bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1489: Vitamin D Receptor Activation Modulates the Effect of Uremia on Aortic Gene Expression and Endothelial Function
Authors: Wu-Wong Jinshyun R , Campbell Thomas J , Kroeger Paul E , Noonan William , Segreti Jason , Ruan Xiaoan , Nakane Masaki , Brooks Kristin A , Polakowski James S , Cox Bryan F
Published: July 2021

Vitamin D receptor (VDR) activation therapy is associated with cardiovascular/survival benefit in chronic kidney disease (CKD), but the mechanism of action is not well understood. The aim of this study is to investigate how uremia affects gene expression in aorta and whether VDR activation modulates the uremic effect. DNA microarray technology was used to assess the gene expression profile in aorta prepared from SHAM and 5/6 nephrectomized (NX) rat, a model of Stage 4/5 CKD, treated with or without 0.17 μg/kg paricalcitol, a VDR activator. As expected, paricalcitol at 0.17 μg/kg after two weeks of treatment effectively suppressed serum parathyroid hormone (PTH); no significant difference was observed in ionized calcium or serum phosphorus. Uremia exhibited a significant effect on the gene expression pattern in the aorta, affecting 468 sequences ( ≥1.5-fold changes with p<0.01 vs. SHAM). When the criteria were tightened to ≥ 2-fold changes with p<0.01, uremia still affected 135 target sequences in the rat aorta with 63 up-regulated and 72 down-regulated. Target genes fell into various categories including metabolism and cellular metabolism. Paricalcitol treatment normalized 95 out of the 135 sequences affected by uremia; many of the genes were related to mitochondrial function and oxidative stress. As a follow-up to the microarray analysis, endothelial function was examined. Uremia significantly affected aortic relaxation (−50.0 ± 7.4% in NX rats vs. −96.2 ± 5.3% in SHAM at 30 μM acetylcholine). The endothelial-dependent relaxation response to acetylcholine (Ach) at 30 μM was improved to −58.2 ± 6.0%, −77.5 ± 7.3% and −90.5 ± 4.0% in NX rats treated with paricalcitol at 0.021, 0.042 and 0.083 μg/kg for two weeks, respectively, while blood pressure and heart rate were not changed. PTH suppression alone didn’t improve endothelial function since cinacalcet, a calcimimetic targeting calcium sensing receptor, suppressed PTH without affecting endothelial-dependent vasorelaxation. In conclusion, VDR activation by paricalcitol modulates the effect of uremia on aortic gene expression and endothelial function independent of PTH and blood pressure control, which may be one of the mechanisms responsible for paricalcitol’s cardiovascular benefit in CKD.
Abstract 1492: Molecular Imaging Of Occult Kidney Inflammation In ApoE −/− Mice Using
19
F MRI Nanobeacons For VCAM-1
Authors: Kaneda Megan , Southworth Richard , Chen Junjie , Zhang Lei , Zhang Huiying , Lanza Gregory , Wickline Samuel
Published: July 2021

Although renal disease has been identified as a comorbidity for late-stage atherosclerosis, the early involvement of renal inflammation in the pathophysiology of progressive atherosclerosis has not yet been defined. To elucidate the impact of hyperlipidemia on incipient kidney disease, we deployed a VCAM-1-specific nanobeacon for magnetic resonance molecular imaging that emits a unique fluorine (
19
F) signature without any background signal (Figure

). A dose of 1ml/kg was given intravenously to 6 cholesterol-fed ApoE −/− and 6 C57BL/6 mice, and VCAM -1 expression was measured after 2 hours at 11.7T
ex vivo
. Marked diffuse expression of VCAM-1 and macrophage infiltration was observed by immunocytochemistry in glomerular structures, especially in efferent and afferent arterioles.
19
F imaging confirmed widespread involvement in the renal cortex (Figure

). Magnetic resonance spectroscopic quantification of renal VCAM-1 expression in control (C57BL/6) mice was significantly lower (36.5±8.8 vs. 9.3±2.2x10
8
NP/g kidney) than that of ApoE−/− mice (p<0.05). These data indicate that
19
F-based molecular MRI nanobeacons can detect and quantify renovascular disease in its earliest preclinical stages, and serve as a tool for both prognosis and management of generalized atherosclerosis.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 1572: Single Histidine Button in Cardiac Troponin I Sustains Heart Performance in Response to Severe Hypercapnic Respiratory Acidosis In Vivo
Authors: Palpant Nathan J , D’Alecy Louis G , Metzger Joseph M
Published: July 2021

Intracellular acidosis is a profound negative regulator of myocardial performance. The purpose of this study was to determine whether titrating myofilament calcium sensitivity could protect the whole animal physiological response to acidosis
in vivo.
We hypothesized that single histidine substituted cardiac troponin I (A164H) transgenic (Tg) mice would confer cardio-protection during a severe respiratory acidosis challenge
in vivo
. Severe hypercapnic acidosis was induced in mice by ventilation with 40% CO
2
. Echocardiographic analysis showed that systolic function and ventricular geometry were maintained in Tg mice. By contrast, nontransgenic (Ntg) littermates experienced marked deterioration in systolic performance and significant LV dilation during this same challenge (ejection fraction during hypercapnia: [Ntg, n =5] 23.6 ± 4.9 vs. [Tg, n=7] 67.8 ± 4.5% P < 0.05; volume at systole during hypercapnia: [Ntg, n = 5] 40.6 ± 5.1 vs. [Tg, n = 7] 14.4 ± 3.4 mm
3
P < 0.05). Using radio-telemetry to monitor blood pressure in freely moving animals, Tg mice maintained significantly higher peripheral arterial blood pressures compared to Ntg mice during a long term acidosis challenge (change in mean arterial pressure: [Ntg, n = 4] −81.5 ± 18.4 vs. [Tg, n = 7] −37.7 ± 6.3 mmHg P < 0.05). For detailed hemodymanic assessment, pressure-conductance analysis was performed. To specifically isolate cardiac performance, animals were treated with a beta blocker, esmolol, during a 40% CO
2
challenge. Survival and load independent measures of contractility were significantly greater in Tg vs. Ntg mice (preload recruitable stroke work: [Ntg, n = 6] 78.6 ± 9.5 vs. [Tg, n = 7] 147.2 ± 9.6 P < 0.05). Furthermore, hemodynamic analysis during beta blockade and hypercapnia showed that Ntg mice underwent marked cardiac decompensation and death within five minutes of acidosis compared to Tg mice that maintained contractility and survived the challenge (survival curve [Ntg, n = 6; Tg n = 7], P < 0.0002). This study shows that, independent of any beta adrenergic compensation, myofilament-based molecular manipulation of inotropy by histidine-modified troponin I maintains contractile function and improves survival during severe hypercapnic acidosis
in vivo
.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 1580: Phosphorylation of Myosin Binding Protein C Contributes to the Force-Frequency Response and Beta-Adrenergic Enhancement of Cardiac Contraction
Authors: Tong Carl W , Wu Xin , Muthuchamy Mariappan , Scherman Joseph A , Valdivia Hector H , Moss Richard L
Published: July 2021

