Takeshi Karino

Honeycomb-patterned thin films of amphiphilic polymers as cell culture substrates

Abstract Recently, we have found honeycomb patterns with sub-micron line width that form during the non-equilibrium process of cast film formation. The honeycomb-patterned films were fabricated using four macromolecular compounds (amphiphilic copolymers containing lactose units or carboxyl groups as side-chains and polyion complexes composed of anionic polysaccharides). The specific binding of lactose by lectin confirmed that the lactose moieties contained in the honeycomb films work as biologically active ligands. Bovine serum albumin (BSA) labeled with fluorescein was covalently attached to the honeycomb films using water-soluble carbodiimide (WSC) as an activator. Using fluorescence imaging of the modified film, we could show that the proteins are immobilized on the honeycomb patterns. Adhesion of bovine aorta endothelial cells (ECs) to the honeycomb films indicates that the honeycomb structure works as an adhesive site for the cells.

Mesoscopic patterning of cell adhesive substrates as novel biofunctional interfaces

In this report, cell adhesion to honeycomb-patterned films is described with respect to the dimensions of the honeycomb structure. The honeycomb-patterned films can be fabricated by casting a dilute solution of amphiphilic polymers on solid substrates. The honeycomb structure is not homogeneous in all dimensions. Analysis of distribution of the honeycomb hole sizes demonstrates a gradual decrease in honeycomb hole diameter along the radius of the cast area. The largest holes were located near geometric center of the cast area. The diameter of the largest honeycomb holes in the cast area could be controlled by varying the cast volume of the polymer solution. Cell cultures on the honeycomb films demonstrated that cell adhesion could be inhibited at the outer region of the cast area. The extent of the inhibition of cell adhesion was influenced by the chemical properties of the polymers constituting the honeycomb films.

Theoretical prediction of low-density lipoproteins concentration at the luminal surface of an artery with a multiple bend.

AbstractTo elucidate the mechanisms of localization of atherosclerotic lesions in man, the effects of various physical and hemodynamic factors on transport of atherogenic low-density lipoproteins (LDL) from flowing blood to the wall of an artery with a multiple bend were studied theoretically by means of a computer simulation under the conditions of a steady flow. It was found that due to a semipermeable nature of an arterial wall to plasma, flow-dependent concentration polarization of LDL occurred at the luminal surface of the vessel, creating a region of high LDL concentration distal to the apex of the inner wall of each bend where the flow was locally disturbed by the formation of secondary and recirculation flows and where wall shear stresses were low. The highest surface concentration of LDL occurred distal to the acute second bend where atherosclerotic intimal thickening developed. At a Re0=500, the values calculated using estimated diffusivities of LDL in whole blood and plasma were respectively 35.1% and 15.6% higher than that in the bulk flow. The results are consistent with our hypothesis that the localization of atherosclerotic lesions results from the flow-dependent concentration polarization of LDL which creates locally a hypercholesterolemic environment even in normocholesterolemic subjects, thus augmenting the uptake of LDL by vascular endothelial cells existing at such sites.

Particle flow behavior in models of branching vessels. II. Effects of branching angle and diameter ratio on flow patterns.

To further elucidate the role of fluid mechanical factors in the localization of atherogenesis and thrombogenesis, we have studied the 3-dimensional flow patterns in square T-junctions with branching angles theta from 30 degrees to 150 degrees and diameter ratios d/D (side: main tube) from 1.05/3.0 to 1.0. Cine films of the motions of tracer microspheres in dilute suspensions were taken at inflow Reynolds numbers from 15 to 400 and flow ratios (main: side tube) from 0.1 to 4.0. Flow patterns with suspension entering through the main tube were similar to those previously described in uniform 3 mm diameter T-junctions: paired vortices (spiral secondary flows) symmetrical about the common median plane formed at the entrances of the main and side daughter tubes. Particles circulated through the main vortex, some crossing above and below the mainstream into and through the side vortex. At the geometrical flow ratio, the main vortex became smaller and smaller as the branching angle (theta less than 90 degrees) and diameter ratio decreased, and was confined to a thin side tube was a minimum. In obtuse angle T-junctions the stagnation point shifted from the flow divider into the side tube, enhancing the flow disturbance there. The velocity distributions in main and side tubes were skewed towards the inner walls close to the flow divider. When flow entered through the side tube, a pair of recirculation zones formed in the main tube at the inner wall of the bend with a sharper angle.

