tbuckley/cabot-search huggingface dataset:
Abstract 5658: Immune Reconstitution Prevents Autoimmune Inflammation and Right Ventricular Remodeling in Immunodeficiency-Associated Pulmonary Hypertension
Authors: Rasa Tamosiuniene, Carlin Long, Kelly Ambler, Yon Sung, Lajos Gera, Lijuan Wang, Mark Nicolls
Journal: Circulation (2009)

Pulmonary arterial hypertension (PH) is a frequently lethal condition commonly associated with autoimmune conditions. Because autoimmunity is closely linked to immunodeficiency, we utilize athymic (AT) nude rats lacking normal T cells to demonstrate how PH is accentuated by a lack of normal regulatory T cell activity. The purpose of this study was to determine whether immune reconstitution (IR) prevents autoimmune inflammation (AI), right ventricular (RV) remodeling and PH using inbred AT rats reconstituted with euthymic spleen cells. Methods and Results: Inbred WAG AT nude rats were given a single injection of vascular endothelial growth factor receptor (VEGFR) antagonist SU5416 (20mg/kg sc), AT+SU group. Control AT was treated with vehicle only (DMSO). In adoptive transfer IR experiments, AT rats received unfractionated 20×10 6 euthymic rat spleen cells, CD4+T cells, CD8+T cells, CD4+CD25+T cells isolated with magnetic cell sorting before or after SU5416 administration. 2D-Echo, pulmonary hemodynamics and RV/LV+S ratio were assessed at 10d and 21d. Echo revealed decreased RV fractional shortening (p&lt;0.05) and PA acceleration time (p&lt;0.005) in AT+SU compared with IR and controls. IR prevented increase of RVSP compared to AT+SU (P&lt;0.05) and of RV/LV+S (P&lt;0.001). RV fetal cardiac gene expression (a-MHC, b-MHC, SERCA, BNP, SkACT, ANP) was consistent with pathologic stress pattern injury pattern and was significantly attenuated by IR. Furthermore, IR reduced expression of Th1, TH2 cytokines and pulmonary infiltration of macrophages, mast cells, B cells, IgG deposition and up-regulated lung BMPR2, FOXP3+, c-kit+, HOX11+ expression with increased c-kit+ and BMPR2 expression being noted in the pulmonary vascular endothelium. Conclusions: This study demonstrates that T cell deficiency is permissive for PH development and that IR prevents hemodynamic RV remodeling, attenuates AI and limits PH. Interestingly, that PH prevention is associated with increased regenerative HOX11+, C-kit stem cell populations.
Abstract 5651: Apelin Deficient Mice Demonstrate Worsening Pulmonary Hypertension in a Nitric Oxide Dependent Manner
Authors: Suparna Chandra, Hedi Razavi, Rani Agrawal, Uditha S Adurkar, Ramendra Kundu, Vinicio A de Jesus Perez, Roham T Zamanian, Marlene Rabinovitch, Thomas Quertermous, Hyung J Chun
Journal: Circulation (2009)

Recent studies have shown high expression of the GPCR APJ and its ligand apelin in the pulmonary vasculature. Given its known vasodilatory properties and pulmonary expression, we hypothesize that disruption of this pathway leads to worsening of pulmonary hypertension in the murine hypoxia model, and that the pathway is abnormally regulated in patients with pulmonary arterial hypertension (PAH). Right ventricular systolic pressures were assessed in apelin deficient (KO) mice after 2 weeks of hypoxia. Lungs were perfused with contrast agent (Microfil), and imaged using micro-computed tomography (CT) imaging. We measured NO levels in the serum and evaluated the lungs for expression of endothelial nitric oxide synthase (eNOS) using quantitative real-time polymerase chain reaction (RT-PCR) and western blots. We found that apelin KO mice developed more severe pulmonary hypertension in response to hypoxia compared to wildtype mice (34.1 vs. 28.3 mmHg, p&lt;0.001). Micro-CT of the lungs demonstrated vascular abnormalities in the apelin KO mice including pruning of smaller vessels and increased tortuosity of the larger vessels. Apelin KO mice had decreased levels of serum NO (p=0.02) and had lower expression of eNOS in the lungs, as determined by RT-PCR and western blots. We also performed ELISA assays to determine apelin levels in patients with PAH. Apelin levels from PAH patients were significantly lower compared to healthy controls (1.25 vs. 0.89 ng/mL, p=0.037). These data suggest that disruption of apelin signaling can exacerbate pulmonary hypertension, and identify this pathway as a potential therapeutic target for treatment of this refractory human disease.
