Adelina Munteanu

Vitamin E Mediates Cell Signaling and Regulation of Gene Expression

Abstract: α‐Tocopherol modulates two major signal transduction pathways centered on protein kinase C and phosphatidylinositol 3‐kinase. Changes in the activity of these key kinases are associated with changes in cell proliferation, platelet aggregation, and NADPH‐oxidase activation. Several genes are also regulated by tocopherols partly because of the effects of tocopherol on these two kinases, but also independently of them. These genes can be divided in five groups: Group 1. Genes that are involved in the uptake and degradation of tocopherols: α‐tocopherol transfer protein, cytochrome P450 (CYP3A), γ‐glutamyl‐cysteine synthetase heavy subunit, and glutathione‐S‐transferase. Group 2. Genes that are implicated with lipid uptake and atherosclerosis: CD36, SR‐BI, and SR‐AI/II. Group 3. Genes that are involved in the modulation of extracellular proteins: tropomyosin, collagen‐α‐1, MMP‐1, MMP‐19, and connective tissue growth factor. Group 4. Genes that are connected to adhesion and inflammation: E‐selectin, ICAM‐1 integrins, glycoprotein IIb, IL‐2, IL‐4, IL‐1b, and transforming growth factor‐β (TGF‐β). Group 5. Genes implicated in cell signaling and cell cycle regulation: PPAR‐γ, cyclin D1, cyclin E, Bcl2‐L1, p27, CD95 (APO‐1/Fas ligand), and 5a‐steroid reductase type 1. The transcription of p27, Bcl2, α‐tocopherol transfer protein, cytochrome P450 (CYP3A), γ‐glutamyl‐cysteine sythetase heavy subunit, tropomyosin, IL‐2, and CTGF appears to be upregulated by one or more tocopherols. All the other listed genes are downregulated. Gene regulation by tocopherols has been associated with protein kinase C because of its deactivation by α‐tocopherol and its contribution in the regulation of a number of transcription factors (NF‐κB, AP1). A direct participation of the pregnane X receptor (PXR) / retinoid X receptor (RXR) has been also shown. The antioxidant‐responsive element (ARE) and the TGF‐β‐responsive element (TGF‐β‐RE) appear in some cases to be implicated as well.

Anti-atherosclerotic effects of vitamin E--myth or reality?

Atherosclerosis and its complications such as coronary heart disease, myocardial infarction and stroke are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. Several epidemiological studies and intervention trials have been performed with vitamin E, and some of them showed that it prevents atherosclerosis. For a long time, vitamin E was assumed to act by decreasing the oxidation of LDL, a key step in atherosclerosis initiation. However, at the cellular level, vitamin E acts by inhibition of smooth muscle cell proliferation, platelet aggregation, monocyte adhesion, oxLDL uptake and cytokine production, all reactions implied in the progression of atherosclerosis. Recent research revealed that these effects are not the result of the antioxidant activity of vitamin E, but rather of precise molecular actions of this compound. It is assumed that specific interactions of vitamin E with enzymes and proteins are at the basis of its non‐antioxidant effects. Vitamin E influences the activity of several enzymes (e.g. PKC, PP2A, COX‐2, 5‐lipooxygenase, nitric oxide synthase, NADPH oxidase, superoxide dismutase, phopholipase A2) and modulates the expression of genes that are involved in atherosclerosis (e.g. scavenger receptors, integrins, selectins, cytokines, cyclins). These interactions promise to reveal the biological properties of vitamin E and allow designing better strategies for the protection against atherosclerosis progression.

