Pre-natal exposure of mice to bisphenol A elicits an endometriosis-like phenotype in female offspring.

Endometriosis is a chronic gynecological disease characterized by the growth of endometrial tissue outside the uterine cavity. Exposure to endocrine disruptors during critical period of development causes long-lasting effects, being the genital system one of the targets. This study describes the effects on female genital system caused by developmental exposure to the endocrine-disrupting chemical bisphenol A (BPA) during pre- and peri-natal development in mice. To this end, timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with BPA (100, or 1000 microg/kg/day). After delivery, pups were held for 3 months; then, pelvic organs were analyzed in their entirety and livers of both pups and moms were studied for the presence of BPA. We found in the adipose tissue surrounding the genital tracts of a consistent number of treated animals, endometriosis-like structure with the presence of both glands and stroma and expressing both estrogen receptor and HOXA-10. Moreover, cystic ovaries, adenomatous hyperplasia with cystic endometrial hyperplasia and atypical hyperplasia were significantly more frequent in treated animals respect to the controls. Finally, BPA was found in the livers of exposed moms and female offspring. In conclusion, we describe for the first time an endometriosis-like phenotype in mice, elicited by pre-natal exposition to BPA. This observation may induce to thoroughly reconsider the pathogenesis and treatment of endometriosis, considering the high incidence of endometriosis and the problems caused by associated infertility.

Molecular analysis of the apoptotic effects of BPA in acute myeloid leukemia cells.

BACKGROUND: BPA (bisphenol A or 2,2-bis(4-hydroxy-phenol)propane) is present in the manufacture of polycarbonate plastic and epoxy resins, which can be used in impact-resistant safety equipment and baby bottles, as protective coatings inside metal food containers, and as composites and sealants in dentistry. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA. Thus, it is necessary to investigate the cytotoxicity and apoptosis-inducing activity of this compound. METHODS: Cell cycle, apoptosis and differentiation analyses; western blots. RESULTS: BPA is able to induce cell cycle arrest and apoptosis in three different acute myeloid leukemias. Although some granulocytic differentiation concomitantly occurred in NB4 cells upon BPA treatment, the major action was the induction of apoptosis. BPA mediated apoptosis was caspase dependent and occurred by activation of extrinsic and intrinsic cell death pathways modulating both FAS and TRAIL and by inducing BAD phosphorylation in NB4 cells. Finally, also non genomic actions such as the early decrease of both ERK and AKT phosphorylation were induced by BPA thus indicating that a complex intersection of regulations occur for the apoptotic action of BPA. CONCLUSION: BPA is able to induce apoptosis in leukemia cells via caspase activation and involvement of both intrinsic and extrinsic pathways of apoptosis.

Beneficial effects of autologous bone marrow cell infusion and antioxidants/L-arginine in patients with chronic critical limb ischemia.

BACKGROUND: Short-term (within 6 weeks follow-up) clinical studies indicate that implantation of bone marrow cells (BMCs) into ischemic limbs may improve peripheral ischemia. Here, the long-term safety and feasibility of intraarterial autologous BMCs with oral treatment with antioxidants and L-arginine were investigated in patients with critical ischemia on account of advanced atherosclerotic peripheral arterial disease (PAD). METHODS: Eighteen patients with PAD (advanced III/IV Fontaine stages) were enrolled in this study (NCT00306085). An additional group of 18 patients taking maximal drug therapy that refused BMC therapy served as control. The BMC-treated group received two doses of BMCs in the leg arteries (time 0 and 45 days). After 30 days from the first BMC dose, patients received daily antioxidants, and L-arginine. Therapeutic neoangiogenesis was estimated by angiography and laser Doppler\capillaroscopy. RESULTS: Ankle brachial index improvement (DeltaABI: >0.1) was seen in 10 patients at 3 months and in 12 patients at 12-18 months. Ischemic ulcers improved in 13 patients (after 6-12 months). Although two patients underwent amputation, the mean maximum walking distance significantly increased at 3 months and was sustained up to 18 months. Among conservative patients, 10 underwent amputation in comparison with two BMC-treated patients (55.6 vs. 13.3%; P=0.014). CONCLUSION: This small study shows that intraarterial autologous BMC and antioxidants and L-arginine therapy is safe and effective in patients with advanced atherosclerotic PAD with positive effects until 18 months.

