Search

The pathogenic mechanisms by which physical exercise influences atherosclerotic lesion formation remain poorly understood. Because vigorous physical training increases oxidative stress, this study tested the hypothesis that graduated and moderate physical exercise together with metabolic intervention ( l -arginine and antioxidants) may contribute to increased vascular protection. Exercise training in mice was induced by graduated swimming. In hypercholesterolemic male mice on an atherogenic high-cholesterol diet, graduated and moderate exercise lowered plasma cholesterol and decreased atherosclerotic lesions compared with sedentary control mice. Antioxidants (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water) and l -arginine (6% in drinking water) supplementation to exercising hypercholesterolemic mice further and synergistically reduced atherosclerosis compared with untreated exercised mice. Arterial oxidation-specific epitopes and systemic oxidative stress were reduced by metabolic intervention. Graduated chronic exercise elicited an increase in production of nitric oxide through increased endothelial nitric oxide synthase expression and ameliorated scavenger activities. Thus, metabolic intervention with l -arginine and antioxidants together with graduated and moderate exercise training reduce atherosclerotic lesion formation.

Yin Yang 1 (YY1) is a member of polycomb protein family involved in epigenetic modifications and transcriptional controls. We have shown that YY1 acts as positive regulator of tumor growth and angiogenesis by interfering with the VEGFA network. Yet, the link between polycomb chromatin complex and hypoxia regulation of VEGFA is still poorly understood. Here, we establish that hypoxia impairs YY1 binding to VEGFA mRNA 3′UTR (p

In this chapter we will describe the use of the tubular network assay as a useful tool in the evaluation of cancer neoangiogenesis (as reported by Arnaoutova et al. [Angiogenesis 12:267–274, 2009]). Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. A detailed working protocol applicable to the study of any type of endothelial cells is provided below and in addition, details on data interpretation analysis and troubleshooting.

We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated ‘SIR-T8’, was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1. British Journal of Cancer (2002) 86, 917–923. DOI: 10.1038/sj/bjc/6600156 www.bjcancer.com © 2002 Cancer Research UK

Restenosis after percutaneous transluminal coronary angioplasty is caused by neointimal hyperplasia, which involves impairment of nitric oxide (NO)-dependent pathways, and may be further exacerbated by a concomitant aging process. We compared the effects of NO-releasing-aspirin (NCX-4016) and aspirin (ASA) on experimental restenosis in both adult and elderly rats. Moreover, to ascertain the efficacy of NCX-4016 during vascular aging, we fully characterized the release of bioactive NO by the drug. Sprague–Dawley rats aged 6 and 24 months were treated with NO releasing-aspirin (55 mg/kg) or ASA (30 mg/kg) for 7 days before and 21 days after standard carotid balloon injury. Histological examination and immunohistochemical double-staining were used to evaluate restenosis. Plasma nitrite and nitrate and S -nitrosothiols were determined by a chemiluminescence-based assay. Electron spin resonance was used for determining nitrosylhemoglobin. Treatment of aged rats with NCX-4016 was associated with increased bioactive NO, compared with ASA. NO aspirin, but not ASA, reduced experimental restenosis in old rats, an effect associated with reduced vascular smooth muscle cell proliferation. NCX-4016, but not ASA, was well tolerated and virtually devoid of gastric damage in either adult or old rats. Thus, impairment of NO-dependent mechanisms may be involved in the development of restenosis in old rats. We suggest that an NCX-4016 derivative could be an effective drug in reducing restenosis, especially in the presence of aging and/or gastrointestinal damage.

A growing body of evidence indicates that c-Myc can play a pivotal role both in neoplasia and cardiovascular diseases. Indeed, alterations of the basal machinery of the cell and perturbations of c-Myc-dependent signaling network are involved in the pathogenesis of certain cardiovascular disorders. Down-regulation of c-Myc induced by intervention with antioxidants or by antisense technology may protect the integrity of the arterial wall as well as neoplastic tissues. Further intervention studies are necessary to investigate the effects of tissue-specific block of c-Myc overexpression in the development of cardiovascular diseases.

The effects of chronic treatment with the new sulfhydryl angiotensin-converting enzyme (ACE)-inhibitor, zofenopril, in comparison with the classical sulfhydryl ACE-inhibitor captopril or enalapril or placebo on the development of atherosclerosis were determined in apolipoprotein-E knockout (apoE(-/-)) mice. Groups of 2-month-old male mice received either placebo (N=10), 0.05 mg/kg/day of zofenopril (N=10), 1 mg/kg/day of zofenopril (N=10), 5 mg/kg/day of captopril (N=10) or 0.5 mg/kg/day of enalapril (N=8). After 29 weeks of treatment, computer-assisted imaging analysis revealed that zofenopril reduced the aortic cumulative lesion area by 78% at 0.05 mg/kg/day and by 89% at 1 mg/ml/day of zofenopril compared to that of the placebo (P

Increased oxidative stress plays an important role in vascular dysfunction and atherogenesis. Both systemic factors, such as hypercholesterolemia and hyperglycemia, and local factors, such as activation of macrophages and T cells, may contribute to oxidative stress. Oxidation of lipids in lipoproteins and cell membranes leads to functionally important modifications of proteins that affect their recognition by cell surface receptors and protein-protein interactions within the cell, including DNA binding. Oxidized LDL and extracellular oxidation modulate oxidation-sensitive signaling pathways, but it is not clear to what extent this results from receptor-mediated activation or from direct effects on the intracellular redox-balance. Extensive evidence indicates that reactive oxygen species (ROS) regulate gene expression by modulating a large number of transcription factors, including the nuclear transcription factor kappa B (NFkappaB), the peroxisome proliferator activated receptorgamma (PPARgamma), and pathways linked to apoptosis. It is also increasingly recognized that cell differentiation and proliferation, cytokine expression, and programmed cell death are determined by the interactions between oxidation-sensitive regulatory pathways previously thought to lead to distinct outcomes. Because hypercholesterolemia exerts pro-oxidant effects both intra- and extracellularly and because increased ROS formation affects vascular reactivity and atherogenesis by modulating multiple signaling pathways and transcriptional events, future investigations of its atherogenic mechanisms should place greater emphasis on the net effect of such modulation on the expression of a large spectrum of genes. One way of doing this will be by defining clusters of genes responding to hypercholesterolemic stimuli--or interventions with structurally unrelated antioxidants--in analogous ways, irrespective of what regulatory pathway they are controlled by. Microarray technologies that allow simultaneous assessment of large numbers of genes may provide a tool for this approach.

