bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4940: Inflammatory Thrombotic Interactions in Young Type 1 Diabetes Subjects: Effects of Glycaemic Control
Authors: Hess Katharina , Mathai Mathew , Koko Thet , Standeven Kristina F , Holland Philip , Carter Angela M , Phoenix Fladia , Grant Peter J , Ajjan Ramzi A
Published: April 2024

The inflammatory and thrombotic pathways have a crucial role in the development of coronary artery disease, and inflammatory thrombotic markers predict cardiovascular events. Although the atherosclerotic process in type 1 diabetes (T1DM) starts at an early age, little is known about inflammatory markers and clot structure in this population. The aims of this study were to investigate

C-reactive protein (CRP), complement C3 (C3) and fibrin clot structure/fibrinolysis in children and young adults with T1DM,

the effects of improving glycaemic control on these parameters and

complement/fibrin interactions affecting clot structure/function.

In 47 children (14±3yrs) C3 levels were higher in T1DM than controls (1.13±0.24 versus 0.83±0.24mg/ml respectively; p<0.001), whereas CRP levels were no different. Clot final turbidity (FT), an indicator of clot density, was higher in diabetes children compared with controls, and lysis time longer (599±18 and 516±17 secs, respectively, p=0.02). In 18 young adults with T1DM (23±5yrs) a 1.3% reduction in HbA1c was associated with a decrease in C3 levels from 1.09±0.05 to 0.96±0.05mg/ml (p<0.05), with no effect on CRP levels. Improving glycaemic control reduced FT and shortened lysis time from 601±21 to 550±19 secs (p=0.02). Clot FT and lysis correlated positively with C3 plasma levels (r=0.39 and r= 0.585, respectively; p<0.01). C3 incorporation into the plasma clot was 26% and 19% in T1DM and controls respectively, a difference that failed to reach statistical significance. However, clots made from plasma-purified fibrinogen diabetes patients exhibited a 71% increase in lysis time in the presence of purified C3, whereas this increase was only 41% using fibrinogen purified from controls (p=0.03). These results demonstrate that young children with T1DM already have evidence of inflammatory thrombotic processes as indicated by elevated C3 levels, a denser clot structure and prolonged clot lysis times. Interactions between C3 and fibrin occur in non-diabetic subjects, but are significantly enhanced in the presence of diabetes. Improving glycaemic control is associated with reduction in C3 levels and favourable changes in clot structure/function, which is potentially due to a direct effect of C3 on clot lysis.
Abstract 4947: Maintenance Treatment With Ticagrelor Yields Greater and More Consistent Inhibition of Platelet Function Compared to Clopidogrel in Patients With Acute Coronary Syndromes: Results From the PLATO PLATELET Substudy
Authors: Storey Robert F , Angiolillo Dominick J , Patil Shankar B , Desai Bhaloo , Husted Steen , Emanuelsson Håkan , Wallentin Lars
Published: April 2024

Background: The PLATO study compared the efficacy of ticagrelor (T), an oral reversible P2Y
12
receptor antagonist, and clopidogrel (C) in patients with non ST elevation ACS or primary PCI-treated STEMI patients. In the PLATO PLATELET substudy, we assessed the extent of inhibition of platelet function in PLATO study patients.

Methods and Results: 69 patients randomised in the PLATO study to receive maintenance therapy with either C 75mg qd (n=32) or T 90mg bid (n=37) underwent platelet function testing after at least 28 days of treatment. Light transmittance aggregometry (LTA) with ADP (5 and 20 uM), VerifyNow P2Y12 (VN) and VASP assays were performed prior to maintenance dose (‘trough’) and 2– 4h after dosing (‘peak’) in all patients. Mean % maximum LTA response induced by ADP 20 uM at trough was 48±14 and 36±12% (p<0.001) and at peak was 44±15 and 28±10% for C and T respectively (p<0.0001)(Figure

). Similar patterns were seen for ADP 5 uM. VN values at trough were 214±79 and 79±78 PRU for C and T and at peak were 196±95 and 31±32 PRU respectively (both p<0.0001)(Figure

). PRI values at trough were 55±22 and 27±21% for C and T and at peak were 44±21 and 14±13% respectively (both p<0.0001). VN and VASP at peak particularly showed more consistency and less variability in response with T compared to C.

Conclusion: Maintenance therapy with ticagrelor 90mg bid in patients with ACS achieves greater and more consistent inhibition of platelet function compared to standard clopidogrel therapy.
Abstract 4950: Loss of SM22 Couples Arterial Inflammation With Osteochondrogenesis in a Murine Artery Injury Model
Authors: Shen Jianbin , Yang Maozhou , Ju Donghong , Jiang Hong , Zheng Jianpu , Li Li
Published: April 2024

Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and calcification are present. However, the role of this down-regulation in arterial pathogenesis is unknown.

Hypothesis
: Down-regulation of SM22 may actively contribute to arterial pathogenesis.

Methods
: Five
Sm22
knockout (
Sm22
−/−
) mice and their wild type littermates were subjected to carotid denudation, an artery injury model. Analyses were conducted on carotids two weeks after injury. SM22 knockdown was performed in triplicate using siRNA in a VSMC line, PAC1, followed by downstream experiments 3 days after transfection.

Results
:
Sm22
−/−
mice developed both enhanced arterial inflammatory response and prominent medial osteochondrogenesis along with remarkable NF-
κ
B activation compared to their wild-type littermates. The inflammation was evidenced by excessive artery swelling, macrophage infiltration and high induction of pro-inflammatory molecules including COX-2, MCP-1, VCAM-1, ICAM-1 and CX3CL1. The medial osteochondrogenesis was characterized by augmented expression of type II collagen, aggrecan, osteopontin, BMP2, SOX9 and RUNX2. In accordance with the
in vivo
findings, expression of the aforementioned pro-inflammatory genes and two key osteochondrogenic transcription factors
Sox9
and
Runx2
was up-regulated after SM22 knockdown in PAC1 cells. Interestingly, SM22 knockdown also led to NF-
κ
B activation, and inhibition of NF-
κ
B pathway reversed the up-regulation of both pro-inflammatory genes and osteochondrogenic genes. As an initiator of NF-
κ
B activation, reactive oxygen species (ROS) production was boosted after SM22 knockdown, and ROS scavenging effectively blocked NF-
κ
B activation and induction of these genes. Further, the compromised actin cytoskeleton after SM22 knockdown might contribute to the elevated ROS.

