a gradual increase in VEGF serum concentration in children without ROP, whereas in children with ROP requiring treatment the levels remained low. CONCLUSIONS: Based on our observations and previously published data, the association of the VEGF gene promoter polymorphisms and the risk of advanced ROP is weak. VEGF serum concentration assessment as early as on the 20th day of life appears to be a promising approach to recognize newborns at risk of the development of advanced ROP.     

Role for extracellular signal-responsive kinase-1 and -2 in retinal angiogenesis

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent mitogen for micro- and macrovascular endothelial cells (ECs). Evidence points to a possible role for two mitogen-activated protein (MAP) kinases, the extracellular-signal responsive kinases (ERK)-1 and -2, in VEGF signaling in ECs. This study was undertaken to begin to define the precise role of MAP kinases in VEGF signal transduction related to angiogenesis. METHODS: Bovine retinal microvascular endothelial cells (BRMECs) and a well-established rat model of retinopathy of prematurity (ROP) were used to investigate the role of ERK-1/2 in EC proliferation and tube formation and in retinal angiogenesis in vivo. RESULTS: Administration of VEGF to BRMEC cultures increased ERK-1/2 phosphorylation, cell proliferation, and tube formation in a dose-dependent manner. Phosphorylation of retinal ERK-1/2 also was increased in the ROP model. An inhibitor of ERK, AG126, and an inhibitor of ERK kinase (MEK), PD98059, exhibited a dose-dependent reduction of ERK phosphorylation and EC proliferation, but not tube formation, in VEGF-stimulated BRMECs. In the ROP model, intravitreous injection of 10 micro M AG126 or PD98059 reduced the retinal neovascular area by 71% and 48%, respectfully. No effect was seen on intraretinal blood vessel growth. CONCLUSIONS: These experiments point to a critical role for ERK and MEK in proliferation of ECs, but not in tube formation. Furthermore, inhibition of either of these two signal intermediates can significantly retard retinal neovascularization. This suggests that the MAPK pathway may provide rational targets for therapeutic intervention in ocular and other diseases with an angiogenic component.     

Vitreous levels of angiopoietin 2 and vascular endothelial growth factor in patients with proliferative diabetic retinopathy

PURPOSE: To investigate the levels of angiopoietin-2 (Ang2) and vascular endothelial growth factor (VEGF) in the vitreous fluids of patients with proliferative diabetic retinopathy (PDR) and to ascertain their involvement, if any, in angiogenesis of PDR. DESIGN: Retrospective case-control study. METHODS: Forty-one eyes of 41 patients with proliferative diabetic retinopathy and 18 eyes of 18 patients with nondiabetic ocular diseases (control group). Nondiabetic control eyes included 11 with idiopathic macular hole and 7 with idiopathic epiretinal membrane. Vitreous fluid samples were obtained at vitrectomy, and the levels of Ang2 and VEGF were measured by enzyme-linked immunosorbent assay. RESULTS: Vitreous level (mean +/- SD) of Ang2 was significantly higher in patients with PDR (1,753 +/- 3,213 pg/ml) than in control patients (112 +/- 113 pg/ml) (P < .0001). The vitreous concentration of VEGF was also significantly higher in patients with PDR (812 +/- 1,108 pg/ml) than in control patients (1.7 +/- 4.4 pg/ml) (P < .0001). Both Ang2 and VEGF levels in eyes with active PDR were significantly higher than in those with inactive PDR. The vitreous concentration of Ang2 correlated significantly with that of VEGF in eyes with proliferative diabetic retinopathy ([correlation coefficient] rho = 0.497, P = .001). CONCLUSIONS: These data demonstrate an increase of Ang2 in the vitreous fluid of patients with PDR and suggest an association of Ang2 and VEGF with angiogenic activity in PDR.     

