Expression of angiogenic and fibrogenic factors in proliferative vitreoretinal disorders

Purpose To investigate the expression of connective tissue growth factor (CTGF) in the retina of human subjects with diabetes mellitus, and CTGF, CD105, and gelatinase B in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. Methods Twelve donor eyes from six subjects with diabetes mellitus, 10 eyes from five nondiabetic subjects, 14 PDR membranes, and five PVR membranes were studied by immunohistochemical techniques. In situ zymography was used to examine gelatinolytic activity in four PDR membranes. Results In nondiabetic retinas, there was no immunoreactivity for CTGF. Diabetic retinas showed immunoreactivity for CTGF in ganglion cells and microglia. Vascular endothelial cells in PDR membranes expressed CTGF, CD105, and gelatinase B in 10 (71.4%), 11 (78.6%), and 5 (35.7%) membranes, respectively. Myofibroblasts in PDR membranes expressed CTGF, and gelatinase B in 14 (100%), and 6 (42.9%) membranes, respectively. There was a significant correlation between the number of blood vessels expressing the panendothelial marker CD34 and the number of blood vessels expressing CTGF (r = 0.7884; P = 0.0008), and CD105 (r = 0.6901; P = 0.0063), and the number of myofibroblasts expressing CTGF (r = 0.5922; P = 0.0257). There was a significant correlation between the number of myofibroblasts expressing α-smooth muscle actin and the number of myofibroblaasts expressing CTGF (r = 0.8393; P = 0.0002). In situ zymography showed the presence of gelatinolytic activity in vascular endothelial cells in PDR membranes. Myofibroblasts in PVR membranes expressed CTGF, and gelatinase B. Conclusions These results suggest a possible role of CTGF, CD105, and gelatinase B in the pathogenesis of proliferative vitreoretinal disorders.     

Expression of advanced glycation end products and related molecules in diabetic fibrovascular epiretinal membranes

Purpose: To investigate associations between expressions of advanced glycation end products (AGEs), transforming growth factor‐β (TGF‐β), tumour necrosis factor‐α (TNF‐α) and integrins and correlations between their expression and level of vascularization and proliferative activity in diabetic fibrovascular epiretinal membranes. Methods: Membranes from eight patients with active proliferative diabetic retinopathy and nine patients with inactive proliferative diabetic retinopathy were studied by immunohistochemistry. Results: Blood vessels expressed AGEs, TGF‐β, TNF‐α and α_vβ₃ integrin in 5, 13, 8 and 8 membranes, respectively. Stromal cells expressed AGEs, TNF‐α and α_vβ₃ integrin in 15, 13 and 3 membranes, respectively. There was no immunoreactivity for α_vβ₅, α₅β₁ and α₂β₁ integrins. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing AGEs (r_s = 0.496; P = 0.043), TGF‐β (r_s = 0.777; P < 0.001) and TNF‐α (r_s = 0.699; P = 0.002). There were significant correlations between number of blood vessels expressing AGEs and number of blood vessels expressing TGF‐β (r_s = 0.532; P = 0.028) and TNF‐α (r_s = 0.626; P = 0.007). The correlation between number of blood vessels expressing TNF‐α and α_vβ₃ integrin was significant (r_s = 0.617; P = 0.008). Number of blood vessels expressing CD34 (P = 0.001), TGF‐β (P = 0.006) and TNF‐α (P = 0.002) and stromal cells expressing AGEs (P = 0.001) and TNF‐α (P = 0.004) were significantly higher in active membranes than in inactive membranes. Conclusion: Interactions of AGEs, TGF‐β, TNF‐α and α_vβ₃ integrin might be involved in pathogenesis of proliferative diabetic retinopathy fibrovascular proliferation.     

