Fayet Cristina, Bendeck Michelle P, Gotlieb Avrum I
Toronto General Research Institute and Department of Pathology, University Health Network, Toronto, Ontario, Canada M5G 2C4.
Cardiovasc Pathol. 2007 Jul-Aug;16(4):203-11. doi: 10.1016/j.carpath.2007.02.008. Epub 2007 May 11.
Fibronectin, an extracellular matrix protein, is associated with the general process of tissue repair and is present in heart valves. In order to understand the cellular mechanisms of heart valve repair, we hypothesized that fibronectin is produced and secreted by valvular interstitial cells (VICs), and when up-regulated in VICs involved in active repair, it is associated with prominent fibrillar adhesions composed of tensin and alpha(5)beta(1) integrin. We investigated the interaction of porcine mitral VICs with the underlying fibronectin matrix and the formation and localization of focal and fibrillar adhesion complexes in an in vitro wound model.
Confluent monolayers of VICs were wounded with a 1-mm-wide cell scraper, maintained in standard media and 10% fetal bovine serum, and fixed at various time points after wounding. Immunohistochemistry was used to localize fibronectin, paxillin, tensin, and alpha(5)beta(1) integrin. F-actin was localized with an Alexa-Fluor-568-labeled phalloidin. Cells were examined with a scanning confocal laser microscope.
In response to in vitro mechanical wounding, migrating VICs at the wound edge expressed cytoplasmic fibronectin compared to nonwounded confluent monolayers. Over 24 to 48 h, fibrils were deposited into the subcellular space. Coincident with this, staining for alpha(5)beta(1) appeared, and tensin redistributed from focal adhesions to fibrillar adhesions, which colocalized with alpha(5)beta(1).
Fibronectin in association with fibrillar adhesions is a component of the matrix that may be secreted by migrating VICs to regulate repair at sites of valve injury.
纤连蛋白是一种细胞外基质蛋白，与组织修复的一般过程相关，且存在于心脏瓣膜中。为了了解心脏瓣膜修复的细胞机制，我们推测纤连蛋白由瓣膜间质细胞（VICs）产生并分泌，并且在参与积极修复的VICs中上调时，它与由张力蛋白和α(5)β(1)整合素组成的显著纤维状黏附相关。我们在体外伤口模型中研究了猪二尖瓣VICs与下层纤连蛋白基质的相互作用以及局部黏附复合体和纤维状黏附复合体的形成与定位。
用1毫米宽的细胞刮铲刮伤汇合的VICs单层，将其置于标准培养基和10%胎牛血清中培养，并在刮伤后的不同时间点固定。采用免疫组织化学法定位纤连蛋白、桩蛋白、张力蛋白和α(5)β(1)整合素。用Alexa-Fluor-568标记的鬼笔环肽定位F-肌动蛋白。用扫描共聚焦激光显微镜检查细胞。
与未受伤的汇合单层相比，体外机械损伤后，伤口边缘迁移的VICs表达细胞质纤连蛋白。在24至48小时内，纤维沉积到亚细胞空间。与此同时，出现了α(5)β(1)的染色，张力蛋白从局部黏附重新分布到与α(5)β(1)共定位的纤维状黏附。
与纤维状黏附相关的纤连蛋白是基质的一个组成部分，可能由迁移的VICs分泌，以调节瓣膜损伤部位处的修复。
