bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5356: Brca1 Is An Essential Regulator Of Cardiac Function
Authors: Shukla Praphulla C , Singh Krishna K , Lovren Fina , Pan Yi , Wang Guilin , Quan Adrian , Teoh Hwee , Jurasz Paul , Leong-Poi Howard , Errett Lee , Brezden-Masley Christine , Stanford William L , Schneider Michael D , Parker Thomas G , Verma Subodh
Published: July 2021

INTRODUCTION:
Preservation of structure and function of the myocardium is critically dependent upon improving the survival of existing cardiomyocytes (CM), through strategies that limit CM apoptosis and DNA damage. BRCA1 is a tumor suppressor gene which functions to promote DNA repair, and protect cells against oxidative and genotoxic stress. We hypothesized that BRCA1 is a novel cellular target to limit CM apoptosis, and prevent aberrant cardiac remodeling.

METHODS AND RESULTS:
Experimental MI in mice caused a profound 16-fold upregulation in BRCA1 expression, which peaked at 72 hours (p<0.01).
In vitro
gain-of-function experiments demonstrated that Ad-BRCA1 overexpression protected neonatal rat CM against doxorubicin- and H
2
O
2
-induced apoptosis, as assessed by FACS (p<0.01) and activated caspase-3. Ad-BRCA1-expressing CM exhibited a profound reduction in p53 expression in response to doxorubicin and H
2
O
2
. Co-immunoprecipitation studies demonstrated a distinct physical interaction of BRCA1 with p53. Inhibition of p53, with pifithrin-alpha, blocked doxorubicin-induced CM apoptosis in a manner similar to BRCA1, but BRCA1-overexpressing CM, when treated with doxorubicin did not show further reduction with pifithrin-alpha, indicating an essential requirement of BRCA1 to modulate p53.
In vivo
gain-of-function studies demonstrated that systemic Ad-BRCA1 delivery completely prevented doxorubicin-induced cardiac dysfunction in mice (echocardiography, p<0.01).
In vivo
loss-of-function studies were performed in CM -specific BRCA1-KO mice (developed using Cre-lox
P
technology), which demonstrated marked cardiac dysfunction and mortality in response to doxorubicin administration (p<
0.01 vs. WT
+
Dox).

CONCLUSIONS:
We report for the first time an essential role of BRCA1 to limit CM apoptosis, and improve cardiac function in response to genotoxic and oxidative stress. Heart specific deletion of BRCA1 promotes severe systolic dysfunction, and limits survival. In addition to the immediate implications for cardiovascular repair, these data may have ramifications for individuals with BRCA1 mutations or cancer syndromes, particularly in the setting of adjuvant chemotherapy.
Abstract 5361: PDK1 Plays Crucial Roles in β-Adrenergic Response and Cell Survival in the Heart
Authors: Ito Kaoru , Akazawa Hiroshi , Yasuda Noritaka , Nakaya Haruaki , Komuro Issei
Published: July 2021

Background:
The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a homeostatic role in the regulation of cellular function in multiple organs, by working downstream of phosphatidylinositol-3 kinase (PI3-K) through activating several kinases including Akt and p70 S6 kinase. We found that myocardial expression of PDK1 was decreased in murine models of heart failure. Although previous studies have reported that PDK1 is important for the regulation of cardiomyocyte size, the precise role of PDK1 in the heart remains to be fully characterized.

Methods and Results:
To elucidate the roles of PDK1 in the postnatal heart, we generated tamoxifen-inducible heart specific PDK1 knockout (PDK1-KO) mice. Deletion of PDK1 at the age of 10 weeks caused severe heart failure. At 1 week after
Pdk1
gene disruption, left ventricular systolic function was already deteriorated without reduction in cardiomyocyte size. Langendorff-perfused hearts from PDK1-KO mice exhibited impaired responsiveness to isoproterenol in spite of preserved responsiveness to forskolin. In PDK1-KO hearts, PI3-K γ activity was enhanced and the expression levels of β1-adrenergic receptor (β1AR) in membrane fraction were decreased. Overexpression of PIK-domain of PI3-K γ blocked β1AR internalization by competitively inhibiting the association between PI3-K γ and G-protein coupled receptor kinase 2, and improved cardiac function in PDK1-KO hearts. In addition, we found that cardiomyocyte apoptosis was significantly increased 1 week after
Pdk1
gene disruption. PDK1-KO hearts showed reduction of Akt and SGK activities, upregulation of Bax protein and endoplasmic reticulum stress signals, which were the potential causes of cardiomyocyte apoptosis. Overexpression of Bcl-2 in PDK1-KO hearts prevented cardiomyocyte apoptosis and partially improved cardiac function in PDK1-KO mice.

Conclusions
: These results suggest that PDK1 is essential for normal cardiac function by not only preserving responsiveness to β-adrenergic stimulation but also preventing cardiomyocyte apoptosis. Since PDK1-KO mice reproduces many aspects of human heart failure, we propose that PDK1 may be a promising molecule targeted for the treatment of heart failure.
Abstract 5379: Increased Myocardial Fatty Acid Uptake by Acyl-CoA Synthetase 1 Impairs Mitochondrial Function and Dynamics
Authors: Bugger Heiko , Hu Xiao Xuan , Tuinei Joseph , Theobald Heather , Holland William L , Sparagna Genevieve , Summers Scott A , Schaffer Jean E , Abel E. Dale
Published: July 2021

