Peculiar flow patterns of RBCs suspended in viscous fluids and perfused through a narrow tube (25 microm).

Red blood cells (RBCs) generally deform to adopt a parachute-like, torpedo-like, or other configuration to align and flow through a capillary that is narrower than their major axis. As described herein, even in a narrow tube (25 microm) with diameter much larger than that of a capillary, flowing RBCs at 1 mm/s align axially and deform to a paraboloid shape in a viscous Newtonian fluid (505 kDa dextran medium) with viscosity of 23.4-57.1 mPa.s. A high-speed digital camera image showed that the silhouette of the tip of RBCs fits a parabola, unlike the shape of RBCs in capillaries, because of the longer distance of the RBC-free layer between the tube wall and the RBC surface ( approximately 8.8 microm). However, when RBCs are suspended in a "non-Newtonian" viscous fluid (liposome-40 kDa dextran medium) with a shear-thinning profile, they migrate toward the tube wall to avoid the axial lining, as "near-wall-excess," which is usually observed for platelets. This migration results from the presence of flocculated liposomes at the tube center. In contrast, such near-wall excess was not observed when RBCs were suspended in a nearly Newtonian liposome-albumin medium. Such unusual flow patterns of RBCs would be explainable by the principle; a larger particle tends to flow near the centerline, and a small one tends to go to the wall to flow with least resistance. However, we visualized for the first time the complete axial aligning and near-wall excess of RBCs in the noncapillary size tube in some extreme conditions.

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties.

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.

Prevention of ischemic neuronal death by intravenous infusion of a ginseng saponin, ginsenoside Rb(1), that upregulates Bcl-x(L) expression.

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

Triglyceride in plasma: prospective effects on microcirculatory functions.

The effect of triglyceride in plasma on RBC aggregation was examined, and the prospective influence on the flow of RBCs in microcirculation and the O2 release was discussed. To minimize the individual differences, blood samples were collected from one subject 2 hrs after high-fat and low fat meals. Triglyceride content in plasma was measured by an enzymatic method, and the rate of rouleaux formation was measured with a low shear rheoscope. The rate of rouleaux formation was increased with the increase of triglyceride concentration. Our previous findings suggested some functional impairment in microcirculation. (1) The enhanced RBC aggregation tends to reduce flow resistance in arterioles, but results in inhomogeneous flow of RBCs in capillaries. (2) The sclerotic change of microvessels alters flow behavior of RBCs, and thereby flow resistance is increased. (3) The enhanced RBC aggregation reduces O2 release from RBCs flowing in microvessels. In conclusion, high triglyceride level in plasma not only changes flow behavior of RBCs in microcirculation and thus increases flow resistance, but also prevents homogeneous tissue oxygenation.

Connective tissue growth factor is released from platelets under high shear stress and is differentially expressed in endothelium along atherosclerotic plaques.

Connective tissue growth factor (CTGF) is overexpressed in atherosclerotic blood vessels. To further investigate the role of CTGF in atherosclerosis, we examined whether CTGF is released from platelets by high shear stress, and whether the expression of CTGF along the atherosclerotic lesions depends on local hemodynamic conditions. Human platelets were subjected to 10 dyn/cm2 or 120 dyn/cm2 and analysed by Western blotting. Furthermore, longitudinal sections of 25 carotid plaques were immunohistochemically analysed for the endothelial expression of CTGF. A very low CTGF amount was secreted from platelets at low shear stress (11.4 +/- 3.9% of total CTGF in platelets). On the contrary, high shear stress caused a markedly increased CTGF release from platelets (29 +/- 13.8%, p = 0.07 vs low shear stress, n = 4). Immunohistochemical analyses showed that the mean numbers of CTGF-positive endothelial cells were significantly higher up-stream as compared with down-stream regions of the luminal surface of atherosclerotic vessels (21.3 +/- 3.6 vs 13.9 +/- 2.8 down-stream, p < 0.001). Moreover, in plaques undergoing intimal neovascularization, newly formed vessels accumulated particularly in up-stream parts of the lesions. In conclusion, this study demonstrated that CTGF is released from platelets by high shear stress. Furthermore, disturbed flow along atherosclerotic vessels may induce endothelial CTGF expression and contribute to the progress of atherosclerotic lesions.

Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3beta cascade.

Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease pathology. Mice lacking Reelin (Reln), double-knockouts lacking the VLDL receptor (VLDLR) and ApoE receptor2 (ApoER2), and mice lacking disabled-1 (Dab1) display increased levels of phosphorylated tau. Because Reln binds to recombinant ApoE receptors, assembly of a Reln/ApoE-receptor/Dab1 (RAD) complex may initiate a signal transduction cascade that controls tau phosphorylation. Conversely, disruption of this RAD complex may increase tau phosphorylation and lead to neurodegeneration. To substantiate this concept, we mated Reln-deficient mice to ApoE-deficient mice and found that in the absence of Reln, tau phosphorylation increased as the amount of ApoE decreased. Paralleling the change in tau phosphorylation levels, we found that GSK-3beta activity increased in Reln-deficient mice and further increased in mice lacking both Reln and ApoE. CDK-5 activity was similar in mice lacking Reln, ApoE, or both. GSK-3beta and CDK-5 activity increased in Dab1-deficient mice, independent of ApoE levels. Further supporting the idea that increased tau phosphorylation results primarily from increased kinase activity, the activity of two phosphatases was similar in all conditions tested. These data support a novel, ligand-mediated signal transduction cascade--initiated by the assembly of a RAD complex that suppresses kinase activity and controls tau phosphorylation.

Suppressive effects of phosphodiesterase type IV inhibitors on rat cultured microglial cells: comparison with other types of cAMP-elevating agents.

We investigated the effects of inhibitors of cAMP-specific phosphodiesterase type IV (PDE IV) on cultured rat microglial cells. Microglial cells expressed mRNA encoding PDE IV. Rolipram and RO-20-1724, specific inhibitors of PDE IV, elevated the intracellular cAMP level much higher than the other types of PDE inhibitors. cAMP in astrocytes but not in cerebrocortical neurons was similarly increased in response to treatment with PDE IV inhibitors examined. The PDE IV inhibitors, a beta-adrenergic agonist isoproterenol and an adenylyl cyclase stimulant forskolin suppressed the proliferation of microglial cells as revealed by PCNA-immunocytochemical staining. The PDE IV inhibitors suppressed release of TNF alpha and nitric oxide (NO) from lipopolysaccharide-activated microglial cells in pure culture, while they did not affect NO release from microglial cells in neuron-microglia coculture. The PDE IV inhibitors also suppressed superoxide anion production by phorbol ester-treated microglial cells. Isoproterenol and forskolin similarly suppressed the macrophage-like functions of activated microglial cells. However, the PDE IV inhibitors displayed novel effects distinct from those of isoproterenol, forskolin and 8Br-cAMP, regarding expression of mRNAs encoding PDE IV, metallothionein-1 and hemeoxigenase-1. The present data showed that the PDE IV inhibitors can be available to control microglial function and that their effects on glial cells should be taken into account when PDE IV inhibitors are used for treatment of brain diseases, such as multiple sclerosis.

Effects of norepinephrine on rat cultured microglial cells that express alpha1, alpha2, beta1 and beta2 adrenergic receptors.

Microglial cells rapidly become activated in response to even minor damage of neurons, suggestive of the intimate interactions between neurons and microglial cells. Although mediators for microglia-neuron interactions have not been well identified, neurotransmitters are possible candidates transmitting signals from neurons to microglial cells. Among the neurotransmitters, we focused on the effects of norepinephrine and other adrenergic agonists on the functions of rat cultured microglial cells. Reverse transcriptase polymerase chain reaction studies revealed that microglial cells expressed mRNAs encoding alpha1A, alpha2A, beta1 and beta2 receptors. Norepinephrine and a beta2 adrenergic agonist terbutaline elevated intracellular cAMP level of microglial cells. Norepinephrine, an alpha1 agonist phenylephrine, a beta1 agonist dobutamine and terbutaline suppressed the expressions of mRNAs encoding pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor alpha. Release of tumor necrosis factor alpha and nitric oxide was suppressed by norepinephrine, phenylephrine, dobutamine and terbutaline. An alpha2 agonist clonidine and dobutamine upregulated the expression of mRNA encoding catechol-O-methyl transferase, an important enzyme to degrade norepinephrine. Norepinephrine, dobutamine and terbutaline upregulated the expressions of mRNA encoding 3-phospshoglycerate dehydrogenase, an essential enzyme for synthesis of L-serine and glycine, which are amino acids necessary for neuronal survival. Clonidine upregulated the expression of mRNA encoding an anti-apoptotic factor Bcl-xL. These results suggest that norepinephrine participates in the regulation of brain function at least partly by modulating the functions of microglia.

Reduced oxygen release from erythrocytes by the acceleration-induced flow shift, observed in an oxygen-permeable narrow tube.

