bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3665: Aggf1 Is Essential for Embryonic and Pathological Angiogenesis
Authors: Zhang Teng , He Ping , Wang Qing
Published: July 2021

Backgroud: AGGF1 is a newly identified angiogenic factor associated with vascular disease Klippel-Trenaunay syndrome. AGGF1 has been shown to promote angiogenesis as potently as VEGF in a chicken embryo angiogenesis assay; however, the physiological role of AGGF1 is unknown.

Methods and Results: We explored the physiological function of AGGF1 in vivo by employing the gene-trapping strategy in mice. AGGF1−/− mice died by E8.5, suggesting that AGGF1 is essential for embryogenesis. AGGF1 heterozygosity resulted in 31% embryonic lethality. Further macroscopic analysis of embryos at different stages revealed growth retardation and defective vascular development in AGGF1+/− embryos. The major vascular phenotypes included failed development of large vessels and interconnected yolk sac vasculature. Interestingly, decreased phosphorylation of VEGFR2 (Tyr951) was detected in AGGF1+/− embryos compared to the wild type littermate controls, suggesting that AGGF1 may function through VEGFR2-mediated signaling during angiogenesis. Moreover, vascular pathologies of various internal organs were detected in older AGGF1+/− mice. Hemorrhages were frequently noted in brain, spleen and lung, and a chronic inflammatory change was readily detected in lungs in AGGF1+/− mice compared to the littermate controls. Permeability assay further revealed increased vascular permeability in old AGGF1+/− mice (50 – 60 weeks) compared to age- and sex-matched wild-type controls. A tumor angiogenesis model induced by two melanoma cell lines was employed to further assess the angiogenic function of AGGF1 in vivo. Tumor growth was significantly inhibited in AGGF1+/− mice. Histological examinations of the tumor sections revealed markedly reduced microvessel formation in tumors grown in AGGF1+/− mice. Moreover, vascular density quantified after PECAM1 immunostaining showed significant reduction in tumors grown in AGGF1+/− mice.

Conclusions: AGGF1 is an important regulator for embryonic angiogenesis and vascular integrity. AGGF1 is also involved in tumor growth and pathological angiogenesis. These results provide for the first time the in vivo experimental evidence that AGGF1 is a potent angiogenic factor essential for both embryonic and pathological angiogenesis.
Abstract 3678: Protein Kinase C-zeta (PKCζ) Is A Novel Mediator Of Endothelial Dysfunction By Inhibiting The ERK5/ Kruppel-like Factor 2 (KLF2) Pathway
Authors: Nigro Patrizia , Woo Chang Hoon , Belisario Maria Antonietta , McClain Carolyn , Abe Jun-ichi , Berk Bradford C
Published: July 2021

Background:
Shear stress-induced activation of ERK5 and KLF2 leads to eNOS expression maintaining normal endothelial cell (EC) function. PKCζ has emerged as a pathologic mediator of EC dysfunction based on 1) a positive correlation between PKCζ activity and disturbed flow pattern; and 2) PKCζ activation is essential for TNFα stimulation of JNK, caspase-3 and EC apoptosis. Therefore we hypothesized that TNFα and its pathologic mediator ONOO
−
would inhibit ERK5 transcriptional activity and eNOS expression induced by flow.

Results:
TNFα and ONOO
−
significantly inhibited ERK5 transcriptional activity, KLF2 promoter activity, and eNOS expression induced by flow (shear stress = 12 dyn/cm2, 24 hrs). Both TNFαand ONOO
−
increased PKCζ activity. Transfection of wild type PKCζ (WT-PKCζ) and catalytic domain of PKCζ (CATζ) significantly inhibited ERK5 transcriptional activity (38±1.4% and 57±2.8% respectively; p<0.01, p<0.005). Also, transfection of PKCζ siRNA reversed TNFα-mediated inhibition of ERK5 transcriptional activity (control vs PKCz siRNA, 28±2.5% vs 9±0.3%, p<0.05), suggesting a critical role for PKCζ in ERK5 transcriptional repression. Surprisingly, TNFα and ONOO
−
did not significantly decrease ERK5 phosphorylation, suggesting that inhibition occurred downstream of ERK5 phosphorylation. Previously we reported that ERK5-SUMOylation inhibited flow-mediated eNOS expression, but we could not detect increased ERK5-SUMOylation in WT-PKCζ or CATζ transfected cells. Importantly, we found that ONOO
−
significantly increased PKCζ-ERK5 interaction. PKCζ is known to contain a PB1 domain, a well studied protein-protein interaction domain. However, mutational analysis demonstrated that the ERK5 binding site in PKCζ was within the catalytic domain of PKCζ, not the PB1 domain. These data suggest that the PKCζ-ERK5 interaction likely inhibits ERK5 transcriptional activity by direct phosphorylation of ERK5 by PKCζ kinase.

Conclusion:
PKCζ is a novel mediator of TNFα and ONOO
−
induced endothelial dysfunction by inhibiting ERK5 transcriptional activity independent of kinase activity and ERK5-SUMOylation.
Abstract 3699: Efficacy and Safety of Colesevelam HCl in Pediatric Patients with Heterozygous Familial Hypercholesterolemia
Authors: Stein Evan A , Marais David , Szamosi Tamas , Raal Frederick , Schurr Daniel , Urbina Elaine M , Hopkins Paul N , Karki Sulekha , Xu Jianbo , Melino Michael
Published: July 2021

