tbuckley/cabot-search huggingface dataset:
Abstract 5277: Central Role of Early Endosomal Antigen 1 (EEA1) in Organization of Angiotensin II Signaling Leading to Akt Activation in Early Endosome
Authors: Rafal R Nazarewicz, Gloria Salazar, Nikolay Patrushev, Lula Hilenski, Alejandra S Martin, Shiqin Xiong, Masuko Ushio-Fukai, R Wayne Alexander
Journal: Circulation (2009)

Akt activation is a major output of Angiotensin II (AngII) signaling. Abundant evidence indicates that AngII signaling occurs in multiple signaling compartments. The mechanisms organizing post-membrane events are incompletely understood. Akt activation requires EGFR transacti-vation at the cell membrane and also requires the endocytotic machinery. We hypothesized that Akt activation appears within the endosomal compartment. In vascular smooth muscle cells, inhibition of endocytosis by phenylarsine oxide (10 μ M), concanavalin A (0.3 mg/ml) or overexpression of dynamin K44A mutant, reduce by 90% AngII-induced Akt phosphorylation at Thr308 and Ser473. Cell fractionation shows that Akt phosphorylation occurs mainly in early endosomal fractions after 3 min of AngII (0.1 μ M). We posited that the early endosomal marker and scaffolding protein, early endosome antigen 1 (EEA1), is crucial for organizing early endosomal AngII-Akt signaling. Cell fractionation, immunostaining and co-immunoprecipitation studies show that AngII recruits EEA1, AT1R, EGFR, PDK1, PAK1, PKC-alpha and Akt into early endosomes to form a signaling complex. We demonstrate that PAK1 and PKC-alpha are essential components of the EEA1-binding complex required for Akt activation. siRNA knockdown of EEA1 decreases AngII-induced ( 0.1 μ M, 3 and 5 min) PDK1, PKC-alpha and Akt phosphorylation by 90%. AngII-induced (0.1 μ M, 5 min) EGFR transactivation or EGFR degradation after 30 and 60 min of EGF stimulation (50 ng/ml) was unaffected. Further, downregulation of EEA1 prevents Akt activation by mislocation of critical components of the signaling complex. Taken together, we demonstrate a novel function for EEA1 itself as a central organizer of AngII-Akt signaling in early endosomes.
Abstract 5335: Significant Decline in the Age Adjusted Mortality From Acute St Segment Elevation Myocardial Infarction in the United States Regardless of Race or Gender With Persistent Higher Mortality Rate for Female and African American Population
Authors: Mohammad Reza Movahed, Jooby John, M Mazen Jamal, Mehrnoosh Hashemzadeh, Mehrtash Hashemzadeh
Journal: Circulation (2009)

Back ground: Treatment of acute ST segment myocardial infarction (STEMI) has improved over the last 2 decades. The goal of this study was to determine trends in the mortality of patients with acute STEMI in the United States over the years based on gender and race. Method: The Nationwide Inpatient Sample (NIS) database was used to analyze the age adjusted mortality rates for STEMI from 1988 to 2004 for inpatients above the age of 40. ICD-9-CM codes consistent with acute STEMI were utilized. Results : A total of 163,915 hospital deaths occurred during the study period. From 1988 the age-adjusted mortality rate declined regardless of gender or race. However, women and African Americans had higher rate of acute STEMI related mortality in comparison to men and Caucasians over the years studied (For 1988 age adjusted mortality rate from STEMI for women was 508 per 100,000 vs. 371 per 100,000 in men. For 2004 age adjusted mortality rate from STEMI for women was 347 per 100,000 vs. 262 per 100,000 in men p&lt; 0.001). For 1988, age adjusted mortality rate from STEMI for Caucasians was 412 per 100,000 vs. 521 per 100,000 in African Americans. For 2004, age adjusted mortality rate from STEMI for Caucasians was 277 per 100,000 vs. 447 per 100,000 in African Americans, p&lt; 0.001, see figures ). Conclusion: Age adjusted mortality from acute STEMI has significantly declined over the last decade regardless of gender race with persistent higher mortality rate in women and African Americans over the years.
