bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5267: The Annexin A5 Homodimer Diannexin Protects Against Ischemia Reperfusion Injury Through Phosphatidylserine Binding
Authors: Wever Kimberley E , Claassen Karina , Frielink Cathelijne , Boerman Otto , Scheffer Gert-Jan , Allison Anthony , Wagener Frank , Masereeuw Rosalinde , Rongen Gerard A
Published: April 2024

Renal ischemia/reperfusion injury (IRI) frequently complicates cardiac and aortic surgery and has prognostic significance. The translocation of phosphatidylserines (PS) to cell surfaces is an important pro-inflammatory signal for cell-stress after IRI. We hypothesized that shielding of exposed PS by the annexin A5 homodimer Diannexin protects against renal IRI. Mice (n =8 – 10) underwent 20 min bilateral clamping of the renal pedicle, followed by 3 or 8 days of reperfusion. Either 200 ug/kg Diannexin or vehicle was injected
i.v.
20 min prior to ischemia. Renal injury peaked on day 3 post-op. When compared to vehicle, Diannexin treatment reduced the IRI-induced glucosuria, prevented the increase in urine flow, reduced the proximal tubule damage and reduced the expression and/or transcription of the renal injury markers Neutrophil Gelatinase Associated Lipocalin (NGAL) and Kidney Injury Molecule-1 (KIM-1; see table

). Reduced KIM-1 expression persisted on day 8 after Diannexin treatment [0.23±0.02
vs.
0.36±0.06; p<0.05]. To confirm PS as the target for Diannexin, mice (n=2– 6) underwent unilateral IRI of the hind limb. Upon reperfusion, 4.5 ug Tc-99m-Diannexin was injected
i.v.
. Diannexin targeted specifically to the ischemic limb when compared to the control limb [4.0±0.60
vs.
0.2±0.02 %ID/g; p<0.001]. Targeting could be reduced by a preceding injection of 500 ug unlabeled annexin A5 [1.5±0.3
vs.
0.7±0.2 %ID/g], suggesting that Diannexin targets to ischemic tissues via PS binding. This study indicates the importance of PS exposition in IRI. We conclude that Diannexin protects against renal IRI by binding to PS, making it a promising tool to prevent IRI in a clinical setting.

Table:

effects of Diannexin on functional, histological and injury marker parameters after renal IRI
Abstract 5299: Release of Sphingosine-1-Phosphate From Platelets Requires Thromboxane Synthesis and Thromboxane Receptor Activation
Authors: Böhm Andreas , Ulrych Thomas , Rosenkranz Anke C , Daum Guenter , Geisslinger Gerd , Hohlfeld Thomas , Schrör Karsten , Rauch Bernhard H
Published: April 2024

The sphingosine-derived lipid signaling molecule sphingosine-1-phosphate (S1P) is an important mediator of vascular homeostasis. S1P signaling has recently been shown to be a downstream signaling component of the receptors for thrombin, the protease-activated receptors (PARs). The present study investigates the requirement of thromboxane formation for the release of S1P induced by the prototypic thrombin receptor PAR1 from human platelets and the consequences for human monocyte and endothelial cell migration. S1P was detected by thin layer chromatography in [3H]sphingosine-labeled platelets and by mass spectrometry. Release of S1P was stimulated about 5 fold (0.3±0.1 vs. 1.6±0.5 nmol/1×106 platelets) by the selective PAR1-activating-peptide (PAR1-AP, 100
μ
M). Acetylsalicylic acid (ASA) or the reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500 mg single dose) attenuated S1P release from platelets in healthy volunteers ex vivo which was paralleled by inhibition of TX formation. S1P release was increased by the thromboxane receptor (TXR) agonist U46619, and inhibited by the TXR antagonist ramatroban. In platelets from TXR-deficient mice, thrombin-induced release of S1P was attenuated and not affected by ASA. Supernatants from PAR1-AP-stimulated platelets increased human umbilical vein endothelial cell (HUVECs) migration and the chemotactic capacity of human peripheral blood monocytes (2- and 3-fold, respectively). These effects were inhibited when platelets were pretreated with ASA. S1P receptor-specific antagonists revealed that the chemotactic responses in HUVECs were mediated by the S1P receptor-1 (S1P1) and by S1P1 and S1P3 in monocytes. These data suggest that S1P release from platelets after thrombin receptor activation requires TX synthesis and TXR activation. This novel pathway likely contributes to the regulation of vascular homeostasis during platelet activation and inflammatory processes. Inhibition of S1P may be involved in the anti-inflammatory actions of ASA in vivo, for example by affecting recruitment of endothelial cells and monocytes to sites of vascular injury.
Abstract 5314: A Critical Role for TGF-
β
Activity in the Protection Against Aortic Aneurysm Formation and Complications in Adult Mice
Authors: Wang Yu , Ait-Oufella Hafid , Herbin Olivier , Bonnin Philippe , Ramkhelawon Bhama , Taleb Soraya , Huang Jin , Offenstadt Georges , Combadière Christophe , Rénia Laurent , Johnson Jason L , Tharaux Pierre-Louis , Tedgui Alain , Mallat Ziad
Published: April 2024

