bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5161: Accelerated Telomere Shortening in CD8+ T-lymphocytes From Patients With Coronary Heart Disease Links Cytomegalovirus-Induced T Cell Response and Deterioration of Myocardial Function
Authors: Hoffmann Jedrzej , Brümmendorf Tim , Aicher Alexandra , Zeiher Andreas M , Dimmeler Stefanie M , Spyridopoulos Ioakim
Published: April 2024

Shortening of mean telomere length (TL) in white blood cells is correlated with the development of coronary heart disease (CHD) as well as with increased mortality due to infectious disease. The goal of this study was to investigate whether TL shortening in CHD is restricted to specific peripheral blood lymphocyte and/or myeloid cell subpopulations. Results were correlated to TL in CD34+ hematopoietic peripheral blood stem and progenitor cells (PBPC) obtained from the same patients. TL was measured by Multicolor Flow-FISH in 12 leukocyte subpopulations following immunomagnetic bead sorting. We investigated TL in 14 young (mean age 25 years) and 13 old healthy male volunteers (65 years), as well as in 25 age-matched patients (65 years) suffering from CHD. We show that TL in granulocytes and monocytes mirrors TL of CD34+ PBPC extremely well (r=0.95; p<0.0001), in patients as well as in healthy adults. TL was approximately 500 base pairs shorter in leukocytes from patients with CHD compared to their age-matched controls. This difference was identical for CD34+ PBPC, monocytes, granulocytes, B-lymphocytes and CD4+ T-cells. Surprisingly, only in cytotoxic CD8+ T lymphocytes (CTL), we found a substantially increased TL deficit of 1000 bp (p<0.001 vs. CD4+ cells) in CHD patients as opposed to controls. Further analysis revealed that TL shortening was particularly pronounced in CD8+CD28− T-cells obtained from cytomegalovirus (CMV) seropositive CHD patients, whereas such a difference was not observed in healthy CMV positive as opposed to CMV negative controls. Moreover, TL shortening of CD8+CD45RA+ T-cells from CMV seropositive patients correlated with the decrease of left ventricular function in CHD patients (r=0.635, p=0.001). In contrast, CMV seropositivity did not have any influence on TL in myeloid cells or PBPC.

Conclusion: Our data suggest that TL shortening in CHD is inherited, thus affecting the baseline TL of all peripheral blood cell populations including PBPC. In addition, CMV seropositive patients exhibit further shortening of their CTLs. Surprisingly, telomere length shortening of CTLs in CMV seropositive individuals demonstrated a very strong correlation with cardiac dysfunction, suggesting a mechanistic link between CHD and immunosenescence.
Abstract 5214: Microvascular Obstruction as Prognostic Marker for Long-Term Outcome After ST-Elevation Myocardial Infarction Compared to Traditional Prognostic Markers
Authors: de Waha Suzanne , Zachrau Johannes , Eitel Ingo , Gutberlet Matthias , Schuler Gerhard , Thiele Holger
Published: April 2024

Background
Microvascular obstruction (MO) assessed by magnetic resonance imaging (MRI) has been shown to be a prognostic marker for combined clinical endpoints (including unstable angina and repeat target vessel revascularization) after ST-elevation myocardial infarction (STEMI) in a very limited number of patients. If MO gives also prognostic information for “hard” endpoints and whether MO adds information independent of traditional prognostic markers and scores has not been investigated, yet.

Methods and Results
STEMI patients reperfused by primary PCI (n=408) underwent contrast-enhanced-MRI 4±7 days after the index event. Clinical follow-up was conducted after a mean of 18±11 months. The primary endpoint was defined as composite of death, non-fatal myocardial reinfarction and congestive heart failure. Secondary outcomes included the individual components of the composite endpoint. In 71% (n=286) MO was present. The composite clinical endpoint occured more frequently in patients with MO compared to patients without MO (19.6 vs. 6.7%, p=0.001). MO presence showed a trend towards higher occurrence of death (7.0 vs. 2.5%, p=0.09), reinfarction (5.6 vs. 1.7%, p=0.09) and congestive heart failure (7.0 vs. 2.5%, p=0.09). In a multivariate cox regression analysis including all parameters significant in univariate analysis (TIMI risk-score, Killip-class, ST-resolution, post-PCI TIMI-flow, infarct size, left-ventricular ejection fraction, endsystolic volume, age and diabetes mellitus), MO remained significantly associated with the occurrence of the composite endpoint (p=0.04). Apart from MO only post-PCI TIMI-flow (p=0.03), diabetes (p=0.03) and age (p=0.05) remained significant predictors for the combined clinical endpoint. The strongest predictor for the occurrence of the combined clinical endpoint was MO (OR 2.5; 95%CI 1.1–5.9), followed by post-PCI TIMI-flow (OR 2.3 95%CI 1.1– 4.9), diabetes (OR 1.8 95%CI 1.1–3.1) and age (OR 1.8 95%CI 1.0 –3.2).

