tbuckley/cabot-search huggingface dataset:
Abstract 5645: Novel Cross-Talk Between Oxidized LDL, Angiotensin II and Oxidative Stress After AT1 Blockade and AT1a/AT1b Double Knockout
Authors: Henning Morawietz, Rusan A Catar, Claudia Goettsch, Ashraf Taye, Gregor Muller, Susann Lehmann, Christian G Ziegler, Stefan R Bornstein, Alexander W Krug, Thomas Walther
Journal: Circulation (2009)

Oxidized low-density-lipoprotein (oxLDL) and angiotensin II (Ang II) can increase formation of reactive oxygen species in endothelial cells. However, the potential cross-talk between both risk factors, the impact of Ang II receptor 1 (AT 1 ) blockade on lipoprotein-induced oxidative stress and endothelial function and of AT 1a /AT 1b double knockout on oxidative stress is not well understood. First, we studied the impact of native and oxidized LDL (nLDL, oxLDL) on Ang II receptor expression and formation of reactive oxygen species in primary cultures of human umbilical artery endothelial cells (HUAEC). Native and oxLDL (max. 100 μ g/mL) induced after 1 h AT 1 (3.3- and 3.7-fold) and AT 2 (1.8- and 2.8-fold) receptor mRNA expression in HUAEC. Increased AT 1 and AT 2 mRNA and protein expression could be observed for up to 24 h. Both lipoproteins increased intracellular AT 1 receptor immunofluorescence in HUAEC. Native LDL and oxLDL activated MEK/ERK and p38 MAPK pathways (ELISA, Western blot) in HUAEC. OxLDL induced oxLDL receptor LOX-1 expression in HUAEC (RT-PCR, Western blot). Induction of both Ang II receptors and of LOX-1 by oxLDL was reduced by AT 1 receptor antagonist candesartan. OxLDL induced in contrast to native LDL superoxide anion formation in HUAEC (chemiluminescence). AT 1 receptor blockade prevented lipoprotein-induced oxidative stress in HUAEC like SOD. Furthermore, AT 1 blockade prevented impaired endothelial function (increase of log EC 50 values and impaired max. relaxation by oxLDL in phenylephrine-preconstricted vessels). In aortic rings of wild-type mice, oxLDL-induced vascular superoxide anion formation was reduced by AT 1 blockade. Finally, deletion of AT 1 receptor subtypes 1a and 1b in AT 1a /AT 1b double knockout mice resulted in the aorta in significant downregulation of protein expression of LOX-1, Nox subunits Nox2, p47phox and p22phox, compared with wild-type mice. In conclusion, augmented vascular oxidative stress and endothelial dysfunction in response to lipoproteins involves induction of the AT 1 receptor. Double knockout of AT 1 receptors reduce LOX-1 and NAD(P)H oxidase expression. Our data suggest a cross-talk between oxidized LDL, angiotensin II and oxidative stress after AT 1 blockade and AT 1a /AT 1b double knockout.