Increased uptake and oxidation of fatty acids (FA) in diabetic hearts may contribute to mitochondrial dysfunction by promoting oxidative stress. To test this hypothesis, we investigated mice with cardiomyocyte-restricted overexpression of long-chain acyl-CoA synthetase 1 (MHC-ACS). In vivo PET studies revealed increased myocardial FA uptake (+120%; p<0.05) in MHC-ACS. Unexpectedly, FA oxidation and myocellular triglyceride content were unchanged, and PPARα-regulated FAO genes and PGC-1α-regulated OXPHOS genes were unaltered. However, cardiac content of the phospholipid precursors ceramide (+260%; p<0.05) and diacylglycerol (+40%; p<0.05) were increased. Mitochondrial cardiolipin content was decreased (−25%; p<0.05) and remodeled with substitution of 18:2 FA chains by unsaturated 22:6 FAs. Both ADP-stimulated mitochondrial O2 consumption (14.3±0.9 vs. 16.8±0.5 nmol/min/mgdw; p<0.05) and ATP synthesis (24.2±1.4 vs. 31.8±1.6 nmol/min/mgdw; p<0.05) were decreased in MHC-ACS in saponin-permeabilized cardiac fibers using palmitoyl-carnitine as a substrate. Mitochondrial superoxide production was increased by 75% (p<0.05). Electron microscopy in 3 to 24 week-old mice revealed increased mitochondrial number, which was highest at 3 weeks (3wk +210%, 12wk +120%, 24wk +130%; all p<0.05), and reduced mitochondrial size. Translocation of the mitochondrial fission protein dynamin-related protein 1 (Drp1) from cytosol to mitochondria was absent (p<0.05) in MHC-ACS, but not in controls. Mitochondrial membrane content of the fission protein fission 1 and the fusion proteins mitofusin 1 and 2 were unchanged. These morphological changes are consistent with increased mitochondrial fission and reduced Drp1 likely represents an adaptive response. Thus remodeling of the myocardial lipid pool and mitochondrial membrane lipid composition are associated with impaired mitochondrial dynamics and represents a novel mechanism for lipid-induced mitochondrial dysfunction in the heart.
Abstract 5384: Acute Lipid Infusion Causes Macrophage Infiltration, Local Inflammation, and Suppresses AMPK in Heart
Authors: Ko Hwi Jin , Jung Dae Young , Kim Jason K
Published: July 2021

Obesity and hyperlipidemia are major risk factors of heart disease. Recent studies showed that inflammation is associated with obesity and plays a role in the pathogenesis of diabetic complications. In this report, we examined the effects of lipid on cardiac inflammation and metabolism. Intralipid (2.5 ml/kg/hr; triglyceride emulsion) and heparin (6 U/hr) or glycerol (control) were intravenously infused for 5 hrs to raise circulating fatty acids (FFA) levels in awake C57BL/6 mice (n=10~11). Plasma FFA levels were raised by 3.5-fold over the glycerol-infused groups, and heart samples were taken at the end of experiments. Acute lipid infusion caused cardiac inflammation and increased local macrophage-specific CD68 levels in heart (Fig. 1

; *
P
<0.05). Local levels of cytokines (IL-6 and TNF-α) were elevated by ~2-fold following lipid infusion (Fig. 2

). Heart expression of the C-C motif chemokine receptor-2 (CCR2), which binds the MCP-1 was elevated by 70% following lipid infusion, and this was associated with increased levels of MyD88 in heart, supporting the role of TLR4 signaling in lipid-induced cardiac inflammation. Lipid-induced elevation of local cytokine levels resulted in increased cardiomyocyte expression of SOCS3 (Fig. 3

). Further, acute lipid infusion reduced total AMPK protein levels and Thr
172
phosphorylation of AMPK in heart (Fig. 4

). These data are consistent with the role of inflammation in the regulation of myocardial AMPK. Our findings indicate that fatty acids and nutrient stress are involved in obesity-induced cardiac inflammation and alterations in myocardial glucose metabolism.
Abstract 5391: Combined Ablation of Junctin and Triadin is Associated with Leaky Ryanodine Receptors and Depressed Cardiac Contractile Function
Authors: Han Peidong , Zhou Xiaoyang , Zhao Wen , Chen Guoli , Chen Shan , Figueira Sarah , Fan Guochang , Allen Paul D , Kranias Evangelia G
Published: July 2021

Junctin (JCN) and triadin (TRD) share similar structures and they both function to anchor calsequestrin (CSQ) to the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR) quaternary Ca-release complex. In failing human hearts, JCN and TRD protein levels are markedly decreased, implicating alterations in SR Ca-cycling and contractility. To address the role of combined JCN and TRD down-regulation in cardiac function, we generated and characterized a murine model deficient in both JCN and TRD. The double-knockout (DKO) mice presented lower body weight, poor fertility, and an apparent cardiac hypertrophy. In addition, deficiency of both JCN/TRD was associated with decreased peak Ca transient amplitude (34%), prolonged transient decay time (48%) and reduced caffeine-induced SR Ca-release. This depressed contractility was also confirmed at the intact organ (Langendorff perfusion) and whole animal (by echocardiography and catheterization) levels. Furthermore, examination of the properties of Ca sparks, which are informative of ryanodine receptor (RyR) gating , revealed increased frequency in the DKO myocytes, which indicated a larger opening probability of RyR. These findings suggest that the combined JCN/TRD-deficiency is associated with leaky RyR and depressed cardiac Ca-cycling, which may contribute to the progression of heart failure.

This research has received full or partial funding support from the American Heart Association, AHA National Center.

Table 1.

Hemodynamic parameters from echocardiography and catheterization
Abstract 5417: Inducible 20S β Subunit LMP-2 is Down-Regulated in db/db Mouse Hearts with Diabetic Cardiomyopathy
Authors: Zu Lingyun , Bedja Djahida , Becker Lewis C , Cai Zheqing P
Published: July 2021

Background: The ubiquitin-proteasome system (UPS) plays an important role in cell growth, differentiation, and survival. Low molecular mass polypeptide (LMP)-2 is an inducible 20S beta subunit and its role in diabetic cardiomyopathy has not been previously studied. We investigated the hypothesis that LMP-2 is down-regulated in diabetic hearts and is involved in diabetic cardiomyopathy.

