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.
Keywords: Fuchs corneal endothelial dystrophy; cell culture; corneal endothelium; extracellular matrix; fibronectin; gene profiling; immunolocalization; integrins; tissue engineering.