Abstract 5648: Epigenetic Basis of Nitrate Tolerance: Fundamental Role of Protein Acetylation
Authors: Carlo Gaetano, Female Claudia Colussi, Male Alessandro Scopece, Stefania Mattiussi, Serena Vitale, Jessica Rosati, Barbara Illi, Maurizio C Capogrossi
Journal: Circulation (2009)

INTRODUCTION : We recently demonstrated that nitric oxide (NO) plays a key role regulating epigenetic enzymes, including histone acetylases (HAT) and deacetylases (HDAC). In this context, we hypothesized that during nitrate tolerance the HDAC/HAT balance could be altered and that drugs capable of modulate HDAC and/or HAT function could have an impact on this alteration. METHODS &amp; RESULTS : Adult male rats (n=6, each group) were treated for 72 hours with subcutaneous nitroglycerin (NTG) alone (50 mg/kg) or in combination with different HDAC inhibitors (DI) including trichostatin A (TSA, 0.6 mg/kg), suberoylanilide hydroxamic acid (SAHA, 0.6 mg/kg), MS-27–275 (0.6 mg/kg) and valproic acid (VPA, 5 mg/kg). Other animals received the HAT inhibitor anacardic acid (ANAC, 2.5 mg/kg) or equivalent amounts of solvent. Ex vivo experiments performed on phenylephrine-contracted aortic rings (AR) showed that the induction of tolerance (-logEC=5.9±0.2 in tolerant vs -logEC=7.6±0.1 in control) was reversed by TSA, SAHA (-logEC=7.6±0.1) and VPA (-logEC=7.4±0.2). ANAC treatment displayed value similar to nitrotolerant (-logEC=6.2±0.1). Moreover, our data revealed that in AR from controls animals a single NTG administration induced relaxation paralleled by a rapid increase in HAT activity. Nitrate tolerant rings, however, exhibited a higher HDAC activity and failed to up-regulate HATs’ function. Remarkably, in AR from animals treated with DI in combination with NTG, nitrate tolerance was prevented while animals treated with ANAC developed resistance to both arterial relaxing or contracting stimuli suggesting that the intracellular level of HAT activity is important for arterial adaptive responses. Mechanistically, in isolated smooth muscle cells the NTG increased myosin light chain (MLC) and smooth muscle actin (SMA) acetylation paralleled by a decreased MLC phosphorylation and contractile protein association. These effects were rescued to normal by DI treatment. CONCLUSIONS : an alteration in HAT/HDAC functional balance occurs during nitrate tolerance in which the content of acetylated contractile proteins is markedly reduce. DIs, preventing this effect, may be envisaged as novel tools for the treatment of nitrate tolerance.
Abstract 5687: Identification of HGF as a Novel Vascular ADAM Metalloprotease Substrate by Phage Display
Authors: Sadaharu Higuchi, Keita Kimura, Akira Takaguri, Akinari Hinoki, Kunie Eguchi, Gerald D Frank, Satoru Eguchi
Journal: Circulation (2009)

Activation of a metalloprotease, ADAM17, and subsequent EGF receptor transactivation via proteolytic cleavage of its pro-ligand has been implicated in vascular remodeling induced by angiotensin II (AngII). The known substrates of ADAM17 include proHB-EGF and proTNF-alpha. However, whether other unidentified ADAM17 substrates are involved in AngII-induced vascular remodeling remains unanswered. To search for such substrates, we made a rat aortic cDNA phage library that has a hexa-histidine tag. After Ni-NTA agarose binding, the library phage was incubated with recombinant ADAM17 and only the cleaved phages were rescued and amplified (bio-panning). Upon repeating the panning 3 times, we obtained about 4000 phage colonies which potentially displayed ADAM17 substrates. From 128 colonies, we identified 71 candidate proteins in 88 readable sequences. Among these proteins, eleven proteins were known as ADAM17 substrates including (amphiregulin, Notch etc.) while six proteins were other protease substrates indicating strong reliability and provability of the technique. Among the novel substrate candidates, we focused on hepatocyte growth factor (HGF), a multifunctional growth factor with a proteolytic mechanism of activation. Upon transfection with a shedding assay vector encoding proHGF with its N terminus fused with ALP, the ALP activity in the condition medium was significantly reduced in ADAM17-null MEF cells compared to the control MEF cells. Moreover, the reduction was rescued with adenovirus vector encoding ADAM17. In rat VSMCs transfected with the assay vector, AngII markedly enhanced the ALP activity in the conditioned medium that was completely blocked with a MMP/ADAM inhibitor, BiPS. Also, AngII stimulated secretion of HGF-alpha in VSMCs. Recombinant HGF markedly stimulated ERK1/2 activity via the Met receptor in VSMCs. It also stimulated proliferation of VSMCs. In conclusion, we have established a novel phage display system for identification of a protease substrate with high probability. HGF was identified as a novel ADAM17 substrate potentially involved in vascular remodeling induced by AngII. This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5636: Time From Onset of Acute Anterior Myocardial Infarction Negatively Influences Bone Marrow Cell Function: Preliminary Data From the NHLBI CCTRN Ancillary Studies
