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Abstract: Estrogens exert rapid, non-genomic effects, which are mediated by plasma membrane-associated estrogen receptors (mER) mERα and mERβ, and the intracellular transmembrane G protein-coupled estrogen receptor (GPER). Membrane-initiated responses contribute to transcriptional activation, resulting in a complex interplay of nuclear and extra-nuclear mechanisms that mediate the acute physiological responses to estrogens. Non-genomic estrogen signaling also activates a variety of intracellular estrogen signaling pathways that regulate vascular function and cell growth involving rapid but also long-term effects. This review discusses recent advances in understanding of the mechanisms of non-genomic estrogen receptor signaling in the vascular wall. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology in obesity. In this context, whether ET-1 modulates hydroxyl radical (.OH) formation and the function of ROS/·OH in obesity is not known. In the present study, formation and function of ROS, including ·OH, were investigated in the aorta of lean and leptin-deficient obese ob/ob mice. Hydroxyl radical formation was detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS in ET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination of α-tocopherol and ascorbic acid. Generation of either ·OH, ·O2-, and H2O2 was strongly inhibited by EPC-K1 (all P < 0.05). In obese mice, basal vascular ·OH formation and ROS activity were reduced by 3-fold and 5-fold, respectively (P < 0.05 vs. lean). ET-1 markedly enhanced ·OH formation in lean (6-fold, P < 0.05 vs. untreated) but not in obese mice. Obesity increased ET-1-induced contractions (P < 0.05 vs. lean), and ROS scavenging further enhanced the response (P < 0.05 vs. untreated). Exogenous ROS, including ·OH caused stronger vasodilation in obese animals (P < 0.05 vs. lean), whereas endothelium-dependent relaxation was similar between lean and obese animals. In conclusion, we present a sensitive method allowing ex vivo measurement of vascular ·OH generation and provide evidence that ET-1 regulates vascular ·OH formation. The data indicate that in obesity, vascular formation of ROS, including ·OH is lower, whereas the sensitivity to ROS is increased, suggesting a novel and important role of ROS, including ·OH in the regulation of vascular tone in disease status associated with increased body weight. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Angiotensin II (Ang II), endothelin-1 (ET-1) and reactive oxygen species (ROS) have been implicated in the development of pathologic changes associated with obesity including hypertension and atherosclerosis. The aim of this study was to investigate the effects of dietary fat content on vasoreactivity and receptor expression at the level of gene and protein expression. Methods: C57BL/6 mice were fed diets of normal (Control, 12.3% kcal from fat), high (HF, 41% kcal from fat) and very high (VHF, 58% kcal from fat) fat content for 15 weeks. Glucose tolerance tests were performed, and aortic rings were exposed to ET-1 (0.01–300 nM) and Ang II (100 nM) in the presence of l-nitro-arginine-methyl ester (l-NAME; 300 μM). Gene and protein expressions of angiotensin and endothelin receptors were examined by real-time PCR and immunoblotting, respectively. The effects of diet on responses to acetylcholine (ACh 0.1–300 μM), in the absence or presence of l-NAME, and to exogenous ROS/lso investigated. Results: Both high fat diets similarly impaired glucose tolerance (P <0.05). Increasing dietary fat augmented contractions to Ang II in a step-wise manner (P <0.05). Conversely, increasing dietary fat had no effect on contractions to ET-1. Exposure to ROS/ed in a rapid vasodilation that was markedly augmented in a step-wise manner with increasing dietary fat (P <0.05). Endothelium-dependent relaxation to ACh was unaffected whereas vasoconstriction to high concentrations of ACh was enhanced in VHF animals (P <0.05 vs. control). Gene expression of the AT1B receptor was increased in the aorta of VHF mice, and aortic ETA receptor protein expression was increased after both high fat diets. Conclusions: These findings demonstrate that changes in dietary fat intake modulate vascular reactivity in response to Ang II and ROS, as well as expression of vascular angiotensin and endothelin receptors. Dietary fat intake may thereby directly affect cardiovascular risk. [Copyright &y& Elsevier]

