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.     

Production of collagen by cells isolated from a retrocorneal fibrous membrane rabbit model

The morphological and biosynthetic characteristics of cells from an experimentally induced rabbit retrocorneal fibrous membrane (RCFM) model were investigated. By transmission electron microscopy the cells within the RCFM demonstrated overlapping cytoplasmic processes and intercellular junctions, neither of which are fibroblast-like characteristics. The extracellular matrix within the RCFM had a fibrillar and amorphous component. Collagenous biosynthetic products of primary cultures of RCFM cells were compared to normal corneal endothelial cells, which produce mainly type IV collagen and a small amount of type V collagen, and fibroblasts, which produce types I, III and V collagens. The collagenous components produced by the RCFM cells were a combination of types I, V and low molecular weight fragments of type IV collagen. Therefore, these morphological and biochemical data suggest that RCFM cells are a type of modified corneal endothelial cell that produce collagens distinctly different from normal corneal endothelial cells.     

Colchicine reverts cell shape but not collagen phenotypes in corneal endothelial cells modulated by polymorphonuclear leukocytes

The authors have shown previously that polymorphonuclear leukocytes (PMN) modulate rabbit corneal endothelial cells (CEC) into cells that irreversibly acquire the characteristics of fibroblasts, including multilayering of spindle-shaped cells and deposition of interstitial extracellular matrix composed predominantly of type I collagen. In an attempt to determine if the changes in cell shape caused by the disruption of cytoskeleton are correlated with the alteration of collagen phenotypes in fibroblastic corneal endothelial cells (FCEC), colchicine and cytochalasin B (CB) were used. A series of dose-response studies were performed, and correlated with exposure time. When cells were exposed to the drugs (ranging from 0.01-4.0 micrograms/ml) 24 hr after plating, the majority of cells treated with colchicine dramatically changed from fibroblastic to polygonal shape: cells became flattened and cytoplasmic processes disappeared. Conversely, no apparent changes were observed in the CB-treated cells. On removal of colchicine, the cells resumed fibroblastic morphology within 24 hr; most of the cells again developed cytoplasmic processes. When collagen phenotypes were analyzed by electrophoresis, types I, III, and V collagen were present in either the colchicine or CB-treated cells, regardless of the concentration of drug used. However, synthesis of type I trimer and type III collagen was significantly increased in the cells treated with colchicine at concentrations greater than or equal to 1.0 microgram/ml; the alpha 1:alpha 2 ratio was approximately 4.5, and type III accounted for 35-40% of the total collagen. CB did not induce a similar alteration. These observations indicate that changes in cell shape are not related to the switch of collagen phenotypes in FCEC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Stability of collagen phenotype in morphologically modulated rabbit corneal endothelial cells

Primary monolayer cultures of rabbit corneal endothelial cells maintained characteristic polygonal morphology and synthesized type IV collagen as a major collagenous peptide. Upon serial passage, [3H]-proline incorporation into proteins gradually decreased from the secondary subculture to a low of 71% of that in the primary culture in the fourth subculture, while type IV collagen synthesis significantly decreased from the tertiary subculture, and the fourth subculture retained only 22% of the primary culture value. When collagen molecules synthesized by the fourth subculture progeny were analyzed by SDS electrophoresis, no alteration was noted. The fourth subculture progeny continued to synthesize the characteristic type IV collagen, which migrates slightly faster than the beta 12 (I) band on SDS electrophoresis. However, the cellular morphology was changed dramatically from the characteristic polygonal shape to enlarged cells in the fourth subculture and mitotic activity was no longer apparent. Immunofluorescence studies showed that the enlarged fourth subculture progeny stained with anti-IV collagen antibodies, as did the primary polygonal endothelial cells. These findings confirmed type IV collagen synthesis in the enlarged endothelial cells. In addition, the morphologically altered cells continuously deposited Descemet's membrane-like extracellular matrices between the basal cell layer and the plastic substratum. These results suggest that endothelial cells lose their proliferative capacity as they age, but compensate for the loss of cell numbers by enlargement while maintaining a normal collagen phenotype.     

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.     

Localization of collagen types I and IV mRNAs in human optic nerve head by in situ hybridization

Using in situ hybridization, individual cells expressing mRNAs for collagen types I and IV were localized in fixed-tissue sections of adult and fetal human optic nerve heads. Astroglial cells lining the cribriform plates and cells inside the cribriform plates of the lamina cribrosa had mRNA for collagen type IV. Cells in the glial columns, pial septa, and vascular wall also contained mRNA collagen type IV. Collagen type I mRNA was expressed by cells of the cribriform plates of the lamina cribrosa of adults. Few cells in the glial columns, pial septa, and blood vessels had mRNA for collagen type I. Scleral fibroblasts contained mRNA for collagen type I. These results indicated that the expression of mRNA for both collagen types I and IV paralleled the localization of these extracellular matrix proteins in the optic nerve head and suggested that both collagen types were synthesized in this tissue throughout life.     