Abstract 5685: TGF-beta Signaling and Kruppel-like Factor 10 Regulate Bone Marrow-derived EPC Differentiation, Function, and Neovascularization
Authors: Akm Wara, ShiYin Foo, Basak Icli, Zhuoxiao Cao, Fehim Esen, Malayannan Subramaniam, Thomas C Spelsberg, Anthony Rosenzweig, Mark W Feinberg
Journal: Circulation (2009)

Background: Endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of contributing to cardiovascular regeneration. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Hematopoiesis consists of a cascade by which hematopoietic stem cells (HSCs) give rise to either a common myeloid progenitor (CMPs) or common lymphoid progenitor (CLPs). The CMP cells can give rise to either granulocyte-monocyte progenitors (GMPs) or megakaryocyte-erythrocyte progenitors (MEPs). Methods and Results: Herein, we identify that CMPs and GMPs preferentially differentiate into CD34+KDR+ EPCs as measured by FACS, uptake of DiIacLDL and U.lex Lectin, matrigel studies, and endothelial markers. Co-culture studies in matrigel demonstrate increased network formation and incorporation when HUVECs were co-incubated with CMPs and GMPs only. Treatment of CMPs or GMPs with IFN-g blocked EPC differentiation, whereas treatment with TGF-b1 potently induced EPC differentiation. Conversely, EPC formation was markedly impaired using blocking antibodies or TGF-b1 deficient CMPs or GMPs, whereas IFN-g deficient mice have increased EPC differentiation. Functionally, EPCs derived from CMPs and GMPs more potently adhered to fibronectin-coated plates, migrated faster in transwell Boyden chambers, and released VEGF nearly ~2 to 2.5-fold higher than EPCs from MEPs and HSCs. Finally, we identified that a TGF-b1-responsive Kruppel-like transcription factor, KLF10, as robustly expressed ~60-fold in EPCs derived from CMPs and GMPs, compared to hematopoietic progenitors lacking EPC markers. Consistently, KLF10−/− mice have marked defects in EPC differentiation (−23.6% vs. WT, P&lt;0.002), TGF-b1 responsiveness (completedly abolished), and impaired blood flow recovery in response to hindlimb ischemia (−23.8% vs. WT, P&lt;0.008), an effect associated with reduced levels of circulating EPCs (−28.2% vs. WT, P&lt;0.05). Conclusion: Among bone marrow progenitors, a hierarchy exists for EPC differentiation. Targeting KLF10 may alter EPC differentiation from bone marrow-derived CMPs and GMPs and influence cardiovascular repair.
