Injured vitreous stimulates DNA synthesis in retinal pigment epithelial cells in culture and within the vitreous

Cultures of rabbit retinal pigment epithelium (RPE) were exposed to normal vitreous and to vitreous injured by intravitreal injection of foreign particles. Counts of labeled RPE nuclei after incubation with³H-thymidine in vitro indicated an increase in DNA synthesis with exposure to normal vitreous and an even greater increase with exposure to injured vitreous. Fractionation of injured vitreous demonstrated that the apparent proliferation stimulus resided in the cell-free supernate. The data suggest that normal vitreous contains a humoral factor that stimulates RPE proliferation and that levels of an active agent increase after vitreal injury. RPE injected into the vitreous also responds by increased DNA synthesis to subsequent vitreal injury. This observation implies that foreign substances in the vitreous, as after vitreal hemorrhage, promote development of extraretinopathies involving RPE by stimulating intravitreal proliferation of invasive RPE cells.     

Vitreous macrophage elicitation: generation of stimulants for pigment epithelium in vitro

Vitreous and macrophage samples were tested for the ability to stimulate proliferation and cell migration in cultured rabbit retinal pigment epithelium (RPE). A macrophage invasion was elicited by the intravitreal injection of latex particles in rabbits and after 3 days the vitreal macrophages were collected. The macrophages themselves, macrophage-conditioned culture medium, and macrophage-incubated vitreous had modest effects on RPE proliferation, but significantly stimulated RPE migration. A portion of the migration activity may be due to macrophage-derived proteases acting on normal vitreous. Mitogenic and additional migration-stimulating activity may also arise from adjacent tissues or from a breakdown of the blood-vitreous barrier that accompanies a macrophage invasion. A macrophage ingress into the vitreous may provide part of the stimulation for the migration and proliferation of RPE in conditions such as proliferative vitreoretinopathy.     

Cytotoxic effects of daunomycin on retinal pigment epithelium in vitro

Proliferative vitreoretinopathy (PVR), the most severe complication of retinal detachment surgery and posterior segment ocular trauma, is characterized by the intraocular proliferation of non-neoplastic cells with formation of vitreal and periretinal membranes resulting in renewed traction retinal detachment. As retinal pigment epithelial (RPE) cells have been shown to play an essential role in the development of PVR, we investigated the acute and chronic effects of daunomycin on the morphology and viability of porcine RPE cells in vitro. The intense and complete inhibition of cell proliferation reported in this study adds to previous evidence that daunomycin may be useful for pharmacological treatment of human PVR.This study was supported by the Retinovit Foundation and the Forschungsförderung NRW     

17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells.

PURPOSE: Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gonads has been reported. The goal of this study was to determine whether estrogen promotes the proliferation of retinal microvascular endothelial cells and to explore the mechanism of it. METHODS: DNA was quantitated using primary cultures of bovine retinal endothelial cells that were incubated with different doses of 17 beta-estradiol (E2), VEGF, or both. The changes in expression level of VEGF and VEGF receptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E2. The presence of estrogen receptors in the endothelial cells was studied by immunohistochemistry and western blot analysis. RESULTS: 17 Beta-estradiol (E2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E2-induced DNA synthesis in BRECs. Ten nanomole E2 augmented vascular endothelial growth factor (VEGF)-induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E2 also increased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 hours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E2 (P < 0.05) and that of VEGF reached maximum with 1 nM E2 (15 times, P < 0.001). VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRECs, and western blot analysis indicated the presence of a 67-kDa protein that was compatible with the estrogen receptor. CONCLUSIONS: These findings suggest that E2 may stimulate BREC growth by the receptor-mediated pathway and that E2 may augment the VEGF-dependent angiogenesis partly through the upregulation of VEGFR2.     

In vitro stimulation of retinal pigment epithelium proliferation by taurine.

Taurine is an amino acid that is essential for retinal integrity and function. Although it has been suggested that the ratio of melatonin to taurine in the interphotoreceptor matrix may regulate the phagocytosis of outer segments by retinal pigment epithelial (RPE) cells, the effect of taurine on the RPE has not been studied. Using cultured RPE cells, we found that in vitro taurine specifically stimulated proliferation of human and rabbit RPE, but had only minimal effect on cultured scleral fibroblasts. The RPE proliferation was due to more cells entering into S-phase and thus an increase in DNA synthesis, was not dependent upon cell density, and was most pronounced in the presence of a low concentration of fetal bovine serum.     

Observations on the retinal pigment epithelium and retinal macrophages in experimental retinal detachment.