Conclusion
Compared to traditional prognostic markers and scores, MO is the strongest predictor for the occurrence of death, non-fatal myocardial reinfarction and congestive heart failure after STEMI. Thus, cardiac MRI adds important information regarding post-infarction prognosis.
Abstract 5281: Novel Role of p66shc in ROS-dependent VEGF Signaling and Cell-Cell Adhesions in Endothelial Cells
Authors: Oshikawa Jin , McKinney Ronald , Fukai Tohru , Ushio-Fukai Masuko
Published: April 2024

p66shc is an adaptor protein and functions as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. We demonstrated that VEGF stimulation rapidly increases ROS via activation of NADPH oxidase, which are involved in VEGF receptor 2 (VEGFR2) autophosphorylation as well as endothelial cell (EC) proliferation and migration. However, the role of p66shc in VEGF signaling has not been investigated. Here we show that VEGF stimulation increases serine phosphorylation of p66shc at Ser36 in ECs (3.1-fold at 5mins, 5.4-fold at 15mins). Adenovirus-mediated transfer of mutant p66shc (S36A) or knockdown of p66shc with siRNA decreases both basal- and VEGF-stimulated ROS levels, which results in inhibiting VEGF-induced VEGFR2 autophosphorylation (43.6% and 64.9%, respectively) as well as phosphorylation of p38MAPK (20.2% and 58.5%) and Akt (57.7% and 76.8%). Functionally, p66shc siRNA or overexpression of p66shc (S36A) inhibits VEGF-induced EC migration (58.6% and 50.9%) and proliferation (86.2% and 100%). Of note, immunofluorescence analysis reveals that p66shc is colocalized with and binds to VE-cadherin/beta-catenin complex at cell-cell contacts in confluent ECs, which is required for stabilizing cell-cell adhesions. Depletion of p66shc with siRNA disrupts VE-cadherin-beta-catenin complex, thereby reducing VE-cadherin localization at adherens junction and increasing endothelial permeability (40.8% increase), as measured by transendothelial electric resistance. Moreover, p66shc siRNA markedly reduces basal- and VEGF-stimulated stress fiber formation, which may contribute to a mislocalization of VE-cadherin. In summary, p66shc functions as a key regulator for ROS-dependent VEGF signaling linked to angiogenesis and actin reorganization as well as stabilizing cell-cell junctions. Thus, p66shc is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases.

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 5310: Re-Endothelialization of Decellularized Heart Reduces Vascular Thrombogenicity
Authors: Robertson Matthew J , Kren Stefan M , Taylor Doris A
Published: April 2024

Perfusion decellularization of cadaveric hearts generates a cell-free extracellular matrix (ECM) that has similar architectural, mechanical, and chemical properties to the native tissue. These scaffolds retain their vascular conduits giving them the potential for anastomosis and transplantation. However, because the matrix is denuded, vascular clotting occurs after transplant even under an anti-coagulation (Coumadin) regimen. To create a less thrombogenic and thus more transplantable decellularized construct, we re-lined vessel conduits throughout the heart with endothelial cells and quantified their functional ability to alter clotting. Cadaveric rat hearts were decellularized by retrograde aortic detergent perfusion. Labeled rat aortic endothelial cells (RAECs) were re-introduced following two different protocols: 40 million RAECs were delivered by arterial (Ar) infusion only, or 20 million RAECs delivered by Ar infusion followed with an additional 20 million RAECs delivered by venous (Ve) infusion. Re-endotheliazed heart constructs were then maintained in a bioreactor under retrograde aortic perfusion for 1 week. Thrombogenicity of the constructs was measured by an
in vitro
thrombomodulin assay of the media flowing through the heart vasculature followed by histologic analysis. Heterotopic transplantation of scaffolds was performed in Rowett Nude rats, and hearts were examined 1 week post transplant. Either delivery route resulted in a whole heart (base to apex) distribution of RAECs with the retention of the endothelial phenotype (e.g. flattened morphology, proliferative (PCNA
+
), eNOS
+
and vWF
+
).
In vitro
thrombogenicity was reduced regardless of the delivery protocol, though there was a trend towards improved performance when cells were delivered via both Ar+Ve routes (6 and 8 fold decrease vs. controls for Ar only or Ar+Ve delivery respectively, N=8 for each condition, p<0.05). After heterotopic transplantation of the re-endothelialized ECM, clotting and cellular invasion were reduced. With this advance we move closer to having a suitable heart construct for transplant while also developing a novel
in vitro
system for use in studies such as examining stem/progenitor cell differentiation in decellularized heart.
Abstract 5315: Receptors for Advanced Glycation Endproducts Cause Decreased Collateral Vessel Formation in the Face of Uncontrolled Diabetes
Authors: Gupta Divya , Weiss Daiana , Landazuri Natalia , Norton Catherine , Guldberg Robert E , Taylor W R
Published: April 2024

Introduction:
Diabetes Mellitus (DM) is associated with the development of poor collateral vasculature. The precise cellular and molecular processes are not well understood, but monocytes (MO) play a critical role. Chronic DM leads to the formation of advanced glycation endproducts (AGEs) which alter molecular functions through the Receptor for Advanced Glycation Endproducts (RAGE). The major goal of this study was to explore the contribution of RAGE to impaired collateral growth in the setting of DM, specifically when absent from MO.

Hypothesis:
Decreased RAGE on MO leads to improved collateral vasculature in the setting of hyperglycemia.

Methods
: Four groups of mice on a C57/Bl6 background were created: Wild type (WT), WT + DM, RAGE Knockout (RAGE KO), RAGE KO + DM. Streptozocin was given intraperitoneally to induce DM. After 4 weeks of DM, the left femoral artery was ligated for a hind limb ischemia model. Laser Doppler Perfusion Imaging (LDPI) was used for qualitative evaluation of collateral vessel development. Alpha-actin smooth muscle staining was used for quantitative anatomic evaluation of neovascularization. Spontaneous running duration was employed as a functional endpoint. Adoptive transfer was used to assess MO involvement. WT + DM mice were injected with RAGE KO MO and RAGE KO + DM mice were injected with WT MO on post-op days 0,2,5,7. LDPI was performed on post-op days 1 and 8

Results
: WT + DM mice had the lowest level of blood flow recovery of all three groups, with a significant difference when compared to RAGE KO + DM (34% +/− 6.3%, 80.8 %+/− 12%; p< 0.001). These significant differences between WT + DM and RAGE KO + DM mice were also present when assessing data obtained from the running wheels (463m +/− 34m, 40, 938m +/− 342m; p<0.001) and histology when assessing the number of new vessels(v) (−9.6v +/− 38.2v, 409.1v +/− 86.9v; p< 0.05). Perfusion ratios were increased in WT + DM mice treated with RAGE KO MO by LDPI on post-op day 8 when compared to perfusion ratios seen in WT + DM mice without RAGE KO MO injection when normalized to the perfusion ratios obtained on post-op day1 (2.3% +/− 0.6% v. 1.04% +/− 0.09%; p< 0.05).

