Biomedical subjects

W F Patton

Publications and source records attributed to W F Patton.

At least 37 records · Page 2Linked to original sources

PubMed1998

Metabolites of the phospholipase D pathway regulate H2O2-induced filamin redistribution in endothelial cells.

Hypoxia/reoxygenation injury to cultured endothelial cells results in cytoskeletal rearrangement and second messenger activation related to increased monolayer junctional permeability. Cytoskeletal rearrangement by reactive oxygen species may be related to specific activation of the phospholipase D (PLD) pathway. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) or metabolites of the PLD pathway for 1-60 min. Changes in cAMP levels, Ca2+ levels, PIP2 production, filamin distribution, and intercellular gap formation are then quantitated. H2O2-induced filamin translocation from the membrane to the cytosol occurs after 1-min H2O2 treatment, while intercellular gap formation significantly increases after 15 min. H2O2 and phosphatidic acid exposure rapidly decrease intracellular cAMP levels, while increasing PIP2 levels in a Ca2+-independent manner. H2O2-induced cAMP decreases are prevented by inhibiting phospholipase D. H2O2-induced cytoskeletal changes are prevented by inhibiting phospholipase D, phosphatidylinositol-4-phosphate kinase, phosphoinositide turnover, or by adding a synthetic peptide that binds PIP2. These data indicate that metabolites produced downstream of H2O2-induced PLD activation may mediate filamin redistribution and F-actin rearrangement.

Calcium↗

PubMed1998

Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes.

Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.

Amino Acid Sequence↗

PubMed1998

Platelet-derived lysophosphatidic acid decreases endothelial permeability in vitro.

We previously reported that platelets release a soluble factor that decreases the solute permeability of cultured bovine aortic endothelial monolayers. This factor was characterized as heat stable, tryspsin sensitive, and not serotonin, adenosine, ADP, or ATP [F. R. Haselton and J. S. Alexander. Am. J. Physiol. 263 (Lung Cell Mol. Physiol. 7): L670-L678, 1992]. We now report its identity as lysophosphatidic acid (LPA). Endothelial permeability decreases rapidly, reversibly, and repeatedly when exposed to platelet supernatants. Continuous exposure produces a sustained decrease in permeability. Methanol extracts of platelet supernatants also decrease endothelial permeability. Treatment of methanol extracts of platelet supernatants with phospholipase B or alkaline phosphatase, which modify the structure of LPA, abolishes the permeability-decreasing activity. However, activity is unaffected by treatment with phospholipase A2. This pattern of enzyme inactivation is consistent with the structure of LPA. Furthermore, synthetic 1-oleoyl-LPA rapidly and significantly decreases endothelial permeability in a concentration-dependent manner. Platelet activation does not appear to be required to produce activity in supernatants from platelet isolations, since P-selectin expression is not increased and thromboxane B2 is < 14 pg/6,000 platelets. Our data show that platelets release a methanol-extractable compound with an enzyme degradation profile consistent with LPA, which decreases the permeability of endothelial monolayers in vitro. In vivo, LPA derived from platelets may be an important mediator of the transport barrier formed by the vascular endothelium.

Alkaline Phosphatase↗

PubMed1998

Selective propagation of retinal pericytes in mixed microvascular cell cultures using L-leucine-methyl ester.

Endothelial cell (EC) propagation has been simplified by developing cell-specific selection criteria. Methods commonly used for selectively isolating EC include: (i) differential sieving of disaggregated tissue, (ii) differential plating of cells on extracellular matrices, (iii) lectin affinity isolation of cell populations and (iv) fluorescence-activated cell sorting of cells labeled with a carbocyanine dye of acetylated low-density lipoprotein (DiI-Ac-LDL). Few criteria for selectively propagating pericytes (PC) are currently available. Nonspecific esterases exhibit a high degree of multiplicity when compared with other mammalian isozymes and may be suitable for the identification and selective propagation of cells of the microvasculature. Evaluation of esterase isotype expression in PC and EC by zymography indicates PC contain alpha-naphthyl acetate and alpha-naphthyl butyrate hydrolyzing esterases as well as dipeptidyl peptidase I, while EC only contain alpha-naphthyl acetate esterase. The cytotoxic response of PC and EC to various amino acid esters is assessed by monitoring vital dye uptake and by light microscopy. Several amino acid esters are cytotoxic to both cell types, whereas 50 mM L-leucine methyl ester (L-Leu OMe) is toxic to EC but not to PC. This amino acid ester is also toxic to mesothelial and retinal pigmented epithelial cells, other common contaminants of PC cultures. Analysis of protein composition by two-dimensional gel electrophoresis indicates that L-Leu OMe does not stimulate expression of stress response proteins in PC. Thus, L-Leu OMe can be utilized to cultivate PC selectively from mixed cell populations.