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. NixA was identified as a specific nickel uptake system in this organism. Addition of small amounts of nickel to media strongly stimulates urea hydrolysis. On the other hand, high nickel concentrations are deleterious to cell growth. As a possible protective reaction, nickel uptake seems to be reduced in H. pylori grown in nickel-rich media. These observations led to investigations of regulation of the expression of the nickel permease NixA. We found that increasing the nickel concentration in media reduced the amount of NixA. In order to address the question of whether this phenomenon was subject to transcriptional or translational regulation, we quantified nixA mRNA from H. pylori by real-time PCR. The amount of nixA mRNA was gradually reduced five- to sevenfold in a time- and concentration-dependent manner. Repression could be measured as soon as 5 min after nickel addition, and the maximum repression occurred after 20 to 30 min. The maximum repression was obtained with an external nickel concentration of 100 µM. The observed nickel repression of NixA was dependent on nikR encoding the nickel-responsive regulatory protein NikR. In conclusion, we demonstrated that synthesis of the NixA nickel permease of H. pylori shows nickel-responsive regulation mediated by NikR to maintain the balance between effective nickel acquisition and a toxic overload. [ABSTRACT FROM AUTHOR]

Background: PIM1 is a constitutively active serine-threonine kinase regulating cell survival and proliferation. Increased PIM1 expression has been correlated with cancer metastasis by facilitating migration and anti-adhesion. Endothelial cells play a pivotal role in these processes by contributing a barrier to the blood stream. Here, we investigated whether PIM1 regulates mouse aortic endothelial cell (MAEC) monolayer integrity. Methods: Pim1-/-MAEC were isolated from Pim1 knockout mice and used in trypsinization-, wound closure assays, electrical cell-substrate sensing, immunostaining, cDNA transfection and as RNA source for microarray analysis. Results: Pim1-/-MAEC displayed decreased migration, slowed cell detachment and increased electrical resistance across the endothelial monolayer. Reintroduction of Pim1- cDNA into Pim1-/-MAEC significantly restored wildtype adhesive characteristics. Pim1-/--MAEC displayed enhanced focal adhesion and adherens junction structures containing vinculin and β-catenin, respectively. Junctional molecules such as Cadherin 13 and matrix components such as Collagen 6a3 were highly upregulated in Pim1-/- cells. Intriguingly, extracellular matrix deposited by Pim1-/- cells alone was sufficient to induce the hyperadhesive phenotype in wildtype endothelial cells. Conclusion: Loss of Pim1 induces a strong adhesive phenotype by enhancing endothelial cell-cell and cell-matrix adhesion by the deposition of a specific extracellular matrix. Targeting PIM1 function therefore might be important to promote endothelial barrier integrity. [ABSTRACT FROM AUTHOR]

In adipose tissue mTOR complex 2 (mTORC2) contributes to the regulation of glucose/lipid metabolism and inflammatory molecule expression. Both processes display diurnal variations during the course of the day. RICTOR and mSIN1 are unique and essential components of mTORC2, which is activated by growth factors including insulin. To assess whether mTORC2 components display diurnal variations, we analyzed steady state mRNA expression levels of Rictor , mSin1 , and mTor in various adipose tissues during a 24 h period. Diurnally regulated expression of Rictor was detected in brown adipose tissues displaying highest mRNA expression levels at the beginning of the 12 h light period (zeitgeber time 2, ZT2). Gene expression patterns of mSin1 and mTor displayed a similar diurnal regulation as Rictor in PVAT while smaller changes were detected for these genes in aorta during the course of the day. Basal mTORC2 activity was measured by phosphorylation of protein kinase C (PKC) α at serine 657 was higher at ZT14 as compared with ZT2 in PVAT. In line, gene expression of inflammatory molecules nitric oxide synthase 2 and tumor necrosis factor α was lower at ZT 14 compared to ZT2. Our findings provide evidence for a diurnal regulation of expression of mTORC2 components and activity. Hence, mTORC2 is possibly an integral part of diurnally regulated signaling pathways in PVAT and possibly in other adipose tissues. [ABSTRACT FROM AUTHOR]

