Gangliosides GM1 and GM2 induce vascular smooth muscle cell proliferation via extracellular signal-regulated kinase 1/2 pathway.

Gangliosides, sialic acid-containing glycophospholipids, accumulate in atherosclerotic vessels and appear to regulate the proliferation of various cell types. Furthermore, vascular smooth muscle cell (VSMC) proliferation is associated with the development and progression of cardiovascular diseases. To demonstrate whether gangliosides are able to modulate the VSMC growth, the effect of gangliosides GM1, GM2, and GM3 on cell DNA synthesis and cell number has been examined. Moreover, we investigated possible intracellular mechanisms by which GM1 and GM2 elicit their mitogenic effects. Stimulation of VSMCs with GM1 and GM2 resulted in a dose-dependent increase in DNA synthesis and cell number, whereas GM3 caused a decrease in DNA synthesis. GM1 and GM2 (50 micromol/L) stimulate phosphorylation of extracellular signal-regulated kinases (ERKs) 1 and 2 and phosphorylation of the c-Jun N-terminal kinase (JNK), with a maximum at 15 minutes, but they do not have an effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). GM3 (50 micromol/L), on the other hand, does not stimulate any of the 3 aforementioned MAPKs. Pretreatment of the cells with 20 micromol/L PD 098,059 caused a complete inhibition of ERK1/2 and JNK MAPK, whereas pretreatment with a Ras (farnesyl transferase) inhibitor did not abrogate the GM1- and GM2-induced ERK1/2 phosphorylation. Furthermore, GM1 and GM2 did not activate Raf-1 kinase. Interestingly, pretreatment of VSMCs with 100 nmol/L pertussis toxin resulted in a complete inhibition of the ERK1/2 phosphorylation. Finally, the GM1- and GM2-induced increase in cell number was significantly inhibited by PD 098,059. We may conclude that GM1 and GM2 stimulate ERK1/2 via a pertussis toxin-sensitive G(i)-coupled receptor through a Raf-1 kinase-independent pathway. Moreover, the GM1- and GM2-induced VSMC growth is ERK1/2 dependent.

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.

Troglitazone and rosiglitazone inhibit the low density lipoprotein-induced vascular smooth muscle cell growth.

Troglitazone (TRO) and rosiglitazone (RSG) belong to the thiazolidinedione class (insulin-sensitizing agents) and exert many of their metabolic effects as peroxisome proliferator-activated receptor gamma (PPARgamma) ligands. In the present study we examined the effects of TRO and RSG on LDL-induced VSMC growth. Pretreatment of VSMC with 1 microM TRO or 0.1 microM RSG completely blocked the LDL-induced cell proliferation as measured by [3H]thymidine incorporation into DNA and by determination of the cell number. We then examined with Western blotting whether these growth suppressing effects are mediated through the mitogen-activated protein kinase (MAPK) pathway, a common signaling pathway activated by growth factors. TRO and RSG had no effect on the LDL-induced stimulation of the MAP kinases ERK1/2, p38 and SAP/JNK. We conclude that thiazolidinediones are potent inhibitors of LDL-induced VSMC growth acting downstream of the cytoplasmic activation of MAPK.

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 [(3)H]-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 microg 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 EC(50) of oleic, linoleic, myristic and palmitic acid were 4.6+/-1.3, 2.4+/-0.2, 116+/-10 and 287+/-30 microM, respectively. The estimated EC(50) 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.

Troglitazone and rosiglitazone induce apoptosis of vascular smooth muscle cells through an extracellular signal-regulated kinase-independent pathway.

Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands thiazolidinediones have been reported to induce apoptosis in macrophages and in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis in VSMC, cultured rat VSMC were treated with increasing doses of the thiazolidinedione analogues troglitazone (TRO) and rosiglitazone (RSG). Both ligands induced cell death in a concentration-dependent manner (EC50 12.1+/-3.3 microM and 1.43+/-0.39 microM, respectively), causing almost complete cell death at the highest concentrations (100 microM and 10 microM for TRO and RSG, respectively), along with an expected parallel decrease in [3H]thymidine uptake into cell DNA (EC50 6.7+/-2.4 microM and 0.75+/-0.19 microM, respectively). The cell count was determined by the coulter counter principle. Furthermore two apoptotic markers were measured, the caspase 3 activity and the cytoplasmic histone-associated DNA fragments, both of which were significantly increased when the aforementioned high concentrations were used. This indicates that apoptosis is involved in the TRO- and RSG-induced VSMC growth suppression. The same concentrations of TRO and RSG caused an unexpected stimulation of the extracellular signal-regulated response kinases 1 and 2 (ERK1/2) and stimulated the p38 mitogenic-activated protein (MAP) kinase as determined by Western blotting. In order to establish whether the proapoptotic effects of TRO and RSG are mediated through ERK1/2 activation, we used the selective MAP kinase kinase (MEK) inhibitor PD98059 (20 microM), which suppressed the TRO- and RSG-induced ERK1/2 activation but did not abolish their proapoptotic effects. We conclude that the thiazolidinedione analogues TRO and RSG induce cell death due to apoptosis in VSMC through an ERK1/2-independent pathway.

Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis.

The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor beta-receptor (PDGF-Rbeta). The effect of PDGF-BB on VSMCs growth was assessed by [(3)H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rbeta, PLC-gamma1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm alpha-actin filaments, paxillin and PDGF-Rbeta was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm alpha-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rbeta level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rbeta, PI 3'-K, PLC-gamma1 and ERK1/2 indicating an action of cyclic AMP on PDGF-beta receptor. We conclude that although cyclic AMP attenuates the PDGF-Rbeta mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs.

Green tea compounds inhibit tyrosine phosphorylation of PDGF beta-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 beta-receptor (PDGF-Rbeta) and on the anchorage-independent growth of A172 glioblastoma cells in semisolid agar has been investigated. Treatment of A172 glioblastoma with 50 microM CG, ECG, EGCG and 25 microM 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-Rbeta. The PDGF-Rbeta downstream intracellular transduction pathway including tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) and phosphatidylinositol 3'-kinase (PI 3'-K) was also inhibited. Spheroid formation was completely inhibited by 50 microM ECG, CG, EGCG and by 25 microM 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-Rbeta.

Short-term stimulation of vascular smooth muscle cells with platelet-derived growth factor (PDGF)-BB and angiotensin II induces.

Growth factors such as the platelet-derived growth factor (PDGF)-BB and angiotensin II (Ang II) have been shown to induce vascular smooth muscle cell (VSMC) proliferation after long stimulation periods. Little is known though, about the effects of PDGF-BB and Ang II on VSMC proliferation after short stimulation periods. The purpose of our study was to examine whether a short term (3-60 min) stimulation of VSMC with PDGF-BB or Ang II is sufficient to induce cell proliferation. Incubation of VSMC with Ang II (100 nM) or PDGF-BB (50 ng/ml) caused a significant increase in [3H]thymidine incorporation starting after a 3-min stimulation, while the cell counts required 32 and 8 h of stimulation, respectively. Mitogen-activated protein kinase activation reached a maximum at 5-10 min of PDGF-BB or Ang II stimulation. This study demonstrates that the growth-promoting effects of PDGF-BB and Ang II are strongly dependent on the length of the stimulation period and that while prolonged stimulation periods (>8-32 h) result in VSMC proliferation, short ones (3-60 min) result only in [3H]thymidine incorporation without an increase in cell count, a fact of considerable pathophysiological significance, considering that the time kinetics of growth factors in the VSMC microenvironment have not as yet been clarified.

Early intracellular signalling pathway of ethanol in vascular smooth muscle cells.

1. 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 (p44(mapk) and p42(mapk)) 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. 2. Measurement of cytosolic Ca(2+) 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 [(3)H]-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-(3)H]-inositol and separation of inositolphosphates by HPLC. 3. Ethanol (0.3 - 1.0% v v(-1), 17 - 170 mM) induced a dose-dependent maximal stimulation of p44(mapk)/p42(mapk) 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 [Ca(2+)](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. 4. 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.

Evidence that lipoproteins are carriers of bioactive factors.

