bluuebunny/crossref_metadata_2025 huggingface dataset:
Very Long-Term Results (More Than 20 Years) of Valve Repair With Carpentier’s Techniques in Nonrheumatic Mitral Valve Insufficiency
Authors: Braunberger E. , Deloche A. , Berrebi A. , Fayssoil A. , Celestin J.A , Meimoun P. , Chatellier G. , Chauvaud S. , Fabiani J.N. , Carpentier A.
Published: July 2021

Background

Mitral valve repair is considered the gold standard in surgery of degenerative mitral valve insufficiency (MVI), but the long-term results (>20 years) are unknown.

Methods and Results

We reviewed the first 162 consecutive patients who underwent mitral valve repair between 1970 and 1984 for MVI due to nonrheumatic disease. The cause of MVI was degenerative in 146 patients (90%) and bacterial endocarditis in 16 patients (10%). MVI was isolated or, in 18 cases, associated with tricuspid insufficiency. The mean age of the 162 patients (104 men and 58 women) was 56±10 years (age range 22 to 77 years). New York Heart Association functional class was I, II, III, and IV in 2%, 39%, 52%, and 7% of patients, respectively. The mean cardiothoracic ratio was 0.58±0.07 (0.4 to 0.8), and 72 (45%) patients had atrial fibrillation. Valve analysis showed that the main mechanism of MVI was type II Carpentier’s functional classification in 152 patients. The leaflet prolapse involved the posterior leaflet in 93 patients, the anterior leaflet in 28 patients, and both leaflets in 31 patients. Surgical technique included a Carpentier’s ring annuloplasty in all cases, a valve resection in 126 patients, and shortening or transposition of chordae in 49 patients. During the first postoperative month, there were 3 deaths (1.9%) and 3 reoperations (2 valve replacements and 1 repeat repair [1.9%]). Six patients were lost to follow-up. The remaining 151 patients with mitral valve repair were followed during a median of 17 years (range 1 to 29 years; 2273 patient-years). The 20-year Kaplan-Meier survival rate was 48% (95% CI 40% to 57%), which is similar to the survival rate for a normal population with the same age structure. The 20-year rates were 19.3% (95% CI 11% to 27%) for cardiac death and 26% (95% CI 17% to 35%) for cardiac morbidity/mortality (including death from a cardiac cause, stroke, and reoperation). During the 20 years of follow-up, 7 patients were underwent surgery at 3, 7, 7, 8, 8, 10, or 12 years after the initial operation. Valve replacement was carried out in 5 patients, and repeat repair was carried out in 2 patients. At the end of the study, 65 patients remained alive (median follow-up 19 years). Their median age was 76 years (age range 41 to 95 years). All except 1 were in New York Heart Association functional class I/II.

Conclusions

Mitral valve repair using Carpentier’s technique in patients with nonrheumatic MVI provides excellent long-term results with a mortality rate similar to that of the general population and a very low incidence of reoperation.
Abstract 583: RNAi Therapeutics For The Lowering Of Cholesterol
Authors: Fitzgerald Kevin , Frank-Kamenetsky Maria , Zimmermann Tracy S , Horton Jay , Akinc Akin , Bramlage Birgit , Bumcrot David , Harborth Jens , John Matthias , Kotelianski Victor , Manoharan Muthiah , Maier Martin , Nechev Lubomir , Racie Timothy , Roehl Ingo , Seiffert Stephan , Shanmugam Sumi , Soutschek Jurgen , Toudjarska Ivanka , Vornlocher Hans-Peter , Zedalis William
Published: July 2021

Delivery of small interfering RNAs (siRNAs)
in vivo
, using clinically relevant modes of administration, is critical for the advancement of RNA interference (RNAi) therapeutics. In this work, we demonstrate systemic delivery of siRNAs and potent
in vivo
down-modulation of two important disease targets, apolipoprotein B (apoB) and proprotein convertase subtilisin kexin 9 (PCSK9). A single injection of liposomal siRNA resulted in >90% silencing of apoB mRNA expression in the liver 48 h after administration. The effect was demonstrated to occur through cleavage of the apoB mRNA at precisely the site predicted for the RNAi mechanism. Reductions in apoB protein, cholesterol, and low-density lipoprotein (LDLc) levels were observed in 48 hours that lasted for at least 23 days, thus demonstrating an immediate, potent and durable biological effect. In addition to apoB we have also demonstrated the ability to down-modulate other important liver targets such as PCSK9. PCSK9 has been closely implicated in LDLc regulation. We have demonstrated PCSK9 down-modulation in several animal models including, mouse, humanized mouse, rat, and non-human primate. Down-modulation of PCSK9 levels resulted in significant lowering of cholesterol (20 – 60%) in all animal models tested. These findings strongly support the potential of RNAi therapeutics as a new class of drug for metabolic and cardiovascular diseases. Our next steps include selecting the most potent lead molecule and moving it into GLP safety studies.
Abstract 591: Leptin receptor long Isoform is Increased and Positively Correlated with Augmented Macrophage Infiltration and Plaque Neovascularization in Carotid Plaques from Neurologically Symptomatic Patients
Authors: Schneiderman Jacob , Simon Amos J , Schroeter Marco R , Engelberg Isaac , Flugelman Moshe Y , Konstantinides Stavros , Shaefer Katrin
Published: July 2021

Objectives:
Symptomatic carotid plaques are frequently characterized by increased inflammation and neovascularization. As leptin promotes plaque angiogenesis and activates inflammatory cells, and since the leptin receptor (ObR) antigen was demonstrated in advanced atherosclerotic plaques, we undertook a systematic analysis of carotid symptomatic and asymptomatic plaques, quantitatively assessing ObR mRNA expression and immunoreactivity. Moreover, parallel analyses of VEGF
165
, and Mac-1 transcript were performed, to account for plaque angiogenesis and macrophage density , respectively.

