bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 585: Endothelial Cells from Adipose Tissue of Obese Humans Display Mesenchymal Characteristics
Authors: Haynes Bronson A , Lehrer Eric J , Bhatt Giann J , Huyck Ryan W , James Ashley N , Dobrian Anca D
Published: June 2021

The mechanisms underlying vascular dysfunction in adipose tissue (AT) in obesity are not clearly understood. Our hypothesis is that in response to pro-inflammatory cytokines (PIC) present in obese AT, endothelial cells (EC) can de-differentiate and acquire a mesenchymal-like phenotype (EndoMT) that leads to endothelial dysfunction. To test our hypothesis, we measured endothelial and mesenchymal markers of CD31
+
CD34
+
EC isolated from omental (OM) and subcutaneous (SC) AT of bariatric subjects (BAMVEC) using RT-PCR and western blot. Permeability and oxidative metabolism were determined by ECIS and Seahorse analyzer XF
e
24, respectively. BAMVEC isolated from both OM and SC fat showed very low protein expression of vWF and VE-Cadherin (EC markers) and abundantly expressed αSMA and the EMT transcription factor twist-1. To determine effects of PIC on EndoMT, commercially available primary endothelial cells from AT (HAMVEC) were treated in vitro with PIC (2.5ng/mL TNFα, IFNγ and TGFβ) for 1, 3 or 6 days. We found progressive down-regulation by >2-fold (p<0.001) of the EC markers vWF, VE-Cadherin, and Occludin compared to controls. As early as 1 day of PIC treatment twist-1 (p<0.001) and snail1 (p<0.05) showed an increase by >2-fold. Similarly, OM and SC BAMVEC expressed >2-fold increase in the mesenchymal genes twist-1, FSP1, αSMA, and snail1 compared to untreated HAMVEC. Metabolically, BAMVEC had increased ATP production and maximal respiration compared to HAMVEC suggesting increased oxidative phosphorylation, a marker of mesenchymal-like cells. PIC stimulation of HAMVEC yielded significant increases in endothelial permeability and motility (p<0.001). Notably, there were no significant differences in any of the markers between OM and SC BAMVEC. These results show that EC in obese AT exhibit a mesenchymal-like phenotype which may account for functional changes such as increased permeability and migration and are not depot specific. Using primary EC from human AT we showed that prolonged exposure to PIC induces a phenotype similar to CD31+CD34+ EC from obese AT. This supports the concept that AT inflammation can promote EC de-differentiation in vivo and our in vitro model is suitable for future studies to uncover the relevant mechanisms.
Abstract 596: Increased Migration of Neutrophils Across Endothelial Cells from Patients with Pulmonary Arterial Hypertension is Related to Reduced Platelet Endothelial Cell Adhesion Molecule -1 and is Prevented by the Neutrophil Elastase Inhibitor Elafin
Authors: Taylor Shalina , Moonen Jan-Renier , Miyagawa Kazuya , Gu Mingxia , Sa Silin , Hennigs Jan , Wang Lingli , Rabinovitch Marlene
Published: June 2021

Pulmonary arterial hypertension (PAH) is a devastating progressive disease associated with a high mortality despite current vasodilator therapies. Perivascular inflammation and high levels of neutrophil elastase are thought to play a pivotal role in the adverse vascular remodeling of small pulmonary arteries that causes PAH. Despite this, the function of neutrophils and in particular, their interaction with the pulmonary arterial endothelium has not been studied in PAH. We hypothesized that neutrophil functions such as adhesion to a substratum or to endothelial cells and transmigration across a substratum or trans-endothelial migration (TEM) are abnormal in PAH on the basis of dysfunction of both cell types. Using a neutrophil like cell line dHL-60 and isolated human neutrophils from donors and PAH patients, we demonstrated no significant difference in adhesion to PAH vs. control PAECs when stimulated with TNF-α (100μg/mL). However, TEM of both IL-8 (100ng/ml) and fMLP (100nM) stimulated dHL-60 cells and donor neutrophils was enhanced in PAH vs. control PAECs (p<0.01) likely related to reduced expression of Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1) in PAH PAECs (p <0.001). We therefore further hypothesized that inhibition of neutrophil elastase via recombinant human elafin would be sufficient to reverse TEM in both PAH and control PAEC by reducing neutrophil adhesion, a feature known to be elastase-dependent. Administration of elafin (1μg/mL) attenuated fMLP and IL-8 induced dHL-60 and neutrophil adhesion to fibrinogen and fibronectin (p<0.05 for both) as well as transmigration across both substrates (p<0.05 for both). Elafin reduced adhesion of neutrophil similarly in PAH and control PAEC (p <0.05 for both). Furthermore, in a dose dependent manner, elafin inhibited TEM in PAH PAEC by 40% in control PAECs, by 20% (p<0.001 and p<0.01 respectively) bringing values to within the same range of suppressing but not eliminating TEM. We therefore conclude that despite the reduction in PECAM-1 in PAH PAEC, inhibition of neutrophil adhesion with the elastase inhibitor elafin is sufficient to prevent the enhanced TEM. Elafin may therefore be of benefit in suppressing the deleterious impact of neutrophil in enhanced inflammation seen in PAH.
Abstract 597: Diet-induced Obesity Requires Signalling Through Tumor Necrosis Factor Receptor-associated Factor 1 (TRAF-1) in Adipocytes
Authors: Wolf Dennis , Anto Michel Nathaly , Hilgendorf Ingo , Bode Christoph , Zirlik Andreas
Published: June 2021

Background:
Accumulation of inflammatory leukocytes is a prerequisite of adipose tissue inflammation during cardio-metabolic disease. We recently reported that a genetic deficiency of the intracellular signalling adaptor TRAF-1 attenuates inflammatory cell recruitment and vascular inflammation in atherosclerosis. Here, we tested the contribution of TRAF-1 to diet-induced obesity (DIO) in mice.

