bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 489: The Role of Hemodynamics in Modulating p120 and Kaiso Expression and Activity in Atherogenesis
Authors: Weinberg Devin L , Pryor Andrew W , Hamilton Christin A , Blackman Brett R
Published: July 2021

Introduction.
The generation of atherosclerosis has been shown to be a non-random process, with endothelial cells (ECs) in regions of disturbed hemodynamics developing an atheroprone (pro-inflammatory) phenotype. While a great deal of research has focused on hemodynamic regulation of EC phenotype, the mechanisms that mediate ECs’ response to local shear stress environment remain elusive. p120-catenin (p120) and its transcription factor binding partner Kaiso are expressed in ECs and possess a variety of functions that potentially contribute to the hemodynamic regulation of EC phenotype in atherosclerosis. Specifically, p120 has been shown to regulate adherens junction stability and regulate gene transcription through interaction with Kaiso.

Hypothesis.
We hypothesize hemodynamic forces modulate the activity of p120 and Kaiso in a manner that alters local endothelial susceptibility to atherogenesis.

Methods.
Atheroprone/protective hemodynamic profiles obtained by human carotid artery imaging were applied to human ECs
in vitro
. Protein/gene expression was then analyzed by Western blot, RT-PCR, and imaging. To determine Kaiso-dependent regulation of target gene expression, siRNAs were used to knockdown Kaiso expression. For
in vivo
validation, vasculature from Apolipoprotein E
-/-
mice was isolated, fixed, and stained.

Results.
In vitro,
atheroprotective flow upregulates p120 expression and increases its junctional continuity and nuclear accumulation. There is a flow-dependent increase in Kaiso expression over time in both flow conditions, but p120/Kaiso interaction is increased under atheroprone flow. Kaiso knockdown under protective flow results in decreased expression of the anti-inflammatory genes eNOS (0.49 fold) and KLF2 (0.68 fold) and increased expression of the inflammatory marker VCAM (1.44 fold), suggesting a previously unknown anti-atherogenic function of Kaiso. In preliminary mouse studies, Kaiso is expressed in ECs overlying both healthy and diseased regions, as well as being highly expressed within the lesions.

Conclusions.
p120 and Kaiso expression are regulated in a flow-dependent manner and are potentially a mechanism by which hemodynamics modulate susceptibility to atherogenesis.
Abstract 492: Arachidonic Acid Induces Activation of Platelet PF4 and Par-1 mRNA, Which Is Attenuated by Aspirin
Authors: Hu Liang , Nardi Michael A , Merolla Michael , Suarez Yajaira , Berger Jeffrey
Published: July 2021

Arachidonic acid (AA) is converted to thromboxane A2 via the cyclooxygenase pathway; however its exact mechanism of platelet activation is uncertain. Inhibition of this pathway via aspirin highlights the importance of this pathway in decreasing thrombotic events.

In the present study, we investigate the effect of AA on platelet activity indicators (leukocyte- and monocyte-platelet aggregation [LPA, MPA] and reticulated platelets [RP]), as well as the expression (mRNA and protein) of platelet markers PF4 and Par-1, previously well established platelet transcripts with quantitative determinations. To this end, whole blood was incubated with AA (150mM) for 30 min at room temperature in the absence or presence of aspirin (1mM) prior to addition of antibodies for platelet activity indicators, and isolating platelets for mRNA and protein expression.

LPA and MPA were significantly increased after AA stimulation in a dose dependent manner, and were inhibited by aspirin treatment. AA significantly increased PF4 and Par-1 protein level as determined by flow cytometry and western blot assays. Pretreatment with aspirin also attenuated this increase in protein levels. Surprisingly, AA stimulation significantly increased thiazole orange staining (a measure of nucleic acids), another marker of increased platelet activity. Importantly, these results suggest that AA-mediated platelet activation produced an overall increase in platelet total RNA content. To confirm these findings, we analyzed the mRNA expression of PF4 and Par-1 by quantitative real time PCR from platelets treated with AA. Interestingly, AA significantly up-regulated the platelet mRNA transcripts of PF4 and Par-1 by 40% to 60%, and pretreatment with aspirin completely attenuated this effect supporting the specificity of the AA effect on platelet RNA.

Altogether, these data suggest that platelet mRNA is affected by AA stimulation, which is attenuated by pretreatment with aspirin. However, the mechanisms responsible for the increased mRNA levels and expression of PF4 and Par-1 (processing of pre-RNA to mRNA) require further investigation. Importantly, our findings provide novel insight regarding platelet activation and a better understanding of mediators in the processes of thrombosis and hemostasis.
Abstract 5: Sortilin Regulates Hepatic VLDL Secretion and LDL Uptake in a Lysosome-Dependent Manner
Authors: Strong Alanna , Ding Qiurong , Edmondson Andrew , Khetarpal Sumeet , Morales Carlos , Lund-Katz Sissel , Phillips Michael , Musunuru Kiran , Rader Daniel
Published: July 2021

