R. Fumagalli

HMG-CoA Reductase Inhibitors Reduce MMP-9 Secretion by Macrophages

-Macrophages secrete matrix metalloproteinases (MMPs) that may weaken the fibrous cap of atherosclerotic plaque, predisposing its fissuration. The 92-kDa gelatinase B (MMP-9) has been identified in abdominal aortic aneurysms and in atherosclerotic tissues. Fluvastatin, through the inhibition of the isoprenoid pathway, inhibits major processes of atherogenesis in experimental models (smooth muscle cell migration and proliferation and cholesterol accumulation in macrophages). We studied the effect of fluvastatin on the activity of MMP-9 in mouse and human macrophages in culture. Conditioned media of cells treated for 24 hours with fluvastatin were analyzed by gelatin zymography. In mouse macrophages, fluvastatin (5 to 100 micromol/L) significantly inhibited in a dose-dependent manner MMP-9 activity from 20% to 40% versus control. The drug, at a concentration as low as 5 micromol/L, inhibited MMP-9 activity ( approximately 30%) in human monocyte-derived macrophages as well. Phorbol esters (TPA, 50 ng/mL) stimulated MMP-9 activity by 50%, and fluvastatin inhibited this enhanced activity up to 50% in both mouse and human macrophages. The above results on the secretion of MMP-9 were confirmed by Western blotting and ELISA. The inhibitory effect of fluvastatin was overcome by the simultaneous addition of exogenous mevalonate (100 micromol/L), a precursor of isoprenoids. Fluvastatins effect was fully reversible, and the drug did not cause any cellular toxicity. The statin did not block directly the in vitro activation of the secreted protease. Similar data were obtained with simvastatin. Altogether, our data indicate an inhibition of MMP-9 secretion by the drug. This effect is mediated by the inhibition of synthesis of mevalonate, a precursor of numerous derivatives essential for several cellular functions.

Non-Lipid-Related Effects of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors

With the increasing knowledge of the pathogenesis of atherosclerosis, it appears that in the future the prevention of cardiovascular disease will involve not only risk factor correction, but also direct pharmacological control of processes occurring in the arterial wall. Among these, a pivotal role is played by smooth muscle cell (SMC) migration and proliferation, which, together with lipid deposition, are prominent features of atherogenesis and restenosis after angioplasty. Mevalonate and other intermediates of cholesterol synthesis (isoprenoids) are essential for cell growth, hence drugs affecting this metabolic pathway are potential antiatherosclerotic agents. Recently, we provided in vitro and in vivo evidence that fluvastatin, simvastatin and lovastatin, but not pravastatin, decrease SMC migration and proliferation dose dependently, independently of their hypocholesterolemic properties. The in vitro inhibition of cell migration and proliferation induced by simvastatin and fluvastatin (70-90% decrease) was prevented completely by the addition of mevalonate, and partially prevented by farnesol and geranylgeraniol (80%), confirming the specific role of isoprenoid metabolites in regulating these cellular events, probably through prenylated protein(s). The in vivo antiproliferative activity of fluvastatin on neointimal hyperplasia in normocholesterolemic rabbits was also prevented fully by the local delivery of mevalonate, by means of an Alzet pump. Fluvastatin and simvastatin also inhibited cholesterol esterification and deposition induced by acetylated LDL in cultured macrophages. This effect was fully prevented by the addition of mevalonate or geranylgeraniol. Taken together, these results suggest that, beyond their effects on plasma lipids, HMG-CoA reductase inhibitors exert a direct antiatherosclerotic effect on the arterial wall, probably through local inhibition of isoprenoid biosynthesis.

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.

Effect of the new calcium antagonist lercanidipine and its enantiomers on the migration and proliferation of arterial myocytes

The in vitro effects were investigated of the new dihydropyridine calcium antagonist (CA) lercanidipine and its enantiomers on arterial myocyte (smooth muscle cell; SMC) migration and proliferation as related to L-type calcium channel inhibition. Lercanidipine and its enantiomers inhibited the replication and migration of arterial myocytes in concentration ranging from 10 to 50 microM. The antiproliferative effect of lercanidipine, evaluated as cell number, was dose dependent, with a potency similar to that of lacidipine and nifedipine, and was unrelated to the stereoselectivity of enantiomers to bind L-type calcium channels. The cell doubling time increased with drug concentration < or = 122 versus 38 h for controls. The cell growth inhibition induced by lercanidipine and its enantiomers was reversible. Lercanidipine dose dependently decreased [3H]thymidine incorporation into DNA; the (R)-enantiomer, displaying the lowest CA activity, was the most potent in this respect. The tested compounds were able to inhibit fibrinogen-induced myocyte migration in a dose-dependent manner, with the (R)-enantiomer showing the more pronounced effect. To directly rule out the role of calcium channels in the antiatherosclerotic properties of lercanidipine, we examined the effect of the compounds on serum-stimulated calcium influx in SMC. Fluorimetry of Fluo 3 was used to measure changes in free cytosolic Ca2+ concentration ([Ca2+]i) in SMC after long-term preincubation (24 h) with the tested CA. Lercanidipine and its enantiomers (25 microM) decreased the serum-induced elevation of [Ca2+]i in SMC with the (S)-enantiomer (69% inhibition) 2.4-fold more active than the counterpart and the racemate (29% inhibition). In conclusion, our in vitro results suggest that lercanidipine may directly interfere with events involved in atherogenesis. The studies performed with enantiomers of lercanidipine suggest that the observed effects are not related to the blockade of voltage-dependent Ca2+ channels and confirm at least in vitro a pharmacologic potential of the compound to negatively influence the process of atherogenesis.

