bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 339: Long-term AAV6-βARKct Myocardial Gene Therapy Improves Cardiac Function Via Restoration Of β-Adrenergic Receptor Signaling And Neurohormonal Status Of The Failing Heart
Authors: Rengo Giuseppe , Lymperopoulos Anastasios , Zincarelli Carmela , Donniacuo Maria , Soltys Stephen , Rabinowitz Joseph E , Koch Walter J 
Published: July 2021

BACKGROUND: The up-regulation of G protein-coupled receptor kinase-2 (GRK2) that is present in compromised myocardium contributes to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function in heart failure (HF). The peptide βARKct, which inhibits the activation of GRK2, has been shown in acute gene transfer experiments to rescue HF. This study was designed to evaluate chronic βARKct expression in post-myocardial infarction (MI) induced HF using stable myocardial gene delivery with adeno-associated virus serotype-6 (AAV6).
METHODS AND RESULTS: In 12 week post-MI HF rats, we delivered βARKct or as a control, Green Fluorescent Protein, via direct intramyocardial injection. We also treated groups with concurrent administration of metoprolol. We found robust and long-lasting (up to 12 weeks post-delivery) transgene expression in the left ventricle (LV) and βARKct expression resulted in significantly improved global heart function as LV ejection fraction and ±dP/dt were increased, whereas LV end diastolic diameter and pressure were decreased. At the molecular level, cardiac βAR density and cAMP accumulation significantly improved over control groups. Fibrotic and hypertrophy markers, as well as heart-to-body weight ratio were markedly decreased by βARKct gene therapy indicating active reversal of adverse LV remodeling. For the first time, we found that chronic βARKct expression and normalization of cardiac βAR signaling led to a reduction of circulating levels of cardiotoxic neurohormones (catecholamines and aldosterone) demonstrating a potential additive mechanism of GRK2 inhibition. Concomitant metoprolol administration preserved the gain in inotropy achieved by βARKct, suggesting compatibility of these two therapeutic modalities, however, metoprolol alone only prevented the deterioration of cardiac function in HF.

CONCLUSIONS
: Chronic cardiac βARKct gene therapy for HF treatment via AAV6-mediated intracardiac gene delivery is feasible and results in improved cardiac function accompanied by restoration of βAR molecular abnormalities and amelioration of neurohormonal status of HF. These findings suggest βARKct gene therapy might be clinically applicable and of significant value for human HF treatment.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 403: BMPER Is an Angiogenic Modulator that is Regulated by KLF-15 and FoxO3A and Controls Bone Morphogentic Protein Activity
Authors: Helbing Thomas , Heinke Jennifer , Volkmar Franziska , Wehofsits Leonie , Baar Kim-Miriam , Diehl Philipp , Bode Christoph , Patterson Cam 
Published: July 2021

BMPER (bone morphogenetic protein [BMP] endothelial precursor cell derived regulator) is an extracellular protein, that interacts with BMPs and thereby modulates BMP dependent vasculogenesis and angiogenesis. Our previous observations suggest a complex regulation of BMPER expression. During embryogenesis BMPER is expressed at the time and at sites of vasculogenesis, whereas in the adult organism it is expressed in heart, lung and skin.
Methods and Results: We have cloned the mouse BMPER promoter and appropriate deletion constructs into pGL3 to regulate luciferase expression. As predicted in silicio, we found that Sp1 and Sp1-like transcription factors such as the krueppel-like factors (KLFs) regulate BMPER transcription. KLF-15 resulted in a 4.5 fold upregulation. Accordingly, BMPER expression was inhibited by the Sp-1/SP-1 like inhibitor mitramycin A. Site specific mutation of a proximal KLF-15 binding site reduced the effect of KLF-15 on BMPER expression. Along the same lines, knock down of KLF-15 in HUVEC by siRNA reduced BMPER expression. The transactivating effect of KLF-15 could be competed away by coexpression of Sp-1 suggesting that both factors may compete for the same binding site in the BMPER promoter. In EMSA, an oligo representing a well characterized KLF-15 binding site in the AceCs2 promoter but not an oligo encoding for a NFkappa-B site competed with the oligo coding for the KLF-15 site in the BMPER promoter. In contrast FoxO3A, a member of the FoxO family of transcription factors, serves as an inhibitor of BMPER expression, as shown by gain and lack of FoxO3A experiments. Additionally, we found that BMPER stimulates angiogenesis in a BMP-4 dependent manner in several in vitro and in vivo assays. Vice versa, BMPER is necessary for BMP-4 to exert is angiogenic activity on endothelial cells.
Conclusion: BMPER is upregulated by KLF-15 and inhibited by FoxO3a. BMPER has angiogenic activity and is a key modulator of the BMP pathway.
Abstract 424: Vascular Smooth Muscle Cell Expression of S100a12 in Vivo Induces Enhanced Oxidative Stress & Vascular Remodeling
Authors: Bowman Marion Hofmann , Wilk Jeannine , Kim Gene , Zhang Yanmin , Rehman Jalees , McNally Elizabeth 
Published: July 2021

