Anacardic acid and thyroid hormone enhance cardiomyocytes production from undifferentiated mouse ES cells along functionally distinct pathways.

The epigenetics of early commitment to embryonal cardiomyocyte is poorly understood. In this work, we compared the effect of thyroid hormone and that of anacardic acid, a naturally occurring histone acetylase inhibitor, or both in combination, on mouse embryonic stem cells (mES) differentiating into embryonal cardiomyocyte by embryoid bodies (EBs) formation. Although the results indicated that anacardic acid (AA) and thyroid hormone were both efficient in promoting cardiomyocyte differentiation, we noticed that a transient exposure of mES to AA alone was sufficient to enlarge the beating areas of EBs compared to those of untreated controls. This effect was associated with changes in the chromatin structure at the promoters of specific cardiomyogenic genes. Among them, a rapid induction of the transcription factor Castor 1 (CASZ1), important for cardiomyocytes differentiation and maturation during embryonic development, was observed in the presence of AA. In contrast, thyroid hormone (T 3) was more effective in stimulating spontaneous firing, thus suggesting a role in the production of a population of cardiomyocyte with pacemaker properties. In conclusion, AA and thyroid hormone both enhanced cardiomyocyte formation along in apparently distinct pathways.

P300/CBP associated factor regulates nitroglycerin-dependent arterial relaxation by N(ε)-lysine acetylation of contractile proteins.

OBJECTIVE: To address the role of epigenetic enzymes in the process of arterial vasorelaxation and nitrate tolerance, in vitro and in vivo experiments were performed in the presence or absence of glyceryl trinitrate (GTN) or histone deacetylases/histone acetylases modulators. METHODS AND RESULTS: In vitro single GTN administration rapidly increased cGMP synthesis and protein N(ε)-lysine acetylation in rat smooth muscle cells, including myosin light chain and smooth muscle actin. This phenomenon determined a decrease in myosin light chain phosphorylation and actomyosin formation. These effects were abolished by prolonged exposure to GTN and rescued by treatment with trichostatin A. In vivo, adult male rats were treated for 72 hours with subcutaneous injections of GTN alone or in combination with the histone deacetylases inhibitors trichostatin A, suberoylanilide hydroxamic acid, MS-27-275, or valproic acid. Ex vivo experiments performed on aortic rings showed that the effect of tolerance was reversed by all proacetylation drugs, including the p300/CREB binding protein-associated factor activator pentadecylidenemalonate 1b (SPV106). Any response to GTN was abolished by anacardic acid, a potent histone acetylases inhibitor. CONCLUSIONS: This study establishes the following points: (1) GTN treatment increases histone acetylases activity; (2) GTN-activated p300/CREB binding protein-associated factor increases protein N(ε)-lysine acetylation; (3) N(ε)-lysine acetylation of contractile proteins influences GTN-dependent vascular response. Hence, combination of epigenetic drugs and nitroglycerin may be envisaged as a novel treatment strategy for coronary artery disease symptoms and other cardiovascular accidents of ischemic origin.

Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: protective role of p21(waf1,cip1,sdi1).

BACKGROUND: In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear. METHODS AND RESULTS: To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 microg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm(2) did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function. CONCLUSIONS: This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.

Endothelial NOS, estrogen receptor beta, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer.

The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor beta (ERbeta). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERbeta/eNOS, ERbeta/HIF-1alpha, or ERbeta/HIF-2alpha combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERbeta and nuclear eNOS plus HIF-2alpha being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERbeta, and HIF-2alpha expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases.

Inhibition of Poly(ADP-ribose)polymerase impairs Epstein Barr Virus lytic cycle progression.