We recently demonstrated that the mitogenic effect of LDL (100 microg/mL) as well as its early intracellular signaling pathway are mediated by a pertussis-toxin (PTX)-sensitive G(i) protein-coupled receptor that is independent from its classical receptor and involves activation of extracellular response kinases (ERK1/2) (also known as p44(mapk)/p42(mapk)). In the present study we examined whether LDL-adherent factors may be responsible for some of the effects of LDL. The term "signaling activity" is used to characterize fractions that cause an increase in intracellular free Ca(2+) concentration or stimulate ERK1/2 and c-fos mRNA expression. LDL, HDL, and VLDL stimulate ERK1/2 with the following order of potency: LDL>HDL>VLDL. After delipidation of LDL with chloroform/methanol/water mixtures a PTX-sensitive signaling activity was found in one fraction arbitrarily called LDL-F. After further analysis of LDL-F compounds by high pressure liquid chromatography, a PTX-sensitive signaling activity was detected only in the fraction with a retention time of 33 minutes (arbitrarily called LDL-F33). Similarly, after separation of sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) by high pressure liquid chromatography, a PTX-sensitive signaling activity was found in the fractions 33 and 33 to 35, respectively. These findings demonstrate that the effects of LDL-F33 are mimicked by similar fractions collected from SPP/SPC, hence suggesting that these LDL-adherent molecules are possibly closely related to SPP/SPC. A PTX-sensitive signaling activity was also detected in HDL and HDL-F33. Therefore, LDL and other lipoproteins may function as carriers for bioactive phospholipids thereby contributing to the development of coronary artery disease. Our findings support a new research concept that may contribute in elucidating cellular mechanisms promoting coronary artery disease.

Lipid-induced changes in vascular smooth muscle cell membrane fluidity are associated with DNA synthesis.

In the present study, we examined whether changes in the membrane fluidity of vascular smooth muscle cells (VSMCs) alter their DNA synthesis. For this purpose, the membrane fluidity of the cells was modulated after treatment of VSMCs with 1,2-dioleoyl phosphatidylcholine (PC). Treatment of VSMCs with 1,2-dioleoyl PC-rich medium containing 10% heat-inactivated human serum and 3 mg/ ml 1,2-dioleoyl PC for 24 h resulted in an increase in VSMC membrane fluidity at all temperatures from 15 degrees to 40 degrees C as well as a 51% inhibition of DNA synthesis, compared with untreated cells. Remarkably, enrichment of VSMCs with 1,2-dioleoyl PC/cholesterol-rich medium containing 10% human serum, 3 mg/ml 1,2-dioleoyl PC and 2 mg/ml cholesterol restored both membrane fluidity and DNA synthesis to the levels of untreated cells. The present findings show an inverse association between increased membrane fluidity and cellular DNA synthesis.

Lysophosphatidic acid stimulates protein kinase C isoforms alpha, beta, epsilon, and zeta in a pertussis toxin sensitive pathway in vascular smooth muscle cells.

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 alpha, beta, epsilon, and zeta 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 microg/mL LPA for 10 min increased the amount of PKC alpha, beta, epsilon, and zeta in the particulate fraction by 689%, 285%, 424%, and 510%, respectively, and returned to control level after 30 min. Correspondingly, the amount of PKC alpha, beta, epsilon, and zeta 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 alpha, beta, epsilon, and zeta isoforms was PTX sensitive. Incubation of VSMC with nonspecific PKC inhibitor 19-27 (10 micromol/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 alpha, beta, epsilon, and zeta in a PTX-sensitive manner. Furthermore stimulation of PKC might be critically involved in LPA-induced mitogenesis in VSMC.

Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

Role of mitogen-activated protein kinase in the angiotensin II-induced DNA synthesis in vascular smooth muscle cells.

The activation of mitogen-activated protein (MAP) kinase and increase in intracellular free calcium concentration ([Ca2+]i) are discussed in reference to activation of different protein kinases and growth of vascular smooth muscle cells (VSMCs). The aim of the present study was to investigate the role of angiotensin (Ang) II-induced increase in [Ca2+]i for activation of 44-kD/42-kD MAP kinase (p44mapk/p42mapk) and DNA synthesis in VSMCs. Experiments were performed by chelation of [Ca2+]i by the intracellular chelator 1,2-bis-(o-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). Ca2+ was measured by the fura 2 method. MAP kinase activation was determined by the Western blotting method. DNA synthesis was determined by measurement of [3H]thymidine incorporation into the cell DNA. Treatment of VSMCs with 20 micromol/L MAPTAM for 30 minutes resulted in a complete abolishment of the maximal Ang II-induced increase at 10 seconds. Ang II phosphorylated the p44mapk/p42mapk in a time-dependent manner, showing a maximum at 3 minutes. In MAPTAM-treated cells, the maximal phosphorylation of MAP kinase isoforms was shifted to 5 minutes, and dephosphorylation was delayed compared with untreated cells. In concordance with this finding, the induction of the MAP kinase phosphatase-1 was markedly impaired in MAPTAM-treated cells. Ang II induced a 2.3-fold increase in [3H]thymidine incorporation into DNA synthesis in untreated cells. This effect was not reduced in MAPTAM-treated cells. Treatment of the cells with PD 98059 (10 micromol/L), a MAP kinase kinase inhibitor, caused 85% inhibition of the Ang II-induced activation of MAP kinases but did not inhibit the Ang II-induced DNA synthesis. In conclusion, the Ang II-induced stimulation of the MAP kinase is a Ca2+-dependent process. Furthermore, blockade of the Ang II-induced stimulation of the early intracellular events, such as increase in [Ca2+]i or phosphorylation of the MAP kinase, is not accompanied by an inhibition of the Ang II-induced DNA synthesis.