S100A12 is a small calcium binding protein that is a signal transduction ligand of the receptor for advance glycation endproducts (RAGE). S100A12, like RAGE, is expressed in the vessel wall of atherosclerotic vasculature, particularly in smooth muscle cells (SMC). While RAGE has been extensively implicated in inflammatory states such as atherosclerosis, the role of S100A12 is less clear. We tested the hypothesis that expression of human S100A12 directly exacerbates vascular inflammation. Several lines of Bl6/J transgenic mice (tg) expressing human S100A12 in SMC under control of the SM22a promoter were generated. Primary aortic SMC from tg and wild type (wt) littermates were isolated and analyzed for (i) proliferation using MTS/Formazan Assay and BrdU incorporation, (ii) oxidative stress using using flow cytometry with MitoSOX antibody, oxidative DNA damage using immunofluorescence microscopy with anti-8-oxo-dG antibody, and NF-kB activation measured by EMSA and (iii) cytokine expression measured by IL-6 ELISA. Furthermore, the aortas from tg and wt mice were examined.
Results: Tg but not wt SMC expressed S100A12 protein. Tg SMC had a significant 1.9 to 2.7 fold increase in conversion of MTS into Formazan at 24–96 hours likely reflective of increased metabolic activity since BrdU incorporation into DNA was less in tg compared to wt SMC (4% vs 21% positive BrdU nuclei, p <0.05). Tg SMC showed significantly higher levels of mitochondrial generated ROS, nuclear staining for oxidative DNA damage which was not detected in the nuclei of wt SMC’s, and a 2.5 fold increase in NFkB activity. IL-6 production at baseline was higher in tg SMC’s (615 vs 213 pg/ml, p< 0.05) and increased dramatically after LPS treatment (10 ng/ml) in tg SMC’s (2130 vs 415 pg/ml). Histologic examination of the thoracic aorta at 10 weeks of age revealed increased collagen deposition in the aortic media with fragmentation and disarray of elastic fibers. In vivo ultrasound revealed a progressive dilation of the aortic arch from age 10 weeks to 16 weeks of age (1.27 to 1.60 mm, p<0.05) in tg but not in wt littermate mice (1.30 to 1.33 mm, p=0.1). These data reveal the novel finding that targeted expression of human S100A12 in SMC modulates oxidative stress, inflammation and vascular remodeling.
Abstract 484: Large Tumor Suppressor 2, A Downstream Target of Mammalian Sterile 20 Like Kinase 1, Is an Endogenous Regulator of Myocyte Size and Apoptosis in the Heart
Authors: Matsui Yutaka , Nakano Noritsugu , Takagi Hiromitsu , Shao Dan , Gao Shumin , Hong Chull , Zhai Peiyong , Yabuta Norikazu , Nojima Hiroshi 
Published: July 2021

