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     

缺氧诱导因子-2α在增生型糖尿病视网膜病变新生血管形成中的作用

目的观察HIF-2α在增生型糖尿病视网膜病变(PDR)纤维血管膜中的表达,初步探讨HIF-2α在PDR新生血管形成中的作用。方法回顾性临床研究。2014年7月至2015年7月于青岛大学附属医院眼科行玻璃体切割手术的PDR患者57例60只眼纳入研究。根据手术前是否行玻璃体腔注射抗VEGF药物治疗将患眼分为PDR未注药组和PDR注药组,分别为30例32只眼、27例28只眼。PDR注药组患眼手术前2~7 d玻璃体腔注射10 mg/ml雷珠单抗0.05 ml(含雷珠单抗0.5 mg)。选取同期特发性黄斑前膜患者18例18只眼作为对照组。于玻璃体切割手术中获得PDR纤维血管膜、黄斑前膜病理标本。免疫组织化学染色法及荧光定量PCR(RT-PCR)检测各组病理标本中HIF-2α、Delta样配体4(Dll4)、VEGF的表达。多组间相关因子表达差异比较采用Kruskal-Wallis检验。两变量间关系行Spearman相关性分析。结果免疫组织化学染色结果显示,PDR未注药组、PDR注药组纤维血管膜中HIF-2α、Dll4、VEGF蛋白表达均呈阳性。PDR未注药组HIF-2α、Dll4、VEGF蛋白相对表达量均高于PDR注药组,差异均有统计学意义(t=4.36、6.01、4.82,P=0.000、0.008、0.016)。RT-PCR检测结果显示,PDR未注药组、PDR注药组、对照组纤维血管膜、黄斑前膜中HIF-2α、Dll4、VEGF mRNA相对表达量比较,差异均有统计学意义(H=18.81、19.60、20.50,P=0.000、0.000、0.000)。其中,PDR未注药组、PDR注药组HIF-2α、Dll4、VEGF mRNA相对表达量均显著高于对照组;与PDR未注药组比较,PDR注药组HIF-2α、Dll4、VEGF mRNA相对表达量均降低。Spearman相关性分析结果显示,HIF-2α与Dll4、VEGF mRNA相对表达量呈显著正相关(r=0.95、0.87,P<0.05)。结论PDR患眼纤维血管膜中HIF-2α表达高于对照组,且与DLL4、VEGF的表达均呈显著正相关。     

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.     

Clinical and histological features of epiretinal membrane after diabetic vitrectomy

Purpose

To investigate the clinical, histopathological, and surgical features of the epiretinal membrane (ERM) after diabetic vitrectomy (DV).

Methods

From August 2007 to January 2010, clinical charts of consecutive proliferative diabetic retinopathy (PDR) cases with significant post-DV ERM, defined as thickened membrane causing macular distortion and vision decrease, were enrolled as the study group; PDR cases without post-DV ERM in 24 months follow-up served as the control group. Factors associated with post-DV ERM formation, morphological and visual changes before and after ERM surgery, and histopathological features were analyzed.

Results

Sixteen eyes were in the ERM group, while 60 eyes were in the control group. Active PDR (p?<?0.001), fibrovascular proliferation (FVP) grade (p?=?0.001), post-DV hemorrhage (p?=?0.012), and residual fibrovascular stump (p?=?0.002) were factors significantly associated with post-DV ERM. Most membranes (87.5 %) developed within 12 months, were widespread beyond the arcade (81.3 %), and connected with retinal vessels (87.5 %). After surgery, significant VA improvement was achieved. ERM recurrence was noted in six eyes (37.5 %). Histopathological examinations of ERMs in six cases showed abundant collagen fibers with epithelial cells. Immunohistochemical staining with CD 34 demonstrated the presence of vascular endothelium in two of the six specimens.

Conclusion

The post-DV ERM is a complex tissue with variable vascularity that often presents with widespread distribution, rapid progression, and causes macular distortion. Associated risk factors include active PDR, high FVP grade, post-DV hemorrhage, and residual fibrovascular stumps. Membrane removal surgery may be beneficial in selected cases, but recurrence is not uncommon.     

PDR患者玻璃体手术前后房水中VEGF水平的变化

目的：检测增殖性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)患者玻璃体手术前后房水中血管内皮生长因子(vascular endothelial growth factor, VEGF)的浓度变化,研究VEGF在PDR发病机制中的作用。

方法：收集30例PDR患者玻璃体手术前后的房水标本,以30例无糖尿病的白内障患者作为正常对照组,用酶联免疫吸附分析(enzyme-linked immunosorbent assay, ELISA)方法检测VEGF的浓度。

结果：PDR患者与正常对照组相比,房水中VEGF浓度显著升高,有统计学差异(P<0.05)。PDR患者玻璃体视网膜手术后与手术前相比,房水中VEGF浓度显著降低,有统计学差异(P<0.05)。

结论：VEGF积极参与了PDR的发生发展,并与PDR后期新生血管形成有着密切的关系。     

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     

Outcome of vitreous surgery and the balance between vascular endothelial growth factor and endostatin

PURPOSE: To predict the results of vitreous surgery in patients with proliferative diabetic retinopathy (PDR), the correlation between vitreous fluid levels of vascular endothelial growth factor (VEGF) or endostatin and the postoperative outcome was investigated. METHODS: VEGF and endostatin levels in vitreous fluid specimens obtained during vitreous surgery were measured by enzyme-linked immunosorbent assay. Expression of VEGF and endostatin in epiretinal membranes was assessed immunohistochemically. Patients were prospectively followed up for 6 months. RESULTS: No improvement and/or progression of PDR was seen in 11 (25%) of 44 eyes (progression group). The vitreous fluid level of VEGF was significantly higher in the progression group than in the regression group (P = 0.0023). Conversely, the vitreous fluid level of endostatin was significantly higher in the regression group than in the progression group (P = 0.0299). Eyes with a high vitreous fluid level of VEGF and a low endostatin level had a significantly greater risk of progression of PDR after vitreous surgery than did eyes with low VEGF and high endostatin levels (odds ratio = 10.00, P = 0.047). VEGF and endostatin were detected immunohistochemically in the fibrovascular epiretinal membranes resected from the subjects. CONCLUSIONS: In this study both VEGF and endostatin were expressed in eyes with PDR. VEGF and endostatin levels in the vitreous fluid correlated with the outcome of vitreous surgery for PDR. Therefore, the outcome of PDR surgery can probably be predicted by measuring cytokines and/or growth factors in the vitreous fluid, with VEGF and endostatin being good candidates.     

Decreased prothrombin time after intravitreal bevacizumab in the early period in patients with proliferative diabetic retinopathy

Purpose: To evaluate the effect of intravitreal bevacizumab (Avastin; Genentech, Inc., San Francisco, CA, USA) (IVB) injection on prothrombin time (PT) in patients with proliferative diabetic retinopathy (PDR) and nondiabetic patients. Methods: Eighty‐nine patients received primary IVB (1.25 mg) in one eye were investigated. The patients were divided into three groups: 34 PDR (diabetic group), 26 nondiabetic (nondiabetic group) patients received IVB and 29 PDR patients without IVB (control group). The levels of PT were detected before and after IVB in study groups and at corresponding time‐points in the control group. Paired samples t‐test was conducted to compare the statistical differences of PT in each group. Results: Significant difference (p < 0.001) of the levels of PT before and after IVB was observed in diabetic group in the early period, and the PT changes (11.0 ± 0.56 before and 10.6 ± 0.45 after IVB) were in fact within the normal range. There were no significant differences in other groups and other time‐points (all p > 0.05). Conclusions: Intravitreal bevacizumab may lead to a decrease in the levels of PT in the early period after IVB in PDR patients, suggesting a temporarily potential effect of IVB on the extrinsic clotting pathway of blood coagulation cascade in diabetic patients.     

