bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 211: Long-Term Mortality in Diabetic Patients with Non-Obstructive Coronary Disease Undergoing Coronary Angiography/Cardiac Catheterization
Authors: Bottinor Wendy , Chawla Raveen , Patel Kalpesh , Turlington Jeremy S , Danyi Peter , Jovin Ion S
Published: June 2021

Background:
Diabetes mellitus is associated with poorer outcomes in patients with cardiovascular disease and patients with diabetes are at increased risk of death. Several studies with coronary computed tomography have shown that, for equal numbers of severely diseased coronary arteries, the outcomes of patients with diabetes is worse compared to patients without diabetes. However, it is unclear if the outcomes of diabetic patients with non-obstructive coronary disease is different than the outcomes of patients without diabetes and non-obstructive disease.

Methods and Results:
We studied 901 consecutive, predominantly (98%) male patients undergoing coronary angiography/cardiac catheterization at a veterans’ administration medical center who had normal coronaries or non-obstructive coronary artery disease and we assessed the association between diabetic status and mortality. The mean age of the patients was 64.4 years. One third of the patients (n=311, 34.5%) were diabetic. Diabetic patients were the same age, and had the same baseline creatinine, but were more often hypertensive, and were more often on statins, angiotensin receptor blockers, angiotensin conversion enzyme inhibitors, calcium channel blockers and aspirin therapy compared to the non-diabetic patients. Contrast-induced nephropathy (CIN) occurred in 8 (2.57%) diabetic patients and in 11 (1.86%) non-diabetic patients at 72 hours after the procedure (odds ratio [OR] 1.39; 95% confidence interval [CI] 0.55 - 3.49; P= 0.48). At 3 months, renal dysfunction was seen in 34 (10.93%) diabetic patients versus 32 (5.42%) of the non-diabetic group (OR 2.14, CI 1.29 - 3.54; P=0.0032). After a median follow up of 67 months 59 patients (18.97 %) of the diabetic group had died versus 72 (12.20 %) of the non-diabetic group (OR 1.68, CI 1.16 - 2.45; P=0.0007). On multivariate analysis, after adjustment for age, comorbidities, medical therapy, baseline creatinine and the incidence of CIN, the presence of diabetes was significantly associated with the 5 year mortality (OR= 2.10, 95% CI 1.44 - 3.85, P=0.0006).

Conclusion:
In this cohort of patients undergoing coronary angiography who had normal coronaries or non-obstructive coronary disease, diabetes was associated with an increased mortality.
Abstract 212: Epigenetically Altered TLR4 Expression May Contribute to Increased Inflammation and Impaired Wound Healing in a Murine Model of Diabetes
Authors: Kimball Andrew , Joshi Amrita , Schaller Matthew , Carson Beau , Singer Kanakadurga , Bermick Jennifer , Ramani Shreyas , Henke Peter , Kunkel Steven , Gallagher Katherine
Published: June 2021

Background:
Wound healing is impaired in diabetes due to dysregulated inflammation. Previous studies by our group and others have demonstrated that inflammatory mediators are significantly increased in diabetic wounds, however, the mechanisms behind this hyper-inflammatory response remain largely unknown. As there is known to be high levels of endotoxin in type 2 diabetic (T2D) wounds, we aimed to assess the role of high fat diet (HFD) on Toll-Like Receptor 4 (TLR4) expression. We hypothesized that HFD/T2D would alter histone methylation, leading to increased receptor expression and enhanced inflammation.

Methods:
C57/BL6 mice were subjected to either normal or HFD for 12-16 weeks to induce the diet-induced obese (DIO) model of physiologic T2D. 4mm hindlimb wounds were created and healing was monitored daily using NIH ImageJ software. Bone marrow-derived macrophages (BMDM) were cultured and TLR4 expression measured by qPCR. Myeloid TLR4 protein expression was assessed in peripheral blood and wound cell isolates by analytical flow cytometry. Macrophages (NK1.1-/Ly6G-/CD11b+) were isolated using magnetic sorting and inflammatory cytokine expression determined with qPCR and bioplex. Chromatin Immunoprecipitation (ChIP) analysis at the NF-kB binding site on the TLR4 promoter was performed using antibodies to trimethylated Histone 3 Lysine 4 (H3K4me3). (N=~5 mice per group for all studies).

