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     

Glutamine synthetase in the developing rat retina: an immunohistochemical study.

The distribution of glutamine synthetase (GS) was studied in rat retinas from the day of birth to adulthood by means of immunohistochemistry. GS was present in the pigment epithelium in the newborn rat, and over the next few days it was demonstrated around blood vessels and within small glial cells. The enzyme was first detected in perikarya and processes of Müller cells on day 5. The adult GS pattern was acquired by day 12, except for persistence of GS in the pigment epithelium. GS in the pigment epithelium gradually diminished to trace amounts between day 12 and 2 months. Additionally, GS was found in the inner epithelium of the ciliary body and posterior epithelium of the iris during the developmental period and in adults. Our results indicate that the dramatic rise in retinal GS biochemically demonstrated by other workers occurs exclusively in Müller cells. Moreover, the complete acquisition of GS in rat retina corresponds chronologically with certain maturational events including the onset of the electroretinographic response. The role of GS in the pigment epithelium and extraretinal structures is uncertain but it may denote their involvement in mucopolysaccharide metabolism.     

Effect of triiodothyronine on glutamine synthetase in the developing rat retina.

It has been previously demonstrated that triiodothyronine (T3) causes a precocious increase in glutamine syntehtase (GS) activity in the developing retina as measured by the glutamyltransferase (GT) reaction. To determine its distribution and the mechanism of its increased activity an immunohistochemical study was performed in retinas of 1 to 24-day-old rats given a subcutaneous injection of T3 on the day of birth. No difference was seen between T3-treated rats and controls on postnatal days 1 and 2. However, there was significantly less stain in T3-injected animals than in control animals especially in the pigment epithelium on day 5 and in Müller cells on days 8. By days 12 and 24 no difference was observed between experimental and control rats. We were thus unable to demonstrate increased synthesis of GS to correlated with its increased activity following T3 administration. On the contrary, we obtained evidence of decreased GS synthesis. It is suggested that T3 either causes both increased GT activity and decreased GS synthesis or that the T3-induced elevation of GS activity results in decreased synthesis of GS presumably through end-product inhibition.     

槲皮素对糖尿病大鼠视网膜单核细胞趋化蛋白-1表达的影响

目的 探讨槲皮素干预糖尿病大鼠视网膜病变的机制.方法 清洁雄性Wistar大鼠,随机分为正常对照组和糖尿病动物组,将成模大鼠随机分为糖尿病组,导升明组,槲皮素组.治疗20周.观察糖尿病大鼠一般情况变化,视网膜血管铺片观察毛细血管内皮细胞数(E)和周细胞数(P),并计算E/P比值,免疫组织化学法观察MCP-1在视网膜的表达,ELISA法检测玻璃体内MCP-1浓度.结果 20周时,与糖尿病阴性对照组相比,槲皮素治疗组体重明显增加,周细胞数明显增多,E/P值明显降低.与导升明阳性对照组相比,槲皮素治疗组体重、周细胞数和E/P值无明显差异.20周时,正常对照组视网膜血管未见MCP-1阳性细胞表达糖尿病组走形迂曲的视网膜毛细血管壁上有大量MCP-1阳性细胞表达,而槲皮素和导升明组视煳膜血管铺片MCP-1表达情况与糖尿病组相比,无明显差别.20周时,正常大鼠玻璃体内存在少量MCP-1,糖尿病组、导升明组和槲皮素组大鼠玻璃体内含有大量MCP-1,相互之间无明显差异.结论 槲皮素对实验性大鼠视网膜病变有一定的治疗作用,但其作用不是通过抑制炎症介质MCP-1表达实现的.     

Hypoxia Induces Galectin-1 Expression Via Autoinduction of Placental Growth Factor in Retinal Pigment Epithelium Cells

