bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3407: The Transcriptional Mediator Complex Is Essential For Cardiac Valve Formation
Authors: Just Steffen , Berger Ina , Meder Benjamin , Hassel David , Hess Alexander , Katus Hugo A , Rottbauer Wolfgang
Published: July 2021

The genetic causes of congenital heart diseases especially cardiac valve disorders are mostly unknown. During the last decade, the zebrafish became an excellent and established model organism (1) to uncover these genetic defects and (2) to elucidate the underlying molecular pathomechanisms. We recently isolated the zebrafish mutation
ping pong
(
png

m683

) in a large-scale ENU-mutagenesis screen for recessive lethal mutations that perturb cardiac function.
png
mutant zebrafish embryos show pathologically developed cardiac valves. Due to malformation of the cardiac AV valves,
png
mutant zebrafish embryos exhibit vigorous regurgitation of blood between the atrium and the ventricle. Furthermore, as a result of the cardiac valve malformation and cardiac dysfunction
png
mutants die at day 6 post fertilization. Expression of several factors known to be crucial for the proper development and formation of the atrio-ventricluar canal (e.g. notch1b, bmp4 or versican) is significantly altered in
png
mutant zebrafish hearts. By a positional cloning approach we demonstrate that the
ping pong
phenotype is caused by a promotor mutation in a zebrafish gene encoding for a novel component of the “transcriptional mediator complex”. This mediator complex is a multi-protein complex that acts as a transcriptional coactivator and transduces informations from transcription factors to the RNA polymerase II.
png
is strongly expressed in zebrafish as well as human cardiomyocytes. Furthermore, sequence alignments demonstrate the evolutionary conservation of the
ping pong
gene product. Gene specific knock-down studies by means of modified antisense oligonucleotides reveal a phenocopy of the
png
mutant phenotype whereas injection of the gene-specific mRNA in
png
mutant embryos restores the mutant phenotype indicating that
png
is indeed responsible for the observed phenotype. The zebrafish evolved as an excellent model organism to study the molecular signalling pathways involved in cardiac valve formation. By detailed characterization of the zebrafish line
ping pong
we will obtain new insights into these molecular mechanisms especially the transcriptional control of valve formation and therefore the pathomechanisms of human cardiac valve disorders.
Abstract 3421: Sox2 Converts Human Mesoangioblasts into Nanog-Expressing Multipotent Cells with Enhanced Cardiac Repair
Authors: Koyanagi Masamichi , Yoon Chang-Hwan , Iwasaki Masayoshi , Boeckel Jes-Niels , Trauth Janina , Rupp Stefan , Schranz Dietmar , Zeiher Andreas M , Dimmeler Stefanie
Published: July 2021

Induced pluripotent cells (iPS) were generated by overexpression of four factors (Oct3/4, Klf4, c-myc and Sox2) in fibroblasts and showed similar characteristic as embryonic stem (ES) cells. In contrast to iPS and ES cells, adult stem cells have a reduced plasticity. Therefore, we investigated whether reconstitution of the four pluripotency genes in adult progenitor cells improves their therapeutic capacity. Circulating mesoangioblasts (cMAB) have been recently identified in peripheral blood and showed distinct marker expression to bone marrow-derived stem cells and endothelial progenitor cells. cMAB are clonally expandable and exhibit high telomerase activity. cMAB expressed the pluripotency genes Oct3/4, KLF4, and c-Myc, whereas Sox2 and another important stemness gene, Nanog was not expressed. Since only Sox2 was absent out of the four pluripotency genes, we hypothesized that Sox2 transduction may be sufficient to induce pluripotency. Sox2 was overexpressed by lentiviral vector. Interestingly, Sox2-tranduced cMAB not only express Sox2 but also re-express the stem cell gene Nanog. Controls confirmed that the constitutively expressed genes, Oct3/4, KLF4, and c-myc, remained present after Sox2 transduction. Sox2-transduced cMAB formed embryoid body-like structures in culture. In order to test whether Sox2-transduced cells may exhibit improved therapeutic potential, we injected Sox2-transduced cMABs into nude mice after acute myocardial infarction. Four weeks after cell therapy with Sox-overexpressing cells, cardiac function was improved compared to mice treated with control cells (LVEDV/weight; 1.34 ± 0.14 vs 1.88 ± 0.02 μl/g, LVEDP; 13.2 ± 1.0 vs 18.7 ± 2.6 mmHg, dp/dt max; 7989 ± 1030 vs 6331 ± 149 mmHg/s, dp/dt min; 6583 ± 683 vs 4792 ± 514 mmHg/s, and Tau; 9.2 ± 0.8 vs 11.6 ± 1.8 ms, respectively). In summary, the complementation of Sox2 in Oct3/4-, Klf4-, and c-myc-expressing cMABs induced Nanog expression and improved the cardiac regeneration capacity of these circulating adult stem cells.
Abstract 3434: The Role Of Notch Signaling In The Adult Myocardium: Control Of Myocyte Hypertrophy And Differentiation
Authors: Nemir Mohamed , Lepore Mario , Croquelois Adrien , Domenighetti Andrea , Radtke Freddy , Pedrazzini Thierry
Published: July 2021

Background
. Notch signaling is essential during development and in homeostasis of several organ systems. Mutations in Notch genes cause cardiac malformations. However the role of this pathway in the adult heart has not been addressed.

Methods
. We investigated Notch signaling in models of cardiac hypertrophy and failure. Notch activation was analyzed by immunostaining and quantitative RT-PCR. Cardiac response to stress was investigated in mice lacking Notch1 in the heart and in transgenics overexpressing Jagged1 in the myocardium.

