Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells.

The underlying mechanism of the antiproliferative effect of S (simvastatin), a HMG-CoA reductase inhibitor, in vascular smooth muscle cells (SMC) is still poorly understood. In the present study, we used synchronized human SMC, isolated from left interior mammary artery, as an in vitro model to test the effects of S on platelet-derived growth factor (PDGF)-induced DNA synthesis, extracellular-regulated kinase 1/2 (ERK1/2), p38/stress-activated protein kinase 2 (SAPK2), RhoA and Rac1 activation. ERK1/2 phosphorylation was triggered within 2 min of PDGF stimulation (early G1 phase) and was blocked by PD98059, a specific inhibitor of the ERK1/2 pathway, which also strongly inhibited PDGF-induced DNA synthesis (IC(50) = 10 micromol/L). PDGF quickly induced p38 phosphorylation (early G1 phase) and SB203580, a specific inhibitor of the p38/SAPK2 pathway, also blocked PDGF-induced DNA synthesis (IC(50) = 0.3 micromol/L). Translocation to the plasma membrane of small GTPases, such as RhoA and Rac1, could not be detected within 15 min of stimulation with PDGF or lysophosphatidic acid (LPA) (early G1 phase), but occurred after 24 hr of PDGF stimulation (late G1/S phase). S inhibited PDGF-induced DNA synthesis (IC(50) = 3.5 micromol/L), and this effect was dependent on intracellular mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate availability. The critical time period for the reversal of the S effect by mevalonate comprised both the early and late G1 phase of the SMC cycle. PDGF-induced ERK1/2 phosphorylation and PDGF-induced p38 phosphorylation were not markedly affected by S during the whole G1 phase. However, S treatment blocked the PDGF- and LPA-induced membrane translocation of RhoA that occurred during the late G1/S phase. In the case of Rac1, the same process was also inhibited by S treatment. We concluded from these results that, in SMC, the early events associated with ERK1/2 and p38 signal transduction pathways, recruited for PDGF-mediated DNA synthesis, were insensitive to S action, whereas the mevalonate-dependent, posttranslational modification of RhoA and Rac1 molecules, required for PDGF-induced membrane translocation, was blocked by this drug. These results suggest that the antiproliferative effect of S can be explained not only by the blockage of RhoA-mediated signaling events but also by Rac1-mediated signaling events.

Simvastatin improves disturbed endothelial barrier function.

BACKGROUND: Recent clinical trials have established that inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) reduce the risk of acute coronary events. These effects of statins cannot be fully explained by their lipid-lowering potential. Improved endothelial function may contribute to the positive effects of statin treatment. METHODS AND RESULTS: In the present study, we report that simvastatin reduces endothelial barrier dysfunction, which is associated with the development of atherosclerosis. Treatment of human umbilical vein endothelial cells for 24 hours with 5 micromol/L simvastatin reduced the thrombin-induced endothelial barrier dysfunction in vitro by 55+/-3%, as assessed by the passage of peroxidase through human umbilical vein endothelial cell monolayers. Similar effects were found on the thrombin-induced passage of (125)I-LDL through human aortic endothelial cell monolayers. This reduction in barrier dysfunction by simvastatin was both dose and time dependent and was accompanied by a reduction in the thrombin-induced formation of stress fibers and focal adhesions and membrane association of RhoA. Simvastatin treatment had no effect on intracellular cAMP levels. In Watanabe heritable hyperlipidemic rabbits, treatment for 1 month with 15 mg/kg simvastatin reduced vascular leakage in both the thoracic and abdominal part of the aorta, as evidenced by the Evans blue dye exclusion test. The decreased permeability was not accompanied by a reduction of oil red O-stainable atherosclerotic lesions. CONCLUSIONS: These data show that simvastatin, in a relatively high concentration, improves disturbed endothelial barrier function both in vitro and in vivo. The data also support the beneficial effects of simvastatin in acute coronary events by mechanisms other than its lipid-lowering effect.

Inhibition of human smooth muscle cell proliferation in culture by farnesyl pyrophosphate analogues, inhibitors of in vitro protein: farnesyl transferase.