糖尿病视网膜病变患者血清VEGF、ES、TKLK、TSP、sICAM-1水平的变化及意义

目的:探讨血清血管内皮细胞生长因子(VEGF)、内皮抑素(ES)、血小板反应蛋白(TSP)、组织激肽释放酶(TKLK)及可溶性细胞间黏附分子-1(sICAM-1)水平在糖尿病视网膜病变(DR)患者血清中的变化及其临床意义.方法:选取我院2014-01/2016-12收集的无眼底病变的糖尿病患者60例(DM组)、非增殖性糖尿病视网膜病变患者60例(NPDR组)、增殖性糖尿病视网膜病变患者60例(PDR组)和同期健康体检对象60例(对照组),检测四组患者血清VEGF、ES、TSP、TKLK、sICAM-1水平并进行比较分析.结果:PDR组患者的血清VEGF、TKLK、sICAM-1水平均显著高于NPDR组、DM组、对照组(P<0.05);PDR组患者的血清ES水平均显著低于NPDR组、DM组、对照组(P<0.05).NPDR组患者的血清VEGF、TKLK、ES水平均显著高于DM组和对照组(P<0.05).NPDR组患者的血清VEGF与ES、TKLK、sICAM-1水平均呈显著的正相关关系(P<0.05).PDR组患者的血清VEGF与TKLK、sICAM-1水平均呈显著的正相关关系(P<0.05),PDR组患者的血清VEGF与ES、TSP相关性不显著(P>0.05).结论:DR患者血清ES、TSP、TKLK、sICAM-1水平均发生显著改变,通过调节VEGF水平影响DR进程.     

Expression of integrins in human proliferative diabetic retinopathy membranes

Background: The process of identifying molecules that regulate angiogenesis is critical to the success of candidate therapies for ocular neovascular disease. The purpose of the study was to determine the pattern of expression for integrins and their colocalization with endothelium in membranes from proliferative diabetic retinopathy (PDR).Methods: Clinically categorized membranes were collected from vitreoretinal surgery. A double immunohistochemical staining procedure was used to identify the presence and colocalization of integrins and endothelium. Five integrins were examined.Results: Endothelial markers were robust in all 4 active-stage PDR membranes but absent in the fibrotic-stage PDR membrane. The expression of ανβ3 and β3 integrins on endothelial cells was observed with low to moderate intensity. The expression of α|β| and βa2β| was moderate but was not colocalized with endothelial cells in active-stage PDR membranes. Integrin ανβ5 was not evident in any of the samples used in this study.Interpretation: The results suggest an essential role of integrins ανβ3 and β3 in the pathogenesis of PDR. It is suggested that ανβ3 and β3 are preferred candidate targets for therapeutic development.     

Circulating bone‐marrow‐derived endothelial precursor cells contribute to neovascularization in diabetic epiretinal membranes

Purpose: The role of vasculogenesis, recruitment and differentiation of circulating bone‐marrow‐derived endothelial precursor cells into mature endothelium in proliferative diabetic retinopathy (PDR) remains undefined. We investigated the presence of bone‐marrow‐derived endothelial precursor cells and the expression of the chemotactic pathway SDF‐1/CXCL12?CXCR4 in PDR epiretinal membranes. Methods: Membranes from eight patients with active PDR and nine patients with inactive PDR were studied by immunohistochemistry using antibodies against CD133, vascular endothelial growth factor receptor‐2 (VEGFR‐2), CD14, SDF‐1 and CXCR4. Results: Blood vessels expressed CD133, VEGFR‐2, CD14, SDF‐1 and CXCR4 in 10, 10, 10, seven and seven out of 17 membranes, respectively. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing CD133 (r_s = 0.646; p = 0.005), VEGFR‐2 (r_s = 0.704; p = 0.002), CD14 (r_s = 0.564; p = 0.018) and SDF‐1 (r_s = 0.577; p = 0.015). Stromal cells in close association with blood vessels expressed CD133, VEGFR‐2, CD14 and CXCR4 in 10, 12, 13 and 14 membranes, respectively. The number of blood vessels expressing CD133 (p = 0.013), VEGFR‐2 (p = 0.005), CD14 (p = 0.008) and SDF‐1 (p = 0.005), and stromal cells expressing CD133 (p = 0.003), VEGFR‐2 (p = 0.013) and CD14 (p = 0.002) was significantly higher in active membranes than in inactive membranes. Conclusion: Bone‐marrow‐derived CD133⁺ endothelial progenitor cells and CD14⁺ monocytes may contribute to vasculogenesis in PDR.     