PurposeGalectin-1/LGALS1, a β-galactoside-binding protein, contributes to angiogenesis and fibrosis in various ocular diseases. Hypoxia-dependent and -independent pathways upregulate galectin-1/LGALS1 expression in Müller glial cells. Here, we present novel findings on the galectin-1/LGALS1 regulatory system in human retinal pigment epithelium (RPE) cells, the major cellular participant in the pathogenesis of neovascular age-related macular degeneration (nAMD).MethodsHuman RPE cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and immunoblot analyses, respectively. The promoter and enhancer regions of LGALS1 were analyzed by reporter assay and chromatin immunoprecipitation. Immunofluorescence analysis of nAMD patient specimens was used to confirm the in vitro findings.ResultsHypoxia induced galectin-1/LGALS1 expression via binding of hypoxia-inducible factor 1α (HIF-1α) to hypoxia-responsive elements in the LGALS1 promoter region. Blockade of vascular endothelial growth factor receptor 1 (VEGFR1) partially decreased hypoxia-induced galectin-1/LGALS1 expression. Among several VEGFR1 ligands induced by hypoxia, placental growth factor (PlGF)/PGF alone upregulated galectin-1/LGALS1 expression via phosphorylation of activator protein 1 (AP-1) subunits following AKT and p38 mitogen-activated protein kinase (MAPK) activation. An AP-1 site in the LGALS1 enhancer region was required for PlGF-induced galectin-1/LGALS1 expression in RPE cells. PlGF application upregulated PGF expression via extracellular signal-regulated kinase 1 and 2, AKT, and p38 MAPK pathways. nAMD patient specimens demonstrated co-localization of galectin-1 with HIF-1α, PlGF, and VEGFR1 in RPE cells.ConclusionsOur present findings implicate the significance of hypoxia as a key inducer of galectin-1/LGALS1 in RPE cells and the autoinduction of hypoxia-induced PlGF as a vicious cycle amplifying the pathogenesis of nAMD.     

Soluble IL-6 Receptor in Vitreous Fluid of Patients with Proliferative Diabetic Retinopathy

Purpose

To identify the biological reaction of soluble interleukin-6 receptor (sIL-6R) in the vitreous of patients with proliferative diabetic retinopathy (PDR).

Methods

The subjects were 45 patients (45 eyes) with vitreoretinal diseases. The patients were divided into three groups: the PDR group comprised 28 patients (28 eyes) with PDR; the pre-proliferative diabetic retinopathy (PPDR) group comprised seven patients (seven eyes) with PPDR combined with diabetic macular edema; and the nondiabetic group comprised ten patients (ten eyes) with idiopathic macular hole or idiopathic epiretinal membrane. Vitreous samples were obtained at vitrectomy. sIL-6R, vascular endothelial growth factor (VEGF), and protein concentration in vitreous samples were determined by enzyme-linked immunosorbent assay (ELISA). sIL-6R levels in serum were also determined by ELISA in nine of the 28 patients with PDR and in six healthy volunteers as controls.

Results

In vitreous fluid, the levels of sIL-6R in the PDR group, PPDR group, and nondiabetic group were 612.7 ± 233.8 (mean ± SD), 746.3 ± 523.1, and 215.4 ± 98.3?pg/ml, respectively. Vitreous levels of sIL-6R in the PDR and PPDR groups were significantly higher than those in the nondiabetic group (PDR group, P < 0.0001; PPDR group, P < 0.01). In serum, the levels of sIL-6R were 39.38 ± 9.43?ng/ml in the PDR group and 22.84 ± 5.32?ng/ml in the control group. sIL-6R levels in serum in the PDR group were significantly higher than those in the control group (P < 0.01). A partial correlation analysis showed a significant correlation between the levels of sL-6R and VEGF in the vitreous in the PDR group (r = 0.34, P < 0.05).

Conclusions

We conclude that the level of sIL-6R in vitreous fluid can be considered as a biomarker of PDR.?Jpn J Ophthalmol 2007;51:100–104 © Japanese Ophthalmological Society 2007

     

CD146/Soluble CD146 Pathway Is a Novel Biomarker of Angiogenesis and Inflammation in Proliferative Diabetic Retinopathy

PurposeInflammation, angiogenesis and fibrosis are pathological hallmarks of proliferative diabetic retinopathy (PDR). The CD146/sCD146 pathway displays proinflammatory and proangiogenic properties. We investigated the role of this pathway in the pathophysiology of PDR.MethodsVitreous samples from 41 PDR and 27 nondiabetic patients, epiretinal fibrovascular membranes from 18 PDR patients, rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy analysis. Blood-retinal barrier breakdown was assessed with fluorescein isothiocyanate-conjugated dextran.ResultssCD146 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic patients. In epiretinal membranes, immunohistochemical analysis revealed CD146 expression in leukocytes, vascular endothelial cells and myofibroblasts. Significant positive correlations were detected between numbers of blood vessels expressing CD31, reflecting angiogenic activity of PDR, and numbers of blood vessels and stromal cells expressing CD146. Western blot analysis showed significant increase of CD146 in diabetic rat retinas. sCD146 induced upregulation of phospho-ERK1/2, NF-κB, VEGF and MMP-9 in Müller cells. The hypoxia mimetic agent cobalt chloride, VEGF and TNF-α induced upregulation of sCD146 in HRMECs. The MMP inhibitor ONO-4817 attenuated TNF-α-induced upregulation of sCD146 in HRMECs. Intravitreal administration of sCD146 in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, intercellular adhesion molecule-1 and VEGF in the retina. sCD146 induced migration of HRMECs.ConclusionsThese results suggest that the CD146/sCD146 pathway is involved in the initiation and progression of PDR.     

