bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5303: Tenascin-c Supports Human Blood Platelet Adhesion and Activation Under Both Static and Flow Conditions
Authors: Schaff Mathieu , Bourdon Catherine , Receveur Nicolas , Cazenave Jean-Pierre , Gachet Christian , Lanza François , Mangin Pierre
Published: April 2024

Targeting platelet activating components preferentially expressed in atherosclerotic plaques could represent an attractive anti-thrombotic strategy. Tenascin-C (TN-C) is an interesting candidate due to its high level of expression in atherosclerotic plaques while it is absent from healthy vessels. The aim of our study was to evaluate the capacity of TN-C to support platelet adhesion and activation. Under static conditions, washed platelets adhered to purified TN-C (1,923±206 platelets/mm
2
) as efficiently as to fibrinogen (2,010±100 platelets/mm
2
), a well characterized platelet adhesive protein. Perfusion of hirudinated blood through TN-C-coated (50
μ
g/mL) micro-capillaries under various shear conditions (300 s
−1
to 1,500 s
−1
) led to efficient capture of individual platelets which did not form aggregates in contrast to the response on a collagen matrix. Interestingly, the number of adherent platelets to TN-C was 3.7 and 1.8 higher than onto fibrinogen and von Willebrand factor (VWF) coated surfaces, respectively. In addition, video-microscopy analysis showed that the vast majority of attached platelets displayed a stationary behavior onto TN-C (86.9±4,1%) whereas they rolled over a VWF matrix (78.7±2,4%) and rapidly detached from fibrinogen (83.1±3,2%). These results suggest that TN-C has higher adhesive properties than VWF and fibrinogen under flow. Adhesion to TN-C under flow required the GPIb-IX complex and integrins
α
IIb
β
3
and
β
1
as shown by inhibition with blocking antibodies and pharmacological inhibitors. Platelets adherent to TN-C under both static and flow conditions, changed their shape and extended filopodia indicating their activation, which was further confirmed by the observation of a sustained intracellular calcium increase. In conclusion, we provide evidence for the ability of TN-C to support platelet adhesion and activation under static and flow conditions indicating a novel putative role in thrombosis. Its presence in atherosclerotic plaques and its implication in platelet recruitment suggest that TN-C could represent a target for anti-thrombotic drugs.
Abstract 5304: Ectonucleoside Triphosphate Diphosphohydrolase-1 (ENTDP-1/CD39) Over-Expression Delays
in vivo
Carotid Arterial Thrombosis in Mice
Authors: Nickoli Michael S , Milks Michael W , Wheeler Debra G , Dwyer Karen M , Cowan Peter J , Robson Simon C , d’Apice Anthony J , Gumina Richard J
Published: April 2024

Background:
Vascular injury results in release adenosine diphosphate (ADP), activating platelets leading to further release of ADP from platelet granules. ADP release results in platelet aggregation and cross-linking. Ectonucleoside Triphosphate Diphosphohydrolase-1 (ENTDP-1/ CD39), an endothelial cell-expressed diphosphonucleotidase, rapidly converts ATP and ADP to AMP which is further degraded by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic, vasodilatory and cardioprotective compound. We hypothesized that transgenic CD39 over-expression would increase the time to thrombosis in an
in vivo
ferric chloride-induced carotid artery thrombosis model.

Methods:
Mice, 8 –12 week old human CD39 overexpressing (hCD39-OE) and wildtype littermates, were anesthetized using 55 mg/kg ketamine and 15 mg/kg xylazine, intubated, and ventilated with room air using a mouse respirator (Harvard Apparatus). A midline incision was made and the left carotid artery was exposed. Baseline carotid blood flow was obtained using a Doppler flow probe (Model 0.5 VB, Transonics Systems). Thrombosis was induced by placing filter paper saturated with 10% FeCl3 on the carotid artery for 3 minutes. Carotid blood flow was monitored continuously and time to cessation of flow analyzed.

Results:
In response to FeCl3 induced injury, wild-type mice demonstrated a consistent time to occlusion of 8±1 minute. In contrast, in animals overexpressing hCD39, the time to occlusion following FeCl3 induced injury was profoundly increased to 60±2 minutes (>7-fold). (Figure

).

Conclusion:
CD39 over-expression significantly delays the time to arterial thrombosis.
Abstract 5326: SIRT1-Dependent and -Independent Effects of Resveratrol on Cardiovascular Oxidative Stress
Authors: Xia Ning , Daiber Andreas , Habermeier Alice , Spanier Gerrit , Lu Qing , Oelze Matthias , Closs Ellen I , Munzel Thomas , Forstermann Ulrich , Li Huige
Published: April 2024