Abstract 5280: Comparing Epigenetic Modifications of Adult Progenitor Cells
Authors: Jes-Niels Boeckel, Masamichi Koyanagi, Kisho Ohtani, Christine Mummery, Stefan Momma, Andreas M Zeiher, Stefanie Dimmeler
Journal: Circulation (2009)

Cell therapy is a promising option for treatment of cardiovascular diseases. Recent studies demonstrate that pluripotency can be induced in somatic cells by a combination of genes such as Oct4, Klf4, c-Myc and Sox2. To address whether adult progenitor cells might express some of the pluripotency genes and therefore might be more easily reprogrammable, we analyzed expression and accessibility of the promoters of pluripotency genes in adult progenitor cells. Oct4 mRNA was expressed at low level (0.2–0.5% compared to human embryonic stem cells (ESC)) in peripheral blood derived early endothelial progenitor cells (EPC), bone marrow-derived mesenchymal stem cells (MSC) and CD34+ cells, whereas neuronal stem cells (NSC) did not express Oct4. Klf4 mRNA was highly expressed in EPC, MSC and CD34+ cells but not in NSC. Interestingly, Sox2 was selectively expressed in NSC (71,65±13,01%) but was low in EPC, MSC and CD34+ (&lt; 4% of hESC). To investigate whether epigenetic mechanisms account for the differential expression of the pluripotency genes, we analyzed promoter DNA methylation by bisulfite sequencing with 2 different primers. The Oct4 promoter showed high incidence of methylated CpG islands in Region A (EPC:82%; MSC:87%; CD34+ 71% compared to ESC: 5 %). Interestingly, the DNA methylation of the Nanog promoter in Region B was low in EPC (37%), MSC (8%), and ESC (9 %) in contrast to CD34+ cells (97%). At the level of histones, chromatine immunoprecipitation experiments demonstrated that the active marks, acetylated-histone H3 (H3Ac) and trimethylation of H3 lysine 4 (H3K4m3) were increased at the Nanog promoter in EPC and MSC. The Sox2 promoter showed a bivalent modification with active H3K4me3 and repressive H3K27m3 marks in EPC, MSC and CD34+. The Klf4 promoter exhibits high levels of active H3Ac and H3K4m3 but no repressive modification in EPC, MSC and CD34+ cells. These data indicate that adult progenitor cells express variable levels of the pluripotency genes. Modification of the epigenetic histone code, e.g. by histone demethylases, might facilitate the induction of pluripotency by remodeling the accessibility of the promoter regions. This may provide an attractive tool to reprogram adult progenitor cells to generate patient-specific stem cells.
