M.R. Soma

Relationship between mevalonate pathway and arterial myocyte proliferation: in vitro studies with inhibitors of HMG-CoA reductase

The role of mevalonate and its products (isoprenoids) in the control of cellular proliferation was examined by investigating the effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (vastatins) on growth and on cholesterol biosynthesis of cultured arterial myocytes (SMC). Simvastatin (S) and fluvastatin (F), but not pravastatin (P), decreased the rate of growth of rat vascular SMC. The inhibition, evaluated as cell number, was dose-dependent with IC50 values of 2.8 and 2.2 microM for S and F, respectively; P (1-500 microM) was inactive. The inhibition of cell growth induced by 3.5 microM S (70% decrease) was prevented completely by the addition of 100 microM mevalonate, partially (70-85%) by the addition of 10 microM geraniol, 10 microM farnesol and 5 microM geranylgeraniol, but not by the addition of squalene, confirming the specific role of isoprenoid metabolites in regulating cell proliferation. All the tested vastatins inhibited the incorporation of [14C]acetate into cholesterol but P had 800 times lower potency than S and F. Similar results were obtained in SMC from human femoral artery. At least 80% inhibition of cholesterol synthesis was necessary to induce a decrease in SMC proliferation. To further investigate the relationship between cholesterol synthesis and cell growth, two enantiomers of F were investigated. The enantiomer more active on HMG-CoA reductase was 70- and 1.6-fold more potent on arterial myocyte proliferation than its antipode and the racemic mixture, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol and mevalonic acid modulation in cell metabolism and multiplication

Cholesterol in animals is a major structural component of cell membranes. It may therefore play a functional role in the modulation of cell osmolarity, the process of pinocytosis and the activities of membrane-associated proteins such as ionic pumps, immune responses, etc. A major relationship exists between the cell-growth processes and the cholesterol biosynthetic pathway. The cholesterol needed for new membranes may be derived either from endogenous synthesis or from exogenous sources, principally plasma low-density-lipoproteins (LDL) which enter the cells by receptor-mediated endocytosis. Both these pathways are enhanced in rapidly growing cells. Conversely, if synthesis is inhibited and no exogenous cholesterol is available, cell growth is blocked. The 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase (the rate-limiting reaction in cholesterol biosynthesis) is the enzyme which catalyzes the conversion of HMGCoA to mevalonic acid. It has been suggested that mevalonate may play an important role in cell proliferation. All cells need at least two products synthesized from mevalonate in order to proliferate, and the only one yet identified is cholesterol. Other melavonate-derived potential candidates as cell-cycle and cell-survival products include the dolichols ubiquinone side chains, isopentenyladenosine derivatives, etc. Furthermore, it has recently been shown that membrane association appears to be an important function in mevalonate-derive modifications of several important proteins such as cellular membrane G proteins, those coded for by oncogenes (ras proteins) and lamins (nuclear proteins). In recent years the development of cholesterol-synthesis-inhibiting drugs, for lowering plasma cholesterol levels has mainly been centred on the control of HMGCoA reductase activity (vastatins). However, because mevalonic acid is the precursor of numerous metabolites, any reduction of such activity may potentiate pleiotropic effects. Vastatins are now, therefore, receiving increased attention as potential pharmacological tools for the control of abnormal cell growth in pathological situations, i.e. tumours and vascular smooth muscle cell proliferation under atherogenic conditions. In our laboratories, we have demonstrated that simvastatin can prevent arterial myocyte proliferation both in vivo and in vitro. Simvastatin can also inhibit in vitro the rate of human glioma cell growth, since it shows a strong synergistic inhibitory effect on cell proliferation when used in association with anticancer agents such as Carmustine or beta-interferon. Both simvastatin-induced cell growth inhibition and the synergy observed with these drugs can be completely reversed by incubating cells with mevalonate. This shows that the effect of simvastatin of cell proliferation is due to its specific inhibitory activity on intracellular mevalonate synthesis.