In this study, it was investigated whether and how inhibitors of protein:farnesyl transferase (PFT) can inhibit the proliferation of human smooth muscle cells (HSMC) in culture. Several farnesyl pyrophosphate (FPP) analogues were synthesized and tested in vitro for their specificity in inhibiting squalene synthase (SS), PFT, or protein:geranylgeranyl transferase-1 (PGGT-1) activities (the latter was determined using a newly designed assay). One of these compounds appeared to be a strong PFT inhibitor (IC50 value: 340 nM) and a weak inhibitor in the other two enzyme assays. This compound (designated as TR006) inhibited the farnesylation of Ras in a Ha-ras transfected cell line (Cohen et al., Biochem. Phamacol. 49: 839-845, 1995) and concomitantly slowed down the growth of these cells. Twenty-five microM of TR006 inhibited the proliferation of HSMC isolated from left internal mammary artery, as measured by counting the cells over a period of three cell cycles (10 days). A structurally related compound (TR007), a specific SS inhibitor, did not influence HSMC proliferation under the same conditions. The inhibition by TR006 was concentration-dependent. In HSMC, synchronized by serum depletion, platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF)-induced DNA synthesis was decreased by a 29-hr pretreatment with 100 microM of TR006, indicating that this inhibitor acted in an early phase of the cell cycle, probably by preventing protein isoprenylation. Some other FPP analogues with comparable IC50 values in the in vitro PFT assay were also able to decrease bFGF-induced DNA synthesis without affecting cell viability. A more negatively charged member of this group, TR018, did not influence the growth factor-induced DNA synthesis, probably due to an impaired uptake into the cells. However, the pivaloyloxomethyl derivative of this compound, which is uncharged, and is thought to be converted into TR018 within the cells, showed a strong decrease in bFGF-induced DNA synthesis in HSMC. These data suggest that the compounds investigated may be developed further for treatment of conditions in which undesirable proliferation of smooth muscle cells plays an important role.

Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitors; comparison with other human cell types.

The effects of 6 HMG-CoA reductase inhibitors: pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin were analyzed in cultured human smooth muscle cells, fibroblasts, endothelial cells and myoblasts. In vascular smooth muscle cells, pravastatin was a much weaker inhibitor of cholesterol synthesis than the 5 other drugs which displayed equally strong inhibitory potency. The anti-proliferative effects of these 6 drugs were analyzed by measuring cell number and mitochondrial dehydrogenase activity (MTT assay) after 3 days of incubation. IC25 values for inhibition of proliferation were very similar among the 4 cell types and were in the following order of magnitude: pravastatin << lovastatin = simvastatin = atorvastatin = fluvastatin << cerivastatin. Only in the case of pravastatin was proliferation inhibited at lower concentration in smooth muscle cells than in the other cell types. Proliferation was also assessed by measuring DNA synthesis in these cells. A 3 day-incubation with 1 microM of pravastatin had no effect on this parameter in all 4 cell types. However, 1 microM of simvastatin or lovastatin caused either an inhibition (in smooth muscle cells and endothelial cells) or stimulation (in fibroblasts) of this process. The effects of simvastatin on cell number, mitochondrial dehydrogenase activity and DNA synthesis were counteracted by simultaneous mevalonate addition. Simvastatin treatment was also associated with a change in the post-translational modification of the ras protein in smooth muscle cells, probably by inhibition of its farnesylation. Moreover, simvastatin treatment blocked the PDGF and bFGF-induced DNA synthesis in synchronized smooth muscle cells, whereas it does not affect the fetal calf serum-induced DNA synthesis in synchronized fibroblasts, suggesting that simvastatin blocks various steps of the cell cycle and that this effect depends on the cell type and the growth signalling pathway activated.

Characterization of phospholipase A2 activity enriched in the nerve growth cone.

Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A2 activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well-characterized cytosolic phospholipase A2 (mol wt 85,000) reveals only trace amounts of this PC- and PE-selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A2 activity elutes essentially as two peaks of high molecular mass, at approximately 65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A2 species. The PI-selective enzyme, the predominant phospholipase A2 activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A2 is resistant to reducing agents and is found in the cytosol as well as membrane-associated in the presence of Ca2+. However, its catalytic activity is Ca(2+)-independent regardless of whether the enzyme is associated with pure substrate or mixed-lipid growth cone vesicles. The PE-selective phospholipase A2 in growth cones was studied in less detail but shares with the PI-selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane-associated forms, and Ca2+ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A2 that share many properties with known, Ca(2+)-independent cytosolic phospholipase A2 species but that appear to be monoselective for PI and PE, respectively. In particular, the PI-selective enzyme may represent a new member of the growing family of cytoplasmic phospholipases A2. The enrichment of the PI-selective phospholipase A2 in growth cones suggests it plays a major role in the regulation of growth cone function.

Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle.

Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 +/- 6 nM and 4.0 +/- 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 +/- 38 nM). Through inhibition of mevalonate production, these compounds have a distinct inhibiting effect on cell proliferation. Because proliferation of myoblasts is important in the repair of damaged skeletal muscle, experiments were performed to investigate the effect of lovastatin, simvastatin, and pravastatin on cell proliferation and cell viability. The more potent inhibitors of sterol synthesis, lovastatin, and simvastatin, were able to inhibit the proliferation of these cells during 3 days of incubation with drug concentrations of 1 microM for lovastatin and 0.1 microM or 1 microM for simvastatin. DNA synthesis was decreased by more than 80% in the presence of 1 microM of lovastatin or simvastatin. In contrast, under these conditions, pravastatin had no influence on cell proliferation or DNA synthesis, which is probably related to the lack of inhibition of sterol synthesis by pravastatin on extended incubation. The three 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors did not disturb cell viability because mitochondrial dehydrogenase activity and ATP content remained proportional to the number of cells in the culture at any concentration used.