The oxygen release from flowing erythrocytes under accelerational force (0-4 g) was examined using an oxygen-permeable, fluorinated ethylenepropylene copolymer tube (25 microm in inner diameter). The narrow tube was fixed vertically on the rotating disk of a new centrifuge apparatus, and erythrocyte suspension was perfused in the direction of Earth gravity. The accelerational force was applied perpendicularly to the flow direction of cells by centrifugation. The microscopic images of the flowing cells obtained at five different wavelengths were analyzed, and marginal cell-free layer and oxygen saturation of the cells were measured. By lowering oxygen tension around the narrow tube, erythrocytes were deoxygenated in proportion to their traveling distance, and the deoxygenation was enhanced with decreasing flow velocity and hematocrit. With increase of the g-value, the shift of flowing erythrocyte column to the centrifugal side was increased, the column was compressed, and the oxygen release from the cells was suppressed. Qualitatively, similar results were obtained by inducing erythrocyte aggregation with Dextran T-70 (MW = 70,400), without accelerational force. These results conclude that both the accumulation of erythrocytes under accelerational force and the enhancement of erythrocyte aggregation by macromolecules lead to the reduction of oxygen release from the flowing cells.

Reelin signals survival through Src-family kinases that inactivate BAD activity.

Reelin plays an important role in the migration of embryonic neurons, but its continuing presence suggests additional functions in the brain. We now report a novel function where reelin protects P19 embryonal cells from apoptosis during retinoic acid-induced neuronal differentiation. This increased survival is associated with reelin activation of the phosphatidyl-inositol-3-kinase (PI3 K)/Akt pathway. When PI3 K was inhibited with LY294002, reelin failed to protect against this retinoic acid-induced apoptosis. The protective effect of reelin includes activating the Src-family kinases/PI3 K/Akt pathway which then led to selective phosphorylation of Bcl-2/Bcl-XL associated death promoter (BAD) at serine-136, while the phosphorylation-incompetent mutation of BAD (S136A) suppressed this protection. These and additional studies define a novel pathway where reelin binds apoE receptors, significantly activates the PI3 K/Akt pathway causing phosphorylation of BAD which helps to protect cells from apoptosing, thus serving an important role in promoting the survival of maturing neurons in the brain.

Neutrophil-activating activity and platelet-activating factor synthesis in cytokine-stimulated endothelial cells: reduced activity in growth-arrested cells.

The reactivity of endothelial cells (ECs) to proinflammatory cytokines is critically important for the pathogenesis of vascular diseases. Here, we studied functional alterations of human ECs during culture under a confluent condition; i.e., the alterations of neutrophil-activating activity, platelet-activating factor (PAF) synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in cytokine-stimulated ECs. Human umbilical vein-derived ECs exhibited the increased activity in neutrophil activation, PAF synthesis, and GM-CSF production when stimulated by proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). The activity of cytokine-stimulated ECs to stimulate superoxide release in human neutrophils and to produce PAF declined markedly in parallel as ECs became growth-arrested during culture under a confluent condition. By contrast, GM-CSF production induced by cytokine stimulation was modestly increased, and up-regulation of intercellular adhesion molecule-1 (ICAM-1) and activation of mitogen-activated protein kinases were not altered. The neutrophil-activating activity of cytokine-stimulated ECs was dependent on PAF synthesis and GM-CSF production from ECs. These findings indicate that the reduced neutrophil-activating activity in growth-arrested ECs may be, at least in part, ascribed to down-regulation of PAF synthesis.

Changes of RBC aggregation in oxygenation-deoxygenation: pH dependency and cell morphology.

The effects of the oxygenation-deoxygenation process on red blood cell (RBC) aggregation were examined in relation to morphological changes in RBCs and the contribution of CO(2). A low-shear rheoscope was used to measure the rate of rouleaux (one-dimensional aggregate) formation in diluted autologous plasma exposed to gas mixtures with different Po(2) and Pco(2). RBC indexes and RBC suspension pH were measured for the oxygenated or the deoxygenated condition, and the cell shape was observed with a scanning electron microscope. In the oxygenation-deoxygenation process, the rate of rouleaux formation increased with rising pH of the RBC suspension, which was lowered in the presence of CO(2). The rate increased with increasing mean corpuscular hemoglobin concentration (thus the cells shrank), which increased with rising pH and decreased in the presence of CO(2). With rising pH, cell diameter increased and cell thickness decreased (thus the cell flattened). In addition, slight echinocytosis was induced in the presence of CO(2), and the aggregation was reduced by the morphological change. In conclusion, RBC aggregation in the oxygenation-deoxygenation process is mainly influenced by the pH-dependent change in the surface area-to-volume ratio of the cells, and the aggregation is modified by CO(2)-induced acidification and the accompanying changes in mean corpuscular hemoglobin concentration and cell shape.

