bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5561: Remote Postconditioning May Attenuate Ischemia-reperfusion Injury in a Murine Hind Limb Through Activation of Adenosine Receptors
Authors: Tsubota Hideki , Marui Akira , Miwa Senri , Nakata Tomohiro , Minakata Kenji , Yamazaki Kazuhiro , Ikeda Tadashi , Sakata Ryuzo
Published: April 2024

Introduction:
Preconditioning is a potent protection from ischemia-reperfusion injury (IRI), but its clinical application has been limited because preconditioning must be established before unanticipated ischemic events. Remote postconditioning (RPC), an application of brief ischemia in remote organs just before reperfusion of ischemic target organ, is a novel approach to attenuate IRI. However, its efficacy and possible mechanisms in IRI of ischemic limb has not been elucidated.

Methods:
Right hind limb ischemia was induced using a rubber tourniquet, and reperfusion was initiated by releasing the tourniquet. RPC was established by 5 minutes’ ischemia followed by 5 minutes’ reperfusion in the left leg just before reperfusion of the right leg. Control mice received ischemia for 3 hours followed by 24 hours reperfusion; RPC mice received ischemia for 3 hours followed by 24 hours reperfusion accompanying with two sets of RPC. After 24 hours of reperfusion, wet/dry ratio of skeletal muscle (degree of tissue edema), myeloperoxidase (MPO) activity (accumulation of neutrophil), and nitro blue tetrazolium (NBT) reduction (tissue necrosis) were evaluated. In addition, 8-sulfophenyltheophylline (SPT), an inhibitor of adenosine receptors, was intraperitonealy injected to mice with or without RPC. NBT reduction was measured to evaluate possible mechanisms.

Results:
Wet/dry ratio, MPO activity and tissue necrosis were significantly lower in RPC group than control group, and injection of SPT impaired the protective effect of RPC (table

).

Conclusion:
RPC attenuated IRI in a murine hind limb ischemia, possibly through the endogenous activation of adenosine receptors. RPC may be promising therapeutic option to treat serious limb ischemia.
Abstract 5564: Circulating Endothelial Progenitor Cells Co-Expressing an Osteobastic Phenotype Are Increased in Patients With Severe Calcific Aortic Valve Stenosis
Authors: Goessl Mario , Moedder Ulrike I , Lerman Lilach O , Sarano Maurice E , Khosla Sundeep , Lerman Amir
Published: April 2024

Objective
To test the hypothesis that the numbers of circulating endothelial progenitor cells (EPC) co-expressing an osteogenic phenotype (EPC-OC) are increased in patients with clinically severe calcific aortic valve stenosis (AoC).

Background
AoC is one of the most common valvular heart diseases. Recent studies have shifted the old paradigm of AoC being a passive, degenerative process to an active biological process associated with an osteoblast-like phenotype. We have recently identified circulating EPC-OC which are significantly increased in patients with severe coronary artery disease (CAD).

Methods and Results
Of 38 patients, 14 underwent aortic valve replacement due to AoC with only mild angiographic CAD. Patients with normal aortic valves and coronary arteries (n=12) or severe CAD without aortic valve disease (n=12) served as controls (Table

). Peripheral blood mononuclear cells were analyzed using flow cytometry following staining for EPC markers (CD133, CD34, KDR) and the osteoblastic marker, osteocalcin (OC). Patients with AoC had significantly higher fractions of OC co-expression on EPC of different maturity than normal patients, similar to patients with CAD (Figure

).

Conclusions
Circulating EPC co-expressing an osteoblastic phenotype are increased and may potentially play a role in the active process of aortic valve calcification.

Table.

Clinical Data.
Abstract 5565: Smoking-Induced Long-lasting Modifications Through a Monoamine Oxidase Epigenetic Regulation
Authors: RENDU Francine , Del Pino Muriel , Chironi Gilles , Callebert Jacques , Peoc’h Katell , Simon Alain , Launay Jean-Marie
Published: April 2024

Objectives:
Smoking is a major cardiovascular risk factor. We postulated that serotonin, released from activated platelets, could be involved in smoking-induced vascular modifications.

Patients & Methods:
We studied 115 men with no history of cardiovascular disease and no diabetes, distributed as smokers (S), former smokers (FS) who had stopped smoking for a mean of 13 years, and never smokers (NS). There was no difference in age, BMI, blood pressure or lipid status between the 3 groups. Serotonin, monoaminoxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA) - the serotonin/MAO catabolite - in plasma samples. Genomic sequencing of the MAO gene promoter was performed on peripheral blood monocytic cells (PBMC).

Results:
Both platelet serotonin and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Unexpectedly, the platelet serotonin content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme amount found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B a study of the methylation of its gene promoter in a supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity.

Conclusions:
This is the first report that smoking induces an epigenetic modification lasting over ten years after quitting. A better understanding of the epigenome may help to further elucidate the development of smoking-induced vascular complications. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent serotonin catabolism is also involved in addiction and lung cancer.
Abstract 5567: The
Ldlr
−/−
Mouse as a Novel Model of Aortic Calcification: Application of Micro-Computerized Tomography
Authors: Awan Zuhier , Denis Maxime , Prat Annik , Goltzman David , Seidah Nabil G , Genest Jacques
Published: April 2024

BACKGROUND
: Patients with familial hypercholesterolemia (FH) due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR) gene exhibit an age-dependent and LDLR gene dosage effect on aortic calcification. We have previously documented that subjects with homozygous FH show significant aortic calcification by their teens, an effect delayed by 20 years in heterozygotes and observed much later in control subjects. The degree of calcification is independent of initial and life long burden of low-density lipoprotein cholesterol concentration. This clinical observation suggests a potential role for the LDLR in osteoblast signalling pathways. In order to better study this observation, we established an animal model of aortic calcification and a novel technique; based on micro-computerized tomography (CT) to examine vascular calcifications in mice.