Simvastatin but not pravastatin inhibits the proliferation of rat aorta myocytes

The in vitro effect of simvastatin and pravastatin, two competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, on the proliferation of rat aortic smooth muscle cells was investigated. Simvastatin, but not pravastatin, inhibited the replication of arterial myocytes in concentrations ranging between 0.01 microM and 10 microM. The inhibition, evaluated as cell number and nuclear incorporation of [3H]thymidine, was dose-dependent and completely prevented by addition of mevalonate (100 microM), confirming the role of mevalonate or its products in regulating cell division and growth. The present results provide evidence that simvastatin, in addition to its effect on cholesterol biosynthesis, interferes in vitro with other processes involved in atherogenesis.

Pathogenesis of atherosclerosisand the role of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors

Atherosclerosis is a complex multifactorial process resulting from an excessive inflammatory/fibroproliferative response to various forms of injurious stimuli to the arterial wall. The potential interactions of cells, cytokines, and growth-regulatory molecules among the different cells in the atherosclerotic lesion present numerous opportunities for modulating lesion formation and progression. Smooth muscle cell (SMC) migration and proliferation, together with lipid deposition, are now recognized as the major phenomena occurring within the arterial wall, and thus these phenomena serve as targets for pharmacologic intervention in the process of atherogenesis. Migration and proliferation of SMC are key events in atherosclerosis--and in restenosis after angioplasty. An understanding of the factors that induce such events is important for the prevention and treatment of these diseases. Mevalonate and other intermediates of cholesterol synthesis (isoprenoids) are essential for cell proliferation; hence drugs affecting this metabolic pathway are potential antiatherosclerotic agents. Recently, this group provided in vitro and in vivo evidence of decreases in SMC proliferation by fluvastatin and simvastatin, but not pravastatin, independent of their cholesterol-lowering properties. The in vitro inhibition of cell migration and proliferation induced by simvastatin and fluvastatin (70-90% decrease) was completely prevented by the addition of mevalonate, and partially prevented (70-80%) by farnesol or geranylgeraniol. This confirms the specific role of isoprenoid metabolites--most probably geranylgerylated protein(s)--in regulating cell migration and proliferation. The inhibitory effect of fluvastatin and simvastatin on cholesterol esterification induced by acetyl low density lipoprotein in macrophages was also prevented by the addition of geranylgeraniol.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein(a) and other lipids after oophorectomy and estrogen replacement therapy

Objective To assess the effect of surgical menopause and subsequent estrogen replacement therapy (ERT) on lipoprotein(a) [Lp(a)] and common lipids. Methods In 24 healthy premenopausal women, lipids (total cholesterol, low-density lipoprotein [LDL] and highdensity lipoprotein [HDL] cholesterol, and triglycerides) and gonadotropins (FSH, LH) were measured the day before hysterectomy with bilateral oophorectomy and then after 1, 2, and 3 months. Blood was also drawn after 2, 4, 6, and 15 days to exclude the effect of surgery on Lp(a). In 19 women who volunteered for ERT, the lipid profile was assayed again after 3, 6, and 12 months of treatment. Results Lipoprotein(a) levels rose significantly over the 3 months after surgery, from a mean ± standard deviation (SD) 5.7 ± 6.1 mg/dL to 10.4 ± 9.2 mg/dL. Total cholesterol and LDL cholesterol levels increased significantly over the first 2 months; HDL cholesterol decreased significantly during the 3 months of follow-up (by 10, 17, and 20%) (P < .001). Plasma triglycerides did not change after surgery. Three months following ERT, Lp(a) and total cholesterol were significantly decreased (28 and 11%, respectively), as was LDL cholesterol (33%) after 6 months. High-density lipopromein cholesterol increased by 24% after 6 months of treatment, and triglycerides rose significantly in the year of therapy (37%). Conclusion These findings suggest that surgical menopause induces atherogenic changes in the lipid profile in 3 months and that ERT soon reverses them.