Methods:
Carotid endarterectomy (CEA) plaques were collected from 26 patients undergoing surgery for hemispheric cerebro-vascular symptoms (13), or progressive asymptomatic internal carotid stenosis (13). A representative sample collected from each plaque was used in real-time quantitative (RQ) PCR analysis for ObRl (long), ObRc (common) isoforms, VEGF
165
, and Mac-1 transcripts. All plaques were classified histologically according to the AHA guidelines. Also, immunohistochemistry for von Willebrand factor (vWF) and ObR antigen were performed.

Results:
All plaques exhibited advanced atherosclerosis, and AHA class V through VI were equally diagnosed in both groups. Interestingly, ObRl transcript levels were preferentially elevated in symptomatic plaques (P<0.03). A similar upregulation was demonstrated for VEGF
165
(P<0.017), and Mac-1 (P<0.013). Immunohistochemical analyses also demonstrated that ObR antigen was significantly augmented in symptomatic plaques (P<0.05), and frequently colocalized with abundance of inflammatory cells. Neovascularization was evident in all plaques, and marginally increased in symptomatic plaques.

Conclusions:
ObR-1 gene is preferentially upregulated, in clinically symptomatic carotid plaques, and positively correlated with simultaneous augmentation of VEGF
165
and Mac-1 transcript. These data suggest that ObR-1 may be linked with histological features of plaque instability, like neovascularization and macrophage infiltration, shown to be associated with ischemic symptoms from carotid unstable plaques.
Abstract 602: FoxO3 Mediates Urotensin-II-induced MMP2 Expression In Pulmonary Artery Smooth Muscle Cells
Authors: Diebold Isabel , Petry Andreas , Burger Max , Djordjevic Talija , Hess John , Görlach Agnes
Published: July 2021

Vascular remodeling in pulmonary hypertension (PH) results from persistent vasoconstriction, extracellular matrix degradation, cell migration and proliferation of vascular cells. However, the mechanisms linking these events are not well understood. Human urotensin-II (hU-II) is a newly identified vasoactive peptide which by binding to a G-protein-coupled receptor termed GPR-14, acts as the most potent vasoconstrictor known. Although hU-II has been associated with vascular remodeling in PH, its precise role in regulating remodeling processes is not well understood. Since degradation of the extracellular matrix plays a pivotal role in pulmonary vascular remodeling, we investigated whether hU-II is linked to expression and activity of matrix metalloproteinases (MMP) in pulmonary artery smooth muscle cells (PASMC). By immunohistochemistry we found high levels of GPR-14 and MMP2 in the media and intima of pulmonary vessels in tissue samples from patients with pulmonary vascular remodeling. In vitro experiments confirmed that hU-II time-dependently enhanced MMP2 mRNA and protein levels as well as MMP2 activity determined by zymography in PASMC. Actinomycin D inhibited hU-II-stimulated MMP2 expression indicating the involvement of a transcriptional mechanism. Bioinformatic analysis of the MMP2 promoter revealed the presence of a consensus site for FoxO3. Reporter gene assays confirmed that hU-II can increase the activity of FoxO transcription factors. Downregulation of FoxO3 by siRNA inhibited hU-II-induced MMP2 expression. Conversely, overexpression of wild type or constitutively active FoxO3 increased MMP2 expression. Consistently, hU-II increased FoxO3 levels in PASMC. FoxO3 was also strongly expressed in the media and intima of pulmonary vessels in tissue samples from patients with pulmonary vascular remodeling. In summary, this study demonstrates that hU-II induces MMP2 via FoxO3. Since the hU-II receptor GPR-14, MMP2 and FoxO3 colocalized in the media and intima of pulmonary vessels from patients with pulmonary vascular remodeling, these findings suggest an important role of this pathway in promoting pulmonary vascular remodeling and implicate FoxO3 as a potential therapeutic target for combating vascular remodeling in PH.
Abstract 604: Targeting Cx43 Prevents Growth Factor-Induced Phenotypic Change in Pig Coronary Artery Smooth Muscle Cells
Authors: Chadjichristos Christos E , Morel Sandrine , Derouette Jean-Paul , Brisset Anne , Roth Isabelle , Sutter Esther , Bochaton-Piallat Marie-Luce , Kwak Brenda R
Published: July 2021

Percutaneous coronary intervention (PCI) is commonly used to treat atherosclerotic coronary arteries, but its efficacy is limited by restenosis at the site of the intervention. We reported previously that reducing the expression of the gap junction protein connexin43 (Cx43) in mice restricted neointima formation after acute vascular injury by limiting the inflammatory response as well as the proliferation and migration of smooth muscle cells (SMCs) towards the damaged site. SMC populations isolated from the pig coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening suggesting that they participate in the intimal thickening. Here, we further investigate the relationship between connexin expression and SMC phenotype using the distinct types of pig coronary artery SMCs. We show that Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was down-regulated and Cx43 was markedly up-regulated in SMCs of stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. Cx43 expression was increased in R-SMCs and these cells no longer expressed Cx40. When S-SMCs were treated with 10 ng/ml platelet-derived growth factor (PDGF-BB) they acquired a rhomboid phenotype and their migratory activity increased (from 40.3±5.7 to 185.9±27.3 migrating cells; mean±SEM, N=4, P<0.01). These changes were accompanied by an increase in Cx43 and loss of Cx40 expression. Importantly, PDGF-BB-induced phenotypic change of S-SMCs was prevented by reducing Cx43 expression with 100 uM antisense for Cx43. Thus, Cx43 antisense-treated SMCs retained their typical elongated appearance and the expression of some SMC differentiation markers, such as alpha-SM actin, whereas the appearance of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in SMCs prevents growth factor-induced changes towards a deleterious phenotype. Our findings suggest that Cx43 might be an additional target for local delivery strategies aimed at reducing restenosis after PCI.
Abstract 629 Secreted Frizzled Related Protein 2 Protects Cells from Apoptosis by Blocking the Effect of Canonical Wnt3a
Authors: Zhang Zhongyan , Deb Arjun , Pachori Alok , He Wei , Guo Jian , Zhang Zhiping , Pratt Richard E , Dzau Victor J
Published: July 2021