SIRT1 is a NAD+-dependent class III histone deacetylase. Recent studies point to SIRT1 as a key regulator of angiogenesis, vascular tone and endothelial function. The aim of the present study was to investigate the role of SIRT1 in regulating oxidative stress in the cardiovascular system. Atherosclerotic apolipoprotein E knockout (ApoE-KO) mice were treated with a SIRT1 activator, resveratrol (30 or 100 mg/kg/day for 7 days via gavage). Resveratrol treatment significantly reduced the levels of 3-nitrotyrosine, malondialdehyde and superoxide in the heart, which was associated with an upregulation of superoxide dismutase isoforms (SOD1–3), glutathione peroxidase 1 (GPx1), and catalase. In parallel, mRNA expression of NADPH oxidase subunits NOX2 and NOX4 was reduced. Resveratrol also inhibited the translocation of p47phox and Rac1 and decreased the activity of NADPH oxidase complex in heart membrane fraction. The cofactor of endothelial nitric oxide synthase (eNOS), (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), is synthesized from GTP via a de novo pathway by the rate-limiting enzyme GTP cyclohydrolase I (GCH1), and essential for eNOS functionality. ApoE-KO mice showed increased oxidative stress and eNOS-mediated superoxide production (i.e. eNOS uncoupling). Treatment of ApoE-KO mice with resveratrol enhanced the expression of GCH1 and increased the cardiac levels of BH4. This was associated with reduced superoxide production from eNOS. To find out whether the observed effects of resveratrol were SIRT-dependent, human endothelial EA.hy 926 cells were treated with resveratrol. Also in these cells, resveratrol treatment led to an upregulation of SOD1–3, GPx1, catalase, GCH1, and a downregulation of NOX4. SIRT1 inhibition with sirtinol or SIRT1 knockdown by siRNA reduced the effects of resveratrol on SOD1, SOD2, GPx1, GCH1, but not those on SOD3, catalase, or NOX4. These data indicate that resveratrol reduces cardiovascular oxidative stress by decreasing superoxide production from NADPH oxidases and uncoupled eNOS, and by enhancing superoxide inactivation by SOD isoforms and catalase. Part of the resveratrol effects are SIRT1-dependent. Thus, SIRT1 is likely a novel target for treatment of cardiovascular disease.
Abstract 5335: Significant Decline in the Age Adjusted Mortality From Acute St Segment Elevation Myocardial Infarction in the United States Regardless of Race or Gender With Persistent Higher Mortality Rate for Female and African American Population
Authors: Movahed Mohammad Reza , John Jooby , Jamal M Mazen , Hashemzadeh Mehrnoosh , Hashemzadeh Mehrtash
Published: April 2024

Back ground:
Treatment of acute ST segment myocardial infarction (STEMI) has improved over the last 2 decades. The goal of this study was to determine trends in the mortality of patients with acute STEMI in the United States over the years based on gender and race.

Method:
The Nationwide Inpatient Sample (NIS) database was used to analyze the age adjusted mortality rates for STEMI from 1988 to 2004 for inpatients above the age of 40. ICD-9-CM codes consistent with acute STEMI were utilized.

Results
: A total of 163,915 hospital deaths occurred during the study period. From 1988 the age-adjusted mortality rate declined regardless of gender or race. However, women and African Americans had higher rate of acute STEMI related mortality in comparison to men and Caucasians over the years studied (For 1988 age adjusted mortality rate from STEMI for women was 508 per 100,000 vs. 371 per 100,000 in men. For 2004 age adjusted mortality rate from STEMI for women was 347 per 100,000 vs. 262 per 100,000 in men p< 0.001). For 1988, age adjusted mortality rate from STEMI for Caucasians was 412 per 100,000 vs. 521 per 100,000 in African Americans. For 2004, age adjusted mortality rate from STEMI for Caucasians was 277 per 100,000 vs. 447 per 100,000 in African Americans, p< 0.001, see figures

).

Conclusion:
Age adjusted mortality from acute STEMI has significantly declined over the last decade regardless of gender race with persistent higher mortality rate in women and African Americans over the years.
Abstract 5414: Serum Uric Acid Levels versus the Abdominal Obesity in Relation to Incident Hypertension in a Prospective Study in a Chinese Community
Authors: Zhang Weili , Sun Kai , Yang Ying , Zhang HongYe , Hui Rutai
Published: April 2024

BACKGROUND:
Most Caucasian-based epidemiological data have shown that hyperuricemia is associated with the risk of hypertension, but whether this association is independent of adiposity and other vascular risk factors remains unclear. Therefore, we evaluated the associations of serum uric acid versus abdominal obesity with incident hypertension in a large Chinese Han population.

METHODS:
We conducted a community-based prospective cohort study comprising 7220 participants (mean age 37 years; 74% men) in the Qingdao Health and Nutrition Examination Survey in China, who were free from hypertension at study entry in 1999–2000. During 4-year follow-up, 1370 men (19.0%) and 208 women (11.0%) had developed hypertension.

RESULTS:
After adjustment for age, body mass index, waist circumference, and other covariates, the relative risks (RR) of developing hypertension according to uric acid quartiles were 0.97, 1.23, and 1.39 [95% confidence interval (CI), 1.16–1.68;
P
trend=0.008] for men and 1.49, 1.58, 1.85 (95%CI, 1.06–3.24;
P
trend=0.007) for women. The associations of hypertension with serum uric acid increased with the component number of metabolic syndrome (none, one, two, or more), which is defined by the criteria of the modified Adult Treatment Panel III and an Asian-specific waist circumference cut-off point (≥90 cm in men, ≥80 in women). Given that abdominal obesity is an important underlying risk factor for metabolic syndrome, in joint analyses of waist circumference and serum uric acid, participants with abdominal obesity had a consistently higher risk than those without. Compared with those in the lowest uric acid quartile and without abdominal obesity, participants who were in the highest quartile and also had abdominal obesity had a 3.0-fold and 3.4-fold greater risk of incident hypertension in men (95%CI, 1.56–2.97) and in women (95%CI, 2.10–3.81), respectively. To minimize the bias from reverse causality due to subclinical hypertension, analyses after excluding the first 2 years of follow-up showed similar results.

CONCLUSIONS:
These data suggest a positive association between serum uric acid and incident hypertension during short-term follow-up in a Chinese population. The association was partly mediated through abdominal obesity. 1
Abstract 5426: Identification of Selective Small Molecule Inhibitors of Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Protein (BMP) Signaling Using Zebrafish-based in vivo Structure Activity Relationship Studies
Authors: Hao Jijun , Ho Joshua N , Daniels Richard N , Karim Kaleh A , Murphy Clare K , Hopkins Corey R , Lindsley Craig , Hong Charles C
Published: April 2024