Flow visualization in isolated transparent natural blood vessels

A novel technique to prepare isolated transparent natural blood vessels was developed and used to study the detailed flow patterns in some regions of the circulation. It was found that paired spiral secondary flows and recirculation zones form downstream of venous valves, at the arterial T-junctions in the dog abdominal aorta, and in the human carotid sinus over a wide range of geometrical and flow conditions, including the time-averaged mean values of the physiological flow rates.

Flow patterns and distributions of fluid velocity and wall shear stress in the human internal carotid and middle cerebral arteries.

BACKGROUND The aim of this study is to elucidate the relationship between the flow patterns and the preferred sites of the development of atherosclerotic lesions and cerebral aneurysms in the human ICA and MCA. METHODS Five isolated transparent arterial trees containing the ICA and MCA with a sufficient length of the carotid siphon were prepared from humans postmortem, and flow patterns and distributions of fluid velocity and wall shear stress in these vessels were studied in detail using flow visualization and high-speed cinemicrographic techniques. RESULTS In the carotid siphon that contained several acute bends, due to the impingement and deflection of the flow at the bends, a strong and complex helicoidal flow formed. As a result, the approaching velocity profile was flattened at the terminal bifurcation of the ICA, but it was sharpened at the first bifurcation of the MCA. Thus, at this latter bifurcation, fluid elements impinged on the vessel wall around the flow divider with much larger velocity than that at the preceding terminal bifurcation of the ICA. Throughout the entire arterial tree, atherosclerotic lesions were found almost exclusively in regions of low wall shear stress. CONCLUSIONS The carotid siphon provided a flattened approaching velocity profile at the terminal bifurcation of the ICA, making the hemodynamic stresses (pressure, tension, and shear stress) exerted on the vessel wall much lower than that at the bifurcation of the MCA where the approaching velocity profile was sharpened. This may account for the relatively low incidence of aneurysm formation at this site.

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.

Roles of protein kinase C delta in the accumulation of P53 and the induction of apoptosis in H2O2-treated bovine endothelial cells.

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H 2 O 2 and investigated the roles of protein kinase C i (PKC i ) and Ca 2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H 2 O 2 caused the accumulation of p53, which was inhibited by rottlerin (a PKC i inhibitor) but not by BAPTA-AM (an intracellular Ca 2+ chelator). PKC i itself was activated through the phosphorylation at tyrosine residues. H 2 O 2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKC i contributes to the accumulation of p53 and that Ca 2+ plays a role in downstream signals of p53 leading to apoptosis in H 2 O 2 -treated BAEC.

Flow patterns and preferred sites of intimal thickening in end-to-end anastomosed vessels.

BACKGROUND Fluid mechanical factors are suspected to be involved in the pathogenesis and localization of intimal hyperplasia in anastomosed vessels. However, no direct correlation has been established between the flow and the exact sites of intimal hyperplasia. Hence we have studied the relationship between the flow and preferred sites of wall thickening in 90-degree- and 45-degree-cut and end-to-end anastomosed vessels. METHODS Twenty-five 90-degree and twenty-five 45-degree end-to-end anastomoses were performed on the femoral arteries of 17 adult mongrel dogs. The vessels were harvested at 3 months after operation, fixed at 100 mm Hg, dehydrated with ethanol, and rendered transparent by immersing them in methyl salicylate. Exact locations and sizes of intimal thickening and characteristics of the flow prevailing at sites of anastomoses were studied in detail by means of flow visualization and cinemicrographic techniques. RESULTS It was found that a perfect correlation exists between the preferred sites of intimal thickening and the regions of slow recirculation flows with low wall shear stresses. In both 90-degree and 45-degree anastomosed vessels, intimal thickening developed only in those vessels in which formation of slow recirculation flows was observed. It was also found that although a pronounced and localized intimal thickening developed in 45-degree anastomosed vessels, the degree of circumferential constriction caused by both surgical procedures and development of intimal thickening was much milder in 45-degree than 90-degree anastomosed vessels. CONCLUSIONS The results suggest that key hemodynamic factors involved in the localization of intimal thickening in end-to-end anastomosed vessels are low velocity of flowing blood and the resultant low shear stresses acting on the vessel wall.

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.