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Biomedical subjects

G A Truskey

Publications and source records attributed to G A Truskey.

45 records · Page 3Linked to original sources
PubMed1990

The effect of fluid shear stress upon cell adhesion to fibronectin-treated surfaces.

Cell attachment to and spreading upon a surface is mediated by adhesion molecules, such as fibronectin. The role of fibronectin in maintaining cell adhesion was examined by measuring cell attachment following exposure of cells to laminar flow in a parallel-plate flow channel. 3T3 fibroblasts were allowed to adhere to glass slides with or without preadsorbed fibronectin for 2 h before exposure to shear stresses ranging from 5 to 140 dyne/cm2. For cells which adhered to glass surfaces, cell loss was biphasic with a significant loss of cells during the first 2 min of flow, followed by a much slower decline in the number of attached cells with time. Following exposure to shear stresses greater than 5 dyne/cm2, the number of attached cells decreased exponentially as the shear stress increased. The distribution of adhesive stresses among the population of cells was log-normal with a median of 50 dyne/cm2, a mean of 82 dyne/cm2 and a standard deviation of 108 dyne/cm2. After exposure to flow for 2 h, the adhesive stress of the remaining cells decreased to a mean value of 50 dyne/cm2. Cell adhesion after exposure to flow was increased by preadsorbing fibronectin to the glass surface. The initial loss of cells from fibronectin-treated glass following exposure to flow correlated with the degree of cell spreading. Preadsorbed fibronectin resulted in a greater number of bonds between the surface and the cell, which in turn promoted cell spreading and increased the adhesive strength of the cell.

Adsorption↗
PubMed1990

Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence.

Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.

Cell Adhesion↗
PubMed1985

Effects of ammonium ion derived from bovine endothelial cells upon low density lipoprotein degradation in cultured vascular smooth muscle cells.

Low density lipoprotein (LDL) metabolism in bovine arterial smooth muscle cells (SMC) was increased upon exposure to endothelial cell conditioned medium. The mass of LDL degraded in the SMC lysosomal system was increased, and kinetic analysis demonstrated that the rate constant for LDL degradation arising from receptor-mediated endocytosis was unchanged. The effects on LDL metabolism were accompanied by stimulation of DNA synthesis in the SMC. These results are in contrast to reports concerning a porcine endothelial cell system where LDL degradation was inhibited by endothelial-derived NH4+. We show that bovine endothelial cells produce insufficient NH4+ to inhibit LDL degradation and conclude that endothelial cell-derived NH4+ is unlikely to be a factor affecting LDL metabolism in the bovine vascular cell culture system.

Ammonia↗
PubMed1985

Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism.

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.

Animals↗
PubMedDate not supplied

Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis.

By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.

Cell Adhesion↗
PubMedDate not supplied

Altered distribution of mitochondria and actin fibers in 3T3 cells cultured on microcarriers.

The mitochondria and actin fibers of 3T3 fibroblasts cultured on microcarriers in spinner flasks were visualized using fluorescent stains. In contrast to cells grown on planar surfaces under static or steady laminar flow conditions, cells exposed to higher levels of turbulent agitation do not form actin stress fibers. Greater agitation also leads to a more diffuse appearance of the mitochondria and a wider distribution of them throughout the cytoplasm. This response may indicate damaged mitochondria, as similar results have been reported for chemical toxins.

3T3 Cells↗
PubMedDate not supplied

Imaging of cell/substrate contacts on polymers by total internal reflection fluorescence microscopy.

A simplified model of total internal reflection fluorescence (TIRF) emission of fluorescently labeled cell membranes [Reichert, W. M.; Truskey, G. A. J. Cell Sci. 1990, 96, 219-230] was used to determine the topography of the cell membrane in apposition to a polymer-coated surface. The homopolymer substrates were spun cast films of hydrophilic poly(hydroxyethyl methacrylate) (polyHEMA) or hydrophobic poly(ethyl methacrylate) (polyEMA). Bovine aortic endothelial cells (BAEC) on preadsorbed fibronectin polymer substrates were either plated for 24 h, fixed, labeled, and examined by TIRF microscopy (TIRFM) and phase-contrast microscopy or plated for 2 h and tested for their adhesion strength in a parallel-plate flow chamber. BAEC attached to polyHEMA showed no evidence of focal contact formation. However, BAEC attached to polyEMA were well spread and showed an array of focal contacts. TIRFM data were transformed to construct a detailed topographical map of relative cell/substrate separation distances. Virtually all of the BAEC plated to polyHEMA were sheared from the surface when subjected to a 50 dyn/cm2 burst of laminar flow, whereas only 10% of the BAEC were sheared from the polyEMA surface. These data suggest that the polyHEMA and polyEMA surface properties (e.g., hydrophobicity) correlate with the presence of BAEC focal contacts and the BAEC attachment strength.

Animals↗
PubMedDate not supplied

Short-term cell/substrate contact dynamics of subconfluent endothelial cells following exposure to laminar flow.

The manner in which fluid stresses are transmitted from the apical to the basal surface of the endothelium will influence the dynamics of cell/substrate contacts. Such dynamics could be important in the design of synthetic vascular grafts to promote endothelial cell adhesion. To examine whether the initial response of cell/substrate contact sites to flow depends on the magnitude of the applied shear stress, subconfluent monolayers of endothelial cells were exposed to flow at 10, 20, and 30 dyn cm-2 wall shear stresses for 20 min. Cell/substrate contact sites were visualized with total internal reflection fluorescence microscopy. Flow induced a rapid fluctuation in the membrane topography, which was reflected in dynamic changes in cell/substrate contacts. Exposure to flow caused marked changes in contact area. Contact movement occurred normal and parallel to the direction of flow. Contact sites demonstrated significant variability in contact area at 30 dyn cm-2 during the experiment but no significant movement of the contact sites in flow direction after 20 min of flow. Mean square displacements of the contact center of mass were described in terms of a directed diffusion model. Prior to onset of flow, contact movement was random. Flow induced a significant convective component to contact movement for 300-600 s, followed by reestablishment of diffusive growth and movement of contacts. These results suggest that fluid stresses are rapidly transmitted from the apical to the basal surface of the cell via the cytoskeleton.

Animals↗