Methods: db/db mice and control db/+ littermates were studied at 20 weeks of age. Cardiac function was measured by echocardiography. Chymotryptic-like activity of the 20S proteasome was analyzed by fluorogenic assay using the substrate Suc-LLVY-aminomethycoumarin (-AMC).

Results: db/db mice had a severe increase in blood glucose and a decrease in heart weight compared with db/+ mice (135±6 vs. 168±7 mg, n = 4, p < 0.01). Left ventricular function was decreased with a lower fractional shortening in db/db mice (48.7±3.2 vs. 60.7±0.4 %, n = 4, p < 0.05). LMP-2 was constitutively expressed in both db/+ and db/db mouse hearts, but its level was severely depressed in the db/db hearts (78±11 vs. 245±18 AU, n = 4, p < 0.01). Consistent with the change in LMP-2, chymotryptic-like activity was also significantly down-regulated in these diabetic hearts (3286±112 vs. 3598±52 AU, n = 3, p < 0.05). Furthermore, the role of LMP-2 in regulating 20S proteasome function and cardiac mass was determined in LMP-2 knockout and wildtype mice. Chymotryptic-like activity was significantly decreased in LMP-2 knockout mouse hearts (11156±360 vs. 18280±454 AU, n = 4, p < 0.01), and cardiac atrophy was found in these knockout mice (147±7.1 vs 178±10.5 mg, n=10; p < 0.01). Taken together, these results suggest that LMP-2 is necessary for normal proteasome function in the heart and its down-regulation may contribute to the development of diabetic cardiomyopathy.
Abstract 5423: rAAV-Mediated Gene Therapy with Inducible Nitric Oxide Synthase (iNOS) Affords Permanent Cardioprotection (1 Year) without Adverse Functional Consequences
Authors: Li Qianhong , Guo Yiru , Wu Wen-Jian , Ou Qinghui , Sanganalmath Santosh K , Tan Wei , Zhu Xiaoping , Mu Jingyao , Dawn Buddhadeb , Bolli Roberto
Published: July 2021

The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with iNOS is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects of iNOS gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS) which enables long-lasting transgene expression. Mice received injections in anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+ 2.9-fold vs. LacZ group, n=6, P<0.05; Fig

) and iNOS activity (+ 3.3-fold vs. LacZ group, n=4, P<0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5+/−2.2%, n=12, vs. 42.9+/−2.6%, n=12, in LacZ group; Fig

). The infarct-sparing effects of iNOS gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (16.3+/−2.3%, n=8; Fig

). Importantly, compared with the LacZ group (n=11), iNOS gene transfer (n=10) had no effect on LV dimensions or function for up to 1 year (at 1 year: LVEDD 4.4+/−0.1 vs. 4.2+/−0.2 mm; LVESD 2.9+/−0.1 vs. 2.9+/−0.2 mm; FS 34+/−1.8 vs. 32+/−2.6%; EF 56+/−2.3 vs. 60+/−2.9%) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year) cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy.
Abstract 5430: Obstructive Sleep Apnoea Exacerbates Muscular Sympathetic Nervous Activity in Patients with Metabolic Syndrome
Authors: Trombetta Ivani C , Nunes Cristiane M , Drager Luciano F , Fraga Raffael F , Alves Maria Janieire N , Rondon Maria Urbana P , Bechara Maita G , Dias Edgar T , Braga Ana M , Laterza Mateus C , Lorenzi-Filho Geraldo , Negrao Carlos E
Published: July 2021

Obstructive sleep apnoea (OSA) and metabolic syndrome (MetS) independently increases muscular sympathetic nervous activity (MSNA). Unknown is whether OSA has an additive effect on MSNA in patients with MetS. We tested the hypothesis that: OSA would have an additive effect on MSNA in patients with MetS. In addition, we studied whether the increase in MSNA in patients with MetS is associated with alteration in arterial baroreflex sensitivity (BRS). Twenty four patients with MetS diagnosed according ATP-III were divided in two groups:

MetS+OSA (n=14) and

MetS (n=10).

They were matched for age, body mass index, waist circumference, and metabolic profile: OSA was defined by an apnoea/hypopnoea index (AHI)>15 events/hour by polysonography. MSNA was recorded directly from the peroneal nerve using the technique of microneurography. Blood pressure (BP) was monitored on a beat-by-beat basis (Finapress) and heart rate by ECG. BRS was analyzed by spontaneous BP and heart rate fluctuations. AHI was higher (42±9 vs. 7±1 events/h,
P
=0.0001) and minimum oxygen saturation lower (77±2 vs. 87±1 %,
P
=0.001) in MetS+OSA patients. Patients MetS+OSA had higher MSNA (55±3 vs. 43±2 bursts/100 beats,
P
=0.01) and systolic BP (158±4 vs.144±3 mmHg,
P
=0.01) when compared with patients with MetS without OSA. Further analysis showed that AHI and minimum oxygen saturation have significant correlation with MSNA (r=0.65;
P
=0.001 and r=−0.48;
P
=0.017, respectively). Patients with MetS+OSA had lower BRS for increases (7.8±0.9
vs.
13.4 ± 1.4 msec/mmHg,
P
=0.01) and decreases (7.2±0.9
vs.
13.2 ± 2.0 msec/mmHg,
P
=0.03) in blood pressure than patients with MetS without OSA. MSNA significantly correlated with BRS during spontaneous increases in blood pressure (r=−0.56,
P
=0.01). OSA exacerbates MSNA in patients with MetS. In addition, the augmented MSNA in patients with MetS+OSA is associated with reduced BRS. These findings suggest that OSA increases the risk for cardiovascular disease in patients with MetS.
Abstract 5432: Mutation of Three Amino Acids in the Disulfide-Ring of a CNP Based Chimeric Natriuretic Peptide Alters its Vascular Properties
Authors: Chen Horng H , Huntley Brenda K , Lee Candace Y , Martin Fernando L , Schirger John A , Burnett John C
Published: July 2021