Amino Acids↗

PubMed1997

A luminescent europium complex for the sensitive detection of proteins and nucleic acids immobilized on membrane supports.

Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. Detecting the absorbance of colorimetric stains is limited by the molar extinction coefficient of the product, however. Development of light-emitting complexes should improve detection sensitivity, but fluorescent labels described to date modify free amino, carboxyl, or sulfhydryl groups often rendering proteins unsuitable for further analysis. Bathophenanthroline disulfonate (BPSA) forms a luminescent europium (Eu) complex that reversibly binds to proteins and nucleic acids. Analysis of charge-fractionated carrier ampholytes and synthetic polymers of different L-amino acids indicates that protein binding is chiefly through protonated alpha- and epsilon-amino side chains. Proteins or nucleic acids immobilized to a nitrocellulose or polyvinyl difluoride membrane by electroblotting, dot-blotting, or vacuum slot-blotting are incubated with the lanthanide complex at acidic pH. Membranes are rinsed, illuminated with UV light and the phosphorescence of BPSA-Eu is measured at 590 to 615 nm using a CCD camera or spectrofluorimeter. The linear dynamic range of the stain is 476- and 48-fold for protein and DNA, respectively. A strong chelating agent such as ethylenediaminetetraacetic acid combined with a shift to basic pH (PH 8-10) elutes BPSA-Eu from the membrane. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, lectin staining, and mass spectrometry.

Animals↗

PubMed1997

A novel anti-inflammatory peptide inhibits endothelial cell cytoskeletal rearrangement, nitric oxide synthase translocation, and paracellular permeability increases.

The endothelial cell (EC) membrane-cytoskeletal interface in part maintains plasma membrane integrity and promotes cell-cell apposition. Nonmuscle filamin (ABP-280), an actin crosslinking protein, promotes orthogonal branching of F-actin and is the major protein that links the peripheral actin network to the plasma membrane through its C-terminal glycoprotein binding site. In response to bradykinin, filamin translocates from the cell periphery to the cytosol within 1 min. A synthetic peptide, corresponding to filamin's C-terminal calcium/calmodulin-dependent protein kinase II phosphorylation site (CaM peptide), prevents calcium-activated filamin translocation in permeabilized bovine pulmonary artery EC. The myristoylated permeable form of this peptide inhibits bradykinin-induced filamin translocation and F-actin rearrangement in cultured intact ECs. In addition, bradykinin-induced paracellular gap formation is significantly attenuated by CaM peptide, which suggests that the presence of a filamin-based peripheral F-actin network is essential for maintaining EC barrier function. Moreover, CaM peptide reduces wound-induced EC migration rate by 40%, which indicates that F-actin rearrangement is required for efficient cell motility. The CaM peptide affects other bradykinin-induced inflammatory responses. EC nitric oxide synthase (eNOS) translocates from the cell membrane to the nuclear fraction within 1-2 min of bradykinin treatment. Pretreatment with CaM peptide inhibits eNOS translocation. However, the peptide has no effect on bradykinin-induced von Willebrand Factor release. In summary, the CaM peptide exhibits several anti-inflammatory properties that include maintaining EC junctional stability and inhibiting eNOS translocation.

Actins↗

PubMed1997

H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway.

Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) for 1-60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H2O2. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H2O2 treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H2O2-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway.

Adenylyl Cyclases↗

PubMed1997

Human omental microvascular endothelial and mesothelial cells: characterization of two distinct mesodermally derived epithelial cells.