Conclusions
: These findings suggest that loss of SM22 in VSMCs coupled injury induced arterial inflammation with osteochondrogenesis in part via ROS induced NF-
κ
B activation and that SM22 plays both anti-inflammatory and anti-osteochondrogenic roles in arterial diseases partly by maintaining actin cytoskeleton integrity.
Abstract 4951: A Genetic Model of Vascular Inflammation: Switchable Expression of Hypoxia Inducible Factor1 Alpha (HIF1a) Produces Multistage Targetable Endothelial Activation
Authors: Arbeit Jeffrey M , Santeford Andrea C , Oladipupo Sunday S , Kaneda Megan M , Lanza Gregory M , Wickline Samuel A
Published: April 2024

Angiogenesis is a linchpin of survival and tissue restoration following ischemia, but also a prerequisite for atherosclerosis. Angiogenesis is induced by a complex interplay of tissue hypoxia, acidosis, and inflammation; conditions coordinated by a myriad of molecular regulators. We have created a switchable transgenic model of microvascular network elaboration allowing day-to-day kinetic analysis. Doxycycline induction of a Tet-regulated constitutively active hypoxia-inducible factor-1a mutant transgene, HIF-1a ^Pro402/564A/Asn803A was targeted to the skin (TetOn-HIF-1a mice). These mice first demonstrate microvascular dilatation on Dox d1, a burst of endothelial proliferation on d3, followed by an exponential decline by d14 –28. Plasma levels of the bone marrow mobilization factors VEGF, PlGF, and SDF1 spike on d3, while tissue levels of these recruitment and retention factors peak at d14. HIF-1 conditioned stroma and activated endothelium recruit large numbers of bone marrow-derived cells of multiple monocytic lineages. There is a striking, day/stage specific decoration of TetOn-HIF-1a microvessels by targeted nanoparticles. AlphaVbeta3 integrin-targeted nanoparticle binding is evident on d3, but absent by d14. VCAM-1-targeted nanoparticles display similar kinetics peaking on d3 (see Figure

); falling to background levels by d14. Thus, a monogenetic perturbation of a master regulatory transcription factor recapitulates the complex multitissue compartmental contributions to ischemic angiogenesis, providing a platform for functional investigation of the biology and therapeutic efficacy of inflammation with vascular-targeted nanoparticles.

Figure 1.

Decoration of the d3 TetOn-HIF- 1a microvasculature by Alexa Fluor 594 loaded, VCAM targeted, PFOB nanoparticles.
Abstract 4954: A Novel Role of CCN3 in Regulating Endothelial Inflammation
Authors: Lin Zhiyong , Natesan Viswanath , Shi Hong , Hamik Anne , Kawanami Daiji , Hao Caili , Mahabaleshwar Ganapati H , Atkins G. Brandon , Rittié Laure , Firth Sue M , Perbal Bernard , Jain Mukesh K
Published: April 2024

Background
: The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. However, the molecular mechanisms regulating endothelial homeostasis remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. While CCN3 expression in endothelial cells has been described, its function in this cell type has yet to be elucidated.

Methods and Results
: A detailed immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, as well as veins. Likewise, CCN3 expression is observed in cultured human endothelial cells derived from a variety of vascular beds. CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) by ~27-fold. Similarly, treatment of HUVEC with HMG CoA-reductase inhibitors (statins) induced CCN3 expression by ~32-fold. A combination of promoter deletion and mutational analyses coupled with chromatin immuno-precipitation studies identified the Kruppel-like factor 2 as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines such as TNF
α
reduced CCN3 expression. Functionally, adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of VCAM-1. Consistent with this observation, CCN3 overexpression significantly reduced monocyte adhesion. Conversely, siRNA mediated “knockdown” of CCN3 protein in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Mechanistically, gain and loss-of-function studies demonstrate that CCN3 negatively regulates NF-
κ
B activity by reducing its translocation into nucleus and DNA binding to VCAM-1 promoter, suggesting that CCN3’s anti-inflammatory effects occur secondary to inhibition of NF-
κ
B nuclear accumulation.

Conclusions:
This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.
Abstract 4963: Cerebral Microvascular Damage in Elderly Depressed Patients is Associated With Structural and Functional Abnormalities of Subcutaneous Small Arteries
Authors: Greenstein Adam , Paranthaman Raghu , Malik Rayaz , Heagerty Anthony M , Jackson Alan , Burns Alistair , Baldwin Robert C
Published: April 2024

Introduction
: Late-life depression is increasingly viewed as a vascular illness with patients exhibiting characteristic white matter brain lesions and in-vivo endothelial dysfunction. Although the ‘vascular depression’ hypothesis pertains to the microvasculature, this circulation has yet to be studied.