Abstract —Maternal hypercholesterolemia during pregnancy is associated with a marked increase in aortic fatty streak formation in human fetuses and faster progression of atherosclerosis during normocholesterolemic childhood. However, the mechanisms responsible are unknown, and the contribution of genetic differences is difficult to assess in humans. The goal of this study was to determine whether maternal hypercholesterolemia per se may cause enhanced fatty streak formation in offspring and whether interventions during pregnancy can reduce it. During pregnancy, 1 group of New Zealand White rabbits was fed control chow and 8 groups were fed hypercholesterolemic diets Chol 1 (yielding plasma cholesterol of 153 mg/dL) or Chol 2 (yielding 359 mg/dL) without or with cholestyramine, vitamin E, or both. Offspring (n=15 to 25 per group) were killed at birth. Maternal hypercholesterolemia enhanced mean lesion size in the aorta of their offspring at birth from 44±18×10 3 μm 2 per section in controls to 85±26×10 3 in Chol 1 and 156±49×10 3 in Chol 2 groups ( P

The prodromal stage of atherosclerotic lesions (known as lipidotic fatty streak accumulation) is already formed during human fetal development.1 Fatty streaks containing characteristic accumulations of lipids, lipid peroxidation products, and macrophages occur in the aorta of premature fetuses. Intimal thickening is also observed in fetal coronary arteries.2,3 During infancy, fatty streaks become increasingly prevalent, and some of them progress to advanced stages of atherosclerotic lesions.4–7 Once initiated, the progression of atherosclerosis is influenced by classic risk factors that promote vascular inflammation and plaque rupture. The observation that maternal hypercholesterolemia is associated with greatly enhanced fatty streak formation in fetal arteries1 indicates that hypercholesterolemia may play a pathogenic role in early fetal atherosclerotic lesions. Maternal hypercholesterolemia is also able to influence fetal sterol metabolism during pregnancy in animal models.8,9 Consistently, direct evidence for a causal role of maternal hypercholesterolemia and the involvement of oxidative stress has been obtained in a rabbit model10,11 and in LDL receptor–deficient mice.12 From a molecular standpoint, many signaling pathways are affected by increased oxidation of LDL or the intracellular formation of reactive oxygen species, and this phenomenon can be exacerbated by concomitant hypercholesterolemia.13 Interestingly, deletion of the p66 shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a very highly hypercholesterolemic diet.14 Finally, growing evidence in the literature suggests that vascular damage occurs early and is mediated by …

Mediator complex (MED) is an evolutionarily conserved multiprotein, fundamental for growth and survival of all cells. In eukaryotes, the mRNA transcription is dependent on RNA polymerase II that is associated to various molecules like general transcription factors, MED subunits and chromatin regulators. To date, transcriptional machinery dysfunction has been shown to elicit broad effects on cell proliferation, development, differentiation, and pathologic disease induction, including cancer. Indeed, in malignant cells, the improper activation of specific genes is usually ascribed to aberrant transcription machinery. Here, we focus our attention on the correlation of MED subunits with carcinogenesis. To date, many subunits are mutated or display altered expression in human cancers. Particularly, the role of MED1, MED28, MED12, CDK8 and Cyclin C in cancer is well documented, although several studies have recently reported a possible association of other subunits with malignancy. Definitely, a major comprehension of the involvement of the whole complex in cancer may lead to the identification of MED subunits as novel diagnostic/prognostic tumour markers to be used in combination with imaging technique in clinical oncology, and to develop novel anti-cancer targets for molecular-targeted therapy. © 2013.

Oxidation and glycation enhance foam cell formation via MAPK/JNK in euglycemic and diabetic subjects. Here, we investigated the effects of glycated and oxidized LDL (glc-oxLDL) on MAPK-ERK and JNK signaling pathways using human coronary smooth muscle cells. Glc-oxLDL induced a broad cascade of MAPK/JNK-dependent signaling transduction pathways and the AP-1 complex. In glc-oxLDL treated coronary arterioles, tumor necrosis factor (TNF) a increased JNK phosphorylation, whereas protein kinase inhibitor dimethylaminopurine (DMAP) prevented the TNF-induced increase in JNK phosphorylation. The role of MKK4 and JNK were then investigated in vivo, using apolipoprotein E knockout (ApoE-/-) mice. Peritoneal macrophages, isolated from spontaneously hyperlipidemic but euglycemic mice showed increases in both proteins and phosphorylated proteins. Compared to streptozotocin-treated diabetic C57BL6 and nondiabetic C57BL6 Wt mice, in streptozotocin-diabetic ApoE-/- mice, the increment of foam cell formation corresponded to an increment of phosphorylation of JNK1, JNK2, and MMK4. Thus, we provide a first line of evidence that MAPK-ERK/JNK pathways are involved in vascular damage induced by glycoxidation. J. Cell. Physiol. 227: 36393647, 2012. (C) 2012 Wiley Periodicals, Inc.

CXCL12, also known as SDF-1, is the single natural ligand for chemokine receptors CXCR4 and CXCR7. CXCL12 has angiogenic properties in normal endothelial tissue and is involved in the outgrowth and metastasis of CXCR4 expressing tumors. Recent investigations have indicated that CXCL12 levels increase after chemo- and anti- VEGF therapy, favouring recurrences. The blockade of CXCL12/CXCR4 axis has emerged as a potential additional or alternative target for neo-adjuvant treatments. We have reviewed recent patent applications between 2008 and 2011 in tumor angiogenesis and the most clinical data supporting the potential use of anti-CXCR4 agents in this field. Among these, AMD3100, also known as Plerixaform (Mozobil® by Genzyme), is approved for stem cell mobilisation in patients with leukaemia, while BKT140 (Emory University), POL6326 (Polyphor Ag) and TG-0054 (ChemoCentryx) are currently in clinical trials in combination with chemotherapy for multiple myeloma and leukaemia. The aptamer Nox-A12 (Noxxon) is in trials for chronic lymphatic leukaemia treatment. MSX-122 (Metastatix) is in Phase I trials for solid tumor treatment, while CXCR7-specific inhibitor CCX2066 (ChemoCentryx) is still in preclinical studies. We have also considered other strategies, such RNA interference and miRNA, which could be tested for solid tumor adjuvant therapy.

Background The polycomb transcription factor Yin Yang 1 (YY1) overexpression can be causally implicated in experimental tumor growth and metastasization. To date, there is no clinical evidence of YY1 involvement in outcome of patients with osteosarcoma. Prognosis of osteosarcoma is still severe and only few patients survive beyond five years. We performed a prospective immunohistochemistry analysis to correlate YY1 immunostaining with metastatic development and survival in a selected homogeneous group of patients with osteosarcoma. Methods We studied 41 patients suffering from osteosarcoma (stage II-IVa). Multivariate analysis was performed using Cox proportional hazard regression to evaluate the correlation between YY1 expression and both metastasis development and mortality. Results YY1 protein is not usually present in normal bone; in contrast, a high number of patients (61%) showed a high score of YY1 positive cells (51-100%) and 39% had a low score (10-50% positive cells). No statistical difference was found in histology, anatomic sites, or response to chemotherapy between the two degrees of YY1 expression. Cox regression analysis demonstrated that the highest score of YY1 expression was predictive of both low metastasis-free survival (HR = 4.690, 95%CI = 1.079-20.396; p = 0.039) and poor overall survival (HR = 8.353, 95%CI = 1.863-37.451 p = 0.006) regardless of the effects of covariates such as age, gender, histology and chemonecrosis. Conclusion Overexpression of YY1 in primary site of osteosarcoma is associated with the occurrence of metastasis and poor clinical outcome.