Authors: Christopher R Cogle, Rodney Pettway, Claudia Zierold, Amy Meacham, Gerard J Madlambayan, Marjorie A Carlson, Udo Obodo, John Petersen, Sarah Baraniuk, Lemuel A Moye’, Jay Traverse, Stephen Ellis, Doris A Taylor, Carl J Pepine
Journal: Circulation (2009)

Bone marrow (BM) contributes to tissue repair in a time dependent fashion. Impaired BM may lead to decreased response to acute myocardial infarction (AMI) but this hypothesis has been examined only from measurements of peripheral blood (PB) cells among heterogeneous patients. Methods: We investigated the association between BM function and time after AMI in a group of patients (mean age 62 years) with recent, initial anterior STEMI, treated by PCI and drug-eluting stenting plus guideline recommended care who had post reperfusion EF ≤ 45% enrolled in CCTRN TIME and Late-TIME trials. BM and PB harvesting were performed at either ≤ 7 days or 14 –21 days after AMI and also from healthy donors. BM and PB endothelial progenitor cells (EPCs) were analyzed by FACS for cell surface phenotyping and by endothelial colony forming cell (ECFC) proliferation. Results: Healthy BM generated proliferative ECFC colonies. In contrast, BM from ≤7 day AMI patients showed a trend towards decreased ECFC proliferation and from 14 –21 day AMI patients generated less ECFC colonies ( P &lt;0.05) with decreased growth rates ( P &lt;0.05). To relate results to EPC mobilization, we quantified EPCs in BM and PB and found no differences in number of EPCs in BM among all groups. However, in 14 –21 day AMI subjects there was a decrease in PB circulating EPCs ( P &lt;0.05). Decrease in PB EPCs post-AMI positively correlated with decrease in BM-derived ECFC proliferation (r=0.5515, P =0.0268). Conclusions: These data are consistent with a time-dependent decrease in EPC proliferative potential after initial anterior reperfused STEMI, suggesting AMI events may preface impaired BM-derived vascular progenitor activity. Another possible explanation for the differences seen is subject differences that guided recruitment into the two studies. These results support the rationale of concentrating angiogenic cells for direct application to sites of myocardial injury.
Abstract 5681: GPR30 is Required for the Pro-apoptotic Effects of Estrogen in Vascular Smooth Muscle Cells
Authors: Robert Gros, Qingming Ding, Jozef Chorazcyzewski, Ross D Feldman
Journal: Circulation (2009)

Rapid and non-genomic effects of estradiol (EST) may be mediated through the G-Protein-coupled receptor dubbed GPR30 receptor. The present studies tested the hypothesis that GPR30 expression, which we demonstrate is readily detectable in freshly isolated vascular tissue but is decreasingly detectable in cultured VSMCs, mediates the effects of EST to alter ERK phosphorylation status and apoptosis. In freshly isolated aortic tissue, EST-stimulated ERK phosphorylation whereas in cultured VSMCs, EST inhibits ERK phosphorylation in a dose-dependent manner. However, gene transfer of GPR30 resulted in EST-mediated stimulation of ERK phosphorylation (138±4%, n=10), which is in contrast to the effects of EST in GFP-expressing primary culture of VSMCs (68±3%, n=14). EST-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin-sensitive and PI3 kinase-dependent; under these conditions, EST also inhibited PKA activation. In contrast, in the absence of GPR30 expression in cultured VSMC, EST stimulated PKA activity and inhibited ERK phosphorylation. We also assessed the impact of GPR30 expression on EST-mediated regulation of VSMCs apoptosis. In cultured VSMC transduced with GFP vector (control conditions), EST (10 nM) mediated a decrease in VSMC apoptosis (80±2%, n=7), an effect that was marginally but significantly enhanced by gene transfer of ER α (71±3%, n=7). The anti-apoptotic effect of EST was attenuated by co-incubation with the PKA inhibitor Rp-cAMP. However, PI3 kinase inhibition did not affect estradiol’s anti-apoptotic actions. In contrast, gene transfer of GPR30 into the VSMC resulted in enhancement of apoptotic rates following EST treatment (169±2%, n=7), and this GPR30-mediated pro-apoptotic effect of EST was attenuated by the PI3 kinase inhibitor, but not by Rp-cAMP. Thus, the effect of EST on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression and/or activity may be an important determinant of the effects of estradiol in the vasculature.