BACKGROUND:
C-type natriuretic peptide (CNP) is a 22-amino-acid peptide produced mainly in the endothelium with potent cardiac unloading and modest blood pressure lowering actions, but minimal renal actions. Based on our previous knowledge, we recently fused a 6 aa sequence from BNP to the C-terminus and a 5 aa sequence from ANP to the N-terminus of CNP. This novel hybrid peptide, CBA-NP, has cardiac unloading actions and mild hypotensive effects similar to CNP. Importantly however, the N and C terminus alterations resulted in potent renal excretory actions. here we test the hypothesis that the 3 aa GSM
15–17
in the disulfide-ring mediate the vascular and hypotensive actions. We therefore mutated GSM
15–17
to REA
15–17
, which we named ABC-NP and compared its
in vivo
and
in vitro
actions to CBA-NP.

METHODS:
We determined the cardiorenal and humoral actions of intravenous bolus administration of CBA-NP (n=5) and ABC-NP (n=5) at 25 microgram/Kg in 2 separate group of normal anesthetized dogs. We also assessed the cGMP response of both peptides in human aortic endothelial cells (HAEC), human cardiac fibroblast (HCF) and isolated canine glomeruli. * p<0.05

RESULTS:
IV bolus administration of CBA-NP and ABC-NP resulted in diuresis* and natriuresis*. There was a significant decrease in mean arterial blood (MAP) pressure with CBA-NP (126±6 to113±7 mmHg*) but no change with ABC-NP(126±8 to126±8 mmHg) . In addition, the reduction in pulmonary capillary wedge pressure (PCWP) and right atrial pressure (RAP) was significantly greater with CBA-NP as compared to ABC-NP. cGMP generation in HAEC and HCF was minimal with ABC-NP and was significantly higher with CBA-NP*. In contrast, cGMP generation was similar in isolated glomeruli between the two peptides.

CONCLUSION:
Our studies demonstrates that mutation of three amino acid (aa) residues within the CNP ring of CBA-NP from GSM
15–17
to REA alters the vascular but not the renal excretory properties. Hence by this strategic mutation within the ring of CBA-NP, we have designed a renal specific peptide ABC-NP resulting in new sequence specific functional information which can be used to design organ specific therapeutic peptides with unique properties tailored for a specific disease state.
Abstract 5456: Vascular Endothelial Cells Endothelin-1 Deficiency Decreased Vascular Protection in Atherosclerosis Mouse Model
Authors: Anggrahini Dyah Wulan , Emoto Noriaki , Nakayama Kazuhiko , Widyantoro Bambang , Miyagawa Kazuya , Nonaka Hidemi , Anggraeni Vita Yanti , Kisanuki Yaz Y. , Yanagisawa Masashi , Hirata Kenichi
Published: July 2021

Endothelium plays important role in protective mechanism of vascular wall. The balance between endothelin-1 (ET-1) and nitric oxide provide endothelial barrier to lipoprotein retain and macrophage recruitment. In contrasts, ET-1 is also a strong vasoconstrictor. In this study, we aim to determine the role of vascular endothelial cells-derived ET-1 in the development of atherosclerosis. For that purpose, we crossbred Vascular Endothelial Cells-specific ET-1 Knockout (VEETKO) mice to ApoEKO mice. ApoE/VEET-DKO exhibited significantly lower ET-1 plasma and mRNA level as compared to ApoEKO mice. No significant differences of blood pressure, plasma cholesterol or lipid profiles were observed in both mice. Surprisingly, after 8 weeks of western diet, we found that the atherosclerotic lesion was exaggerated in the aortic sinus and brachiochepalic artery of ApoE/VEET-DKO mice (n=7) as compared to those of ApoEKO mice (n=7) (ratio/vessel wall, 0.93±0.13vs.0.49±0.09, p<0.05). We further showed the increase in macrophage plaque content and peritoneal macrophage recruitment in DKO mice. To understand the mechanism of vascular protection, we found lower eNOS mRNA level in DKO mice despite only lower tendency of ETB receptor expression. Functionally, the mice lacking ET-1 in endothelial cells showed impaired NO-mediated endothelial function. Decreased vascular protection further led to increase plaque instability in DKO mice. Here we showed that plaque of DKO mice was more lipid enrich as compared to that of ApoEKO (ratio/lesion, 0.56±0.03vs.0.42±0.04, p<0.05). Moreover, lack of ET-1 significantly reduced matrix synthesis following lower SMCs accumulation in the lesion (ratio/vessel wall, 0.28±0.06vs.0.57±0.08, p<0.05), which was mediated by TGFβ. Interestingly, despite similar advance-typed lesion formed, 15% of DKO mice exhibited plaque hemorrhage in brachiochepalic artery. In conclusion, we demonstrated the increase in atherosclerosis and plaque instability in our model. This further suggests that ET-1 produced from vascular endothelial cells is required for protective mechanism in vascular wall in balance with nitric oxide production. Our data imply for the careful monitoring in the use of ET receptor antagonist in clinical setting.
Abstract 5466: In Vivo Molecular Imaging Revealed Chronic Inflammation and Increased Adhesion Molecules in Obese Adipose Tissue in Mice
Authors: Nishimura Satoshi , Nagasaki Mika , Manabe Ichiro , Seo Kinya , Kadowaki Takashi , Sugiura Seiryo , Nagai Ryozo
Published: July 2021