Targeting neoangiogenesis is a well-established anticancer strategy, however, one of the major problems in angiogenesis research, both at the basic and applied levels, remains the development of suitable in vivo methods for assessing and quantifying the systemic angiogenic response. Therefore, there is an urgent need to adopt technically simple and reproducible methodologies which allow to easily quantify neoangiogenesis independently of morphological parameters. Recently, a reproducible and quantitative method was developed, the directed in vivo angiogenesis assay (DIVAA) consisting of the subcutaneous implantation of surgical grade silicone cylinders closed at one end, called angioreactors, into the dorsal flanks of nude mice. In the past few years, DIVAA has been successfully used in evaluating the inhibition and or enhancement of systemic perturbation of angiogenesis by several molecules. Thus, DIVAA studies systemic angiogenesis and its therapeutic modulation associated to cancer progression and metastasis.

BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and Objective: Peripheral arterial disease (PAD) is a major health problem especially when associated to concomitant diabetes and hypercholesterolemia. Hyperglycemia with an overwhelming generation of oxygen radicals and formation of glycation end-products exacerbates oxidation-sensitive mechanisms activated by tissue ischemia. Administration of autologous bone marrow cells (BMC) is an increasing notable intervention to induce therapeutic angiogenesis, ameliorated by metabolic intervention (MT). Recently, hemangioblasts (HS) with functional properties were isolated. Methods: The effects of integrate regimen with intravenous BMC, HS, and MT (1.0% vitamin E, 0.05% vitamin C, and 6% l-arginine) were examined in the ischemic hindlimb of ApoE-/- diabetic and non-diabetic. Blood flow ratio was monitored by use of a laser Doppler blood flowmeter. Capillary density was determined in sections of the adductor and semimembranous muscles with antibody against CD31. Results: BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and, more consistently, in combination with MT ameliorated functional activity via decreased cellular senescence and increased telomerase and chemokine CXCR4 activities. Telomerase activity was also increased by HS alone or HS. +. MT and, more consistently, by BMC. +. HS alone or in combination with MT. Conclusions/interpretation: Intravenous autologous BMC and HS intervention together with MT increased therapeutic angiogenesis in the ApoE-/- diabetic mouse hindlimb. © 2009 Elsevier Ireland Ltd.

Tumor growth requires neoangiogenesis. VEGF is the most potent proangiogenic factor. Dysregulation of hypoxia-inducible factor (HIF) or cytokine stimuli such as those involving the chemokine receptor 4/stromal-derived cell factor 1 (CXCR4/SDF-1) axis are the major cause of ectopic overexpression of VEGF in tumors. Although the CXCR4/SDF-1 pathway is well characterized, the transcription factors executing the effector function of this signaling are poorly understood. The multifunctional Yin Yang 1 (YY1) protein is highly expressed in different types of cancers and may regulate some cancer-related genes. The network involving CXCR4/YY1 and neoangiogenesis could play a major role in cancer progression. In this study we have shown that YY1 forms an active complex with HIF-1α at VEGF gene promoters and increases VEGF transcription and expression observed by RT-PCR, ELISA, and Western blot using two different antibodies against VEGFB. Long-term treatment with T22 peptide (a CXCR4/SDF-1 inhibitor) and YY1 silencing can reduce in vivo systemic neoangiogenesis ( P < 0.01 and P < 0.05 vs. control, respectively) during metastasis. Moreover, using an in vitro angiogenesis assay, we observed that YY1 silencing led to a 60% reduction in branches ( P < 0.01) and tube length ( P < 0.02) and a 75% reduction in tube area ( P < 0.001) compared with control cells. A similar reduction was observed using T22 peptide. We demonstrated that T22 peptide determines YY1 cytoplasmic accumulation by reducing its phosphorylation via down-regulation of AKT, identifying a crosstalk mechanism involving CXCR4/YY1. Thus, YY1 may represent a crucial molecular target for antiangiogenic therapy during cancer progression.

We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth. [Cancer Res 2008;68(6):1797–808]

Previous studies showed that p66(Shc-/-) mice on a very-high-fat diet (HFD) had reduced oxidative stress, foam cell, and early atherosclerotic lesion formation. Here, the authors have used hypercholesterolemic apolipoprotein E (ApoE(-/-)) mice to investigate the role of p66Shc deletion in advanced atheroma. The authors generated mice deficient of both ApoE and p66Shc genes (ApoE(-/-) /p66(Shc-/-)). They used microsatellite polymerase chain reaction (PCR) analysis to analyze the genetic background and considered only animals with a constant percentages of C57B6L and 129SV background strands (it was obtained the 50.3% +/- 6.4% of C57B6L background). Computer-assisted analysis revealed that advanced atherosclerotic lesions in ApoE(-/-)/p66(Shc+/+) were significantly larger than those observed in ApoE(-/-)/p66(Shc-/-). Accordingly, the lipid-laden macrophage foam cells and oxidation-specific epitopes in ApoE(-/-)/p66(shc+/+) HFD-treated groups were higher than those observed in normal diet (ND)-treated groups. Thus, p66(Shc-/-) plays an important protective role also against advanced atherosclerotic lesion formation. Finally, the authors have used microarray to investigate major changes in gene expression in aortas of mice with ApoE(-/-)/p66(Shc-/-) background treated with a very HFD in comparison to ApoE(-/-)/p66(Shc+/+) (these data have been confirmed by by real-time PCR and immunohistochemistry). DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis revealed that CD36 antigen (CD36), tissue inhibitor of metalloproteinase 2 (TIMP2), apolipoprotein E (ApoE), acetyl-coenzyme A acetyltransferase 1 (ACAT1), and thrombospondin 1 (THBS1) can be involved in p66 deletion-dependent vascular protection through the adipocytokine/lipid signaling pathway.

