Biomedical subjects

Shu Chien

Publications and source records attributed to Shu Chien.

At least 37 records · Page 2Linked to original sources

PubMed2006

Biochemical and biophysical studies on the precursor cells of mouse erythrocytes at different stages.

The aim of this research is to study the biochemical and biophysical properties of the precursor cells of mouse erythrocytes at different stages and the molecular mechanisms of their regulation. We investigated the degree of terminal differentiation of splenic erythroblasts obtained from mice during the acute phase of disease caused by the anemia-inducing FVAstrain of Friend virus. We analyzed the transcription and protein levels of alpha-globin, beta-globin (erythroid special protein) and GATA-1 (a special erythroid transcription factor). We also have examined the Ca2+ concentration, the distribution and amount of F-actin, important cellular components such as nucleic acids, lipids, and proteins, and the adhesion of precursor cells of RBC at different stages to vascular endothelium. Our results indicated that Ca2+ concentration and the distribution and structure of F-actin changed with the development of proerythroblasts, and that the adhesion rate between the precursor cells and endothelial cells can be correlated with the expression levels of ICAM-1 and P-selectin. These alternations caused changes in biophysical properties of the cell, such as membrane fluidity and deformability.

Actins↗

PubMed2006

Chronotropic response of cultured neonatal rat ventricular myocytes to short-term fluid shear.

Ventricular myocytes are continuously exposed to fluid shear in vivo by relative movement of laminar sheets and adjacent cells. Preliminary observations have shown that neonatal myocytes respond to fluid shear by increasing their beating rate, which could have an arrhythmogenic effect under elevated shear conditions. The objective of this study is to investigate the characteristics of the fluid shear response in cultured myocytes and to study selected potential mechanisms. Cultured neonatal rat ventricular myocytes that were spontaneously beating were subjected to low shear rates (5-50/s) in a fluid flow chamber using standard culture medium. The beating rate was measured from digital microscopic recordings. The myocytes reacted to low shear rates by a graded and reversible increase in their spontaneous beating rate of up to 500%. The response to shear was substantially attenuated in the presence of the beta-adrenergic agonist isoproterenol (by 86+/-8%), as well as after incubation with integrin-blocking RGD peptides (by 92+/-8%). The results suggest that the beta-adrenergic signaling pathway and integrin activation, which are known to interact, may play an important role in the response mechanism.

Action Potentials↗

PubMed2006

Molecular basis of rheological modulation of endothelial functions: importance of stress direction.

Vascular endothelial cells (EC) play significant roles in regulating circulatory functions. Shear stress and stretch can modulate EC functions by activating mechano-sensors, signaling pathways, and gene and protein expressions. Laminar shear stress with a significant forward direction causes transient activations of monocyte chemotactic protein-1 (MCP-1), sterol response element binding protein (SREBP), and proliferative genes. Sustained laminar shear stress down-regulates these genes and up-regulates genes that inhibit EC growth. In EC subjected to complex flow patterns with little forward direction, activations of MCP-1, SREBP, and proliferation genes become sustained, and mitosis and apoptosis are enhanced. Cyclic uniaxial stretch causes actin stress fibers to orient perpendicular to stretch direction, with a consequent reduction of intracellular stress, transient JNK activation, and protection of EC against apoptosis. Cyclic biaxial stretch without a preferred direction has opposite effects. In the straight part of arterial tree, laminar shear stress with a net forward direction and uniaxial strain in the circumferential direction have anti-atherogenic effects. At vascular branch points, the complex shear flow and mechanical strain with little net direction are atherogenic. Therefore, the direction of stress has important influences on the biorheological effects of flow and deformation on vascular endothelium in health and disease.