Heterozygous familial hypercholesterolemia (heFH) is a genetic disorder resulting in elevated LDL-C, which confers a high risk for a coronary event. Colesevelam HCl (COL), a non-absorbable bile acid sequestrant, is approved to lower LDL-C in adults with primary hypercholesterolemia. This is the first data demonstrating the efficacy and safety of COL in pediatric pts with heFH. This 32wk multicenter, randomized, double-blind (DB), placebo (PLA)-controlled study included: a 4wk PLA run-in (to measure compliance); an 8wk DB period (pts randomized 1:1:1 to PLA, 1.875 g/d COL, or 3.75 g/d COL); an 18wk open-label (OL) treatment to goal (LDL-C <110 mg/dL) wherein all pts received COL 3.75 g/d (and were eligible to receive a statin); and a 2wk follow-up. Males and females, aged 10 –17yrs, with either genetic diagnosis of heFH or history of untreated LDL-C >160 mg/dL combined with familial dyslipidemia in a first degree relative, who had a baseline LDL-C >160 mg/dL (if naíve to lipid-lowering therapy) or >130 mg/dL (if on a statin [≥6wks]) and following a NCEP step 1 diet were included. Additional inclusion criteria were TG <250 mg/dL, ≥Tanner stage 2, and compliance ≥75% during the PLA run-in. Primary efficacy parameter was % change in LDL-C from baseline/Day 1 to Wk 8. The ITT population (randomized pts with a baseline and ≥1 post-baseline measurement) was used to evaluate efficacy parameters. Of the 194 pts randomized, 95.9% and 89.2% completed the DB and OL periods, respectively. Approximately 25% were on a statin at entry into the DB period; a further 10% added a statin during the OL period. At wk 8, LDL-C, TC, and apoB significantly decreased while HDL-C and apoA-I significantly increased with COL 3.75 g/d (
P
<0.01 vs PLA for all; Table

). Adverse events were as expected; no choking was recorded. No effects were noted on growth, sexual maturation, hormone levels, absorption of fat-soluble vitamins, or clotting parameters. In summary, COL lowered LDL-C and was well tolerated in pediatric pts.
Abstract 3701: Vitamin D Deficiency is Associated With Myalgias in Hyperlipidemic Subjects Taking Statins
Authors: Duell P. Barton , Connor William E
Published: July 2021

Myalgias are a frequent symptom among patients taking statins, but it is sometimes uncertain whether myalgias are drug-related. Since vitamin D deficiency is common in the general population, and can be associated with reversible myalgias, we hypothesized that vitamin D deficiency may contribute to symptoms in some patients. This hypothesis was tested in 99 patients referred to our tertiary care academic Lipid Disorders Clinic with a diagnosis of hyperlipidemia. Mean age 58.7 ± 11.6 yr (range 20 – 84), 43% men, mean serum 25-hydroxy vitamin D (vit D) level 26.7 ± 12.5 ng/ml (range 5– 64). Statin-associated myalgias were present in 38.8% of patients. Patients with statin-associated myalgias were the same age as patients without myalgias (59.3 ± 10.4 vs 58.4 ± 12.3, P=NS), but had 32% lower mean vit D (20.5 ± 10.0 vs 30.1 ± 12.5 ng/ml, P=0.0003) and trended toward being female (68% vs 49%, P=0.095). Vit D was similar in men and women (24.3 ± 10.1 vs 28.8 ± 14.0, P=0.09). In the entire cohort, vit D deficiency was prevalent: 62.6% were < 30 ng/ml; 31.9% were < 20 ng/ml. Patients with myalgias were more likely to have vit D < 30 ng/ml (81.3% vs 52.5%, P < 0.01) and < 20 ng/ml (62.5% vs 18.6%, P < 0.01). Among patients with vit D < 20 ng/ml, 62.1% had statin-associated myalgias vs 17.6% of patients with vit D ≥ 30 (P<0.01). About 1/3 of patients reported fewer statin-associated myalgias after unblinded treatment with high dose ergocalciferol for 8 –12 weeks, but most subjects also changed to a different statin. In summary, (1) vit D deficiency is common in this group of patients referred for hyperlipidemia, (2) mild and severe vit D deficiency were significantly more common among patients with statin-associated myalgias; mean vit D level was 32% lower, (3) vit D < 20 was associated with 4-fold higher rates of myalgias vs vit D ≥ 30, (4) reduced myalgias were anecdotally related to treatment with vit D in some patients. In conclusion, vit D deficiency appears to be prevalent among patients with hyperlipidemia, but is significantly more likely among patients with statin-associated myalgias. It is possible that vit D deficiency potentiates statin-induced myalgias, or may cause drug-unrelated myalgias in a subset of statin-treated patients. Further studies are required to elucidate these relationships.
Abstract 3702: Serum Adipocyte Fatty Acid-Binding Protein Levels are Independently Associated with Coronary Atherosclerosis
Authors: Miyoshi Toru , Hirohata Atsushi , Usui Shinichi , Yamamoto Keizo , Hina Kazuyoshi , Hirohata Satoshi , Kusachi Shozo , Ninomiya Yoshifumi , Kusano Kengo F
Published: July 2021

Background
: Adipocyte fatty acid-binding protein (A-FABP) has been shown to play an important role in atherosclerosis in animal models. However, the clinical relevance of these findings is still unknown. We investigated the association between serum A-FABP level and coronary atherosclerosis.

Methods
: 221 patients with stable angina were enrolled after coronary angiography. The plaque volume in the non-culprit vessel (without angiographically documented coronary stenosis [<50%]) was determined with intravascular ultrasound, when these patients underwent percutaneous coronary intervention. The serum A-FABP and adiponection levels were measured by enzyme-linked immunosorbent assays.

Results:
Serum A-FABP levels and adiponectin levels in patients with coronary artery disease were correlated with percent plaque volume (r=0.414, p=0.001, and r=−0.328, p=0.01, respectively). Other proinflammatory markers such as tumor necrosis factor-alpha, interleukin-6 and high sensitive C-reactive protein were not associated with percent plaque volume. Serum A-FABP levels were correlated with serum adiponectin levels (r=−0.318, p=0.002) and tended to be correlated with body mass index (r=−0.218, p=0.08), but not with other inflammatory markers. Stepwise multiple regression analysis showed that serum A-FABP level was selected as a factor associated with coronary percent plaque volume (p<0.001).

