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Biomedical subjects

S Chien

Publications and source records attributed to S Chien.

At least 55 records · Page 3Linked to original sources
PubMed1999

Effect of seeding duration on the strength of chondrocyte adhesion to articular cartilage.

Chondrocyte adhesion to cartilage may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. The objective of this in vitro study was to determine the effect of the duration of seeding time on the ability of chondrocytes to resist detachment from cartilage when subjected to mechanical perturbation (fluid-induced shear stress). Suspensions of adult bovine articular chondrocytes were prepared from primary, high-density monolayer cultures and infused into a parallel-plate shear-flow chamber where they settled onto 50-microm-thick sections of bovine articular cartilage at a density of approximately 20,000 cells/cm2. The chondrocytes were seeded and allowed to attach to the cartilage surface for specific durations (5-40 minutes) in medium including 10% serum at 22 degrees C, after which the cells were exposed to fluid flow-induced shear stresses (6-90 Pa). The fraction of detached cells at each shear stress was calculated from microscopic images. Shear stress was applied for 1 minute because this length of time was sufficient to induce steady-state cell detachment. Increasing the duration of cell seeding led to a more firm attachment of chondrocytes to cartilage. After 9 minutes of seeding, 50% cell detachment was induced by gravitational force alone. After 40 minutes of seeding, 50% detachment required 26 Pa of shear stress. Extrapolation of the data to account for the effect of repeated applications of cell suspensions to an individual cartilage substrate indicated that for a freshly prepared cartilage section, 50% detachment was induced by gravity after 25 minutes of seeding and by 2.3 Pa of shear stress after 40 minutes of seeding. The increase in resistance to shear stress-induced cell detachment with increasing seeding duration suggests that it may be beneficial to allow chondrocytes to stabilize in the absence of applied load for some time after chondrocyte transplantation for cartilage repair in vivo.

Animals↗
PubMed1999

Heat shock-induced necrosis and apoptosis in osteoblasts.

Damage to bone tissue due to heat shock is one of the main causes of the failure of osseointegration at the bone-implant interface. To investigate the effect of heat shock on regeneration of bone tissue, osteoblasts were exposed to heat shock for 10 minutes at 42, 45, or 48 degrees C or kept at 37 degrees C as a control. After 10 minutes of heat shock, disruption of actin filaments was seen in the cells and the degree of disruption increased with the temperature. The cytoskeleton reassembled after a 12-hour incubation at 37 degrees C in the cells treated at 42 or 45 degrees C, but this reversible recovery did not occur in the cells treated at 48 degrees C. Flow cytometric analysis showed that heat shock at 48 degrees C increased the number of necrotic cells to 15-20% within minutes (p < 0.05 compared with 37 degrees C). Apoptosis, evidenced by annexin V staining, DNA laddering, and caspase 3 activation, started after 6-8 hours of incubation, reached a peak at 12 hours, and gradually declined (p<0.05). Pretreatment with the antioxidant N-acetyl-L-cysteine reduced the necrosis induced at 48 degrees C of heat shock by one-half (p<0.05) but had no significant effect on caspase 3 activation induced by heat shock, suggesting that reactive oxygen species were critical in heat shock-induced necrosis but not in apoptosis. Heat shock at 48 degrees C induced a sustained translocation of p53 into the nucleus and a sustained activation of c-jun N-terminal kinase, whereas that at 42 and 45 degrees C induced only transient p53 translocation and c-jun N-terminal kinase activation. These results suggest that the sustained activation of p53 and c-jun N-terminal kinase pathways may contribute to heat shock-induced apoptosis. On the other hand, heat shock protein 70 increased dramatically in the cells treated at 45 or 48 degrees C, suggesting that the protecting mechanism in the cells was also activated. Such protection was able to prevent apoptosis in cells treated at 45 degrees C but not in those treated at 48 degrees C.

Actins↗
PubMed1999

Enhancement of hypothermic heart preservation with fructose 1, 6-diphosphate.