We have demonstrated that mesenchymal stem cells (MSCs) overexpressing the survival gene Akt can confer paracrine protection to ischemic myocytes both in vivo and in vitro through the release of secreted frizzled related protein 2 (Sfrp2). However, the mechanisms mediating these effects of Sfrp2 have not been fully elucidated. In this study, we test the hypothesis that Sfrp2 exerts anti-apoptotic effect by antagonizing pro-apoptotic properties of specific Wnt ligands. To explore this possibility, we studied rat cardiomyoblasts subjected to hypoxia reoxygenation (HR) injury. We examined the effect of Wnt3a and Sfrp2 on HR-induced apoptosis. We choose to study Wnt3a because its expression is up-regulated in response to hypoxia. Wnt3a (3nM) significantly increased cellular caspase activities (35% increase, p<0.05, n=3) and TUNEL staining (38% increase) in response to HR. Sfrp2 attenuated significantly Wnt3a-induced caspase activities in a concentration dependent fashion (0nM, 8.50±0.47; 3nM, 7.28±1.03; 30nM, 7.45±0.49; 300nM, 5.65±0.35), achieving 36% inhibition at a concentration of 300nM. Using a solid phase binding assay, our data demonstrates that Sfrp2 physically binds to Wnt3a. In addition, we observed that 600nM Sfrp2 inhibits 47% of the beta-catenin / TCF transcriptional activities induced by Wnt3a(0.55±0.07 vs 1.03±0.04). Impressively, Dickkopf-1, a protein that binds to the Wnt coreceptor LRP, significantly inhibited the Wnt3a-activated caspase and transcriptional activities by 31% and 77% respectively (caspase activities: 8.62±0.05 vs12.54±0.49; transcriptional activities: 0.41±0.03 vs 1.77±0.01) Similarly, siRNA against beta-catenin inhibited the Wnt3a-activated caspase activities by 59% (6.44±0.92 vs 15.71±0.67). Consistent with this, significantly fewer TUNEL positive cells were observed in siRNA transfected cells than in control cells (43% decrease). Together, our data provide strong evidence to support the notion that Wnt3a is a canonical Wnt with pro-apoptotic action whose cellular activity is prevented by, at least in part, Sfrp2 through direct binding of these molecules. These results can explain the in vivo protective effect of Sfrp2 and highlight its therapeutic potential for the ischemic heart.
Abstract 647: Uncoupling Protein 3 Does Not Regulate Myocardial Fatty Acid Oxidation Rates But Regulates ROS Production and Cardiac Efficiency
Authors: Boudina Sihem , Tuinei Joseph , Theobald Heather , Sena Sandra , Abel E. Dale
Published: July 2021

The biological functions of uncoupling protein 3 (UCP3) in cardiac muscle are incompletely understood. These studies were designed to investigate the role of UCP3 in cardiac muscle energy metabolism under normal and high fat diet (HFD) conditions. Mice lacking UCP 3 (UCP3KO) and wildtype (WT) littermate controls were fed normal chow (NC) or HFD for 10 weeks. Palmitate oxidation rates (POX), myocardial oxygen consumption (MVO2), cardiac efficiency (CE) and cardiac power were determined in isolated working hearts perfused with 5mM glucose and 0.4mM palmitate. Mitochondrial respiration and ATP synthesis were determined in saponin-permeabilized cardiac fibers. ROS levels were determined using the dye dichlofluorescein (DCF). HFD increased UCP3 protein levels by 2-fold in WT (p<0.005). On NC, POX rates were similar between UCP3KO and WT and increased similarly with HFD to 726.5 ± 41.8 and 731.7 ± 7.1 nmol/min/mg DHW, respectively. MVO2 increased by 21% in WT on HF (P < 0.05) but did not increase in HFD UCP3KO. On the HFD, cardiac efficiency (16.3±1.9 vs. 20.4±1.2) and cardiac power (38.1±1.5 vs.45.8±2.6, mW/g) were lower in WT vs. UCP3KO (p<0.05). Mitochondrial state 3 respirations were similar in WT and UCP3KO under NC or HFD conditions. However, ATP synthesis rates were 28% higher in HF UCP3KO vs. HF WT. HFD increased ROS generation by 49% (P < 0.005) in WT hearts. However, ROS generation increased to a greater extent in UCP3KO on HF when compared to WT on HF (212.1 ± 16.2 versus 177.8 ± 9.1, P < 0.05). Thus the efficiency of ATP production was increased in UCP3KO hearts despite higher levels of ROS. In conclusion, UCP3 mediates the increase in MVO2 that accompanies HF feeding without influencing the increase in FA Oxidation. UCP3 limits FA-induced ROS accumulation, by reducing membrane potential and mitochondrial ATP generation and regulates the efficiency of ATP generation in the heart.
Abstract 663: Loss of Peroxisome Proliferator-Activated Receptor-gamma in Endothelial Cells Causes Pulmonary Arterial Hypertension in Transgenic Mice
Authors: Guignabert Christophe , Alastalo Tero-Pekka , Wang Lingli , Rabinovitch Marlene
Published: July 2021

Introduction:
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear receptor expressed in endothelial cells (EC) and known to regulate enzymes important in vascular dilatation and angiogenesis such as endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1). Expression of PPAR-gamma is reduced in the pulmonary arteries (PA) of patients with severe PA hypertension (PAH).

Objective:
We therefore investigated whether loss of PPAR-gamma in EC might cause PAH under normoxic conditions or might worsen the severity of PAH induced by chronic hypoxia.

Materials and Methods:
We generated transgenic mice with targeted deletion of PPAR-gamma in ECs by breeding a mouse expressing Cre under the regulation of the Tie2 promoter with a mouse in which critical exons of PPAR-gamma were flanked by LoxP sites. We compared right ventricular systolic pressure (RVSP), RV hypertrophy (RV/LV+S), in Tie2CrePPAR-gamma
flox/flox
and wild type littermate controls (WT) in normoxia and following 3 weeks of chronic hypoxia (CH) (10 mice per group, 5 per gender) and also assessed muscularization of distal pulmonary vessels and number of peripheral arteries in CH.

