IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro

Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-beta/ml over a 7-day culture period. We found that IGF-II and TGF-beta, alone or combined, increased both (3)H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to alpha-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no alpha-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-beta expressed alpha-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-beta, however, expressed alpha-SM actin, fibronectin, SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-beta mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-beta.     

Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.

PURPOSE: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS: Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS: Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS: A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.     

Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum

PURPOSE: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium. METHODS: Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC. RESULTS: Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels. CONCLUSIONS: The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.     

Effect of growth factors on collagen lattice contraction by human keratocytes.

A three-dimensional gel contraction model was used to evaluate interactions between human keratocytes and different kinds of collagen in the presence or absence of various growth factors. Bovine collagen type I or human placental copolymerized collagen type I/III was used to create the lattices. Normal keratocytes from neonatal, aged, and insulin-dependent diabetic donors, as well as abnormal keratocytes from a donor with macular corneal dystrophy, were cultured. Growth factors included epidermal growth factor (EGF), basic fibroblastic growth factor (FGF), insulin-like growth factor (IGF-I), and platelet-derived growth factor homodimer beta beta (PDGF). Gel area and optical transmittance were determined from computerized measurements. Dose-response experiments (0.01-100 ng/ml) demonstrated that PDGF at 10 ng/ml (P less than 0.005) and EGF at 1 and 10 ng/ml (P less than 0.0001) were the most effective in promoting gel contraction, compared to IGF-I and FGF. Comparison of cell strains revealed different dose-response profiles. Cells from insulin-dependent diabetics and cells from a donor with macular dystrophy contracted lattices more rapidly than cells from normal neonates (P less than 0.0001). Lattices of copolymerized human collagen type III/I demonstrated significantly reduced contraction rates (P less than 0.0001) and increased optical transmittance, compared to bovine collagen type I lattices. Ultrastructural studies revealed that keratocytes extend processes to form a network within the collagen lattice. Specialized intercellular junctional complexes were observed by transmission electron microscopy. This model provides a useful in vitro corneal stroma-equivalent for the study of keratocyte, extracellular matrix, and growth factor interactions.     

The effects of organ-culture on the density of keratocytes and collagen fibers in human corneas

PURPOSE: Keratocytes are important in regaining corneal transparency during wound healing after surgery or trauma. Hitherto, there are still controversies concerning the effects of organ culture on the density and integrity of keratocytes and collagen fibers. The current study aimed at a systematic analysis of the effects of organ-culture on the morphology and density of keratocytes and collagen fibers. METHODS: Human corneas were organ-cultured in MEM for 7 (n = 17, 3 pairs), 14 (n = 18, 9 pairs) and 21 days (n = 18, 9 pairs). Of the pairs one cornea was processed in swollen condition and the fellow cornea after reversal of swelling in MEM plus Dextran. Eleven post-mortem corneas (PM) and 11 fresh corneas obtained from melanoma patients were used as controls. Stromal thickness, number of keratocyte profiles (corrected for swelling), number and diameter of collagen fibers were measured in light microscopical sections and electron micrographs. RESULTS: Stromal swelling due to organ-culture resulted in large keratocyte profiles with many vacuoles and large distances between collagen fibers in the posterior stroma. In contrast both keratocytes and distances between collagen fibers were not affected in the anterior stroma. After reversed-swelling the posterior corneal stroma was similar to that in fresh controls, indicating that the swelling process is largely reversible. The initial decrease in keratocyte density (18%) in the early post-mortem period did not progress during 21 days of organ culture. CONCLUSION: With respect to the morphology and density of keratocytes and collagen fibers it can be concluded that donor corneas remain suitable for transplantation up to at least 21 days after organ-culture.     

Effects of riboflavin/UVA corneal cross‐linking on keratocytes and collagen fibres in human cornea

Purpose: To evaluate the effects of corneal cross‐linking on keratocytes and collagen fibres in human corneas. Methods: Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty and five of them were treated with cross‐linking 6 months before penetrating keratoplasty. Five normal corneal buttons from healthy donors were used as controls. All samples were prepared for TUNEL assay and Western blot analysis for the detection of keratocyte apoptosis and immunohistochemical analysis for the morphological evaluation of keratocytes and collagen fibre diameter. Results: Normal corneas exhibited no TUNEL‐positive keratocytes and keratoconic and cross‐linked corneas showed moderate apoptotic cells mainly in the anterior part of the stroma. This apoptotic trend was confirmed by the cleavage of poly (ADP‐ribose) polymerase assessed using Western blot. The Ki‐67 staining showed a significant increase in the keratocyte proliferation in cross‐linked corneas compared with normal and keratoconus. In cross‐linked corneas CD34‐positive keratocytes were regularly distributed throughout the whole corneal stroma as in the control, and keratoconus was associated with patchy loss of immunoreactivity. The immunohistochemical analysis of collagen type I showed a significant increase in fibre diameter of cross‐linked corneas compared with control and keratoconus. Conclusion: Corneal cross‐linking leads to keratocyte damage; after 6 months a repopulation by proliferating cells, a distribution of CD34‐positive keratocytes as in control and an increase in collagen fibre diameter were observed. These modifications are the morphological correlate of the process leading to an increase in biomechanical stability.     

ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse

Corneal epithelial abrasion elicits an inflammatory response involving neutrophil (PMN) recruitment from the limbal vessels into the corneal stroma. These migrating PMNs make surface contact with collagen and stromal keratocytes. Using mice deficient in PMN integrin CD18, we previously showed that PMN contact with stromal keratocytes is CD18-dependent, while contact with collagen is CD18-independent. In the present study, we wished to extend these observations and determine if ICAM-1, a known ligand for CD18, mediates PMN contact with keratocytes during corneal wound healing. Uninjured and injured right corneas from C57Bl/6 wild type (WT) mice and ICAM-1^−/− mice were processed for transmission electron microscopy and imaged for morphometric analysis. PMN migration, stromal thickness, and ICAM-1 staining were evaluated using light microscopy. Twelve hours after epithelial abrasion, PMN surface contact with paralimbal keratocytes in ICAM-1^−/− corneas was reduced to  ˜ 50% of that observed in WT corneas; PMN surface contact with collagen was not affected. Stromal thickness (edema), keratocyte network surface area and keratocyte shape were similar in ICAM-1^−/− and WT corneas. WT keratocyte ICAM-1 expression was detected at baseline and ICAM-1 staining intensity increased following injury. Since ICAM-1 is readily detected on mouse keratocytes and PMN-keratocyte surface contact in ICAM-1^−/− mice is markedly reduced, the data suggest PMN adhesive interactions with keratocyte-stromal networks is in part regulated by keratocyte ICAM-1 expression.     

Localization of collagen (I) and collagenase mRNA by in situ hybridization during corneal wound healing after epikeratophakia or alkali-burn.

The expression and localization of type I collagen and collagenase gene were studied by in situ hybridization using rabbit cornea during wound healing following epikeratophakia or alkali-burn. In corneas 24 days after epikeratophakia, alpha 1(I) collagen mRNA was detected in keratocytes which had migrated from the host cornea into the keratolens. In contrast, collagenase mRNA was detected in cells which seemed to be inflammatory cells around the suture between the host stroma and the keratolens. The increase of alpha 1(I) collagen mRNA in keratocytes was observed in corneas 94 days after epikeratophakia and in alkali-burned corneas 1-2 months after the burn. These results provide evidence that keratocytes synthesize collagen and that this synthesizing activity lasts for a long period during corneal wound healing.     

Keratocyte activity in wound healing after epikeratophakia in rabbits

Epikeratophakia is a refractive surgical procedure for the correction of aphakia, high myopia, or keratoconus. To solve clinical problems associated with epikeratophakia, a basic knowledge of its postoperative healing process is needed. The authors investigated keratocyte activities, particularly cell proliferation and collagen synthesis, during wound healing after epikeratophakia in rabbits. Epikeratophakia was done on rabbit corneas with a homologous cryolathed keratolens. Ten, 16, 28, 45, 63, 90, 254, and 360 days after the operation, the corneas were excised, labeled with either 3H-thymidine (10 microCi/ml) or 3H-proline (10 microCi/ml) for 4 hr and examined histologically and by autoradiography. Keratocytes in keratolenses were killed during the freezing process. On postoperative day 10, a few keratocytes migrated to the edge of the keratolens from the host stroma. On days 16 and 28, keratocytes in the keratolens and host stroma near the junction between the host and the keratolens incorporated 3H-thymidine, suggesting active proliferation. The proliferating activity was no longer seen after day 45. The repopulation of keratocytes was almost complete on day 90 and gradually returned to normal through day 360. Keratocytes in the keratolens and host stroma beneath the keratolens showed a higher 3H-proline incorporation than the control from days 16-254 with the highest activity at around 4-9 weeks after surgery. These results suggest that remodeling of collagen fibers continues for a long postoperative period after epikeratophakia.     