Levels of Interleukin 27 and Interleukin 35 in the Serum and Vitreous of Patients with Proliferative Diabetic Retinopathy

Purpose: To examine the role of interleukin 27(IL-27) and interleukin 35 (IL-35) in diabetic retinopathy (DR).

Methods: Patients with diabetes mellitus were divided into three groups: diabetes without retinopathy (DWR), non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR). Patients with idiopathic macular epiretinal membrane (IMEM) were included as a control group. The serum and vitreous levels of IL-27 and IL-35 were measured using ELISA.

Results: The serum levels of IL-27 (median 240.900 pg/mL, range 42.224 – 617.810 pg/mL; p < 0.001) and IL-35 (median 11.875 ng/mL, range 8.640 – 19.340 ng/mL; p < 0.001) were significantly decreased in PDR patients compared to controls (median 2712.310 pg/mL, range 1005.375–5786.877 pg/mL and median 25.185 ng/mL, range 22.845 – 29.590 ng/mL, respectively). The vitreous levels of IL-35 were significantly decreased in PDR patients (16.32 ± 3.24 ng/mL) compared to controls (24.54 ± 5.86 ng/mL, p < 0.001).

Conclusions: Serum and vitreous levels of IL-35 and serum level of IL-27 may be associated with the pathogenesis of PDR.     

增殖性糖尿病视网膜病变玻璃体SDF-1和VEGF的含量分析

研究增殖性糖尿病视网膜病变患者玻璃体基质细胞衍生因子（Stromalcell—derivedfactor-1。SDF-1）和血管内皮生长因子（Vascularendothelialgrowthfactor，VEGF）的浓度，及其相互作用关系。方法：酶联免疫吸附法（Enzyme-linkedimmunosorbentassay，ELISA）检测玻璃体内SDF-1和VEGF的含量，每个标本重复3次。实验组为增殖性糖尿病视网膜病变（Proliferativediabeticretinopathy，PDR）的住院患者30例，对照组为同期行玻璃体切除术的特发性黄斑裂孔患者12例。结果：PDR患者玻璃体VEGF的平均浓度为（2865．87±387．85）pg／ml，明显高于特发性黄斑裂孔组[（142．42±21．03）pg／ml，P〈0．0001]。增殖性糖尿病视网膜病变患者玻璃体SDF-1的含量平均为（298．40±24．57）pg／ml，对照组为（86．9l±15．89）pg／ml，两组的差异具有统计学意义（P〈0．0001）。在30例PDR患者玻璃体内VEGF和SDF-1的含量表现为正相关（Pearson相关系数r=0．62，P〈0．001）。结论：增殖性糖尿病患者玻璃体SDF-1和VEGF的含量均高于非糖尿病患者，提示SDF-1和VEGF共同参与了增殖性糖尿病视网膜病变患者病理性新生血管的形成过程。     

Sleep-Disordered Breathing Is a Stronger Risk Factor for Proliferative Diabetic Retinopathy than Metabolic Syndrome and the Number of Its Individual Components

Purpose: To evaluate whether the features of sleep-disordered breathing (SDB) are stronger independent factors for proliferative diabetic retinopathy (PDR) compared to the incidence of metabolic syndrome (MetS) and the number of its individual components.

Methods: We studied a cross-sectional total of 132 patients with type 2 diabetes. Thirty-nine patients had non-proliferative diabetic retinopathy (NPDR) and 93 patients had PDR. Pulse oximetry was conducted, and the patients’ mean oxygen saturation (mean SpO2%) and 4% oxygen desaturation index (4% ODI times/hour) were evaluated. We compared the SDB and MetS variables between the NPDR and PDR patients. A logistic regression analysis was used to determine the independent factors for the diagnosis of PDR.

Results: The MetS diagnosis was made significantly more often in the PDR group (p = 0.04). The number of individual MetS components was significantly greater in the PDR group compared to the NPDR group (p = 0.01). The mean SpO2 of the NPDR group was not significantly different from that of the PDR group. The 4% ODI in the NPDR group was significantly lower than that in the PDR group (p = 0.01). The logistic regression analysis using the prevalence of MetS and the number of MetS components revealed that younger age and high 4%ODI value were independent factors contributing to the diagnosis of PDR.