Results
. The wild-type heart responded to hemodynamic stress by an upregulation of the Notch ligand Jagged1 on cardiac myocytes and an activation of Notch1. Immunostaining showed that Notch1 activation occurred predominantly in cardiomyocytes. Nkx2.5- and GATA4-positive cardiac precursor cells also activated Notch. In cultured cardiomyocytes, Notch signaling interfered with terminal differentiation. In vitro, proliferating Nkx2.5-positive precursor cells displayed intense NIC staining. Under differentiation conditions, these cells downregulated the Notch pathway and produced cardiomyocytes. Pharmacological inhibition of the Notch pathway in vivo exacerbated cardiac hypertrophy and was detrimental to the adaptive response of the heart to stress. Similarly, mice with cardiac-specific deletion of Notch1 exhibited cardiac fibrosis, impaired cardiac function and decreased survival. To investigate the effects of chronic Notch activation in the heart, we generated transgenics with cardiac Jagged1 overexpression. A large proportion (40 –50%) of these mice demonstrated atrial enlargement, impairment of ventricular function, and die at 3 months of age.

Conclusions
. Notch signaling is crucial for maintaining cardiac integrity in the adult mammalian heart. Activation of the Notch pathway relies on stimulated Jagged1 expression. In cardiomyocytes, Notch regulates the extent of the hypertrophic response and probably contributes to cell survival. In cardiac precursors, Notch controls cardiogenic differentiation. Importantly, both chronic inhibition and activation are detrimental to cardiac function, suggesting that fine tuning of Notch signaling in the heart is mandatory for proper cardiac homeostasis.
Abstract 3437: Integrin-linked-kinase Binding Proteins Are Essential Components Of The Cardiac Mechanical Strech Sensor
Authors: Hartkopf Andreas , Huttner Inken , Dahme Tillman , Meder Benjamin , Vogel Britta , Hess Alexander , Katus Hugo A , Rottbauer Wolfgang
Published: July 2021

The cardiac strech sensor enables the heart to adapt its force of contraction to continually changing demands. We recently identified in the zebrafish mutant
main squeeze (msq)
Integrin-Linked-Kinase (ILK) as a component of this strech sensor. Mutations in zebrafish
ilk
lead to down-regulation of stretch responsive cardiac genes and cause progressive heart failure. Antisense-mediated abrogation of zebrafish β-parvin, which forms complexes with ILK, phenocopies
ilk
mutant zebrafish. We here describe two novel ILK-binding-proteins, acting in concert with ILK to control cardiac contractility. In a yeast-to-hybrid-screen with a cardiac specific library we identified two ILK-binding proteins (ILK-bp-1 and ILK-bp-2). Just as we have recently shown for ILK, ILK-bp-1/2 are highly expressed in the zebrafish heart and localize at the sarcomeric Z-disc. To reveal the
in vivo
function of ILK-bp-1/2 we performed knock-down studies on zebrafish with antisense oligonucleotide injection. Similar to the phenotype of ILK-deficient zebrafish, the knock down of ILK-bp-1/2 leads to loss of cardiac contractility and, as shown by in-situ-hybridisation, to down-regulation of the stretch responsive gene
anf.
We analysed the ILK-bp-1/2 morphant hearts structurally and ultra-structurally. Just as we have recently shown for
ilk
deficient hearts, we found that their morphogenesis and myofibrillogenesis are normal, indicating that knock-down of ILK-binding-proteins does not induce a structural but rather a functional heart defect. Taken together, these results indicate, that next to β-parvin other ILK-binding proteins play important roles in the control of heart contractility. ILK and ILK-bp-1/2 act in concert to enable the heart to adapt itself to continually changing demands.
Abstract 3445: Notch1 Represses Bmp-Dependent Aortic Valve Calcification in a Cell-Autonomous Manner
Authors: Nigam Vishal , Srivastava Deepak
Published: July 2021

Calcific aortic stenosis is the third leading cause of adult heart disease and the most common form of acquired valvular disease in developed countries. However, the molecular pathways leading to calcification are poorly understood. We recently reported two families in which heterozygous mutations in NOTCH1 that resulted in premature stop codons caused bicuspid aortic valve and severe calcification of the aortic valve. Notch1 is a part of a highly conserved signaling pathway involved in cell fate decisions, cell differentiation, and cardiac valve formation. We found that mice heterozygous-null for Notch1 had over four-fold more aortic valve calcification compared to age and sex matched littermates (p<0.03). To determine what cell type is involved in the Notch1 related calcification, Notch1+/− bone-marrow was transplanted into wild-type mice. Six months after transplant, there was no difference in the amount of aortic valve calcification between the recipients of Notch1+/− bone-marrow versus recipients of control bone-marrow. In contrast, cultured sheep aortic valve interstitial cells (AVICs) treated with a chemical inhibitor of Notch signaling developed over five-fold more calcification (p<0.03). We found that expression of Bmp2, which has been implicated in human aortic valve disease, was 3-fold higher in aortic valve leaflets of Notch1+/− mice compared to wild type littermates (p<0.02). Furthermore, AVICs treated with the Notch inhibitor had increased Bmp2 protein levels and siRNA-mediated knock-down of Bmp2 expression in the AVICs blocked the calcification process induced by Notch inhibition. These findings suggest that Notch1 signaling within aortic valve cells is required for repression of Bmp2-dependent calcification pathways and may provide novel approaches to inhibit the progression of calcific aortic stenosis.
Abstract 3492: Increased Late I
Na
in Left Atrial Myocytes of Left Ventricular Hypertrophy Rabbits and its Role in Genesis of Atrial Early Afterdepolarizations
Authors: Guo Donglin , Jiao Zhen , Malhottra Binu , Patel Chinmay , Kowey Peter R , Yan Gan-Xin
Published: July 2021