Results:
DIO mice demonstrated significantly delayed wound healing as compared to littermate controls and BMDM isolated from these mice revealed increased TLR4 gene expression. Correspondingly, peripheral blood and wound monocyte/macrophages showed increased TLR4 receptor levels by flow cytometry. ChIP analysis at the TLR4 promoter revealed significantly increased H3K4me3, a gene activating mark, in the HFD BMDM. Finally, macrophage wound isolates from DIO mice had elevated levels of inflammatory gene expression and cytokines.

Conclusion:
Enhanced H3K4me3 at the TLR4 promoter in diabetic macrophages may increase receptor expression and contribute to the hyper-inflammatory response seen in T2D. Further mechanistic studies are needed in order to examine the role of TLR4 in wound healing.
Abstract 215: Fenugreek Seeds Inhibit Hepatic VLDL Overproduction by Attenuating Metabolic Inflammatory Stress
Authors: Shen Jing , Pan Kaichao , Santa Dipak , Su Qiaozhu
Published: June 2021

Fenugreek (
Trigonella foenum-gracecum L
), an annual legume, has been well known for its medicinal properties in treating metabolic diseases. It is a new crop in Nebraska. Its seed contains a number of health-enhancing bioactive compounds, including diosgenin, galactomannan, and 4-hydroxyisoleucine. These compounds synergistically exert multiple health effects on anti-obesity and type 2 diabetes. In this study, we characterized the underlying molecular mechanisms of fenugreek seeds in anti-hyperlipidemia and anti-hyperglycemia. Using a genetic induced hyperlipidemia and a high fat diet (HFD) induced insulin resistance mouse models, we found that a diet rich in fenugreek seeds (2%) was able to lower hypertriglyceridemia induced by depletion of the cyclic AMP responsive element H (CREBH). This phenotype was accompanied by reduced levels of plasma apolipoprotein B100 (apoB) and inflammatory cytokine TNFα. Fenugreek seed feeding inhibited biosynthesis of apoB, and the assembly and secretion of very low density lipoprotein (VLDL) in the liver of CREBH-null mice. Fenugreek seed feeding also induced expression of mitochondrial fatty acid β-oxidation genes, such as PPARα and CPT-1α. Treatment with fenugreek seeds also attenuated ER stress and metabolic inflammation in CREBH-null hepatocytes, indicated by less expression of GRP78 protein, reduced phosphorylation of eIF2α and JNK. Furthermore, fenugreek seed feeding improved insulin sensitivity in a HFD induced insulin resistance mouse model as evidenced by the lower blood glucose levels in a glucose tolerance test and an insulin tolerance test compared to the paired fed-untreated control mice. Fenugreek seeds exerted anti-diabetic effect by enhancing glucose uptake in adipose tissue and liver. Taken together, our study provided new mechanistic insight into the triacylglycerol lowering properties and anti-diabetic nature of fenugreek seeds. This suggests therapeutic potential of fenugreek seed against metabolic disorders and may increase value of Nebraska-grown fenugreek.
Abstract 218: Endothelial Fc gamma Receptor IIB Activation Blunts Insulin Transport to Skeletal Muscle to Cause Insulin Resistance in Mice
Authors: Tanigaki Keiji
Published: June 2021

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance, and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. Having determined that FcγRIIB is expressed in endothelium, here we investigated how endothelial FcγRIIB influences glucose homeostasis. Floxed FcγRIIB mice were crossed with VECadherin-driven Cre recombinase and CRP transgenic mice to yield mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated skeletal muscle insulin delivery. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and endothelial NO synthase (eNOS) antagonism. Furthermore, in knock-in mice harboring constitutively-active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin transport to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin transport has been identified that may contribute to type 2 diabetes pathogenesis.
Abstract 223: Optimization of Apolipoprotein A-1 Mimetic Peptides for Lipid Binding and Lecithin-cholesterol Acyltransferase Activation
Authors: Acevedo-Mariani Frances M , Kang Jukyung , Yuan Wenmin , Pogozheva Irina , Schwendeman Anna
Published: June 2021

Introduction:
Apolipoprotein A-I (apoA-I) mimetic peptides present an exciting avenue in the clinical development of synthetic HDL (sHDL), offering a more feasible and economical approach in sHDL production. One such example is ETC-642, or 22A—a 22 amino acid ApoA-I mimetic peptide tested in single and multiple dose clinical trials. The goal of this study is to understand how specific amino acid substitutions affect physico-chemical properties of 22-mer peptides and correlate them with the ability to form stable nano-sized sHDL particles and promote LCAT activity.

