Keratocyte Cultures Research Articles

Serum-Free Spheroid Culture of Mouse Corneal Keratocytes

To develop a serum-free mass culture system for mouse keratocytes. Corneas of C57BL6/J mice were enzyme digested after the epithelium and endothelium were removed. Stromal cells were cultured in serum-free DMEM/F12 (1:1) containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and B27 supplement. Primary spheres were dissociated by trypsin and subcultured as suspended secondary spheres. Cells from postnatal day (P)6 to P10 spheres were subcultured onto plastic dishes or type I collagen gels for phenotype analysis. The expression of the keratocyte markers keratocan, aldehyde dehydrogenase (Aldh), and CD34, were analyzed by RT-PCR, and vimentin and alpha-smooth muscle actin (alpha-SMA) were examined by immunocytochemistry. Primary keratocytes formed spheres, which were cultured for over 12 passages. Suspended sphere cells expressed vimentin, keratocan, CD34, and lumican, but were negative for cytokeratin K12 (K12) and Pax6. Sphere cells subcultured on plastic exhibited a dendritic morphology characteristic of keratocytes, and maintained keratocan, Aldh, and CD34 expression in serum-free medium. Sphere cells subcultured with 10% serum became fibroblastic, and expressed alpha-SMA when stimulated by transforming growth factor (TGF)-beta. alpha-SMA-positive cells demonstrated contractile properties on collagen gels, compatible with the myofibroblast phenotype. The phenotype of mouse keratocytes can be maintained in vitro for more than 12 passages by the serum-free sphere culturing technique.

MMP-type endopeptidase activity in the cornea. Its evolution during organ culture storage at the Eye Bank. Effect of hyaluronan

About 46% of total corneas obtained from donors in the French Eye Bank cannot be grafted for several reasons as loss of endothelium or other. Corneal cells express proteolytic enzymes, essentially matrix metallo-proteinase MMP-2 and MMP-9. In presence of hyaluronan and some other GAG-s their activity increases as could be shown on keratocyte cultures. Hyaluronan concentration increases during in vitro preservation and can represent a serious hasard for corneal conservation. The control of MMP-release and activation might well be one of the factors involved in graft deterioration. We could show however that only a slight fraction (≤12%) of total, relatively high endopeptidase activity of the cornea is released in the media during storing. It appears therefore that most of the proteolytic activity determined in corneal extracts remains confined to the stroma and might not represent an important risk for preservation, at least for the endothelium.

TGF-beta signal transduction in corneal wound healing as a therapeutic target.

This article reviews recent progress in research on the role of Smad signaling in corneal wound healing. Smad2 and Smad3 are key signaling molecules downstream of the cell surface receptor of transforming growth factor-beta (TGF-beta) or activin. On ligand binding to the receptor, Smads2/3 undergo phosphorylation, form complexes with Smad4, and thence convey signaling. TGF-beta isoforms have been detected in corneal epithelium and are also deposited in wounded stroma, suggesting their participation in the wound-healing process in corneal tissue. Human or mouse uninjured healthy corneal epithelium shows nuclear accumulation of Smads3/4, indicating active Smad signaling in this tissue. Migrating corneal epithelium lacks nuclear Smad accumulation with up-regulation of Smad7, but p38MAPK is activated. Organ-culture experiments show that p38MAPK activation depends on endogenous TGF-beta and that activation of p38MAPK results in cell proliferation cessation with a reduction of Erk activation and acceleration of cell migration in healing corneal epithelium. These findings indicate that during healing of corneal epithelial defects, endogenous TGF-beta activates p38MAPK for cell migration and suppression of cell proliferation and up-regulates Smad7 for inhibition of Smad2 and Smad3 signaling, resulting in rapid initial resurfacing of the epithelium. Such involvement of p38MAPK in cell migration has been reported in many cell types and observed in keratocyte culture. Possible benefits of preserving non-Smad cascades in treating problems in corneal wound healing by manipulating TGF-beta signals have been suggested.

Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea.

To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. Murine Genome_U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts. Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing. The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.