METHOD AND RESULT:
We examined C57BL/6 wild-type (wt) and
Ldlr
−/−
mice at 12 and 18 months. The
Ldlr
−/−
mice develop spontaneous atherosclerosis in normal chow-fed older mice. Vascular calcification has not been previously examined in this model. Under isoflurane anesthesia, the aorta was perfused
in-situ
with saline. The aorta was then carefully dissected and fixed
ex-vivo
. The tissue was embedded in paraffin block and calcium content was determined by digital x-ray imaging, dual X-ray densitometry, and micro-CT scanning of the entire aorta and aortic valve. The paraffin blocks were then sectioned and Alizarin Red stain was used to further quantify calcified tissue. Analysis of 12 and 18 month wt and
Ldlr
−/−
mice confirmed our observations in human, with aortic calcifications identified at areas of shear stress.

CONCLUSIONS:
The
Ldlr
−/−
mouse model develops aortic calcification even when fed a normal chow diet. Calcifications at areas of shear stress suggest that calcific aortic stenosis and arterial wall calcifications may share many similar pathophysiological features. This animal model of aortic calcifications, and the CT imaging technique used in the present study, may provide a better understanding of the potential links between FH and calcium deposition in the vascular wall. This model has the potential to study aortic valve calcifications as well.
Abstract 5568: Impact of Angiotensin II Receptor Blockers on Regression of Coronary Atherosclerotic Plaque -OVATION Study
Authors: Ishii Hideki , Kobayashi Masakazu , Ichimiya Satoshi , Kanashiro Masaaki , Kurebayashi Nobutake , Sone Takahito , Tsuboi Hideyuki , Mukawa Hiroaki , Amano Tetsuya , Uetani Tadayuki , Marui Nobuyuki , Matsubara Tatsuaki , Murohara Toyoaki
Published: April 2024

OBJECTIVES:
Coronary intravascular ultrasound (IVUS) can provide an imformation of coronary atherosclerosis. Recent reports suggest that coronary plaques can be reduced by some medications. On the other hand, angiotensin II receptor blockers (ARBs) improve clinical outcomes in patients with hypertension. The aim of this multi-center study was to investigate whether blood pressure-lowering therapy with ARBs had efficacies regarding regression of coronary atherosclerosis.

Methods and Results:
Seventy one hypertensive patients with stable angina pectoris who underwent elective PCI were enrolled. They received one of two ABRs (olmesartan 20mg/day or valsartan 80 mg/day) after PCI. Non-target coronary lesions with mild to moderate stenosis were measured by volumetric IVUS at both baseline and 6-month follow-up period in those patients. Systolic blood pressure was significantly decreased by 16.7% after treatment with ARBs (P < 0.01 vs baseline). There were no significant differences in vessel volume (103.9±58.4 mm
3
at baseline and 98.7±57.6 mm
3
at 6-month follow-up) and lumen volume (57.2±33.6 mm
3
at baseline and 57.0±34.2 mm
3
at 6-month follow-up) between baseline and 6-month ARB treatment. However, statistically significant reduction in plaque volume was observed in examined coronary arteries (46.7±31.8 mm
3
at baseline and 41.7±28.4 mm
3
at 6-month follow-up, 10.2% decrease from baseline, P = 0.01). Interestingly, serum adiponectine levels increased from 9.0±4.2
μ
g/ml to 10.7±5.4
μ
g/ml (an increase of 19.4 %, P < 0.01), and serum MDA-LDL cholesterol levels decreased from 107.8±46.4 IU/l to 95.5±28.0 IU/l (a decrease of 11.4 %, P < 0.01).

Conclusions:
Blood pressure-lowering therapy with ARBs or 6 months significantly reduced the plaque volume at non-target coronary lesions in patients with stable angina pectoris. Furthermore, a significant increase in serum adiponectine levels and a significant decrease in serum MDA-LDL cholesterol levels were also observed after 6-month ARBs treatment.
Abstract 5578: Cell Polarity Factor Par3 Binds SPTLC1 Modulating Monocyte/Macrophage Serine Palmitoyltransferase Activity, De Novo Ceramide Synthesis and Chemotaxis
Authors: Tamehiro Norimasa , Mujawar Zahedi , Fitzgerald Michael L
Published: April 2024

Elevated sphingolipids associate with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and 2 subunits, and we have identified a novel protein-protein interaction between SPTLC1 and the PDZ protein Partitioning Defective Protein 3 (Par3). Mammalian SPTLC1 orthologs have a highly conserved C-terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with a SPTLC1 C-terminal peptide we found it bound the 3
rd
PDZ domain of Par3. Overlay and immuno-precipitation assays confirmed this interaction and indicate Par3 associates with the SPTLC1/2 holoenzyme by binding the C-terminal PDZ motif of SPTLC1, and this binding can re-localize SPTLC1 to the periphery of the cell at sites of cell-cell contact and in cell protrusions. The SPTLC1/2-Par3 complex was detected at endogenous expression levels in mouse liver and primary macrophages demonstrating its physiologic existence, and siRNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and the de novo synthesis of ceramide and sphingomyelin by nearly 40%. Given monocyte recruitment into inflamed vessels promotes atherosclerosis, and since Par3 and sphingolipids have been associated with polarized cell migration, we tested and found that Par3 and SPTLC1 are required for the ability of THP-1 monocytes to migrate towards monocyte chemoattractant protein 1 (MCP-1). Knockdown of Par3 significantly reduced MCP-1 induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity. Migration in response to other inflammatory G-protein coupled chemokines including fractalkine and fMLP was also inhibited in these cells and in primary mouse macrophages treated with the anti-atherosclerotic SPT inhibitor myriocin. In conclusion, we have identified a novel SPTLC1-Par3 interaction that promotes monocyte serine palmitoyltransferase activity and chemotaxis towards inflammatory chemokine signals.
Abstract 5588: Identification of Aria as a Novel Factor Regulating Endothelial Cell Apoptosis and Angiogenesis in vivo
Authors: Koide Masahiro , Ikeda Koji , Uraoka Maki , Nakagawa Yusuke , Akakabe Yoshiki , Matsubara Hiroaki
Published: April 2024