Mammalian sterile 20-like kinase 1 (Mst1) plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Mst1 forms a complex with hWW45 and Large tumor suppressor 2 (Lats2), thereby activating Lats2. Lats2 dose dependently increased apoptosis in cultured cardiac myocytes and transgenic mice with cardiac specific expression of Lats2 exhibited a cardiac dysfunction, suggesting that stimulation of Lats2 leads to increased cell death and cardiac dysfunction. In order to elucidate the function of endogenous Lats2 in the heart, we generated adenovirus harboring dominant-negative Lats2 (Ad-DN-Lats2) and transgenic mice with cardiac specific expression of dominant-negative Lats2 (Tg-DN-Lats2). Ad-DN-Lats2 prevented induction of apoptosis and inhibition of hypertrophy by Mst1 in cultured cardiac myocytes, suggesting that Lats2 mediates the function of Mst1. In Tg-DN-Lats2, both LV weight/tibia length (LVW/TL; 4.34 vs 3.68, N=9, N=7, p<0.01) and RV weight/tibia length (1.15 vs 0.88, N=9, N=7, p<0.01) were greater than in NTg. Echocardiographically determined LV function was normal in Tg-DN-Lats2 at baseline except that LV wall thickness was greater in Tg-DN-Lats2 than in NTg (LV posterior wall thickness; 0.97 vs 0.79 mm, N=10, N=7, p<0.05). Since expression of Lats2 is increased in response to pressure overload (8 fold), we examined whether upregulation of endogenous Lats2 during pressure overload works as a negative feedback mechanism for cardiac hypertrophy. Tg-DN-Lats2 mice exhibited greater LVW/TL than NTg after transverse aortic constriction (TAC) (5.9 vs 4.7 mm, N=9, N=7, p<0.05). Increases in LVW/TL (+37% vs 27%, N=9, N=7, p<0.05) and LV cross sectional area (+25% vs +16%, N=5, N=5, p<0.05) after TAC were greater in Tg-DN-Lats2 than those in NTg. Moreover, Tg-DN-Lats2 exhibited significantly lower cardiac myocyte apoptosis than NTg after TAC. These results suggest that Lats2 is a negative regulator of cardiac hypertrophy at baseline and in response to pressure overload, and a positive mediator of cardiac myocyte apoptosis under pressure overload. In conclusion, Lats2 is an evolutionarily conserved negative regulator of organ size: namely inhibiting hypertrophy while stimulating apoptosis in the heart.
This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 1443: Systems-Based Analysis of HDL Metabolism in a Mouse Intercross between Strains CAST and C57BL6/J (CxB)
Authors: Mehrabian Margarete , Farber Charles , Langfelder Peter , Ghazalpour Anatole , Zhou Zhiqiang , Aten Jason , Castellani Lawrence , Drake Thomas , Schadt Eric , Lusis Aldons 
Published: July 2021

A recent meta-analysis of three large genome-wide association studies for HDL cholesterol levels revealed several highly significant associations, but altogether these explained less than 10% of the population variance of HDL. Since HDL levels are highly heritable, with heritability estimated at 50–70% in many studies, there are clearly many additional genes, and probably complex genetic and environmental interactions, involved in HDL metabolism. Thus, if “personalized medicine” is to become a reality, these complex factors must be addressed. Combined genetic-genomic approaches have rejuvenated the analysis of complex traits using mouse models, and here report an integrative genomic study of HDL in a large mouse cross. We previously reported the identification of loci associated with HDL cholesterol concentrations using a CXB F2 intercross. We have now generated a much larger CXB cross, consisting of 438 mice, and have integrated genome wide gene expression analysis of liver and adipose with quantitative trait locus (QTL) mapping and causality modeling. These studies were carried out on mice fed a low fat, chow diet and then switched to a high fat, ’Western’ diet. QTL analysis on the clinical traits using R/QTL (http://cran.r-project.org/) revealed a complex inheritance pattern with significant LOD scores at 9 loci, on chromosomes 1,2,4,5,8,9,10,16,18. Of these loci, 6 (chr: 1,4,5,10,16,18) were seen to be involved in genetic-dietary regulation of HDL cholesterol. Expression QTLs (eQTL) were determined using Agilent microarrays for 23,624 transcripts. Genes expressed within a 1-LOD support interval or correlated with HDL (p<2.7E-11) in both adipose and liver were identified. Using Network Edge Orienting (NEO) methods, causal relationships between the identified genes, related QTL peak markers and HDL levels were accessed. The genes were then ranked based on the NEO scores. In liver the highest ranked genes were associated with mitochondrial, ER and golgi trafficking. In adipose, on the other hand, pathways associated with cell signaling, transcription regulation and protein ubiquitation were predicted to be causal for HDL levels. In conclusion, our results reveal a large number of novel pathways and candidate genes for plasma lipid metabolism.
This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells
Authors: van Vliet Piet , de Boer Teun P , van der Heyden Marcel A , Sluijter Joost P , Doevendans Pieter A , Goumans Marie-Jose 
Published: July 2021

