bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5430: Statins Exert Pleiotropic Effects by Enhancement of BMPER Expression via the Rho/Rock Pathway in Endothelial Cells
Authors: Helbing Thomas , Rothweiler Rene , Heinke Jennifer , Goetz Lena , Diehl Philipp , Patterson Cam , Bode Christoph , Moser Martin
Published: April 2024

Background
Statins exert pleiotropic effects on endothelial cells besides cholesterol lowering. Bone morphogenetic proteins (BMPs) have recently been implicated in vascular inflammation and disease. We set out to investigate the effect of statins on BMPER, a novel member of the BMP pathway.

Methods and Results
Mevastatin or simvastatin enhanced BMPER expression in endothelial cells in a time and concentration dependent manner as determined by immunocytochemistry, RT-PCR, and western blotting. Actinomycin D chase analysis and BMPER promoter studies with reporter assays revealed that this is predominantly a post-transcriptional effect resulting in a prolonged BMPER RNA half-life. Rescue experiments using downstream metabolites of the HMG-CoA pathway (mevalonate, GPP, GGPP) suggested that the RhoA/Rock/actin pathway would be involved in BMPER regulation. Indeed we confirmed that the RhoA/Rock/actin pathway is essential for BMPER regulation using the specific pathway activator CNFY. CNFY prevented the upregulation of BMPER by mevastatin whereas pathway inhibitors (C3-toxin, RhoAN19 mutant, fasudil, cytochalasin D) enhanced BMPER expression. As reflected by ICAM-1 regulation BMPER has anti-inflammatory features. Increasing concentrations of BMPER reduced ICAM-1 expression while silencing of BMPER increased ICAM-1 in endothelial cells. Remarkably, mevastatin reduced the expression of proinflammatory BMP4, a direct interaction partner of BMPER.

Conclusion
Mevastatin modulates the BMP pathway by enhancing BMPER expression via the RhoA/Rock/actin pathway as well as by inhibiting BMP4 expression. BMP4 down- and BMPER upregulation contribute to the anti-inflammatory pleiotropic effects of statins and represent a novel pleiotropic mechanism.
Abstract 5431: Activation of Myocardin Function by an Ubiquitin E3 Ligase, EDD
Authors: Hu Guoqing , Wang Xiaobo , Saunders Darren , Henderson Michelle , Russell Amanda , Herring Brian , Zhou Jiliang
Published: April 2024

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of contractile proteins. The expression of these proteins is markedly attenuated during the de-differentiation of smooth muscle that occurs under many pathological conditions. SRF is a transcription factor that plays a central role in the expression of these smooth muscle-specific genes through physical association with various cell-restricted or signal dependent accessory factors. Of the SRF-associated proteins identified, myocardin is the most potent for stimulating expression of smooth muscle-specific genes. Although several proteins have been identified that can modify myocardin function, the mechanism of how myocardin regulation is still poorly understood. To identify additional myocardin associated proteins that are required for myocardin-dependent activation smooth muscle-specific genes we performed a yeast 2-hybrid screen of mouse embryo cDNA library using myocardin as bait. From this screen we identified a HECT domain containing protein EDD as a myocardin binding protein. Previous studies have shown that HECT domain containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. EDD has however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that the amino-terminal domain of myocardin bound to the HECT domain of EDD. We also show that EDD specifically enhanced trans-activation of smooth muscle-specific promoters by myocardin. Furthermore, EDD significantly augmented myocardin’s ability to induce expression of endogenous SMC marker genes, and these effects of EDD were independent on its E3 ligase function. Conversely, depletion of endogenous EDD in fibroblast cells by RNAi attenuated myocardin-induced smooth muscle-specific gene expression, and EDD knockdown in smooth muscle cells resulted in down-regulation of smooth muscle-specific genes. Ongoing experiments are investigating the mechanism by which EDD activates myocardin function. This study reveals an unexpected role for the ubiquitin E3 ligase EDD as an activator of smooth muscle differentiation through its ability to potentiate myocardin function.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5432: Inhibition of Hif Prolyl 4-hydroxylases Protect Against Reoxygenation Injury in Endothelial Cells
Authors: Dimitrova Tatyana , Haertel Frauke V , Muhammad Aslam , Reis Anna , Piper Hans M , Noll Thomas
Published: April 2024

Background: Hypoxia inducible factor-1 (Hif-1
α
) modulates expression of genes essential for cell survival under hypoxia. The oxygen-sensing enzyme prolyl 4-hydroxylase2 (PHD2) targets Hif-1
α
for destruction under normoxia. Hypoxia/Reoxygenation is associated with p53 activation, a master regulator of apoptosis in many type of cells. We hypothesized that preservation of Hif-1
α
beyond the period of hypoxia protects endothelial cells from reperfusion injury. To analyze the protective effect of Hif-1
α
, human umbilical veins endothelial cells were exposed to 1 h of hypoxia (Po2<5mmHg) followed by 24 hrs of reoxygenation (Reox.; Po2=140mmHg). Exposure to hypoxia causes a increase in Hif-1
α
, p53, and Mdm2 (E3 ubiqitin ligase) by 3.1±0.2, 2±0.3, and 1.9±0.2-fold compared to control, respectively. During Reox. Hif-1
α
and Mdm2 declined towards basal level, while p53 remained unaltered. Under the same conditions endothelial apoptosis was increased to 47±3% (annexin V staining). Silencing of PHD2, (53% reduction of PHD2 content) lead to an increase of the Hif-1
α
content during hypoxia and maintained it at that level during Reox. Hif-1
α
stabilization was accompany by an increase in the Mdm2 content and phosphorylation, whereas expression of p53 was reduced. PHD2 silencing reduced apoptosis to half. Additon of DMOG (1mM dimethyloxalylglycine), a PHD inhibitor, at the onset of Reox had the same effect. Reduction of p53 content was restored when the proteosome inhibitor MG-132 was added. Interaction of Mdm2 and P53 (co-immunoprecipitation) was increased compare to Reox. Downregulation of Hif-1
α
by siRNA (64% reduction of Hif-1
α
content) increased apoptosis to 66±5% and abrogate Mdm2-p53 complex formation. Downregulation of Mdm2 by siRNA (38% reduction of content) had no effect on Hif-1
α
but increased p53 level.