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. Here, we identified a novel gene (ARIA) that regulates endothelial apoptosis and angiogenesis. ARIA was expressed in a variety of human cultured endothelial cells as well as in blood vessels of e9.5d mouse embryos. Knockdown of ARIA in HUVEC using siRNA significantly attenuated endothelial apoptosis, but not affected cell migration or proliferation. This anti-apoptotic effect by ARIA-knockdown was attributable to the enhanced expressions of inhibitor of apoptosis (cIAP)-1 and cIAP-2. cIAP-1 and cIAP-2 play as ‘guardians’ of the cell death machinery by directly binding to and neutralizing the caspases as well as by modulating the death signaling through TNF receptors. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the anti-apoptotic effect of ARIA-knockdown. On the other hand, yeast two-hybrid screening identified the 20S proteasome
α
subunit-7 as a binding partner of ARIA. We then found that cIAP-1 and cIAP-2 are targeted by the proteasome, and that ARIA regulates the proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.
In vivo
angiogenesis was studied by Matrigel-plug assay and mouse ischemic retinopathy model, in which ARIA was knocked down by mixing siRNA in the plugs and intravitreal injection of siRNA respectively. In both the models, ARIA-knockdown significantly enhanced the
in vivo
angiogenesis through attenuating the endothelial apoptosis. To further study ARIA function in vivo, we have generated the ARIA knockout mice. Tumor xenograft growth was significantly enhanced in ARIA knockout mice than that in wildtype mice. The number of microvessels was significantly greater with appearance of less apoptotic endothelial cells in the tumor xenografts implanted into ARIA knockout mice than in wildtype mice. Taken together, ARIA is a novel factor regulating endothelial apoptosis as well as angiogenesis through modulating the proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells. Since ARIA is preferentially expressed in endothelial cells, ARIA is an attractive new target for pharmacotherapeutic agents to treat ischemic cardiovascular diseases.
Abstract 5591: KLF2 Restores Neovascularization Capacity of Aged Proangiogenic Cells
Authors: Boon Reinier A , Urbich Carmen , Fischer Ariane , Fontijn Ruud D , Seeger Florian H , Koyanagi Masamichi , Horrevoets Anton J , Dimmeler Stefanie
Published: April 2024

Coronary artery disease (CAD) patients have less circulating endothelial progenitor cells (EPCs), which exhibit impaired neovascularization properties. Inverse correlations were also found between EPC function and risk factors like age. Krüppel-like factor 2 (KLF2) is expressed by mature endothelial cells (ECs), is induced by both shear stress and statins and provokes endothelial functional differentiation. Here, we describe that early EPCs, also known as proangiogenic cells (PACs), express KLF2 at a comparable level to mature ECs and that senescence decreases KLF2 levels. To study the effects of aging on KLF2 levels, we compared progenitor cells of 4 weeks and 16 –18 months old C57BL/6 mice. In addition to the 2.5-fold reduction of circulating Sca1+/c-Kit+/Lin- progenitor cells and the 40% reduction of Sca1+/Flk1+ endothelial committed progenitor cells, the spleen-derived PACs isolated from aged mice showed a 60% lower level of KLF2 when compared to PACs isolated from young mice. Moreover, PACs isolated from peripheral blood of aged CAD patients were shown to contain 3-fold less KLF2 than PACs isolated from healthy young controls. Lentiviral overexpression of KLF2 increased human PAC numbers by 60% during in vitro culture. Endothelial lineage-specific KLF2 overexpression in aged bone marrow-derived mononuclear cells strongly augments neovascularization in vivo in a murine hind-limb ischemia model. These results imply that KLF2 is an attractive novel target to rejuvenate PACs before autologous administration to CAD patients.
Abstract 5593: Toll-like Receptor 2/6 Stimulation Promotes Angiogenesis via Induction of Endothelial GM-CSF Expression
Authors: Grote Karsten , Schuett Harald , Salguero Gustavo , Mühlradt Peter , Schieffer Bernhard
Published: April 2024

Background:
Toll-like receptors (TLRs) are known as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiological and pathological processes has been reported. Since many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the specific TLR2/6 agonist MALP-2, a common bacterial lipopeptide.

Methods and Results:
MALP-2 induced angiogenesis
in vivo
(Matrigel implants, hemoglobin content, H&E staining, P<0.01, n=4–8) and tube formation
in vitro
(Matrigel, P<0.05, n=3–5) which could be inhibited with neutralizing antibodies against TLR2/6. Moreover, endothelial cells responded to MALP-2 with enhanced proliferation and migration (BrdU-incorporation, transwell, P<0.01, n=5–7). Protein array revealed a strong secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) from endothelial cells which was confirmed by ELISA (P<0.01, n=4) and could be inhibited with antibodies against TLR2/6. By contrast, smooth muscle cells and fibroblasts did not secrete GM-CSF in response to MALP-2 (ELISA, n=3–6). In addition, GM-CSF release from isolated vascular segments following MALP-2 stimulation was completely prevented when the endothelium was removed mechanically (ELISA, P<0.01, n=4). Capillaries generated by MALP-2 treatment contained erythrocytes as well as CD45
+
cells (Matrigel implants, CD45 immunostaining, n=4). Following MALP-2 stimulation CD45
+
cells showed likewise enhanced migration (transwell, P<0.05, n=5) and GM-CSF release (ELISA, P<0.01, n=5) however to a lower extent compared to endothelial cells. Finally, inhibition of GM-CSF by siRNA
in vitro
(Matrigel, P<0.05, n=4) or by antibodies
in vivo
(Matrigel implants, P<0.01, n=4–8) inhibited the MALP-2 induced angiogenesis.