Methods/Results:
For this study, we used the 22A peptide (PVLDLFNELLNELLEALKQKLK or ETC-642) and designed 10 variants comprised of a single amino acid substitution, denoted 22A-1-10. We used computational programs FMAP (Folding of Membrane-Associated Peptides), PPM2.0 and FRAMEWORK to select peptides that are predicted to form α-helices, have good membrane penetration (>7.8 Å) with low transfer energy to membranes (ΔG
transfer
<-16.1 kcal/mol), and form stable α-helices (ΔG
helix
<-23.5 kcal/mol) with strong intermolecular LCAT interactions. The calculated ΔG
helix
varied from -18.4 to -24.4 kcal/mol, and G
transfer
varied from -11.1 to -17.2 kcal/mol for the synthesized peptides. The percent peptide α-helicity in lipid bound form was measured by circular dichroism spectroscopy and was found to vary dramatically between 19 and 82%. The lipid solubilization kinetics were determined using UV absorbance to measure turbidity. The particle size and stability of resulting HDL particles were measured by dynamic light scattering and gel permeation chromatography. The peptides with low predicted energy of lipid binding exhibited high helicity and formed stable HDL particles with sizes less than 20 nm. The HDLs formed from apoA-I peptides with high helicity were also able to activate LCAT and facilitate sterol esterification with a K
cat
of 5 to 30 min
-1
.

Conclusions:
Our data shows that that minimalistic changes in primary structure of ApoA-1 peptide can lead significant changes in physico-chemical properties. Peptides with degree of α-helicity exhibited superior lipid binding and formed stable HDL particles of small size able to interact with LCAT.
Abstract 225: Anti-atherosclerotic Effects of Plaque Macrophage Targetable Nanoparticle-mediated PPAR-γ Agonist Delivery in Atherogenic Mice
Authors: Choi Jah Yeon , Ryu Jiheun , Song Joon Woo , Jeon Joo Hee , Kim Hyun Jung , Oh Dong Joo , Oh Wang-Yuhl , Gweon DaeGab , Yoo Hongki , Park Kyeongsoon , Kim Jin Won
Published: June 2021

Introduction:
Atherosclerosis is a process of lipid accumulation and inflammation. In particular, plaque macrophages are associated with fibrous cap destabilization and rupture. We hypothesized that the specific delivery of a PPAR-γ agonist to inflamed plaques via nanoprobe (NP) targeting macrophage mannose receptors could reduce plaque burden and inflammation.

Methods and Results:
Macrophage mannose receptor targetable nanoprobe (MMR-NP) was fabricated by chemically conjugating thiolated glycol chitosan with mannose-PEG-maleimide, followed by incorporating PPAR-γ agonist (lobeglitazone) into this nanoscale delivery system (MMR-Lobe). For in vivo monitoring of therapeutic response, near-infrared fluorescence NP was prepared by conjugation of Cy5.5 (ext/emi 675/694 nm) with MMR-NP. In cellular uptake study, the MMR-NP showed high affinity to mannose receptors on macrophages. MMR-Lobe attenuated LPS-induced inflammatory cytokines such as TNF-α, IL-6, and MMP-9 in RAW264.7 cells. Additionally, MMR-Lobe increased expression of ABCA1, ABCG1 and LXR-α, known as PPAR-γ regulator genes involved in cholesterol efflux, in RAW264.7 cells. Using a customized high-resolution intravital fluorescence microscope, the in vivo serial imaging of carotid atheroma in apoE-/- mice injected with MMR-Lobe (7 mg/kg, twice weekly for 4 weeks) revealed a significant decrease in plaque burden and inflammation (Figure) as compared to baseline, or non-treated controls, or p.o. Lobe treated mice (p<0.01 respectively, 6 mice in each group). En face imaging, FM, and immunostainings corroborated the in vivo findings.