Conclusions: siRNA downregulation as well as pharmacological inhibition of PDH2, manoeuvre which inhibit Hif-1
α
degradation and maintained it at that level during Reox. protect against reperfusion injury. This effect of Hif-1
α
involves stimulation of Mdm2 induced degradation of the proapoptotic p53.
Abstract 5442: Oxidative Stress Regulates Cardiac Stem Cell Differentiation in a MicroRNA-Mediated Mechanism
Authors: Maiellaro-Rafferty Kathryn , Davis Michael E
Published: April 2024

Oxidative stress, specifically hydrogen peroxide (H
2
O
2
) is significantly increased following myocardial infarction and its effect on adult cardiac stem cell (CSC) differentiation is largely unknown. To promote endogenous and implanted CSC differentiation into functional cardiac cells in ischemic tissue, mechanisms of deleterious redox signaling on cardiomyogenesis must be determined. Our data demonstrate that H
2
O
2
time- and dose-dependently inhibits Nkx2.5 expression in CSCs exposed to differentiating medium. Since overexpression of microRNA (miR)-1 increases embryonic stem cell cardiac differentiation, we hypothesized that H
2
O
2
regulates CSC differentiation in a miRNA-mediated mechanism. CSCs exposed to H
2
O
2
(1–100
μ
M) in differentiating media demonstrated time-dependent, decreased expression of miR1 compared to cells exposed to differentiating media alone (1
μ
M: 37±7.6% decrease from Day 5 control; 100
μ
M: 47±11% decrease from Day 8 control). MiR133a, essential for normal cardiac development, was not time-dependently regulated by H
2
O
2
, suggesting it is not redox sensitive. Since

cardiac differentiation in
Drosophila
is regulated by miR-1 binding to the Notch ligand Delta and

Notch activation attenuates embryonic stem cell cardiogenesis, we next examined the effect of miR1 on the Notch pathway in CSCs.

Delta1 expression increased 24±5% above the time-matched control exposed to differentiating media alone after 8 days exposure to H
2
O
2
(0.1
μ
M). Since miR1 exhibited the inverse response to H
2
O
2
, the data support Delta1 as a target for miR1-mediated silencing in CSCs. In summary, our data suggest a mechanism for redox-mediated attenuation of CSC differentiation. H
2
O
2
decreases miR1 expression, which prevents normal Delta1 silencing by miR-1 that occurs during differentiation. This failure of Delta1 degradation increases Notch activation, which subsequently suppresses cardiogenic differentiation. This study underscores the importance of H
2
O
2
scavenging following cardiac injury to promote CSC differentiation, and suggests that targeting anti-cardiogenic miRNA regulation may be a promising strategy for successful CSC-based therapy.
Abstract 5457: Angiotensinogen Hypomorphic Mice Have Markedly Reduced Hypercholesterolemia-induced Atherosclerosis
Authors: Lu Hong , Zhao Mingming , Cassis Lisa A , Daugherty Alan
Published: April 2024

Background and Objective:
The renin angiotensin system exerts profound effects on experimental atherosclerosis. Although multiple enzymes are proposed to generate the increasingly complex array of angiotensin peptides, angiotensinogen is the unique precursor of all angiotensin peptides. Angiotensinogen deficiency in mice leads to poor neonatal survival rate and multiple organ dysfunctions that present barriers to atherosclerosis studies. Therefore, we generated a hypomorphic angiotensinogen (hypoAGT) mouse model to determine the effects of reduced angiotensinogen on atherosclerosis in LDL receptor −/− mice.

Methods and Results:
hypoAGT mice were developed by insertion of a neomycin resistance cassette in the second intron of the angiotensinogen gene. Although hypoAGT mice have almost undetectable plasma angiotensinogen concentrations and markedly higher renin concentrations compared to the wild type control, these mice have normal neonatal survival rate and grossly normal growth development. To determine the contribution of angiotensinogen to atherosclerosis, male wild type and hypoAGT mice in an LDL receptor −/− background were fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for 12 weeks. Body weight was monitored weekly. hypoAGT mice had modestly lower body weight at the initiation of the study. When fed the saturated fat-enriched diet for 12 weeks, wild type mice increased body weight by 49% compared to a 6% increase in hypoAGT mice. Systolic blood pressure (SBP) was measured using a non-invasive tail-cuff system on conscious mice. hypoAGT mice had lower SBP at baseline and when fed the saturated fat-enriched diet. Both wild type and hypoAGT mice were hypercholesterolemic, although hypoAGT mice had lower plasma cholesterol concentrations (wild type versus hypoAGT: 2789±115 versus 1647±106 mg/dl). Atherosclerotic lesion areas were measured on the intima of the aortic arch by the en face technique. hypoAGT mice had a virtual ablation of the development of atherosclerotic lesions (wild type versus hypoAGT: 20.5±2.4 versus 1.1±0.8%).

Conclusion:
Profound reduction of angiotensinogen strikingly attenuates development of hypercholesterolemia-induced atherosclerosis in LDL receptor −/−mice.
Abstract 5463: Cyclophilin A Mediates Endothelial Damage and Promotes Recruitment of Inflammatory Cells and Atherosclerosis
Authors: Nigro Patrizia , Satoh Kimio , O’Dell Michael R , Soe Nwe Nwe , Cui Zhaoqiang , Mohan Amy , Abe Junichi , Alexis Jeffrey , Goldman Bruce I , Sparks Janet D , Berk Bradford C
Published: April 2024

Background-

Cyclophilin A (CyPA) is a proinflammatory cytokine that is highly expressed in vascular smooth muscle cells (VSMC) in mice and humans and is also secreted from VSMC in response to reactive oxygen species (ROS). Development of atherosclerosis involves endothelial cell (EC) dysfunction and activation, vascular inflammatory cytokine release, macrophage migration/activation, and differentiation into foam cells. Based on involvement of CyPA in ROS signaling, we hypothesized that CyPA expression would be proatherogenic.