Sortilin, the protein product of the
SORT1
gene, is a multi-ligand sorting receptor involved in Golgi to lysosome and plasma membrane to lysosome protein trafficking. Genome wide association studies for lipid traits have identified the 1p13 locus harboring the
SORT1
gene as strongly associated both with plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) risk in humans. Adeno-associated virus (AAV)-mediated hepatic sortilin overexpression in LDL receptor deficient mice reduced plasma cholesterol by 30% at two weeks (
n
= 6 mice per group,
P
= 0.02), with a concomitant reduction in LDL-C.
In vivo
VLDL production studies demonstrated a 50% reduction in the VLDL triglyceride secretion rate (
P
= 0.007) and a 50% reduction in apoB secretion (
P
= 0.02) with sortilin overexpression.
In vivo
LDL turnover studies demonstrated a 3-fold increase in the LDL fractional catabolic rate (FCR) with sortilin overexpression (
n
= 6 mice per group,
P
= 0.00002). Sortilin deficiency both alone and on an LDL receptor deficient background led to a 40% and 50% reduction in FCR (
n
= 6 mice per group,
P
= 0.002 and
P
= 0.01). The effect of sortilin on both VLDL secretion and LDL turnover is dependent on the ability of sortilin to traffic to the lysosome, as sortilin mutants that cannot traffic to the lysosome do not affect VLDL secretion or LDL uptake
in vivo
or
in vitro
. Surface plasmon reasonance demonstrated a high affinity interaction between sortilin and the apoB in LDL particles at physiological pH with a K
d
of ∼2 nM, and this affinity virtually disappears at the acidic lysosomal pH. In sum, these data are consistent with a model in which sortilin binds apoB-containing lipoprotein particles in the Golgi apparatus and at the plasma membrane and traffics them to the endolysosomal compartment for degradation, thereby reducing VLDL secretion and facilitating LDL uptake, explaining the strong association of hepatic sortilin overexpression in humans with reduced plasma cholesterol.
Abstract 500: Assessing the Impact of Cardiovascular Disease-Related Genetic Variation on Clinical Cognitive Impairment
Authors: Dube Joseph B , Johansen Christopher T , Robinson John , Lindsay Joan , Hachinski Vladimir , Hegele Robert A
Published: July 2021

Introduction:
“Cognitive impairment, no dementia” (CIND) is a prodromal stage of cognitive decline which marks the onset of dementia and is due most commonly to 1) Alzheimer disease (AD) or 2) vascular dysfunction. In order to assess the genetic component of CIND susceptibility, we investigated cardiovascular disease (CVD) and AD-associated genetic variation in CIND patients, hypothesizing that the genetic variation affecting CVD and AD susceptibility is also associated with CIND susceptibility.
Methods:
Our study cohort was taken from the Canadian Study of Health and Aging (CSHA) and was comprised of patients >65 years old with CIND (n=274) and matched normal controls (n=301). We genotyped ∼200,000 CVD-related SNPs using the Cardio-Metabochip genotyping array (Illumina). We also genotyped a panel of the top 11 AD-associated single nucleotide polymorphisms (SNPs) and APOE isotype. We tested for association between CIND status and genotypes using a logistic regression model adjusted for appropriate covariates. Genetic risk scores (GRSs) evaluated associations between CIND status and the accumulation of multiple genetic markers.
Results:
From our Cardio-Metabochip analysis, we identified 5 novel CIND susceptibility loci, with rs16901621 in
FLJ22536
as our most significantly associated SNP (P=1.05E-06, OR=2.51, 95%CI=1.73-3.63). APOE ε4 isotype was modestly associated with CIND status (P<0.05), while AD SNP risk alleles were not associated (P>0.1).
Conclusion:
Using a high-throughput CVD microarray, we found novel genetic markers for CIND approaching genome-wide levels of significance. In contrast, known genetic markers for AD, such as APOE ε4 showed only modest associations in this cohort. Follow-up of variants in a CSHA replication cohort (n=370) is currently underway.
Abstract 501: Nuclear Factor-κB Is Involved in Platelet CD40 Signaling and Activation
Authors: Hachem Ahmed , Yacoub Daniel , Zaid Younes , Mourad Walid , Merhi Yahye
Published: July 2021

Introduction and hypothesis:
CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to influence platelet activation. We have previously shown that upon ligation, CD40 potentiates platelet activation and aggregation via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In B lymphocytes, CD40 induces activation and nuclear translocation of nuclear factor kappa B (NF-κB), which is dependent on the phosphorylation and dissociation of the inhibitor of kappa B α (IκBα). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling.

Methods and results:
In human platelets, sCD40L induced association of tumor necrosis factor receptor associated factor 2 to CD40, and a time-dependant phosphorylation of IκBα, which is indicative of NF-κB activation. Activation of NF-κB in platelets treated with sCD40L was abolished by CD40L blockade. Pretreatment of platelets with the IκBα inhibitor, BAY 11-7082, reversed IκBα phosphorylation induced by sCD40L, without affecting p38 MAPK activation. On the other hand, pretreatment of platelets with the p38 MAPK phosphorylation inhibitor, SB203580, had no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. Finally, inhibition of IκBα phosphorylation by either BAY 11-7082 or the IκB kinase inhibitor VII reversed sCD40L induced platelet activation, as measured by P-selectin expression, and the potentiation of platelet aggregation induced by a priming dose of collagen.

Conclusion:
This study demonstrates the implication of NF-κB in platelet signaling downstream of CD40, where it plays a role in platelet activation and aggregation upon sCD40L stimulation.
Abstract 508: Plasma Kinetics of 3H-Cholesterol from Labeled Macrophages in Hamsters Treated with Dalcetrapib, Torcetrapib and Anacetrapib
Authors: Niesor Eric J , Maugeais Cyrille
Published: July 2021

Introduction:
A model using hamsters injected intraperitoneally with cholesterol-labeled macrophages has been developed for the study of reverse cholesterol transport following treatment with compounds affecting CETP activity e.g., the CETP modulator dalcetrapib or the CETP inhibitors torcetrapib and anacetrapib.

Hypothesis:
Contrary to torcetrapib and anacetrapib, dalcetrapib allows CETP-induced remodeling of HDL and the formation of pre-β-HDL
in vitro
; these compounds may differentially alter the kinetics of efflux of
3
H-cholesterol (
3
H-C) from macrophages to plasma HDL and elimination from plasma HDL.