Blood Circulation↗

PubMed2005

Phosphatidylinositol 3-kinase/Akt pathway is involved in transforming growth factor-beta1-induced phenotypic modulation of 10T1/2 cells to smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) is known to induce phenotypic modulation of mesenchymal cells to SMCs. However, the intracellular signals regulating induction of the SMC phenotype of mesenchymal cells have not been fully clarified. In the present study, we examined the role of the mitogen-activated protein kinase (MAPK) superfamily and phosphatidylinositol 3-kinase (PI3K)/Akt in the TGF-beta1-mediated phenotypic modulation of 10T1/2 mesenchymal cells to SMCs characterized by the expression of SMC-specific markers, including smooth muscle alpha-actin (SMalpha-actin), myosin heavy chain (SM-MHC), and protein 22-alpha (SM22alpha). The results showed the following: (1) TGF-beta1 induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells in a time-dependent manner. (2) TGF-beta1 induced biphasic increases in extracellular signal-regulated kinase (ERK), p38 MAPK, c-Jun-NH2-terminal kinase (JNK), and Akt phosphorylation. (3) The inhibitor for PI3K/Akt (i.e., LY294002), but not those for MAPKs (i.e., SB203580, PD98059, and SP600125), attenuated the TGF-beta1-induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells; in addition, transfection of 10T1/2 cells with the Akt-specific small interfering RNA (siRNA) significantly reduced their SMalpha-actin and SM-MHC expressions. (4) LY294002 and the Akt-specific siRNA inhibited the TGF-beta1-induced SM22alpha gene expression and promoter activity, suggesting that the TGF-beta1-induced gene expression was mediated by PI3K/Akt at the transcriptional level. (5) LY294002 inhibited the TGF-beta1-induced gene expression and DNA binding activity of serum response factor (SRF). These results indicate that TGF-beta1 is capable of inducing the SMC phenotype of 10T1/2 cells and that this induction is mediated through the PI3K/Akt signaling pathway.

Actins↗

PubMed2005

Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow.

Atherosclerosis develops at regions of the arterial tree exposed to disturbed flow. The early stage of atherogenesis involves the adhesion of leukocytes (white blood cells [WBCs]) to and their transmigration across endothelial cells (ECs), which are located in close proximity to smooth muscle cells (SMCs). We investigated the effects of EC/SMC coculture and disturbed flow on the adhesion and transmigration of 3 types of WBCs (neutrophils, peripheral blood lymphocytes [PBLs], and monocytes) using our vertical-step flow (VSF) chamber, in which ECs were cocultured with SMCs in collagen gels. Such coculture significantly increased the adhesion and transmigration of neutrophils, PBLs, and monocytes under VSF, particularly in the reattachment area, where the rolling velocity of WBCs and their transmigration time were decreased, as compared with the other areas. Neutrophils, PBLs, and monocytes showed different subendothelial migration patterns under VSF. Their movements were more random and shorter in distance in the reattachment area. Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines, which contributed to the increased WBC adhesion and transmigration. Our findings provide insights into the mechanisms of WBC interaction with the vessel wall (composed of ECs and SMCs) under the complex flow environments found in regions of prevalence for atherogenesis.

Atherosclerosis↗

PubMed2005

Cooperative effects of Rho and mechanical stretch on stress fiber organization.

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Delta1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional approximately 3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Delta1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.

Animals↗

PubMed2005

Molecular basis of mechanical modulation of endothelial cell migration.

Vascular endothelial cells (ECs) play important roles in the regulation of vascular functions. Loss of endothelial integrity can lead to vascular diseases such as stenosis resulting from atherosclerosis. The migration of ECs into wounded area in the vessel wall is required for the restoration of its integrity and functions. EC migration results from a balance of externally applied forces (e.g. shear stress), intracellular forces (e.g., those generated by contractile and cytoskeletal proteins), adhesion force between ECs and extracellular matrix (ECM) proteins, and the force of EC-EC coupling through junction proteins. Shear stress modulates EC migration through the regulation of multiple signaling pathways, gene expression, and the reorganization of cytoskeleton, focal adhesion sites, and cell junctions. Investigations of EC migration under shearing can provide valuable knowledge on vascular remodeling process under physiological and pathological conditions.

Animals↗

PubMed2005

Visualizing the mechanical activation of Src.

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

Actin Cytoskeleton↗

PubMed2005

Shear stress inhibits smooth muscle cell-induced inflammatory gene expression in endothelial cells: role of NF-kappaB.

OBJECTIVES: Vascular endothelial cells (ECs) are influenced by shear stress and neighboring smooth muscle cells (SMCs). We investigated the inflammation-relevant gene expression in EC/SMC cocultures under static condition and in response to shear stress. MATERIALS AND METHODS: Under static condition, DNA microarrays and reverse-transcription polymerase chain reaction identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by coculture with SMCs, with 18 upregulated and 5 downregulated. Application of shear stress (12 dynes/cm2) to the EC side of the coculture for 6 hours inhibited most of the proinflammatory gene expressions in ECs induced by coculture with SMCs. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced EC expression of proinflammatory genes, indicating that the NF-kappaB binding sites in the promoters of these genes play a significant role in their expression as a result of coculture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-kappaB recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-kappaB activation in ECs and monocytic THP-1 cell adhesion to ECs. CONCLUSIONS: Our findings suggest that shear stress plays an inhibitory role in the proinflammatory gene expression in ECs located in close proximity to SMCs.