Effects of oral and transdermal hormone replacement therapy on lipoprotein(A) and lipids: A randomized controlled trial

Objective:Our purpose was to compare the effect of oral and transdermal hormone replacement therapy on lipoprotein(a) and other plasma lipids in healthy postmenopausal women. Design:A total of 120 postmenopausal women were enrolled in a prospective randomized controlled study, and allocated either to transdermal 17β-estradiol (50 μg/day) or to oral conjugated estrogen (0.625 mg/day). Forty-one age-matched women were used as the reference group. Plasma lipids and lipoproteins were determined every 3 months and differences were sought by statistical analysis. Results:Plasma lipoprotein(a) dropped after 3 months of treatment either with transdermal estradiol (p < 0.01) or oral estrogen (p < 0.01). Lipoprotein(a) was reduced by 12% and 22%, respectively. No further decreases were seen later on. Plasma total and low-density lipoprotein (LDL) cholesterol concentrations were decreased significantly with both treatments after 3 months of therapy. No difference was seen in the lowering effect on lipoprotein(a), LDL and total cholesterol concentrations between regimens. Plasma high-density lipoprotein (HDL) cholesterol and triglyceride concentrations increased throughout the study only in patients treated with oral estrogen. Conclusions:These data demonstrate that hormone replacement therapy reduces the concentration of lipoprotein(a) when given both orally and transdermally. The lowering effect is achieved quickly because the maximal effect is observed after 3 months of therapy.

Effect of lercanidipine and its (R)-enantiomer on atherosclerotic lesions induced in hypercholesterolemic rabbits.

1 The in vivo antiatherogenic activity of the calcium antagonist lercanidipine and its (R)‐enantiomer was investigated in two different types of atherosclerotic lesions (hyperplastic and fatty‐streak lesions) in rabbits. 2 Lercanidipine (0.3, 1, and 3 mg kg−1 week−1) as well as its (R)‐enantiomer at 3 mg kg−1 week−1 were given by subcutaneous injection for 10 weeks to White New Zealand rabbits, with cholesterol feeding beginning at week 2. The hyperplastic lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty streak lesions were induced by cholesterol feeding. In untreated animals (n=5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries without collar showed a intima/media (I/M) ratio of 0.03±0.02, whereas in carotids with a collar the ratio was 2±0.42. In lercanidipine‐treated animals a significant and dose‐dependent effect on intimal hyperplasia was observed. I/M ratios were 0.73±0.4, 0.42±0.1, 0.32±0.1 for 0.3, 1, and 3 mg kg−1 week−1, respectively (P<0.05). The lercanidipine enantiomer (3 mg kg−1 week−1) was as effective as the racemate (0.41±0.11). Proliferation of smooth muscle cells, assessed by incorporation of BrdU into DNA, was reduced by about 50%, 70%, 85%, and 80% by lercanidipine (0.3, 1, and 3 mg kg−1 week−1) and its (R)‐enantiomer, respectively. 3 The area of fatty‐streaks in the aorta (n=11–15) was significantly reduced by lercanidipine (3 mg kg−1 week−1, 16% vs 27%, P<0.05), a trend was observed also with lower doses. When different segments of the aorta were considered (arch, thoracic, abdominal) a significant and dose‐dependent effect in the thoracic and abdominal aorta was observed also at lower doses. The (R)‐enantiomer was as effective as lercanidipine. 4 These results suggest a direct antiatherosclerotic effect of lercanidipine, independent of modulation of risk factors such as hypercholesterolemia and/or hypertension as demonstrated by the absence of stereoselectivity.

