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)     

Distribution of types I, II, III, IV and V collagen in normal and keratoconus corneas

By using type-specific antibodies to types I, II, III, IV and V collagens, distribution of distinct types of collagen in normal human cornea as well as keratoconus cornea were examined by indirect immunofluorescence microscopy. In normal human cornea, immunohistochemical evidence supported the previous biochemical finding that type I collagen was the major type of collagen in human corneal stroma. No reaction was observed to anti-type II collagen antibody in the whole cornea. Anti-type III collagen antibody reacted with the corneal stroma in a similar fashion as that of anti-type I collagen antibody. Type IV collagen was observed in the basement membrane of the corneal epithelium and in Descemet's membrane. Anti-type V collagen antibody also reacted with the corneal stroma diffusely. Bowman's membrane was strongly stained only with he anti-type V collagen antibody. For further details of the distribution of type I, type III and V collagens in human corneal stroma, immunoelectron microscopic study was undertaken. The positive reaction products of anti-type I and anti-type III collagen antibodies were located on the collagen fibrils, while that of anti-type V collagen antibody was either on or close to collagen fibrils. In keratoconus cornea, no difference was observed in terms of the distribution of type I, III and V collagens, while the disruptive and excrescent distribution of type IV collagen was noted in the basement membrane of the corneal epithelium.     

Comparative studies of collagens in normal and keratoconus corneas

In this paper we present strong evidence that the aberrations in keratoconus corneas are not directly related to alterations in collagen composition and distribution. This conclusion is based on comparative studies of collagen types I, III, IV, V and the recently described collagen types VI and VII in keratoconus and normal corneas. The data are derived from biochemical analysis of collagen fractions sequentially extracted with pepsin and sodium-dodecylsulphate, from amino acid analysis of hydrolysates of entire corneal tissues as well as from immunoblotting of the extracted collagens with specific antibodies. These antibodies were also used to examine the distribution of the collagens in immunofluorescence experiments on corneal sections. The yields of the collagen extractions were demonstrated to be age dependent but were not altered in keratoconus samples. Apart from one case associated with osteogenesis imperfecta type I, comparative studies of keratoconus and normal corneas showed no differences in collagen composition of the extracts. This was confirmed by amino acid analysis of tissue-hydrolysates. The distributions of collagen types I, III, IV, V, VI and VII in corneal sections were in general unchanged in keratoconus corneas, the only differences being in scar tissues observed in the Bowman layer of some keratoconus samples.     

Corneal endothelial cell abnormalities in an early stage of the iridocorneal endothelial syndrome.

A corneal disc, obtained from a 52-year-old woman suffering from an early stage of the iridocorneal endothelial syndrome (ICE), was investigated by various morphological techniques to analyse the structural variations in the endothelial cells and to identify the collagen types within the abnormal layer of Descemet's membrane. Scanning electron microscopy of the posterior corneal surface revealed a mosaic of (a) flat hexagonal cells resembling irregular but normal endothelial cells, and (b) rounded hexagonal (ICE) cells with numerous surface microvilli. Degenerative changes were present in each cell type, but were more common in the flat hexagonal cells which contained intracytoplasmic spaces. By transmission electron microscopy the flat hexagonal cells exhibited many of the features of normal endothelial cells in terms of organelles and intercellular attachments, but lateral invaginations were absent. The ICE cells differed in that the apical surface was covered by microvilli and the cytoplasm contained tonofilaments, which were also observed by light microscopic immunocytochemical staining. Most commonly, intercellular attachments were rudimentary in both types of cell and intercellular spaces were dilated, but desmosomes were sometimes prominent in the ICE cells where interdigitations were pronounced. In some sectors, the basal surface of the ICE cells was indented by deposition of clumps of fibrillar collagenous material. An immunocytochemical study of the abnormal posterior deposits localised type IV collagen to the amorphous matrix and collagen types III and V, but not type I, to the collagen fibril bundles. Mononuclear inflammatory cells were identified between the ICE cells in the monolayer. The evidence suggests that some of the flat hexagonal cells were undergoing a degenerative change while others were transforming into ICE cells.     

