bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5533: Gender Differences in Outcome During Antihypertensive Therapy in Relation to Regression of Electrocardiographic Left Ventricular Hypertrophy: The LIFE Study
Authors: Okin Peter M , Gerdts Eva , Hille Darcy A , Kjeldsen Sverre E , Edelman Jonathan M , Dahlöf Björn , Devereux Richard B
Published: April 2024

Background
: Men and women have well-established differences in the magnitude of ECG amplitudes and durations and women have less regression of ECG left ventricular hypertrophy (LVH) in response to blood pressure lowering, even after adjusting for gender differences in baseline severity of LVH, treatment effects and blood pressure changes. However, whether women experience less prognostic benefit from their lesser regression of ECG LVH has not been examined.

Methods and Results
: Cardiovascular (CV) outcomes were examined in relation to regression of Sokolow-Lyon voltage (SLV) in 4963 women and 4230 men randomly assigned to losartan- or atenolol-based treatment. ECGs were performed at baseline, 6 months and then yearly until study end. Regression of LVH was dichotomized using sex-specific median decreases in SLV ≥3.0 mm in women and ≥4.5 mm in men. Women had significantly lower 5-year event rates for CV death (3.8 vs 6.2%, p<0.0001), MI (3.3 vs 6.0%, p<0.0001), stroke (5.5 vs 7.3%, p=0.003) and the LIFE study composite endpoint of these three events (9.8 vs 15.6%, p<0.0001). In Cox regression analyses performed separately in women and men, women and men had similar reductions in risk of all events associated with regression of SLV defined by decreases in SLV exceeding the sex-specific median entered as time-varying covariates (Table

). The absence of gender differences in predictive value of regression of SLV LVH was confirmed by the absence of any significant interaction term between gender and change in SLV, with analyses performed treating SLV as either a sex-specific median decrease or as a continuous variable with no adjustment for gender.

Conclusions
: Despite lesser regression of ECG LVH than men, women have lower event rates than men and appear to derive the same degree of risk reduction as men from greater regression of SLV, independent of treatment effects, in-treatment blood pressure and the impact of possible gender differences in other risk factors on outcomes.

Outcomes in Relation to Sex-Specific Median Regression of Sokolow-Lyon Voltage LVH
Abstract 5543: Carbon Nanotubes Allow Fluorescence Imaging of Macrophages in Mouse Carotid Atherosclerosis
Authors: Kosuge Hisanori , Terashima Masahiro , Sherlock Sarah , Kitagawa Toshiro , Dai Hongjie , McConnell Michael V
Published: April 2024

Introduction: Macrophages are important imaging targets for characterizing and monitoring atherosclerotic lesions. Carbon nanotubes show promising properties as vehicles for cellular imaging and therapy.

Methods:
In vitro
: mouse macrophage cells (RAW) were incubated for 24 hours with single-walled carbon nanotubes (NT; diameter 1.2nm, concentration 2.5mg/L) labeled with a near infrared fluorophore Cy5.5. NT uptake was examined using confocal microscopy.
In vivo
: FVB mice (N=6) had a macrophage-rich carotid atherosclerotic lesion created through high fat diet for 4 weeks and diabetes induction by 5 daily injections of streptozotocin, followed 2 weeks later by ligation of the left carotid artery (N=4) or sham operation (N=2). Two weeks post operation, all mice were given NT-Cy5.5 (8nmol of Cy5.5) via tail vein and scanned serially over 48 hours by
in-vivo
fluorescent imaging (CRI, Woburn, MA). Then both
in situ
and
ex vivo
imaging were performed followed by histological evaluation.

Results: NT were taken up by 92% of macrophages
in vitro. In vivo
imaging showed limited carotid signal, however, both
in situ
and
ex vivo
imaging showed high signal from the ligated left carotid arteries (upper figure

, graph). No accumulation of NT was seen in non-ligated right carotid arteries or sham-operated left carotid arteries (left carotid - yellow arrows, right carotid - red arrows). Immunofluorescence microscopy of the ligated carotid lesions (lower figure

) showed colocalization of NT-Cy5.5 and macrophages labeled with Mac3/FITC.

Conclusions: NT accumulate in atherosclerotic macrophages
in vivo
and provide a multi-functional platform for plaque imaging and potentially therapy.

This research has received full or partial funding support from the American Heart Association, Western States Affiliate (California, Nevada & Utah).
Abstract 5548: Blockade of KCa3.1 Calcium-activated Potassium Channels Prevents Endothelial Inflammation
Authors: Ohta Masanori , Nandedkar Sandhya , Wulff Heike , Jo Hanjoong , Miura Hiroto
Published: April 2024

Introduction

We previously reported that blockade of the intermediate-conductance calcium-activated potassium channel (KCa3.1) prevents the development of atherosclerosis in apoE
−/−
mice. Endothelial cell (EC) surface CD73, ecto-5′-nucleotidase, extracellularly produces adenosine and prevents EC inflammation. Therefore we hypothesized that KCa3.1 blockade suppresses EC inflammation by upregulating CD73.

Methods

Human coronary artery ECs were preconditioned by 24-hour exposure to laminar shear stress (LS; 15 dyn/cm
2
), and were treated with tumor necrosis factor-
α
(TNF-
α
, 1 ng/ml) for an additional 4 hours under LS in the presence or absence of the KCa3.1 blocker TRAM-34 (100 nM) or the KCa2.3 blocker apamin (100 nM). Expression of KCa3.1, KCa2.3, CD73 and vascular cell adhesion molecule-1 (VCAM-1) transcripts was determined by real-time PCR.