Complicated abdominal aortic aneurysm is a major cause of mortality in elderly men. Recent studies unexpectedly showed that increased angiotensin II (AII)-dependent transforming growth factor (TGF)-
β
activity promotes aortic aneurysm progression in a mouse model of Marfan syndrome. However, the direct role of TGF-
β
activity in other forms of abdominal aortic aneurysm has not been assessed. Here, we show that systemic neutralization of TGF-
β
activity using two different antibodies, including the one previously shown to inhibit aneurysm formation in Marfan syndrome, breaks the resistance of normo-cholesterolemic C57BL/6 mice to AII-induced abdominal aortic aneurysm formation and markedly increases their susceptibility to the disease (from ~ 10% to 80–95%, P<0.0001). These aneurysms display a large spectrum of complications on echography, including fissuration, false channel formation and rupture, leading to death from aneurysm complications. The disease and its complications are refractory to inhibition of IFN-
γ
, IL-4, IL-6 or TNF-
α
signaling. Genetic deletion of T and B cells or inhibition of CX3CR1 chemokine pathway resulted in partial protection. Interestingly, neutralization of TGF-
β
activity led to enhanced monocyte invasiveness, and monocyte depletion markedly inhibited aneurysm progression and complications. Finally, neutralization of TGF-
β
activity led to substantial increase in MMP-12 activity, and MMP-12 deficiency completely prevented aneurysm rupture. These results clearly identify a critical role for TGF-
β
in the taming of the innate immune response and the preservation of vessel integrity in adult normal mice, which contrasts with its reported pathogenic role in Marfan syndrome.
Abstract 5325: Inherited Human Nox2 Deficiency is Associated With Impaired Isoprostane Formation and Reduced Platelet Recruitment
Authors: Pignatelli Pasquale , Carnevale Roberto , Di Santo Serena , Bartimoccia Simona , Panarelli Annarita , Sanguigni Valerio , Finocchi Andrea , Plebani Alessandro , Violi Francesco
Published: April 2024

Platelet isoprostane PGF2
α
-III, a pro-aggregating molecule, is believed to derive from non enzymatic oxidation of arachidonic acid. We assessed the hypothesis that PGF2
α
-III derives also from gp91phox activation and contributes to the phenomenon of platelet recruitment. We studied PGF2
α
-III in platelets from 8 male patients with hereditary deficiency of gp91phox (Nox2) the catalytic subunit of NADPH oxidase, and 8 male controls. Gp91phox deficency is a very rare human disease(prevalence 1:1000.000) characterized by life-threatening infections. Patients were a subgroup from a multicentre study in collaboration with the Italian Primary Immunodeficiency Network (IPINET). Upon stimulation platelets from healthy subjects produced PGF2
α
-III, that was inhibited 8% by aspirin and 43% by an inhibitor of gp91phox. Also, in platelets incubated with a gp91phox inhibitor or with SQ29548, a thromboxane A2 receptors inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 52% and 84% respectively; a lower effect (−13%) was seen with aspirin. Incubation of normal platelets with PGF2
α
-III dose-dependently(1–100 pM) increased platelet recruitment by mobilizing platelet Ca2+ and activating gpIIb/IIIa. Platelets from patients with gp91phox hereditary deficiency had normal thromboxane A2 formation and 75% PGF2
α
-III reduction compared to controls. In gp91phox-deficient patients agonist-induced platelet aggregation was within the normal range while platelet recruitment was reduced compared to controls. Incubation of gp91phox-deficient platelets with PGF2
α
-III (1–100 pM) partially restored platelet recruitment. In conclusion this study provides the first evidence that platelet PGF2
α
-III maximally derives from Nox2 activation and contributes to platelet recruitment via activation of gpIIb/IIIa.
Abstract 5338: Universal Definition of Myocardial Infarction Classification and the Risk of Cardiovascular Death: Observations From the TRITON-TIMI 38 Trial
Authors: Bonaca Marc P , Wiviott Steven D , Braunwald Eugene , Murphy Sabina A , Ruff Christian T , McCabe Carolyn H , Antman Elliott M , Morrow David A
Published: April 2024

The prognostic implications of new or recurrent MI after an acute coronary syndrome have not been completely defined in the era of the Universal Definition of Myocardial Infarction and newer more sensitive biomarker assays. Therefore, we investigated the association between new or recurrent MI by MI Type and the risk of cardiovascular death among 13,608 patients with ACS undergoing coronary intervention in the TRITON-TIMI 38 Trial.

METHODS
: MI events were categorized on the basis of subtypes as defined by the AHA/ACC/ESC/WHF Task Force for the Redefinition of MI. The risk of CV death was evaluated by landmark analysis starting at the time of the new MI through the end of follow-up and adjusted for important clinical covariates. Outcomes were adjudicated by a blinded CEC.

RESULTS
: Pts who experienced a MI during follow up were at higher risk of CV death at 6 months than patients who did not experience a MI (6.5% vs 1.3% vs, p<0.001). This association was present across all subtypes of MI with the highest rate among Type 4b (stent thrombosis, 15.4%) and the lowest among Type 4a (non-stent thrombosis PCI related) (figure

). After adjusting for important clinical covariates, the occurrence of any MI was associated with a 4 fold higher risk of death at 6 months (95% CI 3.3–5.6) with similar increased risk across subtype (figure

).

CONCLUSIONS
: New or recurrent MI after ACS is associated with a significantly increased risk of CV death. This pattern is consistent across all types as defined by the Universal Classification System, including peri-procedural MI. These findings underscore the clinical relevance of these events and therapies aimed at attenuating this risk.
Abstract 5415: Improvement in Diastolic Function With Anti-Hypertensive Therapy: An Analysis From the Combined VALIDD-EXCEED Dataset
Authors: Shah Amil M , Verma Anil , Desai Akshay S , Rocha Ricardo A , Hilkert Robert , Izzo Joseph , Oparil Suzanne , Pitt Bertram , Thomas James D , Zile Michael R , Aurigemma Gerard P , Solomon Scott D
Published: April 2024

Background:
Hypertension is a risk factor for diastolic dysfunction (DD) and HF with preserved EF. We quantified the relationship between blood pressure (BP) reduction and improvement in DD across a broad range of BP.

Methods:
Baseline and follow-up BP and mitral annular early relaxation velocity (E′) were analyzed in 337 subjects with mild HTN (SBP 147±16 mmHg) in the VALIDD trial (38 week f/u) and 190 subjects with more severe HTN (SBP 164±16 mmHg) in the EXCEED trial (24 week f/u), all with DD assessed by reduced E′ and no heart failure. Both trials tested different BP reduction strategies for improvement in DD.