Nebivolol is a cardioselective beta-blocker (BB) currently used for the treatment of hypertension. It has mild vasodilating properties attributed to its interaction with the L-arginine/nitric oxide pathway, a property not shared by other BBs. Carvedilol is a nonselective ß-adrenergic receptor antagonist that also blocks α1-adrenergic receptors and is a potent antioxidant. Anthracyclines (ANTs), daunorubicin and doxorubicin, are commonly used in the treatment of several tumours, but their cardiac toxicity prevents their use at maximum myelotoxic doses, representing an important problem. In this study, we have evaluated the role of these BBs administered in combination with ANTs (daunorubicin and doxorubicin) on a reduction in cardiac toxicity. The combination of BB and ANTs has reduced the release of GSSG and GSH; in particular, co-treatment with nebivolol to ANTs has shown a significant reduction. The total integrated creatine kinase and troponin T activities were improved by BB and ANTs co-treatment. A significant reduction of their release was observed when hearts were treated with nebivolol. Cardiac tissue activity of gluthatione reductase was not significant and similar among experimental groups. In contrast, gluthatione peroxidise, Mn-superoxide dismutase and nitrite/nitrate release were increased after co-treatment with nebivolol. Finally, three parameters have been used to evaluate the cardiac toxicity of ANTs: the left ventricular pressure developed under a constant perfusion pressure (LVDP), the rate of variation of this parameter during systole (contractility) (LV/dt)max and during diastole (relaxation) (LV(dP/dt)min. Combination with BB has shown a reduction in cardiac toxicity; in particular, nebivolol has exerted the most significant cardioprotective effect. © 2007 Elsevier Ltd. All rights reserved.

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Despite advances in imaging, understanding the underlying pathways, and clinical translation of animal models of disease there remains an urgent need for therapies that reduce brain damage after stroke and promote functional recovery in patients. Blocking oxidant radicals, reducing matrix metalloproteinase-induced neuronal damage, and use of stem cell therapy have been proposed and tested individually in prior studies. Here we provide a comprehensive integrative management approach to reducing damage and promoting recovery by combining biological therapies targeting these areas. In a rat model of transient cerebral ischemia (middle cerebral artery occlusion) gene delivery vectors were used to overexpress tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP1 and TIMP2) 3 days before ischemia. After occlusion, autologous bone marrow cells alone or in combination with agents to improve NO bioavailability were administered intraarterially. When infarct size, BrdU incorporation, and motor function recovery were determined in the treatment groups the largest beneficial effect was seen in rats receiving the triple combined therapy, surpassing effects of single or double therapies. Our study highlights the utility of combined drug, gene, and cell therapy in the treatment of stroke. © 2007 by The National Academy of Sciences of the USA.

Metabolic syndrome includes most widely distributed clinical conditions such as obesity, hypertension, dislipidemia, and diabetes. Pomegranate fruit extract (PFE), rich in polyphenolic antioxidants, reduces the expression of oxidation-sensitive genes at the sites of perturbed shear-stress. The aim of this study was to evaluate the effect of PFE in comparison to regular pomegranate juice (PJ) and seed oil on the biological actions of nitric oxide (NO) and the arterial function in obese Zucker rats, a model of metabolic syndrome. Our results indicated that supplementation with PFE or PJ significantly decreased the expression of vascular inflammation markers, thrombospondin (TSP), and cytokine TGFbeta1 (P

Lower-limb ischemia is a major health problem especially when associated to hypercholesterolemia. Because of the absence of effective treatment in the advanced stages of the disease, amputation is undertaken to alleviate unbearable symptoms. Since tissue ischemia and hypercholesterolemia are associated with an overwhelming generation of oxygen radicals, metabolic intervention with antioxidants and l-arginine can induce beneficial effects beyond those achieved by a novel therapeutic approach represented by the use of autologous bone marrow cells (BMCs). The protective effect of BMCs and vascular protection by metabolic cotreatment (1.0% vitamin E added to the chow, 0.05% vitamin C and 6% l-arginine added to the drinking water) were examined in ischemia-induced angiogenesis in the hypercholesterolemic mouse hindlimb. Intravenous BMC therapy improved blood flow and increased capillary densities. This beneficial effect was amplified by metabolic cotreatment, an intervention inducing vascular protection, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage activation.

BACKGROUND: Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases the expression of oxidation-sensitive responsive genes (such as ELK-1 and p-CREB). Polyphenolic antioxidants contained in the juice derived from the pomegranate contribute to the reduction of oxidative stress and atherogenesis during disturbed shear stress. AIM OF THE STUDY: To evaluate the effects of intervention with the Pomegranate Fruit Extract (PFE) rich in polyphones (punicalagin, which is a potent antioxidant) on ELK-1, p-CREB, and endothelial nitric oxide synthase (eNOS) expression induced by high shear stress in vitro and in vivo. RESULTS: At the doses used in the study, both the PFE and the regular pomegranate juice concentrate reduced the activation of ELK-1 and p-CREB and increased eNOS expression (which was decreased by perturbed shear stress) in cultured human endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice. PFE and pomegranate juice increased cyclic GMP levels while there was no significant effect of both compounds on the conversion of L-arginine to L-citrulline. Administration of these compounds to hypercholesterolemic mice significantly reduced the progression of atherosclerosis and isoprostane levels and increased nitrates. This protective effect was relevant with PFE. Vasomotor reactivity was improved and EC(25) values in response to Ach and NONOate were significantly increased in treated mice in comparison to controls. CONCLUSION: This study indicates that the proatherogenic effects induced by perturbed shear stress can be also reversed by chronic administration of PFE.

Peripheral arterial disease (PAD) is a major health problem especially when associated to diabetes. Administration of autologous bone marrow cells (BMC) is emerging as a novel intervention to induce therapeutic angiogenesis in experimental ischemic limb models and in patients with PAD. Since tissue ischemia and diabetes are associated with an overwhelming generation of oxygen radicals and detrimental effects due to formation of glycosylation end-products, metabolic intervention with antioxidants and L-arginine can confer beneficial effects beyond those achieved by BMC alone. The effects of cotreatment with intravenous BMCs and metabolic vascular protection (1.0% vitamin E, 0.05% vitamin C, and 6% L-arginine) were examined in the ischemic hindlimb of diabetic and non diabetic mice. BMC therapy increased blood flow and capillary densities and Ki67 proliferative marker, and decreased interstitial fibrosis. This effect was amplified by metabolic cotreatment, an intervention inducing vascular protection, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress, and macrophage activation.

Cancer and vascular diseases remain the predominant causes of morbidity and mortality in industrialized countries worldwide. The course of atherosclerosis with initiation, progression, and complication parallels the three stages of carcinogenesis with induction, growth, and invasion of tissue and neoangiogenesis. Within this framework, the oncogene c-Myc and growth factors pathways are acquiring increasing importance. Insulin-like growth factor-1 (IGF-1) pathway emerges among them for its versatile pleiotropic actions. A number of genes that permit extensive communication between IGF-1-AKT, p53, and mammalian target of rapamycin (mTOR) pathways have been identified. In turn these pathways lead to p53 transcriptional program, the forkhead transcriptional programs, autophagy, and translational controls, which determine cell growth or arrest, cell survival or death. The increased understanding of the extensive communication and coordination between all these pathways may enable to targeting these events and to prevent neoplastic and vascular diseases. Great effort has been focused on the development of new agents designed to target various steps of c-Myc, Ras, and IGF cascade. However, what have we recently learned about their safety and effectiveness? Here, we review the very recent advances in the identification of novel inhibitors as well as antisense oligonuleotides (ASOs) and siRNA that are proving their usefulness in ongoing clinical trials both in terms of toxicity and specificity.

