Department of Biochemistry, Boston University School of Medicine, 80 E. Concord St., Boston, MA, 02118, USA.
Department of Ophthalmology, Schepens Eye Research Institute/Mass. Eye and Ear, Harvard Medical School, 20 Staniford Street, Boston, MA, 02114, USA.
Exp Eye Res. 2018 May;170:127-137. doi: 10.1016/j.exer.2018.02.021. Epub 2018 Feb 27.
Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.
基质蛋白在发育和修复过程中的沉积对角膜的透明性至关重要。许多细胞对肿瘤中可能发生的缺氧状态有反应，但角膜在睁眼前的发育过程中和昼夜睡眠周期中会缺氧，此时氧气水平可从 21%降至 8%。在这项研究中，我们使用了两种三维（3-D）模型来研究基质细胞如何应对急性缺氧状态。第一种模型是基质构建模型，是一种类似于基质的 3-D 结构，由人角膜成纤维细胞（HCFs）组成，这些细胞受到稳定形式的抗坏血酸刺激 1、2 和 4 周，以自我组装自己的细胞外基质。第二种模型是角膜器官培养模型，是一种角膜伤口愈合模型，由成年大鼠角膜伤口组成，这些伤口被取出并置于培养中以进行愈合。两种模型都分别在常氧或缺氧条件下暴露于不同的时间段，并评估了基质蛋白的表达和/或定位。未检测到与 I 型胶原纤维相关的 V 型胶原发生明显变化；然而，小富含亮氨酸的重复蛋白聚糖和较大的硫酸乙酰肝素蛋白聚糖（perlecan）的表达都发生了显著变化。此外，缺氧降低了沿胶原纤维的 Cuprolinic blue 阳性糖胺聚糖链和硫酸酯酶 1 的数量，硫酸酯酶 1 通过去除 6-O-硫酸基团来调节硫酸乙酰肝素的作用。在基质构建模型中，纤维连接蛋白发生了类似于发育和损伤后的变化。损伤后纤维连接蛋白的这些变化伴随着蛋白聚糖的变化。这些发现表明，急性缺氧变化改变了角膜的生理学，这些模型将使我们能够操纵细胞外环境的条件，以研究角膜发育和创伤。