Metabolic syndrome is a major risk factor of cardiovascular events, and obese visceral adipose tissue remodeling and malfunctioning based on chronic inflammation plays a central role. To assess dynamic multi-cellular interplay, a novel ex vivo (a–c) and in vivo (d–f) adipose tissue imaging method was developed. We found close spatial and temporal interrelationships between angiogenesis and adipogenesis, and both were augmented in obese adipose (c,arrow). In addition, we found increased leukocyte-platelet-endothelial cell interactions in the micro-circulation of obese visceral adipose that were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation (e,f). Both macrophages and endothelial cells showed increased adhesion molecules including ICAM-1 and Selectin families, and P-selectin positive platelets were increased locally in obese adipose. Up-regulated expression of adhesion molecules on multiple cell types suggests their increased interactions contribute to local activation of inflammatory processes within visceral obese adipose tissue. Interestingly, the heightened leukocyte-platelet-endothelial interactions were not observed in obese subcutaneous fat pads. Our results demonstrated the power of our imaging technique to analyze complex inflammatory cellular interplays in vivo and to evaluate new therapeutic interventions against them. Results also indicate that visceral adipose tissue obesity is an inflammatory disease.
Abstract 5476: Hepatic Over-Expression of Cholesteryl Ester Hydrolase Enhances Cholesterol Elimination and in vivo Reverse Cholesterol Transport
Authors: Zhao Bin , Song Jingmei , Ghosh Shobha
Published: July 2021

Neutral Cholesteryl ester hydrolase (CEH) mediated hydrolysis of cellular cholesteryl esters (CE) is required not only to generate free cholesterol for efflux from macrophages but also to release FC from lipoprotein delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH will regulate the hydrolysis of lipoprotein derived CE and enhance reverse cholesterol transport (RCT). Adenoviral mediated CEH over-expression led to a significant increase (>3 fold) in bile acid output (see Figure

). To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol loaded and [
3
H]-cholesterol labeled J774 macrophages were injected intraperitoneally into mice and appearance of [
3
H]-cholesterol in gall bladder bile and feces over 48 hours was quantified. Mice over-expressing CEH had significantly higher [
3
H]-cholesterol in bile and feces and it was associated with bile acids. This CEH-mediated increased movement of [
3
H]-cholesterol from macrophages to bile and feces was significantly attenuated in SR-BI−/−mice. These studies demonstrate that similar to macrophage CEH that rate limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.

This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).
Abstract 5495: Role of SIRT1 in Smooth Muscle Cell Function and Vascular Remodeling
Authors: Sedding Daniel , Vogel Sabina , Tillmanns Harald
Published: July 2021

Background:

The class III histone deacetylase SIRT1 has been identified as a key regulator of ageing and longevity in model organisms such as S. cerevisiae and C. elegans, which regulates cellular functions such as differentiation, senescence and metabolism. However, the role of SIRT1 for Smooth muscle cell (VSMC) function and vascular homeostasis or during vascular remodelling remains unknown.

Methods and Results:

Here, we show that SIRT1 is highly expressed in intact blood vessels in vivo as well as in cultured VSMC. Stimulation of SIRT1 activity by either treatment with the SIRT1 activator resveratrol or adenoviral overexpression of wild type SIRT1 but not with an inactive SIRT1 mutant attenuated serum-induced VSMC proliferation in a dose dependent manner in vitro. In contrast, treatment of VSMC with the small molecule weight inhibitors of SIRT1, nicotinamide and sirtinol, augmented the proliferative and migratory activity of VSMC. Consistent with these data, MEF cells isolated from SIRT
−/−
mice showed an augmented proliferative response to serum stimulation but were also more resistant to starving-induced apoptosis compared to WT-MEF cells. Silencing of endogenous SIRT1 using siRNA resulted in an increased proliferation, migration and apoptosis of VSMC. In vivo, following arterial injury of the mouse femoral artery, SIRT1 was downregulated in the developing neointima. Adenoviral-mediated reconstitution of wild type SIRT1 but not of the inactive SIRT1 mutant prevented neointima formation in vivo.

Conclusion:

Thus, these data identify SIRT1 as a key regulator of vascular proliferative disease processes and indicate that SIRT1 plays an essential role in proliferative migratory and apoptotic processes which regulate vascular homeostasis and remodeling.
Abstract 5523: High Glucose Promotes Intracellular Lipid Accumulation by Impairing the Balance between CD36 and ATP-Binding Cassette Transporter G1 in Vascular Smooth Muscle Cells
Authors: Xue Jia H , Yuan Zu Y , Wu Yue , Zhao Yan , Zhang Wei P , Tian Yu L , Liu Wei M , Kishimoto Chiharu
Published: July 2021

Objective:
Foam cell formation is a characteristic of atherosclerotic lesions. It’s known that high glucose promotes macrophage-derived foam cell formation involved in increased influx or reduced efflux of lipids. The aim of this study is to investigate the influence of hyperglycemia on foam cell transformation of vascular smooth muscle cells (VSMCs) and possible mechanisms contributing to these effects.

Methods and Results:
The results showed that high glucose in cultured human aortic SMCs increased the mRNA and protein expressions of CD36, a regulator of lipid influx, and suppressed the mRNA and protein expressions of ATP binding cassette (ABC) transporters ABCG1, a regulator of cholesterol efflux to HDL, in a dose- and time-dependent manner. However, the ability of cholesterol efflux to lipid-free apoAI was not impaired. VSMCs exposed to high glucose were easily developed into lipid-loaded cells as demonstrated by oil red O staining. Meanwhile, it had a maximum 2.3-fold increase in accumulation of esterified cholesterol compared to VSMCs cultured in normal glucose. Additionally, there was no change found in either liver X receptor (LXR)α or LXRβ, suggesting that high glucose-induced down-regulation of ABCG1 was LXR-independent. Down-regulation of ABCG1 induced by high glucose was almost totally reversed by the NF-κB inhibitors BAY 11–7085, tosyl-phenylalanine chloromethyl-ketone (TPCK) and by the antioxidant N-acetyl-L-cysteine(NAC). This reversal was accompanied by reduced intracellular lipid content. Furthermore, we also demonstrated that high glucose enhanced the binding of nuclear proteins extracted from human VSMCs to the NF-κB regulatory elements. This effect was abrogated by NAC and NF-κB inhibitors.

