Effect of L-ascorbic acid 2-phosphate on cultured rabbit keratocytes (the third report). The effect on production and secretion of type I collagen]

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting phosphate derivative of L-ascorbic acid, on intracellular distribution and production of type I collagen in cultured rabbit keratocytes by an immunohistochemistry and enzyme immunoassay. Exposure of 0.1 mM P-Asc for 10 hours decreased a type I collagen immunoreactivity of the cytoplasm as stained in fine granular materials. 0.1 mM P-Asc induced increase of type I collagen level in the medium. The results suggested that 0.1 mM P-Asc might increase the biosynthesis and secretion of type I collagen in keratocytes and have a therapeutic effect on corneal stromal damage.     

Effect of L-ascorbic acid 2-phosphate on cultured rabbit keratocytes (the second report)

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the proliferation of cultured rabbit keratocytes. P-Asc is a derivative of L-ascorbic acid and it yields more prolonged effects of vitamin C in solution than L-ascorbic acid. The proliferation of cultured rabbit keratocytes was promoted by the presence of P-Asc in culture medium for 10, 20, and 30 days. Transmission electron microscopic observations revealed that the cells were more multilayered in the presence of P-Asc (0.1mM) for thirty days than those in the absence of P-Asc. Moreover, this effect of P-Asc was attenuated by azetidine 2-carboxylic acid which is an inhibitor of collagen synthesis. Hence, it is suggested that the promotive effect of P-Asc on the growth of cultured keratocytes is related to the synthesis of collagen. Based on our observations, P-Asc may have a therapeutic effect on corneal stromal damages such as a corneal chemical burn and surgical trauma.     

l-Ascorbic acid 2-phosphate,a phosphate derivative of l-ascorbic acid,enhances the growth of cultured rabbit keratocytes

We examined the effect of l-ascorbic acid 2-phosphate (P-Asc) on the proliferation of cultured rabbit keratocytes. P-Asc is a phosphate derivative of l-ascorbic acid and has more prolonged vitamin C activity in solution than does l-ascorbic acid. The proliferation of cultured keratocytes was promoted by the presence of P-Asc in culture medium. Transmission electron microscopic observations revealed that cells were more multilayered after culture in the presence of P-Asc (0.1 mM) for 30 days than were those cultured in the absence of P-Asc. The effect of P-Asc was abrogated by l-azetidine 2-carboxylic acid, which is an analogue of proline that inhibits the production and secretion of collagen. Our observations support a therapeutic role for P-Asc in the repair of corneal stromal damage such as that caused by a corneal chemical burn. Offprint requests to: S. Saika     

Ascorbic acid phosphate ester and wound healing in rabbit corneal alkali burns: Epithelial basement membrane and stroma

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the healing of alkali-burned corneas in rabbits. Round filter paper containing 1 N NaOH was applied to the central cornea for 60 or 120 s to produce the alkali burn. Animals were treated with topical saline, 10% ascorbate, or 6.5% P-Asc applied on the cornea. The corneas were then examined histologically. Burned stroma showed no toluidine blue staining, indicating a loss of glycosaminoglycan. In the 60-s burn group, P-Asc reduced the size of the unstained area as compared with the control. Transmission electron microscopy showed basal lamina under new epithelia in the corneas treated with ascorbate or P-Asc, but not in controls. These observations support the theory that P-Asc may have a therapeutic role in the repair of corneal alkali burns.     

Ultrastructural effect of L-ascorbic acid 2-phosphate on cultured keratocytes.

I studied the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting derivative of L-ascorbic acid, on the fine structures of cultured rabbit keratocytes. The results showed that cells cultured with 0.1 mM P-Asc for 30 days were more markedly multilayered than those grown without P-Asc. Dilation of the cisternae of the endoplasmic reticulum in the control cells indicated an accumulation of protein (probably procollagen). In addition, many lysosome-like structures, which may degrade underhydroxylated procollagen, were observed in the cytoplasm of the control cells. A slight increment of free ribosomes was also found within the control cells. P-Asc enhanced the multilayerization of cultured keratocytes. We conclude that the changes seen in the cytoplasm are due to the effect of P-Asc acting as vitamin C.     

