Biomedical subjects

N Chung-Welch

Publications and source records attributed to N Chung-Welch.

10 recordsLinked to original sources

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

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

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↗

PubMed1993

Ambient oxygen tension modulates endothelial fibrinolysis.

PURPOSE: Vascular procedures reoxygenate ischemic endothelial cells (EC) and arterialize saphenous vein (HSV) EC. The balance between the EC-derived fibrinolytic components, plasminogen activator (tPA), and plasminogen inhibitor (PAI-1) contributes to maintaining thromboresistance. This balance also affects proteolysis through plasmin generation, mediating matrix metabolism endothelial migration, angiogenesis, and theoretically affecting the development of intimal hyperplasia. METHODS: To explore the impact of varying oxygen tensions on EC fibrinolysis, HSV and human umbilical vein (HUV) were subjected to Po2 of 40 mm Hg for 24 hours with restoration of Po2 to 150 mm Hg for 24 hours. The tPA and PAI-1 antigen and tPA/PAI-1 antigen ratio in conditioned media (CM), expressed as increases or decreases % change, normalized for cell count, versus controls, were analyzed by enzyme-linked immunosorbent assay. Cellular tPA and PAI-1 mRNAs were assessed by Northern analysis. RESULTS: The tPA but not PAI-1 was significantly decreased after the first 24 hours in HSVEC and significantly decreased after 48 hours in both HUVEC and HSVEC when compared with controls. Messenger RNA for tPA was unchanged but PAI-1 mRNA increased significantly for HSVEC and HUVEC after 24 hours of Po2 of 40 mm Hg, returning to baseline within 24 hours of Po2 to 150 mm Hg restoration. CONCLUSIONS: These data support the hypothesis of a fibrinolytic shift after altered ambient O2 tensions exposure in endothelium and demonstrate that HSVEC are more sensitive to altered O2 tension than HUVEC. Altered O2 tensions depress EC fibrinolysis in this model.

Atmospheric Pressure↗

PubMed1991

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.

Adipose Tissue↗

PubMed1991

Tris-tricine and Tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system.

Human mesothelial cells contain a number of well defined intermediate filament proteins (IFs) that have been completely sequenced including vimentin and the cytokeratins (K7, K8, K18, and K19). The electrophoretic migration of these IFs was monitored as a function of second dimension gel buffer composition using various systems including Tris-glycine (pH 8.3 or 9.2), Tris-glycine with 20% methanol, Tris-borate, Tris-tricine, and sodium phosphate. All of the second dimension buffer chemistries yielded patterns of sufficient resolution to identify the major cytoskeletal proteins but differed in the relative mobilities of the IFs. Using gene sequence calculated molecular weight data, the major cytoskeletal polypeptides of human mesothelial cells were ranked from highest molecular weight to lowest molecular weight. This rank order of sequence calculated molecular weights was then compared to the rank order determined form the actual migration of the polypeptides in the different gel systems. With the Tris-tricine and the Tris-borate gel systems as well as gene sequence data, KS = vimentin greater than beta-tubulin = K7 greater than K18 greater than K19 greater than actin. With the pH 8.3 and 9.2 Tris-glycine systems, as well as the sodium phosphate gel system, the rank order of the polypeptides did not correspond to gene sequence data. Adding 20% methanol to the Tris-glycine system resulted in IF migration that more closely corresponded to the gene sequence derived data. Migration position of the IFs depended upon the temperature of the second dimension separation as well. In mesothelial cells, the migration of a total of 15-25% of the polypeptides was influenced by differing buffer systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Boric Acids↗

PubMed1990

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.

Animals↗

PubMed1989

Phenotypic comparison between mesothelial and microvascular endothelial cell lineages using conventional endothelial cell markers, cytoskeletal protein markers and in vitro assays of angiogenic potential.

Endothelial and mesothelial cells are mesodermally derived simple squamous epithelial cells. A controversy concerning the ontogenetic origin of neoplasms derived from these cell types, commonly cited in the literature, is whether Kaposi's sarcoma is a mesothelioma or an angioma. To assess the similarities and differences between these cell types, pulmonary microvascular endothelial cells (PMVEC) and pericardial mesothelial cells (PMC) were cultured in vitro. PMVEC and PMC were found to be difficult to distinguish from one another by histological criteria alone. Both cell types formed contact-inhibited, and 'cobblestone', monolayers typical of simple epithelial cells. PMVEC and PMC demonstrated positive immunoreactivity to Factor VIII-related antigen and angiotensin-converting enzyme (ACE) antigen. They also showed uptake of 1,1'-dioctacecyl-1,3,3,3',3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) in 4 h. Both PMVEC and PMC expressed low ACE activities when compared to macrovessel endothelial cells. PMVEC and PMC shared similar isoform profiles for vimentin and actin. Both cell types expressed the simple epithelial keratins, cytokeratins 8 and 19, though PMC contained 50% more cytokeratins than PMVEC. Additionally, PMC contained cytokeratin 18, an intermediate filament protein not detectable in PMVEC. PMC formed 15 times as many epithelial ringlets or "stomata" as PMVEC. PMVEC but not PMC could be induced in vitro to differentiate into branching tube-like structures in response to their culture environment. Reorganization of PMVEC into vessel-like structures was more rapid and complete than PMC when embedded in three-dimensional collagen I lattices, cultured on Matrigel or exposed to a shaped-pulsed electromagnetic field. The angiogenic response of PMVEC to specialized culture conditions in vitro may reflect their phenotypic differentiation state characterized by anastomosing vascular structures in vivo, whereas PMC remain differentiated into monolayer sheet-like structures.

Actins↗

PubMed1988

Prostacyclin and prostaglandin E2 secretions by bovine pulmonary microvessel endothelial cells are altered by changes in culture conditions.

The isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established. Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma-derived serum, bovine retinal growth extract (1%), and heparin (90 micrograms/ml) on gelatin coated plates. A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma-derived serum. Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages. MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII-related antigen and uptake of 1,1'-dioctacecyl-1,3,3,3'3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (Dil-Ac-LDL). MVE cells were also positive for angiotensin-converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence. MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 +/- 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGI2) and thromboxane (Tx) A2 (316 +/- 43 and 588 +/- 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay. However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187. In contrast, MVE cells cultured without heparin and growth factor secreted more PGI2 than PGE2 (862 +/- 84 and 89 +/- 12 respectively). Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGI2 and PGE2 production (P less than 0.01). Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGI2. These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.

Animals↗