Conclusions:
MMR-Lobe was able to selectively target atheroma macrophages, and effectively reduce both plaque burden and inflammation as assessed by serial intravital optical imaging. We suggest that nanoprobe-mediated PPAR-γ agonist delivery targeting plaque macrophages holds a promising theranostic approach for high-risk atheromata.
Abstract 229: Correlation of
in vitro
Studies and Human Drug Interaction Studies with Gemcabene
Authors: McShane Margaret , Radulovic Louis , Bisgaier Charles L
Published: June 2021

Background:
Gemcabene is a novel lipid-regulating compound being developed as an adjunct to diet and statin therapy for dyslipidemia treatment. Patients with dyslipidemia typically take many medications often including statins and it is essential to understand potential risk of drug-drug interactions (DDI) to minimize the risk of adverse drug reactions. In the best circumstances, drugs entering the market will provide metabolic or transport pathways that do not interfere with commonly co-administered drugs. The current studies provide the analysis of potential drug interactions with gemcabene both
in vitro
and
in vivo
.

Methods:
Caco-2 cells were used to assess the potential P-gp substrate and inhibitor interaction and the major drug-metabolizing CYP450 isozymes and FMO-3 were used to assess the potential CYP450 and FM0-3 metabolism interaction. The results from the
in vitro
P-gp and CYP450 studies was correlated with the results of three DDI clinical studies with digoxin, atorvastatin and simvastatin.

Results:
In an open-label, multiple-dose study in 12 healthy subjects, gemcabene (900 mg) did not significantly affect the exposure (Cmax and AUC
0-24
) of digoxin (0.25 mg). Specifically, the 90% confidence interval for digoxin AUC
(0-24)
ratios were within the 80% to 125% range, thus confirming the
in vitro
results of no DDI with a P-gp substrate. In two open-label, multiple-dose studies in healthy volunteers, gemcabene (900 mg) did not significantly affect the exposure (Cmax and AUC
0-24
) of atorvastatin (80 mg) or simvastatin (80 mg) thus confirming the
in vitro
results of no DDI with CYP450 (see Figure below).

Conclusion:
These results suggest gemcabene is unlikely to elicit a metabolic (i.e., CYP450 or FMO3) or P-gp-mediated drug interaction. Gemcabene (900 mg) was well-tolerated in combination with highest dose of atorvastatin and simvastatin.

Clinical Implications:
Understanding potential for drug interactions minimizes the risk of adverse drug reactions.
Abstract 236: Identifying Factors Influencing Low Density Lipoprotein Lowering Effect of Ezetimibe in Japanese Subject
Authors: Yamaguchi Satoshi , Arasaki Osamu
Published: June 2021

Background:
Ezetimibe inhibits cholesterol absorption at the small intestine brush border. Ezetimibe lowers low-density lipoprotein (LDL) level. In acute coronary syndrome patients, ezetimibe reduces risk of myocardial infarction and stroke as a secondary prevention. Japanese food has lower fat content than American and European foods. In Japanese individuals, whether ezetimibe has an effect on lowering LDL levels is not well understood. In a clinical setting, the LDL-lowering effect varies slightly. To prevent cardiac events, patients with high sensitivity to ezetimibe need to be identified. The factors influencing the LDL-lowering effect of ezetimibe have not been identified. We investigated the factors that affect ezetimibe sensitivity in Japanese patients.

Methods:
Our study design was a single center retrospective observational study. The inclusion criteria were ezetimibe naive subjects and patients being administered ezetimibe. Primary outcome was the decrease in the rate of LDL at the 365 days, as calculated by the reduced LDL level divided by the baseline level. The secondary outcome was multi-linear regression for the decreasing rate of LDL.

Results:
Fifty-two patients were analyzed. The primary outcome, the decreasing rate of LDL, was -18±17%. LDL decreased significantly analyzed by Wilcoxon signed test (P <0.0001)(Figure1). The positive predictive factors were age, statin level, and baseline LDL level. Diabetes mellitus does not influence the LDL-lowering effect of ezetimibe.

