Gopalakrishnan Adhithya Subramanian, Sirajudeen Sumaiya, Banu Nasrin, Nunes Jessica, Rajendran Divya T, Yadav Seema, Prajna Namperumalsamy Venkatesh, Williams Rachel, Kuppamuthu Dharmalingam, Giridhara Gopalan Ramprasad Obula
Aravind Medical Research Foundation, Madurai, Tamil Nadu, India.
Aravind Eye Hospital and Post-graduate Institute of Ophthalmology, Madurai, Tamil Nadu, India.
Exp Eye Res. 2025 Feb;251:110208. doi: 10.1016/j.exer.2024.110208. Epub 2024 Dec 15.
The need for better and simpler alternative crosslinking strategies to treat keratoconus (KC) is becoming essential as there is only a single approved way to treat it. Recently, conventional UV-A Riboflavin crosslinking is proven to have some disadvantages such as causing damage to the corneal endothelium and inducing keratocyte apoptosis. A chemical cross-linker (CXL) using carbodiimide chemistry and an octanedioic acid spacer is found effective in stiffening the cornea and has the potential to be developed as an alternative therapy to halt KC progression. In order to investigate the molecular changes induced by the cross-linker, we have analyzed the effect of the cross-linker on the activity of matrix metalloproteases (MMPs) in epithelial and stromal layers of KC corneas and in vitro cellular systems to determine its role in stiffening the KC cornea. At well-optimized concentration, KC corneal buttons were treated with the CXL and the stiffening of the cornea was measured. The collagen fibril assembly in the stroma was analyzed using transmission electron microscopy and the activity of MMPs 2 and 9 were visualized using gelatin zymography. KC corneal fibroblasts in culture and tumor necrosis factor-α (TNF-α) induced human corneal epithelial (HCE) cell line were treated with CXL and secretion of MMPs 1, 2, 3 and 9 were analyzed by enzyme-linked immunosorbent assay (ELISA). We found that the CXL stiffened the KC corneas comparable to the normal corneas, with very less cytotoxicity. The collagen fiber assembly was reorganized in an orderly fashion and fibril density and diameter increased after CXL treatment. The activity of MMPs and cathepsin G in the epithelial and stromal layers of KC tissues decreased post-treatment. Secretion and activity of MMPs from the corneal epithelial and stromal cells after CXL treatment were significantly reduced while the epithelial lysyl oxidase activity increased. The CXL, intended to stop the KC progression, modified the extracellular matrix collagen assembly in the stroma and decreased the secretion of a group of metalloproteases and their activity. We have demonstrated a set of molecular changes effected by the CXL, which might aid in the stiffening of the KC cornea.
由于治疗圆锥角膜（KC）只有一种获批的方法，因此寻求更好、更简便的替代性交联策略来治疗圆锥角膜变得至关重要。最近，传统的紫外线A核黄素交联已被证明存在一些缺点，如对角膜内皮造成损伤和诱导角膜细胞凋亡。一种使用碳二亚胺化学和辛二酸间隔基的化学交联剂（CXL）被发现对角膜硬化有效，并且有潜力被开发为阻止KC进展的替代疗法。为了研究交联剂诱导的分子变化，我们分析了交联剂对KC角膜上皮和基质层以及体外细胞系统中基质金属蛋白酶（MMPs）活性的影响，以确定其在KC角膜硬化中的作用。在优化的浓度下，用CXL处理KC角膜植片并测量角膜的硬度。使用透射电子显微镜分析基质中的胶原纤维组装，并使用明胶酶谱法观察MMPs 2和9的活性。用CXL处理培养的KC角膜成纤维细胞和肿瘤坏死因子-α（TNF-α）诱导的人角膜上皮（HCE）细胞系，并通过酶联免疫吸附测定（ELISA）分析MMPs 1、2、3和9的分泌情况。我们发现CXL使KC角膜硬化程度与正常角膜相当，且细胞毒性非常低。CXL处理后，胶原纤维组装以有序方式重新组织，纤维密度和直径增加。KC组织上皮和基质层中MMPs和组织蛋白酶G的活性在处理后降低。CXL处理后，角膜上皮和基质细胞中MMPs的分泌和活性显著降低，而上皮赖氨酰氧化酶活性增加。旨在阻止KC进展的CXL改变了基质中的细胞外基质胶原组装，并降低了一组金属蛋白酶的分泌及其活性。我们已经证明了CXL引起的一系列分子变化，这可能有助于KC角膜的硬化。