Abstract 5676: Free Fatty Acids Induce Dynamic Inflammatory Processes in Pancreatic Islets That Lead to <i>β</i> Cell Dysfunction
Authors: Kosei Eguchi, Ichiro Manabe, Hua Shen, Ryozo Nagai
Journal: Circulation (2009)

Metabolic syndrome is a major risk factor of atherosclerotic cardiovascular disease and previous studies have suggested the pivotal involvement of inflammation in its development. Serum levels of free fatty acids are increased in obese subjects and have been implicated in pathogenesis of both cardiovascular and metabolic diseases. We found that palmitate, the major free fatty acid in blood, when administered to mice markedly exacerbated neointima formation. Interestingly, a continuous infusion of palmitate induced β cell dysfunction. Because neointima formation is an inflammatory process, we addressed if inflammation plays a role in the palmitate-induced islet pathology. In the islets of mice infused with palmitate, the reduction of mRNA of β cell functional markers was accompanied by induction of chemokines including MCP-1 and MIP-1 α , and inflammatory cytokines including IL-1 β and TNF- α . Flow cytometric analysis showed that palmitate infusion induced rapid accumulation of CD11b hi F4/80 + Ly-6C + M1 macrophages in the islets. When macrophages were depleted in mice, the macrophage accumulation was suppressed and the induction of cytokines and chemokines, and β cell dysfunction induced by palmitate was ameliorated, indicating the causative involvement of macrophages in β cell dysfunction. We further analyzed the signaling mechanisms that controls response to palmitate and found that Toll-like receptor 4 (TLR4) signaling mediates chemokine production in β cells. As expected, palmitate-induced M1 macrophage recruitment and β cell dysfunction were markedly suppressed in TLR4 knockout (KO) and Myd88 KO mice. Finally, to confirm the relevance of these phenomena, the islets of db/db and db/+ mice were analyzed. In the islets of db/db mice, β cell dysfunction was accompanied by induction of cytokines and chemokines, and accumulation of CD11b hi F4/80 + Ly-6C + M1 macrophages were observed. Taken together, results of the present study demonstrate that palmitate induces inflammatory processes in pancreatic islet by recruiting Ly-6C + inflammatory monocytes/macrophages, which leads to β cell dysfunction. In conclusion, chronic inflammation induced by free fatty acids appears to play important roles in both cardiovascular and metabolic diseases.
Abstract 5649: Human Alanine: Glyoxylate Aminotransferase 2 Lowers ADMA and Protects From ADMA-induced Impairment in Nitric Oxide Production
Authors: Roman N Rodionov, Daryl J Murry, Steven R Lentz
Journal: Circulation (2009)

Elevated blood concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are found in association with diabetes, hypertension, congestive heart failure and atherosclerosis. ADMA levels are controlled by dimethylarginine dimethyl-aminohydrolases, cytosolic enzymes which hydrolyze ADMA to citrulline and dimethylamine. ADMA also has been proposed to be regulated through an alternative pathway by alanine-glyoxylate aminotransferase 2 (AGXT2), an aminotransferase expressed primarily in the kidney. Little is known about the intracellular localization or activity of human AGXT2. The goal of this study was to define the subcellular localization of human AGXT2 and test the hypothesis that overexpression of human AGXT2 protects from ADMA-induced inhibition in nitric oxide (NO) production. AGXT2 was cloned from a human kidney cDNA library and overexpressed with a C-terminal FLAG epitope tag in COS-7 cells. Mitochondrial localization of human AGXT2 was demonstrated by confocal microscopy. A 41 amino acid N-terminal mitochondrial cleavage sequence was delineated by N-terminal sequencing of the mature protein. To determine the effect of AGXT2 on NO metabolism, human AGXT2 was overexpressed in MS-1 murine endothelial cells. Overexpression of AGXT2 protected MS-1 cells from ADMA-mediated inhibition of NO production. To determine the effect of AGXT2 on ADMA metabolism in vivo , human AGXT2 was overexpressed in the liver of C57BL/6 mice using an adenoviral expression vector. Four days after intravenous injection of the AGXT2 or control adenoviral vectors, mice receiving the AGXT2 vector had significantly lower levels of plasma and tissue ADMA compared with mice receiving the control vector (P&lt;0.05). We conclude that mitochondrially localized human AGXT2 lowers ADMA and protects from ADMA-induced impairment in NO production.