Conclusion: Our findings confirmed that compared to MetS and the number of its individual components, SDB may be a factor contributing to the progression to PDR. However, further careful longitudinal validation studies are needed.     

糖尿病视网膜病变玻璃体切割术前康柏西普玻璃体腔注射时机对VEGF表达水平和视力恢复的影响

目的：探讨糖尿病视网膜病变（DR）玻璃体切割术前康柏西普玻璃体腔注射时机对新生血管膜血管内皮生长因子（VEGF）表达水平及患者视力恢复的影响。方法：前瞻性临床研究。将2015 年5 月至2017 年5 月重庆市开州区人民医院眼科收治的109 例DR患者按随机数余数法分为A组（32 例）、B组（43 例）、C组（34 例）,3 组均行玻璃体切割术,A组术前不予玻璃体腔内注射康柏西普,B组术前3 d术眼玻璃体腔注射康柏西普,C组术前5 d术眼玻璃体腔注射康柏西普,观察3 组手术情况,记录手术时间、术中出血量及医源性裂孔发生情况,所有患者术中留取视网膜下新生血管膜标本,测定VEGF表达水平;观察各组并发症发生率,比较手术前后不同时间患者最佳矫正视力（BVCA）的变化情况。 数据采用单因素方差分析进行比较。结果：B、C组手术时间及术中出血量均少于A组（P<0.05）,术中电凝止血率、医源性视网膜裂孔率均低于A组,术后玻璃体内出血发生率较A组低（P<0.05）,术后角膜水肿、高眼压、前房炎性反应发生率均低于A组（P<0.05）,但B、C组组间比较差异无统计学意义（P>0.05）。B、C组新生血管膜VEGF阳性率低于A组,但B、C组组间比较差异无统计学意义（P>0.05）。术后3、6个月,3组BCVA均上升（P<0.05）,B、C组术后3、6个月BCVA高于A组（P<0.05）,但组间比较差异无统计学意义（P>0.05）。结论：DR患者术前玻璃体腔内注射康柏西普均可缩短手术时间,降低术中、术后并发症发生率,降低新生血管膜VEGF表达水平,促进患者术后视力恢复,但术前3 d或5 d注射效果并无明显差别。     

Elevated Vascular Endothelial Growth Factor Levels in the Vitreous of Patients With Proliferative Diabetic Retinopath

Objective: To detect the levels of vascular endothelial growth factor (VEGF) in the vitreous of patients with proliferative diabetic retinopathy (PDR) and to investigate the possible role of VEGF in the development of neovascularization in PDR. Methods ; Undiluted vitreous samples and fasting venous blood samples were obtained from 27 patients with PDR and 14 subjects with idiopathic macular hole who underwent pars plana vitrectomy. The concentration of VEGF was determined by quantitative enzyme - linked immunosorbent assay (ELISA).Results: The level of vitreous VEGF in patients with PDR (median 0. 41ng/ml, range 0. 09- 11. 56ng/ml) was significantly elevated when compared with that in control subjects (median 0.017ng/ml, range 0.008-0.04ng/ml)(P<0. 001). The median of PDR patients' serum VEGF concentration was 0.19ng/ml (0. 090. 46ng/ ml) which was far lower than vitreous VEGF concentration (P<0. 05). Vitreous VEGF concentration was higher in PDR patients with retinal detachment than that in patient wi     

单点与多点扫描模式激光治疗糖尿病视网膜病变疗效比较

目的：:分析比较单点与多点扫描模式全视网膜激光光凝术（PRP）对非增殖性糖尿病视网膜病变（NPDR）患者的疗效及对视网膜结构和功能的影响。方法：:回顾性系列病例研究。选择2019年1月至2020年7月在青岛市市立医院被确诊为重度NPDR后行PRP治疗且随访6个月以上的患者57例（93眼）。其中27例（46眼）行单点扫描...     