Methods and Results-

Using the ApoE
−/−
mouse model, we compared atherosclerosis development following genetic deletion of CyPA (ApoE
−/−
CyPA
−/−
) in mice fed a high fat diet for 16 wks. Atherosclerosis was assessed by oil-red O (ORO) staining and was significantly greater in ApoE
−/−
mice compared with ApoE
−/−
CyPA
−/−
mice (% ORO, en face aorta; 19.3±7.5 vs. 8.2±2.0,
P
<0.01). Immunostaining of aortic cusp revealed significant reduction of CD45
+
cells, CD3
+
cells, and Mac3
+
cells in ApoE
−/−
CyPA
−/−
compared with ApoE
−/−
mice, supporting the role of CyPA in inflammatory cell recruitment. En face aortic TUNEL staining revealed significant reduction of EC apoptosis in ApoE
−/−
CyPA
−/−
compared with ApoE
−/−
mice (count/area; 23.1±5.5 vs. 6.8±3.2,
P
<0.01). Moreover, ApoE
−/−
CyPA
−/−
mice had reduced expression of vascular cellular adhesion molecule 1 (VCAM-1) and increased expression of eNOS compared to ApoE
−/−
mice, suggesting a key role for CyPA in EC inflammation. These features were not altered by reconstitution of bone marrow cells from ApoE
−/−
CyPA
−/−
mice, suggesting that vascular-derived CyPA plays a crucial role in progression of atherosclerosis. Mechanistic experiments revealed that CyPA may be important for MCP-1 secretion from VSMC. These data are consistent with findings in humans where expression of CyPA, migration of inflammatory cells, and adhesion molecule expression are significantly augmented in ruptured atherosclerotic plaque areas of patients with acute coronary syndromes.

Conclusion-

CyPA is a novel mediator of atherogenesis by promoting EC damage, adhesion molecule expression, and inflammatory cell migration.
Abstract 5488: Comparison of Primary With Fibrinolytic-based Facilitated PCI and Comparison of Fibrinolytic-based PCI With Fibrinolysis Followed by Standard Care for STEMI: Updated Quantitative Review of Randomized Trials
Authors: Thiele Holger , Eitel Ingo , Desch Steffen , Schuler Gerhard
Published: April 2024

Background: Facilitated PCI for STEMI is defined as the use of pharmacological substances before a planned immediate intervention (< 3h), to improve coronary patency. We undertook an updated meta-analysis of randomized controlled trials to compare

fibrinolytic-based facilitated and primary PCI and

fibrinolytic-based facilitated PCI and fibrinolysis followed by standard care.

Methods: We identified 12 trials of patients with STEMI assigned to

facilitated (n=2498) or primary (n=2488) PCI and

8 trials with comparison facilitated PCI (n=969) vs fibrinolysis with standard care (n=955).

Outcomes of death, stroke, non-fatal reinfarction, urgent TVR, and major bleeding were assessed.

Results: The facilitated approach resulted in a greater than two-fold increase in the number of patients with initial TIMI-3-flow compared with primary PCI approach (p<0.001); however, final rates did not differ (p=n.s.). Significantly more patients assigned to facilitated PCI than those assigned to primary PCI died (5.3% vs 3.9%; RR 1.36; 95% CI 1.04; 1.77; p=0.02), had higher non-fatal reinfarction rates (3.3% vs 1.7%; RR 1.99; 95% CI 1.36; 2.92; p<0.001), higher urgent TVR rates (3.3% vs 1.3%; RR 2.48; 95% CI 1.57; 3.97; p<0.001), and higher stroke rates (1.3% vs 0.5%; RR 2.76; 95% CI 1.38; 5.64; p=0.002). Facilitated PCI was also associated with higher rates of major bleeding (6.4% vs 3.9%; RR 1.62 95% CI 1.26; 2.09; p<0.001). In the 2nd comparison, facilitated PCI did not result in a mortality difference (5.1% vs 5.7%; RR 0.89; 95% CI 0.60; 1.33; p=0.63) and no difference in non-fatal reinfarction (4.2% vs 5.2%; RR 0.81; 95% CI 0.52; 1.25; p=0.37). However, there was a difference in non-fatal reinfarction in the stenting era (4.0% vs 7.0%; RR 0.57; 95% CI 0.34; 0.95; p=0.03). There was no difference in major bleeding and stroke in the comparison of facilitated PCI vs fibrinolysis with standard care.

Conclusions:
Fibrinolytic-based facilitated PCI has substantial harm in comparison to primary PCI. In comparison to fibrinolysis with standard care facilitated PCI leads to a small reduction in reinfarction but has no beneficial effect on mortality. A strategy of primary PCI should therefore be implemented for all STEMI patients.
Abstract 5490: Contemporary Risk of In-Hospital Events in Dialysis Patients Undergoing Percutaneous Coronary Interventions: Insights From the NCDR Cath-PCI Registry
Authors: Tsai Thomas T , Maddox Thomas M , Roe Matthew T , Dai David , Casserly Ivan P , Ho P. Michael , Messenger John C , Nallamothu Brahmajee K , Peterson Eric D , Rumsfeld John S
Published: April 2024

Background:
While chronic renal insufficiency is associated with poor outcomes in patients undergoing percutaneous coronary intervention (PCI), little is known about the in-hospital outcomes of chronic dialysis patients due to very small numbers of patients.

Methods:
1,361,183 consecutive patients underwent PCI at 896 sites participating in the Cath-PCI registry from January 2004-September 2008. Patients were stratified by creatinine clearance (CrCl) into 4 groups: Normal renal function (CrCl ≥60 ml/min); Mild to moderate renal insufficiency (31≤CrCl<60 ml/min); severe renal insufficiency (CrCl≤30 ml/min) and dialysis dependent. Using multivariable regression analysis with generalized estimating equations, the associations between renal insufficiency and in-hospital bleeding, vascular complications and death were explored.

