ABSTRACT

Purpose

The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting.

Materials and methods

Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring β–tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients.

Results

Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs.

Conclusion

Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR.

中文翻译：

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PDGF 作为增殖性糖尿病视网膜病变中与纤维血管膜相关的神经突生长的重要引发剂

摘要

目的

纤维血管膜 (FVM) 的形成是增殖性糖尿病视网膜病变 (PDR) 的严重威胁视力的并发症，可能导致视网膜脱离和最终失明。在这些膜的形成过程中，神经突/突起生长在视网膜神经元和神经胶质细胞中，它们既可以作为支架，也可以起到引导或调节作用。为了进一步了解这一过程，我们研究了先前确定的候选蛋白质，来自具有 FVM 的 PDR 患者的玻璃体，是否可以在实验环境中诱导神经突生长。

材料和方法

出生后第 3 天 (P3) 的 C57BL6/N 小鼠幼崽的视网膜外植体在聚-L-赖氨酸和层粘连蛋白涂层培养皿中培养。在添加潜在的神经突生长诱导剂的情况下进行生长刺激实验。通过使用 Image J 测量 β-微管蛋白免疫阳性神经突来进行神经突生长的自动分析。通过 RT-PCR 在 PDR 患者的 FVM 中定量 PDGF 受体的表达。

结果

血小板衍生生长因子 (PDGF) 以浓度依赖性方式诱导神经突生长，而神经调节蛋白 1 (NRG1) 和结缔组织生长因子 (CTGF) 则没有。当比较三种不同的 PDGF 二聚体时，用 PDGF-AB 处理导致最高的神经突诱导，其次是 PDGF-AA 和 -BB。此外，与来自黄斑裂孔患者的非糖尿病对照玻璃体相比，将视网膜外植体与来自 PDR 患者的玻璃体一起孵育导致神经突向外生长的显着诱导，这可以通过添加 CP673451（一种有效的 PDGF 受体 (PDGFR)）来预防抑制剂。在 FVM 中检测到 PDGF 受体的丰富表达。

结论

我们的研究结果表明，PDGF 可能参与视网膜神经突的生长，这与 PDR 中 FVM 的形成有关。