Conclusion:
These results demonstrate a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2 which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.
Abstract 5596: Differential Paracrine Dysfunction and Hypoxic Desensitization in Diabetic CD34+ and CD14+ Bone Marrow Derived Cells (BMDC)
Authors: Jarajapu Yagna P , Thampi Prajitha , Caballero Sergio , Boulton Michael E , Grant Maria B
Published: April 2024

Purpose: To test the hypothesis that BMDC of diabetic (D) origin have impaired responsiveness to the hypoxic microenvironment and ineffective paracrine signaling.

Methods: FACS sorted CD34+ and CD14+ cells from D and non diabetic (n=10) (ND) individuals (n=10) were exposed to normoxia (pO2=40 mmHg) or hypoxia (pO2=5 mmHg). Levels of HIF1-
α
and surface expression of VEGFR1, VEGFR2 and CXCR4 were evaluated. CD34+ and CD14+ cells were placed in culture (24 hr) and conditioned-medium (CM) was concentrated and analyzed for cytokines and growth factors. The effects of CM were assessed on CD34+ cell and CD14+ cell migration, and proliferation. Streptozotocin-induced diabetic mice of 11 months duration or age matched controls received intravitreal injection of fluorescently labeled ND or D CD34+ cells or CD14+ cells. Cells were localized 48 hrs post-injection and the degree of vascular homing to acellular capillaries was evaluated immunohistologically and quantitated.

Results: With hypoxia (4hr), expression of VEGFR2 (not VEGFR1), CXCR4 and HIF1-
α
were significantly increased in ND CD34+ cells but this response was markedly blunted in D CD34+ cells. In contrast, both ND and D CD14+ cells showed decreases in surface expression for all 3 receptors following hypoxia. TNF
α
, MCP1 and TGF
β
were 10-fold higher and SCF and HGF were 3-fold lower in CM from D CD34+ cells compared to ND CD34+ cells. CM from D CD34+ cells increased migration of ND CD14+ cells but not their proliferation (P<0.01). D CD14+ cells did not secrete SCF and Flt-3L and did not support proliferation. ND CD14+ secreted higher levels of IL6, IL1
β
, IL8 and TGF
β
1 compared to D CD34+. ND CD34+ and both ND and D CD14+cells, but not DM CD34+ cells migrated through the vitreous and homed to areas of injured vasculature in diabetic mice, but not control mice (P<0.05).

Conclusions: D CD34+ are unresponsive to hypoxia and cannot home to areas of retinal injury, whereas D CD14+ remain hypoxia responsive, can home as efficiently as ND CD14+ and ND CD34+ and can support migration but not proliferation. Our studies show complex paracrine interactions between BMDC and distinct desensitization of CD34+ cells to hypoxia which supports their observed in vivo dysfunction.
Abstract 5606: Outcomes of Patients With Acute Coronary Syndromes and Chronic Renal Insufficiency: Prognostic Importance of Troponin Elevation and Role of Adjunctive Antithrombotic Therapy (Analysis From the Acuity Trial)
Authors: Acharji Subasit , Nikolsky Eugenia , Mehran Roxana , Lansky Alexandra J , Dangas George D , Feit Frederick , Manoukian Steven , Kirtane Ajay J , Caixeta Adriano , Kesanakurthy Vijaya , Stone Gregg W
Published: April 2024

Objective:
To assess the prognostic value of elevated troponin in patients with acute coronary syndromes (ACS) and baseline chronic renal insufficiency (CRI) and impact of different antithrombotic regimens on outcomes of these pts.

Background
: Elevated troponin levels are useful in establishing diagnosis and prognosis in patients with ACS. However the role of troponin elevation in patients with CRI has been disputed.

Methods and Results:
In the ACUITY trial, among a total of 13,819 patients with ACS, baseline CRI defined as creatinine clearance <60mL/min was present in 2179 pts (15.7%) of whom 1291 pts (59.2%) had troponin elevation. Patients with vs. without elevated troponin had significantly higher rates of 30-day mortality (4.7% vs. 1.0%, p<0.0001), MI (8.3% vs. 5.0%, P=0.003) and composite ischemia (13.6% vs. 7.9%, p<0.0001). The same was true with respect to 1-year rates of mortality (10.7% vs. 6.8%, p=0.0005), MI (13.3% vs. 7.3%, P<0.0001) and composite ischemia (25.2% vs. 20.6%, p=0.002). By multivariable analysis, troponin elevation in patients with baseline CRI was an independent predictor of mortality at 30 days (OR [95%CI] = 4.75 [2.34, 9.65], P<0.0001) and at 1 year (odds ratio [95%CI] = 1.58 [1.12, 2.23], P=0.009) as well as of MI at 1 year (OR [95%CI] = 1.54 [1.05, 2.25], P=0.02). Outcomes in patients with elevated troponin stratified by antithrombotic regimen are presented in the Table

.

