bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 287: Plasma PCSK9 Concentrations in Critically Ill Patients
Authors: Cariou Bertrand , Le Bras Maëlle , Roquilly Antoine , Feuillet Fanny , Sebille Véronique , Costet Philippe , Asehnoune Karim
Published: July 2021

Objective.
Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that impairs LDL clearance by promoting the LDL receptor (LDLR) degradation. Plasma lipid parameters (LDL-C and HDL-C) are related to severity of illness and survival in intensive care patients. Here, we aimed to determine whether circulating PCSK9 concentrations were correlated to the severity of illness in patients with multiple trauma.

Methods/Results.
Plasma PCSK9 were measured, at day 0 and 8 after admission, by ELISA in 111 patients hospitalized in ICU for severe trauma. Patients were included in the HIPOLYTE study and were randomly assigned to hydrocortisone therapy or placebo. Plasma PCSK9 levels were significantly increased by 161% at day 8 compared to day 0 (481 ± 227 vs 231 ± 117 ng/ml, P=0.0001). Hydrocortisone therapy did not alter PCSK9 concentrations at day 8 compared to placebo (451 ± 216 vs 511 ± 239ng/ml, P=0.33). In the whole population of the study, PCSK9 was positively associated with LDL-C (Pearson coefficient : 0.26, p=0.007) at day 0, but not with markers of severity illness. At day 8, an inverse correlation was found between PCSK9 and HDL-C (β =-653 ; P=0.004). Interestingly, PCSK9 concentrations at day 8 were related to markers of severity illness as injury severity score (β =6.17 ; P=0.0007), length of stay in ICU (β =6.14 ; P=0.0001), duration of both mechanical ventilation (β =8.26 ; P=0.0001) and cathecolamines infusion (β =18.57; P=0.015).

Conclusion.
PCSK9 appears as a late biomarker of severity illness in patients hospitalized for multiple trauma in ICU. These results open new perspectives for a role of PCSK9 in critical illness.
Abstract 289: Chronic Systemic Insulin Treatment Attenuates Atherosclerotic Lesion Development in Cholesterol-Fed ApoE
-/-
Mice.
Authors: Chiang Simon , Britto Joanne , Giacca Adria
Published: July 2021

Diabetes mellitus and metabolic syndrome, which are states of insulin resistance, are both well-known to increase the risk of atherosclerosis. However, it is still debated whether this increased risk is due to excess or lack of insulin action. Here, we present evidence that insulin is anti-atherogenic and decreases atherosclerosis independent of its effects on glycemia. ApoE
-/-
mice were fed a 1.25% (w/w) high cholesterol diet for 1 week pre-treatment then implanted with either control pellets or insulin pellets (0.05U/day which achieves a mild 4-fold elevation of circulating insulin without significantly lowering plasma glucose) subcutaneously for 12 weeks thereafter. At the end of the treatment, the mice were perfused and the aortas were harvested. Using en face Oil-Red O staining, the atherosclerotic plaque in the descending aorta was evaluated by measuring lesion area over total area. Insulin-treated mice (n=7) showed a significant decrease in atherosclerotic plaque (9.0 ± 2%) compared to control (n=11; 17.6 ± 2.5%). There was no significant difference in glycemia between the two groups. Based on our results, insulin is anti-atherogenic in the context of euglycemia.
Abstract 297: Increased ETA Receptor-Mediated Contractility of Thoracic Aorta by Endothelin-1 in High-Fat-Diet--Fed Insulin-Resistant Rats
Authors: Poduri Ramarao , Gupta Payal , Gulati Anil
Published: July 2021

Abstract:
Increased incidences of cardio-vascular complications in diabetic conditions are a major concern. Endothelin-1 (ET-1) is an important regulator of vascular contractility, and its effects are mediated through ETA and ETB. Previous data from the lab indicates that GPCR mediate contractile responses are enhanced and the present study is aimed to extend the hypothesis that ETA receptor mediated contractile responses of ET-1 under insulin-resistant condition are enhanced.

Methods:
Male Sprague-Dawley (SD) rats were kept on high-fat diet (HFDIRR, 8 weeks) for inducing insulin-resistance. Further, b-cell specific toxin streptozotocin (STZ, 50 mg/kg; i.p.) was used to induce hypoinsulinemia in rats. The responses of ET-1, ACh, KCl and Ang -II were recorded in the concentration-dependent manner in the thoracic aorta of rats. Specific ETA receptor antagonist, BMS182874 was used to confirm the findings. Specific binding of [3H]-BQ123 was performed to determine the characteristics of ETA receptors. Biochemical parameters were measured and induction of insulin-resistance was confirmed by intra-peritoneal glucose tolerance test (IPGTT).

Results:
ET-1 mediated contractile response (4541 ± 274 mg/mm2 (mean ± s.e.m. (n=8)) was significantly higher in insulin-resistant rats, while that of KCl unchanged. Tempol (100μM) restored the ET-1 mediated contractions in HFDIRR (4541 ± 274 vs 3406 ± 252.8 mg/mm2; p < 0.001; (n=5)). BMS182874 restored NO-mediated endothelium-dependent ACh relaxation in HFDIRR. The specific binding of [3H]-BQ123 to ETA receptors (112.35 ± 5.19 vs. 39.74 ± 4.04 f mol/mg; P < 0.001; (n=5)) was increased in HFDIRR.