Methods and Results:
To test the association of TRAFs and obesity we screened for expression of different TRAFs in adipose tissue. We found an up-regulation of TRAF-1 mRNA in obese mouse and human adipose tissue, resulting from higher gene expression in adipocytes, but not in adipose tissue macrophages. To test a functional relevance of TRAF-1 signalling in obesity, WT or TRAF-1
-/-
mice consumed a high fat diet HFD for 20 weeks. Surprisingly, genetic deficiency of TRAF-1 abolished diet-induced weight gain by supressing peripheral fat depositions. Consequently, TRAF-1
-/-
mice demonstrated ameliorated glucose levels after glucose and insulin tolerance tests and dampened insulin signalling. Consistently, we also found reduced accumulation of adipose tissue macrophages. Mechanistically, TRAF-1
-/-
mice demonstrated no differences in basic energy metabolism, such as in energy expenditure. However, TRAF-1
-/-
adipocytes had higher expression of Adipose Triglyceride Lipase (ATGL) and Hormone-sensitive Lipase (HSL), suggesting increased lipid breakdown in adipocytes. In accord, plasma levels of free fatty acids were higher, while leptin levels were reduced in TRAF-1
-/-
mice. Finally, in a collective of patients with a high prevalence of the metabolic syndrome, TRAF-1 expression correlated with the metabolic syndrome, suggesting clinical relevance of our findings.

Conclusion:
We present the novel finding that the signalling adapter TRAF-1 correlates with obesity in mice and humans. Genetic deficiency of TRAF-1 attenuates diet-induced obesity by increasing lipolysis in adipocytes. These findings identify TRAF-1 as a novel therapeutic target in obesity and adipose-tissue inflammation.
Abstract 598: Puerarin Inhibits Oxldl-mediated Proinflammatory and Proatherogenic Effects in Human Macrophages
Authors: Zhang Heng , Zhai Zhenhua , Li Yao , Shi Yueping , Zhou Ming-sheng
Published: June 2021

Puerarin, a major isoflavonoid compound isolated from the Chinese herb Kudzu roots, has been a long history and widely used for treatment of cerebral and cardiovascular diseases in China and other Asian countries, including coronary artery disease, heart failure and myocardial infarction. However, the underlying mechanisms by which puerarin inhibits vascular atherogenic diseases are largely unknown. The activated macrophages release of inflammatory cytokines and uptake of oxLDL forming foam cells are critical for the development of atherosclerotic plaque. The present study investigated whether puerarin inhibited atherogenic lipid oxLDL-mediated macrophage activation and foam cell formation in human THP1 macrophage. The Cells were cultured and differentiated into macrophages by pre-incubation with 100 ng/ml PMA. Treatment with oxLDL (50 μg/ml) significantly increased the mRNA expression of proinflammatory cytokines tumor necrosis factor α(TNFα, 160%) and interleukin (IL)1β(13 fold) accompanied by upregulation of toll-like receptor 4 (TLR4, 165%) and the ratio of phospho-I
κ
Bα/I
κ
Bα in THP1 macrophage. Puerarin dose-dependently prevented an increase in oxLDL-induced proinflammatory gene expression with downregulation of TLR4 and the ratio of phospho-I
κ
Bα/I
κ
Bα. Furthermore, puerarin prevented oxLDL-mediated lipid deposition and foam cell formation associated with downregulation of scavenger receptor CD36. Flow cytometry analysis showed that puerarin reduced the number of early apoptotic cells of macrophages induced by oxLDL. Our results show that puerarin has anti-inflammatory and anti-atherogenic effects in vitro, the underling mechanisms may involve the inhibition of TLR4/NF
κ
B pathway and downregulation of CD36 expression. The results from the present study provide scientific evidence and may expand our armamentarium to use puerarin for prevention and treatment of cardiovascular and atherosclerotic diseases.
Abstract 6: The RNA-binding Protein ADAR2 Controls Interleukin-6-induced Endothelial Cell Pro-inflammatory Response by Regulating MicroRNA Biogenesis
Authors: Gatsiou Aikaterini , Lunella Federica F , Amrhein Carolin , Guenther Stefan , Gonzalez Claudia Garcia , Schneider Andre , Braun Thomas , Zeiher Andreas M , Dimmeler Stefanie , Stellos Konstantinos
Published: June 2021

Background:
RNA binding proteins (RBPs) are key players in posttranscriptional regulation of mRNAs. Adenosine deaminase acting on RNA-2 (ADAR2) enzyme binds to double-stranded RNAs and controls brain development. The role of ADAR2 in endothelial cell function has not been described so far.

Methods and Results:
ADAR2 is expressed in human and murine endothelial cells and is 2-fold induced by hypoxia
or
hind limb ischemia in mice (P<0.05 for all). ADAR2 deficiency resulted in 73±12% impairment of leukocyte infiltration, in 53±4% reduced neovascularization, and a 40±6% decreased blood-flow recovery of ischemic muscle tissues in a hindlimb ischemia mouse model (P<0.001 for all). Mechanistically, ADAR2 knockdown causes a 2-fold downregulation of 522 transcripts, whereas 113 transcripts are found to be upregulated in RNA-seq experiments. Among the endothelial cell enriched, highly ADAR2-regulated transcripts was interleukin-6 signal transducer (IL6ST also known as glycoprotein-130, gp130), the receptor of interleukin-6 (IL-6). Silencing of ADAR2 resulted in a downregulation of gp130 mRNA and protein expression in endothelial cells by 65±5% and 50±5%, respectively (P<0.001 for both). Similarly, the expression of gp130 mRNA was decreased by 50±25% (P<0.05) in murine lung endothelial cells, which derived from transgenic ADAR2-null mice. Silencing of ADAR2 reduced by at least 2-fold the IL-6-mediated STAT3 phosphorylation, the mRNA expression of the IL-6-induced downstream genes MCP-1, VCAM-1 and E-selectin, and the IL-6-induced platelet and leukocyte adhesion on endothelial cells and endothelial cell network formation (P<0.05 for all). Of interest, ADAR2 regulated gp130 mRNA stability. Co-silencing of ADAR2 and Drosha completely restored the ADAR2-regulated gp130 mRNA expression indicating that ADAR2 regulates the maturation of specific microRNAs (miRs). Indeed, ADAR2 regulates by at least 2-fold the expression of 20 microRNAs, including of miR-579 and miR-320e, which are predicted to bind to the 3’-untranslated region of IL6ST mRNA.