Conclusions:
In patients with ACS and CRI, troponin elevation is a marker of worse 30-day and 1-year outcomes. In high-risk pts with CRI and troponin elevation, bivalirudin monotherapy compared to heparin plus GPI resulted in significantly reduced rates of major bleeding without significant increase in 30-day and 1-year rates of composite ischemia.
Abstract 5615: Dual Role of the Small G Protein Rac-1 in the Regulation of Hypoxia-Induced STAT3 Activation in Endothelial Cells
Authors: Labazi Mohamed , Lamoke Folami , Marcus Dennis M , Caldwell Ruth B , Bartoli Manuela
Published: April 2024

The Signal Transducer and Activator of Transcription 3 (STAT3) is a critical regulator of Vascular Endothelial Growth factor (VEGF) expression and activity in cardiac muscle and vascular endothelial cells. We have previously shown that hypoxia-induced activation of STAT3 is a redox-dependent event and can be blocked by inhibitors of NAD(P)H oxidase activity. Here we investigated the specific role of the small G protein Rac-1 in the regulation of hypoxia-induced STAT3 activation in endothelial cells. Human umbilical vein endothelial cells (HUVEC) and human coronary endothelial cells in culture were subjected to 2% oxygen levels for different times (10min, 30min). Rac-1 inhibition was achieved by transfection of ECs with specific siRNA or by treatments of the cells with 75
μ
M of the Rac-1 inhibitor
NSC23766
(EMD Biosciences). Chromatin immunoprecipitation (ChIP) was conducted to assess STAT3 transcriptional activity. STAT3 phosphorylation and protein-protein association was measured by Western blotting and immunoprecipitation analysis (respectively). We found that inhibition of Rac-1 activity by siRNA or by treatment with
NSC23766
blocks STAT3 binding to VEGF promoter in hypoxic conditions compared to treatment of the cells with scramble siRNA or vehicle only. This effect correlated with blockade of hypoxia-induced STAT3 tyrosine phosphorylation at tyrosine 705 and at serine 727. In addition, hypoxia-stimulated Rac-1 binding to STAT3 at 30 minutes and inhibition of Rac-1 activity stimulated STAT3 binding to Protein Inhibitor of STAT3 (PIAS3) responsible for its sumoylation/inactivation. Taken together our results suggest that Rac-1 activity is associated with hypoxia-stimulated STAT3 activation and STAT3-dependent VEGF by regulating its phosphorylation/activation as well its sumoylation via blockade of STAT3 binding to PIAS3. These data disclose a previously uncovered activity of Rac-1 in the regulation of STAT3 inactivation by sumoylation.
Abstract 5626: Incidence of Thromboembolic Stroke and of Major Bleeding in Patients With Chronic Kidney Disease Treated With and Without Warfarin
Authors: Aronow Wilbert S , Lai Hoang M , Kalen Phoenix , Adapa Sreedhar , Patel Kaushal , Goel Arvind , Vinnakota Ravi , Chugh Savneek , Garrick Renee
Published: April 2024

Background:
The incidence of thromboembolic (TE) stroke and of major bleeding in patients with chronic kidney disease (CKD) and atrial fibrillation (AF) have not been investigated in controlled clinical trials.

Purpose:
To investigate the incidence of TE stroke and of major bleeding in patients with CKD and AF treated with and without warfarin.

Methods:
We investigated the incidence of TE stroke and of major bleeding in 399 unselected patients with chronic CKD and AF treated with warfarin to maintain an International Normalized Ratio between 2.0 and 3.0 (n = 232) and without warfarin (n = 167). Of the 399 patients, 93 (23%) were receiving hemodialysis, and 132 (33%) had an estimated glomerular filtration rate of <15 mL/min/1.73 m
2
.

Results:
New TE stroke occurred in 21 of 232 patients (9%) treated with warfarin at 31-month follow-up and in 43 of 167 patients (26%) treated without warfarin at 23-month follow-up (p<0.001). Of the 21 TE strokes on warfarin, 9 (43%) occurred when the International Normalized Ratio was <2.0. Stepwise Cox regression analysis using 19 variables showed that significant independent variables for predicting TE stroke were prior stroke or transient ischemic attack (odds ratio = 2.9; 95% CI, 1.2–6.7) and use of warfarin (odds ratio = 0.28; 95% CI, 0.16–0.50). In hemodialysis patients, new TE stroke occurred in 5 of 51 patients (10%) treated with warfarin and in 16 of 42 patients (38%) not treated with warfarin (p<0.005). In patients with an estimated glomerular filtration rate <15 mL/min/1.73 m
2
, new TE stroke occurred in 8 of 78 patients (10%) treated with warfarin and in 20 of 54 patients (37%) not treated with warfarin (p<0.001). Major bleeding occurred in 32 of 232 patients (14%) treated with warfarin and in 15 of 167 patients (9%) not treated with warfarin (p not significant).

Conclusions:
CKD patients with AF treated with warfarin to maintain an International Normalized Ratio between 2.0 and 3.0 had a significant reduction in TE stroke and an insignificant increase in major bleeding.
Abstract 5659: Heterozygous Mutation in the Carboxyl-Terminal Domain of the Type 2 Bone Morphogenetic Protein Receptor Leads to Ligand-Specific Gain of Function
Authors: Leyton Patricio , Beppu Hideyuki , Yu Paul B , Bloch Kenneth D
Published: April 2024