Conclusions:
Insulin-resistance up-regulates ETA receptor may be responsible enhanced ET-1 mediated contractility of the thoracic aorta.
Abstract 321: LRP6 Regulates LDL Receptor Internalization and LDL Uptake
Authors: Mani Arya , Go Gwang-Woong , Ye Zhi-jia , Singh Rajvir
Published: July 2021

Genetic variations in
LRP6
gene are associated with high serum LDL cholesterol levels and atherosclerosis. We examined the role of LRP6 in LDL receptor (LDLR) mediated LDL uptake. LDL uptake was increased when LRP6 was overexpressed and reduced when it was knocked down in LDLR deficient CHO cells. Interestingly, LRP6 knockdown in wildtype CHO cells resulted in a much greater decline in LDL uptake compared to ldlA7 cells. This finding suggested interaction between LRP6 and other proteins involved in LDL uptake. Strikingly, LDL receptor internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further investigations showed that LRP6 forms a complex with the LDL endocytic machinery including LDLR, clathrin and ARH and undergoes endocytosis after stimulation with LDL. LDLR internalization was defective in skin fibroblasts of the LRP6
R611C
mutation carriers. LDLR and LRP6 internalizations as well as LDL uptake were significantly impaired in wildtype CHO cells expressing LRP6
R611C
mutation(figa,b). These studies introduce LRP6 as a critical modulator of receptor-mediated LDL endocytosis and identify a mechanism by which variation in LRP6 may contribute to high serum LDL levels and atherosclerosis.
Abstract 353: Mechanical (or “Gravitational”) Unloading Reduces Inflammatory and Cell Adhesion Molecule Gene Expression in Human Endothelial Cells
Authors: Grenon S Marlene , Aguado-Zuniga Jesus , Conte Michael , Hughes-Fulford Millie
Published: July 2021

Objectives:
Mechanical forces including gravity affect mechanotransduction and subsequent cell function. The goal of this study was to investigate the impact of mechanical unloading (MU) and loading (ML) of endothelial cells (ECs) with microgravity and hypergravity respectively, with the hypothesis that MU alters expression of inflammatory and adhesion molecule gene expression and these changes are reversed by ML.

Methods:
Human umbilical vascular endothelial cells (HUVECs) grown to confluency were studied. A desktop random positioning machine and a gravitational cell-loading apparatus provided MU and ML conditions, respectively. The experimental conditions included: 1) controls exposed to 1-gravity environment for 24 h (CL), 2) MU for 24 hours, 3) MU for 24 hours with three 30-minutes periods of ML of 12-gravity (MU/ML). Gene expression was studied with reverse transcription followed by real-time quantitative polymerase chain reaction (qRTPCR).

Results:
MU led to a significant decrease in gene expression of the adhesion molecules ICAM-1, VCAM-1, E-Selectin, as well as TNF-α, IL-6 and VEGF. In contrast, NOS-3, Caveolin-1 and -2 were significantly increased with MU. The changes observed in gene expression with MU were reversed by gravitational mechanical loading (MU/ML).

Conclusions:
Gravitational MU decreases inflammatory and adhesion molecule gene expression and these changes are reversed by short periods of ML. This points towards the importance of gravitational loading in ECs function and cellular interactions.
Abstract 361: Cyclophilin A Mediates Angiotensin II--Stimulated NADPH Oxidase Activation in Vascular Smooth Muscle Cells
Authors: Soe Nwe Nwe , Sowden Mark , Nigro Patrizia , Berk Bradford C
Published: July 2021

Objective:
Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses PPIase activity and scaffold function. CyPA regulates Angiotensin II (Ang II) induced reactive oxygen species (ROS) production in vascular smooth muscle cells. However, the mechanism of this CyPA regulation remains unclear. We hypothesized that CyPA regulates plasma membrane translocation of NADPH oxidase cytosolic subunit, p47phox, which is required for NADPH oxidase structural organization and activity.

Methods and results:
Immunofluorescence studies in rat aortic smooth muscle cells revealed that CyPA translocated from the cytosol to the plasma membrane in response to Ang II in a time dependent manner with a peak at 10min (46.4±5.4 fold increase). Mouse Aortic Smooth Muscle Cells (MASM) were isolated from mice lacking CyPA (CyPA-/-) and wild type controls (WT), treated with Ang II (100nM) and immunofluorescence analysis was performed. Ang II induced p47phox plasma membrane translocation at 10min in WT mice. However, p47 phox translocation was significantly inhibited in CyPA
-/-
MASM. CyPA and p47phox colocalized at the plasma membrane in response to Ang II. Further analysis using subcellular fractionation studies confirmed that Ang II induced p47phox plasma membrane translocation was inhibited in CyPA
-/-
MASM compared to WT (1.2±2.7 vs 4.3±3.4 fold increase). Coimmunoprecipitation analyses confirmed that Ang II increased CyPA association with p47phox in a time dependent manner (2.5±3.4 fold increase at 10min). Finally, pretreatment with the PPIase activity inhibitor, cyclosporine A (1uM), could not inhibit CyPA association with p47phox and CyPA mediated p47phox translocation to the plasma membrane.

Conclusion:
These data suggest that Ang II promotes an association between CyPA and p47phox that enhances plasma membrane translocation of p47phox. This is proposed to increase the NADPH oxidase activity thereby increasing cellular ROS production. This process is independent of the PPIase activity of CyPA. Therefore, inhibition of the CyPA and p47phox association could be a future therapeutic target for Ang II induced ROS regulated cardiovascular diseases such as atherosclerosis and abdominal aortic aneurysm formation.
Abstract 374: Epigenetic Mechanisms Regulate TNF-α-Induced Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Biology
Authors: Ali Muhammad S , Jabbour Pascal M , Tjoumakaris Stavropoula I , Gonzalez L Fernando , Rosenwasser Robert H , Koch Walter J , Dumont Aaron S
Published: July 2021

Objectives:
Inflammation and cerebral Vascular Smooth Muscle Cell (VSMC) phenotypic modulation appear to be involved in cerebral aneurysm formation. Tumor Necrosis Factor-alpha (TNF-α) is found in increased quantity in ruptured and un-ruptured cerebral aneurysms. Our previous work demonstrates that TNF-α potently induces cerebral VSMC phenotypic modulation . We explored the underlying mechanisms involved in these changes.