Cell Adhesion↗

PubMed2005

An extracellular matrix microarray for probing cellular differentiation.

We present an extracellular matrix (ECM) microarray platform for the culture of patterned cells atop combinatorial matrix mixtures. This platform enables the study of differentiation in response to a multitude of microenvironments in parallel. The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. To demonstrate its utility, we applied this platform to study the effects of 32 different combinations of five extracellular matrix molecules (collagen I, collagen III, collagen IV, laminin and fibronectin) on cellular differentiation in two contexts: maintenance of primary rat hepatocyte phenotype indicated by intracellular albumin staining and differentiation of mouse embryonic stem (ES) cells toward an early hepatic fate, indicated by expression of a beta-galactosidase reporter fused to the fetal liver-specific gene, Ankrd17 (also known as gtar). Using this technique, we identified combinations of ECM that synergistically impacted both hepatocyte function and ES cell differentiation. This versatile technique can be easily adapted to other applications, as it is amenable to studying almost any insoluble microenvironmental cue in a combinatorial fashion and is compatible with several cell types.

Animals↗

PubMed2005

Effects of flow patterns on the localization and expression of VE-cadherin at vascular endothelial cell junctions: in vivo and in vitro investigations.

Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to fluctuating shear stress and have high endothelial permeability. Endothelial permeability is modulated by intercellular adhesion molecules such as VE-cadherin. This study was designed to elucidate the effects of different flow patterns on the localization and expression of VE-cadherin in endothelial cells (ECs) both in vivo and in vitro. VE-cadherin staining at EC borders was much stronger in the descending thoracic aorta and abdominal aorta, where the pulsatile flow has a strong net forward component than in the aortic arch and the poststenotic dilatation site beyond an experimental constriction, where the flow near the wall is complex and reciprocating with little net flow. With the use of flow chambers the effects of pulsatile flow (12 +/- 4 dyn/cm2 at 1 Hz) and reciprocating flow (0.5 +/- 4 dyn/cm2 at 1 Hz) on VE-cadherin organization in endothelial monolayers were studied in vitro. VE-cadherin staining was continuous along cell borders in static controls. Following 6 h of either pulsatile or reciprocating flow, the VE-cadherin staining at cell borders became intermittent. When the pulsatile flow was extended to 24, 48 or 72 h the staining around the cell borders became continuous again, but the staining was still intermittent when the reciprocating flow was similarly extended. Exposure to pulsatile or reciprocating flow for 6 and 24 h neither change the expression level of VE-cadherin nor its distribution between membrane and cytosol fractions as determined by Western blot and compared with static controls. These findings suggest that the cell junction remodeling induced by different flow patterns may result from a redistribution of VE-cadherin within the cell membrane. Both the in vivo and in vitro data indicate that pulsatile and reciprocating flow patterns have different effects on cell junction remodeling. The lack of junction reorganization in regions of reciprocating flow in vivo and in vitro may provide a mechanistic basis for the high permeability and the preferential localization of atherosclerosis in regions of the arterial stress with complex flow patterns and fluctuating shear stress.

Animals↗

PubMed2005

Shear stress regulates gene expression in vascular endothelial cells in response to tumor necrosis factor-alpha: a study of the transcription profile with complementary DNA microarray.

We investigate the role of shear stress in regulating the gene expression in endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNF-alpha). ECs were kept in static condition or pre-exposed to a high level (HSS, 20 dynes/cm2) or a low level of shear stress (LSS, 0.5 dynes/cm2) for 24 h, and TNF-alpha was added under static condition for 4 h. In static ECs, DNA microarray showed that TNF-alpha caused a significant increase in expression of 102 genes and a significant decrease in expression of 12 genes. Pre-shearing of ECs decreased the TNF-alpha-responsiveness of many pro-inflammatory, pro-coagulant, proliferative, and pro-apoptotic genes, whereas it increased the responsiveness of some antioxidant, anti-coagulant, and anti-apoptotic genes. LSS showed less regulatory effects than HSS on EC gene expression in response to TNF-alpha. The microarray data were confirmed by reverse-transcription polymerase chain reaction for 64 selected genes. Pre-shearing of ECs at HSS significantly inhibited the TNF-alpha-induced p65 and p50 mRNA expressions and nuclear factor-kappaB (NF-kappaB)-DNA binding activity. Inhibition of NF-kappaB activity with the p65-antisense or lactacystin under static condition blocked the expression of most of the genes that are TNF-alpha-inducible and shear stress-down-regulated. Our findings suggest that laminar shear stress serves protective functions against atherogenesis.