Expression of thrombospondin‐2 as a marker in proliferative diabetic retinopathy

Purpose: To determine the expression of the endogenous anti‐angiogenic and pro‐fibrotic matricellular protein thrombospondin (TSP)‐2 and its receptors CD36 and CD47 in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of TSP‐2 in the retinas of diabetic rats. Methods: Epiretinal membranes from 14 patients with PDR and nine patients with proliferative vitreoretinopathy were studied by immunohistochemistry. Vitreous samples from 30 PDR and 25 nondiabetic patients were studied by enzyme‐linked immunosorbent assay. Vitreous samples and retinas of rats were examined by Western blotting. Results: In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed TSP‐2, CD36 and CD47. In PDR membranes, significant correlations were observed between numbers of blood vessels expressing the panendothelial cell marker CD34 and numbers of blood vessels and stromal cells expressing TSP‐2, CD36 and CD47. The numbers of blood vessels and stromal cells expressing CD34, TSP‐2, CD36 and CD47 were significantly higher in membranes with active neovascularization when compared with those with quiescent disease. Thrombospondin‐2 levels in vitreous samples from PDR patients were significantly higher than those in control patients without diabetes (p < 0.001). Western blot analysis revealed a significant increase in the expression of intact and cleaved TSP‐2 in vitreous samples from PDR patients and in the retinas of diabetic rats compared to nondiabetic controls. Conclusions: Upregulation of TSP‐2 may be a protective mechanism against inflammation and angiogenesis associated with PDR.     

Expression of autotaxin and acylglycerol kinase in proliferative vitreoretinal epiretinal membranes

Purpose: Lysophosphatidic acid (LPA)/LPA₁ receptor pathway is involved in inflammation, angiogenesis and fibrosis. This study was conducted to analyse the expression of LPA‐producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK) and LPA₁ receptor, in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. Methods: Nine active and 13 inactive membranes from patients with PDR and 21 membranes from patients with PVR were studied by immunohistochemistry. Results: In PDR membranes, vascular endothelial cells expressed ATX and AGK in 16 and 19 membranes, respectively. Stromal cells expressed ATX and AGK in 19 and 22 membranes, respectively. Immunoreactivity for LPA₁ receptor was noted in vascular endothelial cells and stromal cells in the five membranes stained for LPA₁ receptor. Numbers of blood vessels and stromal cells expressing CD34, ATX and AGK were significantly higher in active membranes than in inactive membranes. Significant correlations were detected between number of blood vessels expressing the panendothelial cell marker CD34 and number of blood vessels and stromal cells expressing ATX and AGK. In PVR membranes, spindle‐shaped myofibroblasts expressing α‐smooth muscle actin co‐expressed ATX, AGK and LPA₁ receptor. Conclusions: The LPA/LPA₁ receptor pathway may be involved in inflammatory, angiogenic and fibrotic responses in proliferative vitreoretinal disorders.     

Expression of myofibroblast activation molecules in proliferative vitreoretinopathy epiretinal membranes

Purpose: Fibrotic disorders are associated with activation of fibroblasts into extracellular matrix‐secreting myofibroblasts expressing α‐smooth muscle actin (α‐SMA). Myofibroblasts are the predominant cellular component of proliferative vitreoretinopathy (PVR) epiretinal membranes. We investigated the expression of molecules involved in myofibroblast activation, migration and proliferation in PVR epiretinal membranes. Methods: Fifteen membranes were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against snail, fibroblast activation protein (FAP), CD44, hydrogen peroxide‐inducible clone‐5 (Hic‐5), galectin‐3, interleukin‐13 receptor α2 (IL‐13Rα2) and receptor for advanced glycation end products (RAGE). Results: Myofibroblasts expressing α‐SMA were present in all membranes. Myofibroblasts expressed nuclear immunoreactivity for Snail and Hic‐5, cytoplasmic immunoreactivity for FAP, IL‐13Rα2 and RAGE and membranous immunoreactivity for CD44. There was no immunoreactivity for galectin‐3. The number of cells expressing α‐SMA correlated significantly with the number of cells expressing Snail (r = 0.56; p = 0.03), Hic‐5 (r = 0.526; p = 0.044), IL‐13Rα2 (r = 0.773; p = 0.001) and RAGE (r = 0.734; p = 0.002). Conclusions: Snail, FAP, CD44, Hic‐5, IL13Rα2 and RAGE may be involved in proliferative events occurring in PVR.     

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.     

孔源性视网膜脱离并发脉络膜脱离患者的角膜内皮形态学参数

目的：探讨孔源性视网膜脱离并发脉络膜脱离(RRD-CHD)患者的角膜内皮细胞形态学参数变化。

方法：连续收集RRD-CHD患者70例70眼,分为无高度近视组(A组)38眼38眼、高度近视组(B组)32例32眼,另收集正常组(C组)36例36眼。使用角膜内皮镜检测各组角膜内皮细胞最小面积(S_min)、最大面积(S_max)、平均面积、平均密度(CD)、细胞面积标准差(SD)、变异系数(CV)及六角形细胞比例(HG)。

