VEGF localisation in diabetic retinopathy

AIM—To determine the staining pattern of vascular endothelial growth factor (VEGF) at different stages of diabetic retinopathy (including post-laser photocoagulation) and to compare staining in excised fibrovascular and fibrocellular (non-diabetic) preretinal membranes.
METHODS—Immunohistochemical localisation of VEGF, using antibodies raised against VEGF₁₆₅ and VEGF_121,165,189, was carried out on specimens of normal human retina (n=15), diabetic retinas ((a) with no overt retinopathy (n=19), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n=6), (c) with active proliferative retinopathy (n=6), (d) with no residual proliferative retinopathy after photocoagulation therapy (n=15)), excised diabetic fibrovascular membranes (n=19), and non-diabetic fibrocellular membranes (n=7). The degree and pattern of immunostaining was recorded.
RESULTS—In general, VEGF was absent from the majority of normal retinas. VEGF staining was apparent in most diabetic tissues but the staining pattern was dependent on both the specificity of the antibody used and the category of tissue. Staining with the VEGF₁₆₅ antibody was generally confined to endothelial cells and perivascular regions while the VEGF_121,165,189 antibody was also associated with extravascular components of the inner retina. Intensity of immunostaining of diabetic eyes was dependent on the severity of retinopathy being least in diabetics with no overt retinopathy and greatest in retinas with proliferative retinopathy. Interestingly, the intensity of immunostaining in diabetic retinas which had undergone laser surgery for proliferative retinopathy was reduced to basal levels. Moderate to intense immunostaining was observed in all fibrovascular and fibrocellular membranes examined.
CONCLUSIONS—This study supports a circumstantial role for VEGF in the pathogenesis of both the preclinical and proliferative stages of diabetic retinopathy.

Keywords: vascular endothelial growth factor; VEGF; diabetes; diabetic retinopathy     

Expression of Pigment Epithelium-Derived Factor and Vascular Endothelial Growth Factor in Fibrovascular Membranes from Patients with Proliferative Diabetic Retinopathy

Purpose

The expression of pigment epithelium-derived factor (PEDF), a strong inhibitor of angiogenesis, has not been examined in human ocular fibrovascular membranes, to the best of our knowledge. The purpose of this study was to determine whether PEDF is expressed in the fibrovascular membranes in eyes of patients with proliferative diabetic retinopathy (PDR), and to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF).

Methods

The expression of PEDF and VEGF in the fibrovascular membranes excised during vitreous surgery in eight cases of PDR was determined by immunohistochemistry.

Results

VEGF was strongly expressed in the endothelial cells of newly formed vessels in the fibrovascular membranes. In contrast, PEDF was weakly expressed in the endothelial cells and was prominently expressed in the extracellular matrix and fibrous tissue surrounding the new vessels.

Conclusions

Our results suggest that PEDF, along with VEGF, may modulate the formation of fibrovascular membranes in patients with PDR.?Jpn J Ophthalmol 2006;50:116–120 © Japanese Ophthalmological Society 2006     

VEGF与糖尿病视网膜病变早期的关系

目的：研究糖尿病大鼠视网膜病变早期与VEGF的关系。方法：建立糖尿病大鼠模型，随机分为正常对照组(M)，糖尿病一月组(M1)、三月组(M3)、五月组(M5)。分别在视网膜石蜡切片上行VEGF原位杂交、免疫组化、并分别对视网膜血管进行透射电镜观察。结果：石蜡切片中VEGF原位杂交：仅M5表达为67％。VEGF免疫组化：M3表达为34％，M5表达为89％。血管的透射电镜观察：M未见异常改变，从M1开始出现异常改变，以M5最明显。表现为基底膜节段性增厚、断裂缺失，内皮细胞肿张变形，向管腔内指状突起，异染色质浓集居边，周细胞核异染色质浓集居边，线粒体肿胀变性，甚至呈空泡状。结论：糖尿病视网膜病变时血管器质性改变在先，而生长因子表达在后。     