Genetic polymorphisms and retinopathy of prematurity

PURPOSE: Retinopathy of prematurity (ROP) is a major problem among very preterm survivors of neonatal intensive care. Neovascularization of the retina is prominent in the proliferative stages of ROP and is under the control of several factors such as vascular endothelial growth factor (VEGF). This study was undertaken on the hypothesis that genetic polymorphisms of VEGF, transforming growth factor (TGF)-beta1, and tumor necrosis factor (TNF)-alpha would occur more frequently in preterm infants with progressive ROP than in those with mild or no disease. METHODS: The frequencies of VEGF -634 G-->C, VEGF *936 C-->T, TNF-alpha -308 G-->A, and TGF-beta -509 C-->T were determined in DNA from 91 infants who had received treatment for threshold ROP and 97 comparison infants. RESULTS: The frequencies of the VEGF *936 C-->T, TNF-alpha -308 G-->A and TGF-beta -509 C-->T polymorphisms were similar in both groups. The distribution of alleles at VEGF -634 was significantly different between the two groups (P = 0.03). Homozygotes for the G allele, associated with higher VEGF production were twice as likely to have threshold ROP. CONCLUSIONS: The progression of ROP to threshold ROP in very preterm infants may be influenced by genetic differences in VEGF production. Future efforts at prevention of threshold ROP may be directed toward blocking excess production of VEGF.     

Mechanism of angiostatin induced reduction of vascular leakage in retina and iris of rats with retinopathy of prematurity

AIM: To study the effect of an intravitreal injection of angiostatin on vascular leakage in the retina and iris of oxygen-induced retinopathy of prematurity (ROP). METHODS: Brown Norway rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O₂) for 5 days (P7-12) and then returned to normoxia to induce retinopathy. Angiostatin was reconstituted in sterile Phosphate Buffered Saline (PBS) and diluted to desired different concentrations. Angiostatin solution was injected into the vitreous of the right eye of the ROP rats at P14 and the age-matched normal rats through pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. Vascular permeability was quantified at 1, 2 and 3 days after the injection by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method and normalized by total protein concentrations. The expression of vascular endothelial growth factor (VEGF) in retina was evaluated using the Western Blot analysis and immunohis- tochemistry 24 hours following the injection. RESULTS: ROP rats showed significant increases of vascular permeability in the retina and iris (P <0.01). Angiostatin reduces vascular permeability in a dose-dependent manner in the retina of ROP rats. The reduction showed a time course trend. Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2-fold at P15 (P <0.05) and P16 (P < 0.01), respectively. Angiostatin injection significantly reduced VEGF levels in the retina of ROP rats but did not affect retinal VEGF levels in normal rats. CONCLUSION: Angiostatin significantly decreases patholog- ical vascular permeability in the retina and iris of ROP rats but not in normal rats. Angiostatin down-regulates VEGF expression in retina of ROP rats. These results suggest that angiostatin may have a therapeutic potential in the treatment of ROP and other diseases with vascular leakage.     

胎盘生长因子在早产儿视网膜病变纤维血管膜中的表达

目的　研究胎盘生长因子（ｐｌａｃｅｎｔａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＰＬＧＦ）在早产儿视网膜病变（ｒｅｔｉｎｏｐａｔｈｙｏｆｐｒｅｍａｔｕｒｉｔｙ,ＲＯＰ）纤维血管膜的血管内皮细胞、周细胞、巨噬细胞和胶质细胞中的表达和分布。方法　玻璃体切割术中取ＲＯＰ患者视网膜表面纤维血管膜保存备用,冰冻切片,ＨＥ染色、免疫组织化学染色和免疫荧光染色观察ＰＬＧＦ在纤维血管膜上的表达和分布。结果　ＨＥ染色及免疫组织化学染色结果显示ＰＬＧＦ在纤维血管膜中可见阳性表达。免疫荧光双标记法实验结果显示纤维血管膜的ＰＬＧＦ可能由血管内皮细胞、周细胞、巨噬细胞和胶质细胞表达。结论　ＰＬＧＦ可能参与了ＲＯＰ的发病过程,ＰＬＧＦ在ＲＯＰ发病中的作用及其分子机制需要进一步研究。     

