Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro

Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both in vitro and ex vivo. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) in vitro. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O2) or hypoxic (2% O2) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by HKCs, which may have consequential effects on ECM structure in the context of KC.

圆锥角膜（Keratoconus，KC）是一种以中央角膜变薄为特征的进行性角膜扩张症。硬性隐形眼镜、透气性硬性角膜接触镜（rigid gas permeable lenses）与巩膜镜是圆锥角膜早中期阶段的主要治疗手段，用于矫正因角膜结构不规则引发的屈光不正与散光。此类治疗手段存在显著弊端，包括会减少泪膜及氧气向角膜上皮与基质的递送。然而，目前仍不清楚缺氧是否会影响圆锥角膜病理进程中的角膜完整性。多项研究已在体外与离体实验中证实，氧化应激水平升高与圆锥角膜存在关联。本研究提出假设：与健康对照组相比，源自圆锥角膜患者的角膜成纤维细胞更易受缺氧诱导的氧化应激影响，进而加剧圆锥角膜患者的角膜变薄进程。本研究在体外环境下探究了缺氧对健康人角膜成纤维细胞（healthy controls，HCFs）与圆锥角膜患者来源角膜成纤维细胞（KC patients，HKCs）的细胞外基质（ECM）分泌、组装以及基质金属蛋白酶（matrix metalloproteinase，MMP）表达的影响。将HCFs与HKCs在三维构建体中培养3周，随后分别维持于常氧（21% O₂）环境或转移至缺氧（2% O₂）环境，继续培养1周。在培养至第4周时，分离三维构建体，检测其中I型胶原、III型胶原、V型胶原、角膜蛋白聚糖（keratocan）以及MMP-1、MMP-2、MMP-3、MMP-9、MMP-13的表达水平，同时检测缺氧标志物缺氧诱导因子-1α（hypoxia inducible factor-1α）与乳铁蛋白（lactoferrin）的含量。同时收集条件培养液，通过蛋白质印迹法（Western blot）检测其中I型胶原、III型胶原与V型胶原的表达。采用免疫荧光显微镜法检测HCFs与HKCs所组装的ECM厚度。结果显示，缺氧可显著降低HKCs的I型胶原分泌量，并上调MMP-1与MMP-2的表达水平，但对MMP-3、MMP-9及MMP-13的表达无显著影响。在低氧环境中培养1周后，两种细胞所组装的ECM厚度均出现下降。本研究表明，缺氧可调控HKCs的胶原与MMP表达，这可能对圆锥角膜背景下的ECM结构产生实质性影响。