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The design, fabrication, and properties of one of a new class of gradient-index lenses are reported. The lens is an f/2.25 GRIN singlet based on a nanolayered polymer composite material, designed to correct for spherical aberration. The light gathering and focusing properties of the polymer lens are compared to a homogeneous BK7 glass singlet with a similar f-number. The modulation transfer function of the polymer GRIN lens exceeded that of the homogeneous glass lens at all spatial frequencies and was as much as 3 times better at 5 cyc/mm. The weight of the polymer lens was approximately an order of magnitude less than the homogeneous glass lens.

Purpose: Recent studies indicate that transforming growth factor (TGF)beta is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, alpha-actin (alpha-SMA). Although TGFbeta is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating alpha-SMA gene expression and ultimately myofibroblast development., Methods: Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGFbeta (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 microM), Gly-Arg-AL-Asp-Ser-Pro (GRADSP; 100 microM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for alpha-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit alpha-SMA. All experiments were repeated at least three times., Results: Keratocytes exposed to TGFbeta showed expression of alpha-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and alpha-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGFbeta-associated tyrosine phosphorylation of paxillin, pp125fak, p130, PLCgamma, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGFbeta showed a dose-dependent loss of alpha-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly., Conclusions: The data suggest that TGFbeta-mediated alpha-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.

Previous studies have shown that TGF beta 1 induces activation and myofibroblast transformation of cultured rabbit corneal keratocytes. To determine whether TGF beta has a similar function in vivo, we evaluated the effect of TGF beta-blocking antibodies on corneal fibrosis after lamellar keratectomy (LK) in the rabbit. A total of 51 rabbits received standard LK wounds, and eyes were treated with 50 microliters of Celluvisc/PBS, containing 10, 50, or 100 micrograms of 1D11, a mouse monoclonal anti-TGF beta-blocking antibody. Control wounds received either 100 micrograms of an irrelevant mouse monoclonal antibody or vehicle alone. At days 14, 28, 42, and 56, eyes were evaluated by in vivo confocal microscopy (CM) and the mice were killed for light microscopy (LM) and immunostaining with antibodies to human fibronectin. In vivo CM of LK wounds clearly identified a disorganized layer that contained irregularly arranged fibroblasts and reflective extracellular matrix overlying normal corneal stroma. In a subset of 11 eyes stained with 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) immediately after injury, the thickness of the disorganized layer identified by in vivo CM significantly correlated with both anterior corneal fibrosis (r = 0.627; p < 0.025) and depth of keratocyte activation (r = 0.8980; p < 0.0005), indicating that in vivo CM can be used quantitatively to assess anterior stromal fibrosis. In eyes treated with an irrelevant monoclonal antibody, in vivo corneal fibrosis averaged 100 +/- 26 microns thick at day 14, whereas treatment with 10, 50, and 100 micrograms anti-TGF beta significantly reduced (p < 0.0005) the anterior disorganization in a dose-dependent fashion to 101 +/- 32, 45 +/- 11, and 56 +/- 18 microns, respectively. Semiquantitative measurement of anti-fibronectin staining within the wound revealed that anti-TGF beta significantly reduced the intensity of anti-fibronectin staining in the anterior 50 microns of the corneal stroma (p < 0.003). These findings indicate that TGF beta plays an important in vivo role in keratocyte activation and myofibroblast transformation. Furthermore, the in vivo use of TGF beta-blocking antibody effects may allow modulation of corneal fibrosis after refractive surgery.

The process of corneal wound healing involves the transformation of adjacent corneal keratocytes to myofibroblast-like cells characterized by the development of prominent microfilament bundles containing alpha-smooth muscle-specific actin (alpha-SM), a contractile protein thought to be important in mediating wound contraction. Recent studies have shown that the expression of alpha-SM in cultured corneal keratocytes can be induced by serum and TGF beta 1. To study the cellular and molecular mechanisms underlying this transformation process and to begin to identify the role of alpha-SM in wound contractile events, we generated immortalized rabbit corneal cell strains with extended life by using SV40 transfection. Two unique strains were isolated (TRK-36 and TRK-43). TRK-36, which appears similar to normal corneal keratocytes, maintains a stellate, keratocyte morphology when grown in the absence of serum and transforms to a myofibroblast-like cell when treated with TGF beta 1 (1 ng/ml), as indicated by the induced expression of alpha-SM actin. TRK-43 exhibits features characteristic of myofibroblasts in that it constitutively expresses alpha-SM actin under serum-free conditions. Both strains show in vitro contraction of collagen gels < or = 80% in 24 h in serum-containing medium. Interestingly, under serum-free conditions, TRK-43 cells showed significantly greater contraction of collagen gels compared with those of TRK-36. Overall, the establishment and further study of these cell strains may provide important insights into the molecular mechanisms underlying myofibroblast transformation.

The effects of serum, transforming growth factor (TGF) beta 1, bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF beta 1 (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed alpha-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 is a potent modulator of myofibroblast transformation under serum-free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF beta 1-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.

Purpose: Previous studies have suggested that the disappearance of anterior keratocytes after injury to the overlying epithelium is mediated by apoptosis. The authors examined the expression of the apoptosis-related modulators, Fas (receptor), Fas ligand, Bax, Bcl-2, Bcl-XL, and interleukin-1 beta converting enzyme (ICE) in corneal cells as candidate mediators of this response and tested the effect of Fas receptor-stimulating antibody on corneal stromal fibroblast cells in vitro., Methods: Reverse-transcription-polymerase chain reaction was used to detect FAS, FAS ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNA expression in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells. Immunohistochemistry was applied to detect Fas and Fas ligand proteins in fresh-frozen sections of normal human cornea. The effect of FAS-stimulating monoclonal antibody on first-passage stromal fibroblasts was studied using a DNA fragmentation assay, the live-dead assay with fluorescent microscopy, toluidene blue staining with light microscopy, and electron microscopy., Results: FAS, Fas ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNAs are expressed in all three major cell types of the cornea. Fas protein is expressed in corneal epithelial, keratocyte, and endothelial cells in fresh-frozen human cornea. Fas ligand protein, however, was detected in corneal epithelial and endothelial, but not keratocyte, cells. Fas-stimulating antibody induced first-passage stromal fibroblast cell death with morphologic changes and DNA fragmentation consistent with apoptosis., Conclusions: The Fas system (Fas and Fas ligand) modulators and final common pathway mediators of apoptosis are expressed in corneal cells. The distribution of Fas (epithelial, keratocyte, and endothelial cells) and Fas ligand (epithelial and endothelial cells) protein expression in fresh-frozen corneal tissue suggests that Fas ligand expressed in corneal epithelial and endothelial cells modulates functions in keratocyte cells and, possibly, autocrine-juxtacrine functions in epithelium and endothelium. The Fas-Fas ligand system is expressed in the cornea and could have important functions in normal corneal physiology and in the pathophysiology of corneal disease, including modulation of keratocyte apoptosis after epithelial injury.