Monoclonal antibody (3G5)-defined ganglioside: cell surface marker of corneal keratocytes.

To evaluate the anti-ganglioside monoclonal antibody 3G5 as a marker of corneal keratocytes. 3G5 expression on keratocytes was investigated by immunofluorescence microscopy. Studies were performed on frozen sections of normal human, bovine, porcine, rabbit, rat, and mouse corneas and on repairing rabbit cornea. In vitro studies were performed on tissue-cultured human, bovine, porcine, mouse, and rabbit keratocytes. 3G5 stained frozen sections of human, bovine, porcine, rat, and rabbit cornea but not mouse cornea and the staining pattern followed the distribution of stromal keratocytes but did not stain epithelium or endothelium. Subconfluent human and bovine keratocyte cultures were 3G5 negative. Almost 100% of the human and bovine cells that were maintained at confluence without replacement of serum-containing culture medium for 2 weeks became 3G5 positive. The 3G5 antigen was constitutively expressed on cultured rabbit and porcine keratocytes under all conditions examined. Mouse keratocyte cultures did not express 3G5. The 3G5 antigen was not present on myofibroblastic cells in the repairing area of a full-thickness wound in rabbit cornea that had been healing for 20 days. The area surrounding the healing wound expressed 3G5 antigen in an altered distribution, whereas 3G5 antigen was distributed in the expected pattern in areas that were distant from the wound. When rabbit keratocytes were induced to express the myofibroblast marker alpha-smooth muscle actin by treatment with TGFbeta1 in vitro, the pattern of 3G5 staining was altered. The 3G5 antigen is a useful marker for the identification of corneal keratocytes and for documenting their response to environmental stimuli associated with wound repair.

Tropoelastin Biosynthesis by Corneal Cells

The vertebrate cornea is an avascular tissue and does not contain elastic fibers. We tested the capacity of corneal epithelial cells and stromal keratocytes to synthesize tropoelastin. Explant cultures and cell cultures were obtained from these two cell types in standard culture conditions. Their elastin-synthetic activity was compared to skin explant cultures and to dermal fibroblast cell cultures. Both corneal cell types synthesized tropoelastin as shown by the incorporation of a radioactive precursor followed by immunoprecipitation of tropoelastin. When serial cultures of keratocytes were tested, tropoelastin biosynthesis strongly increased after the 3rd passage and was at the 9th passage more than the double of that of the first passage. When cocultures were studied with or without cell contact, epithelial cells partially inhibited tropoelastin biosynthesis by keratocytes. This inhibition was somewhat stronger (–36%, p < 0.005) with cell-to-cell contact than keeping separate epithelial cells and keratocytes bathing in the same medium (–18%, p < 0.005). When human skin fibroblasts were substituted for keratocytes with cell-to-cell contact, their tropoelastin biosynthesis was also inhibited by corneal epithelial cells (–42%, p < 0.005), to the same extent as for keratocytes. In Transwell culture, this inhibition was again somewhat lower (–36%, p < 0.005). Some diffusible factor produced by epithelial cells is apparently involved. The epithelial inhibition of tropoelastin biosynthesis by stromal keratocytes might represent one of the mechanisms keeping corneal stroma exempt of elastin fibers.

Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing.

The physiologic mechanisms involving growth factors, including PDGF-BB, EGF, and TGF-beta 1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF-BB, EGF, TGF-beta 1, keratocytes (1 x 10(5)/well) and epithelial cells (2 x 10(5)/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF-BB between 0 and 75 ng/mL; and TGF-beta 1 between 0 and 10 ng/mL. Cell proliferation was measured using 3H-thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05). Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF-BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF-beta 1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL. Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF-BB and EGF stimulate, while TGF-beta 1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.

