Proteomics Studies Suggest That Nitric Oxide Donor Furoxans Inhibit In Vitro Vascular Smooth Muscle Cell Proliferation by Nitric Oxide-Independent Mechanisms.

Physiologically, smooth muscle cells (SMC) and nitric oxide (NO) produced by endothelial cells strictly cooperate to maintain vasal homeostasis. In atherosclerosis, where this equilibrium is altered, molecules providing exogenous NO and able to inhibit SMC proliferation may represent valuable antiatherosclerotic agents. Searching for dual antiproliferative and NO-donor molecules, we found that furoxans significantly decreased SMC proliferation in vitro, albeit with different potencies. We therefore assessed whether this property is dependent on their thiol-induced ring opening. Indeed, while furazans (analogues unable to release NO) are not effective, furoxans' inhibitory potency parallels with the electron-attractor capacity of the group in 3 of the ring, making this effect tunable. To demonstrate whether their specific block on G1-S phase could be NO-dependent, we supplemented SMCs with furoxans and inhibitors of GMP- and/or of the polyamine pathway, which regulate NO-induced SMC proliferation, but they failed in preventing the antiproliferative effect. To find the real mechanism of this property, our proteomics studies revealed that eleven cellular proteins (with SUMO1 being central) and networks involved in cell homeostasis/proliferation are modulated by furoxans, probably by interaction with adducts generated after degradation. Altogether, thanks to their dual effect and pharmacological flexibility, furoxans may be evaluated in the future as antiatherosclerotic molecules.

Nitric oxide-donating atorvastatin attenuates neutrophil recruitment during vascular inflammation independent of changes in plasma cholesterol.

PURPOSE: Polymorphonuclear neutrophils, the first leukocytes to infiltrate the inflamed tissue, can make important contributions to vascular inflammatory processes driving the development of atherosclerosis. We herein investigated the effects of atorvastatin and NCX 6560 (a nitric oxide (NO)-donating atorvastatin derivative that has completed a successful phase 1b study) on neutrophilic inflammation in carotid arteries of normocholesterolemic rabbits subjected to perivascular collar placement. METHODS: Atorvastatin or NCX 6560 were administered orally (5 mg/kg/day or equimolar dose) to New Zealand White rabbits for 6 days, followed by collar implantation 1 h after the last dose. Twenty-four hours later carotids were harvested for neutrophil quantification by immunostaining. RESULTS: Treatment with NCX 6560 was associated with a lower neutrophil infiltration (-39.5 %), while atorvastatin did not affect neutrophil content. The result was independent of effects on plasma cholesterol or differences in atorvastatin bioavailability, which suggests an important role of NO-related mechanisms in mediating this effect. Consistent with these in vivo findings, in vitro studies showed that NCX 6560, as compared to atorvastatin, had greater inhibitory activity on processes involved in neutrophil recruitment, such as migration in response to IL-8 and IL-8 release by endothelial cells and by neutrophils themselves. Pretreatment with NCX 6560, but not with atorvastatin, reduced the ability of neutrophil supernatants to promote monocyte chemotaxis, a well-known pro-inflammatory activity of neutrophils. CONCLUSION: Experimental data suggest a potential role of NO-releasing statins in the control of the vascular inflammatory process mediated by polymorphonuclear neutrophils.

Targeting of the Lipid Metabolism Impairs Resistance to BRAF Kinase Inhibitor in Melanoma.

Drug resistance limits the achievement of persistent cures for the treatment of melanoma, in spite of the efficacy of the available drugs. The aim of the present study was to explore the involvement of lipid metabolism in melanoma resistance and assess the effects of its targeting in cellular models of melanoma with acquired resistance to the BRAF-inhibitor PLX4032/Vemurafenib. Since transcriptional profiles pointed to decreased cholesterol and fatty acids synthesis in resistant cells as compared to their parental counterparts, we examined lipid composition profiles of resistant cells, studied cell growth dependence on extracellular lipids, assessed the modulation of enzymes controlling the main nodes in lipid biosynthesis, and evaluated the effects of targeting Acetyl-CoA Acetyltransferase 2 (ACAT2), the first enzyme in the cholesterol synthesis pathway, and Acyl-CoA Cholesterol Acyl Transferase (ACAT/SOAT), which catalyzes the intracellular esterification of cholesterol and the formation of cholesteryl esters. We found a different lipid composition in the resistant cells, which displayed reduced saturated fatty acids (SFA), increased monounsaturated (MUFA) and polyunsaturated (PUFA), and reduced cholesteryl esters (CE) and triglycerides (TG), along with modulated expression of enzymes regulating biosynthetic nodes of the lipid metabolism. The effect of tackling lipid metabolism pathways in resistant cells was evidenced by lipid starvation, which reduced cell growth, increased sensitivity to the BRAF-inhibitor PLX4032, and induced the expression of enzymes involved in fatty acid and cholesterol metabolism. Molecular targeting of ACAT2 or pharmacological inhibition of SOAT by avasimibe showed antiproliferative effects in melanoma cell lines and a synergistic drug interaction with PLX4032, an effect associated to increased ferroptosis. Overall, our findings reveal that lipid metabolism affects melanoma sensitivity to BRAF inhibitors and that extracellular lipid availability may influence tumor cell response to treatment, a relevant finding in the frame of personalized therapy. In addition, our results indicate new candidate targets for drug combination treatments.

