Regulation of cell-mediated collagen gel contraction in human retinal pigment epithelium cells by vascular endothelial growth factor compared with transforming growth factor-β2

Background: To explore the potential role of vascular endothelial growth factor compared with transforming growth factor‐β2 in the regulation of human retinal pigment epithelium cell‐mediated collagen gel contraction. Methods: The retinal pigment epithelium cell mediated type I collagen gel contraction assay was performed to evaluate and compare the effect of vascular endothelial growth factor and transforming growth factor‐β2. The number of viable retinal pigment epithelium cells in the gel and the expression of α‐smooth muscle actin were analysed. Results: Both vascular endothelial growth factor and transforming growth factor‐β2 caused a time dependent gel contraction, associated with over expression of α‐smooth muscle actin in retinal pigment epithelium cells undergoing a fibroblast like transformation. The decrease in volume of the collagen gel and increase in α‐smooth muscle actin expression were more significant in the transforming growth factor‐β2‐treated group than in vascular endothelial growth factor‐treated group beginning at day 2, and the growth of retinal pigment epithelium cells was significantly more inhibited in the transforming growth factor‐β2‐treated group compared with the vascular endothelial growth factor‐treated group after day 1 (P < 0.05). Transforming growth factor‐β2 stimulation increased both vascular endothelial growth factor mRNA expression and secretion. The α‐smooth muscle actin expression and the change in volume of collagen gel were significantly positively correlated in both experimental groups. Conclusions: Both vascular endothelial growth factor and transforming growth factor‐β2 can cause induction of retinal pigment epithelium cell‐mediated collagen gel contraction in vitro via partial upregulation of α‐smooth muscle actin expression.     

Glucosamine inhibits epithelial‐to‐mesenchymal transition and migration of retinal pigment epithelium cells in culture and morphologic changes in a mouse model of proliferative vitreoretinopathy

Purpose: To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)‐β signalling and several processes involved in proliferative vitreoretinopathy (PVR). Methods: We evaluated the surface levels of TGF‐β receptor and its binding of TGF‐β in ARPE‐19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Results: Glucosamine reduced the surface levels of TGF‐β receptor and the ability of ARPE‐19 cells to bind TGF‐β. In ARPE‐19 cells, TGF‐β1 plus epidermal growth factor (EGF) or TGF‐β2 increased the expression of alpha‐smooth muscle actin (α‐SMA) and decreased the expression of zona occludens protein (ZO‐1). Transforming growth factor‐(β2) also caused the release of platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet‐derived growth factor and CTGF stimulated cell migration, and TGF‐β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Conclusions: Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF‐β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial–mesenchymal transition and PVR.     

The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells

Purpose  

Transforming growth factor (TGF)-β is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-β induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-β2-induced collagen expression in human retinal pigment epithelial cells.     

Fibronectin synthesis in subretinal membranes of proliferative vitreoretinopathy.

In situ hybridisation and immunohistochemical studies were conducted on six surgically excised subretinal membranes of proliferative vitreoretinopathy to investigate whether displacement of retinal pigment epithelial and glial cells to subretinal membranes was associated with fibronectin production by the subretinal membrane cells. Fibronectin messenger RNA (mRNA) and fibronectin immunoreactivity were observed in some cells in all of the subretinal membranes studied and up to 30% of the cells in individual specimens showed intense labelling for fibronectin mRNA. The results support the concept that the cells in subretinal membranes produce fibronectin. Locally produced fibronectin may play a role in subretinal membrane cohesion, and displacement of retinal pigment epithelial and glial cells from their normal location may induce the cells to manufacture fibronectin. Fibronectin production may be more prominent in migrating subretinal cells.     

增生性玻璃体视网膜病变膜剥离标本的免疫组化研究

目的 探讨不同增生性玻璃体视网膜病变(PVR)的增殖特征。方法采用5种特异性抗体对12例PVR膜样本进行免疫组织化学研究。结果成纤维细胞、神经胶质细胞为参与PVR膜的主要细胞成分,视网膜色素上皮细胞(RPE)、巨噬细胞、纤维连接蛋白和新生血管也参与了PVR的病理过程。结论新生血管主要参与了增生性视网膜血管病变的病理过程。增殖膜中增殖的细胞、细胞外基质和血管成分参与了PVR的病理过程并起着不同的作用。     

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.     

Variation in epiretinal membrane components with clinical duration of the proliferative tissue.

Immunohistochemical investigations were conducted on surgically excised epiretinal membranes to determine how cellular and extracellular components of proliferative vitreoretinopathy membranes change with time. Specimens of less than four months' duration contained a significantly higher proportion of retinal pigment epithelial cells than later membranes. No association was found between membrane duration and the content of collagen subtypes I to IV and laminin, but 'early' specimens contained significantly more fibronectin than did 'late' membranes. Fibronectin and collagens I, III, and IV showed a variable relationship with glial cells and were most consistently associated with retinal pigment epithelial and fibroblast-like cells. These observations may explain some of the surgical features of epiretinal membranes.     