Conclusions:
This study shows for the first time that ADAR2 controls miR biogenesis and thus IL-6/STAT3 signalling in endothelial cells.
Abstract 60: Shear Stress Maintains Endocardial Phenotype in Ipsc Derived Endocardial Cells
Authors: Vander Roest Mark , Johnson Camryn , Baldwin H. Scott , Merryman W. David
Published: June 2021

Objectives:
Specialized endocardial cells are responsible for the development of heart valves
in utero
. During a highly regulated morphogenetic process, these endocardial cells undergo endothelial-to-mesenchymal transformation (EMT) to become valve interstitial cells (VICs) and reorganize the extracellular matrix to form the structure of the valves. Potentially, induced pluripotent stem cells (iPSCs) may be coaxed into endocardial cells, then to VICs, to yield a suitable cell source for tissue engineered heart valves. Unfortunately, no method to generate iPSC derived endocardial cells exists. Current biochemical strategies utilize static culture, which does not represent the dynamic mechanical environment of the developing heart, which is known to affect differentiation and function of endocardial cells.

Methods and Results:
Human iPSCs were differentiated and purified to endothelial progenitors (CD34+), seeded onto collagen IV coated plates, and grown to confluency. Using a FlexCell system and a custom-built fluid shear device
(A,B)
, mechanical strain and shear stress were administered to the maturing cells independently, which were then assayed via qPCR for changes to endocardial and EMT markers. Bidirectional shear stress
(C)
was found to downregulate endothelial markers CD31, VE-cadherin and VEGFR2, as well as endocardial specific gene Nfatc1, yet increased expression of EMT markers BMP2 and Snai2. Conversely, unidirectional shear
(D)
increased Nfatc1 while causing lower expression of BMP2 and Snai2 than bidirectional shear. Cyclic strain decreased both endocardial and EMT markers
(E)
.

Conclusions:
These data suggest that unidirectional shear stress maintains an endocardial phenotype, while bidirectional shear stress induces EMT, promoting an interstitial cell phenotype. These stimuli may be utilized to maintain and expand patient-specific endocardial and valve interstitial cells for the creation of tissue engineered heart valves.
Abstract 609: Plasma Carnitine is Associated with Gut Microbiome Composition, Diet, and Markers of Cardiometabolic Health
Authors: Smith Holly M , Reilly Muredach P , Ferguson Jane F
Published: June 2021

Cardiometabolic health is influenced by both diet and gut microbiome composition, however mechanisms remain unclear. The dietary-derived metabolite carnitine has been of particular interest for its potential gut microbial-mediated relationship to atherosclerosis. Using plasma carnitine as an intermediate probe, we examined the relationship between diet, gut microbiome composition, circulating metabolite levels, and measurements of cardiometabolic health. Samples (blood, stool) and data (diet, anthropometrics) were collected from 136 healthy subjects. Purified stool 16S V4 DNA was sequenced (Illumina MiSeq, 300bp paired-end reads, ~150,000 reads/sample). Plasma carnitine was analyzed by mass spectrometry. There were several dietary components significantly associated with plasma carnitine, with an overall pattern of a diet rich in animal products and refined carbohydrates (dairy, processed meats, non-whole grains and starchy vegetables) associated with higher carnitine, while monounsaturated fat intake was associated with lower carnitine. Plasma carnitine was significantly negatively correlated with several bacterial genera including
Blautia
(r=-0.3 p=0.001),
Parabacteroides
(r=-0.2, p=0.03), and
Coprococcus
(r=-0.389, p<0.001). Carnitine levels above the median were associated with increases in cardiometabolic risk factors including higher systolic blood pressure (SBP, 118 vs 111 mmHg, p=0.014), BMI (27 vs. 24 kg/m
2
, p=0.002), waist-hip ratio (WHR, 0.85 vs 0.8, p=0.001) as well as higher levels of blood components associated with cardiovascular risk, including circulating monocytes (p=0.007) and hemoglobin (p=0.006). Both diet and microbiome composition also associated with several risk markers (WHR, SBP, hemoglobin), albeit to a lesser extent than plasma carnitine. In conclusion, we provide evidence for inter-related relationships between diet, microbiome composition, circulating metabolites, and markers of cardiometabolic health.
Abstract 610: Ineffective Suppression of Adipocyte Lipolysis in Metabolic Syndrome: An
in vivo
Test for Adipocyte Insulin Resistance
Authors: Ford Jennifer L , Boston Raymond C , Walker Rachel E , Shearer Gregory C
Published: June 2021

Background:
Insulin resistance is a major contributor to metabolic syndrome, disrupting both glucose and non-esterified fatty acid (NEFA) dynamics through ineffective glucose clearance and decreased suppression of lipid droplet lipolysis. The minimal model of glucose dynamics is used for glycemic insulin sensitivity however it does not measure adipocyte insulin sensitivity, the primary determinant of plasma NEFA. An in-vivo approach to measuring adipocyte insulin sensitivity using NEFA is employed, comparing healthy and metabolic syndrome subjects. Both the models are employed to estimate insulin sensitivity and validate the NEFA approach.

Objective:
To test the use of NEFA kinetics to measure adipocyte insulin sensitivity compared to the glucose minimal model.

Approach and results:
Metabolic syndrome (n=56) and optimally healthy (n=14) subjects underwent a frequently sampled intravenous glucose tolerance test, and plasma analyzed for insulin, glucose, and NEFA. Insulin sensitivity (
S
I
) and glucose effectiveness (
S
G
) were calculated from the glucose minimal model.
S
I
was 1.7 (mU/L)
-1
min
-1
and 0.40 (mU/L)
-1
/min
-1
and
S
G
was 0.027 min
-1
and 0.017 min
-1
for the healthy and metabolic syndrome groups, respectively, indicating substantial glycemic insulin resistance in the latter. A model using glucose as the driver for NEFA kinetics was then applied. We found the initial rate of NEFA utilization by tissues (NU) was less, but the threshold glucose (tG) and glucose concentration required for a unit change in lipolysis inhibition (
G
i
) were greater in metabolic syndrome verses healthy (NU: 0.050[0.045, 0.057] vs. 0.068[0.054, 0.086] p=0.03; tG: 6.7[6.2, 7.2] vs. 5.0[4.3, 5.9] p=0.001;
G
i
: 0.30[0.25, 0.35] vs. 0.17[0.07, 0.27] p=0.02). No differences were found in initial rate of NEFA production or glucose utilization.

