Motoki Tagami

Sphingosine 1‐phosphate induces the production of glial cell line‐derived neurotrophic factor and cellular proliferation in astrocytes

Sphingosine 1‐phosphate (S1P) is a platelet‐derived bioactive sphingolipid that evokes a variety of biological responses. To understand the role of S1P in the central nervous system, we have examined the effect of S1P on the production of glial cell line‐derived neurotrophic factor (GDNF) and growth regulation of cortical astrocytes from rat embryo. Moreover, we examined the possibility that the expression of GDNF is regulated differently in cultured astrocytes from the stroke‐prone spontaneously hypertensive rat (SHRSP) than in those from Wistar kyoto rats (WKY). The mRNA expression was quantitated by RT‐PCR based on the fluorescent TaqMan methodology. A new instrument capable of measuring fluorescence in real time was used to quantify gene amplification in astrocytes. GDNF protein was investigated by enzyme‐linked immunosorbent assay. S1P induced the expression of GDNF mRNA and the production of GDNF protein in a dose‐dependent manner in WKY astrocytes. Moreover, S1P increased cell numbers and induced the proliferation of astrocytes. In addition, the level of mRNA expression and protein production of GDNF was significantly lower in SHRSP than WKY astrocytes following exposure to S1P. These findings revealed that S1P augments GDNF protein production and cellular growth in astrocytes. Also, our results indicate that production in SHRSP astrocytes was attenuated in response to S1P compared with that observed in WKY. We conclude that S1P specifically triggers a cascade of events that regulate the production of GDNF and cell growth in astrocytes. Our results also suggest that the reduced expression of GDNF caused by S1P is a factor in the stroke proneness of SHRSP. GLIA 41:199–206, 2003.

Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis.

DETAILED EXAMINATION OF CARTILAGE FORMATION and ENDOCHONDRAL OSSIFICATION USING HUMAN MESENCHYMAL STEM CELLS

1. Cartilage formation is one of the most complex processes in biology. The aim of the present study was to produce a simplified in vitro system to resolve its complexities.

Dietary polyphenols regulate endothelial function and prevent cardiovascular disease

Vascular endothelial cell (EC) dysfunction strongly induces development of cardiovascular and cerebrovascular diseases. Epidemiologic studies demonstrated a preventative effect of dietary polyphenols toward cardiovascular disease. In studies using cultured vascular ECs, polyphenols were recognized to regulate nitric oxide and endothelin-1 (ET-1) production. Furthermore, epigallocatechin-3-gallate inhibited the expression of adhesion molecules by a signaling pathway that is similar to that of high-density lipoprotein and involves induction of Ca(2+)/calmodulin-dependent kinase II, liver kinase B, and phosphatidylinositol 3-kinase expression. The effects of polyphenols on ECs include antioxidant activity and enhancement of the expression of several protective proteins, including endothelial nitric oxide synthase and paraoxonase 1. However, the observed effects of dietary polyphenols in vitro do not always translate to an in vivo setting. As such, there are many questions concerning their physiological mode of action. In this review, we discuss research on the effect of dietary polyphenols on cardiovascular disease and their protective effect on EC dysfunction.

Hypoxia-induced changes in tight junction permeability of brain capillary endothelial cells are associated with IL-1beta and nitric oxide

We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.

Differential regulation of glial cell line-derived neurotrophic factor (GDNF) mRNA expression during hypoxia and reoxygenation in astrocytes isolated from stroke-prone spontaneously hypertensive rats.

Glial cell line‐derived neurotrophic factor (GDNF) plays several important roles in the survival and recovery of mature neurons during ischemia. We examined the possibility that the expression of GDNF mRNA and the release of GDNF protein are regulated differentially in cultured astrocytes from the stroke‐prone spontaneously hypertensive rat (SHRSP) compared with those from Wistar Kyoto rats (WKY) during hypoxia and reoxygenation (H/R) and after exposure to glutamate and hydrogen peroxide (H2O2). The mRNA expression was quantitated by reverse transcription‐polymerase chain reaction (RT‐PCR) based on the fluorescent TaqMan methodology. A new instrument capable of measuring fluorescence in real‐time was used to quantify gene amplification in astrocytes. GDNF protein was investigated by enzyme‐linked immunosorbent assay (ELISA). GDNF mRNA expression and GDNF protein release at normoxia were greater in SHRSP than in WKY astrocytes. During H/R, however, the mRNA expression and protein release tended to be reduced in SHRSP compared with WKY. Glutamate and H2O2 induced the expression of GDNF mRNA and the release of GDNF protein in both WKY and SHRSP in a dose‐dependent manner. Levels of GDNF mRNA and protein in SHRSP were significantly lower than in WKY. These findings indicate that GDNF production in SHRSP astrocytes was low in response to H/R, glutamate, and H2O2, compared with that observed in WKY. We conclude that the attenuated production of GDNF in astrocytes is involved in neuronal vulnerability in SHRSP during H/R, as GDNF production, which is stimulated by glutamate and H2O2, is closely related to the protective effect against H/R‐mediated neurotoxicity. GLIA 37:1–7, 2002.

Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen

SummaryIn this study bovine aortic endothelial cells were co-cultured with astrocytes from fetal Wistar Kyoto rats. Endothelial cells growing on type-I collagen, development. Although some cells appeared to be mature, horseradish peroxidase penetrated within 1 min of incubation through the intercellular junctions of these endothelial elements maintained on type-I collagen. In contrast, endothelial cells on type-IV collagen, co-cultured with astrocytes, were well developed; their intercellular junctions were well established, and plasmalemmal vesicles reduced in number. As a result, horseradish peroxidase was unable to penetrate through the endothelial cells grown on type-IV collagen and co-cultured with astrocytes because of the reduced extent of the junctional and vesicular transport. These findings reveal that (1) type-IV collagen is essential for the differentiation of endothelial cells, (2) endothelial cell-astrocyte interactions occur during co-culture, and (3) endothelial permeability depends on astrocyte-produced factors, in addition to type-IV collagen.

The study of metal ion release and cytotoxicity in Co–Cr–Mo and Ti–Al–V alloy in total knee prosthesis – scanning electron microscopic observation

We surgically retrieved two cobalt(Co)–chromium(Cr)–molybdenum(Mo) and five titanium(Ti)–aluminum(Al)–vanadium(V) alloy knee prostheses from patients because of mechanical failure and pain. We examined the distribution of the small particles which were released from the Co–Cr–Mo and Ti–Al–V alloys using a backscattered scanning electron microscopy (SEM). In addition we analyzed the metals in the artificial knee joints and the tissues adjacent to them using energy dispersive X-ray spectroscopy (EDS). We demonstrated that a myriad of fine particles, produced by the abrasion of both Co–Cr–Mo and Ti–Al–V alloys, accumulated in the synovial cells. As Co–Cr–Mo alloys disintegrate easily in the cells, Co dissolves from the peripheral areas of them, although Cr remains within the cells. In contrast Ti–Al–V alloys are very stable in the synovial cells. From these findings we conclude that the Co–Cr–Mo alloys are hazardous to the body as the alloys release Co which enters the body. In contrast the Ti–Al–V alloys are very stable and are patently safer. Artificial joints, however, are still in considerable need of improvement.

Altered gene expressions during hypoxia and reoxygenation in cortical neurons isolated from stroke-prone spontaneously hypertensive rats

The expressions of Bcl-2, thioredoxin (TRX) and cytochrome c oxidase III (CO III) mRNAs after hypoxia and reoxygenation (H/R) were examined by quantitative reverse transcription-polymerase chain reaction using cultured cortical neurons isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY). The differences in gene expressions of Bcl-2, TRX and CO III mRNA between SHRSP and WKY were most remarkable at 30 min of oxygen stimulation, and the expressions of these genes were significantly lower in SHRSP compared with those in WKY. These findings pointed out that redox regulatory function and energy metabolism in SHRSP neurons were markedly reduced by oxygen stimulation after hypoxia, and such changes may be involved in neuronal vulnerability.

Studies of hypertension-induced vascular hypertrophy in cultured smooth muscle cells from spontaneously hypertensive rats.

Mechanisms of vascular hypertrophy induced by hypertension were studied in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) and compared with those from normotensive Wistar-Kyoto (WKY) rats. Fetal calf serum-stimulated ornithine decarboxylase (ODC) activity of cultured smooth muscle cells was greater in SHR and SHRSP than in WKY. Beta- but not alpha-adrenergic agonist stimulated ODC activity acutely in cultured smooth muscle cells from WKY, and isoprenaline-induced activation was blocked by the beta-blocker, propranolol, and enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. These results indicate that cultured vascular smooth muscle cells from SHR and SHRSP are more prone to increase the protein synthesis than those from WKY through the trophic induction of ODC activity and that the regulation of ODC activity by catecholamines is mediated through beta-agonistic effect in cultured smooth muscle cells.