Results

Preconditioning ECs with LS resulted in increased KCa3.1 and KCa2.3 expression (6.6±1.8 and 3.3±0.4 fold increase vs. static condition, p<0.05, n=4 –10, respectively). The LS preconditioning also increased CD73 expression (2.4±0.7 fold p<0.05, n=4 –10), whereas it decreased VCAM-1 expression (0.6±0.2 fold, p<0.05, n=7). TNF-
α
increased VCAM-1 expression (23.5±3.2 fold, p<0.001) with no change in CD73 expression (1.0±0.3 fold, p=ns) under static conditions. LS decreased TNF-
α
-induced increase in VCAM-1 expression and increased CD73 expression (17.0±1.1 and 1.9±0.5 fold, n=4, respectively). Addition of TRAM-34 enhanced the LS-induced changes in VCAM-1 and CD73 expressions with markedly lower VCAM-1 (9.0±3.0 fold, p<0.05 vs static + TNF-
α
) and much higher CD73 expression (3.1±1.0 fold). In contrast, addition of apamin markedly exaggerated the VCAM-1 expression, while it increased CD73 expression as much as TRAM-34 did.

Conclusions

KCa3.1 blockade suppresses TNF-
α
-induced EC inflammation by increasing CD73 expression and decreasing VCAM-1 expression. EC KCa3.1 might therefore be one of the targets of anti-atherosclerosis therapy with KCa3.1 blockers.
Abstract 5551: Apelin is Necessary for the Maintenance of Insulin Sensitivity
Authors: Yue Patrick , Jin Hong , Aillaud-Manzanera Marissa , Deng Alicia C , Azuma Junya , Kundu Ramendra K , Reaven Gerald M , Quertermous Thomas , Tsao Philip S
Published: April 2024

BACKGROUND:
The recently discovered cardioactive peptide apelin has been shown to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role and molecular mechanism of action.

METHODS:
Insulin (0.75 U/kg) tolerance testing (ITT) was performed on mice with generalized deficiency of the apelin gene (APKO). Additionally, fasting glucose, insulin, free fatty acid (FFA), and adiponectin levels were determined. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 weeks. Following the infusion, ITTs were repeated and the animals sacrificed. In selected animals, insulin (0.75 U/kg) was delivered 30 minutes prior to sacrifice; soleus muscles were then harvested, homogenized in lysis buffer, and insulin-induced Akt phosphorylation determined by Western blotting. Apelin-13 infusion, ITTs, and serum insulin, glucose, FFA, and adiponectin measurements were also performed in obese diabetic
db/db
mice. To probe the underlying mechanism for apelin’s effects, apelin-13 (1
μ
M) was also delivered to cultured C2C12 myotubes. 2-[
3
H]-deoxyglucose uptake and Akt phosphorylation were assessed.

RESULTS:
APKO mice had significantly decreased insulin sensitivity by ITT, increased insulin (0.70±0.033 vs. 0.55±0.098 pg/mL; p < 0.05) and FFA (231±11 vs. 172±14
μ
mol/L; p < 0.01) levels, and decreased adiponectin levels (64±3.3 vs. 80±8.0
μ
g/mL; p < 0.05). Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to both APKO and
db/db
mice resulted in improvements in insulin sensitivity. No changes in body weight were observed in either animal model with or without apelin. In C2C12 myotubes, apelin increased glucose uptake (10.6±0.70 vs. 6.01±0.18 pmol/mg protein/min; p < 0.01) and Akt phosphorylation. These events were fully abrogated by inhibitors of G
i
(pertussis toxin) and AMPK (Compound C), but only partially by phosphoinositide 3-kinase inhibition (LY294002).

CONCLUSIONS:
Apelin is necessary for the maintenance of insulin sensitivity
in vivo
. Apelin’s effects on glucose uptake and Akt phosphorylation may be mediated by a G
i
and AMPK-dependent pathway.
Abstract 5560: Association of 47 Candidate Proteomic Markers of Vascular Disease With the Ankle Brachial Index: The Mayo Clinic Proteomic Markers of Arteriosclerosis Study
Authors: Kim Charles X , Klee George G , Mosley Thomas H , Kullo Iftikhar J
Published: April 2024

Objectives:
We investigated the association of 47 candidate proteomic markers in pathways of inflammation, thrombosis, lipoprotein metabolism, vascular remodeling, and vasoregulation, with the ankle-brachial index (ABI) and peripheral arterial disease (PAD) in a bi-ethnic cohort of African Americans (AA) and non-Hispanic Whites (NHW).

Methods:
Multiple assay platforms were used to measure 47 candidate proteomic markers in 1324 AA and 1237 NHW participants belonging to hypertensive sibships. ABI was measured using a standard protocol, with values >1.4 excluded and PAD defined as ABI <0.9. Multivariable regression analyses (with generalized estimating equations) were employed to identify markers independently associated with ABI and PAD after adjusting for age, BMI, smoking, hypertension, diabetes, history of heart attack and stroke, medication use, total and HDL cholesterol, and estimated GFR as well as physical activity, alcohol consumption and education level.

Results:
Higher circulating levels of CRP, myeloperoxidase, NT-proBNP, osteoprotegerin, D-Dimer and fibrinogen were each associated with lower ABI in both AA and NHW. Additional markers significantly associated with lower ABI were; in AA - serum amyloid A, ICAM, IL-6, TNFR-I, TNFR-II, MCP-1, Lp(a), pro-atrial natriuretic peptide, pro-arginine vasopressin, pro-adrenomedullin, pro-endothelin, osteonectin, Factor VIII; and in NHW- Hsp27, ox-LDL and resistin. Markers associated with presence of PAD were: in AA - CRP, NT-proBNP, Lp(a), pro-arginine vasopressin and osteoprotegerin; in NHW -CRP, NT-proBNP, Hsp27, myeloperoxidase, oxidized-LDL, Factor VIII, and D-Dimer. When the significant markers were included in a final model, the R
2
for ABI increased from 15.9 to 19.4 for AA and from 18.1 to 22.1 for NHW; the c-statistic for PAD increased from 0.791 to 0.833 inAA and from 0.841 to 0.866 in NHW.

