Colocalization of increased transforming growth factor-β-induced protein (TGFBIp) and Clusterin in Fuchs endothelial corneal dystrophy

PURPOSE: To investigate the differential expression of TGFBIp in normal human and Fuchs endothelial corneal dystrophy (FECD) endothelial cell-Descemet’s membrane (HCEC-DM) complex, and to asses the structural role of TGFBIp and clusterin (CLU) in guttae formation. METHODS: HCEC-DM complex was dissected from stroma in normal and FECD samples. Proteins were separated by 2-D gel electrophoresis and subjected to proteomic analysis. N-terminal processing of TGFBIp was detected by Western blot analysis with two separate antibodies against the N- and C-terminal regions of TGFBIp. Expression of TGFBI mRNA was compared by using real-time PCR. Subcellular localization of TGFBIp and CLU in corneal guttae was assessed by fluorescence confocal microscopy. RESULTS: A major 68-kDa fragment and a minor 39-kDa fragment of TGFBIp were identified on 2-D gels. Western blot analysis revealed an age-dependent proteolytic processing of the TGFBIp N terminus resulting in the increased formation of 57-kDa (P = 0.04) and 39-kDa (P = 0.03) fragments in older donors. FECD HCEC-DM showed a significant increase in the 68-kDa (P = 0.04), 57-kDa (P = 0.01), and 39- kDa (P = 0.03) fragments of TGFBIp. Real-time PCR analysis revealed that TGFBI mRNA was significantly increased (P = 0.04) in FECD samples. TGFBIp formed aggregates at the lower portions of guttae, next to Descemet’s membrane, whereas CLU localized mostly on top of the TGFBIp-stained areas at the level of the endothelial cell nuclear plane. CONCLUSIONS: The overexpression of proaggregative protein CLU, and proadhesive protein TGFBIp, have been colocalized in the guttae. Such findings provide us with a better understanding of the major contributors involved in the aberrant cell-extracellular matrix interactions seen in the guttae of patients with FECD

Fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 are strongly expressed in the epithelium of human granular and lattice type I corneal dystrophies

Purpose: To determine the extracellular matrix proteins involved in the formation of human granular and lattice type I corneal stromal dystrophies, the expression patterns of fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 were compared in human corneal stromal dystrophy samples. Methods: Ten cases of granular dystrophy, 7 cases of lattice dystrophy, and 6 normal corneal buttons collected during corneal transplantation were examined for their expression patterns of fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 by immunohistochemistry. Results: Highly elevated fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 were observed in the epithelial layer of both granular and lattice type I dystrophies. Fibrillin-2, tenascin-C, and matrilin-4 in the granular dystrophy and all antibodies in the lattice dystrophy showed statistically significant staining in the corneal stroma (p<0.05). Interestingly, fibrillin-2, matrilin-2, and matrilin-4 stained significantly in amyloid plaques of lattice type 1 dystrophy. Conclusions: Fibrillin-2, tenascin-C, matrilin-2, and matrilin-4 may be markers of the pathogenesis of either granular or lattice type I corneal dystrophy, as revealed by immunohistochemical analysis. Each molecule seems to be involved in the regeneration and reorganization of the corneal matrix in granular and lattice type I dystrophies

Galectin-3, IL-1A, IL-6, and EGF levels in corneal epithelium of patients with recurrent corneal erosion syndrome

Purpose: To determine the galectin-3 (Gal3), interleukin-1 (IL-1), interleukin-6 (IL-6), and epidermal growth factor (EGF) levels in corneal epithelium of patients with recurrent corneal erosion (RCE) syndrome and compare them with healthy controls. Methods: In this prospective interventional case control study, 32 eyes of 32 patients with RCE syndrome who had corneal epithelial erosions and 28 eyes of 28 healthy participants scheduled for photorefractive keratectomy (control group) were included. Exclusion criteria included corneal dystrophies, ectasia, dry eye, previous ocular surgery or topical medications, and systemic diseases. Epithelial samples were obtained during epithelial debridement in the study group and mechanical epithelial keratectomy in the control group. Galectin-3 levels were studied by the chemiluminescent microparticle immunoassay method. IL-1, IL-6, and EGF levels were determined using corresponding ELISA kits. Results: The median Gal3 levels were 132.25 ng/mL in the study group and 106.50 ng/mL in the control group. The median IL-1 and IL-6 levels were 6.24 pg/mL and 10.16 pg/mL, respectively, in the study group which were higher than that in the control group. The median EGF level in the study group was lower than that the control group with 1.30 pg/mL versus 2.67 pg/mL. In the control group, there was a significant positive correlation between EGF and IL-6 (r = 0.554; P = 0.040). A similar correlation was not observed in patients with RCE (r = -0.071; P = 0.794). Conclusions: The lack of increased EGF expression and the imbalance between growth factors, adhesion molecules, and interleukins may be the reason for the impaired wound healing response in RCE syndrome

Cornea organoids from human induced pluripotent stem cells.

