Regulation of calpain 2 expression by miR-223 and miR-145.

Calpain 2 (CAPN2) is a Ca2+-dependent cysteine-protease that is involved in different cellular processes. Despite its important role, little is known about how CAPN2 expression is regulated. This study addressed the potential regulation of CAPN2 by microRNAs (miRNAs) in human endothelial cells. Two miRNAs were found to regulate CAPN2 expression by two distinct mechanisms, one direct and the other indirect. MiR-223 directly targeted CAPN2 by binding to the CAPN2 3'-untranslated region. Mir-223 overexpression decreased CAPN2 protein levels in cultured cells and in mice miR-223 antagonism led to an increase in CAPN2 protein in lung tissue. MiR-145 overexpression also decreased CAPN2 expression but did not affect a CAPN2 luciferase construct, indicating that the effect was indirect. MiR-145 targets histone deacetylase (HDAC) 2, and HDAC inhibition transcriptionally regulated CAPN2 expression by hyperacetylation of the promoter of CAPN2 gene and a subsequent decrease in polymerase 2 binding. Indeed, down regulation of HDAC2 by miR-145 not only decreased CAPN2 protein expression and calpain activity, but also protected paxillin against calpain-dependent degradation. Thus, protein levels of CAPN2 are regulated by miR-223, acting directly on the 3'-untranslated region as well as by miR-145, which acts via an increase in HDAC2. ENZYMES: Calpain 2 (EC 3.4.22.53), histone deacetylase 2 (EC 3.5.1.98).

Myeloid-Specific Deletion of the AMPKα2 Subunit Alters Monocyte Protein Expression and Atherogenesis.

The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.

Epigenetic control of the angiotensin-converting enzyme in endothelial cells during inflammation.

The angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system, which is involved in the regulation of blood pressure. Alterations in ACE expression or activity are associated with various pathological phenotypes, particularly cardiovascular diseases. In human endothelial cells, ACE was shown to be negatively regulated by tumor necrosis factor (TNF) α. To examine, whether or not, epigenetic factors were involved in ACE expression regulation, methylated DNA immunoprecipitation and RNA interference experiments directed against regulators of DNA methylation homeostasis i.e., DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs), were performed. TNFα stimulation enhanced DNA methylation in two distinct regions within the ACE promoter via a mechanism linked to DNMT3a and DNMT3b, but not to DNMT1. At the same time, TET1 protein expression was downregulated. In addition, DNA methylation decreased the binding affinity of the transcription factor MYC associated factor X to the ACE promoter. In conclusion, DNA methylation determines the TNFα-dependent regulation of ACE gene transcription and thus protein expression in human endothelial cells.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase.

OBJECTIVE: To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). METHODS & RESULTS: A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. CONCLUSIONS: Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Maternal treatment of spontaneously hypertensive rats with pentaerythritol tetranitrate reduces blood pressure in female offspring.

Pentaerythritol tetranitrate is devoid of nitrate tolerance and shows no reproductive or developmental toxicity in animal studies. Recently, pentaerythritol tetranitrate has been demonstrated to reduce the risk of intrauterine growth restriction and the risk of preterm birth in women with abnormal placental perfusion. This study was conducted to test the perinatal programming effect of pentaerythritol tetranitrate in spontaneously hypertensive rats, a rat model of genetic hypertension. Parental spontaneously hypertensive rats were treated with pentaerythritol tetranitrate (50 mg/kg per day) during pregnancy and lactation periods; the offspring received standard chow without pentaerythritol tetranitrate after weaning. Maternal treatment with pentaerythritol tetranitrate had no effect on blood pressure in male offspring. In the female offspring, however, a persistent reduction in blood pressure was observed at 6 and 8 months. This long-lasting effect was accompanied by an upregulation of endothelial nitric oxide synthase, mitochondrial superoxide dismutase, glutathione peroxidase 1, and heme oxygenase 1 in the aorta of 8-month-old female offspring, which was likely to result from epigenetic changes (enhanced histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation) and transcriptional activation (enhanced binding of DNA-directed RNA polymerase II to the transcription start site of the genes). In organ chamber experiments, the endothelium-dependent, nitric oxide-mediated vasodilation to acetylcholine was enhanced in aorta from female offspring of the pentaerythritol tetranitrate-treated parental spontaneously hypertensive rats. In conclusion, maternal pentaerythritol tetranitrate treatment leads to epigenetic modifications, gene expression changes, an improvement of endothelial function and a persistent blood pressure reduction in the female offspring.

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity.

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.

Dexamethasone upregulates Nox1 expression in vascular smooth muscle cells.