Sphere formation from corneal keratocytes and phenotype specific markers

The keratocytes are specialized mesenchymal cells that produce and maintain the extracellular matrix of the corneal stroma. With a typical dendritic and flattened appearance, these cells can morph into fibroblasts and myofibroblasts upon injury, and produce abnormal or fibrotic extracellular matrices detrimental to corneal transparency. Insights into mechanisms that regulate these phenotypic switches and optimal culture conditions that preserve the keratocyte phenotype are important for tissue engineering of the corneal stroma. Like other cell types with self-renewing capacity, keratocytes can form spheres in culture. Here we investigated human and bovine keratocytes with respect to their sphere forming capabilities, and sought to identify potentially distinguishing markers for the keratocyte and fibroblast phenotypes. Keratocytes, isolated from bovine and human corneas, cultured in serum-free medium supplemented with insulin, selenium and transferrin, assumed typical keratocyte morphology, converted to fibroblasts in serum-containing medium and reverted to keratocytes after serum-deprivation. The bovine keratocytes produced spheres under adherent or low attachment conditions, while the human keratocytes produced spheres under low attachment conditions only. The primary keratocytes and fibroblasts expressed vimentin, confirming their mesenchymal origin. Keratocan, considered to be a marker for keratocytes, was also detected in early passage bovine fibroblasts. BMP3 was expressed in keratocytes and keratocyte-derived spheres, while cadherin 5 in keratocytes only, suggesting these as potential keratocyte markers.     

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.     

Increased stromal extracellular matrix synthesis and assembly by insulin activated bovine keratocytes cultured under agarose

Previously, pharmacological levels of insulin have been shown to stimulate the synthesis of normal corneal stromal collagen and proteoglycans by bovine keratocytes in culture. Here we compared insulin to physiological levels of IGF-I and found that IGF-I also stimulated the synthesis of these extracellular matrix components, but less than that of insulin. Keratocytes in monolayer culture secreted most of the collagen synthesized into the media in the form of procollagen, a precursor of collagen. We found that an overlay of 3% agarose on the keratocytes in culture enhanced the conversion of procollagen to collagen and increased the deposition of collagen and proteoglycans into the cell layer. The extracellular matrix associated with the keratocytes cultured under agarose exhibited a corneal stromal-like architecture. These results suggest that enhancing the conversion of procollagen to collagen is a key step in the formation of extracellular matrix by keratocytes in vitro. Agarose overlay of insulin activated keratocytes in culture is a useful model for studying corneal stromal extracellular matrix assembly in vitro.     

Increased SPARC accumulation during corneal repair

Keratocytes can become fibroblasts and myofibroblasts during corneal injury and wound healing. We used the in vitro bovine keratocyte repair model system, which involves culturing collagenase-isolated keratocytes in serum-free media and then adding serum or serum plus TGF-beta to the culture media to induce the fibroblast and myofibroblast phenotypes, respectively, to evaluate the synthesis of secreted products by the cells. Serum and serum plus TGF-beta rapidly induced the fibroblast morphology and alpha smooth muscle actin, a marker of myofibroblasts. Keratocytes cultured in serum and serum plus TGF-beta also increased the synthesis of several high molecular weight products (approximately 100kD and larger) and the accumulation of a 43kD protein shown to be osteonectin/SPARC by both sequencing tryptic peptides from the protein and by reaction with antisera to osteonectin/SPARC. Immunohistochemical staining of mouse corneas with antisera to SPARC seven days post-wounding also demonstrated an increased accumulation of SPARC in the regions undergoing repair. These results indicate SPARC accumulation is a marker for stromal repair.     

The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

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.     

兔角膜基质细胞在壳聚糖胶原共混膜体外培养研究

目的　探讨壳聚糖 胶原共混膜作为载体体外培养兔角膜基质细胞的可行性。方法　先同时消化角膜上皮及内皮层 ,然后刮去上皮、内皮、前弹力层和后弹力层 ,将剩余的基质层剪碎消化 ,接种在壳聚糖 胶原共混膜上 ,通过间接免疫荧光细胞化学染色对培养的细胞进行鉴别。结果　角膜基质细胞应用消化培养法 4h后部分基质细胞与壳聚糖 胶原共混膜有贴壁现象出现 ,细胞呈梭形。培养 2 4h后 ,基质细胞呈纺锤形且透明 ,此后细胞分裂增殖越来越多并向周围延伸 ,培养第 6天细胞已经达到完全融合状态 ,呈梭形 ,排列比较整齐。在 6d左右达到 10 0 %融合状态 ,间接免疫荧光细胞化学染色、Vim染色、胞浆染色阳性。结论　传代的细胞具有角膜基质细胞的生物特性 ,壳聚糖 胶原共混膜适合角膜基质细胞传代培养     

Keratocyte activity in wound healing process following epikeratophakia in rabbits