Expression of vascular endothelial growth factor and its receptors in rosacea

BACKGROUND: Rosacea is a common chronic disease of unclear pathogenesis, characterised by inflammation and vascular abnormalities of the facial skin and ocular surface. Recognising that vascular endothelial growth factor (VEGF) is vasoactive and has inflammatory activities, the expression of this molecule and its receptors, VEGF-R1 and VEGF-R2, in rosacea was investigated. METHODS: Formalin-fixed, paraffin wax-embedded sections of skin obtained from 20 patients with rosacea were immunostained to detect expression of VEGF, VEGF-R1 and VEGF-R2, using an indirect methodology incorporating antigen retrieval. Adjacent sections were stained with haematoxylin and eosin. RESULTS: Biopsy specimens were characterised by perivascular and perifollicular lymphohistiocytic infiltration and dilated vascular channels. In addition to keratinocyte and epithelial staining, which was also noted in normal skin, vascular endothelium frequently stained positive for VEGF-R1 (14/20, 70%) and VEGF-R2 (20/20, 100%), but infrequently for VEGF (2/20, 10%). In most specimens, infiltrating leucocytes, including lymphocytes, macrophages and plasma cells, expressed VEGF (17/20, 85%), VEGF-R1 (20/20, 100%) and VEGF-R2 (20/20, 100%). CONCLUSION: Expression of VEGF receptors, both by vascular endothelium and infiltrating mononuclear cells, is observed in rosacea. Although not expressed by endothelium, VEGF is present in epidermis and epithelium, and is expressed by infiltrating cells. VEGF receptor-ligand binding may contribute to the vascular changes and cellular infiltration that occurs in rosacea.     

Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey

PURPOSE: The vascular endothelial growth factor (VEGF) family is involved in vascular leakage and angiogenesis in diabetic retinopathy (DR) in the eye, but may also have physiological functions. Based on the hypothesis that differential VEGF receptor (VEGFR) expression in the retina is an important determinant of effects of VEGF, this study was conducted to investigate VEGFR expression in the diabetic retina and in an experimental monkey model of VEGF-A-induced retinopathy. METHODS: In retinas of 27 eyes of diabetic donors, 18 eyes of nondiabetic control donors, and 4 monkey eyes injected with PBS or VEGF-A, expression patterns of VEGFR-1, -2, and -3 in relation to leaky microvessels, as identified by the marker pathologische anatomie Leiden-endothelium (PAL-E) were studied by immunohistochemistry. RESULTS. In control human retinas and retinas of PBS-injected monkey eyes, all three VEGFRs were expressed in nonvascular areas, but only VEGFR-1 was constitutively expressed in retinal microvessels. In diabetic eyes, increased microvascular VEGFR-2 expression was found in association with PAL-E expression, whereas microvascular VEGFR-3 was present in a subset of PAL-E-positive cases. In VEGF-A-injected monkey eyes, VEGFR-1, -2, and -3 and PAL-E were expressed in retinal microvessels. CONCLUSIONS: The VEGFR-1, -2, and -3 expression patterns in control retinas suggest physiological functions of VEGFs that do not involve the vasculature. Initial vascular VEGF signaling may act primarily through VEGFR-1. In diabetic eyes, expression of retinal VEGFR-2 and -3 is increased, mainly in leaky microvessels, and VEGF-A induces vascular expression of the VEGF-A receptor VEGFR-2 and the VEGF-C/D receptor VEGFR-3. These findings indicate a dual role of VEGFs in the physiology and pathophysiology of the retina and suggest that microvascular VEGFR-2 and -3 signaling by VEGFs occurs late in the pathogenesis of DR, possibly initiated by high levels of VEGF-A in established nonproliferative DR.     

Captopril and vascular endothelial growth factor in a mouse model of retinopathy

PURPOSE: Angiotensin converting enzyme (ACE) inhibition has been shown in animal models of retinopathy and in patients with diabetes to improve retinal neovascularization. The mechanism is not clearly identified, but could potentially be mediated via vascular endothelial growth factor modification. The objective of this study was to determine the effect of captopril, an angiotensin converting enzyme (ACE) inhibitor, on retinal VEGF, VEGF-R1, and VEGF-R2 expression in a mouse model of oxygen induced retinopathy (OIR). METHODS: A mouse model of OIR was used and retinal tissue was obtained at P7, prior to oxygen exposure, at P12, just after oxygen exposure, and at P17, the time of maximal retinal neovascularization for VEGF, VEGF-R1 and VEGF-R2 assessment. A group of animals were treated with captopril (0.5 mg/kg/d SC from P7 for five days). RESULTS: Captopril plus OIR treated animals had higher levels of retinal VEGF mRNA and protein at P12 (p < 0.05) and lower levels at P17 (p < 0.05) than OIR animals. VEGF-R1 mRNA expression increased 16 fold from P7 to P17 (p < 0.05) in room air reared animals. VEGF-R1 mRNA expression was unaffected by OIR and/or captopril treatment. VEGF-R2 mRNA expression decreased from P7 to P17 by 1.5-fold in room air reared animals (p = 0.001). Retinal VEGF-R2 mRNA and protein expression were significantly higher at P12 in OIR plus captopril treated animals than OIR animals (p = 0.01). CONCLUSIONS: In summary, captopril maintains VEGF and increases VEGF-R2 expression during the period of hyperoxia when VEGF expression is normally suppressed. Captopril treatment during oxygen exposure is associated with a reduction in the angiogenic response at day 17 as manifested by decreased VEGF and VEGF-R2 expression in retinal tissue. Angiotensin converting enzyme inhibition is associated with changes in expression of VEGF and VEGF-R2 in the evolution of retinal neovascularization in the mouse model of retinopathy.     