Beta-adrenergic agonists enhance cardiac function by increasing contractility, lusitropy and heart rate. We propose that phosphorylation of myosin binding protein C (cMyBP-C), a thick filament accessory protein, plays a key role in adrenergic modulation of function by regulating crossbridge interaction kinetics. To test this idea, we developed transgenic mice expressing similar levels of either cMyBP-C with
Ser
to
Ala
mutations at 3 known protein kinase A (PKA) sites (t3SA mice) or wild type cMyBP-C (tWT mice) on the cMyBP-C knock-out (KO) background. Using echocardiography, t3SA hearts were found to have concentric hypertrophy and exhibited diastolic and systolic dysfunction, but tWT hearts were normal. We recorded twitch force and intracellular Ca
2+
([Ca
2+
]
in
) in intact papillary muscles to assess
in vivo
myofilament function. At 1 Hz stimulation, t3SA and tWT exhibited similar twitch forces and [Ca
2+
]
in
. Increasing the frequency to 3 Hz produced a smaller force increase in t3SA but similar increases in [Ca
2+
]
in
compared to tWT. t3SA myocardium exhibited a smaller gain in function with increased frequency of stimulation, i.e., a smaller increase in twitch force
vs.
change in [Ca
2+
]
in
and smaller increases in (+dF/dt)
Max
, and (−dF/dt)
Max
. These results suggest that basal levels of phosphorylation of cMyBP-C in tWT myocardium contribute to the positive force frequency relationship in myocardium. For both t3SA and tWT, 1 μM dobutamine further increased twitch force and [Ca
2+
]
in
at 3 Hz. Phosphoprotein staining showed that t3SA and tWT troponin I (TnI) phosphorylation levels were initially similar and increased to similar levels with dobutamine; therefore, dobutamine effects may involve TnI phosphorylation. These results can be explained by

a frequency dependent recruitment of myofilament functional groups that involves cMyBP-C phosphorylation and

further effects of PKA phosphorylation of cMyBP-C and TnI to accelerate crossbridge turnover kinetics.
Abstract 1599: Hydrogen Sulfide Mediates Myocardial Preconditioning via Upregulation of Antioxidant and Anti-Apoptotic Signaling Pathways
Authors: Calvert John W , Jha Saurabh , Gundewar Susheel , Duranski Mark R , Lefer David J
Published: July 2021

The emergence of hydrogen sulfide (H
2
S) as a potent cardioprotective mediator necessitates the elucidation of its cytoprotective mechanisms. Therefore, we evaluated potential mechanisms of H
2
S-mediated cardioprotection using an
in vivo
model of preconditioning (PC). The H
2
S donor, IK1001 (100 μg/kg), or vehicle was administered to mice via an intravenous injection 24 hours prior to left coronary artery occlusion and reperfusion. Mice were subjected to 45 min of myocardial ischemia followed by reperfusion for up to 24 hr, during which time the extent of myocardial infarction was evaluated, serum troponin-I levels were measured, and the degree of oxidative stress was assessed. In separate studies, mice were treated with IK1001 and myocardial tissue was collected during both the early (30 min and 2hr) and late (24 hr) PC periods to evaluate potential cellular targets of H
2
S. IK1001 provided profound protection against ischemic injury as evidenced by significant decreases in infarct size, serum troponin-I levels, and oxidative stress. Additional studies revealed that during the early PC period H
2
S increased the nuclear localization of Nrf-2, a transcription factor that regulates the gene expression of a number of antioxidants, and increased the nuclear localization of phosphorylated STAT-3. During the late PC period, H
2
S increased the expression of the antioxidants, Trx-1 and Trx-2, increased the expression of HSP90, HSP70, Bcl-2, and Bcl-xL and also inactivated the pro-apoptogen Bad. Our results reveal that the cardioprotective effects of H
2
S are mediated in large part by a combination of antioxidant and anti-apoptotic signaling.
Abstract 1603: Statins Pleiotropically Mitigate LOX-1 and Cav-1 by Upregulating Hemeoxygenase-1/HSP-90 for e-NOS Phosphorylation and Reduced Ventricular Remodeling during Hypercholesterolemia
Authors: Penumathsa Suresh Varma , Koneru Srikanth , Otani Hajime , Maulik Gautam , Maulik Nilanjana
Published: July 2021