Abstract 5282: RAPL, a New Mediator of Angiogenesis, Promotes Integrin Endocytosis in Endothelial Cells
Authors: Guillaume Carmona, Stefanie Dimmeler, Emmanouil Chavakis
Journal: Circulation (2009)

The molecular mechanisms regulating angiogenesis are not entirely understood. Integrins are critical mediators of angiogenesis. In our previous work, we identified the small GTPase Rap1 as a mediator of integrin affinity in endothelial cells (EC) and angiogenesis. RAPL is an effector protein of Rap1 promoting integrin activation and adhesion in lymphocytes. However, the role of RAPL for integrin regulation in EC and angiogenesis is unclear. Silencing of RAPL with siRNA significantly decreased angiogenic sprouting of HUVEC in a 3-dimensional spheroidal system (83 % inhibition) and blocked VEGF-induced invasion (68 % inhibition) in comparison to scrambled transfected EC. However, while silencing of Rap1 reduced HUVEC adhesion, silencing of RAPL unexpectedly significantly increased adhesion suggesting distinct roles for RAPL and Rap1 in integrin regulation in EC. In line with these results, silencing of RAPL significantly increased surface expression of β 1-integrin as assessed by FACS, while not affecting β 1-integrin mRNA and total protein levels in HUVEC. It is conceivable that the enhanced surface expression of β 1-integrins upon silencing of RAPL is caused by a redistribution of the integrin from an intracellular pool to the cell surface. Therefore, we studied the role of RAPL for the endocytosis (internalization) of β 1-integrins in HUVEC. Strikingly, silencing of RAPL blocked β 1-integrin endocytosis by 43±6 % as determined by confocal microscopy. Additionally, silencing of RAPL reduced internalization of β 1-integrin as assessed by a FACS-based endocytosis assay. Interestingly, RAPL co-immunoprecipitated with GEP100, a guanine nucleotide exchange factor (GEF) for Arf6, which mediates β 1-integrin internalization in HeLa cells. Furthermore, silencing of GEP100 significantly impaired angiogenic sprouting of HUVEC (55 % inhibition) and VEGF-induced invasion. In conclusion, our results demonstrate for the first time that RAPL mediates, in association with GEP100, β 1-integrin internalization thereby promoting angiogenic sprouting and migration. Taken together, our data identify a new angiogenic signaling pathway and shed light on the relevance of β 1-integrin internalization for angiogenic functions such as sprouting and migration.
Abstract 5314: A Critical Role for TGF- <i>β</i> Activity in the Protection Against Aortic Aneurysm Formation and Complications in Adult Mice
Authors: Yu Wang, Hafid Ait-Oufella, Olivier Herbin, Philippe Bonnin, Bhama Ramkhelawon, Soraya Taleb, Jin Huang, Georges Offenstadt, Christophe Combadière, Laurent Rénia, Jason L Johnson, Pierre-Louis Tharaux, Alain Tedgui, Ziad Mallat
Journal: Circulation (2009)

Complicated abdominal aortic aneurysm is a major cause of mortality in elderly men. Recent studies unexpectedly showed that increased angiotensin II (AII)-dependent transforming growth factor (TGF)- β activity promotes aortic aneurysm progression in a mouse model of Marfan syndrome. However, the direct role of TGF- β activity in other forms of abdominal aortic aneurysm has not been assessed. Here, we show that systemic neutralization of TGF- β activity using two different antibodies, including the one previously shown to inhibit aneurysm formation in Marfan syndrome, breaks the resistance of normo-cholesterolemic C57BL/6 mice to AII-induced abdominal aortic aneurysm formation and markedly increases their susceptibility to the disease (from ~ 10% to 80–95%, P&lt;0.0001). These aneurysms display a large spectrum of complications on echography, including fissuration, false channel formation and rupture, leading to death from aneurysm complications. The disease and its complications are refractory to inhibition of IFN- γ , IL-4, IL-6 or TNF- α signaling. Genetic deletion of T and B cells or inhibition of CX3CR1 chemokine pathway resulted in partial protection. Interestingly, neutralization of TGF- β activity led to enhanced monocyte invasiveness, and monocyte depletion markedly inhibited aneurysm progression and complications. Finally, neutralization of TGF- β activity led to substantial increase in MMP-12 activity, and MMP-12 deficiency completely prevented aneurysm rupture. These results clearly identify a critical role for TGF- β in the taming of the innate immune response and the preservation of vessel integrity in adult normal mice, which contrasts with its reported pathogenic role in Marfan syndrome.
