bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4176: No Protection Against New Cardiac Events of a High Omega-3 Index in Patients Presenting with an Acute Coronary Syndrome
Authors: Aarsetoey Hildegunn , Pönitz Volker , Grundt Heidi , Harris William S , Nilsen Dennis W
Published: April 2024

Objectives:
Previous studies evaluating the association between n-3 fatty acids (FAs) and coronary heart disease suggest that their main effect might be a reduction in the risk of fatal myocardial infarction (MI) and sudden death. This protective effect seems to be related to a direct stabilizing effect of n-3 FAs on the myocardium itself. Red blood cell EPA + DHA (expressed as percent of total FAs; the Omega-3 Index) has recently been found to be a good surrogate for the cardiac content of omega-3 FAs and may serve as an independent risk factor for cardiac events. We examined the long-time prognostic value of the Omega-3 Index in a coastal population surviving an acute coronary syndrome (ACS).

Methods:
The Omega-3 Index was measured at admission in 466 patients hospitalized with ACS. During a follow-up time of two years, recurrent MI (defined as a TnT > 0.05 with a typical MI presentation) and all-cause mortality was registered. Odds ratios (OR) for new events were computed across quartiles of the Omega-3 Index; Q1 < 5.24 %, Q2 [5.24- 6.385 %], Q3 [6.385–7.89 %] and Q4 ≥ 7.89%.

Results:
Follow-up event-rates are given in Table 1

. Comparing Q4 to Q1 the univariate analysis gives an OR of 1.03 (p = 0.787) for re-MI, 0.90 (p= 0.328) for all-cause mortality and 0.95 (p= 0.556) for the combined endpoint of re-MI and/or death after two years of follow-up.

Conclusion:
A high Omega-3 Index had no protective effect against new cardiac events and/ or death in this cohort of Norwegian ACS patients. This might be related to the very high intake of fish in this costal population, with the level of n-3 FAs exceeding a threshold limit beyond which no further protection can be demonstrated.

Table 1
Abstract 574: Protein-Protein Interactions between Components of the Cardiac Ryanodine Receptor Complex and their Impairment by Genetic Defects Linked to Sudden Cardiac Death
Authors: Terentyev Dmitry , Vedamoorthyrao Srikanth , Terentyeva Radmila , Williams Simon C , Gyorke Sandor
Published: April 2024

Intracellular Ca release in cardiac muscle is mediated by a mutimolecular complex composed among other proteins of the ryanodine receptor (RyR) channel, the integral proteins triadin (TRD) and junctin and the Ca binding proteins calsequestrin (CASQ2), which is also known to form polymers required for high capacity-intrastore Ca binding. Mutations of these proteins result in disregulated myocyte SR Ca release, cardiac arrhythmia and sudden death. We used fluorescence resonance energy transfer (FRET) and fusion constructs utilizing the cyan and yellow fluorescent proteins (CFP and YFP, respectively) as donor-acceptor pairs to investigate interactions between TRD and CASQ2 and between CASQ2 monomers in a heterologous expression system (3T3 cells). All fusion constructs were localized to the stores of the endoplasmic reticulum. When coexpressed, CFP-TRD and YFP-CASQ2 exhibited FRET the efficiency of which depended on the Ca loading state of the intracellular Ca stores. FRET efficiency was high when the stores were depleted and low when stores were Ca loaded, thus revealing a negative dependency of TRD-CASQ2 interaction on [Ca]. Cotransfection of CFP-CASQ2 and YFP-CASQ2 resulted in FRET with an efficiency that increased at high Ca store loads and decreased at low store loads, consistent with positive dependence of CASQ2-mulimerization. Expression of two different CASQ2 mutants (R33Q and G112+5X) linked to sudden cardiac death specifically affected the ability of CASQ2 either to interact with TRD or to polymerize. These results indicate that functionally- relevant interactions exists between components of the multimolecular RyR Ca release channel complex. These interactions could provide a molecular basis for the dynamic control of SR Ca release by intra-store Ca and for defective SR Ca release caused by mutations of CASQ2 to cause CPVT.
Abstract 576: Defective Domain-Domain Interaction Between C-Terminal and Central Regions of Ryanodine Receptor as a Critical Cause of Diastolic Ca
2+
Spark and Delayed Afterdepolarization
Authors: Tateishi Hiroki , Yano Masafumi , Yamamoto Takeshi , Xiaojuan Xu , Ono Makoto , Uchinoumi Hitoshi , Mochizuki Mamoru , Noma Toshiyuki , Kobayashi Shigeki , Ohkusa Tomoko , Matsuzaki Masunori
Published: April 2024

Ryanodine receptor-2 (RyR2) central domain peptide, DPc10 (Gly
2460
-Pro
2495
), was found to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between N-terminal
1– 600
and central domains
2000 –2500
. Using the domain peptide approach, here, we investigated how single point mutation at C-terminal region in CPVT influences on intracellular Ca
2+
handling.

Methods and Results:

Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (n= 5), then fluorescently labeled with methylcoumarin acetate (MCA) using DP
4090 – 4123
, a synthetic domain peptide corresponding to Pro-Glu (mutable domain in CPVT), as a site-directing carrier; the carrier was removed after MCA labeling. The DP
4090 – 4123
mediated a specific MCA fluorescence labeling of RyR2. After tryptic digestion, the major MCA-fluorescently labeled fragment of RyR2 (150 kD) was detected by an antibody (Ab) against central region (Ab
2163
), but not by Ab
2808,
N-terminal (Ab
10
) and C-terminal region (Ab
5030
). Addition of DP
4090 – 4123
to the MCA-labeled SR induced domain unzipping between C-terminal and its putative counter domains (EC50=30μM), as confirmed by a marked quenching of the MCA fluorescence by a large-size fluorescence quencher. The DP
4090 – 4123
induced a prominent SR Ca
2+
leak (EC50=30μM). In DP
4090 – 4123
-incoporated cardiomyocytes (by protein delivery kit), diastolic Ca
2+
spark frequency was markedly increased (4.0±1.0 s
-1
100ìm
-1
, p<0.01), compared to normal cardiomyocytes (1.4±0.4 s
-1
100ìm
-1
), and after addition of 100nM forskolin both Ca2+ wave and delayed afterdepolarization-mediated Ca
2+
transient (DAD-CaT) were also frequently observed (but not in normal cardiomyocytes). All of these effects of DP
4090 – 4123
were abolished either by addition of 10 μM tetracaine or by Asn-to-Lys mutation made in DP
4090 – 4123
, mimicking human CPVT mutation.