Conclusion:
Our results indicate that suppression of lipid-droplet lipolysis requires greater stimulus in metabolic syndrome compared to insulin sensitive adipocytes. Further, the rate of NEFA removal is less in metabolic syndrome. These results reveal components of insulin sensitivity not demonstrated by the glucose model. The NEFA model provides a measurement of adipocyte insulin sensitivity not captured by glycemic indices.
Abstract 618: Targeted Delivery of microRNA-146a/-181b Protects Against Endothelial Dysfunction and Prevents Atherosclerosis
Authors: Wong Wing Tak , Ma Shuangtao , Huang Yu , Tian Xiaoyu
Published: June 2021

Objective:
Atherosclerosis, which underlies most cardiovascular diseases (CVD), is a leading cause of mortality and disability in developed countries. The endothelium is an important site of vascular regulation, and its dysfunction initiates atherosclerosis and supports its progression. Current drug therapies, percutaneous angioplasty, and stenting alleviate symptoms but do not reverse atherosclerosis. Thus, more effective strategies that prevent or reverse atherosclerosis via improved endothelial function are needed.

Approach:
E-selectin serves as a surface marker of inflamed endothelial cells (ECs). We attempted to deliver microRNA (miR)-146a and miR-181b to inflamed endothelium covering atherosclerotic plaques with an E-selectin-targeting multistage vector (ESTA-MSV to inhibit atherosclerosis. Cy5-conjugated miR-146a and miR-181b were packaged in polyethylene glycol-polyethyleneimine (PEG/PEI) nanoparticles and then loaded into ESTA-MSV microparticles. Male apolipoprotein E-deficient mice were fed with Western diet and injected intravenously with the particles prepared as above biweekly for 12 weeks.

Results:
Atherosclerotic plaque size decreased with treatment of miRs packaged in ESTA-MSV but not in PEG/PEI. Moreover, vascular inflammation markers such as macrophages in aortic root lesion and expression of chemokines in aortic tissues were decreased while the vascular smooth muscle cells and collagen were increased in plaques from ESTA-MSV/miRs-treated mice compared with vehicle-treated group. Systemic delivery of miR-146a/-181b significantly improved the ACh-induced relaxation of aortas and carotid arteries and inhibited ACh-induced endothelium-dependent contraction of carotid arteries of ApoE-/- mice fed with Western diet for 12 weeks

Conclusions:
Our data demonstrate that the ESTA-MSV microparticle-mediated delivery of miR-146a/-181b ameliorates the endothelial dysfunction and prevents atherosclerosis.
Abstract 62: Selenoprotein P Promotes Vascular Smooth Muscle Cell Proliferation and Pulmonary Hypertension -A Possible Novel Therapeutic Target-
Authors: Kikuchi Nobuhiro , Satoh Kimio , Omura Junichi , Satoh Taijyu , Kurosawa Ryo , Nogi Masamichi , Otsuki Tomohiro , Numano Kazuhiko , Kozu Katsuya , Suzuki Kota , Sunamura Shinichiro , Tatebe Shunsuke , Aoki Tatsuo , Sugimura Koichiro , Shimokawa Hiroaki
Published: June 2021

Background:
Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is the main characteristics in pulmonary arterial hypertension (PAH); however, its mechanism still remains unclear.

Methods & Results:
To explore a novel therapeutic target in PAH, we performed a gene expression microarray screening using PASMCs from patients with PAH (PAH-PASMCs) and those from controls and found up-regulation of selenoprotein P (SeP) in PAH-PASMCs (>5-fold, P<0.01). SeP is a widely expressed extracellular protein, which transports selenium to whole body. Immunostaining showed strong expression of SeP in thickened pulmonary arteries (PA) in PAH. Chronic exposure of mice to hypoxia (O
2
10%) increased SeP expression in the lung. In PAH-PASMCs, hypoxia (O
2
2%, 24 hours) increased SeP expression compared with normoxic controls (O
2
21%) (1.6-fold, P<0.05). Treatment with human recombinant SeP (10μg/mL) up-regulated pro-proliferative genes, activated signals associated with cell-cycle, and increased PAH-PASMCs proliferation (P<0.05, n=8 each). SeP siRNA suppressed pro-proliferative signals with a resultant reduction in PASMCs proliferation. Next, SeP-deficient mice (

SeP
-/-

) were exposed to hypoxia (O
2
10%) for 4 weeks, which resulted in suppression of right ventricular systolic pressure (RVSP), RV hypertrophy and PA remodeling compared with controls (all P<0.05, n=11 each). PASMCs proliferation was reduced in

SeP
-/-

PASMCs compared with

SeP
+/+

PASMCs (P<0.05).

SeP
-/-

PASMCs showed ERK1/2 inactivation, AMPK activation, and reduced secretion of pro-inflammatory cytokines. SMC-targeted deletion of SeP suppressed the development of hypoxia-induced PH compared with controls (all P<0.05, n=10). In contrast, liver-specific deletion of SeP did not affect the development of hypoxia-induced PH (all P>0.05, n=8-9). In the clinical study, serum SeP levels were significantly elevated in PAH patients (n=203) compared with controls (n=20) (P<0.001). Event-free curve revealed that high plasma SeP (≥27μg/ml) predicted poor outcome in PH patients (death or lung transplantation, P<0.01).

Conclusions:
These results indicate that SeP, especially in PASMCs, promotes PA remodeling, suggesting that it is a novel therapeutic target for PAH.
Abstract 623: Effect of High-intensity Statin Therapy on High-density Lipoprotein (HDL) Subfractions and Regression of Coronary Atheroma: The SATURN Trial
Authors: Hoogeveen Ron C , Shao Mingyuan , Wolski Kathy , Puri Rishi , Ballantyne Christie M , Nicholls Stephen J
Published: June 2021

Aim:
Statin therapy can slow the progression of coronary atherosclerosis. However, the clinical factors underlying the beneficial effects of statin therapy on disease progression remain poorly understood. We examined the relationship of circulating HDL subfractions with measures of coronary atheroma following long-term high-intensity statin therapy.