Immunohistologic study of proliferative vitreoretinopathy

An immunohistologic study was performed on pars plana specimens obtained by biopsy in ten patients with rhegmatogenous retinal detachment, with or without proliferative vitreoretinopathy. Using immunofluorescence or immunoperoxidase procedures, linear deposits of IgG, IgA, and complement components were found in the eight cases of retinal detachment with proliferative vitreoretinopathy at the basal pole of the pigment epithelial cells and within the stroma. In contrast, these deposits were absent from the normal pars plana and from the retinal detachment without proliferative vitreoretinopathy. Moreover, pigment and nonpigment epithelial cells were found to express HLA-DR and HLA-DQ determinants in six of the eight patients with proliferative vitreoretinopathy. Our results are similar to those obtained in a previous study on proliferative diabetic retinopathy, which suggests the involvement of autoimmune phenomena in proliferative diseases and eventual interactions between the immune system and peptide growth factors. However, whether or not this immune reaction functions in the initiation or extension of intraocular proliferative syndromes remains to be determined.     

凝血酶对体外培养的视网膜色素上皮细胞生长的影响

目的 探讨凝血酶对视网膜色素上皮细胞的影响。方法 利用体外培养的视网膜色素上皮细胞，观察不同浓度的凝血酶对视网膜色素上皮细胞的影响。结果 各浓度的凝血酶对体外培养的视网膜色素上皮细胞具有促进增殖的作用。且40U/ml尤为明显。结论 凝血酶对体外培养的视网膜色素上皮细胞具有促进增殖的作用。这为探讨增生性玻璃体视网膜病变的提供了实验依据。     

Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor

AIMS: To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation. METHODS: RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and alpha smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay. RESULTS: Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of alpha smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1beta, transforming growth factor beta1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction. CONCLUSIONS: These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.     

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.     

Immunocytology of cellular components in vitreous and subretinal fluid from patients with proliferative vitreoretinopathy.

Proliferative vitreoretinopathy accounts for most of failures in retinal detachment surgery. It results from the formation of membranes spreading onto inner and outer surfaces of the detached retina and within the vitreous body, but the nature of the growing cells and the mechanisms of proliferation remain speculative. A cytological study was thus undertaken on 35 specimens of vitreous and subretinal fluid obtained surgically in patients with proliferative vitreoretinopathy. Various types of cells were identified: typical pigment epithelial cells, lightly pigmented and large totally unpigmented macrophage-resembling cells, smaller unpigmented cells and lymphocytes. Immunocytological procedures with 10 different monoclonal antibodies directed against different markers of epithelial and immunocompetent cells showed the epithelial nonmacrophagic origin of the intravitreal and subretinal cells, as most of these cells were positive for cytokeratin but remained negative for macrophage markers. Examination of intravitreal pigment granules, using autofluorescence analysis by epi-illumination and toluidine blue staining, showed two distinct populations of pigmented cells, one containing melanin and the other lipofuscin, suggesting that pigmented cells could originate from the retinal and ciliary pigment epithelia. As concerns lymphocyte identification, only B cells were seen, whereas no T lymphocyte could be found. Fibronectin was found on a minority of cells in 4 vitreous specimens, but cells positive for glial fibrillary acidic protein could not be seen. These results confirm the involvement of pigment epithelial cells and the strong morphological changes they undergo during the course of proliferative vitoretinopathy, but the mechanisms of proliferative phenomena after retinal detachment remain to be determined.     

Gelatinase B in proliferative vitreoretinal disorders

Purpose: To investigate whether gelatinases A and B are involved in the pathogenesis of proliferative vitreoretinal disorders.Methods: In a prospective study of 101 consecutive patients, vitreous and paired serum samples were obtained from 38 patients with rhegmatogenous retinal detachment complicated by proliferative vitreoretinopathy, 25 patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy, and 38 patients with proliferative diabetic retinopathy. Gelatinase activities were determined by quantitative zymography.Results: All vitreous samples contained comparable levels of the constitutive gelatinase A. Inducible gelatinase B was detected in eight (32%) of 25 vitreous samples from patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy (mean ± SD, 319.5 ± 521.0 scanning units), in 17 (44.7%) of 38 vitreous samples from patients with proliferative vitreoretinopathy (560.6 ± 718.9 scanning units), and in 34 (89.5%) of 38 vitreous samples from patients with proliferative diabetic retinopathy (1,707.2 ± 1,220.3 scanning units). The incidence of detection of gelatinase B in proliferative diabetic retinopathy cases was significantly higher than it was in rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and proliferative vitreoretinopathy cases (P < .001). Gelatinase B levels in the vitreous samples of patients with proliferative diabetic retinopathy were higher than the levels found in patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and in patients with proliferative vitreoretinopathy (P = .0152). Gelatinase A was detected in all the tested sera, whereas none of the tested paired serum samples contained detectable gelatinase B activity.Conclusions: Gelatinase B may play an important role in extracellular matrix degradation associated with neovascularization in proliferative diabetic retinopathy.     

Homologous serum-stimulated fibronectin synthesis in human retinal pigmented epithelial cells.

