Noriko Goda

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.

Evaluation of micromotion of vascular endothelial cells in electrical cell-substrate impedance sensing (ECIS) method using a mathematical model

We proposed a mathematical model for the microdynamics of cultured cells measured with ECIS (Electrical Cell-substrate Impedance Sensing) system that can separately evaluate cell-to-cell and cell-to-substrate gaps. Our mathematical model is composed of culture medium impedance between cells (Zsol), cell impedance (Zc), and polarization impedance of the electrode (Zp). Zsol consists of the resistance between cells (Rsol) and the capacitance between cells (Csol) of the culture medium. In particular, Rsol is the resistance component related to the cell-to-cell distance (A). Zc consists of capacitance of the cell membrane (Cc) and resistance of the cell membrane (Rc). Zp depends on the cell-to-substrate distance (h) because of the shielding effect of cells to the electrode. The shielding effect is defined as shielding coefficient (Sk). We examined the changes in the impedance of the electrode without or with cells in various conditions. The electrical characteristics of the electrode with or without cells agreed well with those measured in ECIS system. It was found that whether A or h caused the changes in the impedance could be determined based on the changes in the total resistance and reactance (capacitance); A mainly affects the total resistance value, and h mainly affects total capacitance value. Therefore, we can simply estimate the changes in cell-to-cell and cell-to-substrate gaps with measured total resistance and reactance (capacitance). Based on these results, when the cultured endothelial cells (HUVEC) were stimulated with estrogen for 40 hours, it was proved that the cell-to-cell distances decreased, even though the cell-to-electrode distances slightly increased. This result suggests that the barrier function of endothelium is fortified by estrogen.

Quantitative Evaluation of Micro-motion of Vascular Endothelial Cells in Electrical Cell-substrate Impedance Sensing (ECIS) Method Using a Precision Mathematical Model

We constructed a precision mathematical model for quantitative evaluation of the micro-dynamics of cultured cells measured with ECIS (electrical cell-substrate impedance sensing) system that could separately estimate cell-to-cell distance and cell-to-substrate distance. For wide applications of this method, we constructed mathematical models describing for some kinds of cell and some kinds of confluent conditions. The mathematical models of these impedances could be fit well especially from 1 kHz to 10 kHz, which were most interesting frequency range for micro-dynamic analysis in ECIS methods

Evaluation of micromotion of vascular endothelial cells with estrogen in electrical cell-substrate impedance sensing (ECIS) method using a mathematical model cell-to-cell distance and cell-to-substrate distance

We proposed a mathematical model for the micro-dynamics of cultured cells measured with ECIS (Electrical Cell-substrate Impedance Sensing system. Our mathematical model is composed of culture medium impedance between cells (Z/sub sol/), cell impedance (Z/sub c/), and polarization impedance of the electrode (Z/sub p/). It was found that whether cell-to-cell or cell-to-substrate gaps caused the changes in the impedance could be determined based on the changes in the total resistance and reactance (capacitance). Cell-to-cell gaps mainly affects the total resistance value. Cell-to-substrate gaps mainly affects total capacitance value. Based on these results, when the cultured endothelial cells (HUVEC) were stimulated with estrogen for 40 hours, it was proved that the cell-to-cell distances decreased, even though the cell-to-substrate distances slightly increased. This result suggests that the barrier function of endothelium is fortified by estrogen.

Evaluating object and region of concentric electrode in bio-electrical impedance measurement

Concentric electrode is easy to use and used widely for measuring bio-electrical impedance. But, its evaluating region was not investigated in detail. Then, the characteristics of concentric electrode were studied from various points of view. In case of use without electrode paste, impedance is determined with the contacting condition between electrode and skin surface over all frequency range. In case of use with electrode past, impedance is composed of stratum corneum in the frequency range of 20 Hz-1 kHz and is mainly composed of subcutaneous tissue in the range of 200 kHz-1 MHz. In the high frequency range, evaluating region of concentric electrode is the area less than the radius or the gap of center electrode.

Evaluation of the electrical cell-substrate impedance sensing system (ECIS) method using a mathematical model

We produced a mathematical model for the electrical characteristics of the culture solution, cells and electrode interface according to measurements of the ECIS electrode system, and with this method, the impedance without cells and the impedance with cells in various confluent stages could be accurately reproduced by selecting parameters in this model.

Quantitative evaluation of nano-order micromotion of cultured cells using electric cell-substrate impedance sensing method

Electric cell-substrate impedance sensing (ECIS) system can detect electrically micro-motion of the cell cultured in the gold electrode, originally developed by I. Giaever et al. The ECIS system makes data continuously on resistance and capacitance values change in electric impedance by the cell micro-motion at real time. Human umbilical vein endothelial cells were cultured on the ECIS electrode, and measured after chemical substance (estrogen) stimulation electric impedance over 40 hours. A parameter analyzed cellular motion by using the precise mathematical model simulated cell electric impedance based on the Cole-Cole model. This mathematical model agreed with the experiment value over wide frequency range 25 Hz to 60 kHz. Impedance of the mathematical model in the ECIS system varied with the change in cell-cell distance A and cell-substrate distance h in the motion of the cultured cell. The parameter for the distance of A and h was defined as SA and Sh to do the simple and easy evaluation of the cell micro-motion. Furthermore, the actual dimension evaluation of A, h was tired from these parameters. A change in impedance following estrogen stimulation was analyzed by using the mathematical model, and changes in A and h were estimated.

Quantitative Evaluation of Effect for Radiation Exposure to Cultured Cells Using Electrical Cell-substrate Impedance Sensing (ECIS) Method

We proposed a precision mathematical model for quantitative evaluation of micro-dynamics of cultured cells measured with ECIS (Electrical Cell-substrate Impedance Sensing) based on equivalent electrical circuit that could separately estimate cell-to-cell distance and cell-to-substrate distance. The model was composed of three parts, the cell impedance, culture medium electrolyte impedance between cells in perpendicular direction to electrode, and polarization impedance of the electrode. The cell impedance, mainly cell membrane impedance is formed by the equation of Cole-Cole dispersion. We measured the frequency characteristics 25 Hz to 60 kHz of impedance of BAEC (bovine aortic endothelial cell), in full confluent condition of each cell with ECIS. The mathematical models of these impedances could be fit well especially from 1 kHz to 10 kHz, which were most interesting frequency range for micro-dynamic analysis in ECIS method. Based on this precision model, we could propose the evaluation method of the cell-to-cell distance A and the cell-to-substrate distance h. Namely, in order that the micro-motions of A and h can be analyzed easily by vector impedance change based on the mathematical model, we introduced new parameters S A and S h. If vector impedance value is obtained in each condition, parameter values correspond to the vector impedance variation can be instantly determined and evaluations of micro-motion of the cells can be performed. If A decreases, S A decreases because culture medium resistance R sol increases and if h decreases, S h decreases because polarization impedance Z 0 increases. We investigated the effect of X-ray radiation exposure from 1 Gy to 100 Gy to the cultured cell BAEC by ECIS. The impedance change could be confirmed from just after X-ray exposure. The X-ray stimulation of 100 Gy resulted in the large scale of increase in the cell-to-cell distance A and the slight decrease in the cell-to-electrode distances h.