Abstract 5310: Re-Endothelialization of Decellularized Heart Reduces Vascular Thrombogenicity
Authors: Matthew J Robertson, Stefan M Kren, Doris A Taylor
Journal: Circulation (2009)

Perfusion decellularization of cadaveric hearts generates a cell-free extracellular matrix (ECM) that has similar architectural, mechanical, and chemical properties to the native tissue. These scaffolds retain their vascular conduits giving them the potential for anastomosis and transplantation. However, because the matrix is denuded, vascular clotting occurs after transplant even under an anti-coagulation (Coumadin) regimen. To create a less thrombogenic and thus more transplantable decellularized construct, we re-lined vessel conduits throughout the heart with endothelial cells and quantified their functional ability to alter clotting. Cadaveric rat hearts were decellularized by retrograde aortic detergent perfusion. Labeled rat aortic endothelial cells (RAECs) were re-introduced following two different protocols: 40 million RAECs were delivered by arterial (Ar) infusion only, or 20 million RAECs delivered by Ar infusion followed with an additional 20 million RAECs delivered by venous (Ve) infusion. Re-endotheliazed heart constructs were then maintained in a bioreactor under retrograde aortic perfusion for 1 week. Thrombogenicity of the constructs was measured by an in vitro thrombomodulin assay of the media flowing through the heart vasculature followed by histologic analysis. Heterotopic transplantation of scaffolds was performed in Rowett Nude rats, and hearts were examined 1 week post transplant. Either delivery route resulted in a whole heart (base to apex) distribution of RAECs with the retention of the endothelial phenotype (e.g. flattened morphology, proliferative (PCNA + ), eNOS + and vWF + ). In vitro thrombogenicity was reduced regardless of the delivery protocol, though there was a trend towards improved performance when cells were delivered via both Ar+Ve routes (6 and 8 fold decrease vs. controls for Ar only or Ar+Ve delivery respectively, N=8 for each condition, p&lt;0.05). After heterotopic transplantation of the re-endothelialized ECM, clotting and cellular invasion were reduced. With this advance we move closer to having a suitable heart construct for transplant while also developing a novel in vitro system for use in studies such as examining stem/progenitor cell differentiation in decellularized heart.
Abstract 5279: GIT1 is Required for Neointima Formation by Regulating Notch1 Signaling Pathway
Authors: Jinjiang Pang, Ryan hoefen, Min Hur, Heidi Michaloski, Amy Mohan, Bradford C Berk
Journal: Circulation (2009)

Notch1 is a critical regulator of cell fate, angiogenesis and cardiovascular development. Upon ligand (Delta and Serrate/Jagged) binding, Notch1 is proteolytically cleaved by γ -secretase to generate the Notch intracellular domain (N1-ICD). N1-ICD translocates to the nucleus to stimulate target gene expression. Recently it was shown that Notch1 mediates vascular smooth muscle cell (VSMC) proliferation and neointimal formation after vascular injury. G-protein-coupled receptor-kinase-2 interacting protein 1 (GIT1), is a multi-domain scaffold protein, that plays an important role in angiotensin II (AngII) induced Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII) activation. It has been shown that AngII stimulates VSMC growth through CamKII-Akt pathway. Activation of Akt increases γ -secretese activity and subsequently increases Notch1 cleavage. Our hypothesis is that knockdown of GIT1 will impair the activation of CamKII-Akt pathway, which will decrease Notch1 cleavage, and subsequently decrease neointima formation. Stimulation of VSMC with AngII