Regulation of gene expression by α-tocopherol

Abstract Several genes are regulated by tocopherols which can be categorized, based on their function, into five groups: genes that are involved in the uptake and degradation of tocopherols (Group 1) include α-tocopherol transfer protein (α-TTP) and cytochrome P450 (CYP3A); genes that are associated with lipid uptake and atherosclerosis (Group 2) include CD36, SRBI and SRAI/II. Genes that modulate the expression of extracellular proteins (Group 3) include tropomyosin, collagenα1, MMP-1, MMP-19 and connective tissue growth factor (CTGF). Genes that are related to inflammation, cell adhesion and platelet aggregation (Group 4) include Eselectin, ICAM-1, integrins, glycoprotein IIb, Il-2, IL-4 and IL-β. Group 5 comprises genes coding for proteins involved in cell signaling and cell cycle regulation and consists of PPARγ, cyclin D1, cyclin E, Bcl2-L1, p27 and CD95 (Apo-1/Fas ligand). The expression of P27, Bcl2, α-TTP, CYP3A, tropomyosin, Il-2, PPAR-γ, and CTGF appears to be up-regulated by one or more tocopherols whereas all other listed genes are down-regulated. Several mechanisms may underlie tocopherol-dependent gene regulation. In some cases protein kinase C has been implicated due to its deactivation by α-tocopherol and its participation in the regulation of a number of transcription factors (NFκB, AP-1). In other cases a direct involvement of PXR/ RXR has been documented. The antioxidant responsive element (ARE) appears in some cases to be involved as well as the transforming growth factor β responsive element (TGF-β-RE). This heterogeneity of mediators of tocopherol action suggests the need of a common element that could be a receptor or a co-receptor, able to interact with tocopherol and with transcription factors directed toward specific regions of promoter sequences of sensitive genes. Here we review recent results of the search for molecular mechanisms underpinning the central signaling mechanism.

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma.

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by α-tocopherol. We show here that α-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, α-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3′-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by α-tocopherol. However, α-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-γ (PPARγ; troglitazone), indicating that this pathway is susceptible to α-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and α-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARγ element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARγ and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARγ signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Characterization of three human sec14p-like proteins: α-Tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled alpha-tocopherol to mitochondria in the same order of magnitude as the human alpha-tocopherol transfer protein (alpha-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Modulation of Cell Proliferation and Gene Expression by α‐Tocopheryl Phosphates: Relevance to Atherosclerosis and Inflammation

Abstract: The effect of a mixture of α‐tocopheryl phosphate plus di‐α‐tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP‐1 monocytic leukemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which α‐tocopherol was equally inhibitory. TPm led in nonstimulated THP‐1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low‐density lipoprotein surface binding and oxLDL uptake. In nonstimulated THP‐1 cells, α‐tocopherol had only very weak effects on these events.

HIV Protease Inhibitors‐induced Atherosclerosis: Prevention by α‐Tocopherol

Prolonged treatments with inhibitors of human immunodeficiency (HIV)‐encoded protease (ARPI) have been reported to induce early atherosclerotic events. Our in vitro study indicates that α‐tocopherol may prevent drug‐induced premature atherosclerosis since it interferes with CD36 scavenger receptor over‐expression induced by ritonavir in monocytes. The mechanism of CD36 upregulation by ritonavir involves inhibition of the ubiquitin‐proteasome system and α‐tocopherol is able to normalize proteasome activity. These findings suggest that ARPI combined with early α‐tocopherol supplementation may decrease the drug‐induced atherosclerotic risk. IUBMB Life, 56: 629‐631, 2004

Exposure of Human Endothelial Progenitors to Sevoflurane Improves Their Survival Abilities

Abstract Endothelial progenitor cells (EPCs) have prominent roles in vessel and tissue repair; however, their regenerative efficacy is diminished due to the poor survival in the hostile microenvironment of the injured organs. Recent data suggest a promising potential of volatile anesthetics for improving stem cell biology. Thus, we hypothesized that exposure to sevoflurane could stimulate growth and viability of cultured EPCs. Total mononuclear cells were isolated from human umbilical cord blood by gradient centrifugation. After five days in culture, the cells were exposed for one or two hours to sevoflurane 2% or 4% in air/5% CO2, or only to air/5% CO2 (sham control) in a sealed modular chamber. 24 or 48 hours post-exposure, viability, proliferation and apoptosis were assessed using lactate dehydrogenase (LDH) leakage assay, a methyl tetrazolium salt (MTS) assay and FITC-annexin V/ propidium iodide (PI) staining, respectively. LDH leakage was discretely lowered, whereas the levels of formazan were significantly increased (p < 0.05 for 1 h incubation with 4% sevoflurane at 24 hrs post-exposure, and with 2% sevoflurane at 48 h post-exposure) in the preconditioned cultures, proving no cytotoxic effects and increased proliferation in treated cells versus control samples. Early (p < 0.05) and late apoptosis (p < 0.05 only for 2% sevoflurane) were diminished following the procedure. Thus, the commonly used sevoflurane anesthetic has protective effects on viability and proliferation of human early endothelial progenitor cells in vitro, suggesting a promising potential of anesthetic preconditioning for improving the regeneration of ischemic tissues.