Biomedical subjects

E P Kay

Publications and source records attributed to E P Kay.

28 records · Page 2Linked to original sources

PubMed1990

Corneal endothelium modulation factor released by polymorphonuclear leukocytes. Partial purification and initial characterization.

Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified corneal endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first-passaged cells contained type IV collagen RNA in an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells.

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PubMed1989

Expression of types I and IV collagen genes in normal and in modulated corneal endothelial cells.

Expression of type I and type IV collagen genes was studied in normal endothelial cells and in modulated endothelial cells (formerly designated as fibroblastic corneal endothelial cells: FCEC). There appear to be distinct collagen phenotypes in these cells; normal cells produce type IV collagen as a major type, while the modulated cells produce predominantly type I collagen. The absence of type I synthesis in normal cells, and absence of type IV collagen in FCEC were confirmed by immunoblot analysis of the accumulated collagen in the cellular and medium layers in cultures of these cells. When collagen mRNAs were translated in a cell-free system, the results indicate that neither type I collagen RNAs in the normal cells, nor type IV collagen RNAs in the modulated cells were translated. Using cloned cDNA probes, the relative quantities of transcripts of these collagens were determined by slot blot hybridization; normal cells, which synthesize no detectable type I collagen, were found to contain type I collagen RNAs in similar amounts as did the modulated cells, while the levels of type IV RNAs in modulated cells were as high as those in normal cells, in spite of the absence of their translational products. Northern blot analysis demonstrated that there are no distinct differences in the size of type I collagen RNAs in the normal and modulated cells. In contrast, type IV collagen RNA in the modulated cells was much smaller than the normal cells, suggesting that the alpha 1(IV) RNA is readily degraded.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1988

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)

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PubMed1987

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)

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PubMed1986

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.

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PubMed1985

Type I collagen synthesis by corneal endothelial cells modulated by polymorphonuclear leukocytes.

When primary corneal endothelial cells were grown in polymorphonuclear leukocyte (PMN)-conditioned medium, a minor population of cells acquired fibroblastic morphology. Such modulated endothelial cells supported by PMN-conditioned medium grew much faster than the major nonresponding polygonal endothelial cell. Upon serial passages, the modulated endothelial cells became the dominant cell type and eventually formed a homogeneous fibroblastic culture. At the same time, phenotypic changes of collagen were observed. The primary endothelial cells grown in PMN-conditioned medium, consisting of responding elongated cells and nonresponding polygonal endothelial cells, produced predominantly type IV collagen with type III collagen as a minor component. As cells were subcultured and fibroblastic cells became dominant, type IV collagen synthesis was dramatically decreased and type I collagen synthesis was increased in parallel. When they reached the fully modulated stage, the cultures synthesized types I and III collagen, with type I accounting for 75-85% of the total. Type I collagen synthesized by the fibroblastic endothelial cells shared common characteristics with known type I collagen, such as migration behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CNBr peptide profiles, and immunologic identity. Thus, PMNs apparently contribute to the modulation of corneal endothelial cells, causing them to acquire characteristics of fibroblasts, cell multilayering, and deposition of interstitial extracellular matrix composed predominantly of interstitial type I collagen.

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PubMed1984

Effect of monensin on secretion of basement membrane collagen by cultured rabbit corneal endothelial cells in comparison with rabbit tendon fibroblasts.

Monensin, a monovalent ionophore, affects differently the secretion of basement membrane collagen and interstitial procollagen. At 1 X 10(-8) M, monensin inhibited only slightly the secretion of procollagen synthesized by tendon cells while secretion of basement membrane collagen synthesized by endothelial cells was inhibited by 43% after 2 hours of labelling. At higher concentration (1 X 10(-6) M), the secretion of type I procollagen by tendon cells was inhibited after two h (36%) or 24 h (24%) of labelling. The pattern of basement membrane collagen secretion varied from inhibition to stimulation depending on the labeling time. No changes in the phenotypic expression of collagen were observed in either the monensin-treated tendon cultures or endothelial cells. Phase-contrast microscopy revealed differential responses of cells to monensin: tendon cells appeared spindle-shaped or rounded-up, in contrast to the enlarged shape displayed by endothelial cells.

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PubMed1984

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.

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PubMed1984

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.

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PubMed1982

Basement membrane collagen synthesis by rabbit corneal endothelial cells in culture. Evidence for an alpha chain derived from a larger biosynthetic precursor.

Corneal endothelial cells in culture synthesize basement membrane collagen and secrete it into the medium. This collagen sediments faster than interstitial collagen by velocity sedimentation and is disulfide-bonded. After reduction, two biochemically distinct chains can be determined by cyanogen bromide peptide mapping. These chains migrate close to each other and immediately below beta 12(I) components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with pepsin gives rise to a major band which still retains interchain disulfide bonds and which will not convert to components with the mobility of interstitial alpha chain by reduction. However, an alpha chain and three minor collagenase-sensitive and pepsin-resistant peptides are generated if the molecule is reduced and alkylated under nondenaturing conditions prior to pepsin treatment. When collagen which accumulates in the media over a long period of time is compared to the newly synthesized molecules, there is an apparent differential resistance to limited pepsin treatment. However, the products which are generated in both cases share electrophoretic identity.

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