Methods and Results
: 25 patients aged 72.6±5.4 years with late-life depression were compared with 21 control participants aged 72.4±6.5 years. The groups were matched for blood pressure, glycaemia, lipids, co-morbidities and treatment with statins and agents modifying the renin-angiotensin axis. Patients underwent MRI brain scan and a matched subset underwent subcutaneous gluteal fat biopsy (15 patients and 15 control participants) from which small arteries were isolated and studied using pressure myography. Depressed patients showed evidence of cerebral microvascular damage (MRI scan): Virchow Robin Scores- Patients:3.88±1.45 vs Controls:2.76±1.55, p=0.01, White Matter Lesions- Patients:342±748.1 vs Controls:202±881, p=ns. Small artery studies demonstrated endothelial dysfunction (Percentage relaxation: Patients:82±4% vs Controls:96±1.5%, p<0.05) which was not completely explained by reduced nitric oxide synthase bioavailability (Relaxation after LNMMA incubation: Patients:67±5.6% vs Controls:83±11%, p<0.05). Contractility to Nor-adrenaline was equivalent between the groups. There was also abnormal hypertrophic wall growth in depressed patients (Medial Cross sectional area: Patients:13294
μ
m
2
vs Controls:10555
μ
m
2
, p<0.05, Growth Index: 26%), but no difference in arterial distensibility.

Conclusions
: Despite identical cardiovascular profiles, patients with late-life depression show profound abnormalities in both structure and function of small arteries, suggestive of deficiencies in local autoregulation. Although the arteries taken were from subcutaneous fat, our findings are in support of cerebral microvascular pathology in this condition.
Abstract 4965: High Mobility Group Box 1 Protein Expression is Associated With Expansion and Rupture of Human Abdominal Aortic Aneurysm
Authors: Kohno Takashi , Anzai Toshihisa , Kaneko Hidehiro , Shimizu Hideyuki , Shimoda Masayuki , Miyasho Taku , Okamoto Minoru , Yokota Hiroshi , Yamada Shingo , Yoshikawa Tsutomu , Okada Yasunori , Ishizaka Akitoshi , Yozu Ryohei , Ogawa Satoshi
Published: April 2024

Background: Abdominal aortic aneurysm (AAA) expansion is characterized by widespread transmural inflammation and extracellular matrix degradation. High mobility group box1 protein (HMGB1) is one of the recently defined damage-associated molecular pattern molecules derived from necrotic and activated inflammatory cells, and functions as a proinflammatory mediator. The aim of this study was to elucidate the clinical significance of HMGB1 expression in patients with AAA.

Methods and Results: Serum HMGB1 concentration was measured by enzyme-linked immunosorbent assay in 40 patients with infrarenal AAA (6 patients with ruptured and 34 patients with nonruptured AAA), who underwent surgical repair of AAA. Serum HMGB1 level on admission did not differ between the presence or absence of atherosclerotic risk factors, including smoking, hypertension, hyperlipidemia, age and sex, and cardiovascular medications. These variables were similar between patients with ruptured and nonruptured AAA. Serum HMGB1 level was significantly higher in patients with ruptured AAA compared with nonruptured AAA (4.9±3.4 vs 1.9±2.9 ng/ml, p<0.05). Thirty-eight AAA tissues taken during surgical repair were examined for immunoblotting (4 ruptured and 34 nonruptured AAA). The HMGB1 level in aneurysmal tissue was positively correlated with AAA diameter (r=0.43, p<0.05). In addition, HMGB1 expression was higher in patients with ruptured AAA than those with nonruptured AAA (p<0.05). Gelatin zymography showed that matrix metalloproteinase (MMP)-2 and -9 levels were significantly elevated in the ruptured AAA compared with nonruptured AAA (all p<0.05). Patients with MMP-2 level>median value had a higher HMGB1 level in aneurysmal tissue than those without (p<0.05). Immunohistochemistry demonstrated that HMGB1 was highly expressed in macrophages and neutrophils within the degenerating aortic media and periadvential fat tissues of AAA. HMGB1 was also expressed in smooth muscle cells and fibroblasts within the degenerating aortic media.

Conclusions: Elevation of HMGB1 level in serum and aneurysmal tissue was observed in patients with expansion and rupture of AAA, in association with activation of MMPs. Serum HMGB1 could be a useful marker of the development of AAA rupture.
Abstract 5006: Aspirin Non-Response as Determined by Multiple Platelet Function Assays
Authors: McCall Meadhbh , Peace Aaron , Tedesco Tony , Conroy Ronan , Foley David , Cox Dermot
Published: April 2024

Background.
While low dose aspirin has become the standard of care for the prevention of cardiovascular events there is evidence that enteric aspirin may not be as effective as plain aspirin.

Methods.
We screened 138 patients with cardiovascular disease who were taking aspirin and measured their aspirin response with three platelet function assays: Light Transmission aggregometry (LTA) with arachidonic acid, the VerifyNow™-Aspirin and Thromboxane B
2
(TxB
2
) ELISA. Resistance was defined as >20% aggregation by LTA, >550 ARU in the VerifyNow™ and >10 ng/ml by TxB
2
ELISA. Patients aspirin preparations were altered and dosages increased to a maximium of 300mg plain aspirin if patients showed lack of response to their aspirin by any one assay.

Results.
24% of patients were non-responders in at least one assay. There was 13% agreement between LTA and TxB
2
, 9% between TxB
2
and VerifyNow™ and 19% between VerifyNow™ and LTA. Only 9% of patients defined as resistant by one assay were resistant to all three. Upon repeat measurement, with compliance encouraged (through education) and assured (directly observed) only 6.5% of patients were still non-responders in at least one assay. Compliant non-responding patients were heavier than the responding population (98.5kg versus 77.9kg). Only 2.7% (105kg) of the cohort remained non-responders after swtiching their aspirin preparations from enteric-coated to plain. Increasing aspirin dosage to 150mg plain aspirin resulted in 0.4% non-response.