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation.
Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway.
Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.
Abstract 1633: RhoA and Rho Kinase Activation in Human Pulmonary Hypertension - Role of 5-HT Signaling
Authors: Guilluy Christophe , Eddahibi Saadia , Agard Christian , Savale Laurent , Fadel Elie , Adnot Serge , Pacaud Pierre , Loirand Gervaise 
Published: July 2021

Background- The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations. Here, we investigated possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH.
Methods and Results- Lungs, platelets, and quiescent cultured pulmonary-artery smooth-muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) were characterized by marked elevations in RhoA and Rho kinase activity and by a strong increase in serotonin binding to RhoA, compared to controls. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT-induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT-induced proliferation of PA-SMCs from iPH patients, that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22–5-HTT+ mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22–5-HTT+ mice with either fasudil or fluoxetine limited both PH progression and RhoA/Rho kinase activation.
Conclusions- RhoA and Rho kinase activities are increased in iPH, as a result of transglutaminase-mediated transamidation of RhoA by 5-HT internalized via 5-HTT. Direct involvement of this pathway in 5-HT-mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.
Abstract 1637: Difference of Pulmonary Arterial Microvasculature in Patients with Pulmonary Hypertension Using Scanning Electron Microscope
Authors: Miura Aya , Nakamura Kazufumi , Kusano Kengo F , Matsubara Hiromi , Miura Daiji , Miura Ryuji , Ogawa Aiko , Ikeda Tetsuya , Fujio Hideki , Miyaji Katsumasa , Nishii Nobuhiro , Morita Hiroshi , Oka Takefumi , Ohtsuka Aiji , Date Hiroshi , Ohe Tohru 
Published: July 2021

Background and Objectives: Pulmonary vascular diseases (pulmonary arterial hypertension: PAH, pulmonary veno-occlusive disease: PVOD and pulmonary capillary hemangiomatosis: PCH) are well-known diseases that cause pulmonary hypertension (PH). However, the differences of structural characteristics of pulmonary microvasculature in these diseases have not been elucidated enough. We examined the morphological differences among these diseases by methacrylate resin (Mercox) perfusion using scanning electron microscope.
Methods and Results: Ten lung samples were 8 patients with PH and 2 control subjects without PH. These were obtained at lung transplantations or autopsies. Microvascular corrosion replicas were made by Mercox perfusion from the pulmonary artery. Pathological diagnosis revealed 5 PAH, 2 PVOD and 1 PCH in patients with PH. In PAH samples, small arteries or arterioles showed twisted and tortuous courses, but such vessels were not observed in samples from control, PVOD and PCH. Binary branching was observed in control, but not in PVOD and PCH. In PVOD and PCH, several branching was commonly observed. Moreover, the loss of capillary network and the disruption of arterioles were more severe in samples from PVOD and PCH patients than in samples from PAH patients.
Conclusion: Pulmonary microvascular morphology is seriously different among the type of underlying diseases in PH patients.
Abstract 3377: Modulation of Vessel Stability by the ELR-negative Chemokine IP-10
Authors: Bodnar Richard J , Yates Cecelia C , Du Xiaoping , Wells Alan 
Published: July 2021