Human omental microvascular endothelial (HOME) and mesothelial (MESO) cells share many phenotypic properties, but can be characterized from one another based upon a comprehensive panel of endothelial and mesothelial markers. Traditional cell markers such as von-Willebrand factor, DiI-Ac-LDL, and Ulex europaeus I lectin are not sufficient to distinguish between HOME and MESO cells. Furthermore, immunoreactivity to a panel of endothelial cell-specific monoclonal antibodies, including representatives from the known clusters of differentiation (CD), indicate that some of these antigens are coexpressed in HOME and MESO cells. In distinguishing between the two cell types, HOME and not MESO cells express E-selectin, E/P-selectin, P-selectin (CD62), Le-y, and VLA-6 (CDw49f*). Moreover, HOME cells and not MESO cells form tube-like structures when cultured on Matrigel. MESO cells differ from HOME cells based upon (1) the expression of cytokeratins; (2) their rapid proliferation in response to platelet-derived growth factor; and (3) a change from an epitheliod to fibroblast-like morphology in response to tumor necrosis factor and epidermal growth factor. Both HOME and MESO cells express tissue plasminogen activator and plasminogen activator inhibitor, but urokinase activity is only expressed by MESO cells. As there is no one universal endothelial or mesothelial cell marker that can specifically confirm the identity of these cells, it appears necessary to employ a comprehensive panel of cell markers to rule out the possibility of misidentifying a cell culture.

Antibodies, Monoclonal↗

PubMed1997

Two-stage isolation procedure for obtaining homogenous populations of microvascular endothelial and mesothelial cells from human omentum.

The human omentum is a highly vascularized tissue often advocated as a source of human microvascular endothelial (HOME) cells. The omentum also contains mesothelial (MESO) cells and isolation protocols published to date do not describe a separation of the two cell populations. Using a two-stage collagenase digestion procedure, homogenous populations of HOME and MESO cells are obtained from the same omental tissue sample. HOME and MESO cells are both simple squamous epithelial cells and consequently are often difficult to discriminate between based on morphology and reactivity with many of the conventional endothelial and mesothelial cell markers. Both HOME and MESO cells form typical cobblestone, contact-inhibited monolayers, metabolize DiI-Ac-LDL, and are immunoreactive to von Willebrand Factor and Ulex europeaus I lectin. However, MESO cells are distinguishable from HOME cells based upon their expression of cytokeratins. Moreover, HOME cells and not MESO cells form capillary-like structures when cultured on Matrigel. It appears that HOME and MESO cells share many phenotypic properties, but are distinguishable from one another based upon a comprehensive panel of endothelial and mesothelial markers. Both cell types should be useful for studying the biology and pathology of the human microvasculature in vitro.

Cells, Cultured↗

PubMed1997

Filamin redistribution in an endothelial cell reoxygenation injury model.

Ischemia-reperfusion injury increases vascular permeability in part by generating reactive oxygen species that disassemble the endothelial cell actin dense peripheral band. This is followed by an increase in the number and diameter of intercellular gaps. Millimolar concentrations of reactive oxygen metabolites lead to nonspecific endothelial cell injury, but micromolar concentrations activate inflammatory second messenger cascades which produce distributional changes in endothelial cell cytoskeletal proteins. H2O2 (100 microM) causes translocation of filamin, from the membrane to the cytosol within 1 min. Subsequently, gap formation occurs within 10-25 min, which is attributed to rearrangement of the dense peripheral band of F-actin. Plasma membrane blebbing occurs after 90 min and decreases in mitochondrial activity occur after 1-2 h. Deferoxamine (iron chelator) and TEMPO (nonspecific free radical scavenger) inhibit these changes. H2O2 (100-1000 microM) does not increase endothelial cell intracellular Ca2+ through 30 min and pretreating cells with a Ca2+-calmodulin kinase inhibitor or an intracellular Ca2+ chelator does not prevent filamin translocation. Filamin redistribution and actin rearrangement are early events in H2O2-mediated endothelial cell injury that appear to occur through Ca2+-independent pathways.