Translation of type IV procollagen messenger RNA from cultured cat retinal pigment epithelial cells

The activity of type IV procollagen mRNA was detected in the total cytoplasmic RNA prepared from normal feline retinal pigment epithelial cells in culture. The translation products contained two distinct bands in the pro alpha chain region on SDS-PAGE, which were sensitive to collagenase digestion. Corresponding bands were identified in the immunoprecipitate of the translation products after reaction with an anti-type IV collagen antiserum and separation by protein A-Sepharose column chromatography.     

Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix.

PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.     

Effects of intraocular irrigating solutions on the spreading of rabbit corneal endothelial cells on extracellular matrices

The effects of four commercially available irrigating solutions on the spreading of rabbit corneal endothelial cells on various extracellular matrices were studied. Cultured rabbit corneal endothelial cells, suspended in one of the following intraocular irrigating solutions, Opeguard MA, BSS, BSS Plus, lactated Ringer solution (Lactec) or physiological saline, were placed on uncoated tissue culture plates or on plates coated with extracellular matrices (fibronectin, laminin, collagen type I, or collagen type IV). The cell area was measured after 45 minutes' incubation. The cells spread on all of the extracellular matrices examined but not on the uncoated tissue culture plates. On the fibronectin or laminin matrix, the cell area was significantly greater with Opeguard MA or BSS Plus. On laminin and collagen type IV, the cell area was the greatest with Opeguard MA. On collagen type I, the cell area was significantly greater with Opeguard MA, BSS, or BSS Plus. These results demonstrated that the rabbit corneal endothelial cells responded to the extracellular matrices, and that Opeguard MA or BSS Plus provided more favorable conditions for the spreading of these cells. These results indicated that both Opeguard MA and BSS Plus might aid the spreading of corneal endothelial cells during wound-healing immediately after intraocular surgery.     

Combined effect of extracellular matrices and growth factors on bovine corneal endothelial cells in culture

PURPOSE: First, to confirm that corneal endothelial cells in the confluent state have the capability to form cellular covering. Second, to establish a method to study the combined effect of extracellular matrices (ECMs) and growth factors on the biological response in corneal endothelial cells in culture. METHODS: Bovine corneal endothelial cells were cultured inside a cylinder set on a plastic dish. They formed a confluent cell nest on the dish coated with type I or type IV collagen, laminin, or fibronectin. After the removal of the cylinder, hepatocyte growth factor (HGF), epidermal growth factor, transforming growth factor-alpha or transforming growth factor-beta(1) was added to the cultures. Each confluent cell nest enlarged outward, and its increased area size was measured. Cellular response in the nest, including cellular proliferation, was analyzed. RESULTS: The size of the increased area of the culture on type IV collagen plus HGF was the largest of all the combinations of ECMs and growth factors. The responses of component cells in the increased area consisted of cellular hypertrophy, proliferation, migration and giant cell formation. The treatment with type IV collagen plus HGF clearly promoted all the above responses. CONCLUSIONS: The biological response of corneal endothelial cells was regulated by ECMs and growth factors.     

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.     

Iris vasculopathy in exfoliation syndrome. An immunocytochemical study.

Samples of control iris tissue obtained from seven enucleated eyes and nine exfoliative iridectomy specimens were prepared for an immunocytochemical study of the matrix in the walls of iris vessels. The distribution of collagen types I, IV and laminin was studied in normal and exfoliative vessels. Laminin was an integral component of exfoliation material and was present mainly in the matrix of the outer surface of normal vascular cohort cells. The laminin content in the latter location was reduced in vessels in which exfoliation aggregates were not visible. Collagen type IV was absent from exfoliation material. While type I was present in the deposits, it was considered to represent a residue of native normal tissue. Exfoliation deposits appeared to stimulate the synthesis of collagen types I and IV at an early stage of the disease.     

The extracellular matrix composition of the monkey optic nerve head

The presence and distribution of laminin, heparan sulfate proteoglycan and collagen types I, III and IV were immunohistologically determined in cynomolgus monkey optic nerve heads using the avidin-biotin-peroxidase complex technique. Collagen types I and III were detected within the collagenous plates of the scleral lamina cribrosa, in the septa and pia mater of the postlaminar optic nerve and in blood vessel walls in all regions of the optic nerve head. Collagen type IV, laminin and heparan sulfate proteoglycans were all localized to the margins of the collagenous laminar plates of the scleral lamina cribrosa and along the margins of the optic nerve septa and the pia mater. All three components also appeared beneath the blood vessel endothelium throughout the optic nerve head. Within the lamina cribrosa, collagen types I and III occupy the core of the scleral laminar plates and may provide structural support for optic nerve bundles exiting the eye. The distribution of collagen type IV, laminin and heparan sulfate proteoglycan corresponds to basement membranes from two sources: vascular endothelial cells and glial cells lining the axonal bundles. Abnormalities of these substances may influence optic nerve function and susceptibility to elevated intraocular pressure by altering their mechanical support functions within the nerve head, by interfering with axonal nutrition, or both.