BACKGROUND/AIM: It has been demonstrated that dexamethasone-induced hypertension can be prevented by the NADPH oxidase inhibitor apocynin. The effect of dexamethasone on NADPH oxidase, however, is unknown. The present study was conducted to investigate the effect of dexamethasone on the gene expression of Nox1, the major NADPH oxidase isoform in vascular smooth muscle cells. RESULTS: Oral treatment of Wistar-Kyoto rats with dexamethasone (0.03 mg/kg/day) for 12 days led to an upregulation of Nox1 mRNA expression in the aorta. In cultured A7r5 rat aortic smooth muscle cells, dexamethasone increased Nox1 mRNA expression in a concentration- and time-dependent manner. The upregulation of Nox1 mRNA expression was completely prevented by the glucocorticoid receptor antagonist mifepristone. The effect of dexamethasone on Nox1 expression was likely to be indirect as it could be largely blocked by cycloheximide, an inhibitor of protein biosynthesis. Dexamethasone increased Nox1 mRNA stability as well as Nox1 transcription. The dexamethasone-induced Nox1 expression was completely prevented by scriptaid, a pan-inhibitor of histone deacetylases (HDAC), indicating a crucial role for HDAC enzymes. In total, A7r5 cells expressed 8 HDAC isoforms, with HDAC1, 5, 6 and 7 being the most abundant ones. Knockdown of these 4 individual HDAC enzymes did not prevent the effect of dexamethasone on Nox1 expression, although HDAC5 knockdown markedly reduced basal Nox1 expression. CONCLUSION: Dexamethasone upregulates Nox1 expression in vascular smooth muscle cells. This effect involves the glucocorticoid receptor and HDAC enzymes.

Endothelial dysfunction in tristetraprolin-deficient mice is not caused by enhanced tumor necrosis factor-α expression.

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP(-/-) mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP(-/-) mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP(-/-) mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP(-/-) mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP(-/-) animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP(-/-) animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.

Role of SIRT1 and FOXO factors in eNOS transcriptional activation by resveratrol.

Many of the cardiovascular protective effects of resveratrol are attributable to an enhanced production of nitric oxide (NO) by the endothelial NO synthase (eNOS). Resveratrol has been shown to enhance eNOS gene expression as well as eNOS enzymatic activity. The aim of the present study was to analyze the molecular mechanisms of eNOS transcriptional activation by resveratrol. Treatment of human EA.hy 926 endothelial cells with resveratrol led to a concentration-dependent upregulation of eNOS expression. In luciferase reporter gene assay, resveratrol enhanced the activity of human eNOS promoter fragments (3500, 1600, 633 and 263bp in length, respectively), indicating that the proximal promoter region is required for resveratrol-induced eNOS transcriptional activation. Knockdown of the NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1) by siRNA prevented the upregulation of eNOS mRNA and protein by resveratrol. Forkhead box O (FOXO) transcription factors are established downstream targets of SIRT1. siRNA-mediated knockdown of FOXO1 and FOXO3a abolished the effect of resveratrol on eNOS expression, indicating the involvement of these factors. Resveratrol treatment enhanced the expression of FOXO1 and FOXO3a in EA.hy 926 cells. Reporter gene assay using promoter containing forkhead response elements showed increased FOXO factor activity by resveratrol. In electrophoretic mobility shift assay, the enhanced binding of nuclear proteins to the eNOS promoter regions by resveratrol could be blocked by antibodies against FOXO1 and FOXO3a. In conclusion, resveratrol enhances the expression and activity of FOXO transcription factors. The SIRT1/FOXO factor axis is involved in resveratrol-induced eNOS transcriptional activation.

Transcriptional regulation of Nox4 by histone deacetylases in human endothelial cells.

Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production mainly depends on the expression levels of the enzyme. The present study was aimed to investigate the mechanisms of Nox4 transcription regulation by histone deacetylases (HDAC). In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, treatment with the pan-HDAC inhibitor scriptaid led to a marked decrease in Nox4 mRNA expression. A similar down-regulation of Nox4 mRNA expression was observed by siRNA-mediated knockdown of HDAC3. HDAC inhibition in endothelial cells was associated with enhanced histone acetylation, increased chromatin accessibility in the human Nox4 promoter region, with no significant changes in DNA methylation. In addition, we provided evidence that c-Jun played an important role in controlling Nox4 transcription. Knockdown of c-Jun with siRNA led to a down-regulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding of c-Jun to the Nox4 promoter region was reduced despite the open chromatin structure. In parallel, the binding of RNA polymerase IIa to the Nox4 promoter was significantly inhibited as well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC inhibition decreases Nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the Nox4 promoter, most likely because of a hyperacetylation-mediated steric inhibition.