BACKGROUND: We hypothesized that the addition of fructose 1, 6-diphosphate (FDP) to a hypothermic heart preservation solution could improve metabolic recovery because it has several beneficial effects. MATERIALS AND METHODS: Twenty adult Sprague-Dawley rats were used to study hypothermic heart preservation. The hearts were removed under general anesthesia and preserved at 4 degrees C in Euro-Collins solution (30 ml/kg) for 8 h. In the study group (N = 10), FDP (5 mM) was added to the Euro-Collins solution. In the control group (N = 10), no FDP was added. Heart function was studied after preservation using a working heart model. The ability of various concentrations of fructose 1,6-phosphate to passively diffuse through an egg phosphatidylcholine multilamellar vesicle (MLV) membrane bilayer was examined. RESULTS: Cardiac output ranged from 17.0 +/- 1.9 to 24.9 +/- 1.6 ml/min in the study group vs 2.0 +/- 1.0-12.3 +/- 1.7 ml/min for controls, average aortic flow was 10. 8 +/- 1.4 ml/min in the study group vs -1.3 +/- 1.6 ml/min for controls, and maximum LV generated power was 22.8 +/- 1.7 J/min vs 10.1 +/- 1.6 J/min for controls. Coronary flow, left ventricular stroke volume and stroke work, and myocardial oxygen consumption were much higher in the study group than in the control group. Coronary vascular resistance was lower in the study group than in the control group. Electron microscopic study indicated that many myocytes displayed patches of swollen mitochondria in the control group, but was rarely observed in the study group. The addition of 50 mM FDP caused substantial changes in MLV permeability. No dose of sucrose buffers outside the vesicles resulted in a significant changes of MLV permeability. CONCLUSIONS: Our results indicate that the addition of FDP to Euro-Collins solution significantly improves hypothermic rat heart preservation, and FDP appeared to cross the membrane bilayer.

Animals↗
PubMed1999

Strain distribution in the layered wall of the esophagus.

The function of the esophagus is to move food by peristaltic motion, which is the result of the interaction of the tissue forces in the esophageal wall and the hydrodynamic forces in the food bolus. To understand the tissue forces in the esophagus, it is necessary to know the zero-stress state of the esophagus, and the stress-strain relationships of the tissues. This article is addressed to the first topic: the representation of zero-stress state of the esophagus by the states of zero stress-resultant and zero bending moment of the mucosa-submucosa and the muscle layers. It is shown that at the states of zero stress-resultant and zero bending moment, these two layers are not tubes of smaller radii but are open sectors whose shapes are approximately cylindrical and more or less circular. When the sectors are approximated by circular sectors, we measured their radii, opening angles, and average thickness around the circumference. Data on the radii, thickness-to-radius ratios, and the opening angles of these sectors are presented. Knowing the zero-stress state of these two layers, we can compute the strain distribution in the wall at any in vivo state, as well as the residual strain in the esophageal wall at the no-load state. The results of the in vivo states are compared to those obtained by a conventional approach, which treats the esophageal wall as a homogeneous material, and to another popular simplification, which ignores the residual strains completely. It is shown that the errors caused by the homogeneous wall assumption are relatively minor, but those caused by ignoring the residual strains completely are severe.

Animals↗
PubMed1999

Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress.

The disruption of microtubules by treating bovine aortic endothelial cells with 10(-7)-10(-5) M colchicine caused apoptosis, as evidenced by DNA laddering and TdT-mediated dUTP nick end labeling fluorescence staining. Colchicine treatment also induced a sustained activation of c-Jun NH(2)-terminal kinase (JNK) that lasted for >/=12 h. The blockade of JNK activity by using the negative interfering mutant JNK(K-R) markedly decreased the apoptosis induced by colchicine. Exposure of bovine aortic endothelial cells to laminar shear stress (12 dyn/cm(2)) caused a transient (<2 h) activation of JNK, and there was no induction of apoptosis. The sustained activation of JNK may play a significant role in the apoptosis induced by colchicine.

Animals↗
PubMed1999

Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress.

Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress-induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress-induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate-responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress-induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

Animals↗
PubMed1998

Fluid shear stress activation of IkappaB kinase is integrin-dependent.