Results:
Tie2CrePPAR-gamma
flox/flox
vs. WT mice had mildly elevated RVSP (24.6 vs 21.56mmHg) and RVH during normoxia (P <0.01 for both) with similar systemic blood pressures. However the RVSP following CH was similar in Tie2CrePPAR-gamma
flox/flox
and WT mice (28.1mmHg vs 28.5mmHg) as was RVH and muscularizaton of distal vessels, and number of arteries relative to alveoli.

Conclusions:
Thus loss of PPAR-gamma in ECs appears to result in mild pulmonary vasoconstricton, and while not exacerbated during chronic hypoxia, this feature may have an impact on recovery during return to normoxia, if EC production of eNOS and HO-1 are necessary for regression of vascular disease.
Abstract 665: Hypertension Is Associated With Phenotypic Changes Of Peripheral Blood Monocytes.
Authors: Guzik Tomasz J , Bzowska Malgorzata , Czesnikiewicz-Guzik Marta , Jasiewicz-Honkisz Barbara , Adamek-Guzik Teresa , Korbut Ryszard , Pryjma Juliusz
Published: July 2021

While immunopathogenesis of atherosclerosis is well defined, immunological mechanisms of hypertension (HTN) and its link to atherosclerosis remain unclear. We hypothesized, that primary HTN is associated with a pro-inflammatory and pro-atherogenic phenotype of circulating monocytes, characterized by co-expression of the Fc-gamma receptor (CD16+) and LPS receptor (CD14+high).

Methods: 132 subjects (74M and 58F) with typical clinical risk factors of atherosclerosis were studied. HTN (85%) was diagnosed based on blood pressure monitoring and use of anti-HTN drugs. Monocyte characteristics in the peripheral blood were determined by flow cytometry and plasma cytokine levels by ELISA.

Results: CD16+CD14+ cells were more prevalent in subjects with HTN and were highest in subjects whose blood pressure was poorly controlled (Figure

). A significant correlation between blood pressure levels at the time of study (JNC7) and prevalence of CD14+CD16+ monocytes was found (Figure

). These relationships remained significant in multi-variate analysis (p = 0.01; type 3 sum of squares ANOVA) taking into account other major risk factors for atherosclerosis or treatments. Further analysis of subpopulations showed that levels of CD16 +CD14+ (high) monocytes, rather than CD16 + CD14(dim) revealed a strong relationship to hypertension. These monocytes showed significantly higher expression of activation markers HLA-DR and CD45RA and TNF-α production(p<0.05). These results provide a novel marker of inflammation in hypertension and may provide a link between hypertension and the development of atherosclerosis, which has been clinically well defined, but mechanistically unclear.
Abstract 724: Synectin is Required for Normal PDGF Secretion and Smooth Muscle Cell Basic Science Recruitment
Authors: Paye Julie M , Chittenden Thomas W , Phng Li-Kun , Gerhardt Holger , Simons Michael
Published: July 2021

Background: Synectin, a ubiquitously expressed scaffold and PDZ protein, has been shown to be a key regulator in the formation of arterial vasculature. Primary arterial endothelial cells isolated from synectin−/− mice exhibited defects in PDGF signaling that were not present in arterial synectin+/+ or either venous synectin+/+ or synectin−/− endothelial cells.

Methods: To address the role of synectin in mural cell recruitment, retinas from synectin−/− (KO) and synectin+/+ (WT) mice were stained for isolectin and alpha-smooth muscle actin. To determine the mechanisms responsible for defective smooth muscle cells recruitment, primary arterial and venous endothelial (EC) and smooth muscle (SMC) cells were isolated from synectin+/+ and synectin−/− mice.

Results: Examination of the retinal vasculature in synectin−/− mice demonstrated poor smooth muscle cell coverage of the forming arterial tree with a number of SMC cells sitting some distance away from the vessels, a defect reminiscent of retinal abnormalities observed in PDGF-B−/− mice. Western blot analysis of lysates from primary KO SMC demonstrated levels of PDGFR-β analogous to WT, although expression of PDGF-B was markedly reduced in lysates from arterial, but not venous, primary KO endothelial cells. Transduction of KO EC with an adenoviral synectin construct was sufficient to restore PDGF-B protein levels. Further, qPCR was used to determine whether defects in PDGF-B protein production were at the transcription or translation level. Interestingly, there was only a minor decrease in the amount of PDGF-B mRNA in KO compared to WT EC, suggesting that synectin deficiency has selectively affected post-transcriptional regulation of PDGF-B expression. Migration of WT and KO SMC in response to PDGF-B or conditioned media from WT and KO endothelial cells was also assessed to determine the cell type specificity of PDGF defects in KO mice.

Conclusions: Synectin is required for post-transcriptional regulation of PDGF-B in endothelial cells. Deficits in PDGF-B secretion by synectin−/− endothelial cells result in inadequate smooth muscle recruitment and coverage of arterial vessels.
Abstract 725: Protein Kinase C delta Inhibits Type I Collagen Basal Expression through CCAAT-binding Factor and c-Krox in Vascular Smooth Muscle Cells
Authors: Kamiya Kentaro , Zohlman Andrew , Rapp Amy , Wang Chunjie , Kent K. Craig , Liu Bo
Published: July 2021