Conclusions:

Domain-domain interaction between C-terminal and central domains (within 1200 –2850) seems to play an important role in channel gating of RyR2, and its defectiveness by point mutation may induce diastolic Ca
2+
spark and delayed afterdepolarization that triggers lethal arrhythmia.
Abstract 582: Cardiac Fibroblast Bridges Conduct Electrical Signals between Myocyte Islands.
Authors: Mitchell Ponthetta , Borg Thomas K , Gourdie Robert G , Gao Bruce Z
Published: April 2024

In this study, the electrophysiological properties of fibroblasts cocultured with cardiomyocytes were characterized using a novel cell micropatterning system. In the infarct area of a heart, the cardiac fibroblasts are known to increase collagen deposition leading to fibrosis. The fibrous tissue acts as an insulator in the damaged area and thus modulates local electrical signal conduction. This is fibroblast’s “passive role” in myocardium signal conduction. However, studies have shown that when placed in physical contact with viable myocytes, fibroblasts can form functional gap junctions and conduct electrical signal. This coupling phenomenon has been suggested as playing a role in re-entrant arrhythmias that occur in the infarct to healthy tissue border zone but there is still little known about the actual involvement fibroblasts have in the heart’s electrical communication. Our interest is in designing an in vitro model using cell micropatterning to study and characterize the electrophysiological properties of fibroblasts cocultured with myocytes. In this study we evaluate the hypothesis that fibroblast will conduct electrical signal between myocytes but this conduction will be restricted by the number of fibroblast involved. A combination of photolithography and laser micropatterning is used to achieve cell micropatterning. Polydimethyl siloxane (PDMS) stencils, created by photolithography, are used to form the myocyte islands and laser micropatterning is used to form the fibroblast cell bridge connecting the myocyte islands. Various PDMS stencils which varied the distance between the myocyte islands were made enabling us to form fibroblast bridges of various lengths (50 um to 500 um). Dual patch clamp experiments were performed to evaluate action potential propagations along the fibroblast bridge and characterize its electrical capabilities. With our laser micropatterning we were able to form fibroblast conducting bridges up to 390 um. In conclusion, fibroblast bridges can conduct electrical signals over short distances however with the increase of the fibroblast bridge length, signal propagation delay also increased. This conduction delay phenomenon observed in vitro could explain the cause of reentrant arrhythmias in vivo.
Abstract 592: Erythropoietin Attenuates the Development of Experimental Autoimmune Myocarditis
Authors: Takahashi Masafumi , Hirose Sho-ichi , Ogawa Ryo , Morimoto Hajime , Izawa Atsushi , Ise Hirohiko , Ise Hirohiko , Ise Hirohiko , Hongo Minoru , Ikeda Uichi
Published: April 2024

Background:
Myocarditis is a major cause of dilated cardiomyopathy (DCM) and end-stage heart failure; however, no effective treatment has been established. Recent evidence indicates that autoimmunity plays an important role in the development of myocarditis and DCM. Since erythropoietin (EPO) has been shown to not only have cardioprotective effects but also attenuate autoimmune diseases, we investigated whether EPO attenuates the development of a rat experimental autoimmune myocarditis (EAM) model.

Methods and Results:
Male Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. Rats were received human recombinant EPO (EPO group: 6000 IU/kg, 3 days/week, day 0 to 20, n = 14) or saline (control group, n = 15) subcutaneously, and evaluated on day 21. In EPO group, the myocarditis-affected area and heart/body weight ratio was significantly attenuated compared with those in control group (area: 7.7 ± 2.5 vs. 27.4 ± 4.3%, p<0.001; ratio: 3.79 ± 0.8 vs. 4.81 ± 1.4, p<0.05). Echocardiogram and blood pressure (BP) study demonstrated significant improvement of left ventricular contraction and systolic BP in EPO group (%FS: 45.1 ± 1.8 vs. 38.5 ± 2.8%, p< 0.05; BP: 113.4 ± 4.7 vs. 97.8 ± 2.4 mmHg, p<0.001). Immunohistochemistry showed that EPO treatment decreased the infiltration of macrophages (ED1: 30.2 ± 10.7 vs. 122.9 ± 3.3/HPF, p<0.01) and T cells (OX-38: 10.6 ± 2.7 vs. 41.6 ± 2.9/HPF, p<0.01), and %degranulation of mast cells (27.5 ± 7.5 vs. 62.5 ± 7.5%, p<0.05). Real-time RT-PCR analysis revealed that EPO treatment decreased the expression of inflammatory cytokines, such as IFN-γ (p<0.01), MCP-1 (p<0.01), and osteopontin (p<0.01), but not TNF-α and IL-10, in the myocardium. We further tested the effect of EPO on cardiac myosin-induced lymphocyte proliferation and cytokine expression in the splenocytes isolated from EAM rats in vitro. Treatment with cardiac myosin significantly stimulated proliferation and the expression of IFN-γ and TNF-α; however, EPO failed to inhibit them.