Results:
Baseline SBP was 154.0±18.3 mmHg (IQR 141–165) and baseline E′ was 7.6±1.2 cm/sec (IQR 6.7–8.5). At follow-up, SBP decreased by 16.5±20.8 mmHg, E′ increased by 0.9±1.5 cm/sec, mean achieved SBP was 137.2±15.4 mmHg (IQR 127–145), and mean final E′ was 8.4±1.8 cm/sec (IQR 7.1–9.7). BP reduction was significantly associated with increase in E′, even after adjustment for age, gender, race, smoking, baseline SBP, DBP, HR, trial enrollment, treatment assignment, and baseline E′ (adjusted P=0.0002). Achieved SBP was significantly predictive of final E′, independent of baseline SBP or E′ (adjusted p=0.0005).

Conclusions:
Among a broad range of hypertensives with diastolic dysfunction, degree of BP reduction is directly related to extent of improvement in diastolic function, with patients achieving SBP < 130 mm Hg demonstrating the greatest overall improvement. These data underscore an important load-dependence of tissue Doppler E′.
Abstract 5423: Adaptation to Oxygen Deprivation in Human Pluripotent Stem Cells, Endothelial Progenitors, and Endothelial Cells
Authors: Abaci Hasan E , Truitt Rachel , Luong Eli , Drazer German , Gerecht Sharon
Published: April 2024

Hypoxia plays an important role during vascular development and regeneration through accumulation of hypoxia-inducible factor 1 alpha (HIF-1alpha) and activation of downstream pathways. We sought to explore the in vitro response of cultures of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), human endothelial progenitor cells (hEPCs), and human umbilical cord vein endothelial cells (HUVECs) to normoxic and hypoxic oxygen tensions. We first measured cell growth and dissolved O2 (DO) in media of adherent cultures in atmospheric oxygen, physiological oxygen (5% O2) and hypoxic conditions (1% O2). Higher aerobic metabolism was observed in both hESCs and iPSCs cultured in atmospheric oxygen, while hESCs and iPSCs cultured in 5% and 1% O2 had slower or no cell growth, respectively. At each oxygen tension, feeder-free cultured iPSCs were found to consume higher amount of oxygen than feeder-free cultured hESCs. Transport analysis revealed that the oxygen uptake rate (OUR) of hESCs, iPSCs and HUVECs was low as DO availability decreased, whereas the OUR of hEPCs was found to be low only in 1% O2. Next, we examined HIF-1alpha accumulation and the expression of target genes, including VEGF and angiopoietins (angiogenesis), Glut-1 (metabolism), Bnip3 (autophagy) and Bnip3L (apoptosis). Accumulation of HIF-1alpha was detected in all four cell lines cultured in 1% O2. Corresponding upregulation of VEGF, ANGPT2 and GLUT-1 was observed in response to HIF-1alpha accumulation, while upregulation of ANGPT1 was detected only in hESCs. Upregulation of BNIP3 and BNIP3L was detected in hESCs after being cultured for five hours in 5% and 1% O2, which levels corresponded to decreased and no cell growth, respectively. Flow cytometric analysis revealed changes in cell cycle in response to 5% and 1% oxygen tensions. These results demonstrate different cell responses to physiological and hypoxic environments, recognizing that continuous on-line measurements and control over oxygen environments during SC cultures will make it possible to derive desirable cellular responses.
Abstract 5425: Human CD34+ Cell Differentiation toward the Endothelial Lineage in the Zebrafish Embryo
Authors: Pozzoli Ombretta , Vella Pietro , Iaffaldano Grazia , Lacovich Marta , Devanna Paolo , Lamia Carla Lora , Cotelli Franco , Fascio Umberto , Biondi Andrea , Capogrossi Maurizio C , Pesce Maurizio
Published: April 2024

Background.
The zebrafish embryo provides a number of advantages for
in vivo
studies of vertebrate angiogenesis, including the possibility to observe in real-time the development of the embryonic vasculature. Furthermore, cell transplantation into the zebrafish embryo is an interesting model system for the study of stem cells differentiation.

Objective.
The objective of the present work is to determine whether human CD34
+
progenitor (hCD34
+
) cells differentiate toward the endothelial lineage and contribute to new blood vessels development after transplantation into the zebrafish embryo.

Methods and Results.
hCD34
+
cells were isolated from cord blood, labelled and injected into the zebrafish blastula (400 cells). Human CD14
−
cells were used as controls. Flow cytometry analysis of injected embryos dissociated 24- and 44-hours after transplantation showed endothelial differentiation of hCD34
+
cells, as demonstrated by increased expression levels of endothelial markers (KDR, CD105, CD146), whereas hematopoietic lineage markers were not affected. Further, we showed KDR
+
and Endothelin
+
human cells localized in the dorsal artery and in the cardinal vein. hCD34
+
and hCD14
−
cells (500–1000) were injected into the sinus venosus of developing Tg(
fli1
:EGFP) transgenic embryos (48hpf), prior to immune system development. Time-lapse confocal analysis performed 2h after transplantation showed circulating hCD34
+
cells in developing vessels. At 12h after injection, some hCD34
+
cells had incorporated into the vessel wall. One day after transplantation, hCD34
+
- injected embryos exhibited altered blood vessels sprouting in the growing tail vasculature as well as enhanced angiogenesis at the level of the subintestinal vein; these results raised the possibility that hCD34
+
cells may synthesize angiogenic factors. In agreement with this hypothesis it was found that hCD34
+
cell injection into the zebrafish blastula rescued the vascular phenotype caused by
Vegfc
knock-down. The above results were not observed in hCD14
−
cell - injected zebrafish.

Conclusions.
The present study demonstrates the evolutionary conservation of hCD34
+
cells differentiation mechanisms toward the endothelial lineage and their angiogenic properties in the zebrafish embryo.
Abstract 5495: More Rapid and Consistent Inhibition of Platelet Aggregation is Achieved by Loading With Ticagrelor Compared to Clopidogrel in Patients With Acute Coronary Syndromes: Results From the PLATO PLATELET Substudy
Authors: Storey Robert F , Patil Shankar B , Ecob Rosemary , Angiolillo Dominick J , Wallentin Lars
Published: April 2024

Background: Ticagrelor (T) is an oral reversible P2Y
12
receptor antagonist that does not require conversion to an active metabolite for antiplatelet activity, unlike clopidogrel (C). The PLATO study compared the efficacy of T and C in patients with a broad range of acute coronary syndromes (ACS). In this substudy (PLATO PLATELET), we assessed the onset and extent of inhibition of platelet function in PLATO study patients.