Protein Kinase C delta (PKCδ) regulates multiple functions in vascular smooth muscle cells (VSMCs) including proliferation, migration, and apoptosis. In previous studies, we have demonstrated that PKCδ activity is necessary for TGFβ-induced fibronectin production in VSMCs. The purpose of the current study is to understand the role of this kinase in regulation of type I collagen synthesis. Using A10 VSMCs derived from rat thoracic aorta, we showed that TGFβ (5ng/ml x 48 hours) increased type I collagen expression. Pretreatment of A10 with Rottlerin (2uM, 1 hour), a selective inhibitor of PKCδ, dramatically increased type I collagen basal expression independent of TGFβ. To understand the mechanism involved with PKCδ, we used a Luciferase reporter that contains the human COL1A2 promoter (−772 to +58). Rottlerin stimulated the Luciferase reporter activity by 3.53±0.13 fold (n=3, p<0.01). Moreover, inhibition of PKCδ with a dominant negative mutant led to upregulation of reporter activity (1.77±0.15 fold induction, n=3, p<0.01), while activation of PKCδ with a constitutive active mutant reduced collagen reporters (0.19±0.02 fold reduction, n=3, p<0.01). Using a series of 5′ deletions of COL1A2/Luciferase constructs (−772, −353, and −108), we narrowed the location of the PKCδ-response element to the -108/+58 region of the COL1A2 promoter, which is distinct from the TGFβ response element that is further upstream. This region contains a CCAAT motif and a c-Krox motif that have been previously implicated in collagen expression. Using a gel shift assay, we found that inhibition of PKCδ with Rottlerin or the dominant negative mutant increased the binding of CCAAT-binding Factor (CBF) to the CCAAT motif but decreased the binding at the c-Krox site. Taken together, our data indicate that PKCδ inhibits type I collagen basal expression by affecting its gene transcription through a mechanism involving CBF and c-Krox in VSMCs. We believe that PKCδ is one of the key players in mediating extracellular matrix production and could contribute to pathogenesis of vascular diseases.
Abstract 741: Atrial Fibrillation Begets Atrial Fibrillation: Acute Autonomic Remodeling Leads to Enhanced Inducibility for Atrial Fibrillation
Authors: Lu Zhibing , Scherlag Benjamin J , Niu Guo-Dong , Lin Jiaxiong , Ghias Muhammad , Lazzara Ralph , Jackman Warren M , Po Sunny S
Published: July 2021

Introduction:
There have been many forms of remodeling reported to play a role in the concept of “atrial fibrillation (AF) begets atrial fibrillation.” The role of intrinsic cardiac nervous system (ICNS) in this remodeling process was evaluated in this study.

Methods:
Continuous rapid pacing (1200 bpm, 2× threshold, TH) was performed at the left atrial appendage to maintain AF. Group 1 (N=7): 6-hours of pacing followed by ganglionated plexi (GP) ablation, both left and right side; Group 2 (N=7): GP ablation followed by 6-hours of pacing. At 2x and 10x TH, the effective refractory period (ERP) and window of vulnerability (WOV), i.e., longest-shortest coupling of premature stimulus which induced AF in milliseconds (ms), were measured at the left atrium, right atrium and pulmonary veins (PVs) every hour during the 6-hours of pacing before and after GP ablation.

Results:
In group 1 (rapid pacing with GP intact) ERP progressively shortened in the first two hours and then stabilized both at 2×TH and 10×TH (Figure 1A

), however, WOV progressively widened throughout the time period (Figure 1B

). After GP ablation, ERP was significantly longer than prior to ablation and AF could not be induced (WOV=0, p<0.01) at any pacing site at either 2×TH or 10×TH (Figure 1B

). In group 2 (initial GP ablation), ERP exhibited a slightly increasing trend as the pacing time increased. AF could not be induced in 6/7 dogs during the 6-hour pacing, and was inducible in 1/7 with a cumulative WOV of only 10 ms.

Conclusion:
The ICNS is crucial for the process of “AF begets AF” in the acute stages of remodeling by rapid atrial pacing.
Abstract 751: Integrated Genetic Linkage Analysis and Expression Profiling in the Rat Heart to Identify Primary Drivers of Cardiac Hypertrophy
Authors: Sarwar Rizwan , Petretto Enrico , Lu Han , Schroen Blanche , Kumaran Mande K , Grieve Ian C , Fischer Judith , Hubner Norbert , Mangion Jonathan , Pinto Yigal M , Pravenec Michal , Aitman Timothy J , Cook Stuart A
Published: July 2021

Intro:
Although up to 60% of left ventricular mass (LVM) can be accounted for by extra-cardiac factors, the cause of remaining variance is uncharacterised.

Hypothesis:
Cardiac gene expression is under genetic control and these genetic effects account, at least in part, for the uncharacterised component of LVM.

Method:
We combined genetic linkage analysis with genome-wide expression profiling in a recombinant inbred (RI) rat strain panel to map the genetic determinants of cardiac gene expression, taking into account naturally occurring variation in blood pressure. Cardiac gene expression in 29 RI strains was quantified with 128 Affymetrix 230 2.0 microarrays, and linkage analysis of gene expression was performed with correction for multiple testing. Candidate genes for LVM were defined as gene colocalised with regions of the rat genome previously associated with LVM. Candidate genes identified in the rat were prioritised by assessing whether their human orthologues were dynamically regulated in heart biopsies from patients with cardiac hypertrophy undergoing surgery for aortic stenosis (
n
=20) as compared to controls (
n
=7), as determined with Affymetrix U133 microarrays.

Results:
We showed that genetic regulation of cardiac transcription is predominant when compared to extra-cardiac effects. This enabled us to determine the major control points of cardiac gene expression in the rat (
n
=3,744, genome-wide
P
<0.05). A subset of 50 genes that mapped to themselves and colocalised with regions of the rat genome known to regulate LVM were identified. One of these 50 rat genes was mimecan or osteoglycin precursor (
Ogn
), whose orthologue showed the highest correlation with LVM out of the 22,284 probesets used in the human microarray analysis (
r
=0.62,
P
=0.0008). We went on to refine the rat QTL associated with
Ogn
(peak LOD 4), and identified sequence variations that might be causative. We then showed that cardiac protein levels of OGN are increased in both rat and human hypertrophy.