Antioxidants↗

PubMed1997

Activation of endothelial cell kinin receptors leads to intracellular calcium increases and filamin translocation: regulation by protein kinase C.

Membrane-associated cytoskeletal proteins provide support for endothelial cell (EC) junctional cell adhesion molecules. Nonmuscle filamin is a dimeric actin cross-linking protein that interacts with F-actin and membrane glycoproteins. Both bradykinin and des-Arg9-bradykinin cause filamin redistribution from the plasma membrane to the cytosol of confluent EC. Kinin-induced filamin translocation parallels the dynamics of intracellular Ca2+ increases. Pretreatment with kinin receptor antagonists blocks the Ca2+ response as well as filamin translocation induced by kinins. Protein kinase C activation prior to kinin stimulation attenuates intracellular Ca2+ increases and filamin translocation. BAPTA, a cell-permeable Ca2+ chelator, attenuates bradykinin-induced intracellular Ca2+ increases and filamin translocation. This study demonstrates that bovine pulmonary artery ECs express both kinin B1 and B2 receptors, and that activation of either receptor leads to intracellular Ca2+ increases. This Ca2+ signalling, which is downregulated by protein kinase C activation, is essential for kinin-induced filamin translocation.

Animals↗

PubMed1996

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases.

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.

Animals↗

PubMed1996

Pyrogallol red-molybdate: a reversible, metal chelate stain for detection of proteins immobilized on membrane supports.

Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. The metal chelates form protein-dye complexes in the presence of metal ions at acidic pH but are eluted from the proteins by immersing membranes in a solution of basic pH that contains other chelating agents. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, N-terminal and internal protein sequencing. Among the metal complexes evaluated to date, the triazine dye-ferrous complexes (ferene S, ferrozine) and the ferrocyanide-ferric complexes provide the most sensitive detection of proteins immobilized on membranes. While the pyrogallol red-molybdate complex is commonly used in solution-based total protein assays, its utility as a reversible stain for proteins immobilized on membranes has not been reported. Pyrogallol red-molybdate complexes readily stain proteins on nitrocellulose and polyvinyl difluoride membranes with similar sensitivity as ferrozine-ferrous complexes. Analysis of charge-fractionated carrier ampholytes and synthetic polymers of different L-amino acids indicate that binding is prominently via protonated alpha and epsilon-amino side chains. Carbamylation of amino groups in bovine serum albumin substantially diminishes pyrogallol red-molybdate binding to the protein. The stain is reversible, resistant to chemical interference, and compatible with immunoblotting.

Chelating Agents↗

PubMed1996

Solid-phase metal chelate assay for quantifying total protein: resistance to chemical interference.

Recently, we developed reversible metal chelate stains that are fully compatible with immunoblotting and protein sequencing. Membrane supports are incubated in Ferrozine/ferrous complex followed by ferrocyanide/ferric complex (double-metal chelate [DMC] stain). Proteins are quantified by computerized densitometry. In this study, the metal chelate stains are used for routine protein quantitation. Manually applying samples to membranes leads to variable spot spreading. Better results are achieved using a slot-blot apparatus to maintain a constant application area. The Ferrozine/ferrous and DMC assays are compared to colloidal gold and bicinchroninic acid (BCA) assays with respect to chemical interference, protein-to-protein variation, dynamic linear range and sensitivity. The DMC assay provides a superior linear range (100-fold range) and BCA assays (47-fold). Though the colloidal gold assay is more sensitive, it suffers from poor reproducibility, high protein-to-protein variation and lower tolerance to interfering agents. The BCA assay has the least protein-to-protein variation but is also least sensitive and most susceptible to interfering agents.

Chelating Agents↗

PubMed1995

Components of the protein synthesis and folding machinery are induced in vascular smooth muscle cells by hypertrophic and hyperplastic agents. Identification by comparative protein phenotyping and microsequencing.