Contrasting effect of estrogen on VEGF induction under different oxygen status and its role in murine ROP

PURPOSE: It has been reported that 17beta-estradiol (E2) may enhance the proliferation of bovine retinal vascular endothelial cells (BRECs) by increasing the expression of VEGFR-2 and VEGF. The hypothesis in the current study was that estrogen may contribute to fetal vascular development and the cessation of exposure to estrogen of premature infants on birth may have an inhibitory effect on retinopathy of prematurity (ROP). Because ROP is thought to develop under relative hypoxia after exposure to high-dose oxygen, this study was conducted to investigate how estrogen modulates hypoxia-induced VEGF in BRECs and mouse ROP. METHODS: Gene expression of VEGF and hypoxia-inducible factor (HIF)-1alpha were studied in BRECs, with or without E2, under normoxia and hypoxia (1% O2). A binding assay was performed to determine whether estrogen interferes with HIF-1-mediated induction of VEGF. In a mouse ROP model, effects of E2 were evaluated by avascular area, subsequent extraretinal neovascularization, and retinal expression of the VEGF gene, by administering E2 during hyperoxia (75% O2) and/or after exposure to room air. RESULTS: Hypoxia-induced VEGF mRNA in BRECs was reduced dose dependently by 1 to 100 nM E2. E2 reduced hypoxia-induced binding of HIF-1 to the VEGF promoter site and reduced the HIF-1alpha mRNA level. In mouse ROP, injection of E2 during hyperoxia increased retinal VEGF mRNA and reduced the retinal avascular area at the end of hyperoxia. E2 treatment during the normoxia that followed reduced VEGF mRNA and extraretinal neovascularization. Treatment with E2 throughout both periods significantly improved retinopathy. CONCLUSIONS: Estrogen may function as a significant modulator of the level of VEGF mRNA under different oxygen conditions and could serve as a prophylactic agent for ROP.     

血管抑素降低ROP幼鼠视网膜及虹膜血管渗漏性的作用机制研究

目的：探讨血管抑素（angiostatin）玻璃体注射对氧诱导的早产儿视网膜病变（retinopathy of prematurity，ROP）的视网膜及虹膜血管渗漏的作用及其机制。方法：将出生7d（P7）的Brown Norway鼠置高氧环境（750mL／L O2）5d后再置正常氧环境诱导ROP，建立ROP动物模型，并以年龄相匹配的正常鼠作为正常对照。所有ROP鼠（P14）及正常鼠均右眼玻璃体腔注射血管抑素，左眼注射相同剂量的PBS（磷酸盐缓冲生理盐水）作为对照。用Evans蓝微血管渗透性检测法及总蛋白标准化分别于注射后1，2和3d检测视网膜和虹膜的血管渗透性；用Western blot蛋白印迹分析和免疫组化方法检测注射24h后血管内皮生长因子（vascular endothelial growth factor，VEGF）在视网膜的表达。结果：ROP鼠视网膜及虹膜的血管渗透性明显增加（P〈0．01）；中剂量（3．75ug/眼）和高剂量（7．5ug/眼）血管抑素降低ROP鼠视网膜血管渗透性（P〈0．05，P〈0．01），而低剂量组（1．88ug／眼）没有引起明显改变，呈现剂量依赖型；三种不同剂量的血管抑素玻璃体注射后ROP鼠的虹膜均未发生明显的血管渗透性的改变。血管抑素注射后第1d和第2d视网膜血管渗透性明显降低（P〈0．05。P〈0．01），而第3d无明显降低，其作用呈现出时间进程。Western blot蛋白印迹和免疫组化分析表明血管抑素显著降低了ROP鼠视网膜的VEGF水平，但对正常鼠无影响。结论：血管抑素可以降低ROP鼠视网膜的病理性血管渗漏，其血管渗透性下降可能与血管抑素下调VEGF的表达有关。血管抑素可能对ROP等其他视网膜血管渗漏性疾病具有潜在的治疗作用。