Conc:
Combined linkage and expression studies provide a new and powerful systems approach to dissecting the pathophysiology of genetically complex traits. These data implicate
Ogn
as a primary genetic driver and biomarker of cardiac hypertrophy and warrant further functional testing.
Abstract 804: Noninvasive Integrated SPECT/CT Molecular Imaging of Activated Factor XIII Activity in Thrombosis
Authors: Jaffer Farouc A , Figueiredo Jose L , Wojtkiewicz Gregory , Zhang Hanwen , Patel Purvish , Nahrendorf Matthias , Tung Ching-Hsuan , Weissleder Ralph
Published: July 2021

Background
: Activated factor XIII (FXIIIa) is a blood transglutaminase that mediates fibrinolytic resistance and is a hallmark of acute thrombi. Noninvasive molecular imaging of FXIIIa may offer a novel approach to identify acute thrombi and to gauge fibrinolytic resistance in vivo. Here we developed and validated a FXIIIa thrombosis imaging strategy using noninvasive integrated SPECT/CT.

Methods:
A FXIIIa-targeted peptide agent (F13) was synthesized using NQEQVSPLTLLK chelated to DOTA and then labeled with
111
InCl
3
. A control agent (C13,
111
In-NAEQVSPLTLLK) was analogously synthesized. In vitro validation of the F13 agent was performed in human plasma clots. Next, the in vivo blood-half life of F13 was determined in mice (n=4). In vivo thrombosis studies (n=15 mice) were then performed using 10% ferric chloride jugular venous thrombi aged 1 hour or 16 hours. Mice were intravenously injected with 200 μCi of F13 or C13. After 4 hours, mice underwent integrated CT angiography (72 μm isotropic resolution) and SPECT imaging (32 minute acquisition). In situ thrombi were then resected for radioactivity and weight measurements.

Results
: Human plasma clots incubated with F13 showed 280–740% greater counts per minute (CPM) than controls (p<0.01). F13 binding was dose-dependent and >90% inhibited by pretreatment with iodoacetamide, an alkylating agent. The blood half-life of F13 was calculated to be 16 minutes. In one hour thrombi, in vivo SPECT/CT imaging revealed strong focal F13 SPECT signal in the co-registered ipsilateral venous thrombi but not the contralateral normal jugular vein. One hour thrombi in the F13 group had 15-fold greater radioactivity than the C13 group (4.6±3.6% vs. 0.3±0.2% injected dose per gram tissue, IDGT, p<0.01). Compared to 1 hour thrombi, 16 hour old thrombi had 4-fold less F13 radioactivity (1.1%±0.1% IDGT, p<0.05).

Conclusions
: Blood transglutaminase FXIIIa can be noninvasively detected using a FXIIIa-sensitive and specific imaging agent for integrated SPECT/CT. The current in vivo results further validate that activated factor XIII is a hallmark of acute thrombi and declines in activity over time. This clinically translatable imaging strategy could permit visualization of FXIIIa in patients with thrombotic syndromes.
Abstract 810: Recruitment of the Inflammatory Subset of Monocytes to Sites of Ischemia Induces Angiogenesis in a Monocyte Chemoattractant Protein-1 Dependent Fashion
Authors: Capoccia Ben , Gregory Alyssa , Link Daniel
Published: July 2021

Our lab has previously shown that mobilization of monocytes from the bone marrow to the blood by G-CSF and/or AMD3100 administration stimulated angiogenesis and enhanced revascularization following the induction of hindlimb ischemia in mice. Recent data suggest that monocytes can be divided into two distinct subsets based upon the expression of the fractalkine receptor CX3CR1. The inflammatory subset of monocytes express low levels of CX3CR1 but high levels of CCR2 and Gr-1. In contrast, the resident subset of monocytes express high levels of CX3CR1 but no CCR2 or Gr-1. Using transgenic mice expressing GFP under the control of the 2.1kb CX3CR1 promoter, we sorted inflammatory and resident monocytes and adoptively transferred them into mice 24 hours after the induction of hindlimb ischemia. Whereas inflammatory monocytes markedly improved revascularization, no effect was observed with resident monocytes. These data suggest that the inflammatory monocyte subset may be an attractive cell population to study in the therapeutic setting. The mechanism by which monocytes stimulate angiogenesis at sites of inflammation is not fully understood. Herein, we show that adoptively transferred CFSE-labeled monocytes are rapidly and specifically recruited from the blood to the ischemic hindlimb. These recruited donor monocytes induce a massive secondary recruitment of host monocytes that is associated with local production of MCP-1. To further explore the role of MCP-1 in this process, we repeated these adoptive transfer experiments in MCP-1
−/ −
mice. Adoptive transfer of wild type monocytes into wild type mice markedly improved revascularization while no angiogenic effect was observed after adoptive transfer into MCP-1
−/ −
mice. Moreover, whereas recruitment of adoptively transferred CFSE-labeled monocytes into the ischemic limb in MCP-1
−/ −
mice was normal, the induction of MCP-1 expression and secondary recruitment of endogenous monocytes from the blood was significantly attenuated. Furthermore, MCP-1
−/ −
mice showed no enhancement of angiogenesis following mobilization with G-CSF and AMD3100. These data suggest that MCP-1 plays a key role in the inflammatory angiogenic response by recruiting host monocytes from the blood into the ischemic tissue.
Abstract 820: Autoregulation of CGRP Expression via β
2
-Adrenoreceptor Signaling in Intrinsic Cardiac Adrenergic Cells
Authors: Nguyen Vince , Zarraga Ignatius Gerardo , Rastogi Saurabh , Birnbaum Yochai , Uretsky Barry , Huang Ming-He
Published: July 2021

Background
: Calcitonin gene-related peptide (CGRP) is a neuropeptide with broad salutary cardiovascular effects. Mechanisms underlying cardiac CGRP regulation are poorly understood. The intrinsic cardiac adrenergic (ICA) cell is a novel cardiac neuroendocrine cell that expresses the δ-opioid receptor. We have shown that δ-opioid stimulation of ICA cells induces epinephrine liberation exerting an infarct-size limiting effect via β
2
-adrenoreceptor (β
2
-AR) stimulation. In this study we hypothesize that ICA cells synthesize and release CGRP which is involved in myocardial function and that CGRP gene expression can be autoregulated by epinephrine released from the ICA cell or regulated exogenously via β
2
-AR agonist.