Angiogenesis, a physiological process of forming new blood vessels from pre-existing ones, plays a vital role in organogenesis, tissue response to ischemia, and malignant tumor growth. Angiogenesis is a complex process, requiring a finely tuned balance between various stimulatory and inhibitory signals. Vascular endothelial growth factors (VEGFs) are a major class of proangiogenic mediators that fuction by binding and activating the VEGF receptor tyrosine kinases, including VEGFR2/KDR/Flk-1. Additionally, bone morphogenetic proteins (BMPs) play a critical role in modulating angiogenesis via cross talks between the BMP and VEGF signaling at variety of levels. We recently identified dorsomorphin, the first known small molecule BMP signaling inhibitor, based on its ability to perturb zebrafish embryonic dorsoventral axis, which is normally established by a BMP signaling gradient. Here we show that dorsomorphin can also disrupt angiogenic intersomitic vessel (ISV) formation and has significant off-target activity against the type-2 VEGF receptor (Flk1/KDR). To determine whether dorsomorphin’s anti-angiogenic effects in vivo are due to its effects on BMP, VEGF, or both signals, we conducted a structure activity relationship (SAR) studies of over 70 dorsomorphin analogs purely based on their in vivo effects on zebrafish embryos. We found that dorsomorphin analogs which caused embryonic dorsalization, but had no effect on ISV formation were potent BMP inhibitors but not VEGF/PDGF inhibitors. Conversely, analogs that disrupt angiogenesis but not dorsoventral axis were found to be potent VEGF inibitors, but not BMP inhibitors. These results show that BMP signaling is not required for ISV formation in zebrafish. Moreover, this effort has led to the identification of exquisitely selective BMP and KDR inhibitors with superior in vivo bioavailability and efficacy. To our knowledge, this is the first large-scale SAR study performed in whole animals, and demonstrate the potential of zebrafish chemical embryogenesis for drug development.
Abstract 5428: Nuclear Factor Kappa B Mediates Postnatal Alveolarization by Promoting Pulmonary Angiogenesis via Vascular Endothelial Growth Factor Receptor-2 Regulation
Authors: Alvira Cristina M , Alastalo Tero P , Chen Chihhsin , Lyu Shu-Chen , Ahn Yong-Tae , Cornfield David N
Published: April 2024

Vascular development drives alveolarization via vascular endothelial growth factor receptor-2 (VEGFR-2) signaling. Chronic lung disease of infancy, bronchopulmonary dysplasia (BPD) results from arrested alveolar and vascular development. However, the transcription factors that control alveolarization are unknown. Recent data identified a novel, developmentally regulated, anti-inflammatory role for the transcription factor nuclear factor kappa B (NF
κ
B) in the neonatal lung, as opposed to its recognized, pro-inflammatory role in the mature lung. Thus, we hypothesized that NF
κ
B is essential for alveolarization. To test this hypothesis, neontal mice were treated with the NF
κ
B inhibitor, BAY 11–7082 (BAY), or vehicle (PBS). At 8h, lung was procured for gene and protein expression. At 24h, lungs were fixed at 25 cm H2O pressure for morphometric analysis, determination of apoptosis (TUNEL), and immunohistochemistry (IHC) to detect proliferating cell nuclear antigen (PCNA) and capillary density (CD31). Chromatin immunoprecipitation (ChIP) was performed on lung tissue to evaluate if the NF
κ
B subunit, p65, binds the VEGFR2 promoter. Pulmonary endothelial cells (EC) were isolated using CD31 coated beads to determine VEGFR2 expression and VEGF-mediated survival and proliferation. At 8h, NF
κ
B blockade (BAY) decreased VEGFR2 mRNA and protein in the lung (p<0.01, versus PBS). At 24h, lungs of BAY treated mice had increased alveolar area (p<0.05) and decreased radial alveolar counts (p<0.01), in association with decreased capillary density (p<0.01), reduced proliferation (p<0.001), and increased apoptosis (p<0.05). ChIP demonstrated that, in control lungs, p65 binds the VEGFR2 promoter. Furthermore, NF
κ
B blockade with BAY caused a dose-dependent reduction of VEGFR2 mRNA and protein in pulmonary EC, and impaired VEGF mediated EC survival and proliferation. In summary, NF
κ
B blockade disrupts postnatal alveolarization by decreasing VEGFR2, demonstrating that NF
κ
B promotes alveologenesis through VEGFR2 mediated pulmonary EC survival and proliferation. We speculate that during lung development:

NF
κ
B promotes lung angiogenesis; and

impaired NF
κ
B activity may play a causal role in the arrest of alveolar formation characteristic of BPD.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5436: Generation of Induced Pluripotent Stem Cells From Peripheral Blood of Coronary Artery Disease Patients
Authors: Kim Sinae , Park Changwon , Han Ji Woong , Kim Sung-Whan , Kim Hyongbum , Choi Yong Jin , Lee Eugine , Patel Riyaz , Park In-Hyun , Daley George Q. , Quyyumi Arshed , Yoon Young-sup
Published: April 2024

Background.
Studies have demonstrated that ectopic expression of defined transcription factors can reprogram somatic cells into induced pluripotent stem cells (iPS). However, the sources of patient-derived iPS cells are limited to fibroblasts or mesenchymal cells, which require invasive procedures and long-term manipulation. Accordingly, in this study we aimed to generate human iPS cells from peripheral blood mononuclear cells (PB-MNCs) of coronary artery disease (CAD) patients.

Methods and Results.
We recruited one healthy volunteer and three male patients aged 58, 62, and 77, respectively, who have CADs confirmed by coronary angiography. PB-MNCs obtained by density gradient centrifugation were cultured in an adherent fashion. The adherent cells were infected with the lentiviral supernatant for the four transcription factors of OCT4, SOX2, KLF4 or c-MYC. We used third generation lentiviral vectors which do not have infectivity. At 10 days post-infection, colonies showing hES cell-like morphology were selected and transferred to feeder layers for expansion. After 3 to 4 passages, we observed tightly packed and flat colonies of cells characterized by large nuclei and scant cytoplasm, highly suggesting successful generation of iPS cells from PB-MNCs of all the patients (HiPS-ECP1, HiPS-ECP2 and HiPS-ECP3) and a healthy volunteer (HiPS-ENB). A series of assays including cytochemistry, real-time RT-PCR and teratoma formation assay revealed identical pluripotency of these iPS cells to human ES cells (H1). Bisulfite sequencing of the
OCT4
and
NANOG
promoters in the iPS cells showed profound demethylation similar to hES cells. We next examined the differentiation capacity of the iPS cells. The iPS cells readily form EBs at 10 days in suspension culture. Upon further cultivation of EBs for another 10 days, these iPS cells spontaneously differentiated into cell types of all 3 embryonic germ layers.

