Synthesis and Characterization of a Trigonal Layered Compound AgInS2.

Depending on thermal and pressure conditions, AgInS2 exhibits various crystal structures. In this study, we synthesized a high-purity polycrystalline sample of trigonal AgInS2, which is a layered compound, using a high-pressure synthesis technique. The crystal structure was investigated by synchrotron powder X-ray diffraction and the Rietveld refinement. On the basis of band calculation, X-ray photoelectron spectroscopy, and electrical resistance measurements, we found that the obtained trigonal AgInS2 is a semiconductor. Temperature dependencies of electrical resistance of AgInS2 were measured by a diamond anvil cell up to 31.2 GPa. Although semiconducting behavior was suppressed with pressure, metallic behavior was not observed within the pressure range investigated in this study.

Synthetic Route of Layered Titanium Nitride Chloride TiNCl Using Sodium Amide.

A synthetic route in a closed system for layered titanium nitride chloride TiNCl has been developed using sodium amide NaNH2 as a nitrogen source. A highly crystalline sample is obtained by an appropriate thermal decomposition of aminated titanium chloride. The obtained TiNCl was also characterized using electronic resistivity measurement and photoemission spectroscopy. TiNCl showed hopping conduction compatible with an in-gap state revealed by photoelectron spectroscopy. However, it appeared highly electron-doped, albeit without showing superconductivity. Comparison with the spectrum of superconducting sodium-doped samples suggests the presence of the microstructure required to exhibit superconductivity.

Soft x-ray irradiation induced metallization of layered TiNCl.

We have performed soft x-ray spectroscopy in order to study the photoirradiation time dependence of the valence band structure and chemical states of layered transition metal nitride chloride TiNCl. Under the soft x-ray irradiation, the intensities of the states near the Fermi level (EF) and the Ti3+component increased, while the Cl 2pintensity decreased. Ti 2p-3dresonance photoemission spectroscopy confirmed a distinctive Fermi edge with Ti 3dcharacter. These results indicate the photo-induced metallization originates from deintercalation due to Cl desorption, and thus provide a new carrier doping method that controls the conducting properties of TiNCl.

Mechanical Role of Nesprin-1-Mediated Nucleus-Actin Filament Binding in Cyclic Stretch-Induced Fibroblast Elongation.

The intracellular mechanical link tethering the nucleus to the cytoskeleton has been suggested to be the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Previous studies have reported that knockdown of nesprin-1, a component of the LINC complex that directly binds to actin filaments, suppresses cellular morphological response to mechanical stimuli. The relation between nesprin-1 knockdown and cellular morphological changes, however, remains unclear. In this study, we examined the mechanical role of nucleus-actin filament binding in morphological changes of fibroblasts exposed to cyclic stretching. After exposure to 10% cyclic stretching for 6 h, fibroblasts transfected with nesprin-1-specific small interfering RNA showed fewer elongated shapes compared with non-transfected cells. To further examine the mechanical role of the nucleus and nucleus-bound actin filaments, we applied cyclic stretching to fibroblasts treated with Trichostatin A (TSA), which decreases nuclear stiffness and thereby reduces nucleus-binding actin filament tension. TSA-treatment was found to induce more rounded cellular shapes than those of non-treated cells under both static and cyclic stretching conditions. These results suggest that the tension of nucleus-bound actin filaments plays an important role in the formation of elongated fibroblasts under cyclic stretching and that nesprin-1 knockdown causes a decrease of tension in nucleus-associated actin filaments.

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain.

Accumulating evidence implicates the significance of the physical properties of the niche in influencing the behavior, growth and differentiation of stem cells. Among the physical properties, extracellular stiffness has been shown to have direct effects on fate determination in several cell types in vitro. However, little evidence exists concerning whether shifts in stiffness occur in vivo during tissue development. To address this question, we present a systematic strategy to evaluate the shift in stiffness in a developing tissue using the mouse embryonic cerebral cortex as an experimental model. We combined atomic force microscopy measurements of tissue and cellular stiffness with immunostaining of specific markers of neural differentiation to correlate the value of stiffness with the characteristic features of tissues and cells in the developing brain. We found that the stiffness of the ventricular and subventricular zones increases gradually during development. Furthermore, a peak in tissue stiffness appeared in the intermediate zone at E16.5. The stiffness of the cortical plate showed an initial increase but decreased at E18.5, although the cellular stiffness of neurons monotonically increased in association with the maturation of the microtubule cytoskeleton. These results indicate that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. Taken together, our method profiles the stiffness of living tissue and cells with defined characteristics and can therefore be utilized to further understand the role of stiffness as a physical factor that determines cell fate during the formation of the cerebral cortex and other tissues.