Atrial fibrillation is the most common sustained arrhythmia affecting more than 2 million people annually in USA. Previous studies have shown that left ventricular hypertrophy (LVH) results in an increase in the late I
Na
that plays an important role in genesis of ventricular arrhythmias. We tested the hypothesis that LVH, which is associated with elevated pressure in the left atrium, could enhance the late I
Na
in left atrial (LA) myocytes, leading to increased trigger activities. Rabbit LVH, which exhibited a significantly greater left ventricle to body mass ratio, was induced using the renovascular hypertension model. Interestingly, early afterdepolarizations (EADs) at action potential phase 2 and 3 occurred in 6 of 10 LA myocytes isolated from 5 LVH rabbits at a pacing cycle length of 2000 ms, whereas EADs were elicited in none of 10 cells isolated from 5 control rabbits (
p
<0.01). Spontaneously activities (SA) were observed in 6 of 10 LA myocytes from five LVH rabbits at the pacing rate of 8000 ms. The density of the late I
Na
was significantly larger in LA myocytes of LVH rabbits than that recorded in control rabbits (0.59±0.02 pA/pF in LVH versus 0.42±0.05 pA/pF in control, n=6,
p
<0.01). Ranolazine, a late I
Na
blocker, exerted a concentration-dependent blocking effect on the late I
Na
in LA myocytes of the rabbits (IC
50
=15.7±0.6 μM) and abolished all of atrial EADs and SA of the LVH rabbits at 30 μM. Our results demonstrate that LVH results in a significant increase in the late I
Na
in the LA myocytes that may render these cells susceptible to genesis of EADs. The late I
Na
is a potentially useful ionic target by antiarrhythmic drugs for the treatment of atrial fibrillation in the setting of LVH.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3499: Extracellular Loop (Domain I, S5-S6) of α1D L-type Ca Channel is an Antigenic Site in Autoimmune Associated Congenital Heart Block
Authors: Karnabi Eddy , Qu Yongxia , Wadgaonkar Raj , Mancarella Salvatore , Yue Yunkun , Chahine Mohamed , Clancy Robert , Buyon Jill P , Boutjdir Mohamed
Published: July 2021

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have established that anti-SSA/Ro -SSB/La autoantibodies inhibit L-type α
1D
Ca current, I
Ca-L
and cross-react with the α
1D
Ca channel protein. This study aims at identifying the possible binding sites on α
1D
protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four α
1D
Ca channel protein domains I–IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera from 118 mothers with CHB children and 28 control healthy sera were used in this study. Seventeen of 118 (14.4%) maternal CHB sera reacted with the extracellular loop of Domain I S5-S6 region (E1). In contrast, 2 of 28 (7%) healthy control sera reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D. reading of the positive sera establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera demonstrated inhibition (44.1%) of the α
1D
I
Ca-L
expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 extracellular loop fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel α
1D
subunit as a target for autoantibodies from mothers with CHB children. This novel finding provides insights into the development of therapeutic peptides that could bind to the pathogenic antibodies.
Abstract 3500: Rescue and Worsening of Electrocardiographic Abnormalities in Two Experimental Transgenic Murine Models of Congenital Heart Block
Authors: Karnabi Eddy , Qu Yongxia , Boutjdir Mohamed
Published: July 2021

Congenital Heart Block (CHB) is a passively acquired autoimmune disease associated with the transfer of maternal autoantibodies anti-SSA/Ro -SSB/La through the placenta causing AV block and sinus bradycardia. We previously established a murine model for CHB in which pups born to wild type (WT) mothers immunized with Ro and/or La recombinant proteins developed conduction abnormalities similar to those seen in the clinical settings and inhibited L-type Ca channels (LTCC) responsible for electrogenesis at the AV and SA node. Here, we hypothesized that overexpression of cardiac LTCC should prevent or reduce the electrocardiographic abnormalities seen in the pups, whereas LTCC knockout should exhibit severe conduction abnormalities and increased mortality. Transgenic (TG) mice overexpressing LTCC and LTCC knockouts were immunized with Ro and La antigens. Following the immunization and boosters, a high antibody titer was demonstrated both in mothers and pups. ECG parameters were measured from pups born to immunized and to WT mothers. Ca channel overexpressing pups had significantly lesser sinus bradycardia and AV block compared to their non-TG (NTG) litter mates and WT pups. Sinus bradycardia was seen in 10% (n= 90) of TG pups v.s. 19.7% in WT (n=76, p<0.05) vs. 22% NTG littermates (n=109, p<0.05) and AV block in 8.8% of TG (n=90) v.s. 25% in WT (n= 76, p<0.05) v.s. 27.5% in NTG (n=109, p<0.05). On the other hand, Ca channel knockout pups had a significantly higher sinus bradycardia and a higher percentage of AV bock; 100% in knockout pups (n=16, p<0.05), 42.3% in heterozygotes (n=52, p<0.05), and 18.2% in NTG (n=33, p<0.05). No electrocardiographic abnormalities were observed in control WT pups (nonimmunized). Cardiac specific overexpression of LTCC significantly rescues the incidence of AV block and sinus bradycardia in pups exposed to circulating maternal anti-Ro/La autoantibodies. Whereas exposure of LTCC knockout pups to anti-Ro/La autoantibodies significantly worsened the conduction abnormalities. This finding support the hypothesis that maternal antibodies’ inhibition of LTCC contributes to the development of CHB. Altogether the results are relevant for future novel therapies for CHB.
Abstract 3551: Cyclophilin a Promotes Angiotensin II-Induced Inflammation and Cardiovascular Hypertrophy in Mice
Authors: Satoh Kimio , Casey Liam , O’Dell Michael R , Nigro Patrizia , Mohan Amy , Lu Yan , Alexis Jeffrey , Yan Chen , Blaxall Burns C , Berk Bradford C
Published: July 2021

