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Abstract-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 [ 3 H]-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 mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased Key Words: mineralocorticoids Ⅲ aldosterone T he utility of mineralocorticoid receptor (MR) antagonists in renal injury has been suggested in preclinical and clinical studies. 1-12 MR blockade had no effect on systemic blood pressure but markedly ameliorated glomerular injury in stroke-prone spontaneously hypertensive rats 3 and rats treated with angiotensin II (Ang II) and an NO synthase inhibitor, 4 cyclosporine A 5 or radiation. 6 In patients with chronic renal failure 7 and early diabetic nephropathy, 8 addition of a nonselective MR antagonist, spironolactone, to angiotensinconverting enzyme (ACE) inhibitors had no hemodynamic effects but markedly reduced the urinary protein excretion rate (U protein V). For hypertensive patients, it has also been indicated that monotherapy with spironolactone 9 or a selective MR antagonist, eplerenone, 10 is more effective than ACE inhibitors in reducing U protein V. Furthermore, White et al 11 showed that in hypertensive patients, eplerenone has a similar blood pressure-lowering effect to a calcium antagonist, amlodipine, but reduced the urinary albumin-to-creatinine ratio to a greater extent than amlodipine. Thus, these observations support the notion that MR blockade has renoprotective effects through mechanisms that cannot be simply explained by hemodynamic changes.We demonstrated recently that chronic administration of aldosterone to rats induced glomerular injury characterized by mesangial matrix expansion and cell overgrowth. 12 We also observed that the aldosterone-induced glomerular injury was prevented by treatment with eplerenone. These results in...

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.

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An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

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The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-tosubstrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1␤-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.A t the early stage of atherosclerosis, an aggregation of lipid-rich macrophages that were derived from monocytes was observed in the intima (1). Adhesion of monocytes to the arterial endothelial cells and their migration into the arterial intima are the earliest key events in atherogenesis (1). Previous studies demonstrated that leukocytes could migrate throughout the endothelial monolayer via not only paracellular but also transcellular pathways in vivo and in vitro (2-5). The endothelial cell micromechanics that are involved in endothelial cell micromotion and the mechanical properties of endothelial cells may be key factors in these processes. Earlier studies reported the importance of adhesion molecules in the adhesion and migration of monocytes to endothelial cells (6, 7). We previously demonstrated that the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) induces the decrease in the amount of focal adhesion kinase (p125 FAK ) with reduction of the density of F-actin stress fibers in HUVEC (8). These results suggest that the adhesion of monocytes induces changes of the adhesiveness of endothelial cells to the substrate and the mechanical properties of endothelial cells. However, the accompanying micromechanics and micromotion of endothelial cells were little understood.For the micromotion measurement of the cultured cells, Giaever and Keese (9) developed a morphological biosensor, the electric cell-substrate impedance sensing (ECIS) system. The advantage of this system is that quantitative estimation of cell-to-cell and cell-to-substrate distances can be performed separately and in real time. Atomic force micros...

The effect of flow direction on the morphology of cultured bovine aortic endothelial cells is studied. Fully confluent endothelial cells cultured on glass were subjected to a fluid-imposed shear stress of 2 Pa for 20 min and 24 h using a parallel plate flow chamber. Experiments on shear flow exposure were performed for (i) one-way flow, (ii) reciprocating flow with a 30 min interval and (iii) alternating orthogonal flows with a 30 min interval. After flow exposure, the endothelial cells were fixed and F-actin filaments were stained with rhodamine phalloidin. Endothelial cells were observed and photographed by means of a microscope equipped with epifluorescence optics. The shape index (SI) and angle of cell orientation were measured, and F-actin distributions in the cells were statistically studied. Endothelial cells under the one-way flow condition showed marked elongation (SI = 0.39 +/- 0.16, mean +/- S.D.) and aligned with the flow direction. In the case of the reciprocating (SI = 0.63 +/- 0.14) and the alternating orthogonal flows (0.64 +/- 0.14), cells did not elongate so strongly as in the case of one-way flow. Although most cells in the reciprocating flow aligned with the flow direction, the cell axes in the alternate orthogonal flow distributed around a mean value of -45.1 degrees with a large S.D. value. Endothelial cells can be expected to recognise the flow direction, and change their shape and F-actin structure.