Rabbit corneal endothelial cells modulated by polymorphonuclear leukocytes are fibroblasts. Comparison with keratocytes

The authors previously reported that polymorphonuclear leukocytes modulate rabbit corneal endothelial cells into fibroblasts, which acquire the characteristics of fibroblasts. The progeny of the fibroblastic corneal endothelial cells (FCEC) were further studied to compare the characteristics of the fibroblast with those of keratocytes as a function of culture age. During 11 days in culture, FCEC showed 32 population doublings, whereas keratocytes underwent 10 population doublings. When collagen phenotypes of both cultures were analyzed as a function of culture age, labeled collagens in both cultures were fractionated into types I, III, and V. The proportion of each collagen was relatively unchanged in keratocytes regardless of culture age: type I accounted for 92-96%, type III for 2-6% and type V for 2-5%. In contrast, the profiles were significantly changed in FCEC: at day 2, type I accounted for 57%, type III for 37.5%, and type V 5.5%. Over the following 2 days, type I increased to approximately 75%, whereas type III collagen decreased to approximately 20%. As FCEC multilayered, type I collagen synthesis reached a stationary level of 80%, with 12% of type III. When the stoichiometry of type I collagen was compared, the alpha 1/alpha 2 ratio was 6.2 in FCEC and the ratio was 3.5 in keratocytes at day 2. The ratio reached a normal value at day 7 in FCEC and at day 3 in keratocytes. The synthesis of type I trimer and transient alteration of type I/III and the rapid growth rate at early stages of growth, indicate that FCEC behave like cells seen in wound healing or other rapidly growing tissues, in contrast to the stabilized keratocytes.     

Distribution of specific collagen types and fibronectin in normal and keratoconus corneas

The distribution of five types of collagen and fibronectin in 6 normal and 9 keratoconus corneas was examined, using immunofluorescent staining and the enzyme-labeled antibody method. Types I, III and V collagens were detected in the corneal stroma. There was essentially no difference between normal and keratoconus corneas in their distribution. Type IV collagen and fibronectin were detected in the basement membrane of the normal corneal epithelium, while in the keratoconus corneas the disruption of the basement membrane as well as the excrescence of basement membrane materials was observed. The abnormal distribution of the type IV collagen and fibronectin was also observed in the anterior stromal area of keratoconus corneas.     

Collagens of the retinal microvascular basement membrane and of retinal microvascular cells in vitro

We have analysed the collagens present in vascular basement membranes isolated from bovine retinal and cerebral microvessels and bovine renal glomeruli, and from the non-vascular basement membrane of bovine lens capsule. These are compared with the collagens produced by cultured bovine retinal microvascular pericytes and lens epithelial cells, and by canine retinal microvascular endothelial cells, in vitro. Biochemical and immunocytochemical analyses indicate that all of the vascular basement membrane preparations have an identical collagenous composition, consisting of the same polypeptides present in lens capsule (primarily type IV collagen), together with other polypeptides that are identified as type I, and a small amount of type III collagen. Identification of the latter is based on two-dimensional gel electrophoresis in the presence and absence of a reducing agent. Immunocytochemical studies, however, demonstrate type I, type IV and some type V collagen in the basement membranes of the isolated microvessels. The cultured microvascular cells produce predominantly type I collagen molecules, but they also produce other collagen peptides that appear to be type IV, and, at least in some experiments, small amounts of type III collagen. The biochemical identification of collagens type I and IV is confirmed by immunocytochemistry. However, results with anti-type I collagen and procollagen antibodies in cultured pericytes vary with antibodies from different sources. The quantities of the type IV peptides produced by the cultured cells also vary in different experiments.     