Vascular endothelial cells (ECs), forming a boundary between the circulating blood and the vessel wall, are constantly subjected to fluid shear stress due to blood flow. The aim of this study was to determine the role of the recently identified IkappaB kinases (IKKs) in shear stress activation of NF-kappaB and to elucidate the upstream signaling mechanism that mediates IKK activation. Our results demonstrate that IKKs in ECs are activated by shear stress in a rapid and transient manner. This IKK activation is followed by IkappaB degradation and NF-kappaB translocation into the nucleus. Transfection of plasmids encoding catalytic inactive mutants of IKKs, i.e. hemagglutinin (HA)-IKKalpha(K44M) and HA-IKKbeta(K44A), inhibits shear stress-induced NF-kappaB translocation. In addition, constructs encoding antisense IKKs, i.e. HA-IKKalpha(AS) and HA-IKKbeta(AS), attenuate shear stress induction of a promoter driven by the kappaB enhancer element. Preincubation of the EC monolayer with a monoclonal anti-alphavbeta3 integrin antibody (clone LM609) attenuates shear stress induction of IKK. Inhibition of tyrosine kinases by genistein causes a similar down-regulating effect. These results suggest that the integrin-mediated signaling pathway regulates NF-kappaB through IKKs in ECs in response to shear stress.

Animals↗
PubMed1998

Estimation of oxygen delivery in newborns with a univentricular circulation.

BACKGROUND: The management of neonates with complex congenital anomalies depends on careful interpretation of arterial blood gas values. Improved interpretation of these oxygen parameters may allow clinicians to avoid unexpected cardiovascular events. This study examined whether systemic oxygen delivery (DO2) can be maximized by the use of indices derived from oxygen saturation measurements in neonates with hypoplastic left heart syndrome. METHODS AND RESULTS: For the single-ventricle heart with both circulations in parallel, we used a previously developed computer simulation to obtain DO2 as a function of systemic arterial (SaO2) and venous (SvO2) oxygen saturation, arteriovenous oxygen difference (Sa-vO2), or pulmonary-to-systemic flow ratio (Qp/Qs). We also examined the oxygen excess factor, SaO2/Sa-vO2 (Omega). We found that (1) slight increases in SaO2 may be associated with large decreases in DO2. (2) Low values for SvO2 indicate low values for DO2. (3) Curves for Sa-vO2 and Qp/Qs are redundant in the data provided. (Qp/Qs, however, provides these data in more physiologically relevant terms.) (4) High values for Qp/Qs (>4) are associated with low DO2. (5) Estimating Qp/Qs from oxygen saturation measurements may result in errors when pulmonary venous oxygen saturation is not available. (6) Maximizing DO2 is extremely difficult using SaO2, SvO2, and Qp/Qs. (7) A linear relationship exists between Omega and DO2, and this linear relationship is not altered by changes in cardiac output. CONCLUSIONS: Patients with low SvO2 values require attention. Ideally, after reducing Qp/Qs to <1.5, Omega might be a better index to guide further therapy and maximize DO2. Interventions that increased Omega would be considered beneficial, whereas interventions that decreased Omega would be considered detrimental.

Cardiac Output↗
PubMed1998

Shear stress induces spatial reorganization of the endothelial cell cytoskeleton.

The morphology of endothelial cells in vivo depends on the local hemodynamic forces. Cells are polygonal and randomly oriented in areas of low shear stress, but they are elongated and aligned in the direction of fluid flow in regions of high shear stress. Endothelial cells in vitro also have a polygonal shape, but the application of shear stress orients and elongates the cells in the direction of fluid flow. The corresponding spatial reorganization of the cytoskeleton in response to the applied hemodynamic forces is unknown. In this study, we determined the spatial reorganization of the cytoskeleton throughout the volume of cultured bovine aortic endothelial cells after the cells had been exposed to a physiological level of shear stress for 0, 1.5, 3, 6, 12, or 24 h. The response of the monolayer to shear stress was not monotonic; it had three distinct phases. The first phase occurred within 3 h. The cells elongated and had more stress fibers, thicker intercellular junctions, and more apical microfilaments. After 6 h of exposure, the monolayer entered the second phase, where the cells exhibited characteristics of motility. The cells lost their dense peripheral bands and had more of their microtubule organizing centers and nuclei located in the upstream region of the cell. The third phase began after 12 h of exposure and was characterized by elongated cells oriented in the direction of fluid flow. The stress fibers in these cells were thicker and longer, and the heights of the intercellular junctions and microfilaments were increased. These results suggest that endothelial cells initially respond to shear stress by enhancing their attachments to the substrate and neighboring cells. The cells then demonstrate characteristics of motility as they realign. The cells eventually thicken their intercellular junctions and increase the amount of apical microfilaments. The time course of rearrangement can be described as a constrained motility that produces a new cytoskeletal organization that alters how the forces produced by fluid flow act on the cell and how the forces are transmitted to the cell interior and substrate.

