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Biomedical subjects

S Chien

Publications and source records attributed to S Chien.

At least 109 records · Page 6Linked to original sources
PubMed1993

Increased aortic endothelial death and enhanced transendothelial macromolecular transport in streptozotocin-diabetic rats.

Hypertension, cigarette smoking and diabetes mellitus are well-known risk factors for atherosclerosis and coronary heart disease. Repeated endothelial cell injury and increased lipid entry have been suggested as initiating events in atherogenesis. Our previous studies have demonstrated that the frequency of endothelial cell death and associated endothelial permeability were significantly increased in the aorta of spontaneously hypertensive rats and chronic oral nicotine-treated rats. In the present investigation, we examined the hypothesis that diabetes also increases the frequency of arterial endothelial cell death and hence transendothelial macromolecular transport, which may have some implications in increasing lipid entry and thus accelerating atherogenesis. Diabetes was induced in 15 male Sprague-Dawley rats by intraperitoneal injection of 60 mg streptozotocin per kg body weight. The duration of diabetes was 6 weeks. A group of 15 age-matched rats, injected only with the buffer and maintained over the same time period, served as the controls. In en face preparations of the thoracic aorta, IgG-containing dead endothelial cells were identified by an indirect immunoperoxidase method, and endothelial leakage to Evans blue-albumin complexes was quantified by fluorescence microscopy. Diabetic rats, compared to control rats, had significantly higher values for the frequency of endothelial cell death (0.77 +/- 0.10% vs 0.38 +/- 0.04%; p < 0.005 by two-tailed, unpaired Student's t-test) and the number density of Evans blue-albumin leaky foci (4.33 +/- 0.48/mm2 vs 2.99 +/- 0.38/mm2; p < 0.05 by two-tailed, unpaired t-test) in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Let our voices be heard and amplified. The time is now!

The future health and well-being of all our citizens is at stake. We must make our voices heard, we must amplify their effect by mobilizing our entire society. We are doing this not for ourselves, but for the country, for all our citizens, and for our future generations. As citizens, we are obliged to help our administration and Congress set their agenda. If there was ever a time to voice the need to support biomedical research, it is now. Please give this your highest priority. If we do not take up this responsibility, we will have no one else to blame if medical progress is retarded due to inadequate funding. Rise to the occasion and meet the challenge. Let our voice be amplified and be heard, loudly and clearly. Do not wait; do not postpone even for a moment. NOW.

Budgets↗
PubMed1993

Active motion of polymorphonuclear leukocytes in response to chemoattractant in a micropipette.

A novel experimental method of producing and observing the active motion of polymorphonuclear leukocytes (PMNs) using a micropipette technique has been recently developed (Usami et al., 1992). The present paper develops a quantitative theory for the chemoattractant gradients and cell locomotion observed in these experiments. In previous experimental methods (e.g., the Boyden chamber, the Zygmond chamber and the Dunn chamber) for study chemotaxis of leukocytes, fibroblasts, and PMNs, the exact nature of the concentration gradient of the chemoattractant is unknown. The cells may themselves modify the local gradient of the chemoattractant. In experiments using the micropipette, an internal source of chemoattractant provides well-defined boundary and initial conditions which allow the computation of the chemoattractant concentration gradient during the active locomotion of the PMNs. Since the cell completely fills the pipette lumen, convection is limited to the motion of the cells themselves. In coordinates moving with cell, it is assumed that diffusion is the only mechanism of mass transport of the chemoattractant (fMLP). Computations of the fMLP concentration during locomotion of the cell were carried out for a range of rates of fMLP binding by the receptors expressed on the front face of the cell membrane. The results show that the front face of the cell is subjected to increasing fMLP concentration during the cell motion. The sequence of events involve receptor binding of fMLP, signal transduction, polymerization of the cell cytoskeleton at the membrane of the front face, spatially dependent adhesion to the pipette wall, and localized contraction of the cytoskeleton. This sequence of events leads to the steady locomotion of the leukocytes in the micropipette. The computation of the distribution of the fMLP concentration during cell locomotion with constant velocity in micropipette experiments shows that the cell is exposed to increasing concentration of fMLP. This suggests that chemotaxis maybe induced by temporal gradient of an attractant.