Methods:
Male Golden Syrian hamsters were fed a chow diet and received dalcetrapib 150 mg/kg bid, n=10; torcetrapib 30 mg/kg qd, n=10; anacetrapib 30 mg/kg qd, n=10; or vehicle (0.5% HPMC), n=8, for 10 days. On Day 7, all animals were administered ip
3
H-C-labeled J774 macrophages and treated as above for a further 3 days. The appearance of
3
H-C in plasma HDL (24, 48 and 72 h), liver and in feces was measured and
3
H-C specific activity (SA) calculated.

Results:
Compared to vehicle, torcetrapib and dalcetrapib raised plasma total cholesterol by 14% and anacetrapib by 21% with a similar trend for HDL-C. At 24, 48 and 72 h, HDL
3
H-C SA was -22.3, +4.9 and -0.5% (p<0.01, NS, NS) for torcetrapib, -16.7, -1.7 and -3.0% (p<0.05, NS, NS) for anacetrapib and +35.3, +36.4 and +8.7% (p<0.007, 0.012, NS) for dalcetrapib compared to vehicle. At sacrifice the percentage of injected dose in liver did not differ between treatment and vehicle for torcetrapib, anacetrapib and dalcetrapib. The SA of
3
H-C in fecal cholesterol was +19.8, +0.5 and +44.8% (NS, NS, p<0.05 vs vehicle) and in fecal bile acids was +49.7, +7.8 and +56.0% (p<0.03, NS, NS vs vehicle) respectively.

Conclusions:
The SA of HDL
3
H-C from labeled macrophages is significantly higher for dalcetrapib at 24 and 48 h but lower for torcetrapib and anacetrapib at 24 h in treated hamsters compared to vehicle. A comparable SA to vehicle at later time points suggests an active mechanism of
3
H-C removal from macrophages and elimination for dalcetrapib, while
3
H-C for torcetrapib and anacetrapib is initially diluted in the HDL-C pool followed by attainment of equilibrium in this pool.
Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis
Authors: Chatterjee Ishita , Wary Kishore K
Published: July 2021

Rationale:
A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood.

Goal:
To address the endothelial cell (EC) specific function of Lpp3 in mice.

Results:
Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3.

Conclusion:
These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.
Abstract 533: Shear-Induced Collateral Artery Growth Modulated by Endoglin but Not by ALK1
Authors: de Vries Margreet R , Seghers Leonard , Hoefer Imo E , Pardali Evangelia , Hierck Beerend P , Ten Dijke Peter , Goumans Marie José , Quax Paul H
Published: July 2021

Introduction:
TGF- β stimulates both shear stress induced arteriogenesis and ischemia induced angiogenesis, by signaling through different receptors. In this study the role of TGF-β receptors ALK1 and endoglin is assessed in a murine model for hind limb ischemia induced neovascularization.

Methods and Results:
Femoral artery ligation was performed in mice heterozygous for either endoglin, or ALK1 and littermate controls. Blood flow recovery, monitored by laser doppler perfusion imaging, was significantly hampered in both endoglin and ALK1 heterozygous mice compared to controls by maximal 40% and 49%, respectively.

Collateral artery size was significantly reduced in endoglin heterozygous mice compared to controls, but not in ALK1 heterozygous mice. Capillary density in ischemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared to controls. Furthermore, both endoglin and ALK1 heterozygous mice showed significant more erythrocyte extravasation near the capillaries than their littermate controls, pointing towards less mature capillaries. Murine endothelial cells were exposed to shear stress in vitro to study the differential role of endoglin and ALK1 in shear induced neovascularization. This resulted in high levels of endoglin messengerRNA, but not of ALK1.

Conclusion:
Here we demonstrated that both endoglin and ALK1 play a crucial role in blood flow recovery. Importantly, endoglin is essential in both shear induced collateral artery growth and in ischemia induced angiogenesis, whereas ALK1 is only involved in ischemia induced angiogenesis.
Abstract 65: Interleukin-6 Signaling and Abdominal Aortic Aneurysm
Authors: Harrison Seamus C , Smith Andrew J , Jones Gregory T , Holmes Michael V , Swerdlow Daniel , Folersen Lasse , Cereceda Anders F , Bown Matthew J , Rampuri Riaz , Agu Obi , De Borst Gert J , van der Graaf Yolanda , Proce Jackie , Baas Annette F , van Rijj Andre M , Eriksson Per , Casas Juan P , Asselbergs Folkert W , Hingorani Aroon D , Humphries Steve E
Published: July 2021

Introduction

Abdominal aortic aneurysm (AAA) is a common disease that predisposes to aortic rupture, which has a high mortality. The only intervention that is proven to reduce rupture is invasive surgery. Understanding causal disease pathways may allow development of non-surgical therapies to target AAA formation, progression or rupture. In this study we investigate the role of interleukin-6 and AAA.

Methods

We performed systematic review and meta-analysis of the published literature in order to determine the association between circulating interleukin-6 (IL-6) and presence of AAA. We then investigated the association between a common non-synonymous variant in the interleukin-6 receptor gene (IL6R) and presence of AAA, performed in vitro functional analyses of this variant, and expression studies in aortic tissue.