Conclusions:
These results suggested that hyperglycemia-induced foam cell formation in VSMCs was related to the imbalanced lipid flux by increasing CD36 mediated modified LDL uptake and reducing ABCG1 regulated intracellular cholesterol efflux. Moreover, this effect was associated with activated NF-κB pathway signaling.
Abstract 5554: Runx2 Represses TGFβ/Smad-Mediated Connective Tissue Growth Factor Gene Expression in Vascular Smooth Muscle Cells
Authors: Ohyama Yoshiaki , Tanaka Toru , Shimizu Takehisa , Sato Hiroko , Koitabashi Norimichi , Doi Hiroshi , Iso Tatsuya , Arai Masashi , Kurabayashi Masahiko
Published: July 2021

[Background] Runx2, a key transcription factor for osteoblastic differentiation, is expressed in calcified atherosclerotic plaques. We recently reported that Runx2 represses vascular smooth muscle cells (VSMC) differentiation and promotes its osteogenic differentiation. Connective tissue growth factor (CTGF) has been implicated in the progression to vulnerable plaque by inducing mononuclear cell chemotaxis and VSMC apoptosis despite of its potent stimulatory effect on synthesis of extracellular matrix. In this study, we investigated the regulatory mechanism of CTGF gene expression by Runx2 in VSMC.

[Methods and Results] RT-PCR analyses showed that adenovirally overexpressed Runx2 significantly repressed the basal expression of the CTGF gene in human aortic SMCs (HASMCs). Consistent with this, knockdown of the Runx2 expression in HASMCs by small interfering RNA (siRNA) increased CTGF mRNA levels. Luciferase assays showed that Runx2 reduced the transcriptional activity of the CTGF promoter. Transfection of a series of 5′-deletion constructs revealed that Runx2 inhibited CTGF expression through the sequence element located at 5′ untranslated region of CTGF mRNA. We next examined the effects of Runx2 on the TGFβ-induced CTGF expression. Runx2 overexpression significantly attenuated the TGFβ-mediated induction of CTGF expression in HASMCs, and knockdown of Runx2 by siRNA enhanced the induction of CTGF expression in response to TGFβ. Runx2 repressed TGFβ-induced CTGF promoter activity through the sequence containing Smad binding element (SBE), and luciferase assay using SBE-specific mutation construct showed that Runx2 repressed CTGF promoter activity in an SBE-dependent manner. Overexpression of Runx2 significantly reduced TGFβ and Smad3-mediated luciferase activity of 4xSBE-tkLuc, which contains four copies of SBE. Co-immunoprecipitation showed that Runx2 formed a complex with Smad4.

[Conclusion] These data demonstrate that Runx2 represses basal and TGFβ-induced CTGF gene expression in VSMC, and thus suggest that besides the potential for inducing vascular calcification, Runx2 may affect plaque stability by modulating extracellular matrix synthesis through inhibiting CTGF gene expression and TGFβ signaling.
Abstract 552: Posterior Involvement Attenuates Predictive Value of ST-Segment Elevation in Lead V4R for Right Ventricular Involvement in Inferior Acute Myocardial Infarction
Authors: Kosuge Masami , Kimura Kazuo , Ebina Toshiaki , Hibi Kiyoshi , Tsukahara Kengo , Okuda Jyun , Iwahashi Noriaki , Ishikawa Toshiyuki , Uchino Kazuaki , Umemura Satoshi
Published: July 2021

ST-segment elevation (ST ↑) ≥1.0 mm in lead V4R is considered a reliable marker of right ventricular involvement (RVI) in inferior acute myocardial infarction (IMI). However, the impact of posterior involvement (PI) on the relation between RVI and ST ↑ in lead V4R is unknown. We studied 267 patients with a first IMI who had total occlusion and TIMI 3 flow of the right coronary artery within 6 h after the onset. A 12-lead ECG, lead V4R, and leads V7–9 were recorded on admission. RVI was defined as occlusion proximal to the first right ventricular branch. The perfusion territory was assessed by angiographic distribution score, and PI was defined as a score of ≥0.7. Myocardial blush grade was assessed immediately after reperfusion. Patients were stratified according to the presense or absense of PI and RVI. Times to admission and reperfusion were similar in the 4 groups. RVI was associated with higher peak creatine kinase and higher rates of impaired myocardial reperfusion (blush grade 0/1) and congestive heart failure during hospitalization in the presense or absense of PI, especially the former. RVI was associated with a higher rate of ST ↑ in lead V4R in the absence, but not in the presence, of PI. ST ↑ in lead V4R identified RVI with sensitivities of 34% and 96% (p<0.001) and specificities of 83% and 82% (NS) in the presence and absence of PI, respectively. In patients with reperfused IMI, RVI is associated with a larger infarct size and impaired myocardial reperfusion. However, the incidence of RVI diagnosed by ST ↑ in lead V4R was underestimated in the presence of posterior involvement. ST ↑ in lead V4R caused by RVI might be attenuated due to a reciprocal change in posterior ST ↑.
Abstract 558: Association of Chronic Kidney Disease and Diabetes Mellitus with 5-Year Outcomes after Acute Myocardial Infarction
Authors: Dai Kazuoki , Ishihara Masaharu , Inoue Ichiro , Kawagoe Takuji , Shimatani Yuji , Kurisu Satoshi , Nakama Yasuharu , Maruhashi Tatsuya , Kagawa Eisuke , Matsushita Junichi , Ikenaga Hiroki
Published: July 2021