Elevated protein carbonyl and HIF‐1α levels in eyes with proliferative diabetic retinopathy

Purpose: To evaluate the role of protein carbonyls and hypoxia inducible factor‐1α (HIF‐1α) in diabetic eyes with proliferative diabetic retinopathy (PDR). Methods: Prospective consecutive controlled observational study was performed. Vitreous samples were collected at the start of the 3‐ppp vitrectomy. Protein carbonylation analysis was performed by Western blotting with antibody against 2,4‐Dinitrophenol (anti‐DNP), following derivatization of protein carbonyls with 2,4 Dinitrophenylhydrazine (DNHP). Protein carbonylation was quantified by scanning densitometry analysis and relativized to the total amount of protein into the ponceau staining of membranes. Vitreous HIF‐1 α was determined with ELISA in a subgroup of the samples. Thirty‐one eyes were operated due to PDR (study group). Of the 189 controls, 39 had nonproliferative diabetic retinopathy (non‐PDR), 111 retinal detachment (RD) and 39 macular hole/pucker (MH). Results: Comparison of eyes with PDR with controls revealed that the mean vitreous concentrations of protein carbonyls were significantly higher in the eyes affected with PDR being 242 ± 130 (SD) compared with non‐PDR controls 180 ± 142, nondiabetic eyes affected with RD 175 ± 131 and MH/pucker 140 ± 95 (p = 0.008, one‐way anova ). Mean HIF‐1α values were higher in eyes with PDR compared with controls (RD, MH/pucker); the values being 0.53 ± 0.34 (SEM; n = 4) and 0.13 ± 0.04 (SEM; n = 19), respectively (p = 0.009). Conclusions: Protein carbonyl and HIF‐1 α levels were significantly increased in the vitreous fluid of surgically treated eyes with PDR. Our findings suggest an association between increased intravitreal levels of protein carbonyls and the pathogenesis of PDR.     

VEGF在PDR中的表达及作用的研究

目的:研究增殖性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)患者血液、房水、玻璃体中血管内皮生长因子(vascular endothelial growth factor,VEGF)含量的变化,探讨VEGF与PDR的关系,为抗VEGF药物治疗的给药途径及剂量等提供理论依据。方法:采用双抗体夹心酶联免疫吸附测定法定量检测无糖尿病视网膜病变(NDR)组,单纯性糖尿病视网膜病变(BDR)组,增殖性糖尿病视网膜病变(PDR)组患者和正常对照组血浆中VEGF含量,还检测PDR患者房水、玻璃体中和正常对照组房水、玻璃体中VEGF含量,并进行综合分析。试剂盒购自美国R&D公司,其质量和灵敏度相对较高。结果:PDR组房水中VEGF含量有增高趋势,但与正常对照组比较,无统计学差异(P>0.05)。PDR患者玻璃体中VEGF含量明显增高,与正常对照组比较差异非常显著(P<0.01)。PDR组自身血浆、房水、玻璃体中VEGF含量比较有逐渐增高趋势,三者之间有显著性差异(P<0.01)。正常对照组血浆、房水、玻璃体中VEGF含量三者之间无显著性差异(P>0.05)。血浆VEGF含量在正常对照组中最高,而玻璃体中VEGF含量在PDR患者中最高。结论:PDR患者眼内尤其是玻璃体中VEGF含量大幅度增高,可能对促进DR发展恶化起了关键性的作用。在正常人,VEGF更多地存在于血浆中发挥其生物学效应。在严重DR患者中,玻璃体中异常地出现大量VEGF,推测来自缺血缺氧的视网膜,并可能有向眼前段扩散的趋势。     

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...     