Methods and Results: 24 C-naive patients with NSTE ACS or STEMI undergoing primary PCI and sequentially randomised in a double blind, double dummy design to C (300mg [n=7] or 600 mg load [n=5]) or T (180mg load [n=12]) in the PLATO study in a single centre were entered into the first phase of PLATO PLATELET. Light transmittance aggregometry (LTA) with ADP (5 and 20 uM) and whole blood single platelet counting aggregometry (WBSPC) using ADP 1–100 uM was performed prior to study drug administration and 1, 2, 4, 8 and 12 hours after study drug. VerifyNow P2Y12 (VN) and VASP assays were also performed pre and 4h post study drug. Data obtained after abciximab administration was excluded except for VASP data. There was more rapid and greater inhibition of platelet aggregation with T vs C, with most T-treated patients showing a substantial effect at 1h post dose that was sustained until 12h post dose (Figure

). VN data at 4h were 273±124 and 68±97 PRU for C and T respectively (p<0.01). VASP data showed a similar trend. C 300 vs 600mg doses showed the same patterns of LTA response.

Conclusion: T 180mg loading dose has a faster onset of action with greater inhibition of platelet function compared to C in patients with ACS. WBSPC and VN can rapidly assess the early effects of P2Y
12
antagonists.
Abstract 5501: Enlarged Ischemic Damage and Decreased SDF-1a Expression in the Brain of db/db Diabetic Mice
Authors: Das Avik , Chen Shuzhen , Zhang Wenfeng , Wang Jinju , Morris Mariana , Chen Yanfang
Published: April 2024

Introduction:
Stromal cell derived factor-1a (SDF-1a) and its specific receptor Cysteine-X-Cysteine receptor 4 (CXCR4) together forms an important regulatory axis of angiogenesis and tissue repair after ischemia. Emerging evidence suggests that dysfunction of SDF-1a/CXCR4 axis may be responsible for impairment of angiogenesis and delayed wound healing in type 2 diabetes mellitus. The type 2 diabetes mellitus is a major risk for ischemic stroke. However, there are questions as to whether SDF-1a/CXCR4 axis is involved in ischemic stroke in diabetes. In this study, we determined the effect of diabetes on ischemic stroke and regulation of SDF-1a in brain.

Methods and Results:
Male db/db mice (8 weeks) and age-matched db/+ control mice were used (glucose 524.1±10.2 vs. 179.7±16.5 mg/dl, db/db vs. db/+, n=16, p<0.01). Focal ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery and the cerebral damage was determined 48 hrs after surgery by triphenyltetrazolium chloride (TTC) staining. Cerebral microvascular density in the frontal cortex was measured by immunostaining for CD31. The expression of SDF-1a in ischemic (48 hrs after MCAO) and non-ischemic (contra-lateral to MCAO side) brain was determined using western blot and real-time RT-PCR methods. We found:

Ischemic damage volume was much increased in db/db mice (28.6±2.2% and 16.5±1.5%, db/db vs. db/+, n=6, p< 0.01);

Microvascular density in the cortex of db/db mice was reduced as compared with db/+ mice (338±29 vs. 416±32 capillaries/mm
2
, db/db vs. db/+, n=4, p< 0.05);

SDF-1a expression was down-regulated in the non-ischemic brain of db/db mice (db/db vs. db/+, real-time RT-PCR: 63±9% vs. 100±16%, n=5, p< 0.01; western blot: 0.15±0.02 vs. 0.19±0.02, n=6, p< 0.05);

There was also less up-regulation of SDF-1a in the ischemic brain of db/db mice (db/db vs. db/+, real-time RT-PCR:175±24% vs. 400±43%, n=5, p< 0.01; western blot: 0.23±0.02 vs. 0.29±0.02, n=6, p< 0.05).

Conclusion:
Impaired SDF-1a regulation and reduced microvascular density may be linked to enlarged ischemic cerebral damage and a potential therapeutic target for ischemic stroke in diabetes.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5526: Maternal Smoking and Smoking Cessation in Early Pregnancy and Endothelium-Dependent Bradykinin-Mediated Relaxation of Uterine Small Arteries
Authors: Andersen Malene R , Uldbjerg Niels , Stender Steen , Sandager Puk , Aalkjćr Christian
Published: April 2024

Background -

Impaired endothelium-dependent vasodilatation in pregnant smokers may reduce the uterine blood flow to the fetus and result in retarded fetal growth. The present study aimed at investigating endothelium-dependent bradykinin-mediated vasodilation in uterine small arteries from pregnant non-smokers, smokers, and ex-smokers who quitted smoking early in pregnancy.

Methods and Results -

Of 29 healthy, singleton pregnancies scheduled for elective cesarean section; 12 were non-smokers, 9 were smokers, and 8 were ex-smokers. Serum cotinine verified self-reported smoking. Newborns of smokers were shorter and had a smaller head circumference compared to those of non-smokers (P<0.05), but newborns of ex-smokers were similar to those of smokers and non-smokers. Uterine arteries were dissected from myometrial biopsies and vascular function was assessed on a small vessel myograph for isometric recordings. Preconstricted arteries from non-smokers had a better bradykinin-induced relaxation than arteries from smokers and ex-smokers (P<0.05). In the presence of L-N
G
-nitroarginine (nitric oxide (NO) synthase inhibitor), arteries from smokers and ex-smokers did not relax to bradykinin, while arteries from non-smokers relaxed at high bradykinin concentrations (P<0.05). In the presence of L-N
G
-nitroarginine and indomethacin (cyclooxygenase inhibitor) arteries from non-smokers no longer responded to bradykinin. Relaxations to sodium nitroprusside (NO donor) were similar in arteries from non-smokers and smokers, but were better in arteries from ex-smokers (P<0.05).