Background -

Cyclophilin A (CyPA) is a chaperone protein secreted from vascular smooth muscle cells (VSMC) in response to reactive oxygen species (ROS). We have recently demonstrated that extracellular CyPA stimulates at least 3 signaling pathways (ERK1/2, Akt and JAK) and mediates numerous cellular effects of ROS. Angiotensin II (Ang II) induces ROS through NADPH oxidases and activates matrix metalloproteinase (MMP) in VSMC. ROS and MMPs have been demonstrated to mediate cardiac hypertrophy and remodeling. We hypothesized that VSMC-derived CyPA contributes to AngII-induced cardiovascular hypertrophy in vivo due to its proinflammatory properties.

Methods and Results -

ApoE
−/−
and ApoE
−/−
CyPA
−/−
mice were treated with AngII (1000 ng/min/kg for 4 weeks) to induce cardiac hypertrophy. Long-term infusion of AngII significantly increased heart/body weight ratio in ApoE
−/−
mice, which was significantly less in ApoE
−/−
CyPA
−/−
mice (6.6±1.0 vs. 4.8±0.7,
P
<0.01). Echocar-diography confirmed a significantly greater increase in LV mass in ApoE
−/−
mice compared to ApoE
−/−
CyPA
−/−
mice (112% vs. 47%). Perivascular accumulation of inflammatory cells and cardiac myofibroblasts in ApoE
−/−
mice was significantly greater than in ApoE
−/−
CyPA
−/−
mice. Consequently, coronary artery ROS production (DHE fluorescence) and MMP activation (in situ zymography) were markedly increased by AngII in ApoE
−/−
mice compared to ApoE
−/−
CyPA
−/−
mice. To determine the source of CyPA, bone marrow cells (BMCs) transplantation was performed. The heart/body weight ratio was still higher in ApoE
−/−
mice compared with ApoE
−/−
CyPA
−/−
mice after reconstitution with GFP
+
CyPA
+/+
BMCs (6.7±0.6 vs. 5.6±0.9,
P
<0.01). Recruitment of GFP
+
BMCs to the heart in chimeric ApoE
−/−
mice was significantly greater than the chimeric ApoE
−/−
CyPA
−/−
mice (count/area; 218±63 vs. 109±43,
P
<0.01). To prove a vascular source of CyPA was essential, VSMC-specific CyPA overexpressing mice were generated. In these mice there was a significant increase in cardiac MMP activity after AngII infusion (VSMC-Tg > WT > CyPA
−/−
).

Conclusion -

CyPA is a novel mediator of AngII-induced cardiac hypertrophy by stimulating vascular ROS production, MMP activation, and inflammatory cell recruitment.

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 3552: Pharmacological Inhibition or Genetic Disruption of Protein Tyrosine Phosphatase 1B Attenuates both Myocardial and Endothelial Dysfunction in Mice with Heart Failure
Authors: Vercauteren Magali , Kurtz Baptiste , Gomez Elodie , Henry Jean-Paul , Bauer Fabrice , Hooft Rob , Tremblay Michel L , Thuillez Christian , Richard Vincent
Published: July 2021

We have shown previously that acute,
in vitro
inhibition of protein tyrosine phosphatase 1B (PTP1B) improved endothelial function of peripheral resistance arteries in mice with chronic heart failure (CHF), as demonstrated by the restored flow-mediated, NO-dependent vasodila-tation (FMD). This is most likely due to increased tyrosine phosphorylation pathways involved in shear stress-induced activation of eNOS. The present study evaluates the impact of
chronic
pharmacological inhibition or genetic disruption of PTP1B, on cardiac and endothelial dysfunction in CHF mice. CHF was induced by coronary ligation, either in C57BL/6 mice, or in wild type (WT) or PTP1B-deficient (PTP1B−/−) BALB/c mice. CHF mice (either C57BL/6 or BALB/c WT) were untreated or treated with the PTP1B inhibitor AS279 (60 mg/kg/day) for 2 months. Mice were then anesthetized with isoflurane for echocardiographic evaluation of left ventricular (LV) function and remodeling. After euthanasia, small mesenteric artery segments were isolated and mounted in an arteriograph for the evaluation of FMD. Echocardiographic results are shown in the Table

. In parallel,
in vitro
vascular studies showed that chronic AS279 restored FMD both in C57BL/6 (max FMD: control: 17±2, n=9; CHF untreated: -1±1, n=9; CHF + AS279: 14±2%, n=19; p<0.01 vs. CHF untreated) and BALB/c CHF mice (Control: 29±4, n=16; CHF untreated: 7±1, n=5; CHF + AS279: 24±6%, n=7; p<0.01 vs. CHF untreated). Compared to CHF WT, FMD was also increased in PTP1B−/− CHF mice (WT: 7±1, n=5, PTP1B−/−: 16±6%, n=6, p<0.05). Additionally,
in vitro
downregulation of PTP1B (by a 3 day incubation with shRNA) also increased FMD in arteries isolated from CHF mice (max FMD: untreated: 6±2; scrambled shRNA: 7±2; shRNA PTP1B: 27±2%, p<0.01). Thus, chronic pharmacological inhibition or genetic disruption of PTP1B both restores endothelial function and improves cardiac dysfunction and remodeling, suggesting that this enzyme may be a new target for the treatment of CHF.