Methods:
Serial coronary intravascular ultrasound (IVUS) was utilized in SATURN to monitor changes in atheroma burden [percent atheroma volume (PAV)] in 915 patients with coronary artery disease, treated with rosuvastatin (40 mg) or atorvastatin (80 mg) daily for 24 months.

Results:
Baseline levels of total HDL-C, apo E-containing HDL-C (apoE HDL-C), HDL
3
-C and HDL
2
-C did not differ significantly between treatment groups. Compared with the atorvastatin-treated group, rosuvastatin-treated patients demonstrated greater increases in levels of total HDL-C (4.4% vs. -1.8%, p<0.001), apoE HDL-C (8.5% vs. -3.3%, p<0.001), HDL
3
-C (3.3% vs. -2.7%, p<0.001) and HDL
2
-C (7.0% vs. -0.7%, p<0.001). The alterations in apo E HDL-C and HDL
3
-C levels were associated with PAV regression in rosuvastatin-treated patients (β=-0.84, p=0.018 and β=-1.10, p=0.056, respectively) but not in the atorvastatin-treated group (β=0.41, p=0.22 and β=0.71, p=0.22 for apo E HDL-C and HDL
3
-C, respectively).

Conclusions:
Rosuvastatin therapy resulted in favorable changes in HDL subfractions and on-treatment levels of apoE HDL-C and HDL
3
-C were associated with greater disease regression.
Abstract 630: Novel Chimeric Anti-proteoglycan Antibody Inhibits Arterial Retention of Proatherogenic Lipoproteins in a Rat Model of Insulin Resistance
Authors: Soto Yosdel , Mangat Rabban , Vázquez Ana M , Proctor Spencer D
Published: June 2021

Background:
The response-to-retention hypothesis for atherosclerosis describes subendothelial retention of apolipoprotein B-containing lipoproteins mediated by proteoglycans (PG). Further we know that diabetes is also associated with both increased circulating chylomicron remnants and remodeling of proatherogenic PGs. We have recently reported antiatherogenic properties of a novel chimeric monoclonal antibody (chP3R99) that recognizes PG sulfated molecules.

Hypothesis:
chP3R99 monoclonal antibody may interfere with the interaction of atherogenic lipoproteins with arterial sulfated PGs during insulin resistance.

Methods and Results:
chP3R99 antibody recognized sulfated glycosaminoglycans by ELISA showing a preferential binding to chondroitin sulfate. Also, chP3R99 blocked the interaction of proatherogenic lipoproteins with this glycosaminoglycan
in vitro
in a dose-dependent manner and its intravenous injection into healthy Sprague-Dawly rats (n=6, 1 mg/animal) inhibited LDL (4 mg/kg; intraperitoneally) aortic retention. To further assess this property in an insulin resistant condition, carotid arteries from control and JCR:LA-cp rats (n=4) were perfused
ex vivo
with apoB48 containing remnant lipoproteins (prepared via rabbit hepatectomy procedure), with or without Cy3-LDL (150 μg/mL) for 20 minutes. Confocal microscopy analysis revealed an increased arterial retention of both remnants (3.6 fold) and LDL (2.8 fold) in carotid vessels from insulin resistant rats relative to control. However, chP3R99 pre-perfusion resulted in decreased retention of remnants (-30%) and LDL (-60%) associated arterial cholesterol. Data suggests that the chP399 antibody may interfere with the arterial attachment of both remnants and LDL
in vivo,
but with differential efficacy.

Conclusions:
Relative to LDL, remnant lipoproteins had preferential accumulation in arterial vessels from insulin resistant rats
ex vivo
, which could then be inhibited by acute pre-exposure to the chP3R99 antibody. These
in vivo
data support the concept for an innovative approach to target the retention of proatherogenic lipoproteins in a pre-clinical setting.
Abstract 633: Macrophage SR-BI Suppresses Atherosclerosis by Modulation of Autophagy via VPS34/Belclin-1 Pathway
Authors: Tao Huan , Yancey Patricia G , Blakemore John L , Zhang Youmin , Ding Lei , Jerome W. Gray , Vickers Kasey C , Linton MacRae F
Published: June 2021

Background:
Autophagy modulates vascular cell lipid metabolism, lipid droplet turnover, foam cell formation, cell survival and death, and inflammation. Scavenger receptor class B type I (SR-BI) deficiency causes impaired lysosome function in macrophages and erythrocytes.

Methods and Results:
Bone marrow transplantation studies were performed in ApoE and LDLR deficient mice to examine the effects of hematopoietic SR-BI deletion on atherosclerotic lesion autophagy. In addition, in vitro studies compared WT versus SR-BI
-/-
macrophages. Under conditions of cholesterol induced stress, the mRNA and protein levels of critical autophagy players including ATG5, ATG6/Belcin-1, ATG7 and LC3II were decreased by 37.8% to 84.6% (P<0.05 to 0.01) in SR-B1
-/-
macrophages and atherosclerotic aortic tissue compared to controls. Electron microscopic analysis showed that SR-BI
-/-
versus WT macrophages had 80% fewer (P<0.05) autophagsomes in response to cholesterol enrichment. Macrophage SR-BI deficiency led to 1.8-fold (P<0.05) more lipid deposition and 2.5-fold more (P<0.01) apoptosis in response to oxidized LDL. Furthermore, hematopoietic SR-BI deletion caused 2.3 fold (P<0.05) more cell death in aortic atherosclerotic lesions compared to the WT control. Pharmacologic activation of autophagy did not reduce the levels of lipid droplets or cell apoptosis in SR-BI null macrophages vs WT control. WT peritoneal macrophages were used to examine SR-BI subcellular distribution and its interaction with VPS34/Beclin-1. In response to induction of autophagy, macrophage SR-BI was expressed in lysosomes and co-localized with LC3-II. Furthermore, we found that SR-BI directly interacted with the VPS34/Beclin-1 complex.