Methods:
Rat cerebral VSMCs were treated with TNF-alpha at 50 ng/ml (based on preliminary data) for 2, 6 and 24 hours. Cells at 80-90% confluence were fixed with formaldehyde. DNA was sheared using sonication. Chromatin immune-precipitation was performed using the following antibodies: 1) anti-KLF4 (Krupple Like Factor 4), 2) anti-HDAC2 (Histone Deacetylase 2), 3) anti-H3K9Ac (Histone 3 Lysine 9 Acetylation), 4) anti-H3K27triMe (Histone 3 Lysine 27 Tri-Methylation) and 5) anti-H4Ac (Histone 4 Acetylation). qPCR was performed with primers specific to CArG containing promoter regions of differentiation marker genes {(alpha-Actin and Myosin Heavy Chain (MHC)} and Myocardin. Results were normalized against input DNA. As part of in vivo experiments Pluronic Gel (40% w/v) containing TNF-alpha at 30-60 ug/ml was applied to rat carotid vessels for 6-8 hours. Vessels were harvested and frozen in liquid nitrogen and chromatin immune-precipitation was performed.

Results:
TNF-alpha stimulation promoted profound phenotypic modulation of cerebral VSMCs. We demonstrated an increased percent enrichment of alpha-actin, MHC and Myocardin promoters with KLF4, HDAC2, and H3K27triMe antibodies and decreased enrichment with H3K9Ac antibody. H4Ac antibody showed decreased enrichment of alpha-Actin and MHC promoter in vivo. Hence, there is evidence of direct binding of the transcription factor, KLF4, to marker gene promoter regions. Along with direct effects, KLF4 exerts indirect effects via binding with HDAC2 which deactylates H3 and H4 histones. Although, KLF4 demonstrated increased binding at 24 hours but HDAC2 did not. HDAC2 binding was more significant at early time points. These effects promote heterochromatin formation and decrease SRF binding to VSMC gene CArG box chromatin which in turn results in decreased transcription of marker genes and phenotypic modulation.

Conclusions:
We have demonstrated underlying molecular mechanisms involved in repression of cerebral VSMCs marker genes following exposure to TNF-alpha. TNF-alpha has a role in inflammation, matrix degradation and phenotypic modulation. This suggests a novel mechanism whereby epigenetic control of chromatin structure may play an important role in regulating VSMC gene expression in response to inflammatory cytokines like TNF-alpha which has implications for the pathogenesis of cerebral aneurysms.
Abstract 383: Characterization of Inflammatory Cytokine Effects on the Blood-Brain Barrier Using an in Vitro Human Brain Microvascular Endothelial Cell Model
Authors: Rochfort Keith D , Martin Fiona A , Murphy Ronan P , Cummins Philip M
Published: July 2021

Introduction:
The blood-brain barrier (BBB) represents a restrictive interface between the circulation and CNS. Within the multicellular neurovascular unit (NVU), the microvascular endothelium resides at the forefront of this dynamic barrier in direct contact with blood. Following cerebrovascular injury (e.g. ischemic stroke, trauma, infection), inflammatory cytokines are released by NVU cells (neurons, astrocytes, pericytes, endothelial cells) and circulating immune cells, often leading to elevated microvessel permeability and pathology. Much has yet to be uncovered however about how cytokines specifically impact endothelial BBB phenotype at the molecular level. In the present study, we investigated how TNF Alpha and IL-6 affect adherens/tight junction assembly using a human brain microvascular endothelial cell (HBMVEC) model
in vitro
.

Methods:
Cultured HBMVECs were exposed to either TNF Alpha or IL-6 over a range of doses (1-100 ng/ml) and times (6 and 18 hours). Cytokine levels were monitored by both standard and multiplex ELISA. Endothelial barrier phenotype was routinely monitored with respect to paracellular permeability (transwell monolayer assay) and junctional protein expression (qRT-PCR & Western blotting).

Results:
(i) Both TNF Alpha and IL6 treatments increased HBMVEC permeability in a dose- and time-dependent manner. Treatments also increased cell proliferation whilst having minimal impact on cell viability (as monitored by FACS, MTS, LDH and xCelligence assays); (ii) In parallel with permeability increases, cytokine treatments dose-/time-dependently decreased mRNA and protein levels for tight junction-associated occludin, zonnula occludens-1 and claudin-5, as well as for adherens junction-associated VE-cadherin; (iii) TNF Alpha treatment was also seen to dose-/time-dependently induce IL-6 expression and release from HBMVECs.

Conclusions:
Inflammatory cytokines (TNF Alpha and IL-6) reduce microvascular endothelial BBB integrity in-part through a mechanism involving reduced synthesis of junctional proteins. Cytokine-mediated post-translational modification of junctional proteins (e.g. occludin phosphorylation and ubiquitination) is also suspected in these events. Moreover, the TNF Alpha-stimulated production/release of IL-6 may reflect a putative mechanism through which the former cytokine elicits BBB compromise.
Abstract 388: TXNIP Knockdown Rescues Diabetes-Related Impairment in Endothelial Progenitor Cell-Mediated Angiogenesis
Authors: Dunn Louise , Simpson Philippa , Lecce Laura , Liu Richard , Levitt Joseph , Lim Patrick , Bursill Christina , Ng Martin K
Published: July 2021

Introduction:
Endothelial progenitor cell (EPC) participation in neovascularization of ischemic tissue is impaired in diabetes mellitus. Thioredoxin interacting protein (TXNIP) is strongly induced by glucose. TXNIP is overexpressed in tissues of diabetes sufferers and is associated with impaired angiogenesis. We hypothesized that gene-silencing of TXNIP in diabetic EPC would ameliorate impaired EPC angiogenic function.

Methods and results:
EPC (CD34+/CD45+/KDR+) isolated from young Type 1 diabetes patients were markedly reduced, which was associated with induction of TXNIP and reduced expression of VEGF compared to age- and sex-matched healthy controls. We further found that high glucose (15-25 mmol/L) impaired EPC angiogenic processes in vitro, including cell proliferation, migration and vascular network formation. This was associated with induction of TXNIP and a reduction in VEGF. However, EPC transfected with siRNA to TXNIP were protected from high-glucose mediated impairment of angiogenic processes and had normal VEGF production. Next, diabetic and healthy EPC were treated with TXNIP siRNA and xenotransplanted into a normoglycemic Balb/c nude mouse model of unilateral hindlimb ischemia. Xenotransplanted diabetic EPC were associated with reductions in blood flow recovery (laser Doppler perfusion imaging) and increased tissue ischemia/amputations. However, gene silencing of TXNIP in diabetic EPC restored blood flow recovery, with reductions in tissue ischemia/amputations, to levels seen in mice xenotransplanted with healthy control EPC.