Animals↗
PubMed1998

Temperature transitions of protein properties in human red blood cells.

Human red blood cells (RBC) undergo a sudden change from blocking to passing through 1.3 +/- 0.2-micrometer micropipettes at a transition temperature (Tc) of 36.4 degrees C. For resealed RBC ghosts this transition occurs at 28.3 degrees C (Tg). These findings are attributed to an elastomeric transition of hemoglobin from being gel-like to a fluid and to an elastomeric transition of membrane proteins such as spectrin. Spectrin shows a uniform distribution along the aspirated RBC tongue above Tg in contrast to the linear gradient below Tg.

Biophysical Phenomena↗
PubMed1998

Effects of disturbed flow on endothelial cells.

Atherosclerotic lesions tend to localize at curvatures and branches of the arterial system, where the local flow is often disturbed and irregular (e.g., flow separation, recirculation, complex flow patterns, and nonuniform shear stress distributions). The effects of such flow conditions on cultured human umbilical vein endothelial cells (HUVECs) were studied in vitro by using a vertical-step flow channel (VSF). Detailed shear stress distributions and flow structures have been computed by using the finite volume method in a general curvilinear coordinate system. HUVECs in the reattachment areas with low shear stresses were generally rounded in shape. In contrast, the cells under higher shear stresses were significantly elongated and aligned with the flow direction, even for those in the area with reversed flow. When HUVECs were subjected to shearing in VSF, their actin stress fibers reorganized in association with the morphological changes. The rate of DNA synthesis in the vicinity of the flow reattachment area was higher than that in the laminar flow area. These in vitro experiments have provided data for the understanding of the in vivo responses of endothelial cells under complex flow environments found in regions of prevalence of atherosclerotic lesions.

Arteriosclerosis↗
PubMed1998

Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells.

The aim of this study was to elucidate the upstream signaling mechanism that mediates the fluid shear stress activation of mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs), in vascular endothelial cells (ECs). Our results indicate that p60src is rapidly activated by fluid shear stress in bovine aortic endothelial cells (BAECs). Shear stress induction of the hemagglutinin (HA) epitope-tagged HA-JNK1 and the Myc epitope-tagged Myc-ERK2 was significantly attenuated by v-src(K295R) and c-src(K295R), the kinase-defective mutants ofv-src and c-src, respectively. HA-JNK1 and Myc-ERK2 were activated by c-src(F527), a constitutively activated form of p60src, and the activation was abolished by RasN17, a dominant-negative mutant of p2lras. In contrast, although HA-JNK1 and Myc-ERK2 were also activated by RasL61, an activated form of p21ras, the activation was not affected by v-src(K295R). These results indicate that p60src is upstream to the Ras-JNK and Ras-ERK pathways in response to shear stress. The shear stress inductions of the promoters of monocyte chemotactic protein-1 (MCP-1) and c-fos, driven by TPA-responsive element (TRE) and serum-responsive element (SRE), respectively, were attenuated by v-src(K295R). This attenuation is associated with decreased transcriptional activities of c-Jun and Elk-1, the transcription factors targeting TRE and SRE, respectively. Thus, p60src plays a critical role in the shear stress activation of MAPK pathways and induction of Activating Protein-1 (AP- 1)/TRE and Elk-1/SRE-mediated transcription in ECs.

Animals↗
PubMed1998

Effects of mechanical forces on signal transduction and gene expression in endothelial cells.