After experimental retinal detachment in rabbits macrophages are a prominent feature in the subretinal space or within the retina. Two sources for these macrophages are identified. The retinal pigment epithelium (RPE) may undergo metaplasia and actively 'bud'; the evolving macrophage is then formed by a vitreal protrusion of the cytoplasm of an RPE cell which is 'nipped off' by lateral protrusions from adjacent cells. In addition, in regions of RPE proliferation, blood-borne cells were found in Bruch's membrane and among the mass of proliferated RPE cells, suggesting that blood-borne cells may pass from the choroidal circulation through Bruch's membrane and the RPE layer.     

生长因子诱导的血管内皮细胞增殖作用的研究

目的 研究生长因子对血管内皮细胞增殖活动的诱导和刺激作用。以进一步探讨生长因子在视网新生血管疾病中所起的作用。方法 采用血管内皮生长因子（VEGF）和碱性成纤维生长因子（bFGF）诱导离体培养的血管内皮细胞的增殖。研究VEGF及bFGF对血管内皮细胞DNA合成、细胞生长和细胞周期的调控，以及两种因子的协同效应。结果 VEGF及bFGF均可显著刺激血管内皮细胞的增生，其刺激效应与生长因子浓度呈剂量依赖关系。共同作用时，具有协同效应。两种因子均可显著增加^3H-TdR的掺入量，其刺激作用与浓度呈剂量依赖关系，共同作用时亦具有协同效应。VEGF和bFGF均可显著地产加S期细胞的比例。结论 VEGF及bFGF均可显著地刺激内皮细胞的增殖，促进细胞DNA的合成。两种因子联合应用时，具协同效应。提示：两种生长因子在视网膜新生血管的发生和发展中可能具有一定作用，且存在因子间的协同效应。     

Glycosaminoglycan metabolism of cultured fibroblasts from bovine vitreous

Glycosaminoglycan metabolism of cultured vitreal cells of bovine origin was investigated with the following results:1. Cultured vitreal cells distributed the synthesized macromolecules after [³⁵S] sulfate incorporation into the culture medium (extracellular pool), the membrane-bound proteoglycans (pericellular pool), and within the cells (intraocular pool). No extracellular plateau was reached after 3 days; the pericellular and intracellular pools showed a steady state of radioactivity after 48 and 24 h, respectively.2. Pulse-chase experiments exhibited a rapid turnover of intracellular polysaccharides and a slower metabolism of pericellular polymers. Pinocytosis, adsorption and degradation of vitreal and corneal cell secretion by vitreal cells were observed to a comparable extent.3. Chondroitin sulfate isomers and heparan sulfate could be detected in minor proportions.The relevance of glycosaminoglycan metabolism in the replacement of cultured vitreal cells into the vitreous cavity is discussed.     

Pathophysiology of Proliferative Vitreoretinopathy in Retinal Detachment

Because proliferative vitreoretinopathy cannot be effectively treated, its prevention is indispensable for the success of surgery for retinal detachment. The elaboration of preventive and therapeutic strategies depends upon the identification of patients who are genetically predisposed to develop the disease, as well as upon an understanding of the biological process involved and the role of local factors, such as the status of the uveovascular barrier. Detachment of the retina or vitreous activates glia to release cytokines and ATP, which not only protect the neuroretina but also promote inflammation, retinal ischemia, cell proliferation, and tissue remodeling. The vitreal microenvironment favors cellular de-differentiation and proliferation of cells with nonspecific nutritional requirements. This may render a pharmacological inhibition of their growth difficult without causing damage to the pharmacologically vulnerable neuroretina. Moreover, reattachment of the retina relies upon the local induction of a controlled wound-healing response involving macrophages and proliferating glia. Hence, the functional outcome of proliferative vitreoretinopathy will be determined by the equilibrium established between protective and destructive repair mechanisms, which will be influenced by the location and the degree of damage to the photoreceptor cells that is induced by peri-retinal gliosis.     

Transferrin and transferrin receptor expression in intraocular proliferative disease. APAAP-immunolabeling of retinal membranes and ELISA for vitreal transferrin

Transferrin (TF) is the major transport protein involved in human iron metabolism. The expression of the cell-surface TF receptor is associated with cellular proliferation, the dominant feature of proliferative vitreoretinal disorders with traction retinal detachment. A total of 14 retinal membranes from patients with different clinical diagnoses contained immunoreactive TF. Expression of the cell-surface TF receptor was confirmed by a monoclonal anti-human TF-receptor antibody label in 11 of the 14 specimens. We developed a noncompetitive enzyme-linked immunosorbent assay (ELISA) for TF and found it to be a significant component of vitreal protein, with a level of 65.7 ± 33.9 mg/l. Vitreal TF as a major iron acceptor probably has a protective function, but its interaction with macrophages and its growth-promoting activity may subsequently stimulate the proliferation of fibroblasts and retinal pigment epithelial cells.This study was supported by the Retinovit Foundation     

