Choroidal endothelial cells transmigrate across the retinal pigment epithelium but do not proliferate in response to soluble vascular endothelial growth factor
Choroidal endothelial cells transmigrate across the retinal pigment epithelium but do not proliferate in response to soluble vascular endothelial growth factor
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DOI：
10.1016/j.exer.2005.08.021
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发表时间：
2006-04-01
影响因子：
3.4
通讯作者：
Hartnett, ME
中科院分区：
文献类型：
--
作者：
Geisen, P;McColm, JR;Hartnett, ME
本研究的目的是研究可溶性VEGF对人脉络膜内皮细胞（CEC）跨RPE单层迁移的影响，因为它与AMD中的脉络膜新生血管形成有关。然后将原代人CEC接种到插入物中。在生长72小时后对Transwell插入物中的CEC进行计数。在ARPE-CEC共培养条件培养基中或在添加有外源性VEGF（121）和/或VEGF（165）的培养基中培养CEC后，也测量CEC增殖。在具有8.0 μ m孔的Transwell上进行重复迁移测定：将绿色标记的CEC接种在Transwell插入物中，在插入物的下侧接种或不接种红色标记的ARPE。在一些迁移测定中，将ARPE铺在威尔斯孔中以提供CEC迁移的趋化梯度。接种CEC 72小时后，将孔培养基内的绿色细胞计数为穿过上皮单层的CEC，或将插入物下侧的细胞计数为穿过Transwell插入物但不超过ARPE单层的CEC。在将CEC接种到插入物中时，将VEGF的中和抗体加入到trans威尔斯孔的威尔斯孔中，并对迁移的CEC进行计数。与对照组相比，CEC在共培养条件培养液（P=0.001）或共培养试验（P < 0.001）中的增殖显著增加。然而，当VEGF（121）、VEGF（165）或两者同时加入单个CEC时，对CEC增殖没有影响。VEGF抗体不能降低共培养条件培养液对CEC的增殖作用。与孔中没有ARPE的迁移测定相比，在孔中铺板的ARPE显著增加CEC迁移（p < 0.001）。在孔内具有ARPE的迁移测定的孔培养基中测量的VEGF蛋白显著大于孔内没有ARPE的测定（P < 0.004）。外源性VEGF中和抗体可显著降低内皮细胞的迁移率，且这种作用具有剂量依赖性，VEGF可为内皮细胞跨ARPE单层迁移提供趋化梯度。中膜中VEGF的中和部分减少了移行。而可溶性VEGF并不增加单个CEC的增殖，共培养条件培养基增强增殖，表明VEGF以外的生长因子引起CEC增殖。这些发现可能与AMD中神经感觉视网膜内隐匿性CNV向CNV的转化有关。(c)2005爱思唯尔有限公司保留所有权利。
The purpose of this study was to investigate the effects of soluble VEGF on human choroidal endothelial cell (CEC) transmigration across an RPE monolayer as it relates to choroidal neovascularization in AMD.In coculture assays, ARPE-19 (ARPE) was plated on the undersides of Transwell inserts having 0.4 mu m pores. Primary human CECs were then plated into the insert. CECs in the Transwell inserts were counted after 72 hr of growth. CEC proliferation was also measured after culturing CECs in ARPE-CEC coculture-conditioned media or in media with exogenous VEGF(121) and/or VEGF(165) added. Transmigration assays were performed on Transwells with 8.0 mu m, pores: green-labelled CECs were plated in Transwell inserts with or without red-labelled ARPE plated on the undersides of the insert. In some transmigration assays, ARPE was plated into the wells to provide a chernotactic gradient for CEC transmigration. After 72 hr CECs were plated, green cells were counted either within the well media as CECs that transmigrated the epithelial monolayer, or on the underside of the insert as CECs that transmigrated the Transwell insert to but not beyond the ARPE monolayer. A neutralizing antibody to VEGF was added to the wells of Transwells at the time the CECs were plated in the insert and transmigrated CECs were counted. VEGF protein was measured in the conditioned media of ARPE and CEC coculture and in transmigration assays.Compared to control, CEC proliferation significantly increased when CECs were cultured in coculture conditioned media (P=0.001) or in coculture assays (p < 0.001). However, there was no effect on CEC proliferation when VEGF(121), VEGF(165), or both were added to solo CECs. Antibody to VEGF did not reduce the proliferative effects of coculture conditioned media on CEC. ARPE plated in the well significantly increased CEC transmigration (p < 0.001) compared to transmigration assays without ARPE in the well. VEGF protein measured in the well media of transmigration assays having ARPE within the well was significantly greater than in the assays without ARPE within the well (P < 0.004). Exogenous neutralizing antibody to VEGF significantly reduced transmigration, and this effect was dose-dependent.VEGF provides a chernotactic gradient for human CECs to transmigrate across a monolayer of ARPE. Neutralization of VEGF in the media partially reduces transmigration. Whereas soluble VEGF does not increase proliferation of solo CECs, coculture conditioned media enhances proliferation, suggesting that growth factors other than VEGF cause CEC proliferation. These findings may have relevance to the transformation of occult CNV into CNV within the neurosensory retina in AMD. (c) 2005 Elsevier Ltd. All rights reserved.