Heterozygous mutations in the gene encoding the bone morphogenetic protein (BMP) type II receptor (BMPR2) are associated with familial and idiopathic pulmonary arterial hypertension. These mutations are found to affect all domains of the protein, including the amino-terminal ligand-binding domain, the intracellular kinase domain, and the long cytoplasmic carboxyl-terminal (CT) domain. Binding of BMP ligands to BMPR2 and BMP type I receptors leads to phosphorylation of SMAD 1, 5, and 8 and expression of genes including Id1. In pulmonary artery smooth muscle cells (PaSMC) isolated from mice with a heterozygous nonsense mutation in the BMPR2 amino-terminal domain (BMPR2
+/−
), BMP4 and BMP7 signaling is reduced, whereas deletion of both BMPR2 alleles paradoxically augments BMP7 signaling. However, the impact of heterozygous mutations in the BMPR2 CT domain on BMP signaling is still unknown. To characterize the function of the BMPR2 CT domain, we generated a mouse (BMPR2
SG/+
) in which BMPR2 exon 12 (encoding the CT domain) was deleted, and a sequence specifying green fluorescent protein (GFP) was inserted in frame after exon 11, resulting in a BMPR2
s
hort form-
G
FP fusion protein (SG). Using qRT-PCR and primers for exons 4 and 5, similar levels of BMPR2 transcripts were detected in PaSMC isolated from wild-type (WT) and BMPR2
SG/+
mice. In contrast, using primers for exon 12, there was a 50% reduction of BMPR2 transcripts in BMPR2
SG/+
PaSMC. Similarly, immunoblots performed with a CT-specific antibody revealed that WT BMPR2 protein levels were 50% less in BMPR2
SG/+
than in WT PaSMC. SG protein expression was confirmed using an anti-GFP antibody. Induction of SMAD1/5/8 phosphorylation and Id1 gene expression in response to BMP4 (10 ng/ml) did not differ between WT and BMPR2
SG/+
PaSMC. However, the induction of SMAD1/5/8 phosphorylation and Id1 gene expression by BMP7 were both more than 2-fold greater in BMPR2
SG/+
than WT PaSMC. On the other hand, BMPR2
+/−
PaSMC had reduced SMAD1/5/8 activation and Id1 transcription in response to both BMP4 and BMP7. In conclusion, our findings suggest that heterozygous mutations in the BMPR2 CT domain may lead to gain of signaling for a subset of BMP ligands, including BMP7.
Abstract 5674: Toll-like Receptor-2 and -4 Signaling Regulates Inflammation and Matrix Degradation in the Human Atherosclerotic Plaque
Authors: Gregan Scott M , Navin Tina J , Georgiou Ektoras , Foxwell Brian M , Davies Alun H , Feldmann Marc , Monaco Claudia
Published: April 2024

Background
The current consensus view of the mechanisms leading to plaque rupture has assigned a central role to inflammation and matrix degradation. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions but their functional role in matrix degradation in human atherosclerosis is unknown.

Methods
We explored the effects of blocking TLR-2, -4 and the downstream signaling adaptors myeloid differentiation primary response gene 88 (MyD88, shared by most TLRs and Trif-related adaptor molecule (TRAM; used by TLR-4) in an
in vitro
model of human atherosclerosis. Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation in order to obtain a single cell suspension of mixed cell types. Cells were cultured in the presence or absence of TLR signaling blockers immediately after isolation. Phenotyping by flow cytometry showed that macrophages are the most abundant cells in the mixture, being approximately double the proportions of smooth muscle cells and lymphocytes.

Results
A dominant-negative form of MyD88 (MyD88DN) decreased the production of MCP-1/CCL2 (P=.015), IL-8/CXCL8 (P =.002), IL-6 (P =.001), MMP-1) (P =.016), and MMP-3 (P =.0001), as well as nuclear factor kappaB (NFkB) activation (P= .0001) in atheroma cell cultures. TLR-2 neutralizing antibodies inhibited NFkB activation (P = .025), and significantly reduced MCP-1/CCL2 (P=.0001), IL-8/CXCL8 (P=.007), IL-6 (P=.0001), and MMP-1, -2, -3, -9 production (P=.0001). In contrast, a dominant negative form of the adaptor TRAM (TRAM
DN
), which is used by TLR-4, significantly reduced NF
κ
B activity, MMP-3 (
P
=.049) and MMP-1, but had no effect on gelatinases MMP-2 and -9 or inflammatory mediators.

Conclusions
Our data indicates that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. However, we also uncovered a role for TLR-4/TRAM-dependent signaling in MMP production, suggesting that the MyD88-independent pathway may play a role in mechanisms related to matrix degradation and plaque vulnerability. TLR blockade may represent a therapeutic strategy for atherosclerosis and its complications.
Abstract 5683: Insights From Network Analysis: Deficiency of Glutathione Peroxidase-1 Increases In-stent Stenosis
Authors: Ali Ziad , Raiesdana Azad , Qu Xiumei , Ali Ayyaz , Pavlovic Aleksandra , Wheeler Matthew , Channon Keith , Quertermous Thomas , Ashley Euan
Published: April 2024

In-stent stenosis (ISS) is the principal cause of PCI failure. We used a novel genome-wide gene expression network approach in human atherectomy samples (n=89) to identify signaling networks critical to the process of ISS. We found that a subnetwork with glutathione peroxidase-1 (GPx1) as its hub was among the most significantly differentially regulated between de novo atherosclerosis and ISS (Fig 1

, bar thickness denotes association strength). We investigated the importance of GPx1 in ISS using a novel model of balloon angioplasty and stenting (BAS) and a knockout mouse with constitutive deficiency of GPx1. Thoracic aortic segments underwent BAS before being grafted onto the carotid artery of isogenic recipients. Groups included donor GPx1-KO mice and their littermate controls (n=5/group). Despite equal stent expansion and injury scores, ISS was increased by 45% (P<0.001) in GPx1-KO mice 28 days following stenting, confirming the observational data from the human studies (Fig 2

, white arrows denote neointima). To investigate a mechanism for these findings, we quantified superoxide production (O2-). O2- production was significantly increased in GPx1-KO mice measured on both aortic sections using oxidative confocal microtopography (~2.8-fold, P<0.001) and whole aorta by lucigenin enhanced illuminescence (~2.2-fold, P<0.001). Further, studies aimed at establishing a therapeutic role for the isoflavone genistein in modulating GPx1 activity demonstrated increased enzymatic activity in coronary endothelial cells (~78%, P=0.01). Taken together, these findings establish an important role for GPx1 network signaling in vascular wall redox processes critical to ISS.