Results

Meta-analysis of seven case-control studies of AAA (infra-renal aortic diameter ≥ 3cm), pooling data from 869 cases and 851 controls demonstrated consistently higher levels of circulating IL-6 in AAA compared to controls (standardised mean difference (SMD) = 0.42 units, 95% CI = 0.32-0.52, I2 = 69.4%, P for fixed effects = 1.29 x 10-16, P for random effects = 1.2 x10-5). There was less heterogeneity when comparing large AAA (>5cm, n = 547), to disease free controls (n = 712) (SMD = 0.46 units, 95% CI 0.34-0.57, I2=31.5, P = 2.2 x 10-14). The rare allele of rs7529229, which tags the non-synonymous variant (p.Asp358Ala, rs2228154) was consistently associated with reduced risk of AAA in meta-analysis of 4 studies, pooling data from 4,708 cases and 15,816 (per allele odds ratio 0.84, 95% CI 0.80-0.89, P = 2.7 x 10-11, I2=0). This variant was also associated with reduced risk incident AAA endpoints (rupture +/- repair) in prospective analysis of 7,888 individuals with known arterial disease (hazard ratio = 0.81, 95% CI 0.67 - 0.99, P = 0.043). Expression studies revealed strong correlation between IL-6 expression target genes (STAT3, MYC, ATF3, BCL3 and ICAM1) in aortic adventitia. In vitro analyses demonstrate that in lymphoblastoid cells, there is a genotype specific reduction in expression of downstream mediators of IL-6 signalling in response to IL-6 stimulation.

Conclusions

These data indicate that signalling via the IL-6 receptor is likely to be causal in the development of AAA. This suggests that this pathway may represent a novel target for pharmacological treatment of AAA with biological agents such as Tocilizumab, and clinical trials to assess this may be warranted.
Abstract 68: Reducing Hepatic Very-Low-Density Lipoprotein Production by Knockdown of Microsomal Triglyceride Transfer Protein Decreases Transintestinal Cholesterol Excretion
Authors: Marshall Stephanie M , Kelley Kathryn L , Davis Matthew , Wilson Martha D , Rudel Lawrence L , Brown John M , Temel Ryan E
Published: July 2021

Atherosclerotic coronary vascular disease (ASCVD) remains the number one killer of Americans. A way to reduce the low-density lipoprotein cholesterol (LDLc), the primary risk factor of ASCVD, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both biliary and non-biliary pathways. The lipoprotein that delivers cholesterol from the liver through the plasma to the small intestine (SI) for transintestinal cholesterol excretion (TICE) is not yet known. We hypothesized that cholesterol leaves the liver via very low-density lipoproteins (VLDL) for TICE. We assessed this hypothesis by using antisense oligonucleotides (ASO) to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol (0.2% wt/wt) diet for six weeks, wild type (WT) mice and hepatic Niemann-Pick C1-like 1 transgenic mice (L1Tg), which predominantly excrete cholesterol via TICE, were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased hepatic MTP protein expression by 60% and increased hepatic total cholesterol concentration 3.5 fold. Regardless of treatment with control or MTP ASO, L1Tg mice had an 80% reduction in biliary cholesterol compared to WT mice. However, L1Tg mice treated with MTP ASO displayed a 60% reduction in fecal neutral sterol (FNS) excretion. In summary our data show that when VLDL production is decreased in mice with reduced biliary cholesterol secretion capacity, FNS excretion is impaired. These data support our conclusion that cholesterol leaves the liver through VLDL for TICE.
Abstract 92: A Novel Role for Dipeptidyl Peptidase-4 and Adenosine Deaminase in Regulating Macrophage and Dendritic Cell Function: Implications for Treatment of Type 2 Diabetes
Authors: Zhong Jixin , Rao Xiaoquan , Deiuliis Jeffrey , Needleman Bradley , Rajagopalan Sanjay
Published: July 2021

Introduction:
In addition to its powerful effects on glycemic control via enzymatic degradation of incretins, Dipeptidyl peptidase-4 (DDP4) also exerts non-metabolic functions. Despite the ubiquitous use of DPP4 inhibitors in the treatment of Type 2 diabetes (T2D), their effects on immune cell function are poorly understood.

Hypothesis:
In this study, we investigated the hypothesis that DPP4 may play a critical role in modulating T cell proliferation through its expression on dendritic cells (DCs) and further characterized its role in adipose inflammation in patients with morbid obesity.

Methods:
Monocytes were isolated from peripheral blood of healthy donors. DCs were induced from monocytes by rhGM-CSF and rhIL-4. DPP4 expression and T-cell proliferation were detected by flow cytometry. DPP4 knockdown was accomplished by transient transfection with DPP4-specific siRNA. Human visceral adipose tissues were used for stromal vascular fraction isolation.

Results:
7.5±3.1% (n=10) of peripheral monocytes expressed DPP4. DPP4 dramatically increased when monocyte was differentiated to DC/Macrophage or activated by LPS. Interestingly, DPP4 expression was much higher on the DC/Macrophage in adipose tissue than that of peripheral blood. Furthermore, an increase of DPP4 on adipose tissue macrophages in obese patients was observed (13.2±2.4 vs. 7.9±1.6 for obese vs. lean, n=5). And there was a correlation between DPP4 expression and insulin level (y = 0.9094x-3.6088, R
2
=0.6943). Cell surface DPP4 was able to bind adenosine deaminase (ADA). The binding enhanced the capability of DCs to stimulate T-cell proliferation by relieving the suppressive effect of adenosine on T-cell proliferation. Knockdown of DPP4 on DCs by siRNA attenuated their ability to stimulate T-cell proliferation without influence on the secretion of pro-inflammatory cytokines by DCs.

Conclusions:
DPP4 on DCs mediates binding of ADA and may re-create a microenvironment suitable for T-cell proliferation. This interaction in DCs between ADA and DPP4 may promote adipose T-cell inflammation and play facilatatory role in T2D. Pro-inflammatory effects of DPP4 and the elucidation of its enzymatic v.s. non-enzymatic contributions may be of fundamental importance in the understanding of T2D.
Abstract 1: Molecular Basis of Hypertriglyceridemia Associated with the APOA5 c553.G>T SNP
Authors: Sharma Vineeta , Forte Trudy M , Ryan Robert O
Published: July 2021

Objectives

Apolipoprotein (apo) A-V is a low abundance protein with profound effects on plasma triglyceride (TG) levels. Several APOA5 SNPs correlate with hypertriglyceridemia (HTG). The c.553 G>T SNP, substituting a Cys for Gly at position 162 of mature apoA-V, is prevalent in Asian populations and correlates with HTG. To investigate the mechanism underlying this association, gene transfer studies were performed in apoa5-/- mice.