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.     

糖尿病大鼠视网膜中缺氧诱导因子-1α和血管内皮生长因子表达的研究

目的:检测缺氧诱导因子-1α(HIF-1α)与血管内皮生长因子(VEGF)在糖尿病大鼠视网膜中的表达,探讨其在糖尿病视网膜病变(DR)发生发展中的作用。方法:SD大鼠120只随机分为对照组与DR组,DR组采用链脲佐菌素(STZ)一次性建立糖尿病模型。应用免疫组化SP法和RT-PCR技术分别于1,3,6mo检测视网膜中HIF-1α与VEGF蛋白及其mRNA的表达变化。结果:HIF-1α与VEGF蛋白及其mRNA在对照组视网膜中不表达,在DR组中呈进行性表达增强(P<0.05);DR组中HIF-1α与VEGF的表达呈正相关(r=0.605,P<0.05)。结论:DR视网膜中HIF-1α与VEGF表达增强,与DR的发生发展密切相关。     

Colocalization of Tie2, angiopoietin 2 and vascular endothelial growth factor in fibrovascular membrane from patients with retinopathy of prematurity

PURPOSE: The tyrosine kinase receptor Tie2 and its ligands, the angiopoietins (Angs), play important roles in vascular integrity and neovascularization, modulating vascular endothelial growth factor (VEGF) activity. To elucidate the potential role of Angs and the Tie2 system in retinopathy of prematurity (ROP), we have investigated the expression of Angs, Tie2 and VEGF within fibroproliferative membranes in ROP. METHODS: Fibroproliferative membranes were obtained from 38 cases with stage 5 ROP at the time of vitrectomy. Membranes were fixed in formalin and embedded in paraffin. Each specimen was serially sectioned for immunohistochemistry. Polyclonal antibodies specific for Ang1, Ang2, Tie2 and VEGF were used for immunostaining. Immunoreactivity for von Willebrand factor (factor VIII) was also assessed to confirm the identity of vascular endothelial cells. RESULTS: Positive staining for Tie2 was observed in 23 of 38 specimens (60.5%). Tie2 was localized in vascularized regions of fibrovascular membranes and was co- expressed with VEGF and factor VIII. Ang2 stained positively in 18 of 38 (47.3%) serial sections where Tie2 was present, and was also co-expressed with VEGF and factor VIII. Ang1 was not generally observed in these specimens (3/38). CONCLUSIONS: VEGF and Ang2-Tie2 interactions may play an important role in the pathogenesis of ROP.     

Expression and effect of proline hydroxylase domain 2 in retina of diabetic rats

AIM: To observe the expression of proline hydroxylase domain 2 (PHD2) in the retina of diabetic rats and investigate the relationship between PHD2 and relevant intraocular vascular proliferation factors. METHODS: Sixty male specific pathogen free (SPF) Sprague-Dawley (SD) rats were randomly divided into two groups: the diabetic group and the control group. The rats in the diabetic group were intraperitoneally injected with 60 mg/kg (0.60 mL/100g) of streptozotocin to induce a diabetic rat model. The rats in the control group were injected with an equal volume of sodium citrate buffer solution by the same method. Hematoxylin-eosin (HE) staining and immumofluorescence (IF) method were adopted to observe the pathological changes of retinal tissues and the expression of PHD2, glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF) by 8wk. RT-PCR method was applied to detect the expressions of mRNA of PHD2, VEGF and GFAP. The relationship between PHD2 and other vascular proliferation factors was analyzed. RESULTS: HE staining showed that there was the retinal tissue edema in the diabetic group, and the arrangement was in disorder, and proliferation could be seen. IF staining: in the retina of normal rats, PHD2 was not expressed, GFAP and VEGF were mainly expressed in astrocytes; while in the diabetic rats, PHD2, GFAP and VEGF staining showed strong positivity in all retinal layers, mainly in neurogliocytes. PHD2 was co-expressed with VEGF and GFAP. The mRNA expression levels of PHD2, GFAP and VEGF in the diabetic group were obviously higher than that in the control group,respectively 1.83 times, 1.75 times and 2.08 times. The difference had statistical significance (P<0.01). CONCLUSION: The high expression of PHD2 in the retina of early-stage diabetic rats might result from secretion of neurogliocytes induced by local high-concentration blood glucose, thus promoting the expression of VEGF and GFAP. PHD2 plays an important role during the occurrence of diabetic retinopathy.     

