Herpes simplex virus-mediated gene delivery to the rodent visual system

PURPOSE: To determine the types of cells in the visual system of the mouse and rat that can express a transgene delivered by an attenuated replication competent Herpes simplex virus-1 (HSV-1) vector. METHODS: C57/BL6 x BALB/C mice and Albino rats were treated with 1 x 10(7) pfu of the HSV-1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. The hrR3 virus was delivered by topical application to the cornea, intravitreal (IV) injection, intracameral injection (IC), or stereotactic injection into the visual cortex (VC). At specified times postinfection, animals were killed and tissues were removed, fixed, sectioned, and stained with X-gal or hematoxylin and eosin for histochemical and histopathologic examination. RESULTS: Topical delivery after corneal scarification in both mouse and rat resulted in lacZ expression in 25% of the corneal epithelial cells and 25% of the retinal pigment epithelium (RPE) cells. Topical application without concurrent scarification also resulted in transgene delivery to 20% of the RPE cells of the rat but not the mouse. IV injection resulted in expression primarily in RPE cells, with up to 100% of the cells expressing lacZ in the mouse and rat. Other cells expressing the transgene included ciliary body (CB) and optic nerve cells. Up to 25% of the retinal ganglion cells in the rat expressed lacZ, but only rarely in mice. IC delivery in rats resulted in expression in trabecular meshwork, CB cells, RPE, and iris epithelium. Injection into area 17 of the rat VC resulted in efficient labeling of the VC neurons and neurons in the lateral geniculate nucleus without any evident pathology or inflammation. Neither inflammation nor disease pathology was observed in either the mouse or rat after any route of delivery. CONCLUSIONS: It was demonstrated that the hrR3 HSV-1 virus can deliver a functioning gene to several cell types in the eye and neurons in the VC and that the virus can move via retrograde transport to nuclei that project to the VC.     

Herpes simplex virus mediated gene transfer to primate ocular tissues.

We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and beta-galactosidase activity was measured histochemically. Six cynomolgus monkey eyes received viral injections into the anterior chamber (2 x 10(7) pfu) and/or the vitreous (5 x 10(7) pfu), and the distribution of cells expressing lacZ was evaluated. In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent beta-galactosidase activity. In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells. Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively. We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis. This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys. Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable.     

Culture of retinal pigment epithelial cells from subretinal fluid

Culture of cells from subretinal fluid (SRF) was performed using 29 SRF samples obtained at retinal reattachment surgery. Proliferating cells were found in 58.6% of the samples studied. The cells were of retinal pigment epithelial (RPE) origin, as evidenced by their brown pigmentation in primary culture and their positive immunostaining for cytokeratins 8/18. The age of the patients did not affect the proliferative capacity of the cells. Proliferating cells were present in all samples from eyes with proliferative vitreoretinopathy (PVR) of grade C1 or more. In primary culture the cells had a fibroblast-like morphology, resembling that of ordinary RPE cells exposed to the vitreous. We conclude that the SRF of many patients with PVR contains viable proliferating RPE cells and that SRF offers a new source of RPE cells for studies on the pathogenesis of PVR.     

Enhanced expression of the complement factor H mRNA in proliferating human RPE cells

Background  

RPE cells are a major player in various diseases of the retina and choroid. Proliferating RPE cells are thought to be an initiating factor in proliferative vitreoretinopathy (PVR); the aging RPE cells are important in age-related macular degeneration (AMD). Early passages of cultured human retinal pigment epithelial cells were used as a model system to identify differentially expressed genes in proliferating retinal pigment epithelial (RPE) cells.     

EIU in the rat promotes the potential of syngeneic retinal cells injected into the vitreous cavity to induce PVR

PURPOSE: To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR). METHODS: Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed. RESULTS: The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye. CONCLUSIONS: In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.     