BACKGROUND: Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins involved in several cellular events as well as in processes that characterize the infective cycle of some viruses. In the present study, we investigated the role of poly(ADP-ribosylation) on Epstein-Barr Virus (EBV) lytic cycle activation. RESULTS: Inhibition of PARP-1 by 3-aminobenzamide (3-ABA) during EBV induction, diminished cell damage and apoptosis in the non-productive Raji cell line while markedly reducing the release of viral particles in the productive Jijoye cells. Furthermore, incubation with 3-ABA up-regulated the levels of LMP1 and EBNA2 latent viral proteins. At the same time, it slightly affected the expression of the immediate early BZLF1 gene, but largely down-regulated the levels of the early BFRF1 protein. The modulation of the expression of both latent and lytic EBV genes appeared to be post-transcriptionally regulated. CONCLUSION: Taken together the data indicate that PARP-1 plays a role in the progression of EBV lytic cycle and therefore, PARP inhibitors might represent suitable pharmacological adjuncts to control viral spread in EBV productive infection.

Papilloma protein E6 abrogates shear stress-dependent survival in human endothelial cells: evidence for specialized functions of paxillin.

BACKGROUND: To investigate how endothelial cells transduce intracellular signals in response to laminar shear stress (SS), we made use of the papilloma virus oncoprotein E6 which interacts with and induces degradation of numerous cellular proteins including p53 and members of the PDZ-domain family. E6 also recognizes paxillin (PXN), a fundamental component of focal adhesions, interfering with its association to focal adhesion kinase (FAK). METHODS AND RESULTS: Human umbilical vein endothelial cells, expressing E6 or its mutated variant DeltaE6(105-110) (DeltaE6) which does not inactivate p53, were cultured under static conditions or exposed to a laminar SS of 12 dyn/cm(2) for 16h. In response to SS, cells expressing E6 or DeltaE6 failed to synthesise nitric oxide and directionally remodel their cytoskeleton, as indicated by morphology and phalloidin staining of actin microfilaments. Under these conditions, PXN association with FAK, its localization to the plasma membrane, and its phosphorylation on tyrosine-31, which partially encompasses the PXN/FAK docking site, were severely compromised. These alterations were paralleled by the impairment of important SS-dependent endothelial functions, including nitric oxide production and survival upon serum deprivation. The direct targeting of PXN expression by RNA interference partially reproduced the E6 phenotype, impairing flow-dependent cell orientation and survival but not nitric oxide production. CONCLUSIONS: These results provide evidence that papilloma virus E6 protein interferes with the function of the SS-mechanosensor and suggests a potential a role for PXN in this process.

p21(Waf1/Cip1/Sdi1) mediates shear stress-dependent antiapoptotic function.

OBJECTIVE: The antiapoptotic effect of p21(Waf1/Cip1/Sdi1) (p21) was examined in human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress (SS) or to the nitric oxide donor sodium nitroprusside (SNP) and in a mouse model of hindlimb ischemia. METHODS: In vitro: Cells were cultured without serum and in the presence of cobalt chloride to simulate hypoxia for 12 h (T0). Shear stress was applied to endothelial cells for additional 12 h. In vivo: Hindlimb ischemia was realized in mice by femoral artery ligation. SNP was acutely administered by subcutaneous injection or by Alzet osmotic pumps for a longer treatment. RESULTS: At T0, HUVEC were either exposed to SS (15 dyn/cm2/s(-1)), treated with SNP or kept in static condition (ST) for 1-12 h; after additional 12 h in ST, 30-35% of cells still alive at T0 had died. In this condition, both SS and SNP treatments markedly increased p21 levels and reduced apoptosis in HUVEC. Recombinant adenoviruses carrying p21 (AdCMV.p21) or antisense p21 (AdCMV.ASp21) cDNA revealed that AdCMV.p21-infected HUVEC were protected from death while AdCMV.ASp21 reduced SS- and SNP-dependent protection from apoptosis. In mice, apoptosis was detected in endothelial cells of ischemic hindlimbs as early as 8 h after femoral artery ligation. Treatment with SNP enhanced p21 expression and protected ischemic tissue from damage. Remarkably, direct in vivo injection of AdCMV.p21 significantly reduced the number of apoptotic nuclei in the presence of ischemia. CONCLUSIONS: The present study establishes that, under our experimental conditions, (a) p21 plays an important role in SS and nitric oxide antiapoptotic effect in vitro, and (b) p21 gene transfer prevents apoptosis in vitro and in vivo, following acute interruption of blood flow.