Type IV collagen and corneal epithelial adhesion and migration. Effects of type IV collagen fragments and synthetic peptides on rabbit corneal epithelial cell adhesion and migration in vitro

Type IV collagen, a 500-kilodalton (alpha 1)2(alpha 2)1 heterotrimer with noncollagenous domains (NC1) is the major molecule in most basement membranes in the body. In addition to its structural role as scaffolding, type IV collagen is involved in promoting adhesion and migration of various cell types in vitro, including rabbit corneal epithelial cells. This study assessed the effect of purified proteolytic fragments of type IV collagen and selected synthetic peptides derived from the alpha 1 and alpha 2 chains that are related to the adhesion and directed migration of dissociated primary cultured rabbit epithelial cells. Two homologous peptides (HEP-1 and HEP-2) derived from alpha 1 and alpha 2 NC1 regions were found to promote epithelial cell adhesion. A peptide (HEP-3) derived from an interruption of the triple helix of type IV collagen was effective in promoting corneal epithelial cell migration in both chemotaxis and haptotaxis assays. The helical fragment of type IV collagen promoted both directed migration and ample adhesion, indicating that there may be at least another moiety in the helical region responsible for cell adhesion. The results with these peptides revealed to some extent how corneal epithelial cells react at the molecular level with type IV collagen. They could serve as the basis for therapeutic agents to modify corneal epithelial behavior in situations of perturbed wound healing.     

Distribution and isoform characterization of type XII collagen in bovine cornea

Streptavidin peroxidase immunohistochemistry and immunoelectron microscopy were used to determine the localization of type XII collagen in sections from bovine corneas. Type XII collagen extracted from bovine cornea and skin was assayed by Western blotting. Immunohistochemical experiments showed that type XII collagen was restricted to the corneal stroma; it was not present in corneal epithelium, epithelial basement membrane, Descemet's membrane or endothelium. Type XII collagen was distributed throughout the corneal stroma, and it was prominently localized at the superficial stroma. Immunoelectron-microscopic examination demonstrated that type XII collagen was regularly found along the surface of banded collagen fibrils with a periodic distribution. By Western blot analysis, we observed that extracts from bovine cornea contained both the long and short isoforms of type XII collagen, whereas extracts from bovine skin contained only the short isoform. The homogeneous distribution and/or presence of the long isoform of type XII collagen may be related to the characteristically regular arrangement of collagen fibrils and thereby the transparency of corneal tissue.     

Protein expression pattern of collagen type XV in mouse cornea

Purpose To examine the alteration of protein expression pattern of collagen type XV in cornea during embryonic development and adult tissue repair. Collagen type XV is a basement membrane collagen of a subfamily of multiplexins (multiple triple helix domains and interruptions). Its COOH-terminal peptide has an anti-angiogenic effect and its distribution in avascular tissue of cornea is of interest.Methods Eyes of mouse embryos [day (E) 10.5–18.5] and healing adult mouse corneas following either débridement injury or incision were embedded in paraffin. Deparaffinized sections were processed for immunofluorescent staining with anti-collagen XV antibody.Results At E14.5 embryonic corneal epithelium, as well as fibroblasts in eyelids, began to express this collagen type very faintly, and at E18.5, besides corneal epithelial expression, epidermis, palpebral conjunctiva, and keratocytes started to express collagen type XV. In adult mouse cornea, collagen type XV was observed in basal and suprabasal epithelial cells and stroma, but not in the subepithelial basement membrane. Healing epithelial cells following débridement or incision injury down-regulated its protein expression.Conclusions Mouse embryonic corneal epithelium and keratocytes begin to express collagen type XV before birth. Healing murine corneal epithelium down-regulates collagen XV expression. The presence of collagen XV in corneal stroma may play a part in avascularity.     