Echocardiographic results in mice with 2 months CHF
Abstract 3598: Critical Role of Th17-CD4
+
T cells in Angiogenic Response to Hindlimb Ischemia in Mice
Authors: Hata Takeki , Takahashi Masafumi , Kawaguchi Masanori , Kashima Yuichiro , Shiba Yuji , Ise Hirohiko , Hida Shigeaki , Iwakura Yo-ichiro , Taki Shin-suke , Ikeda Uichi
Published: July 2021

Background:
Accumulating evidence indicates that CD4
+
T cells contribute to the development of collateral vesssels in ischemic tissue; however, little is known about the responsible subset of CD4
+
T cells in the induction of angiogenesis. Th17 cells are recently identified as a new subset of CD4
+
T cells and have been associated with the pathogenesis of certain autoimmune diseases. Th17 cells specifically secrete interleukin-17 (IL-17) and regulate various biological functions. The purpose of this study is to investigate the role of CD4
+
T and Th17 cells in angiogenic response to hindlimb ischemia.

Methods and Results:
Unilateral hindlimb ischemia was produced in wild-type (WT: C57BL/6, 8- to 10-week-old) mice treated with or without a neutralizing antibody against CD4. Blood flow perfusion and capillary formation were assessed by using a laser Doppler perfusion imaging (LDPI) and CD31 immunostaining, respectively. Well-developed collateral vessels and capillary formation were observed in WT mice in response to hindlimb ischemia. Treatment with a neutralizing anti-CD4 antibody resulted in almost complete CD4
+
T cell depletion (flow cytometry analysis, control: 45.4% vs. antibody: 1.0%) and a significant decrease in angiogenesis after the induction of hindlimb ischemia (LDPI, 21 days, control: 0.61 ± 0.1 vs. antibody: 0.41 ± 0.1, p<0.05). IL-17-deficient (IL-17
−/−
) mice also showed a significant reduction of blood flow perfusion, compared with WT mice (LDPI, day 14: 0.56 ± 0.3 vs. 0.31 ± 0.2, p<0.05; day 21: 0.66 ± 0.3 vs. 0.37 ± 0.3, p=0.05). IL-17
−/−
mice had severe ischemic damage of the limb and resulted in a 25% incidence of autoamputation by day 21 (no limb loss in WT mice). Furthermore, capillary formation was also decreased significantly in IL-17
−/−
mice (692.9 ± 165.6/mm
2
vs. 1223.3 ± 267.3/mm
2
, p<0.01).

Conclusion
: These findings demonstrate that Th17 cells, a new subset of CD4
+
T cells, contribute to the angiogenic response to hindlimb ischemia and provide new insights into the mechanism by which T cells promote collateral development and angiogenesis.
Abstract 3635: Inhibition of Inhibitor Kappa B Kinase Beta Augments Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production in Aortic Endothelial Cells
Authors: Mohan Sumathy , Konopinski Ryzard , Natarajan Mohan
Published: July 2021

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases (inhibitor kappa B kinases α, β and γ) in non-vascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90 and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (4.02 ± 0.81-folds) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.04 ± 0.06-folds). Another important and novel finding is that both IKKβ and eNOS could be co-immunoprecipitated with Hsp-90 (Figures A & B

) thus indicating the possible existence of a complex of IKKβ and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2μM) or Radicicol (20μM) mitigated (0.45 ± 0.04 -fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.416-fold). Blocking of IKKβ expression by IKK inhibitor II (15μM wedelolactone) or siRNA improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.
Abstract 3659: High-Mobility Group Box 1 Restores Cardiac Dysfunction after Myocardial Infarction in Transgenic Mice
Authors: Kitahara Tatsuro , Takeishi Yasuchika , Shishido Tetsuro , Suzuki Satoshi , Kato Shigehiko , Sasaki Toshiki , Bilim Olga , Ishino Mitsunori , Kubota Isao
Published: July 2021

High-mobility group box 1 (HMGB1) is a nuclear DNA-binding protein and is released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis. To test the hypothesis that HMGB1 enhances angiogenesis and restores cardiac dysfunction after myocardial infarction, we generated transgenic mouse with cardiac specific overexpression of HMGB1 (HMGB1-Tg) using α-myosin heavy chain (MHC) promoter. The left anterior descending coronary artery was ligated in HMGB1-Tg and wild-type littermate (Wt) mice. After coronary artery ligation, HMGB1 was released into circulation from the necrotic cardiomyocytes of HMGB1 overexpressing hearts. The size of myocardial infarction was smaller in HMGB1-Tg than in Wt mice (figure

). Echocardiography and cardiac catheterization demonstrated that cardiac remodeling and dysfunction after myocardial infarction were prevented in HMGB1-Tg mice compared to Wt mice. Furthermore, survival rate after myocardial infarction in HMGB1-Tg mice was higher than that in Wt mice (figure

). Immunohistochemical staining revealed that capillary and arteriole formations after myocardial infarction were enhanced in HMGB1-Tg mice. We demonstrated the first in vivo evidence that HMGB1 enhances angiogenesis, restores cardiac dysfunction, and improves survival after myocardial infarction. These results may provide a novel therapeutic approach for left ventricular dysfunction after myocardial infarction.
Abstract 3667: Hypoxic Preconditioning of Mononuclear Cells Enhances Angiogenesis by Increasing Their Retention after Implantation into Ischemic Tissue
Authors: Kubo Masayuki , Li Tao-Sheng , Kamota Takahiro , Ohshima Mako , Qin Shu-Lan , Hamano Kimikazu
Published: July 2021

Background

: Cell-based angiogenesis is a promising treatment for ischemic diseases, but the poor retention of implanted cells in targeted tissue is a major drawback. We tested the hypothesis that hypoxic preconditioning increased retention and angiogenic potency of cells after implantation into ischemic tissues.