In conclusion.
This study provides the first evidence that iPS cells can be successfully generated from PB of CAD patients, which have almost identical characteristics to iPS cells derived from healthy volunteers or human ES cells. This advance will facilitate the use of iPS cells for autologous cell therapy for ischemic cardiovascular diseases.
Abstract 5440: Forward Programming of Pluripotent Stem Cells Towards Distinct Cardiovascular Cell Types
Authors: David Robert , Stieber Juliane , Fischer Evelyn , Brunner Stefan , Brenner Christoph , Pfeiler Susanne , Schwarz Florian , Franz Wolfgang-Michael
Published: April 2024

Aims:
The proliferative potential of pluripotent stem cell derived cardiomyocytes is limited and reasonable yields for novel therapeutic options have yet to be achieved. In addition various clinical applications will require the generation of specific cardiac cell types. Whereas early cardiovascular precursors appear to be important for novel approaches such as reseeding decellularized hearts, direct cell transplantation may require ventricular cells. Our recent work demonstrated that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in pluripotent cells. This led to our hypothesis that “forward programming” towards specific subtypes may be feasible via overexpression of distinct early cardiovascular transcription factors.

Methods and results:
Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells. In comparison to control transfected mES cells a 5 fold increased appearance of beating foci was observed as well as upregulated mRNA and protein expression levels. In contrast to MesP1 no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. Likewise Flk-1, the earliest known cardiovascular surface marker was not induced via Nkx2.5 as opposed to MesP1. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies (EBs) but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes in comparison to control transfected ES cells whereas Nkx2.5 led to preferentially differentiated ventricular cells.

Conclusion:
Our findings show proof of principle for cardiovascular subtype specific programming of pluripotent stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with differentiation factors such as Nkx2.5 further downstream.
Abstract 5443: Osteopontin Stimulates Secretion of Angiogenic Proteins in Endothelial Progenitor Cells
Authors: Vaughan Erin E , Duffy Angela , O’Brien Timothy
Published: April 2024

Patients with Type 1 Diabetes Mellitus (T1DM) often demonstrate an ineffective angiogenic response to ischemia. This may be due to a decrease in the number and function of their endothelial progenitor cells (EPC). EPC are promising cells for autologous cell transplantation in the treatment of T1DM hence it is imperative to understand the underlying cause of their dysfunction. Therefore, we identified proteins that are dysregulated in EPC from patients with T1DM. Osteopontin (OPN), a secreted protein involved in cell migration, cell survival, and tumour growth was found to be down regulated. Thus, we hypothesized that OPN down regulation in T1DM EPC contributes to a decreased angiogenic response. To elucidate the role of OPN in EPC, EPC were isolated from OPN knockout (KO) mice and wild type mice (WT) for a matrigel tubule assay to assess
in vitro
angiogenic potential. KO EPC induced significantly less tubule formation than WT EPC (p<.05, n=3). Knockout EPC that were pre-incubated with recombinant OPN induced tubule formation at levels similar to WT and significantly higher than KO cells that were not incubated with OPN (p<.05, n=3). Further, conditioned media (CM) from WT cells induced tubule formation to the same levels as the EPCs themselves suggesting that secreted proteins are responsible for the angiogenic effect. Interestingly, when KO EPC were pre-incubated with OPN, washed, and fed with new media the CM media induced tubule formation to WT levels(n=3), even though there was no OPN directly in the media. Hence, we further hypothesized that OPN is acting on EPC to induce the secretion of angiogenic cytokines. To test this, a chemi-array was performed on EPC from WT, KO and KO exposed to OPN. WT EPCs expressed FGF
α
at a much higher level than KO cells. Expression of FGF was restored to WT levels when KO cells were pre-incubated with OPN. Further, WT cells expressed Il-6 and TGF-
α
whereas KO cells did not express these proteins at detectable levels. Interestingly, when KO cells are exposed to OPN both IL-6 and TGF-
α
are expressed at WT levels. Taken together, this data suggests that OPN expression increases the angiogenic potential of EPCs via an autocrine mechanism whereby OPN is secreted by the EPC and subsequently induces the expression of a variety of angiogenic proteins.
Abstract 5452: Consistency of Detection of Abnormal Late Enhancement in Left Ventricular Myocardium by Multislice CT in Myocardial Disease in Comparison With Cardiac Magnetic Resonance Using Segment by Segment Analysis
Authors: Takaoka Hiroyuki , Funabashi Nobusada , Uehara Masae , Daimon Michiko , Ueda Marehiko , Murayama Taichi , Komuro Issei
Published: April 2024

Purpose: To evaluate the capability of multislice CT (MSCT) to detect abnormal late enhancement (LE) in the left ventricular (LV) myocardium in myocardial disease, we compared MSCT with cardiac magnetic resonance (CMR) using segment by segment analysis and summarized the merit of performing late phase acquisition of MSCT. Materials and

Methods: 79 subjects (61 male, 56.9±15.6 years) with suspected LV myocardial abnormalities (24 hypertrophic cardiomyopathy, 13 dilated cardiomyopathy, 5 cardiac sarcoidosis, 4 cardiac amyloidosis, 3 arrhythmogenic right ventricular cardiomyopathy, and 1 Fabry disease, et al) underwent enhanced MSCT (Aquilion One, or Light Speed Ultra 16) and CMR (Intera Achieva). Images were divided into 8 segments: 6 in the LV and each one in the right ventricle and either right or left atrium for each subject.