Conclusions
: RAGE on MO appears to play a significant role in the hindrance of collateral vessel development in the face of hyperglycemia.
Abstract 5321: Dicer Depletion in Adult Hearts Induces Oxidative Stress, Mitochondrial Dysfunction and Causes Rapid Loss of Cardiac Function
Authors: Roy Sashwati , Banerjee Jaideep , Gnyawali Surya C , Khanna Savita , Sen Chandan K
Published: April 2024

Introduction.
Dicer, an RNAse III endonuclease, plays a key role in the processing of miRNA into their functional mature form. A number of recent reports point towards a central role of miRNA in cardiac development and function. However, the short-term effects of dicer deficiency on the function of the adult heart and underlying mechanisms remain to be elucidated. Oxidative stress and dysregulated mitochondrial function are known to play a major role in acute cardiac pathologies.

Hypothesis
. Arrest of miR maturation by heart-specific dicer knock-out in adult animals result in rapid loss of cardiac function caused by mitochondrial dysfunction and oxidative stress.

Methods
. Mice homozygous for Dicer-floxed alleles (Dicer
fl/fl
) and transgenic Myh6-cre/Esr1 mice were crossed to generate double-transgenic (Myh6-cre/Esr1-Dicer
fl/fl
) mice. At 8 weeks of age Myh6-cre/Esr1-Dicer
fl/fl
mice were treated with vehicle or tamoxifen (tam; 20 mg/kg per day) daily ip injections for five consecutive days. To determine the acute functional consequences of Dicer depletion, heart function was determined using gated cardiac MRI and echocardiography. Myocardial miR profiling was performed using a bead array system. Candidate miRs were verified using quantitative PCR.

Results
. Tam injection for 5 days effectively depleted Dicer protein in myocardium. miRNA array data show significant (p< 0.05, n=3) decrease in the levels of a number of abundant mature miRNA including miR-1, miR-133 a&b. Following 5 days of Tam injection, a significant (p< 0.05, n=5) decrease in ejection fraction (32±9%↓), stroke volume (23±7%↓) and cardiac output (28.3±6%↓) was noted in Dicer
−/−
compared to Dicer
+/+
mice. Studies with isolated mitochondria showed a significant decrease in energy dependent pore opening and increase in uncoupled mitochondria in the heart of Dicer
−/−
mice. Such compromise in mitochondrial function was associated with structural disintegration and increased (30±7.5%↑; p<0.05, n=3) tissue oxidative stress.

Conclusions
. This study provides first evidence demonstrating that depletion of dicer in adult mice results in mitochondrial dysfunction and oxidative stress in the myocardium which is followed by acute loss of cardiac functions.
Abstract 5334: Association Between Hospital Process Performance and 1-Year Post-discharge Mortality in Patients With Non-ST-elevation Myocardial Infarction: Results From 36,262 Patients
Authors: Shah Bimal R , Roe Matthew T , Chen Anita Y , Alexander Karen P , Wang Tracy Y , Ohman E. Magnus , Gibler W. Brian , Peterson Eric D
Published: April 2024

Background: While hospitals’ adherence to performance measures for evidence-based NSTEMI care has been associated with better in-hospital outcomes, its association with long-term mortality is unknown.

Methods: Clinical data from CRUSADE were matched to Medicare claims, creating a longitudinal record of outcomes for patients aged >=65 years. Cox proportional hazard modeling was used to examine the association between 1-year post-discharge mortality and hospital composite adherence to 9 Class I ACC/AHA guideline therapies (antiplatelet, beta-blocker, any heparin, and GP IIB/IIIA within 24 hours; antiplatelet, beta-blocker, clopidogrel, ACEI/ARB, and lipid-lowering agent at discharge).

Results: Between February 19, 2003, and December 31, 2006, 36,262 NSTEMI patients treated at 268 CRUSADE hospitals were linked to Medicare data. The median hospital composite performance adherence score was 81% and varied widely (lowest to highest quartile: 73%, 79%, 83%, and 88%). Greater composite performance adherence was strongly associated with reduction in 1-year mortality. The observed 1-year mortality rate was 21%. Unadjusted mortality rates were 24%, 22%, 19%, and 19% in the lowest to highest hospital adherence score quartiles. This association persisted after adjusting for baseline clinical factors and additionally for revascularization (p<0.001 between highest and lowest hospital adherence score quartiles for all hazard ratios) (Figure

).

Conclusion: Hospital adherence to ACC/AHA AMI guideline therapies is strongly associated with improved long-term mortality, further supporting the use of these metrics for measuring and improving AMI. care.
Abstract 5427: Regulation of Arterial Branching Morphogenesis by Notch Ligand Delta-like 1
Authors: Limbourg Florian P , Woiterski Jeanette , Augustynik Michael , Napp L C , Limbourg Anne , Drexler Helmut
Published: April 2024

Arterial development and homeostasis are regulated by genetic factors and hemodynamic forces but how hemodynamic forces shape arterial branching morphogenesis and whether there is cross-talk to genetic programms is largely unknown. Here we show that the Notch ligand Delta-like 1 (Dll1) is regulated by hemodynamic forces to regulate arterial branching morphogenesis and adult arterial phenotype in vivo.

