Extracellular Matrix and Integrin Expression Profiles in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Model

Abstract

Primary corneal endothelial cell (CEC) cultures and 3D-engineered tissue models were used to study the aberrant deposition of extracellular matrix (ECM) in a vision impairing pathology known as Fuchs endothelial corneal dystrophy (FECD). CECs were isolated from excised Descemet membranes of patients with end-stage FECD. CECs isolated from healthy corneas served as controls. Microarray gene profiling was performed on postconfluent cultures of healthy and FECD cells. Protein expression analyses were conducted on tissue models that were engineered by seeding an endothelium on previously devitalized human stromal carriers. The engineered endothelia were kept in culture for 1–3 weeks to reform the endothelial monolayer. Protein expression of integrin subunits α4, α6, αv, and β1, as well as laminin, type IV collagen, fibronectin, clusterin, and transforming growth factor β-induced protein (TGFβIp) was then assessed by immunofluorescence. Microarray analysis showed nonstatistical twofold downregulation of collagen-coding genes (COL4A4, COL8A2, and COL21A1) and a twofold upregulation of the COL6A1, laminin α3 gene LAMA3, and integrin subunit α10 gene ITGA10 in FECD cells. Fibronectin type III domain containing 4 (FNDC4) and integrin β5 (ITGB5) genes was significantly upregulated in FECD cells. Immunostainings demonstrated that the protein expression of the integrin subunits α4, α6, αv, and β1, type IV collagen, as well as laminin remained similar between native and engineered endothelia. TGFβIp expression was found on the stromal side of both FECD and healthy Descemet's membrane, and only one out of three FECD specimens was positive for the clusterin protein. Interestingly, the ECM protein fibronectin was also found to have a stronger presence on engineered FECD tissues, a result consistent with the native FECD specimens. To conclude, this study allowed to identify fibronectin deposition as one of the first steps in the pathogenesis of FECD, as defined by our engineered tissue model. This opens the way to an entirely new perspective for in vitro pharmacological testing of new therapies for FECD, the leading indication for corneal transplantation in North America.

Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

“…The goal of these experiments was to ascertain whether CECs from normal human corneas can be stimulated to proliferate in organ culture where the low basal proliferation may better reflect the in vivo situation 34 compared with dissociated culture systems that produce high levels of proliferation of normal and dystrophic/FECD CECs. 7,8,[44][45][46] In this human corneal organ culture model, unstimulated CECs in the mid zone had very few cells incorporating EdU, indicating that this model is more reflective of the in vivo situation where the number of cells changes only slowly.…”

Placenta Transcriptome Profiling in Intrauterine Growth Restriction (IUGR)

“…Revealed underexpression (log2FC = −2.07) of IL7R could have contributed to the immunological imbalance in IUGR-affected samples. Also, fibronectin type III domain containing 4 ( FNDC4 ) dampen macrophage activation [57,58], and its robust upregulation results in the reduction of inflammation [59]. Considering mentioned functions, the identified underexpression of FNDC4 (log2FC = −2.38), in the growth-restricted placentas, may be associated with progression of pro-inflammatory state contributing to the severity of the disease [57,59].…”

A fine-tuned β-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis

“…Moreover, the majority of cells in culture were shown to downregulate various proteins involved in β-catenin activation. Of those proteins, Endoglin 43 , ITGB5 78 , and SPARC 79 were previously found to be expressed in CEnC (Supplementary Table S2 ). β-catenin is indeed generally fast degraded once in the cytoplasm 75 , and the condition captured by the proteomic analysis showed how β-catenin may have been already digested by the proteasome or moving back to the cell membrane (Fig.…”