Several studies have shown that both chronic kidney disease (CKD) and diabetes mellitus are risk factors for mortality in patients with acute myocardial infarction (AMI). This study was undertaken to investigate influence of CKD on the prognostic significance of diabetes in patients with AMI. Between January 1996 and December 2005, 888 patients with AMI underwent coronary angiography within 24 hours after the onset of chest pain. CKD was difined estimated glomerular filtration rate (eGFR) of less than 60.0 ml/minute/1.73 m
2
of body-surface area (stage3–5). Kaplan-Meier method was used to compare 5-year survival of diabetic and non-diabetic patients, in the presence (n=337) or absence (n=551). Kaplan-Meier curves for 5-year survival rate are shown in Figure

. In the absence of CKD, there was no significant difference in 5-year survival rate between patients with diabetes and those without (93 % v.s. 94 %, p=0.82). In patients with CKD, however, diabetes was associated with lower 5-year survival rate (65 % v.s. 87 %, p<0.001). Multivariate analysis showed that diabetes was an independent predictor for 5-year survival in patients with CKD (OR 3.2, 95%CI 1.8–5.8, p=0.0002), but not in patients without CKD (OR 1.1, 95%CI 0.4–2.5, p=0.82). Diabetes mellitus was an independent predictor for death after AMI in patients with CKD. Aggressive treatment after AMI should be advocated in diabetic patients with CKD.
Abstract 559: Elevated Level of sCD40L During Acute Coronary Syndrome Leads to Systemic MIP-2 Release
Authors: Stadlbauer Thomas , Molitor Benedikt , Jochen Wilhelm , Harwaldt Petra , Schaub Meike , Hoelschermann Hans , Tillmanns Harald , Hofmann Wolf Karsten
Published: July 2021

Purpose:
Acute coronary syndromes (ACS) have been associated with sCD40 Ligand (sCD40L) release. Endothial cells are located at the interface between the blood and parenchymal cells and take active part in many physiological and pathologic processes, including inflammation. It has been suggested that CD40L is a key player in the pathogenesis of atherosclerosis and ACS, originating from endothelial cells. When sCD40L is present in human plasma during ACS, it is mainly derived from activated platelets. This may lead to activation of endothelial cells resulting in atherosclerosis and eventually atherothrombosis. Therefore, the aim of the present study was to investigate differential gene expression profiles of endothelial cells after stimulation with sCD40L.

Methods:
Human endothelial cells (HUVEC) were stimulated with sCD40L for 2 hours. Differential gene expression patterns were evaluated with Oligonucleotide microarray hybridization (UG-U133A, Affymetrix Inc, Santa Clara, CA). Significantly up-regulated gene expression was confirmed and quantitated by Real-time-PCR as well as ELISA. In serum from ACS patients expression of sCD40L was correlated with these up-regulated genes products.

Results:
Stimulation of HUVEC with sCD40L leads to a profound release of proinflammatory mediators like IL-8, ICAM1, ISGF3 as well as MCP-1 on a transcriptional level. In addition, gene expression profiling revealed a highly significant increase in expression of the macrophage inflammatory protein 2 (MIP-2), also known as GRO-beta or CXCL2. This was quantitated as approximately 30 fold up-regulation by real-time-PCR as well as in ELISA analysis (8 fold at 8 to 24 hours) derived from supernations. In clinical serum samples from ACS patients there was a highly significant correlation between sCD40L and MIP-2 levels (r = 0.88, p < 0.01)

Conclusion:
Proinflammatory mediators as well as MIP-2 play an important role in the chemoattraction of macrophages, which underlines the key role of CD40L as a link between thrombotic events, e.g ACS, and inflammatory processes, e.g. atherosclerosis. Therefore targeting MIP-2 up-regulation might be a potential approach to prevent the progression of atherosclerotic diseases as well as a new therapeutic approach to treat ACS.
Abstract 569: NT-proBNP is an Important Independent Predictor of Clinical Events after Primary PCI for STEMI
Authors: Ezekowitz Justin A , Bakal Jeffery A , Huber Kurt , Theroux Pierre , James Stefan K , Betriu Amadeo , Weaver W. Douglas , Granger Christopher B , Armstrong Paul W
Published: July 2021

Acute myocardial infarction with ST-segment elevation (STEMI) remains a major global public health issue. Despite advances in therapy, patients remain at risk for death, repeat myocardial infarction (MI) shock and heart failure (HF). Novel markers that predict those at risk are needed. We studied 903 STEMI patients in The Assessment of Pexelizumab in Acute Myocardial Infarction trial (which enrolled STEMI patients presenting < 6 hrs of symptom onset who were to undergo primary PCI) in a case-control design (cases selected based on the trial’s primary composite outcome - death, shock or HF - and matched on age, gender and infarct location to controls). NT-proBNP (pg/ml) was measured at randomization and 24 hrs. Outcomes (individually and the composite) of death, shock, and HF at 90 days were examined by quartiles of NT-proBNP. A CART model was used to categorize adjusted risk. NT-proBNP was higher in patients who had events. Patients with higher NT-proBNP levels at baseline (median symptom onset to randomization 2.7 hrs) and 24 hrs had more events (composite p<0.001; death p<0.0001; HF p<0.0001; shock p=0.05) - See figure

. Using the CART model (adjusted for age, gender and infarct location), baseline Killip class and NT-proBNP could further subcategorize patients into 90 day mortality categories 4%, 10%, 30%, and 53%. In fact, only 4 patients (1%) with a 24 hour NT-proBNP <999 pg/ml had any event in the next 90 days. Although the overall prognosis in STEMI patients undergoing primary PCI is good, NT-proBNP performed early and at 24 hrs provides important prognostic information for predicting negative outcomes i.e. shock, heart failure and death.

Figure.