Cell Movement↗
PubMed1993

Lipid transport aspects of atherogenesis.

In this review we shall examine the current understanding of events that lead to the incipient formation of the early foam cell lesion in atherogenesis and its localization. Particular emphasis will be placed on the intimal transport mechanisms that lead to the growth of extracellular lipid liposomes in the intima, since there is now substantial evidence that this growth is the triggering event in the complex sequence of processes that leads to the recruitment of blood borne monocytes into the subendothelial intima and their subsequent conversion to macrophages. The role of the endothelium, intimal proteoglycans and internal elastic lamina (IEL) in modulating the transport of low density lipoproteins (LDL) in the subendothelial space will be analyzed and a new hypothesis for the co-localization of liposome formation, cellular level endothelial leakage and monocyte entry described. The possible modifications of LDL in the liposomes that facilitate the conversion of monocytes into foam cells is summarized. We also discuss the fluid dynamic aspects of intimal transport and the relationship of fluid shear stress to the localization of cellular level endothelial leakage of LDL. The effect of fluid shear on other endothelial cell functions has been recently reviewed in [1].

Animals↗
PubMed1993

Human monocyte colony-stimulating factor stimulates the gene expression of monocyte chemotactic protein-1 and increases the adhesion of monocytes to endothelial monolayers.

The stimulation of the human umbilical vein endothelial cell (HUVEC) with recombinant human monocyte-derived colony-stimulating factor (MCSF) increased the gene expression of monocyte chemotactic protein (MCP-1). Northern blot analysis indicated that 50 U/ml of MCSF is the optimal concentration for this effect. The elevation of MCP-1 mRNA started as early as 1 h after stimulation and was maintained for at least 8 h. An increased MCP-1 level in MCSF-treated HUVEC was also demonstrated at the protein level by immunocytochemical staining using a polyclonal MCP-1-specific antibody. HUVEC activated by 50 U/ml of MCSF for 5 h showed a stronger immunofluorescence staining than control cells. Micropipette separation of THP-1 monocytes from HUVEC showed that the activation of both THP-1 and endothelium by MCSF led to an increase in the separation force by more than three times (36.2 +/- 6.7 x 10(-4) vs. 9.6 +/- 3.6 x 10(-4) dyn). An increased adhesiveness was also observed after MCSF activation of peripheral blood monocytes and HUVEC (16.7 +/- 2.7 x 10(-4) vs. 5.2 +/- 0.9 x 10(-4) dyn). The increased adhesive force in both systems was blocked by the use of anti-MCP-1 (5.5 +/- 0.8 x 10(-4) and 6.8 +/- 1.1 x 10(-4) dyn). Similar results were obtained in experiments in which only HUVEC, but not monocytes, were activated by MCSF. This increased adhesion of untreated monocytes to MCSF-activated HUVEC was also blocked by the addition of anti-MCP-1. In contrast, experiments in which only THP-1 or peripheral blood monocytes, but not HUVEC, were treated with MCSF did not show a significant increase of adhesion between these cells. These results indicate that MCSF augments monocyte-endothelium interaction primarily by its action on the endothelial cell and that this function is probably mediated through an increased expression of MCP-1. The MCSF/MCP-1-dependent adhesive mechanism might be operative in the arterial wall in vivo to lead to the trapping of the infiltrated monocyte-macrophage in the subendothelial space during atherogenesis.

Antibodies↗
PubMed1993

Mechanical models of pseudopod formation.

The active locomotion of polymorphonuclear leukocytes into a glass pipette has been recently reported using formyl-methionyl-leucyl-phenylalanine (fMLP) as a chemoattractant. The frontal portion of the leukocyte appears clear and free of granules as observed in pseudopod formation. Three possible mechanisms for pseudopod formation are considered: (1) pressure-flow generated by actin-myosin contraction at the rear of the cell or at the base of the pseudopod; (2) osmotic pressure generated at the cell membrane, interior to the cell; and (3) actin polymerization of the cell membrane at the leading edge of the pseudopod. Experimental data on the movement of F-actin toward the rear of the cell, away from the front, favors polymerization of G-actin to F-actin at the leading edge. The active role of osmotic pressure and contraction at the base of the pseudopod are possible but not yet proven.