Methods and Results
: In situ hybridization coupled with immunofluorescent double labeling localized CGRP mRNA expression exclusively to ICA cells in explanted human left ventricular tissue. To determine whether δ-opioid-enhanced epinephrine release from ICA cells autoregulates CGRP gene expression, isolated and cultured (for 2-weeks) adult rat ICA cells were treated with δ-opioid agonist DPDPE (0.1 μM for 1h) in the absence and presence of the β
2
-AR antagonist ICI-118551 (ICI, 10 μM). DPDPE induced 4-fold increase in CGRP mRNA at 6h post DPDPE-treatment by real-time PCR. This effect was abolished in the presence of ICI. To confirm the epinephrine-β
2
-AR signaling pathway, exogenous epinephrine was applied (0.1 μM for 1h) to ICA cells in the absence and presence of ICI (10 μM). Epinephrine induced 16-fold increase in CGRP mRNA levels at 6h post-treatment. This effect was completely abolished in the presence of ICI. To determine whether endogenous CGRP release from ICA cells exerts tonic modulation on myocyte contractile function, we studied the interaction between ICA cells and myocytes in ICA cell-myocyte co-culture model using a ratio fluorescent spectrometer. Application of CGRP-receptor antagonist CGRP
8 –37
(0.2 μM) reduced myocyte [Ca
2+
]i transients by 30 ± 5%.

Conclusion
s: ICA cells exclusively express CGRP gene in human and animal hearts. Robust and sustained CGRP gene-expression can be induced via β
2
-AR signaling through autocrine epinephrine positive feedback or pharmacological β
2
-AR stimulation.
Abstract 189: Extended-Release Niacin Improves Endothelial Function, Restores Re-Endothelialization Capacity of Endothelial Progenitor Cells and Augments Vasoprotective Properties of HDL in Patients with Metabolic Syndrome
Authors: Sorrentino Sajoscha A , Besler Christian , Bahlmann Ferdinand H , Meyer Martin , Mueller Maja , Horváth Tibor , Heinemann Mariko , Flemmer Stella , Bahr Matthias J , Haller Hermann , Drexler Helmut , Landmesser Ulf
Published: July 2021

Background:
High density lipoprotein (HDL)-targeted therapies are currently evaluated as a novel therapeutic strategy for cardiovascular disease. However, recent data suggest that HDL may become dysfunctional in metabolic syndrome (MetS). We therefore evaluated the effect of extended-release niacin (ERN) on endothelial function, re-endothelialization capacity of endothelial progenitor cells (EPC) and HDL quality, i.e. the capacity of HDL to stimulate endothelial nitric oxide (NO) production and reduce superoxide (O
2
.−
) production.

Methods and Results:
Thirty MetS patients were randomized to 3 month treatment with ERN (1500 mg/d) or placebo. Flow-dependent, endothelium-mediated vasodilation (FDD) was characterized by high-resolution ultrasound, EPCs were cultured and HDL was isolated by ultracentrifugation before and after therapy.
In vivo
re-endothelialization capacity of EPCs was tested by transplantation of EPCs (5x10
5
cells) into nude mice using a carotid injury model. Furthermore, the effects of HDL on endothelial cell superoxide (O
2
. −
) and NO production were determined by electron spin resonance spectroscopy. ERN therapy significantly raised HDL levels (36.4±4.2 vs. 43.4±6.9 mg/dl, P <0.01), whereas no change was observed after placebo (35.9±6.2 vs. 33.7±6.1 mg/dl, P n.s.). FDD was improved by ERN (5.2±1.8 vs 9.8±2.2%; P<0.01), but not after placebo therapy (4.9±1.5 vs. 4.3±0.9 %; P n.s.). Moreover, the re-endothelialization capacity of EPCs was markedly increased after ERN, but not after placebo (REA-ERN: 34±9%;
P<
0.001 vs. placebo). Importantly, HDL isolated after ERN, but not after placebo therapy stimulated endothelial cell NO production and inhibited endothelial cell O
2
. −
production, suggesting that improved vasoprotective properties of HDL contributed to beneficial effects of ERN therapy on endothelial function.

Conclusions:
The present study demonstrates that extended-release niacin improves endothelium-dependent vasodilation, restores re-endothelialization capacity of EPCs and importantly improves vasoprotective functions of HDL from patients with metabolic syndrome. These data suggest that extended-release niacin therapy has a beneficial effect on HDL vasoprotective qualities.
Abstract 846: Gsk3β Inhibition Is Required For Postconditioning To Modulate Mitochondrial Permeability Transition Pore Opening
Authors: Gomez Ludovic , Gharib Abdallah , Ovize Michel
Published: July 2021

Introduction:
Activation of the PI3kinase pathway and subsequent Inhibition of GSK3β by phosphorylation at serine-9 residue has recently been shown to protect against myocardial lethal reperfusion injury.

Hypothesis:
We hypothesized that GSK3β may play a key role in protection afforded by postconditioning. We used transgenic mice (Tg) that express a constitutively active (i.e. that cannot be inactivated by phosphorylation at serine 9) form of GSK-3β.

Methods:
Anesthetized WT and Tg mice underwent 60 minutes of coronary artery occlusion followed by 24 hrs of reperfusion (n=9/gr). At reflow, mice received either no intervention (controls: C-WT and C-Tg), postconditioning (postC) by 3 cycles of [1 min ischemia / 1 min reperfusion], or an IV bolus injection (1 minute before reflow) of either 10 mg/kg of cyclosporine A (CsA), a potent inhibitor of the mitochondrial permeability transition pore (mPTP) opening, or 70μg/kg of SB 216763 (SB), a specific inhibitor of GSK3β. Infarct size (%of area at risk) was measured by TTC staining. CRC, an index of Ca
2+
-induced mPTP opening in isolated mitochondria was measured by a fluorimetric approach.