Conclusions -

The findings suggest that maternal smoking reduces endothelium-dependent bradykinin-mediated relaxation in uterine small arteries contributing to retarded fetal growth, and suggest that smoking cessation early in pregnancy does not abolish this effect, but improves smooth muscle sensitivity to NO.
Abstract 5529: Excessive Inotropic Response During Exercise Reflects Cardiovascular Stiffening in Newly Diagnosed Essential Hypertensives
Authors: Tsiachris Dimitris , Tsioufis Costas , Syrseloudis Dimitris , Mazaraki Anastasia , Tzamou Vanessa , Andrikou Ioannis , Chatzis Dimitris , Stefanadis Christodoulos
Published: April 2024

Introduction:
Exaggerated blood pressure response during exercise (EBPR) is associated with high risk for worsening hypertension, while arterial stiffness and left ventricular (LV) diastolic dysfunction have both been related with adverse cardiovascular outcomes.

Hypothesis:
EBPR may be associated with arterial stiffness and LV diastolic dysfunction in a large cohort of hypertensives.

Methods:
222 untreated non-diabetics (152 males, mean age 54 years) with stage I – II EH underwent treadmill exercise testing and were classified as hypertensives with EBPR (n=75) based on the systolic BP elevation ≥210mmHg for men and ≥190mmHg for women, at peak exercise. The remaining patients were classified as normal inotropic responders (n=147). In all subjects carotid to femoral pulse wave velocity (PWV) was measured by means of Complior SP, while LV diastolic function was estimated by pulsed Tissue Doppler Imaging (TDI), averaging diastolic mitral annular velocities (Em, Am) from 4 separate sites of measurement.

Results:
Hypertensives with EBPR compared to normal inotropic responders had increased 24-hour systolic BP (137.4±10 vs 134±9mmHg, p=0.013) and PP (52.6±8 vs 49±7mmHg, p=0.001), while there was no difference in terms of age, sex and body mass index. Hypertensives with EBPR exhibited also greater relative wall thickness (0.47±0.06 vs 0.44±0.07, p=0.014), E/Em (9.45±3.1 vs 8.35±3.3, p=0.034) and PWV (9.1±1.6 vs 8.5±1.3m/s, p=0.007), as well as significantly lower values of Em (8.5±2.5 vs 9.4±2.9cm/s, p=0.046), while they did not differ in LV mass index and transmitral flow Doppler parameters (p=NS for all). Exercise capacity was also significantly deteriorated in hypertensives with EBPR based on the lower values of achieved METs (9.95±2.3 vs 11±2.3, p=0.003). Peak systolic BP at exercise was correlated with 24-hour systolic BP (r=0.240, p<0.001), 24-hour PP (r=0.218, p=0.001), PWV (r=0.152, p=0.046) and E/Em (r=0.150, p=0.049). Multiple logistic regression analysis revealed that PWV was the only independent predictor of EBPR (OR=1.31, p=0.047).

Conclusions:
EBPR constitutes a sign of cardiovascular stiffening in the setting of newly diagnosed EH, further elucidating the adverse prognostic role of abnormal systolic BP elevation at peak exercise.
Abstract 5534: Critical Role of CuZnSOD in Regulating Renal Microcirculation and in Angiotensin II-Induced Hypertension
Authors: Ma Zufu , Steege Andreas , Patzak Andreas , Lai En Yin , Eriksson Ulf J , Lundberg Jon , Persson Erik G , Carlström Mattias
Published: April 2024

Superoxide (O
2
−
), main reactive oxygen species in the vasculature, plays a major role in both renal hemodynamic and blood pressure control. O
2
−
levels are limited by superoxide dismutase (SOD) isoforms. Their functional significance in renal and systemic hemodynamics is not clear. The role of SOD1 in afferent arteriolar responsiveness and in angiotensin II (Ang II)-induced hypertension was investigated in SOD1-deficient (SOD1-ko), SOD1-transgenic (SOD1-tg) mice and in littermate controls (wild-type). Arteriolar constrictions to Ang II (10
−14
–10
−6
mol/l) were weaker in SOD1-tg (−14%) and stronger in SOD1-ko (−89%) compared with wild-types (−41%). Unspecific nitric oxide synthase (NOS) inhibition with N

ω

-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 10
−4
mol/l) reduced basal diameters in wild-types by −8%, in SOD1-ko by −2%, and in SOD1-tg by −38%. Simultaneous application of L-NAME and Ang II caused a similar response in all groups. SOD-mimetic (Tempol; 10
−4
mol/l) had no significant effect in wild-types or SOD1-tg, but caused vasodilatation in SOD1-ko (11%). Simultaneous application of tempol and Ang II attenuated the contractile response only in SOD1-ko (−46%), compared with Ang II alone. Basal blood pressures were similar; however the hypertensive response to chronic Ang II infusion was exaggerated in SOD1-ko (27%) compared with wild-types (9%). The different SOD1-levels were not associated with changes in nitrate/nitrite (NOx) excretion or in renal mRNA expression for NOS-, NADPH oxidase-, SOD-isoforms, or Ang II receptors. In conclusion, SOD1 plays an important role in the control of afferent arteriolar tone, mainly by modulating the NO bioavailability. SOD1-deficiency aggravates Ang II-induced hypertension, which underscores the importance of oxidative stress in this model of hypertension.
Abstract 5537: Inhibition of Pancreatic Lipase and the Renin Angiotensin System Synergistically Decreases Body Fat
Authors: Li Feng , Takahashi Nobuyuki
Published: April 2024