We examined the hypothesis that pomegranate juice (PJ) can revert the potent downregulation of the expression of endothelial nitric-oxide synthase (NOSIII) induced by oxidized low-density liporotein (oxLDL) in human coronary endothelial cells. Western blot and Northern blot analyses showed a significant decrease of NOSIII expression after a 24-h treatment with oxLDL. Accordingly, we observed a significant dose-dependent reduction in nitric oxide bioactivity represented by both basal and bradykinin-stimulated cellular cGMP accumulation. These phenomena were corrected significantly by the concomitant treatment with PJ. Our data suggest that PJ can exert beneficial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the NOSIII bioactivity.

Nitric oxide (NO) is a molecule that has gained recognition as a crucial modulator of vascular disease. NO has a number of intracellular effects that lead to vasorelaxation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet adhesion. Endothelium damage induced by atherosclerosis leads to the reduction in bioactivity of endothelial NO synthase (eNOS) with subsequent impaired release of NO together with a local enhanced degradation of NO by increased generation of reactive oxygen species with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Many commonly used vasculoprotective agents have their therapeutic actions through the production of NO. L-Arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. Finally, eNOS gene polymorphism might be an additional risk factor that may contribute to predict cardiovascular events. However, further studies are needed to understand the possible clinical implications of these correlations.

Moderate physical exercise (PE) combined with metabolic treatment (MT) (antioxidants and l -arginine) are well known to reduce atherosclerotic lesion formation in hypercholesterolemic mice. However, the long-term beneficial effects on unstable atheroma remain poorly understood. We started early PE training in large groups of 6-week-old hypercholesterolemic mice (by graduated swimming) alone or in combination with nutritional supplementation (1.0% vitamin E added to the chow and 0.05% vitamin C and 6% l -arginine added to the drinking water). Inactive controls did not receive PE. The spontaneous development of atherosclerotic plaque rupture (associated with advanced atherosclerosis) and survival rates were evaluated. Moderate PE elicited an increase in plasma levels of nitric oxide. Early combined treatment with PE and MT in the hypercholesterolemic mice significantly reduced lesions (also detected noninvasively at 10 months) and spontaneous atherosclerotic plaque rupture and prolonged survival more effectively than each intervention alone. Thus, early concerted actions of MT and PE improve the natural history of atherosclerotic lesions and reduce the plaque instability in hypercholesterolemic mice.

Pomegranate juice (PJ), which is a rich source of potent flavonoid antioxidants, was tested for its capacity to protect nitric oxide (NO) against oxidative destruction and enhance the biological actions of NO. Employing chemiluminescence headspace analysis, PJ was found to be a potent inhibitor of superoxide anion-mediated disappearance of NO. PJ was much more potent than Concord grape juice, blueberry juice, red wine, ascorbic acid, and DL-alpha-tocopherol. As little as 3 microl of a 6-fold dilution of PJ, in a reaction volume of 5000 microl, produced a marked antioxidant effect, whereas 300 microl of undiluted blueberry juice or nearly 1000 microl of undiluted Concord grape juice were required to produce similar effects. PJ and other antioxidant-containing products were found to augment the anti-proliferative action of NO (DETA/NO) on vascular smooth muscle cell (rat aorta) proliferation. PJ was much more effective than the other products tested and elicited no effects when tested alone in the absence of added NO. Similarly, neither PJ nor the other products enhanced the anti-proliferative action of alpha-difluoromethylornithine, a stable substance that inhibits cell growth by NO-independent mechanisms. In order to determine whether PJ is capable of increasing the production of NO by vascular endothelial cells, PJ was tested for its capacity to upregulate and/or activate endothelial NO synthase (eNOS) in bovine pulmonary artery endothelial cells. PJ elicited no effects on eNOS protein expression or catalytic activity. Moreover, PJ did not enhance promoter activity in the eNOS gene (COS-7 cells transfected with eNOS). These observations indicate that PJ possesses potent antioxidant activity that results in marked protection of NO against oxidative destruction, thereby resulting in augmentation of the biological actions of NO.

New approaches to atherosclerosis-related diseases include novel uses of proven treatments and development of innovative agents. Several commonly used cardiovascular drugs such as dihydropyridine calcium antagonists, ACE inhibitors containing the sulphydryl group, or highly lipophilic beta-blockers have some anti-atherosclerotic activities. Moreover, new clinical trials suggesting that additional reduction of low-density lipoprotein cholesterol levels with statin therapy results in additional benefit in coronary heart disease prevention. Notably, new cholesterol transport or bile acid transport inhibitors have been found to produce significant reductions in intestinal cholesterol absorption and experimental atherosclerosis. Inhibitors of acyl coenzyme A:cholesterol acyltransferase, which can reduce cholesterol storage in macrophages and in arterial lesions, have also been developed. Finally, newer therapeutical strategies against atherogenesis may include the use of antioxidants and cholestyramine during pregnancy or the development of metalloproteinase inhibitors.

Lower-limb ischemia is a major health problem. Because of the absence of effective treatment in the advanced stages of the disease, amputation is undertaken to alleviate unbearable symptoms. Novel therapeutic approaches include the intramuscular use of autologous bone marrow cells (BMCs). Because tissue ischemia is associated with an overwhelming generation of oxygen radicals and negative effects due to perturbed shear-stress, metabolic intervention with antioxidants and l -arginine could potentially induce beneficial effects beyond those achieved by BMCs. The protective effect of autologous BMCs and vascular protection by metabolic cotreatment (1.0% vitamin E added to the chow and 0.05% vitamin C and 6% l -arginine added to the drinking water) were examined in ischemia-induced angiogenesis in the mouse hindlimb, a model of extensive acute peripheral arterial occlusion. i.v. BMC therapy improved blood flow and increased capillary densities and expression of Ki-67, a proliferation-associated protein. This beneficial effect was amplified by metabolic cotreatment, an intervention inducing vascular protection, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress, and macrophage activation. Therefore, although a cautious approach is mandatory when experimental findings are extended to human diseases, autologous BMCs together with metabolic intervention could be an effective clinical treatment for peripheral arterial disease.

