重组人血管抑制因子Vasostatin对视网膜血管内皮细胞增殖的影响

目的 探讨重组人血管抑制因子Vasostatin对体外培养的恒河猴视网膜血管内皮细胞(RF／6A)增殖的影响。方法 以bFGF刺激增殖，体外培养恒河猴视网膜血管内皮细胞(RF／6A)至4代，分别加入不同质量浓度的重组人Vasostatin蛋白，并采用MTT法、^3H-TdR法检测2、4、6d时其对血管内皮细胞增殖抑制的作用，同时利用流式细胞仪对重组人Vasostatin蛋白是否诱导细胞凋亡进行了探讨。结果 MTT法、^3H-TdR两种方法均显示重组人Vasostatin蛋白以质量浓度依赖的方式抑制bFGF刺激的恒河猴视网膜血管内皮细胞(RF／6A)的增殖。流式细胞仪检测显示加入重组人Vasostatin蛋白后，G0／G1期无明显的亚二倍体核形峰出现，其不是通过诱导细胞凋亡的方式来抑制细胞增殖的。结论 重组人Vasostatin蛋白在体外具有抑制bFGF诱导的血管内皮细胞增殖的作用，其有可能成为一种有效的血管新生抑制剂。     

bFGF对兔视网膜Müller细胞表达VEGF的影响

血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）是视网膜细胞分泌的两种最重要的血管生成因子。二者有协同作用，bFGF还可以诱导内皮细胞表达VEGF。视网膜Müller细胞是视网膜中分泌VEGF的主要细胞之一，为了进一步探讨Müller细胞在糖尿病视网膜病变（DR）中的作用，本研究观察了bFGF对体外培养免视网膜Müller细胞表达VEGF的影响。     

bFGF对兔视网膜Müller细胞表达VEGF的影响

血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)是视网膜细胞分泌的两种最重要的血管生成因子.二者有协同作用,bFGF还可以诱导内皮细胞表达VEGF.视网膜Müller细胞是视网膜中分泌VEGF的主要细胞之一,为了进一步探讨Müller细胞在糖尿病视网膜病变(DR)中的作用,本研究观察了bFGF对体外培养兔视网膜Müller细胞表达VEGF的影响.     

血管内皮生长因子与眼内新生血管

眼内新生血管是多种致盲眼病的病理生理基础,新生血管形成受多种因子调控,其中血管内皮生长因子(VEGF)是最重要的细胞因子。它与特异性受体结合后通过复杂机制促进血管内皮细胞增殖、迁移和通透性增加。缺氧可诱导VEGF表达,VEGF通过NO的介导作用发挥促血管新生的作用。还有多种抑制和促进血管因子共同作用于血管内皮细胞。目前,人们通过基因手段抑制VEGF及其受体达到抑制血管新生的作用,为眼内新生血管性疾病开辟前景。     

缺氧、bFGF和VEGF对牛视网膜血管内皮细胞和周细胞增殖的影响

目的　探讨缺氧和细胞生长因子对视网膜血管内皮细胞和周细胞增殖的影响。方法　 ( 1)采用细胞克隆、机械除杂法培养视网膜血管内皮细胞和周细胞。 ( 2 )四唑盐比色试验 (MTT)和细胞计数法描绘正常和缺氧状态下牛视网膜血管内皮细胞和周细胞的增殖生长曲线。 ( 3 )加入碱性成纤维细胞生长因子 (bFGF)和血管内皮细胞生长因子(VEGF)后用MTT法观察它们对内皮细胞增殖的影响。结果　 ( 1)缺氧组牛视网膜血管内皮细胞在第 4、5d数目较正常组显著增加 (P <0 0 5 ) ,周细胞则在 4～ 7d数目明显增多。 ( 2 )加入VEGF可使四甲基偶氮唑盐 [3 ( 4 ,5 dimethylthia zolzyl) 2 ,5 diphenyltetrazo diumbromide ,MTT]值达到 2 1786,加入bFGF可使MTT值达到 2 9897,加入VEGF和bFGF使MTT值达到 2 943 1,而对照组仅为 1 0 13。结论　 ( 1)缺氧环境能刺激培养的牛视网膜血管内皮细胞和周细胞的增殖。( 2 )VEGF和bFGF能促进视网膜血管内皮细胞增殖 ,且bFGF促增殖的能力更强 ,但两者相加并不能增加促增殖作用。     