Results:
Overall, 24,711 (1.8%) patients were dialysis dependent. In-hospital bleeding increased with worsening renal function (1.5% vs 2.8% vs 5.5% vs 3.7%, p<0.001) as did vascular complications (0.6% vs 0.9% vs 1.4% vs 1.3%, p<0.001) and in-hospital death (0.5% vs 1.6% vs 5.3% vs 4.7%, p<0.001). After multivariable adjustment, patients with worsening levels of renal insufficiency had progressively higher odds of in-hospital bleeding, vascular complications and death (
Table

). Patients on dialysis represented the highest risk group overall.

Conclusion:
Approximately 2% of PCI patients are on chronic dialysis and have high rates of in-hospital adverse events. Compared to patients with normal renal function, dialysis is strongly associated with increased bleeding, vascular complications and death. Strategies to improve the care and outcomes of dialysis patients receiving PCI are warranted.

Table:

Adjusted Odds Ratio of In-Hospital Events By Creatinine Clearance (CrCl) in Patients Who Receive PCI
Abstract 5494: Pharmacokinetics and Pharmacodynamics of Ticagrelor in Patients With Stable Coronary Artery Disease
Authors: Husted Steen E , Gurbel Paul , Storey Robert F , Bliden Kevin , Høimark Lene , Antonino Mark J , Mahla Elisabeth , Tantry Udaya , Butler Kathleen , Wei Cheryl , Teng Renli
Published: April 2024

Background:
Ticagrelor (T), the first reversibly binding oral P2Y
12
receptor antagonist, has been evaluated in patients with ACS. Reversible inhibition is desirable for patients who experience bleeding complications or require invasive procedures.

Methods:
Two studies examined the pharmacokinetics (PK) of T in patients with stable coronary artery disease (CAD). In the multicenter, randomized, double-blind parallel group ONSET-OFFSET study, patients with stable CAD currently receiving aspirin (75–100 mg) were randomized to doses of T (180 mg load, 90 mg bid maintenance [n=57]), clopidogrel (C) (600 mg load, 75 mg qd maintenance [n=54]) or placebo (n=12) for 6 weeks. PK of T and AR-C124910XX (AR), an active metabolite, were evaluated following loading and last maintenance doses. In RESPOND (2-way crossover design), patients with stable CAD classified as C non-responders (n=41) and responders (n=57) were randomized to 14 days treatment with C 600 mg load/75 mg qd or T 180 mg loading/90 mg bid with no washout between treatments. On Days 1 and 14, t
max
, C
max
and AUC
0 – 8
of T and AR were evaluated.

Results:
In ONSET-OFFSET, C
max
, t
max
and t
1/2
values of T (733 ng/mL, 2 h, and 10.2 h, respectively) and AR (210.3 ng/mL, 2.1 h, and 12.8 h) at 90 mg bid were comparable to those seen previously in healthy subjects. E
max
estimates of final extent IPA (%) vs plasma concentration curves of T and AR were 98.6% and 91.8%, respectively. Trough plasma T (304.6 ng/mL) and AR (120.7 ng/mL) concentrations were 4 and 6 times higher than the respective EC
50
estimates (58.6 ng/mL and 15.7 ng/mL, respectively). In RESPOND, mean C
max
and AUC
0 – 8
of T following 2-week maintenance doses were similar between C responders (724.2 ng/mL and 3982.7 ng·h/mL) and non-responders (764.4 ng/mL and 3985.2 ng·h/mL). PK of T were not affected when T was administered 24 h post C dosing.

Conclusions:
This dosing regimen of T and AR effectively inhibited platelet aggregation; trough plasma levels of T (90 mg bid) and AR were sufficient to achieve high IPA. The pharmacodynamic response followed PK. Importantly, PK of T administered after crossover from C were unchanged and not affected by patient responsiveness to C.
Abstract 5497: Platelet Activation in Patients Undergoing Carotid Artery Stenting Procedure
Authors: Camera Marina , Bogani Paola , Ghulam Ali S , Galli Stefano , Tirloni Ezio , Canzano Paola , Ravagnani Paolo , Brambilla Marta , Tremoli Elena , Montorsi Piero
Published: April 2024

Background
. Carotid Artery Stenting (CAS) is an evolving method to treat carotid stenosis. A limitation of CAS is the periprocedural risk of temporary or permanent ischemic neurological deficits resulting from local thrombosis and distal embolization. Platelet activation in CAS occurs as a result of vessel wall damage and subendothelium exposure. The dual antiplatelet regimen (aspirin+thienopyridine) has a significant impact on reducing adverse neurological outcomes.

Aim
. To analyze platelet activation during dual antiplatelet therapy and two months after thienopyridine discontinuation in patients who underwent CAS procedure.

Methods
. We enrolled 40 patients with high-grade carotid artery stenosis (≥ 70% NASCET criteria). Blood was withdrawn 1 day before (T0), 1 (T1) and 30 days (T2) after CAS and 2 months after thienopyridine discontinuation (T3). Platelet activation markers (PAC1, CD62 and Tissue Factor [TF] and the percentage of monocyte-platelet aggregates [MPA]) were assessed by whole blood flow cytometry in resting conditions and upon in vitro ADP stimulation and reported as %UR. As a comparison, platelet activation in 16 healthy subjects was also analyzed (Controls).

Results
. In resting conditions, CD62 and PAC1 positive platelets and MPA were constant throughout the time points examined (average T0-T3: 0.22±0.02, 0.16±0.01, 9.51±0.68 respectively) and similar to those found in Controls. In vitro ADP-induced CD62, PAC1 and MPA levels were significantly higher at T3 when thienopyridine was discontinued compared to T0, T1 and T2 and similar to that observed in Controls. Levels of TF positive platelets were constant, in resting conditions, throughout the time points examined (average T0-T3: 5.04±0.43), but 3 fold higher that those found in Controls (1.95±0.56, p<0.01). Drug treatment does not inhibit ADP-induced TF expression being the values similar to those found in Controls.