L-serine-mediated release of apolipoprotein E and lipids from microglial cells.

Apolipoprotein E (ApoE), one of the genetic risk factors for Alzheimer's disease, is considered to have a critical role in transporting lipids in the brain. In the present study, we investigated ApoE release in primary rat microglial cultures. Microglial cells released ApoE in response to L-Ser in culture medium, and ApoE-immunoreactivity was detected in granules in the cell periphery and in perinuclear structures. Immunocytochemical studies, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR) results all supported the notion that microglial cells are the potential source of ApoE in the brain. L-Ser enhanced ApoE release in a concentration-dependent manner without upregulating ApoE mRNA expression. Astrocytes presumably enhanced production and release of ApoE by microglial cells through secretion of L-Ser. As revealed by gel chromatography, ApoE was secreted as a component of lipoproteins, and L-Ser enhanced release of cholesterol and triglycerides together with ApoE. Activation of microglial cells by lipopolysaccharides and serum resulted in an overall decrease of the ApoE release. These findings suggest that microglial cells are a significant source of lipoproteins containing ApoE in the brain under physiological conditions, and that L-Ser is an important mediator of the neuron-astrocyte-microglia network in the brain.

O2 release from Hb vesicles evaluated using an artificial, narrow O2-permeable tube: comparison with RBCs and acellular Hbs.

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5'-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2 carrying ability to plasma expanders. The O2 release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 microm) exposed to a deoxygenated environment. Measurement of O2 release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2 release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2 release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2 release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2 transport.

NFkappaB regulates plasma apolipoprotein A-I and high density lipoprotein cholesterol through inhibition of peroxisome proliferator-activated receptor alpha.

The levels of plasma HDL cholesterol and apoA-I in NFkappaB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFkappaBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFkappaB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IkappaBalpha super-repressor ameliorated the reduction. This IkappaBalpha-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), suggesting that NFkappaB inactivation induces apoA-I through activation of PPARalpha. To further support this idea, the expression of IkappaBalpha increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARalpha-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFkappaB increases apoA-I and HDL cholesterol through activation of PPARalpha in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease.

Testosterone up-regulates aquaporin-4 expression in cultured astrocytes.

Aquaporin-4 (AQP4) is located on astrocyte endfeet that face blood vessels in the brain and in the pia. It is thought to play a crucial role in the development of brain edema. To confirm the notion that sex steroids and dexamethasone influence brain edema through AQP4 regulation, we investigated the effects of 17beta-estradiol, testosterone, and dexamethasone on the expression of AQP4 in cultured astrocytes. Testosterone significantly up-regulated AQP4 at the level of both protein and mRNA. At a concentration of 100 nM, testosterone significantly increased AQP4 protein levels and ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Moreover, this effect was attenuated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate, which can rapidly decrease AQP4 mRNA expression, indicating that the response was specific. These results indicate that AQP4 can alter the osmotic fragility of astrocytes and that testosterone can influence brain edema through AQP4 regulation, whereas 17beta-estradiol and dexamethasone cannot.

Two populations of microglial cells isolated from rat primary mixed glial cultures.

Because of variations in the morphology and function of microglial cells, it has often been claimed that microglial cells should be classified into two or more subtypes. However, such subtypes have not fully been characterized. In the present study, we isolated microglial cells expressing microglia-markers CD11b and CD68 from rat mixed glial cultures on the fifth and on the thirteenth days in vitro (DIV 5 and 13) and demonstrate that these two populations of microglial cells have distinct morphology and function. Microglial cells isolated on DIV 5, which we have termed immature cells, are characterized by the presence of large somata, large peroxidase- and alkaline phosphatase-positive granules, and high proliferative activity and suppressed responsiveness to lipopolysaccharide (LPS). In contrast, the microglial cells isolated on DIV 13, which we have termed mature cells, are devoid of granules, appear to be in a state of cell cycle arrest, and respond to LPS by the induction of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha, and interleukin-6. Isolated immature cells maintained in pure culture failed to express iNOS in response to LPS. However, if these cells were cultured on astrocyte-derived extracellular matrix (AsECM) or pure laminin, the cells exhibited an induction of iNOS in response to LPS. AsECM and laminin were also able to induce a state of cell cycle arrest in cultured isolated immature cells. Thus, classification into two types of microglial cells is possible, but both types are in the same cell lineage, because the immature cells can differentiate into mature microglial cells in the presence of laminin or AsECM. Therefore, "microglioblasts" may be the appropriate term for the immature cells.