Maurizio Soma

Inhibition of isoprenoid biosynthesis and arterial smooth-muscle cell proliferation.

Recently, we provided in vitro and in vivo evidence that several vastatins with different potencies decrease arterial smooth-muscle cell (SMC) proliferation independently of their hypocholesterolemic properties. In this study, the in vivo dose-dependent antiproliferative activity of fluvastatin on neointimal formation induced by the insertion of a collar around one carotid artery was investigated in normocholesterolemic rabbits (five animals per treatment group). Intraperitoneal fluvastatin treatment progressively inhibited intimal to medial tissue ratios (I/M) by 5, 48, and 64% versus controls at doses of 3, 5, or 10 mg/kg/day, respectively. Local arterial delivery by an Alzet pump of mevalonate (8 mg/kg/day) at the site of collar placement fully prevented a fluvastatin (5 mg/kg/day) inhibitory effect on both I/M and SMC proliferation, as assessed by direct incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The results suggest that vastatins exert a direct antiproliferative effect on intimal myocytes beyond their effects on plasma lipids, probably through local inhibition of isoprenoid biosynthesis.

Cholesterol metabolism in hypercholesterolemia-resistant rabbits.

Normal rabbits typically respond to a diet high in cholesterol with a large increase in the concentration of plasma cholesterol. We have previously described the breeding and partial characterization of a variant rabbit which does not respond to a high cholesterol diet with changes in plasma cholesterol concentration. In the present report we have characterized three components involved in cholesterol homeostasis: the B/E (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (HMG-CoA reductase, EC 1.1.1.34) and acyl-coenzyme A: cholesterol acyltransferase activity (ACAT, EC 2.3.1.26) in the livers of the hypercholesterolemia-resistant rabbits. Using normal cholesterol-fed rabbit [125I] beta-VLDL as a ligand, liver membranes prepared from resistant rabbits fed a low-cholesterol diet had 70% higher binding capacity than membranes from normal rabbits fed the same diet. Similar experiments demonstrated that the resistant rabbits had a 240% higher B/E receptor binding capacity compared to normal animals when liver membranes were prepared from animals fed a 0.25% cholesterol-enriched diet. No difference in the binding affinity of [125I]beta-VLDL was detected in membranes prepared from normal or resistant animals. When fed a low-cholesterol diet, the resistant rabbits had approximately 2-fold higher hepatic HMG-CoA reductase activity (97.4 +/- 3.5 pmol product/mg/min in resistant animals compared to 45 +/- 1.1 pmol product/min/mg in normal animals). The difference was exaggerated in animals fed the 0.25% cholesterol-enriched diet, 73.3 +/- 5.5 vs 2.4 +/- 0.56 pmol product/min/mg for resistant and normal membranes respectively. The basal activity of ACAT in hepatic membranes was significantly lower in the resistant rabbits compared to normal rabbits (138 +/- 11 vs 268 +/- 19 pmol cholesteryl ester/min/mg in resistant and normal rabbits respectively); when fed a 0.25% cholesterol-enriched diet, the enzyme was induced 6-fold in normal animals but was increased only 2-fold in the resistant animal. These biochemical data suggested that the resistant rabbit maintained low intracellular cholesterol even when fed a cholesterol-enriched diet. Direct measurement of cellular cholesterol and cholesteryl esters demonstrated that the concentration of these lipids was significantly lower in the resistant animal than in normal animals with the largest differences found in the cytoplasmic rather than the membrane compartment. These studies demonstrate that the resistant rabbit manifests several quantitative differences in cholesterol metabolism and in the regulation of cholesterol metabolism; but these studies do not directly explain the underlying cause of the resistance to hypercholesterolemia in the resistant rabbit.

Plasma catabolism of human apolipoprotein E isoproteins: Lack of conversion of the doubly sialilated form to the asialo form in plasma

Apolipoprotein E (apoE) circulates as a mixture of sialylated and asialylated forms. In this study the catabolic fate and the plasma turnover rate of the different apoE forms have been investigated in vivo in humans. Asialo apoE (E) and doubly sialylated apoE (Ess) were isolated by preparative isoelectrofocusing from the VLDL of subjects homozygous for the E3 allele. 131E3 and 125E3ss were injected simultaneously into three hypertriglyceridemic subjects, and plasma samples were collected up to the sixth day. VLDL were isolated by ultracentrifugation, and the apoE forms were separated by isoelectrofocusing. Gel bands corresponding to E3 and E3ss were cut out and counted for the associated radioactivity. Residence times in plasma for 131E3 and 125E3ss were 0.95 +/- 0.16 and 0.74 +/- 0.16 days, respectively. As determined from the gel count distribution up to 24 hours, no conversion of the injected sialylated form to the correspondent asialylated form was detected.

Rapid modulation of rat adipocyte lipoprotein lipase: effect of calcium, A23187 ionophore, and thrombin

The effect of calcium ions on the lipoprotein lipase (LPL) activity in rat adipocytes has been investigated. Incubation of the cells in the absence of extracellular calcium produced a rapid decline of LPL activity in the cells. The enzyme, however, could be immediately reactivated in less than 3 min by the addition of calcium. The degree of reactivation was proportional to the concentration of extracellular calcium. alpha 1 agonists phenylephrine and methoxamine affected LPL activity only slightly, as did vasopressin and angiotensin II. In contrast, calcium ionophore A23187 elicited a quick and transient enzyme activation which reached its peak 4 min after the addition of the drug. Thrombin (0.1 U/ml) produced the most rapid and intense response. The effect of thrombin was already evident 10 s after its addition, and the enzyme activity almost doubled above the basal level. Extracellular calcium was necessary to achieve thrombin activation. Contrary to previous thought, these data support the conclusion that LPL may undergo rapid activation, and that calcium ions are critically involved in this activation process. Thrombin rapidly raises LPL activity and may be one of its physiological activators in vivo.

Estrogen Effect on Brain Biology and Cognition

The central nervous system (CNS) is an important target for the activity of sex steroids. Several studies have demonstrated that estrogen receptors are expressed in different brain areas, although the mechanism through which estrogens act on the CNS are not yet fully understood and may involve both direct actions on the CNS as well as systemic actions on vasculature or metabolism.