Department of Nanobiotechnology, KNBIRVO Block, Vision Research Foundation, Sankara Nethralaya, 18/41, College Road Nungambakkam, Chennai, Tamil Nadu, India.
School of Chemical and Biotechnology, SASTRA, Deemed University, Tanjore, Tamil Nadu, India.
Sci Rep. 2020 Dec 7;10(1):21382. doi: 10.1038/s41598-020-77138-3.
Keratoconus (KC) is a corneal dystrophy characterized by progressive ectasia that leads to severe visual impairment and remains one of the leading indications for corneal transplantation. The etiology is believed to be multifactorial and alterations have been documented in the biomechanical, biochemical and ultrastructural characteristics of the cornea. While the exact site of disease origin is still debated, changes in the corneal epithelium are believed to occur even before the disease is clinically manifested. In this study we investigate the possible role of β-catenin as mechanotransducer in KC corneal epithelium. The sheets of corneal epithelium removed from keratoconic eyes when they underwent collagen crosslinking as a therapeutic procedure were used for this study. The healthy corneal epithelium of patients undergoing Laser Refractive Surgery for the correction of their refractive error, served as controls. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyse the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, and Rac123. Co-immunoprecipitation of β-catenin followed by mass spectrometry of KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. Histology data reported breaks in the Bowman's layer in KC patients. We hypothesize that these breaks expose the epithelium to the keratoconic corneal stroma, which, is known to have a decreased elastic modulus and that β-catenin acts as a mechanotransducer that induces structural changes such as loss of polarity (Syntaxin3) and barrier function (ZO1) through membrane delocalization. The results of our study strongly suggest that β-catenin could be a putative mechanotransducer in KC epithelium, thus supporting our hypothesis.
圆锥角膜（KC）是一种角膜营养不良，其特征是进行性扩张，导致严重的视力损害，仍然是角膜移植的主要适应症之一。病因被认为是多因素的，并且已经在角膜的生物力学、生化和超微结构特征方面记录了改变。虽然疾病的确切起源部位仍有争议，但即使在疾病临床表现之前，角膜上皮的变化也被认为会发生。在这项研究中，我们研究了β-连环蛋白作为 KC 角膜上皮机械转导子的可能作用。当它们接受胶原交联作为治疗程序时，从圆锥角膜眼中取出的角膜上皮片用于这项研究。接受激光屈光手术矫正其屈光不正的健康患者的角膜上皮作为对照。对 KC 上皮进行免疫印迹和组织免疫荧光研究，以分析β-连环蛋白、E-钙粘蛋白、ZO1、α-连环蛋白、细胞周期蛋白 D1、α-辅肌动蛋白、RhoA 和 Rac123 的表达和定位。对 KC 上皮进行β-连环蛋白的共免疫沉淀，然后进行质谱分析，以鉴定其相互作用的伙伴。这通过使用在不同刚度的支架上生长的上皮组织进一步验证。组织学数据报告圆锥角膜患者的 Bowman 层破裂。我们假设这些破裂使上皮暴露于圆锥角膜基质，已知该基质的弹性模量降低，并且β-连环蛋白作为机械转导子，通过膜定位丧失诱导结构变化，如极性丧失（Syntaxin3）和屏障功能丧失（ZO1）。我们的研究结果强烈表明，β-连环蛋白可能是 KC 上皮中的一种潜在机械转导子，从而支持我们的假设。