Conclusions:
Novel protein markers significantly add to prediction of ABI and PAD in both AA and NHW and may have clinical utility in diagnosis and prognosis of PAD as well as in identifying new therapies for atherosclerotic vascular disease.
Abstract 5562: Renal Resistive Index is Strongly Associated With Poor Outcome After Coronary Bypass Surgery: A Prospective Study
Authors: Aboyans Victor , Tanguy Bénédicte , Bonnet Vincent , Lacroix Philippe , Laskar Marc
Published: April 2024

Backgound:
The presence of peripheral artery disease and renal failure are known as poor prognostic markers in patients undergoing coronary revascularization. While the presence of renal artery stenosis (RAS) is frequent in patients with coronary artery disease, the prognostic value of this lesion has never been assessed in the setting of coronary revascularization.

Hypothesis:
We hypothesized that renal vascular disease is associated with poorer postoperative outcome after coronary bypass surgery (CABG), independent of renal function.

Methods:
We prospectively assessed by Duplex the renal arteries of 401 consecutive patients prior to CABG. We defined RAS by the presence of at least 2 out of the 3 following criteria: maximal velocity >1.8 m/s, reno-aortic velocity ratio >3.5, reno-renal velocity ratio >2.7. Additionally we defined high renal arterial resistance (HRAR) in case of resistive index >0.80. The 30-days post-operative outcome was composite, combining death, stroke or myocardial infarction. Logistic regression models were used to assess associations.

Results:
Among the 68 women and 333 men enrolled (age 67.6±10.7 years), RAS and HRAR were found in 33 (8.2%) and 45 (11.2%) cases, respectively. Adjusted for age, sex, glomerular filtration rate, CVD risk factors, treatments, the presence of peripheral artery disease, and the presence of chronic pulmonary disease, we did not find any significant association between RAS and the composite outcome (OR=0.90, 95%CI:0.28 –2.95, p=ns). Conversely, in the same model, HRAR was strongly associated with the primary composite outcome (OR=3.97, 95%CI:1.36 –11.02, p=0.01).

Conclusion:
Beyond the glomerular filtration rate and other usual prognostic markers, renal artery ultrasound provides additional prognostic information. While the presence of renal artery stenosis is not found of any post-operative prognostic value, a high renal arterial resistance is strongly and independently associated with short-term cardiovascular outcomes.
Abstract 5563: Pioglitazone Improves Endothelium-Dependent Vasodilation in Hypertensive Patients With Impaired Glucose Tolerance Through a Decrease in Oxidative Stress
Authors: Takayuki Hidaka , Higashi Yukihito , Soga Junko , Fuii Yuichi , Hata Takaki , Idei Naomi , Fujimura Noritaka , Yohsizumi Masao , Kihara Yasuki
Published: April 2024

Objective
: The aim of this study was to determine long-term pioglitazone treatment restores endothelial function in hypertensive patients with impaired glucose tolerance (IGT) and to elucidate the roles of oxidative stress and insulin resistance in endothelial function/

Research Design and Methods
: We evaluated the effects of pioglitazone treatment for 12 weeks on forearm blood flow (FBF) responses to acetylcholine (Ach), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in 34 hypertensive patients with IGT who were randomly divided into a pioglitazone group (n=17) and a conventional therapygroup (control group) (n=17) and in 34 age- and gender-matched healthy subjects. FBF was measured by using a strain-gauge plethysmography.

Results
: FBF response to ACh was greater in healthy subjects than in hypertensive patients with IGT, although the FBF responses to SNP were similar in the two groups. Pioglitazone increased the FBF responses to ACh from 16.3±6.8 to 23.6±7.1 mL/min per 100 mL tissue (P<0.05) and decreased urinary excretion of 8-hydroxy-2′-deoxyguanosine, an indez of oxidative stress. Pioglitazone did not alter SNP-stimulated vasodilation. Administration of N

G

-monomethyl-L-arginine, a nitric oxide (NO) synthase inhibitor, abolished the augmentation of FBF responses to ACh correlated with the decrease in urinary extretion of 8-hydroxy-2′-deoxyguanosine but not with changes in other parameters (r=−0.36, P<0.01).

Conclusion
: These findings suggest that pioglitazone improves endothelial function in hypertensive patients with IGT through an increase in NO bioavailability by, at least in part, a decrease in oxidative stress.
Abstract 5572: RAGE Signaling in Macrophages Contributes to Atherosclerosis in ApoE Null Mice
Authors: Shen Xiaoping , Rosario Rosa , Lu Yan , Yan Shi Fang , Schmidt Ann Marie
Published: April 2024

Macrophage-derived foam cell formation is an essential process in atherosclerosis. RAGE (Receptor for Advanced Glycation Endproducts) and its ligands play a role in atherosclerotic lesion formation, especially in diabetes. RAGE is expressed in monocytes/macrophages and previous studies revealed key roles for RAGE in macrophage migration and production of cytokines. To test the role of RAGE signaling in monocytes/macrophages, we developed transgenic mice expressing cytoplasmic domain-deleted RAGE (dominant-negative RAGE (DN-RAGE) in macrophages under control of the Macrophage Scavenger Receptor-type A promoter to study the role of RAGE in atherosclerosis (noted as MSR-DN-RAGE). Four groups of mice, all in apoE−/− background and fed with normal chow diet, were tested under diabetic (D) and non diabetic ND) conditions. At age 24 weeks, lesion areas by
en
face analysis of entire aorta and at the aortic sinus were significantly reduced in MSR-DN-RAGE+/apoE−/− vs. apoE−/− mice (in both ND and D) (Table

). Immunohistochemistry of lesions demonstrated significantly reduced % of macrophages/lesion area in both ND and D MSR-DN-RAGE+/apoE−/− vs. apoE−/− mice, and in D mice, significantly lower % smooth muscle cells/lesion area in MSR-DN-RAGE+/apoE−/− vs. apoE−/− mice (Table

). The % T cells/lesion area were not different among the ND and D groups (Table

). We conclude that macrophage RAGE signaling critically contributes to atherosclerosis in apoE−/− mice irrespective of the diabetic state.
Abstract 5573: Critical Role of Notch1 in Bone Marrow-derived Macrophages in Mediating Atherosclerosis
Authors: Zhou Qian , Wang Hong-Wei , Boisvert William A , Liao James K
Published: April 2024

Background: Notch receptors regulate binary cell fate determination and are critical for embryonic development. However, the role of Notch signaling in the postnatal period remains to be determined. Although Notch receptors and ligands are expressed in atherosclerotic lesions, its functional role in macrophages in mediating atherosclerosis is unknown.