Profile of intraocular tumour necrosis factor‐α and interleukin‐6 in diabetic subjects with different degrees of diabetic retinopathy

Purpose: To assess and correlate the levels of inflammatory mediators in the eyes from non‐diabetic and diabetic subjects without retinopathy (NDR), with non‐proliferative diabetic retinopathy (NPDR) or with proliferative diabetic retinopathy (PDR) to corresponding erum levels. Methods: The levels of interleukin 1β, interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) were analysed by an ELISA‐mimicking technique in the vitreous from 26 diabetic subjects with active PDR and 27 non‐diabetic subjects, or by a multiplex bead assay in the aqueous humour from 35 diabetic subjects with NDR/NPDR and 40 non‐diabetic subjects. Intraocular protein production was estimated in vitreous specimens by calculating a vitreous/serum ratio. Results: In the vitreous, IL‐6 was higher in diabetic [157.5 (25.0–1401.0) pg/ml; median (min–max)] than in non‐diabetic subjects [44.0 (5.0–4425) pg/ml; p = 0.021]. The vitreous/serum ratio was high (55.5:1 and 16:1, respectively), suggesting intraocular production. TNF‐α was lower in diabetic [18.0 (8.0–46.0) pg/ml] than in non‐diabetic subjects [22.0 (13.0–47.0) pg/ml; p = 0.034], but the vitreous/serum ratio was elevated in both groups (2:1 and 3.4:1, respectively). TNF‐α levels were higher in serum from diabetic subjects [9.0 (5.0–53.0) pg/ml versus 6.7 (3.0–11.0) pg/ml; p < 0.001]. Aqueous levels of inflammatory mediators did not differ between diabetic subjects with NDR/NPDR and non‐diabetic subjects despite elevated TNF‐α in serum [27.8 (6.8–153.7) pg/ml versus 16.4 (4.1–42.4) pg/ml; p = 0.021]. Conclusion: Intraocular inflammation seems to be involved in PDR but does not seem to be prominent in early retinopathy stages, i.e. NDR or NPDR. Diabetic subjects have an overall increased inflammatory activity compared to non‐diabetic subjects, as demonstrated by increased serum levels of TNF‐α.     

链脲佐菌素诱导的小鼠糖尿病视网膜病模型及促血管新生分子的表达

目的：：建立链脲佐菌素( streptozotocin, STZ)诱导的小鼠增殖性糖尿病视网膜病( proliferative diabetic retinopathy, PDR)动物模型,并观察在增殖性糖尿病视网膜病发生、发展过程中血管内皮生长因子( vascular endothelial growth factor, VEGF)及其受体(VEGFR1, VEGFR2),金属基质蛋白酶(matrix metalloproteinase, MMP)2, MMP9表达的变化。方法：C57 BL/6 J小鼠连续5 d腹腔注射STZ (55 mg/kg )。末次注射后7d检测血糖浓度。糖尿病诱导成功的小鼠和正常小鼠继续饲养3~5 mo。实验结束后进行视网膜病理组织观察,并利用CD31免疫荧光染色检测视网膜血管的分布情况。采用实时定量荧光 PCR 分析检测 VEGF, VEGFR1, VEGFR2, MMP2, MMP9的基因表达。结果：视网膜组织病理观察和CD31免疫荧光染色实验结果均表明5月龄的糖尿病小鼠视网膜组织中血管数目明显比同月龄正常小鼠多。同时,与同月龄正常小鼠相比,5月龄糖尿病小鼠视网膜组织中 VEGF, VEGFR1, VEGFR2, MMP2, MMP9的基因表达也明显增加。结论：本研究表明STZ诱导的糖尿病小鼠在5月龄时发生     

