Yasuyuki Fujiwara

Identification and Functions of Chondroitin Sulfate in the Milieu of Neural Stem Cells

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase β, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.

Cell Density-dependent Regulation of Proteoglycan Synthesis by Transforming Growth Factor-β1 in Cultured Bovine Aortic Endothelial Cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-β1(TGF-β1) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-β1on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-β1 also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [3H]glucosamine, [35S]sulfate, or35S-labeled amino acids in the presence of TGF-β1. The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M rand composition were analyzed by Sepharose CL-6B chromatography, and the core protein M r was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-β1 on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-β1 induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of ∼50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of ∼400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-β1 cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-β1 promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.

Phosphodiesterase-III inhibitor prevents hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA.

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA.

Stimulatory effect of lead on the proliferation of cultured vascular smooth-muscle cells

The effect of lead nitrate on the DNA synthesis of cultured bovine aortic smooth-muscle cells has been studied. Lead at 0.5-10 microM stimulated the incorporation of [3H]thymidine into the acid-insoluble fraction of the cells in a dose-dependent manner. However, other heavy metals including zinc, copper, manganese and nickel did not show such a stimulatory effect. Stimulation of [3H]thymidine incorporation by basic fibroblast growth factor was additive to that by lead. However, stimulation by either platelet-derived growth factor or acidic fibroblast growth factor was reduced by the metal. The leakage of lactate dehydrogenase into the medium from smooth-muscle cells, a marker of cell death, was not changed by the metal. Calcium ionophore A23187 inhibited [3H]thymidine incorporation in vascular endothelial cells but stimulated it in vascular smooth-muscle cells. These results suggest that lead may stimulate the proliferation of cultured vascular smooth-muscle cells probably via a calcium-dependent pathway; the metal may mimic calcium within the cells.

Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan, a novel sulfated polysaccharide isolated from Spirulina platensis.

Calcium spirulan (Ca-SP) is a novel sulfated polysaccharide isolated from a blue-green alga Spirulina platensis. Ca-SP inhibits thrombin by activation of heparin cofactor II. Therefore, it could serve as an origin of anti-atherogenic medicines. Since maintenance of vascular endothelial cell monolayers is important for prevention of vascular lesions such as atherosclerosis, the effect of Ca-SP at 20 microg/ml or less on the repair of wounded bovine aortic endothelial cell monolayers in culture was investigated in the present study. When the monolayers were wounded and cultured in the presence of Ca-SP, the polysaccharide inhibited the appearance of the cells in the wounded area. The inhibition was also observed even when the repair was promoted by excess basic fibroblast growth factor, which is one of the autocrine growth factors that are involved in the endothelial cell monolayer maintenance. On the other hand, Ca-SP inhibited the cell growth and the incorporation of [3H]thymidine into the acid-insoluble fraction of proliferating endothelial cells, suggesting that Ca-SP inhibits endothelial cell proliferation. From these results, it is concluded that Ca-SP may retard the repair process of damaged vascular endothelium through inhibition of vascular endothelial cell proliferation by induction of a lower ability to respond to stimulation by endogenous basic fibroblast growth factor.

Inhibitory effect of lead on the proliferation of cultured vascular endothelial cells

We investigated the effect of lead nitrate (0.5, 1.0, 2.0 or 5.0 microM) on the proliferation of cultured bovine aortic endothelial cells. After exposure to lead, the number of cells and the incorporation of [3H]thymidine into the acid-insoluble fraction of the cells were reduced in parallel in a concentration-dependent manner. Histologically, lead treatment resulted in a decrease in the cell number accompanied by a change in the cell shape from polygonal to spindle; however, no degenerative change was observed except in 5.0 microM lead-treated cells. Furthermore, stimulation of [3H]thymidine incorporation by either basic or acidic fibroblast growth factor was significantly reduced by lead. However, the leakage of lactate dehydrogenase into the medium from the cells, a marker of nonspecific cell damage, was not changed by lead. From these results, it was revealed that lead inhibits the proliferation of cultured vascular endothelial cells without nonspecific cell damage. Although lead does not destroy the monolayer of endothelial cells, the metal may exhibit its noxious effect in the repair process of the vascular endothelium.

Promotion of cultured vascular smooth muscle cell proliferation by low levels of cadmium.