Conclusions
. Significant higher levels, compared to Controls, of TF-positive platelets circulating in peripheral blood of CAS patients. Dual antiplatelet therapy inhibits the expression of all ADP-induced platelet activation markers, but TF. This prothrombotic platelet phenotype may have implications for CAS complications.
Abstract 5498: The Cumulative Risk of Stroke and Death From Carotid Endarterectomy Followed by Coronary Artery Bypass Surgery in Patients With Concomitant Critical Coronary and Carotid Stenosis
Authors: Rana Zeshan A , Tahsin Saif , Mustafa Nowwar , Weintraub William S , Rahman Ehsanur
Published: April 2024

Background:
Coexisting critical coronary artery disease (CAD) requiring coronary artery bypass grafting (CABG) and critical carotid stenosis requiring carotid endarterectomy (CEA) is becoming increasingly common. The reported incidence of stroke and death as perioperative complications of CABG range from 1.1 to 3.8% and 1 to 3%, respectively. The cumulative risk of stroke and death in patients undergoing a CEA followed by CABG in a high-risk subset, as described above, is not well defined.

Objective:
To determine the cumulative risk of in-hospital stroke and death when CEA precedes CABG, and also if a preceding history of transient ischemic attack (TIA)/cerebrovascular accident (CVA) increases the risk of in-hospital stroke and death.

Methods:
Using ICD-9 codes we identified 114 consecutive patients in our institution who were scheduled to undergo a staged CEA followed by CABG. Records of all patients were reviewed to identify comorbidities and confirm the incidence of all-cause mortality and stroke. The patients were further divided into two groups based on the presence or absence of a prior history of TIA/CVA.

Results:
Three patients died after CEA, 111 patients proceeded to CABG.

Conclusion:
Cumulative risk of stroke and death from both procedures should be considered in these patients. Despite CEA procedure aimed at reducing the risk of perioperative stroke from CABG, 3.6% of the patients in our study had a nonfatal CVA after CABG. Patients with a prior history of TIA/CVA did not show a significant increase in periprocedural risk of stroke and death.

Table 1

Cumulative risk of nonfatal CVA and Death

Table 2

Risk of nonfatal CVA and Death based on a history of TIA/CVA
Abstract 5504: Transient Decrease in Circulating Dendritic Cell Precursors After Acute Stroke - Potential Recruitment Into the Brain
Authors: Yilmaz Atilla , Fuchs Tanja , Dietel Barbara , Altendorf Regina , Cicha Iwona , Stumpf Christian , Schellinger Peter , Blümcke Ingmar , Schwab Stefan , Daniel Werner G. , Garlichs Christoph D. , Kollmar Rainer
Published: April 2024

Background: The role of dendritic cells (DC) as potent mediators of inflammation has not been sufficiently investigated in stroke.

Methods: Circulating myeloid (mDCP), plasmacytoid (pDCP), and total DCP (tDCP) were flow cytometrically analyzed in

healthy controls (n=29), patients with

asymptomatic A. carotis interna stenosis (ACI-S, n=46),

transient ischemic attack (TIA, n=39),

acute ischemic stroke (AIS, n=73), and

acute hemorrhagic stroke (AHS, n=31).

The National Institutes of Health Stroke Scale (NIHSS) and the infarction size in CT scan were evaluated after stroke. In a patient subgroup, postmortem immunohistochemical brain analyses were performed to detect mDC (CD209), pDC (CD123), T cells (CD3), and HLA-DR.

Results: In AIS and AHS, circulating mDCP, pDCP, and tDCP (each p<0.005) were significantly reduced. A significant inverse correlation was found between the NIHSS and circulating DCP (p<0.02), as well as between hsCRP and circulating DCP (p<0.001). Patients with large stroke size in CT scan had significantly lower mDCP, pDCP, and tDCP (each p<0.01) than those with smaller stroke. Follow-up analysis showed a significant recovery of circulating DCP in the first days after stroke. In the infarcted brain, a dense infiltration of mDC colocalized with T cells, single pDC, and high HLA-DR expression were observed.

Conclusions: Acute stroke leads to a decrease in circulating DCP. Potentially, circulating DCP are recruited from the blood into the infarcted brain, and probably trigger cerebral immune reactions there.
Abstract 5506: Warfarin Effectiveness and Stroke Rate in Ambulatory Patients With Non-valvular Atrial Fibrillation Managed in Community-based Primary Care Practices
Authors: Ewen Edward , Elliott Daniel J , Simon Teresa A , Ramdas Shyamkrishnan , Jackson Joseph , Weintraub William S
Published: April 2024

Background:
Despite the known efficacy of warfarin for the prevention of stroke in non-valvular atrial fibrillation (NVAF), its effectiveness in primary care practice is less clear. This study examined the use of warfarin, the time in therapeutic range, and the incidence rate of first-time stroke in NVAF patients in a community-based primary care population.

Methods:
We performed a retrospective evaluation of patients with NVAF from 1998–2009 in a large health system with a shared electronic medical record. The presence of NVAF, warfarin use, INR values, and stroke events were collected through a review of office and hospital records, laboratory values, and medication use. Exposure was categorized into four categories: no warfarin, warfarin with INR < 2, warfarin with INR 2–3, and warfarin with INR > 3. Person-time in days was computed for each category and incidence rates for stroke by warfarin exposure were computed.

Results
: A total of 840 NVAF patients qualified; with a mean age of 71 years, 49% female, and 14% African-American. Of these, 511 were exposed to warfarin, of whom 207 (40.5%) had complete INR records available. During 885 person-years of follow-up for these 207 patients, warfarin was continued through only 48.9% (432 person-yrs.). The INR was maintained at or above therapeutic range for 60.3% (260 person-yrs.) of the time for patients on warfarin. There were 18 strokes in the 207 with complete INR values, of which 11 (61%) occurred while off anticoagulation. Effective anticoagulation was associated with lower stroke rates (see table

).