Results: Among the 632 segments, LE was detected in 127 segments in MSCT, 137 segments in CMR, and 100 segments in both MSCT and CMR. LE was detected in 27 segments in only MSCT, and in 37 segments in only CMR. LE was not detected in 468 segments in either MSCT or CMR. In comparing CT with CMR for detecting LE, the sensitivity, specificity, positive and negative predictive values, and consistency were 73.0, 94.5, 78.7, 92.7 and 89.9%, respectively.

Conclusions: The consistency of detection of LE in LV myocardium by MSCT in myocardial disease compared with CMR was 89.9% in spite of poor contrast resolution of CT. MSCT should be used to evaluate LV myocardium in subjects with suspected LV myocardial abnormalities by adding late phase acquisition despite increments of radiation exposure, and even though late phase acquisition is more desirable than pre enhanced scanning.
Abstract 5460 Heat Shock Protein 27 Over-Expression in Hematopoietic Cells is Atheroprotective and Anti-Inflammatory
Authors: Seibert Tara A , Chen Yong-Xiang , McNulty Melissa , Zhoa XiaoLing , Sun Kevin , Rayner Katey , O’Brien Edward R
Published: April 2024

Previously, we demonstrated that Heat shock protein 27 (HSP27) shows diminished expression with progression of human coronary atherosclerosis [ATVB 2005]. Moreover, we have shown that global over-expression of HSP27 (HSP27
o/e
) protects against the development of atherosclerosis in ApoE
−/−
mice in part via binding to Scavenger Receptor-A [Circ Res 2008]. More recent experiments reveal that macrophage apoptosis is less frequent in advanced (12 wk) lesions from apoE
−/−
HSP27
o/e
mice and highlight macrophage specific anti-inflammatory protective mechanisms. The purpose of this study was to determine if blood-borne cells (e.g., mononuclear cells) are sufficient and required for the HSP27 atheroprotective phenotype in female ApoE
−/−
mice.

Methods/Results: Female ApoE
−/−
mice (~10 Weeks old and ≥ 18.5 g), underwent
μ
-irradiation to eliminate endogenous bone marrow before receiving bone marrow from either female ApoE
−/−
or ApoE
−/−
HSP27
o/e
donors. After 6 weeks of recovery recipients were placed on a high fat/cholesterol diet for 4 weeks to induce atherosclerosis before being euthanized. ApoE
−/−
mice receiving bone marrow from ApoE
−/−
HSP27
o/e
had detectable levels of HSP27 mRNA in liver and spleen. In addition, the HA tag of the over-expression construct was detectable in liver sections and HSP27 was measured in the serum from mice receiving the ApoE
−/−
HSP27
o/e
bone marrow. Over-expression of HSP27 in cells of hematopoietic origin resulted in a 52% reduction in aortic
en face
lesion burden (p=0.005) and a 32% reduction in the lesion area found in aortic sinus cross-sections (p=0.02). The macrophage content of lesions in both murine populations was similar. In vitro bone marrow derived macrophages from ApoE
−/−
HSP27
o/e
mice had a 2.7-fold decrease in oxidized LDL uptake after 24 hrs (p=0.002) and were less adherent to LPS activated HUVECs (p<0.05) when compared to ApoE
−/−
derived macrophages. In conclusion, the over-expression of HSP27 in bone marrow derived cells is sufficient and necessary to provide atheroprotection in ApoE
−/−
mice and points to a central role for HSP27 in reducing the inflammatory milieu of atherosclerotic lesions.
Abstract 5462: Dual Effects of Nox2-NADPH Oxidase During Atherosclerotic Plaque Development in Apolipoprotein E-Null Mice
Authors: Judkins Courtney P , Diep Henry , Broughton Brad R , Mast Anja E , Hooker Elizabeth U , Miller Alyson A , Selemidis Stavros , Dusting Gregory J , Sobey Christopher G , Drummond Grant R
Published: April 2024

The reactive oxygen species (ROS) -producing NADPH oxidases are implicated in atherogenesis, although the relative contribution of the different isoforms of these enzymes to plaque development has not been established. We investigated the role of the ‘classical’ Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE
−/−
) mice. Nox2 expression (Western blotting) and localisation (immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (isometric contractions to N

γ

-nitro-L-arginine methyl ester) and atherosclerotic lesion development along the luminal surface of the aorta and in the aortic sinus, were measured in 12, 19 and 26 week-old wild-type, ApoE
−/−
and Nox2
−/−
/ApoE
−/−
mice maintained on a Western style diet (21% fat, 0.15% cholesterol). Nox2 expression was elevated in the endothelium of ApoE
−/−
mice prior to lesion formation and this was associated with elevated ROS production. In developing plaques, macrophages were also a source of Nox2. The absence of Nox2 in 12 and 19 week-old Nox2
−/−
/ApoE
−/−
mice had minimal effects on plasma lipids but was associated with a marked reduction in vascular ROS production and an increase in NO bioavailability.
En face
lesion coverage of the aorta from the arch to the iliac bifurcation was also markedly reduced in 12 and 19 week-old Nox2
−/−
/ApoE
−/−
versus ApoE
−/−
mice. However, in 26 week-old mice, differences in
en face
lesion area were no longer apparent between strains and, surprisingly, intimal thickening in the aortic sinus was more pronounced in the Nox2
−/−
/ApoE
−/−
mice. Thus, we have described, for the first time, that Nox2-NADPH oxidase plays dual roles in atherosclerotic plaque development in ApoE
−/−
mice, being pro-atherogenic in early stages, and possibly protective in later stages of the disease.
Abstract 5466: Nuclear Receptor Nurr1 in Vascular Smooth Muscle Cells is Protective in In-stent Restenosis
Authors: Bonta Peter I , Pols Thijs W , van Tiel Claudia M , Bot Ilze , de Winter Robbert , Pannekoek Hans , Biessen Erik A , de Vries Carlie J
Published: April 2024

Introduction

Coronary atherosclerosis is treated by percutaneous coronary intervention (PCI) in combination with stent placement. The major drawback of this intervention is (in-stent) restenosis, which is characterized by activation and excessive proliferation of smooth muscle cells (SMCs). In previous studies we have shown that nuclear receptor Nurr1 inhibits the inflammatory response of macrophages and reduces lipid loading of these cells. In this study we investigated nuclear receptor Nurr1 in SMCs in human and experimental vascular restenosis.