The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions

Neurotrophic factors and corneal nerve regeneration

The cornea has unique features that make it a useful model for regenerative medicine studies. It is an avascular, transparent, densely innervated tissue and any pathological changes can be easily detected by slit lamp examination. Corneal sensitivity is provided by the ophthalmic branch of the trigeminal nerve that elicits protective reflexes such as blinking and tearing and exerts trophic support by releasing neuromediators and growth factors. Corneal nerves are easily evaluated for both function and morphology using standard instruments such as corneal esthesiometer and in vivo confocal microscope. All local and systemic conditions that are associated with damage of the trigeminal nerve cause the development of neurotrophic keratitis, a rare degenerative disease. Neurotrophic keratitis is characterized by impairment of corneal sensitivity associated with development of persistent epithelial defects that may progress to corneal ulcer, melting and perforation. Current neurotrophic keratitis treatments aim at supporting corneal healing and preventing progression of corneal damage. Novel compounds able to stimulate corneal nerve recovery are in advanced development stage. Among them, nerve growth factor eye drops showed to be safe and effective in stimulating corneal healing and improving corneal sensitivity in patients with neurotrophic keratitis. Neurotrophic keratitis represents an useful model to evaluate in clinical practice novel neuro-regenerative drugs

Unravelling Molecular Mechanisms Underlying Inherited Corneal Endothelial Disease

Fuchs endothelial corneal dystrophy (FECD) and posterior polymorphous corneal dystrophy (PPCD) are clinically distinct heritable conditions associated with corneal endothelial barrier dysfunction that ultimately result in loss of corneal clarity and subsequent visual impairment. FECD is a common age-related corneal dystrophy that, in up to 80% of patients, is associated with a trinucleotide repeat expansion (termed CTG18.1) within an intronic region of the transcription factor encoding gene TCF4. PPCD is a rare autosomal dominant corneal dystrophy attributed to mutations in three distinct transcription factor encoding genes, (OVOL2 [PPCD1], ZEB1 [PPCD3] and GRHL2 [PPCD4]) that are all established regulators of epithelial-mesenchymal transition (EMT), suggesting a shared mechanisms of dysregulation may underlie distinct genetic subtypes of this disease. In this thesis I present the use of established patient-derived corneal endothelial cell (CEC) culture techniques in combination with next generation sequencing (NGS) based technologies to probe the genetic aetiologies and transcriptomic signatures of dysregulation underlying these diseases. Specifically, a novel amplification-free approach was developed, utilised, and refined to enable the CTG18.1 repeat expansions to be interrogated at the nucleotide level within a FECD patient cohort. This approach revealed striking levels of repeat length instability and mosaicism are associated with CTG18.1 expansion, advancing our understating of FECD pathophysiology in addition to more broadly illustrating the power of this long-read non-amplification dependant sequencing methodology to study repetitive genomic regions. RNA-seq data was generated from PPCD patient- and control-derived CEC cultures to define mechanism of transcriptomic dysregulation underlying disease and advance our understanding of the pathophysiology of this genetically heterogenous disease. Bioinformatic interrogation of these data highlighted dysregulated expression of the PPCD-associated OVOL2/ZEB1/GRHL2 axis and EMT-associated genes, and ectopic expression of corneal progenitor epithelium cell-type markers within the PPCD1 and PPCD3 corneal endothelium. Furthermore, epithelial cell-type- specific gene isoforms were upregulated in PPCD1 and PPCD3 corneal endothelium including targets of the epithelial splicing regulator protein, ESRP1. Over-expression of ESRP1 was subsequently modelled in immortalised endothelial cell line (HCEC12). Consequently, an upregulation of ESRP1 target gene epithelial-cell-type specific isoforms and corneal progenitor epithelium markers was discovered, suggesting a major role of ESRP1 in PPCD pathogenicity. Finally, a refined cohort of genetically unresolved PPCD patients recruited at Moorfields Eye Hospital (MEH) and General University Hospital (GUH), Prague, was established to identify additional genetic causes of PPCD