Abstract 5684: A Sex-Specific Role for Androgens in Angiogenesis
Authors: Daniel P Sieveking, Patrick Lim, Renée W Chow, Louise L Dunn, Shisan Bao, Kristine C McGrath, Alison K Heather, David J Handelsman, David S Celermajer, Martin K Ng
Journal: Circulation (2009)

The role of androgens in cardiovascular disease remains controversial. Although men tend to develop CVD earlier than women, consistent with increased atherosclerosis, repair/adaptation mechanisms may be enhanced in males. For example, male gender is associated with increased collateralization in patients with severe coronary artery lesions. As angiogenesis, is critical to cardiovascular repair/regeneration, we hypothesized that androgens may modulate this process. We studied the effects of androgens on angiogenesis using human endothelial cells (ECs) and two distinct in vivo models. Dihydrotestosterone (DHT), a potent natural androgen specific for the androgen receptor (AR) augmented key angiogenic events in vitro , including EC migration, proliferation and tubulogenesis. Strikingly, this occurred in male but not female ECs. AR antagonism and siRNA-mediated knockdown abrogated all DHT effects in male ECs, while AR overexpression conferred androgen sensitivity in female ECs. In vivo , castration dramatically reduced vascularization of matrigel plugs in male and female mice. This reduction was fully reversed with DHT treatment in male but not female mice (Fig. 1 ). Additionally, male castration impaired neovascularization and blood flow recovery in hindlimb ischemia, a finding that was rescued with DHT treatment. These findings were also mirrored in hypoxia inducible factor-1 α and stromal cell-derived factor-1 α expression, and the mobilzation of various angiogenic/progenitor cells. Our findings suggest endogenous androgens play an important role in angiogenesis in males but not females, with implications for the role of androgen replacement in men. Fig. 1
Abstract 5647: Impact of Growth Factors, Shear Stress and Transcription Factor COUP-TFII on the Notch Pathway in Human Endothelial Cells
Authors: Henning Morawietz, Coy Brunssen, Corina Gryczka, Slobodanka Korten, Winfried Goettsch
Journal: Circulation (2009)

Arterial and venous endothelial cells can differ in vascular function thus leading to vessel-specific diseases. The molecular mechanisms for differentiation and maintenance of the phenotype of arterial and venous endothelial cells are not well understood. Using primary cultures of human umbilical artery (HUAEC) or vein (HUVEC) endothelial cells we identified by DNA microarrays Hey2 as arterial and COUP-TFII as venous marker gene. Differential expression of these genes and preferential arterial expression of further genes of the Notch signalling pathway (Dll4, Notch4, Hey1, Hey2) were confirmed by RT- and real-time PCR in independent HUAEC and HUVEC preparations. Differential protein expression of Hey2 and COUP-TFII was confirmed as well. We further showed exclusive expression of Hey2 and COUP-TFII in HUAEC and HUVEC over several passages (up to passage 12). Next, we analyzed the impact of growth factors on the preferential arterial genes of the Notch pathway. In medium supplemented with different growth factors (IGF-1, hEGF-1, PDGF-BB, bFGF-2 or VEGF-A) only VEGF-A was able to induce expression of Dll4, Notch4 and Hey1 in venous endothelial cells. A further increase of the high arterial Dll-4 and Hey1 expression was achieved by VEGF-A incubation in HUAEC. This increased expression of