Agnese Granata

Everolimus Inhibits Monocyte/Macrophage Migration in Vitro and Their Accumulation in Carotid Lesions of Cholesterol-Fed Rabbits

Monocytes/macrophages recruited into the arterial wall during atherogenesis are crucial in the initiation and progression of atherosclerosis and play a fundamental role in the destabilization process that is the main causal event of acute coronary syndromes. In the present study, we investigated the effect of the mammalian target of rapamycin inhibitor everolimus on macrophage accumulation within carotid lesions elicited by perivascular collar placement in cholesterol-fed rabbits. Everolimus (1.5 mg/kg given 1 day before collaring followed by 1 mg/kg/day for 14 days, administered by oral gavage) markedly decreased lesion macrophage content as compared with vehicle control (-65%; p < 0.01). This effect was associated with a reduction in intimal thickening and occurred in the absence of changes in plasma cholesterol concentrations. To gain insights on the potential mechanism(s) underlying this effect, we investigated the influence of everolimus on chemoattractant-induced migration of human monocytes in vitro. Pretreatment with therapeutic concentrations of everolimus (10 nM) significantly lowered monocyte chemotaxis in response to various chemotactic factors (i.e., monocyte chemoattractant protein-1/CCL2, fractalkine/CX3CL1, interleukin-8/CXCL8, complement fragment 5a, or N-formyl-Met-Leu-Phe) without inducing monocyte cell death. These results suggest that everolimus may favorably influence the atherosclerotic process by affecting the recruitment of monocytes into early lesions.

Effect of S(-) perillic acid on protein prenylation and arterial smooth muscle cell proliferation.

A number of proteins post-translationally modified by the covalent attachment of mevalonate-derived isoprene groups farnesol (FOH) or geranylgeraniol (GGOH), play a role in cell proliferation. For this reason, protein farnesyltransferase (PFTase) and protein geranylgeranyltransferases (PGGTases) I and II have gained attention as novel targets for the development of antiproliferative agents. Monoterpenes [limonene, perillic acid (PA) and its derivatives] have been shown to inhibit cell growth and protein prenylation in cancer cells. In the present study, we evaluated the effect of S(-) PA on diploid rat aorta smooth muscle cell (SMC) proliferation as related to protein prenylation. S(-) PA (1-3.5 mM) decreased, in a concentration-dependent manner, rat SMC proliferation as evaluated by cell counting and DNA synthesis. Morphological criteria and flow cytometry analysis excluded the induction of apoptosis as a potential antiproliferative mechanism of S(-) PA on SMC and confirmed a block of the cell cycle progression in G(0)/G(1) phase. The antiproliferative effect of S(-) PA could not be prevented by the addition of mevalonate, FOH, and GGOH to the culture medium and was independent of cholesterol biosynthesis. Densitometric analysis of fluorographed gels, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell lysates, further supported that S(-) PA (1-3.5 mM), under the same experimental conditions, concentration-dependently inhibited FOH (up to 70%) and GGOH (up to 70%) incorporation into cellular proteins. We provide evidence that S(-) PA affects protein prenylation, an effect that may contribute to its inhibition of SMC proliferation.

Mevalonate pathway and isoprenoids regulate human bronchial myocyte proliferation

The role of mevalonate and geranylgeraniol in the control of cellular proliferation of cultured human bronchial myocytes was examined by investigating the effect of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in mevalonate synthesis. Simvastatin inhibited the rate of growth of human bronchial smooth muscle cells in a concentration-dependent manner, with an IC50 value of 0.97 +/- 0.1 microM. Mevalonate (100 microM), as well as geranylgeraniol (5 microM), at their highest non-toxic concentrations, restored cell proliferation to control levels.

Fluvastatin synergistically improves the antiproliferative effect of everolimus on rat smooth muscle cells by altering p27kip1/cyclin E expression

Multiple intracellular signaling pathways stimulate quiescent smooth muscle cells (SMCs) to exit from G0 and re-enter the cell cycle. Thus, a combination of two drugs with different mechanisms of action may represent a suitable approach to control SMC proliferation, a prominent feature of in-stent restenosis. In the present study, we investigated the effect of everolimus, a mammalian target of rapamycin inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. The antiproliferative action of everolimus was amplified by 2.5-fold by the addition of subliminal concentrations of fluvastatin (5 × 10-7 M), lowering the IC50 value from 2.5 × 10-9 to 1.0 × 10-9 M. The increased antiproliferative effect of everolimus by fluvastatin was prevented in the presence of mevalonate, farnesol, or geranylgeraniol, suggesting the involvement of prenylated proteins. Cell cycle analysis and [3H]thymidine incorporation assay demonstrated that the two drugs synergistically interfered with the progression of G1 phase. In particular, the drug combination significantly up-regulated p27Kip1 levels by 47.0%, suppressed cyclin E by 43.0%, and it reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared with everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of rat SMCs to the antiproliferative action of the drug combination, measured by cell counting, [3H]thymidine incorporation, and cell cycle analysis, with higher levels of hyperphosphorylated form of Rb. Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27kip1, which affects Rb phosphorylation and leads to G1 phase arrest.