Methods: To determine the role of macrophage Notch receptors in atherosclerosis, we transplanted bone marrow (BM) from wild-type (WT), Notch1
+/−
and Notch3
−/−
mice into irradiated recipient LDLr
−/−
mice. Atherosclerosis was induced with a 16-week diet containing 15.8% wt/wt fat and 1.25% cholesterol. The degree of atherosclerosis in the
en face
aorta and subaortic sinus was evaluated using Oil Red O staining. Macrophage recruitment and chemotaxis were evaluated by thioglycollate-induced peritonitis and in a modified Boyden chamber, respectively. Statistical analysis was performed using Mann-Whitney
U
-test. A
p
value <0.05 was considered significant.

Results: Physiological parameters after BM transplantation (BMT) and following atherogenic diet were not different between the 3 groups. Compared to WT BMT mice, Notch1
+/−
BMT mice showed substantially less atherosclerotic lesions in the aorta (9.7% vs. 13.6%,
p
<0.05) and subaortic sinus (515 ×10
3
μ
m
2
vs. 696 ×10
3
μ
m
2
,
p
<0.05). In contrast, Notch3
−/−
BMT mice developed similar degree of atherosclerosis compared to WT BMT mice (aortic lesion: 14.7%; subaortic sinus lesion: 670 ×10
3
μ
m
2
;
p
<0.05 for both compared to WT BMT mice). This correlated with decreased recruitment of macrophages in response to thioglycollate-induced peritonitis in Notch1
+/−
BMT mice, but not in Notch3
−/−
or WT BMT mice. Indeed, Notch1
+/−
macrophage exhibited decreased chemotaxis to monocyte chemotactic protein (MCP)-1. The decreased chemotaxis in Notch1
+/−
macrophages was associated with decreased extracellular regulated kinase (ERK) phosphorylation.

Conclusions: Our findings indicate that Notch1 in BM-derived macrophages contributes to atherosclerosis by mediating macrophage chemotaxis, in part, through the ERK signaling pathway and suggest that inhibition of Notch1 signaling in macrophages may have therapeutic benefits in preventing atherosclerosis.
Abstract 5590: Therapeutic Neovascularization by AAV 2/9-based Gene Transfer of hVEGF-A and hPDGF-b in Chronic Ischemia (Pig and Rabbit Model)
Authors: Pfosser Achim , Hinkel Rabea , Thormann Michael , Lebherz Corinna , Wuchrer Alexander , Globisch Franziska , El-Aouni Chiraz , Banfi Andrea , Boekstegers Peter , Kupatt Christian
Published: April 2024

In patients suffering from ischemic cardiomyopathy or peripheral artery disease, lacking interventional or surgical treatment options, induction of neovascularization via vascular growth factor application is a novel therapeutic concept. Here we tested an approach by combining hVEGF-A with hPDGF-B and prolonging the expression period by using a AAV as viral vector.

Methods:
In rabbits (n=5 per group), 0.5×10
11
rhAAV-VEGF-A with or without 1×10
12
rAAV-hPDGF-B were retroinfused at d7 of femoral artery excision. At d7 and 35 angiography of both hindlimbs was performed for collateral score and frame count score (cinedensitometry, % d7). Capillary/muscle fiber ratio (C/MF) was determined at d35. In pigs, a reduction stent graft was implanted into the circumflex artery (Cx), leading to complete occlusion at day 28, when retroinfusion of rAAV-VEGF-A/PDGF-B (
2×1012
particles each) or rAAV-LacZ (4×10
12
particles) was performed. Global myocardial function (EF and LVEDP) and regional myocardial perfusion (fluorescent microspheres) were assessed at d28 and d56, were as regional myocardial function was ovbtained at day 56.

Results:
In rabbits, rAAV-hVEGF-A strongly induced angiogenesis (C/MF 1,32±0,07 vs. 0.96±0,08 in LacZ-controls), but not collateral growth (125±7% d7 vs. 95±6% d7, p=0.09) or perfusion (136±12% vs. 107±9%, p=0.07). hVEGF-A/hPDGF-B co-transfection, however, enhanced collateral growth (146±9%, p=0.01) and perfusion (163±8%, p<0.05) at a similar capillary density (1,44±0,10 CM/F). In the pig model, retroinfusion of rAAV-hVEGF-A/hPDGF-B increased regional myocardial blood flow (1,5±0,1ml/g vs. 1,0±0,1ml/g in mock-transfected controls, p<0.05), based on growth of collaterals (9,5±0,3 vs. 5,5±0,5 in controls, p<0.01) and capillary density (146±8 vs. 66±6/field in controls). rAAV-hVEGF-A/hPDGF-B improved global myocardial function (EF: 48±1% vs. 33±1% in control; LVEDP: 14.7±0.9 vs. 19.6±0.8mmHg in controls, p<0.05) accompanied by an improved regional myocardial function in the RCx area (11±3 % SES vs 2±6 %, controls).

Conclusions:
Longterm overexpression of hVEGF and PDGF substantially enhanced angiogenesis and arteriogenesis in the ischemic region followed by an improved function, wich is superior to hVEGF alone.
Abstract 5604: Refining Clinical Trial Composite Outcomes With Investigator-Assessed Weights: An Application to the ASSENT-3 Trial
Authors: Armstrong Paul W , Westerhout Cynthia M , Van de Werf Frans , Califf Robert M , Wallentin Lars , Welsh Robert C , Wilcox Robert G , Bakal Jeffrey A
Published: April 2024

Background:
Declining cardiac mortality and rising costs of clinical trials places a new priority on efficient use of all patient outcomes. Whereas traditional time-to-event analysis exclusively assigns equal weight to the first event in the composite end point, this is counterintuitive to stakeholders.