Abstract 5683: Insights From Network Analysis: Deficiency of Glutathione Peroxidase-1 Increases In-stent Stenosis
Authors: Ziad Ali, Azad Raiesdana, Xiumei Qu, Ayyaz Ali, Aleksandra Pavlovic, Matthew Wheeler, Keith Channon, Thomas Quertermous, Euan Ashley
Journal: Circulation (2009)

In-stent stenosis (ISS) is the principal cause of PCI failure. We used a novel genome-wide gene expression network approach in human atherectomy samples (n=89) to identify signaling networks critical to the process of ISS. We found that a subnetwork with glutathione peroxidase-1 (GPx1) as its hub was among the most significantly differentially regulated between de novo atherosclerosis and ISS (Fig 1 , bar thickness denotes association strength). We investigated the importance of GPx1 in ISS using a novel model of balloon angioplasty and stenting (BAS) and a knockout mouse with constitutive deficiency of GPx1. Thoracic aortic segments underwent BAS before being grafted onto the carotid artery of isogenic recipients. Groups included donor GPx1-KO mice and their littermate controls (n=5/group). Despite equal stent expansion and injury scores, ISS was increased by 45% (P&lt;0.001) in GPx1-KO mice 28 days following stenting, confirming the observational data from the human studies (Fig 2 , white arrows denote neointima). To investigate a mechanism for these findings, we quantified superoxide production (O2-). O2- production was significantly increased in GPx1-KO mice measured on both aortic sections using oxidative confocal microtopography (~2.8-fold, P&lt;0.001) and whole aorta by lucigenin enhanced illuminescence (~2.2-fold, P&lt;0.001). Further, studies aimed at establishing a therapeutic role for the isoflavone genistein in modulating GPx1 activity demonstrated increased enzymatic activity in coronary endothelial cells (~78%, P=0.01). Taken together, these findings establish an important role for GPx1 network signaling in vascular wall redox processes critical to ISS. Fig. 1 Fig. 2
Abstract 5656: Anticentromere Antibody Increases ICAM-1 Expression and Monocyte Adhesion in Pulmonary Endothelial Cells: Implications for Scleroderma-Associated Pulmonary Hypertension
Authors: Shirley Nakhla, Rahn Ilsar, Alison K Heather, David S Celermajer
Journal: Circulation (2009)

BACKGROUND: Scleroderma is characterized by the presence of high circulating levels of anticentromere antibody (ACA) and is commonly complicated by pulmonary hypertension (PHT). We hypothesized that ACA may alter pulmonary endothelial cell function. METHODS: Human pulmonary microvascular endothelial cells (HPMvECs) were treated with ACA (0.25 – 1.0 mg/mL, equivalent to serum titers of 1:320 – 1:2560) and IgG controls. Monocyte adhesion to HPMvECs was measured by automated cell count and cell adhesion molecule expression (ICAM-1 and VCAM-1) was assessed by flow cytometry (protein) and RT-PCR (mRNA). Promoter activity was assessed by luciferase reporter vector in HPMvECs and human umbilical vein endothelial cells (HUVECs). ACA uptake into HPMvECs was assessed by fluorescent microscopy. RESULTS: Fluorescently labeled ACA was shown to penetrate HPMvECs within 10 min. ACA increased ICAM-1 protein levels in a dose-dependent manner (118±11, 132±8 &amp; 162±7% increase vs control (100%) for 0.50, 0.75 &amp; 1.0 mg/mL respectively, P &lt; 0.0001, n=5; Fig ). No effect was observed for VCAM-1. ICAM-1 increases were associated with enhanced monocyte adhesion to endothelial cells (211±9%, 1.0 mg/mL vs control, P &lt; 0.0001, n=3). ICAM-1 mRNA levels were markedly increased by ACA (745±113%, 1.0 mg/mL vs control, P &lt; 0.0001, n=4). ACA did not affect ICAM-1 mRNA stability but increased ICAM-1 promoter activity (136±9% &amp; 148±12%, 1.0 mg/mL vs control for HPMvECs &amp; HUVECs respectively, P &lt; 0.01, n=3). CONCLUSION: ACA increases HPMvECs ICAM-1 expression and monocyte cell adhesion. These findings may implicate a role for ACA in the development of PHT in scleroderma.