Immunolocalization of type VI collagen in developing and healing rabbit cornea

We have localized type VI collagen in normal developing and corneal scar tissue. Indirect immunofluorescence showed that type VI collagen was distributed throughout the normal stroma and most of the scar. No fluorescence was detected along the posterior margin of the scar and in a retrocorneal membrane continuous with the scar. Since the corneal endothelium in rabbits contributes to the formation of scar tissue and retrocorneal membrane, our observations suggest that the endothelium does not synthesize type VI collagen. Indirect immunoelectron microscopy showed that type VI collagen was located abundantly between collagen fibrils as fine filamentous structures containing beads with a periodicity of 100 nm, consistent with published observations of other tissues. Because these filaments are more prominent when stained with ruthenium red, and predigestion of tissue with Chondroitinase ABC enhances binding of monoclonal antibody to type VI collagen, proteoglycans probably are associated with this collagen in the cornea. Ultrastructural observations supported by previous biochemical analyses show that the proportion of type VI collagen to fibrillar collagen is smaller in scar tissue compared with fetal cornea. The abundance of type VI collagen and its distribution and association with proteoglycans in rabbit corneal tissues suggest that this macromolecule plays a role in the tensile strength and transparency of the stroma.     

Modulation of type III collagen synthesis in bovine corneal endothelial cells

Bovine corneal endothelial cells in culture synthesize predominantly type III collagen, unlike rabbit corneal endothelial cultures which synthesize type IV collagen. In an attempt to document whether this type III collagen synthesis by bovine cells is a tissue culture-specific phenomenon, collagens synthesized by organ culture of bovine Descemet's membrane/corneal endothelium complex were compared with those of subsequent tissue culture cells, up to the eighth passage. The biosynthetically labeled collagens were analyzed on SDS electrophoresis. The soluble fractions of tissues extracted with neutral salt followed by pepsin digestion contained only type I collagen; no other radiolabeled collagens were detected in organ culture. When pepsin treatment was eliminated, type IV collagen was identified in the tissue extract by immunoblot analysis using monoclonal antibody; type III collagen failed to show a positive band by immunoblot analysis. The pepsin-treated medium fraction of the primary culture contained types I, III and V collagen; type IV collagen was identified by either the characteristic electrophoretic mobility or by immunoblot analysis only prior to the proteolysis step. The subsequent subcultures continued to synthesize types I, III and V collagen, but type IV collagen was no longer detectable from the third passage on. No substantial quantitative changes in the expression of individual collagens were observed during subculture. From the primary culture, type I collagen accounted for 30%, type III for 60% and type V for 10%. Enhanced expression of type III collagen was observed in the eighth passage and in primary cultures grown on type I collagen matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of elasmobranch corneal and scleral collagens

Corneas, dissected from young and adult spiny dogfish sharks (Squalus acanthias), were prepared for transmission electron microscopy and immunofluorescence. In the latter case, tissues were fixed in formaldehyde solutions, sectioned with a cryostat, incubated with antibodies specific for collagen types I and II, and examined by indirect immunofluorescence. Collagen α- and β- chains were separated by sodium dodecylsulfate-slab gel electrophoresis and characterized by two-dimensional mapping of ¹²⁵I-labeled peptides generated by tryptic and chymotryptic digestion.The corneal stroma, the sutural fibers which span the stroma, and the surrounding limbus were positive for type I collagen, as judged by immunofluorescence. The corneal stroma was negative for type II collagen. Scleral cartilage matrix was intensely positive for type II collagen, but was negative for type I. In addition, the perichondrium of the scleral cartilage was positive for type I collagen. In confirmation of these results, slab gel electrophoresis revealed α1, and α2-like bands from shark corneal stroma, but only an α1-like band from shark cartilage collagen. Two-dimensional peptide mapping revealed some degree of resemblance between the α1 band of shark corneal stroma and the α1 band of chick type I collagen. Likewise, the α1 band of shark cartilage collagen somewhat resembled the α1 band of chick type II collagen. The α2-like band of shark corneal stroma did not closely resemble the α2 band of chick type I collagen. The most prominent β band of shark corneal stroma appeared to be a dimer composed of one α1 chain and one α2 chain. The collagen of shark corneal stroma was very susceptible to degradation by pepsin, whereas that from shark cartilage was much less susceptible.     