Fluid shear stress and circumferential stretch play important roles in maintaining the homeostasis of the blood vessel, and they can also be pathophysiological factors in cardiovascular diseases such as atherosclerosis and hypertension. The uses of flow channels and stretch devices as in vitro models have helped to elucidate the mechanisms of signal transduction and gene expression in cultured endothelial cells in response to shear stress, which is a function of blood flow and vascular geometry, or mechanical strain, which is a function of transmural pressure and the mechanical properties and geometry of the vessel. Shear stress has been found to increase the activities of a number of kinases to modulate the phosphorylation of many signaling proteins in endothelial cells, eg, the proteins in focal adhesion sites and the proteins in the mitogen-activated protein kinase pathways. Downstream to such signaling cascades, multiple transcription factors such as AP-1, NF-kappaB, Sp-1, and Egr-1 are activated. The actions of these transcription factors on the corresponding cis-elements result in the induction of genes encoding for vasoactivators, adhesion molecules, monocyte chemoattractants, and growth factors in endothelial cells, thus modulating vascular structure and function. Some of the effects of mechanical strain on endothelial cells are similar to those by shear stress, eg, the signaling pathways and the genes activated, but there are differences, eg, the time course of the responses. Studies on the effects of mechanical forces on signal transduction and gene expression provide insights into the molecular mechanisms by which hemodynamic factors regulate vascular physiology, and pathophysiology.

Animals↗
PubMed1998

An animal model to study erythrocyte senescence with a narrow time window of erythrocyte production.

Using the method of inducing spherocytic anemia in the rabbit with antibody serum, we have developed an animal model in which red blood cells (RBCs) can nearly grow synchronously. With this model, we determined that the surface charge density on the RBC membrane decreased with cell aging. The change was not linear, being much more profound in the latter half of RBC life span. There was a positive correlation between the mean RBC density and its "age" (r = 0.847, p < 0.01). However, the density distribution of the RBCs at the same "age" showed a broad range, and the density values for RBC groups with different ages showed considerable overlap. This indicates that the density gradient technique can be used to separate RBC population into fractions with different mean ages, but has a low resolving power for obtaining individual RBCs of a given "age".

Anemia↗
PubMed1998

An animal model to study erythrocyte senescence with a narrow time window of erythrocyte production: alterations in osmotic fragility and deformability of erythrocytes during their life span.

Using the model in which the entire RBC population was nearly synchronously produced following the induction of spherocytic anemia in the rabbit with antibody serum, we determined the changes of RBC osmotic fragility and deformability with aging. The results showed that the osmotic fragility increased with the RBC aging process in a nonlinear manner, being much more profound in the later part of the RBC life span. The RBC deformation index (DI) was measured by an ektacytometry. It is found that the DI decreased with RBC aging in a nonlinear fashion, with increasingly greater changes in the later part of the RBC life span. The alterations of RBC mechanical properties with aging may be attributable to a number of factors, including changes of RBC size and shape, and the viscoelasticity of the cytoplasm and membrane.

Anemia, Hemolytic↗
PubMed1997

Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases.

Shear stress, the tangential component of hemodynamic forces, activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signal transduction pathways in cultured vascular endothelial cells to induce the transcriptional activation of many immediate early genes. It appears that integrins, protein-tyrosine kinases, and the structural integrity of actin are important factors involved in these shear stress-induced responses. The underlying molecular events were investigated by the application of a shear stress of 12 dyn/cm2 on bovine aortic endothelial cells (BAEC). We found that such a shear stress increased the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK) and its association with growth factor receptor binding protein 2 (Grb2) in a rapid and transient manner, suggesting that FAK may be linked to these mitogen-activated protein kinase signaling pathways through a Grb2. Son of sevenless (Sos) complex. FAK(F397Y), which encodes a dominant negative mutant of FAK, attenuated the shear stress-induced kinase activity of Myc epitope-tagged ERK2 and hemagglutinin epitope-tagged JNK1. DeltamSos1, encoding a dominant negative mutant of Sos in which the guanine nucleotide exchange domain has been deleted, also attenuated shear stress activation of Myc-ERK2 and hemagglutinin-JNK1. Pretreating the confluent BAEC monolayers with a blocking type anti-vitronectin receptor monoclonal antibody had similar inhibitory effects in these shear stress-activated ERKs and JNKs. Confocal microscopic observation further demonstrated that FAK tended to cluster with vitronectin receptor near the abluminal side of the sheared BAEC. These results demonstrate that FAK signaling is critical in the shear stress-induced dual activation of ERK and JNK.

Actins↗