time- dependently caused Notch1 cleavage, N1-ICD formation, and Hey1 expression. VSMC treated with AngII and GIT1 siRNA showed dramatic decreases in Notch1 cleavage and Akt activation. Since Notch 1 is important for cell proliferation and survival, knockdown of GIT1 may inhibit cell proliferation and increase cell apoptosis. Serum-stimulated growth was decreased by 60% in GIT1 KO VSMC compared to WT VSMC. Moreover, apoptosis induced by serum derivation in GIT1 KO VSMC increased 2-fold compared with WT VSMC. Cyclin D1, which is a key regulator of cell cycle, was significantly decreased in GIT1 KO VSMC. Finally to confirm the physiological role of GIT1 in VSMCs in vivo, complete carotid ligation was performed in GIT1 WT and KO mice. After carotid ligation, GIT1 expression in vessels of WT mice increased. Intimal and medial hyperplasia after 4 weeks was markedly decreased in GIT1 knockout mice. In conclusion, GIT1 plays a crucial role in VSMC proliferation and survival by regulating Notch1 activation, thereby regulating flow-mediated vascular remodeling and intima formation. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 5278: Essential Role of the Rho Exchange Factor Arhgef1 for Angiotensin II-induced RhoA/Rho Kinase Activation and Arterial Tone Regulation
Authors: Christophe Guilluy, Jérémy Brégeon, Gilles Toumaniantz, Malvyne Rolli-Derkinderen, Elizabeth Scalbert, Antoine Bril, Pierre Pacaud, Gervaise Loirand
Journal: Circulation (2009)

Rho exchange factors (Rho-GEFs) are signaling molecules responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although over-activation of the small G protein RhoA and its effector Rho kinase is recognized as a critical component of the pathogenesis of hypertension in experimental models and in human, the molecular mechanisms and the RhoA-GEF(s) involved in this activation of RhoA signaling in vascular smooth muscle cells (VSMCs) are still unknown. Nevertheless, angiotensin II (Ang II) has been shown to play a key role in the over-activation of RhoA in hypertension. Here we identify the RhoA-GEF and the molecular mechanism responsible for the activation of RhoA and Rho kinase in response to Ang II type 1 receptor activation (AT1R) in rat aortic VSMCs. Systematic functional analysis by siRNA-mediated silencing of the 28 RhoA GEFs indicated that only Arhgef1 (p115, lsc) is involved in Ang II (0.1 μ M)-mediated RhoA/Rho kinase activation. Knock-down of Arhgef1 had no significant effect on the activation of RhoA/Rho kinase induced by the thromboxan analog U46619, endothelin-1 and noradrenaline. The potency and efficacy of noradrenaline, phenyl-ephrine, U46619 and endothelin-1 to elicit contraction of rat aortic rings were not modified by Arhgef1 silencing whereas the contractile effect of Ang II was abolished. Ang II-induced Arhgef1 activation was further confirmed by in vitro guanine nucleotide exchange assay on RhoA, and we demonstrated by the transfection of Arhgef1 mutants that Ang II-induced Arhgef1 activation was due to its phosphorylation on Tyr738 by the tyrosine kinase Jak2. To confirm the functional role of Arhgef1 in Ang II-mediated regulation of vascular tone and hypertension, we then determined the activity of Arhgef1 in arteries from Ang II treated rats by pull-down assay. Chronic infusion of Ang II (250 ng/kg/min, 14 days) through an osmotic minipump induced an increase in arterial pressure associated with enhanced RhoA/Rho kinase activity and activation of Arhgef1 in arteries. Our results thus show that Arhgef1 activation and its downstream RhoA signaling are essential for Ang II-induced contraction and identify Arhgef1 as a potential specific target for the treatment of Ang II-dependent hypertension.