Effects of a pomegranate fruit extract rich in punicalagin on oxidation-sensitive genes and eNOS activity at sites of perturbed shear stress and atherogenesis.

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.

Feijoa sellowiana derived natural Flavone exerts anti-cancer action displaying HDAC inhibitory activities.

Curative properties of some medicinal plants such as the Feijoa sellowiana Bert. (Myrtaceae), have been often claimed, although the corresponding molecular mechanism(s) remain elusive. We report here that the Feijoa acetonic extract exerts anti-cancer activities on solid and hematological cancer cells. Feijoa extract did not show toxic effects on normal myeloid progenitors thus displaying a tumor-selective activity. In the Feijoa acetonic extract, fractionation and subsequent purification and analyses identified Flavone as the active component. Flavone induces apoptosis which is accompanied by caspase activation and p16, p21 and TRAIL over-expression in human myeloid leukemia cells. Use of ex vivo myeloid leukemia patients blasts confirms that both the full acetonic Feijoa extract and its derived Flavone are able to induce apoptosis. In both cell lines and myeloid leukemia patients blasts the apoptotic activity of Feijoa extract and Flavone is accompanied by increase of histone and non-histone acetylation levels and by HDAC inhibition. Our findings show for the first time that the Feijoa apoptotic active principle is the Flavone and that this activity correlates with the induction of HDAC inhibition, supporting the hypothesis of its epigenetic pro-apoptotic regulation in cancer systems.

Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture.

Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischaemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells are important for developing targeted cellular therapies and reproducibility of data are the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133(+) cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23% +/- 4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133(+) enriched cells was 50% +/- 7% (P < 0.01). These data were obtained through a novel and a more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 subfields). When stimulated with tumour necrosis factor alpha (TNF)-alpha and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133(+) enriched cells. However, both cultured total PBMCs and CD133(+) enriched cells respond similarly to TNF-alpha or glucose-induced p38-phosphorylation. EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133(+) enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133(+) expression was observed (P < 0.01) This may have relevance during intervention studies using cultured EPCs.

The role of oxidative stress in adult critical care.

Oxidative stress defines an imbalance in production of oxidizing chemical species and their effective removal by protective antioxidants and scavenger enzymes. Evidence of massive oxidative stress is well established in adult critical illnesses characterized by tissue ischemia-reperfusion injury and by an intense systemic inflammatory response such as during sepsis and acute respiratory distress syndrome. Oxidative stress could exacerbate organ injury and thus overall clinical outcome. We searched MEDLINE databases (January 1966 to June 2005). For interventional studies, we accepted only randomized trials. Several small clinical trials have been performed in order to reduce oxidative stress by supplementation of antioxidants alone or in combination with standard therapies. These studies have reported controversial results. Newer large multicenter trials with antioxidant supplementation should be performed, considering administration at an early stage of illness and a wider population of critically ill patients.

Expression of transcription factor Yin Yang 1 in human osteosarcomas.

The transcription factor Yin Yang 1 (YY1) is known to be present in some human cancer cell lines and its expression correlates with immune-mediated apoptosis. By using Western blot analysis, we have shown that the YY1 protein is strongly expressed in human osteosarcoma cells and localised mainly in the nucleus. Moreover, by using immunohistochemistry and RT-PCR techniques, we have analysed the expression of YY1 protein in biopsies from human osteosarcomas. The YY1 protein was not detectable by immunohistochemistry in osteoid tissue. However, its expression was restricted to osteosarcoma tissues. These data were confirmed by densitometric analysis of RT-PCR for YY1 expression. Thus, YY1 gene activation appears to be an early event in the process of osteoblastic transformation and its detection may represent, together with the analysis of other established markers, a useful diagnostic tool in human osteosarcomas.

