Abstract
Purpose: :
Epithelial-to-mesenchymal transition (EMT) is a pathological process leading to the formation of anterior subcapsular cataracts (ASC). Mediated by transforming growth factor beta (TGFβ), EMT involves the transformation of the monolayer of lens epithelial cells (LECs) into "spindle shaped" myofibroblasts, resulting in multilayered plaque formation beneath the lens capsule. Our lab has previously demonstrated that TGFβ treatment, and subsequent EMT of rat lenses, involved the secretion of matrix metalloproteinases (MMPs) -2 and -9. Co-treatment of this in vitro lens cataract model with TGFβ and a MMP2/9 inhibitor suppressed ASC formation. Having identified MMP-2 and -9 as participants in ASC, the unique roles of MMP-2 and -9 in TGFβ-induced EMT in ex vivo mouse LEC explants was determined.
Methods: :
Lens epithelial explants, with epithelia attached to their native lens capsule, were isolated from MMP-2 and -9 WT and KO mice and maintained in culture. Confluent LEC explants were then stimulated with TGFβ for 72 hrs, fixed and stained with αSMA (a marker of EMT), and E-cadherin.
Results: :
In the ex vivo mouse LEC explants, TGFβ triggered a transformation of LECs from a tightly packed cuboidal monolayer to an elongated mesenchymal phenotype, which included expression of αSMA. In the absence of MMP-2, TGFβ was still able to induce filamentous αSMA expression and a concurrent loss of E-cadherin, similar to WT explants (n= 5). In contrast, LEC explants from MMP-9 KO mice treated with TGFβ did not acquire a characteristic spindle-like mesenchymal phenotype and showed substantially less αSMA expression compared to their WT littermate explants (n=4).
Conclusions: :
These studies indicate that, in contrast to MMP-2, MMP-9 appears to be the more critical mediator of EMT in mouse LEC explants following TGFβ treatment. These results are in line with previous findings from our laboratory demonstrating that while TGFβ delivery via adenoviral injection led to ASC formation in MMP-2 KO mice, MMP-9 KOs were resistant to EMT and ASC formation (ARVO abstract, 2011). Future studies in determining how the absence of MMP-2 and -9 differentially affect the actin cytoskeleton and the presence of focal adhesions in these mouse LEC explants will help to clarify the manner in which either MMP is involved in the EMT process.
Keywords: EMT (epithelial mesenchymal transition) • cataract • cytokines/chemokines