Methods and Results

: Peripheral blood mononuclear cells (PBMNCs) were isolated from C57BL/6 mice. Hypoxic preconditioning of PBMNCs was done by 24 hours of culture at 33°C in 2% O
2
. We used normoxia-cultured or freshly collected PBMNCs as controls. Compared with the normoxia-cultured PBMNCs, the hypoxia-preconditioned PBMNCs increased the adhesion capacity (75.1 ± 11.5/field vs. 39.7 ± 5.1/field, P<0.01). Furthermore, RT-PCR analysis showed that the expression of integrin μM and CXCR4 was significantly higher in the hypoxia-preconditioned PBMNCs than in the normoxia-cultured PBMNCs (P<0.05). Interestingly, immunohistochemistry revealed that the expression of ICAM-1, a ligand of integrin μM, and SDF-1, a chemokine for CXCR4, were remarkably increased in the ischemic hindlimbs. Three days after the intramuscular implantation into ischemic hindlimbs, the retention of the hypoxia-preconditioned PBMNCs (36.7 ± 4.2/field, P<0.01) was significantly higher than that of the normoxia-cultured (23.6 ± 1.3/field) or freshly collected PBMNCs (23.4 ± 2.6/field). We also noted higher microvessel density and better blood flow in the ischemic hindlimbs implanted with the hypoxia-preconditioned PBMNCs than in those implanted with the normoxia-cultured or freshly collected PBMNCs, 28 days after treatment; at 1023 ± 59/mm
2
vs. 767 ± 77/mm
2
and 775 ± 50/mm
2
, respectively (P<0.01) and 77.2 ± 5.5% vs. 63.6 ± 4.3% and 64.3 ± 2.5%, respectively (P<0.05) in the hypoxia, normoxia, and fresh groups.

Conclusion

: These results show that hypoxic preconditioning of PBMNCs is a feasible method of enhancing therapeutic angiogenesis by increasing their retention within ischemic tissues after implantation. Further studies are ongoing to elucidate whether the angiogenic potency of cells derived from the diseased animals is enhanced similarly to that of cells derived from healthy animals, after hypoxic preconditioning.
Abstract 3669: Transcoronary Gene Transfer of Serca2a Enhances Coronary Blood Flow through an Increase of eNOS Activity in Endothelial Cells
Authors: Hadri Lahouaria , Lipskaia Larissa , Kawase Yoshiaki , Clement Nathalie , Plenge Tobias , Lebeche Djamel , Lompré Anne-Marie , Hajjar Roger J
Published: July 2021

Congestive heart failure (CHF) is associated with impaired endothelium-dependent nitric oxide (NO)-mediated vasodilatation. Clinical trials using AAV1-SERCA2a myocardial gene delivery in CHF patients are currently underway. We hypothesized that transcoronary SERCA2a gene transfer can increase NO production by endothelial cells (EC). To assess the effects of SERCA2a gene transfer on coronary flow and endothelial function in vivo, we used a preclinical porcine volume-overload heart failure model (severe mitral valve regurgitation (MR)). Two months after MR creation, animals underwent intracoronary injection of AAV1-SERCA2a or saline. Four months post MR creation, the average coronary flow peak velocity (APV) was measured in the mid portion of the left anterior descending artery (LAD), and adjusted to the diameter of coronary artery and the weight of the left ventricle. Compared to saline treated animals, SERCA2a has significantly increased the adjusted coronary flow (ml/min/g) (0.467 ± 0.09, n=6 vs. 0.886 ± 0.18, n=7; P<0.01) but no difference was observed compared to sham operated animals (1.003 ± 0.2137, n=4; P=NS). Confocal immunofluorescence of swine coronary artery sections demonstrated that SERCA2a is expressed in endothelial and vascular smooth muscle cells; however, eNOS was expressed only in EC in all animal groups. Western blot analyses showed down-regulation of SERCA2a and eNOS expression in LAD, LCA and RCA from failing saline-injected animals. In coronary arteries from SERCA2a-transduced animals, expression levels of SERCA2a and eNOS were similar to those in sham-operated animals. To further investigate the effect of SERCA2a gene transfer on endothelial function we used cultured human coronary artery EC. RT-PCR analysis, western blot and e-NOS promoter/ luciferase reporter assay have shown that SERCA2a gene transfer increased expression and Ser-1177 phosphorylation of e-NOS. Furthermore, cGMP production was increased in SERCA2a-infected EC, confirming its possible functional effect on eNOS activity. In conclusion, our results demonstrate that transcoronary SERCA2a gene transfer improves endothelial function in failing hearts through regulation of eNOS expression and activity.
Abstract 3689: Loss of Myeloid Related Protein-8/14 Exacerbates Cardiac Allograft Rejection
Authors: Shimizu Koichi , Libby Peter , Rocha Viviane Z , Folco Eduardo J , Shubiki Rica , Croce Kevin J , Hogg Nancy , Mitchell Richard N , Simon Daniel I
Published: July 2021

Background: The S100 calcium-binding proteins MRP-8 (S100A8) and MRP-14 (S100A9) heterodimerize to form MRP-8/14 complexes that regulate myeloid cell function, control inflammatory responses, and serve as an early serum marker for monitoring acute allograft rejection. Despite functioning as a putative pro-inflammatory mediator, the pathophysiological role of MRP-8/14 in cardiovascular disease remains unknown. This study used murine cardiac transplantation to investigate the role of MRP-14 in cardiac allograft rejection using MRP-14-deficient mice (MRP14−/−) lacking MRP-8/14 complexes.