Conclusions:
A-FABP is independently associated with coronary atheromatous plaque burden. Our results have provided clinical evidence supporting the role of A-FABP in coronary atherosclerosis.
Abstract 3718: MMP-12 Inhibitor Reduces Severity of ANGII-induced Aortic Abdominal Aneurysms in apoE−/− Mice
Authors: Savio Dawn A , Halpern Anita R , Wu Yuchuan , Li Wei , Sypek Joseph , Quinet Elaine M
Published: July 2021

Abdominal aortic aneurysm (AAA) is an inflammatory disorder characterized by local connective tissue degradation, macrophage recruitment and infiltration leading to aortic dilation and rupture. Aneurysms of the abdominal aorta represent a significant cardiovascular risk for which inflammation plays an integral role in the defined pathology. Genetic ablation of metalloprotease-12 (MMP-12) eliminates metalloelastase activity and attenuates aneurysm formation in apoE−/− mice. In the current study, a selective MMP-12 inhibitor, WAY-644 was evaluated in the well-established murine model of ANGII-induced aneurysm formation. This inhibitor displays activity for murine MMP-12, IC50 = 6.3 nM by FRET analysis, with low crossreactivity for other MMPs (exception MMP-8), and has established
in vivo
efficacy in inflammation models. Coadministration of WAY-644 to hyperlipidemic apoE−/− mice during ANGII infusion (1.44 mg/kg) for 28d alters the severity of AngII-induced AAAs as measured by changes in abdominal aortic wet weights and typical AAA classification. As expected, plasma MMP-12 protease activity measured by FRET analysis was inhibited. RNA profiling of abdominal aortic aneurysm tissue characterizes ANGII-induced AAA expansion driven by macrophage infiltration, destructive MMP production and attenuation by MMP-12 inhibition. The transcription of a subset of proinflammatory genes activated with ANGII treatment was repressed by the inhibitor. These genes include quantitative markers of macrophage accumulation in the vessel wall, CD68, MCP1/CCL2, CCR2, MMP-12, and Csf1. Associated reductions in gene markers for inflammation and oxidative stress, ie., heme oxidase (HO), nitric oxide synthase (nos2), Ikbkb, and Stat3 also correlate with MMP-12 antagonism. These changes occur in the absence of lipid changes (TC or TG), or quantitative changes in aortic arch lesions in the ANGII-infused animals. The findings support a mechanism whereby MMP-12 metalloelastase inactivation reduces macrophage recruitment to aneurysmal lesion sites, to lessen activated-macrophage expression of proinflammatory cytokines that figure prominently in vascular wall destruction and the pathogenesis of AAAs.
Abstract 3722: Deficiency Of Hck And Fgr Src Kinases Leads To Delayed Atherosclerosis In LDLr
−/−
Mice.
Authors: Medina Indira , Biessen Erik A.
Published: July 2021

Hck and Fgr are members of the Src family of tyrosine kinases involved in regulation of immune activation and leukocyte function. In macrophages, deficiency of Hck and Fgr leads to impaired integrin signal transduction, altered cytoskeleton and reduced motility, while loss of Hck alone was seen to skew monocyte differentiation towards an M2 macrophage phenotype. In this study we evaluated the involvement of Hck and Fgr in atherosclerosis progression and composition. Female LDLr−/− mice were lethally irradiated and reconstituted one day later with bone marrow from either Hck/Fgr deficient or wild-type mice. After six weeks of recovery, the animals were fed an atherogenic diet during 13 weeks and then sacrificed for lesion analysis and ex-vivo experiments. Blood leukocyte counts were not different between groups. Peritoneal macrophages from the Hck/Fgr deficient mice exhibited a reduced migratory capacity (17 fold decrease compared to control, P=1E10 – 6) with altered formation of phylopodia. In agreement with these findings, reconstitution with Hck/Fgr deficient bone marrow resulted in 36% reduction in lesion size (P=0.02, n=10). Whereas necrotic core size did not differ between groups, fibrous caps and collagen content were markedly smaller in plaques of Fgr/Hck deficient chimeras, both as total area and as percentage of plaque area, suggesting a reduced stability (fibrous cap: −73% P=0.002 (absolute), 13 vs. 30%, P=0.0002 (relative); collagen: −87%, P=0.0001 (absolute); 2 vs. 11% P=0.0005 (relative)). In conclusion, deficiency of Hck and Fgr Src kinases leads to reduced atherosclerosis accompanied by reduced fibrosis and collagen contents, which may be partly attributable to impaired migration of monocytes to the plaque.
Abstract 3727: Beta-1 Adrenergic Antagonism Inhibits Cardiovascular Adaptation to Hemodilution Causing Cerebral Hypoxia
Authors: Hare Gregory M , Ragoonanan Tenille E , Mazer C David , Leong-Poi Howard , Wilson David F , Beattie W Scott
Published: July 2021