Conclusion:
Ezetimibe lowers LDL level in Japanese patients. Elderly individuals and subjects with higher LDL levels are sensitive to ezetimibe.
Abstract 237: Evaluating CETP Inhibition in Context: CETP Inhibition Modifies High-density Lipoprotein Composition in CETP-expressing Transgenic Mice Differently Depending on Diet
Authors: Zhu Lin , Emfinger Christopher , Palmisano Brian , Stafford John M
Published: June 2021

CETP inhibition has been used as a strategy to raise HDL cholesterol, but trials with two CETP inhibitors torcetrapib and dalcetrapib failed to prevent cardiovascular events for high-risk individuals. Of note, approximately 2/3 of patients in these clinical trials were obese. Obesity and fatty liver are known to significantly impact aspects of liver cholesterol metabolism and HDL biology. We propose that high-fat diet feeding (HFD) might adversely impact the effects of CETP inhibition on HDL composition and function. Mice naturally lack CETP expression, so we used CETP transgenic mice to evaluate the effects of administering the CETP inhibitor torcetrapib on HDL composition in chow- and HFD-fed CETP-transgenic mice. Because of the very short half-life of torcetrapib we used acute intravenous infusion of torcetrapib. In both diet groups torcetrapib infusion suppressed plasma CETP activity and raised HDL cholesterol content. In chow-fed CETP mice we found that torcetrapib resulted in changes in HDL composition that would be predicted to be favorable, including increases in apoA1, apoM and apoE protein levels for 2.5-fold (
P
<0.01), 1.8-fold (
P
<0.05) to 2-fold (
P
<0.05), respectively. By contrast, in HFD-fed CETP mice those effects of torcetrapib were diminished, torcetrapib had no effect on apoA1, apoM or apoE. In addition, the acute phase protein SAA1 in HDL was increased significantly by torcetrapib only in HFD-fed mice. Our results show that the effect of pharmacologic CETP inhibition on HDL composition is context-specific to the diet. These findings may be helpful to understanding why CETP inhibition, while beneficial in early animal trials, failed to reduce cardiovascular events in clinical trials where most individuals were obese.
Abstract 252: Interleukin-27 Signaling Regulates Myeloid Cells Activation in Atherosclerosis
Authors: Peshkova Iuliia , Fatkhullina Aliia , Koltsova Ekaterina
Published: June 2021

Atherosclerosis is a lipid-driven inflammatory disease characterized by the progressive plaque growth in the vessels. Cytokines are important mediators of inflammation and atherosclerosis. While pro-inflammatory cytokines were extensively investigated, little is known about the role of anti-inflammatory cytokines as to their ability to control vascular inflammation. We tested whether immunoregulatory IL-27R signaling is important to control inflammation in mouse models of atherosclerosis. We found that atherosclerosis-prone mice with hematopoietic deficiency of IL-27R (

Ldlr
-/-

mice reconstituted with bone marrow from
Il27ra
-/-
) or global deficiency (

Il27ra
-/-

x

Apoe
-/-
)

developed significantly larger atherosclerotic lesions compared to controls. Atherosclerotic lesions in IL-27R deficient mice contained more CD45
+
leukocytes and CD4
+
T cells, which produced pro-atherogenic cytokines IL-17A and TNF-α. These cytokines normally suppressed by IL-27, regulated the expression of CCL2 and other chemokines, which in turn led to accumulation of myeloid CD11b
+
and CD11c
+
cells in atherosclerotic aortas. Using two-photon microscopy, we found enhanced interactions between antigen presenting cells and T cells in the aortas of IL-27R deficient mice accompanied by enhanced CD4 T cell proliferation. Moreover, macrophages in

Il27ra
-/-

aortas also demonstrated enhanced ability to produce pro-inflammatory cytokines, including IL-1. The blockade of IL-1R signaling, however, strongly suppressed atherosclerosis progression in IL-27R deficient but not control mice, suggesting an important role of IL-27 in the regulation of IL-1 production in atherosclerosis. Overall, our data demonstrate that IL-27R signaling in atherosclerosis is required to control function of antigen presenting cells modulating subsequent T cell activation in the aortas. Moreover, it controls macrophage activation and pro-inflammatory myeloid cell-derived cytokine production. These mechanisms altogether curb pathogenic T cell lineage differentiation and, thus, atherosclerosis, suggesting potent anti-atherogenic role of IL-27.
Abstract 253: Di-n-butyl Phthalate: An Indoor Pollutant Induces Murine Macrophages Oxidative Stress and Inflammation, a Potential Atheropathogenesis Modulator
Authors: Mazhar Zahra , Alsayrafi Dhuha , Wu Tong , Garelnabi Mahdi
Published: June 2021

