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Biomedical subjects

S Chien

Publications and source records attributed to S Chien.

At least 19 recordsLinked to original sources
PubMed2002

ERK activation and alpha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts.

Osteoblast growth and differentiation are critical processes for bone development and maintenance, and are regulated by both humoral and mechanical factors. Humoral (hormonal) factors can affect gene transcription via MAPkinases, e.g., extracellular signal-regulated kinase (ERK). We studied whether the ERK pathway is also involved in processing mechanical inputs in human bone cells. Exposing MG63 cells to physiologically relevant levels of fluid flow resulted in ERK phosphorylation. Genistein blocked this response, indicating that it is dependent on tyrosine phosphorylation. Furthermore, alpha v beta 3 integrins were activated in response to fluid flow, as shown by recruitment of adaptor molecule Shc and clustering of alpha v beta 3 in focal adhesion-like structures. Antibodies blocking formation of beta 1 or beta 3 integrin-matrix interactions or RGD peptides could not inhibit fluid flow-induced ERK phosphorylation, suggesting that formation of new integrin-matrix interactions is not essential for this response and that other upstream mechanosensors regulate induction of ERK phosphorylation in response to fluid flow in human bone cells.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Changes of biophysical behavior of k562 cells for p16 gene transfer.

p16 gene was transferred into human erythroleukemia cell line K562 that had been subjected to p16 deletion. The changes of biophysical behavior of K562 cells after gene transfer were studied with the micropipette technique. The micropipette data were analyzed with a standard solid viscoelastic model. In comparison to untransfected control K562 cells and the cells transfected with an empty vector, the p16-transfected K562 cells showed an increase in the elastic element K(1), which is inversely proportional to the maximum deformation over a long period of time, whereas the viscous element mu and the other elastic element K(2) showed no significant difference. The results indicate that K562 cells became more rigid after p16 transfer. The p16-transfected K562 cells also had a higher surface charge density. This study contributes to our knowledge about the suppression effect of p16 gene on tumor cell migration and about its use in gene therapy.

Biophysical Phenomena↗
PubMed2001

DNA microarray analysis of gene expression in endothelial cells in response to 24-h shear stress.

The recently developed DNA microarray technology provides a powerful and efficient tool to rapidly compare the differential expression of a large number of genes. Using the DNA microarray approach, we investigated gene expression profiles in cultured human aortic endothelial cells (HAECs) in response to 24 h of laminar shear stress at 12 dyn/cm(2). This relatively long-term shearing of cultured HAECs led to the modulation of the expression of a number of genes. Several genes related to inflammation and EC proliferation were downregulated, suggesting that 24-h shearing may keep ECs in a relatively noninflammatory and nonproliferative state compared with static cells. Some genes were significantly upregulated by the 24-h shear stress; these includes genes involved in EC survival and angiogenesis (Tie2 and Flk-1) and vascular remodeling (matrix metalloproteinase 1). These results provide information on the profile of gene expression in shear-adapted ECs, which is the case for the native ECs in the straight part of the aorta in vivo.

Adult↗
PubMed2001

Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment.

Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin alpha(v)beta3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear-induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of flow. The results therefore define the role of integrins and Rho in a pathway leading to endothelial cell adaptation to flow.

Animals↗
PubMed2001

Effects of flow patterns on endothelial cell migration into a zone of mechanical denudation.

Vascular endothelial cells (ECs) in vivo are subject to different flow conditions due to the variation in vessel geometry. The aim of this study is to elucidate the effects of different flow conditions on EC monolayer migration into a mechanically denuded zone and their underlying mechanisms. Both laminar and disturbed flows significantly enhanced EC migration. EC migration speed was the fastest under laminar flow, which preferentially promoted directional EC migration from the upstream side of the wounded monolayer. C3 exoenzyme (a Rho inhibitor) inhibited EC migration under static and flow conditions, and markedly reduced the effects of flow on EC migration. These results indicate that flow promotes EC migration through the Rho signaling pathway. Genistein (a tyrosine kinase inhibitor) selectively retarded EC migration under disturbed flow, suggesting that tyrosine phosphorylation may play a role in EC migration under disturbed flow. This study has demonstrated that different flow patterns differentially affect EC monolayer migration into the denuded zone involving multiple mechanisms.

ADP Ribose Transferases↗
PubMed2001

Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands.