Statin based lowering of low density lipoprotein (LDL) cholesterol has been considered as one of the major factors of cardiovascular risk reduction. Independent of lipid lowering, statins up-regulates the endothelial nitric oxide production through caveolin-1 (Cav-1) downregulation. The novel receptor, lectin like Ox-LDL receptor-1 (LOX-1) is known to bind, internalize and degrade Ox-LDL for its action. This study was designed to investigate the molecular mechanism of statin mediated Cav-1 & LOX-1 regulation. Rats were randomized into: control (normal diet); HC (5% high cholesterol diet for 8 weeks) and HC + Pravastatin (S) (1mg/kg) administered orally for 2 weeks (HCS). Statin reduced the cholesterol, LDL & increased the HDL levels compared to non-treated HC. Hearts were subjected to permanent left anterior descending coronary artery occlusion (MI). LV functions by echocardiography was examined 30 days after MI. Decreased LV inner diameter (7.4 vs 9 mm), increased ejection fraction (47 vs 39 %) and fractional shortening (25 vs 20 %) was observed in HCS compared to HC. Cav-1, LOX-1 protein expression was upregulated in HC (2, 1.8 fold) compared to control which were decreased to 1.2 and 1.1 fold on statin treatment. We have observed in HCS increased Heme Oxygenase-1 (HO-1, 1.9 fold), HSP-90 expression (1.7 fold) and phosphorylation of eNOS (p-eNOS) by 1.6 fold whereas in HC 0.4, 0.5 and 0.4 fold respectively as compared to control. Over expression of HO-1 and HSP-90 by statin treatment might have facilitated for the stimulated displacement of eNOS from Cav-1 for its activation. Further to examine how HO-1 and HSP-90 regulate the eNOS activation we have generated cardiac specific HO-1 over expressed mice. Immunohistochemical analysis of HO-1 mice clearly demonstrated decreased Cav-1-eNOS interaction and increased p-eNOS (1.8 fold) as shown by immunoblot analysis compared to wild type. Moreover, we observed increased HSP-90 (2 fold) which is known for the serine phosphorylation of eNOS. These results indicate for the first time; a novel mechanism of action by statins in regulation of the caveolar platform by Cav-1, LOX-1, HO-1/HSP-32 & HSP-90 for eNOS activation which would be a novel treatment strategy to prevent HC mediated cardiovascular complications.
Abstract 1612: Pharmacological Postconditioning Effect of G-csf Is Mediated through Activation of Pi3k-akt-enos Pathway and Opening the Mitochondrial Katp Channels in a Rabbit Model of Myocardial Infarction
Authors: Sumi Shohei , Iwasa Masamitsu , Kobayashi Hiroyuki , Yasuda Shinji , Yamaki Takahiko , Narantuoya Bau , Minatoguchi Shinya
Published: July 2021

It has been reported that granulocyte-colony-stimulating factor(G-CSF) improves cardiac function after myocardial infarction (MI). However, its direct effect on the myocardium and its signaling pathway remain unclear. We examined the acute beneficial effect of G-CSF on myocardial infarct size and its precise mechanisms in a rabbit model of myocardial infarction. In 80 Japanese white rabbits, MI was induced by 30 min of ischemia and 48 hours of reperfusion. Rabbits were intravenously injected with 10 μg/kg of G-CSF (G-CSF group, n=10) or saline (control group, n=10) immediately after reperfusion. The G-CSF+5HD group (n=10) was injected with 5-HD(5-hydroxydecanoate, a mitochondrial KATP channel blocker) 5 min before G-CSF injection. The G-CSF +wortmaninn group (n=10) was injected wortmaninn (0.6mg/kg) 5 min before G-CSF injection. G-CSF+L-NAME group (n=10) was injected with L-NAME (10mg/kg) 5 min before G-CSF injection. The 5HD alone (n=10), wortmannin alone (n=10), L-NAME alone (n=10) groups were respectively injected 5HD, wortmannin and L-NAME immediately after reperfusion. Myocardial infarct size was calculated as a percentage of the risk area of the left ventricle. Western blot analysis was performed to examine the Akt and phospho-Akt and phospho-eNOS in the ischemic myocardium at 48 hours of reperfusion. The infarct size was significantly smaller in the G-CSF group (26.7±2.7%) than in the control group (42.3±4.6%). The infarct size-reducing effect of G-CSF was completely blocked by 5-HD (42.5±1.66%), wortmaninn(44.7±4.81) and L-NAME (42.1±4.2%). Wortmannin, L-NAME or 5HD alone did not affect the infarct size. Western blotting showed higher expression of phospho-Akt and phosho-eNOS in the infarct area in the G-CSF group than in the control group. G-CSF administered immediately after reperfusion reduces myocardial infarct size via activation of PI3K, Akt, eNOS and opening the mitochondrial KATP channels.
Abstract 1616: The Role For Caveolin And Caveolae In Delayed Protection From Ischemia-reperfusion Injury
Authors: Tsutsumi Yasuo M , Ishikawa Yoshihiro , Roth David M , Patel Hemal H
Published: July 2021

Introduction:
Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. We have recently shown that both caveolin-1 and caveolin-3 knockout (Cav-1 KO and Cav-3 KO, respectively) mice are unable to be protected from myocardial ischemia-reperfusion injury by acute treatment with volatile anesthetics. Therefore, we tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolin-dependent.

Methods:
Biochemical assays were performed in excised hearts. Electron microscopy was used to assess caveolae formation. An
in vivo
mouse model of ischemia-reperfusion injury with delayed anesthetic preconditioning (delayed APC) was tested in wild-type (WT), Cav-1 KO, and Cav-3 KO mice. Mice were exposed to 30 min isoflurane or oxygen and allowed to recover for 24 h. After 24 h recovery, mice underwent 30 min left anterior descending coronary artery occlusion, followed by 2 h of reperfusion at which time infarct size was determined.

Results:
To elucidate a role for caveolins in delayed APC, wild-type mice were exposed to delayed APC and hearts were fractionated on a discontinuous sucrose gradient to isolate buoyant caveolar membranes. Delayed APC increased the amount of Cav-3 protein but not Cav-1 protein in buoyant fractions. Glucose transporter-4 (GLUT-4), known to interact with Cav-3 and affect cardiac protection, was also increased in buoyant fractions after APC. Microscopically distinct caveolae were observed in WT and Cav-1 KO mice but not Cav-3 KO mice. We assessed the impact of caveolae formation in induction of delayed APC. Infarct size as a percent of the area at risk was reduced by isoflurane in WT (24.0 ± 2.5% vs. 45.1 ± 2.9%, p < 0.05) and Cav-1 KO mice (27.2 ± 4.4%). Cav-3 KO mice did not show delayed APC (41.5 ± 2.2%).