培养人视网膜色素上皮细胞中β-半乳糖苷酶活性研究

目的　观察培养人视网膜色素上皮 (RPE)细胞中复制衰老相关的 β 半乳糖苷酶活性。 方法　在培养的RPE细胞中 ,以传代次数 ( 5、10、2 0 )和取材供体的年龄 ( 2 6、44、5 7岁 )分组 ,进行标准化的 β 半乳糖苷酶活性和细胞增殖能力检测。结果　培养RPE细胞中 β 半乳糖苷酶活性阳性细胞数量随传代次数的增加而增加 ,在 2 0代的细胞中 ,阳性细胞的数量为 87%± 1 2 % ,明显高于第 5代和第 10代细胞 (P <0 0 5 )。而细胞的增殖能力明显下降 (P <0 0 5 )。结论　培养RPE细胞复制衰老的发生与细胞传代次数和取材年龄相关 ,其相关研究可能对了解年龄相关性视网膜疾病的发病机制有帮助     

Increased soluble interleukin-6 receptor in vitreous fluid of proliferative vitreoretinopathy

PURPOSE: To clarify the effect of the interaction between interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on retinal pigment epithelial (RPE) cell proliferation in proliferative vitreoretinopathy (PVR). METHODS: Concentrations of IL-6 and sIL-6R molecules in vitreous fluids were measured in eyes with PVR and idiopathic macular hole (MH), and the localization of IL-6 and IL-6R on the PVR fibrous membrane was studied. Production of IL-6 and sIL-6R by cultured RPE cells and the effect of the IL-6/sIL-6R complex on growth of cultured RPE cells were analyzed. RESULTS: Positive staining of IL-6 and IL-6R was observed in proliferating membranes in all PVR cases. IL-6 and sIL-6R concentrations in vitreous fluid from eyes with PVR were significantly higher than in eyes with MH (p < 0.05). A time-dependent increase in IL-6 molecules was identified in the culture medium of RPE cells, although sIL-6R was not detected. Dose-dependent growth of RPE cells was observed in the three concentrations (50, 100, and 500 ng/ml) of sIL-6R used. CONCLUSION: IL-6 derived from blood during the breakdown of the blood-retinal barrier (BRB) and produced by RPE cells and hematogenous sIL-6R cause RPE proliferation.     

Mutant herpes simplex virus-mediated suppression of retinoblastoma.

PURPOSE: To test the ability of a mutant herpes simplex virus (HSV) hrR3 to inhibit growth of Y79 human retinoblastoma in vitro and in vivo. METHODS: Cultured Y79 cells were infected with multiplicities of infection (MOI) ranging from 0.004 to 0.1 of hrR3. Surviving cells were counted using trypan blue dye exclusion. Using X-gal staining, expression of the lacZ gene was examined in vitro on day 3 postinfection to evaluate viral replication. Nude mice harboring Y79 tumors subcutaneously received an intraneoplasmic injection of 5 x 10(7) plaque-forming units of hrR3. The tumor sizes were measured weekly. Expression of the lacZ gene was also examined on one week postinfection. RESULTS: There are 31% and 13% cells surviving in cultured Y79 cells infected by hrR3 at an MOI of 0.1 on days 3 and 5 postinfection respectively compared to those of mock-infected cells. Also more than 70% of Y79 cells were stained with X-gal at an MOI of 0.1 which demonstrated active viral replication in vitro. Virus-treated subcutaneous tumors were smaller than control tumors (p<0.05, Student's t-test) on days 14, 21, and 28 postinfection. Positive X-gal staining was also observed in the tumor nodule which was challenged with this viral vector. CONCLUSIONS: We have demonstrated that hrR3 is capable of inhibiting Y79 tumor growth both in cell culture and in nude mice. These data suggest that gene therapy using this mutant HSV vector can be a new supplementary therapeutic modality for retinoblastoma.     