Abstract 5299: Release of Sphingosine-1-Phosphate From Platelets Requires Thromboxane Synthesis and Thromboxane Receptor Activation
Authors: Andreas Böhm, Thomas Ulrych, Anke C Rosenkranz, Guenter Daum, Gerd Geisslinger, Thomas Hohlfeld, Karsten Schrör, Bernhard H Rauch
Journal: Circulation (2009)

The sphingosine-derived lipid signaling molecule sphingosine-1-phosphate (S1P) is an important mediator of vascular homeostasis. S1P signaling has recently been shown to be a downstream signaling component of the receptors for thrombin, the protease-activated receptors (PARs). The present study investigates the requirement of thromboxane formation for the release of S1P induced by the prototypic thrombin receptor PAR1 from human platelets and the consequences for human monocyte and endothelial cell migration. S1P was detected by thin layer chromatography in [3H]sphingosine-labeled platelets and by mass spectrometry. Release of S1P was stimulated about 5 fold (0.3±0.1 vs. 1.6±0.5 nmol/1×106 platelets) by the selective PAR1-activating-peptide (PAR1-AP, 100 μ M). Acetylsalicylic acid (ASA) or the reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500 mg single dose) attenuated S1P release from platelets in healthy volunteers ex vivo which was paralleled by inhibition of TX formation. S1P release was increased by the thromboxane receptor (TXR) agonist U46619, and inhibited by the TXR antagonist ramatroban. In platelets from TXR-deficient mice, thrombin-induced release of S1P was attenuated and not affected by ASA. Supernatants from PAR1-AP-stimulated platelets increased human umbilical vein endothelial cell (HUVECs) migration and the chemotactic capacity of human peripheral blood monocytes (2- and 3-fold, respectively). These effects were inhibited when platelets were pretreated with ASA. S1P receptor-specific antagonists revealed that the chemotactic responses in HUVECs were mediated by the S1P receptor-1 (S1P1) and by S1P1 and S1P3 in monocytes. These data suggest that S1P release from platelets after thrombin receptor activation requires TX synthesis and TXR activation. This novel pathway likely contributes to the regulation of vascular homeostasis during platelet activation and inflammatory processes. Inhibition of S1P may be involved in the anti-inflammatory actions of ASA in vivo, for example by affecting recruitment of endothelial cells and monocytes to sites of vascular injury.
Abstract 5313: Reduction of S100A12 in Marfan Syndrome Aortic Smooth Muscle Cells Attenuates Inflammatory and Apoptotic Pathways
Authors: Marion Hofmann Bowman, Joseph Gawdzik, Jeremy Nicolarsen, Jai Raman, Lisa Dellefave, Elizabeth McNally
Journal: Circulation (2009)

Marfan syndrome (MFS) is associated with thoracic aortic aneurysms. S100A12 has been associated with initiation of inflammatory pathways via activation of the receptor for advance glycation endproducts (RAGE) and is up regulated in smooth muscle cells of ruptured human coronary artery plaques. We tested the hypothesis that S100A12 is expressed in SMC in human aortic aneurysms of MFS, and that downregulation of S100A12 may suppress inflammatory and apoptosis pathways. Methods: Aortic tissue from a patient undergoing surgery for MFS aneurysm and control aortic tissue were compared and several cell lines of primary SMC’s were established. SMC that carry a mutation in the FBN-1 gene (Arg529Term) and control SMC were each transfected with siRNA-S100A12-GFP and siRNA-control-GFP. GFP positve SMC were isolated by FACS, followed by qRT-RCR using arrays for inflammation and apoptosis pathways (RT2 profiler PCR Array, SA Biosciences). Results: Immunohistochemistry of aortic tissue from MFS revealed expression of S100A12 in SMC, but not in SMC of control aortic tissue. S100A12 was expressed strongest in SMC near the focal areas of medial degeneration. Cultured SMC that carry the FBN-1 mutation strongly expressed S100A12 protein. S1090A12 expression was completely abolished by transfection with siRNA-S100A12-GFP construct but not with siRNA control-GFP construct. S100A12 reduction in FBN-1 mutant SMC suppressed at least two fold the mRNA of 80% of genes on the inflammation array and of 91% of the genes on the apoptosis array. The death receptor Fas, caspase 10 and effector caspases 3 and their substrate DFFA were probed using semi-quantitative immunoblotting. FBN-1 mutant SMC showed approximately 2-fold increase for Fas, and two fold increase in Casp3 and DFFA compared to control SMC. Treatment with Fas CH11 activating monoclonal antibody induced Casp3 approximately 12 fold in FBN-1 mutant SMC compared to a two fold induction in control SMC (p&lt; 0.001). Reduction of S100A12 in FBN-1 mutant SMC reversed this pattern with s significant reduction in Fas, caspase 10 &amp; 3, and DFFB. Summary: S100A12 is upregulated in SMC in thoracic aneurysms and reduction of S100A12 reduces Fas-mediated apoptosis in aortic SMC in MFS. 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).