Methods

Adeno-associated virus (AAV2/8) harboring the coding sequence for wild type apoA-V (AAV2/8-apoA-V), G162C apoA-V (AAV2/8-G162C) and LacZ (AAV2/8-LacZ) were injected (1x10e12 virus genome) into the tail vein of 8 apoa5-/- mice per group. Blood samples were collected weekly for 4 weeks and TG and apoA-V levels measured. FPLC was performed on plasma obtained from AAV2/8-apoA-V and AAV2/8-G162C mice. VLDL, LDL, HDL and the lipoprotein-free region were characterized.

Results

Compared to AAV2/8-LacZ mice, AAV2/8-apoA-V mice had significantly lower plasma TG levels (50% ±5). Unlike AAV2/8-apoA-V, mice injected with AAV2/8-G162C displayed little or no reduction in TG despite similar amounts of plasma apoA-V protein. Immunoblot analysis of FPLC fractionated plasma revealed that, whereas wild-type apoA-V was lipoprotein associated (VLDL and HDL), G162C apoA-V was largely recovered in the lipoprotein-free fraction. Immunoblot analysis following SDS-PAGE under non-reducing conditions revealed that lipoprotein-associated wild type apoA-V and G162C apoA-V are monomeric; by contrast, the electrophoretic mobility of G162C apoA-V recovered in the lipoprotein-free fraction was retarded.

Conclusions

Gene transfer of wild type apoA-V induces a significant reduction in plasma TG levels of apoa5-/- mice. By contrast, G162C apoA-V failed to induce a corresponding decrease in plasma TG, recapitulating effects observed in human populations harboring this SNP. The propensity of G162C apoA-V to form a disulfide bond with one or more plasma proteins interferes with its lipoprotein binding ability, resulting in loss of function. The results provide a molecular explanation for HTG associated with a common APOA5 SNP. The gene transfer strategy employed provides a platform for studies of other common apoA-V SNPs.
Abstract 125: Weight Loss Selectively Increases High-density Lipoprotein without apoC-III, a Subfraction with Protective Association Against Coronary Heart Disease.
Authors: Wang Liyun , Talayero Beatriz G , Furtado Jeremy D , Bray George A , Sacks Frank M
Published: July 2021

Human high-density lipoprotein (HDL) particles are highly heterogeneous in composition, and in relation to coronary heart disease (CHD). Specifically, HDL containing apoC-III predicts
increased
incidence of CHD, whereas HDL that does not contain apoC-III predicts
decreased
CHD incidence. Recently, we found that obese subjects had a higher concentration of HDL with apoC-III and a lower concentration of HDL without apoC-III than normal weight controls. However, the effects of weight loss on HDL with or without apoC-III are not known.

We studied 39 obese participants (mean BMI of 34) in a weight-loss trial assigned to a high fat 40%/high protein 25%/low carbohydrate 35% diet for 2 years. We chose this diet group because HDL-cholesterol (-C) levels increased the most compared to the other diet groups with more carbohydrate and less fat. Participants with at least 8% loss of initial body weight at 2 years were assigned to the weight loss group (n=21), while those without a change in weight comprised the no-weight loss group (n=18). Subjects were matched by age (mean 51 years), gender, and race. HDL without apoC-III and HDL with apoC-III were separated from plasma by sequentially using anti-apoA-I and anti-apoC-III immunoaffinity chromatography.

For both groups, the mean baseline HDL-C and total plasma apoA-I concentrations were 48 and 119mg/dL, respectively. Weight loss increased HDL-C concentration by an average of 25% (p<0.01 for changes between groups), and total apoA-I by 9% (p<0.02). In the weight-loss group, mean apoA-I concentrations of HDL without apoC-III increased by12% (from 99 to 111 mg/dL) compared to a decrease of 6% in the no-weight loss group (from 107 to 101mg/dL) (p<0.02). The apoA-I concentration of HDL with apoC-III, 15 mg/dL, remained unchanged.

In conclusion
, moderate weight loss selectively increased the concentration of HDL without apoC-III, the predominant HDL type that predicts reduced incidence of CHD. In contrast, weight loss did not affect HDL with apoC-III, an HDL type associated with increased CHD risk. This represents a partial normalization of HDL subtypes by moderate weight loss. This effect on HDL subpopulations defined by absence of apoC-III suggests a novel mechanism by which weight loss may reduce cardiovascular disease risk.
Abstract 159: Comparing Blood Pressure Control in Diabetic vs Non-Diabetic Veterans following Percutaneous Coronary Intervention
Authors: Blumenkrants Dmitry , Siddiqui Saifullah M , Challa Karthik , Ladani Amit , Shroff Adhir
Published: July 2021

Background

As per the US Joint National Committee VII (JNC-7) recommendations, patients with known underlying coronary artery disease and diabetes should have goal blood pressures (BP) of systolic (SBP) <130 and diastolic (DBP) <80 to decrease morbidity and mortality associated with cardiovascular disease. In addition to lifestyle modification, these goals can be attained by use of multiple classes of drugs including beta-blockers (BB), angiotensin agonists (ACE-I/ARB), calcium channel blockers (CCB), diuretics and nitrates.