Conclusion:
In this community-based setting, of the patients with complete INR records, effective warfarin use was associated with a 37.5% reduction in stroke risk; however, in these patients anticoagulation was attempted less than 50% of the time and therapeutic range achieved less than 25% of this time. Our data suggest further inquiry is needed into the reasons for failing to achieve adequate anticoagulation in actual practice.

Stroke Rates by Treatment and Effectiveness*
Abstract 5524: Progenitor Cell Mobilization is FGF-2 Dependant and Results in the Activation of Osteoclasts
Authors: Steinmetz Martin , Pelster Bastian , Arnal Jean-Francois , Nickenig Georg , Werner Nikos
Published: April 2024

Background
Fibroblast growth factor-2 (FGF2) regulates proliferation of bone marrow (BM) stromal cells and has pro-angiogenic effects regulating endothelial cell growth, migration, and reendothelialization. Here we determine the underlying molecular mechanisms of FGF2-dependent statin-mediated progenitor cell mobilization.

Methods and Results
To determine the molecular mechanisms of FGF2-mediated progenitor cell mobilization, FGF2−/− and wild type (wt) mice were treated with atorvastatin (10mg/kg body weight s.c. per day) and placebo. In contrast to wt mice, the number of Sca1/flk-1 positive EPC in peripheral blood (flow cytometry) of atorvastatin treated FGF2−/− mice did not increase. This was due to an accumulation of Sca1/flk-1 positive cells within the BM. Inhibition of EPC mobilization in FGF2−/− mice was associated with a delay in reendothelialization in a mouse model of a focal endothelial cell denudation and a severe reduction in neoangiogenesis despite statin treatment. BM transplantation experiments demonstrated that reconstitution of FGF2−/− mice with wt stem cells completely restored statin-mediated EPC mobilization while wt mice reconstituted with FGF2−/− stem cells developed a severe mobilization defect associated with an impaired endothelial dependent vasorelaxation (organ chamber) and enhanced neointima formation. In wt mice, statin treatment was associated with an increased number of activated osteoclasts compared to placebo treated mice which are known to play a pivotal role in stem cell mobilization. Treatment with osteoclast-activating RANKL induced an effective mobilization of Sca1/flk1 and Sca1/CD117 positive progenitor cells and enhanced the number of early outgrowth EPC.

Conclusion
Medullary FGF2 is essential in the statin-mediated mobilization of EPC from BM into peripheral blood. A reduction in the number and activation status of osteoclasts may account for the impairment in EPC mobilization.
Abstract 5527: Double-Stranded RNA Causes Leakage in Human Pulmonary Artery Endothelial Cell Monolayer
Authors: Bálint Zoltán , Zabini Diana , Végh Gergely A , Váró György , Wilhelm Imola , Krizbai István A , Kónya Viktória , Heinemann Ákos , Olschewski Horst , Olschewski Andrea
Published: April 2024

Proper function of endothelial cells is necessary to maintain vessel integrity and to regulate the uptake of circulating substances. Increased levels of circulating RNA may result from excessive cell damage or cancer. We investigated the effect of double-stranded RNA (dsRNA) on monolayer permeability, mechanical properties and intercellular junctions of primary human pulmonary artery endothelial cells (hPAECs). Primary hPAECs were used to investigate shape and mechanical changes caused by Poly I:C (synthetic dsRNA) and dsRNA incubation by means of
in situ
atomic force microscopy in time. To assess junctional changes, vascular endothelial cadherin staining was performed on control and 24h Poly I:C or dsRNA treated hPAECs. For visualizing cytoskeletal rearrangements, the F-actin network was stained with phalloidin. The cellular barrier properties of hPAECs, as a read-out for cell function, were assessed by measuring changes in the trans-endothelial electric resistance. The
in situ
atomic force microscopy topographies show a remarkable cell shape change after 2 hours of incubation with Poly I:C. The cell height is increased and they become more elongated. Furthermore, after 6 hours the cell-cell contacts are disrupted and the surface of the culturing dish appeared on the topographies. The local force measurements showed a time dependent stiffening of hPAECs under Poly I:C incubation. Parallel to this effect a 2-fold increase in the Young moduli of the cells was observable, which was further raised to 5-fold increase compared to the initial state, at 24 hours of incubation. The 24h Poly I:C incubated cells presented a decrease in VE-cadherin staining, pointing to loosened cell-cell contacts. The phalloidin staining showed an increased signal at the cell boundaries, confirming the cytoskeletal rearrangement. The electrical resistance of the monolayer was decreased by 50% after 24h Poly I:C or dsRNA treatment, whereas the first, significant drop in the resistance was already observed after 2 hours. In conclusion, our data suggest that double-stranded RNA causes endothelial leakage in human pulmonary endothelial cell monolayer and contributes to barrier disruption as well as endothelial dysfunction.
Abstract 5528: Novel Role of PKC
ζ
-ERK5 Complex in TNF
α
-mediated eNOS Protein De-stabilization
Authors: Nigro Patrizia , Abe Jun-ichi , Woo Chang-Hoon , Satoh Kimio , McClain Carolyn , O’Dell Michael R , Lee Hakjoo , Lim Jae-Hyang , Li Jian-Dong , Heo Kyung-Sun , Fujiwara Keigi , Berk Bradford C
Published: April 2024

Background:

Laminar steady (s−) flow-induced activation of ERK5 leads to KLF2 activation and subsequent eNOS expression, thus maintaining normal endothelial cell (EC) function. In contrast, PKC
ζ
has emerged as a pathological mediator of EC dysfunction based on

a positive correlation between PKC
ζ
activity and disturbed (d−) flow; and

the essential role of PKC
ζ
activation in TNF
α
stimulation of JNK, caspase-3 and EC apoptosis.