RESULTS: The onset of Dll1 expression was observed in fetal arteries at embryonic day E12.5, concurrent with the establishment of proper arterial circulation. Dll1 expression was restricted to endothelial cells (EC) of arteries and absent in veins or capillaries. Arterial branching morphogenesis in the developing retinal vasculature was significantly reduced in Dll1+/− mice, which lacked arterial branches forming the secondary vascular plexus. To test the influence of hemodynamic forces on Dll1 expression we exposed human aortic EC to cyclic stretch in vitro (10 and 15%), which upregulated Dll1 mRNA and protein levels and activated Notch target gene expression of Hes1 in a time-dependent manner. Dll1 regulation by hemodynamic forces had a strong impact on the branching phenotype of adult coronary arteries. Whole-mount ink perfusion and SMA-stained ventricular cross sections demonstrated significant rarefication of small, medium and large coronary arteries of Dll1+/− mice (p<0.01), while the number of microvessels was significantly increased (p<0.01). Quantification of the perfusion territory of the left descending coronary artery (LAD) by Evans blue perfusion revealed a significantly reduced perfusion territory in Dll1+/− mice (37±5% of wt, p<0.01), consistent with a regulation of coronary branching and extension by Dll1. Notably, while body size was normal, heart weight and LV dimensions were reduced in Dll1+/− mice, but EF was increased to maintain a normal stroke volume and cardiac output. To test whether hemodynamic forces regulate Dll1 expression in vivo we occluded the LAD and performed Dll1 stainings in sections 24hrs after occlusion, which revealed downregulation of EC Dll1.

CONCLUSION: Hemodynamic forces regulate Dll1 which regulates arterial branching morphogenesis during development and postnatal life.
Abstract 5439: Human Induced Pluripotent Stem Cell-derived Endothelial Cells Exhibit Heterogeneity
Authors: Jalil Rufaihah A , Nguyen Ha N , Huang Ngan F , Hunter Arwen L , Pera Renee R , Cooke John P
Published: April 2024

Vascular endothelial cells derived from pluripotent stem cells are useful in various therapeutic strategies for repair and revascularization of ischemic tissue in patients with vascular defects. One potential source for these therapeutic endothelial cells (ECs) is the induced pluripotent stem cells (iPSCs). In the present study, we generated iPSCs from human adult dermal fibroblasts using retrovirus-mediated transduction of Oct-4, Sox-2, Klf-4 and c-Myc. Differentiation to endothelial lineage was initiated by growing iPSCs in suspension for 4 days to form embryoid bodies (EB) and seeding on gelatin-coated dishes in
α
MEM and 20% defined FBS supplemented with 50ng/ml VEGF, bFGF and BMP4 for 10 days. iPSC-ECs were then isolated using phycoerythrin-conjugated CD31 antibody and were FACS sorted. Real time gene expression analysis and immunocytochemical staining of EC, pluripotency and transgene markers were performed. Further in vitro functional characterization such as matrigel tube formation, BrdU proliferation and cell migration assay in response to angiogenic growth factors were also carried out. All iPSC-ECs exhibited cobble-stone morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of iPSC-ECs revealed positive staining for EC markers such as CD31, vascular endothelial cadherin (CD144), endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The iPSC-ECs proliferated well in growth factor-supplemented culture media and exhibited angiogenic behavior, forming capillary-like structures within 24 hours when placed in Matrigel. Intriguingly, we observed variations in the efficiency of endothelial generation between iPSC lines derived from different individuals, with EC yields ranging from 7±3.2% to 13±5.5% of total EB-derived cells. In addition, we noted that iPSC-ECs expressed lower levels of CD144, eNOS and vWF by rtPCR and immunohistochemistry when compared to human microvascular ECs. The results in this study demonstrated the feasibility of differentiating and isolating ECs from iPSCs. They also outline differences that exist between iPSC-EC lines and mature ECs. These differences may have an impact on functional capabilities of iPSC-ECs in cellular therapies.
Abstract 5441: Critical Role of Increased MicroRNA-126 Expression for the Pro-angiogenic Capacity of Autologous Human CD34+ Stem/Progenitor Cells
Authors: Mocharla Pavani , Briand Sylvie , Giannotti Giovanna , Doerries Carola , Lüscher Thomas F , Landmesser Ulf
Published: April 2024

Circulating, bone-marrow derived CD34+ progenitor cells have been shown to promote ischemia-induced neovascularisation, and are currently explored as a potential novel therapeutic strategy in ischemic heart disease. By using culture approaches “early” endothelial progenitor cells (EPCs) are thought to be derived from CD34+/CD14+ cells, whereas “late outgrowth” EPCs are thought to be derived from CD34+/CD14− cells. The aim of the present study was to determine the expression of pro- and anti-angiogenic microRNAs in CD34+ cells and to characterize their role for endothelial growth promoting effects.

Methods: We examined the pro- and anti-angiogenic microRNA expression profile, of circulating CD34+14+ and CD34+14− stem/progenitor cells, as compared to CD34−14+ (monocytes) and CD34−14− (lymphocytes) isolated by MACS sorting from mononuclear cells, by using quantitative RT-PCR. Furthermore, pro-angiogenic effects of these cell populations were examined by tubulogenesis in co-cultures with differentiated endothelial cells (HAECs). In vivo endothelial repair capacity was determined by cell transplantation (200,000 cells) into nude mice with carotid endothelial injury. The functional role of differentially regulated miRs was characterized by transfection with anti-miRs and miR mimics.

Results: Only CD34+14+ and CD34+14−, but not CD34−14+ or CD34−14− cells exerted a pro-angiogenic effect, paralleled by a substantially increased expression of the pro-angiogenic miR126 and miR130a. Similarly, in vivo endothelial repair was enhanced by CD34+ cells with no significant difference between CD34+14+ and CD34+14− cells. CD34+ cell transfection with anti-miR-126 abolished the pro-angiogenic effects of CD34+ cells, whereas forced expression of miR126 (miR mimic) markedly enhanced their pro-angiogenic effects.