Conclusions:
These results demonstrate that isoflurane-induced delayed preconditioning involves translocation of Cav-3 and GLUT-4 to caveolae and the presence of microscopically distinct caveolae (dependent on Cav-3 expression) are a requisite for induction of delayed protection in the myocardium.

This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 1618: Role of Nitric Oxide in Regulation of Myocardial Glucose Metabolism; A Potential Mechanism of Cardioprotection
Authors: Kaiserova Karin , Guo Yiru , Bolli Roberto , Bhatnagar Aruni
Published: July 2021

Ischemia induces a switch in myocardial substrate utilization from fatty acids to glucose and stimulates nitric oxide (NO) production. Although increased NO synthesis can ameliorate ischemic injury, mechanism by which NO confers cardioprotection remains unclear. Based on our recent observation that increased NO production contributes to ischemic activation of aldose reductase (AR), we tested the hypothesis that NO prevents ischemic injury by increasing reduction of glucose to sorbitol via polyol pathway over its oxidation to lactate in glycolysis, that could prevent acidosis. Our data show that in isolated mouse hearts subjected to 30 min of ischemia, sorbitol levels increased from 43.2 ± 2.4 to 68.7 ± 4.6 nmol/mg (n=6; P < 0.05) and returned to pre-ischemic levels after 30 min of reperfusion. Treatment with eNOS agonist, bradykinin (10 μM), prior to ischemia, increased AR activity from 27.5 ± 0.7 to 35.9 ± 1.4 mU/mg (n=6; P < 0.01) and decreased lactate levels from 3.7 ± 0.6 to 1.5 ± 0.4 mM (n=6; P < 0.05) upon reperfusion. Bradykinin also decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release by 65 ± 7% and 85 ± 9%, respectively. In contrast, NOS inhibitor, L-NAME (0.1 mM) decreased AR activity (22.4 ± 2.0 mU/mg), but increased the activity of glyceraldehyde-3-phosphate dehydrogenase (from 2.4 ± 0.1 to 3.5 ± 0.2 U/mg) and lactate production (5.8 ± 0.9 mM). AR inhibitor, sorbinil (0.1 mM) increased lactate production (5.9 ± 0.1 mM) and exacerbated ischemic injury. Pretreatment with bradykinin normalized lactate levels and reduced injury caused by sorbinil. To examine whether upregulation of AR is sufficient to confer cardioprotection, cardio-specific AR transgenic mice were generated. Echocardiography confirmed, that these mice had normal myocardial function and when subjected to 30 min of coronary occlusion followed by 24 h of reperfusion displayed reduced myocardial infarct size (49.6 ± 4.1
vs
64.6 ± 3.5 % of the area at risk; transgenic
vs
wild type; n=7; P < 0.01). Collectively, these results show that NO mediates cardioprotection by increasing glucose flux via polyol pathway, which may represent an adaptive mechanism protecting the heart from ischemic injury.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 1665: Angiotensin II Inactivates eNOS Through Tyrosine Phosphorylation
Authors: Loot Annemarieke E , Fisslthaler Beate , Fleming Ingrid
Published: July 2021

We recently reported that in endothelial cells, the proline-rich tyrosine kinase PYK2 is activated by fluid shear stress and phosphorylates eNOS on tyrosine 657. Tyrosine phosphorylation of eNOS
in vitro
completely inhibits enzyme activity and limits NO production in response to shear stress. Overexpression of PYK2 in isolated arteries abrogated NO-mediated vasodilatation. In atherosclerotic lesions, eNOS is partially inactive even in the presence of sufficient co-factors. Since PYK2 is also known to be activated by angiotensin (Ang) II and reactive oxygen species, we speculated that this mechanism plays a role in the loss of NO bioavailability in pathologies associated with increased Ang II and H
2
O
2
levels. In native porcine aortic endothelial cells and in freshly isolated murine aortae, Ang II and H
2
O
2
both enhanced the phosphorylation of eNOS on Tyr657. This effect was correlated with increased PYK2 phosphorylation and the association of PYK2 with eNOS as shown by co-immunoprecipitation. Furthermore,
in vivo
oxidative stress induced by endotoxins also increased p-Tyr657eNOS levels in mouse aortae. In cultured endothelial cells, H
2
O
2
had a dual effect on eNOS phosphorylation and activity. At low concentrations (1 − 100 μmol/L) H
2
O
2
stimulated the phosphorylation of Tyr657 without affecting that of either Ser1177 or Thr495. At these concentrations, H
2
O
2
also limited basal NO production and markedly impaired NO production in response to bradykinin and ionomycin. Furthermore, low (30 μmol/L) concentrations of H
2
O
2
impaired acetylcholine-induced vasodilatation in murine aorta. At higher concentrations (300–500 μmol/L) of H
2
O
2
, increased intracellular Ca
2+
elicited the phosphorylation of Ser1177 and increased NO production. Taken together, PYK2 activation under pathological conditions, i.e. increased Ang II and H
2
O
2
causes the phosphorylation of eNOS on Tyr657 impeding NO production and endothelium dependent vasodilatation. This mechanism, in addition to eNOS uncoupling, may underlie the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin-angiotensin system and elevated redox stress.
Abstract 1667: Angiotensin II and Free Cholesterol Synergistically Impaired eNOS Bioavailability via Lipid Raft
Authors: Amiya Eisuke , Maemura Koji , Takeda Norihiko , Saito Tetsuya , Shiga Taro , Hosoya Yumiko , Nakao Tomoko , Imai Yasushi , Nagai Ryozo
Published: July 2021

