Coordinated Regulation of Palladin and α-Smooth Muscle Actin by Transforming Growth Factor-ß in Human Corneal Fibroblasts.

PURPOSE: To investigate the role of palladin in the cornea, we examined expression of this actin assembly-related protein in normal, diseased, or injured corneal tissue as well as in cultured corneal fibroblasts. METHODS: Expression of palladin and α-smooth muscle actin (α-SMA) in the rat cornea with an incision wound, in the normal and diseased human cornea, and in cultured human corneal fibroblasts was examined by immunofluorescence or immunoblot analysis. RESULTS: The expression of both palladin and α-SMA was detected at the lesion site during wound healing in the rat cornea. Whereas neither palladin nor α-SMA was detected in the normal human cornea, the colocalization of both proteins was detected in diseased human corneas with underlying conditions characterized by the presence of fibrosis. The expression of both palladin and α-SMA in cultured human corneal fibroblasts was increased by transforming growth factor-ß (TGF-ß) in a manner sensitive to inhibition by blockers of Smad or mitogen-activated protein kinase (MAPK) signaling. Finally, RNA interference-mediated depletion of palladin attenuated the TGF-ß-induced upregulation of α-SMA expression in human corneal fibroblasts as well as TGF-ß-induced collagen gel contraction mediated by these cells. CONCLUSIONS: Palladin is expressed in the rat and human cornea in association with scar formation. Expression of palladin in human corneal fibroblasts is increased by TGF-ß in a manner dependent on Smad and MAPK signaling and is required for the TGF-ß-induced upregulation of α-SMA.

Quantitative analysis of collagen lamellae in the normal and keratoconic human cornea by second harmonic generation imaging microscopy.

PURPOSE: To characterize the structural properties of collagen lamellae in the normal and keratoconic human corneal stroma, we measured their width and angle relative to Bowman's layer (BL). METHODS: Thirteen normal and four keratoconic corneas were examined. Collagen lamellae in tissue blocks from the central cornea were visualized by second harmonic generation imaging microscopy. Images obtained in 1-µm steps from BL to Descemet's membrane (DM) were subjected to three-dimensional reconstruction. The reconstructed data sets were divided into 10 layers of equal depth (L1-L10) for analysis. The width of lamellae adherent to BL (L0) was also determined. RESULTS: For the normal cornea, the width (mean ± SD) of collagen lamellae was 6.5 ± 1.7 µm at L0, decreased to 4.3 ± 1.3 µm at L1, and then increased gradually with progression toward DM to 122.2 ± 34.5 µm at L10, whereas the angle of lamellae was 20.9° ± 5.4° at L1 and decreased initially to 10.6° ± 3.2° at L2 before declining gradually to 2.7° ± 2.2° at L10. The width and angle of collagen lamellae in the keratoconic cornea were significantly larger and smaller, respectively, relative to those in the normal cornea. CONCLUSIONS: In the normal human cornea, collagen lamellae adjacent to BL are narrow and form a steep angle with BL, whereas they increase in width and their angle relative to BL flattens with progression toward DM. These properties of collagen lamellae are altered in keratoconus and are likely related to abnormalities of corneal shape.