A recent clinical study (POISE) demonstrated an increased incidence of stroke in surgical patients treated with a beta-1 adrenergic antagonist. This stroke risk may be attributed to reduced cerebral perfusion in treated patients who are exposed to hemodynamic stress during surgery, including acute blood loss and fluid resuscitation. We tested the hypothesis that acute beta-1 adrenergic antagonism attenuates cardiovascular responses to acute hemodilution causing cerebral tissue hypoxia. After ACC approval, anesthetized rats (1–2% isoflurane) were treated with metoprolol (3 mg kg
−1
i.v.) or saline vehicle (n=8/outcome). Acute hemodilution was then performed by exchanging 50% of estimated blood volume (30 ml kg
−1
) with pentastarch (1:1). Experimental outcomes included heart rate (HR), mean arterial blood pressure (MAP), cardiac output (CO, echocardiography), carotid and cerebral tissue blood flow (CBF, Doppler), and hippocampal and kidney tissue oxygen tension (P
Br
O
2
, P
Ki
O
2
, phosphorescence quenching). Arterial blood gases (ABGs), and hemoglobin concentration (Co- oximetry) were also measured. Data was analyzed by two-way ANOVA (mean ± SD). Acute beta blockade reduced baseline HR (334 ± 31 vs. 291 ± 22 bpm, p<0.05). No differences in ABG and co-oximetry values were observed between groups and a target hemoglobin concentration near 50 g.L
−1
was achieved. MAP was maintained near 70 mmHg and was not different between groups at any time. After hemodilution CO and CBF increased, P
Br
O
2
was maintained and P
Ki
O
2
decreased in control rats. In beta blocked rats, the CO and CBF responses were greatly attenuated leading to a significant reduction in P
Br
O
2
(15 ± 4 vs. 30 ± 12 mmHg, p<0.05). P
Ki
O
2
was reduced comparably to the control group. Acute metoprolol treatment attenuated the cardiovascular response to acute hemodilution resulting in cerebral tissue hypoxia. Acute beta-1 adrenergic blockade may increase the risk of cerebral hypoxia in surgical patients who are exposed to blood loss and fluid resuscitation (CAS and PSI support).
Abstract 3730: Celecoxib Does Not Deteriorate Antiplatelet Effects of Aspirin and Clopidogrel in Healthy Volunteers
Authors: Lee Wonjae , Kwon Dong-A , Cho Hyun-Ju , Suh Jung-Won , Choi Su-Yeon , Kim Sang-Hyun , Kim Chul-Ho , Oh Byung-Hee , Park Young-Bae , Kim Hyo-Soo
Published: July 2021

Background: Celecoxib, cyclooxygenase (COX)-2 inhibitor, has been reported to inhibit neointimal hyperplasia in animal studies and to reduce restenosis after coronary stenting in COREA-TAXUS clinical trial. The main concern is that celecoxib would increase thrombogenecity by inhibiting the synthesis of prostacyclin in endothelial cells. However, it is not known whether the administration of celecoxib would weaken antiplatelet effects of aspirin and clopidogrel which are used after stenting.

Methods: We recruited healthy volunteers (n=40) and randomized them into five subgroups. Each subject received the medication for 6 days and blood samples were taken day 0 and 7. Celecoxib 200mg twice a day, and/or aspirin 100mg daily, and/or clopidogrel 75 mg daily was administered. We compared platelet function by using light transmittance aggregometry and arachidonic acid metabolite assay among subgroups.

Results: Celecoxib single treatment did not significantly affect platelet aggregation. Dual asprin and Plavix treatment inhibited platelet aggregation by 53%, which was not affected by addition of celecoxib showing 52% inhibition (t-test: p = 0.873). The changes of prostacyclin level did not differ among each treatment group (ANOVA: p=0.193). Although statistically not significant, celecoxib seems to lower the thromboxane level further when added to antiplatelet agents.

Conclusion: Celecoxib treatment did not interfere with antiplatelet effect of aspirin or clopidogrel, suggesting that celecoxib as anti-restenosis agent would be administered safely during coverage of dual antiplatelet therapy in patients with coronary stenting without increasing thrombogenecity.
Abstract 3757: Targeted Inactivation of Receptor for Advanced Glycation Products Reduced Oxidative LDL-Induced Inflammation and Atherosclerosis in Non-Diabetic LDL Receptor−/− Mice
Authors: Sun Li , Ishida Tatsuro , Kuriyama Masamitsu , Yasuda Tomoyoki , Hara Tetsuya , Hirata Kenichi , Hayashi Yoshitake
Published: July 2021

Receptor for advanced glycation products (RAGE) is a multiligand receptor on vascular cells and plays an important role in the progression of diabetic complications. To define if RAGE modulates atherosclerosis under non-diabetic conditions, we examined the effect of RAGE deficiency in non-diabetic mice with hyperlipidemia. RAGE knockout mice (RAGE−/−) were bred with low-density lipoprotein receptor knockout (LDLr−/−) mice to generate the double knockout (DKO) mice. The mice were fed with western diet for 12 weeks, and aortic atherosclerotic lesions were analyzed histologically. Although there was no difference in body weight, fasting blood glucose and serum AGE levels between DKO and LDLr−/− mice, DKO mice exhibited a significant decrease in the size and macrophage content in atherosclerosis lesions compared with LDLr−/− mice. Expressions of endothelial adhesion molecules such as VCAM-1 and ICAM-1 in the aorta were lower in DKO mice than those in LDLr−/− mice. Chemiluminescence and fluorescence-based assays revealed that DKO mice showed lower oxidative stress in the vessel wall than LDLr−/− mice. Complementary in vitro studies revealed that peritoneal macrophages isolated from RAGE−/− mice exhibited a decrease in oxidative LDL (oxLDL)-induced ERK activation and proliferation when compared to RAGE+/+ macrophages. In contrast, overxpression of RAGE in COS7 cells resulted in the augmented ERK activation in response to oxLDL, which is abolished by the co-incubation with anti-RAGE blocking antibodies. The results indicated that oxLDL activated RAGE and increased oxidative stress via NADH/NADPH oxidase pathway. OxLDL may serve as a new ligand for RAGE. RAGE inactivation inhibits atherosclerosis by reducing pro-inflammatory and oxidative stress under the hyperlipidmic, non-diabetic condition.
Abstract 3789: Induction of Murf-1 is Required for Tnf-alpha Induced Loss of Skeletal Muscle Function
Authors: Adams Volker , Magner Norman , Linke Axel , Gasch Alexander , Hirner Stephanie , Gielen Stephan , Labeit Siegfried , Schuler Gerhard
Published: July 2021

Background:
Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are known to impair skeletal muscle (SM) function. Furthermore, TNF-α induces the expression of atrogin-like muscle specific ubiquitin E3-ligases, presumed to mediate muscle atrophy. The relative contributions of respective ubiquitin ligases, like MuRF1 for the TNF-α induced reduction in muscle function are not known.

Methods:
TNF-α or saline was injected either into C57Bl6 or MuRF1
−/−
mice. After 16 –24h the expression of MuRF1 in the SM was quantified by qRT-PCR and western blot. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, 2D-gel electrophoresis was performed.