We have recently demonstrated that mice lacking renin are lean, and excrete more fat in the feces, which is associated with less than 20 % expression of pancreatic lipase and colipase. Treating wild type (WT) mice with an angiotensin receptor blocker losartan (ARB) or an angiotensin converting enzyme (ACE) inhibitor enalapril does not affect body weight, body fat, fecal fat excretion and pancreatic lipase expression, although their colipase expression in the pancreas is decreased to the low levels of the
Ren1c
−/− mice. Because colipase is necessary for activation of lipase, we hypothesized that inhibition of both pancreatic lipase and colipase by combination of ARB or ACE inhibitor and lipase inhibitor (orlistat) more effectively decreases body weight and body fat than either of them alone. Treating WT mice with both losartan (0.45g/L in drinking water) and orlistat (200 mg/kg diet) for two weeks reduced the body fat significantly more than mice treated with losartan alone or orlistat alone (control 12.5±1.42 %, losartan 8.7±1.9 %, orlistat 3.8±0.8 %, losartan + orlistat 0.2±2.5 %). The fecal fat content in mice treated with losartan and orlistat is also significantly higher than that of mice treated with losartan alone or orlistat alone (control 46.1±6.2 mg/day, losartan 32.0±4.0 mg/day, orlistat 137.1±9.6 mg/day, losartan + orlistat 186.0±39.6 mg/day). We conclude that inhibiting pancreatic lipase and colipase by using combination of lipase inhibitor and ARB or ACE inhibitor is a promising treatment of the metabolic syndrome.

This research has received full or partial funding support from the American Heart Association, Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).
Abstract 5542: Annexin A5 Reduces Inflammation Mediated Vascular Remodelling and Post-interventional Atherosclerosis and Improves Vascular Function in Hypercholesterolemic Mice
Authors: Ewing Mark M , de Vries Margreet , Nordzell Mariette , Jukema J W , Frostegard Johan , Pettersson Knut , Quax Paul
Published: April 2024

Annexin A5 is abundant in atherosclerotic plaques, has anti-thrombotic properties, and it may reduce ischaemia-reperfusion injury. We showed that annexin A5 could reduce neointima development in mouse models of vein graft restenosis and cuff induced neointima formation in hypercholesterolemic ApoE3*Leiden mice. These effects were associated to a reduction in vessel wall leucocytes, indicating an anti-inflammatory effect. Here we further investigated if annexin A5 possess anti-inflammatory properties, and can affect functional consequences of inflammation in the vessel wall.

Methods. Plasma cytokines were measured by multiplex immunoassay in hypercholesterolemic ApoE3*Leiden mice 28 days after the placement of a vein graft in the carotid artery. Mice were treated with 1 mg/kg bw of annexin A5 or placebo once daily by intraperitoneal injection. In a second experiment, ApoE
(−/−)
mice were given a western diet for 16 weeks, after which they were treated with annexin A5 or placebo for 3 days, as above. They were then anaesthetised (isoflurane) and a Samba Preclin pressure transducer was introduced into the aortic arch through a carotid artery. Endothelium mediated dilatation was induced by intraperitoneal injection of 3
μ
g metacholine.

Results. Plasma levels of G-CSF, IFN
γ
and MIP-1
β
/CCL4 were significantly reduced by annexin A5 28 days after surgery. Vein graft remodelling was reduced by 48±12% in the annexin A5 treated mice. In apoE
(−/−)
mice, diastolic and systolic blood pressure were not significantly different. Placebo treated mice responded to metacholine with a minor reduction in diastolic (−1 mmHg at 3 minutes after injection) and systolic blood pressure (−5 mmHg). In contrast, annexin A5 treated mice responded with a pronounced and more long-lasting reduction in diastolic (− 11 mmHg) and systolic (−22 mmHg) blood pressures, p < 0.05 for both pressures.

Conclusions. Annexin A5 has systemic anti-inflammatory properties, as shown by reduced plasma cytokine levels, as well as it (as previously shown) blocks vascular inflammation in disease models. It can also normalise disease related impairment of endothelium mediated dilatation. Annexin A5 may have therapeutic benefits in inflammation associated vascular disease.
Abstract 5545: An Experimental Study With Microwave Thermography for the Assessment of Atherosclerotic Vascular Disease
Authors: Toutouzas Konstantinos , Synetos Andreas , Drakopoulou Maria , Tsiamis Eleftherios , Karanasos Antonis , Michelongona Archontoula , Grassos Haris , Stefanadi Elli , Siores Elias , Stefanadis Christodoulos
Published: April 2024

Background: Intravascular thermography (IVT) is the only method currently available, which can measure heat generation from atheromatic plaques indicating local inflammation. Thermal heterogeneity of the atheromatic plaque has been correlated with mophological characteristics of the culprit lesion in patients with coronary artery disease. Microwave radiometry (MR) is a new non-invasive method. We investigated whether thermal heterogeneity measured by MR is correlated with IVT findings in an experimental model of atherosclerotic disease.

Methods: Microwave radiometry (RTM-01-RES system) was applied noninvasively in 18 atherosclerotic segments of the aorta (3 segments, 5cm long in 6 rabbits) that were clearly separated from each other, as well as in 18 control segments. Microwave radiometry measures natural electromagnetic radiation from the internal tissues at microwave frequencies that is proportional to the temperature of tissue. The system consists of an internal temperature sensor with the antenna, a skin temperature sensor and the data processing unit. The accuracy of measuring the internal temperature of the device is±0.2°C and the depth of temperature detection is 1–7 cm. Thereafter, IVT was performed in the same segments. For both techniques temperature difference (ΔT) was calculated by subtracting the mean temperature of proximal healthy vessel wall from the maximal temperature of each segment.

Results: Thermal heterogeneity was detected by both methods in the rabbits with the high atherogenic diet. Microwave radiometry detected that ΔT of the atherosclerotic aortas was significantly higher compared to the ΔT of the controls (0.98±0.27 °C versus 0.40±0.14°C, p<0.001). These findings were confirmed by the measurements acquired by IVT. Mean temperature difference was 0.07±0.01 °C for the atherosclerotic aortas versus 0.02±0.01°C, (p<0.001) for the control group. The segments that had the highest ΔT were those that were recognized in angiography as having significant atheromatosis.