Induction of proliferative vitreoretinopathy by a unique line of human retinal pigment epithelial cells

BACKGROUND: The most widely used models of proliferative vitreoretinopathy (PVR) rely on injection of cells into the vitreous of animals. Using retinal pigment epithelial (RPE) cells from human PVR membranes may produce a more accurate model of human PVR. We performed a study to determine whether human RPE cells derived from a single epiretinal membrane (ERM) are capable of inducing the same disease in the rabbit eye, and whether the induced ERMs had cellular components similar to those of human PVR membranes. METHODS: Cells were harvested from a human ERM obtained at surgery for PVR. RPE cells were cultured from the membrane and injected into the right eye of 24 New Zealand albino rabbits. The left eyes served as controls. The eyes were examined by indirect ophthalmoscopy over 4 weeks. The enucleated eyes were then examined by means of microscopy and histochemical analysis. RESULTS: By day 7, PVR had developed in all but 1 of the 24 experimental eyes, with 8 progressing to localized tractional retinal detachment. By day 21, localized tractional retinal detachment had developed in 17 eyes; 1 eye progressed to extensive tractional retinal detachment by day 28. Immunostaining showed that mostly RPE cells, but also myofibroblasts, glial cells and collagen, were present in the newly formed rabbit PVR membranes. INTERPRETATION: Human RPE cells cultured from a PVR membrane appear to be capable of inducing PVR in rabbits. The resultant ERMs are similar to those formed in human PVR and consist mainly of RPE cells.     

Myofibroblast and extracellular matrix origins in proliferative vitreoretinopathy

Background

To evaluate origins of the fibrocontractive cell populations and their relation to collagens I and II in proliferative vitreoretinopathy (PVR).

Methods

Human PVR membranes were evaluated by indirect immunofluorescence for GFAP, cytokeratin-18 (CK-18), α-smooth muscle actin (αSMA), collagens I and II. Collagen expression by porcine Müller and retinal pigment epithelial cells (RPE) was evaluated using RT-PCR of RNA harvested from freshly isolated primary and proliferating cultures.

Results

Collagen I was detected in all PVR samples and was widely distributed in the extracellular matrix. In contrast, collagen II was present in only two of the ten samples and was localized to thin, acellular bands near the border of the tissues. Using cell type-specific markers CK-18 and GFAP, RPE and glia were localized to the collagen I-rich matrices. Cells positive for GFAP and CK-18 can also co-express αSMA. Normal and proliferating RPE express collagen I, but Müller cells show no evidence of collagen I expression until they proliferate in culture. In contrast, normal RPE and Müller cells contain message for collagen II which is lost shortly after introduction into culture.

Conclusions

Collagen I appears to be the predominate fibrillar collagen in human PVR membranes and collagen II a comparatively minor component. Müller cells and RPE are physically associated with the collagen I matrix and are capable of expressing this protein suggesting that they are the origin. It also appears that the majority of myofibroblasts in PVR membranes are derived from either RPE or Müller cells suggesting that they play a major role in membrane development.     

Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy

AIMS: To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors.Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes. RESULTS: In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-beta. The TGF-beta is activated, upregulating expression of CTGF. Under the influence of TGF-beta and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury. CONCLUSIONS: HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.     

早期视网膜内增生性玻璃体视网膜病变的发病机制

增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)是视网膜重建的非特异性组织修复过程中的一部分.它发生在视网膜脱离后,视网膜外层缺血,光感受器细胞逐渐死亡(主要是凋亡),神经元丢失并刺激胶质细胞(主要是Müller细胞)肥大,视网膜也开始重建及保护余下的神经元,但这一系列反应如果过度,胶质组织就会代替神经元,视网膜也会受到牵拉而缩短,发生早期的PVR改变.整个过程中,胶质细胞起始了PVR,之后视网膜色素上皮细胞发生去分化,转化为巨噬细胞或成纤维细胞样形态,之后细胞或纤维膜发生收缩,阻止了视网膜的复位,PVR随之发生.这个过程我们称之为早期视网膜内增生性玻璃体视网膜病变.     