Conclusions.
These data show that whilst aspirin non-response is a problem, there is significant inter-assay agreement making it difficult to define. However, over 2/3 (69.6%) of non-responding patients became responders after encouraging compliance. The remaining patients were heavier patients and switching to plain aspirin which has a higher bioavailability improved their response to aspirin. Only 1 patient could be detected who did not respond to aspirin suggesting that aspirin resistance is very rare and most cases are due to poor compliance or underdosing in heavier patients exacerbated by the lower bioavailability of enteric-coated aspirin. While there was poor agreement between assays they were effective at identifying non-compliant patients.
Abstract 5010: Is Impact of Proton Pump Inhibitor on Platelet Reactivity to Clopidogrel A Class Effect?
Authors: Hokimoto Seiji , Ogawa Hisao , Yamamoto Koichiro , Kaikita Koichi , Ono Takamichi , Nakagawa Kazuko
Published: April 2024

Background: Concomitant administration of clopidogrel and proton pump inhibitor (PPI) is associated with the reduced effect on platelet response to clopidogrel, and increased risk of adverse outcome after stent placement in patients with acute coronary syndrome. However, it remains unknown whether all PPIs may reduce platelet inhibitory effects of clopidogrel. The aim of this study was to examine the impact of rabeprazole, which is one of PPIs and mostly not metabolized by CYP2C19, on platelet response to clopidogrel.

Methods: Adenosine diphosphate (ADP) induced platelet aggregation (PA) using light transmission aggregometer (MCM HEMA TRACER 313) was measured as response to clopidogrel in 127 patients (male 64%, age68 years) with coronary artery disease (CAD), and CYP2C19 polymorphism, which is involved with metabolism of clopidogrel and PPI, is also examined using PCR.

Results: CYP2C19 genotypes were divided into 3 groups such as homozygous extensive metabolizer (homoEM, n=46, CYP2C19*1/*1), heterozygous EM (heteroEM, n=52, *1/*2, *1/*3), and poor metabolizer (PM, n=29, *2/*2, *2/*3, *3/*3). In contrast to distribution of CYP2C19 polymorphism in Caucasian, the rate of CYP2C19 loss of function was higher in Japanese. ADP induced PA decreased significantly in homoEM, subsequently heteroEM, and was not well inhibited in PM (PA; 3250+/−1800, 4250+/−1300, and 5010+/−1370, respectively). However, ADP induced PA was not significantly different between patients treated with (n=29; PA=4637 +/−1800) and without rabeprazole (n=98; PA=3980 +/−1159). Moreover, there were no differences in ADP induced PA between groups with and without rabeprazole, in each subgroup categorized according to the genotype of CYP2C19. As to clinical events, the intake of PPI was not associated with increased risk of mortality and stent thrombosis in patients treated with stent delivery.

Conclusion: In contrast to the reported negative PPI-clopidogrel drug interaction, the intake of rabeprazole is not associated with impaired response to clopidogrel and increased risk of mortality and stent thrombosis in patients with CAD.
Abstract 5013: Body Mass Index is the Main Determinant of High On-treatment Platelet Reactivity and of Failed Dose Adjustment According to Platelet Reactivity Monitoring
Authors: Bonello N , Armero S , Paganelli F , Mancini J , Maillard L , de Labriolle A , Barragan P , Bonello Caroline , Dignat-George F , Camoin-Jau L , Bonello Laurent
Published: April 2024

High on-treatment platelet reactivity(HTPR) after clopidogrel loading dose(LD) predicts the risk of thrombotic events after percutaneous coronary intervention(PCI). We have demonstrated that HTPR could be overcome in the majority of cases using dose-adjustment according to PR monitoring resulting in an improved clinical outcome. However this strategy failed in 10% of patients with HTPR. Cytochromes(CYP)2C19 polymorphism is a major determinant of the response to clopidogrel and could be responsible for dose-adjustment failure. We aimed to determine predictors of a failure of the dose-adjustment strategy. Seventy-three patients undergoing PCI were included in this prospective multicentre study. A VASP index ≥ 50% after a 600mg LD of clopidogrel defined HTPR. Dose-adjustment was performed according to PR monitoring to reach a VASP index < 50%. Genetic polymorphism of CYP2C19 was determined by direct sequencing. Clinical predictors of HTPR were BMI(p=0.01), diabetes(p=0.03) and acute coronary syndrome(p=0.02). The mutant 2* allele of CYP2C19 681A>G loss of function polymorphism was also significantly associated with HTPR(p=0.04). The rate of successful dose adjustment was similar in carriers of the CYP2C19*2 allele and carriers of the wild-type allele. The only independent predictor of a failed dose-adjustment was a high BMI(p=0.01). In conclusion, high BMI, acute coronary syndrome, diabetes and CYP2C19*2 are associated with HTPR after a 600mg LD of clopidogrel. Dose-adjustment overcomes HTPR in carriers of the CYP2C19*2 allele. BMI is the only independent predictor of failed dose adjustment. Thus drug under-dosage seems to be the main determinant of HTPR.
Abstract 5033: Safety and Efficacy of Carotid Stenting in the Very Elderly
Authors: Grant Arthur , White Christopher , Ansel Gary , Bacharach Michael , Metzger Christopher , Velez Carlos
Published: April 2024

Background: Carotid artery stenting (CAS) has emerged as an alternative to carotid endarterectomy (CEA) in patients at high-risk for complications from surgery. The very elderly (> 79 years old) are one subgroup of patients identified as being at increased risk for carotid surgery. However, there is concern that the very elderly are also at increased risk for complications of CAS. A stroke and death rate of 12% was reported in very elderly patients during the roll-in phase of Carotid Revascularization Endarterectomy versus Stent Trial (CREST). We are reporting on a large clinical series of CAS with independent neurological assessment in the very elderly.

Methods: Between 1994 and 2008, a consecutive series of 418 CAS patients (> 79 years old) were treated at four high-volume centers with extensive CAS experience. Independent neurologic assessment was performed after CAS procedures. Thirty-day follow-up information was available in 389 patients.