Emerging evidence demonstrates the reduction of eNOS expression in d-flow area, but the molecular mechanisms remain unknown.

Methods and Results:

First, both TNF
α
and d-flow activated PKC
ζ
, and TNF
α
inhibited s-flow-induced eNOS protein expression in EC. Interestingly, the reduction of eNOS expression by TNF
α
was abolished by adenoviral infection of a dominant negative PKC
ζ
(Ad.DN-PKC
ζ
). However, Ad.DN-PKC
ζ
could not reverse the TNF
α
-mediated reduction of KLF2/eNOS mRNA expression. These data portend a post-translational downregulation of eNOS expression via PKC
ζ
. Pretreatment with MG132 (proteosome inhibitor) inhibited TNF
α
-mediated reduction of eNOS protein, suggesting involvement of the ubiquitin-proteosome system. In vitro run-off assays showed that TNF
α
de-stabilized eNOS, and transduction of Ad.DN-PKC
ζ
stabilized eNOS, which suggested a key role of PKC
ζ
activation on eNOS stability. Using a mammalian two-hybrid assay we found a direct association between PKC
ζ
and ERK5. Further, we found that the catalytic domain of PKC
ζ
(aa 405–591) and the C-terminal region of ERK5 (aa 571– 806) appeared to be the domains required for this direct interaction. In vitro kinase assays revealed that Ser 486 of ERK5 is a novel phosphorylation site of PKC
ζ
. Then, we investigated the contribution of both PKC
ζ
-ERK5 binding and phosphorylation on eNOS stability. First, overexpression of the ERK5 fragment (aa 571– 806) inhibited PKC
ζ
-ERK5 binding and PKC
ζ
-mediated reduction of eNOS. Second, we found that an ERK5S486A mutant (Ad.ERK5 S486A) significantly inhibited eNOS de-stabilization compared with ERK5 wild type (Ad.ERK5-WT).

Conclusion:

These data suggest a key role of PKC
ζ
- ERK5 complex in TNF
α
-mediated eNOS protein de-stabilization and subsequent EC dysfunction.
Abstract 5536: Strain-dependent Susceptibility for Hypertension in Mice Resides in the Natural Killer Gene Complex
Authors: Taherzadeh Zhila , VanBavel Ed , de Vos Judith , Matlung Hanke , van Montfrans Gert , Brewster Lizzy , Seghers Leonard , Quax Paul H.A. , Bakker Erik N.T.P.
Published: April 2024

Hypertension is accompanied by vascular remodeling and inflammation. We tested the hypothesis that the sensitivity to develop hypertension and vascular remodeling depends on the immunological background.

Methods: Blood pressure, vascular remodeling and expression of inflammatory cytokines were determined in mice with a genetically determined difference in immune background: C57BL/6 mice and BALB/c mice; and additionally BALB.B6-Cmv1r mice, a congenic strain where the natural killer (NK) gene complex of C57BL/6 mice is introduced in BALB/c background. Mice were treated for 4 weeks with L-NAME (NG-nitro-L-arginine methyl ester). Systemic and local inflammatory responses were quantified in spleen and mesenteric arteries respectively.

Results: L-NAME treatment induced a significant increase in mean arterial blood pressure in C57BL/6 mice, from 101±4 mmHg (n=7) to 154±5 mmHg (n=5, P<0.001). This was accompanied by a highly significant increase in vessel wall-to-lumen ratio, from 0.104±0.002 (n= 6) to 0.126±0.003 in L-NAME treated mice (n=6, P<0.001). In BALB/c mice the blood pressure (117±3 mmHg for untreated vs. 126±6 mmHg for L-NAME treated mice) and wall to lumen ratio (0.104±0.003 in untreated vs. 0.113±0.003 in L-NAME treated mice) were not altered after 4 weeks of L-NAME treatment. The congenic BALB.B6-Cmv1r mice showed a similar sensitivity to develop hypertension and vascular remodeling as C57Bl/6 mice: blood pressure increased from 111±11 mmHg to 155±7 mmHg (P<0.01). The wall to lumen ratio increased from 0.107±0.003 to 0.125±0.008 (P<0.05). Expression of inflammatory markers such as IFN
γ
, IL1, IL10 and CD68 (macrophages) was similar in C57BL/6 and BALB/c, with exception of CD11b, which increased in vessels of C57BL/6 mice only. Confocal microscopy showed that CD11b co-localized with NK1.1, and reflected the recruitment of natural killer cells in C57BL/6 mice.

Conclusions: These data show that vascular remodeling and hypertension are strain dependent. We have identified the NK gene complex as important determinant in the genetically determined sensitivity to develop hypertension in mice.
Abstract 5544: ED-B Fibronectin Spect Imaging Detects Aortic Plaque Formation in vivo
Authors: Dietrich Thore , Berndorff Dietmar , Stepina Elisaveta , Hauff Peter , Dinkelborg Lutger , Neumann Marleen , Atrott Kirstin , Bouayrou Riad , Fleck Eckart , Graf Kristof
Published: April 2024

The extracellular matrix protein ED-B fibronectin (ED-B) is upregulated in inflammatory atherosclerotic lesions and is associated with local macrophage accumulation. In the present study we evaluated whether a conjugate of 99mTc with the single chain antibody against ED-B (99mTC-anti-ED-B) can be used for in vivo detection of atherosclerotic plaque lesions in apolipoprotein E- deficient mice (apoE
−/−
) compared to wildtype control mice (apoE
+/+
).

Methods:
12 month old apoE
−/−
and apoE
+/+
mice were studied by SPECT 4 hours after injection of 148 MBq of 99mTC-anti-ED-B. Finally mice were sacrificed and analyzed by autoradiography, histology, immunohistochemistry (IHC) and morphometry.