Angiogenesis plays a critical role in tissue development and wound repair. Regulation of the vascular tree is critical to organogenesis and neo-organogenesis. The initial vigorous vessel proliferation and arborization that occurs during tissue development and wound repair must be pruned to form mature tissue. The signals that trigger this vascular regression are still ill-defined. Mitotic endothelial cells express the CXC receptor 3 (CXCR3), which binds the ELR-negative chemokines IP-10 (CXCL10), platelet factor 4 (PF4, CXCL4), IP-9/I-TAC (CXCL11), and MIG (CXCL9), with IP-9 and IP-10 being expressed during the later remodeling phases of wound repair when vascular involution occurs. We hypothesized that not only does this signaling system limit angiogenesis, but that CXCR3 signaling also compromises endothelial cell tube integrity contributing to regression. Treatment of newly formed tubes/cords on Matrigel (in vitro) or vessels in a subcutaneous Matrigel plug (in vivo) with IP-10 in either presence or absence of VEGF caused a dissociation of the tubes and subsequent vascular involution. This followed CXCR3 induced cleavage of β3 integrin in endothelial cells. CXCR3 triggering of mu-calpain activity caused cleavage of the cytoplasmic tail of human β3 integrins at the calpain cleavage sites NPLY747 and TSTF754. IP-10 stimulation was also found to activate caspase 3, blockage of which prevented cell death but not tube dissociation. Thus, CXCR3 activation in newly formed vessels results in endothelial dissociation from matrix, followed by anoikis. This is the first direct evidence for an extracellular signaling mechanism through CXCR3 causing the dissociation of newly formed blood vessels.
This study was supported by funds from VA Medical Research Program and National Institute of General Medical Sciences (NIH, USA).
Abstract 3385: Cardiac Stem Cell Survival During DNA Damage Is Regulated By Modulation Of Histone Acetyl Transferase p300
Authors: Jain Sumit , Rodrigues Claudia , Yuan Huijun , Liu Jing , Webster Keith A , Bishopric Nanette H 
Published: July 2021

Background: Many sources of oxidative stress, including ischemia/reperfusion and chemotherapy, can induce DNA damage in the heart, leading to loss of cardiomyocytes and development of heart failure. Cardiac stem cells (CSCs) may serve to regenerate injured myocardium. However, ischemic injury has been associated with senescence and apoptosis in CSCs, potentially limiting their contribution to tissue repair. The histone acetyltransferase p300 has been shown to be an important regulator of genome stability and is involved in cell survival in response to DNA damage. We hypothesized that p300 could play a role in CSC response to oxidant stress.
Methods: c-kit+, sca-1+ CSCs were isolated and cloned by serial dilution from adult mouse hearts. CSC clones were stably transfected with lentivirus vectors carrying shRNAmir against p300 or a non-silencing RNA (NS) and maintained under puromycin selection prior to treatment. CSCs were exposed to doxorubicin (DOX), a DNA-damaging agent, for defined intervals. Levels of p300 were monitored by Western analysis and expression of DNA damage-regulated genes was assessed using multiplex realtime QPCR. CSCs were treated with 1 uM DOX and survival was assessed over a period of 72h.
Results: Levels of p300 increased rapidly in CSCs within 4h after exposure to doxorubicin and were sustained for at least 24h. CSCs expressing p300 shRNAmir (p300 KD) contained ~20% of the amount of p300 in CSCs transfected with the NS sequence (NS CSCs). QPCR analysis of DNA repair and cell cycle control genes indicated that 44 were significantly expressed in CSCs; 13 of these were up-regulated by p300 loss, and 5 were down-regulated, indicative of a DNA damage response to p300 loss.
Both cell proliferation rates and progression through the cell cycle were slower in p300 KD vs. NS CSCs. Surprisingly, following 48h treatment with DOX, p300 KD CSCs had 32% improved survival vs. NS CSCs.
Conclusions: Under acute genotoxic stress, p300 accumulates in CSCs. Reducing p300 levels induces the expression of genes involved in the DNA damage response, probably as a compensatory response, and slows CSC proliferation. Loss or inhibition of p300 is likely to cause multifactorial growth arrest in CSCs and thereby reduce their vulnerability to genotoxic agents.
This research has received full or partial funding support from the American Heart Association, AHA National Center.