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JV Jester, J Huang; TGFß Induces Rabbit Corneal Keratocyte Proliferation and Myofibroblast Differentiation Through a PDGF Autocrine Loop . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2932.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In the cornea, there is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes that putatively undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast, which is in part TGFß dependent. The purpose of this study was to further define the cellular and molecular mechanism of keratocyte activation and myofibroblast differentiation by evaluating the effects of PDGF, a regulator of TGFß induce cell proliferation in other systems. Methods: Primary rabbit corneal keratocytes were initially grown using a serum-free culture system that maintains a normal keratocyte phenotype. Cells were then exposed to TGFß (1 ng/ml), blocking antibodies to PDGF (5-25 µg/ml), PDGF (100 ng/ml) and/or GRGDdSP (RGD, 50-100 µM) at various times and combinations. Keratocyte activation and myofibroblast differentiation was assessed by immunostaining for the presence of Ki67, a nuclear cell-cycle associated protein, expression of α-smooth muscle actin (α-SMA) and tyrosine phosphorylation using immunostaining and western blotting. Results: The addition of PDGF blocking antibodies to TGFß treated cells inhibited 80% of the cell cycle entry (p<0.005) as well as myofibroblast differentiation and α-SM expression, while the addition of PDGF to anti-PDGF reversed this effect. Interestingly, PDGF treatment alone induced keratocyte differentiation to fibroblastic, spindle shaped cells as differentiated from myofibroblastic, TGFß treated cells. However, TGFß treatment of PDGF stimulated cells showed no temporal differences in myofibroblast differentiation compared to TGFß treatment of naïve, serum-free cultured cells. Furthermore, PDGF had no effect on the blocking of TGFß induced myofibroblast differentiation by RGD, which inhibits integrin-fibronectin interactions, and release of blocked cells through removal of RGD showed no difference to that of naïve cells in the temporal tyrosine phosphorylation cascade leading to myofibroblast differentiation. Conclusion: Activation of quiescent keratocytes by TGFß requires the induction of a PDGF autocrine loop. However, myofibroblast differentiation appears separate and distinct from the fibroblast differentiation pathway and requires the synergistic interactions of growth factors and integrin receptor signaling.
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