Differential Expression of Proteoglycans by Corneal Stromal Cells in Keratoconus.
- 1Instituto Universitario Fernández-Vega, Universidad de Oviedo & Fundación de Investigación Oftalmológica, Oviedo, Spain 2Department of Functional Biology, University of Oviedo, Oviedo, Spain.
- 2Instituto Universitario Fernández-Vega, Universidad de Oviedo & Fundación de Investigación Oftalmológica, Oviedo, Spain 3Department of Morphology and Cell Biology, University of Oviedo, Oviedo, Spain.
- 3Instituto Universitario Fernández-Vega, Universidad de Oviedo & Fundación de Investigación Oftalmológica, Oviedo, Spain.
- 4Instituto Universitario Fernández-Vega, Universidad de Oviedo & Fundación de Investigación Oftalmológica, Oviedo, Spain 2Department of Functional Biology, University of Oviedo, Oviedo, Spain 4Department of Microbiology, Hospital Universitario Central de A.
Keratoconus is a heterogeneous disease associated with a range of pathologies, including disorders that involve proteoglycans (PGs). Some PG alterations, mainly in keratan sulfate (KS), occur in keratoconus. In this article, we studied the differential expression of the genes encoding PGs in cells isolated from keratoconus patients and healthy controls, as well as in corneal sections.
Human central corneal tissue was obtained from cadaver donors and patients undergoing penetrating keratoplasty surgery. A transcriptomic approach was used, employing quantitative PCR, to analyze both the expression of the enzymes involved in glycosaminoglycan (GAG) biosynthesis and the PG core proteins. The expressions of the differentially expressed core proteins and of the GAG chains were also analyzed by immunocytochemistry in the cultured cells, as well as by immunohistochemistry in corneal sections.
The mRNA levels of most major matrix PG mRNAs in the cultured keratoconic stromal cells decreased except collagen XVIII, which increased. At keratocyte surfaces, some heparan sulfate PGs were down-regulated. With respect to GAGs, there were changes in gene expression for the polymerization of the GAG chains, mainly KS and chondroitin sulfate, and in the modification of the saccharidic chains, pointing to an alteration of the sulfation patterns of all GAG species.
Most of the PG core proteins underwent significant changes in cultured keratoconic cells and corneas. With regard to GAG chains, the polymerization of the chains and their chemical modification was modified in way that depended on the specific type of GAG involved.
- PMID: 27258434
Keratoconus is a corneal ectasia that results in the cornea taking on a conical shape, which prevents light entering the eye from being focused correctly on the retina, thus causing distortion of vision. It causes severe astigmatism, scarring, and ultimately loss of vision in one out of five patients, necessitating corneal transplant. Keratoconus is a multifactorial, multigenetic disorder with complex inheritance patterns, though environmental factors like wearing contact lenses, chronic eye rubbing or atopy of the eye probably play an equally important role in disease causation. Histologically, keratoconus displays many abnormal features that affect different layers of the cornea, including abnormal epithelial and stromal keratocyte shapes, local thickening of the epithelium, Bowman’s layer breakage, thinning of the stroma, and iron deposits in the basal layer of the corneal epithelium.
The central stroma constitutes 90% of corneal depth, and consists of an extracellular matrix (ECM) interspersed with keratocytes, which occupy about 10% of the substantia propia. The corneal stroma is composed primarily of collagen fibrils, each of which comprises a core type V collagen coated with type I collagen, which is coated in turn by different proteoglycans (PGs). PGs are a diverse group of glycoconjugates composed of various core proteins post-translationally modified with linear, anionic polysaccharides called glycosaminoglycans (GAGs), which consist of repeating disaccharides (Figure 1). It has been proposed that ECM PG core proteins bind around the collagen fibrils while the GAG chains stick out laterally away from the sides of these fibrils, forming an exterior hydrophilic shell (Figure 1). The current molecular model of the corneal stroma suggests that PGs play a pivotal role in the establishment and maintenance of the arrangement of the regularly-spaced and uniformly-thin collagen fibrils, which allows light to pass through the cornea.
Various eye diseases appear to be related to changes in PGs and GAGs, as is the case in the progressive opacification of the cornea and excessive storage of GAGs characteristic of mucopolysaccharidosis, or the corneal dystrophies resulting from the failure of normal KS production: cornea plana and macular corneal dystrophy. Interestingly, some connective tissue disorders that may appear in association with keratoconus involve molecular alterations in PGs and GAGs, for example Ehlers-Danlos syndrome, osteogenesis imperfecta and Crouzon’s syndrome.
This work carried out, for the first time, a transcriptomic study of the genes encoding the PG core proteins located in the keratoconus corneal stroma and compared them to healthy controls, as well as of the enzymes involved in the biosynthesis of the different GAG chains. The analysis of differential expression of the genes involved in the biosynthesis of PGs in cultures of keratoconic stromal cells indicated that a percentage of the genes experienced significant changes in their transcript levels relative to healthy cells. These changes were evident at the level of certain core proteins, as well as in their GAG chains, resulting in altered levels of transcription of many of the enzymes involved in the polymerization of the chains and chemical modifications including epimerizations, N-sulfations and various O-sulfations, depending on the specific type of GAG involved (Figure 1). These changes at the molecular level may explainof the alterations observed in the corneal structure of patients with keratoconus.
Differential expression of proteoglycans in keratoconus corneal stroma.
A: Section of a keratoconic cornea.
B: Stromal matrix; model of the interaction between collagen fibrils (blue circles) and proteoglycans (black lines), including keratan sulfate proteoglycans (lumican, keratocan, osteoglycin) and chondroitin sulfate proteoglycans (decorin, biglycan).
C: Keratan sulfate disaccharide unit.
D: chondroitin sulfate disaccharide unit.
E: Corneal keratinocytes. F: cell-surface heparan sulfate proteoglycan. G: Heparan sulfate disaccharide unit.
The genes for enzymes that catalyze specific reactions in the biosynthesis of the different GAG saccharide units (C, D, G) are highlighted in red.