Abstract 5674: Toll-like Receptor-2 and -4 Signaling Regulates Inflammation and Matrix Degradation in the Human Atherosclerotic Plaque
Authors: Scott M Gregan, Tina J Navin, Ektoras Georgiou, Brian M Foxwell, Alun H Davies, Marc Feldmann, Claudia Monaco
Journal: Circulation (2009)

Background The current consensus view of the mechanisms leading to plaque rupture has assigned a central role to inflammation and matrix degradation. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions but their functional role in matrix degradation in human atherosclerosis is unknown. Methods We explored the effects of blocking TLR-2, -4 and the downstream signaling adaptors myeloid differentiation primary response gene 88 (MyD88, shared by most TLRs and Trif-related adaptor molecule (TRAM; used by TLR-4) in an in vitro model of human atherosclerosis. Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation in order to obtain a single cell suspension of mixed cell types. Cells were cultured in the presence or absence of TLR signaling blockers immediately after isolation. Phenotyping by flow cytometry showed that macrophages are the most abundant cells in the mixture, being approximately double the proportions of smooth muscle cells and lymphocytes. Results A dominant-negative form of MyD88 (MyD88DN) decreased the production of MCP-1/CCL2 (P=.015), IL-8/CXCL8 (P =.002), IL-6 (P =.001), MMP-1) (P =.016), and MMP-3 (P =.0001), as well as nuclear factor kappaB (NFkB) activation (P= .0001) in atheroma cell cultures. TLR-2 neutralizing antibodies inhibited NFkB activation (P = .025), and significantly reduced MCP-1/CCL2 (P=.0001), IL-8/CXCL8 (P=.007), IL-6 (P=.0001), and MMP-1, -2, -3, -9 production (P=.0001). In contrast, a dominant negative form of the adaptor TRAM (TRAM DN ), which is used by TLR-4, significantly reduced NF κ B activity, MMP-3 ( P =.049) and MMP-1, but had no effect on gelatinases MMP-2 and -9 or inflammatory mediators. Conclusions Our data indicates that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. However, we also uncovered a role for TLR-4/TRAM-dependent signaling in MMP production, suggesting that the MyD88-independent pathway may play a role in mechanisms related to matrix degradation and plaque vulnerability. TLR blockade may represent a therapeutic strategy for atherosclerosis and its complications.
Abstract 5661: Akt/mTOR is an Essential Downstream Pathway of Platelet-Derived Growth Factor Signaling in Pulmonary Artery Smooth Muscle Cells
Authors: Aiko Ogawa, Amy L Firth, Jason X Yuan
Journal: Circulation (2009)

Background: Platelet-derived growth factor (PDGF) is known to be a potent mitogen involved in the development and progression of pulmonary vascular remodeling. Recently, PDGF has attracted attention due to the inhibition of its receptor having a positive effect on hemodynamics, remodeling, and survival. Indeed, a PDGF receptor inhibitor is now undergoing clinical trials. It is, however, unclear which downstream pathway is important in pulmonary vascular remodeling. The Akt/mTOR pathway is central to many biological responses including cell growth, proliferation, survival, and metabolism. The involvement of the Akt/mTOR pathway in PDGF signaling was evaluated, in addition to a potential role in store-operated Ca 2+ entry (SOCE), which we previously reported to be important in cell proliferation and vascular remodeling in idiopathic and thromboembolic pulmonary hypertension. Methods and Results: In human pulmonary artery smooth muscle cells (PASMC) and fibroblasts, PDGF transiently phosphorylated Akt, mTOR and p70S6K (24.8-, 3.6-, 8.1-fold increase in 15 minutes, P&lt;0.01, 0.001, 0.01, respectively). Interestingly, PDGF failed to induce Akt phosphorylation in pulmonary artery endothelial cells. Chronic treatment with PDGF (10 ng/ml) significantly enhanced the increase in cytosolic Ca 2+ due to cyclopiazonic acid (10 microM)-induced Ca 2+ mobilization (from 0.75±0.02 to 0.88±0.03 arbitrary unit (a.u.), P&lt;0.001) and SOCE (from 1.03±0.02 to 1.51±0.05 a.u., P&lt;0.001). When treated with rapamycin (10 nM), an inhibitor of mTOR, or Akt inhibitor (1 microM), SOCE was significantly reduced (to 1.28±0.05, 0.60±0.02 a.u., respectively, P &lt;0.001). Conclusions: The data suggest that Akt/mTOR signaling is an important pathway in the regulation of SOCE in PASMC. A greater understanding of this pathway may enable the development of novel therapeutic approaches in patients with pulmonary vascular disease.
