Claudia Seul

Green tea compounds inhibit tyrosine phosphorylation of PDGF β-receptor and transformation of A172 human glioblastoma.

The effect of the green tea compounds 2‐(3,4‐dihydroxyphenyl)‐3,4‐dihydro‐2H‐1‐benzopyran‐3,5,7‐triol (catechin), epicathechin (EC), epigallocathechin‐3 gallate (EGCG), epicathechin‐3 gallate (ECG) and catechin‐3 gallate (CG) on the tyrosine phosphorylation of PDGF β‐receptor (PDGF‐Rβ) and on the anchorage‐independent growth of A172 glioblastoma cells in semisolid agar has been investigated. Treatment of A172 glioblastoma with 50 μM CG, ECG, EGCG and 25 μM Tyrphostin 1296 resulted in an 82±17%, 77±21%, 75±8% and 55±11%, respectively (mean±S.D., n=3) inhibition of the PDGF‐BB‐induced tyrosine phosphorylation of PDGF‐Rβ. The PDGF‐Rβ downstream intracellular transduction pathway including tyrosine phosphorylation of phospholipase C‐γ1 (PLC‐γ1) and phosphatidylinositol 3′‐kinase (PI 3′‐K) was also inhibited. Spheroid formation was completely inhibited by 50 μM ECG, CG, EGCG and by 25 μM Tyrphostin 1296. We conclude that catechins of the green tea possessing the gallate group in their chemical structure act as anticancer agents probably partly via their ability to suppress the tyrosine kinase activity of the PDGF‐Rβ.

Lysophosphatidic acid and intracellular signalling in vascular smooth muscle cells

Growth of vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of atherosclerosis and hypertension. Lysophosphatidic acid (LPA), a natural phospholipid is thought to be an important VSMC mitogen and has recently been suggested to play an important role in the development of vascular disease. In the present study, we describe the effects of LPA on intracellular signalling pathways in VSMC. LPA (5 micrograms/ml) induced an increase of cytosolic free calcium concentration ([Ca2+]i) in the presence and absence of extracellular Ca2+ and markedly stimulated the Na+/H+ exchanger. LPA dose-dependently caused a stimulation of the 42-kDa mitogen-activated protein kinase (MAP kinase) isoform with a maximum at 5 min. Also, LPA induced a 5-fold increase in [3H]thymidine incorporation into cell DNA above the basal value, as well as a 42% increase in cell number. Pretreatment of VSMC with pertussis toxin (PTX) (100 ng/ml) for 24 h markedly blunted the LPA-dependent intracellular signalling transduction including the increase in [Ca2+]i, activation of the Na+/H+ exchanger, activation of MAP kinase and the increase in cell DNA synthesis. These findings demonstrate that the effects of LPA on intracellular signalling transduction pathway as well as on VSMC growth are mediated by PTX-sensitive guanosine triphosphate (GTP) binding protein (Gi protein).

Lysophosphatidic acid stimulates protein kinase C isoforms α, β, ε, and ζ in a pertussis toxin sensitive pathway in vascular smooth muscle cells

Abstract The natural phospholipid lysophosphatidic acid (LPA) has been characterized as an important vascular smooth muscle cell (VSMC) mitogen whose effects are mainly mediated by pertussis toxin (PTX)-sensitive guanosine triphosphate (GTP)-binding protein (Gi-protein). Protein kinase C (PKC) isoforms play an important role in intracellular signaling cascades and in growth of VSMC. In the present study we investigated the effect of LPA on activation of PKC isoforms α, β, e, and ζ in VSMC by Western blot of cytosolic and membrane fractions. Furthermore, we examined the role of PKC activation on LPA-induced growth of VSMC using PKC inhibitor 19-27. Stimulation of VSMC by 5 μg/mL LPA for 10 min increased the amount of PKC α, β, e, and ζ in the particulate fraction by 689%, 285%, 424%, and 510%, respectively, and returned to control level after 30 min. Correspondingly, the amount of PKC α, β, e, and ζ in the cytosolic fraction decreased by 32%, 94%, 44%, and 95%, respectively, compared to control. Furthermore, we could show that LPA-induced activation of PKC α, β, e, and ζ isoforms was PTX sensitive. Incubation of VSMC with nonspecific PKC inhibitor 19-27 (10 μmol/L) for 24 h resulted in a 30% inhibition of LPA-induced DNA synthesis as measured by [3H]thymidine incorporation. In conclusion, in VSMC LPA stimulated translocation of PKC isoforms α, β, e, and ζ in a PTX-sensitive manner. Furthermore stimulation of PKC might be critically involved in LPA-induced mitogenesis in VSMC.