Immunohistochemistry and electron microscopy of retrocorneal scrolls in syphilitic interstitial keratitis

PURPOSE: To describe the immunohistochemical and electron microscopic characteristics of retrocorneal scrolls in syphilitic interstitial keratitis. METHODS: Five eyes of five patients with congenital syphilitic interstitial keratitis who underwent keratoplasty for corneal opacities and corneal edema were studied. The corneal buttons were processed for histologic examination with hematoxylin and eosin staining and underwent immunohistochemistry stainings for collagen types I, III, IV, V, VI, VIII, fibronectin, laminin, and decorin. The corneal buttons were also processed for transmission electron microscopy and immunoelectron microscopy. RESULTS: Light microscopy revealed that the retrocorneal scrolls had a multilayered, amorphous, acellular matrix. All scrolls were lined with attenuated corneal endothelial cells. The Descemet membranes in all specimens had areas of irregular thickening with attenuated endothelium. Immunohistochemical assessment of the scrolls showed positive staining for collagens I, III, IV, VI, VIII, fibronectin, laminin, and decorin but not for alpha -SMA. Immunoelectron microscopy confirmed these findings. Transmission electron microscopy showed multilaminar disorganized structures in scrolls composed of long- and short-fiber collagens. CONCLUSIONS: We confirmed the presence of collagens I, III, IV, VI, VIII and proteoglycans in the retrocorneal scrolls lined with attenuated endothelium. Our findings may provide further insight into the pathogenesis of keratopathy in syphilitic interstitial keratitis.     

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.     

Immuno-electron labelling of matrix components in congenital hereditary endothelial dystrophy

Two corneal buttons were obtained from a patient with congenital hereditary endothelial dystrophy (CHED) at the ages of 2.5 years (right eye) and 14 years (left eye) and were studied by light and electron microscopy including immunogold labelling for collagen types I -V and laminin. The posterior collagenous layer (PCL) of Descemet's membrane contained collagen types I, III-V, and laminin: the latter was also localised to finebanded and granular material in the posterior non-banded zone (PNBZ). Comparison of the endothelium at 2.5 years and 14 years revealed occasional dystrophic changes in the former and extensive dystrophic changes in the latter. The distribution of collagen types I, III and V within the PCL supports previous morphological observations of fibroblast-like change of the endothelium in CHED. Persisting endothelial properties were manifest as positive labelling of type IV collagen and laminin. An excessive amount of laminin found in PNBZ and PCL is another stress-related endothelial reaction.Parts of this publication were presented at the First Annual Meeting of the European Community Ophthalmic Research Association (ECORA), 1993     

Modulation of endothelial cell morphology and collagen synthesis by polymorphonuclear leukocytes

Primary corneal endothelial monolayers exposed to polymorphonuclear leukocytes undergo a series of morphologic alterations. Elongation occurred in foci within 3 days after removal of polymorphonuclear leukocytes, the modulated endothelial foci grew into fibroblastic colonies, and the fibroblastic cells eventually overgrew the endothelial cells. Control cultures of endothelial cells originated from confluent monolayers became enlarged, attenuated and lost their characteristic polygonal shape within 10 days following postconfluency , but no fibroblastic changes were seen. "Wounding" the endothelial monolayer with a focal freeze resulted in death of cells with slow regeneration. In the presence of polymorphonuclear leukocytes, cell migration into the wound was enhanced, and there was selective proliferation of fibroblastic cells. Indirect immunofluorescent studies showed that anti-type I collagen antibodies stained the fibroblastic foci in the polymorphonuclear leukocyte-treated endothelial cells and the fully modulated endothelial cells. The fully modulated cells also showed loss of contact inhibition leading to mutilayering of cells and extracellular matrices, which accumulated not only between the basal cell layers and plastic substratum but also in the cellular interstices. When collagen phenotype was analyzed by SDS electrophoresis in comparison with corneal endothelial phenotypes (type IV collagen), type I procollagen synthesis became evident in the secondary subculture originated from polymorphonuclear leukocyte-treated endothelial cells. Limited pepsin treatment gave rise to type I collagen as a major collagenous peptide. Polymorphonuclear leukocytes, thus, apparently contribute to the modulation of endothelial cells into fibroblastic cells, which also switch their collagen phenotype from type IV to type I collagen synthesis.     