Conclusion:
Hyperglycemia-mediated induction of TXNIP impairs EPC angiogenic function in vitro and in vivo. Inhibition of TXNIP in diabetic EPC rescues impaired angiogenesis, with implications for therapeutic modulation of diabetic vascular disease.
Abstract 394: E2F1 Suppresses Cardiac Neovascularization by Downregulating VEGF and PlGF Expression
Authors: Wu Min , Zhou Junlan , Cheng Min , Boriboun Chan , Biyashev Dauren , Wang Hong , Kishore Raj , Qin Gangjian
Published: July 2021

Objective:
The E2F transcription factors are best characterized for their roles in cell-cycle regulation, cell growth, and cell survival. Here we investigated the potential role of E2F1 in cardiac neovascularization.

Methods and Results:
Myocardial infarction (MI) was induced in WT and E2F1
-/-
mice. Compared to observations in WT mice, cardiac function, capillary density, and endothelial-cell (EC) proliferation were greater (P<0.01), infarct sizes were smaller (P<0.01), apoptotic ECs were less common (P<0.01); border-zone levels of vascular endothelial-cell growth factor (VEGF) (P<0.05) and placental growth factor (PlGF) (P<0.01) were higher; and border-zone p53 levels were lower (P<0.01); in E2F1
-/-
mice. Blockade of VEGF receptor 2 (VEGFR2) signaling with the selective inhibitor SU5416 or with the VEGFR2-blocking antibody DC101 abolished the differences between E2F1
-/-
mice and WT mice in cardiac function, infarct size, capillary density, EC proliferation, and EC apoptosis. Hypoxia-induced VEGF and PlGF upregulation was significantly greater in E2F1
-/-
than in WT cardiac fibroblasts, and E2F1 overexpression suppressed PlGF upregulation in both WT and p53
-/-
cells; however, VEGF upregulation was suppressed only in WT cells. E2F1 interacted with and stabilized p53 under hypoxic conditions, and both E2F1:p53 binding and the E2F1-induced suppression of VEGF promoter activity were absent in cells that expressed an N-terminally truncated E2F1 mutant.

Conclusions:
E2F1 limits cardiac neovascularization and functional recovery after MI by suppressing VEGF and PlGF upregulation through p53-dependent and -independent mechanisms, respectively.
Abstract 406: Acrolein, a Component of Tobacco Smoke and Age-Related Oxidative Stress Disrupts Structure and Function of Apolipoprotein E
Authors: Tran Tuyen , Irvine Ken , Kim Sea , Bielicki John , Narayanaswami Vasanthy
Published: July 2021

Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a critical role in regulating plasma cholesterol homeostasis and in lowering cholesterol/triglycerides level. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. ApoE mediates this function via essential lysine residues that interact with the LDLr. Our preliminary study shows that rats exposed to second hand smoke displayed oxidative modification of apoE by acrolein, a highly reactive aldehyde, and dissociation of lipoprotein-bound apoE. From the in vivo study, we hypothesize that modification of apoE by acrolein at lysine residues will alter its conformation and disrupt its ability to interact with lipids, heparin and the LDLr. The objective of this project is to study the effect of oxidative stress mediated in vitro modification on recombinant rat apoE. To accomplish our objective, we: (i) over-expressed, isolated and purified recombinant rat apoE; (ii) carried out dose-dependent chemical modification of purified apoE with acrolein followed by mass spectrometry; (iii) performed chemical denaturant-induced unfolding to obtain information on the conformation of acrolein-modified apoE; (iv) performed lipid and heparin binding assays and assessed LDLr binding capability of modified protein. Acrolein modification of apoE was confirmed by Western blot analysis using an antibody specific for acrolein-modified lysines. Mass spectrometric analysis indicates modification by acrolein leading to FDP-lysine formation. Chemical denaturant-induced unfolding studies revealed that the overall fold of acrolein-modified apoE was impaired. Modified apoE also demonstrated a decrease in binding affinity for heparin and decreased lipid binding ability. Lastly, the LDLr binding ability of acrolein-modified apoE was significantly disrupted. Overall, we conclude that acrolein disrupts the structural and functional integrity of apoE, which is likely to affect its role in maintaining plasma cholesterol homeostasis. Our data provide a molecular basis for the potential role of oxidative stress (due to environmental factors or aging) mediated modification of apoE in altering lipoprotein metabolism, with direct implications in cardiovascular disease.
Abstract 410: 3-Dimensional Conformation of HDL ApoA-I Generated by ABCA1
Authors: Sorci-Thomas Mary , Fulp Brian , Shah Dharika , Jerome Gray W , Zabalawi Manal , Thomas Michael
Published: July 2021