表皮生长因子、碱性成纤维细胞生长因子对人视网膜色素上皮细胞DNA合成的影响

目的探索表皮生长因子(epidermal growth factor,EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对培养的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞促进DNA合成的最佳刺激浓度，并对两种因子的协同作用进行探讨。方法培养的人RPE细胞第6代用于本实验。应用氚标胸腺嘧啶核苷(³H-thymidine, ³H-TdR)掺入试验及放射自显影检测EGF、bFGF对RPE细胞的促DNA合成作用。结果EGF、bFGF均可引起剂量依赖的促有丝分裂作用。在含2%血清的培养液中,EGE、bFGF作用最佳浓度为1ng/ml,明显低于无血清培养液中EGF、bFGF作用的最佳浓度(10ng/ml)。联合应用10ng/mlEGF、10ng/mlbFGF约提高RPE细胞合成DNA能力2.96倍。结论EGF、bFGF对培养的人RPE细胞具有促进DNA合成作用，且两者可产生协同效应。(中华眼底病杂志,1998,14:98-100)     

血管内皮生长因子与视网膜新生血管性疾病

血管内皮生长因子（ＶＥＧＦ）是新近确定的一种特异性刺激血管内皮细胞增殖及新生血管形成的生长因子。视网膜血管内皮细胞存在ＶＥＧＦ高亲和受体，而且受体数目较其它组织内皮细胞多。ＶＥＧＦ的特点是它的表达受局限部氧浓度的调节。视网膜血管内皮细胞、色素上皮细胞、周皮细胞、Ｍｕｅｌｌｅｒ细胞均能合成并分泌ＶＥＧＦ，缺氧可上调其基因表达。ＶＥＧＦ既可刺激视网膜血管内皮细胞增殖及移行，也可诱导视网膜新生血管形成。     

白藜芦醇对视网膜血管内皮细胞增殖及VEGF表达的影响(英文)

目的:探讨白藜芦醇对缺氧诱导的视网膜血管内皮细胞增殖的影响。方法:用100μmol/LCoCl2模拟缺氧环境,以体外培养的人视网膜血管内皮细胞为模型,采用MTT法测定细胞增殖、SABC法检测血管内皮生长因子的表达,并以计算机图像分析仪进行分析,观察研究白藜芦醇对CoCl2诱导的视网膜血管内皮细胞增殖的影响。结果:白藜芦醇对CoCl2诱导的视网膜血管内皮细胞增殖具有显著抑制作用(P<0.01),且呈时间和剂量依赖关系;同时,各给药组均抑制VEGF的表达(P<0.01)。结论:白藜芦醇能抑制CoCl2诱导的视网膜血管内皮细胞增殖及VEGF的表达,将为视网膜新生血管疾病的药物治疗提供一条新思路。     

血管内皮生长因子受体与眼新生血管及增生性病变

血管内皮生长因子（VEGF）是一种特异性刺激血管内皮细胞增殖和新生血管形成的生长因子,是最直接的血管内皮细胞促分裂素,与血管内皮生长因子受体（VEGFR）结合而发挥各项功能。在视网膜细胞、色素上皮细胞、表皮细胞、M(?)ller细胞内均有VEGFR表达。缺氧可上调其基因表达,直接造成视网膜新生血管形成及增生性玻璃体视网膜病变。对VEGFR结构、功能、与新生血管的关系,VEGFR表达增高所导致的病理变化,VEGFR高表达的调节因素,如何抑制VEGFR阻断VEGF/VEGFR信号传导途径抑制VEGF作用等方面进行了较为全面的综述。     

血管内皮生长因子与视网膜新生血管性疾病

血管内皮生长因子（ＶＥＧＦ）是新近确定的一种特异性刺激血管内皮细胞增殖及新生血管形成的生长因子。视网膜血管内皮细胞存在ＶＥＧＦ高亲和受体，而且受体数目较其它组织内皮细胞多。ＶＥＧＦ的特点是它的表达受局部氧浓度的调节。视网膜血管内皮细胞、色素上皮细胞、周皮细胞、Ｍüｌｅｒ细胞均能合成并分泌ＶＥＧＦ，缺氧可上调其基因表达。ＶＥＧＦ既可刺激视网膜血管内皮细胞增殖及移行，也可诱导视网膜新生血管形成。视网膜新生血管性疾病患者玻璃体ＶＥＧＦ含量增高。组织学定位表明ＶＥＧＦ的表达主要在内核层、神经节细胞层、ＲＰＥ细胞及Ｍüｌｅｒ细胞。阻断ＶＥＧＦ的产生及生物活性，有助于治疗视网膜新生血管性疾病     

Growth factor combinations modulate human retinal pigment epithelial cell proliferation