Conclusion: Microwave radiometry is the first non-invasive method for detection of local inflammatory activation in arterial segments in an experimental model. Further studies in humans should be performed for the evaluation of this method.
Abstract 5549: Bone Morphogenetic Protein-2 (BMP-2) and BMP-7 Act as Instructive Factors to Specify Notch Signaling Controlling Smooth Muscle Cell Phenotype
Authors: Shimizu Takehisa , Tanaka Toru , Iso Tatsuya , Kawai-Kowase Keiko , Kurabayashi Masahiko
Published: April 2024

Background:

Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity including synthetic, contractive and osteogenic phenotypes in response to environmental cue. Bone morphogetic proteins (BMPs) and Notch signaling are essential determinants of cellular identity, but their mode of action to specify SMC phenotype is poorly defined. Here we investigate the cooperation between BMPs and Notch signaling in cultured VSMC.

Methods and Results:

We have recently shown that overexpression of Notch1 intracellular domain (N1-ICD) in human aortic SMC (HASMC) induced expression of both SMC and osteoblast marker genes. Interestingly, BMP-2, an osteogenic BMP implicating in vascular calcification, markedly enhanced N1-ICD-induced expression of osteoblast marker genes. Consistently, ALP activity as well as matrix mineralization were also synergistically induced by Notch and BMP-2 signaling. A specific silencing using siRNA confirmed that Msx-2 was responsible for the enhanced osteogenic phenotype induced by Notch and BMP-2. Experiments using site-specific mutation constructs of Msx2 promoter and ChiP assay revealed that the Msx-2 promoter contains functional RBP-Jk-binding site at −3770, and both this site and Smad-binding site are necessary for synergistic activation of the Msx-2 gene expression by Notch and BMP-2. In contrast, BMP-7 markedly augmented N1-ICD-induced expression of SMC marker genes, whereas expression of osteoblast marker genes was unchanged. Of note, BMP-7 failed to enhance N1-ICD-induced SMC marker gene expression in cells deficient of myocardin, indicating myocardin mediates the crosstalk between Notch and BMP-7 signaling.

Conclusion:

These results suggest that by BMP-2 and BMP-7 play an instructive role in specifying the Notch/RBP-Jk pathway which induces either differentiated phenotype or osteogenic phenotype of SMC Collectively, this study provides, at least in part, a mechanistic basis for the “context dependency” characteristic of Notch signaling.
Abstract 5556: Immunoglobulin-like Cell Adhesion Molecule Necl-5 Regulates Vascular Endothelial Growth Factor-Induced Angiogenesis by Controlling the Interaction Between VEGFR2 and Integrin
α
v
β
3
Authors: Kinugasa Mitsuo , Rikitake Yoshiyuki , Amano Hisayuki , Satomi-Kobayashi Seimi , Tawa Hideto , Kureha Fumie , Kurogane Yusuke , Nagamatsu Yuichi , Miyoshi Jun , Takai Yoshimi , Hirata Ken-ichi
Published: April 2024

Background
Peripheral arterial disease (PAD) is highly prevalent worldwide. PAD is a major cause of lower limb amputation, which is associated with a poor quality of life and prognosis. Vascular endothelial growth factor (VEGF) is a key pro-angiogenic cytokine proposed as therapeutic angiogenesis for PAD. However, more investigation is needed to better understand VEGF-induced angiogenesis and its application to PAD. The activity of VEGF receptor 2 (VEGFR2) is regulated by direct interactions with VEGF and other cell surface proteins, including integrin
α
v
β
3. However, the regulation mechanism of VEGFR2 and integrin
α
v
β
3 interaction has not been established. Nectin-like molecules (Necls) are immunoglobulin-like cell adhesion molecules (CAMs) that regulate cell movement, proliferation and survival. Here we show that Necl-5, a member of Necls that binds integrin
α
v
β
3, regulates the interaction between VEGFR2 and integrin
α
v
β
3 in vascular endothelial cells (ECs) and that this CAM plays an important role in angiogenesis.

Methods and Results
Immunohistochemistry revealed that Necl-5 was preferentially expressed in ECs including mouse hind limb capillary endothelium. Flow cytometry showed that Necl-5 was expressed in various cultured ECs. Although Necl-5-knockout mice displayed no obvious defect in vascular development, these mice exhibited impaired adaptive angiogenesis following hind limb ischemia. To clarify the regulation mechanism of angiogenesis by Necl-5, we knocked down Necl-5 by siRNA and studied the role of Necl-5 in angiogenesis
in vitro
. VEGF-induced tube formation on Matrigel, migration, and proliferation were significantly inhibited in Necl-5-knockdown ECs. Conversely, serum deprivation-induced apoptosis was enhanced in Necl-5-knockdown ECs. Knockdown of Necl-5 prevented VEGF-induced interaction of integrin
α
v
β
3 with VEGFR2, and then suppressed the VEGFR2-mediated activation of Akt, a critical signal for angiogenesis and cell survival.

Conclusions
These findings indicate that Necl-5 regulates VEGF-induced angiogenesis by controlling the interaction between VEGFR2 and integrin
α
v
β
3, and the VEGFR2-mediated signaling. Therapeutic intervention of Necl-5 may represent a novel strategy in the patients with PAD.
Abstract 5584: Extra Domain A of Fibronectin (EDA) is Critically Involved in Collateral Artery Growth
Authors: Groot Daphne , Grundmann Sebastian , de Kleijn Dominique P.V. , Pasterkamp Gerard , Hoefer Imo E.
Published: April 2024

Background
Shear stress induced arterial remodeling leading to collateral artery formation (arteriogenesis) is an important protective mechanism in cardiovascular disease, redirecting blood flow around arterial stenoses. Toll Like Receptors (TLRs), part of the innate immune system, play an important role in arterial remodeling, indicating the presence of a yet unknown endogenous ligand responsible for TLR mediated arteriogenesis.