Role of oxidative stress in experimental sepsis and multisystem organ dysfunction.

Massive increase in radical species can lead to oxidative stress, promoting cell injury and death. This review focuses on experimental evidence of oxidative stress in critical illnesses, sepsis and multisystem organ dysfunction. Oxidative stress could negatively affect organ injury and thus overall survival of experimental models. Based on this experimental evidence, we could improve the rationale of supplementation of antioxidants alone or in combination with standard therapies aimed to reduce oxidative stress as novel adjunct treatment in critical care.

Antioxidant intervention attenuates myocardial neovascularization in hypercholesterolemia.

BACKGROUND: Hypercholesterolemia (HC) and atherosclerosis can elicit oxidative stress, coronary endothelial dysfunction, and myocardial ischemia, which may induce growth-factor expression and lead to myocardial neovascularization. We tested the hypothesis that chronic antioxidant intervention in HC would attenuate neovascularization and preserve the expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF). METHODS AND RESULTS: Three groups of pigs (n=6 each) were studied after 12 weeks of normal or 2% HC diet or HC+antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily). Myocardial samples were scanned ex vivo with a novel 3D micro-CT scanner, and the spatial density and tortuosity of myocardial microvessels were determined in situ. VEGF mRNA, protein levels of VEGF and VEGF receptor-1, HIF-1alpha, nitrotyrosine, and superoxide dismutase (SOD) were determined in myocardial tissue. The HC and HC+antioxidant groups had similar increases in serum cholesterol levels. HC animals showed an increase in subendocardial spatial density of microvessels compared with normal (160.5+/-11.8 versus 95.3+/-8.2 vessels/cm2, P<0.05), which was normalized in HC+antioxidant (92.5+/-20.5 vessels/cm2, P<0.05 versus HC), as was arteriolar tortuosity. In addition, HC induced upregulation of VEGF, HIF-1alpha, and nitrotyrosine expression and decreased SOD expression and activity, all of which were preserved by antioxidant intervention. CONCLUSIONS: Changes in myocardial microvascular architecture invoked by HC are accompanied by increases in HIF-1alpha and VEGF expression and attenuated by antioxidant intervention. This underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early atherogenesis.

Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis.

Increased generation of oxidants, resulting from disruption of aerobic metabolism and from respiratory burst, is an essential defense mechanism against pathogens and aberrant cells. However, oxidative stress can also trigger and enhance deregulated apoptosis or programmed cell death, characteristic of atherosclerotic lesions. Oxidation-sensitive mechanisms also modulate cellular signaling pathways that regulate vascular expression of cytokines and growth factors, and influence atherogenesis, in particular when increased levels of plasma lipoproteins provide ample substrate for lipid peroxidation and lead to increased formation of adducts with lipoprotein amino acids. In some cases, increased oxidation and apoptosis in a group of cells might be beneficial for survival and function of other groups of arterial cells. However, overall, oxidation and apoptosis appear to promote the progression of diseased arteries towards a lesion that is vulnerable to rupture, and to give rise to myocardial infarction and ischemic stroke. Recent rapid advances in our understanding of the interactions between oxidative stress, apoptosis and arterial gene regulation suggest that selective interventions targeting these biological functions have great therapeutic potential.

Oxidative stress-related increase in ubiquitination in early coronary atherogenesis.