Methods and results

In a prospective cohort of 601 patients undergoing PCI including stent-placement, we report a strong association of Nurr1 haplotypes with angio-graphic and clinical in-stent restenosis risk (Hazard ratio (HR) 3.1; 95%CI:1.9–5.1 and HR 3.4; 95%CI 1.6–7.1, respectively). Furthermore, Nurr1 is expressed in human in-stent restenosis and induced in cultured human SMCs in response to serum or tumor necrosis factor (TNF)-
α
-mediated activation. Lentivirus-mediated gain and loss of function experiments in SMCs demonstrated that overexpression of Nurr1 inhibited proliferation, consistent with increased expression of the key cell-cycle inhibitor p27
Kip1
, whereas Nurr1 silencing enhanced SMC growth. The TNF
α
-induced pro-inflammatory response of SMCs is inhibited by Nurr1 as reflected by reduced interleukin-1
β
, TNF
α
and monocyte chemo-attractant protein-1 expression. In subsequent mouse experiments, Nurr1 was locally expressed in the carotid artery of ApoE−/− mice after wire-induced injury, and we revealed that Nurr1 inhibits SMC-rich lesion formation. In line with this observation, silencing of murine Nurr1 in the vessel wall increased lesion formation.

Conclusion
Nurr1 haplotypes associate with human in-stent restenosis risk and Nurr1 is expressed in in-stent restenosis. In addition to the anti-inflammatory function of Nurr1 in macrophages, we show that Nurr1 inhibits proliferation and inflammatory responses in SMCs, which explains inhibition of SMC-rich lesion formation in mice. Since small-molecule drugs that enhance the activity of Nurr1 have been identified, we propose this nuclear receptor Nurr1 as an attractive novel target for (local) intervention in restenosis.
Abstract 5480: Laminin Receptor Activation Inhibits Endothelial Tissue Factor Expression
Authors: Holy Erik W , Stämpfli Simon F , Akhmedov Alexander , Camici Giovanni G , Lüscher Thomas F , Tanner Felix C
Published: April 2024

Background.
Tissue factor (TF) is an important trigger of arterial thrombosis. The extracellular matrix glycoprotein laminin is a major compound of the basement membrane contributing to vessel wall integrity. The green tea catechin epigallocatechin-3-gallate (EGCG) is a ligand of the 67 kDa laminin receptor (67-LR). This study investigates the effect of 67-LR activation by laminin and EGCG on endothelial TF expression.

Methods and Results
. Immunofluorescence demonstrated that human aortic endothelial cells expressed 67-LR abundantly. Western blot analysis demonstrated that cells grown on laminin expressed less TF in response to TNF-
α
than those grown on fibronectin (n=6;
p
<0.001). EGCG inhibited TNF-
α
and histamine induced TF expression and activity in endothelial cells in a concentration-dependent manner (1–30
μ
M) resulting in a 87% reduction of TF expression (n=5;
p
<0.001). In contrast, expression of tissue factor pathway inhibitor was not affected (n=4;
p
=NS). Real-time PCR and promoter studies revealed that inhibition of TF expression occurred at the transcriptional level. EGCG (30
μ
M) impaired activation of the mitogen-activated protein (MAP) kinase c-Jun terminal NH2 kinase (JNK), but not ERK or p38. The JNK inhibitor SP600125 (1
μ
M) reduced TF promoter activity (n±4,
p
±0.001) and protein expression (n=4;
p
<0.001). 67-LR blocking antibodies blunted the inhibitory effect of EGCG on JNK activation and TF expression. Expression of Vascular cell adhesion molecule 1 (VCAM-1) was not regulated by JNK, and its expression was not affected by 67-LR activation.

Conclusions
. Laminin receptor activation inhibits endothelial TF expression by impairing JNK phosphorylation. This study identifies a new mechanism by which the basement membrane contributes to hemostatic balance of the endothelium in intact vessels and suggests 67-LR as a potential target for the development of novel anti-thrombotic therapies.
Abstract 5500:
PDGFD
Variation is Associated With Increased Susceptibility to First-ever Non-hypertensive Intracerebral Hemorrhage Whereas Predicts Decreased Risk of Recurrence
Authors: Bai Yongyi , Chen Jingzhou , Sun Kai , Wang Yibo , Xu Xiqi , Hui Rutai
Published: April 2024

Background

Platelet-derived growth factor D (PDGF-D) is a most recently discovered member of the PDGF family and plays an important role in angiogenesis, vessel remodeling and repairing in response to injury. We hypothesized that genetic variation in
PDGFD
gene might alter the susceptibility to stroke.

Methods

We determined the genotypes of a single nucleotide polymorphism (SNP) (−858A/C, rs3809021), the only identified tag-SNP in the promoter region of
PDGFD
from the HapMap CHB sample, in 1484 patients with stroke (654 cerebral thrombosis, 419 lacunar infarction, 411 intracerebral hemorrhage [ICH]) and 1528 control subjects from an unrelated Chinese Han population and followed the stroke patients up for a median of 4.5 years (range 0.1 to 5.4). Functions of −858 variant were studied by using luciferase reporter assays.