Effects of 26-Aminocholesterol, 27-Hydroxycholesterol, and 25-Hydroxycholesterol on Proliferation and Cholesterol Homeostasis in Arterial Myocytes

The major relation existing between cell growth and cholesterol homeostasis prompted us to investigate the effect of 26-aminocholesterol (26-NH2), 27-hydroxycholesterol (27-OH), and 25-hydroxycholesterol (25-OH) on these cellular events. To test this relation, we incubated human and rat arterial myocytes with the sterols for 72 hours. All the tested compounds (0.5 to 7.5 mumol/L) inhibited rat and human myocyte proliferation and cholesterol biosynthesis in a dose-dependent manner. 26-NH2 was more potent than oxysterols in inhibiting human myocyte proliferation but equieffective in rat cells; 27-OH and 25-OH displayed similar activity in both cell lines. Inhibition of nuclear incorporation of thymidine in rat myocytes is consistent with decreased cell count. The antiproliferative effect of the tested sterols was reversible. The high inhibition (80%) of cholesterol biosynthesis necessary to induce a decrease in myocyte proliferation suggests a causal relation between the cholesterol synthetic pathway and these cellular processes. In addition, all the tested sterols were able to inhibit hydroxymethyl glutaryl-coenzyme A reductase activity in intact myocytes but not in cell-free extracts. The finding that 26-NH2 but not 27-OH or 25-OH does not suppress LDL receptor activity in either human or rat myocytes supports the achievement of selectivity over the coordinately regulated LDL receptor gene. The ability of 26-NH2 to interfere with myocyte proliferation and cholesterol synthesis without affecting the LDL receptor pathway confers at least in vitro a pharmacological interest on the compound in the process of atherogenesis.

Investigation of the biogenetic reaction sequence of cholesterol in rat tissues, through inhibition with AY-9944

An inhibitor of Δ7-reductase, AY-9944 (trans-1,4-bis(2-dichlorobenzylaminomethyl cyclohexane dihydrochloride), was used to investigate the last steps of cholesterol formation in brain and liver of adult and newborn rats. The accumulation of different sterols in the two tissues of the same animals was observed. Δ5,7-Cholestadien-3β-ol, Δ7,24-cholestadien-3β-ol and Δ5,7,24-cholestatrien-3β-ol, which are not present in detectable amounts in control brains, were identified in brains of growing rats treated with AY-9944. An accumulation of Δ5,7-cholestadien-3β-ol only was found in adult rat tissues.These differences in sterol accumulation are discussed in relation with the possible in vivo pathways of cholesterol biosynthesis.

Calcium Antagonists and Low Density Lipoprotein Receptors

The effect of different calcium antagonists on receptor-mediated LDL catabolism by human cells in culture was investigated. The calcium antagonists have been recently classified in six types, based on their pharmacological activities. The three types selective for the slow calcium channels (types I, II, and III), and the nonselective type IV have been investigated in respect to LDL metabolism. Calcium antagonists of type I (verapamil-related compounds) and type III (diltiazem) induce an increase of receptor-mediated uptake of human LDL. In contrast, calcium antagonists of type II (nifedipine-related compounds) and type IV (flunarizine) are inactive. Verapamil and diltiazem stimulate LDL receptor activity in normal fibroblasts, in fibroblasts obtained from a hypercholesterolemic type IIa heterozygous patient, in the human hepatoma cell line HepG2, but not in receptor-negative cells. The stimulatory effect depends on drug concentrations in the culture medium. Cycloheximide and alpha-amanitin prevent the stimulating effect of calcium antagonists on LDL uptake. The possible mechanisms of this action of calcium antagonists and the relationship between the in vitro stimulation of LDL receptor activity and the in vivo inhibition of lipid deposition in the arterial wall elicited by calcium antagonists are discussed. Calcium antagonists may exert part of their antiatherosclerotic activity by counteracting the inhibition of receptor-mediated lipid metabolism induced by calcium deposition in the cellular components of the arterial walls.

Ability of the LDL receptor from several animal species to recognize the human apo B binding domain: studies with LDL from familial defective apo B-100

To verify whether the LDL receptors from different animal species recognize the binding domain of human apo B-100 we studied the interaction of LDL from control and familial binding defective apo B-100 (FDB) with cultured cells. Human, monkey, bovine, guinea pig and rabbit LDL receptors distinguish between normal and binding defective LDL with a displacement ratio (defective/normal) of 3.3, 2.6, 3.4, 3.1 and 2.0, respectively. Guinea pig and rabbit receptors, however, showed affinities 2-3-fold lower than the human receptor. Hamster, rat and mouse cells failed to differentiate between normal and FDB LDL with a ratio of 1.2, 0.8, and 1.4; the apparent affinities were 4-8 times lower than that of the human receptor. The data from the latter species suggest that the LDL receptor recognizes an area of human apo B different from the human receptor binding domain. The ability of antibody Mb47 to inhibit the binding of human LDL to human, rabbit and guinea pig but not to mouse cells further stresses this concept. Moreover, in 17 alpha-ethinyl estradiol-treated rats the rate of disappearance from plasma of FDB and control 125I-labelled LDL was identical, thus confirming the in vitro observations. These data suggest that the binding domain of the LDL receptor is functionally conserved in man, monkey, cow, rabbit and guinea pig, but is quite distinct in rat, mouse and hamster.

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.