Results:
WT animals injected with TNF-α had higher MuRF1 mRNA (saline: 56.6±12.1 vs. TNF-α: 133.6±30.3 arb. Units; p<0.05) and protein expression (saline: 0.38±0.11 vs. TNF-α: 1.07±0.25 arb. Units; p<0.05) as compared to saline injected littermates. However, TNF- α was unable to induce MurRF1 expression in MuRF1
−/−
mice. Furthermore, TNF- α reduced force development at 150Hz by 25% in C57Bl6 animals (saline: 2412±120 vs. TNF-α: 1799±114 g/cm2; p<0.05), but not in MuRF1
−/−
mice (saline: 2424±198 vs. TNF-α: 2431±180 g/cm2; p=NS). The proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T (TNNT3) in WT animals treated with TNF- α as compared to MuRF1
−/−
mice receiving TNF-α . In addition, TNF-α injection into C57Bl6 animals resulted in a down-regulation of eEF1γ ( WT: 0.60±0.02 vs. WT+TNF-α: 0.39±0.05 arb. Units; p<0.05). This reduction was not seen in MuRF1
−/−
mice receiving TNF- α (KO: 0.59±0.03 vs. KO+TNF-α: 0.68±0.01 arb. Units; p<0.05)

Conclusion:
The results of this study demonstrate for the first time, that the TNF- α induced reduction in SM force development depends on the induction of the atrophy related E3-ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-α-MuRF1-mediated down-regulation of TNNT3 and the elongation factor eEF1γ.
Abstract 3933: Local Cytokine Release is Increased in Less Mature Collateral Arteries and Reflects Collateral Arterial Oxygen Pressure in Man
Authors: Schirmer Stephan H , van Royen Niels , Moerland Perry D , Fledderus Joost O , Baan Jan , Henriques José P , van der Schaaf René J , Vis Marije M , Piek Jan J
Published: July 2021

Background: The molecular background of the maturation of the human collateral circulation in response to coronary narrowings is poorly understood, particularly because of the complexity to evaluate coronary collateral growth in-vivo. Local sampling of collateral blood in patients could provide valuable insights in this process. We sought to determine metabolic parameters and plasma cytokine profiles in the human coronary collateral circulation in relation to collateral maturation.

Methods and Results: We used a Proxis® proximal embolic protection device (St. Jude Medical) to perform blood sampling directly from the coronary collateral circulation in 60 patients with non-total (n=25) or total coronary occlusions (n=35). Coronary collateral flow index (CFI) was assessed by intracoronary pressure measurements. Oxygenation and lactate content was measured as well as 30 cytokines potentially involved in collateral artery growth, using a custom-made multiplex assay. Oxygen gradient between systemic and collateral arterial correlated inversely with CFI (R=−0.63, p<0.001). No rise in lactate or change in pH was found in collateral blood. Locally increased plasma levels were found for bFGF, Eotaxin, MIF, MCP-1, VEGF and TGFbeta, while SCF was significantly decreased. The highest cytokine gradients were found in patients with the least developed collateral circulation. The majority of cytokines correlated more strongly with pO2 extraction across the collateral bed than with CFI.

Conclusion: Upregulation of cytokines at the site of coronary collateralization and in collateral vessels with enhanced oxygen gradient between systemic and collateral arterial blood demonstrate increased inflammatory activity in arteries where oxygen delivery is insufficient. Less pronounced cytokine gradients in collateral arteries in CTOs indicate reduced arteriogenic activity in these vessels. Determining oxygen gradient may be a more sensitive tool for the assessment of the functional capacity of the collateral circulation in patients than measurements of CFI.
Abstract 3937: Generation and Analysis of Neural Crest- and Endothelial-specific Mutant Mice for Foxc1
Authors: Seo Seungwoon , Hayashi Hisaki , Kume Tsutomu
Published: July 2021

The forkhead transcription factor Foxc1 has been implicated in craniofacial, ocular and cardiovascular development. Foxc1 is expressed in mesoderm- and neural crest (NC)-derived cells, including endothelial cells (ECs) of the heart and blood vessels and vascular smooth muscle cells. However, the precise role of Foxc1 in specific cell types still remains unclear. Therefore, to define the distinct function of Foxc1 in NC and EC, we have generated conditional mutant mice for Foxc1 crossed with either Tie2-Cre or Wnt1-Cre mice. EC-specific Foxc1 mutants survive until adulthood with no apparent embryonic defects, although they show vascular abnormalities in the adult. By contrast, NC-specific Foxc1 mutants die perinately with haemorrhagic hydrocephalus, rudimentary frontal bones, and abnormal patterning of the aortic arch. NC-derived cells also give rise to the stroma and endothelium of the cornea, an avascular organ whose transparency is critical for vision. Importantly, we found that NC-specific Foxc1 mutants show failure of the formation of the anterior chamber and corneal endothelium in the eye. Mutant corneal stroma is much thicker than normal with increased cell proliferation. Most Intriguingly, NC-specific Foxc1 mutants exhibit ectopic neovascularization in the cornea with significant upregulation of Mmp9, sFlt1, Flt1 and Tek at E15.5, while Vegfa and Fgf expression is not changed. By contrast, the cornea of EC-specific Foxc1 mutants is normally formed and avascular. These data suggest that the cell-autonomous function of Foxc1 in the neural crest is essential for craniofacial and cardiovascular development and that Foxc1 plays an important role in inhibition of vascular formation in the cornea.
Abstract 3956: CD11b Positive Monocytes Are Required for Endothelial Regeneration
Authors: Werner Nikos , Haghighat Bita , Engel Daniel , Kurts Christian , Nickenig Georg , Wassmann Sven
Published: July 2021