Obesity involves hypoxic adipose tissue and low-grade chronic inflammation. We investigated the impact of hypoxia on inflammatory response to TNF-α in white and brown adipocytes. In response to TNF-α, the expression of the inducible enzymes iNOS and COX-2 was prominently and selectively potentiated during hypoxia while only moderately under normoxia. Levels of their products, nitrite and prostaglandinE 2 were elevated accordingly. NS398, a selective COX-2 inhibitor, reduced nitrite levels. The expression of PGC-1α, a transcriptional co-activator involved in mitochondrial biogenesis, and PPARγ, a transcription factor involved in adipocyte homeostasis, was reduced by TNF-α during hypoxia. These results suggest that hypoxia potentiates the inflammatory response by TNF-α in both white and brown adipocytes and downregulates the transcription factors involved in adipocyte function. [ABSTRACT FROM AUTHOR]

Abstract: Activation of mitogen-activated protein kinases (MAPKs) is important for vascular contraction. Decreased nitric oxide availability combined with activation of MAPKs contributes to an increase in vascular tone. In this study, we have determined the involvement of extracellular signal-regulated kinases1/2 (ERK1/2) and c-Jun N-terminal kinases (JNKs) in reactivity of mouse aortae in the absence of nitric oxide. Additionally, we have examined the contribution of these kinases to endothelium-dependent and prostaglandin F2α (PGF2α)-induced contractions. Precontracted aortic rings were treated with MAPK/ERK kinase1/2 (MEK1/2) inhibitor U0126 or JNKs inhibitor SP600125 to determine reactivity after inhibition of nitric oxide synthase using organ bath chambers. Additionally, rings were pretreated with or without these inhibitors to assess PGF2α- and acetylcholine-induced, endothelium-dependent contractions. Specificity of the inhibitors was evaluated in each aortic ring by determining the phosphorylation levels of ERK1/2 and c-Jun using Bio-Plex™ phospho-protein detection kit. In the absence of nitric oxide both inhibitors caused relaxation, and the dilator response was increased by 2.5-fold using SP600125 in comparison with U0126. Transient endothelium-dependent contractions were blocked by U0126, whereas SP600125 strongly attenuated sustained PGF2α-induced contractions. U0126 inhibited only phosphorylation of ERK1/2, while SP600125 at higher concentrations not only inhibited phosphorylation of c-Jun but also ERK1/2 phosphorylation. In conclusion, the present study demonstrates that in aortae inhibition of activated ERK1/2 and JNKs mediates vascular relaxation, even in the absence of nitric oxide. Activation of ERK1/2 contributes predominantly to transient endothelium-dependent contractions while JNKs, possibly synergistically with ERK1/2, leads to sustained PGF2α-induced contractions. [Copyright &y& Elsevier]

Abstract: Endothelin (ET) and bone morphogenic proteins (BMP) have been implicated in the development of micro- and macrovascular complications of type 2 diabetes mellitus due to atherosclerosis. This study investigated vascular BMP-expression during early development of experimental autoimmune diabetes mellitus and whether ETA receptors are involved in its regulation, using the selective ETA receptor antagonist BSF461314. Specificity of BSF461314 was confirmed through ET-mediated p44/42 mitogen-activated protein kinase (ERK1/2) phosphorylation experiments. For animal studies, non-obese diabetic (NOD) and control mice at 16 weeks of age were treated with BSF461314 for 6 weeks. Plasma glucose levels were measured before and after treatment and vascular gene expression of BMP-2, BMP-7, and BMP-type II receptor was determined in the aorta by quantitative real-time polymerase chain reaction analysis. At the beginning of the study in all animals, plasma glucose levels were within the normal range. After 6 weeks gene expression of vascular BMP-2, BMP-7 and BMP-type II receptor was almost doubled in NOD mice compared with non-diabetic controls (p <0.05). Concomitant treatment with BSF461314 significantly reduced expression of all BMPs and lowered plasma glucose levels in NOD mice close to controls (all p <0.05 versus untreated). In conclusion, vascular BMP-2, BMP-7, and BMP-type II receptor expression is upregulated in early stages of autoimmune diabetes mellitus. The data further indicate that ETA receptors inhibit diabetes-associated activation of vascular BMPs and regulate plasma glucose levels suggesting that ETA receptors might provide a new therapeutic target to interfere with the early development of atherosclerosis in patients with type 1 diabetes mellitus. [Copyright &y& Elsevier]