Conclusions:
These findings suggest that EPO attenuated inflammatory cell infiltration and cytokine expression, and improved cardiac function in EAM; thus, EPO has a therapeutic potential for the treatment of myocarditis.
Abstract 593: A Novel PGE2 Receptor 4-Selective Agonist Suppresses Acute Rejection via Inflammatory Factor Regulation in Murine Cardiac Allografts
Authors: Ogawa Masahito , Suzuki Jun-ichi , Takayama Kiyoshi , Isobe Mitsuaki
Published: April 2024

Background: PGE2 exerts an anti-inflammatory action by ligation of the PGE2 receptor subtype EP4 among the four of the receptor subtypes in inflammatory diseases. Because little is known about the effect of EP4R in transplantation, we examined the anti-inflammatory effect of EP4 using the murine cardiac transplantation model.

Methods and Results: We developed a novel EP4 selective agonist, EP4RAG, and performed subcutaneous injection of EP4RAG (3mg/kg) into recipient mice. We demonstrated that EP4RAG treatment prolonged graft survival (13.5+/−1.4 days, P<0.05, n=7) compared to that of the vehicle treated group (7.3+/−0.2 days, n=7). Allografts were harvested on day 7 for pathological and molecular examination. While the vehicle treated group showed severe myocardial cell infiltrations (38.4+/−2.5 %, n=10), the EP4RAG treated group attenuated the development (16.7+/−3.4 %, P<0.05, n=8). EP4RAG suppressed various inflammatory factors such as cytokines (e.g. IFN-g, IL-6), chemokines (e.g. IP-10, MIP-1) and adhesion molecules (e.g. ICAM-1) compared to the vehicle treated group in mRNA and protein levels. In vitro assay, we demonstrated that EP4RAG suppressed T lymphocyte proliferation (0.067+/−0.001 OD, P<0.05) compared to the control group (0.105+/−0.003 OD) by MLR. We also showed that EP4 mainly attenuated the activation of macrophages by co-culture. EP4RAG inhibited the activation of NF-kB compared to the control group in macrophages with IL-1beta stimulation.

Conclusions: An EP4R agonist, EP4RAG is potent for the suppression of acute rejection through proinflammatory factor regulation.
2006 Thomas W. Smith Memorial Lecture—From a Girl to a Mouse: A Journey in the Rhythm of the Heart
Authors: Priori Silvia
Published: April 2024

In the past 10 years, impressive advancements have occurred in the understanding of genetic bases of cardiac arrhythmias. This field has evolved into truly “translational” science in which the flow of data from bench to bedside has promoted advancements in basic electrophysiology and in clinical arrhythmology alike. As a paradigm of the fruitful integration between basic and clinical science in this area, I will present the story of the discovery of the molecular basis of catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare arrhythmogenic disorder characterized by adrenergically induced polymorphic ventricular tachycardia and sudden death in children. After being profoundly involved in the management of a little girl who experienced cardiac arrest, we diagnosed CPVT and initiated the search for the gene of this disease. We discovered that CPVT is caused by mutations in the cardiac ryanodine receptor’s gene (RyR2), a key protein implicated in the control of intracellular calcium. In vitro functional characterization demonstrated that the mutant RYR2 releases calcium from the sarcoplasmic reticulum during diastole, thus causing life-threatening arrhythmias. We developed a knock-in mouse model of CPVT that carries the same mutation identified in our patients, and this model is now an important source of data contributing to the understanding of CPVT and helping in the assessment of novel therapeutic strategies. Thus, the little girl is now hoping that the mouse will help doctors find a better treatment for her disease.
2006 Katharine A. Lembright Award—“The Thin Stream of Fear Trickling Through the Mind”: Impact of Anxiety in Cardiac Patients
Authors: Moser Debra K.
Published: April 2024

Anxiety has both functionally appropriate and pathological consequences. Among people with cardiac disease, anxiety can be functionally appropriate when it triggers an individual to quickly seek treatment for acute cardiac symptoms. Anxiety, however, may have pathological consequences when it is persistent or severe. These consequences may include the following: difficulty adhering to prescribed treatments and making recommended lifestyle changes; adoption of or failure to change risky behaviors; increased risk for acute cardiac events; and increased risk for in-hospital complications after admission for acute coronary syndrome. Nevertheless, because anxiety is a universal emotion that is managed without consequence by many people, its significance is often ignored by healthcare providers. The purpose of this presentation is to describe findings from a biobehavioral program of research in which the impact of psychosocial (with a major emphasis on anxiety) and behavioral variables on biological outcomes has been systematically examined. In this program of research we have (1) examined and compared the intensity of anxiety in international samples of various cardiac patient groups; (2) determined gender differences in the expression of anxiety; (3) investigated factors predicting anxiety levels, including perceived control; (4) studied healthcare providers’ knowledge of anxiety assessment and their practices with regard to the assessment and management of anxiety; (5) determined the impact of anxiety on clinical outcomes in cardiac patients and elucidated mechanisms for the linkage; and (6) tested interventions to decrease anxiety and improve morbidity and mortality. We hope to improve patient outcomes on a widespread basis by placing anxiety in the forefront of clinical cardiac practice.
Abstract 4934: CLI-Bright: A Phase I Randomized Blinded Clinical Trial Comparing the Effect of Intramuscular Injection of Autologous Bone Marrow Derived Aldehyde Dehydrogenase Bright Cells and Mononuclear Stem Cells in Patients With Critical Limb Ischemia
Authors: Perin Emerson C , Silva Guilherme V , Chronos Nicolas , Mitchell Robert G , Mendelsohn Farrell O , Gahremanpour Amir , Zheng Yi , Canales John , Fernandes Marlos R , Shaw Sandi , Smith Deirdre , Cabreira Maria G , Keller Laurence H , Willerson James T , Annex Brian H
Published: April 2024

Background:
Critical limb ischemia (CLI) is an advanced stage of peripheral arterial disease (PAD) associated with high morbidity and mortality. Functional improvement and lower amputation rates have been reported in clinical studies using intramuscular (IM) injection of autologous bone marrow mononuclear cells (ABMMNC) into ischemic limbs. Aldehyde Dehydrogenase-Bright (ALDH
br
) cells are a highly potent subpopulation of ABMMNC and preclinical studies suggest that ALDH
br
cells may contribute more effectively to angiogenesis. Therefore, we investigated the safety and efficacy of ALDH
br
compared to ABMMNC in CLI patients who were not candidate for revascularization.