Results:
In vivo SPECT imaging of apoE
−/−
mice demonstrated a significant signal activity in the thoracic area which colocalizes with the aortic arch and the supraaortic arteries. The activity corresponded to aortic lesion formation was 52.2±40.6 (1/1*ccm). Low activity was observed in apoE
+/+
9.4±4.9 (1/1*ccm), which had no significant lesion formation. The strongest signals were detected in the aortic root, the aortic arch and along the abdominal part of the aorta from apoE
−/−
. No vascular signal activity was seen in apoE
+/+
after injection of 99mTC-anti-ED-B. The autoradiographic analysis of aortas from apoE
−/−
and apoE
+/+
, confirmed the in-vivo observation and demonstrated signal localization in typical plaque lesions, which significantly correlated with ED-B immunoreactivity, plaque lesion (r = 0.82, P<0.01) and local macrophage accumulation (IHC with Mac3 r = 0.89, P<0.01). There was a significant correlation between ED-B-radioactivity and macrophages (r= 0.93, P<0.01).

Conclusion:
SPECT imaging using a 99mTc labelled antibody against ED-B fibronectin is a robust method to detect and assess inflammatory plaque lesion formation in-vivo and correlates with local macrophage accumulation.
Abstract 5553: S100A1 Modulates Endothelial Nitric Oxide Availability by Co-ordinate Regulation of Nitric Oxide Synthase Phosphorylation and Gene Expression of Related Signaling Molecules
Authors: Teichert-Kuliszewska Krystyna , Desjardins Jean-Francois , Parker Thomas G
Published: April 2024

Background:
S100A1, a Ca
2+
-sensor protein in endothelial cell, regulates endothelial function. S100A1 knockout mice (KO) are hypertensive with impaired nitric oxide (NO) production and endothelial-dependent vasodilatation. We examine the impact of S100A1 on phosphorylation of Ser
1177
and Thr
495
, positive and negative regulatory residues, respectively, in endothelial nitric oxide synthase (eNOS) and on expression of diverse genes involved in eNOS signaling.

Methods and Results:
Endothelial cells (EC) were isolated and from pulmonary vasculature of wild-type (WT) and KO mice. WT EC exhibited higher proliferative rates than KO measured by WST-method, and expressed as absorbance/5000 cells/well (0.8923±0.091 vs. 0.268±0.055, p<0.05, n=3) and higher NO production (2214.00±1016.17 vs. 566.75±243.79 nM, p<0.05, n=6). Lower eNOS activity in KO was associated with higher and lower basal phosphorylation at Thr
495
and Ser
1177
, respectively. Bradykinin (100 nM) resulted in rapid (1 minute) 3-fold higher maximal phosphorylation of Ser
1177
in WT vs. KO with equivalent rapid dephosphorylation of Thr
495
in both cell types. By contrast, acetylcholine (10
μ
M) at 30 seconds produced more robust dephosphorylation of Thr
495
in WT compared to KO, with comparable effects on Ser
1177
phosphorylation. In KO versus WT EC, PCR microarray analysis demonstrated greater than 2-fold increase in mRNA expression of caveolin-1, NADPH oxidase 1, glutathione peroxidase, and calmodulin-1. KO EC exhibited a 5-fold reduction in eNOS mRNA, confirmed by a reduction in eNOS protein (4.02±0.19 fold, n=3).

Conclusion:
Absence of S100A1 in endothelial cells results in impaired proliferation, decreased NO availability, and regulation of gene expression that contributes or, alternately, compensates for this phenotype. Lack of S100A1 also impairs co-ordinated eNOS phosphorylation at baseline, and in response to agonists, that augments NO production. Thus, S100A1 plays a multifactorial and novel role in controlling NO synthesis and the endothelial phenotype.
Abstract 5559: Lack of Sequestosome1 Accelerates Neointimal Hyperplasia and Carotid Artery Remodeling
Authors: Sakai Satoshi , Warabi Eiji , Sugimoto Rika , Katayanagi Satoru , Yanagawa Toru , Aonuma Kazutaka , Ishii Tetsuro
Published: April 2024

The cytoplasmic signal adaptor protein designated sequestosome1 (SQSTM1) (A170 in mouse, ZIP in rat or p62 in human) plays a key role in modulating signal transduction via membrane receptors. SQSTM1 has a UBA ubiquitin-binding domain in the C-terminus and participates in the assembly of ubiquitinated protein aggregates termed sequestosome and modulation of ubiquitination pathways involved in NF-
κ
B activity and receptor trafficking. We have found that deficiency of SQSTM1 in mice exhibits mature-onset obesity accompanied by insulin and leptin resistance. We previously established that redox sensitive transcription factor Nrf2 upregulates SQSTM1 expression in response to atherogenic stimuli or laminar shear stress in vascular cells, and here examine role of SQSTM1 in neointimal hyperplasia and vascular remodeling in vivo following carotid artery ligation. Neointimal hyperplasia was markedly enhanced at proximal sites of ligation after 3 weeks in SQSTM1−/− (n=10) compared with wild type mice (n=10). The intimal area and stenotic ratio were 2.1- and 1.7-fold higher in SQSTM1−/− mice, respectively, indicating enhanced proliferation of vascular smooth muscle cells (SMCs). When aortic SMCs were isolated from wild type and SQSTM1−/− mice and cultured in vitro, we found that SQSTM1−/− SMCs proliferated more rapidly in response to fetal calf serum (FCS) and attained 2–3-fold higher cell densities compared to wild type SMCs. Moreover, FCS caused a rapid transient down regulation of SQSTM1 in wild type SMCs. Early and late phases of p38MAPK activation in response to FCS stimulation were more enhanced in SQSTM1−/− SMCs, and inhibitors of p38 and ERK1/2 signaling pathways significantly attenuated SMCs proliferation. In summary, SQSTM1−/− mice exhibit enhanced neointimal hyperplasia and vascular remodeling following arterial ligation in vivo. The enhanced proliferation of SQSTM1−/− aortic SMCs in vitro highlights a novel role for SQSTM1 in suppressing smooth muscle proliferation following vascular injury.