Actins↗
PubMed1992

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Amino Acid Sequence↗
PubMed1992

Deformability measurements on individual sickle cells using a new system with pO2 and temperature control.

Although the rheologic behavior of sickle erythrocytes (SS cells) is highly dependent on oxygen tension (pO2) and temperature, very little data exist regarding the effects of deoxygenation and reoxygenation on the rheology of "individual" SS cells at body temperature. We have devised and assessed a new experiment system, in which micropipette aspiration can be performed on individual cells in a constant-temperature chamber that has ports for changing media with different pO2 (effected in 30 to 120 seconds) and sensing probes for monitoring pO2 and temperature. This system enabled us to simultaneously alter and monitor pO2 at 37 +/- 0.5 degrees C, and to monitor and study a single cell under microscopic observation. The static rigidity (E) and dynamic rigidity (eta) of individual SS cells were determined by repeated aspirations of the same cell under various pO2. With stepwise reductions in pO2, E and eta showed no significant changes before sickling, but once sickled, their values markedly increased by 10(2)- to 10(3)-fold concomitantly with morphologic alteration of the cell. Thus, the deformability of a single SS cell behaves in an "all or none" manner at a critical pO2, and earlier studies on the effect of deoxygenation on the rheology of SS cell suspensions probably reflect the overall behavior of SS cells with widely distributed critical pO2.

Anemia, Sickle Cell↗
PubMed1992

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E1 (PGE1) on platelets is mediated through the PGE1 receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE1 and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 microns. The time course of the extension length (Dp, in microns) of the platelets in response to aspiration with a negative pressure (delta P) of 5 cm H2 O (delta P x Rp = 0.15 dynes/cm) was analyzed. PGE1 treatment (0.1 microM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE1-treated)/Dp/Rp (control), was significantly reduced to 0.90 +/- 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE1-treated platelets had a higher [cAMP]i than controls. Platelets treated with PGE1 or dbcAMP showed a reduced [Ca2+]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE1 and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]i and low [Ca2+]i after PGE1 treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.

Alprostadil↗
PubMed1992

Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein.

Human erythrocyte tropomodulin is a novel tropomyosin regulatory protein that binds to the end of erythrocyte tropomyosin and blocks heat-to-tail association of tropomyosin along actin filaments. It has been proposed to play a role in modulating the association of tropomyosin with the spectrin-actin complex in the erythrocyte membrane skeleton. Immunoscreening of a human fetal liver cDNA expression library in lambda gt11, followed by 5'-end extension by polymerase chain reaction from the same library, yielded a composite cDNA sequence of 2665 base pairs (bp). It contains a 34-bp 5'-untranslated region, a 1.6-kilobase (kb) 3'-untranslated region, and a complete open reading frame of 1077 bp that encodes a protein of 359 amino acids with a calculated molecular mass of 40.6 kDa and a pI of 4.8. Authenticity of the tropomodulin cDNA was confirmed by a complete sequence match of 49 predicted amino acids with the sequences of three tryptic peptides of the erythrocyte tropomodulin. The sequence has no internal repeats and no significant homology with any known proteins. Secondary structure predictions indicate that tropomodulin may consist of a series of seven or eight short alpha-helical segments and fold into a somewhat compact shape. The tropomyosin binding activity has been mapped to an N-terminal region containing residues 39-138. Nine independent PCR clones, five from a human reticulocyte cDNA library and four from the fetal liver cDNA library, revealed identical N-terminal 103 amino acids, suggesting that the sequence reported here may also be of erythrocyte tropomodulin. Northern analysis of human reticulocyte RNA showed two hybridizing bands of 2.7 and 1.6 kb, indicating that the 2665-bp cDNA sequence reported here was that of the longer transcript.

Amino Acid Sequence↗
PubMed1992

The Benjamin W. Zweifach Award Lecture. Blood cell deformability and interactions: from molecules to micromechanics and microcirculation.