Abstract 5646: Endothelial Intrinsic NF- <i>κ</i> B Signaling Regulates iNOS And eNOS Expression <i>in vivo</i> in Endotoxemic Mice
Authors: Xiaobing Ye, Dongmei Song, Shu Fang Liu
Journal: Circulation (2009)

We have previously demonstrated that endothelial NF- κ B blockade mitigates septic shock and septic vascular dysfunction ( Circulation 118: S570, 2008). Since endothelial and inducible nitric oxide synthase (iNOS and eNOS) are known to be involved in vascular dysfunction, this study examined the contribution of endothelial intrinsic NF- κ B signaling in LPS induced alterations in iNOS and eNOS expression. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I- κ B α selectively on endothelium were injected with saline (1 ml/kg, i.p.) or E Coli LPS (10 mg/kg, i.p.). Aortic levels of iNOS and eNOS proteins were determined by Western blot. The iNOS and eNOS bands were quantified using densitometry and expressed as arbitrary optical density units. Plasma and aortic levels of nitrite/nitrate, and aortic level of TNF- α were measured using commercial kits. Aortic level of iNOS protein (O.D units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 0±0, 0±0, 935±107 and 257±58 (p &lt; 0.01, WT-LPS vs other 3 groups). Plasma and aortic levels of nitrite/nitrate, for WT-Con, TG-Con, WT-LPS and TG-LPS groups, were 4.4±0.7, 3.9±0.4, 19.3±3.4 and 6.9±1.3 μ M in plasma, and 5.8±1.9, 5.8±0.9, 40.2±3.9 and 19.9±2.0 nmole/mg protein in aorta (p &lt; 0.01, WT-LPS vs other 3 groups). Aortic level of eNOS protein (O.D units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 896±54, 799±61, 586±46 and 838±49 (p &lt; 0.05, WT-LPS vs other 3 groups). Down-regulation of eNOS expression was associated with increased aortic TNF- α expression. Aortic levels of TNF- α (pg/mg protein) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 68±26, 23±12, 753±132 and 369±54 (p &lt; 0.01, WT-LPS vs other 3 groups). IHC staining of aortic sections showed that TG-LPS mice had a significantly reduction in LPS-induced iNOS or TNF- α expression in both endothelial and smooth muscle cells, indicating a paracrine interaction between the two cells. These results suggest blockade of endothelial NF- κ B pathway mitigates septic vascular dysfunction by inhibiting vascular iNOS expression and by preventing eNOS down-regulation. (Supported by NIH GM063907)
Abstract 5680: Serum and Glucocorticoid-Inducible Kinase 1 Regulates Sirtuin 3, the Mitochondrial Deacetylase via Phosphorylation and Sumoylation Mechanisms in Rat Aortic Smooth Muscle Cells
Authors: Wei Zhong, Winston E Thompson, Sharon Francis-David
Journal: Circulation (2009)

Serum and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase whose activity and expression is up-regulated in vascular neointimal lesions in association with restenosis. Our previous studies show that vascular smooth muscle cell (VSMC) proliferation is augmented upon activation of SGK1. However, the molecular mechanism(s) was not fully examined. Thus, to identify proteins that participate in SGK1-mediated stimulation of VSMC proliferation, we performed a kinase substrate protein array with activated SGK1. We identified sirtuin3 (Sirt3), a class III histone deacetylase as a novel putative substrate for SGK1 in this screen. Preliminary studies from two-dimensional SDS-PAGE indicate that expression of active SGK1 (S422D-SGK1) is associated with a shift in Sirt3 to a more acidic profile, consistent with an increase in phosphorylation. Moreover, S422D-SGK1 expression resulted in a 2- and 1.6-fold increase in Sirt3 mRNA