Biotransformation of glyceryl trinitrate by blood platelets as compared to vascular smooth muscle cells

The present study investigated the metabolism of glyceryl trinitrate by washed human platelets as compared to that by rat vascular smooth muscle cells. Possible changes in metabolism after induction of nitrate tolerance were also studied in both systems. Incubation of the cells with glyceryl trinitrate (0.1 mM) resulted in a time-dependent release of nitrite (NO2-) amounting to 6.30 +/- 0.63 nmol mg protein-1 h-1 in vascular smooth muscle cells and 0.61 +/- 0.08 nmol mg protein-1 h-1 for platelets, respectively. The nitric oxide (NO) scavenger, oxyhemoglobin (10 microM), significantly reduced NO2- generation in both cell types studied. Nitrate tolerance was induced by incubation of the cells with glyceryl trinitrate (2 mM) for 2 h. In tolerant vascular smooth muscle cells as well as in tolerant platelets, NO2- release was significantly reduced. The inhibitory capacity of glyceryl trinitrate on ADP (6 microM)-induced platelet aggregation and on intracellular Ca2+ signals was significantly reduced in tolerant platelets. The data show a direct metabolism of glyceryl trinitrate by human blood platelets which is subject to a type of tolerance development similar to that in vascular smooth muscle cells.

Early intracellular signalling pathway of ethanol in vascular smooth muscle cells

ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 (p44mapk and p42mapk) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects on growth and on early intracellular events normally induced by growth factors in VSMCs. Measurement of cytosolic Ca2+ and pH in cell monolayers was performed using fura‐2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [3H]‐thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c‐fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo‐[2‐3H]‐inositol and separation of inositolphosphates by HPLC. Ethanol (0.3–1.0% v v−1, 17–170 mM) induced a dose‐dependent maximal stimulation of p44mapk/p42mapk at 30 min and expression of c‐fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca2+]i, activation of the Na+/H+ exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3–5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol.

The role of platelet-derived growth factor-BB-induced increase in cytosolic free Ca2+ in activation of mitogen-activated protein kinase and DNA synthesis in vascular smooth muscle cells

Background Platelet derived growth factor (PDGF)-BB is an important vascular smooth muscle cell (VSMC) mitogen. PDGF-BB induces an increase in intracellular free calcium concentration ([Ca2+]i), an activation of mitogen-activated protein (MAP) kinase and an increase in DNA synthesis. The increase in [Ca2+]i is thought to be an important second messenger in the intracellular signalling cascade, leading to growth of VSMC. Objective The aim of the present study was to elucidate the role of the PDGF-BB-induced increase in [Ca2+]i in the activation of MAP kinase and increase in DNA synthesis. Binding of [Ca2+]i was performed by the intracellular chelator bis-(2-amino-5-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). Methods Ca2+ levels were measured by the Fura-2 method. MAP kinase activation was determined by Western blotting. DNA synthesis was determined by measurement of incorporation of [3H]-thymidine into the cell DNA. Results Administration of 50 ng/ml PDGF-BB induced an increase in [Ca2+]i, an activation of MAP kinase and an increase in DNA synthesis. In bis-(2-amino-5-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester (MAPTAM)-treated cells the PDGF-BB-induced effect on [Ca2+]i was totally blunted, whereas no effect on MAP kinase activation and DNA synthesis could be observed. Conclusions These findings show that the effect of PDGF-BB on MAP kinase activation is independent of calcium level. [Ca2+]i might be implicated in the PDGF-BB-induced mitogenic process only in conjugation with other signalling components.

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.

Effects of authentic and VLDL hydrolysis‐derived fatty acids on vascular smooth muscle cell growth