Both hypercholesterolemia and angiotensin II (Ang II) are potent factors inducing endothelial dysfunction that leads to atherosclerosis. It has been reported that coexistence of hypercho-lesterolemia and Ang II accelerates atherogenesis markedly. Several studies showed that elevated cellular cholesterol affected cellular function by modifying lipid raft. We investigated the contribution of lipid raft to bridging hypercholesterolemia and Ang II in endothelial cells. Free cholesterol (Chol) loading induced 2–3 fold increase in cellular cholesterol content. Co-treatment of Ang II and Chol markedly impaired eNOS phosphorylation and dimerization than Chol or Ang II loading alone. This inactivation of eNOS corresponded to eNOS translocation to Chol-rich fractions in density centrifugation. In this fraction, eNOS phosphorylation and dimerization were markedly suppressed. Olmesartan or ROS inhibitor suppressed this eNOS trafficking and reversed the impairment of eNOS bioavailability. In immunohistochemistry, the target of this eNOS trafficking was identified as Chol-rich vesicle structure at the perinuclear area and those were fused with lysosomal membrane associated protein-1 (LAMP-1), a lysosome marker, labeled endosome. These results suggested that in cholesterol enriched condition, AngII induced eNOS trafficking to Chol-rich vesicles that were fused with lysosome at the perinuclear area and keeps eNOS inactive. We next investigated the association between these Chol-rich vesicles and lipid raft. GM1, phospho-caveilin1 and Rac, which are all lipid raft actors, also shifted to Chol-rich fractions in density centrifugation and assembled at LAMP-1 labeled endosome at the perinuclear area after Ang II and Chol loading. Therefore, LAMP-1 labeled endosome was considered to be the intracellular target of lipid raft in that condition. In electron microscopy, Chol-rich vesicles corresponded to multivesicular bodies and these converged to lysosome with Ang II addition, which were consistent with immunohistochemical findings. These studies show the important crosstalk between AngII and Chol signaling leading to endothelial dysfunction and provide a novel insight into the mechanisms of endothelial dysfunction by hypercholesterolemia.
Abstract 1683: Ca2+/Redox-Sensitive Tyrosine Kinase PYK2 Promotes Atherogenesis through OxLDL/ROS/p21-Dependent Premature Senescence and Cytokine Induction in Endothelium
Authors: Katsume Asako , Okigaki Mitsuhiko , Matsui Akihiro , Yamaguchi Shinichiro , Kishita Eigo , Yamada Hiroyuki , Matsubara Hiroaki
Published: July 2021

Background: Calcium/redox-sensitive tyrosine kinase PYK2 plays crucial roles in cell migration. Analysis of PYK2-deficient macrophages has been reported that their directional migration was impaired due to dysfunction of PI3K, small GTPase Rho, and Ca (2+)-mobilization. Since monocyte/macrophage plays crucial role in atherogenesis, we newly generated the PYK2-knock-out mice and investigated the role of PYK2 in atherogenesis.

Methods: PYK2−/− mice (P-KO) were crossbred with ApoE−/− mice (E-KO), and PYK2−/−/ApoE−/− double knockout (D-KO) mice were established. Four-week-old mice were fed with high-fat-diet for 8 weeks, and the thoracic aorta and aortic root were histologically analyzed. Then we studied the mechanisms of the difference in atherogenesis.

Results: At the 8th week, atherosclerotic area of thoracic aorta in D-KO was less than E-KO (~54%, P < 0.01, n = 15, respectively). Numbers of infiltrating macrophage in D-KO mice was also reduced (~52%, P < 0.01, n = 15). We also verified that no significant difference in atherosclerotic area between E-KO recipient with D-KO bone marrow and D-KO recipient with E-KO bone marrow. At the 7th day, the expression of VCAM1, and MCP-1 in the thoracic aorta in D-KO mice was inhibited (~43 to 58%, n = 5, P < 0.01, respectively). Inflammatory cells were barely detected in the region at that time. TNF-alpha and MCP-1 mRNA of peripheral blood-leukocytes in D-KO was also 38% lower (P < 0.05, n = 5 each). In the primary-cultured P-KO endothelial cells, TNF-alpha-induced expressions of VCAM-1 and MCP-1 was decreased to 31 and 32% respectively, compared to the wild-type (WT) (each n = 5, P < 0.01). Lysophosphatidylcholine (LPC), the component of Oxidized LDL, -induced Reactive Oxygen Species (ROS)-production was attenuated to 54% of the WT (p < 0.05) using DCF staining, and the high cholesterol diet induced ROS-production in endothelium in D-KO were decreased by 8-OHDG staining. Senescence-associated-beta-gal-stained area and ROS-dependent expression of p21 in the thoracic aorta in D-KO-mice was diminished. In P-KO ECs, LPC-induced p21 expression via Ets-1 or STAT1 was also decreased markedly.

Conclusion: PYK2 promotes atherogenesis by inducing ROS/p21-dependent premature senescence and cytokine induction in endothelium.
Abstract 1709: Transforming Growth Factor Beta Inhibition Increases Matrix Metalloproteinase-9 Activity and Enhances Elastin Degradation in a Murine Model of Kawasaki Disease
Authors: Alvira Cristina M , Kim Yu-Mee , Guignabert Christophe , Wang LingLi , Rabinovitch Marlene
Published: July 2021

Background: Kawasaki disease (KD) is a leading cause of acquired heart disease in children. However, the molecular mechanisms leading to coronary aneurysms, and the host factors determining high-risk patients are unknown. We have previously shown that mice haploinsufficient for tropoelastin (EL ±), develop more severe destruction of the coronary elastin compared to wild type (WT) mice after injection with Lactobacillus casei wall extract (LCWE), an inducer of coronary arteritis. In contrast to the beneficial effect of TGFβ blockade on the aneurysms that form in Marfan syndrome (MS), we hypothesized that TGFβ protects against aneurysm formation in this inflammatory model by:

enhancing tropoelastin; and

suppressing proteolytic activity.

Methods: WT and EL ± mice were given either TGFβ neutralizing antibody (TGFβ-Nab) or control IgG in addition to LCWE. At 14d, tropoelastin and plasminogen activator inhibitor-1 (PAI-1) protein was determined, and matrix metalloproteinase (MMP)-2 and -9 and serine elastase activity was measured. Elastin degradation and total medial elastin content was assessed by morphometric analysis at 42d.