The aim of this study is to elucidate the role of integrins in transducing fluid shear stress into intracellular signals in vascular endothelial cells, a fundamental process in vascular biology. We demonstrated that shear stress activates specific integrins in endothelial cells plated on substrates containing the cognate extracellular matrix (ECM) ligands. The shear stress-induced mechanotransduction, as manifested by integrin-Shc association, was abolished when new integrin-ECM ligand interactions were prevented by either blocking the integrin-binding sites of ECM ligands or conjugating the integrins to immobilized antibodies. Our results indicate that the dynamic formation of new connections between integrins and their specific ECM ligands is critical in relaying the signals induced by shear stress to intracellular pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Inhibition of neointimal formation in porcine coronary artery by a Ras mutant.

BACKGROUND: Therapeutic approaches to reduce the neointimal formation caused by balloon injury have been focused mainly on experimental models of restenosis in the rat carotid artery. However, restenosis in rat carotid artery may not replicate the coronary arterial responses to injury in larger animals and humans. METHODS: In this study, we used pig coronary arteries as an animal model to evaluate the preventive effects of a virus-mediated dominant negative mutant RasN17 on balloon injury-induced restenosis. The viral particles were delivered to the balloon-injured coronary arteries via a dispatch catheter to keep the virus in a confined arterial segment for 10 min to reach optimal transfection. Six weeks after balloon injury, the pigs were sacrificed and the left anterior descending arteries were isolated for histological analysis. RESULTS: Neointima formation was prominent in the group receiving balloon injury as compared with the uninjured controls. A remodeling process with migration of collagen was also found in the injured coronary arteries. The application of AdRasN17 led to a 56% decrease in neointima formation and a 75% increase in lumen size, as compared with the balloon-injured vessels treated with AdLacZ control. CONCLUSIONS: These results suggest that AdRasN17 is an effective therapeutic gene in preventing balloon injury-induced neointimal formation in pig coronary arteries.

Animals↗
PubMed2001

Dynamic contact forces on leukocyte microvilli and their penetration of the endothelial glycocalyx.

We develop a theoretical model to examine the combined effect of gravity and microvillus length heterogeneity on tip contact force (F(m)(z)) during free rolling in vitro, including the initiation of L-, P-, and E-selectin tethers and the threshold behavior at low shear. F (m)(z) grows nonlinearly with shear. At shear stress of 1 dyn/cm(2), F(m)(z) is one to two orders of magnitude greater than the 0.1 pN force for gravitational settling without flow. At shear stresses > 0.2 dyn/cm(2) only the longest microvilli contact the substrate; hence at the shear threshold (0.4 dyn/cm(2) for L-selectin), only 5% of microvilli can initiate tethering interaction. The characteristic time for tip contact is surprisingly short, typically 0.1-1 ms. This model is then applied in vivo to explore the free-rolling interaction of leukocyte microvilli with endothelial glycocalyx and the necessary conditions for glycocalyx penetration to initiate cell rolling. The model predicts that for arteriolar capillaries even the longest microvilli cannot initiate rolling, except in regions of low shear or flow reversal. In postcapillary venules, where shear stress is approximately 2 dyn/cm(2), tethering interactions are highly likely, provided that there are some relatively long microvilli. Once tethering is initiated, rolling tends to ensue because F(m)(z) and contact duration will both increase substantially to facilitate glycocalyx penetration by the shorter microvilli.

Animals↗
PubMed2001

Temperature transition of human hemoglobin at body temperature: effects of calcium.

We studied the effects of calcium ion concentration on the temperature dependence of rheological behavior of human red blood cells (RBCs) and concentrated hemoglobin solutions. Our previous study (G. M. Artmann, C. Kelemen, D. Porst, G. Büldt, and S. Chien, 1998, Biophys. J., 75:3179-3183) showed a critical temperature (Tc) of 36.4 +/- 0.3 degrees C at which the RBCs underwent a transition from non-passage to passage through 1.3 microm micropipettes in response to an aspiration pressure of -2.3 kPa. An increase in intracellular Ca2+ concentration by using the ionophore A23187 reduced the passability of intact RBCs through small micropipettes above T(c); the micropipette diameter needed for >90% passage increased to 1.7 microm. Viscometry of concentrated hemoglobin solutions (45 and 50 g/dl) showed a sudden viscosity transition at 36 +/- 1 degrees C (Tc(eta)) at all calcium concentrations investigated. Below Tc(eta), the viscosity value of the concentrated hemoglobin solution at 1.8 mM Ca(2+) was higher than that at other concentrations (0.2 microM, 9 mM, and 18 mM). Above Tc(eta), the viscosity was almost Ca2+ independent. At 1.8 mM Ca2+ and 36 +/- 1 degrees C, the activation energy calculated from the viscometry data showed a strong dependence on the hemoglobin concentration. We propose that the transition of rheological behavior is attributable to a high-to-low viscosity transition mediated by a partial release of the hemoglobin-bound water.

