Koichi Niwa

The Effects of a Shear Flow on the Uptake of LDL and Acetylated LDL by an EC Monoculture and an EC-SMC Coculture

To elucidate the mechanisms of localized genesis and development of atherosclerosis and anastomotic intimal hyperplasia in man, a coculture of bovine aortic endothelial cells (ECs) and smooth muscle cells (SMCs) was prepared, and the effects of a shear flow on the uptake of lipoproteins by the cells was studied by incubating the EC–SMC coculture as well as an EC monoculture with a culture medium containing either DiI-LDL or DiI-Ac-LDL and subjecting to a laminar shear flow. It was found that in both the presence and absence of a shear flow that imposed the ECs an area mean shear stress of 13.3 dynes/cm2, the uptake of LDL by an EC–SMC coculture was much greater than that by an EC monoculture, whereas that of Ac-LDL was almost the same. The uptake of LDL by an EC monoculture increased slightly by being exposed to a shear flow, whereas that by an EC–SMC coculture did not. In contrast to this, the uptake of Ac-LDL by both an EC monoculture and an EC–SMC coculture decreased drastically by a shear flow, suggesting that the action of a shear flow on the uptake of Ac-LDL by vascular cells is very different from that of LDL.

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not.

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H2O2-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H2O2 at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H2O2-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H2O2-treated cells in Western blot analysis. An H2O2-induced increase of the intracellular Ca2+ concentration ([Ca2+]i) was also observed and an intracellular Ca2+ chelator (BAPTA-AM) significantly inhibited the H2O2-induced increase of permeability. However, it showed no inhibitory effects on the H2O2-induced phosphorylation of p38 MAPK and ERK. The H2O2-induced increase of [Ca2+]i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca2+]i are essential for the H2O2-induced increase of endothelial permeability and that ERK is not.

Study on mechanism of cell damage caused by microbubbles exposed to ultrasound

To elucidate the mechanisms of cell damage caused by microbubbles exposed to ultrasound, two series of experiments were carried out. In one series of experiments, microbubbles and cells were exposed to 1-MHz pulsed ultrasound and observed using a high-speed camera. In another series of experiments, endothelial cells were exposed to ultrasound with and without microbubbles, and the viabilities of the cells were evaluated by fluorescent staining. In the high-speed images, generation of a small stream of the surrounding liquid caused by nonuniform contraction of a microbubble and deformation of a cell beside the bubble were observed. In the cell viability test, the percentage of damaged cells in the presence of microbubbles that had been exposed to ultrasound was significantly higher than that of cells without microbubbles that had been exposed to ultrasound. These results indicated that bubbles exposed to ultrasound causes mechanical stress to act on cells and that this mechanical stress may cause cell injury.

A basic study on sonoporation with microbubbles exposed to pulsed ultrasound

PurposeSonoporation is an ultrasound technique that enables large molecules that normally do not penetrate the cell membrane to pass through it. Recent studies show that pulsed ultrasound in the presence of microbubbles increases the permeability of the cell membrane. However, the mechanism and basic properties of this sonoporation remain unclear. We thus investigated the mechanism of generation and frequency of occurrence of sonoporation, as well as the repair of a cell membrane damaged by microbubbles.MethodsThe spatial relationship between microbubbles and cells was observed microscopically when cells were sonicated with pulsed ultrasound. Effects of microbubbles on the cells were observed with a high-speed camera, and the ratio of cell membrane damage and repair was examined using fluorescent microscopy.ResultsDamage to the cell membrane, caused mainly by mechanical effects of the expansion and contraction of microbubbles, significantly increased the permeability of the cell membrane. The frequency of cell membrane damage was closely associated with the presence of microbubbles and increased with increase in acoustic pressure. The ratio of repair of damaged cells was about 70% during 3u2009min after a single shot of pulsed ultrasound, indicating that repair of damaged cell membranes requires little time.ConclusionWe examined the frequency of occurrence of cell membrane damage and repair in sonoporation using pulsed ultrasound and microbubbles. Our results should prove useful for improving pulsed-ultrasound sonoporation.

Temperature Dependence of Processes Proximal and Distal to the Glucose-Induced [Ca2+]i Rise in Stimulus-Secretion Coupling in Rat Pancreatic Islets

Cooling is known to inhibit glucose-induced insulin secretion from pancreatic islets, but temperature-dependent processes in stimulus-secretion coupling remain unclear. In the present study, we examined the effects of cooling on the glucose-induced increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and concomitant insulin secretion in rat pancreatic islets to analyze the temperature dependence of processes proximal and distal to the Ca2+ signal in stimulus-secretion coupling. Rat pancreatic islets were isolated and perifused. [Ca2+]i was measured using fura-2. Glucose (15 mM) caused a triphasic [Ca2+]i response in single islets at 35 degrees C: an initial decrease and a transient increase followed by a gradual increase, on which series of Ca2+ transients were frequently superimposed. Cooling to 30 and 25 degrees C caused slower and smaller [Ca2+]i responses with a Q10 (temperature coefficient) of 1.8. Glucose caused biphasic insulin secretion at 35 degrees C, which was inhibited by cooling, with a Q10 of 11.6. The ratio of glucose-induced insulin secretion to [Ca2+]i rise (IS/Ca) was calculated to represent the efficiency of Ca2+ to cause exocytosis. The Q10 value of the ratio of IS/Ca was 6.6. The Q10 values of the ratio of IS/Ca in the responses to high K+ (30 mM), carbamylcholine (100 microM) and glibenclamide (2 microM) were 5.6, 3.8, and 13.0, respectively. These values were greater than the Q10 values of corresponding [Ca2+]i responses: 1.2, 1.4, and 1.8, respectively. From these results, we conclude that cooling inhibits not only the glucose-induced [Ca2+]i rise but also Ca(2+)-activated exocytosis, and that the latter is much more sensitive to cooling than the former.