Results: TGFβ blockade worsened the LCWE-mediated fragmentation of the coronary elastin, with areas of complete dissolution of the external lamina in WT mice, and even more severe features in EL ± mice. Tropoelastin protein was 40% lower in the EL ± vs. WT mice after LCWE (P < 0.05), and TGFβ blockade further exaggerated this discrepancy. TGFβ-Nab markedly reduced PAI-1 in the WT mice after LCWE (P < 0.05), and produced a similar pattern in the EL ± mice, and induced a four-fold induction of MMP-9 activity in both genotypes. This effect was associated with increased elastin fragmentation (P < 0.05), and decreased total medial elastin content (P < 0.05), despite inhibition of the LCWE-mediated induction of serine elastase activity.

Conclusions: We conclude that:

diminished tropoelastin expression worsens elastin destruction in the setting of acute coronary arteritis; and

TGFβ protects the vasculature in this model by maintaining PAI-1 levels and suppressing activation of MMP-9.

Thus strategies to block TGFβ, currently in use to prevent aneurysms in MS, are unlikely to be beneficial in KD, and may be detrimental.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1717: Impact of a 600-mg Loading Dose of Clopidogrel on 30-Day Outcome in Unselected Patients Undergoing Percutaneous Coronary Intervention
Authors: Bonello Laurent , Lemesle Gilles , de Labriolle Axel , Roy Probal , Steinberg Daniel H , Slottow Tina Pinto , Xue Zhenyi , Torguson Rebecca , Kaneshige Kimberly , Suddath William , Kent Kenneth , Satler Lowell , Pichard Augusto , Waksman Ron
Published: July 2021

Background:
Based on biological studies of platelet reactivity (PR), the recent ACC/AHA guidelines recommend a 600-mg loading dose (LD) of clopidogrel in patients undergoing percutaneous coronary intervention (PCI). However, there is lack of studies addressing the clinical impact of such a LD of clopidogrel.

Objective:
We aimed to compare the clinical efficacy and safety of a 600-mg LD of clopidogrel compared to a 300-mg LD in an unselected cohort of patients undergoing PCI.

Methods:
A cohort of 4105 unselected patients undergoing PCI in our institution was included in the study and divided according to the LD used: the high LD group (600mg) included 3146 patients and the low LD (300mg) group included 959. The primary endpoint was the rate of major adverse cardiovascular events (MACE) at 1 month.

Results:
Patients in lthe ow LD group more often had diabetes mellitus and a history of myocardial infarction (36.8 vs 31.9%; p = 0.01). Left ventricular ejection fraction was similar (0.49 ± 0.14 vs 0.48 ± 0.14; p = 0.25). Angiographic and procedural characteristics were identical between the 2 groups. Patients in the high LD group had fewer MACE after 1 month (2.9 vs 5.2%; p < 0.001). In multivariate analysis, a 600-mg LD of clopidogrel was significantly associated with MACE at 1-month follow-up, with an odds ratio of 0.62 (95% CI 0.41– 0.95; p = 0.03).

Conclusions:
A 600-mg LD was associated with a significant decrease in the rate of post PCI-MACE at 1 month without any increase in bleeding complications. The results of our study therefore support the current guideline of a 600-mg LD of clopidogrel in patients undergoing PCI.
Abstract 1718: Combination of Direct Acting FXa Inhibitor and P2Y
12
Antagonist at Non-Effective doses Provides Significant Inhibition of Arterial Thrombosis
Authors: Andre Patrick , Conley Pamela B , Deguzman Francis , Pak Yvonne , Inagaki Mayuko , Sinha Uma , Phillips David R
Published: July 2021

Previous studies have shown that inhibition of FXa and P2Y
12
used individually inhibit arterial thrombosis. The present study was designed to determine the antithrombotic activity achieved by the combination of non-effective doses of PRT054021 (FXa inhibitor) and PRT060128 (P2Y
12
antagonist). Arterial thrombosis was achieved by FeCl
3
injury of mesenteric arteries of the mouse and monitored in real time using intravital microscopy. Antagonism of either FXa or P2Y
12
dose-dependently inhibited early (time for appearance of first thrombus, TFFT) and late (time to vascular occlusion, TTO) phases of thrombosis (see Table

). Maximal effects were achieved at plasma concentrations superior or equal to 1 μg/ml for the two antagonists. Interestingly, the co-administration of non-effective doses of PRT054021 and PRT060128 significantly delayed time for appearance of first thrombus and vascular occlusion, demonstrating potent synergistic antithrombotic activity. PRT054021 and PRT060128 demonstrated a strong synergistic effect when administered in combination at non-effective doses. This combination is of particular relevance in acute settings, when thrombotic risk is at its paroxysm and maximal protection is sought as early as possible. These data also indicate that combined inhibition of FXa and P2Y
12
by reversible antagonists could provide substantial benefits to ACS patients throughout the chronic phase of the disease.
Abstract 1721: Deletion of Both Macrophage ABCG1 and ApoE Independently Promotes Atherosclerotic Lesion Development in LDL Receptor Knockout Mice
Authors: Out Ruud , Lammers Bart , Hildebrand Reeni B. , Quinn Carmel M. , Williamson David , Hoekstra Menno , Meurs Illiana , Van Berkel Theo J.C. , Jessup Wendy , Van Eck Miranda
Published: July 2021

Objective
ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein E (apoE) play a role in macrophage cholesterol efflux and consequently the development of atherosclerosis. Although a possible interaction between ABCG1 and apoE in cholesterol efflux was postulated, the combined action of these proteins in atherosclerosis is still unclear.

Methods and Results
LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCG1/apoE double KO (dKO) mice, their respective single knockouts, and wild-type (WT) controls. After feeding a high-fat/high-cholesterol diet for 6 weeks, no differences were found in serum lipid levels. However, the mean atherosclerotic lesion area in dKO transplanted animals (187 ± 18 × 10
3
μ m
2
) was 1.4-fold (p < 0.01) increased compared to single knockouts (ABCG1 KO: 138 ± 5 × 10
3
μm
2
; apoE KO: 131 ± 7 × 10
3
μm
2
) and 1.9-fold (p< 0.001) as compared to WT controls (97 ± 15 × 10
3
μm
2
). In vitro cholesterol efflux experiments confirmed that combined deletion of ABCG1 and apoE resulted in a larger attenuation of macrophage cholesterol efflux to HDL as compared to single knockouts.