Fig. 1

Fig. 2
Abstract 5684: A Sex-Specific Role for Androgens in Angiogenesis
Authors: Sieveking Daniel P , Lim Patrick , Chow Renée W , Dunn Louise L , Bao Shisan , McGrath Kristine C , Heather Alison K , Handelsman David J , Celermajer David S , Ng Martin K
Published: April 2024

The role of androgens in cardiovascular disease remains controversial. Although men tend to develop CVD earlier than women, consistent with increased atherosclerosis, repair/adaptation mechanisms may be enhanced in males. For example, male gender is associated with increased collateralization in patients with severe coronary artery lesions. As angiogenesis, is critical to cardiovascular repair/regeneration, we hypothesized that androgens may modulate this process. We studied the effects of androgens on angiogenesis using human endothelial cells (ECs) and two distinct
in vivo
models. Dihydrotestosterone (DHT), a potent natural androgen specific for the androgen receptor (AR) augmented key angiogenic events
in vitro
, including EC migration, proliferation and tubulogenesis. Strikingly, this occurred in male but not female ECs. AR antagonism and siRNA-mediated knockdown abrogated all DHT effects in male ECs, while AR overexpression conferred androgen sensitivity in female ECs.
In vivo
, castration dramatically reduced vascularization of matrigel plugs in male and female mice. This reduction was fully reversed with DHT treatment in male but not female mice (Fig. 1

). Additionally, male castration impaired neovascularization and blood flow recovery in hindlimb ischemia, a finding that was rescued with DHT treatment. These findings were also mirrored in hypoxia inducible factor-1
α
and stromal cell-derived factor-1
α
expression, and the mobilzation of various angiogenic/progenitor cells. Our findings suggest endogenous androgens play an important role in angiogenesis in males but not females, with implications for the role of androgen replacement in men.

Fig. 1
Abstract 5751: High Glucose Induces Apoptosis in Endolthelial Cells via Epigenetic Regulation of Insulin-Like Growth Factor Receptor Expression
Authors: Li Yangxin , Song Yao-Hua , Diez Jose G , Elaydar MacArthur A , Wu Hanjing , Geng Yong-Jian
Published: April 2024

High glucose-induced endothelial cell dysfunction is considered to be the main cause of the development of vascular complications in patients with diabetes. In this study, we examined the effect of high glucose (HG) on apoptosis of human umbilical vein endothelial cells (HUVEC) and investigated the molecular mechanisms involved. HUVEC was exposed to HG and apoptosis was assessed by Death ELISA. Our data showed that HG induced significant apoptosis in HUVEC cells after exposing to HG for 5 days (O.D. value = 0.36±0.07 in HG group vs. 0.13±0.05 in control, p<0.05). HG induced apoptosis in HUVEC is associated with decreased of insulin-like growth factor receptor (IGF-1R) mRNA (4.1±0.6 fold at 72 h, p<0.05) as assessed by real-time PCR and IGF-1R protein (3. 1±0.5 fold at 96 h, p<0.05) as assessed by western blot, which were dose- and time-dependent effects (25 mM of glucose rather than 5 mM at 72 h and 96 h). The effects of HG on reduced expression of IGF-1R and apoptosis were blocked by silencing histone deacetylase 1 (HDAC1) with small interference RNA (siRNA-HDAC1) compared to non-targeting scrambled siRNA. Moreover, HG negatively regulated IGF-1R promoter activity as determined by ChIP (chromatin immunoprecipitation) analysis, which was dependent on HDAC1 since siRNA-HDAC1 attenuated the effects of HG on IGF-1R promoter activity. HG also increased the association of IGF-1R promoter with HDAC1, and decreased the association of IGF-1R promoter with acetylated histone-4. Furthermore, trichostatin A, a specific HDAC inhibitor, relieved the repression of IGF-1R and prevented apoptosis of HUVEC following HG state. These results suggest that HG-induced repression of IGF-1R is mediated by the association of HDAC1 with the IGF-1R promoter, and by the subsequent reduced recruitment of chromatin-modifying proteins, such as histone 4 to the IGF-1R promoter. In conclusion, our data demonstrate that high glucose exposure decreases expression of IGF-1R and decreases the association of acetylated histone-4 with the IGF-1R promoter, and suggest that enhancing acetylation of histone 4 and IGF-1R expression may be a useful strategy to prevent diabetic-induced apoptosis of endothelial cells.

This research has received full or partial funding support from the American Heart Association, South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 5752: Role of MicroRNA-503 in Diabetic Endotheliopathy
Authors: Caporali Andrea , Völlenkle Christine , Masson Rachel , Baker Andrew , Agami Reuven , Carmona-Saez Pedro , Martelli Fabio , Emanueli Costanza
Published: April 2024

Diabetes disturbs endothelial function and reparative neovascularisation, thus contributing to limb ischemia. Recently, we showed that the atypical neurotrophin receptor p75
NTR
mediates diabetes-determined impairment of reparative neovascularization. Diabetes induces p75
NTR
expression in microvascular endothelial cells (ECs) of ischemic limb muscles, thus impairing EC survival and functions. Conversely, p75
NTR
inhibition restores proper post-ischemic neovascularization in diabetic mice. Making a step forward into the understanding of the relevance of p75
NTR
in diabetic microangiopathy, here we studied whether p75
NTR
modulates the expression of microRNAs (miRNAs) in ECs. MiRNAs are small non-coding RNAs that act as endogenous regulators of gene expression, by controlling degradation and/or translational repression of target mRNAs. Using microarray analyses of miRNAs expression in human umbilical vein ECs (HUVECs) transduced with
p75

NTR

or control (empty vector), we detected that miRNA-503 (miR-503) was consistently induced in
p75

NTR

-transduced ECs. Moreover, both p75
NTR
and miR-503 expression in HUVECs was increased following culture in high glucose to mimic diabetic hyperglycaemia. Furthermore, miR-503 expression was higher in ischemic muscles of diabetic mice in comparisons to non diabetic mice. Lentivirus-mediated overexpression of miR-503 in HUVECs impaired cell proliferation and decreased
in vitro
capillary-like network formation on matrigel. In order to search for miR-503 target genes, we used the CORNA R package and TargetScan to identify the link between
in silico
predicted target genes of miR-503 and genes downregulated in the Illumina™ microarray of
p75

