The role of alpha V integrins in lens epithelial mesenchymal transition and posterior capsular opacification

Mamuya, Fahmy
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University of Delaware
Posterior Capsular Opacification (PCO) is the major complication after cataract surgery. PCO occurs when residual lens cells (LCs) remaining following cataract surgery undergo a wound healing response producing a mixture of αSMA expressing myofibroblasts and lens fiber cells which impair vision. Prior investigations have proposed that integrins play a central role in PCO while my data indicated that αV integrin and its interacting β-subunits; β1, β5, β6, β8 along with α-smooth muscle actin (αSMA) are upregulated in residual lens epithelial cells in a mouse model of cataract surgery. To test the hypothesis that αV integrins are functionally important in PCO pathogenesis, I created mice lacking the αV integrin subunit in all lens cells using a conditional knockout approach. Adult lenses lacking αV integrins were analyzed for function and cellular organization by grid analysis, conventional light, confocal and scanning electron microscopy. At three months of age, fiber cells were surgically removed from αV integrin null and control lenses and the extent of EMT post-surgery was measured by QRT-PCR and immunofluorescent detection of EMT markers. Lenses lacking αV integrin subunits are transparent and show no apparent morphological abnormalities when compared to control lenses. When challenged with surgery/injury, control lenses developed LC multilayering along with upregulation of αSMA, β1-integrin, fibronectin, vitronectin, tenascin-C and TGF-β induced protein within 48hrs. In contrast, LECs lacking αV integrins showed no increase in LC multilayering with little to no upregulation of αSMA, fibronectin, vitronectin, and TGF-β induced protein 48hrs post-surgery. In addition, I saw a significant increase in the mRNA expression of αSMA, fibronectin, tenascin-C and TGF-β induced protein 24hrs post-surgery in control lenses with no significant increase in lenses lacking the αV integrin subunit. This effect appears to result from the known roles of αV integrins in latent TGF- β activation since αV integrin null lenses do not exhibit detectable SMAD-3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF-β in fibrotic PCO as well as Anterior Subcapsular Cataracts (ASC). To look further into whether αV integrins play a role in the TGF-β associated fibrotic lens EMT, I utilized mice homozygous for a 12 nucleotide deletion in the βB2-crystallin gene (Crybb2 Phil/Phill ), which have been previously demonstrated in our lab to undergo a juvenile lens epithelium EMT as early as four weeks postnatal, leading to ASC which is followed by the development of severe lens abnormalities. At 2 months age, Crybb2 Phil/Phill demonstrates identical EMT characteristics as seen in wildtype mice at 48hrs post-surgery. Their lens epithelium becomes multilayered and upregulates αSMA, fibronectin, vitronectin, TGF-β induced protein and phosphorylated SMAD-3. Moreover, a SuperArray RT-PCR gene expression analysis on mRNA expression of TGF-β associated genes showed that, while all TGF-β ligands 1, 2 and 3 and the vast majority of the down-stream canonical TGF-β signaling associated players, including SMAD, 2, 3 or 4 were not upregulated in Crybb2 Phil/Phill epithelium, TGF-β receptor II, TGF-βi and Follistatin which are genes known to upregulate in the aftermath of TGF-β signaling, were significantly upregulated. These findings suggested that the Crybb2 Phil/Phill epithelium was undergoing an extreme unregulation of TGF-β signaling which seem to be regulated mainly at the TGF-β activation level point. This was attested by an active TGF-β assay which confirmed that Crybb2 Phil/Phill lenses had four times the amount of active TGF-β as compared to wildtype lenses. In addition, αV integrins were found to upregulate at both the mRNA and protein level in Crybb2 Phil/Phill lens epithelium EMT similar to that seen in the lens post-surgery suggesting that βB2-crystallin may have a non-refractive function in maintaining lens epithelial integrity which will be a topic for future investigations. Overall, while αV integrin only subtlety regulates the development and function of the lens, it is involved in regulating the phenotypic alterations in lens epithelial cells that occurs following lens injury/cataract surgery leading to PCO. This is the first study to examine the roles of αV integrins in lens development, refractive functions and the pathogenesis of PCO/ASC. These data suggest that therapeutics antagonizing αV integrin function could be used to prevent TGF-β fibrotic PCO following cataract surgery as well as ASC development.