Prostaglandin I2 induces apoptosis via upregulation of Fas ligand in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Pulmonary vascular remodeling with idiopathic pulmonary arterial hypertension (IPAH) is associated with impaired apoptosis of pulmonary artery smooth muscle cells (PASMCs). We have reported that high-dose prostaglandin I2 (PGI2) therapy markedly improved hemodynamics in IPAH patients. The therapy is thought to reverse vascular remodeling, though the mechanism is unclear. The aim of this study is to assess proapoptotic effects of PGI2 on PASMCs obtained from IPAH patients. METHODS: We investigated proapoptotic effects of PGI2 in PAH-PASMCs by TUNEL assays, caspase-3,-7 assays and transmission electron microscopy. We examined the expression of Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, in PAH-PASMCs. We measured the serum FasL levels in IPAH patients treated with PGI2. RESULTS: TUNEL-positive, caspase-3, 7-active cells and fragmentation of the nucleus were detected in PAH-PASMCs treated with PGI2. The percentage of apoptotic cells induced by PGI2 at a high concentration was higher than that induced by PGI2 at a low concentration. PCR-array analysis revealed that PGI2 upregulated the FasL gene in PAH-PASMCs, and we measured the FasL expression by quantitative RT-PCR and Western blotting. PGI2 significantly increased the mRNA level of FasL by 3.98 fold and the protein level of FasL by 1.70 fold. An IP receptor antagonist inhibited the induction of apoptosis, elevation of cyclic AMP and upregulation of FasL by PGI2. Serum FasL level had a significant positive correlation with PGI2 dose in IPAH patients treated with PGI2. CONCLUSIONS: PGI2 has proapoptotic effects on PAH-PASMCs via the IP receptor and upregulation of FasL.

Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic ß-adrenergic stimulation.

BACKGROUND: Left ventricular (LV) hypertrophy is often present in patients with diastolic heart failure. However, stiffness of hypertrophied cardiomyocytes in the transverse direction has not been fully elucidated. The aim of this study was to assess passive cardiomyocyte stiffness of hypertrophied hearts in the transverse direction and the influence of actin-myosin cross-bridge formation on the stiffness. METHODS AND RESULTS: Wistar rats received a vehicle (control) or isoproterenol (ISO) subcutaneously. After 7 days, compared with the controls, ISO administration had significantly increased heart weight and LV wall thickness and had decreased peak early annular relaxation velocity (e') assessed by echocardiography. Elastic modulus of living cardiomyocytes in the transverse direction assessed by an atomic force microscope was significantly higher in the ISO group than in controls. We added butanedione monoxime (BDM), an inhibitor of actin-myosin interaction, and blebbistatin, a specific myosin II inhibitor, to the medium. BDM and blebbistatin significantly reduced the elastic modulus of cardiomyocytes in the ISO group. X-ray diffraction analysis showed that the reflection intensity ratio (I((1,0))/I((1,1))) at diastole was not different before and after treatment with BDM, which induces complete relaxation, in control hearts, but that I((1,0))/I((1,1)) was significantly increased after BDM treatment in the ISO group, indicating residual cross-bridge formation in hypertrophied hearts. CONCLUSIONS: Passive cardiomyocyte stiffness in the transverse direction is increased in hearts with ISO-induced hypertrophy and this is caused by residual actin-myosin cross-bridge formation.

Live-cell visualization of the trans-cellular mode of monocyte transmigration across the vascular endothelium, and its relationship with endothelial PECAM-1.

In response to atherogenic stimuli, blood monocytes transmigrate across the vascular endothelium not only through endothelial cell-cell junctions (para-cellular) but also through endothelial cells themselves (trans-cellular). The molecular mechanism of the latter is mostly unknown, because it rarely happens, especially in vitro. Although many reports have recognized trans-cellular migration from snapshot images of leukocytes halfway across the endothelium at non-junctional locations, it often produces a false-positive result, because some leukocytes that initiate trans-cellular migration withdraw and return to the apical endothelial surface. Thus, analyzing the entire process is essential. In this study, complete monocyte trans-cellular migration was successfully captured for live cells, with simultaneous visualization of endothelial PECAM-1. We suggest the possible existence of both PECAM-1-related migration at peri-junctional sites and PECAM-1-unrelated migration at sites remote from junctions. This is the first report to describe the entire process of monocyte trans-cellular migration for live cells and its relationship with endothelial PECAM-1.

Monocyte trans-endothelial migration augments subsequent transmigratory activity with increased PECAM-1 and decreased VE-cadherin at endothelial junctions.