Kaplan-Meier curve for the primary composite outcome stratified by baseline NT-proBNP (a), or 24 hour NT-proBNP (b).
Abstract 590: Evaluation of the Effects of Cardiac Arrest and CPR Using Intracardiac Echocardiography
Authors: Burke Martin C , Lim Kiam K , Smelley Matthew , Beshai John F , Kim Susan S , Lin Albert C , Knight Bradley P
Published: July 2021

For every minute of cardiac arrest, mortality increases by 10%. Still, the variablity of meaningful neurologic survival suggests multiple factors involved in the hypoxia and encephalopathy of cardiac arrest (CA). We studied the cardiac chamber environment during ventricular fibrillation and asystole using direct ultrasound visualization using real-time intracardiac echocardiography (ICE). Four pigs were studied under general anesthesia per protocol. Venous access was obtained for catheter placement within the right heart. A bipolar paciing wire was placed into the right ventricle. A 10 French Acuson ICE catheter was placed into the right atrium using fluoroscopy. Baseline images were obtained. Ventricular fibrillation was induced using direct current energy. 30 minutes of ventricular fibrillation and terminal asystole was observed. Chest compressions were delivered intermittently with direct visualization of effects. Intra-chamber thrombus was quantified by chamber and by arrhythmia. All subjects began to form intracardiac thrombus within 1 minute of ventricular fibrillation. The persistence of sinus rhythm during fibrillation prevented thrombus formation in the atria and the basal portions of both ventricles. The thrombus was predominantly in the right heart within the first 4 minutes of CA. ICE documents that chest compressions completely clear the thrombus when delivered within the first five minutes of CA. Asystole led to more solidified thrombus that was more difficult to clear. Thrombus formation during CA is marked and may be a predominant reason for neurologic damage post resuscitation. Asystole was associated with complete chamber thrombus in comparison to ventricular fibrillation.
Abstract 593: Hemodynamic Effect of a δ Opioid Receptor Agonist DADLE on Rats with Hemorrhagic Shock
Authors: Wang Tong , Lin Xiaohong , Wu Haidong , Fu Yue , Huang Zitong
Published: July 2021

Delta opioid receptor agonist can reduce the metabolism and energy demand of ischemic organs in ischemic/reperfusion injury. H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) is a delta opioid receptor agonist. In the present study, we investigated the hemodynamic effect of DADLE on rats with hemorrhagic shock. DADLE can improve the hemodynamics of rats with hemorrhagic shock. Thirty six healthy male SD rats were randomly divided into sham group, control group and DADLE group(1mg/kg group and 5mg/kg group). The control and DADLE animals were bled in 10 minutes by uninterrupted withdrawal from the arterial catheters to set up the model of hemorrhagic shock, then maintained MAP at about 40 mmHg for 60 minutes. All animals were observed for 3 hours after resuscitation with saline injection or DADLE injection and SAP, DAP, MAP, LVSP and ±dP/dtmax were recorded at 30, 60, 90, 120, 180 minutes following injection. After resuscitation, MAP, LVSP and ±dP/dtmax were unchanged in the control group, and remained downtrend. In contrast, significant increases(p<0.01) were found in all hemodynamic data following the administration of DADLE (1mg/kg or 5mg/kg), and the peak values were reached at 30 minutes. There was no significant difference between 1mg/kg and 5mg/kg DADLE groups. DADLE improves the hemodynamics during the course of hemorrhagic shock.
Abstract 637: Utilization of Retrograde Right Bundle Branch Block to Differentiate Atrio Ventricular Nodal from Accessory Pathway Conduction
Authors: Kapa Suraj , Henz Benhur , Dib Chadi , Cha Yong-Mei , Friedman Paul A , Munger Thomas M , Ladewig Dorothy J , Hammill Stephen C , Packer Douglas L , Asirvatham Samuel J
Published: July 2021

Determining whether retrograde ventriculoatrial (VA) conduction is through the AV node (AVN) or an accessory pathway (AP) is critical for successful ablation of supraventricular tachycardia (SVT). With introduction of ventricular extrastimuli (VEST), retrograde right bundle branch block (retro RBBB) may occur and result in abrupt VH interval prolongation. We hypothesized when retrograde conduction is via an AP, VA interval change is less than VH interval change, whereas with retrograde AVN conduction, VA interval change is equal to or greater than VH interval change. We retrospectively reviewed the electrophysiology (EP) studies of patients undergoing ablation for AVNRT (n=55) or AVRT (n=50). The intracardiac electrograms were reviewed for induction of retro RBBB and change in VH and VA intervals during VEST. Parahisian pacing, decremental pacing, arrhythmia diagnosis and ablation outcomes were reviewed. All results were found to be reproducible between two independent observers with inter and intra observer reliability scores of 1.00 for identification of retro RBBB and greater than 0.85 for measurement of VH and VA intervals. Of 105 patients, 84 (80%) had evidence of induced retro RBBB during VEST. The average VH interval increase with induction of retro RBBB was 53.7 ms for patients with retrograde AP conduction and 54.4 ms for patients with AVN conduction (p=ns). The average VA interval increase with induction of retro RBBB was 13.6 ms with AP conduction and 70.1 ms with AVN conduction (P < 0.001). All patients with a greater VH than VA interval change had a final diagnosis of AVRT and those with a VH change less than VA change had AVNRT, yielding sensitivity and specificity of 100%. Using a cutoff of 50 ms for change in VA interval with onset of retro RBBB to diagnose AVNRT accurately identified AVNRT in 100% and AVRT in 95%. Induction of retro RBBB during VEST is common during EP studies for SVT. The relative change in the VH and VA intervals during retro RBBB accurately differentiates retrograde AVN from retrograde AP conduction with strong predictive accuracy. The use of retro RBBB based intervals is a useful technique facilitating the diagnosis of SVT in the EP laboratory, even in the absence of inducible tachycardia.