Conclusions: These findings demonstrate for the first time a differential pro-angiogenic microRNA expression of CD34+ stem/progenitor cells as compared to CD34− mononuclear cells. Importantly expression of miR126 is critical for endothelial growth promoting effects of CD34+ cells, and represents an interesting novel target to enhance the pro-angiogenic capacity of autologous CD34+ stem/progenitor cells.
Abstract 5450: Detection of Intracoronary Thrombus by Magnetic Resonance Imaging in Patients With Acute Coronary Syndrome
Authors: Jansen Christian H , Perera Divaka , Wiethoff Andrea J , Makowski Marcus R , Lockie Timothy , Indermuehle Andreas , Razavi Reza M , Chiribri Amedeo , Nagel Eike , Greil Gerald F , Redwood Simon , Botnar Rene M
Published: April 2024

Background: Persistent intracoronary thrombus following plaque rupture is associated with an increased risk of subsequent myocardial infarction and mortality. Presently, coronary thrombus can only be visualized invasively by X-Ray Angiography (XRA), intravascular ultrasound, or angioscopy. Purpose: The aim of this study was to investigate the use of non-contrast enhanced magnetic resonance imaging for direct thrombus visualization (MRDTI) in patients with a recent ACS (troponin T >1.0).

Method and Results: 14 patients (13 male) underwent MRDTI within 24 –72h of presenting with ACS prior to invasive X-ray angiography (XRA). MRDTI (fig. a

) was performed using a navigator-gated free breathing and cardiac-triggered T1-weighted 3D inversion-recovery black-blood gradient-echo sequence without contrast administration. 7 patients were found to have intracoronary thrombus on XRA ±/− thrombus extraction (2xLAD, 1xLCX, 3xRCA and 1PDA branch of RCA) (fig c

.) and 7 had no visible thrombus. MRDTI (fused with magnetic resonance angiography, fig. b

) correctly identified thrombus in 6 of 7 patients (PDA thrombus not detected) and correctly classified the control group of 7 patients without thrombus formation. Contrast to noise ratio of thrombus as compared to the vessel lumen of non-affected segments was 6-fold increased (67.5±12.2 vs. 10.2±6.6, p <0.01).

Conclusion: MRDTI allows selective visualization of coronary thrombus in patients after myocardial infarction. This technique may be useful for direct coronary thrombus detection in patients with ACS or unstable angina.
Abstract 5473: TGF
β
Induction of Vascular Smooth Muscle Differentiation Markers is Mediated by Nox4 and p38 Mitogen-activated Protein Kinase
Authors: Martin-Garrido Abel , Brown David I , Lyle Alicia N , Dikalova Anna , Griendling Kathy K
Published: April 2024

Vascular smooth muscle cells (VSMCs) undergo phenotypic transformation in response to environmental cues. In response to injury, VSMC switch from a “contractile” to “synthetic” phenotype, which is implicated in restenosis and arteriosclerosis. Thus, accelerating VSMC redifferentiation may be one way to limit lesion development. TGF
β
induces differentiation in VSMC; however, the mechanism is incompletely understood. Previous work has implicated Nox4 in differentiation. In this study, we hypothesized that TGF
β
induces differentiation via Nox4 and p38 mitogen-activated protein kinase (p38) pathways. In VSMCs, treatment with TGF
β
(2 ng/mL) for 24 hours increases reactive oxygen species (ROS) production (146±15% vs control) and Nox4 protein expression (150±12% vs control), both of which are reduced by transfection with small interfering RNA against Nox4 (siNox4). At 48 hours, TGF
β
maximally induces the expression of the differentiation markers smooth muscle alpha actin (SMA) and calponin (CNN) and increased incorporation of SMA into stress fibers. This induction is blunted by preincubation with the flavin oxidase inhibitor diphenylene iodonium (DPI) (90±8% inhibition) or by siNox4 (100% inhibition). To investigate the role of p38, we first demonstrate p38 is activated by TGF
β
at 60 minutes (370±70%) and it is not ROS dependent, because it is not blocked by preincubation with DPI. However, p38 remains activated for up to 24 hours, as measured by continued phosphorylation of its substrates, heat shock protein 27 (650±48%) and the obligatory differentiation transcription factor serum response factor (280±50%). This late activity of p38 is prevented by DPI (68±10% and 57±5% inhibition of HSP27 and SRF). Moreover, the inhibition of p38MAPK partially reverses the upregulation of SMA (60±20% inhibition) and CNN (67%±23 inhibition) by TGF
β
, and acts in cooperation with Rho kinase (87±10% and 97±1% inhibition of SMA and CNN). Thus, we conclude that Nox4 and p38 are critical regulators of TGFâ-induced differentiation in VSMC, and that Nox4 maintains the activity of p38. These data raise the possibility that Nox4 and p38 are potential targets in the treatment of cardiovascular diseases in which VSMC dedifferentiation plays a pivotal role.
Abstract 5479: Endothelial Cell TFPI Regulates Murine Arterial Thrombosis but Not Hemostasis or Development
Authors: White Thomas A , Witt Tyra A , Johnson Tucker F , Pan Shuchong , Sandu Gurpreet S , Mueske Cheri S , Kleppe Laurel S , Holroyd Eric W , Simari Robert D
Published: April 2024

Background:
Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI
Flox
) were crossed with the Tie2 Cre
+
or LysM Cre
+
mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI
Tie2
or TFPI
LysM
).