Methods:
21 patients (mean age 72, 13 men) with ankle-brachial index (ABI) < 0.5 and Rutherford category (RC) 4 – 5, were randomized to receive 10 IM injections of either ALDH
br
cells (n=11) or ABMMNCs (n=10) into the gastrocnemius muscle of the affected limb. Primary outcomes at 3 months were safety and efficacy based on ABI, transcutaneous partial pressure of oxygen (tcPO
2
), RC, and ischemic ulcer grade. Paired t-test or Wilcoxon sign test was used for statistical analysis.

Results:
The mean number of ALDH
br
cells and ABMMNCs implanted were 1.33 × 10
6
and 1.207 × 10
6
. There were no significant clinical differences between the two groups at baseline. There was no adverse reaction to IM injections. One amputation occurred in each group. One death occurred in ALDH
br
patients, which was not related to the therapy. At 3 months in ALDH
br
group, ABI and RC improved 0.14 (p=0.025) and 0.64 (p=0.04), respectively. In the ABMMNC group, there were non significant changes in ABI and RC (0.08 and 0.67, respectively). No significant changes from baseline were noted in tcPO
2
and ischemic ulcer grade in either group.

Conclusion:
IM implantation of ALDHbr cells appears to be safe and our efficacy measures will be the first to compare selected from unselected cells in treatment of critical limb ischemia.

Table:

ABI, RC and tcPO2 in both groups
Abstract 4949: Bleeding and Ischemic Complications in Patients With Acute Coronary Syndromes and Chronic Kidney Disease in Relation to Different Glycoprotein IIb/IIIa Inhibitors: Analysis From the Acute Catheterization and Urgent Intervention Triage strategy (ACUITY) Trial
Authors: Afolabi-Brown Olayinka , Nikolsky Eugenia , Lansky Alexandra J , Mehran Roxana , Fahy Martin , McLaurin Brent T , Cox David A , Feit Frederick , Colombo Antonio , Caixeta Adriano , Stone Gregg W
Published: April 2024

Objectives:
We evaluated outcomes of moderate- to high-risk patients (pts) with acute coronary syndromes (ACS) and chronic kidney disease (CKD) receiving alternative antithrombotic therapy in the ACUITY trial.

Background
: Knowledge of safety and efficacy of different antithrombotic medications is important considering the choice of therapy in pts with ACS having CKD.

Methods:
In the ACUITY trial, 2468(19.1%) pts had CKD (baseline creatinine clearance <60 ml/min) and 10,472 (80.9%) pts had preserved renal function. Pts were randomized to receive 1 of 3 antithrombotic regimens including heparin + glycoprotein IIb/IIIa inhibitors (H + GPI), bivalirudin (Biv) plus a GPI, or Biv alone. Type of GPI (abciximab or a small-molecule GPI [eptifibatide and tirofiban]) was at operator’s discretion. Dosages were adjusted for renal function. Patients treated with Biv + a GPI were excluded from this analysis.

Results
: 30-day outcomes are presented in the table

. Regardless of antithrombotic regimen, CKD was associated with remarkable increase in ischemic events, and more than 2-fold increase in rates of major bleeding. In pts with CKD, there were no significant differences in rates of major bleeding among those who received H +a small-molecule GPIs vs abciximab (p=0.7). In pts with CKD, Biv monotherapy compared to H +a small-molecule GPIs resulted in remarkably lower rates of major bleeding (p=0.003) without increase in rates of ischemic events. Bivalirudin monotherapy resulted in non significantly lower rates of bleeding than H+abciximab (p=0.3).

Conclusions:
CKD is associated with higher rates of 30-day ischemic events and major bleeding. Rates of major bleeding did not differ significantly between small-molecule GPIs and abciximab when used in conjunction with heparin in pts with ACS and baseline CKD. In pts with or without CKD, bivalirudin monotherapy resulted in the lowest rates of hemorrhagic events without increasing risk of ischemic complications.
Abstract 4956: Intravital Microscopic Analysis Revealed Indoxyl Sulfate-induced Leukocyte Recruitment via E-selectin in Mice After Subtotal Renal Ablation
Authors: Ito Shunsuke , Osaka Mizuko , Higuchi Yusuke , Nishijima Fuyuhiko , Ishii Hideto , Yoshida Masayuki
Published: April 2024

[Background]
Despite positive correlation between chronic kidney disease (CKD) and atherosclerosis, a causative role of uremic toxins in vascular inflammation has not been well characterized. Indoxyl sulfate (IS) is one of the uremic toxins increased in CKD patients. However, the effect of IS on inflammation had not been tested. We thus examined an effect of IS on leukocyte-endothelial interaction
in vivo
and
in vitro
.