兔LASEK与PRK术后角膜Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的Western Blot研究

目的:用WesternBlot法比较准分子激光上皮下角膜磨削术(LASEK)与准分子激光角膜切削术(PRK)术后角膜Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的动态变化情况。方法:52只新西兰白兔分为8组,对每只兔右眼行LASEK,左眼行PRK。术后1d、7d、1个月、3个月、4个月、5个月、6个月观察Haze情况,处死动物取角膜行Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的动态变化过程的免疫组织化学检查与WesternBlot检测。结果:经免疫组化和WesternBlot研究发现:LASEK组术后角膜基质中Ⅰ、Ⅲ型胶原开始增生的时间早于PRK组,表达的强度与PRK组有显著差异;两组的Ⅴ、Ⅵ型胶原动态变化曲线相似,达到表达高峰的时间一致,PRK组Ⅴ、Ⅵ型胶原的表达均明显高于LASEK组,术后6个月时LASEK组表达显著弱于PRK组。结论:PRK手术后角膜基质中Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的表达强度、达到高峰及恢复正常的时间与LASEK相比存在显著差异,表明PRK术后基质内有胶原的过量沉积,可能是临床PRK术后Haze严重及屈光回退的组织学基础。WesternBlot法是一种可比较准确地半定量检测胶原含量动态变化的研究方法。     

Extracellular matrix of the human retrobulbar optic nerve

The presence and distribution of laminin, fibronectin and type I through V collagens were determined in the human retrobulbar optic nerve using a immunohistochemical method, the biotin-streptavidin (B-SA) system. Type I and type III collagens, which were interstitial collagens, were detected diffusely within pial septa, pia mater, arachnoid membrane, dura mater and adventitia around the central retinal artery and vein. Type IV collagen and laminin, which were basement membrane materials, were localized linearly along the borders between optic nerve fascicles and pial septa, adventitia, pia mater. Also They were also stained in all vascular walls and the arachnoid membrane. Type V collagen and fibronectin were detected both in the interstitial connective tissues, diffusely, and in the basement membranes, linearly. Fibronectin was stained more intensively in the basement membranes than type V collagen.     

Corneal collagen fibrils: dissection with specific collagenases and monoclonal antibodies

To investigate the relationship of collagen types I and V within corneal fibrils, and the collagenolytic mechanisms potentially involved in corneal development and remodeling, we have incubated cryostat sections of avian corneas with collagenases that specifically degrade collagen types I or V to digest selectively the collagen in situ. These preparations were then analyzed by immunofluorescence histochemistry and immunoelectron microscopy using anti-collagen type-specific monoclonal antibodies. Digestion of corneal sections with the type I-specific collagenase ("I'ase") exposed antigenically masked type V collagen, indicating that epitopes on type V collagen in heterotypic fibrils are inaccessible to the antibody due to their interaction with type I collagen. Sections digested with the type V collagen-degrading enzyme ("V'ase") showed no removal of type V collagen. However, after the fibrillar structure was disrupted by acetic acid treatment before enzyme digestion, the type V collagen was then degraded. Likewise, prior digestion of type I collagen by I'ase also rendered type V collagen susceptible to digestion by V'ase. These results suggest that the cleavage sites on type V collagen also are buried within heterotypic fibrils and therefore inaccessible to the enzyme. They also document, for the first time, V'ase activity against type V collagen in situ. Electron microscopic observations of sections partially digested with the I'ase revealed many short striated fibrillar segments from which smaller filaments protrude. Both the striated regions and some of the filaments were labeled by an antibody against type I collagen; anti-type V antibody reacted preferentially with the thin filaments. Thus avian corneal fibrils contain type I collagen, in which filaments of type V collagen are embedded. Complete removal of the fibrillar stromal matrix in the course of normal or pathological remodeling requires at least two different collagenases acting in concert.     

Types of collagen synthesised by the lens epithelial cells of human cataracts.