Methods

We conducted a retrospective cohort study focusing on the attainment of the JNC-7 guidelines, comparing outcomes between 415 diabetic to 637 non-diabetic patients undergoing PCI between September 2004 and December 2012 at the Jesse Brown Veterans Administration Hospital in Chicago, IL. Blood pressure (BP) measurements and antihypertensive medications pre and post PCI at 6-month follow-up were documented.

Results

Among the diabetic population, the mean SBP decreased from 136 to 131 mmHg (p < 0.0001) and mean DBP decreased from 73 to 70 mmHg (p < 0.0001). In the non-diabetics, the mean SBP decreased from 133 to 127 mmHg (p < 0.0001) and the mean DBP decreased from 74 to 71 mmHg (p < 0.0001).

With regards to JNC-7 guidelines, the percent of diabetics at SBP goal increased from 42% to 49% (p = 0.047) and percent at DBP goal increased from 74% to 82% (p = 0.008). In non-diabetics, percent at goal for SBP increased from 46% to 57% (p < 0.0001) and percent at DBP goal increased from 68% to 76% (p = 0.003). Among diabetics, there was a statistically significant (p <0.0001) increase in use of BB from 77% to 90%. In non-diabetics, there was a statistically significant (p <0.0001) increase in use of BB from 64% to 86% and ACE-I/ARB from 51% to 70%.

Conclusions

In both groups (diabetics and non-diabetics) undergoing PCI, both systolic and diastolic blood pressure improved with more patients achieving JNC-7 targets. Among diabetics, there was a significant increase in utilization of BB. Among non-diabetics, there was a significant increase in utilization of BB and ACE-I/ARB.
Abstract 160: The Role of Inhibitor of Differentiation 3 in the Regulation of Monocyte Chemoattractant Protein-1 Within Epididymal Adipose Tissue During Early Stages of Obesity
Authors: Kaplan Jennifer L , Garmey James C , Harmon Daniel B , Marshall Melissa A , McNamara Coleen A
Published: July 2021

Background

Global deletion of helix-loop-helix (HLH) transcription factor Inhibitor of Differentiation 3 (Id3) attenuates HFD-induced obesity. Specifically,

Id3
-/-

mice have less epididymal adipose tissue (eAT); an effect that may be linked to eAT inflammation. Adipose tissue macrophages play a key role in HFD-induced obesity and are recruited by Monocyte Chemoattractant Protein-1 (MCP-1). MCP-1 is produced by a wide variety of cells.

Id3
-/-

mice have attenuated HFD-induced levels of MCP-1 mRNA and protein in eAT. Consistent with lower levels of MCP-1 in eAT,

Id3
-/-

mice also have decreased numbers of proinflammatory macrophages in eAT after HFD. Id3 is broadly expressed and involved in many disease pathways. It is currently unclear how Id3 is regulating MCP-1 in eAT, or in which MCP-1-producing cell.

Results

Id3
-/-

mice and
C57Bl/6
littermate controls were fed HFD (60% fat) for 1 and 4 weeks. Stromavascular fraction (SVF) cells from eAT were cultured with Brefeldin A to inhibit protein secretion. Intracellular levels of MCP-1 were analyzed via flow cytometry. Both 1 and 4 weeks of HFD increased the number of MCP-1+ cells, compared to chow-fed controls. There were two levels of MCP-1 expression. Adipocyte progenitors, identified as CD31-CD45-Ter119-CD34+CD29+Sca-1+, express high levels, and macrophages, identified as CD45+F4/80+CD11b+, express mid levels of MCP-1. MCP-1 high cells increased after 1 week of HFD, and this increase was amplified after 4 weeks of HFD. MCP-1 mid cells did not increase until 4 weeks of HFD. However, in

Id3
-/-

mice, both populations were decreased in number at both stages of HFD. MCP-1+ cells in

Id3
-/-

mice also had lower mean fluorescence intensity (MFI) compared to WT controls, indicating lower levels of MCP-1 per cell.

Conclusion

Global deletion of Id3 decreases the number of MCP-1 expressing cells in epididymal adipose tissue, as well as MCP-1 levels per cell. These data suggest that Id3 promotes an immediate response to HFD via an increase in the number of MCP-1 producing cells. There is also evidence that Id3 is directly regulating expression of MCP-1 within adipocyte progenitors. Further studies will elucidate the exact mechanism of regulation of both the number of cells and expression of MCP-1 by Id3.
Abstract 179: Vascular Inflammation and Hepatic Steatosis among the Overweight Metabolically Healthy Individuals and Normal Weight with Cardiometabolic Risk Factor Clustering
Authors: Shaharyar Sameer , Karim Adil , Blaha Michael J , Ali Shozab S , Jamal Omar , Agatston Arthur , Blumenthal Roger S , Nasir Khurram , Santos Raul D
Published: July 2021

BACKGROUND

Emerging literature has elucidated a new phenotype, metabolically healthy overweight/obese (MHO) who have a similar CVD risk to normal weight individuals. The aim of the study was to compare the relative risk of both phenotypes with subclinical inflammatory-cardiometabolic disorders.

METHODS

We evaluated 6,464 healthy non diabetic Brazilian subjects (43±10 years, 79% males). Those with <2 metabolic risk factors (MRF)were considered metabolically healthy and those with BMI≥25 kg/m2 as overweight (OW). High sensitivity C reactive protein (hs-CRP) was measured to assess degree of underlying inflammation and hepatic steatosis (HS) was determined via abdominal ultrasound.

RESULTS

Overall, 52% OW individuals were metabolically normal, and 11% normal-weight had≥2 MRF. The respective prevalence for elevated CRP (≥ 3 mg/dl) was 28% vs 16%, p<0.0001) and 71% vs 28% for HS, respectively. As compared to those with normal weight/metabolically benign, the highest risk of elevated CRP & HS was noted among those with overweight/metabolically unhealthy individuals (table). Both MHO individuals & normal weight metabolically unhealthy participants had similar association with elevated CRP & HS.

