Human retinal pigment epithelium proliferation, migration and fluid transport

PDGF-C和-D是人视网膜色素上皮(RPE)表达的主要亚型。具有功能活性的PDGFR和PDGFR主要在顶膜表达。PDGF-BB、-CC、-DD显著促进细胞增殖，PDGF-BB、-AB、-DD显著促进细胞迁移，提示PDGF-BB、-CC、-DD在RPE病理生理过程中起重要作用。促炎细胞因子混合物(肿瘤坏死因子/白介素1/干扰素)通过诱导细胞凋亡、破坏细胞骨架和紧密连接来抑制PDGF诱导的增殖和迁移。 干扰素-γ(IFNG)是最重要的炎症介质之一，在多种视网膜疾病中表达上调。这些威胁视力的视网膜疾病发生在眼后；它们主要影响光感受器和邻近的视网膜色素上皮细胞，这些细胞起到保护神经视网膜的健康和完整性的作用。功能活性的IFNG受体主要定位于人胚胎视网膜色素上皮(HfRPE)的基底膜。该受体的激活显著抑制碱性成纤维细胞生长因子(BFGF)、血小板衍生生长因子(PDGF)和表皮生长因子(EGF)诱导的RPE迁移和增殖。相比之下，I型干扰素(干扰素α和干扰素β)对RPE的增殖无影响。IFNG对hfRPE的抑制作用可被JAK抑制剂I和AG490显著阻断，但不能被JAK3抑制剂阻断。此外，该JAK/STAT通路的激活上调了干扰素调节因子1(IRF-1)，但对IRF-2和ICSBP/IRF-8没有影响。有趣的是，IFNG显著刺激邻近脉络膜组织的细胞增殖。在基础浴液中加入IFNG，但在顶浴中加入IFNG，可显著增加hfRPE单层的液体转运(JV)。IFNG诱导的JV增加可被囊性纤维化跨膜电导调节因子(CFTRinh-172)的特异性抑制剂显著阻断。CFTR蛋白主要定位于hfRPE的基底膜。预先给予环己胺(4或24小时)可阻断IFNG引起的JV增加。IFNG对细胞内cAMP和Ca~(2+)水平无明显影响。然而，IFNG确实增加了P38MAPK的磷酸化。此外，加入P38 MAPK抑制剂SB203580和SB202190可显著降低IFNG引起的JV增加。随后添加的CFTRinh-172进一步减少了合资企业。尽管IFNG对细胞内cAMP无明显影响，但特异性蛋白激酶A抑制剂(H-)和RP-8-BR-cAMP显著抑制IFNG刺激的JV增加，竞争性地阻断cAMP与PKA的结合并阻断cAMP的作用。我们的结论是，IFNG抑制RPE的迁移和增殖，激活依赖CFTR的液体通过RPE的体内外吸收，JAK/STAT1、IRF-1、P38MAPK和PKA都参与了这些反应。这些发现提出了几个治疗目标，用于治疗增生性视网膜疾病，并清除在许多视网膜病变后发生的视网膜下腔积液。

PDGF-C and -D are the major isoforms expressed in human retinal pigment epithelium (RPE). Functionally active PDGFR-α and -β are mainly expressed at the apical membrane. PDGF-BB, -CC, -DD significantly increased proliferation while PDGF-BB, -AB and -DD significantly increased cell migration, suggesting a critical role in RPE pathophysiology. A pro-inflammatory cytokine mixture (TNFα/IL-1β/IFNγ) abrogated PDGF-induced proliferation and migration by inducing apoptosis, and by disrupting the cytoskeleton and tight junctions. Interferon gamma (IFNg) is one of the most important inflammatory mediators which are up-regulated in many retinal diseases. These sight threatening retinal diseases occur in the back of the eye; they mainly affect photoreceptors and the closely adjacent retinal pigment epithelial cells, which serve to protect the health and integrity of the neural retina. Functionally active IFNg receptor is mainly localized at the basolateral membrane of human fetal retinal pigment epithelium (hfRPE). The activation of this receptor significantly inhibits basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) induced RPE migration and proliferation. In contrast, type I IFN (IFN alpha and IFN beta) did not affect RPE proliferation. The inhibitory effects of IFNg on hfRPE were significantly blocked by JAK inhibitor I and by AG490 , but not by JAK3 inhibitor. In addition, activation of this JAK/STAT pathway up-regulates interferon regulatory factor 1 (IRF-1), but has no effect on IRF-2 and ICSBP/IRF-8. Interestingly, IFNg significantly stimulated the proliferation of cells from adjacent choroidal tissue. Addition of IFNg to the basal, but not the apical bath, significantly increased fluid transport (JV) across hfRPE monolayer. The IFNg induced JV increase was significantly blocked by a specific inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) (CFTRinh-172). CFTR protein is expressed and mainly localized on the basolateral membrane of hfRPE. IFNg induced JV increase was blocked by pretreatment with cyclohexamide (4 or 24 hours). IFNg had no measurable effects on intracellular cAMP or Ca2+ levels. IFNg did however increase the phosphorylation of P38 MAPK. In addition, the IFNg induced JV increase was significantly reduced by the addition of P38 MAPK inhibitors, SB203580 and SB202190. A further JV decrease was produced by the subsequent addition of CFTRinh-172. Although IFNg has no measurable effect on intracellular cAMP, IFNg stimulated Jv increase was significantly inhibited by specific protein kinase A (PKA) inhibitor (H-89) and Rp-8-Br-cAMPS, which competitive blocked cAMP binding to PKA and blocked the effect of cAMP. We conclude that IFNg inhibits RPE migration and proliferation, activates CFTR-dependent fluid absorption across RPE in vitro and in vivo, and that JAK/STAT1, IRF-1, P38 MAPK and PKA are all involved in mediating these responses. These finding suggest several therapeutic targets for treating proliferative retinal diseases and removing the fluid accumulation in the subretinal space that occurs following many retinal pathologies.