Results: In major histocompatibility complex class II allomismatched cardiac transplantation (bm12 donor hearts and C57BL/6 recipients), allograft survival averaged 5.9 ± 2.9 weeks (n=10) in MRP14−/ −recipients compared to > 12 weeks (n = 15, p<0.0001) in wild-type (WT) recipients. At 2 week post-transplantation, allografts in MRP14−/ − recipients had significantly higher parenchymal rejection (PR) scores (2.8 ± 0.8, n=8) than WT recipients (0.8 ± 0.8, n=12, p<0.0001). MRP14−/ − recipients had significantly increased allograft accumulation of T cells and macrophages and mRNA levels of IFN-γ and IFN-γ–associated chemokines (IP-10, Mig, and I-TAC) compared to WT recipients. The MRP14−/− recipients also had significantly more lymphocytes in draining lymph nodes than WT recipients (cell number per lymph nodes: 23.7 ± 0.7 x 105 for MRP14−/− vs. 6.0 ± 0.2 x 105 for WT, p < 0.0001). MRP14−/− dendritic cells (DC) stimulated with IFN-γ expressed significantly higher levels of the co-stimulatory molecules CD86 and ICOSL, and mixed leukocyte reaction using allo-EC-primed MRP14−/− DC resulted in significantly higher antigen-presenting function than WT DC.

Conclusion: Taken together, these observations indicate that MRP-8/14 complexes participate in acute allograft rejection by modulating dendritic cell antigen presentation and T cell priming. The unexpected finding that deficiency of MRP-8/14 complexes exacerbates acute allograft rejection indicates that MRP-8/14 has distinct roles in modulating immune cell functions, such as migration and antigen presentation.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3704: Prevalence of Lipid Disorders and Treatment Patterns in Overall and High Risk U.S. Adults with Dyslipidemia in 2003–2004
Authors: Ghandehari Heli , Le Hoangvy , Ambegaonkar Baishali , Sazonov Vasilisa , Wong Nathan
Published: July 2021

Background:

Recent data on the extent of lipid disorders and treatments used among U.S. adults with dyslipidemia are limited; particularly among those with high coronary heart disease (CHD) risk. We therefore examined the prevalence of lipid disorders and utilization of lipid modifying therapies (LMT) to determine any gaps in therapy.

Methods:

We identified 1,111 U.S. adults aged ≥20 years (mean age 56 years, 45% female; representing 126 million US adults) with dyslipidemia (defined as elevated LDL-C as per National Cholesterol Education Program guidelines, or being on LMT) from the National Health and Nutrition Examination Survey 2003–2004. Of these, 609 (representing 19.4 million) adults with dyslipidemia were identified as high risk (i.e. CHD, diabetes or other CHD risk equivalents). LMT included statins, fibrates, niacin, cholesterol absorption inhibitors, bile acid sequestrants, as well as combination therapies. Lipid disorders were defined according to deviation from recommended levels for LDL-C (target level defined according to updated NCEP guidelines by risk group), HDL-C (≥40mg/dL for men and ≥50mg/dL for women) and triglycerides (TG) (<200 mg/dL).

Results:

Of the total population (n=1,111) with dyslipidemia, 85% remained uncontrolled for LDL-C, HDL-C, and or TG; 36% had ≥2 lipid disorders, while 42% had HDL-C and/or TG disorders with or without elevated LDL-C. Among high risk subjects, 88% remained uncontrolled on ≥1 of these lipid parameters; 36% had ≥2 lipid disorders simultaneously while 43% had HDL-C and/or TG disorders irrespective of LDL-C levels. However, only 34% (39% among high risk) were on LMT. Of those on treatment, monotherapy with statins was most common (~80%), followed by statin combination therapy (8 –10% depending on risk status).

Conclusions:

Our cross-sectional assessment of US adults suggests that despite high prevalence of lipid disorders (up to 85%), LMT is utilized by <40% of subjects. Furthermore, there is a large disconnect between high prevalence of HDL and/or TG disorders, with or without elevated LDL-C, and utilization of therapies targeting these lipids (<10% of therapy utilized was non-statins), a particular concern among high risk patients.
Abstract 3705: Pioglitazone Augments Niacin-Induced Increases in Adiponectin in Non-Diabetics with Metabolic Syndrome and Low HDL
Authors: Dunbar Richard L , Gadi Ramprasad , Bloedon LeAnne T , Ramprasad Sushma , Wolfe Megan , Baer Amanda , Rader Daniel J , Samaha Frederick F
Published: July 2021

Background Niacin and pioglitazone (PIO) increase HDL by uncertain mechanisms. Both increase adiponectin, whose effects on carbohydrate and fatty acid metabolism may benefit HDL indirectly. Little is known of the combined effects of the drugs on adiponectin and other adipokines. We hypothesized that PIO would complement changes in adipokines from niacin, and in turn, augment HDL. Methods In an open-label run-in, non-diabetics with low HDL titrated from 0 to 2 g extended-release niacin over 4 weeks, and were randomized to add PIO 30 mg or placebo in a double-blind manner. After 6 weeks, PIO was increased to 45 mg for another 6 weeks. We assessed changes in fasting adiponectin, resistin, complement C3 (the serum-detectable precursor of acylation stimulating protein), free fatty acids (FFA), hydroxy-butyrate (HBA), and triglycerides (TG), and their relationship to changes in HDL. Results Of 72 completers, 34 took niacin and placebo and 38 took niacin and PIO. The table

shows absolute and relative change of each analyte from baseline to 16 weeks. Relative change in adiponectin was strongly correlated with change in HDL (Spearman’s rho +0.49, p<0.0001), as was change in TG (rho −0.50, p<0.0001); the other analytes had little influence. On multivariable regression, change in HDL was predicted by change in adiponectin (p=0.0001) and TG (p<0.0001), which remained significant on adjustment for changes in glucose and insulin.