Blood Viscosity↗
PubMed2001

Role of tensile stress and strain in the induction of cell death in experimental vein grafts.

Tensile stress and strain are known to induce vascular cell proliferation, a process that is physiologically counterbalanced by cell death. Here we investigate whether tensile stress and strain regulate vascular-cell death by using an end-to-end anastomosed rat vein graft model. In such a model, the circumferential tensile stress in the graft wall was increased by approximately 140 times immediately after surgery compared with that in the venous wall. This change was associated with an increase in the percentage of TUNEL-positive cells at 1, 6, 24, 120, 240, and 720h with two distinct peaks at 1 and 24h (10.1+/-3.5 and 14.4+/-3.2%, respectively) compared with that in control jugular veins (0.4+/-0.5 and 0.5+/-0.5% at 1 and 24h, respectively). When tensile stress and strain in the vein graft wall were reduced by using a biomechanical engineering approach, the rate of cell death was reduced significantly (3.6+/-1.1 and 1.6+/-0.5% at 1 and 24h, respectively). Furthermore, DEVD-CHO, a tetrapeptide aldehyde that inhibits the activity of caspase 3, significantly suppressed this event. These results suggest that a step increase in tensile stress and strain in experimental vein grafts induces rapid cell death, which is possibly mediated by cell death signaling mechanisms.

Anastomosis, Surgical↗
PubMed2001

Low viscosity Ektacytometry and its validation tested by flow chamber.

The flow chamber was used to observe the orientation and small deformation of red blood cells (RBCs) in a shear flow of low viscosity. With the aid of computer software, the percentage of RBCs oriented to the C=0 orbit (OI)(F) and the degree of deformation (DI)(F) of such RBCs were calculated by processing the photographs. It was found that these parameters were highly correlated, respectively, to the orientation index (OI)(E) and the small deformation index (DI)(E) obtained by our low viscosity Ektacytometry (LVE). Thus, our flow chamber research has provided direct evidence to validate the use of this low viscosity Ektacytometry. Although there are relative merits for the flow chamber method using low viscosity medium, the LVE is more likely to be applied in clinic for its simplicity and convenience.

Animals↗
PubMed2001

The uptake and metabolism of fructose-1,6-diphosphate in rat cardiomyocytes.

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been theorized to increase the metabolic activity of ischemic tissues. Here we examine the effects of externally applied FDP on cardiomyocyte uptake and metabolism. Adult rat cardiomyocytes were isolated and exposed to varying concentrations (0, 5, 25 and 50 mM) of FDP for either 1, 16 or 24 h of hypoxia (95% N2/5% CO2), each time period followed by a 1 h reoxygenation (95% air/5% CO2). The uptake of FDP by rat cardiomyocytes was more concentration-dependent than time-dependent. Furthermore, the uptake of FDP by the cardiomyocytes was similar in the hypoxia and normoxia treated cells. Alamar Blue, a redox indicator that is sensitive to metabolic activity, was used to monitor the effects of the FDP on cardiomyocyte metabolism. In the 1 h hypoxia or normoxia group, the 5, 10 and 25 mM FDP showed a significant increase in metabolism compared to the control cells. When the length of hypoxia was extended to 16 h, all doses of FDP were greater than control. And at the 24 h hypoxia or normoxia time period, only the 10, 25 and 50 mM FDP groups were greater than control. The results indicate a non-linear trend between the external concentration of FDP and the changes noted in metabolism. The findings from this study indicate that a narrow concentration range between 5-10 mM augments cardiomyocyte metabolism, but higher or lower doses may have little additional affect.

Animals↗
PubMed2001

A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.