AIM: To investigate protease activated receptor 2 (PAR-2) expression in human coronary atherosclerotic lesions because PAR-2 is involved in the modulation of inflammatory events and vascular function. METHODS: An immunohistochemical analysis was performed on serial arterial sections, using the following antibodies: MDA2, a murine monoclonal antibody against malondialdehyde lysine epitopes of oxidised low density lipoprotein (oxLDL); HAM-56, a monoclonal antibody against human macrophages/foam cells; B5, a rabbit polyclonal antibody against PAR-2; and SAM11, a mouse monoclonal antibody against human PAR-2. Sections containing at least one lesion showing substantial immunostaining were counted as positive, and results were expressed as per cent of all sections of the same artery. RESULTS: PAR-2 expression was enhanced in human coronary atherosclerotic lesions. This phenomenon correlated with an increase in oxLDL epitopes in the coronary artery. CONCLUSION: This study shows for the first time that PAR-2 expression is enhanced in human coronary atherosclerotic lesions, and suggests that PAR-2 dependent cellular trafficking may be one of the regulatory signalling responses to vascular injury. Further pharmacological studies will establish whether modulation (and in which direction) of PAR-2 represents a possible therapeutic target for controlling the vascular response to injury.

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l -arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo . Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR −/− ) mice showed increased activities of redox-transcription factors (ELK-1, p -Jun, and p -CREB) and decreased expression of eNOS. Intervention with antioxidants and l -arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo . These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and l -arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations.

Several experimental and clinical studies have shown that oxidized low-density lipoprotein and oxidation-sensitive mechanisms are central in the pathogenesis of vascular dysfunction and atherogenesis. Here, we have used p66 Shc−/− and WT mice to investigate the effects of high-fat diet on both systemic and tissue oxidative stress and the development of early vascular lesions. To date, the p66 Shc−/− mouse is the unique genetic model of increased resistance to oxidative stress and prolonged life span in mammals. Computer-assisted image analysis revealed that chronic 21% high-fat treatment increased the aortic cumulative early lesion area by ≈21% in WT mice and only by 3% in p66 Shc−/− mice. Early lesions from p66 Shc−/− mice had less content of macrophage-derived foam cells and apoptotic vascular cells, in comparison to the WT. Furthermore, in p66 Shc−/− mice, but not WT mice, we found a significant reduction of systemic and tissue oxidative stress (assessed by isoprostanes, plasma low-density lipoprotein oxidizability, and the formation of arterial oxidation-specific epitopes). These results support the concept that p66 Shc−/− may play a pivotal role in controlling systemic oxidative stress and vascular diseases. Therefore, p66 Shc might represent a molecular target for therapies against vascular diseases.

Apoptosis of arterial cells induced by oxidized low-density lipoprotein (oxLDL) is thought to contribute to the progression of vascular dysfunction and atherogenesis. It is well established that diabetes mellitus is accompanied by both glycosylation and oxidation of LDL (glc-oxLDL), but the biological effects of these modified lipoproteins are poorly understood. We demonstrate here for the first time that glc-oxLDL increases TUNEL positivity and caspase-3 activation (by Western blot and immunocytochemistry) of human coronary smooth muscle cells. Overall, these effects induced by glc-oxLDL were greater than those achieved with oxLDL. Thus, glc-oxLDL activated downstream apoptotic signaling. This may influence the evolution of atherogenesis and vascular complications in diabetes.

Background — Maternal hypercholesterolemia during pregnancy is associated with markedly enhanced fatty streak formation in human fetal aortas and accelerated progression of atherosclerosis in normocholesterolemic children. Methods and Results — To establish the causal role of maternal hypercholesterolemia in a genetically homogeneous murine model and to test the hypothesis that pathogenic events during fetal development result in persistent changes in arterial gene expression, female LDL receptor-deficient (LDLR −/− ) mice were fed regular chow or high-fat diets supplemented with 0.075% or 1.25% cholesterol during pregnancy. Lesion sizes were determined in the aortic origin of their chow-fed offspring at 3 months. Maternal hypercholesterolemia more than doubled lesion sizes in male offspring ( P 1.7-fold. Quantitative PCR for one of these genes using RNA from individual mice yielded similar results. Comparative immunostaining for several of the above genes also indicated increased protein content in offspring of hypercholesterolemic mothers. Conclusions — These findings establish an atherogenic effect of maternal hypercholesterolemia in genetically uniform mice and indicate that changes in aortic gene expression persist long after fetal exposure to hypercholesterolemia. In addition to elucidating pathogenic mechanisms initiated during fetal development, this approach may identify genes in morphologically normal arteries that influence the susceptibility to classical risk factors of atherosclerosis.

The effects of chronic treatment with nitric oxide-containing aspirin (NO-aspirin, NCX-4016) in comparison with regular aspirin or placebo on the development of a chronic disease such as atherosclerosis were investigated in hypercholesterolemic low-density lipoprotein (LDL)-receptor-deficient mice. Male mice were assigned randomly to receive in a volume of 10 ml/kg either placebo ( n = 10), 30 mg/kg/day NO-aspirin ( n = 10), or 18 mg/kg/day of regular aspirin ( n = 10). After 12 weeks of treatment, the computer-assisted imaging analysis revealed that NO-aspirin reduced the aortic cumulative lesion area by 39.8 ± 12.3% compared with that of the placebo ( P < 0.001). Regular aspirin did not reduce significantly aortic lesions (−5.1 ± 2.3%) compared with the placebo [ P = 0.867, not significant (NS)]. Furthermore, NO-aspirin reduced significantly plasma LDL oxidation compared with aspirin and placebo, as shown by the significant reduction of malondialdehyde content ( P < 0.001) as well as by the prolongation of lag-time ( P < 0.01). Similarly, systemic oxidative stress, measured by plasma isoprostanes, was significantly reduced by treatment with NCX-4016 ( P < 0.05). More importantly, mice treated with NO-aspirin revealed by immunohistochemical analysis of aortic serial sections a significant decrease in the intimal presence of oxidation-specific epitopes of oxLDL (E06 monoclonal antibody, P < 0.01), and macrophages–derived foam cells (F4/80 monoclonal antibody, P < 0.05), compared with placebo or aspirin. These data indicate that enhanced NO release by chronic treatment with the NO-containing aspirin has antiatherosclerotic and antioxidant effects in the arterial wall of hypercholesterolemic mice.

