Basal mTORC2 activity and expression of its components display diurnal variation in mouse perivascular adipose tissue.

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.

Hypoxia potentiates tumor necrosis factor-α induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in white and brown adipocytes.

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 prostaglandinE2 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.

Rictor in perivascular adipose tissue controls vascular function by regulating inflammatory molecule expression.

OBJECTIVE: Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. APPROACH AND RESULTS: Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictor(ad-/-)). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictor(ad-/-) mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictor(ad-/-) mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1α, and tumor necrosis factor-α in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictor(ad-/-) mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictor(ad-/-) mice with no effect in rictor(fl/fl) mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. CONCLUSIONS: Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Loss of pim1 imposes a hyperadhesive phenotype on endothelial cells.

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 ß-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.

Regulatory role of G protein-coupled estrogen receptor for vascular function and obesity.

We found that the selective stimulation of the intracellular, transmembrane G protein-coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Need for research on estrogen receptor function: importance for postmenopausal hormone therapy and atherosclerosis.

BACKGROUND: Cardiovascular disease is the leading cause of morbidity and mortality in men and women worldwide. Although rare in premenopausal women, its incidence rises sharply after menopause, indicating atheroprotective effects of endogenous estrogens. OBJECTIVE: This review discusses the differential effects of estrogen receptor function on atherosclerosis progression in pre- and postmenopausal women, including aspects of gender differences in vascular physiology of estrogens and androgens. METHODS: Recent advances in the understanding of the pathogenesis of atherosclerosis, estrogen receptor function, and hormone therapy are reviewed, with particular emphasis on clinical and molecular issues. RESULTS: Whether hormone therapy can improve cardiovascular health in postmenopausal women remains controversial. Current evidence suggests that the vascular effects of estrogen are affected by the stage of reproductive life, the time since menopause, and the extent of subclinical atherosclerosis. The mechanisms of vascular responsiveness to sex steroids during different stages of atherosclerosis development remain poorly understood in women and men. CONCLUSION: In view of the expected increase in the prevalence of atherosclerotic vascular disease worldwide due to population aging, research is needed to determine the vascular mechanism of endogenous and exogenous sex steroids in patients with atherosclerosis. Such research may help to define new strategies to improve cardiovascular health in women and possibly also in men.

Fat intake modifies vascular responsiveness and receptor expression of vasoconstrictors: implications for diet-induced obesity.

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 microM). 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 microM), in the absence or presence of L-NAME, and to exogenous ROS/.OH were also 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/.OH resulted 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 AT(1B) receptor was increased in the aorta of VHF mice, and aortic ET(A) 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.

A WD-FYVE protein binds to the kinases Akt and PKCzeta/lambda.

WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.

Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus.

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 ET(A) receptors are involved in its regulation, using the selective ET(A) 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 ET(A) receptors inhibit diabetes-associated activation of vascular BMPs and regulate plasma glucose levels suggesting that ET(A) receptors might provide a new therapeutic target to interfere with the early development of atherosclerosis in patients with type 1 diabetes mellitus.

Deletion of Rictor in brain and fat alters peripheral clock gene expression and increases blood pressure.

The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity.

Endothelial Rictor is crucial for midgestational development and sustained and extensive FGF2-induced neovascularization in the adult.

To explore the general requirement of endothelial mTORC2 during embryonic and adolescent development, we knocked out the essential mTORC2 component Rictor in the mouse endothelium in the embryo, during adolescence and in endothelial cells in vitro. During embryonic development, Rictor knockout resulted in growth retardation and lethality around embryonic day 12. We detected reduced peripheral vascularization and delayed ossification of developing fingers, toes and vertebrae during this confined midgestational period. Rictor knockout did not affect viability, weight gain, and vascular development during further adolescence. However during this period, Rictor knockout prevented skin capillaries to gain larger and heterogeneously sized diameters and remodeling into tortuous vessels in response to FGF2. Rictor knockout strongly reduced extensive FGF2-induced neovascularization and prevented hemorrhage in FGF2-loaded matrigel plugs. Rictor knockout also disabled the formation of capillary-like networks by FGF2-stimulated mouse aortic endothelial cells in vitro. Low RICTOR expression was detected in quiescent, confluent mouse aortic endothelial cells, whereas high doses of FGF2 induced high RICTOR expression that was associated with strong mTORC2-specific protein kinase Cα and AKT phosphorylation. We demonstrate that the endothelial FGF-RICTOR axis is not required during endothelial quiescence, but crucial for midgestational development and sustained and extensive neovascularization in the adult.

Inhibition of activated ERK1/2 and JNKs improves vascular function in mouse aortae in the absence of nitric oxide.

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 F(2α) (PGF(2α))-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 PGF(2α)- 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 PGF(2α)-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 PGF(2α)-induced contractions.

Non-genomic regulation of vascular cell function and growth by estrogen.

Estrogens exert rapid, non-genomic effects, which are mediated by plasma membrane-associated estrogen receptors (mER) mERalpha and mERbeta, 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.

Distinct roles of estrogen receptors alpha and beta mediating acute vasodilation of epicardial coronary arteries.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy.

Endothelin stimulates vascular hydroxyl radical formation: effect of obesity.

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 alpha-tocopherol and ascorbic acid. Generation of either *OH, *O(2)(-), and H(2)O(2) 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.

Differential effects of 17beta-estradiol on function and expression of estrogen receptor alpha, estrogen receptor beta, and GPR30 in arteries and veins of patients with atherosclerosis.

Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17beta-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17Beta-estradiol caused concentration-dependent, endothelium-independent relaxation in arteries (P<0.05 versus solvent control) but not in veins (P not significant). 17Beta-estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor beta was 10-fold higher than that of estrogen receptor alpha or GPR30 (P<0.05). Expression of all 3 of the receptors was reduced after exposure to 17beta-estradiol in arteries but not in veins (P<0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17beta-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17beta-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy.

Nickel represses the synthesis of the nickel permease NixA of Helicobacter pylori.

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 microM. 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.