Bone marrow (BM)-derived circulating endothelial progenitor cells (EPC) contribute to endothelial cell (EC) regeneration. Systemic transfusion of EPC fosters the restoration of the endothelium after EC damage in mice. Transfusion of mononuclear cells (MNC) leads to an even more pronounced endothelial regeneration compared to EPCs. Here, we determine the role of monocytic cells and their interaction with circulating EPC on EC regeneration. To evaluate the effect of monocytes on re-endothelialization, neointima formation, and endothelial function, wild-type (WT) mice received repetitive intravenous injections of spleen-derived MNC, CD11b-depleted MNC, CD11b+ monocytes, or cell-free vehicle after EC denudation. Endothelial restoration was enhanced and neointima formation reduced after transfusion of MNC and CD11b+ cells. CD11b-depleted cells had no effect. Transfusion of CD11b+ cells in ApoE−/− mice improved endothelial-dependent vasorelaxation while CD11b-depleted cells had no effect. Next, WT mice were treated with clodronate liposomes (10μl/g bw) which induces the ablation of circulating monocytes. Re-endothelialization was diminished in clodronate- compared to vehicle-treated mice. Next, we selectively depleted CD11b+ monocytes using CD11b-DTR mice which express the human diphtheria toxin receptor (DTR) under the control of the CD11b promoter. Treatment with diphtheria toxin (DT) resulted in the complete ablation of circulating monocytes. Re-endothelialization was delayed in DT-treated compared to vehicle-treated mice. Systemic treatment of WT mice with Sca1+ EPC improves endothelial function. Repetitive transfusion of Sca1+ cells enhanced re-endothelialization in vehicle-treated CD11b-DTR mice compared to DT-treated mice. Rescue experiments showed that transfusion of CD11b+ cells in CD11b-DTR mice treated with either vehicle or DT resulted in a comparable enhancement of re-endothelialization. Our results indicate that circulating CD11b+ monocytes contribute to EC regeneration. Ablation of monocytes
in vivo
leads to a delay in re-endothelialization after focal EC damage which can not be rescued by transfusion of Sca1+ cells.
Abstract 5257: Priming with Angiopoietin-1 Augments Angiogenic Potential of Peripheral Blood Stem Cells Mobilized with Granulocyte-Colony Stimulating Factor
Authors: Lee Choon Soo , Kim Min-Seok , Cho Hyun-Jai , Hur Jin , Kwon Yoo-Wook , Choi Su-Yeon , Kang Hyun-Jae , Kim Sang-Hyun , Oh Byung-Hee , Park Young-Bae , Kim Hyo-Soo
Published: July 2021

Background: We introduce the concept of ‘priming’ mobilized peripheral blood stem cells(
mob
-PBSCs) with G-CSF using Angiopoietin-1(Ang-1).

Methods and Results: Ang-1 was administered as a priming agent because it was found that
mob
PBSCs highly express Tie2, Ang-1 receptor [19.2±3% vs. 1.2±0.2%, p<0.01 for
mob
PBSCs vs PBMCs]. We investigated whether the effect of Ang-1 on
mob
PBSCs differentiation is mediated via the transcriptional regulation of ets-1. The gene expression of ets-1 increased in a time dependent manner after Ang-1 stimulation(400ng/ml) in
mob
PBSCs. Compared with vehicle, treatment with Ang-1 for 4 hours resulted in stronger expression of endothelial markers, such as, CD31 and VE-cadherin, which were significantly reduced with antisense ets-1 oligodeoxynucleotides(ODN). These results were also confirmed at the protein level by FACS analysis. We postulated that integrins that contain an ets-1 binding motif in their promoter region may be activated by Ang-1, such as α4β1 and α5β1 integrins. RT-PCR showed increased mRNA expression of α4, α5, and β1 integrins after Ang-1 stimulation, and its attenuation after transfection with antisense ets-1 ODN. These results were also confirmed at the protein level by FACS analysis. To correlate these findings with functional changes in
mob
PBSCs, we performed in vitro adhesion assays. Ang-1 stimulation increased
mob
PBSCs adhesion to HUVECs and fibronectin, while pretreatment with anti-hTie2 or anti-integrin antibody prior to Ang-1 stimulation abrogated these results. And, we checked the effect of Ang-1 in vivo model.
mob
PBSCs were obtained from rabbit and primed with COMP-Ang1(400ng/ml) for 4 hours.
mob
PBSCs were labeled with Tc-99m HMPAO and were injected into a central artery of rabbit ear.
mob
PBSC engraftment was quantified by the ratio of whole rabbit ear radioactivity over total infused
mob
PBSC radioactivity. In contrast, cells primed with COMP-Ang1 showed markedly increased engraftment by 53±33%(
p
<0.05). Finally,
mob
PBSCs primed with Ang-1 enhanced neovascularization in a nude mouse hindlimb ischemia model.

Conclusion: These results suggest that
mob
PBSCs priming with Ang-1 may enhance the efficacy of transplanted
mob
PBSCs in terms of increasing neovascularization in ischemic tissue.
Abstract 5280: Positron Emission Tomography (PET) for
In Vivo
Quantification and Optimization of Acute Cell Retention after Intramyocardial Cardiac-Derived Stem Cell Delivery
Authors: Terrovitis John , Lautamäki Riikka , Bonios Michael , Fox James , Engles James M , Yu Jianhua , Leppo Michelle K , Pomper Martin , Wahl Richard L , Tsui Benjamin M , Bengel Frank M , Abraham M. Roselle , Marbán Eduardo
Published: July 2021

