Alge Claudia S, Priglinger Siegfried G, Kook Daniel, Schmid Holger, Haritoglou Christos, Welge-Lussen Ulrich, Kampik Anselm
Department of Ophthalmology, Ludwig-maximilians-University, Munich, Germany.
Invest Ophthalmol Vis Sci. 2006 Jan;47(1):415-26. doi: 10.1167/iovs.05-0308.
To determine whether the beta-galactoside-binding matricellular protein Gal-1 is expressed in human specimens of proliferative vitreoretinopathy (PVR) and to evaluate its influence on RPE migration.
RT-PCR was used to detect Gal-1-specific transcripts in PVR membranes, and the expression pattern of Gal-1 was examined by immunohistochemistry. Expression of Gal-1 in native, low- and high-density cultured RPE cells was determined by Western blot analysis. Cultured human RPE cells were treated with bFGF, TGF-beta2, PDGF-BB, or HGF. The dose-response of Gal-1 mRNA expression was measured by by real-time quantitative RT-PCR and Northern blot analysis. Induction of Gal-1 protein was confirmed by Western blot analysis. To study the effect of Gal-1 on RPE migration in vitro, Gal-1 expression was silenced by RNA interference. beta-Lactose was used to saturate extracellular galectins. RPE cell migration was assessed by a modified Boyden chamber assay, with HGF as the chemoattractant.
Gal-1 mRNA expression was present in human specimens of PVR membranes, and staining for Gal-1 was distributed throughout the extracellular matrix (ECM) of PVR membranes. Colocalization was found with laminin and fibronectin and cells of epithelial origin. Western blot analysis revealed greater baseline expression levels in low-density cultured RPE cells than in native and high-density cultured RPE cells. Treatment with HGF caused a dose-dependent increase in Gal-1 expression. Low expression levels of Gal-1 correlated with a reduction of RPE migration to 14% of control. beta-Lactose inhibited HGF-induced RPE cell migration to 23% of control.
Gal-1 is present in the extracellular matrix of PVR membranes and may be derived from dedifferentiated RPE cells. The expression level of Gal-1 appears to be related to a migratory RPE phenotype and stimulation by HGF, both conditions implicated in the pathogenesis of early PVR. Furthermore, HGF-induced RPE migration may be dependent, at least in part, on Gal-1- and beta-galactoside-dependent mechanisms.
确定β-半乳糖苷结合基质细胞蛋白Gal-1是否在增殖性玻璃体视网膜病变（PVR）的人体标本中表达，并评估其对视网膜色素上皮（RPE）细胞迁移的影响。
采用逆转录聚合酶链反应（RT-PCR）检测PVR膜中Gal-1特异性转录本，并用免疫组织化学法检测Gal-1的表达模式。通过蛋白质免疫印迹分析确定Gal-1在天然、低密度和高密度培养的RPE细胞中的表达。用碱性成纤维细胞生长因子（bFGF）、转化生长因子-β2（TGF-β2）、血小板衍生生长因子-BB（PDGF-BB）或肝细胞生长因子（HGF）处理培养的人RPE细胞。通过实时定量RT-PCR和Northern印迹分析测量Gal-1 mRNA表达的剂量反应。通过蛋白质免疫印迹分析证实Gal-1蛋白的诱导。为了研究Gal-1对体外RPE细胞迁移的影响，通过RNA干扰使Gal-1表达沉默。用β-乳糖饱和细胞外半乳糖凝集素。以HGF作为趋化因子，通过改良的Boyden小室试验评估RPE细胞迁移。
Gal-1 mRNA表达存在于PVR膜的人体标本中，Gal-1染色分布于PVR膜的整个细胞外基质（ECM）中。发现其与层粘连蛋白、纤连蛋白以及上皮来源的细胞共定位。蛋白质免疫印迹分析显示，低密度培养的RPE细胞中的基线表达水平高于天然和高密度培养的RPE细胞。用HGF处理导致Gal-1表达呈剂量依赖性增加。Gal-1低表达水平与RPE细胞迁移减少至对照的14%相关。β-乳糖将HGF诱导的RPE细胞迁移抑制至对照的23%。
Gal-1存在于PVR膜的细胞外基质中，可能来源于去分化的RPE细胞。Gal-1的表达水平似乎与迁移性RPE表型以及HGF刺激有关，这两种情况都与早期PVR的发病机制有关。此外，HGF诱导的RPE细胞迁移可能至少部分依赖于Gal-1和β-半乳糖苷依赖性机制。