Methods:
We surveyed clinical investigators and asked them to assess the relative severity weights they would place on death, recurrent myocardial infarction (re-MI), cardiogenic shock and congestive heart failure (CHF) in ST-elevation myocardial infarction (STEMI) patients. Their assessment was then applied to a modified survival analysis of selected 30 day endpoints in the ASSENT-3 trial which randomized 6095 STEMI patients and compared three reperfusion strategies:

tenecteplase (TNK) + unfractionated heparin (UH);

TNK + enoxaparin (enox);

TNK + abciximab (abx).

Results:
Respondents (n=23) allocated relative values from a standard point scale which was converted to a single denominator (2.0) resulting in the following weights: death 1.0, shock 0.5, CHF 0.3 & re-MI 0.2. The Table

provides the event rates calculated according to the first event as well as at any time during 30-day follow-up (Panel A). In Panel B traditional first event versus weighted composites using first event and all events in each patient are shown. Note that when the traditional time-to-event analysis is re-examined with a weighted composite there is an attenuation of the excess events initially observed with UH (p=0.05 vs p=0.18). By contrast, when all events from each patient are used in the weighted composite, a trend for benefit with enox was observed (p=0.15).

Conclusions:
This approach adds potential value to traditional analytical techniques by more efficiently incorporating the differential value of all events in each patient thereby providing new options for future trial design and analysis.

Table 1.
Abstract 5608: Recognition of Abnormal Glucose Metabolism and Its Impact on Treatment in Patients With Acute Myocardial Infarction in Germany: Results of the SWEETHEART Registry
Authors: Gitt Anselm K , Towae Frank , Juenger Claus , Zeymer Uwe , Zahn Ralf , Senges Jochen
Published: April 2024

Background: Recent reports from epidemiological studies demonstrated a high prevalence of undiagnosed diabetes in patients with coronary artery disease. These findings led to joint guidelines by the ESC and the EASD on diabetes, pre-diabetes and cardiovascular disease.

Methods: Since 2007, 1,773 consecutive patients with STEMI or NSTEMI were enrolled into the MI-registry SWEETHEART to document patients’ characteristics and treatment in clinical practice with special emphasis on the identification of abnormal glucose metabolism. In patients with previously unknown diabetes, oral glucose tolerance test was performed at day 4 after the acute MI. We examined the prevalence of abnormal glucose metabolism and its impact on treatment in clinical practice.

Results: Out of 1,773 consecutive patients with MI, 919 (52.3%) presented with STEMI and 837 (47.7%) with NSTEMI. Diabetes was already known in 33.0% of patients. Oral glucose tolerance tests identified another 16.1% of patients with manifest diabetes and 22.4% with impaired fasting glucose or impaired glucose tolerance (pre-diabetes). Only 28.5% of the MI population had no signs of abnormal glucose tolerance. Medical treatment for secondary prevention at discharge was quite well according to guidelines for antiplatelet therapy, betablockers, statins, ACE-inhibitors and ARB for all patients. Nevertheless, only 61.1% of patients with newly diagnosed diabetes did receive treatment for diabetes at discharge, in whom 74.0% only received diet.

Conclusion: There was a high prevalence of newly diagnosed diabetes and pre-diabetes in consecutive patients with myocardial infarction in Germany. Despite new guidelines by the ESC and the EASD with clear recommendations for early and aggressive therapy of diabetes in patients with cardiovascular disease, 40% of myocardial infarction patients with newly diagnosed diabetes did not receive diet or an antidiabetic treatment at discharge.
Abstract 5613: Class IIa HDAC6 Contributes to Angiogenesis and Endothelial Cell Function
Authors: Kaluza David , Urbich Carmen , Rössig Lothar , Zeiher Andreas M , Dimmeler Stefanie
Published: April 2024

Acetylation of histone proteins contributes to the regulation of gene expression by alteration of chromatin structure. Histone deacetylases (HDACs) act as modulators of gene expression by deacetylation of histone proteins as well as non-histone proteins. Previous studies identified the class IIa histone deacetylases HDAC5 and HDAC7 as regulators of angiogenesis. The class IIb HDAC6 is the only HDAC that possesses two functional deacetylase domains and is known to be involved in lymphocyte chemotaxis and in cancer development. However, the function of HDAC6 in endothelial cells is unknown. Therefore, we analyzed the effect of HDAC6 for endothelial cell functions during angiogenesis. HDAC6 is mainly localized in the cytoplasm of human umbilical vein endothelial cells. Knockdown of HDAC6 with siRNA oligonucleotides significantly decreased endothelial cell sprouting from spheroids (42%±7, p<0.05, N=5), tube formation in matrigel assay (81%±2, p<0.05, N=3) and endothelial cell migration in a scratched wound assay (65%±5, p<0.05, N=3) without affecting cell viability. These results were confirmed using a second unrelated oligonucleotide. Consistently, overexpression of HDAC6 wild-type increased endothelial cell sprouting (122%±8, N=4), indicating that HDAC6 is a positive regulator of angiogenesis. The catalytic activity of the second HDAC-domain seems to be necessary for the pro-angiogenic effect of HDAC6, because overexpression of a deletion mutant, which lacks the second deacetylase domain, did not affect sprouting (95%±10, N=4). Because the second HDAC-domain is known to be responsible for
α
-tubulin deacetylation and acetylation of
α
-tubulin might play a role in cell migration, we assessed the effect of the HDAC6 specific tubulin deacetylation inhibitor tubacin on endothelial sprout formation. Application of tubacin did not affect sprouting, indicating that the mechanism is independent of
α
-tubulin deacetylation. In summary, we found that the cytoplasmic HDAC6 is necessary for in vitro angiogenesis as assessed by endothelial sprouting, tube formation and migration. Mechanistically, the pro-angiogenic effect of HDAC6 in endothelial cells requires the second deacetylase domain, but is independent of
α
-tubulin deacetylation.
Abstract 5619: Bleeding With Clopidogrel Plus Aspirin in Stroke Prevention in Atrial Fibrillation Compared to Bleeding With Warfarin: Lessons From ACTIVE-W and ACTIVE-A
Authors: Verheugt Freek W , Focks Jeroen Jaspers , Brouwer Marc A
Published: April 2024