The ubiquitin-proteasome system (UPS) is involved in the removal of damaged proteins and the activation of transcription factors, such as nuclear-factor-kappaB. Recent reports, however, questioned the functional activity of the UPS under conditions of increased oxidative stress, such as experimental hypercholesterolemia, which was the objective of our study. Pigs were placed on a normal chow diet (N) or on a hypercholesterolemic diet without (HC) or with vitamin C and E supplementation (HC+VIT) for 12 weeks. Compared with N, plasma concentration of total cholesterol increased in both HC and HC+VIT [76 +/- 21 vs. 400 +/- 148 (P<0.05) and 329 +/- 102 (P<0.05) mg/dL], whereas increase in lipid peroxidation, as assessed by LDL-malondialdehyde plasma concentration, was found in HC but not in HC+VIT [6.6 +/- 0.7 vs. 8.5 +/- 0.3 (P<0.05) and 6.8 +/- 0.7 nmol/mg protein]. In comparison with N, the level of ubiquitin conjugates in the coronary artery, as assessed by immunoblotting, increased by 42% in HC but not in HC+VIT and was localized predominantly to media vascular smooth muscle cells by immunostaining. There was no difference in proteasome proteolytic activity among the study groups. These results demonstrate that the UPS is functionally active in early atherogenesis despite increase in oxidative stress with important repercussions in the pathophysiology and therapy of cardiovascular diseases.

Mechanisms of renal structural alterations in combined hypercholesterolemia and renal artery stenosis.

OBJECTIVE: Atherosclerotic renovascular disease (ARVD) aggravates renal scarring more than other causes of renal artery stenosis (RAS), but the underlying pathogenic mechanisms of this potential profibrotic effect remain unclear. We tested the hypothesis that coexistence of atherosclerosis and RAS interferes with renal tissue remodeling. METHODS AND RESULTS: Single-kidney hemodynamics and function were quantified in vivo with electron-beam computed tomography in 3 groups of pigs (n=7 each): normal pigs, pigs 12 weeks after induction of unilateral RAS (RAS group), and pigs with similar-degree RAS fed a 12-week 2% hypercholesterolemic diet (HC+RAS, simulating early ARVD). Kidneys were studied ex vivo by Western blotting and immunohistochemistry. Renal volume, renal blood flow, and glomerular filtration rate were similarly decreased in RAS and HC+RAS ischemic kidneys, accompanied by similar increased expression of profibrotic factors like transforming growth factor-beta, tissue inhibitor of metalloproteinase-1, and plasminogen activator inhibitor-1. Nevertheless, HC+RAS kidneys showed increased intrarenal fibrosis compared with RAS-only kidneys. Furthermore, expression of nuclear factor-kappaB was increased, expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems was decreased, and apoptosis was blunted. CONCLUSIONS: Diet-induced HC superimposed on RAS accelerates the development of fibrosis in the stenotic kidney by amplifying profibrotic mechanisms and disrupting tissue remodeling. These alterations might contribute to renal disease progression in ARVD and might account for the increased propensity for end-stage renal disease.

New advances in microarrays: finding the genes causally involved in disease.

Much progress has been made in the technologies available to assess global alterations in mRNA levels in clinical research samples. Although such transcript profiling can provide a powerful research tool, the broad range of options can be bewildering for the inexperienced investigator, and more often than not the limitations and pitfalls of this approach are not fully appreciated. It is important to recognize that the major goal of transcript profiling experiments can be to identify novel therapeutic targets or delineate complex patterns of gene expression that provide a potentially pathognomonic phenotype. Microarrays are therefore potentially powerful tools to address the need for high-throughput analysis techniques, but a careful postanalysis follow-up and validation of microarray experiments will be needed soon (1). A projected decrease in cost and complexity may facilitate increased availability of this technology in the physician's office, and future applications may then allow tailoring of clinical diagnosis and targeted therapeutic strategies.

c-Myc oncoprotein: cell cycle-related events and new therapeutic challenges in cancer and cardiovascular diseases.