UNLABELLED: RBC deformability and PMN-endothelial interaction have been used as two examples to illustrate how recent investigations have generated information from molecules to micromechanics and the microcirculation, and how some of the results can be used to understand the physiology and pathophysiology in man. While this presentation on the molecular basis of microcirculatory events is focused on micromechanics, active work is being conducted to establish the molecular basis of many other microcirculatory processes. These include endothelial transport, vascular smooth muscle activity, neurohumoral control of the microcirculation, and angiogenesis, as well as some of the disease states such as ischemia, shock, and cancer. The field is still in its infancy, and we are only seeing the tip of the iceberg. Further developments in this fertile interdisciplinary field will allow us to gain further understanding of the molecular basis of the microcirculatory processes in health and disease. IN CONCLUSION: (i) The deformability and interactions of blood cells play a significant role in microcirculatory dynamics. (ii) Modern biological approaches have provided insights into the molecular bases of blood cell deformability and interactions. (iii) Understanding of the physiology and pathophysiology of the microcirculation requires the application of knowledge derived from molecular and cell biological studies to the in vivo microcirculatory preparations. (iv) Bridging of the new biology and in vivo microcirculatory investigations represents a great challenge and a golden opportunity for microcirculation researchers.

Anemia, Sickle Cell↗
PubMed1992

Locomotion forces generated by a polymorphonuclear leukocyte.

There have been very few studies which have measured the physical forces generated by cells during active movements. A special micropipette system has been designed to make it possible to observe cell motion within the pipette and to apply a pressure to counter the chemotactic migration of the cell. This provides a direct measure of the locomotion force generated by the cell. The average velocity of forward motion is 0.33 microns/s in the absence of counter-pressure. The application of a positive counter-pressure (C-P) causes a decrease in the velocity of the forward motion of the cell. At 17 cm H2O of C-P, the cell velocity drops to zero and even moves backward with a higher C-P. The results show that the decrement of velocity is linearly related to the magnitude of the C-P with a complete stoppage at a pressure of 17 cm H2O which corresponds to a force of 0.003 dyn. The maximum work rate of the cell is approximately 2.5 x 10(-8) erg/s.

Biomechanical Phenomena↗
PubMed1992

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules.

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.

Antigens, CD↗
PubMed1992

Long-term nicotine exposure increases aortic endothelial cell death and enhances transendothelial macromolecular transport in rats.

Repeated endothelial cell injury has been suggested as an initiating factor in atherogenesis. Dying or dead endothelial cells have been shown to make significant contributions to the local enhancement of transendothelial macromolecular transport. Since cigarette smoking is one of the major risk factors for atherosclerosis, we examined the hypothesis that smoking accelerates atherogenesis by increasing the frequency of endothelial cell death and hence transendothelial macromolecular transport. Sixteen male Sprague-Dawley rats were given nicotine at a weight-adjusted dose of 5 mg/kg body wt per day in their drinking water over a period of 6 weeks. A group of 16 age-matched male Sprague-Dawley rats not exposed to nicotine and maintained over the same time period served as the control group. In en face preparations of thoracic aorta, immunoglobulin G-containing dying or dead endothelial cells were identified by the indirect immunoperoxidase method, and endothelial leakage to Evans blue-albumin (EBA) complexes (5 minutes after intravenous injection) was visualized by fluorescence microscopy. The results showed that in nicotine-treated rats, 51% of dead endothelial cells were associated with EBA leakage, which was responsible for 57% of total EBA leaky foci. Both the frequency of endothelial cell death (0.94 +/- 0.11% versus 0.40 +/- 0.04%, p < 0.0001 by two-tailed, unpaired Student's t test) and the number density of EBA leaky foci (6.45 +/- 1.23/mm2 versus 3.30 +/- 0.49/mm2, p < 0.05 by two-tailed, unpaired t test) were significantly greater in nicotine-treated rats than in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Force contribution of the LFA-1/ICAM-1 complex to T cell adhesion.