Rationale
: Many patients with atrial fibrillation (AF) are felt ineligible for warfarin often due to a perceived unacceptable bleeding risk. Aspirin was shown to be an inferior alternative. In warfarin eligible patients in the large ACTIVE-W study stroke prevention with aspirin plus clopidogrel (A+C) was inferior to warfarin, whereas bleeding with A+C was unexpectedly quite similar to that of warfarin. Recently, in patients ineligible for warfarin in the large ACTIVE-A trial A+C was found to be superior to aspirin alone against stroke, but at the cost of a significantly increased risk of bleeding including hemorrhagic stroke. Here we put both trials into perspective with regard to safety, efficacy and trade-off.

Methods
: In ACTIVE-W patients eligible for warfarin (n=6,706) were randomized to warfarin or A+C and followed for 1.8 year (y) when the trial was stopped prematurely. Patients ineligible for warfarin (n=7,554) were randomized in ACTIVE-A to A+C or aspirin alone and followed for 3.6 y. Clopidogrel dose was 75 mg and aspirin dose 75–100 mg daily in both trials.

Results:
Baseline risk was highly comparable in both ACTIVE-trials (table

).

Conclusions
: The lowest stroke rate is seen with warfarin. Major bleeding with A+C is rather similar in both trials and comparable with warfarin. Given the bleeding with A+C in both warfarin eligible and ineligible patients, warfarin has the most favorable trade-off. These results suggest that perceived bleeding risk
per se
does not make AF patients ineligible for warfarin. Thus, warfarin should remain the cornerstone for stroke prevention in AF.
Abstract 5620: Relationship Between Stroke Risk (CHADS
2
Score) and C-reactive Protein in Patients With Non- Rheumatic Atrial Fibrillation
Authors: Maehama Tomoko , Okura Hiroyuki , Imai Koichiro , Obase Kikuko , Miyamoto Yoshinori , Saito Ken , Hayashida Akihiro , Neishi Yoji , Kawamoto Takahiro , Yoshida Kiyoshi
Published: April 2024

Background- CHADS
2
score is used for the stroke risk stratification in patients with atrial fibrillation (AF). Although C-reactive protein (CRP) has been shown as a possible correlate of left atrial (LA) thrombus formation in patients with non-rheumatic AF (NRAF), the relationship between CHADS
2
score and CRP is unclear.

Methods- A total of 165 patients with NRAF (105 male, age 71±10 years) who underwent transesophageal echocardiography (TEE) were enrolled and analyzed. Patients with prosthetic valve, aortic dissection and infectious disease were excluded from this study. The CHADS
2
score was calculated based on a presence or absence of risk factors (
C
ongestive heart failure,
H
ypertension,
A
ge≥75years,
D
iabetes mellitus,
S
troke or transient ischemic attack) as reported previously. Based on the CHADS
2
score, study patients were grouped into low (0 to 1 score), moderate (2 to 3 score) and high (4 to 6 score) risk categories.

Results- Patients at low risk had significantly lower CRP levels than those at moderate or high risk (Low: 0.27±0.46 vs. Moderate: 0.40±0.56 vs. High: 0.93±0.85mg/dl, p<0.01). As expected, incidence of LA spontaneous echo contrast and LA thrombus by TEE increased with increasing CHADS
2
score. Conclusion- In patients with NRAF, CHADS
2
score is related to CRP and LA thrombus formation.
Abstract 5621: A Phase 2 Feasibility Trial of the Oral Direct Thrombin Inhibitor AZD0837 in the Prevention of Stroke and Systemic Embolic Events in Patients With Atrial Fibrillation, Who Are Unable or Unwilling to Take Warfarin Therapy
Authors: Lip Gregory Y , Rasmussen Lars H , Olsson S B , Zetterstrand Sofia , Stahre Christina , Bylock Anders , Aunes-Jansson Maria , Wåhlander Karin
Published: April 2024

Many patients with atrial fibrillation (AF) cannot be treated with vitamin K antagonists (VKA) such as warfarin for various reasons, and will therefore not receive effective thromboprophylaxis. Development of a novel oral anticoagulant with an improved safety profile while maintaining the efficacy of VKA would represent a significant advance in the management of AF. The primary objective of the present Phase 2 trial (
NCT00623779
) was to assess the feasibility of conducting a study in patients with AF unable or unwilling to take warfarin, by evaluation of drop-out rate and compliance with treatment and study procedures. Secondary objectives included assessment of safety/tolerability (including bleeding), and pharmacodynamic properties of AZD0837. Patients were randomised to receive AZD0837 extended-release tablets 150 mg (n=41) or 300 mg (n=42) once daily, or standard therapy (no treatment, ASA 75–325 mg or clopidogrel 75 mg once daily) (n=45). The study was blinded to AZD0837 dose. Treatment duration was between 4 weeks and 6 months (median 6 weeks). Reasons for patients not being treated with warfarin were: permanent cessation or refusal by the patient (62%), physicians assessment that VKA was inappropriate (21%), inability to keep INR between 2.0 –3.0 over a 3-month period (24%), and warfarin allergy (2%). 40% of patients were warfarin-naïve. Compliance with study treatment (mean±SD) was 96.9±16.5% for AZD0837 150 mg and 99.8±11.4% for 300 mg. Compliance with study visits was 93.3±15.0% for AZD0837 150 mg, 95.6±10.4% for 300 mg and 97.5±6.8% for standard therapy. The number of drop-outs were 4, 6 and 3, whilst minor or clinically significant minor bleeds were reported in 0, 5 and 2 with AZD0837 150 mg, 300 mg and standard therapy, respectively. No major bleeding events were reported. Both doses of AZD0837 showed relevant anticoagulation effects in terms of reduced levels of fibrin D-Dimer, and prolongation of ECT and APTT. This Phase 2 trial indicates that a larger study in this group of AF patients in need of effective thromboprophylaxis may be feasible, as judged by compliance and drop-out rates. The oral direct thrombin inhibitor AZD0837 has a favorable safety profile including a low incidence of bleeding events, and seems to offer effective anticoagulation.
Abstract 5629: Time Course Analysis of Bone-marrow Derived Progenitor Cell Transdifferentiation During Neointima Formation
Authors: Daniel Jan-Marcus , Tillmanns Harald , Sedding Daniel G
Published: April 2024