Conclusion Pioglitazone augments niacin-induced changes in adiponectin and complement C3 thought to be favorable, and neutralizes niacin’s unfavorable increase in FFA. Adiponectin strongly predicted HDL after adjustment for TG. Since PIO had no independent effect on TG, we speculate that additional HDL-raising from PIO is mediated by the large increase in adiponectin. In summary, PIO enhances HDL-raising from niacin in proportion to increased adiponectin.
Abstract 3714: Heme Oxygenase 1 Determines the Outcome of Vulnerable Plaque
Authors: Noordeloos Annemarie M , Cheng Caroline , Simoons M L , Pasterkamp G , Duckers H J
Published: July 2021

Plaque vulnerability is an important determinant in the prognosis of CAD. However the mechanism of transition from stable to vulnerable plaque (VP) remains unclear. Heme oxygenase 1 (HO-1) and its metabolites of heme degradation have been implicated in the cytoprotective defense response against oxidative injury. Here, we sought to assess the role of HO-1 in the development of VP in carotid artery disease in human and in a mouse model of VP. Atherectomy biopsies of 109 patients with clinical significant CAD were collected and stratified according to plaque morphology. HO-1 protein expression was determined by ELISA and was correlated with plaque parameters and pro-inflammatory biomarkers.To evaluate the function of HO-1 in VP, HO-1 was induced by CoPPIX injections in a murine model for VP in APOE −/− mice. HO-1 expression correlated closely to characteristics of atheromatous plaque vulnerability (p< 0.005) as indicated by intimal macrophage and lipid accumulation, and inverse correlations coincided with VSMCs and collagen. HO-1 expression levels correlated with proinflammatory biomarker titers including intraplaque MMP9 and IL-8 and IL6. HO-1 was mainly expressed in atheromatous infiltrating macrophages. In line with these observations, HO-1 expression was significantly increased in the VP vs. the stable lesions in our murine VP model. Systemic CoPPIX injections in the murine model resulted in an effective five-fold increase at HO-1 protein level. HO-1 induction did not affect the intima/media ratio, nor the percentage of intimal collagen or macrophages in the intima. However, systemic HO-1 induction significantly increased the percentage of intraplaque VSMCs by two-fold, while decreasing the percentage of lipids by two-fold. HO-1 protein expression closely correlated with histomorphological atherosclerotic plaque vulnerability and with expression levels of MMPs and pro-inflammatory cytokines in CAD patients. HO-1 induction in the murine VP model stabilized plaque morphology by reducing lipid accumulation and increasing VSMCs contect in these lesions. These data indicate that HO-1 expression in VP vs. stable plaques is enhanced as a compensatory atheroprotective response.
Abstract 3719: Deletion of Angiotensin II type 2 Receptor Accelerates Adipogenesis in Murine Mesenchymal Stem Cells with Wnt 10b/beta-catenin Signaling: Potential Link to Obesity and Metabolic Syndrome
Authors: Matsushita Kenichi , Wu Yaojiong , Wang Hao , Pratt Richard E , Dzau Victor J
Published: July 2021

Recent evidence suggests that the reninangiotensin system (RAS) plays a vital role in adipocyte biology and the pathophysiology of metabolic syndrome. Obesity is a major risk factor and mesenchymal stem cells (MSCs) are suggested to be an important source of new fat cells. Previously we reported that MSCs have all the components of the RAS and that angiotensin II (Ang II) can influence adipocyte differentiation. However, the effect of Ang II type 2 receptor (AT2R) deletion on adipogenesis in mouse model is unknown. Accordingly, we employed AT2R-null mice and examined the role of AT2R in the differentiation of MSCs to adipocytes.

Methods and Results: Murine MSCs were isolated from AT2R-null mice and wildtype littermates (WT). Confluent MSCs were differentiated into adipocytes by the established protocol. Adipogenesis was quantitated by assessing lipid droplet accumulation using 2 different methods. Using the fluorescent dye which binds to lipid droplets (AdipoRed), AT2R-null group showed significantly increased total fluorescence (261.6 ± 49.6 % vs. WT) on day 7. Oil red O staining, followed by extraction of the absorbed dye and measurement of the absorbance on day 14, also exhibited that AT2R-null group showed significantly increased lipid droplet accumulation (202.7 ± 14.1 % vs. WT). We next examined the expression of adipocyte-specific genes by real-time RT-PCR. AT2R-null group exhibited a significantly increased expression of PPARγ, fatty acid synthase, and adiponectin (vs. WT). Since Wnt 10b/beta-catenin signaling is reported to play an important role to inhibit adipogenesis, we further examined it by real-time RT-PCR and immunoblotting. The AT2R-null group exhibited significantly decreased Wnt 10b expression accompanied by a decrease of cellular beta-catenin (vs. WT).

Conclusion: Our results demonstrate that the AT2 receptor has an inhibitory effect on adipogenic differentiation in murine mesenchymal stem cells. Moreover, this inhibitory effect is associated with Wnt 10b/beta-catenin signaling which plays an important role to inhibit adipogenesis. These results provide important insights into the pathophysiology of obesity and obesity related consequences such as metabolic syndrome and may identify potential therapeutic targets.