Results: The average age was 83.2±2.8 years. Most patients were male (63.2%) and the target lesion carotid stenosis was asymptomatic in two-thirds (68.2%) of patients. The majority of patients treated with CAS had a history of coronary artery disease (74.4%), hypertension (87.8%) and dyslipidemia (71.1%). One third (30.1%) were diabetic and more than half (56.5%) were current or former smokers. Embolic protection devices (EPD) were used in 78.7% of cases with the CAS procedure being performed prior to EPD availability being the most common reason for not using them. The 30-day incidence of stroke and death was 2.8% (11/389). The cumulative incidence of major cardiovascular events (stroke, death or myocardial infarction) during that time period was 3.3% (13/389).

Conclusion: This large series of CAS with independent neurologic assessment is convincing evidence that the very elderly (> 79 years old) can safely undergo CAS with stroke and death rates comparable to younger patients. The key to obtaining these excellent results is that CAS be performed by high-volume, experienced operators who exercise restraint regarding patient selection.
Abstract 5040: Antioxidative Treatment in Salt Loaded Zucker Diabetic Fatty Rats Prevents Vascular Hypertrophy
Authors: Resch Markus , Fredersdorf Sabine , Birner Christoph , Schmid Peter , Schach Christian , Riegger Günter A , Luchner Andreas , Endemann Dierk H
Published: April 2024

Vascular hypertrophy of resistance arteries is an early form of end-organ damage. In this study we test whether antioxidative treatment by inhibition of distinct sources of oxidative stress (namely NADPH oxidase and mitochondria) improves vascular remodeling and thereby reduces end-organ damage in diabetes mellitus (DM) associated with salt sensitive hypertension. As a model of type 2 DM Zucker Diabetic Fatty (ZDF) rats receiving high salt (5.5%) diet were divided into 3 groups receiving no antioxidative treatment (ZDF+S) or treated with inhibitors of sources of oxidative stress with apocynin (inhibitor of the NADPH oxidase) (ZDF+S+A) or with PJ-34 (PARP-inhibitor) (ZDF+S+PJ) for 6 weeks. Euglycemic Zucker lean (ZDL) rats served as controls. Systolic blood pressure (SBP) was measured weekly by tail cuff method. MRNA expressions of p22phox as a subunit of NADPH oxidase were quantified by RT-qPCR in mesenteric resistance arteries. Vascular hypertrophy (media to lumen ratio, M:L) and passive mechanical stiffness of mesenteric resistance arteries were assessed using a pressurized myograph. SBP was significantly higher in ZDF+S compared to ZDL (ZDF+S: 144±1; ZDL: 134±3 mmHg; p<0.05). There was no relative effect of antioxidative treatment on serum glucose levels and SBP (ZDF+S+A: 142±1; ZDF+S+PJ: 145±2 mmHg). M:L of mesenteric arteries was significantly increased in ZDF+S vs ZDL (5.8±0.3 vs 4.7±0.1; p<0.05) indicating marked vascular hypertrophy. Antioxidative treatment significantly prevented vascular hypertrophy by inhibition of NADPH oxidase with apocynin (M:L in ZDF+S+A: 4.4±0.2; p<0.05 vs ZDF+S) and by PARP-inhibition with PJ-34 (M:L in ZDF+S+PJ: 4.5±0.1; p<0.05 vs ZDF+S). Levels of p22phox were increased in ZDF+S vs ZDL (1.7±0.2 vs 1.3±0.2). Antioxidative treatment decreased the expression of p22phox (ZDF+S+A: 1.3±0.1; ZDF+S+PJ: 1.4±0.1). Antioxidative treatment did not change vascular strain indicating no effect on vascular stiffness. Antioxidative treatment with an inhibitor of NADPH oxidase and with a PARP-inhibitor prevented vascular hypertrophy in salt loaded ZDF rats without changing blood pressure levels. Thus, targeted inhibition of sources of oxidative stress may be of importance to prevent end-organ damage in diabetes.
Abstract 5044: Aldose Reductase-Dependent Effects on Endothelial Dysfunction and Endothelial Progenitor Cells in Diet-Induced Obesity
Authors: Hellmann Jason , Haberzettl Petra , Wheat Laura , Conklin Daniel J , O’Toole Timothy E , Bhatnagar Aruni
Published: April 2024

Chronic diabetes increases cardiovascular disease risk. Long-term hyperglycemia in diabetes increases non-glycolytic consumption of glucose via the polyol pathway, which has been linked to the development of secondary diabetic complications. To examine the contribution of this pathway, wild-type (WT; C57BL/6) mice and mice deficient in aldose reductase (AR-null), an enzyme catalyzing the first step of the pathway, were fed normal chow (NC, 13.5 % kcal fat) or a high-fat diet (HFD, 42% kcal fat) for 12 weeks. In comparison with WT mice (n=12), AR-null (n=12) mice gained significantly more weight (+14.9±0.9 g vs. +20.8±1.1 g; P<0.05) and displayed greater increase in plasma cholesterol (+133±213 mg/dl vs. +194±17 mg/dl; P<0.05) and insulin (+0.40±0.11 vs. +0.57±0.07 ng/ml; P<0.05). Insulin (100 nM) exposure increased Akt phosphorylation in isolated aorta of WT NC mice but not of AR-null mice (see figure

). Aorta isolated from HFD WT and AR-null mice were insulin-insensitive. High-fat feeding decreased acetylcholine-induced relaxation of aorta measured
ex vivo
in AR-null mice by 25±4 % (P<0.05, n=12) but not in WT mice. High fat feeding decreased the levels of Sca
+
/Flk
+
endothelial progenitor cells (EPCs) in the peripheral blood (per 50,000 events: 197±79 vs. 103±19; P<0.05) yet increased EPCs in spleen (812±456 vs. 2088±1173; P<0.05) and bone marrow (432±269 vs. 1031±658; P<0.05) in WT mice. These changes were absent in AR-null mice. Collectively, these data suggest that AR-dependent metabolism during pre-diabetic conditions plays a significant role in the maintenance of endothelial health perhaps through impaired insulin signaling and trafficking of EPCs.
Abstract 5056: Genetic Disruption of the ATP-binding Cassette Transporter BCRP1/ABCG2 Impairs Cardiac Repair After Myocardial Infarction
Authors: Higashikuni Yasutomi , Nakamura Kazuto , Takaoka Minoru , Enomoto Soichiro , Iwata Hiroshi , Sahara Makoto , Tanaka Kimie , Sata Masataka , Nagai Ryozo
Published: April 2024

Introduction
Recently, the ATP-binding cassette transporter BCRP1/ABCG2 has been shown to regulate the function and survival of side population cells, which have been identified in various organs including heart and have stem cell properties. In addition, previous studies have revealed that BCRP1/ABCG2 is also expressed in endothelial cells of capillaries and arterioles in heart. This study was performed to clarify the role of BCRP1/ABCG2 in cardiac repair after myocardial infarction (MI).