Retinal angiomatous proliferation responds safely to a double dose (1.0 mg) of ranibizumab

A 77‐year‐old man presented with sudden foggy central vision in the right eye. The visual acuity (VA) was 6/60 (R) and 6/6 (L). Funduscopy revealed superficial macular haemorrhage in the right eye. Using fluorescein angiography and indocyanine green angiography, retinal angiomatous proliferation was confirmed. Two intra‐vitreal injections of bevacizumab were given but the VA did not improve. Following this, he received an intra‐vitreal injection of ranibizumab. Regression of the retinal angiomatous proliferation was observed and the VA of the right eye returned to 6/10. Simultaneously, his left eye suffered from sudden visual loss and retinal angiomatous proliferation was diagnosed. Three intra‐vitreal injections of ranibizumab were given. Regression of the retinal angiomatous proliferation was observed and the VA of the left eye was stabilised. Another 80‐year‐old man complained of sudden distorted vision in his left eye. Funduscopy and optical coherence tomography (OCT) revealed superficial macular haemorrhage and retinal pigment epithelial detachment (RPED). The VA was 6/12 and retinal angiomatous proliferation was diagnosed. He received an intra‐vitreous injection of bevacizumab followed by photodynamic therapy (PDT). The RPED was resolved; however, the VA dropped to 2/60. Optical coherent tomography, fluorescein angiography and indocyanine green angiography were used to indentify retinal angiomatous proliferation. Intra‐vitreal injection(s) of a double dose (1 mg) of ranibizumab is a worthwhile treatment, as it can stabilise and even improve the VA without significant side effects.     

替德肝素抑制冷冻后视网膜色素上皮细胞分泌肝细胞生长因子的研究

目的探讨替德肝素(tedelparin)在抑制冷冻后的人视网膜色素上皮(humanretinapigmentepithelium,hRPE)细胞分泌肝细胞生长因子(humanhepatocytegrowthfactor,HGF)和生长中的作用。方法体外培养的RPE细胞在-80℃下进行冷冻,冷冻时间分为0、15和60s,随后继续体外培养(体外实验组)和注入正常兔眼玻璃体(体内实验组)并在第6天时取出一些兔眼的玻璃体液加入到正常RPE细胞培养液中孵育48h;每实验组再分为两个亚组替德肝素治疗(终浓度25μl/ml)组和未治疗组,在3d、6d时收集细胞培养上清和玻璃体样本,ELISA法测定HGF含量,MTT法测定48h后RPE细胞的增殖状态。结果在体外实验组,比较未冷冻组,冷冻后的RPE细胞随着冷冻时间延长HGF分泌水平增加(F=2736,P<001),替德肝素干预组HGF分泌水平下降(F=17950,P<001)。在体内实验组(兔玻璃体)随着冷冻时间延长HGF浓度较对照组明显增高(F=624,P<001),当玻璃体液(冷冻15s和60s,第6天)加入到正常RPE细胞培养液48h后刺激细胞增殖(P<001),在替德肝素干预组,细胞增殖明显减弱(F=4490,P<005)。结论冷冻可刺激RPE细胞在体外和体内玻璃体环境中HGF的高分泌,且随着冷冻时间增加更为显著。替德肝素可降低冷冻后RPE细胞分泌HGF水平和抑制促生长环境中的RPE细胞生长,具有预防PVR的作用。     

Development of traction retinal detachments following intravitreal injections of retinal Muller and pigment epithelial cells

We injected varying numbers of retinal Muller glia into the rabbit vitreous in an established model of traction retinal detachment. We used indirect ophthalmoscopy to observe the changes elicited during the following 1 month. Although the severity of the tractional changes increased with increasing numbers of the glial cells, the pathology produced stabilized within the 1st week of injury. Muller glia were less effective at eliciting retinal detachments than retinal pigment epithelial cells (RPE) or mixtures of glia and RPE. Intravitreal tissue membranes derived from the glia differed morphologically from those derived from RPE. The glial membranes had fewer fibroblast-like cells, synthesized less extracellular matrix, and showed lower intravitreal cell proliferation, as determined by³H-thymidine radioautography. Our findings indicate that membranes composed only of Muller glial cells promote less severe retinal pathology than those membranes composed of RPE or mixed cell types.This study was supported in part by National Eye Institute grant EY04799 (J.M.B.), Core Center Grant EY01931 (J.M.B.), an unrestricted grant from Research to Prevent Blindness, Inc., and the Good Samaritan Foundation for Ophthalmic Research (Portland, Oregon)     