AIMS/BACKGROUND--Residual lens epithelial cells (LECs) undergo fibrous proliferation after cataract surgery, resulting in capsular fibrosis. The purpose of this study was to determine the types of collagen produced in cultured LECs derived from human cataract LECs. METHODS--A circular section of the anterior capsule, about 5 mm in diameter, with LECs attached was obtained by anterior capsulotomy during cataract surgery and cultured directly without dispersion of the cells in a well, on the bottom of which a disc-shaped, thin plate of poly(methyl methacrylate) had been placed. At 5 to 6 weeks of culture, the proliferated cells of the culture were stained immunohistochemically with antibodies against human collagens I-VI by the avidin-biotin-peroxidase complex method. RESULTS--Collagens I, IV, V, and VI were positive in the cultured cells. Types IV and V were strongly present in almost all the cells whereas types I and VI were only observed in a few cells. Collagens II and III were negative. CONCLUSIONS--Since the lens capsule is known to be comprised of collagen IV, collagens I, V, and VI seem to be produced newly in culture. The capsular fibrosis seen after cataract surgery in vivo as a wound healing process of the lens capsule, may contain these types of collagens. The present culture model is useful for studying secondary cataract formation.     

Immunohistochemical and ultrastructural analysis of dysplastic epithelium of human ocular surface: basement membrane and intermediate filament.

PURPOSE: The dysplastic corneal epithelium is characterized by the abnormal proliferation of epithelial cells. The phenotypes of these cells have not been elucidated. We investigated whether such epithelium expresses the phenotypes of corneal or conjunctival epithelial cells. METHODS: The corneas and conjunctivae from four normal subjects and from one patient with epithelial dysplasia of the central cornea were immunostained for IV and VII collagens and for cytokeratins. Monoclonal antibodies against collagen IV reacted to the [alpha1(IV)]2alpha2(IV) or alpha5(IV) molecule. Anti-cytokeratin antibodies were used to define epithelial cell types. The ultrastructure of the basement membrane (BM) of each specimen also was examined. RESULTS: Type VII collagen immunoreactivity was detected in all the specimens of epithelial BM. The anti-collagen IV [alpha1(IV)]2alpha2(IV) antibody labeled the conjunctival BMs, not the BMs of the corneal epithelia, of each subject. The normal corneal epithelial BM, not the BM of the conjunctival or dysplastic corneal epithelium, was immunolabeled with anti-alpha5(IV) antibody. The pattern of cytokeratin expression in the corneal epithelial dysplasia resembled that seen in the normal conjunctivae. Small breaks in the BM of dysplastic corneal epithelium were ultrastructurally revealed. The number of hemidesmosomes in the dysplastic corneal epithelium was decreased as compared with that in the normal BM. CONCLUSION: The composition of collagen types within the BM and the cellular phenotype of the dysplastic epithelium in the cornea resembled those of conjunctival epithelium, not of the cornea.     

Immunohistochemical Characterization of Epithelial Cells Implanted in the Flap–Stroma Interface of the Cornea

Purpose To investigate the expression of extracellular matrix collagens and their relationship to corneal opacities after implantation of epithelial cells in the flap–stroma corneal interface.Methods A corneal flap was made on rabbit eyes, and epithelial cells, mechanically scraped from tissue surrounding the flap, were implanted beneath the flap. The corneas were harvested 1, 3, 7, and 30 days following surgery. Histological and immunohistochemical examinations were performed. The expression and localization of types I, III, and IV collagens and gelatinase A were determined.Results Slit-lamp examination showed corneal opacity in the area where the epithelial cells were implanted. Histological study revealed clusters of epithelial cells between the flap and stromal interface. One week and 1 month after the implantation, intense immunoreactivity for collagen type IV was detected at the perimeters of the intrastromal epithelial islands, but not in the interface outside the implanted epithelial cells. Weak positive staining for gelatinase A was detected in the implanted epithelial cells and surrounding keratocytes.Conclusions The heavy deposition of collagen type IV surrounding the implanted epithelial cells indicated that it might be an essential component of the interface haze observed in patients following laser in situ Keratomileusis. Gelatinase A may also play a role in the regulation of stromal remodeling after epithelial ingrowth. Jpn J Ophthalmol 2005;49:79–83 © Japanese Ophthalmological Society 2005