LIPA gene mutations affect the composition of lipoproteins: Enrichment in ACAT-derived cholesteryl esters.

BACKGROUND AND AIMS: Cholesteryl ester storage disease (CESD) due to LIPA gene mutations is characterized by hepatic steatosis, hypercholesterolemia and hypoalphalipoproteinemia, exposing affected patients to an increased cardiovascular risk. Further insights into the impact of LIPA gene mutations on lipid/lipoprotein metabolism are limited. Aim of the study was to investigate the effect of carrying one or two mutant LIPA alleles on lipoprotein composition and function. METHODS: Lipoproteins were isolated from 6 adult CESD patients, 5 relatives carrying one mutant LIPA allele (carriers) and 12 sex/age matched controls. Lipid profile, lipoprotein mass composition and the fatty acid distribution of cholesteryl esters (CEs) were assessed. HDL function was evaluated as the ability to promote nitric oxide release by endothelial cells. RESULTS: Despite the lipid-lowering therapy, total cholesterol, LDL-cholesterol and triglycerides were increased in CESD patients compared to controls, while HDL-cholesterol was reduced. Carriers also displayed elevated total and LDL-cholesterol. Very low and intermediate density lipoproteins from CESD patients and carriers were enriched in CEs compared to the control ones, with a concomitant reduction of triglycerides. Fatty acid composition of CEs in serum and lipoproteins showed a depletion of linoleate content in CESD patients, due to the reduced LCAT activity. In CESD HDL, fatty acid distribution of CEs was shifted towards saturated ones, if compared to control HDL. The changes in HDL composition did not affect HDL ability to promote nitric oxide release by endothelial cells. CONCLUSIONS: LIPA gene mutations significantly affected plasma levels and lipid composition of lipoproteins, likely contributing to the increased cardiovascular risk of affected patients.

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.

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.

Raloxifene inhibits matrix metalloproteinases expression and activity in macrophages and smooth muscle cells.

Secretion of matrix metalloproteinases (MMPs) by macrophages and smooth muscle cells (SMC) may impair atherosclerotic cap integrity leading to atherosclerosis complications. Selective estrogen receptor modulators (SERMs) have favourable impact on plasma lipid levels, but their role in the prevention of atherosclerosis still remains unclear. We investigated the effects of raloxifene, a second generation SERM, on MMP expression and activity in cultured macrophages and SMC, and in rabbit carotid lesions. Human monocyte-derived macrophages were isolated from blood of healthy donors. SMC were isolated from the intima-media layers of collared rabbit carotid arteries. Cells were incubated for 24h with increasing concentrations of raloxifene. Ovariectomized rabbits fed a 1% cholesterol-rich diet were subjected to pericarotid collar placement and treated with or without 10mgkg(-1)d(-1) raloxifene for 2 weeks. In macrophages, raloxifene treatment (0.1-10microM) significantly reduced MMP-9 gelatinolytic potential in a concentration-dependent manner, without affecting MMP-9 activation. This effect was estrogen receptor (ER)-dependent and due to the inhibition of MMP-9 promoter-driven transcription following an interaction with NF-kB pathway. Similarly, in cultured SMC, raloxifene inhibited up to 40% MMP-2 gelatinolytic activity. In vivo, raloxifene decreased the expression of MMP-2, MMP-3, and MMP-9 by intimal cells and the total gelatinolytic activity of collared carotids. These effects were accompanied by reduction of lesion size and inhibition of macrophage accumulation. Overall, results indicate that raloxifene may reduce MMPs expression and activity in macrophages and smooth muscle cells and favourably affect lesion formation.