Background and purposeAtherosclerosis occurs later and is less extensive in intracranial arteries than in extracranial arteries. However, the mechanisms responsible are poorly understood. A previous study has suggested a better antioxidant protection of intracranial arteries.MethodsTo assess the influence of age on arterial activity of antioxidant enzymes and atherogenesis, we compared intracranial and extracranial arteries of humans of different ages who retrospectively lacked confounding classic risk factors (48 premature fetuses aged 6.4+/-0.8 months [mean+/-SD], 58 children aged 7.9+/-3.8 years, 42 adults aged 42.5+/-5.1 years, and 40 elderly subjects aged 71.8+/-3.4 years; all males). Lesions were quantified by computer-assisted imaging analysis of sections of the middle cerebral and basilar arteries, the left anterior descending coronary artery, the common carotid artery, and the abdominal aorta. Macrophages, apolipoprotein B, oxidized LDL, and matrix metalloproteinase-9 in lesions were determined by immunocytochemistry. The effect of aging on atherogenesis was then compared with that on the activity of 4 antioxidant enzymes in the arterial wall.ResultsAtherosclerosis was 6- to 19-fold greater (P

The proteins of the Ets family are transcription factors involved in signal transduction, cell cycle progression, and differentiation. In this study, we report that thyroid cell neoplastic transformation is associated with a dramatic increase in ETS transcriptional activity, which is dependent on the accumulation of Ets-1, Ets-2, and other Ets-related proteins. Inhibition of ETS transactivation activity by the Ets-dominant negative construct (Ets-Z) induced programmed cell death in human thyroid carcinoma cell lines but not in normal thyroid cells. Apoptotic cell death induced by Ets-Z was dependent on the reduction of c-MYC protein levels, because it was prevented by overexpressinn of c-myc. Taken together, these data indicate that the induction of Ets-l and Ets-2 transcription factors plays a pivotal role in thyroid cell neoplastic transformation.

This study tested the hypothesis that c-Myc activation, an oxidation-sensitive transcription factor, and its binding partner Max occurs in coronary arteries of hypercholesterolemic (HC) pigs, and can be attenuated by chronic antioxidant intervention. Coronary arteries were isolated from normal, HC pigs, or HC supplemented with antioxidant vitamins (HC + vitamins). The expression of the c-Myc/Max complex, and its target genes GADD45 and p53, was studied in nonatherosclerotic, early lesions (LL), positively staining for oil-red-O, in adjacent lesion-prone regions (PL), and in healthy segments (HV). The expression of c-Myc and Max in HC was 2- to 3-fold greater in PL, and 4-fold in LL, compared to normal vessels (P < 0.01). The expression of GADD45 was down-regulated, and of p53 increased, in the same regions. These alterations were attenuated in the HC + vitamins. Thus, c-Myc activation is an early atherosclerosis, in both PL and LL coronary arterial regions, and can be blunted by chronic dietary antioxidant intervention.

Apoptosis may play an important role in atherogenesis. Oxidized low-density lipoprotein (oxLDL) promotes apoptosis in the arterial wall in addition to several other proatherogenic effects. Tocopherol supplements have been suggested to protect against coronary heart disease (CHD) in epidemiological studies. The effects of oxLDL and alpha- and gamma-tocopherol on apoptotic signaling pathways are poorly understood. Thus, the goal of the study was to investigate these pathways in the presence of copper-oxidized LDL and tocopherols in human coronary smooth muscle cells (SMC). We showed that oxLDL-mediated apoptosis, assessed by DNA fragmentation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, and caspase activation stimulated several transcription factors and proapoptotic dynamic movements of the Bcl-2 family proteins through the mitogen-activated protein kinase (MAPK) and Jun kinase pathways. alpha-Tocopherol and gamma-tocopherol significantly reduced these molecular events and cell death effectors caspase-3 and -8. Under our experimental conditions, alpha-tocopherol was significantly more effective than gamma-tocopherol, and oxLDL-mediated apoptosis increased c-Jun, cyclic AMP-responsive element-binding, Ets-like element kinase-dependent 7, and activating transcription factor-2 proteins as well as nuclear activity of the activated protein-1 complex in human coronary SMC. Moreover, our results demonstrate that tocopherols may exert their antiatherogenic effects at least in part via reduction of the MAPK and JunK cascade together with a protective profile of apoptotic genes of the Bcl-2 family. These data are consistent with the beneficial effects of tocopherols on atherogenesis seen in experimental studies and on CHD in epidemiological surveys.

Maternal hypercholesterolemia during pregnancy is associated with enhanced fatty streak formation in human fetuses and faster progression of atherosclerosis during childhood even under normocholesterolemic conditions. A causal role of maternal hypercholesterolemia in lesion formation during fetal development has previously been established in rabbits. The same experimental model is now used to establish that maternal hypercholesterolemia or ensuing pathogenic events in fetal arteries enhance atherogenesis later in life. Five groups of rabbit mothers were fed chow, cholesterol-enriched chow, or cholesterol-enriched chow plus 1000 IU vitamin E, 3% cholestyramine, or both during pregnancy. Offspring of all groups (n=136) were fed a mildly hypercholesterolemic diet for up to a year and had similar cholesterol levels. Aortic lesion sizes and lipid peroxidation products in plasma and lesions in offspring were determined at birth, 6 months, or 12 months. Lesion progression in offspring of hypercholesterolemic mothers was greater than in all other groups. At each time point, offspring of hypercholesterolemic mothers had 1.5- to 3-fold larger lesions than offspring of normocholesterolemic mothers ( P P P

Background —Oxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experimental models. OxLDL-mediated effects on c-Myc are poorly characterized, and those on c-Myc nuclear pathways are completely unknown. c-Myc stimulates smooth muscle cell (SMC) proliferation and could be involved in atherosclerosis. We investigated the early effects of oxLDL and α-tocopherol on c-Myc, its binding partner Max, and the carboxy-terminal domain–binding factors activator protein-2 and elongation 2 factor in human coronary SMCs. We also investigated whether 9-week treatment of Watanabe heritable hyperlipidemic (WHHL) rabbits with diet-enriched α-tocopherol reduces c-Myc expression and oxLDL in the left coronary artery. Methods and Results —OxLDL enhanced c-Myc/Max expression and transcription by cotransfection assay and the nuclear activities of E2F and activator protein-2 by binding shift and supershift in coronary SMCs. α-Tocopherol significantly reduced these molecular events. Furthermore, α-tocopherol reduced early lesions, SMC density, and the immunohistochemical presence of c-Myc, which colocalized with oxLDL/foam cells in the coronaries of WHHL rabbits. Conclusions —We provide the first evidence that oxLDL and α-tocopherol may influence c-Myc activation and several c-Myc–dependent signaling pathways in human coronary SMCs. The observation that in vivo, an antioxidant reduces both c-Myc and oxLDL in early coronary lesions of rabbits is consistent with, but does not prove, the hypothesis that c-Myc–dependent factors activated by oxidative processes contribute to atherogenesis and coronary heart disease.