CONCLUSION

Our study highlights that healthy overweight/obesity & normal weight metabolically unhealthy phenotypes are not entirely benign and efforts are needed to reverse these conditions.
Abstract 187: Regulation of Vascular Smooth Muscle Cell (VSMC) Inflammation and Migration by the Pro-resolving Lipid Mediator Maresin-1 (mar1)
Authors: Chatterjee Anuran , Toy Robert , Mottola Giorgio , Chen Mian , Conte Michael S
Published: July 2021

Introduction

Resolution of acute inflammation is regulated by endogenous lipid mediators derived from polyunsaturated fatty acids such as docosahexaenoic acid (DHA), however little is known about mechanisms of resolution in vascular injury. We investigated the effects of the DHA-derived mediator Mar1 on VSMC phenotype responses.

Methods

Primary human VSMCs were obtained from saphenous vein. VSMC were pretreated with Mar1 (10-100nM) then exposed to TNF-α (10ng/ml), and inflammatory responses assessed using a monocyte adhesion (U937) assay, expression of cell adhesion molecules and pro-inflammatory molecules (qPCR, western blot, ELISA), and production of superoxide (DHE). VSMC migration was measured in a transwell assay with PDGF-AB as the agonist, and cyotskeletal changes were assessed by actin-phalloidin staining.

Results

Mar-1 (100 nM) reduced U937 adhesion to TNF-stimulated VSMC, VCAM-1, and pro-inflammatory cytokine (IL-6, IL-8) expression. Superoxide production measured by DHE fluorescence was reduced by 57% (p=0.002) and Nox4 expression was markedly attenuated (43%, p=0.01). Mar-1 (0.01-100nM) induced rapid cytoskeletal changes with increased cell area, and reduced VSMC migration (76%, p=0.004) to PDGF-AB (50ng/ml; Figure).

Conclusions

Mar-1 attenuates TNF-α inflammatory activation of VSMC, with reduction in pro-inflammatory gene expression, oxidant stress, and monocyte adhesion. Mar-1 reduces actin polymerization and inhibits VSMC chemotaxis to PDGF. Pro-resolving mediators may represent a new class of endogenous vascular therapeutics.
Abstract 197: Emerging Role of Toll-like Receptor 4 and Heat Shock Protein 60&70 in Ischaemia-Induced Skeletal Muscle Damage In Vitro
Authors: Navi Ali , Yu Rebekah , Shi-Wen Xu , Shaw Sidney , Baker Daryll , Tsui Janice
Published: July 2021

OBJECTIVES

The innate immune response contributes to the skeletal muscle damage in patients with critical limb ischaemia (CLI); however, the detailed signaling mechanisms are not fully understood. We hypothesized that simulated ischaemia induces inflammatory cytokine release from skeletal myotubes, via a mechanism that involves heat shock protein (HSP) 60&70, known endogenous ligands of Toll-like receptor 4 (TLR4), in vitro.

METHODS

Human gastrocnemius muscle biopsies were taken from patients with CLI undergoing major lower limb amputation and from patients with no peripheral arterial disease (PAD). Human myoblasts were isolated, cultured to myotubes and then pre-treated with TLR4 neutralizing antibody prior to exposure to simulated ischaemia. Fluorescent immunostaining was carried out to confirm cell differentiation; ELISA analysis were carried out to quantify IL6 and TNF-α release; and Western blot was used to assess expression of HSP60&70, TLR4 and cleaved caspase-3 as a marker of apoptosis.

RESULTS

Myotubes from patients with CLI expressed greater levels of cleaved caspase-3 and TLR4 as compared to those from patients with no PAD. When exposed to ischaemic conditions, increased IL6 and TNF-α release and upregulation of HSP60&70, cleaved caspase-3 and TLR4 were observed in myotubes from both groups of patients compared to culturing in normoxic conditions (P<0.05). Pre-treatment of myotubes from patients with CLI with TLR4 neutralizing antibody prior to simulated ischaemia was associated with reduced expression of HSP60&70, IL6, TNF-α and cleaved caspase-3 (P<0.05).

CONCLUSIONS

Increased cytokine release, apoptosis and expression of HSP60&70 and TLR4 occur in ischaemic skeletal muscle in vitro. TLR4 antagonism was associated with reduced apoptosis and inflammatory cytokine release and down-regulation of HSP60&70 expression. This suggests a potential pathway where TLR4 and its endogenous ligands contribute to a positive feedback loop to maintain a proinflammatory environment during ischaemia.
Abstract 204: Brilliant Blue FCF is an Alternative Vein Marking Dye That Restores Functional Viability in Human Saphenous Vien Graft
Authors: Brophy Colleen , Hocking Kyle M , Komalavilas Padmini , Lander Cynthia , Cheung-Flynn Joyce
Published: July 2021

Objective

The human saphenous vein (HSV) is the most effective conduit for infrainguinal peripheral bypass grafting. Optimal vein graft preparation is critical to vein graft patency. Graft marking using surgical skin markers is detrimental to endothelial and smooth muscle function of the graft. The objective of this study was to evaluate the effect of FD&C Brilliant Blue No. 1 (FCF) as an alternative for vein graft marking.

Methods

Segments of HSV were collected after typical harvest and graft preparation. The tissues were cut into rings and either left unmarked or marked with FCF (2.6 mM). The functional viability of the rings was determined in a muscle bath.