We performed a systematic analysis of 929 human disease gene entries associated with at least one mutant allele in the Online Mendelian Inheritance in Man (OMIM) database against the recently completed genome sequence of Drosophila melanogaster. The results of this search have been formatted as an updateable and searchable on-line database called Homophila. Our analysis identified 714 distinct human disease genes (77% of disease genes searched) matching 548 unique Drosophila sequences, which we have summarized by disease category. This breakdown into disease classes creates a picture of disease genes that are amenable to study using Drosophila as the model organism. Of the 548 Drosophila genes related to human disease genes, 153 are associated with known mutant alleles and 56 more are tagged by P-element insertions in or near the gene. Examples of how to use the database to identify Drosophila genes related to human disease genes are presented. We anticipate that cross-genomic analysis of human disease genes using the power of Drosophila second-site modifier screens will promote interaction between human and Drosophila research groups, accelerating the understanding of the pathogenesis of human genetic disease. The Homophila database is available at http://homophila.sdsc.edu.

Amino Acid Sequence↗
PubMed2001

Leukocyte rolling in rat mesentery venules: distribution of adhesion bonds and the effects of cytoactive agents.

A new method of analyzing in vivo measurements of leukocyte WBC rolling along venular endothelium (EC) has been developed to extract insightful information on the dynamics of WBC-EC bond formation and disruption. The rolling velocity of WBCs was obtained by intravital microscopy of rat mesenteric venules. For the "spontaneous" rolling observed following exteriorization of the mesentery, we estimated that the average distance between clusters of adhesion bonds which tether a rolling cell to the venular wall was about 2 microm, and that the average lifetime of a bond cluster at the trailing edge of the rolling cell, from its exposure to the tensile force to its release, was on the order of 0.05 s. Both the inter-cluster distance and the lifetime were significantly reduced by treatments with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine and the cytokine interleukin-1, while the average lifetime of the stretched bond clusters was not significantly changed by treatment with the cytoskeleton-modifying agents cytochalasin B and colchicine. Each of the four treatments significantly reduced the heterogeneity in the cell rolling velocity, presumably by the selective recruitment of WBC subsets from the circulating WBC population or by a reduction in the heterogeneity of endothelial adhesiveness. These results were analyzed in the context of in vitro data in the literature on molecular bonds of cell adhesion. The findings suggest that, in the case of "spontaneous" rolling, there are on average approximately 2-3 clusters of adhesion bonds between a rolling cell and the vessel wall, and approximately five bonds in each cluster.

Animals↗
PubMed2001

Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity.

Blood flow-associated shear stress may modulate cellular processes through its action on the plasma membrane. We quantified the spatial and temporal aspects of the effects of shear stress (tau) on the lipid fluidity of 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC(16)(13)]-stained plasma membranes of bovine aortic endothelial cells in a flow chamber. A confocal microscope was used to determine the DiI diffusion coefficient (D) by fluorescence recovery after photobleaching on cells under static conditions, after a step-tau of 10 or 20 dyn/cm(2), and after the cessation of tau. The method allowed the measurements of D on the upstream and downstream sides of the cell taken midway between the respective cell borders and the nucleus. In <10 s after a step-tau of 10 dyn/cm(2), D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of tau. D returned to near control values within 5 s after cessation of tau. Downstream D showed little secondary changes throughout the 10-min shearing, as well as after its cessation. Further investigations into the early phase, with simultaneous measurements of upstream and downstream D, confirmed that a step-tau of 10 dyn/cm(2) elicited a rapid (5-s) but transient increase in upstream D and a concurrent decrease in downstream D, yielding a significant difference between the two sites. A step-tau of 20 dyn/cm(2) caused D to increase at both sites at 5 s, but by 30 s and 1 min the upstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes in membrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications in shear-induced membrane protein modulation.

Animals↗
PubMed2001

Endothelial cellular response to altered shear stress.

Endothelial cells are normally exposed constantly to mechanical forces that significantly influence their phenotype. This symposium presented recent information concerning endothelial cell responses to shear stress associated with blood flow. Endothelial cell shear stress mechanosensors that have been proposed include membrane receptor kinases, integrins, G proteins, ion channels, intercellular junction proteins, membrane lipids (e.g., those associated with caveolae), and the cytoskeleton. These sensors are linked to signaling cascades that interact with or result in generation of reactive oxygen species, nitric oxide, and various transcription factors among other responses. Endothelial cells adapt to sustained shear stress, and either an increase or decrease from normal shear leads to signaling events. In vitro models for the study of endothelial cell responses must consider the pattern of shear stress (e.g., steady vs. oscillatory flow), the scaffold for cell growth (e.g., basement membrane or other cell types such as smooth muscle cells), and the extent of flow adaptation. These cellular responses have major relevance for understanding the pathophysiological effects of increased shear stress associated with hypertension or decreased shear stress associated with thrombotic occlusion.

Endothelium, Vascular↗