Effects of lacidipine on experimental models of atherosclerosis

Aim: To evaluate the effect of lacipine on the major processes of atherogenesis. Methods: Cell-culture methods were used to study the effect of lacidipine. Low-density lipoprotein (LDL) receptor expression and cholesterol esterification were evaluated in human skin fibroblasts and in mouse peritoneal macrophages, respectively. The effect of lacidipine on cellular proliferation was tested on aortic myocytes cultured from rat aorta. Results: Lacidipine did not affect LDL receptor expression, but it inhibited the ability of acetyl LDL to stimulate cholesterol esterification in macrophages by more than 95%. The drug inhibited cellular proliferation in a dose-dependent manner. This antiproliferative effect was confirmed in human femoral artery myocytes. In accord with the inhibitory effect on cellular growth, preliminary in vivo studies suggest that lacidipine may reduce neointimal formation induced by perivascular manipulation of the carotid artery in hypercholesterolemic rabbit. Conclusions: Our results indicate that lacidipine may be antiatherosclerotic through an effect on the major processes involved in atheroma formation.

Dual effects of the antioxidant agents probucol and carvedilol on proliferative and fatty lesions in hypercholesterolemic rabbits

The in vivo direct antiatherogenic activity of the antioxidant probucol (200 mg/kg per day) or the beta-blocker with antioxidant properties carvedilol (10 and 20 mg/kg per day) was tested in the same animal in two different types of atherosclerotic lesion (proliferative and fatty lesions) induced in cholesterol-fed rabbits (1%). Drugs were given daily mixed with standard diet for 8 weeks; body weight and plasma lipid profile were not different among groups throughout the study. Aortic fatty lesions were induced by cholesterol feeding (n = 25 in each group) and their extent expressed as % of aorta inner surface covered by plaques was significantly reduced by both drugs (28.2+/-9.6%, P <0.05, 19.9+/-6.2%, P <0.01 for low- and high-dose carvedilol, respectively; 22.3+/-7.6%, P <0.01 for probucol, versus 41.6+/-10.7% in control rabbits). Proliferative lesions were obtained by positioning a hollow silastic collar around one carotid artery 6 weeks after dietary and drug treatments started (n = 5 in each group). The neointimal formation, mostly composed by myocytes, was determined by measuring cross-sectional thickness ratio of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals, collared arteries resulted in a significant neointimal cell accumulation compared to the sham (1.10+/-0.14 versus 0.02+/-0.01) without change in medial thickness. I/M ratio was reduced by about 50% in animals treated with probucol (0.51+/-0.1) and carvedilol (0.66+/-0.21 and 0.52+/-0.1 in the low- and high-dose group, respectively). Total plasma TBARS were more than 50% lower in both probucol- and high-dose carvedilol-treated rabbits. Results show that pharmacological pretreatment with antioxidants directly inhibits early atherogenic processes, representing a potentially useful approach in the prevention of atherosclerosis.

Effect of lacidipine on fatty and proliferative lesions induced in hypercholesterolaemic rabbits

1 The in vivo antiatherogenic activity of the calcium antagonist, lacidipine, was investigated in two different types of atherosclerotic lesions (proliferative and fatty lesions) induced in rabbits. 2 The proliferative lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty lesions were induced by cholesterol feeding. Cholesterol (1%) and lacidipine (1,3, and 10 mg kg−1) were given daily mixed with standard diet for 8 weeks to White New Zealand rabbits. The intimal hyperplasia (proliferative lesion) was induced 6 weeks after dietary and drug treatment started. 3 The neointimal formation was determined by measuring cross sectional thickness of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals (n=5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries with no collar (sham) showed an I/M tissue ratio of 0.03±0.02, whereas in the carotid with collar the ratio was 0.62±0.12. In lacidipine‐treated animals a significant and dose‐dependent effect on proliferative lesions at all three doses tested, was observed. I/M ratios were 0.47±0.02, 0.40±0.09, 0.32±0.02 for doses 1, 3, and 10 mg kg−1 day−1, respectively (P<0.05). 4 The fatty lesion extent was significantly reduced by lacidipine at the 10 mg kg−1 day−1 dose, although a trend was also observed with lower dosage. 5 These results suggest a direct antiatherosclerotic effect of lacidipine, independent of modulation of risk factors such as hypercholesterolaemia and/or hypertension. Furthermore, the proliferative lesions are apparently more sensitive to lacidipine than are lipid‐rich lesions.