增生性玻璃体视网膜病变中组织型转谷氨酰胺酶的表达

目的:检测增生性玻璃体视网膜病变(PVR)视网膜前膜和视网膜色素上皮(RPE)细胞中组织型转谷氨酰胺酶(tTG)的表达情况,探讨tTG是否参与PVR的发病过程.方法:PVR患者21例,收集视网膜前膜,C级8例,D级13例,行tTG免疫组织化学染色.另对培养3-5代的人RPE细胞分别用含有1 00mL/L PVR玻璃体液、正常玻璃体液和PBS的DMEM培养液进行孵育24h后行tTG免疫细胞化学染色.光镜下观察,比较C级和D级膜的表达阳性率,显微图像分析测量3组RPE细胞染色的平均灰度值.结果:在PVR视网膜前膜中tTG呈阳性表达(81%),C级和D级膜表达阳性率无差异 (C级88%,D级77%,P＞0.05).培养的人RPE细胞表达tTG,在PVR患者的玻璃体液作用下,tTG表达的平均灰度值为137.0±2.6,与正常玻璃体液组(143.5±2.9)及PBS对照组(143.6±3.0)相比,表达水平升高(P＜0.05).结论:PVR视网膜前膜和培养的人RPE细胞tTG表达阳性,在PVR患者的玻璃体液作用下,RPE细胞的tTG表达水平升高,这表明tTG参与了PVR的发病过程,在PVR视网膜前膜的形成过程中可能有重要作用.     

视网膜下液对培养的人眼视网膜色素上皮细胞和成纤维细胞增殖作用的影响

目的：了解不同程度增生性玻璃体视网膜病变（PVR）的视网膜下液（SRF）对视网膜色素上皮细胞（RPE）、成纤维细胞（FB）增殖的影响。方法：用溴标法和MTT法测定不同程度PVR的SRF对RPE和FB增殖的促进作用。结果：几乎所有的SRF都具有促进细胞增殖的作用,用1：10比例稀释,PVR程度为PVR＜C1,PVRC1,PVR＞C13组。SRF作用24h后RPE的增殖率分别为对照组的128.5％,139.8％,156.8％,FB的增殖率分别为对照组的126.3％,143.1％及172.3％。结论：SRF液促进细胞增殖的作用随着PVR程度的加重而增强。     

Evidence that a-wave latency of the electroretinogram is determined solely by photoreceptors

PURPOSE: To identify the retinal cells that determine the a-wave latency of rats. METHODS: Electroretinograms (ERGs) were recorded from the rod-dominated (0.85% cones) retinas of Long-Evans rats following an intravitreal injection of 1 microL of 40 mM 2-amino-4-phosphonobutyric acid to block the activity of the ON pathway of the second order retinal neurons. ERGs were also recorded following an intraperitoneal injection of sodium iodate to destroy the retinal pigment epithelial (RPE) cells. Damage to a large area of the retina was produced by constant light exposure, and focal damage to the retina was induced by argon laser photocoagulation. The effects of age and anesthesia level on the a-wave latency were also determined. RESULTS: Blocking the activity of the ON pathway of the second order retinal neurons did not alter the a-wave latency, and destroying the RPE cells also did not alter the a-wave latency. Damage to a large area of the retina resulted in prolonging the latency but focal retinal damage did not alter the a-wave latency. The a-wave latency was longer in young rat pups but was adult-like by 18 days. The level of anesthesia had no effect on the latency except at very deep stages. CONCLUSIONS: The a-wave latency is determined solely by the activity of the photoreceptors. A prolonged latency would indicate that the photoreceptors are damaged over a large area of the retina.     

Antiproliferative Wirkung von Genistein auf kultivierte retinale Pigmentepithelzellen vom Schwein

The isoflavonoid genistein is known as a potent inhibitor of proliferation in endothelial cells and in some tumor cell lines in vitro and in vivo. Cell growth arrest is mediated by inhbitor of the tyrosine-kinase receptor, a target for many growth factors such as fibroblast growth factor, platelet-derived growth factor, epidermal growth-factor and vascular endothelial growth factor, which may play a role in the development of proliferative vitreoretinopathy (PVR). In this study we report on the antiproliferative effect of genistein on retinal pigment epithelial cells (RPE), which is the major cell type involved in PVR. METHODS: Cell culture experiments using porcine RPE treated with different concentrations (5 up to 100 microgram/ml) of genistein in minimum essential medium +10% fetal calf serum. Inhibition of proliferation was demonstrated by two different methods: (1) uptake of 5-bromo-2-deoxyuridin (BrdU) in S phase after different exposure times (2) expression of proliferating cell nuclear antigen 72 h after scratching confluent RPE. RESULTS: Exposure of RPE to genistein resulted in a concentration-dependent inhibition of cell proliferation at concentrations over 25 microgram/ml geinistein. BrdU incorporation was reduced to 65% after 24- and 48-hour treatment with 100 microgram/ml genistein in comparison with the untreated cells. Proliferation of RPE growing into the artificial wound was inhibited when cells were exposed for 72 h to more than 25 microgram/ml genistein. CONCLUSIONS: Genistein at concentrations over 25 microgram/ml inhibits RPE proliferation. Further studies will be required to determine the applicability of genistein or other phytoestrogens in order to prevent uncontrolled wound healing processes such as PVR.     

Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells.

PURPOSE: To improve our understanding of how retinal pigment epithelial (RPE) cells behave in vivo and to establish similarities with dedifferentiation and adaptive events observed in RPE cells cultured under simulated intraocular pathologic conditions. At the same time, to examine the origin of epithelioid-shaped and fibroblast/fusiform-shaped cells in epiretinal membranes (ERM) from proliferative vitreoretinopathy (PVR). METHODS: Cells of ERM were studied by electron-immunocytochemical techniques, using simple, double, and triple immunostaining for cytokeratins (CK), vimentin (Vim), and glial fibrillary acidic protein (GFAP). Ultrastructural morphology analysis was also carried out. Adult human RPE cells were obtained and cultured with normal and pathologic vitreous from proliferative vitreoretinal disorders, subretinal fluid aspirates from retinal detachment, and normal human serum. Their cytoskeleton was fractionated at 7 (early cultures) and 24 (late cultures) days of culture, electrophoresed, immunoblotted for intermediate filament proteins, and quantified by densitometric analysis for each condition. Changes in phenotype characteristics were also evaluated. RESULTS: Epithelioid-shaped and fibroblast/fusiform-shaped cells, resembling RPE cells, expressed CK-Vim-GFAP simultaneously as intermediate filament proteins in their cytoskeleton. RPE cells in culture also expressed CK-Vim-GFAP and changed from an epithelial shape to a migratory fibroblast/fusiform-shaped phenotype in the presence of subretinal fluid aspirates and pathologic vitreous from proliferative intraocular disorders. In simulated cultures of proliferative intraocular disorders, cells decreased or retained their CK7, CK8, and CK18, retained Vim, and increased CK19 and GFAP, while their mesenchymal morphology became clearer over time. CONCLUSIONS: Studies of intermediate filament proteins in vivo suggest that dedifferentiation occurs in RPE cells in ERM. Dedifferentiated RPE cells may be responsible for epithelioid-like and fibroblast/fusiform-like cells. Furthermore, changes in intermediate filament protein levels were observed in RPE cells in simulated cultures of proliferative intraocular disorders. These changes were linked to cells acquiring a mesenchymal migratory, phenotype. Results indicate that the dedifferentiation of RPE cells occurs both in vivo and in vitro and that it can be explained as an epithelial-mesenchymal transition.     

Alterations in the structure of the epiretinal membranes in PVR -- assumptions and reality

BACKGROUND: Proliferative vitreoretinopathy (PVR) is known to be the main cause of failure for routine retinal detachment surgery. Our aim was to document the ultrastructural changes in the epiretinal membranes in cases of PVR. MATERIAL AND METHODS: Epiretinal membranes were taken during vitrectomy of 64 patients with different stages of PVR and further analysed using transmission and scanning electron microscopy. RESULTS: In the early stages of the disease mainly retinal pigment epithelial (RPE) cells, fibroblasts and occasional glial cells were found. Moreover, some of the RPE cells showed altered characteristics. In contrast, epiretinal membranes from the developed stages of PVR were comprised mostly of fibroblasts and a considerably diminished number of RPE cells. Cells with marks of degeneration were generally found. CONCLUSIONS: Our results point out that epiretinal membranes in PVR are dynamic structures, constantly changing their cell and matrix composition with the progression of the disease. The morphological changes together with the clinical data are important points to consider when discussing the most appropriate therapeutic approach for each case.