Conclusions:
SR-BI deficiency leads to defective autophagy and accelerates macrophage foam cell formation and apoptosis in experimental mouse atherosclerotic lesions. Macrophage SR-BI regulates expression of critical autophagy players and directly modulates autophagy via the VPS34/Beclin-1 pathway, identifying novel targets for the treatment of atherosclerosis.
Abstract 635: Cholesterol Loading Increases the Size and Cholesterol Content of Nascent HDL Formed via the Interaction of Apo AI With Cellular ABCA1
Authors: Xu Bingqing , Bell James M , Gillard Baiba K , Gotto Antonio M , Pownall Henry J
Published: June 2021

Cardiovascular disease (CVD) is a major cause of mortality and morbidity and development of new therapies that address its underlying causes is an important public health priority. Although CVD negatively correlates with high-density lipoprotein-cholesterol (HDL-C), HDL-raising therapies have not reduced CVD risk, particularly with statin co-therapy. Current data suggests that improving HDL functionality is more important to cardioprotection than increasing plasma HDL-C. Thus, it is important to identify structure-function relationships for various HDL and their precursors, nascent HDL. Nascent HDL is formed via the interaction of apo AI with ATP-binding cassette transporter A1 (ABCA1); rHDL formed via the interaction of apo AI with multilamellar membranes are
in vitro
models of nascent HDL. Both rHDL and nascent HDL are discoidal and their sizes increase with the cholesterol content of membranes from which they are derived. Given that very large rHDL, ~36 nm are formed at high cholesterol concentrations, ~15 mol%, we tested the hypothesis that similar large nascent HDL form via the interaction of apo AI with cells after hyperloading with cholesterol. To test this hypothesis, we incubated BHK-ABCA1 cells with cholesterol-loaded methyl β-cyclodextrin (CDX) for two hours. The free cholesterol (FC) content of the cells increased with increasing CDX:cholesterol concentration, from 29 ± 2 to 52 ± 6 μg FC/ mg cell protein. Concurrently, the size and cholesterol content of the nascent HDL produced by the interaction of apo AI with the cells also increased, with sizes ranging from ~ 7 to ~20 nm. These data suggest that the same membrane qualities and their modification by cholesterol determine the size of both rHDL and nascent HDL. Future studies will identify their structure in greater detail by a combination of physicochemical methods.
Abstract P292: ENPP1-Fc Protein Inhibits Proliferation of Human Vascular Smooth Muscle Cells
Authors: Yan Yan , Shah Anumeha , Saigal Ashmita , Faas Susan , Cheng Zhiliang , Askew Kim , Marozsan Andre
Published: June 2021

Background:
Arterial stenosis leading to hypertension or heart failure is common in patients with Generalized Arterial Calcification of Infancy (GACI), an ultra-rare disease associated with loss of function mutations in ENPP1, an ectonucleotide pyrophosphatase that hydrolyzes extracellular ATP. GACI is characterized by accelerated calcification and severe myointimal proliferation. The role of ENPP1 in myoinitimal proliferation is unknown. Here, we examined the effect of ENPP1 on proliferation of human vascular smooth muscle cells (hVSMCs).

Methods:
ENPP1 expression was assessed in primary hVSMCs using qRT-PCR. ENPP1 expression was silenced using siRNA and its activity was verified by a cell based assay or inorganic pyrophosphate (PPi) assay and its effect on VSMC proliferation was determined by 3H-thymidine incorporation.

Results:
Treatment of primary hVSMCs with siRNA specific to the hENPP1 resulted in ~90% decrease in ENPP1 levels. Cellular enzyme activity correlated with ENPP1 expression in VSMCs. Silencing ENPP1 in hVSMCs led to 1-3 fold increase in proliferation relative to that of cells transfected with control siRNA in 2 out of 2 donors. Peak cell proliferation was observed at 5 days post-transfection. Human iPSC derived VSMCs (iVSMCs) expressed higher levels of ENPP1 than primary hVSMCs. Silencing ENPP1 in iVSMCs resulted in 3-5 fold increase in proliferation relative to that of cells transfected with negative control siRNA in 2 out of 2 donors. Addition of recombinant ENPP1-Fc protein restored ENPP1- associated proliferation in all donors (8099.75 ±134.32 (untreated) vs 1478 ± 55.34 (5ug/ml ENPP1-Fc treated, P<0.001)). In contrast, bisphosphonates, a current off-label treatment for patients with GACI, had no effect on cellular proliferation. Increased levels of PPi were also detected in culture supernatants obtained from cells treated with the ENPP1-Fc protein.

Conclusion:
For the first time, we have demonstrated that ENPP1 knockdown promotes proliferation of human VSMCs, and treatment with a functionally active ENPP1-Fc protein significantly inhibits ENPP1-associated proliferation. These results suggest that ENPP1 enzyme replacement may be a potential strategy to treat myointimal proliferation in patients with GACI disease.
Association of Apparent Treatment-Resistant Hypertension With Differential Risk of End-Stage Kidney Disease Across Racial Groups in the Million Veteran Program
Authors: Akwo Elvis A. , Robinson-Cohen Cassianne , Chung Cecilia P. , Shah Shailja C. , Brown Nancy J. , Ikizler T. Alp , Wilson Otis D. , Rowan Bryce X. , Shuey Megan M. , Siew Edward D. , Luther James M. , Giri Ayush , Hellwege Jacklyn N. , Velez Edwards Digna R. , Roumie Christianne L. , Tao Ran , Tsao Phil S. , Gaziano J. Michael , Wilson Peter W.F. , O’Donnell Christopher J. , Edwards Todd L. , Kovesdy Csaba P. , Hung Adriana M. ,   On behalf of the VA Million Veteran Program
Published: June 2021