Methods & Results
RT-PCR for endogenous TLR ligands was performed in mouse hind limb tissue after femoral artery occlusion (n=10). Hsp60 and EDA, candidate endogenous TLR-4 ligands, were significantly upregulated compared to sham operated hind limbs (EDA: occlusion
0.88
±
0.77 versus sham 0.0096
±
0.012 pg plasmid; HSP60: occlusion 0.0050
±
0.0049 versus sham 0.0026
±
0.0021 pg plasmid).
To address the functional role of the increased EDA expression, EDA −/− (n=22) and corresponding wild type mice (BALB/C, n=22) were subjected to unilateral femoral artery ligation and contra lateral sham operation. Maximum hind limb perfusion restoration was assessed using fluorescent microspheres 7 days after surgery under maximal vasodilation. Perfusion restoration was significantly decreased in EDA −/− mice compared to wild type mice (EDA 22±2.6% vs. BALB/C 31±1.8% of sham operated hind limb perfusion, p=0.011), supporting a critical role of EDA during collateral artery growth.

Conclusion
We demonstrate that the Extra Domain A of fibronectin may be the endogenous TLR ligand critically involved in collateral development. Its expression is enhanced during collateral development while its depletion in mice attenuates perfusion restoration after acute arterial occlusion.
Abstract 5585: Inhibition of Notch Signalling by Soluble Form of Delta-like 4 Converts Reparative Angiogenesis Into Inefficient Inflammatory-like Neovascularization in a Murine Model of Peripheral Ischemia
Authors: Zen Ayman Al Haj , Emanueli Costanza , Madeddu Paolo
Published: April 2024

Notch signalling by the ligand Delta-like 4 (Dll4) was previously shown to be absolutely required for normal embryonic vascular development and is involved in tumour angiogenesis. In this study, we aimed to define the effect of inhibition of Dll4-mediated signalling in ischemia-induced angiogenesis. An adenovirus encoding soluble Dll4 (AdsDll4) or empty vector (control) was injected in murine ischemic limb muscles (n=15 per group). Analysis of AdsDll4 treated muscles by PCR showed that Notch downstream genes were downregulated compared to control. Five days following ischemia induction, whole-mount immunostaining technique was applied to visualize and analyze vessel architecture in 3D by confocal microscopy. In AdsDll4-treated ischemic muscle, neo-vessel network was considerably disrupted and contained more sprouting endothelial cells (Fig

). Interestingly, we observed a strong accumulation of infiltrating inflammatory CD45 positive cells in the AdsDll4-treated ischemic muscle compared to control (958±252 vs 378±131 cell/mm2, p<0.0005). Blood flow recovery was impaired in AdsDll4 treated animals with compared to control measured by Laser Doppler Perfusion Imaging (perfusion ratio: 0.49±0.2 vs 0.74±0.1, p=0.02, 7 days after ischemia). Furthermore, the perfused vessels visualized by lectin infusion of live animals were markedly reduced in AdsDll4 treated group in comparison to control. In conclusion, our findings suggest that Notch/Dll4 signaling could play a crucial role in ischemia-induced angiogenesis not only by regulating vascular sprouting but also coordinating the cross-talk between infiltrating inflammatory cells and endothelial cells.

Fig.

In control Ischemic muscle (Ad-null) the infiltrating cells were aligned along with the long axis of the vessels whereas in AdsDll4-treated ischemic muscle they lost that relationship with vessels and were aggregated indiscriminately in clusters. In the AdsDll4-injected ischemic muscle, sprouting endothelial cells are increased in chaotic manner. CD45 (Pan-leukocyte marker, green), Isolectin B4 (Endothelial cell marker, red) - scale bar=50
μ
m
Abstract 5595: Regulated Expression and Function of c-Myb During Cardiovascular-directed Differentiation of Mouse Embryonic Stem Cells
Authors: Ishida Masayoshi , Kolodziejska Karolina M , Kattman Steven , Keller Gordon , Husain Mansoor
Published: April 2024

Background:

c-Myb plays a pivotal role in hematopoiesis and in proliferation of vascular smooth muscle cells (VSMC). We showed that
c-myb
null (KO) mouse embryonic stem cells (ESC) were unable to form contractile VSMC, but formed contracting cardiomyocytes (CM) in embryoid bodies (EB) with seemingly greater efficiency. This suggested lineage-defining effects of c-Myb during CM and VSMC differentiation.

Methods & Results:

To investigate underlying mechanisms, we first studied c-Myb levels in wild-type (WT) ESC. In serum-conditioned spontaneously differentiating WT-ESC, Western blot revealed high c-Myb levels on days 0 –2, rapid/complete loss of c-Myb on days 2.5 to 3, and subsequent re-expression during days 4 – 6 of differentiation. The loss of c-Myb between days 2.5–3.0 did not occur with any change in mRNA, but was completely blocked by proteosome inhibitor MG132. We next studied ESC-derived cardiovascular progenitor populations by flow cytometry using VEGFR2 and PDGFR
α
makers. By day 3, VEGFR2+/PDGFR
α
- cells were significantly fewer, while VEGFR2-/PDGFR
α
+ and VEGFR2+/PDGFR
α
+ cells were more frequent, in KO vs. WT EB. In
serum-free
cardiac-directed differentiation cultures of WT ESC, Western blot revealed the highest levels of c-Myb expression at day 2, with rapid loss by day 3 in VEGFR2+/PDGFR
α
+ cells. Of interest, VEGFR2+/PDGFR
α
- cells showed the highest levels of c-Myb, while VEGFR2+/PDGFR
α
+ cells expressed the lowest. Subsequently, c-Myb was not detectable in beating CM populations and differentiating CM that emerged under these conditions. Lentiviral constructs enabling inducible expression of a shRNA against c-Myb were used to temporally knock down c-Myb expression and observe corresponding effects on cardiovascular differentiation. Finally, co-IP and mass spectrometry identified TIF1
β
, a known negative regulator of c-Myb, as a putative c-Myb partner proteins affecting c-Myb-modulated cardiovascular lineage specification.

Summary:

These data show that c-Myb is tightly regulated by proteosomal degradation and protein-protein interactions during ESC differentiation, and that it plays a role in the differentiation of VEGFR2+/PDGFR
α
- cells, which may represent the source of the contractile VSMC observed in EBs.