Results

The SNP −858A-bearing
PDGFD
promoter exhibited 3.78-fold increase of transcription activity than the SNP −858C-bearing promoter. Genotype analyses were conducted for both dominant and additive models. The A allele of the −858 locus showed significantly increased risk of ICH (dominant model: odds ratio [OR] 1.29, 95% confidence interval [CI] 1.00 to 1.68,
P
=0.05; additive model: OR 1.24, 95% CI 1.01 to 1.52,
P
=0.04) than wild-type genotype. Further analyses showed that −858 A allele conferred about 2-fold increase in risk of non-hypertensive ICH (dominant model: OR 2.1, 95%CI 1.34 to 3.29,
P
=0.001; additive model: OR 1.75, 95% CI 1.24 to 2.46,
P
=0.001). After a median follow-up of 4.5 years, 52 patients had a recurrent ICH. After adjusting for conventional cardiovascular risk factors, we found that −858 A allele was associated with a reduced risk of ICH recurrence (dominant model: adjusted hazard ratio [HR] 0.09, 95%CI 0.01 to 0.74, P=0.025; additive model: HR 0.21, 95% CI 0.04 to 1.16,
P
=0.073) in non-hypertensive patients. No association was observed between the
PDGFD
variant and hypertensive ICH and atherothrombotic stroke.

Conclusions

The −858A allele was associated with increased first-ever non-hypertensive ICH and was a predictor of lower risk of ICH recurrence. Further studies should be conducted to reveal the role of PDGF-D at various stages of ICH development beneficial, or deleterious.
Abstract 5505: The Impact of Selectively Performed Echocardiography in Patients With Acute Ischemic Stroke or Transient Ischemic Attack
Authors: Yao Gui-Hua , Grigerick Darrel , Camarano Gustavo , Armon Carmel , Jiang Leng
Published: April 2024

Background:
The updated ACC/AHA/ASE guideline in 2003 recommended that echocardiography be performed selectively in patients with acute ischemic stroke or TIA. We aimed to evaluate the diagnostic yield and therapeutic impact of transthoracic echocardiography (TTE) with such an approach.

Methods:
A total of 984 consecutive patients with acute ischemic stroke or TIA were included in the study. Clinical data were systemically reviewed, including the reports of ECG, brain MRI/CT and carotid duplex ultrasound (CDUS). TTE data were retrospectively analyzed.

Results:
TTE tests were performed in 624 (63.4%) patients (mean age 70.9±14.1 years, 47.4% of men). Atrial fibrillation (AF) was present in 116 (18.6%) patients. An overall potential cardiac source of embolism (PCSE) was found in 155 (24.8%). Atrial septal abnormality (PFO± septal aneurysm) and LV dysfunction (EF ≤ 30%± wall motion abnormality/LV aneurysm) were the most frequent PCSE in younger (< 45 years) and older (≥ 45 years) patients, respectively. The overall diagnostic yield of TTE was comparable in both groups (35.5% vs 26.8%,
P
= 0.303). Significant carotid stenosis was found in 27 (7.5%) patients in the older group by CDUS, but none in the younger patients. After excluding patients with AF and significant carotid stenosis, who have independent indication for anticoagulation or intervention, the therapeutic impact of TTE was more significant in younger patients (27.6% vs 11.5%,
P
= 0.019). Numbers needed to test (NNT) for detection of a PCSE was 4 in younger patients and 9 in older patients, the latter being more than 2-fold of the former.

Conclusion:
Selectively performed TTE in patients with ischemic stroke or TIA can provide important information on PCSE. The diagnostic yield and therapeutic impact are much higher in younger patients.

Table.

Potential cardiac source of embolism
Abstract 5514: Laminar Shear Stress Upregulates Endothelial KCa2.3 and KCa3.1 Calcium-activated Potassium Channels via Activating Calmodulin-dependent Protein Kinase Kinase and AMP-activated Protein Kinase
Authors: Ohta Masanori , Nandedkar Sandhya , Jo Hanjoong , Miura Hiroto
Published: April 2024

Aim

Endothelial cell (EC) KCa2.3 and KCa3.1, calcium-activated potassium channels, play a crucial role in NO- and EDHF-mediated regulation of vascular tone by controlling membrane potential and intracellular calcium levels. Since laminar shear stress (LS) is a major determinant of EC function in a calcium-dependent manner, we hypothesized that LS upregulates KCa2.3 and KCa3.1 via activating calcium/calmodulin (CaM) kinase cascade.

Methods

Human coronary artery ECs were exposed to static, LS (15 dyn/cm
2
) or oscillatory shear stress (OS; +/−5 dyn/cm
2
× 1Hz) condition for 24 hours in the absence or presence of STO-609 (CaM-dependent kinase kinase (CaMKK) inhibitor, 10
μ
g/ml), Compound C (AMP-activated protein kinase (AMPK) inhibitor, 10
μ
M), KN-62 (calcium/CaM-dependent kinase (CaMK) inhibitor, 10
μ
M), KG-501 (cAMP response element binding protein (CREB) inhibitor, 25
μ
M), or tumor necrosis factor-
α
(TNF-
α
; 1 or 10 ng/ml). The mRNA expression and CREB phosphorylation were determined by real-time PCR or Western blotting.

Results

LS markedly increased KCa2.3 and KCa3.1 expressions (3.3±0.4 and 6.6±1.8 fold increases vs. static, n=4 –10, p<0.001), whereas OS reduced KCa2.3 and unchanged KCa3.1 expressions (0.4±0.04 and 1.1±0.5 fold, n=3, p=ns). STO-609 significantly inhibited LS-induced increase in KCa2.3 and KCa3.1 expressions (1.8±0.1 fold and 1.5±0.4 fold) and markedly decreased LS-induced CREB phosphorylation (LS; 2.3 fold and LS+STO-609; 1.5 fold vs. static). KCa2.3 and KCa3.1 expressions were also reduced by Compound C (2.2±1.6 and 2.0±1.0 fold, respectively), but not by KN-62 (5.9±1.1 and 6.5±1.2 fold, respectively). KG-501 suppressed LS-induced increase in both channels (KCa2.3; 1.5±0.7 and KCa3.1; 1.9±0.7 fold). TNF-
α
completely inhibited both channel expressions in a dose-dependent manner (1 and 10 ng/ml; KCa2.3; 0.6±0.01 and 0.1±0.1 fold, and KCa3.1; 0.7±0.1 and 0.3±0.03 fold).