氧诱导的血管增生性视网膜病变小鼠模型

目的 建立可量化的血管增生性视网膜病变小鼠模型。方法 将鼠龄为7d的C57BL/6J幼鼠17只暴露于75％氧浓度环境下饲养持续5d,然后回到正常空气中饲养;17只同龄幼鼠置于正常空气环境中饲养作为对照。ADP酶法视网膜铺片了解视网膜血管的改变;用组织切片观察并计数突破视网膜内界膜的内皮细胞核数目;视网膜组织切片用CD31进行免疫组织化学染色。结果 持续高浓度氧使幼鼠视网膜血管收缩、分支闭塞、中央部可见灌注降低,相对低氧使视网膜血管扩张、增生。组织切片可见正常对照组平均每张切片突破内界膜内皮细胞核数目<1个,给氧组平均24个/切片,两组比较差别有显著性意义(P<0.01)。给氧组视网膜组织切片经用CD31抗体处理后显示内界膜玻璃体面细胞染色阳性。结论 该模型具有可重复性强、可定量研究的优点,是进行视网膜新生血管发生机制及药物干预的合适模型。     

Vitreal pathogenic role in optic pit foveolar retinoschisis and central serous chorioretinopathy

Purpose: To expand on current theories concerning the vitreal‐induced mechanism underlying the development of foveolar retinoschisis and macular sensory detachments associated with optic nerve head pits. To propose the notion that vitreal traction may contribute to the pathogenesis of serous detachments in central serous chorioretinopathy (CSC). Reports: We describe two patients, one with macular retinoschisis and the other with central serous detachment. The first patient, a 45‐year‐old Hispanic female, presented with a temporally located optic nerve head pit, foveolar retinoschisis and schisis retinal spaces extending to the surrounding macula and to the disc. The second patient, a 43‐year‐old Haitian male, developed a central serous retinal detachment OS with decreased visual acuity one day following in‐office administration of Apraclonidine (0.5 per cent Iopidine, Alcon) and Dorzolamide‐Timolol Maleate (Cosopt, Merck) to lower elevated intraocular pressure (IOP). Macular retinal pigment mottling and epiretinal membrane sheen OU had been observed on his initial visit. Visual acuity improved within a three day period with resolution of the serous detachment. Conclusion: We suggest that the persistence of Cloquet's canal may permit fluid leakage into the proximal vitreous in cases of congenital optic nerve head pits. Tangential vitreal traction may promote the opening of a fistula at the optic pit and additionally thrust vitreal fluid into the pit and retinal space inducing the formation of schisis spaces, foveolar‐schisis and underlying sensory serous detachment. We question whether a reduction in vitreous volume, induced by initial administration of anti‐glaucoma medications, may contribute to the development and/or recurrence of central serous choroidopathy in predisposed individuals.     

Changes in [K+]0 at the vitreal surface compared with those around receptors in the isolated rabbit retina

Isolated rabbit retinas were superfused from the receptor side with a plasma-saline mixture kept at 35° C. The vitreal side was exposed to an atmosphere of humidified warm oxygen. In one study the second-order neuronal activity was suppressed with aspartate and glutamate; in another study transmission was not blocked. When all neurons were active, [K⁺]₀ around receptors was 4.5 ± 0.4 mM in the dark. During a long (60s) exposure to light stimulus, [K⁺]₀ dropped to 73% of the dark value and reaccumulated to 80%. At the vitreal surface, [K⁺]₀ in the dark was 4.7 ± 0.8 mM. During the 60s light stimulus, [K⁺]₀ increased transiently, dropped to 83% of the dark value, then increased again to 91%. A continuous decrease of [K⁺]₀ at the vitreal surface during long light stimuli concurrent with the increase of [K⁺]₀ around receptors would indicate that the spatial buffering capability of the Müller cells contributes to the reaccumulation of potassium. Such a decrease, however, was not detected. After the blockage of transmission, [K⁺]₀ values did not vary significantly from those after light stimulus in unblocked preparations. In the dark, [K⁺]₀ was 5.2 ± 0.9mM at the vitreal surface and 4.6 ± 0.4 mM around the receptors.     

