The preferred mode of instability was investigated in an axisymmetric air jet of moderate Reynolds number. Natural instabilities are shown to scale with local shear-layer thickness and the preferred mode is shown to be a shear-layer instability. The spatial evolution of the preferred mode was examined by exciting the flow acoustically and then mapping the phase-locked velocity fluctuations. Throughout the potential core region the phase-locked profiles are shown to agree with the eigensolutions of the Orr—Sommerfeld stability equations provided the calculations are based on measured, mean velocity profiles. The excitation intensity was varied from low levels, where the flow was merely tagged, to high levels where the mean flow was substantially distorted, and over that range of excitation there was no apparent deterioration in the agreement with stability predictions.
Many of the segmented polyurethanes currently used in cardiovascular prostheses undergo either modification of their surface structure or are lined with a confluent monolayer of endothelial cells to improve their hemocompatibility. During the establishment of an endothelial cell lining on these biopolymers it is necessary to continually monitor the number of viable cells that are covering the substrate. Yet, not all of the conventional cell enumeration techniques are suitable for assessing the growth of endothelial cells on polyurethanes. Methods, such as direct cell counting, dye uptake, or DNA or protein staining require either a transparent scaffold or lead to termination of the culturing process prior to measurement. In addition, some of the spectroscopic assays are often hampered by interaction of the dyes and/or solubilizers with the various constituents (e.g., catalyzers, antioxidants) and/or functional groups in the polyurethane formulations. In addressing these problems, we adapted a novel, highly reproducible fluorescent assay which is based on reduction by viable cells of an electrochemically sensitive compound, Alamar Blue. The bioreduced product is soluble and stable in culture media and noncytotoxic. In addition, the assay is independent of the geometry or physicochemical properties of the polymeric surfaces. In the present study we focus on the implementation of this assay to monitoring attachment and growth of various endothelial cell types on segmented polyurethanes.
We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the beta-adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G-protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G-proteins, adenylyl cyclase, and phosphodiesterase.
We tested the hypothesis that elevated blood pressure, a known stimulus for vascular remodeling and an independent risk factor for the development of atherosclerotic disease, can modulate basal and cytokine-induced tissue factor (TF; CD 142) expression in cultured human endothelial cells (EC). Using a chromogenic enzymatic assay, we measured basal and tumor necrosis factor-α (TNF-α; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC (HVCEC) cultured at atmospheric pressure and at 170 mmHg imposed pressure for up to 48 h. Basal TF activities were 22 ± 10 U/mg protein for HAEC and 14 ± 9 U/mg protein for HVCEC and were upregulated in both cell types >10-fold by TNF-α. Exposure to pressure for 5 h induced additional elevation of basal TF activity by 47 ± 16% ( P < 0.05, n = 6) for HAEC and 17 ± 5% ( P < 0.05, n = 3) for HVCEC. Pressurization also enhanced TF activity in TNF-α-treated cells from 240 ± 28 to 319 ± 32 U/mg protein in HAEC ( P < 0.05, n = 4) and from 148 ± 25 to 179 ± 0.8 U/mg protein ( P < 0.05, n = 3) in HVCEC. Cytokine stimulation caused an ∼100-fold increase in steady-state TF mRNA levels in HAEC, whereas pressurization did not alter either TF mRNA or cell surface antigen expression, as determined by quantitative RT-PCR methodology and ELISA. Elevated pressure, however, modulated the EC plasma membrane organization and/or permeability as inferred from the increased cellular uptake of the fluorescent amphipathic dye merocyanine 540 (33 ± 7%, P < 0.05). Our data suggest that elevated static pressure modulates the hemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.
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