PURPOSE: Retinal pigment epithelial (RPE) cell proliferation is an important step in the pathogenesis of ocular diseases such as proliferative retinopathy. Growth factors play a major role in modulating the development and progression of these diseases. The aim of this study was to examine the effects of single growth factors as compared to growth factor combinations on the stimulation of RPE cell proliferation. METHODS: Human RPE cells were treated with 10 ng/ml IGF-1, PDGF, bFGF, EGF, VEGF, and TGFbeta(2), or with the combination of each of the growth factors. RPE cell proliferation was determined by [(3)H]-thymidine incorporation after growth factor stimulation for 24 hr. RESULTS: RPE cell proliferation was significantly increased when PDGF and bFGF were combined as compared to the single growth factor (p<0.005). There was a decrease in DNA synthesis for combined growth factors as compared to the single growth factor for IGF-1 plus TGFbeta(2) (p<0.05), IGF-1 plus VEGF (p<0.005), PDGF plus TGFbeta(2) (p<0.005), VEGF plus TGFbeta(2) (p<0.005 as compared to VEGF), and bFGF plus TGFbeta( 2) (p = 0.01 as compared to bFGF). The remaining growth factor combinations showed no significant synergistic effect on human RPE cell proliferation. CONCLUSIONS: These data lead to the implication that DNA synthesis in human RPE cell culture is synergistically affected by certain growth factor combinations, but not by all of them. This observation may be important for a better understanding of growth factor interactions occuring in proliferative diseases in the eye.     

生长因子诱导视网膜色素上皮细胞增殖的协同作用研究

目的从细胞水平探讨生长因子:肿瘤坏死因子-α(tumor necrosis factor,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对视网膜色素上皮(retinal pigment epithelium,RPE)细胞增殖的协同调控作用。方法通过PPE细胞培养，采用氚标胸腺嘧啶核苷(3 H-thymidine,3 H-TdR)掺入测定三种生长因子单用和分别联用诱导RPE细胞DNA合成改变，细胞计数观察RPE细胞生长变化。结果三种生长因子单用均可明显促进RPE细胞DNA合成及细胞数目增加,TNF-α、IL-β和bFGF的3 H-TdR掺入每分钟计数值(counts per minute,cpm)分别是对照组的2.74、2.66和1.69倍(P<0.05)。两种生长因子联用较单用cpm明显提高,其中TNF-α+IL-βcpm是对照组的3.14倍(P<0.05)。三种生长因子联用时cpm是对照组的3.74倍(P<0.05)。结论三种生长因子间存在促RPE细胞增殖的协同作用,这可能是生长因子对RPE细胞增殖的重要调控机制之一。(中华眼底病杂志,1998,14:95-97)     

Effects of angiogenic growth factor combinations on retinal endothelial cells

The aim of this paper was to determine if growth factors, known to be upregulated in proliferative diabetic retinopathy, exerted combined effects on retinal endothelial cells. The authors explored the individual and collective actions of insulin-like growth factor I (IGF-I), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor-2 (FGF-2) and placenta growth factor (PlGF) on several parameters that reflect the angiogenic potential of endothelial cells. The effect of growth factors on cell migration and survival/proliferation was examined using primary cultures of bovine retinal endothelial cells (BREC). The authors also determined the growth factor action on capillary-like tube formation on a reconstituted basement membrane matrix and on the newly described phenomenon of secondary sprouting, in which endothelial cell colonies spontaneously survive, proliferate, migrate and invade the matrix after the original capillary-like tubes have collapsed. Sprouting cells were positive for von Willebrand factor and could aggregate into larger tubes with lumens. Incubation with VEGF+IGF-I or PlGF+FGF-2 enhanced tube stability by 40-50%, more than each growth factor alone or other combinations (5-20%). The concurrent addition of four growth factors did not improve the response seen with growth factor pairs. Surprisingly, PDGF-BB induced tube collapse. IGF-I and FGF-2 mildly enhanced BREC proliferation/survival (5-15%). However, VEGF+IGF-I or PlGF+FGF-2 increased BREC proliferation/survival by 25% under low serum conditions, whereas combinations of all four growth factors exerted a clearly synergistic effect (250% increase). PDGF-BB or FGF-2 stimulated secondary sprouting and were the only factors capable of exerting this effect alone. Even though VEGF, IGF-I or PlGF were not effective, if administered in pairs, they demonstrated increased responses. PDGF-BB was also able to enhance the effect of FGF-2+IGF-I+VEGF on BREC secondary sprouting, but not of any of them individually. No other growth factor tested was able to significantly improve the action of combinations of three other growth factors. VEGF increased cell migration in a wounded monolayer assay two-fold and PDGF-BB, 2.5 times, but other individual growth factors were ineffective. PlGF+FGF-2 enhanced cell migration more than each factor alone. VEGF+IGF-I+PlGF+FGF-2, however, increased cell migration four-fold. In summary, this study indicates that growth factors, overexpressed in diabetic retinopathy eyes, enhance the angiogenic characteristics of cultured cells (tube formation, proliferation, secondary sprouting and migration). Their effects, however, can be greatly augmented by other growth factors that alone exert little or no action. Therefore, diabetic retinal neovascularization may result from the additive or synergistic action of several growth factors.     

Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells

Background

Proliferation and migration of retinal endothelial cells (REC) are associated with the development of proliferative diabetic retinopathy. REC proliferation is stimulated by isoforms of vascular endothelial growth factor-A (i.e., VEGF₁₂₁ and VEGF₁₆₅), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1) of which VEGF₁₆₅ also enhances migration of REC. Effects induced by VEGF-A can be blocked with ranibizumab, a VEGF-binding Fab fragment used in therapy of diabetic macular edema. In this study, we investigated potential angiogenic effects of placental growth factors (PlGF-1, PlGF-2) as other members of the VEGF family and whether the primary action of VEGF₁₆₅ is modulated in the presence of bFGF, IGF-1 and PlGF-1/-2. We also studied how effects of growth factor combinations can be attenuated with ranibizumab.

Methods

Effects of single growth factors or their combinations on proliferation and migration of immortalized bovine retinal endothelial cells (iBREC) were studied with or without ranibizumab or the inhibitor of VEGF receptors KRN951.

Results

Proliferation of iBREC was significantly stimulated by 1–100 ng/ml PlGF-1 or PlGF-2, but additive effects were not observed with various combinations of the tested growth factors. Ranibizumab neutralized VEGF’s effect on proliferation but was not effective when the other growth factors were used in combination with VEGF. bFGF and IGF-1 but not PlGF-1 or PlGF-2 stimulated iBREC migration as single agents, and they further enhanced VEGF-induced migration. The effects of such growth factor combinations including VEGF on migration were efficiently blocked by targeting only VEGF with ranibizumab. Migration induced by VEGF plus bFGF and IGF-1 was also almost completely inhibited by KRN951 interfering with VEGF receptor signalling.

Conclusions

Migration but not proliferation of iBREC induced by combinations of bFGF, IGF-1, PlGF-1 or PlGF-2 together with VEGF is efficiently suppressed by ranibizumab. VEGF-mediated signalling through VEGFR2 seems to control REC migration dominantly in the presence of other growth factors.     

Generation and characterization of a recombinant adenovirus expressing vascular endothelial growth factor for studies of neovascularization in the eye.

There is accumulating evidence that an increased expression of vascular endothelial growth factor from retinal pigment epithelial cells may be important in choroidal neovascularization. In vivo studies have demonstrated that subretinal injection of recombinant adenovirus vectors produces long-term transgene expression specifically within retinal pigment epithelial cells. A recombinant adenovirus encoding of vascular endothelial growth factor (Ad.RSV.VEGF) was therefore produced and characterized in order to determine whether an upregulation of vascular endothelial growth factor expression is sufficient to induce choroidal neovascularization. Ad.RSV.VEGF was produced by homologous recombination and its identity confirmed by restriction enzyme analysis. Ad.RSV.VEGF was characterized in vitro by the transduction of cultured retinal pigment epithelial cells. The in vitro characterization confirmed vascular endothelial growth factor mRNA and protein expression from Ad.RSV.VEGF and demonstrated the biological activity of the vascular endothelial growth factor protein. A preliminary in vivo study suggested that the subretinal injection of Ad.RSV.VEGF induced vascular leakage.     

Angiogenic factors in human proliferative sickle cell retinopathy

BACKGROUND/AIMS: Preretinal neovascular formations called sea fans develop at the border of non-perfused peripheral retina in sickle cell retinopathy. Angiogenic factors which could contribute to their development, however, have not been examined previously. The objective of this study was to determine immunohistochemically if vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) were associated with sea fan formations. METHODS: Immunohistochemistry on cryosections was used to localise bFGF, VEGF, heparan sulphate proteoglycan, human serum albumin, collagens IV and II, and von Willebrand factor in tissue from five sickle cell and one control subject. RESULTS: The greatest immunoreactivity for VEGF and bFGF was in the feeder and preretinal vessels of sea fans (p<0.01). The most prominent reaction product was localised to vascular endothelial cells. In retinal vessels, VEGF and bFGF immunoreactivities were greater in sickle cell subjects (both proliferative and non-proliferative) than in the control subject (p<0.01 and p<0.02 respectively). In the sickle cell retina, no angiogenic factor immunoreactivity was detected in non-perfused periphery and there was no significant difference in bFGF or VEGF immunoreactivity between perfused retina and the border of perfused and non-perfused areas. CONCLUSION: Our results demonstrate for the first time that VEGF and bFGF are associated with sea fan formations in sickle cell retinopathy. Both factors may function in an autocrine manner because immunoreactivity for these factors was greater within the neovascularisation than in adjacent retina.