Epikeratophakia is a refractive surgery for the correction of aphakia, high myopia and keratoconus. Although many clinical studies of epikeratophakia have been performed, its wound healing process is not well understood. In the present study, we investigated keratocyte activities, particularly cell proliferation and collagen synthesis activity, during wound healing following epikeratophakia in rabbit corneas. Ten, 16, 28, 45, 63 and 90 days after the operation, corneas were excised, labeled with either 3H-thymidine (10 microCi/ml) or 3H-proline (10 microCi/ml) in DME for 4 hours and examined histologically, and by autoradiography. Ten days after the operation, almost no cells were seen in the keratolens except for a few keratocytes which had migrated to the edge of the keratolens. At days 16 and 28, keratocytes in the keratolens and in the host stroma near the junction between the host and the keratolens incorporated 3H-thymidine, suggesting active proliferation. The proliferating activity was no longer seen at days 45, 63 and 90. At day 90, almost complete repopulation with keratocytes was observed. Keratocytes in the keratolens and in the host stroma beneath the keratolens showed higher activity of 3H-proline incorporation than those in the control from day 16 through day 90 with the highest activity at day 28.     

Cellular changes in transplanted human corneas

PURPOSE: To measure endothelial cell and keratocyte densities in transplanted corneas and the changes in these densities with time. METHODS: The endothelia of 500 consecutive penetrating corneal transplants were studied longitudinally by specular microscopy for 10 to 20 years. The keratocytes of 36 corneal transplants that varied in postoperative times from 1 month to 20 years were studied cross-sectionally by clinical confocal microscopy. The keratocytes of five transplanted corneas were studied longitudinally by confocal microscopy at 1 day, 1 week, and 1 month postkeratoplasty. RESULTS: Endothelial cell density decreased progressively at an accelerated rate for 20 years after transplantation, with concurrent increases in the coefficient of variation of cell area and corneal thickness and decreases in the percentage of hexagonal cells. Grafts with insufficient endothelial cells developed late endothelial failure, which was the primary cause of graft failure after the first 5 postoperative years. The grafts with late endothelial failure did not lose endothelial cells faster than grafts that did not fail, but instead had fewer cells immediately after transplantation, diminishing to a critically low cell density earlier. The keratocyte density was also decreased in transplanted corneas. Keratocytes became "activated" during the first week after keratoplasty and in grafts with late endothelial failure. CONCLUSION: It should be possible to prevent or delay late endothelial failure, the primary cause of graft failure, by increasing the number of endothelial cells on transplanted corneas. The status of the keratocytes appears to affect corneal transparency and, thus, visual quality in the grafted eye.     

Involvement of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing

PURPOSE: The involvement of downstream messengers of transforming growth factor (TGF)-beta in the differentiation of corneal fibroblasts into myofibroblasts was investigated. The effects of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 upregulated by TGF-beta were examined in human corneal fibroblasts, and the possible involvement of IGF axis components in corneal wound healing was assessed in a mouse model. METHODS: Human corneal fibroblasts were incubated with TGF-beta2 or IGF-I, to investigate IGF-I, IGF-II, IGFBP-3, type I collagen, and alpha-smooth muscle actin (alpha-SMA) mRNA, as well as IGFBP-3 protein expression, during myofibroblast differentiation. DNA synthesis was evaluated with a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. IGFBP-3 mRNA expression, protein expression, and immunolocalization were investigated in mouse corneas after photorefractive keratectomy (PRK). RESULTS: TGF-beta2 treatment induced expression of IGF-I and IGFBP-3 mRNA and of IGFBP-3 protein in human corneal fibroblasts. TGF-beta2 and IGF-I both stimulated expression of type I collagen. TGF-beta2 but not IGF-I potently stimulated alpha-SMA mRNA expression. IGF-I potently stimulated basal DNA synthesis, whereas IGFBP-3 inhibited it. IGF-I potently stimulated proliferation of TGF-beta2-activated myofibroblasts without reversing the activated fibrogenic phenotype, whereas IGFBP-3 suppressed IGF-I-induced proliferation of corneal fibroblasts. IGFBP-3 mRNA and protein increased in mouse corneas soon after PRK, when in vivo immunostaining of the corneas showed expression of IGFBP-3 in the deep layer of the corneal stroma. CONCLUSIONS: These results suggest that during corneal wound healing, TGF-beta stimulates IGF axis components, whereas IGFBP-3 may modulate IGF-I-induced myofibroblast proliferation to suppress corneal mesenchymal overgrowth.     

Matrix metalloproteinase 2: involvement in keratoconus

PURPOSE: The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus. The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas. METHODS: The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents. RESULTS: Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. CONCLUSIONS: The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.