Success of stem cell transplantation for cardiac regeneration is partially limited by low retention/engraftment of the delivered cells. A clinically applicable method for accurate quantification of cell retention would enable optimization of cell delivery. To quantify acute myocardial retention and identify strategies that improve retention following intramyocardial injection of cardiac-derived stem cells (CDCs). Two million CDCs derived from syngeneic, male Wistar Kyoto (WK) rats were labeled with
18
FDG (2μCi/mL) and injected intramyocardially into the ischemic region of female WK rats (n=43) immediately following permanent left coronary artery ligation. The effects of fibrin glue, local 2,3-butanedione-2-monoxime (BDM, an excitation-contraction uncoupler), bradycardia (adenosine) and cardiac arrest were examined.
18
FDG PET images were coregistered with myocardial perfusion
13
NH
3
images for myocardial delineation, and with chest CT images for attenuation correction. Quantitative PCR for the male-specific SRY gene was performed in 6 control animals to validate the PET results. Myocardial cell retention 1 hr after cell delivery in control animals was 17.6±11.5% (n=6) by quantitative PCR, and 17.8±7.3% (n=7) by PET. When CDCs were injected immediately following induction of cardiac arrest (n=4), retention was greatly increased to 75.6±18.6% (
P
<0.001), suggesting a role for cardiac contraction, heart rate and/or coronary flow on CDC retention. Adenosine slowed the ventricular rate and doubled CDC retention (35.4±5.3%, n=4,
P
<0.05). A similar increase in CDC retention was observed following epicardial application of fibrin glue at the injection site (37.5±8.2%; n=8,
P
<0.01). The combination of fibrin glue and adenosine further improved CDC retention to 39.0±11.6% (n=8,
P
<0.001). Local BDM treatment transiently suppressed contractility at the injection site (by visual inspection), but did not affect CDC retention (14.9 ±6.9%,
P
=ns).
In vivo
PET permits accurate measurement of CDC retention early after intramyocardial delivery. Sealing injection sites with fibrin glue may be a clinically translatable method for improving stem cell engraftment in a beating heart.
Abstract 5286: Human AV Junctional Pacemaker Shift Due to Cholinergic and Adrenergic Stimulations: Optical Imaging with a Novel Long Wavelength Voltage-Sensitive Dye
Authors: Fedorov Vadim V , Ambrosi Christina M , Hucker William J , Glukhov Alexey V , Foyil Kelley V , Wuskell Joe , Loew Leslie , Moazami Nader , Efimov Igor R
Published: July 2021

It is well known that under different physiological and pathologic conditions, the atrioventricular junction (AVJ) may become the pacemaker of the heart. However, unlike the well-characterized AVJ pacemaker in animal models, autonomic control of the human AVJ pacemaker has not been studied. Explanted human hearts with different types of cardiomyopathy (n=7) were obtained at the time of cardiac transplantation and perfused with cardioplegic solution. The AVJ was cannulated, isolated from the rest of the heart, immobilized with the excitation-contraction uncoupler blebbistatin (10 μmol/L) and optically mapped using the infrared voltage sensitive dye di-ANBDQBS. Imaging was conducted with 100x100 CMOS camera from endocardial field of view ranging 24x24 to 36x36mm
2
, which allowed to simultaneously map right side of intratrial and intraventricular septa, coronary sinus (CS) and His bundle regions. In control, we found AVJ rhythm of 31+/−15 bpm (2409+/−1264 ms) in all human preparations which originated in compact AV node (N-region, n=4) and/or region between AV node and His bundle (NH-region, n=3). Isoproternol (Iso, 1 μM) induced AVJ rhythm acceleration up to 68+/−12 bpm (913+/−192 ms), temporary pacemaker shift to CS region (n=2) and improved conduction in both slow and fast pathways, which induced reentrant arrhythmias in 3/5 preparations. Acetylcholine (ACh, 1–3 μM) slowed rhythm to 21+/−6 bpm (3130+/−1146 ms) and conduction in both pathways up to complete block, and induced temporary pacemaker shift to CS region in 3/4 preparations. Moreover, we found bifocal activation in 3/4 preparation when two main pacemakers (CS and compact AV node) worked asynchronously with different frequencies, accompanied by the exit block from AV node. We showed by high-resolution optical mapping for the first time that unlike in animal models (rabbit and rat) in isolated coronary perfused human AVJ leading pacemaker localized mostly in the N- or NH-regions. Beta-adrenergic (Iso) and cholinergic (ACh) stimulations of AVJ can significantly accelerate or slow rhythm and conduction, as well as induced pacemaker shift to CS, and result in reentrant arrhythmias.

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 5293: A New Animal Model of Hypercholesterolemia and Atherosclerosis: Mice Deficient in All Nitric Oxide Synthases
Authors: Tsutsui Masato , Yatera Yasuko , Shimokawa Hiroaki , Nakata Sei , Shibata Kiyoko , Morishita Tsuyoshi , Sabanai Ken , Tasaki Hiromi , Nakashima Yasuhide , Yanagihara Nobuyuki , Otsuji Yutaka
Published: July 2021

We have recently developed mice lacking all three nitric oxide synthase (NOS) isoforms: nNOS, iNOS, and eNOS (
PNAS
2005). In this study, we examined the effects of a high-cholesterol (HC) diet on lipid metabolism and vascular lesion formation in those mice. Experiments were performed in 2-month-old male wild-type (WT) and singly, doubly, and triply NOS
−/−
mice (n=6–9). They were maintained on either a regular diet or a HC diet for 3 months. The HC feeding significantly increased plasma levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) in all the genotypes studied as compared on the regular diet (all
P
<0.05). These serum levels of TC and LDL on the HC diet (mg/dl) were both significantly higher in all the singly, doubly, and triply NOS
−/−
genotypes as compared with the WT genotype (singly nNOS
−/−
[371±61 and 205±65], iNOS
−/−
[559±62 and 350±62], eNOS
−/−
[619±22 and 395±25], doubly n/iNOS
−/−
[518±77 and 328±72], n/eNOS
−/−
[635±56 and 458.8±42], e/iNOS
−/−
[480±38 and 260±40], triply n/i/eNOS
−/−
[2316±704 and 1588±715], and WT [326±43 and 244±54]) (all
P
<0.05). Notably, the extent of the dyslipidemia was by far severest in the triply n/i/eNOS
−/−
genotype among the NOS
−/−
genotypes, and intriguingly, the serum levels of TC and LDL in the triply n/i/eNOS
−/−
genotype were equivalent to those in apolipoprotein E
−/−
mice that exhibit severe hypercholesterolemia. Lipid accumulation in the aorta on the HC diet (lipid area, %, oil red O staining) was also significantly more accelerated in all the NOS
−/−
genotypes than in the WT genotype (singly nNOS
−/−
[6.6±1.5], iNOS
−/−
[6.7±2.2], eNOS
−/−
[5.5±2.3], doubly n/iNOS
−/−
[4.7±1.7], n/eNOS
−/−
[6.4±1.4], i/eNOS
−/−
[6.8±1.3], triply n/i/eNOS
−/−
[20.6±1.0], and WT [3.6±1.2]), while the extent of the aortic atherosclerosis was again by far severest in the triply n/i/eNOS
−/−
genotype (all
P
<0.05). These results demonstrate that mice deficient in all NOSs manifest severe hypercholesterolemia and lipid-rich atherosclerotic lesion formation in response to a HC diet, indicating a pivotal role of the whole NOS system in preventing those disorders. Our triply NOS
−/−
mouse is a new experimental model of human hypercholesterolemia and atherosclerosis.
Abstract 5307: Cardiomyocyte Sodium Accumulation Promotes Diastolic Dysfunction in Serca2 Knockout Mice
Authors: Louch William , Hougen Karina , Aronsen Magnus , Mork Halvor K , Sjaastad Ivar , Andersson Kristin B , Christensen Geir , Sejersted Ole M
Published: July 2021