增殖性视网膜病变玻璃体切割物的细胞学及免疫组化研究

目的探讨不同视网膜增殖性疾病细胞增殖的特征及其异同。方法采用３种特异性抗原，即抗人细胞角蛋白（ｃｙｔｏｋｅｒａｔｉｎ，ＣＫ），抗胶质纤维酸性蛋白（ｇｌｉａｌｆｉｂｒｉｌａｒｙａｃｉｄｉｃｐｒｏｔｅｉｎ，ＧＦＡＰ），抗肌动蛋白（Ａｃｔｉｎ）对２８例临床上不同的视网膜疾病的增殖膜及玻璃体切除液样本进行免疫细胞化学研究。结果增殖性玻璃体视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｖｉｔｒｅｏｒｅｔｉｎｏｐａｔｈｙ，ＰＶＲ）的增殖特征以胶质细胞和视网膜色素上皮细胞（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）为主，二者在ＰＶＲ发病中作用不同。增殖性糖尿病视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎａｏｐａｔｈｙ，ＰＤＲ）玻璃体内出现ＲＰＥ细胞可加剧增殖膜收缩。增殖膜血管壁上肌动蛋白含量增多可能对周细胞脱落起促进作用。结论玻璃体内细胞增殖与生长因子水平升高可能是增殖性病变日益加剧的重要机制和原因之一。     

Basic fibroblast growth factor mRNA, bFGF peptide and FGF receptor in epiretinal membranes of intraocular proliferative disorders (PVR and PDR)

Basic fibroblast growth factor (bFGF) has been shown to be involved in epiretinal membrane formation in proliferative vitreoretinal disorders. However, up to now, little knowledge exists, as to the actual cellular source of this potent mitogen.We examined 20 epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) (n = 12) and proliferative vitreoretinopathy (PVR) (n = 8) for the presence of bFGF peptide, fibroblast growth factor receptor-1 (FGFR-1) and bFGF messenger ribonucleic acid (mRNA).Using a specific antibody, we detected bFGF peptide in most (8/10) examined PDR membranes and in all (8/8) PVR membranes. Moreover, we found positive staining for the corresponding receptor.Local production of bFGF in epiretinal membranes was confirmed by nonisotopic in situ hybridisation for bFGF mRNA in some (4/7) examined PDR membranes and some (3/4) examined PVR membranes. All membranes which contained bFGF mRNA were also positive for bFGF peptide.In conclusion, bFGF is produced and stored in epiretinal membranes. Together with the corresponding receptor, bFGF may play a role in the auto- and paracrine control of the proliferative processes at the vitroretinal interface.Abbreviations aFGF acidic fibroblast growth factor - bFGF basic fibroblast growth factor - FGFR-1 fibroblast growth factor receptor-1 - mRNA messenger ribonucleic acid     

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.     

Cellular components of proliferative vitreoretinal membranes.

To understand the pathogenesis of proliferative vitreoretinal membrane formation which occurs in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), etc., accurate identification of the cellular components of the membrane is needed. This study was performed to identify cellular components of the membranes by means of immunohistochemical technique. 11 proliferative vitreoretinal membranes which were surgically obtained from 7 eyes with PVR and 4 eyes with PDR were stained with monoclonal antibodies against cytokeratin, glial fibrillary acidic protein (GFAP), or vimentin using immunoperoxidase technique (ABC method). In the PVR membranes, mean cell positivities for cytokeratin, GFAP and vimentin were 48%, 1% and 92%, respectively and in the PDR membranes, 0%, 5% and 93%, respectively. The above results suggest that retinal pigment epithelial cells and fibroblasts are major cellular components of PVR membranes, and that mesenchymal cells are major cellular components and glial cells are minor cellular components of PDR membranes.