Cell Adhesion↗

PubMed2005

Molecular basis of the effects of shear stress on vascular endothelial cells.

Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells (ECs) are subjected to the shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular functions, e.g., proliferation, apoptosis, migration, permeability, and remodeling, as well as gene expression. The ECs use multiple sensing mechanisms to detect changes in mechanical forces, leading to the activation of signaling networks. The cytoskeleton provides a structural framework for the EC to transmit mechanical forces between its luminal, abluminal and junctional surfaces and its interior, including the cytoplasm, the nucleus, and focal adhesion sites. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows. In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.

Endothelial Cells↗

PubMed2005

Biochemistry and biomechanics of cell motility.

Cell motility is an essential cellular process for a variety of biological events. The process of cell migration requires the integration and coordination of complex biochemical and biomechanical signals. The protrusion force at the leading edge of a cell is generated by the cytoskeleton, and this force generation is controlled by multiple signaling cascades. The formation of new adhesions at the front and the release of adhesions at the rear involve the outside-in and inside-out signaling mediated by integrins and other adhesion receptors. The traction force generated by the cell on the extracellular matrix (ECM) regulates cell-ECM adhesions, and the counter force exerted by ECM on the cell drives the migration. The polarity of cell migration can be amplified and maintained by the feedback loop between the cytoskeleton and cell-ECM adhesions. Cell migration in three-dimensional ECM has characteristics distinct from that on two-dimensional ECM. The migration of cells is initiated and modulated by external chemical and mechanical factors, such as chemoattractants and the mechanical forces acting on the cells and ECM, as well as the surface density, distribution, topography, and rigidity of the ECM.

Actins↗

PubMed2005

TFAR19 gene changes the biophysical properties of murine erythroleukemia cells.

TFAR19 is a novel apoptosis-related gene and can accelerate cell apoptosis in the presence of apoptosis inducements. Here, we studied the effects of TFAR19 on some biophysical properties of mouse erythroleukemia (MEL) cells and their molecular and structural basis. After transfected with TFAR19 and apoptosis inducement, MEL revealed a high cell membrane fluidity, a decrease in resynthesis of phospholipids, an increase in the proteins/nucleic acids ratio, a relatively orderly cytoskeleton network, an impaired deformability, a low integrin aM expression, and a decrease in adhesion to endothelial cells. These findings suggest the potential of TFAR19 for antitumor cell migration, and thus for antitumor gene therapy.

Animals↗

PubMed2004

Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases.

Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm2) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm2). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm2). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes.

Animals↗

PubMed2004

Laminar flow activates peroxisome proliferator-activated receptor-gamma in vascular endothelial cells.

BACKGROUND: Steady laminar flow is atheroprotective, in part because of its antiinflammatory effects on vascular endothelial cells (ECs). We studied the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) in ECs in response to laminar flow and the associated antiinflammatory effect. METHODS AND RESULTS: Using flow channel with cultured ECs, we found that laminar flow activated the PPARgamma-mediated PPAR-responsive element (PPRE) activity and increased the mRNA encoding CD36, a PPARgamma-targeted gene. Analysis of the CD36 promoter revealed that PPRE was required for flow activation. Laminar flow induced the GAL-PPARgamma-LBD fusion protein, which suggests that flow activation of PPARgamma was ligand dependent. The pharmaceutical inhibitors of phospholipase A2 (PLA2) and cytochrome P450 epoxygenases (CYP450s) were able to block the laminar flow-activated PPARgamma. We also showed that lipid extracts from flow media contained ligands for the activation of PPARgamma in other cell types. This paracrine activation exerted antiinflammatory effects in ECs and THP-1 cells, including the suppression of cytokine-induced nuclear factor-kappaB activation and expression of intercellular adhesion molecule-1. CONCLUSIONS: Laminar flow activates endogenous PPARgamma in ECs, which is ligand dependent. The flow production of PPARgamma ligands is through the PLA2-CYP450 pathway, and the induced PPARgamma ligands exert antiinflammatory effects in several types of cells.

Animals↗

PubMed2004

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature.

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3+/-0.2-microm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (Tc = 36.4+/-0.3 degrees C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 degrees C were non-linear and showed accelerated denaturation between 35 and 39 degrees C with a midpoint at 37.2+/-0.6 degrees C. The transition was reversible below 39 degrees C and independent of solution pH (pH 6.8-7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N2 showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.

Body Temperature↗