Isolation of a putative keratocyte activating factor from the corneal stroma

Fetal bovine serum has commonly been used to expand the population of keratocytes in culture. Tissue extracts, however, have also been used to grow other cell types. We prepared a DMEM/F12 extract of corneal stroma and compared the growth and morphology of collagenase-isolated keratocytes cultured in DMEM/F12, or DMEM/F12 containing either stromal extract or fetal bovine serum. Cell proliferation was measured by 3H-thymidine and BrdU incorporation as well as by DNA quantitation. The extract was fractionated by gel filtration. Cell morphology was assessed by phase-contrast microscopy. Culture in both extract and serum stimulated keratocytes to proliferate, but keratocytes cultured in the extract grew more slowly due to a longer cell cycle and to a lower final density because of greater sensitivity to contact inhibition. Keratocytes cultured in serum became fibroblastic while those cultured in extract retained the dendritic morphology of quiescent keratocytes. The stimulating factors in the corneal extract were more sensitive to heat inactivation and of higher molecular weight than the stimulating factors in serum. These results indicate that the mitogenic activity in extract and serum are different and that the phenotypes resulting from growth in serum and extract are also different. Keratocytes cultured at low cell densities in the corneal extract may mimic keratocyte activation, an initial and crucial event for keratocytes during the corneal wound healing process.

EFFECT OF HYALURONAN ON MMP EXPRESSION AND ACTIVATION

Matrix metalloproteases (MMPs) play a crucial role in tissue remodelling in a variety of physiological and pathological processes. Hyaluronan is also involved in the same processes. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMPs. The activity of MMPs has been shown to be regulated at the level of transcription and activation of the zymogen form.In order to explore the possible relationship between matrix components and especially hyaluronan, we studied the effect of hyaluronan on MMP expression (biosynthesis and activation) in the culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate N-Suc(ala)3pNA, we measured elastase-type endopeptidase activity produced by fibroblasts and keratocytes and characterized the MMPs by zymography.Hyaluronan added to fibroblast cultures stimulated the membrane-bound elastase-type endopeptidase activity in a dose dependent fashion. Similar results were obtained with keratocyte cultures. In the presence of 1mg/ml hyaluronan there was an increase in MMP expression and also an activation of latent MMPs both by fibroblasts and keratocytes.

Does MMP-2 expression and secretion change with increasing serial passage of keratocytes in culture?

The effects of ageing on matrix metalloprotease degradation of the extracellular matrix during corneal wound healing are largely unknown. The following study used an in vitro model of ageing to assess changes in MMP-2 RNA expression and protein secretion. Early passage (EP) EK1.BR keratocyte cultures from 14 to 18 cumulative population doublings (cpds) and late passage (LP) cultures from 40 to 47 cpds were used to isolate protein and mRNA samples. Total protein from EP and LP cultures was measured using the Bradford protein assay. Zymographic analysis of EP and LP samples was carried out to compare MMP-2 activity. Northern blot analysis was used to assess changes in MMP-2 mRNA expression by EP and LP cultures, using a digoxigenin (DIG) based chemiluminescent detection system. LP cultures secreted more total protein per cell. MMP-2 but not MMP-9 activity was detected in keratocyte cultures. Densitometric analysis of zymograms and calculation of MMP-2 activity indicated a significant increase in MMP-2 activity per cell (P<0.05, n=11). No difference was observed in the levels of MMP-2 mRNA expressed by EP and LP cultures. An increase in MMP-2 activity per cell by LP cultures suggests that senescent keratocytes increase their degradative capacity. Similar changes in the keratocyte phenotype within the ageing cornea may alter the balanced response necessary for adequate wound healing and may have implications for the therapeutic use of MMP inhibitors in the eye.

Human keratocyte migration into collagen gels declines with in vitro ageing

Although senescence in various cell types has been shown to have detrimental effects on wound repair, the effect of this phenomenon on corneal function with increasing age has yet to be elucidated. This study investigated the effect of in vitro ageing on keratocyte migration into a collagen gel matrix. The keratocyte cell strain EK1.BR was cultured to late passage and a comparison of early passage migration with that of late passage migration was carried out. Early or late passage keratocytes were seeded onto 6 collagen gels (1.75 mg ml −1) for each experiment. Gels were incubated at 37°C for 72 h, stained with calcein AM (0.5 mg ml −1) and assayed for cell migration using fluorescent microscopy. Changes in the effect of EGF on keratocyte migration with age were assessed by the addition of EGF (20 ng ml −1) to 3 of the 6 gels in each experiment. Proliferative lifespan was measured by immunocytochemical detection of Ki67 activity. This study shows for the first time that keratocyte migration, and migration in response to EGF stimulation, significantly declines with increasing age of keratocytes in culture ( P < 0.001). As keratocyte migration in response to cytokine stimulation is vital for corneal repair, the accumulation of senescent keratocytes with age may impair corneal wound healing.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas

To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2). Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting. All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2. The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.