Free cholesterol alters macrophage morphology and mobility by an ABCA1 dependent mechanism

OBJECTIVE To investigate whether the morphological and functional changes typical of cell immobilization induced by free cholesterol (FC) accumulation in macrophages is related to the activity of the ATP-binding cassette transporter (ABCA1). METHODS AND RESULTS FC loading induced actin rearrangement with ruffling and cell spreading in macrophages that normally express ABCA1, but to a significant lesser extent in ABCA1-KO mouse peritoneal macrophages (MPMs) and in normal cells upon pharmacological inhibition of ABCA1 with probucol. In ABCA1-KO MPMs and in probucol-treated J774 cell migration was inhibited to a lower extent by FC as compared to control cells. Similar results were found in stably ABCA1 knocked down J774 (ABCA1-KD-J774) obtained by RNA interference. FC accessible to cholesterol oxidase, a measure of plasma membrane FC content, was significantly higher in FC-loaded WT MPMs and control J774 than in FC-loaded ABCA1-KO MPMs, ABCA1-KD-J774 or probucol-treated J774. In parallel plasma membrane total phospholids and sphingomyelin increased after cholesterol loading in control J774 but not in ABCA1-KD-J774. In addition, apoA-I, that removes FC from ABCA1 specific pool, partially restored chemotactic response in FC-loaded control J774. No effect was observed with HDL(2) that does not interact with ABCA1. Finally, FC-induced Rac activation was more efficient in control J774 than in ABCA1-KD-J774. and was prevented by probucol and apoA-I in control J774. CONCLUSION In macrophages ABCA1 activity mediates FC ability to alter plasma membrane organization, to inhibit cell migration, and to activate a Rac-mediated signaling pathway.

27-Hydroxycholesterol modulation of low density lipoprotein metabolism in cultured human hepatic and extrahepatic cells

27‐Hydroxycholesterol, 25‐hydroxycholesterol and cholesterol suppressed LDL uptake and degradation in human extrahepatic and hepatic cell lines hi a concentration‐dependent manner. Cholesterol was the least potent, and the inhibitory effect of oxysterols was more pronounced in skin fibroblasts and in endothelial cell line EAhy 926 than in hepatoma HepG2 cells. Shorter incubations were required for oxysterols to achieve 50% inhibition of LDL uptake and degradation in all cultured cells. The inhibition of LDL catabolism in extrahepatic cells by 27‐hydroxycholesterol occurred at concentrations close to those observed in human plasma (0.2–0.6 μM). The results support a possible role of 27‐hydroxycholesterol, a physiological oxysterol, in the regulation of cellular cholesterol homeostasis in non‐hepatic tissues.

Progesterone modulates the expression of HDL binding sites in human skin fibroblasts

The aim of this work was to study the effects of progesterone on the expression of high density lipoprotein binding sites by cultured human skin fibroblasts. At concentrations ranging between 10(-6)- and 10(-4) M the hormone showed a dose-dependent induction of the HDL binding sites. The effect was maximal at 48 h. The increased HDL binding was only due to an up-regulation of binding sites, without changes of the apparent Kd. This effect was not related to changes of cellular cholesterol content, and was not affected by inhibition of protein synthesis. These data suggest that the expression of binding sites for HDL can be modulated via a mechanism that does not depend upon cellular cholesterol content.

Trapidil derivatives and low density lipoprotein metabolism by human skin fibroblasts and by human hepatoma cell line Hep G2

The effect of trapidil (RocornalR) and some of its newly developed derivatives (AR 12456, AR 12463, AR 12465, AR 12464) on the receptor-mediated low density lipoprotein (LDL) binding, uptake and degradation was studied in human skin fibroblasts (HSF) and in human hepatoma cell line Hep G2. Compound AR 12456 influenced this pathway in a selective way: it enhanced the uptake and degradation of 125I-LDL by Hep G2 cells in a dose-dependent manner, but inhibited it in HSF. Scatchard analysis of the saturable LDL binding in Hep G2 indicates that the effect of compound AR 12456 is the result of an increased number of LDL binding sites. Compound AR 12465 was less effective on LDL catabolism. Trapidil and the other derivatives were inactive under the same experimental conditions. When Ar 12456 was preincubated with Hep G2 cells and then the incubation medium was transferred to HSF, a stimulation of specific LDL pathway occurred also in this cell line. These findings suggest that a metabolite(s) of AR 12456 might be responsible for the enhanced expression of LDL receptors in cultured human cells.

Monoclonal antibody 5A binds apolipoprotein B-48 and inhibits the low density lipoprotein-receptor interaction.

In a panel of 10 monoclonal antibodies raised against human LDL we detected three antibodies (named 5A, 6B, and 6E) which recognize both apolipoprotein B-100 and B-48. Antibody 5A inhibited, in a dose dependent manner, the interaction of 125I-LDL with their receptor on human skin fibroblasts. Using thrombolytic fragments, the epitope of antibody 5A was mapped to the carboxy terminal region of apo B-48. MAB 5A was equipotent with MAB Mb 47, an inhibitory antibody whose epitope lies near a putative receptor binding domain of apo B in thrombolytic fragment T2. These findings suggest that areas other than the carboxy terminal portion of apo B-100 may participate in the LDL-receptor interaction, either directly or by determining the exposition of high affinity sites of apo B-100.

Causes Underlying the Reduced Response to Simvastatin Treatment in Hypercholesterolemic Patients

Simvastatin, a competitive inhibitor of HMG-CoA reductase, effectively reduces elevated plasma cholesterol levels by up regulating the LDL receptor expression. Therefore in patients whose hypercholesterolemia (HC) is due to a defective LDL-receptor interaction vastatins may not be as effective as expected. To verify this hypothesis we studied the possible causes for the poor response (<15% decrease of LDL cholesterol) to simvastatin (40 mg/die) in 11 HC patients. Biochemical defects were identified in 5 patients. Three patients presented with binding-defective LDL, without the 3500 mutation, and the remaining two had normal LDL but their serum contained factors interfering with the LDL-receptor interaction. From these results we conclude that distinct biochemical defects might contribute to the poor response to simvastatin in hypercholesterolemic patients.