Inhibition of glycosphingolipid synthesis by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and the modulation of IL-1beta-stimulated expression of inducible nitric oxide synthase in rat aortic smooth muscle cells.

1. The composition of glycosphingolipids is altered in atherosclerotic tissue. In order to study the possible modulation of interleukin-1beta (IL-1beta)-induced expression of inducible nitric oxide synthase (iNOS) by endogenously synthesized glycosphingolipids, we investigated rat aortic vascular smooth muscle cells (VSMC) grown in the presence of the inhibitor of glycosphingolipid synthesis, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). 2. Depletion of glycosphingolipids by PDMP (20-30 microM) was demonstrated by thin-layer chromatography of D-[1-(14)C]-galactose- or L-[-U14C]-serine-labelled glycosphingolipids. Nitrite generation was measured by the diaminonaphthalene assay, nitric oxide was determined by the oxyhaemoglobin technique and iNOS protein was detected by immunocytochemistry. 3. In VSMC grown in the presence of PDMP, the glycosphingolipid content was reduced by 30-50%. In PDMP-treated VSMC, IL-1beta (3 micro ml[-1])-stimulated release of nitrite (135 +/- 4 nmol mg(-1) protein 48 h[-1]) was significantly increased as compared to IL-1beta-stimulated control cells (40 +/- 3 nmol mg(-1) protein 48 h(-1); n = 6, P < 0.001). Similarly, IL-1beta (3 micro ml(-1), 36 h)-stimulated release of nitric oxide was higher in PDMP-treated VSMC (6.1 +/- 0.5 nmol mg(-1) protein h[-1]) as compared to untreated cells (2.0 +/- 0.6 nmol mg(-1) protein h(-1); n = 3, P < 0.01). These findings were confirmed by the demonstration of increased expression of iNOS protein (14.9 +/- 1.2% vs 6.4 +/- 0.2%; n = 4, P < 0.001), as shown by immunocytochemistry. 4. Evidence is presented that endogenous glycosphingolipids are important modulators of cytokine-induced iNOS expression. In view of an altered glycosphingolipid profile in atherosclerotic arteries, these mechanisms might be of relevance for the pathogenesis of atherosclerosis and restenosis subsequent to vessel injury.

The growth-promoting effect of low-density lipoprotein may Be mediated by a pertussis toxin-sensitive mitogen-activated protein kinase pathway.

Low-density lipoprotein (LDL) is known to be a mitogenic factor for vascular smooth muscle cells (VSMCs), fibroblasts, and endothelial cells. In the current study, we describe possible intracellular mechanisms by which LDL elicits its mitogenic effects. Stimulation of VSMCs with LDL resulted in a pertussis-toxin (PTX)-sensitive stimulation of the 44-kDa mitogen-activated protein (MAP) kinase (p44(mapk)) and 42-kDa MAP kinase (p42(mapk)) isoforms as well as in a PTX-sensitive increase in intracellular free Ca2+ concentration ([Ca2+]i). Binding of the LDL-induced increase in [Ca2+]i to the intracellular Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester resulted in a 2-fold increase in the phosphorylated p44(mapk) and p42(mapk) isoforms but did not influence the LDL effect of VSMC DNA synthesis. PD 98059, a MAP kinase kinase inhibitor, remarkably attenuated the LDL-induced activation of MAP kinases and DNA synthesis. Treatment of normal human skin fibroblasts and human fibroblasts isolated from patients with familial hypercholesterolemia homozygote class 1 mutations, which are not able to produce the classic LDL receptor, resulted also in a PTX-sensitive increase in cell DNA synthesis and stimulation of the p44(mapk) and p42(mapk) isoforms in both cell types. These results demonstrate that the mitogenic effect of LDL is mediated by a PTX-sensitive Gi-coupled receptor that is independent of its classic receptor and involves activation of MAP kinase isoforms. Furthermore, the mitogenic effect of LDL may be mediated by the activation of the MAP kinase pathway. In contrast, the LDL-induced increase in [Ca2+]i may be implicated in this process only in conjugation with other signaling components.

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).

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.

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.

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 microM) 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.