[Methods and Results]
Pretreatment of HUVEC with IS (0.2 mM to 2.0 mM for 20 h) significantly enhanced THP-1 cell adhesion to TNF-
α
-activated HUVEC under flow condition (p<0.01). IS enhanced expression level of E-selectin, but not ICAM-1 or VCAM-1 in TNF-
α
-activated HUVEC. IS treatment induced phosphorylation of JNK and p65 component of NF-
κ
B in TNF-
α
-activated HUVEC. IS-induced activation of NF-
κ
B was further confirmed by luciferase assay using HUVEC transfected with an NF-
κ
B
firefly
-luciferase cDNA construct. SP600125, a JNK inhibitor, and BAY11–7082, an NF-
κ
B inhibitor, attenuated IS-induced E-selectin expression and subsequent THP-1 cells adhesion under flow. IS increased dihydroethidium-associated oxidative stress in HUVEC, which is stabilized by apocynin, an NAD(P)H oxidase inhibitor. Next, a pathophysiolocgical role of IS in CKD was examined in nephrectomized mice with (Nx+IS) or without IS (Nx). An intravital microscopic analysis revealed a significant induction of leukocyte adhesion to the femoral artery in the Nx+IS group when compared to Nx group (p<0.05). Expression levels of E-selectin and the NAD(P)H oxidase subunits (p47phox and p22phox) were increased in Nx+IS mice. When we monitored inflammatory markers of monocytes, expression of CD11b and production of ROS was significantly increased in Nx mice, which was blunted by an oral absorbent of IS.

[Conclusion]
These results suggest that IS enhances leukocyte-endothelial cell interaction through up-regulation of E-selectin. The underlying mechanisms seem to involve oxidative stress-mediated JNK and NF-
κ
B dependent pathways. These observations provide a novel role for IS in CKD-related vascular inflammation.
Abstract 4957: Robust Transfection of Vascular Endothelium for siRNA Knockdown of Vascular Cell Adhesion Molecule-1 Through a Novel Nanoparticle-Lipid Raft Pathway
Authors: Kaneda Megan M , Lanza Gregory M , Wickline Samuel A
Published: April 2024

Controlling adhesion molecule expression as an antiatherosclerotic therapy is a heretofore untested concept that could modify disease progression and/or interdict acute coronary syndromes.

Methods: To design a synthetic vehicle to deliver vascular cell adhesion molecule-1 (VCAM-1) siRNA to endothelial cells, perfluorocarbon nanoparticles (PFC-NP) were loaded with the cationic lipid 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) in the lipid monolayer to form transfection complexes (10 pM PFC-NP + 5 nM siRNA) with VCAM-1 specific siRNA. Mouse 2F2B endothelial cells were treated with TNF
α
(10 ng/ml) to induce VCAM-1 expression and after 48h, mRNA and protein levels were measured. For endocytosis colabeling studies, transfection complexes were produced with fluorescently labled siRNA (red) and incubated with endothelial cells in the presence of fluorescently labeled pathway specific markers (green, macropinocytosis: dextran, clathrin: transferrin, lipid raft: cholera toxin-B).

Results: PFC-NP mediated siRNA transfection led to a significant decrease in both VCAM-1 mRNA and protein levels(A) . siRNA exhibited little intracellular colocalization with common markers of both macropinocytosis (B) and clathrin mediated endocytosis (C), while abundant membrane colocalization with lipid rafts was observed(D).

Conclusion: Because endothelial cells harbor an excess of lipid rafts in their plasma membranes, the development of an effective targeted nanoparticle carrier for siRNA that utilizes lipid raft transport rather than traditional endocytotic pathways could represent a valuable strategy for controlling vascular inflammation in atherosclerosis.

This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 5023: Correlates of High Sensitivity Troponin T Concentrations in Chest Pain Patients With and Without Acute Coronary Syndrome
Authors: Januzzi James L , Bamberg Fabian , Lee Hang , Truong Quynh A , Nichols John , Karakas Mahir , Mohammed Asim A , Schlett Christopher L , Nagurney John T , Hoffmann Udo , Koenig Wolfgang
Published: April 2024

Background:
For evaluation of chest pain, consensus guidelines recommend use of a cardiac troponin cut-point that corresponds to the 99
th
percentile of a healthy population. “High sensitivity” troponin (hsTn) assays provide good accuracy to this very low level, but cardiac structure and function correlates of hsTn concentrations in patients with acute chest pain are not well understood.

Methods:
377 patients (mean age 53.7 years, 64.2% male) with chest pain and low-to-intermediate likelihood for acute coronary syndrome (ACS) were enrolled in the emergency department. Blood was tested using a pre-commercial hsTnT assay (Roche Diagnostics). Simultaneous cardiac computed tomography (CT) imaging was performed using a 64-slice scanner (Sensation 64, Siemens Medical Solutions).

Results:
37 patients (9.8%) had an acute coronary syndrome (ACS). Using the 99
th
percentile cut-point of 13 pg/mL, hsTnT had 62% sensitivity and 89% specificity for ACS. Predictors of hsTnT values in all subjects included age (
β
= .019; P =.005), number of coronary segments (
β
= .324; P = .03) or coronary vessels (
β
= .634; P = .004) with significant stenosis, LV mass (
β
= .008; P < .001), LV ejection fraction (
β
= .042; P = .04), regional LV dysfunction (
β
= .669; P < .001), and NT-proBNP levels (
β
= .001; P = .009). Among patients without ACS (N=340), an hsTnT above 13 pg/mL identified those with more complex medical histories, more prevalent CAD and abnormalities of cardiac size and function (Table

).