Cadmium is a toxic heavy metal which has been shown to be a possible risk factor of atherosclerosis in epidemiological and experimental studies. Since intimal hyperplasia in vascular tissue is an important component of atherosclerosis, we examined the effect of cadmium on the proliferation of vascular smooth muscle cells cultured in a serum-free medium. It was found that cadmium at 100 nM or less can increase the incorporation of [3H]thymidine into the acid-insoluble fraction of growing bovine and rabbit aortic smooth muscle cells but not of growing bovine aortic endothelial cells. Although vascular smooth muscle cells are sensitive to cadmium cytotoxicity, no increase in the leakage of lactate dehydrogenase from the cells was caused by the metal at 200 nM or less in bovine aortic smooth muscle cells. Intracellular accumulation of radioactive calcium in bovine aortic smooth muscle cells was significantly increased by cadmium. It was therefore suggested that low levels of cadmium may promote the proliferation of vascular smooth muscle cells through intracellular calcium-dependent signalling pathway. The present study supports the hypothesis that cadmium can be a risk factor of atherosclerosis through dysfunction of vascular smooth muscle cells as well as vascular endothelial cells under certain conditions.

Tolerance to cadmium cytotoxicity is induced by zinc through non-metallothionein mechanisms as well as metallothionein induction in cultured cells

To investigate the relationship between metallothionein induction and zinc-induced tolerance to cadmium cytotoxicity at the cell level, Chang liver cells were pretreated with a wide range of concentrations of zinc sulfate and challenged with toxic levels of cadmium after removal of zinc. It was found that zinc-pretreated cells were significantly resistant even when the extracellular level of zinc was low and metallothionein was not induced by the metal. Pretreatment with zinc resulted in a lesser accumulation of cadmium and metallothionein after challenge with cadmium. In porcine kidney LLC-PK1 cells and bovine aortic smooth muscle cells pretreated with low levels of zinc, the intracellular accumulation of cadmium was significantly less and cadmium-induced decrease in intracellular zinc was significantly prevented without a change of metallothionein content; as the result, the toxic effect of cadmium was significantly diminished. It was therefore concluded that a tolerance to cadmium cytotoxicity is induced by zinc via non-metallothionein mechanisms such as decreasing intracellular cadmium as well as metallothionein induction at the cell level. The non-metallothionein mechanism was postulated to be particularly importatnt when the extracellular concentration of zinc is under the threshold for metallothionein induction.

Possible mechanism for lead inhibition of vascular endothelial cell proliferation: a lower response to basic fibroblast growth factor through inhibition of heparan sulfate synthesis

Although lead inhibits the proliferation of vascular endothelial cells, the mechanism has been incompletely understood. A lower response to basic fibroblast growth factor (bFGF) of growing bovine aortic endothelial cells after exposure to lead was investigated using a cell culture system in the present study. It was shown that lead significantly decreased the incorporation of [3H]thymidine into the acid-insoluble fraction of the cells but the inhibition disappeared in the presence of bFGF neutralizing antibody. Pretreatment with lead resulted in a reduction of the stimulation by exogenous bFGF on the [3H]thymidine incorporation. Lead decreased endogenous bFGF bound to cell surface heparan sulfate proteoglycans in a concentration-dependent manner but not the high affinity FGF receptor without a change of the accumulation within the cells. In spite of such a change in the endogenous bFGF distribution, the total amount of the growth factor synthesized was not significantly changed by lead. Although the binding of [125I]bFGF to heparan sulfate proteoglycans can be directly inhibited by lead, the inhibition was not so marked. On the other hand, lead markedly suppressed the incorporation of [35S]sulfate into heparan sulfate accumulated in the cell layer and the conditioned medium, suggesting that the metal inhibited the synthesis of the glycosaminoglycan in growing endothelial cells. Inhibition of the [3H]thymidine incorporation by lead was significantly restored by heparin. Since the binding of bFGF to its receptor is strongly promoted by heparan sulfate, the present data suggest that lead inhibits vascular endothelial cell proliferation by induction of a lower response to endogenous bFGF through a suppression of heparan sulfate synthesis.

Stimulation by zinc of cultured vascular endothelial cell proliferation: Possible involvement of endogenous basic fibroblast growth factor

Vascular endothelial cells from bovine aorta were cultured with zinc sulfate (20 microM or less) for 24 h and labeled with [3H]thymidine or [14C]leucine during the last 3 h of the culture. It was found that the cell number was significantly increased by zinc alone; the incorporation of both [3H]thymidine and [14C]leucine was also stimulated by the metal. The stimulation by zinc occurred in the presence or absence of exogenous basic fibroblast growth factor (bFGF). However, other heavy metals including copper, manganese and nickel did not exhibit such a stimulatory effect, suggesting that zinc plays a particular role in endothelial cell proliferation. Stimulation of the [3H]thymidine incorporation by zinc disappeared in the presence of either cycloheximide or anti-bFGF IgG; in addition, a lipoxygenase inhibitor nordihydroguaiaretic acid diminished the zinc stimulation but a cyclooxygenase inhibitor indomethacin did not exhibit such an inhibitory effect. These results indicated that zinc stimulated the proliferation of endothelial cells through the lipoxygenase pathway which mediates the stimulation by endogenous bFGF. In other words, the metal may amplify the endogenous bFGF-dependent proliferation of the cells.