This report describes the 3-dimensional conformation of nascent HDL apoA-I particles generated via ATP binding cassette transporter A1 (ABCA1). Three main types of particles were consistently obtained from; overexpressing HEK293 cells, bone marrow derived mouse macrophages and HepG2 cells. Particles were separated by FPLC and sized using gel electrophoresis and electron microscopy. Total lipid compositions were obtained from HPLC and mass spectrometry and protein conformation by chemical cross-linking and mass spectrometry (CCLMS). The first particle type corresponded to a lipid-rich nascent HDL (nHDL) between 13-10 nm in diameter, containing 3 molecules of apoA-I, with 45% of their total lipid as cholesterol, 35% as phosphatidylcholine and 20% as sphingomyelin with a total of ∼250 molecules of lipid per particle. The second type corresponded to 7-8 nm diameter particle containing 2 molecules of apoA-I and 26 total lipid molecules, while the third particle type was a monomer of apoA-I with 3 lipid molecules. Three-dimensional information was derived from the 13-10 nm nHDL particles by first crosslinking these purified particles and separating the monomeric, dimeric and trimeric apoA-I bands by SDS-PAGE. These CCL products were subjected to in-gel trypsin digest and LC-MS/MS. Reconstructed MS total-ion chromatograms were evaluated for CCL peptides with identity verified by MS/MS sequencing. Using several lysine-specific crosslinkers with arm lengths from 12Å to 36Å we compiled the distribution of CCLs. The same analysis was performed for the 7-8 nm nHDL that only yielded dimmer and monomer. Several intramolecular, interpeptide and intermolecular crosslinks were identified for each type category of nHDL. CCLs from the 7-8 nm nHDL suggested that the 2 apoA-I molecules assumed antiparallel counter-rotating helices. Tighter bending of the double belt was required to cover the 26 lipid molecules. Crosslinking of the 13-10 nm nHDL suggested that all three molecules were antiparallel to one another. The conformation that fit the CCLs was a belt with the middle apoA-I molecule antiparallel to the two outer apoA-I molecules. Overall, these data suggest that apoA-I adapts its conformation to encompass available lipid, but with larger amounts of lipid a belt conformation is preferred.
Abstract 412: Saturated Fat Ingestion Impairs HDL Function in Diabetes
Authors: Yassine Hussein N , Schall Christina , Sabesitoofyani Arash , Stump Craig S , Jackson Angela M , Borchers Christoph H , Reaven Peter , Lau Serrine S
Published: July 2021

Recent studies have suggested that HDL cholesterol efflux may be an important determinant of atherosclerosis and CVD. We hypothesized that environmental factors such as high saturated fat diets may impair HDL cholesterol efflux, and this may be particularly relevant in conditions associated with HDL dysfunction such as uncontrolled type 2 diabetes (T2DM). We studied the effect of an acute ingestion of saturated fat on the HDL function and composition in diabetes. To test this hypothesis, we recruited 4 non-diabetic controls and 6 subjects with poorly controlled (T2DM). Subjects ingested a cup of whip cream and plasma was obtained fasting and 4 hours after the ingestion. We measured cholesterol efflux on ABCA1 transfected BHK cells from HDL isolated by ultracentrifugation. Plasma prebeta1 HDL was assayed by ELISA. To determine how changes in efflux might relate to HDL composition, we developed an HDL proteomics assay using Multiple Reaction Monitoring (MRM) with 60 HDL protein targets using standard isotope peptides on isolated HDL samples. Plasma HDL-C and ApoA1 did not change significantly after the ingestion of the whip cream. The HDL efflux capacity increased in controls by two fold (P =0.05) while it decreased in T2DM by 0.6 (P=0.035). The change in efflux correlated with the change of prebeta1 HDL in controls (R2=0.88, P=0.04) and in T2DM (R2=0.86, P=0.007). Using MRM, we found that Apolipoprotein E (ApoE) and complement 4 beta chain (C4b) protein content that also correlated with the change in efflux. ApoE decreased in controls (Means ±SE area ratio, Pre /Post) (94 ±11/ 70 ±1, P=0.06), and increased in T2DM (58 ±10/ 87 ±20 P=0.06) while C4b was detected after fat ingestion (0/ 10±4, P=0.05) in controls but was not detectable after the fat ingestion in T2DM (2.7± 1.1/ 0, P=0.07). We conclude HDL from patients with poorly controlled T2DM has an impaired capacity to efflux cholesterol after saturated fat ingestion, possibly through mechanisms that involve ApoE and complement C4.
Abstract 425: Coronary Artery Disease in Patients with Premature Atherosclerotic Peripheral Artery Disease
Authors: Rana Hamza , Andrews Jeanette S , Hansen Kimberley J , Levy Pavel J
Published: July 2021

Objective:
Premature atherosclerotic peripheral artery disease (PAD) is being diagnosed with increasing frequency. Little is known about concomitant coronary artery disease (CAD) in patients with premature PAD. This study examines prevalence, associated clinical characteristics, and predictors of concomitant CAD in young PAD patients.

Methods:
We studied patients with severe atherosclerotic PAD <55 years of age (mean 49.36±6.45 yrs) treated at a single academic Vascular center between 1998 and 2010. Data was collected at the time of initial evaluation. CAD was defined by documented acute coronary syndrome; and/or prior coronary revascularizations. Associations with concomitant CAD were evaluated univariately using chi-square tests for categorical characteristics or t-tests for continuous characteristics, and using multivariable logistic regression.

Results:
Total of 561 patients (46% female, 20% Black) were analyzed. Mean age at diagnosis was 46.64±6.86 years. Risk factors included smoking (97%), hyperlipidemia (67%), hypertension (64%), family history of premature CAD (47%), and diabetes (25%). Aortoiliac disease was present in 77% of patients; 36% were disabled. Overall, 174 (31%) patients had clinical CAD. Patients with premature CAD were less likely to be Blacks (p=0.004), had greater frequency of hypertension, hyperlipidemia, diabetes, family history of premature CAD, polyvascular disease (i.e. cerebral vascular disease [CeVD]) (p<.001 for each), renovascular disease (p=.016) and mesenteric disease (p=.012). Multivariable logistic regression modeling showed higher odds of concomitant CAD for patients with hyperlipidemia (OR 4.71; 95 CI 2.82-7.85; p<.0001), diabetes (OR 2.11; 95% CI 1.28-3.47; p<0.01), family history of premature CAD OR 2.00; 95% CI 1.27-3.14;p<0.01) CeVD (OR 2.15; 95% CI 1.34-3.48;p<0.01), mesenteric vascular disease (OR 2.70;95% CI 1.19-6.14; p=.02). One pack year in smoking increase had 1.01 times odds of concomitant CAD (95% CI 1.001-1.018; p=.02).