Conclusions:
Among patients with chest pain, concentrations of hsTnT identify patients with more significant risk profiles and more extensive structural heart disease, even in patients without the diagnosis of ACS.
Abstract 5055: CC Chemokine Receptor 5 Signaling Suppresses Inflammation and Attenuates Adverse Remodeling Following Myocardial Infarction by Recruiting Regulatory T Cells
Authors: Dobaczewski Marcin , Xia Ying , Frangogiannis Nikolaos G
Published: April 2024

Myocardial infarction triggers an inflammatory reaction that is critically involved in the pathogenesis of cardiac remodeling. Optimal cardiac repair is dependent on regulatory mechanisms that suppress inflammation preventing excessive matrix degradation. Chemokines are rapidly induced in the healing infarct mediating recruitment of leukocyte subsets with distinct properties. CCR5, the receptor for the CC chemokines MIP-1
α
, MIP-1
β
and RANTES plays an important role in immunoinflammatory responses. We hypothesized that CCR5 signaling may regulate the inflammatory response following myocardial infarction playing a role in the development of cardiac remodeling. CCR5 and its ligands MIP-1
α
and MIP-1
β
were markedly induced in reperfused mouse infarcts. Flow cytometry demonstrated that 38.3%+3.75 of the mononuclear cells infiltrating the infarcted myocardium after 24h of reperfusion expressed CCR5. CCR5 −/− mice and WT animals showed comparable macrophage and neutrophil infiltration in the infarct; however, CCR5 absence was associated with marked upregulation of pro-inflammatory cytokine (IL-1
β
, TNF-
α
and IL-6) and chemokine (MIP-2, MIP-1
α
, -
β
and IP-10) mRNA expression in the infarcted myocardium. CCR5+ mononuclear cells isolated from WT infarcts had anti-inflammatory properties exhibiting higher expression of the inhibitory cytokine IL-10 than CCR5- cells. In addition, mononuclear cells isolated from CCR5 −/− infarcts had reduced IL-10 expression. Enhanced inflammation in the absence of CCR5 was associated with impaired recruitment of foxp3+ regulatory T cells (Tregs), a CD4+ lymphocyte subpopulation with suppressive properties (Tregs as a percentage of T cells: WT 13.1%+ 1.1 vs. KO 7.2%+ 1.1; p<0.05). In WT mice the CCR5+ subset of Tregs exhibited enhanced expression of IL-10 reflecting potent anti-inflammatory activity. Enhanced inflammation in CCR5 −/− infarcts was associated with increased MMP and decreased TIMP expression, resulting in accentuated cardiac dilation (LVEDV: WT 52.99+2.43 mm3 vs. KO 65.13+4.53 mm3; p=0.02) without affecting the size of the infarct. CCR5-mediated recruitment of Tregs may restrain post-infarction inflammation preventing excessive matrix degradation and attenuating adverse remodeling.
Abstract 5079: Hypertension in Soluble Guanylate Cyclase
α
1
- Deficient Mice is Associated With Hyperaldosteronism
Authors: Buys Emmanuel S , Raher Michael J , Rauwerdink Kristen M , Sips Patrick Y , Brouckaert Peter , Bloch Kenneth D
Published: April 2024

Introduction
. Hypertension is an important modifiable risk factor for cardiovascular disease. Altered activity of both the renin-angiotensin system (RAS) and the nitric oxide (NO)-cGMP signaling pathway have been associated with hypertension. However, the interaction of the RAS and the NO-cGMP signaling pathway remains incompletely characterized. We previously reported that male mice deficient in the
α
1
subunit of soluble guanylate cyclase (sGC
α
1
−/−
mice), an important NO receptor, develop hypertension. Here, we hypothesized that increased activity of the RAS underlies the hypertension associated with sGC
α
1
-deficiency.

Methods
. Plasma renin activity and aldosterone were measured in the plasma of anesthetized (100 mg/kg ketamine, 5 mg/kg xylazine) 12–14 weeks old male wild-type (WT) and sGC
α
1
−/−
mice by radioimmunoassay. Na
+
and K
+
were analyzed using Spotchem blood chemistry. Blood pressure was measured invasively (Millar pressure volume catheter) in male WT and sGC
α
1
−/−
mice, treated with vehicle or the aldosterone receptor antagonist, Spironolactone (100 mg/kg/day, sc for 7 days).

Results
. PRA was higher in sGC
α
1
−/−
than in WT mice (0.29±0.01 vs 0.23±0.03
μ
g/ml/hr, respectively, P<0.05). Plasma levels of aldosterone were elevated in sGC
α
1
−/−
mice as compared to WT mice (0.47±0.03 vs 0.34±0.03 ng/ml, respectively, P<0.05). Correlating with the hyperaldosteronism seen in sGC
α
1
−/−
mice, plasma K
+
levels were higher (5.4±0.2 vs 4.4±0.2 mEq/L, respectively, P<0.05), and plasma Na
+
levels were lower (144±0 vs 146±1 mEq/L, respectively, P<0.05) in sGC
α
1
−/−
mice than in WT mice. Mean arterial pressure (MAP) was the same in WT mice treated with Spironolactone or vehicle (131±5 vs 137±3 mmHg, respectively), but MAP was less in sGC
α
1
−/−
mice after treatment with Spironolactone than after treatment with vehicle (125±8 vs 173±14 mmHg, respectively, P<0.01).

Conclusions
. Together, these data suggest that altered RAS activity and, more specifically, increased aldosterone signaling, contribute to the hypertension associated with sGC
α
1
-deficiency. These findings highlight the importance of sGC in the regulation of the RAS and suggest that sGC may be a therapeutic target in renin-dependent hypertension.
Abstract 5095: The Role of Circulating Transforming Growth Factor-β in Vascular Ehlers-Danlos Syndrome: Implications for Drug Therapy
Authors: Schoenhoff Florian S , Griswold Benjamin F , Matt Peter , Sloper Leslie J , Yamazaki Michiyo , Carlson Olga D , Dietz Harry C , Van Eyk Jennifer E , McDonnell Nazli B
Published: April 2024

Objective: TGFβ signaling has been shown to play a major role in the pathogenesis of Marfan syndrome (MFS), leading to a novel treatment strategy through TGFβ inhibition using losartan. We hypothesized that circulating TGFβ levels may be elevated in disorders that disrupt arterial wall extracellular matrix, such as the vascular form of Ehlers-Danlos syndrome (VEDS).