Methods and Results
MI was induced in 8- to 12-week-old wild-type (WT) mice (n=51) and Bcrp1/Abcg2 knock-out (KO) mice (n=60) by ligating the left anterior descending artery. At 28 days after MI, the survival rate was significantly lower in KO mice than in WT mice (28.3% versus 74.5%, p=0.0001). The main cause of death in KO mice was cardiac rupture (CR) (67.4%), whereas CR was observed in only 30.8% among WT mice (p=0.019). Echocardiography showed that ventricular remodeling was more deteriorated in KO mice than in WT mice (left ventricular end-diastolic diameter, 5.16±0.31 mm versus 4.47±0.30 mm, p<0.05; ejection fraction, 23.8±4.9% versus 32.9±5.8%, p=0.002), although baseline data did not differ between the two groups. The left ventricular end-diastolic pressure was more elevated in KO mice than in WT mice (10.7±4.1 mmHg versus 5.3±3.0 mmHg, p=0.006). Histological assessment revealed that myocyte hypertrophy and fibrosis in the non-infarcted area were exacerbated in KO mice compared with those in WT mice (myocyte cross-sectional area, 397.6±44.9
μ
m
2
versus 312.7±27.1
μ
m
2
, p<0.05; collagen volume fraction, 3.8±1.2% versus 2.2±0.7%, p=0.008). Capillary density in the peri-infarction area at 5 days after MI was significantly reduced in KO mice than in WT mice as determined by anti-CD31 immunostaining (1033±188/mm
2
versus 1414±214/mm
2
, p=0.008). The number of macrophages (MΦs) and myofibroblasts (MFs), as identified by immunostaining for Mac-3 and
α
-SMA respectively, also decreased in KO mice than in WT mice (MΦs, 1359±251/mm
2
versus 1906±440/mm
2
, p=0.04; MFs, 1780±479/mm
2
versus 2582±330/mm
2
, p=0.007).

Conclusions
Cardiac repair after MI was impaired in KO mice. BCRP1/ABCG2 may play an important role in angiogenesis and in recruitment of MΦs and MFs after MI.
Abstract 5058: Glucose OxidationStimulation Affords Dramatic Protection Against in vivo Myocardial Ischemia Reperfusion Injury
Authors: Ussher John Edward R , Wang Wei , Oka Tatsujiro , Dyck Jason R , Lopaschuk Gary D
Published: April 2024

During reperfusion of the ischemic myocardium, fatty acid oxidation rates quickly recover, while glucose oxidation rates remain low, which decreases cardiac efficiency during reperfusion. Malonyl CoA decarboxylase (MCD) degrades malonyl CoA and increases fatty acid oxidation by relieving malonyl CoA-dependent inhibition of carnitine palmitoyl transferase-1. Studies have shown that MCD inhibition improves reperfusion recovery of ischemic hearts ex vivo by reducing fatty acid oxidation rates and increasing glucose oxidation rates. The objective of this study was to determine whether MCD inhibition would translate into a reduction in infarct size. Wild Type (WT, n = 7) or MCD deficient (MCD−/−, n = 7) mice had their left anterior descending coronary artery ligated for 30 min followed by 24 hr reperfusion. MCD−/− mice demonstrated a dramatic reduction in infarct size compared to WT mice (10.8±3.8 vs. 39.5±4.7 % of area at risk, P < 0.05) despite similar areas at risk (53 vs. 52 % of left ventricle). Furthermore, malonyl CoA levels were higher in the area at risk of hearts from MCD−/− mice (3.9±0.7 vs. 1.9±0.5 nmol/g wet weight, P < 0.05). Because the protection against ex vivo ischemia/reperfusion injury in MCD−/− mice was associated with increased myocardial glucose oxidation rates, secondary to an inhibition of fatty acid oxidation, we next determined whether direct stimulation of pyruvate dehydrogenase (PDH) and subsequent glucose oxidation would also afford protection against myocardial infarction. Infusion of the PDH activator, dichloroacetate (100 mg/kg intraperitoneal injection, followed by hourly injections of 50 mg/kg for the 24 hr duration of reperfusion), demonstrated significant protection against myocardial infarction (36.4±2.4 vs. 50.3±3.2 % of area at risk, P < 0.05). We demonstrate that MCD inhibition is beneficial against ischemia/reperfusion injury, possibly due to its effects on glucose oxidation stimulation, validating the optimization of myocardial metabolism as a novel therapy for ischemic heart disease.
Abstract 5064: Endothelial-Specific Deletion of Cx40 Promotes Atherosclerosis by Increasing CD73-Dependent Leukocyte Adhesion
Authors: Chadjichristos Christos E , Scheckenbach Ludwig , Sarieddine Maya Z Richani , Roth Isabelle , Bacchetta Marc , de Wit Cor , van Veen Toon A , Chanson Marc , Kwak Brenda R
Published: April 2024

Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. Here, we sought to determine whether Cx40 contributes to the development of the disease. Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Tie2CRE+ Cx40fl/fl ApoE−/− mice; EC-Cx40del mice). EC-Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in EC-Cx40del mice after 10 weeks high-cholesterol diet; lipid deposition in the thoracic-abdominal aortas was 21.4±3.5% (N=10, p<0.01) in EC-Cx40del mice as compared to 12.6±1.3% for Tie2CRE+ Cx40wt/wt ApoE−/− and 7.1±1.3% for Tie2CRE- Cx40fl/fl ApoE−/− controls. Moreover, spontaneous lesions were observed in the aortic sinuses of young EC-Cx40del mice without diet. These lesions showed monocyte infiltration into the intima, increased expression of VCAM-1 and decreased expression of the ecto-enzyme CD73 in the endothelium. The pro-inflammatory phenotype of EC-Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. The number of leukocytes that migrated to the alveolar space in response to intratracheal instillation of LPS was markedly enhanced (p<0.001) in EC-Cx40del mice compared to controls, both 3 and 6 hours after the treatment. Endothelial CD73 is known to induce anti-adhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with siRNA or anti-sense decreased CD73 expression and activity, and increased leukocyte adhesion to mouse endothelial cells (p<0.01). These effects were reversed by an adenosine receptor agonist. A control siRNA or Cx40 sense had no effect on leukocyte adhesion. We suggest that Cx40-mediated gap junctional communication contributes to a quiescent non-activated endothelium by propagating adenosine-evoked anti-inflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.
Abstract 5070: Pitavastatin Has Anti-atherosclerotic Effects via Inhibition of TRAIL Expression on CD4 T Cells
Authors: Sato Kayoko , Gomita Keiko , Hagiwara Nobuhisa
Published: April 2024

Background:
Statins reduce cardiovascular-related morbidity and mortality, however their effects on inflammation in atherosclerosis are not fully understood. We investigated whether pitavastatin can modulate cytotoxic functions of CD4 T cells in acute coronary syndrome (ACS) and inhibit the development of atherosclerosis in apoE deficient mice (apoE KO).

Methods and Results:
Fresh CD4 T cells were isolated from blood obtained from 37 patients with ACS without statin treatment on admission and 34 control patients. CD4 T cells derived from ACS patients effectively induced vascular endothelial cell (EC) apoptosis significantly more than controls (
P
<0.0001). CD4 T cells from ACS patients expressed higher levels of TRAIL (
P
<0.002) than those of controls. We further examined whether statin could regulate TRAIL expression and cytotoxicity of CD4 T cells. Pitavastatin (PIT, 10
μ
M) blocked CD4 T cell-mediated EC apoptosis (
P
=0.001) and reduced the expression of TRAIL (
P
<0.01) on CD4 T cells from ACS patients. Finally, to investigate whether TRAIL-expressing CD4 T cells had biological relevance in vivo, 12-week-old ApoE-deficient mice were fed a high-fat diet supplemented with 1mg/kg/day PIT (n= 5), 10mg/kg/day (n= 5), or Vehicle (0.5% carboxy-methyl cellulose, n= 5) for 8 weeks. Sudan IV staining of the aorta and oil red O-staining of the aortic sinus revealed that 1mg/kg/day and 10mg/kg/day PIT significantly suppressed the development of atherosclerotic lesions depending on dose (
P
<0.05,
P
<0.0001, respectively). In addition, PIT suppressed inflammatory infiltrates and CD4 T cells in atherosclerotic plaque by immunohistochemistry. Furthermore, to investigate whether PIT inhibited the expression of TRAIL on CD4 T cells, CD4 T cells were isolated from spleen and analyzed by FACS. The expression of TRAIL was significantly inhibited by 1mg/kg/day and 10mg/kg/day PIT (
P
<0.01,
P
<0.005, respectively).

Conclusions:
TRAIL-expressing CD4 T cells are enriched in ACS patients and apoE deficient mice and induce endothelial injury, which may contribute to the increasing plaque instability. Pitavastatin therapy may prevent T cell-mediated endothelial cell damage and contribute to the prevention of atherosclerotic plaque development and plaque instability.
Abstract 5071: Suppression of Macrophage Recruitment, Foam Cell Formation and Atherogenesis by the Negative Regulator of Cytokine Signaling SOCS-1
Authors: Grothusen Christina , Schuett Harald , Lumpe Stefan , Luchtefeld Maren , Grote Karsten , Drexler Helmut , Schieffer Bernhard
Published: April 2024

Introduction:
Inflammation is the hallmark of atherosclerosis leading to myocardial infarction, sudden cardiac death or stroke. While pro-inflammatory signaling pathways have been studied extensively, the impact of negative regulators of cytokine signalling i.e. suppressor of cytokine signaling (SOCS)-1 remains unclear. We postulated that a systemic SOCS-1 deficiency (socs-1
−/−
) in the atherosclerosis prone low density lipoprotein receptor (ldlr
−/−
) mouse results in a pre-mature, aggravated atherogenesis.

Methods and Results:
Since systemic deletion of SOCS-1 is lethal within 3 weeks post partum due to a systemic inflammation mainly caused by uncontrolled signalling of interferon-
γ
(IFNG), we used mice lacking mature lymphocytes (rag2
−/−
), the major source for interferon-
γ
. Thus, socs-1
−/−
, rag2
−/−
mice were crossed with ldlr
−/−
mice and fed a high-cholesterol western diet for 30 days. Even though serum lipid levels did not differ between the groups, triple-KO mice (s
ocs-1
−/−
, rag2
−/−
, ldlr
−/−
) showed enhanced atherosclerotic plaque formation (en face: oil red O staining/aortic root: oil red O staining, N=8 –15 animals/group, P<0.05) that was mainly due to enhanced accumulation of macrophages (aortic root: MOMA-2 immunohistochemistry, N=6 –15 animals/group, P<0.05). In-vitro, s
ocs-1
−/−
bone-marrow derived macrophages (BMDM) exhibited increased migration towards CCL-2 associated with enhanced secretion of CCL-2 (transwell assay, ELISA, N=8, P<0.05). Furthermore, s
ocs-1
−/−
BMDM showed enhanced foam cell formation when stimulated with oxidized(ox)LDL (oil red O staining, N=8, P<0.05).