Conclusions:
Clinical CAD was prevalent in 1/3 of patients with premature PAD, and those with premature CAD were less likely to be Black. Among patients with premature PAD, higher odds of concomitant clinical CAD were associated with presence of hyperlipidemia, diabetes, family history of premature CAD, polyvascular disease.
Abstract 432: MEK/ERK Inhibition Corrects the Defect in VLDL Assembly and Secretion in HepG2 Cells via Activation of Cell Death--Inducing DFFA-Like Effector B (Cide B), ApoCIII and Lipin-1
Authors: Qiu Wei , Zhang Jing , Wang Huajin , Carman George M , Sundaram Meenakshi , Yao Zemin , Seidah Nabil G , Adeli Khosrow
Published: July 2021

Suppression of MEK/ERK signalling in HepG2 cells promotes the assembly and secretion of VLDL-sized apoB100-containing lipoproteins via an unknown mechanism (Tsai et al,
Arterioscler Thromb Vasc Biol
2007; 27:211-218). In this report, we investigated the mechanisms underlying the profound stimulatory effects of MEK/ERK inhibition on VLDL assembly and secretion in HepG2 cells. Following MEK/ERK inhibition with the inhibitor U0126, the mRNA levels of lipin-1α, -1β, apoCIII and CideB were significantly increased by 1.22, 1.25, 3.6, and 1.9 fold, respectively, all key factors involved in intracellular lipid metabolism. Gain-of-function studies were then used to mimic the effects seen with MEK/ERK inhibition. Upon transient overexpression of lipin-1α, and-1β, secretion of VLDL-apoB was increased by 2.07 and 2.23 fold, respectively. Similarly, overexperession of wild type apoCIII (C3WT) significantly increased the secretion of VLDL-apoB. By contrast, loss-of-function approaches using RNA interference showed that knockdown of apoCIII or CideB significantly decreased secretion of VLDL-apoB by 22% or 69%, respectively. Overexpression of MTP could not block the inhibitory effects of CideB knockdown on VLDL secretion in MEK/ERK-inhibited HepG2 cells. Taken together, our data suggest that increased expression of a number of key protein factors, particularly CideB, may mediate the profound stimulatory effect of MEK/ERK inhibition on VLDL assembly and secretion. Enhanced expression of CideB, apo CIII, and lipin-1 may alter and promote availability of TG pools available for VLDL assembly in a process independent of MTP.
Abstract 434: A Novel 5-Step Model for the Mechanism of ABCA1-Mediated Nascent HDL Biogenesis
Authors: Wang Shuhui , Brubaker Gregory , Gulshan Kailash , Smith Jonathan D
Published: July 2021

Objective—
Lipid-poor apoA-I acts as an acceptor for cell cholesterol and phospholipids via the cell membrane protein ABCA1, generating nascent HDL. However, the mechanism of this process is not understood at the molecular level.

Methods and Results—
We propose a novel five-step model of nascent HDL biogenesis: ABCA1 remodeling of the plasma membrane lipids exposing phosphatidylserine and apoA-I binding to ABCA1 are the first two independent steps; third, ABCA1 facilitates apoA-I partial unfolding; forth, partially unfolded apoA-I inserts into the modified plasma membrane resulting in apoA-I lipidation; and fifth, nascent HDL is released from the cell. We created fluorescent apoA-I indicators that can monitor apoA-I unfolding and lipidation states. In cell free assays of reconstituted HDL (rHDL) generation from apoAI and DMPC liposomes, the fluorescent indicators demonstrated apoA-I unfolding and lipidation concurrent with rHDL formation. Next, HEK293 cells were stably transfected with different ABCA1 vectors encoding wild type (WT) and W590S and C1477R Tangier disease mutation isoforms. WT ABCA1 mediated cholesterol efflux to apoA-I (requires all steps) and sodium taurocholate (NaTC, requires only the membrane remodeling step,). Although neither mutant could efflux cholesterol efficiently to apoA-I, they were blocked at different steps. The W590S mutant bound apoAI but could not efflux cholesterol to NaTC, thus it was blocked at the membrane remodeling step. However, the C1477R mutant could not bind apoAI but could efflux cholesterol to NaTC, thus its activity was blocked at the apoAI binding step. When the lipidation indicator apoA-I was incubated with stably transfected HEK cells, we observed cell associated lipidated apoA-I in cells expressing WT ABCA1, but mostly unlipidated apoA-I was associated with the cells expressing W590S ABCA1.

Conclusion—
Our results support a novel five-step model for nascent HDL biogenesis: 1, 2) ABCA1 remodeling of the plasma membrane and apoA-I binding to ABCA1, which facilitate 3) apoA-I partial unfolding and 4) and lipidation by the remodeled membrane, followed by 5) the release of nascent HDL.
Abstract 436: Assessing Statin Therapy and Lipid Profile Goals 6 Months After Percutaneous Coronary Intervention at a Veterans Hospital
Authors: Ladani Amit , Challa Karthik , McGraw Sloane , Shivaraju Anupama , Shroff Adhir
Published: July 2021

Background:
Lipid lowering therapy is an established core measure of secondary prevention in coronary artery disease (CAD) management. The National Cholesterol Education Program - Adult treatment panel III (NCEP-ATP III) advises a minimum LDL level less than 100 mg/dL in patients with CAD and a recommended LDL less than 70 mg/dL for high risk patients. Statins remain the preferred agent for LDL reduction.

Methods:
We conducted a retrospective, observational study on all patients undergoing PCI at an urban Veterans Hospital from September 2004 to January 2011. Statin use and lipid profiles at 6 months post-PCI were compared to pre-PCI values.

Results:
A total of 933 unique patients had PCI during the study period. The mean total cholesterol decreased 18 mg/dL and the mean LDL decreased 15 mg/dL during 6 months follow-up (p< 0.001) (refer to table). The mean HDL did not differ significantly. The percent of patients at NCEP-ATP III guideline’s goal of LDL < 100 mg/dL increased from 58% pre-PCI to 74% post-PCI. Goal LDL < 70 mg/dL increased from 22 to 31% at 6 months. The use of statins increased from 69% to 89%.