There are contradictory findings regarding the effects of free fatty acids on vascular smooth muscle cell (VSMC) growth. In the present study we investigated the effects of fatty acids released from hydrolysis of human VLDL triglycerides by lipoprotein lipase and of the fatty acids most abundant in the hydrolysed VLDL, namely oleic, linoleic, palmitic and myristic acid, all non albumin‐bound, on VSMC growth. The effect of fatty acids on VSMC growth was assessed by [3H]‐thymidine incorporation, colourimetrically, by cell counting, by determination of the cytoplasmic histone‐associated DNA fragments and the caspase 3 activity. The fatty acid concentrations were determined by gas chromatography‐mass spectrometry. Stimulation of ERK1/2 and p38 was determined by the chemiluminescence Western blotting method. Incubation of VSMC with purified VLDL (100 μg ml−1) and lipoprotein lipase (35 u ml−1) led to almost complete cell death although the ERK1/2 and the p38 MAP kinases were stimulated. The EC50 of oleic, linoleic, myristic and palmitic acid were 4.6±1.3, 2.4±0.2, 116±10 and 287±30 μM, respectively. The estimated EC50 of myristic and palmitic acid when derived from hydrolysed VLDL were 10 and 8 times, respectively, lower than when used alone. Apoptosis was not involved in the fatty acid‐induced VSMC growth suppression/death. We conclude that (a) non albumin‐bound fatty acids cause VSMC necrosis in a dose‐dependent manner with a parallel ERK1/2 and p38 stimulation, (b) unsaturated fatty acids are more toxic to VSMC than saturated, and (c) saturated fatty acids are more toxic to VSMC in the hydrolysed VLDL than when used individually.

Cholesterol Enhances Platelet-Derived Growth Factor-BB-Induced [Ca2+]i and DNA Synthesis in Rat Aortic Smooth Muscle Cells

In the present study, we describe possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular diseases. Treatment of rat aortic smooth muscle cells for 20 hours with cholesterol-rich liposomes (500 micrograms/mL cholesterol, 100 micrograms/mL low-density lipoprotein) resulted in a 76 +/- 12% increase in total cholesterol content. The effects of cholesterol enrichment were examined by determination of changes in cell membrane fluidity. Fluidity of the cholesterol-enriched cell membranes was decreased at all temperatures between 15 degrees C and 40 degrees C. Changes in membrane fluidity in whole cell membranes represented changes in fluidity of microsomal membranes isolated by Percoll gradient ultracentrifugation. The basal [Ca2+]i and the maximal platelet-derived growth factor (PDGF)-BB-induced [Ca2+]i was elevated by 30% and 90% in cholesterol-enriched cells, respectively. In contrast, the resting pH, and the PDGF-BB-induced stimulation of the Na+/H+ exchange were not affected in cholesterol-enriched cells. The effect of PDGF-BB on [3H]thymidine incorporation in cholesterol-enriched cells was elevated by 40% in comparison with untreated cells. Our findings show that cellular cholesterol may be involved in the development of vascular diseases via modulation of the PDGF-induced increase in [Ca2+]i and DNA synthesis in vascular smooth muscle cells.

Effect of the Na+/H+ antiport inhibitor Hoe 694 on the angiotensin II-induced vascular smooth muscle cell growth.

1 Hoe 694 (3‐methylsulphonyl‐4‐piperidinobenzoyl)guanidine methanesulphonate) was characterized as a new, potent, non‐amiloride inhibitor of the Na+/H+ exchanger. In order to elucidate the role of the Na+/H+ exchanger isoform 1 (NHE‐1) in the regulation of vascular smooth muscle cell growth, we investigated the effects of different amiloride analogues and of Hoe 694 on angiotensin II‐induced cell growth. Since intracellular pH, the intracellular free Ca2+ concentration and the expression of the transcription factor c‐fos seem to be involved in the regulation of cell growth, the effects of the amiloride analogues and Hoe 694 on the angiotensin II‐induced changes in these three parameters were examined. 2 Measurement of cytosolic Ca2+ and pH in cell monolayers was performed using fura‐2/AM and BCECF/AM, respectively. The effect of angiotensin II on cell growth was examined using (1) [3H]‐thymidine incorporation, (2) the bromo‐2‐deoxyuridine (BrdU) immunfluorescence assay, (3) the colorimetric determination of cell mitochondrial dehydrogenase activity and (4) determination of cell number. Total RNA was extracted from cells by the guanidinium isothiocyanate/CsCl procedure. The expression of c‐fos was quantitated by Northern blotting. 3 Various amiloride analogues inhibited the angiotensin II‐induced stimulation of the Na+/H+ exchanger, the increase in cytosolic Ca2+ and cell growth but not the induction of c‐fos mRNA. Hoe 694 (1–25 μm) dose‐dependently inhibited the angiotensin II‐induced stimulation of the Na+/H+ exchanger but had no significant effects on cytosolic Ca2+, c‐fos mRNA levels or cell growth. 4 Our findings support the concept that activation of the Na+/H+ exchanger is not essential for angiotensin II‐induced vascular smooth muscle cell growth.