BACKGROUND: Although the importance of monocyte trans-endothelial migration in early atherogenesis is well recognized, it is unclear whether and how one transmigration event affects endothelium to facilitate subsequent ones. In this study, we tested the hypothesis that monocyte transmigration alters endothelial junctional organization to facilitate subsequent transmigration. METHODS AND RESULTS: When human monocytes were added twice at intervals of ≈30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50% of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments). CONCLUSIONS: These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.

Altered nano/micro-order elasticity of pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a disease characterized by progressively increased resistance of pulmonary arteries. In this study, we evaluated the mechanical property of single pulmonary artery smooth muscles cells (PASMC) from patients with IPAH and tested whether the PASMC showed abnormal response to a vasodilator by use of an atomic force microscope (AFM). METHODS: PASMC were isolated and cultured from explanted lungs of 7 patients with IPAH (IPAH-PASMC). Normal vascular specimens from 3 patients with bronchogenic carcinoma were used as normal controls (normal PASMC). The nano/micro-order elasticity of five to ten living PASMC in each sample was measured by parabolic force curves of cantilever deflection/indentation obtained by using an AFM. The elasticity measurements were performed under control conditions and under condition of nitric oxide (NO) treatment (190 and 380 nmol/L). RESULTS: There was no significant difference between nano/micro-order elasticity of normal PASMC and that of IPAH-PASMC under the control conditions. In normal PASMC, NO (190 and 380 nmol/L) significantly reduced (i.e., softened) the nano/micro-order elasticity. However, NO did not reduce elasticity in IPAH-PASMC, indicating higher vasodilator-resistive nano/micro-order rigidity in IPAH-PASMC. CONCLUSION: Nano/micro-order elasticity change in PASMC in response to vasodilation induced by NO is reduced in patients with IPAH.

Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins.

Recently, sphingosine 1-phosphate (S1P) has been highlighted as an endothelial barrier-stabilizing mediator. FTY720 is a S1P analog originally developed as a novel immunosuppressant. The phosphorylated form of FTY720 binds to S1P receptors to exert S1P-like biological effects, suggesting endothelial barrier promotion by FTY720. To elucidate whether FTY720 induces signaling events related to endothelial barrier enhancement under hyperglycemic conditions, human microvascular endothelial cells (HMVECs) preincubated with hyperglycemic (30 mM) medium were treated with 100 nM FTY720 for 3 h. Immunofluorescent microscopy and coprecipitation study revealed FTY720-induced focal adhesion kinase (FAK)-associated adherens junction (AJ) assembly at cell-cell contacts coincident with formation of a prominent cortical actin ring. FTY720 also induced transmonolayer electrical resistance (TER) augmentation in HMVEC monolayers in both normoglycemic and hyperglycemic conditions, implying endothelial barrier enhancement. Similar to S1P, site-specific FAK tyrosine phosphorylation analysis revealed FTY720-induced FAK [Y576] phosphorylation without phosphorylation of FAK [Y397/Y925]. Furthermore, FTY720 conditioned the phosphorylation profile of FAK [Y397/Y576/Y925] in hyperglycemic medium to the same pattern observed in normoglycemic medium. FTY720 challenge resulted in small GTPase Rac activation under hyperglycemic conditions, whereas increased Rho activity in hyperglycemic medium was restored to the basal level. Rac protein depletion by small interfering RNA (siRNA) technique completely abolished FTY720-induced FAK [Y576] phosphorylation. These findings strongly suggest the barrier protective effect of FTY720 on HMVEC monolayers in hyperglycemic medium via S1P signaling, further implying the possibility of FTY720 as a therapeutic agent of diabetic vascular disorder.

Gene expression of endothelial cells due to interleukin-1 beta stimulation and neutrophil transmigration.

During the inflammatory response, endothelial cell (EC) functions and mechanics change dramatically. To understand these responses, the authors analyzed changes in EC gene expression in an in vitro model of inflammation using cDNA microarrays. After interleukin-1 beta (IL1beta) stimulation, over 2500 genes were differentially expressed, of which approximately 2000 had not been previously identified by microarray studies of IL1beta stimulation in human umbilical vein endothelial cells (HUVECs). Functional grouping of these genes according to gene ontologies revealed genes associated with apoptosis, cell cycle, nuclear factor (NF)-kappa B cascade, chemotaxis, and immune response. Interestingly, claudin-1, known to exist in endothelial cell-cell junctions was up-regulated, but claudin-5 and occludin, which also exist in EC junctions, were down-regulated. Pre-b-cell colony enhancing factor (PBEF), a cytokine which may play a role in regulating endothelial permeability, was also up-regulated following IL1beta stimulation. Neutrophil transmigration across IL1beta-stimulated ECs did not induce changes in EC gene expression as strongly as IL1beta stimulation alone. Nineteen genes after 1 h and 22 genes after 3 h of neutrophil application were differentially expressed. These results indicate that, in terms of transcriptional effects on ECs, neutrophil transmigration is a relatively small perturbation in comparison to the background of large scale changes induced in ECs by cytokine stimulation. Supplementary materials are available for this article. Go to the publisher's online edition of Endothelium for the following free supplementary resources: supplementary figures and tables.