Conclusion:
There were significant improvements in total cholesterol and LDL values at six months post-PCI, as well as the percentage of patients who met the NCEP-ATP III recommended goal of LDL cholesterol less than 100 mg/dL and the suggested goal of 70 mg/dL. At six months, there was an increase usage of statin therapy.
Abstract 44: Reactive Oxygen Species Mediate TLR4 MyD88 Independent Signaling in Human Hemoglobin-Associated Macrophages Through TLR4 Lipid Raft Interactions
Authors: Finn Aloke , Nakano Masataka , Polavarapu Rohini , Karmali Vinit , Saeed Omar , Narula Jagat , Kolodgie Frank , Virmani Renu
Published: July 2021

Objectives:
Experimental data indicate an important role for Toll-like Receptor 4 (TLR4) MyD88 independent signaling in upregulating Interferon β (IFN-β) production and driving atherosclerosis. We recently identified a distinct non-foam cell macrophage (M(Hb) or Hb-associated macrophage) in areas of intraplaque hemorrhage characterized by reduced reactive oxygen species (ROS) and pro-inflammatory cytokines. In this study, we investigated the role of iron and ROS in mediating TLR4 MyD88 independent signaling in these cells.

Methods and Results:
Areas rich in M(Hb) in atherosclerotic plaques demonstrated significantly reduced IFN-β expression compared to foam cell areas by immunostaining and quantitative PCR. M(Hb) did not upregulate IFN-β when exposed to ox LDL in contrast to control macrophages, a response which was inhibited in the presence of a TLR4 blocking antibody. To further investigate TLR4 responses in M(Hb), we used the TLR4 activator LPS. LPS produced significant increases in IFN-β in control macrophages but had no effect in M(Hb). This defect could be corrected by raising intracellular iron by pretreating M(Hb) with hepcidin prior to LPS treatment, suggesting redox state mediates this effect. The interaction of TLR4 with TRIF was examined by immunoprecipitation of lysates from control or M(Hb) cells treated with LPS using a TLR4 antibody and immunoblotting for TRIF. LPS treatment of control but not M(Hb) cells resulted in an increase in TRIF. Hepcidin pretreatment of M(Hb) corrected this interaction in response to LPS while differentiating monocytes in superoxide dismutase prevented it. Lastly, the interaction between lipid rafts and TLR4 was examined using FITC-cholera toxin (CTx) and a TLR4 antibody. In control cells the distribution of CTx on the plasma membrane was homogeneous and TLR4 localized to both the membrane and intracellular compartment. After LPS, a large fraction of TLR4 translocated to the plasma membrane, and colocalization of TLR4 and CTx was observed. In M(Hb) the LPS- induced translocation of TLR4 to the membrane rafts was inhibited.

Conclusion:
M(Hb) cells modulate TLR4 MyD88 independent signaling through reducing ROS which inhibits TLR4 lipid raft interactions.
Abstract 444: Angiotensinogen Influences Metabolic Rate and Metabolome in Plasma and Hepatic Tissue
Authors: Wu Congqing , Lu Hong , Cassis Lisa A , Daugherty Alan
Published: July 2021

Background and Objective:
Angiotensinogen(AGT) is the only known substrate of the renin-angiotensin system. We have reported previously that mice with whole body reduction of AGT (hypomorphic AGT mice) resists diet-induced weight gain. We have subsequently demonstrated that hepatocyte-derived AGT is primarily responsible for the observed phenotype. In order to define the potential mechanism, we determined the effects of AGT deficiency on metabolic rate in hepatocyte-specific AGT deficient mice (

Agt
hpt-/-

) and the metabolome of both plasma and liver in hypomorphic AGT mice.

Methods and Results:

Agt
hpt-/-

mice were generated by breeding AGT floxed mice with transgenic mice expressing Cre under the control of albumin promoter. Male

Agt
hpt-/-

mice and the littermates were fed a saturated fat-enriched diet (21% wt/wt milk fat) for 3 months.

Agt
hpt-/-

mice had significantly lower body weight than their littermates (30.9 ± 1.6 versus 41.1 ± 1.9 g, p < 0.01). While two groups exhibited no difference in lean mass,

Agt
hpt-/-

mice had 40% less fat mass compared to their littermates (6.2 ± 1.1 versus 11.9 ± 1.6 g, p < 0.05). This phenotype was not attributed to intestinal fat absorption. To determine metabolic rates,

Agt
hpt-/-

mice were single-housed for 5 consecutive days after 3 days of acclimation in a metabolic phenotyping system (TSE Systems, MO). The following parameters were recorded every 30 minutes: food intake, water consumption, O
2
consumption, CO
2
production and locomotion. Data were normalized to lean mass. Food intake and locomotion were not different between two groups. However, we found that

Agt
hpt-/-

mice had significantly increased O
2
consumption (by 21% at day cycle and 15% at night cycle) and CO
2
production (by 23% at day cycle and 14% at night cycle). While mice with whole body reduction of AGT exhibited a similar lean phenotype, mass spectrometric analyses of the metabolome showed that approximately one third of the 300 metabolites analyzed had significant changes in the liver and plasma in hypomorphic AGT mice when compared to their wild type littermates. These changes ranged from bile acid production to free fatty acid metabolism.