Abstract: This study investigated the role of endothelin-1 for hyperglycemia, vascular, and pancreatic injury in early type I diabetes in non-obese-diabetic (NOD) mice. Endothelium dependent relaxation to acetylcholine and vascular gene expression of endothelin converting enzyme (ECE) isoforms 1 and 2 were studied as indicators of vascular injury. Endothelial NO bioactivity in the aorta was reduced in diabetic NOD mice while vascular expression of ECE-1 and ECE-2 mRNA was increased compared with controls (all p &#60;0.05). Vascular histology was normal in all animals. Unexpectedly, treatment of prediabetic NOD mice for 6 weeks with the orally active ETA receptor antagonist BSF461314 prevented onset of diabetes without affecting insulitis severity. ETA receptor blockade also restored abnormal endothelial NO bioactivity and reduced ECE-1 and ECE-2 gene expression in NOD mice to levels comparable with healthy controls (p &#60;0.05). Moreover, secretion of endothelin-1 in a time-dependent fashion was observed by pancreatic islet β-cells cultured in vitro. These data suggest a critical role for ETA receptor signaling in the development of autoimmune forms of diabetes and the early vascular injury associated with it. [Copyright &y& Elsevier]

The lymphotoxin-β receptor (LTβR) plays critical roles in inflammation and lymphoid organogenesis through activation of NF-κB. In addition to activation of the classical NF-κB, ligation of this receptor induces the processing of the cytosolic NF-κB2/p100 precursor to yield the mature p52 subunit, followed by translocation of p52 to the nucleus. This activation of NF-κB2 requires NIK and IKKα, while NEMO/IKKγ is dispensable for p100 processing. IKKβ-dependent activation of canonical NF-κB is required for the expression but not processing of p100 and for the expression of proinflammatory molecules including VCAM-1, MIP-1β, and MIP-2 in response to LTβR ligation. In contrast, IKKα controls the induction by LTβR ligation of chemokines and cytokines involved in lymphoid organogenesis, including SLC, BLC, ELC, SDF1, and BAFF. [Copyright &y& Elsevier]

Protein kinase Cμ (PKCμ) represents a new subtype of the PKC family characterized by the presence of a pleckstrin homology (PH) domain and an amino-terminal hydrophobic region. In order to analyse the potential role of PKCμ in signal-transduction pathways, stable PKCμ transfectants were established with human and murine cell lines. All transfectants showed a reduced sensitivity to tumor-necrosis-factor (TNF)-induced apoptosis, which correlated with the amount of transgene expressed and with an enhanced basal transcription rate of NF-κB-driven genes including the inhibitor of apoptosis protein 2 (cIAP2) and TNF-receptor-associated protein 1 (TRAF1). Sensitivity to apoptosis induced by the lipid mediator ceramide was unchanged in PKCμ transfectants. In support of a PKCμ action on NF-κB, we show enhancement and downregulation of TNF-induced expression of a NF-κB-dependent reporter gene by transient overexpression of wild-type and kinase-negative mutants of PKCμ, respectively. Interestingly, no significant changes were found in an electrophoretic mobility shift assay, indicative of PKCμ action downstream of IκB degradation, probably by modulation of the transactivation capacity of NF-κB. The dominant negative action of the kinase-negative mutant further suggest a regulatory role of PKCμ for NF-κB-dependent gene expression. [ABSTRACT FROM AUTHOR]