Effect of Hypericin on Confocal Imaging of Ca^2 Signaling in Cultured Human Retinal Pigment Epithelium

Purpose:To investigate the mechanism of the Ca^2 signaling in cultured human retinal pigment epithelial(RPE) cells with the protein kinase C(PKC) specific inhibitor-hypericin stimulation.Methods: Cultured human RPE cells were analyzed using the fluorescence Ca^2 dye fluo-3 AM and laser scanning confocal microscope(LSCM) after stimulation with 100nM phorbol 12-myristate 13-acetate(PMA)and (or)5 concentrations of hypericin(1,2,3,4and 5μM)。Results:The normal fluorescence in RPE cells was strong and distributed throughout the cells,The nucleus appeared to be more fluorescent than the cytoplasm.After stimulation with PMA alone or 5 concentrations of hypericin,a rapid decrease in flurescence intensity was observed.There was no obvious difference in decreased curve among 5 concentrations,However,after stimulation with a 24 hr preincubation of PMA and 5 concentrations of hypericin,a further decrease was not observed.Conclusion:Fluo-3 AM appears to be a good indicator of the change in Ca^2 occurring in RPE cells and hypericin is a strong inhibitor of Ca^2 influx channel,Hypericin has potential as a therapeutic drug for proliferative vitreoretinopathy(PVR),the inhibitory effect on PVR might be caused by blocking the PKC activity and inhibiting Ca^2 influx Pathway.Eye Science 2001;17:148-153.     

Pan-Retinal Argon Laser Photocoagulation in the Treatment of Diabetic Retinopathy – A Review of its Effects on Vision and the Eye

ABSTRACT A summary of major papers that investigate the treatment rationale, the effects on visual function and the complications of argon laser photocoagulation in proliferative diabetic retinopathy is presented. The often confusing and sometimes contradictory results of these studies, particularly in the area of visual function after treatment, suggest a need for further research on the subject.     

白细胞介素-2对高糖状态下人视网膜色素上皮细胞的影响

目的研究白细胞介素-2(IL-2)对高糖状态下人视网膜色素上皮(RPE)细胞的增殖及其分泌和表达血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)的影响。方法MTT自动比色法观察IL-2浓度对高糖状态下RPE细胞增殖的作用;ELISA法测RPE细胞分泌VEGF的变化;免疫组化观察RPE细胞表达VEGF的变化。结果0.1～10μg/L的IL-2均能明显促进高糖状态下RPE细胞的增殖,并能明显提高RPE细胞分泌VEGF的水平及提高VEGF在细胞中的表达。结论高糖状态下,IL-2可明显促进人RPE细胞的增殖并能提高RPE细胞分泌VEGF的水平和提高VEGF在RPE细胞中的表达,IL-2可能在增殖性糖尿病视网膜病变中起一定的作用。     

蒙药明目十一味丸对糖尿病视网膜病变Ⅰ期视网膜功能的影响

评价蒙药明目十一味丸治疗糖尿病视网膜病变（DR）Ⅰ期的临床疗效。方法：前瞻性临床研究。选取2016年7月至2017年3月在内蒙古民族大学附属医院眼科就诊的DRⅠ期患者120例（120眼），通过随机数字表法将其分成蒙药组和对照组各60例，蒙药组加用明目十一味丸，对照组加用羟苯磺酸钙胶囊，一个疗程28 d，共3个疗程。分别在治疗前后对2组患者进行多焦视网膜电图（mfERG）及超广角眼底照相检查，记录N1区域和P1区域的潜伏期、振幅变化及视网膜微动脉瘤和出血点的数目。采用t检验、χ2 检验对数据进行分析。结果：对照组和蒙药组在治疗前后mfERG测得的R1～R5 P1 区域的振幅密度值差异有统计学意义（t对照组=66.86、18.05、36.33、47.43、30.56，P<0.001； t蒙药组=31.52、54.56、101.98、127.02、45.74，P<0.001），N1区域的振幅密度值差异有统计学意义（t对照组=70.18、47.02、78.08、57.44、64.51，P<0.001；t蒙药组=46.09、140.47、145.14、50.46、73.94， P<0.001）。与对照组相比，蒙药组治疗前后R1～R5 P1、R1～R3 N1区域的振幅密度值差值差异有统计学意义（tP1=8.96、23.96、25.10、28.80、13.67，P<0.001；tN1=9.38、30.34、52.06，P<0.001）， R4～R5 N1区域的振幅密度值差值差异无统计学意义。2组治疗前后潜伏期比较，R1～R5 P1区域、N1区域的差异均无统计学意义。2组治疗前后微动脉瘤及出血点数目比较，差异有统计学意义（t=2.08，P=0.042；t=2.07，P=0.043）。眼底照相蒙药组与对照组有效率分别达88%、73%，差异有统计学意义（χ2 =4.36，P=0.037）。结论：蒙药明目十一味丸能明显改善Ⅰ期DR视网膜功能的损伤，疗效显著，可作为治疗DRⅠ期的常规用药。