准分子激光角膜切削术后转化生长因子β及Ⅰ、Ⅲ型胶原的免疫组化观察

目的观察准分子激光角膜切削术（ｐｈｏｔｏｒｅｆｒａｃｔｉｖｅｋｅｒａｔｅｃｔｏｍｙ,ＰＲＫ）术后,猴角膜愈合过程中角膜转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒｂｅｔａ,ＴＧＦβ）的表达,以及Ⅰ、Ⅲ型胶原的表达情况,探讨ＴＧＦβ与ＰＲＫ术后角膜愈合过程中Ⅰ、Ⅲ型胶原合成的关系。方法对３只（６只眼）恒河猴行ＰＲＫ治疗。输入－１０００Ｄ治疗程序,切削深度为１０３μｍ。分别于术后１、３及６个月行光镜、电镜及免疫组化检查。结果术后１个月,ＴＧＦβ在角膜上皮层染色阳性,上皮下也可见少量成纤维细胞染色阳性;３个月,角膜上皮染色减弱;６个月时染色呈阴性;对照组为阴性。Ⅰ、Ⅲ型胶原于术后１、３及６个月时均有较强的阳性染色,对照组Ⅰ型胶原染色阳性,Ⅲ型胶原染色阴性。结论ＴＧＦβ参与ＰＲＫ术后角膜的愈合过程,并且可能与术后角膜上皮下新生胶原中Ⅰ、Ⅲ型胶原的合成有关。     

Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels

PURPOSE: To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. METHODS: Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. CONCLUSION: These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.     

Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats

PURPOSE: To understand the pathophysiology of the corneal basement membrane in diabetes, we compared the localization of laminin and type IV collagen in the epithelial basement membrane during corneal epithelial wound healing in diabetic and nondiabetic rats. METHODS: Streptozotocin was used to induce diabetes in half the rats. Two weeks later, the whole corneal epithelium was debrided. Diabetic and healthy rats (3-5 per group) were sacrificed before debridement and 1, 3, and 7 days and 1 month afterwards. The localization of laminin and type IV collagen was observed in cryosections by epifluorescence microscopy. RESULTS: In unwounded corneas of both diabetic and normal rats, laminin and type IV collagen were localized in the corneal epithelial basement. The intensity of fluorescence, however, was clearly stronger in the diabetic rats. In normal rats, wounding initially removed laminin and type IV collagen, but during healing these two proteins reappeared beneath the resurfacing corneal epithelium. Although similar results were observed in diabetic rats, the expression of laminin and type IV collagen was delayed, and their deposition was fragmented and irregular. CONCLUSIONS: These results suggest that delayed corneal epithelial wound healing in diabetes might involve delayed reappearance and abnormal reformation of epithelial basement membrane proteins.     

A cellular model for the investigation of Fuchs' endothelial corneal dystrophy

Fuchs' Endothelial Corneal Dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' Dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.     

Heterogeneity of collagens in rabbit cornea: type VI collagen

Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These physical characteristics, together with the susceptibility of these polypeptides to collagenase and their amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of [14C] glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI collagen deposited in scar tissue is markedly lower than that found in neonate corneas.     

The immunohistochemical changes of TGF-beta, type I and III collagen in corneal healing after photorefractive keratectomy

OBJECTIVE: To observe the immunohistochemical expression of transforming growth factor-beta (TGF-beta), type I and III collagen in monkey corneal healing after photorefractive keratectomy (PRK) and find whether TGF-beta is involved in the process of corneal healing and correlated with the syntheses of type I and III collagen. METHODS: Three rhesus monkeys (6 eyes) were operated for myopic ablation of 10.0 diopters. The depth of ablation was 103 microm. Microscopy, transmission electron microscopy and immunohistochemistry were performed at 1, 3 and 6 months after PRK. RESULTS: TGF-beta staining was positive in the epithelium and a few fibroblasts under the epithelium at one month after the operation. The staining was negative at 6 months and in the control specimens. Postoperatively, type I and III collagen stainings were evident at 1, 3 and 6 months. Type I collagen staining was positive and type III was negative in the controls. CONCLUSION: TGF-beta is involved in the corneal healing after PRK, and perhaps correlated with the syntheses of type I and III collagen.