The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments.
The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments.
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DOI：
10.1167/iovs.15-16388
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发表时间：
2015-03
影响因子：
4.4
通讯作者：
M. Miron-Mendoza;Eric Graham;Pouriska B. Kivanany;Jonathan Quiring;W. Petroll
中科院分区：
文献类型：
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作者：
M. Miron-Mendoza;Eric Graham;Pouriska B. Kivanany;Jonathan Quiring;W. Petroll
我们先前报道了细胞外基质成分（纤维蛋白与胶原）调节角膜成纤维细胞在三维培养中的扩散和迁移模式。在这项研究中，我们调查凝血酶和细胞收缩性在介导这些差异的细胞行为的作用。方法：将角膜成纤维细胞铺在纤维状胶原和纤维蛋白基质上，以评估细胞铺展。为了评估三维细胞迁移，将接种有角膜成纤维细胞的压实胶原基质包埋在脱细胞胶原或纤维蛋白基质内。构建体在含有血小板衍生生长因子（PDGF）、有或没有凝血酶、Rho激酶抑制剂Y-27632和/或肌球蛋白II抑制剂blebbistatin的无血清培养基中培养。我们使用三维和四维成像来评估细胞的机械行为，连接和细胞骨架组织。结果凝血酶刺激角膜成纤维细胞的收缩性增加。凝血酶也诱导Rho激酶依赖性的细胞群集上的顺应性胶原基质，但不是刚性基板上。相反，纤维蛋白基质上的细胞即使在Rho激酶被抑制时也聚结成簇。在巢状基质中，即使在凝血酶存在下，细胞也总是独立地通过胶原迁移。相反，迁移到纤维蛋白中的细胞形成相互连接的网络。Y-27632和blebbistatin都降低了纤维蛋白中的迁移率，但细胞继续集体迁移。结论：结果表明，虽然凝血酶诱导的肌动球蛋白收缩可以诱导成纤维细胞在顺应性胶原基质上的聚集，但它不会诱导3-D胶原结构内的集体细胞迁移。此外，与纤维蛋白相互作用的角膜成纤维细胞的聚集或集体迁移不需要增加收缩性。
PURPOSE We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior. METHODS To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization. RESULTS Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively. CONCLUSIONS The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin.