Prenylcysteine oxidase 1, an emerging player in atherosclerosis.

The research into the pathophysiology of atherosclerosis has considerably increased our understanding of the disease complexity, but still many questions remain unanswered, both mechanistically and pharmacologically. Here, we provided evidence that the pro-oxidant enzyme Prenylcysteine Oxidase 1 (PCYOX1), in the human atherosclerotic lesions, is both synthesized locally and transported within the subintimal space by proatherogenic lipoproteins accumulating in the arterial wall during atherogenesis. Further, Pcyox1 deficiency in Apoe-/- mice retards atheroprogression, is associated with decreased features of lesion vulnerability and lower levels of lipid peroxidation, reduces plasma lipid levels and inflammation. PCYOX1 silencing in vitro affects the cellular proteome by influencing multiple functions related to inflammation, oxidative stress, and platelet adhesion. Collectively, these findings identify the pro-oxidant enzyme PCYOX1 as an emerging player in atherogenesis and, therefore, understanding the biology and mechanisms of all functions of this unique enzyme is likely to provide additional therapeutic opportunities in addressing atherosclerosis.

Marked neointimal lipoprotein lipase increase in distinct models of proclivity to atherosclerosis: a feature independent of endothelial layer integrity.

Lipoprotein lipase (LPL) in the arterial wall has been proposed to enhance the retention of apoB-containing lipoproteins, an early event in atherosclerosis. As the neointima is considered the primary site of lipid accumulation in atherogenesis, the arterial expression and location of LPL was investigated in distinct experimental models of neointimal formation in normolipidemic rabbits and rats. Neointima elicited by balloon aortic denudation or raised beneath an anatomically intact endothelial layer by placing a silastic collar around the common carotid artery, both showed a striking LPL immunostaining that mostly co-localized with neointimal smooth muscle cells. Besides, increased LPL protein and mRNA in deendothelialized aortas was demonstrated by Western and Northern blot analysis, respectively, suggesting an enhanced expression of LPL in injured arteries. It was concluded that LPL is increased in neointima developed in either denuded vessels or arteries with a preserved endothelium, a finding which suggests that LPL abundance may be an attribute of the neointima, whatever the stimulus that promotes its formation. On the basis of former evidence concerning the role of LPL in lipid retention, this study provides a possible explanation for the injury-induced vessel susceptibility to atherosclerosis, and the particular proneness of the neointimal layer to lipid accretion.

Clinically relevant pleiotropic effects of statins: drug properties or effects of profound cholesterol reduction?

Clinical trials have firmly established that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) can induce the regression of vascular atherosclerosis and reduce cardiovascular-related morbidity and death in patients with and without coronary artery disease. It is usually assumed that these beneficial effects are due to the ability of statins to reduce cholesterol synthesis. However, because mevalonic acid is not only the precursor of cholesterol but also of many non-steroidal isoprenoid compounds, the inhibition of HMG-CoA reductase may lead to pleiotropic effects. As shown by the data reported in this review, some statins can interfere with major events involved in the formation of atherosclerotic lesions, regardless of their hypolipidemic properties. The relevance of these effects in humans remains to be established (particularly in view of the high statin doses required to produce a direct vascular action), thus their contribution to the reduction in cardiovascular events observed in clinical trials has become one of the major challenges for future studies aimed at clarifying the anti-atherosclerotic benefits of statins.

Upregulation and activation of Stat6 precede vascular smooth muscle cell proliferation in carotid artery injury model.

The role of signal transducers and activators of transcription (STAT) proteins in modulating proliferation and differentiation of various cell types in the hematopoietic system and the central nervous system has been well established. In contrast, the pathophysiological role of these proteins in vascular proliferative diseases has remained unproven, despite in vitro observations emphasizing the involvement of the STAT system in mediating vascular smooth muscle cell (VSMC) proliferation. On the basis of our previous observations demonstrating the occurrence of a specific modulation of Stat6 protein during the proliferative, migratory, and differentiation phases of the developing brain, we investigated whether Stat6 protein is present and modulated in arterial tissue challenged by perivascular injury. The time course of expression and localization of Stat6 after arterial injury was analyzed by immunohistochemistry, Western blot analysis, and confocal microscopy. Six hours after injury, the expression of Stat6 was markedly increased. This overexpression preceded the onset of VSMC proliferation and was downregulated starting from 7 days after injury, coincident with the decline of VSMC proliferation. Moreover, early after injury, Stat6 was predominantly localized at the nuclear level, denoting its functional activation. Conversely, Stat6 staining at later time points was largely cytosolic, suggesting silencing effects of this signaling pathway. These data indicate that Stat6 signaling may contribute to the modifications of gene expression underlying VSMC activation in the context of acute vascular proliferative diseases.

New insights into the pharmacodynamic and pharmacokinetic properties of statins.

The beneficial effects of statins are assumed to result from their ability to reduce cholesterol biosynthesis. However, because mevalonic acid is the precursor not only of cholesterol, but also of many nonsteroidal isoprenoid compounds, inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase may result in pleiotropic effects. It has been shown that several statins decrease smooth muscle cell migration and proliferation and that sera from fluvastatin-treated patients interfere with its proliferation. Cholesterol accumulation in macrophages can be inhibited by different statins, while both fluvastatin and simvastatin inhibit secretion of metalloproteinases by human monocyte-derived macrophages. The antiatherosclerotic effects of statins may be achieved by modifying hypercholesterolemia and the arterial wall environment as well. Although statins rarely have severe adverse effects, interactions with other drugs deserve attention. Simvastatin, lovastatin, cerivastatin, and atorvastatin are biotransformed in the liver primarily by cytochrome P450-3A4, and are susceptible to drug interactions when co-administered with potential inhibitors of this enzyme. Indeed, pharmacokinetic interactions (e.g., increased bioavailability), myositis, and rhabdomyolysis have been reported following concurrent use of simvastatin or lovastatin and cyclosporine A, mibefradil, or nefazodone. In contrast, fluvastatin (mainly metabolized by cytochrome P450-2C9) and pravastatin (eliminated by other metabolic routes) are less subject to this interaction. Nevertheless, a 5- to 23-fold increase in pravastatin bioavailability has been reported in the presence of cyclosporine A. In summary, statins may have direct effects on the arterial wall, which may contribute to their antiatherosclerotic actions. Furthermore, some statins may have lower adverse drug interaction potential than others, which is an important determinant of safety during long-term therapy.

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

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. 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. 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. 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.

Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses.

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.

The menopause and lipid metabolism: strategies for cardiovascular disease prevention.

The menopause and its biology are as yet still incompletely understood. Very little is known about biological and molecular changes in cardiovascular target tissues and organs after menopause. Experimental and clinical evidence indicate that prevention of cardiovascular disease by estrogens is aimed both at the correction of risk factors and at the direct control of vessel structure and function. The effects of progestogens on these processes are still debated. Few other medical interventions have as great a potential for affecting morbidity and mortality as does hormone replacement therapy in postmenopausal women. Hormone replacement therapy has produced effects on health risk, some are reduced, some are raised, while some remain uncertain, suggesting that further testing through specific clinical trials are required before confident recommendations can be made about the full range of benefits and risks. Lipid lowering therapy may be an acceptable alternative for postmenopausal women at risk for cardiovascular disease.

Proapoptotic effect of atorvastatin on stimulated rabbit smooth muscle cells.

The in vitro and in vivo activity of atorvastatin and other 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors (fluvastatin, pravastatin and simvastatin) has been investigated. Atorvastatin, fluvastatin, pravastatin and simvastatin caused a significant and dose-dependent (0.1-50 microM) reduction in cell multiplication of vascular smooth muscle cells (SMC) in cultures associated with the retardation of cycling cells in the G1 and G2/M compartments at 24 h, a phenomenon leading to apoptosis (programmed cell death) in several experimental in vitro models. The effects on the cell cycle resulted in a strong inhibition of cell proliferation at 48 h, followed by apoptosis when incubation was prolonged to 72 h as assessed by nuclei morphology and cytofluorimetric analysis of DNA. The apoptotic effect observed for the statins is completely prevented by the addition of exogenous mevalonate at a 100 microm concentration. in vivo SMC proliferation was stimulated by applying a silastic collar to the outside surface of carotid arteries in normocholesterolemic rabbits in the presence of an anatomically intact endothelium. The positioning of the collar promoted apoptosis in control vessels as assessed by Terminal Deoxynucleotidyl Transferase-dUTP-Biotin Nick-End Labeling (TUNEL) assay. The pre-treatment with 5 mg kg-1 per day of atorvastatin before collar insertion strongly increased the number of TUNEL-positive cells, suggesting a pro-apoptotic effect of HMGCoA reductase inhibitors also in vivo, even though cell DNA rearrangement still needs to be excluded. No apoptotic signal was detectable in sham operated arteries with no collar in either control or atorvastatin-treated rabbits. These data indicate that HMGCoA reductase inhibitors effect on the arterial wall may involve the modulation of both cell proliferation and programmed cell deaths supporting a possible role of statins in the prevention of early lesion and restenosis.

Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content.

Plasma-membrane composition plays a crucial role in most of the cellular functions that depend on membrane processes. In virtually all cell types the proteolytic processing of Alzheimer amyloid precursor protein (APP) to generate soluble APP (sAPP) is believed to occur at the plasma membrane or in its immediate proximity. Alteration of this metabolic pathway has been linked to the pathogenesis of Alzheimer's disease. We analysed the effect of membrane cholesterol enrichment on APP metabolism. Incubation of COS cells with increasing concentrations of non-esterified cholesterol carried by rabbit beta-very low-density lipoprotein caused a dose-dependent inhibition of sAPP release: 70% inhibition with 10 microg/ml non-esterified cholesterol. A less pronounced inhibitory effect was observed on treatment with human low-density lipoprotein. Inhibition of sAPP release was independent of receptor-mediated lipoprotein metabolism since simultaneous treatment with chloroquine did not modify the effect of lipoprotein treatment. In addition, treatment with cholesterol dissolved in either ethanol or methyl-beta-cyclodextrin elicited the same effect. Excess non-esterified cholesterol did not cause cell toxicity. Cell cholesterol mass inversely correlated with sAPP release. Progesterone, which inhibits shuttling of non-esterified cholesterol between the plasma membrane and intracellular pools, had no effect on the inhibition of sAPP release from cholesterol-loaded cells, providing indirect evidence that cholesterol may act at the plasma membrane.

In vivo enhanced antitumor activity of carmustine [N,N'-bis(2-chloroethyl)-N-nitrosourea] by simvastatin.

The effects of a combination of simvastatin, a cholesterol-lowering agent, and carmustine (BCNU; N,N'-bis(2-chloroethyl)-N-nitrosourea) on experimental C6 glioma were studied in vitro and in vivo. In vitro simvastatin and BCNU alone inhibited cell proliferation in a dose-dependent fashion. A subliminal concentration of simvastatin (0.1 microM) markedly and synergistically increased the BCNU toxicity to C6 glioma cells. The cytofluorimetric analysis of DNA from simvastatin-treated C6 glioma cells showed, besides the already described arrest in G1, an arrest/retardation in G2-M. Mitotic index from C6 cells incubated with simvastatin (10 microM) decreased by about 90%, indicating a specific C6 arrest/retardation in G2. The drug effects could be completely reversed by simvastatin withdrawal or mevalonate addition to the cultured cells. The combination of simvastatin and BCNU resulted predominantly from the profound retardation of cells in the G2-M compartment of the cell cycle. In vivo simvastatin (administered daily mixed with food) and BCNU (single i.p. injection), when given separately, caused a dose-dependent inhibition of labeling index in C6 glioma homografts (ID50, 61 mg/kg/day and 8.7 mg/kg, respectively). The combination of the lowest doses tested (simvastatin, 25 mg/kg/day and BCNU 0.3 mg/kg) resulted in a significant growth delay (compared to either drug alone) in C6 glioma (P < 0.05). There was no significant increase in toxicity as assessed by myelosuppression (WBC counts and bone marrow labeling index) and body weight. The results provide in vivo support for the combined use of simvastatin, a cholesterol-lowering agent, and BCNU in brain tumor treatment.

PKC activity in rat C6 glioma cells: changes associated with cell cycle and simvastatin treatment.

The parallel effects of simvastatin on cell cycle and PKC activity in rat C6 glioma cells were investigated. Simvastatin, 2.5 microM, for 24 h resulted in cell growth arrest in early G1 phase of the cell cycle and in a significant increase of total PKC activity (283 +/- 42 vs 470 +/- 61 pmoles/min/mg protein p = 0.002 for control cells and simvastatin-treated cells, respectively). The effect of simvastatin was fully prevented by mevalonate. A time dependent increase of PKC activity was observed in control exponentially free-growing C6 cells approaching confluency: a highly significant negative correlation (r = -0.91 p < 0.0001) between PKC activity and growth rate was calculated. PKC activity was high in cells arrested in G0 by serum starvation (0.4%). Following addition of complete medium (17.5% serum) the PKC activity progressively decreased and reached a minimum when cells traversed the G2/M phase, as determined by DNA analysis distribution. PKC activity dropped 30% in simvastatin-arrested early G1 cells; 44% in hydroxyurea-arrested cells at the G1/S boundary; and 73% in Colcemid mitosis-blocked cells. The results show that C6 glioma cell PKC activity is maximal in a G0 quiescent state and varies at different points of the cell cycle.