Summary and Conclusion:
SOCS-1 deficiency combined with a pro-atherogenic background results in increased atherosclerotic plaque formation. The underlying mechanism involves an enhanced oxLDL uptake and migration capacity of s
ocs-1
−/−
macrophages as well as enforced recruitment of inflammatory cells by increased secretion of CCL-2. Thus, SOCS-1 seems to represent a molecular brake controlling macrophage recruitment and foam cell formation in atherogenesis and thereby may evolve as a suitable target for future therapeutic strategies.
Abstract 5087: LDL Lowering Does Not Improve Calf Muscle Perfusion or Energetics or Exercise Performance in Peripheral Arterial Disease
Authors: West Amy M , Anderson Justin D , Epstein Frederick H , Meyer Craig H , Hagspiel Klaus D , Wang Hongkun , Berr Stuart S , Harthun Nancy L , Weltman Arthur L , DiMaria Joseph M , Hunter Jennifer R , Christopher John M , Kramer Christopher M
Published: April 2024

Introduction.
Prior studies suggest benefit of statins on walking performance in patients with peripheral arterial disease (PAD).

Hypothesis.
We hypothesized that LDL reduction in PAD would improve calf muscle perfusion and energetics as assessed by magnetic resonance imaging (MRI) and spectroscopy (MRS).

Methods.
Fifty eight patients with mild-to-moderate symptomatic PAD (mean±SD age 63±10 years, ankle brachial index (ABI) 0.70±0.14) were studied before and 1 year after therapy with simvastatin 40mg or simvastatin 40mg/ezetimibe 10mg (R,n=31) if statin-naive or ezetimbibe 10mg (Z,n=27) if on a statin. Calf muscle phosphocreatine recovery time constant (PCr) was measured using
31
phosphorus MRS immediately after symptom-limited exercise on a 1.5T scanner. Calf muscle perfusion was measured using first-pass contrast-enhanced MRI with 0.1 mM/kg gadolinium infused at peak exercise, (n=44 after excluding those with renal dysfunction). Time intensity curves in calf muscle were normalized to nearby proton density and the upslope was used to assess perfusion. Gadolinium-enhanced MRA was graded for number and degree of stenoses. Patients performed a 6 minute walk and Skinner-Gardner treadmill test with VO
2
measurement. Changes in parameters between groups were analyzed with F-test and between time point with paired t-test.

Results.
LDL at baseline was higher in R than Z (118±36 verus 103±25 mg/dl), p=0.07 and decreased more in R than Z (−42±35 versus −21±32), p=0.02 resulting in similar final LDL. There were no differences in any of the MRI or exercise parameters between treatment groups. See table

for changes in parameters for the study as a whole.

Conclusion.
LDL lowering therapy over 1 year in PAD did not improve calf muscle perfusion, cellular metabolism, or exercise parameters. Statins are known to decrease risk of myocardial infarction, stroke, and death in PAD, but LDL lowering per se does not improve exercise calf muscle physiology or exercise performance.
Abstract 5089: Impaired Calf Skeletal Muscle Glucose Uptake in Subjects With Peripheral Artery Disease and Intermittent Claudication
Authors: Pande Reena L , Park Mi-Ae , Perlstein Todd S , Desai Akshay S , Doyle Jeanne , Copeland-Halperin Robert S , Redline Whitney M , Di Carli Marcelo F , Creager Mark A
Published: April 2024

Objectives
: Severity of claudication symptoms does not correlate well with ankle brachial index (ABI) in patients with peripheral artery disease (PAD), suggesting that non-hemodynamic mechanisms may play an important role. Given the association of PAD with insulin resistance, we hypothesized that local calf muscle glucose uptake is impaired in PAD, and therefore that local insulin resistance may contribute to the pathophysiology of claudication.

Methods:
We studied 37 subjects with PAD (ABI ≤ 0.9) and stable claudication and 11 healthy controls who underwent dynamic positron emission tomography (PET) imaging of the legs with 18-FDG during a hyperinsulinemic-euglycemic clamp (insulin @ 2 mU/kg/min). Using fused PET/CT images, regions of interest were drawn in the primary muscle groups of the lower leg. Average glucose uptake was quantified using the graphical Patlak analysis. Whole body insulin sensitivity was assessed as the glucose disposal rate (M) from the insulin clamp. Data are presented as median [interquartile range, IQR] or mean± standard error (SE).

Results:
Compared to healthy, age-matched controls, PAD subjects were insulin resistant (M = 3.4 mg/kg/min [IQR 2.7– 4.8] vs. 5.0 [3.7– 6.6], p=0.019) even after excluding those with diabetes (24% of PAD subjects), p=0.04. Average calf muscle glucose uptake was significantly lower in PAD subjects (48.6±2.6 mmol/kg/min) compared with healthy controls (62.9±6.5 mmol/kg/min), p=0.009. Similar impairments were observed in the subgroup of PAD subjects without diabetes (49.5±3.1, p=0.04 vs. healthy controls). Whole body insulin sensitivity explained 10% of the variability in local calf muscle glucose uptake (r
2
=0.10, p=0.04).

Conclusions:
We demonstrate that subjects with claudication are insulin resistant and have reduced calf muscle glucose uptake, independent of coexistent diabetes. The poor correlation between whole body insulin sensitivity and lower extremity glucose uptake suggests unique determinants of leg glucose uptake in PAD. These findings support our underlying hypothesis that impaired calf muscle substrate utilization due to local insulin resistance may be a novel mechanism underlying the pathophysiology of intermittent claudication.