Conclusions
Deletion of macrophage ABCG1 or apoE does lead to a moderate increase in atherosclerotic lesion development while combined deletion of ABCG1 and apoE induces a more dramatic increase in atherosclerosis. These results indicate an added, independent effect for both macrophage ABCG1 and apoE in atherosclerosis.
Abstract 1729: PPARγ Regulates ABCG1 Expression in Macrophages via LXR-Driven Dual Promoters
Authors: Ayaori Makoto , Ogura Masatsune , Nakaya Kazuhiro , Hisada Tetsuya , Takiguchi Shun-ichi , Yakushiji Emi , Kondo Harumi , Kusuhara Masatoshi , Ohsuzu Fumitaka , Ikewaki Katsunori
Published: July 2021

ATP binding cassette transporter G1 (ABCG1), which is expressed in macrophages, has been implicated in the efflux of cholesterol to high density lipoprotein. Peroxisome proliferator-activated receptor γ (PPARγ) has been reported to be involved in cholesterol efflux from macrophages, and increased expression of ABCG1 via liver receptor X (LXR)-dependent and independent pathways. However, the mechanisms by which ABCG1 expression is increased by PPARγ have not been fully characterized. We observed that pioglitazone, a PPARγ ligand, increases cholesterol efflux from THP-1 macrophages, as well as ABCG1 mRNA and protein levels. Treatment with actinomycin D abolished the inducible effect of pioglitazone on ABCG1, indicating that pioglitazone transcriptionally activated ABCG1 expression. To clarify how pioglitazone regulates ABCG1 expression, we investigated promoter activity using reporter constructs containing human ABCG1 promoter A and B (located upstream of exon 1 and 5, respectively), with or without mutated LXR-binding sites. The results indicated that pioglitazone activated both promoters in an LXR-dependent manner. We also observed that pioglitazone increased two major transcripts driven by promoter A and B using specific primers for each transcript. To determine whether PPARγ and LXRα were involved in these effects of pioglitazone, we performed siRNA-knockdown of PPARγ and LXRα in macrophages, which resulted in 75% and 91% decreases in PPARγ and LXRα mRNA levels, respectively. PPARγ and LXRα-knockdown, respectively, completely or partially abolished pioglitazone-induced ABCG1 expression. In conclusion, these results suggest that pioglitazone transcriptionally increased ABCG1 expression in macrophages by activating dual promoters in an LXR-dependent manner. Further studies are needed to assess LXR-independent mechanisms for the stimulatory effect of pioglitazone on ABCG1.
Abstract 1733: Tumor Necrosis Factor Receptor-1 Aggravates Aging-Associated Atherosclerosis
Authors: Zhang Lisheng , Peppel Karsten , Brian Leigh , Freedman Neil J
Published: July 2021

Aging is believed to contribute to atherogenesis independently of traditional atherosclerosis risk factors, but the mechanisms responsible for this contribution remain obscure. Although serum tumor necrosis factor (TNF) levels increase with aging, vascular cell TNF receptor-1 (TNFR1) expression does not diminish. For these reasons, we tested the hypothesis that TNFR1 contributes to aging-associated atherosclerosis. To isolate aging of the vascular wall from the effects of prolonged exposure to systemic atherogenic factors, we used carotid arteries from 18-month-old (
aged
) and 8-wk-old (
young
), congenic
tnfr1
−/−
and WT mice as carotid interposition grafts in congenic 8-wk-old
apoe
−/−
mice. Two weeks post-op, WT carotids developed macrophage-rich neointimal lesions. Area dimensions (mm
2
) of aged and young specimens, respectively, were: neointima, 0.020 ± 0.009 and 0.010 ± 0.006 (p < 0.02); media, 0.06 ± 0.02 and 0.03 ± 0.01 (p < 0.01); external diameter, 0.45 ± 0.05 and 0.39 ± 0.06 (p < 0.03). Thus, aged arteries showed greater atherosclerosis than young arteries, even at early stages of atherogenesis. ICAM-1 and MCP-1 expression, and macrophage prevalence in aged specimens were 1.9 ± 0.4-fold, 1.9 ± 0.2- and 2.5 ± 0.5-fold more (p < 0.04), respectively, than in young specimens, assessed by quantitative immunofluorescence. Seven wks post-op, neointimal area, macrophage and apoptotic (cleaved caspase-3
+
) cell prevalence, and MMP-9 expression were greater in aged than in young arteries, by 1.6 ± 0.3-, 2.3 ± 0.3-, 2.1 ± 0.7-, and 1.5 ± 0.2-fold (p < 0.04), while neointimal SMC prevalence and collagen content were 2.0 ± 0.2- and 1.7 ± 0.1-fold less (p < 0.04). In contrast, 7-wk-old aged and young
tnfr1
−/−
carotid grafts had equivalent intimal and medial areas, macrophage and apoptotic (cleaved caspase-3
+
) cell prevalence, VCAM-1 expression, and collagen content. Neointimal and medial areas were less in
tnfr1
−/−
than in WT arteries, by 2.7 ± 0.5-fold and 1.4 ± 0.1-fold, respectively (p < 0.05). We conclude that arterial wall aging contributes to atherosclerosis through mechanisms related to macrophage recruitment, independently of systemic factors, and that TNFR1 signaling in the arterial wall promotes aging-dependent atherosclerosis.
Abstract 1738: The I(
f
)-inhibitor Ivabradine Exerts Vascular Anti-oxidative And Anti-inflammatory Effects Selectively Mediated By Reduction Of Heart Rate
Authors: Custodis Florian , Baumhaekel Magnus , Schlimmer Nils , List Franka , Gensch Christoph , Böhm Michael , Laufs Ulrich
Published: July 2021

Objective:
In epidemiological studies, elevated resting heart rate (HR) is associated with increased cardiovascular morbidity. We therefore characterized the effects of selective heart rate reduction by inhibition of the
I
(f)
-current by ivabradine (IVA) in mice.