卡托普利防治糖尿病小鼠视网膜病变的实验研究

目的:探讨卡托普利对糖尿病视网膜病变发生的防治作用。方法:应用免疫组织化学方法和计算机图象分析技术对糖尿病小鼠未治疗组和卡托普利治疗组视网膜组织AngII和VEGF进行免疫组织化学测定和半定量分析。结果:AngII和VEGF在两组小鼠视网膜神经节细胞和血管内皮细胞均为阳性表达,而在正常对照组两者均为阴性表达。与糖尿病未治疗组相比,卡托普利治疗组的AngII和VEGF的平均灰度值明显增加,而面密度值明显降低,说明治疗组的免疫信号降低,阳性表达细胞数减少。结论:卡托普利可降低糖尿病小鼠视网膜组织AngII和VEGF的阳性表达,推测其对糖尿病视网膜病变具有一定的保护作用。     

玻璃体腔注射抗血管内皮生长因子单克隆抗体bevacizumab对增生型糖尿病视网膜病变纤维血管膜中整合素链接激酶表达的影响

目的 观察玻璃体腔注射抗血管内皮生长因子单克隆抗体bevacizumab(商品名Avastin)对增生型糖尿病视网膜病变(PDR)纤维血管膜中整合素链接激酶(ILK)表达及视网膜血管内皮细胞数量的影响.方法 玻璃体切割手术中取出的24例PDR患者的视网膜前纤维血管膜,其中12例患者手术前1周玻璃体腔单次注射bevaei...     

塞来昔布对糖尿病大鼠视网膜血管内皮生长因子表达的影响

目的 观察塞来昔布对糖尿病大鼠视网膜血管内皮生长因子（VEGF）表达的影响。 方法 将36只大鼠用链脲佐菌素（STZ）腹腔注射制成糖尿病大鼠模型，随机分成糖尿病组(n=18)和塞来昔布灌胃组(n=18)。塞来昔布灌胃组大鼠经口灌胃塞来昔布50 mg/kg；糖尿病组经口灌胃等体积生理盐水。另18只正常大鼠为正常对照组。3个月后处死全部大鼠，应用免疫组织化学技术检测视网膜VEGF蛋白表达，逆转录多聚酶链式反应（RT-PCR）方法检测视网膜VEGF-mRNA和环氧化酶(COX)-2-mRNA的表达。 结果 正常对照组视网膜VEGF-mRNA和COX-2-mRNA表达量少，VEGF免疫组织化学反应弱阳性，VEGF蛋白表达量低；糖尿病组较正常对照组视网膜VEGF-mRNA和COX-2-mRNA表达均上调(P＜0.05)，VEGF免疫组织化学反应呈强阳性，VEGF蛋白表达增高(P＜0.01)；塞来昔布灌胃组较糖尿病组视网膜VEGF mRNA表达显著下降(P＜0.05)，COX-2- mRNA的表达无显著降低(P>0.05)，VEGF免疫组织化学反应阳性减弱，VEGF蛋白表达降低(P＜0.01)。 结论 塞来昔布可通过抑制COX-2的活性进一步抑制STZ诱导的糖尿病大鼠视网膜VEGF-mRNA及蛋白表达。(中华眼底病杂志,2007,23:265-268)     

五苓散对糖尿病视网膜病变大鼠血-视网膜屏障的保护作用

目的：探讨五苓散对糖尿病视网膜病变(DR)大鼠血-视网膜屏障的保护作用及其机制。

方法：SD雄性大鼠50只随机分为空白组、模型组、高剂量组、低剂量组、阳性对照组,每组10只,采用高脂高糖饮食联合腹腔注射链脲佐菌素诱导糖尿病大鼠模型。高剂量组和低剂量组大鼠采用五苓散水煎液灌胃,模型组和空白组大鼠采用等量生理盐水灌胃,阳性对照组大鼠右眼球内注射康柏西普。给药12wk后,采用酶联免疫吸附法(ELISA)检测大鼠血清C反应蛋白(CRP)和可溶性细胞间黏附分子1(sICAM-1)表达水平,HE染色观察视网膜组织结构,伊文思蓝(EB)渗漏试验观察大鼠血-视网膜屏障通透性,免疫组织化学法检测血管内皮生长因子(VEGF)在视网膜中的表达。

结果：模型组大鼠空腹血糖水平和视网膜组织EB渗透量、CRP和sICAM-1表达均较空白组明显升高,高剂量组大鼠视网膜组织EB渗透量、CRP和sICAM-1表达均低于低剂量组和阳性对照组(均P<0.05)。模型组大鼠视网膜神经节细胞层结构紊乱,视网膜水肿,VEGF表达明显升高; 与低剂量组和阳性对照组比较,高剂量组大鼠视网膜组织神经节细胞排列较清晰,水肿减轻。

结论：五苓散能有效降低糖尿病大鼠血清炎症因子水平,降低视网膜组织中VEGF的表达,减轻水肿,保护血-视网膜屏障。