Fibroblast Growth Factor-2 Promotes Keratan Sulfate Proteoglycan Expression by Keratocytes in Vitro

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

IL-4 Induces Eotaxin Production in Corneal Keratocytes but Not in Epithelial Cells

Background: In severe allergic eye diseases, the breakdown of epithelial barrier function can lead to severe corneal damage such as erosions or ulcers which often resist treatment. Although eosinophils are thought to play a crucial role in corneal tissue damage in severe ocular allergy, the mechanisms of eosinophil recruitment to the cornea has not been fully clarified. Eotaxin has been found in tears of severe allergic patients with corneal ulcer. In this study, we investigated whether the Th2 cytokine interleukin-4 (IL-4) induces eotaxin production in human corneal epithelial cells and keratocytes. Methods: Primary cultures of human corneal epithelial cells and keratocytes were incubated with IL-4 and/or TNF-α for 48 h. Released eotaxin was measured by ELISA, and the eotaxin proteins were visualized by immunocytochemistry. Eotaxin mRNA expression in cultured cells was analyzed by RT-PCR. Results: IL-4 induced eotaxin production in keratocytes in a dose- and time-dependent manner which was enhanced by TNF-α. There was no detectable eotaxin produced by corneal epithelial cells (<5 pg/ml). The cytoplasm of keratocytes incubated with IL-4 stained positively against anti-eotaxin antibodies, while eotaxin mRNA was detected in keratocytes incubated with IL-4. Conclusions: Human corneal keratocytes, but not epithelial cells, are capable of producing eotaxin by stimulation with IL-4. Our results suggest that eotaxin production in keratocytes induced by IL-4 may play an important role in eosinophil recruitment to corneal ulcers in allergic ocular disease. Eotaxin production by keratocytes may explain the severity of allergic disease involving the corneal stroma.

The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures.

Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas

Purpose. To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2). Methods. Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting. Results. All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2. Conclusion. The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.

Role of ocular matrix metalloproteinases in peripheral ulcerative keratitis

AIMPeripheral ulcerative keratitis (PUK) is an ocular manifestation of rheumatoid arthritis and other similar systemic diseases. The purpose of this inquiry was to investigate the involvement of matrix metalloproteinases (MMPs)...

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.

IL-4 regulates chemokine production induced by TNF-α in keratocytes and corneal epithelial cells

BACKGROUNDEosinophils are thought to play a major role in the pathogenesis of corneal lesions in ocular allergies. The regulation of chemokine production in corneal cells by the Th2 cytokine, interleukin...

Enhanced Expression of a Transmembrane Phosphotyrosine Phosphatase (LAR) in Keratoconus Cultures and Corneas

The purpose of the study was to identify genes that are differentially expressed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the polymerase chain reaction (PCR) using defined, arbitrary primers. The products were displayed on polyacrylamide gels and bands that were differentially expressed were excised, re-amplified and subcloned. The resulting clones were sequenced and utilized as probes for Northern blots with cultured cell RNA or Southern blots of corneal cDNA. One of the products that appeared to be more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembrane phosphotyrosine phosphatase. Western analyses and immunohistochemistry with monoclonal and/or polyclonal antibodies to LAR were used to examine keratocyte cultures and fresh frozen normal, keratoconus and pseudophakic bullous corneas.We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only at low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluorescence with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells throughout central keratoconus corneas, while no central staining was seen in normal or bullous keratopathy corneas.LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differential display, Northern analyses, immunohistochemistry and Western blotting.