Methods and Results:
The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI
Tie2
and TFPI
LysM
mice were phenotypically similar to TFPI
Flox
littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI
Tie2
mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI
LysM
mice show partial deletion in blood cells and complete deletion in macrophages. TFPI
Tie2
mice had a 60% reduction (P < 0.0001) in circulating TFPI activity compared to TFPI
Flox
littermate controls. TFPI
LysM
mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI
Flox
counterparts. Neither TFPI
Tie2
nor TFPI
LysM
mice had significant changes in hemostasis compared to TFPI
Flox
mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI
Tie2
or TFPI
LysM
and their TFPI
Flox
littermates. In a ferric chloride model of arterial thrombosis, TFPI
Tie2
mice had a significant reduction (P < 0.05) in time to occlusion while TFPI
LysM
mice did not differ compared to TFPI
Flox
mice.

Conclusions:
Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI
Tie2
mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.
Abstract 5485: Potent Antithrombotic Activity of EP224283, a Dual Factor Xa Inhibitor and GPIIb-IIIa Antagonist, in Arterial Thrombosis in Mice
Authors: Hechler Beatrice , Freund Monique , Riehl Nadia , Cazenave Jean-Pierre , Petitou Maurice , Gachet Christian
Published: April 2024

Arterial thrombosis results from combined platelet activation and coagulation at sites of vascular injury. In acute ischemic events, the cornerstone of treatment is the association of antiplatelet drugs with anticoagulants. Here we investigated the effects of EP224283, a molecule combining antithrombin-dependent factor Xa (FXa) and GPIIb-IIIa inhibition, in several thrombosis models including systemic thromboembolism, localized laser-induced thrombosis of mesenteric arterioles and carotid thrombosis after atherosclerotic plaque rupture in ApoE−/− mice. IV infusion of EP224283 produced dose-dependent anti-FXa activity and inhibition of ADP-induced platelet aggregation (PA). Specifically, 10 nmol/kg EP224283 exhibited plasmatic anti-FXa activity of 0.61±0.01 U/ml without detectable inhibition of PA, while 300 nmol/kg EP224283 exhibited both strong anti-FXa activity (7.6±0.5 U/ml) and 90% PA inhibition, 15 min after infusion. In systemic thromboembo-lism induced by collagen+adrenaline, where thrombin generation is minimal, 85% of control mice died. EP224283 (10 nmol/kg) had no effect on mortality, while all mice receiving EP224283 (300 nmol/kg) survived. When thromboembolism was induced by tissue factor, generating high amounts of active thrombin, mice injected with EP224283 (10 nmol/kg) displayed 40% reduced mortality while all mice injected with EP224283 (300 nmol/kg) survived. These results suggest that the antithrombotic effect is due to both the antiplatelet and anticoagulant properties of EP224283. In a model of localized thrombosis of mesenteric arterioles triggered by laser injury, where both platelet activation and thrombin generation are involved, EP224283 (10 nmol/kg) reduced thrombus stability, while EP224283 (300 nmol/kg) profoundly decreased thrombosis. In this model, high dose hirudin or clopidogrel do not completely inhibit thrombus formation. Thus the dual properties of EP224283 explain its efficacy in this model. Finally, in a model of atherosclerotic plaque rupture induced by ultrasounds, EP224283 (10 nmol/kg) reduced thrombosis by 80%. These results suggest that combining antiplatelet and anticoagulant activities in a single compound is a promising strategy in treatment of acute atherothrombosis.
Abstract 5499: Promoter Variant of
Angiopoietin-2
and Plasma Angiopoietin-2 Are Associated With Risk of Stroke Recurrence in Patient With Lacunar Infarction
Authors: Chen Jingzhou , Yu Hui , Sun Kai , Song Weihua , Bai Yongyi , Zhang Weili , Lou Kejia , Song Yan , Zhang Yinhui , Hui Rutai
Published: April 2024

Background-
Risk factors for stroke recurrence are still not fully defined. Angiopoietin-2 is a very important mediator in the vessel development and repair. We hypothesized that altered expression of
angiopoietin-2
will confer risk of stroke recurrence.

Methods -
The association of plasma angiopoietin-2 (determined by using ELISA) and the variants in
angiopoietin-2
promoter with stroke recurrence was tested in 1735 patients with stroke of three subtypes, lacunar infarct (n=475), atherothrombotic (n=794) and hemorrhage (n=466), for a period of following-up 4.5 years (mean), the association was evaluated by using Kaplan-Meier analysis and the Cox regression models.

Results
-We found that angiopoietin-2 levels were associated with risk of stroke recurrence in lacunar infarct patients. Taking the lowest quartile as reference, the adjusted hazard ratio (HR) and 95% confidence intervals (CI) for stroke recurrence was 1.47 (0.75–2.88) for the second quartile, 2.44 (1.31–4.56) for the third and 2.00 (1.05–3.85) for the fourth. Allele T of rs3739391 in
angiopoietin-2
promoter was associated with elevated angiopoietin-2 levels and increased risk of stroke recurrence in patients with lacunar infarct with HR 1.72 (1.09–2.72) relative to the allele C, but neither in those with atherothrombotic nor in those with hemorrhagic stroke.

Conclusion-
Our results indicate that both angiopoietin-2 and allele T of rs3739391 might be the risk marker for stroke recurrence in the patients with lacunar infarction. These findings may help to improve future prevention or therapy strategies for stroke.
Abstract 5530: Associations Between Arterial Blood Pressure and Adipokines Expression in Subcutaneous, Femoral and Pericardial Adipose Tissue in Patients With Coronary Atherosclerosis
Authors: Bakogiannis Constantinos , Antoniades Charalambos , Tousoulis Dimitris , Antonopoulos Alexis S , Ntalarizou Evangelia , Miliou Antigoni , Psarros Costas , Triantafyllou Costas , Lymperiadis Dimitris , Stefanadis Christodoulos
Published: April 2024

Background: Adipokines such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and adiponectin are synthesized in adipose tissue (AT) and may affect the development of hypertension. We characterised the synthesis of these adipokines in subcutaneous, femoral and pericardial AT of patients with advanced atherosclerosis, and we examined their impact on arterial blood pressure.