PURPOSE: Fibronectin expression has been recorded in subretinal membranes from patients with proliferative vitreoretinopathy (PVR). Retinal pigmented epithelial (RPE) cells are a major cellular component of PVR membranes and synthesize fibronectin. We investigated the effects of human serum (as a model of vascular leakage in vivo) on the expression of fibronectin by cultured human RPE cells and compared the responses to those stimulated by fetal bovine serum (FBS). METHODS: Human RPE cells were incubated in M199 with 1, 10 and 40% human adult serum or FBS for 72 h. RESULTS: Retinal pigmented epithelial cells expressed maximum extracellular matrix fibronectin when exposed to 40% human serum using immunohistochemistry. Using ELISA to quantify fibronectin, 10 and 40% human serum significantly increased the total fibronectin (P < 0.01; n = 4), whereas FBS did not affect fibronectin expression. CONCLUSIONS: The results show that human serum contains substances stimulating fibronectin synthesis in human RPE cells.     

Inhibition of migration but stimulation of proliferation of human retinal pigment epithelial cells cultured with uniform vesicles of silicone oil

Background  

It has been hypothesized that emulsification of silicone oil might stimulate preretinal membrane formation and proliferative vitreoretinopathy. This study aimed to test the effects of vesicles of silicone oil (VSO), which were prepared by a novel membrane emulsification technique, on the migration and proliferation of human retinal pigment epithelial (hRPE) cells in vitro.     

视网膜后膜的超微结构观察

在34例视网膜脱离伴增殖性玻璃体视网膜病变的慢性病例中，观察到12例视网膜后膜，占35.3%.其中对1例经玻璃体手术切除的标本进行了透射电镜检查。神经胶质细胞、视网膜色素上皮细胞及大量胶原纤维是后膜的主要细胞或间质成分。 （中华眼底病杂志，1994，10：165-166）     

Effect of tranilast on proliferation,collagen gel contraction,and transforming growth factor beta secretion of retinal pigment epithelial cells and fibroblasts

The efficiency of tranilast for the treatment of proliferative vitreoretinopathy (PVR) was investigated in vitro. A tetrazolium-based colorimetric assay showed that the 300-microM concentration of tranilast inhibited proliferation of bovine retinal pigment epithelial (RPE) cells and rabbit dermal fibroblasts with no toxicity. The contraction of collagen gels embedded with these cells was evaluated in the cultures. Compared with the gel incubated with minimal essential medium and 0.35% bovine serum albumin and/or fetal calf serum, tranilast inhibited gel contraction. Enzyme-linked immunosorbent assay revealed that a 300-microM concentration of tranilast inhibited transforming growth factor-beta(1) (TGF-beta(1)) secretion significantly (p < 0.01). These results suggest that tranilast may inhibit the proliferation of RPE cells and fibroblasts and contraction of intraocular fibrous membranes by suppressing TGF-beta(1) secretion from these cells with a potential to treat PVR.     

Subretinal proliferation

Subretinal proliferation is often present but less often identified as a component of proliferative vitreoretinopathy. Subretinal membranes form from retinal pigment epithelial cells and retinal glial cells that migrate into the subretinal space of eyes with long-standing retinal detachments. The membranes grow as sheets but break up into bands as the cells contract, with the stronger parts remaining intact.     

增生性玻璃体视网膜病变增生膜再塑型机制的研究

目的 观察不同病程增生性玻璃体视网膜病变（PVR）增生膜中不同细胞成分、细胞外基质（ECM）、基质金属蛋白酶（MMPs）及其抑制剂（TIMPs）随病程变化的规律，探讨PVR增生膜的再塑型机制。 方法 病程2个月至8年的孔源性视网膜脱离伴PVR患者的增生膜手术标本16例，用免疫组织化学方法标记增生膜中视网膜色素上皮（RPE）细胞、胶质细胞等不同的细胞成分，纤维连接蛋白（FN）、层粘连蛋白（l aminin）、Ⅰ～Ⅳ型胶原等不同ECM成分，以及MMPs（MMP2、MMP9）和TIMP1，分析不同病程PVR增生膜中各标记成分的变化以及与病程的相关性。 结果 随PVR病程延长，增生膜中RPE细胞、MMP2、FN表达逐渐减少(P=0.014，P=0.001，P=0.008)， 胶质细胞、Ⅰ、Ⅲ型胶原逐渐增多（P=0.022，P=0.001，P=0.008)，层粘连蛋白和Ⅱ、Ⅳ型胶原均有表达，但不随病程变化。RPE细胞、MMP2、纤维连接蛋白的表达与病程呈负相关，胶质细胞、Ⅰ、Ⅲ型胶原的表达与病程呈正相关；MMP2与FN变化呈正相关。MMP9、TIMP1始终都有表达，但不随病程变化。 结论 在PVR增生膜形成和发展的过程中，增生膜中的RPE细胞、胶质细胞、FN、Ⅰ、Ⅲ型胶原、MMP2参与了PVR的再塑型过程。 (中华眼底病杂志， 2006， 22： 308-312）