结果：RRD-CHD患者和正常组的角膜内皮细胞CD、HG均有差异(P<0.001)。A组的CD与B组、C组均有差异(P<0.05),B组与C组有差异(P<0.001)。在A组中,SD、CV与眼轴(r_s=-0.426、0.494, 均P<0.01),CD与眼压、眼轴(r_s=-0.025、0.368, 均P<0.05),HG与病程(r_s=0.552, P<0.05)均相关。在RRD-CHD患者中,SD、CV与眼轴(r_s=0.236、0.159, 均P<0.05),HG与病程(r_s=0.142, P<0.05),S_max与眼压(r_s=-0.314, P<0.01)均相关。

结论：RRD-CHD患者持续低眼压状态下角膜内皮细胞的HG和CD均明显降低,眼轴、病程和眼压可影响角膜内皮的形态学参数。     

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.     

Intraocular expression of thymosin β4 in proliferative diabetic retinopathy

Purpose: To examine an association between thymosin β4 as potentially angioproliferative factor and proliferative diabetic retinopathy. Methods: The clinical study part included 62 patients with proliferative diabetic retinopathy (PDR) (study group) and 24 patients with non‐diabetic pre‐retinal membranes (control group). All patients underwent pars plana vitrectomy. We examined the thymosin β4 concentration in vitreous and plasma; and the expression of thymosin β4, glial fibrillary acidic protein (GFAP) and CD31 (PECAM‐1 or Platelet Endothelial Cell Adhesion Molecule) and the levels of thymosin β4 mRNA and vascular endothelial growth factor (VEGF) mRNA in the excised membranes. The experimental study part consisted of 24 Sprague–Dawley rats with streptozotocin‐induced diabetes mellitus and 24 age‐matched control animals without diabetes. We determined the mRNA concentrations of thymosin β4, VEGF and GFAP in the rat retinas. Results: In the clinical study part, the vitreal and plasma thymosin β4 concentrations were significantly higher in the study group than control group (p = 0.04 and p = 0.01, respectively), and were significantly (p = 0.028) correlated with each other. Co‐expression of thymosin β4 and CD31 was observed in the diabetic fibrovascular membranes. Thymosin β4 mRNA and VEGF mRNA levels were significantly (p < 0.01) higher in diabetic membranes than in non‐diabetic membranes. In the experimental study part, the diabetic retinas showed co‐localization of thymosin β4 and GFAP. The mRNA levels of thymosin β4, VEGF and GFAP were significantly (p < 0.01) higher in diabetic rats than in control animals. Conclusions: Thymosin β4 was produced in intraocular fibrovascular membranes of patients with PDR and in rats with experimental diabetes mellitus. Thymosin β4 may play a role in diabetic retinal neovascularization.     

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.     

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     

Correlation between components of newly diagnosed exudative age‐related macular degeneration lesion and focal retinal sensitivity

Purpose: To analyse lesion components determining retinal sensitivity in microperimetry in eyes with newly diagnosed exudative age‐related macular degeneration (AMD). Methods: Visual acuity, contrast sensitivity, microperimetry, optical coherence tomography (OCT), and fluorescein (FA) and indocyanine green (ICGA) angiographies of 23 eyes of 23 patients were analysed. Central microperimetry grids with 28 test stimulus sites were automatically aligned with three‐dimensional OCTs and manually aligned with angiographies. Thicknesses of the neuroretina, neuroepithelial detachment (NED), retinal pigment epithelial (RPE) elevation and subretinal tissue were measured under the 644 microperimetry stimulus sites. Areas of classic and occult choroidal neovascularizations (CNVs), subretinal and intraretinal haemorrhage, and late hyperfluorescence in ICGA were identified. The impact of the lesion components on retinal sensitivity was evaluated with correlation analysis and multivariate modelling. Results: Decreased retinal sensitivity correlated significantly with the presence of CNV, haemorrhage, subretinal tissue and RPE elevation. Out of the OCT parameters, the most important determinant of sensitivity was the thickness of RPE elevation (Spearman’s rho, r = ?0.202, p < 0.0001). The thicknesses of subretinal tissue (r = ?0.168, p < 0.0001) and NED had weaker effects (r = ?0.147, p < 0.0001), and the neuroretinal thickness remained nonsignificant. In multivariate modelling, RPE elevation and subretinal tissue in OCT, CNV membranes in angiographies and haemorrhage had the strongest impacts on retinal sensitivity. Conclusion: The most important lesion components affecting retinal function were RPE elevation and subretinal tissue in OCT as well as neovascular membranes and haemorrhage in angiographies. NED and neuroretinal thickening remained less significant.     