Advanced stages of both cancer and atherosclerosis are characterized by a local increase in tissue mass that may be hard to control. This increase in tissue mass can be attributed to oxidation-sensitive modification of cell cycle-related events, including cellular proliferation, differentiation, and apoptosis, which could be secondary to alteration in the activity of tumor suppressor gene and oncogene products. The oncogene c-Myc has classically been considered to be involved in carcinogenesis and has more recently been implicated in both endothelial dysfunction and atherogenesis as well. Consequently, inhibition of c-Myc-dependent signaling has become a novel therapeutic opportunity and challenge in atherosclerosis and other cardiovascular diseases. Antioxidant strategies, RNA synthesis inhibitors such as mithramycin, and gene therapeutic approaches with antisense oligonucleotides against c-Myc are some of the promising strategies. In general, the increased biologic understanding of the participation of cell cycle events and targeting these events may enable to attenuate or prevent some of the complications of vascular and neoplastic diseases.

[Childhood infection and endothelial dysfunction: a potential synergistic link in atherosclerosis]. / Infezione e disfunzione endoteliale nell'infanzia: un potenziale sinergismo nell'aterosclerosi.

The prodromal stage of atherosclerotic lesions is already formed during human fetal development. The presence of infections during childhood may increase synergistically the progression of atherogenesis. After delivery, especially those children exposed to severe maternal hypercholesterolemia should be followed up for the onset and development of acute and chronic infections and be included in clinical and noninvasive examinations of vascular function.

Embolization of traumatic and non-traumatic peripheral vascular lesions with Onyx.

PURPOSE: The aim of our study is to verify the feasibility and the efficacy of Onyx as embolization agent in the treatment of traumatic and non-traumatic peripheral vascular lesions. MATERIALS AND METHODS: In the period between September 2006 and March 2012, we treated with Onyx 26 patients (14 males/12 females; age range, 18-85 years old; mean age, 65 years old), 11 of which with traumatic peripheral vascular lesions and 15 with non-traumatic vascular lesions (9 neoplastic hemorrhagic lesions, 3 arteriovenous malformations (AVMs) and 3 aneurysms). Follow-up controls were performed with clinical examination and by multidetector computed tomography (MDCT) imaging 1, 6, and 12 months after the procedure. RESULTS: All peripheral vascular lesions were embolized with Onyx; 3 patients with aneurysms were treated with Onyx associated with endovascular coils. Four elective and 22 emergency embolization procedures were performed. In all patients, we obtained cessation of bleeding and the complete and permanent embolization of all vascular lesions. CONCLUSIONS: Onyx is an effective and safe embolization agent for peripheral vascular lesions.

c-Myc oncoprotein: a dual pathogenic role in neoplasia and cardiovascular diseases?

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.

Statin treatment and the natural history of atherosclerotic-related diseases: pathogenic mechanisms and the risk-benefit profile.

Large-scale intervention trials demonstrate that treatment with statins, the most effective lipid lowering drug class, significantly reduces the risk of coronary heart disease events. Recent evidence suggests that more aggressive LDL cholesterol lowering with newly developed statins may provide greater clinical benefit, even in individuals with moderately elevated serum cholesterol levels. There is increasing evidence that statins exert a myriad of other beneficial pleiotropic effects on the vascular wall, thus altering the course of atherosclerotic disease. In the long-term treatment, non-life-threatening side effects may occur in up to 15% of patients receiving one statin. Significant elevations in the activity of serum aminotransferase and creatine kinase alone or in combination with muscle pain in statin-treated patients should be taken seriously. The combination of the statins with gemfibrozil results in higher rates of drug toxicity. Reports show possible adverse effects of statins on nervous system function including mood alterations, however, statins have also been associated with improvement in central nervous system disorders. Special attention must be paid to the tolerability of the statins in children, elderly and transplant patients. Future clinical studies and surveillance information will warrant long term safety of each member of this class of lipid-lowering agents. New classes of patients with diabetes, metabolic syndrome and renal diseases may have clinical benefits from statins. New upcoming clinical trials will address the fundamental question of whether statin treatment can protect from the natural history of atherosclerotic-related diseases. This will require a more prolonged follow-up (i.e., 10 to 15 years). Finally, the basic understanding of newer pathogenic mechanisms involving the effects of statins on angiogenesis and the nitric oxide pathway should be explored in the clinical setting as well as the study of pathogenic mechanisms by which statins can affect plaque instability.