Methods: Fifty patients undergoing elective CABG (14 hypertensives 36 non-hypertensives) were recruited. Subcutaneous AT was collected from the site of CABG incision, femoral AT was collected from the area surrounding the saphenous vein (perivascular) and pericardial AT was collected from the site close to the right ventricle. AT was transferred to the lab within 30 minutes and was cultured ex-vivo for 4 hours. MCP-1, IL-6 and adiponectin were quantified in AT culture supernatants.

Results: Hypertension was associated with higher IL-6 in pericardial adipose tissue (14.7±1.8 vs 10.0±1.5 pg/mg p<0.05), MCP-1 (7.07±1.2 vs 2.7±0.6 pg/mg p<0.05) and adiponectin (229.5±15.9 vs 168.9±19.3 pg/mg, p<0.05). Importantly, hypertension was not associated with femoral IL-6 (10.9±1.3 vs 11.9±2.2 pg/mg p=NS), subcutaneous IL-6, (6.9±1.0 vs 8.4±2.1 pg/mg p=NS), femoral MCP-1 (5.7±0.8 vs 4.0±1.1 p=NS), femoral adiponectin (174±34 vs 221±26pg/mg p=NS), subcutaneous MCP-1 (2.37±0.29 vs 3.3±1.5 pg/mg p=NS) or subcutaneous adiponectin (175±18 vs 182±23.2 pg/mg p=NS) AT synthesis. Diastolic (but not systolic) blood pressure was strongly correlated with IL-6 in femoral (rho=0.492, p=0.038) and subcutaneous (rho=0.659, p=0.04) but not pericardial (rho=0.386, p=NS) AT.

Conclusions: Adipokines (IL-6, MCP-1 and adiponectin) synthesized in pericardial adipose tissue are closely associated with the presence of arterial hypertension in patients with advanced atherosclerosis. However, adipokines synthesised in femoral and subcutaneous adipose tissue are strongly associated with the variability of diastolic but not systolic blood pressure.
Abstract 5535: Vascular Insulin Resistance in Prehypertensive Rats Contributes to the Development of Hypertension: Role of PI3 Kinase/Akt/Enos Signaling
Authors: Zhang Haifeng , Li Rong , Wang Wenqing , Wang Xiaoming , Huang Chen , Gao Feng
Published: April 2024

Objective:
It is well known that systemic insulin resistance (IR) is closely associated with metabolic syndrome including type 2 diabetes and hypertension. However, it remains unclear whether vascular IR acts as an early etiologic factor for the development of hypertension. The present study aimed to test the hypothesis that vascular IR in the conduit arteries contributes to the pathogenesis of hypertension in hypertension-prone normotensive rats, and to further investigate the underlying mechanism.

Methods:
Male spontaneously hypertensive rats (SHRs) aged 5-weeks (young) and 15-weeks (adult) were studied and vascular IR was assessed as the function of isolated aortic vasodilatory response to insulin
in vitro
.

Results:
Compared with Wistar-Kyoto (WKY) rats, adult SHRs exhibited significant hypertension (systolic blood pressure, 173±4 vs. 121±4 mmHg in adult WKY, n = 8,
P
<0.01) with significantly decreased aortic vasodilatation to insulin (38.7±4.4% vs. 70.9±3.6%, n = 8 –10 aortic segments/group from 5–7 rats,
P
<0.01), whereas young SHRs had normal blood pressure (121±2 vs. 114±4 mmHg in young WKY,
P
>0.05) but exhibited similar vascular IR (42.3±5.2% vs. 68.5±5.9%,
P
<0.01). Both young and adult SHRs showed significant downregulated expression of PI3-Kinase and decreased insulin-stimulated phosphorylations of Akt and eNOS in vascular tissues (n = 3,
P
<0.01). More importantly, treatment with rosiglitazone (RSG), an insulin sensitizer, for 2 wks increased vascular PPAR
γ
expression and restored PI3-Kinase/Akt/eNOS-mediated signaling pathway induced by insulin, thus improved aortic vasodilatory response to insulin in young but not in adult SHRs (
P
<0.05).

Conclusion:
Vascular IR, characterized by the impairment of PI3-Kinase/Akt/eNOS signaling in vascular endothelium, may play a role in endothelial dysfunction and subsequent development of hypertension in normotensive young SHRs.
Abstract 5547: Can IL-6 Exert Atheroprotective Action on Human Macrophages via Activation of ABCA1 Through the Jak-2/Stat3 Pathway?
Authors: Frisdal Eric , Olivier Maryline , Larrède Sandra , Chapman M. J , Guérin Maryse , Le Goff Wilfried
Published: April 2024