Dll-4 and Hey-1 was prevented by VEGF receptor-2 (KDR) inhibitor SU5416. Inhibition of the Notch pathway by gamma-secretase inhibitor X (GSI-X) reduced Dll-4 and Hey-1 expression in venous and arterial endothelial cells without effecting Hey2 expression. In order to reduce arterial Hey2 expression we next applied shear stress (1 dyn/cm 2 or 30 dyn/cm 2 ) to arterial endothelial cells. The main arterial transcription factor Hey2 was not changed. Next, we cloned full-length and terminal deletions of the human Hey2 promoter (−2132/+194; −1080/+194; −418/+194; −54/+194 bp). All fragments showed high basal activity (82-, 84-, 293-, 25-fold). Finally, double transfection with a COUP-TFII overexpression plasmid reduced Hey2 promoter activity of the −2132/+194 fragment by 50%. In conclusion, our data suggest an interaction of the venous marker COUP-TFII and the arterial marker Hey2 at the Hey2 promoter.
Abstract 5682: Novel Role of Copper Transporters CTR1 and ATP7A in PDGF-induced Vascular Smooth Muscle Cell Migration and Vascular Remodeling
Authors: Takashi Ashino, Norifumi Urao, Jin Oshikawa, Huan Wang, Yuqing Huo, Ronal D McKinney, Masuko Ushio-Fukai, Tohru Fukai
Journal: Circulation (2009)

Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration primary through the PDGF receptor-beta (PDGFR), which contributes to vascular remodeling in vivo. Previous study shows that copper chelation inhibits neointimal formation in response to injury. However, underlying mechanisms remain unknown. Intracellular copper levels are tightly regulated by the copper importer CTR1 which localizes at plasma membrane (PM) and the copper exporter ATP7A which traffics between trans-Golgi network (TGN) to PM. Here we show that both CTR1 and ATP7A are expressed in cultured VSMC. Co-immunoprecipitation assay shows that ATP7A binds to PDGFR basally and PDGF stimulation rapidly promotes their association within 5 min, which is prevented by knockdown of CTR1 with siRNA (85%). Proteomic analysis in caveolae/lipid raft (C/LR) fraction reveals that ATP7A is localized in C/LR where PDGFR is found. PDGF stimulation promotes recruitment of ATP7A to C/LR fraction, which is inhibited by CTR1 siRNA (95%). Furthermore, inductively coupled plasma mass spectrometry (ICP-MS) shows that C/LR contains high amount of copper (139.7 (C/LR) vs. 17.9 (non-C/LR) ppb/mg proteins). In response to wound injury or PDGF stimulation, ATP7A translocates from TGN to PM at the leading edge where it colocalizes with PDGFR and the LR marker cholera toxin subunit B in actively migrating VSMCs, which is prevented by CTR1 siRNA or C/LR disruptor methyl-beta cyclodextrin. Note that this ATP7A translocation is associated with decrease in copper levels in C/LR, suggesting increase in copper efflux in C/LR during active VSMC migration. Functionally, PDGF-induced VSMC migration is significantly inhibited by knockdown of CTR1 or ATP7A with siRNAs (84% and 68%, respectively) as well as treatment with cell permeable copper chelator TTM (50.2%) and cell impermeable copper chelator BCS (39.1%). Moreover, ATP7A expression is dramatically increased in the neointima in a mouse wire injury model. In summary, CTR1-dependent translocation of the copper exporter ATP7A to C/LR and its binding to PDGFR plays an important role in PDGF-induced VSMC migration, which may contributes to vascular remodeling in response to injury and development of atherosclerosis. This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota &amp; Wisconsin).