Background: Transdifferentiation of bone marrow derived progenitor cells (BMPC) into smooth muscle cells (SMC) was suggested to contribute to vascular remodeling processes. However, the ability of these cells to account for highly differentiated contractile SMC remains elusive. In this study, we examined the time points of accumulation, differentiation and the long term contribution of BMPC compared to locally derived SMC in the process of neointima formation.

Methods and Results: C57BL/6 mice were lethally irradiated and subsequently rescued by bone-marrow transplantation (BMTx) of eGFP+-BM. Arterial remodeling was induced by wire injury of the femoral artery, and vessels were harvested after 3 days, 1, 2, 3, 4, 6 and 16 weeks (n=10 animals per time point). Neointima/media ratio and the number of eGFP+-cells in the vessel wall were quantified. Following a careful analysis using deconvolution analysis of z-image stacks and confocal microscopy, the expression of
α
SMA in eGFP+-cells occurred to be a very rare event. This was also observed for the co-expression of CD31 in BM-derived cells. Moreover, some cells positive for eGFP and
α
SMA also expressed monocytic lineage markers like CD68 or CD11b. The number of BM derived cells in the neointima constantly declined at later timepoints, and most of the eGFP+-cells were indeed identified as monocytes/macrophages. At 16 weeks after dilatation, highly differentiated SMC were exclusively derived from GFP negative and thus pre-existing resident cells. In addition to these data, selective prevention of resident vascular cell proliferation by irradiation of mice before BMTx inhibited neointima formation in a dose dependent manner.

Conclusions: The contribution of eGFP+-cells to the cellular compartment of the neointimal lesion is limited to a temporary time period of the inflammatory response to the vascular injury. Hence, BM derived cells contribute to arterial remodeling, but these cells do not transdifferentiate into definitive and highly differentiated SMC.
Abstract 5631: Isolation of a Resident and Dynamic Oct4+ Stem Cell Population From Adult Skin
Authors: Limbourg Anne , Lozanovski Vladimir , Schnabel Sabine , Templin Christian , Napp L C , Drexler Helmut , Limbourg Florian P
Published: April 2024

The transcription factor Oct3/4 is a critical regulator of pluripotency in embryonic stem cells and induced pluripotent stem (iPS) cells and iPS generation requires re-activation of endogenous Oct4 expression. Since Oct4 might be a critical regulator of somatic stem cell pluripotency we have employed a genetic, bona-fide reporter strategy to determine the temporo-spatial expression of Oct4 in adulte mice.

RESULTS: Postnatal Oct4 expression was determined in transgenic Oct4::eGFP reporter mice by flow cytometry, for hearts and skin the response to injury was also evaluated (n=9 –28). The fidelity of the Oct4-GFP reporter strain was confirmed by immunofluorescence confocal microscopy and by RT-PCR. No GFP+ cells were detected in bone marrow or spleen, nor in the atria or ventricles of the myocardium under baseline and post-ischemic conditions. Importantly, Oct4+ cells resided in skin, where levels increased postnatally over time until puberty and returned to low levels in the adult (Oct4+/500.000 cells: puberty 400±268; adult 53±23). Furthermore, the Oct4+ cell pool increased robustly after wound injury (289±156, n=9, p<0.05) even in remote skin areas. In situ analysis revealed the existence of GFP+ niches associated with distinct regions of hair follicle. Oct4+ cells after FACS-sorting revealed a phenotype of c-kit lo, sca-1 +, CXCR4 lo, alpha6 integrin lo, beta1 integrin -, and expressed by qRT low levels of Oct4, Sox2, Klf4, and high levels of c-myc, factors involved in iPS induction. The differentiation potential of Oct4+ skin cells was evaluated by transplanting dual reporter cells (GFP+; lacZ+) into nude mice following hindlimb ischemia and analysis of contribution to tissue lineages. 4 weeks after transplantation, LacZ+ cells contributed to muscle cells, adipose tissue and vascular structures.

CONCLUSION: Genetic reporter studies reveal no evidence for Oct4+ cells in bone marrow or heart but, unexpectedly, show persistence of resident Oct4+ stem cells in adult skin, which contribute to mesodermal cell lineage after transplantation. These findings suggest adult skin as an important source for regenerative cell therapy.
Abstract 5632: Cardiac Repair Following Myocardial Injury is Mediated by Notch1 Signaling in Mesenchymal Stem Cells
Authors: Li Yuxin , Hiroi Yukio , Ngoy Souen , Wang Hongwei , Okamoto Ryuji , Noma Kensuke , Wang Chao-Yung , Zhou Qian , Liao Ronglih , Liao James K
Published: April 2024

Background:
Notch receptors regulate binary cell fate determination and are critical for embryonic development. In particular, Notch1 regulates the fate of cardiac progenitor cells and is an important mediator of vasculogenesis. However, it is little known whether Notch1 could contribute to the cardiac repair process following myocardial injury.