Association of preoperative vitreous IL‐8 and VEGF levels with visual acuity after vitrectomy in proliferative diabetic retinopathy

Acta Ophthalmol. 2010: 88: e311–e316

Abstract.

Purpose: To determine whether the vitreous levels of interleukin 8 (IL‐8) and vascular endothelial growth factor (VEGF) of patients with proliferative diabetic retinopathy (PDR) were associated with poor visual acuity after vitrectomy. Methods: Observational cross‐sectional study. Patient clinical characteristics and preoperative eye characteristics (63 eyes): visual acuity, iris neovascularization, vitreous haemorrhage, macular detachment, macular oedema, active retinal neovascularization, neovascularization of the disc, burned out PDR (defined as natural end stage of PDR with inactive membranes without previously performed laser photocoagulation) and panretinal photocoagulation were registered prior to vitrectomy for each patient. Vitreous VEGF and IL‐8 levels were measured using the cytometric bead array method. Poor postoperative visual acuity was defined as visual acuity of <20/200 and was checked 2 years after vitrectomy. Results: Twenty‐one of the 63 eyes (33.3%) had poor visual acuity after vitrectomy. Univariate analysis showed that vitreous levels of IL‐8, the absence of panretinal photocoagulation, preoperative macular detachment and poor preoperative visual acuity were significantly associated with poor final visual acuity after vitrectomy. A stepwise multiple logistic regression analysis showed that elevated vitreous levels of IL‐8 (p < 0.0001), macular detachment (p = 0.011) and the absence of panretinal photocoagulation (p = 0.03) were independent predictors for poor visual outcome. Conclusions: Elevated vitreous IL‐8 level could either be a marker of ischaemic inflammatory reaction, or it could play a role in deteriorating visual acuity by DR progression or both. Further studies are needed to provide better understanding of IL‐8 and inflammation involvement in visual prognosis in PDR.     

Myeloid-Related Protein-14/MRP-14/S100A9/Calgranulin B is Associated with Inflammation in Proliferative Diabetic Retinopathy

Purpose: To investigate the expression of the leukocyte proteins myeloid-related protein (MRP)-8 and MRP-14 in proliferative diabetic retinopathy (PDR) and the effect of MRP-8/MRP-14 (calprotectin) heterodimer on induction of proinflammatory factors in human retinal microvascular endothelial cells (HRMEC).

Methods: Epiretinal membranes from 20 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR), vitreous fluid samples from PDR and non-diabetic subjects and HRMEC were studied by immunohistochemistry and Western blot analysis.

Results: MRP-14 expression was localized in endothelial cells, leukocytes and myofibroblasts in all PDR membranes. MRP-8 expression was limited to intravascular leukocytes in 42% of the studied membranes. In PVR membranes, MRP-14 was expressed in leukocytes and myofibroblasts, whereas MRP-8 immunoreactivity was limited to leukocytes. MRP-14 was significantly upregulated in vitreous from PDR patients. MRP-8/MRP-14 (calprotectin) increased expression of intercellular adhesion molecule-1, but attenuated vascular cell adhesion molecule-1 expression in HRMEC.

Conclusions: Increased MRP-14 levels are associated with inflammation in PDR.     

Unbalanced Vitreous Levels of Osteoprotegerin,RANKL, RANK,and TRAIL in Proliferative Diabetic Retinopathy

Purpose: We investigated the expression of the proinflammatory and proangiogenic factor osteoprotegerin (OPG) and its ligands, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and the receptor RANK in proliferative diabetic retinopathy (PDR).

Materials and methods: Vitreous samples from PDR and nondiabetic control patients and epiretinal membranes from PDR patients were studied by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis.

Results: Vascular endothelial growth factor, OPG, and soluble RANK levels in vitreous samples from PDR patients were significantly higher than that in nondiabetic controls. Soluble TRAIL levels were significantly lower in PDR patients than that in nondiabetic control, whereas soluble RANKL levels did not differ significantly. RANKL, RANK, and TRAIL were expressed in vascular endothelial cells, myofibroblasts, and CD45-expressing leukocytes in PDR epiretinal membranes.

Conclusions: Dysregulated expression of OPG/RANKL/RANK pathway and TRAIL might be related to inflammation and angiogenesis in PDR.     