Conclusions

LS upregulates KCa2.3 and KCa3.1 expressions via CREB phosphorylation through activation of CaMKK and AMPK, a downstream kinase of CaMKK, whereas TNF-
α
suppresses both channel expressions. Thus KCa2.3 and KCa3.1 may play an important role in endothelial adaptation to hemodynamic changes and in the maintenance of EC function.
Abstract 5531: The Impacts of Thin-Slice Contrast-Enhanced Computed Tomography and Rapid Cortisol Measurement During Adrenal Venous Sampling
Authors: Nishioka Kenji , Teragawa Hiroki , Oki Kenji , Fujii Yuichi , Mitsuba Naoya , Fujimura Takenori , Mikami Shinsuke , Noma Kensuke , Ishida Takafumi , Higashi Yukihito , Kihara Yasuki
Published: April 2024

Background: It has been considered that Adrenal venous sampling (AVS) is the golden standard to identify primary aldosteronism (PA). However, there have been technical difficulties in AVS, especially in right AVS. Therefore, we investigated whether the anatomical examination of adrenal vein using thin-slice contrast-enhanced computed tomography (CT) and the monitoring cortisol concentrations during catheterization affect the success rate of AVS in patients with suspected of having PA.

Methods: Forty-eight patients who were suspected of having PA, were enrolled. Thin-slice contrast-enhanced CT with 1.2mm-slice and rapid cortisol assay using electro chemiluminescent Immunoassay (ECLIA) were performed in 18 patients (Group I). Contrast-enhanced CT with 5mm-slice were performed in 20 patients (Group II). Samples were collected from the vena cava as well as from right and left adrenal veins. We evaluated the procedure success rate and the total radiation time in two groups.

Results: Date are expressed as the mean±SEM. The patients characteristics were similar in the two groups. On contrast-enhanced CT, right adrenal vein was detected in all patients in Group I (100%) although it was detected only 11 patients (55%) in Group II (p<0.05). The success rate of AVS was significant higher in Group I (96%), than in Group II (30%, p<0.0001) while the success rate of left AVS was not different in the two groups. The total radiation time was shorter in Group I (22±8 min) than Group II (32±9 min, p<0.05).

Conclusions: Anatomical examination using thin-slice contrast-enhanced CT and intraoperative cortisol assays increase the procedure success rate, and decrease the duration time of radiation. The rapid cortisol assay kit might be valuable and timely information for improving the success rate of adrenal venous sampling.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5532: Apoptosis, Interstitial Collagen and Diastolic Function in Essential Hypertension
Authors: Rivera Miguel , Cortes Raquel , Morillas Pedro , Rosello Esther , Grigorian Lilian , Martinez Luis , Lauwers Catheline , de Burgos Fernando Garcia , Soria Federico , Juanatey Jose Ramon Gonzalez , Paya Rafael , Lozano Teresa , Orosa Placido , Miro Vicente , Antorrena Isabel , de Rivas Beatriz , Mainar Luis , Bosca Jose Leandro Perez , Baron Gonzalo , Climent Vicente , Calabuig Jose Ramon , Jordan Alejandro , Salvador Antonio , Portoles Manuel , Bertomeu Vicente
Published: April 2024

Introduction: Sustained hypertension (HT) leads to left ventricular hypertrophy (LVH) and left ventricular dilatation, processes associated with loss of cardiac myocytes through apoptosis. Nevertheless, the relationship of apoptosis, myocardial fibrosis and diastolic function remains unknown. Therefore, the aims of the present study were to analyze the relationship between aminoterminal propeptide levels of type III procollagen (PIIINP), sFas, soluble TNF receptor 1 (sTNF-R1) and diastolic function in essential HT. Hypothesis: We assessed the hypothesis that increased cardiac collagen turnover would be associated with LVH, diastolic dysfunction and apoptosis even in asymptomatic HT.

Methods: The asymptomatic hypertensive group consisted of 253 Caucasian patients (mean age 60±13 years, 139 males) from 11 hospitals. A routine physical examination, laboratory analyses, and echo-Doppler study were performed.

Results: Serum concentrations of PIIINP were higher in hypertensive patients compared to control group [4.18 (3.55 – 5.04
μ
g/L) vs. 3.54 (2.98 – 4.54
μ
g/L), p=0.006] and also plasma levels of sFas and sTNF-R1 [1.40 (1.13 – 1.69 ng/mL) vs. 1.00 (0.84 – 1.27 ng/mL), p<0.0001; 385 (291 – 545 pg/mL) vs. 236 (194 – 302 pg/mL), p<0.0001]. Log-transformed concentrations of anti-apoptotic cytokines were correlated between them (r=0.404, p<0.0001). Moreover, serum PIIINP concentrations were associated with log-transformed sFas (r=0.386, p<0.0001) and sTNF-R1 (r=0.298, p<0.001). Then, multivariate analyses included sFas (p<0.0001) and sTNF-R1 (p<0.0001) as independent factors of serum procollagen levels. Finally, marker concentrations were significantly correlated with diastolic parameters.

Conclusions: This study showed increased circulating levels of PIIINP, sFas and sTNF-R1 in our group of asymptomatic hypertensive patients. Furthermore, sFas and sTNF-R1 are independent factors of serum type III procollagen, and diastolic parameters showed a highly significant relationship with myocardial fibrosis and anti-apoptotic cytokines.