NTR

-transduced HUVECs and of
p75

NTR

-transduced limb muscles. Functional analysis revealed that several target genes were involved in cell cycle control and angiogenesis. Among this group of genes, we identified CyclinE1, cdc25A and VEGF-A as direct targets of miR-503 in ECs. These results suggest a role of miRNA-503 in diabetes-induced microangiopathy and impaired angiogenesis.
Abstract 5754: Rescue of Diabetes-Related Impairment of Angiogenesis by Gene Silencing of Thioredoxin-Interacting Protein
Authors: Dunn Louise L , Buckle Andrew , Simpson Philippa , Sieveking Daniel , Chow Renee , Lim Patrick , Ng Martin
Published: April 2024

Objectives: Vascular complications of diabetes mellitus (DM) are associated with impaired angiogenesis, the mechanisms of which are poorly understood. The thioredoxin (TRX) system has been implicated in angiogenesis. Interestingly, thioredoxin-interacting protein (TXNIP), antagonist to TRX, is induced by glucose. We hypothesized that hyperglycemic-modulation of TXNIP/TRX plays a role in DM-impairment of angiogenesis.

Methods:

Endothelial cells (ECs) were cultured in 5–25 mM glucose and siRNA used to modulate the TXNIP/TRX system. EC migration, proliferation, tubulogenesis, TRX activity, VEGF, qPCR and protein were examined.

Unilateral hindlimb ischemia was induced in a murine model of Type 1 DM. Txnip siRNA was delivered i.m. and limb perfusion assessed by laser Doppler, with ischemia, foot movement and capillary density also measured.

Results: Hyperglycemia induced a dose-dependent inhibition of key angiogenic events including EC migration, proliferation and tubulogenesis (62.1Â±2.8%, 72.9Â±7.0% and 48.3Â±4.7% at 25 mM vs. control; P < 0.05), with increased TXNIP expression (5.1Â±1.8-fold mRNA; 2.0Â±0.1-fold protein), enhanced TXNIP/TRX interaction and inhibition of TRX activity (59.8Â±4.8%; P<0.001). TXNIP siRNA reversed the inhibitory effects of hyperglycemia on migration, proliferation, tubulogenesis and TRX activity to levels of control. DM was associated with a profound impairment of blood flow recovery following hindlimb ischemia (0.284Â±0.038 vs. 0.582Â±0.021; P<0.001). However, i.m. delivery of Txnip siRNA dramatically increased limb perfusion in DM mice to levels not statistically different from control (0.443Â±0.047). Txnip siRNA also increased capillary density (2.0Â±0.4-fold), while ischemia and foot movement scores significantly improved in DM mice receiving Txnip siRNA (vs. DM control; P<0.05). In both models, hyperglycemia/DM reduced VEGF expression and interfered with VEGF action, while TXNIP siRNA rescued VEGF levels to that of control.

Conclusions: TXNIP plays a key role in angiogenesis and impaired neovascularization in DM, in part via modulation of VEGF. Our findings demonstrate that inhibition of TXNIP can abrogate the deleterious effects of hyperglycemia in angiogenesis.
Abstract 16642: Clinical Outcomes and Improved Survival in Patients With Protein Losing Enteropathy After the Fontan Operation
Authors: John Anitha S , Johnson Jennifer A , Driscoll David J , Warnes Carole A , Cetta Frank
Published: April 2024

Background:
Patients (pts) with protein losing enteropathy (PLE) following the Fontan operation (op) have a reported 50% mortality at 5 years (yrs) post diagnosis. We review our clinical outcomes in this pt population.

Methods:
From 1997 to 2010, 42 pts (55% M) with PLE following the Fontan op were identified from clinical databases. Data were collected by retrospective chart review.

Results:
Mean age at PLE diagnosis was 18.9 yrs + 11 (4.7-57.9 yrs) and initial Fontan op was 10.1 yrs + 10.8 (1.8-37.6 yrs). Mean time from Fontan op to PLE diagnosis was 8.4 yrs + 14.2 (30 days-27yrs). Types of Fontan op included: atriopulmonary connection (28, 67%), lateral tunnel or intraatrial conduit (6, 14%), or extracardiac conduit (8, 19%). Mean time from diagnosis to last follow-up was 8 yrs + 6 (1-22yrs). Survival was 88% at 5 yrs. There were 11 deaths, mean age 32 yrs + 7.7 (22-47 yrs). Mean time interval from diagnosis to death was 7.5 yrs + 5.4 (1-18yrs). Causes of death include: sepsis (7), complications after Fontan conversion (1), and unknown (3). Decreased survival was seen in pts with high Fontan pressure (mean >15mmHg, p=0.04), decreased ventricular function (EF < 55%, p=0.03), and NYHA class >2 at diagnosis (p=0.04). Pts who died had higher pulmonary vascular resistance (3.8 + 1.6 WU vs 2.1 + 1.1 WU, p=0.017), lower cardiac index (1.6 L/min/m
2
+ 0.4 vs 2.7 L/min/m
2
+ 0.7, p<0.0001), and lower mixed venous saturation (53% versus 66%, p=0.01) compared to survivors. All factors were obtained at initial PLE diagnosis. Pts treated with medical therapy only (n=15) resulted in symptomatic improvement in 7 pts (47%), no improvement in 5 (33%), and death in 3 (20%). Pts treated with medical + surgical/interventional catheterization procedures (n=27) resulted in symptomatic improvement in 10 pts (37%), no improvement in 9 (33%), and death in 8 (30%). Treatments used more in survivors included spironolactone (21, 68%), octreotide (7, 21%), sildenafil (6, 19%), fenestration creation (15, 48%), and relief of Fontan obstruction (7, 23%). Of the 31 survivors, 17 pts remain well-controlled.

Conclusions:
PLE remains difficult to treat, but in the current era, survival has improved with advances in treatment. Further study is needed to understand the mechanism of disease and ideal treatment strategy.