Topical Ranibizumab inhibits inflammatory corneal hem‐ and lymphangiogenesis

Purpose: Ranibizumab (Lucentis^®) is a Fab‐Fragment of a recombinant, humanized, monoclonal VEGF (anti‐vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied. Methods: The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme‐linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE‐1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F. Results: An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea. Conclusion: Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications.     

Performance of three biometry devices in patients with different grades of age‐related cataract

Purpose: The new Lenstar biometry device was compared in a typical clinical setting to the IOL‐Master and Visante‐OCT. Methods: Fifty‐one eyes of 51 patients with age‐related cataract were examined with Lenstar LS900 (Haag Streit AG) biometer, IOL‐Master V.5 (Carl Zeiss Meditec AG) and Visante‐OCT (Carl Zeiss Meditec AG) before cataract surgery. Central corneal thickness (CCT), anterior chamber depth (ACD), keratometry readings of flattest and steepest meridian (K), corneal radius (R) and axial length (AL) values were correlated. Cataracts were graded according to the Lens Opacities Classification System III (LOCS) regarding nuclear colour (NC), nuclear opalescence (NO), cortical (C) and posterior subcapsular (P) cataract. Results: Mean values and standard deviations for AL, K and R was 23.66 ± 1.23 mm and 23.67 ± 1.26 mm, 43.24 ± 1.69 dpt and 43.16 ± 1.71 dpt, 7.68 ± 0.29 mm and 7.70 ± 0.28 mm with the IOL‐Master and with the LS900, respectively (r = 0.99 and p = 0.76, r = 0.99 and p = 0.029, r = 0.89 and p = 0.14, respectively). Visante‐OCT demonstrated highest values of three devices regarding to ACD followed by Lenstar LS900 and IOLMaster. Axial length measurements were unfeasible in 10% of the cases (five patients) and this significantly correlated with the presence of posterior subcapsular cataract of LOCS III grade 4.0 or higher. Conclusions: IOL‐Master, Lenstar LS900 and AC–OCT proved to be excellent non‐contact measurement methods in eyes with age‐related cataract. Nevertheless, ultrasound biometry is still required for cases with dense posterior subcapsular cataract.     

Biomechanical profile of the cornea in primary congenital glaucoma

Purpose: The aim of our study was to investigate the biomechanical properties of the cornea in primary congenital glaucoma (PCG) and to identify the potential ocular determinants, which affect the corneal biomechanical metrics. Methods: Corneal hysteresis (CH), corneal resistance factor (CRF) and central corneal thickness (CCT) were measured in 26 patients with PCG (40 eyes) with the aid of ocular response analyser. In vivo laser‐scanning confocal microscopy was used for the estimation of stromal keratocyte density (KD) and the evaluation of corneal endothelium. Twenty normal subjects (40 eyes) served as controls. Student’s t‐test and Pearson’s correlation coefficients were used for statistical analysis. p Values <0.05 were considered statistically significant. Results: Corneal hysteresis, CRF and CCT were significantly reduced in patients with PCG (all p < 0.05). Corneal hysteresis and CRF negatively correlated with the corneal diameter in both groups (r₁ = ?0.53, r₂ = ?0.66, p < 0.001 for CH and r₁ = ?0.61, r₂ = ?0.69, p < 0.001 for CRF). Moreover, we identified a significant correlation between CH and CRF with CCT in both groups (r₁ = 0.51, r₂ = 0.48, p < 0.001 for CH and r₁ = 0.45, r₂ = 0.44, p < 0.001 for CRF). Mean KD was significantly reduced both in the anterior and posterior corneal stroma in patients with PCG (764 ± 162 and 362 ± 112 cells/mm², respectively) compared with controls (979 ± 208 and 581 ± 131 cells/mm², respectively) (p < 0.001). There was no significant correlation between the keratocyte density in anterior and/or posterior stroma and CH or CRF in any group (r₁ = 0.29, r₂ = 0.31, p < 0.06). Mean endothelial cell density was also significantly reduced in PCG group (2920 ± 443 cells/mm²) compared with control group (3421 ± 360 cells/mm²) (p < 0.001). Pleomorphism and polymegalism were significantly increased in corneal endothelium of patients with PCG. Conclusions: Our results showed a significant reduction in CH and CRF in PCG. Both CH and CRF were negatively correlated with corneal diameter. A significant correlation of CH and CRF with CCT was identified in both groups. Keratocyte density was decreased in PCG, but did not have a significant impact on CH and CRF. Mean endothelial density was also decreased in PCG. Our results suggest that reduced CCT and increased corneal diameter are major ocular determinants for the modified corneal biomechanical profile in PCG, while cellular alterations in corneal stroma and endothelium have no significant biomechanical impact.     