Pilocarpine toxicity in retinal ganglion cells

PURPOSE: Muscarinic agents reduce intraocular pressure by enhancing aqueous outflow, probably by stimulating ciliary muscle contraction. However, pilocarpine is a well characterized neurotoxin and is widely used to generate animal seizure models. It was therefore investigated whether pilocarpine was also toxic to retinal ganglion cells. METHODS: Dissociated whole retinal preparations were prepared from postnatal day 16 to 19 rats. Retinal ganglion cells had been previously back-labeled with a fluorescent tracer. Retinal cells were incubated with pilocarpine, lithium, and inositol derivatives, and viability of the retrogradely labeled retinal ganglion cells was assayed after 24 hours. RESULTS: Pilocarpine was toxic to retinal ganglion cells in a dose-dependent fashion. This toxicity was potentiated by lithium and blocked by epi- and myo-inositol. CONCLUSIONS: Pilocarpine is toxic to retinal ganglion cells in a mixed culture assay. This toxicity appears to depend on the inositol pathway and is similar to its mode of action in other neurons. However, 0.4 mM pilocarpine (the lowest concentration that did not affect ganglion cell survival) is roughly 1000-fold higher than the vitreal concentration and 20-fold higher than the scleral concentration that can be obtained with topical administration of 2% pilocarpine in the rabbit eye.     

Antiproliferative drugs in the treatment of experimental proliferative vitreoretinopathy

We used an experimental model of proliferative vitreoretinopathy (PVR) and cell-induced traction retinal detachment to study the therapeutic value of six cytotoxic drugs (actinomycin C, colchicine, cytosine arabinoside hydrochloride, 5-fluorodeoxyuridine, vinblastine sulfate, and daunomycin). Pigmented rabbits were injected with 2.5 X 10(5) cultured homologous dermal fibroblasts. At the same time, cytotoxic drugs were injected into the vitreous in an attempt to inhibit cellular proliferation. The sensitivity of the cells to the drug was also tested in vitro before injection. Daunomycin at a dose of 10 nmol per eye stopped cellular proliferation and subsequent traction retinal detachment in vivo. The electroretinogram (ERG) showed no evidence of drug-induced retinal toxicity with this dose of daunomycin. No alteration in frequency or severity of vitreal membranes and retinal detachment was observed after injection of equivalent doses of the other drugs.     

Evaluation of experimental proliferative vitreoretinopathy in rabbits

We established an effective animal experimental model for proliferative vitreoretinopathy (PVR) by intravitreally injecting cultured cells in order to investigate therapies for PVR, including intravitreal drugs, radiation and hyperthermia. After making posterior vitreal separation by injecting SF6 gas into the rabbit's vitreous body, cultured cells were intravitreally injected. Fundus changes were followed up for 4 weeks and PVR stage was recorded according to the classification described by Hida et al. The cultured cells injected were rabbit dermal fibroblasts (5 x 10(4) and 10 x 10(4) and human retinal pigment epithelial (RPE) cells (5 x 10(4]. A total of 37% of animals reached STAGE 5-7 (traction retinal detachment) in 4 weeks in 41 eyes injected with 5 x 10(4) rabbit fibroblasts and were 86% in 14 eyes injected with 10 x 10(4) rabbit fibroblasts. The 10 eyes injected with human RPE cells showed STAGE 2 or less in 4 weeks. Consequently, we selected the experimental PVR model using injecting of 10 x 10(4) rabbit fibroblasts as the most effective method.     

阿魏酸对培养视网膜神经细胞增殖活性的影响

目的 研究阿魏酸促进视网膜神经细胞活性的可能机制,为治疗退行性视网膜病变提供依据。方法 以7个月人胚胎视网膜、新生小牛视网膜和生后4个月小鼠视网膜为研究对象,以细胞培养、MTT法和氚标记胸苷掺入法等为研究手段,检测阿魏酸、脑源性神经营养因子(BDNF)和葛根素作用72h后,视网膜神经细胞增殖和DNA合成变化。结果 阿魏酸对3种视网膜神经细胞均具有促增殖作用,细胞活性强度呈现剂量依赖性,对于小鼠视网膜神经细胞,阿魏酸在15．6—1000．0mg／L内均具有促细胞增殖作用;对于新生小牛和7个月人胚胎视网膜神经细胞,阿魏酸在125．0～1000．0mg／L内具有促细胞增殖作用。对7个月人胚胎视网膜神经细胞,阿魏酸在62．5—1000．0mg／L内具有促DNA合成作用,最佳效应浓度为500．0mg／L;BDNF在12,5～50．0μg/L内具有促细胞增殖作用,最佳效应浓度为50．0μg／L;葛根素未见明显促增殖效应。结论 阿魏酸对3种视网膜神经细胞的促增殖作用明显强于BDNF。阿魏酸能有效提高视网膜神经细胞活性,有望成为一种新的防治退行性视网膜病变的有效成分。