Patients and Methods: Circulating levels of TGFβ-1 were measured in EDTA plasma of 141 patients with MFS (n=21, mean age 43±16y), VEDS (n=41, 37±13y) and controls (n=79, 48±13y) using an electrochemoluminescence platform (LLOQ 4±2.6pg/ml).

Results: In keeping with prior work, this study revealed increased circulating TGFβ in patients with MFS compared to control patients (9.2±1.2ng/ml vs. 2.5±0.4ng/ml, p<0.0001). Remarkably, the levels seen in patients with VEDS were also significantly higher than controls (8.0±1.0ng/ml vs. 2.5±0.4ng/ml, p<0.0001) and statistically indistinguishable from those seen in MFS (9.2±1.2ng/ml vs. 8.0±1.0ng/ml, p=0.5). In a bivariate logistic regression model, the odds ratio was 7.5 (p=0.02, CI 1.3– 43.3) when comparing quartiles of TGFβ values in VEDS patients with age, sex and BMI matched controls. There was a non-statistically significant trend for higher TGFβ levels in patients without cardiovascular medication (MFS, 11.4±3.0ng/ml; VEDS, 8.5±1.3ng/ml) compared to patients treated with ARBs and/or ACE-Is (MFS, 6.1±1.0ng/ml; VEDS, 4.6±0.8ng/ml), whereas β-blockers appeared to have some impact on TGFβ levels in MFS (9.0±1.7ng/ml) but not in VEDS patients (8.3±1.8ng/ml). The influence of age on TGFβ levels in the controls is statistically significant but weak (r=0.3, p=0.007). In the disease groups, TGFβ is inversely related to age (MFS, r=−0.7, p<0.0001; VEDS, r=−0.1, p=0.6), which is most likely due to ascertainment bias towards a less severe phenotype in the older patients and longer duration of medical therapy, especially in the MFS population.

Conclusion: Further investigation is warranted to establish the role of TGFβ signaling in the pathogenesis of VEDS. The current results may help to provide a rationale for the use of TGFβ lowering drug therapy in this patient population as it is emerging in the MFS patient population.
Abstract 5100: Vulnerability of Atherosclerotic Plaque in Relation to Blood Flow Direction: Mechanisms of Increased Plaque Rupture at the Upstream Region
Authors: Cicha Iwona , Woerner Anja , Osterode Katharina , Raaz Dorette , Daniel WG , Garlichs Christoph
Published: April 2024

Objectives:
Rupture of atherosclerotic plaque, the leading cause of acute coronary syndromes and ischemic strokes, occurs mainly at the upstream (proximal) shoulder of the lesion. We investigated the mechanisms leading to plaque rupture in relation to blood flow direction.

Methods:
The occurrence of ulceration, endothelial erosion, intimal neovascularization, and intraplaque hemorrhage were histologically determined in 70 human carotid specimens. Longitudinal sections of the plaques were immunohistochemically analyzed for cathepsin L, mast cell chymase, 5-lipoxygenase, Bax, VEGF, and connective tissue growth factor (CTGF) using specific monoclonal antibodies.

Results:
In >70% of ulcerated plaques, ulceration/rupture was observed at the upstream region of the plaque. Similarly, the occurrence of active intimal neovascularization and intraplaque hemorrhage was increased in upstream region (p<0.01), whereas endothelial erosion was more frequent downstream. The mean numbers of cells positive for cathepsin L, were significantly higher upstream as compared with downstream regions of the atherosclerotic lesions (58±7 upstream vs 33.1±5 downstream; p<0.001). Similarly, the expression of chymase was markedly increased in the upstream region of the plaque (34.3±5 upstream vs 17.1±4 downstream, p<0.001, n=70). The key enzyme in leukotriene production, 5-lipoxygenase, was abundantly expressed in majority of the analyzed plaques, however, no significant differences were observed in its expression between the two plaque shoulders. Pro-apoptotic protein Bax was detected more frequently at the upstream plaque shoulder, and localized mainly to smooth muscle cells and macrophages. The immunoreactivity of CTGF and VEGF showed a very high degree of variability between different plaques, but the overall statistical analyses demonstrated a significant increase in both CTGF and VEGF expression at the upstream shoulder in majority of the analysed plaques.

Conclusions:
Local hemodynamic forces affect plaque stability. Rupture at the upstream region of atherosclerotic lesion is related to activation of proteolytic and pro-apototic mechanisms, as well as enhanced neovascularization and intraplaque hemorrhage.
Abstract 5152: Normotensive and Hypertensive Obese Patients Show the Presence of Structural and Functional Alterations in Subcutaneous Small Resistance Arteries
Authors: De Ciuceis Carolina , Porteri Enzo , Rizzoni Damiano , Corbellini Claudia , La Boria Elisa , Boari Gianluca E , Pilu Annamaria , Castellano Maurizio , Francesco Mittempergher , Di Betta Ernesto , Salerni Bruno , Tosini Rossella , Rosei Claudia Agabiti , Rosei Enrico Agabiti
Published: April 2024

Structural alterations of subcutaneous small resistance arteries of hypertensive patients, as indicated by an increased media to lumen (M/L) ratio, are frequently present in hypertensive and or/diabetic patients, and may represent the earliest alterations that may be observed. In addition, M/L of small arteries have a strong prognostic significance. However, no data are available about the structure and endothelial function of small resistance arteries of obese patients. Therefore, we have investigated 16 patients with severe obesity. Six of them were normotensive, and 10 hypertensive. We compared results obtained with those observed in 12 normotensive lean controls and in 12 hypertensive lean patients. Systolic and diastolic blood pressure were evaluated by a standard sphygmomanometric approach, using appropriate cuff for obese patients. All patients underwent a biopsy of subcutaneous fat before or during surgical intervention of gastric banding or intestinal derivation. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph, according to Mulvany-Halpern technique, and M/L, media thickness, media cross-sectional area were measured. A concentration-response curve to acetylcholine (10 –9 –10 –5 Mol/l) was performed, in order to evaluate endothelial function. The results are summarized in the Table

. Obese patients, independently from the presence of hypertension, diabetes and dyslipidemia, show the presence of an increased M/L and of an increased media cross-sectional area. Preliminary data suggest the presence of endothelial dysfunction, as indicated by a reduced endothelium-dependent vasodilatation, especially in hypertensive obese patients. In conclusion, our data suggest that the presence of obesity is associated to structural alterations of subcutaneous small resistance arteries, mainly characterized by hypertrophic remodeling.

