Division of Chemistry for Materials, Faculty of Engineering, Graduate School of Mie University, Tsu, Japan.
Perit Dial Int. 2011 Jul-Aug;31(4):477-85. doi: 10.3747/pdi.2010.00166. Epub 2011 Jun 30.
In long-term peritoneal dialysis, myofibroblast-like cells found in the interstitium of the peritoneum are assumed to be a transformed type of mesothelial cell-epithelial-mesenchymal transition-positive [EMT(+)] human peritoneal mesothelial cells (HPMCs)-because they express a mesothelial marker, cytokeratin. However, no direct evidence about how these cells are able to invade from the mesothelium has yet been obtained.
In this study, we aimed to verify whether EMT(+) HPMCs would, in vitro, invade three-dimensionally along certain chemotactic factors.
We used reverse-transcriptase polymerase chain reaction to measure expression of Snail, E-cadherin, α(5)-integrin, and matrix metalloproteinase 2 (MMP2) messenger RNA (mRNA) in HPMCs exposed to 10 ng/mL transforming growth factor β1 (TGFβ1) and how that expression corresponds to cell motility, as represented by a video movie. We used the Transwell (12 μm pore diameter: Sigma-Aldrich, Tokyo, Japan) to construct a three-dimensional (3D) cell migration chamber. In the lower chamber, a concentration gradient of fibronectin (FN) or albumin(Alb) was formed in 0.1% type I collagen by diffusion (C(0)=22 nmol/L; concentration gradient: C/C(0)=0.7). All cells beneath the membrane were counted 72 hours after 5×10(4) EMT(+) HPMCs (HPMCs after a 48-hour exposure to 10 ng/mL TGFβ1) had been spread in the upper chamber.
After 72 hours, the increased motility of HPMCs resulting from their exposure to 10 ng/mL TGFβ1 had returned to baseline, but they retained an elongated morphology. Expression of Snail and MMP2 mRNA reached maximum at 24 hours. Expression of E-cadherin declined, and expression of α(5)-integrin increased continuously. In the 3D invasion study, significantly enhanced invasion by EMT(+) but not EMT(-) HPMCs was clearly seen in the presence of a FN concentration gradient (p<0.01), although invasion by EMT(+) and EMT(-) HPMCs in the absence of a FN concentration gradient was not statistically significantly different. Compared with the EMT(+) control (no concentration gradient), invasion by EMT(+) HPMCs was 2.1 ± 0.5 times (p<0.05) and 1.4 ± 0.4 times (p=nonsignificant) higher along the FN and Alb concentration gradients respectively. Increased invasion along the FN concentration gradient was significantly inhibited (p<0.05) when the HPMCs were pre-incubated with 5 μg/mL RGDS (a blocker for α(5)-integrin to FN).
We conclude that EMT(+) HPMCs invade collagen gel along the FN concentration gradient because of specific binding to RGDS receptors, which bind integrins such as α(5)-integrin, upregulating invasion-related gene expression associated with synthesis of the cytoskeleton protein α smooth muscle actin.
在长期腹膜透析中，腹膜间质中发现的成纤维细胞样细胞被认为是一种转化型间皮细胞-上皮-间充质转化阳性[EMT(+)]人腹膜间皮细胞（HPMCs），因为它们表达间皮标志物细胞角蛋白。然而，尚未获得关于这些细胞如何能够从间皮侵入的直接证据。
本研究旨在验证体外 EMT(+)HPMCs 是否能够沿着某些趋化因子在三维方向浸润。
我们使用逆转录酶聚合酶链反应来测量暴露于 10ng/ml 转化生长因子β1（TGFβ1）的 HPMCs 中 Snail、E-钙粘蛋白、α(5)-整合素和基质金属蛋白酶 2（MMP2）信使 RNA（mRNA）的表达，以及如何对应于视频电影中代表细胞迁移的运动性。我们使用 Transwell（12μm 孔径：Sigma-Aldrich，东京，日本）构建了一个三维（3D）细胞迁移室。在下室中，通过扩散形成了纤维连接蛋白（FN）或白蛋白（Alb）在 0.1%I 型胶原中的浓度梯度（C(0)=22nmol/L；浓度梯度：C/C(0)=0.7）。在将 5×10(4)EMT(+)HPMCs（在暴露于 10ng/ml TGFβ1 48 小时后）铺展在上室 72 小时后，计算穿过膜下的所有细胞。
72 小时后，HPMCs 暴露于 10ng/ml TGFβ1 导致的迁移能力已恢复至基线水平，但仍保持细长形态。Snail 和 MMP2mRNA 的表达在 24 小时达到最大值。E-钙粘蛋白表达下降，α(5)-整合素表达持续增加。在 3D 侵袭研究中，在 FN 浓度梯度存在的情况下，明显增强了 EMT(+)但不是 EMT(-)HPMCs 的侵袭（p<0.01），尽管 EMT(+)和 EMT(-)HPMCs 在不存在 FN 浓度梯度的情况下侵袭没有统计学差异。与 EMT(+)对照（无浓度梯度）相比，EMT(+)HPMCs 在 FN 浓度梯度下的侵袭率分别提高了 2.1±0.5 倍（p<0.05）和 1.4±0.4 倍（p=无意义）。FN 浓度梯度的侵袭明显受到抑制（p<0.05），当 HPMCs 用 5μg/ml RGDS（FN 中α(5)-整合素的阻断剂）预孵育时。
我们得出结论，EMT(+)HPMCs 沿着 FN 浓度梯度侵入胶原凝胶，因为它们与 RGDS 受体特异性结合，该受体与α(5)-整合素等整合素结合，上调与细胞骨架蛋白α平滑肌肌动蛋白合成相关的侵袭相关基因表达。