Visual acuity and microperimetric mapping of lesion area in eyes with inflammatory cystoid macular oedema

Purpose: To evaluate the effect of fluid accumulation on local visual function in inflammatory cystoid‐macular‐edema (ICME). Methods: This cross‐sectional study applied optical‐coherence‐tomography over a 12 × 12 fovea‐centered field in 50 patients with ICME and mapped the extent of fluid‐filled spaces in various retinal layers, of subretinal‐fluid and of diffuse‐edema. Regression analysis examined effect of planimetric fluid‐distribution on best‐corrected‐visual‐acuity (BCVA) and mean microperimetric‐sensitivity. Results: BCVA decreased with increasing central‐neuroretinal‐thickness (r = 0.52, p = 0.001), total central‐retinal‐thickness, including subneuroretinal‐fluid (r = 0.41, p = 0.006), total cystoid‐and‐diffuse edema‐area (r = 0.35, p = 0.036) and cystoid inner‐nuclear‐layer area (r = 0.39, p = 0.02). Mean retinal‐sensitivity decreased with increasing diffuse edema‐area (r = ?0.86, p < 0.0001), total cystoid‐and‐diffuse edema‐area (r = ?0.54, p = 0.001), cystoid inner‐nuclear‐layer area (r = ?0.46, p = 0.008) and cystoid ganglion‐cell‐layer area (r = ?0.6, p = 0.049), central‐neuroretinal‐thickness (r = ?0.42, p = 0.028) and total central‐retinal‐thickness (r = ?0.34, p = 0.039). In multivariate‐analyses BCVA was best described by central‐neuroretinal‐thickness, duration of edema, total cystoid‐and‐diffuse edema‐area and cystoid inner‐nuclear‐layer area (R² = 0.5, p = 0.002). Mean retinal‐sensitivity was best described by diffuse edema‐area, total cystoid‐and‐diffuse edema‐area and central‐neuroretinal‐thickness (R² = 0.75, p < 0.0001). Subretinal‐fluid area and cystoid outer‐nuclear/Henle’s layer area had no effect on either BCVA or microperimetry. Conclusions: Thickening of the neurosensory‐fovea, not subfoveal‐fluid, had major impact on both BCVA and retinal‐sensitivity. The extent of edema in inner retinal layers also had major impact on both of these two functional parameters. Visual‐impairment seems to differ depending on the layers involved, thus different types of fluid accumulation may potentially be given varying treatment priorities.     

Increased intravitreous interleukin-18 correlated to vascular endothelial growth factor in patients with active proliferative diabetic retinopathy

Purpose

To determine the intravitreous levels of interleukin-18 (IL-18) and vascular endothelial growth factor (VEGF) in patients with proliferative diabetic retinopathy (PDR) and to ascertain their association with PDR activity.

Methods

Thirty eyes of 30 diabetic patients with PDR were divided into two groups (active PDR, n?=?17; quiescent PDR, n?=?13). Fifteen eyes of 15 non-diabetic patients (macular hole, n?=?9; epiretinal membrane, n?=?6) served as controls. All vitreous fluid samples were obtained during vitrectomy. IL-18 and VEGF were measured by enzyme-linked immunosorbent assay. Serum glycosylated hemoglobin as well as the basic demographic data was documented.

Results

Both IL-18 and VEGF levels were higher in patients with PDR than control (P?<?0 .01 and P?<?0 .01, respectively). Both IL-18 and VEGF in active PDR were higher than those in quiescent PDR (P?=?0.048 and P?=?0.03, respectively). A significant positive correlation (Spearman rank correlation coefficient (r _s)?=?0.502, P?=?0.005) between IL-18 and VEGF was observed in all PDR patients but not in the control. The correlation between VEGF and IL-18 was even stronger in the subgroup of active PDR (r _s?=?0.684; P?=?0.002), whereas no significant correlation was found in the subgroup of quiescent PDR (r _s?=?0.049; P?=?0.873).

Conclusions

Both intravitreous IL-18 and VEGF were elevated in patients with PDR, which were closely correlated in active PDR. IL-18 may contribute to retinal angiogenesis by acting together with or via VEGF, and become the potential therapeutic target for treatment of PDR.