Table
Abstract 5194: Pioglitazone Attenuates Angiotensin II-induced Atherosclerosis via a Smooth Muscle Cell-Specific PPARgamma Mechanism but Has No Effect on Abdominal Aortic Aneurysms
Authors: Subramanian Venkateswaran , Bruemmer Dennis , Golledge Jonathan , Daugherty Alan
Published: April 2024

Objective:

Chronic infusion of angiotensin II (AngII) augments development of atherosclerosis and induces abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Smooth muscle cells (SMCs) play a pivotal role in the development of these two pathologies. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have previously been shown to attenuate atherosclerosis in male mice. The purpose of the study was to determine the role of SMC-specific PPARgamma in ligand-mediated attenuation of AngII-induced atherosclerosis and AAAs.

Methods and Results:

Pairs of LDL receptor −/− mice with homozygous floxed PPARgamma alleles (PPARgammaf/f) were bred. Cre was expressed under the control of the SMC-specific promoter, SM22. Littermates were generated that did not express Cre (Cre0/0, n = 33) or were hemizygous for Cre (Cre1/0, n = 24). Male mice were fed a fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) with or without the PPARgamma agonist, pioglitazone (Pio - 20 mg/kg/day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. Pio administration attenuated AngII increased systolic blood pressure equivalently in both Cre0/0 and Cre1/0 groups. SMC-specific deficiency of PPARgamma or Pio administration had no effect on serum cholesterol concentrations and lipoprotein-cholesterol distributions. SMC-specific PPARgamma deficiency increased atherosclerosis (Cre1/0 = 11.6±1.5% versus Cre0/0 = 6.7±0.9% P<0.05). Pio administration reduced atherosclerosis in Cre0/0 mice (Pio-Cre0/0 = 2.9±0.4% versus Control-Cre0/0 = 6.7±0.9%, P<0.05, n=7–12 ), but failed to reduce atherosclerosis in mice with SMC-specific PPARgamma deficiency (Control-Cre1/0 = 11.6±1.5% versus Pio-Cre1/0 = 9.9±2.5%, P=NS). SMC-specific PPARgamma deficiency or Pio administration had no effect on AngII-induced AAAs as measured by ex vivo external diameter (Control-Cre0/0 = 2.0±0.2, Control-Cre1/0 = 2.4±0.4 versus Pio-Cre0/0 = 2.2±0.2, Pio-Cre1/0 = 1.9±0.2 mm, P=NS).

Conclusion:

Administration of Pio attenuates AngII-induced atherosclerosis via the activation of SMC-specific PPARgamma but did not influence formation of AAAs in male hypercholesterolemic mice.

This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 5209: Oxidative Stress Uncouples eNOS in a BH4-independent Manner via the Formation of Protein Thiol-radicals
Authors: Chen Chun-An , Druhan Lawrence J , Wang Tse-Yao , Chen Yeong-Renn , Zweier Jay L
Published: April 2024

Imbalance of nitric oxide (NO) and superoxide (·O
2
−
) production in the endothelium contributes to many cardiovascular diseases, including hypertension, atherosclerosis, and heart failure. Under conditions of oxidative stress, NO synthase (NOS) can switch from NO to ·O
2
−
generation. We have demonstrated that endothelial NOS (eNOS) is reversibly S-glutathiolated
in vivo
and
in vitro
, in response to oxidative stress. This redox modification of eNOS leads to uncoupling, with decreased eNOS-derived NO and increased ·O
2
−
. There are several proposed mechanisms that can lead to protein S-glutathiolation: thiol-disulfide exchange with GSSG; formation of thiyl radicals, sulphenic acid, or protein-S-nitrosothiols, which in turn react with GSH. Here we demonstrate the oxidant induced formation of an eNOS protein thiyl radical using immunoblotting, immunostaining, and mass spectrometry. BH
4
-free eNOS was used to self generate ·O
2
−
, which in turn can modify sulfhydryl groups to form protein radicals. We trapped these short lived protein radicals with the spin trap DMPO. Immunoblotting using an anti-DMPO antibody, demonstrated the formation of eNOS protein radicals, which were abolished by SOD and L-NAME, and were Ca
2+
/CaM sensitive, indicating that protein radical formation is due to ·O
2
−
generation from the eNOS heme domain. With BH
4
reconstituted eNOS, which will only generate NO, formation of the eNOS protein radical is completely inhibited. Furthermore, in endothelial cells treated with menadione to trigger cellular ·O
2
−
generation, formation of eNOS protein radical as visualized with confocal microscopy was increased compared to control, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Using mass spectrometric analysis, we identified Cys908 as the only residue involved in protein radical formation. Of note, we have also demonstrated the oxidant-induced S-glutathiolation of Cys908. Thus, eNOS protein radical formation provides the groundwork for a mechanism of ·O
2
−
-directed regulation of eNOS, involving S-glutathiolation; defining a unique pathway for the redox regulation of cardiovascular function.

This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).