Little is known in quantitative terms about forces between cells generated during adhesion and recognition, or about the contribution of any one set of molecular associations to the development of these forces. To determine the forces involved in adhesion dependent on lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), we have measured the junctional avidity between single cell pairs consisting of a cloned T cell that expresses LFA-1 and a fibroblast cell that expresses MHC class II molecules and ICAM-1 after transfection. Micromanipulation was used to induce conjugation of cell pairs and to determine the force required to separate the conjugate. T cell adhesion to three related fibroblast cell lines was compared: the parent line that does not express ICAM-1 or other LFA-1 counter-receptors, and two transfectants that have high and moderate levels of surface ICAM-1 expression. The force needed to separate the conjugates varied with the fibroblast ICAM-1 expression levels. The T cell adhesion to ICAM-1-expressing fibroblasts was strong, and the critical separation stresses measured for the three cell lines were 1.4 x 10(3) dyn/cm2 (1 dyn=10(-5) N) for the ICAM-1-negative fibroblast, 4.98 x 10(3) dyn/cm2 for the fibroblast with a moderate level of ICAM-1 expression, and 6.25 x 10(3) dyn/cm2 for the fibroblast line with the highest ICAM-1 expression. The dependence of adhesion strength on the LFA-1/ICAM-1 complex was confirmed by the use of blocking antibodies, which showed the contribution from the interaction of CD4/MHC class II to be negligible.

Animals↗
PubMed1992

Influence of temperature on rheology of human erythrocytes.

The effect of variation in temperature on the rheological properties of human red blood cells was studied by determining the cell deformation in response to aspiration pressure applied via a micropipette. The time history of the deformation was analyzed by the use of a Voigt viscoelastic model consisting of an elastic element (E) in parallel with a viscous element (eta). Viscosity values were obtained during the initial rapid deformation phase (phase I), the later slow deformation phase (phase II), and the recovery phase. With a rise in temperature from 0 to 45 degrees C, both E and eta values decreased. A thermotropic transition temperature was found at approximately 21 degrees C for phase I viscosity and at approximately 9 degrees C for viscosity in the recovery phase, but not for phase II viscosity. These responses to temperature variations provide insights into the viscoelastic properties of the erythrocyte membrane.

Elasticity↗
PubMed1992

Role of intercellular junctions in the passage of horseradish peroxidase across aortic endothelium.

BACKGROUND: The manner in which molecules are transported across the arterial endothelial layer has been a subject open to much interpretation and controversy. Further elucidation and clarification of these mechanisms are of primary interest. EXPERIMENTAL DESIGN: To investigate the ultrastructural features of arterial endothelial junctions and to evaluate their functional roles as a transendothelial pathway for macromolecular transport, experiments were performed on the thoracic aortae of adult male Sprague-Dawley rats by using the ultrathin serial sectioning technique and horseradish peroxidase (HRP). The aorta was perfusion-fixed with or without prior intravenous injection of HRP. RESULTS: The intercellular clefts exhibited a great deal of variety in shape, being linear, winding, interdigitated, irregular and/or dumbbell-shaped in appearance. Besides the typical 20-nm width encountered at the uniform region of intercellular clefts, local widenings (up to several hundred nm) were quite common. The arterial endothelial junctions were highly organized. Junctional elements, including tight junctions and gap junctions, were frequently present in the same intercellular cleft, even on the same plane of sectioning. Sometimes, gap junctions were found without tight junctions, but the intercellular clefts were rarely obliterated by tight junctions alone. Some intercellular clefts were not obliterated by either gap or tight junctions, and HRP was found to reach the subendothelial space by passing through these junctionless clefts. Densitometric determination of the HRP concentration profile in such junctionless clefts showed a decreasing gradient from the luminal to the abluminal front. The serial sections provided evidence that the apparently free vesicles were actually plasmalemmal membrane invaginations open to the luminal or abluminal front in the arterial endothelium. CONCLUSIONS: The present study showed that the junctionless normal endothelial clefts, in addition to the transiently open junctions surrounding mitotic cells, might provide a significant pathway in the transendothelial transport of macromolecules with the size of HRP.

Animals↗