Results

Marking with FCF did not impair contractility of the smooth muscle (Figure 1A). Contractility was

restored

in HSV segments that had no functional contractile responses after harvest and preparation (Figure 1B). To determine the mechanism of this protective effect of FCF, purinergic receptors were activated with BzATP with and without pre-incubation with FCF (50μM) or a purinergic receptor antagonist (oATP, control) and the rise in [Ca
2+
]
i
was measured. FCF and oATP reduced the BZATP-evoked rise in [Ca
2+
]
i
(Figure 1C), suggesting that the salutary effects of FCF were due to inhibition of a P2X or P2Y receptor.

Conclusions

FCF is a non-toxic alternative to surgical skin markers that antagonizes purinergic receptors in HSV. FCF restored functional viability to injured vascular smooth muscle. FCF may protect HSV against injury-elicited ‘danger’ signals such as extracellular ATP that is released during injury-induced preparation.
Abstract 206: Small GTPase Rap1 Transmits Mechanical Signals to Control Vascular Tone and Blood Pressure
Authors: Lakshmikanthan Sribalaji , Zheng Xiaodong , Zieba Bartosz J , Nishijima Yoshinori , Ge Zhi-Dong , Momotani Ko , Lund Hayley , Maas Jason E , Szabo Aniko , Vasquez-Vivar Jeannette , Mattson David L , Auchampach John A , Somlyo Avril V , Zhang David X , Chrzanowska-Wodnicka Magdalena
Published: July 2021

Smooth muscle (SM)-mediated vessel contraction and endothelium-dependent vessel dilation are major mechanisms controlling basal vascular tone. Increased vascular tone leads to arterial hypertension, a risk factor for cardiovascular diseases. Agonist-induced, Ca2+-dependent SM contraction is modulated by intracellular cAMP, which promotes SM relaxation by inhibition of Rho-mediated myosin regulatory light chain (RLC20) phosphorylation and actomyosin contractility. In response to elevated blood flow, endothelium releases dilator substances that relax vascular SM, and in particular defects in production of nitric oxide (NO) lead to hypertension.

Rap1 is a small GTPase that integrates signals from multiple receptors and two Rap1 isoforms, Rap1a and Rap1b are ubiquitously expressed. We found global Rap1b deletion leads to cardiac hypertrophy and hypertension and examined the effect of Rap1-deficiency on vascular tone. Using SM and endothelium tissue-restricted Rap1 knockout mice we show that deletion of Rap1, via distinct mechanisms in the two tissues, contributes to elevated vascular tone. In SM Rap1 regulates basal contraction level and mediates cAMP-induced desensitization of contraction. Rap1 suppresses RhoA-mediated RLC20 phosphorylation, Ca+2 sensitization and relaxation, signaling that is further enhanced upon cAMP-dependent activation of Rap1 by Rap1 GEF Epac.

In endothelium, we find Rap1 is essential for regulation of NO-dependent vasodilation, as deletion of three of the four Rap1 alleles leads to a significant decrease in NO-dependent vasodilation. Significantly, we find that Rap1 is required for normal shear stress-induced NO release and Rap1-deficency in endothelium leads to elevated blood pressure in mice. Mechanistically, we show that Rap1 is required for transducing signals from the endothelial mechanosensing complex triggering signaling leading to NO production. Because shear stress from flowing blood is the main determinant of NO release, this novel finding positions Rap1 as a key regulator of endothelial function.

In conclusion, Rap1 in SM and endothelium, via distinct mechanisms, plays a key role in maintaining normal vascular tone and blood pressure.
Abstract 22: OSBPL6 is a Novel miR-33 Target Gene Involved in Cellular Cholesterol Efflux
Authors: Hennessy Elizabeth J , Rayner Katey J , Sokolovska Anna , Ouimet Mireille , Esau Christine , Moore Kathryn J
Published: July 2021

Cellular lipid homeostasis is fundamental to human health. Recent studies indicate that microRNAs (miRNAs) play important roles in the post-transcriptional regulation of lipid metabolism genes. This is illustrated by the discovery, by our group and others, of miR-33 as a central regulator of multiple aspects of lipid metabolism, including cholesterol efflux, fatty acid oxidation, and plasma levels of HDL cholesterol and VLDL triglycerides. In a study of miR-33 antagonism in non-human primates, we identified the most highly de-repressed miR-33 target gene as OSBPL6 (oxysterol binding protein like 6). A member of the OSBP family of cytosolic proteins that bind cholesterol/oxysterols, OSBPL6 contains an ER-targeting FFAT motif as well as a predicted plasma membrane-targeting pleckstrin homology domain, suggesting a putative role for OSBPL6 in vesicular traffic between these compartments. Notably, the OSBPL6 gene resides in a chromosome 2 locus associated with a predisposition to CAD in two major human heart disease studies (Family Heart Study, Framingham Heart Study). We therefore sought to further understand the regulation and function of OSBPL6 in cellular lipid homeostasis.

Results

We show that OSBPL6 is induced in macrophages and hepatocytes in response to cholesterol loading in vitro, as well as in the livers of LDLR-/- mice and non-human primates fed a high fat diet. Overexpression of miR-33 reduces OSBPL6-3’UTR activity by 40%, and this repression is relieved by mutation of the putative miR-33 binding site in the 3’UTR, confirming that OSBPL6 is a direct target of miR-33. Accordingly, transfection of HEPG2 cells with miR-33 mimic decreases mRNA expression of Osbpl6, but not the related family member Osbpl1. Notably, siRNA-mediated knockdown of OSBPL6 in cholesterol-loaded HEPG2 cells or THP-1 macrophages impairs cholesterol efflux to both apoA-I and HDL, and conversely, OSBPL6 overexpression enhances cholesterol efflux to these acceptors.

Conclusion

These data identify OSBPL6 as a novel miR-33 target gene in mice and humans that regulates cellular cholesterol trafficking and efflux via the ABC transporters, further highlighting the coordinated regulation by miR-33 of pathways that promote cellular cholesterol clearance.