Apoptosis of arterial cells induced by oxidized low density lipoproteins (OxLDL) is thought to contribute to the progression of atherosclerosis. However, most data on apoptotic effects and mechanisms of OxLDL were obtained with extensively oxidized LDL unlikely to occur in early stages of atherosclerotic lesions. We now demonstrate that mildly oxidized LDL generated by incubation with oxygen radical-producing xanthine/xanthine oxidase (X/XO) induces apoptosis in primary cultures of human coronary endothelial and SMC, as determined by TUNEL technique, DNA laddering, and FACS analysis. Apoptosis was markedly reduced when X/XO-LDL was generated in the presence of different oxygen radical scavengers. Apoptotic signals were mediated by intramembrane domains of both Fas and tumor necrosis factor (TNF) receptors I and II. Blocking of Fas ligand (FasL) reduced apoptosis by 50% and simultaneous blocking of FasL and TNF receptors by 70%. Activation of apoptotic receptors was accompanied by an increase of proapoptotic and a decrease in antiapoptotic proteins of the Bcl-2 family and resulted in marked activation of class I and II caspases. Mildly oxidized LDL also activated MAP and Jun kinases and increased p53 and other transcription factors (ATF-2, ELK-1, CREB, AP-1). Inhibitors of Map and Jun kinase significantly reduced apoptosis. Our results provide the first evidence that OxLDL-induced apoptosis involves TNF receptors and Jun activation. More important, they demonstrate that even mildly oxidized LDL formed in atherosclerotic lesions may activate a broad cascade of oxygen radical-sensitive signaling pathways affecting apoptosis and other processes influencing the evolution of plaques. Thus, we suggest that extensive oxidative modifications of LDL are not necessary to influence signal transduction and transcription in vivo.

Protease-activated receptor-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage whose better known member is the thrombin receptor. The pathophysiological role of PAR-2 remains poorly understood. Because PAR-2 is involved in inflammatory and injury response events, we investigated the role of PAR-2 in experimental myocardial ischemia-reperfusion injury. We show for the first time that PAR-2 activation protects against reperfusion-injury. After PAR-2-activating peptide (2AP) infusion, we found a significant recovery of myocardial function and decrease in oxidation at reflow. Indeed, the glutathione cycle (glutathione and oxidized glutathione) and lipid peroxidation analysis showed a reduced oxidative reperfusion-injury. Moreover, ischemic risk zone and creatine kinase release were decreased after PAR-2AP treatment. These events were coupled to elevation of PAR-2 and tumor necrosis factor α (TNFα) expression in both nuclear extracts and whole heart homogenates. The recovery of coronary flow was not reverted by L-nitroarginine methylester, indicating a NO-independent pathway for this effect. Genistein, a tyrosine kinase inhibitor, did not revert the PAR-2AP effect. During early reperfusion injury in vivo not only oxygen radicals are produced but also numerous proinflammatory mediators promoting neutrophil and monocyte targeting. In this context, we show that TNFα and PAR-2 are involved in signaling in pathophysiological conditions, such as myocardial ischemia-reperfusion. At the same time, because TNFα may exert pro-inflammatory actions and PAR-2 may constitute one of the first protective mechanisms that signals a primary inflammatory response, our data support the concept that this network may regulate body responses to tissue injury.

The effects of angiotensin-converting enzyme (ACE)-inhibition with zofenopril on the development of atherosclerosis and low-density lipoprotein (LDL) oxidation were determined in Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Rabbits received either placebo (n = 6) or 0.5 mg/kg/day of zofenopril (n = 6). After 6 weeks of treatment, the computer-assisted analysis revealed that zofenopril reduced the aortic and common carotid corrected cumulative lesion area by 34% and 39%, respectively (p < 0.05 vs placebo-treated group). The intimal/medial ratio of the largest fatty streaks was 0.426+/-0.158 in the zofenopril-treated group and 0.875+/-0.238 in the placebo-treated group (p < 0.05). Furthermore, we found in the zofenopril-treated group smaller lesions with an intimal/medial ratio of zofenopril also reduced plasmatic LDL oxidation, as shown by significant reduction of malondialdehyde content (p < 0.01) and relative agarose gel mobility (p < 0.05), as well as by the prolongation of the lag-time (p < 0.05). Compared to zofenopril-treated rabbits, arterial sections of the placebo-group had significant increase in the intimal presence of macrophages-derived foam cells (p < 0.05), ox-LDL (p < 0.01), and native LDL (p < 0.01) detected by immunocytochemistry with RAM-11, MDA2 and NP1533975 monoclonal antibodies, respectively. To investigate the amount of platelet accumulation in the atherosclerotic plaque we also measured platelet-associated radioactivity. Autologous platelets were labeled with 111Indiumoxine and injected intravenously. After 2 hours, WHHL were sacrificed and arterial sections were counted for platelet-associated radioactivity. In the placebo-treated group, platelet radioactivity was 0.52+/-0.12 equivalent of radioactivity per mg of tissue in the common carotid and 0.25+/-0.18 in the abdominal aorta; in contrast, rabbits treated by zofenopril had 0.20+/-0.12 in the common carotid and 0.06+/-0.01 in the abdominal aorta. These data indicate that ACE-inhibition with zofenopril has antiatherosclerotic and antioxidant effects in WHHL-rabbits. Our results also shows that these effects could be linked to a reduced wall-associated platelet deposition at the site of atherosclerotic lesions.

Background —Atherosclerotic lesions in intracranial arteries occur later and are less extensive than in extracranial arteries. To investigate potential mechanisms responsible for this difference, in particular the atherogenic response to hypercholesterolemia and LDL oxidation, we compared the extent of fatty streak formation and the composition of these very early lesions in intracranial arteries of human fetuses from normocholesterolemic and hypercholesterolemic mothers with those in extracranial arteries. Methods and Results —Lesions were quantified by computer-assisted image analysis of 30 oil red O–stained sections, each from the middle cerebral, basilar, and common carotid arteries and the abdominal aorta of human fetuses (spontaneous abortions and premature newborns who died within 12 hours of birth; both of fetal age 6.2±1.3 months) from 43 hypercholesterolemic mothers and 34 normocholesterolemic mothers. Macrophages, apolipoprotein B, and 2 epitopes of oxidized LDL in lesions were determined immunocytochemically. Activities of superoxide dismutase, catalase, and glutathione peroxidase in the arterial wall were also determined. Lesion numbers and sizes were dramatically greater in the abdominal aorta (area of the largest lesion per section: 66.5±10.9 ×10 3 μm 2 ) and the carotid (11.6±5.3 ×10 3 μm 2 ) than in the basilar and middle cerebral artery (0.4±0.1 and 0.8±0.2 ×10 3 μm 2 , respectively; P Conclusions —Exposure to hypercholesterolemia during fetal development results in extensive formation of fatty streaks in extracranial but not intracranial arteries. The fact that such a difference in lesion formation occurs in the absence of many other atherogenic risk factors found later in life suggests that differences in the atherogenic response to hypercholesterolemia are an important contributor to the slower onset of the disease in intracranial vessels in adults. Fetal arteries may allow elucidation of the mechanisms responsible, for example, better protection of intracranial arteries against free radical–mediated atherogenic processes.