Axonal origin and purity of growth cones isolated from fetal rat brain.

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of 'growth cone particles' (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Arachidonic acid turnover and phospholipase A2 activity in neuronal growth cones.

We analyzed de novo synthesis and local turnover of phospholipids in the growing neuron and the isolated nerve growth cone. The metabolism of phosphatidylinositol (PI) was studied with regard to the incorporation of saturated and unsaturated fatty acids and inositol. A comparison of de novo phospholipid synthesis in the intact neuron (whole brain, cell cultures) versus local turnover in isolated growth cone particles (GCPs) from fetal rat brain revealed different incorporation patterns and, in particular, high arachidonic acid (AA) turnover in PI of GCPs. These observations, together with elevated levels of free AA (2.5% of total AA content) in GCPs, demonstrate the predominance of acylation/deacylation in the sn-2 position of PI. GCP phospholipase A2 (PLA2) activity was demonstrated using [3H]-or [14C]AA-phosphatidylcholine (PC) or -PI as the substrate in vitro and GCPs or a cytosolic GCP extract as the source of enzyme. In contrast to PC, which is hydrolyzed very slowly, PI is a very good GCP PLA2 substrate. PLA2 activity is much higher in GCPs than that of phospholipase C, as demonstrated by the comparison of AA and inositol turnover, by the low levels of 1,2-diacylglycerol generated by GCPs, and by the resistance of AA release to treatment of GCPs with RHC-80267, a specific inhibitor of diacylglycerol lipase. The predominance of PLA2 activity in GCPs raises questions regarding its regulation and the functional roles of PI metabolites, especially lysocompounds, in growth cones.

Quantitative analysis of fetal rat brain neurons developing in primary cultures. I. Stereological study of the neuronal differentiation.

An ultrastructural stereological analysis was performed to analyze the morphological differentiation of primary cultures of fetal rat brain neurons, growing for two weeks in a serum-free medium. The number of neurons and of gliofibrillary acidic protein (GFAP)-positive glial cells was estimated by light microscopy counting in the culture wells. These cultures provided a quasi-pure neuronal population, since the number of GFAP-positive glial cells was found to be 1% (day 7) and 2% (day 14) respectively of the total number of cultured cells. Cell counts and the stereological measurements were related to the surface area of the culture well. The neuronal differentiation was characterized by an increase in the plasma membrane surface area (x9) and volume (x8) of neurites, contrasting with the decrease in the perikarya surface area and volume. These primary stereological data were combined with the number of neurons to obtain parameters characterizing an average neuron. The increase in membrane surface area of an average neuron was found to be a linear function of time, 29 micron 2 and 445 micron 2 of new membrane being added per day of culture to perikarya and neurites respectively. The number of chemical synapses was also counted and compared to the changes in the plasma membrane surface area. After 7 days in vitro they increased in number more rapidly than the increase in the plasma membrane surface area of neurons.

Quantitative analysis of rat brain neurons developing in primary cultures. II. Changes in the distribution of N-CAM associated to neuronal cell surfaces.

Cultured rat fetal brain cells underwent morphological differentiation, as quantitatively described in the companion paper. In the same system, biochemical and immunolabeling studies were performed to analyze the developmental changes in neural cell adhesion molecule (N-CAM) distribution and quantity at the cell surface of neurons. The cell surface-associated N-CAM, related to the culture protein content, remained stable during the two-week period under study, as demonstrated by 125I-protein A binding assays. Immunogold labeling experiments, both in transmission and scanning electron microscopy, indicated a dramatic decrease in N-CAM site density in each membrane compartment, perikarya and neurites. This temporal variation of N-CAM distribution was not accompanied by differences in N-CAM site density between these two membrane compartments. On the other hand, individual perikarya, observed in scanning electron microscopy, showed various levels of labeling. In addition, immunoblot experiments demonstrated the absence of chemical modulation of N-CAM during the period under study, since the high molecular weight (embryonic) form remained dominant. Moreover, an increase in the total N-CAM amount was detected, contrasting with the stable quantity of cell surface-associated N-CAM. This suggested the existence of an N-CAM intracellular pool in cultured neurons. Finally, since the neurite membrane surface area increased 9-fold (companion paper) and since only a 5-fold decrease in N-CAM site density was observed in this compartment, N-CAM supply to neurite membranes was postulated.