Abstract:Purpose
There is limited information on region-specific gene expression in the human corneal stroma. In this study, we aimed to investigate the expression profile of the extracellular matrix and adhesion molecules in the normal corneal stroma using laser capture microdissection (LCM) and molecular techniques.
Methods
Frozen sections of human cornea without ocular disease were used to isolate the central and peripheral corneal stromal keratocytes by LCM. RNA was extracted from LCM-captured tissues and the RT2… Show more
“…Recently, this perception was supported by in situ hybridization analysis in mice, demonstrating that TGFBI mRNA was restricted to the corneal epithelium (Poulsen et al, 2018). However, in humans, TGFBIp is also expressed by keratocytes, particularly in the peripheral corneal stroma (Liu et al, 2017). We estimated the concentration of TGFBIp in the human cornea using a quantitative western blotting protocol (unpublished data).…”
Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-β-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.
“…Recently, this perception was supported by in situ hybridization analysis in mice, demonstrating that TGFBI mRNA was restricted to the corneal epithelium (Poulsen et al, 2018). However, in humans, TGFBIp is also expressed by keratocytes, particularly in the peripheral corneal stroma (Liu et al, 2017). We estimated the concentration of TGFBIp in the human cornea using a quantitative western blotting protocol (unpublished data).…”
Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-β-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.
“…2g) evident in wild type control animals. The Col6a2 gene is known to be expressed in the central corneal stroma of humans, but the phenotype of the cornea deficient in this protein has not been reported 21 .Fig. 2Corneal abnormalities in Fam20a , Col6a2 , and Nadsyn1 knockout mice.…”
Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
“…Recently, Liu et al . using quantitative PCR showed TGFBI mRNA expression in human keratocytes, however the authors did not compare mRNA expression levels between corneal cell types and the contribution by keratocytes may be significant lower than seen for the epithelium as supported from our in situ hybridization data. Our unpublished observation of human corneas maintained in culture medium where the epithelial layer has detached, shows unusual low levels of TGFBIp in the stroma when analyzed by mass spectrometry than normally seen for corneas without predetachment of the epithelium.…”
TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI À/À mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.Abbreviations ECM, extracellular matrix; iTRAQ, isobaric tags for relative and absolute quantitation; LC-MS/MS, liquid chromatography tandem mass spectrometry; POSTN, periostin gene; SRM, selected reaction monitoring; TEM, transmission electron microscopy; TGFBI, transforming growth factor beta-induced protein.
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