Vascular smooth muscle cells (VSMC) are the principal cellular component of the blood vessel wall. Atherosclerosis, hypertension, and angiogenesis are associated with abnormal VSMC growth. Angiotensin II is hypertrophic for cultured adult rat aortic VSMC, whereas platelet-derived growth factor and serum are hyperplastic. To identify changes in specific proteins associated with either hyperplastic or hypertrophic growth, high resolution two-dimensional gel electrophoresis was performed on extracts from quiescent rat aortic VSMC and from VSMC exposed for 24 h to growth factors (10% fetal calf serum, platelet-derived growth factor, or angiotensin II). 12 proteins were up-regulated and 5 down-regulated by treatment with growth factors. Eight of the up-regulated and one of the down-regulated proteins were identified by internal protein microsequencing from electroblotted two-dimensional gels or by co-electrophoresis of purified proteins in two-dimensional gels. Four of the proteins up-regulated by growth factors were identified as mediators of protein folding. These were heat shock proteins, HSP-60 and HSP-70, protein disulfide isomerase, and protein disulfide isomerase isozyme Q-2. Additional proteins were identified as elongation factor EF-1 beta, a component of the protein synthesis apparatus, and calreticulin, another putative molecular chaperone. Vimentin and actin were also up-regulated, whereas an isoform of myosin heavy chain was down-regulated. Hyperplastic and hypertrophic growth were accompanied by similar changes in protein expression, suggesting that both types of growth require up-regulation of the protein synthesis and folding machinery.

Amino Acid Sequence↗

PubMed1994

Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting.

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.

Amino Acid Sequence↗

PubMed1994

A glutathione S-transferase (GST) isozyme from broccoli with significant sequence homology to the mammalian theta-class of GSTs.

A novel glutathione S-transferase (GST) was purified from broccoli (Brassica oleracea var. italica). Partial amino-acid sequencing indicated that the protein shared significant homology with several different plant GSTs from maize, silene, Dianthus, Nicotiana and Triticum, but little homology to yeast (Issatchenkia) GST. One region of the polypeptide near the N-terminal also shared significant homology to a region of rat 5-5, rat 12-12 and human theta-GST (collectively referred to as the theta-GST-class) but little structural homology to the common mammalian cytosolic GSTs (alpha-, mu- or pi-classes). The broccoli GST was retained on a novel membrane based glutathione affinity matrix and displayed activity towards 1-chloro-2,4-dinitro-benzene (CDNB), a general GST substrate, as well as 4-nitrophenethyl bromide, a marker substrate for the theta-class of GSTs. The characteristics of the broccoli GST potentially define it as a member of the theta-class. This is consistent with the view that the theta-class may have arisen prior to the divergence of animals and plants while the mammalian mu-, pi- and alpha-classes evolved after the two kingdoms were established.

Amino Acid Sequence↗

PubMed1994

Cytokeratin-positive and cytokeratin-negative cultured endothelial cells from bovine aorta and vena cava.

The heterogeneous morphology of microvascular endothelial cells obtained from the bovine corpus luteum was recently attributed to the occurrence of cytokeratin (CK) positive and CK negative endothelial cells. The aim of the present study was to establish comparable differences for bovine macrovascular endothelial cells. For this reason, endothelial cells were scraped from the abdominal aorta as well as the inferior vena cava of cows. At the level of phase contrast microscopy, primary cultures originating from both large vessels could be classified as CK positive or CK negative endothelial cells. After seeding CK positive endothelial cells on Matrigel matrix, a two-dimensional meshwork of so called pseudotubules formed within 2 h. By using immunofluorescence localization CK positive cells were identified by a complex meshwork. It consisted of CK 8, 18 and 19 as displayed by Western blots. The CK negative group showed spindle-shaped or polygonal endothelial cells according to light microscopy. In postconfluent cultures, spindle-shaped cells developed a three-dimensional meshwork of tubules. After seeding spindle-shaped cells on Vitrogen 100 matrix, pseudotubules formed within 1 day. In considering the frequency of occurrence, primary harvests from the vena cava contained less than 1% CK positive cells. With respect to growth, the cell number was two to three times higher for the CK negative group than the CK positive group as judged on day 13 after cell seeding. It is concluded that subpopulations of endothelial cells are derived from large blood vessels.

Animals↗