transforming-growth-factor-beta and Fuchs--Endothelial-Dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease that impacts the structure and stiffness of the Descemet's membrane (DM), the substratum for corneal endothelial cells (CECs). These structural alterations of the DM could contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and transforming growth factor-β (TGF-β) pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid (AA) is found at high concentrations in FECD and its impact on CEC survival has been investigated. However, how TGF-β and AA effect the composition and rigidity of the CEC's matrix remains unknown.. In this study, we investigated the effect of AA, TGF-β1 and TGF-β3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix (ECM) secreted by primary bovine corneal endothelial cells (BCECs).. Immunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-β1 and TGF-β3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-β induces the expression of matrix proteins collagen IV, laminin, and lysyl oxidase homolog 1.. Molecular pathways identified in this study demonstrate the differential role of soluble factors in the pathogenesis of FECD.

Topics: Animals; Cattle; Endothelial Cells; Endothelium, Corneal; Fuchs' Endothelial Dystrophy; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3

Fuchs endothelial corneal dystrophy (FECD) is a slowly progressive bilateral disease of corneal endothelium in which accumulation of extracellular matrix (ECM) and loss of corneal endothelial cells (CECs) are phenotypic features. The corneal endothelium maintains corneal transparency by regulating water hydration; consequently, corneal endothelial dysfunction causes serious vision loss. The only therapy for corneal haziness due to corneal endothelial diseases, including FECD, is corneal transplantation using donor corneas, and no pharmaceutical treatment is available. We provide evidence that the expression levels of transforming growth factor-β (TGF-β) isoforms and TGF-β receptors are high in the corneal endothelium of patients with FECD. A cell model based on patients with FECD shows that TGF-β signaling induced a chronic overload of ECM proteins to the endoplasmic reticulum (ER), thereby enhancing the formation of unfolded protein and triggering the intrinsic apoptotic pathway through the unfolded protein response (UPR). We propose that inhibition of TGF-β signaling may represent a novel therapeutic target that suppresses cell loss as well as the accumulation of ECM in FECD.

Topics: Cell Death; Cell Line; Cornea; Fuchs' Endothelial Dystrophy; Humans; Signal Transduction; Transforming Growth Factor beta; Unfolded Protein Response

Fuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is a major cause of corneal transplantation. It is characterized by abnormal deposition of extracellular matrix (ECM), such as corneal guttae, accompanied by a loss of endothelial cells. Although recent studies have revealed several genomic factors, the molecular pathophysiology of FECD has not yet been revealed. In this study, we establish a cellular in vitro model by using immortalized corneal endothelial cells obtained from late-onset FECD and control patients and examined the involvement of epithelial mesenchymal transition (EMT) on excessive ECM production. We demonstrate that the EMT-inducing genes ZEB1 and SNAI1 were highly expressed in corneal endothelial cells in FECD and were involved in excessive production of ECM proteins, such as type I collagen and fibronectin through the transforming growth factor (TGF)-β signaling pathway. Furthermore, we found that SB431542, a specific inhibitor of TGF-β type I ALK receptors, suppressed the expression of ZEB1 and Snail1 followed by reduced production of ECM. These findings suggest that increased expression levels of ZEB1 and Snail1 in FECD cells were responsible for an increased responsiveness to TGF-β present in the aqueous humor and excessive production of ECM. In addition, these results suggest that the regulation of EMT-related genes by blocking the TGF-β signaling pathway may be a feasible therapeutic strategy for FECD.

Topics: Cell Line, Transformed; Extracellular Matrix; Fuchs' Endothelial Dystrophy; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

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.. 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.. 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.. 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.

Topics: Adolescent; Aged; Aged, 80 and over; Blotting, Western; Child, Preschool; Clusterin; Descemet Membrane; Electrophoresis, Gel, Two-Dimensional; Endothelium, Corneal; Extracellular Matrix Proteins; Female; Fuchs' Endothelial Dystrophy; Humans; Male; Microscopy, Confocal; Middle Aged; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Deposition of abnormal sub-epithelial matrix and posterior collagenous layer by epithelium and endothelium, respectively, in Fuchs' dystrophy gives us the opportunity to determine if these tissues synthesize beta ig-h3.. Immunohisto-/immunocytochemistry of corneas were conducted with rabbit anti-human beta ig-h3 and monoclonal anti-human type VI collagen. Labeled sense and anti-sense beta ig-h3 oligonucleotide probes were used for in situ hybridization.. beta ig-h3-specific fluorescence was found just beneath detached epithelium in the sub-epithelial matrix, abnormal Descemet's membrane and posterior collagenous layer. Type VI collagen co-localized with beta ig-h3 within abnormal sub-epithelial matrix and corneal stroma adjacent to Descemet's membrane. beta ig-h3 mRNA was detected in corneal epithelium of dystrophic corneas.. Expression of beta ig-h3 in sub-epithelial matrix and posterior collagenous layer of Fuchs' dystrophy is consistent with the synthesis of new extracellular matrices by epithelial and endothelial tissues. beta ig-h3 mRNA in corneal epithelium further supports an epithelial source of this protein. Endothelial synthesis of beta ig-h3 is based on circumstantial evidence due to cell loss during surgical and histological procedures. Co-localization of beta ig-h3 with type VI collagen in abnormal sub-epithelial matrix and at the stromal/Descemet's membrane interface suggest this collagen in association with beta ig-h3 interacts with these tissues and anchors them to the adjacent stroma.

Topics: Animals; Collagen; Cornea; Endothelium, Corneal; Epithelium; Extracellular Matrix Proteins; Eye Proteins; Fluorescent Antibody Technique, Indirect; Fuchs' Endothelial Dystrophy; Humans; In Situ Hybridization; Microscopy, Immunoelectron; Rabbits; RNA, Messenger; Transforming Growth Factor beta