Results:
PostC, CsA and SB significantly reduced infarct to 29%, 36% and 34% respectively (p<0.05 vs. C: 56±5%) in WT mice. In contrast, postC failed to significantly reduce infarct size in Tg mice (51±5%), while as expected CsA limited necrosis (36±6%). In WT, CRC was significantly decreased in C versus sham (75±14 nmol Ca
2+
/mg), but improved in postC, CsA and SB mice, averaging 155±31, 267±11 and 183±37 nmol Ca
2+
/mg respectively (p<0.05). In Tg mice, CRC was not significantly changed in postC versus control (139±44 nmol Ca
2+
/mg), but improved in CsA.

Conclusion:
These results suggest that inhibition of GSK3β is involved in postconditioning’s protection against infarction, likely by preventing mPTP opening during the first minutes of reperfusion.
Abstract 855: Circulating Mesoangioblasts Differentiate Into Cardiac Myocytes And Improve Function After Acute Myocardial Infarction
Authors: Iwasaki Masayoshi , Koyanagi Masamichi , Rapp Stefan , Schuetz Corina , Bushoven Philipp , Schranz Dietmar , Galvez Beatriz G , Cossu Giulio , Zeiher Andreas M , Urbich Carmen , Dimmeler Stefanie
Published: July 2021

Mesoangioblasts (MAB) are vessel-associated cells identified during embryonic development. In contrast to hemangioblasts, MAB express mesenchymal (CD73) and endothelial marker, but lack the hematopoietic marker CD45. We recently identified circulating MAB in children. Children-derived MAB showed vigorous proliferation capacity and high telomerase activity. However, the potential of cardiac differentiation in these cells was not elucidated. Therefore, we tested the capacity of children-derived MAB to aquire a cardiomyogenic phenotype. MAB expressed several cardiac transcription factors such as Nkx2.5, GATA4 and MEF2C and the stem cell markers c-kit and islet-1. In order to assess cardiac differentiation capacity, we performed co-culture assays with neonatal rat cardiomyocytes (CM). Immunochemical analysis revealed that MAB expressed cardiac α-sarcomeric actinin 6 days after co-culture. Moreover, human troponin T (TnT) was expressed as demonstrated by human specific RT-PCR. To confirm these data, we examined TnT expression in MAB isolated of a 2 years old patient with a known mutation of TnT. Sequences of the cloned RT-PCR products were identical to human TnT except for the known mutation providing genetic proof of concept for cardiac differentiation. In order to exclude fusion between MAB and CM as a mechanism, we used paraformaldehyde-fixed CM as scaffold. In this assay, human TnT also was detected, indicating that differentiation is sufficient to induce cardiac marker gene expression. Next, we tested the effect of MAB to improve cardiac function. MAB were injected intramuscularly in nude mice after myocardial infarction. Functional analysis using Millar catheter 2 weeks after infarction demonstrated that cell therapy lowered filling pressure and preserved diastolic function when compared to the PBS injected group (LVEDP: −20.3%, tau: −20.6%, vs PBS injected heart). Furthermore, left ventricular volume was also decreased (LVEDV/weight −27.3%). In summary, children-derived MAB express cardiac-specific genes after co-culture with CM and improved cardiac function in vivo. Given that MAB can be easily isolated and expanded from peripheral blood, these cells might be suitable to augment cardiac repair in children with heart failure.
Abstract 865: Gata-4 Is An Angiogenic Survival Factor Of The Infarcted Heart
Authors: Tenhunen Olli , Leskinen Hanna , Serpi Raisa , Rysä Jaana , Pennanen Harri , Soini Ylermi , Nemer Mona , Ruskoaho Heikki
Published: July 2021

Recent data suggest that the cardiac-restricted transcription factor GATA-4 is an anti-apoptotic factor required for adaptive responses as well as a key regulator of hypertrophy and hypertrophy-associated genes in the heart. As a leading cause of chronic heart failure, reversal of post-infarction left ventricular remodeling represents an important target for therapeutic interventions. Here we studied the role of GATA-4 as a mediator of post-infarction remodeling. Rats were subjected to experimental myocardial infarction (MI) by ligating the left anterior descending coronary artery (LAD). Ligation of the LAD decreased the DNA binding activity of GATA-4 by 69 % at day 1 after MI (P<0.001, n=7– 8) as assessed by gel mobility shift assays. At 2 weeks the GATA-4 DNA binding was significantly upregulated (2.4-fold, P<0.05, n=7), and returned to baseline at 4 weeks. To determine the functional role of GATA-4, rats underwent LAD ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing GATA-4. Hearts treated with the GATA-4 gene transfer exhibited significantly increased ejection fraction (58±5% vs. 38±3% in LacZ-treated control animals with MI, P<0.001, n=8 –9) and fractional shortening (28±3% vs. 16±1%, P<0.001, n=8 –9) 2 weeks after MI. Accordingly, the infarct size was significantly reduced (26±4% vs. 45±4%, P<0.01, n=8 –9). To determine the cardioprotective mechanisms of GATA-4, the number of cardiac stem cells, apoptotic cardiomyocytes and capillaries were assessed. The number of capillaries (59±4/field vs. 48±3/field, P<0.051, n=7– 8) and c-kit positive stem cells (13±5 cells vs. 4±2 cells, P<0.05, n=7– 8) were increased in GATA-4 treated hearts, and a tendency to decreased apoptosis was observed in TUNEL-stained histological sections. These results indicate that the reversal of reduced GATA-4 activity prevents adverse post-infarction remodeling through increased angiogenesis, recruitment of cardiac stem cells and anti-apoptosis. GATA-4-based gene transfer may represent a novel, efficient therapeutic approach for heart failure.