Methods and Results:
Male ApoE
−/−
mice fed a high-cholesterol diet were treated with IVA (10 mg/kg/d) or vehicle for 6 weeks (n = 10 per group). IVA reduced HR by 13.4% (472 ± 9 vs 545 ± 11 bpm,
p
< 0.01) but did not alter blood pressure or lipid levels. Endothelium dependent relaxation of aortic rings was significantly improved in IVA fed animals (
p
< 0.01). IVA decreased atherosclerotic plaque size in the aortic root by > 40% and in the ascending aorta by > 70%,
p
< 0.05. HR reduction had no effect on the number of endothelial progenitor cells in the blood and the bone marrow and did not alter aortic eNOS, p-Akt, VCAM-1 or ICAM-1 expression, but decreased MCP-1 mRNA to 26 ± 7% (p < 0.05). IVA reduced vascular NADPH oxidase activity to 48 ± 6% and decreased L-012 chemiluminescence to 24 ± 9% (both p < 0.05). Lipidperoxidation was reduced to 65 ± 8% in the vasculature of the IVA group compared to vehicle treatment (p < 0.05). DHE fluorescence microscopy in aortic sections detected reduction of ROS release to 62 ± 4% in IVA treated mice (p < 0.01). The
in vivo
effects of IVA were absent at a dose that did not lower HR and were absent in aortic rings treated
ex vivo
. Protein expression of p-Akt, eNOS and p-eNOS was not altered in cultured endothelial cells (EC) by increasing doses of Iva. Similarly, NADPH oxidase activity in EC was not changed as well as the Ang II induced free radical release in vascular smooth muscle cells (DCF-fluorescence).

Conclusions:
Selective HR reduction improves endothelial function and reduces atherosclerotic plaque formation in ApoE −/− mice. Those effects are in part mediated by decreased markers of oxidative stress and downregulation of MCP-1. The control experiments show that a direct effect of IVA on vascular cells is unlikely and support the reduction of heart rate as the primary mechanism of action.
Abstract 1872: Transmural Dispersion of Repolarization in Human Ventricular Wall
Authors: Glukhov Alexey V , Fedorov Vadim V , Foyil Kelley V , Moazami Nader , Efimov Igor R
Published: July 2021

Transmural dispersion of repolartization (DR) has been studied in numerous animal models. Some studies presented evidence of midmyocardial population of M-cells that possess distinctly long action potential duration (APD). We aimed to investigate APD and DR in explanted failing human left ventricles (LV). We optically mapped APD in coronary-perfused human LV wedge preparations (n=4) using CMOS camera in presence of blebbistatin (BB, 10
μ
M). Microelectrode recordings were used to validate BB in human LV. RESULTS: During slow pacing (S1S1=2,000ms), APD at the endo-, mid-, and epicardium was 586±27ms, 578±19ms, and 496±14ms, respectively. Maximum APD gradient was 24±5 ms/mm, DR was 35±6 ms. Progressive decrease in S1S1 up to functional refractory period (460±26 ms) induced an inhomogeneous shortening of APD (up to 350±26 ms, 352±36 ms and 346±30 ms, respectively), decrease in APD gradient and DR (4±3 ms/mm and 20±4 ms, respectively, p<0.01) We present for the first time evidence of transmural APD gradient in failing human LV. However, we could not confirm presence of distinct population of M-cells that were found in several animal models.
Abstract 1876: Functional Effect of Chronic Urotensin II Receptor Antagonist on L-Type Ca
2+
Current in Mouse Model of Progressive Heart Failure
Authors: Zhou Peng , Cheng Heng-Jie , Cross Michael , Callahan Michael F , Brosnihan Bridget , Little William C , Cheng Che-Ping
Published: July 2021

Voltage-gated Ca
2+
channels play fundamental roles in the regulation of cardiac function by various neurotransmitters. Recently, we have shown that urotensin II (UII), a potent vasoconstrictor, inhibits L-type Ca
2+
current (I
Ca,L
) and produces negative inotropic action. In heart failure (HF), the UII-mediated pathway is upregulated, suggesting a therapeutic value of UII receptor antagonist (UII-ANT) for HF. However, the role and mechanism of chronic UII-ANT in HF is unclear. We tested the hypothesis that chronic UII-ANT may improve cardiac I
Ca,L
, preventing β-adrenergic deregulation on I
Ca,L
and limit HF progression. We examined plasma levels of norepinephrine (NE), left ventricular (LV) function, and myocyte I
Ca,L
responses to isoproterenol (ISO) in 3 age-matched groups of mice:

HF (n = 7), 2 months after ISO (150 mg/kg sq for 2 days);

HF/UII-ANT (n = 11), 1 month after receiving ISO, then urantide, a potent UII-ANT (10
−5
M/kg/day, sq via implanted osmotic mini pump), given for 1 month; and

Controls (n = 7). I
Ca,L
was measured using whole-cell voltage clamp technique.

Compared with controls, ISO-treated mice progressed to HF with 4.7-fold increase in plasma NE (18975 vs 4066 pg/ml) and LV dilatation associated with increased myocyte length (ML, 155 vs120 μm) and heart-to-body weight ratio (H/BW, 7.6 vs 5.5 g/kg). Stroke volume (SV, 30.3 vs 61.4 μl) and ejection fraction (EF, 39% vs 60%) were decreased. Compared with normal myocytes, in HF myocytes, I
Ca,L
was reduced (50%, 3.7 ± 0.2 vs 7.4 ± 0.2 pA/pF), and I
Ca,L
response to β-AR stimulation (ISO, 10
−8
M) was attenuated (11% vs 35%) (p < 0.01). In HF/UII-ANT mice, plasma NE (5148 pg/ml), SV (57.9 μl), and EF (57%) returned close to control levels with retained normal ML (124 μm) and H/BW (5.9 g/kg). Moreover, compared with controls, in HF/UII-ANT mice, ISO caused similar increases in the peak I
Ca,L
(32% vs 35%). Chronic UII-ANT treatment normalizes LV L-type Ca
2+
channel basal function and β-adrenergic regulation, leading to regression of LV and myocyte dysfunction and remodeling in mice with ISO-induced HF.

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