The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes.

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.

Edaravone mimics sphingosine-1-phosphate-induced endothelial barrier enhancement in human microvascular endothelial cells.

Edaravone is a potent scavenger of hydroxyl radicals and is quite successful in patients with acute cerebral ischemia, and several organ-protective effects have been reported. Treatment of human microvascular endothelial cells with edaravone (1.5 microM) resulted in the enhancement of transmonolayer electrical resistance coincident with cortical actin enhancement and redistribution of focal adhesion proteins and adherens junction proteins to the cell periphery. Edaravone also induced small GTPase Rac activation and focal adhesion kinase (FAK; Tyr(576)) phosphorylation associated with sphingosine-1-phosphate receptor type 1 (S1P(1)) transactivation. S1P(1) protein depletion by the short interfering RNA technique completely abolished edaravone-induced FAK (Tyr(576)) phosphorylation and Rac activation. This is the first report of edaravone-induced endothelial barrier enhancement coincident with focal adhesion remodeling and cytoskeletal rearrangement associated with Rac activation via S1P(1) transactivation. Considering the well-established endothelial barrier-protective effect of S1P, endothelial barrier enhancement as a consequence of S1P(1) transactivation may at least partly be the potent mechanisms for the organ-protective effect of edaravone and is suggestive of edaravone as a therapeutic agent against systemic vascular barrier disorder.

[Confocal microscopy].

Fluorescence-based molecular imaging has long and widely been used in the field of biomedical research, and now has become an indispensable tool. The development of confocal microscopy in the 1980s was a major breakthrough in the field. It can optically slice biological samples along the optical axis, which had been impossible with the conventional fluorescent microscopy. In this review, we first discuss the basic principle, and advantages of the confocal microscopy, and next describe the recent topics on the improved microscopy including high-speed confocal microscopy, FRET(fluorescence resonance energy transfer), FRAP (fluorescence recovery after photobleaching), and two photon excitation microscopy. Finally, we illustrate some examples of the biomedical applications of the confocal microscopy.

Oxidized LDL specifically promotes the initiation of monocyte invasion during transendothelial migration with upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions.

It is poorly understood how oxidized LDL (oxLDL) promotes monocyte dynamics in transendothelial migration (TEM) in atherogenesis. We developed an in vitro 3D-live-single cell TEM assay system with subendothelial oxLDL embedded in ultra-thin collagen gels, mimicking subendothelial oxLDL accumulation in vivo. With dividing monocyte dynamics into three stages (1: adhesion on endothelium, 2: invasion and 3: complete transmigration below endothelium), we analyzed the stage transition dynamics of individual living human monocytes. OxLDL did not enhance initial monocyte adhesion to endothelium (stage 1), but it specifically primed adherent monocytes to start invasion (stage 1-->2). Once invasion started, it had no effect thereafter on monocyte stage transition (stage 2-->3). OxLDL upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions without monocyte addition, both of which could promote monocyte entry by enhanced homophilic binding to monocyte PECAM-1, and by disrupted junctional barrier, respectively. Meanwhile, monocyte speed at neither locomotion on endothelium (stage 1) nor subendothelial migration (stage 3) was altered by oxLDL. These data indicate that before monocyte adhesion, endothelial junctions changed their conformation to more monocyte-acceptable state in response to oxLDL, resulting the stage-specific promotion of monocyte TEM (stage 1-->2; initiation of invasion) with no enhancement of its initial adhesion or migration speed.

Oxidative stress produced with cell migration increases synthetic phenotype of vascular smooth muscle cells.

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle alpha-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased alpha-actin.

Involvement of aldosterone and mineralocorticoid receptors in rat mesangial cell proliferation and deformability.

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [3H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho-ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 micromol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 micromol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.

Direct observation and quantitative analysis of spatiotemporal dynamics of individual living monocytes during transendothelial migration.

OBJECTIVE: To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM). METHODS AND RESULTS: We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30-50 microm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 +/- 5.4 min (mean +/- S.D., n=61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 +/- 3.2 min (mean +/- S.D., n=53 monocytes). CONCLUSIONS: Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.