Single-cell atlas of keratoconus corneas revealed aberrant transcriptional signatures and implicated mechanical stretch as a trigger for keratoconus pathogenesis

Abstract

Keratoconus is a common ectatic corneal disorder in adolescents and young adults that can lead to progressive visual impairment or even legal blindness. Despite the high prevalence, its etiology is not fully understood. In this study, we performed single-cell RNA sequencing (scRNA-Seq) analysis on 39,214 cells from central corneas of patients with keratoconus and healthy individuals, to define the involvement of each cell type during disease progression. We confirmed the central role of corneal stromal cells in this disease, where dysregulation of collagen and extracellular matrix (ECM) occurred. Differential gene expression and histological analyses revealed two potential novel markers for keratoconus stromal cells, namely CTSD and CTSK. Intriguingly, we detected elevated levels of YAP1 and TEAD1, the master regulators of biomechanical homeostasis, in keratoconus stromal cells. Cyclical mechanical experiments implicated the mechanical stretch in prompting protease production in corneal stromal cells during keratoconus progression. In the epithelial cells of keratoconus corneas, we observed reduced basal cells and abnormally differentiated superficial cells, unraveling the corneal epithelial lesions that were usually neglected in clinical diagnosis. In addition, several elevated cytokines in immune cells of keratoconus samples supported the involvement of inflammatory response in the progression of keratoconus. Finally, we revealed the dysregulated cell-cell communications in keratoconus, and found that only few ligand-receptor interactions were gained but a large fraction of interactional pairs was erased in keratoconus, especially for those related to protease inhibition and anti-inflammatory process. Taken together, this study facilitates the understanding of molecular mechanisms underlying keratoconus pathogenesis, providing insights into keratoconus diagnosis and potential interventions.

Unraveling the mechanobiology of cornea: From bench side to the clinic

“…β-catenin in CEpCs acts as a mechano-transducer of substrate stiffness that induces structural changes, such as the disruption of the cytoskeleton (F-actin), loss of polarity (Syntaxin3), and barrier function (ZO-1), by delocalizing from the membrane to the cytosol in KC ( Figure 4 ) ( Amit et al, 2020 ). YAP and its cooperator, TEA domain transcription factor (TEAD), in stromal cells are the mechanotransducers of stretch that prompt protease production (including MMP1, MMP3, cathepsin D, and cathepsin K) in KC ( Figure 4 ) ( Dou et al, 2022 ). Some case reports have indicated that eye rubbing is closely associated with KC ( Table 2 ) ( Bawazeer et al, 2000 ; Weed et al, 2008 ; Kandarakis et al, 2011 ; Panahi-Bazaz et al, 2014 ; Gunes et al, 2015 ; Panikkar et al, 2016 ; Yusuf and Salmon, 2016 ; Bral and Termote 2017 ).…”

“…Different types of cells in the cornea can perceive and transduce mechanical signals in distinct ways, which modulate the expressions of specific genes and influence diverse biological functions. Recent studies have shown that several corneal diseases, including keratoconus ( Amit et al, 2020 ; Dou et al, 2022 ), keratectasia ( Dupps and Wilson 2006 ), dry eye disease ( Yoshioka et al, 2015 ; Yamaguchi and Shiraishi 2018 ), myopia ( Kang et al, 2018 ; Xin et al, 2021 ), and bullous keratopathy after laser iridotomy ( Kaji et al, 2005 ; Yamamoto et al, 2010 ), are closely related to abnormal responses of corneal cells to mechanical forces.…”

Single-cell transcriptomics of the ocular anterior segment: a comprehensive review

“… [ 72 ] Dou et al ScRNA-Seq 10x Genomics Human adult central cornea Keratoconus 20,312 (keratoconus corneas), 18,902 (healthy corneas) 2022 CTSD and CTSK are potential novel markers for keratoconus stromal cells; elevated levels of YAP1 and TEAD1 are found in keratoconus stromal cells. [ 26 ] Wu et al ScRNA-Seq Fluidigm C1 System Murine sclera Myopia 93 (49 from form-deprivation sclera, 44 from control eyes) 2018 Hypoxia-inducible factor-1α (HIF-1α) promotes myopia development. [ 80 ] Alam et al ScRNA-Seq; ATAC-Seq; Bulk RNA Seq 10x Genomics Murine cornea and conjunctiva Dry eye disease 11,165 2022 γδ T cells are the predominant IL-17 expressing conjunctival immune cell population in Pinkie mouse strain with loss of function RXRα mutation.…”

“…Dou et al analysed gene expression patterns of 20,312 cells from keratoconus patient central corneas and similarly found that 340 genes were upregulated and 422 genes were downregulated [ 26 ]. Importantly, the authors identified cathepsin D ( CTSD ), cathepsin K ( CTSK ), YES-associated protein 1 ( YAP1 ), and TEA domain transcription factor ( TEAD1 ) as novel biomarkers in keratoconus stromal cells.…”

“…These studies have made unparalleled contributions to our understanding of limbal epithelial stem cell populations and their role in mediating corneal and conjunctival homoeostasis and wound healing. Additionally, scRNA-seq studies like Dou et al’s study have provided important insights into embryonic development, stem cell differentiation, cell type-specific transcription factors, and signalling pathways involved in disease states such as keratoconus, glaucoma, uveal melanoma, and Fuchs endothelial corneal dystrophy (FECD) [ 26 ].…”