Methods and Results:
We generated mutant mice with cardiac-specific deletion of Notch1 (
α
MHC-Cre/Notch1
flox/flox
mice, C-N1−/−). After left descending artery (LAD) ligation, infarct size, cardiac dimension, and heart function were similar between C-N1−/− mice and control mice. However, global heterozygous Notch1 (N1+/−) mice exhibited larger infarct size, greater LV dimension, and worsening LV function compared to that of WT mice. These findings suggest that non-cardiac Notch1 contributes to cardiac repair. When the bone marrow (BM) of N1+/− mice was transplanted into WT mice, infarct size, and impaired heart function were greater, vascularity in the infarct border area was reduced, compared to WT mice transplanted with WT BM. Alternatively, WT BM transplantation (BMT) could rescue the increased myocardial injury observed in N1+/− mice. Using GFP labeled BM cell, we found more mesenchymal stem cells (MSCs) from WT BM could be recruited to the heart compared to that of N1+/− BM. Notch1 deficient also prevent the differentiation of BM-derived cells into vascular cells. In vitro, compared to WT MSCs, N1+/− MSCs showed less migration and proliferative capacity. When N1+/− MSCs were intramyocardially injected into the infarct heart of WT mice, infarct size was greater and blood vessel density was reduced compared that of WT MSCs. Indeed, N1+/− MSCs showed decreased survival after injection compared to WT MSCs. Furthermore, the expression of Notch1 target genes, Hey1 and HeyL, and cytokine receptors CSF3R and CXCR4 were reduced in N1+/− MSCs. This decrease was reversed by overexpression of Notch1 intracellular domain.

Conclusions:
These findings indicate that Notch signaling in MSC is critical for cardiac repair after myocardial infarction, and suggest that therapeutic strategies that increase Notch1 signaling in MSC could have clinical benefits in patients with ischemic heart disease.
Abstract 5648: Epigenetic Basis of Nitrate Tolerance: Fundamental Role of Protein Acetylation
Authors: Gaetano Carlo , Colussi Female Claudia , Scopece Male Alessandro , Mattiussi Stefania , Vitale Serena , Rosati Jessica , Illi Barbara , Capogrossi Maurizio C
Published: April 2024

INTRODUCTION
: We recently demonstrated that nitric oxide (NO) plays a key role regulating epigenetic enzymes, including histone acetylases (HAT) and deacetylases (HDAC). In this context, we hypothesized that during nitrate tolerance the HDAC/HAT balance could be altered and that drugs capable of modulate HDAC and/or HAT function could have an impact on this alteration.

METHODS & RESULTS
: Adult male rats (n=6, each group) were treated for 72 hours with subcutaneous nitroglycerin (NTG) alone (50 mg/kg) or in combination with different HDAC inhibitors (DI) including trichostatin A (TSA, 0.6 mg/kg), suberoylanilide hydroxamic acid (SAHA, 0.6 mg/kg), MS-27–275 (0.6 mg/kg) and valproic acid (VPA, 5 mg/kg). Other animals received the HAT inhibitor anacardic acid (ANAC, 2.5 mg/kg) or equivalent amounts of solvent. Ex vivo experiments performed on phenylephrine-contracted aortic rings (AR) showed that the induction of tolerance (-logEC=5.9±0.2 in tolerant vs -logEC=7.6±0.1 in control) was reversed by TSA, SAHA (-logEC=7.6±0.1) and VPA (-logEC=7.4±0.2). ANAC treatment displayed value similar to nitrotolerant (-logEC=6.2±0.1). Moreover, our data revealed that in AR from controls animals a single NTG administration induced relaxation paralleled by a rapid increase in HAT activity. Nitrate tolerant rings, however, exhibited a higher HDAC activity and failed to up-regulate HATs’ function. Remarkably, in AR from animals treated with DI in combination with NTG, nitrate tolerance was prevented while animals treated with ANAC developed resistance to both arterial relaxing or contracting stimuli suggesting that the intracellular level of HAT activity is important for arterial adaptive responses. Mechanistically, in isolated smooth muscle cells the NTG increased myosin light chain (MLC) and smooth muscle actin (SMA) acetylation paralleled by a decreased MLC phosphorylation and contractile protein association. These effects were rescued to normal by DI treatment.

CONCLUSIONS
: an alteration in HAT/HDAC functional balance occurs during nitrate tolerance in which the content of acetylated contractile proteins is markedly reduce. DIs, preventing this effect, may be envisaged as novel tools for the treatment of nitrate tolerance.
Abstract 5651: Apelin Deficient Mice Demonstrate Worsening Pulmonary Hypertension in a Nitric Oxide Dependent Manner
Authors: Chandra Suparna , Razavi Hedi , Agrawal Rani , Adurkar Uditha S , Kundu Ramendra , de Jesus Perez Vinicio A , Zamanian Roham T , Rabinovitch Marlene , Quertermous Thomas , Chun Hyung J
Published: April 2024

Recent studies have shown high expression of the GPCR APJ and its ligand apelin in the pulmonary vasculature. Given its known vasodilatory properties and pulmonary expression, we hypothesize that disruption of this pathway leads to worsening of pulmonary hypertension in the murine hypoxia model, and that the pathway is abnormally regulated in patients with pulmonary arterial hypertension (PAH). Right ventricular systolic pressures were assessed in apelin deficient (KO) mice after 2 weeks of hypoxia. Lungs were perfused with contrast agent (Microfil), and imaged using micro-computed tomography (CT) imaging. We measured NO levels in the serum and evaluated the lungs for expression of endothelial nitric oxide synthase (eNOS) using quantitative real-time polymerase chain reaction (RT-PCR) and western blots. We found that apelin KO mice developed more severe pulmonary hypertension in response to hypoxia compared to wildtype mice (34.1 vs. 28.3 mmHg, p<0.001). Micro-CT of the lungs demonstrated vascular abnormalities in the apelin KO mice including pruning of smaller vessels and increased tortuosity of the larger vessels. Apelin KO mice had decreased levels of serum NO (p=0.02) and had lower expression of eNOS in the lungs, as determined by RT-PCR and western blots. We also performed ELISA assays to determine apelin levels in patients with PAH. Apelin levels from PAH patients were significantly lower compared to healthy controls (1.25 vs. 0.89 ng/mL, p=0.037). These data suggest that disruption of apelin signaling can exacerbate pulmonary hypertension, and identify this pathway as a potential therapeutic target for treatment of this refractory human disease.