and protein; respectively, compared to empty vector or a kinase-defective SGK1 mutant (S422A-SGK1). To determine whether Sirt3 expression is regulated by growth factors, we performed timecourse studies with PDGF-BB, a potent inducer of VSMC proliferation as well as SGK1. Quantitative real-time PCR analysis, revealed a modest reduction in Sirt3 mRNA (~25%) after 4 hours of stimulation. However, under the same conditions, PDGF-BB augments cytoplasmic Sirt3 protein expression more than 32-fold, suggesting a posttranslational method of regulation. Notably, sumoylation is known to regulate the activity, stability or subcellular distribution of its substrates and our analysis indicates that Sirt3 contains the core SUMO consensus motif. Thus, we assessed whether Sirt3 can be sumoylated and tested the hypothesis that PDGF acting through SGK1 signaling modulates sumoylation of Sirt3 in VSMC. Indeed, results obtained from an in vitro sumoylation assay indicate that Sirt3 is a target for SUMO-1. Lastly, we found a higher level of SUMO-1 conjugation in Sirt3 immunoprecipitates from VSMC expressing S422D-SGK1 compared to empty vector- or S422A-SGK1-expressing cells. Altogether, our findings implicate Sirt3 as novel substrate and potential mediator of the growth effects of SGK1 in VSMC.
Abstract 5642 Thrombospondin-1, via CD47, Blocks Endothelial-Dependent Arterial Relaxation by Limiting Endothelial Nitric Oxide Synthase Activation
Authors: Jeffrey S Isenberg, Eileen Bauer, Yan Qin, Thomas Miller, Russell Bandle, Gabor Csanyi, Patrick J Pagano, Phillip M Bauer, Jurgen B Schnermann, David D Roberts
Journal: Circulation (2009)

Introduction: The secreted protein thrombospondin-1 (TSP1) is an important negative regulator of nitric oxide/cGMP signaling in vascular cells. TSP1, signaling through its necessary receptor CD47, limits activation of soluble guanylate cyclase and cGMP-dependent protein kinase. Extending these discoveries we recently defined a role for TSP1 in the acute control of blood pressure. However, it is not clear if the inhibitory role TSP-CD47 signaling has on blood pressure is secondary to a direct effect on arterial relaxation. To investigate this we tested the hypothesis that TSP1 directly limits arterial vasorelaxation by limiting activation of endothelial nitric oxide synthase (eNOS). Methods: TSP1-CD47 regulation of eNOS activation was studied in endothelial cells and arteries. Vasorelaxation was assessed ex vivo in arteries from wild type, TSP1 and CD47 null mice. Endothelial dependent changes in blood pressure were studied in transgenic mice. Results: TSP1 and CD47 null arteries demonstrated significantly greater dilation in response to physiologic activation of eNOS by acetylcholine compared to wild type vessels. Treating the luminal endothelial compartment of both wild type and TSP1 null arteries (but not CD47 null) with TSP1 inhibited vessel relaxation to acetylcholine and potentiated vasoconstriction to phenylephrine. TSP1 and a recombinant fragment of the signature domain that specifically binds CD47 potently blocked both acetylcholine-stimulated eNOS activation and changes in intracellular calcium in cultured endothelial cells. In vivo, acetylcholine-stimulated hypotention in TSP1 null mice was blocked by intravenous replacement of TSP1. Conclusion: Circulating TSP1, through CD47, limits endothelial dependent arterial relaxation and demonstrates a role for circulating TSP1 as a hypertensive agent supporting blood pressure through continuous modulation of both endogenous NO production and signaling.