Conclusion:
Angiotensinogen contributes to metabolic rate and influences metabolome in plasma and hepatic tissue in mice.
Abstract 446: Serum Amyloid A Is Proatherogenic
Authors: Thompson Joel C , Brandewie Kyle , Nelson Christina , Wilson Patricia , Tannock Lisa
Published: July 2021

Serum amyloid A (SAA), along with high sensitivity C- Reactive Protein, is a clinical marker of atherosclerosis, a vascular inflammatory disease responsible for half a million deaths per year in the US. Interestingly, SAA is modestly but chronically elevated in several diseases such as diabetes and obesity that confer greater risk to developing atherosclerosis. Recently SAA has been shown to contribute to the progression of atherosclerosis but its role in initiation of atherosclerosis has yet to be determined. As outlined in the response to retention hypothesis of atherosclerosis, proteoglycan mediated lipoprotein retention is thought to be a key initiating step. We previously demonstrated that mice overexpressing SAA had increased vascular biglycan, a small matrix proteoglycan, which was larger and had greater LDL binding affinity than native biglycan. Here we tested the hypothesis that overexpression of SAA would lead to increased atherosclerosis. ApoE
-/-
male mice were injected with an adenovirus carrying the gene for SAA (adSAA), a null control (adnull) or saline and maintained on chow for 16 weeks. SAA was elevated immediately after injection of adSAA but returned to baseline by 10 days; there was no elevation of SAA in adnull or saline injected mice. Although the duration of elevation of SAA was short, mice injected with adSAA had significantly increased atherosclerosis at the aortic sinus (p<0.05) and en face aorta (P<0.0009) compared to controls. SAA, apoB and biglycan co-localized within atherosclerotic lesions. There was no difference in lipid levels, lipoprotein distribution, body weight or glycemic indices between groups. Thus, we now demonstrate that SAA accelerates atherosclerosis initiation, and we propose that SAA’s induction of vascular biglycan leading to increased lipoprotein retention is the mechanism.
Abstract 45: Antiatherosclerotic Effects of miR-33 Inhibition: Increased Reverse Cholesterol Transport and Alternative-Activation (M2) of Macrophages
Authors: Rayner Katey J , Sheedy Frederick J , Esau Christine , Hussain Farah N , Temel Ryan E , Parathath Saj , van Gils Janine , Rayner Alistair , Chang Aaron , Suarez Yajaira , Fernandez-Hernando Carlos , Fisher Edward A , Moore Kathryn J
Published: July 2021

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL and exploit its atheroprotective effects have remained elusive. Recent studies identified miR-33 as an intronic microRNA, located within the SREBF2 gene, that suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and plasma HDL, suggesting that antagonism of miR-33 may be atheroprotective. We hypothesized that systemic delivery of an oligonucleotide inhibitor of miR-33 would increase plasma HDL and promote reverse cholesterol transport (RCT), and therefore have a beneficial impact on atherosclerosis. To test this, we treated Ldlr
-/-
mice with established atherosclerotic plaques with anti-miR33 or a control anti-miR for 4 weeks. Treatment with anti-miR-33 increased circulating HDL levels by 37% and enhanced RCT to the plasma, liver, and feces by up to 80%. Consistent with this, anti-miR33-treated mice showed a marked reduction in plaque size and lipid content, as well as an increase in indicators of plaque stability. Laser capture microdissection of lesional CD68+ cells demonstrated that anti-miR33 oligonucleotides directly targeted the plaque macrophages, where they enhanced ABCA1 expression and cholesterol removal. Moreover, macrophages from anti-miR33-treated mice showed an enrichment in anti-inflammatory M2 markers (Arg1, Il10) and reduced expression of proinflammatory M1 markers (iNos and Tnfa). Notably, overexpression of miR-33 in pMφ in vitro decreases markers of the M2 phenotype, Arg1 and Il-4, and increases the expression of inflammatory cytokines such as Tnfa and Il-1b. In contrast, anti-miR-33 polarizes pMφ to an M2 phenotype (Arg1, Fizz1, Il-10 and Il-4), with an associated downregulation of inflammatory genes (Tnfa, Il-1b). Overall, these results indicate that anti-miR33 has multiple beneficial effects on atherosclerosis, including increasing HDL and RCT, and reducing lesional inflammation by promoting macrophage polarization to the reparative M2 state, highlighting the promise for anti-miR33 therapy for the treatment of cardiovascular disease.
Abstract 469: Vitamin D Deficiency Potentiates Restenosis Following Coronary Angioplasty in Hypercholesterolemic Swine
Authors: Gupta Gaurav K , Agrawal Tanupriya , Del Core Michael G , Hunter William J , Agrawal Devendra K
Published: July 2021

Introduction:
Vitamin D is a sectosteroid that functions through vitamin D receptor (VDR), a transcription factor, which regulates various downstream signaling pathways and controls the transcription of many targets genes. Vitamin D deficiency is associated increased risk of incident congestive heart failure, myocardial infarction, and cardiovascular disease mortality. However, most of the studies which identified an association between lower vitamin D intake or lower 25-hydroxyvitamin D and increased risk of cardiovascular disease are observational or cross-sectional studies. There has been no careful study evaluating the effect of vitamin D supplementation on coronary artery disease outcome following coronary intervention.

Objective:
The purpose of this study was to examine the effect of vitamin D deficiency and vitamin D supplementation on coronary artery restenosis following balloon angioplasty in hypercholesterolemic Yucatan microswine.

Methods:
Twelve female microswine were fed vitamin D-deficient or vitamin D-sufficient high cholesterol diet. At six months, animals underwent coronary angioplasty. Following coronary intervention swine in the vitamin D-sufficient high cholesterol diet group received supplementation of 1,000 IU or 3,000 IU of vitamin D3. Six months later, angiogram was performed followed by optical coherence tomography to monitor the development of intimal hyperplasia and restenosis. At the end of 12 months animals were euthanized, coronary arteries were harvested and morphological and histological studies were performed.

Results:
Findings from the optical coherence tomography and histomorphometric studies showed a significant decrease in neointimal hyperplasia, increase in in-segment lumen diameter, and decrease in the intima-media ratio in coronary arteries following balloon angioplasty in swine with vitamin D supplementation compared to the animals with vitamin D-deficient status. In the in-vitro studies, calcitriol inhibited proliferation of coronary artery smooth muscle cells.

Conclusion:
These data suggest that vitamin D deficiency increases intimal hyperplasia and restenosis following coronary balloon angioplasty in hypercholesterolemic swine. Since vitamin D inhibits smooth muscle cell proliferation, supplementation of non-secosteroidal VDR ligands prior to coronary intervention could help in preventing the neointimal formation and restenosis.