Apparent treatment-resistant hypertension (ATRH) has been linked to end-stage kidney disease (ESKD) and cardiovascular disease. We tested the hypothesis that the effect of ATRH on ESKD is greater in Black patients than in White patients and investigated the effect of ATRH on ESKD independent of
APOL1
genotype. In a retrospective cohort of 139 685 hypertensive veterans (22% Black, 5% women) in the Million Veteran Program, ATRH was defined as failure to achieve outpatient blood pressure <140/90 mmHg with 3 antihypertensives including a thiazide or use of ≥4. Outcomes included incident ESKD, myocardial infarction, and stroke. Poisson models were used to test effect modification by race. Over a median follow-up of 10.3 years (interquartile range, 5.8–11.7), 17 521 incident ATRH cases were observed. Compared with nonresistant hypertension, patients with ATRH had higher incidence rates (per 1000-person-years) of ESKD (4.7 versus 1.6), myocardial infarction (6.7 versus 3.4), and stroke (16.7 versus 8.5). A greater attributable risk of ESKD because of ATRH was observed among Black patients (44.4/1000) compared with White patients (25.5/1000). Black patients with ATRH had a 2.3-fold higher risk of ESKD compared with Black patients with nonresistant hypertension; 3-fold the risk of White patients with ATRH, and 9-fold the risk of White patients with nonresistant hypertension (
P
-interaction<0.001). Among Black patients, ATRH remained associated with a 98% (95% CI, 1.66–2.75) higher risk of ESKD after adjustment for
APOL1
genotype. Patients with ATRH experienced excess ESKD and cardiovascular disease risk. This excess ATRH-related ESKD risk was magnified among Black patients independently of
APOL1
genotype. Targeted treatment of ATRH could curtail ESKD and cardiovascular disease incidence.
Impact of Chronic Hypertension and Antihypertensive Treatment on Adverse Perinatal Outcomes: Systematic Review and Meta‐Analysis
Authors: Al Khalaf Sukainah Y. , O'Reilly Éilis J. , Barrett Peter M. , B. Leite Debora F. , Pawley Lauren C. , McCarthy Fergus P. , Khashan Ali S.
Published: June 2021

Background

Maternal chronic hypertension is associated with adverse pregnancy outcomes. Previous studies examined the association between either chronic hypertension or antihypertensive treatment and adverse pregnancy outcomes. We aimed to synthesize the evidence on the effect of chronic hypertension/antihypertensive treatment on adverse pregnancy outcomes.

Methods and Results

Medline/PubMed, EMBASE, and Web of Science were searched; we included observational studies and assessed the effect of race/ethnicity, where possible, following a registered protocol (CRD42019120088). Random‐effects meta‐analyses were used. A total of 81 studies were identified on chronic hypertension, and a total of 16 studies were identified on antihypertensive treatment. Chronic hypertension was associated with higher odds of preeclampsia (adjusted odd ratio [aOR], 5.43; 95% CI, 3.85–7.65); cesarean section (aOR, 1.87; 95% CI, 1.6–2.16); maternal mortality (aOR, 4.80; 95% CI, 3.04–7.58); preterm birth (aOR, 2.23; 95% CI, 1.96–2.53); stillbirth (aOR, 2.32; 95% CI, 2.22–2.42); and small for gestational age (SGA) (aOR, 1.96; 95% CI, 1.6–2.40). Subgroup analyses indicated that maternal race/ethnicity does not influence the observed associations. Women with chronic hypertension on antihypertensive treatment (versus untreated) had higher odds of SGA (aOR, 1.86; 95% CI, 1.38–2.50).

Conclusions

Chronic hypertension is associated with adverse pregnancy outcomes, and these associations appear to be independent of maternal race/ethnicity. In women with chronic hypertension, those on treatment had a higher risk of SGA, although the number of studies was limited. This could result from a direct effect of the treatment or because severe hypertension during pregnancy is a risk factor for SGA and women with severe hypertension are more likely to be treated. The effect of antihypertensive treatment on SGA needs to be further tested with large randomized controlled trials.
Risk Factors and Outcomes of Recurrent Drug‐Eluting Stent Thrombosis: Insights From the REAL‐ST Registry
Authors: Enomoto Soichiro , Kuramitsu Shoichi , Shinozaki Tomohiro , Ohya Masanobu , Otake Hiromasa , Yamanaka Futoshi , Shiomi Hiroki , Natsuaki Masahiro , Nakazawa Gaku , Ando Kenji , Kadota Kazushige , Saito Shigeru , Tamura Toshihiro , Kimura Takeshi , Toyota Fumitoshi , Otsuji Hideaki , Sugihara Makoto , Serikawa Takeshi , Matsuo Hitoshi , Tanigaki Toru , Noda Toshiyuki , Kato Takashi , Dai Kazuoki , Iwasaki Masamichi , Takaya Tomofumi , Dan Kazuhiro , Okino Hideto , Toyofuku Mamoru , Saito Makoto , Kim Kite , Otake Hiromasa , Nagasawa Akira , Nakayoshi Takaharu , Chibana Hidetoshi , Kimura Takeshi , Shimooka Yoshinori , Shibata Yoshisato , Ogata Kenji , Kurogi Kazumasa , Sakamoto Ryohei , Kataoka Tetsuro , Ishii Mitsuru , Yamamoto Fumi , Kawamoto Hiroyoshi , Yabushita Hiroto , Kozuki Amane , Kobayashi Yohei , Higami Hirooki , Natsuaki Masahiro , Suzuyama Hiroto , Sakakura Kenichi , Watanabe Yusuke , Yamasaki Seiji , Katagiri Yuki , Horie Kazunori , Takii Toru , Saito Shigeru , Yamanaka Futoshi , Tamura Toshihiro , Enomoto Soichiro , Nakazawa Gaku , Matsumoto Shingo , Tanaka Nobuhiro , Nishina Hidetaka , Kakefuda Yuki , Sonoda Shinjo , Anai Reo , Doijiri Tatsuki
Published: June 2021

Background

Stent thrombosis (ST) after drug‐eluting stent (DES) implantation remains a life‐threatening complication. Recurrent ST (RST) is not a rare phenomenon, potentially contributing to high mortality after the index ST events. However, little evidence is available about the incidence, risk factors, and clinical outcomes of definite RST after DES thrombosis.