Cholesterol-laden macrophage foam cells constitute a hallmark of the inflammatory dimension of atherosclerosis. The ATP-binding cassette A1 transporter (ABCA1) promotes macrophage cholesterol efflux to apolipoprotein AI (apoAI), thereby attenuating atherosclerotic plaque progression. Interleukin-6 (IL-6) is a macrophage secretory product which may modulate intraplaque inflammation, but whose precise role in atherogenesis is unclear. We therefore evaluated the impact of IL-6 on cholesterol homeostasis in human monocyte-macrophages and THP-1 cells. ABCA1-mediated cholesterol efflux to apoAI was significantly increased (2.5-fold) by IL-6 (50 ng/mL) subsequent to the induction of ABCA1 mRNA and protein levels. Stimulation of ABCA1-mediated cholesterol efflux was however abolished by a specific inhibitor of Jak-2 or the Jak-2/Stat3 signalling pathway. The implication of Stat3 in the stimulatory effect of IL-6 was confirmed by the use of a Stat3 inhibitory peptide. In addition, ABCA1-mediated efflux of cholesterol, driven by the ingestion of apoptotic, free-cholesterol-loaded THP-1 cells, was equally induced by IL-6 in THP-1 phagocytes. Finally, IL-6 enhanced phagocytosis of apoptotic Jurkat cells in THP-1 macrophages, an effect abolished by either a Jak-2 inhibitor or glyburide, an inhibitor of ABCA1 activity. Thus, induction of ABCA1 expression by IL-6 in human monocyte-macrophages through the Jak-2/Stat3 pathway may favour reduction in both foam cell formation and growth of the necrotic plaque core, potentially contributing to enhanced plaque stability.
Abstract 5570: TRAF5 Deficiency Accelerates Atherogenesis in Mice: A Role for TRAF5 as Protective Factor in Atherosclerosis?
Authors: Missiou Anna , Varo Nerea , Rudolf Philipp , Münkel Christian , Ernst Sandra , Walter Carina , Bode Christoph , Libby Peter , Zirlik Andreas
Published: April 2024

Background: TNF receptor associated factors (TRAFs) function as cytoplasmic adaptor proteins for cytokines of the tumor necrosis factor (TNF) interleukin/toll-like receptor superfamily such as CD40L, TNFalpha, and IL-1beta, established mediators of atherogenesis. We recently reported over-expression of TRAFs in atheromata, TRAF-dependent inflammatory gene expression in atheromata-associated cell types, and limitation of atherogenesis in mice deficient for TRAF1. Here, we test the hypothesis that TRAF5 modulates atherogenesis in vivo.

Methods and Results: TRAF5–/LDLR– mice consuming a high cholesterol diet for 18 weeks developed significantly larger atherosclerotic lesions compared with LDLR– controls. Intimal lesion size increased by up to 328±11% and 174±15% in sections of the aortic arch and aortic root, respectively (n>13 per group, P<0.01 each). Plaques of TRAF5-deficient animals contained up to 149±15% more lipids (P<0.05) and tended to contain more macrophages and less collagen, characteristics associated with vulnerable plaques in humans. Smooth muscle cell content and lymphocyte content remained unchanged. In vitro, TRAF5-deficient endothelial cells expressed higher levels of the adhesion molecule VCAM-1 and the chemokine KC. Furthermore, TRAF5-deficient macrophages took up increased amounts of acethylated LDL (N=3). Finally, patients suffering from stable coronary heart disease and acute coronary syndrome expressed significantly lower amounts of TRAF5 mRNA in blood than healthy controls (0.026±0.0016 vs. 0.023±0.0018 vs. 0.029±0.0017 TRAF5/GAPDH mRNA, N=325, P<0.01) supporting a protective role of TRAF-5 in atherosclerosis.

Conclusions: We present the novel finding that TRAF5 deficiency accelerates atherogenesis in mice. These data identify TRAF5 as potential treatment target for chronic inflammatory diseases such as atherosclerosis.
Abstract 5574: Cooperation of MT1-MMP and LOX-1 for RhoA- and Rac1-dependent Signaling Pathways in Oxidized LDL-mediated Endothelial Dysfunction and Atherosclerosis
Authors: Sugimoto Koichi , Ishibashi Toshiyuki , Sawamura Tatsuya , Uekita Hironori , Kamioka Masashi , Inoue Nobutaka , Shiomi Masashi , Takuwa Yoh , Teramoto Tamio , Takeishi Yasuchika
Published: April 2024

Background:
Membrane type 1-matrix metalloproteinase (MT1-MMP) functions not only as a proteolytic enzyme but also as a signaling molecule. Lectin-like oxidized-LDL receptor-1 (LOX-1) is a major receptor for oxidized LDL (ox-LDL). RhoA and Rac1 play crucial roles in endothelial dysfunction including eNOS downregulation and NADPH oxidase-dependent reactive oxygen species (ROS) generation. Hypothesis: MT1-MMP modifies ox-LDL-triggered RhoA- and Rac1-dependent endothelial dysfunction and atherosclerosis.

Methods:
GTP-loading of RhoA and Rac1 were assessed by pull-down assays. The activity of MT1-MMP was evaluated by fluorescent assay. A neutral antibody to LOX-1 (TS92) was used for inhibition of LOX-1, while MT1-MMP was silenced by siRNA. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence. Fluorescent immunohistochemistry and immunoprecipitation were performed using the cultured endothelial cells (ECs) and the aortae of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits.

Results:
Ox-LDL activated RhoA and Rac1 within 5 and 15 minutes, as well as the MT1-MMP activity of membrane fractions after 15 minutes in ECs, which was inhibited by TS92. Ox-LDL-induced activation of RhoA and the downstream events including endothelial cell invasion and eNOS downregulation were blocked by silencing of MT1-MMP. Knockdown of MT1-MMP also markedly attenuated ox-LDL-triggered Rac1 activation, NADPH oxidase activity and ROS generation. Interestingly, LOX-1 and MT1-MMP were partly co-localized in ECs by fluorescent immunohistochemistry, and immunoprecipitation demonstrated that MT1-MMP formed a complex with LOX-1. Furthermore, immunohistochemistry revealed that the two molecules were co-localized in ECs and in the aortic atherosclerotic lesions of WHHLMI rabbits. In addition, immunoprecipitation demonstrated that MT1-MMP and LOX-1 formed a complex in cultured ECs and in the atherosclerotic aortae of WHHLMI rabbits.

Conclusions:
We show new insights into modulation of endothelial dysfunction and atherosclerosis by an MT1-MMP-LOX-1 axis, suggesting that MT1-MMP is a cell surface modifier of ox-LDL-triggered RhoA and Rac1-activated signaling pathways through LOX-1.