Terminal decompensation during heart failure involves deterioration of diastolic function. We investigated the mechanisms underlying this functional decline in mice with cardiomyocyte-specific, conditional excision of the Serca2 gene (KO). At 4 weeks following gene deletion, SERCA levels in KO cardiomyocytes were reduced by more than 95% from flox-flox (FF) controls. Surprisingly, echocardiographic measurements indicated only moderate impairment of
in vivo
function, as systolic and diastolic tissue velocities were 62% and 72% of FF values, respectively. Diastolic heart failure developed in KO between 6 and 7 weeks, as diastolic tissue velocity rapidly declined to 51% of FF values. We compared cardiomyocyte contractions and Ca
2+
cycling at the 4 and 7 week time points. In KO cells, contractions were reduced between 4 and 7 weeks (from 40% to 14% of FF values), and the rate of relaxation was slowed (from 11% to 3% of FF values). Similar alterations were observed in Ca
2+
transients. Sarcoplasmic reticulum (SR) Ca
2+
content was markedly reduced in 4-week KO, although a minute thapsigargin-sensitive SR Ca
2+
release could be induced. SR content was further decreased in 7-week KO and SR Ca
2+
release was not detectable, although Western blots showed no difference in SERCA levels between 4 and 7 week KO. Ca
2+
influx via Ca
2+
channels was enhanced in KO (integrated current ≈200% of FF) at both time points. However, greater NCX-mediated Ca
2+
extrusion in 4-week KO was partially reversed in 7-week KO due to elevation in cytosolic [Na
+
] (34 mM vs 25 mM in FF). Normalizing cytosolic [Na
+
] using patch clamp increased the rate of decline of the Ca
2+
transient in 7-week KO to 4-week KO values. Thus, KO mice compensate for loss of SR function by increasing trans-sarcolemmal Ca
2+
flux. However, in the longer term, cytosolic Na
+
accumulation impairs NCX-mediated Ca
2+
extrusion, which promotes development of diastolic heart failure.
Abstract 5322: Mutation-Linked, Defective Inter-Domain Interaction within the Cardiac Ryanodine Receptor as a Critical Cause of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
Authors: Uchinoumi Hitoshi , Yano Masafumi , Suetomi Takeshi , Xu Xiaojuan , Ono Makoto , Tateishi Hiroki , Okuda Shinichi , Kobayashi Shigeki , Yamamoto Takeshi , Ikeda Yasuhiro , Ohkusa Tomoko , Matsuzaki Masunori
Published: July 2021

We previously reported that interaction of N-terminal (0–600) and central domains (2000–2500) of the cardiac ryanodine receptor (RyR2), harboring many mutations associated with CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we examined the pathogenic role of the inter-domain interaction within RyR2 in the mutation-linked channel disorder in human CPVT-associated RyR2
R2474S/+
knock-in (KI) mice model. In all KI mice (6/6), bi-directional ventricular tachycardia was observed during or after exercise with treadmill, but not in wild-type (WT) mice (0/7). Sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP
2460–2495
(DPc10), harboring the same CPVT mutation site as KI mice; R2474S, as a site-directing carrier. Only in KI (but not in WT) mice, partial domain unzipping was taken place at baseline, and further domain unzipping occurred in response to cAMP (1 μM), as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In the saponin-permiabilized KI cardiomyocytes; [Ca
2+
] was buffered at 30 nM by 0.5 mM EGTA, the relationship curve between the frequency of Ca
2+
sparks (SpF: s
−1
·100μm
−1
; by Rhod- 2) and SR Ca
2+
content {Ca
2+
con
; by caffeine application}, obtained by incremental addition of cAMP (0.1–1 μM), was markedly shifted to the left (towards lower SR Ca
2+
content) compared to WT cardiomyocytes, although RyR2 2808Ser phosphorylation is similarly increased in WT and KI. For instance, at 1 μM of [cAMP], Ca
2+
con
decreased by 43 % from WT, although SpF was more increased in KI than WT (KI: 19.6±0.5; WT: 14.7±0.4, p<0.01). Interestingly, addition of DPc10 (50 μM), which was found to induce similar domain unzipping (competing with native domain) in WT as KI (+cAMP), to WT cardiomyocytes reproduced the leftward shift of the SpF-Ca
2+
con
curve seen in KI. In conclusion, single point mutation at specific region may causatively induce defective inter-domain interaction between N-terminal and central domains in response to PKA phosphorylation, and in turn enhance the sensitivity of the channel to activation by luminal [Ca
2+
]: i.e. decreased threshold [Ca
2+
]
L
to induce spontaneous Ca
2+
sparks, leading to CPVT.