Methods and Results

From REAL‐ST (Retrospective Multicenter Registry of ST After First‐ and Second‐ Generation DES Implantation), this study evaluated 595 patients with definite ST (first‐generation DES thrombosis, n=314; second‐generation DES thrombosis, n=281). During a median follow‐up of 31 months, we identified 32 patients with definite RST after first‐generation DES thrombosis (n=18) and second‐generation DES thrombosis (n=15). Cumulative incidence of RST was 4.5% and 6.0% at 1 and 5 years, respectively, which did not significantly differ between first‐generation DES thrombosis and second‐generation DES thrombosis. Independent predictors of definite RST were early ST (hazard ratio [HR], 2.38; 95% CI, 1.06–5.35 [
P
=0.035]) and multivessel ST (HR, 3.47; 95% CI, 1.03–11.7 [
P
=0.044]). Definite RST was associated with a 2.8‐fold increased risk of mortality (adjusted HR, 2.78; 95% CI, 1.35–5.73 [
P
=0.006]).

Conclusions

Cumulative incidence of definite RST did not significantly differ between first‐generation DES thrombosis and second‐generation DES thrombosis. Early ST and multivessel ST were risk factors of definite RST. Definite RST significantly increased mortality after DES thrombosis, highlighting the clinical importance of preventing RST to improve outcomes of patients with ST.

Registration

URL:
https://www.clinicaltrials.gov
; Unique identifier: UMIN000025181.
Abstract 116: Minorities, Women, and Stroke Belters Left Behind in t-PA Use Despite Quality Improvement Efforts
Authors: Madsen Tracy E , Melluzzo Shannon , Wira Charles R , Magdon-Ismail Zainab , Day David , Gropen Toby ,   On behalf of The NorthEast Cerebrovascular Consortium Investigators
Published: June 2021

Introduction:
We aimed to describe factors associated with IV tPA use over time, including geography, hospital-related factors, and quality improvement efforts.

Methods:
Acute ischemic stroke (AIS) discharges with available hospital identifiers (2005 to 2011) in the National Inpatient Sample (NIS) were identified using ICD9 codes 433.X1, 434.X1, and 436. IV tPA use was determined using procedure code 9910 and code V4588 for drip and ship patients; patient/hospital characteristics were obtained from the NIS. Stroke center designation and Get With The Guidelines (GWTG) performance award data were publicly available. Logistic regression using patient characteristics only was followed by the addition of hospital characteristics, then state characteristics (Table). Adjusted odds ratios (aOR) with 95% CI were reported.

Results:
There were 563,087 AIS; median age 74 (IQR 62 – 83), 14.9% Black, and 53.3% female. Overall, 3.76% (n=21,172) received IV tPA. In the fully adjusted model (Table), patients that were Black, Hispanic, or female were less likely to receive IV tPA, while those with private insurance were more likely to receive IV tPA. Each subsequent year, patients were 11% more likely to receive IV tPA (aOR 1.11, 95%CI 1.09-1.13). Patients discharged from designated stroke centers, GWTG hospitals with silver or gold awards, or hospitals that were teaching, large, or urban were more likely to receive IV tPA compared to patients discharged from small, critical access, or rural hospitals. Patients discharged in stroke belt states were less likely to receive IV tPA.

Conclusions:
Treatment of AIS with tPA is increasing over time. Discharge from GWTG hospitals with performance achievement awards, stroke centers, or teaching, large, or urban hospitals increased this likelihood. Patients who were black, Hispanic, female, or in the stroke belt were less likely to receive tPA. Further efforts are needed to address these performance gaps.
Abstract 127: Predictors of Good Functional Outcomes in an Acute Ischemic Stroke Cohort with a Large Infarct Core Treated with Mechanical Thrombectomy: Signals to Treat?
Authors: Wagner Jeffrey , Frei Donald , Nogueira Raul , Siddiqui Adnan , Zaidat Osama O , Yoo Albert , Soulimani Ghita , Mualem Elan , Barraza Leticia , Meyer Denise , Ammar Lynne , Stankiewicz Trisha , Liang Franco , Kuo Sophia S , Buell Hope , Bose Arani , Sit Siu Po ,   for the PIVOTAL, PICS, RetroSTART, START, and SEPARATOR 3D Investigators
Published: June 2021

Purpose:
Mechanical thrombectomy has been demonstrated to provide benefits in the treatment of acute ischemic stroke (AIS). But whether to treat AIS patients with a large infarct core remains controversial. Although it is a common practice that patients with large infarct core are not offered endovascular treatment, previous data have consistently shown a proportion of these patients may benefit from IA intervention. The purpose of this study is to identify predictors of good outcomes in an AIS cohort with a large infarct volume previously treated with mechanical thrombectomy.

Hypothesis:
We hypothesize that among AIS patients with a large infarct volume, younger patients (≤66 years) who present with lower NIHSS scores will show good functional outcomes (mRS 0-2) at 90 days if treated with mechanical thrombectomy.

Methods:
Univariable and multivariable analyses were preformed to identify factors that predict good functional outcomes in AIS patients with ASPECTS 0-5 who were treated with the Penumbra System. Five previous prospective, multicenter trials (PIVOTAL, PICS, RetroSTART, START, SEPARATOR 3D) were included in this study. Patients who presented with symptoms of AIS were analyzed for association between presenting demographics and modified Rankin scale (mRS) score at 90 days in univariate and multivariate analyses.

Results:
Data for 614 patients with a median age of 69 years and an NIHSS score of 18 met study criteria. Of these, the 90-day mRS 0-2 rate and mortality were, respectively, 40.23% and 25.41%. Among those with ASPECTS 0-5 (N=93), 17.20% had good functional outcome. An age of ≤66 years was significantly associated with good outcome (p<0.0001) among those with ASPECTS 0-5. Within this age group who had ASPECTS 0-5, a baseline NIHSS score of ≤ 20 (p= 0.0088) with a target vessel location at the MCA (p=0.0210) were also strong predictors of good outcome if treated by mechanical thrombectomy.

Conclusion:
These data demonstrate that age ≤66 years, baseline NIHSS score of ≤ 20 with a target vessel location in the MCA are important predictors of good outcomes in an AIS cohort with a large infarct core who are eligible for mechanical thrombectomy.