Di-n-butyl phthalate (DBP) is used in air fresheners. It is a colorless oily compound which also used for manufacturing bendable plastics. DBP can cause low acute or chronic toxicity; however, the effect of DBP on humans in the form of air fresheners is not well studied. The effect of DBP on the human cardiovascular system has not been studied. Macrophages are involved in atherosclerosis progression and development; murine macrophages (RAW 264.7) were used for this study. The macrophages were treated with 10uM, 20uM and 50uM DBP for 6 hours, 12 hours and 24 hours. Genes involved in inflammation and antioxidant activity like TNF-a, MCP-1, VCAM-1, NF-kB, PON1, SOCS were analyzed after treatment. macrophages showed statistically significant increases in TNF-alpha expression (p≤ 0.05) after 24 hour treatment with DBP. The expression of NF-kB showed a significant increase in response to DBP treatment (p≤ 0.05) at 24 hours. MCP-1 and VCAM-1 gene expressions were also upregulated after exposure to DBP. Interestingly, catalase gene expression was upregulated in all treatments after 24 hours of exposure however PON-1 gene expression showed differential upregulation responses. Our data clearly suggest that DBP induces inflammation in macrophages. PON1 and catalase upregulated gene expression indicative of a compensatory response to oxidative stress associated with the treatment. Oxidative stress and inflammation mediated macrophages responses strongly linked to atherosclerosis pathogenesis.
Abstract 259: Genetic Inactivation of Sirt3 Does Not Alter Endothelial Function or Vascular Compliance in Hypercholesterolemic Mice
Authors: Roos Carolyn M , Smith Leslie A , Zhang Bin , Ribeiro Mauricio S , Miller Jordan D
Published: June 2021

Endothelial dysfunction and arterial stiffening major risk factors associated with progression of atherosclerosis and increased risk of acute coronary events. Recent data suggest sirtuins may play a role in protecting against a variety age-related diseases, and we recently reported that sirtuins are reduced in the aging vasculature. The roles of SIRT3 (a deacetylase that is largely restricted to the mitochondria) in the regulation of vasomotor function and arterial stiffness, however, remains poorly understood. Therefore, we hypothesized that genetic inactivation of SIRT3 in hypercholesterolemic mice will impair endothelial function and reduce vascular compliance compared to hypercholesterolemic mice with normal levels of SIRT3. We used Ldlr
-/-
/ApoB
100/100
(LA) mice that were either wild-type for SIRT3 (LA-SIRT3
+/+
) or null for SIRT3 (LA-SIRT3
-/-
) and fed a western diet for 12 months. At time of sacrifice, aorta was removed for isolated organ chamber baths to measure changes in endothelium-dependent relaxation to acetylcholine (ACH), and carotid arteries were harvested to assess changes in vascular compliance using a pressurized cannulation system. Surprisingly, losses of SIRT3 did not impair relaxation to ACH in aorta compared to LA-SIRT3
+/+
mice (38.2±4.4%, 37.0±4.4%, respectively). Furthermore, reductions in SIRT3 did not impact vascular compliance or distensibility in carotid arteries from the same animals. In conclusion, in contrast to our hypothesis, inactivation of SIRT3 does not impact vasomotor function or conduit vessel compliance in advanced atherosclerosis. Importantly, this and may be explained by a “floor-effect”, and future work examining the effect of SIRT3 overexpression on vascular function and pathology will be instrumental in determining its biological role and ultimate therapeutic utility.
Abstract 260: Apolipoprotein A-I Binding Protein Regulates Macrophage Polarization in Atherosclerosis
Authors: Schneider Dina A , Fang Longhou , Miller Yury I
Published: June 2021

Our laboratory recently demonstrated that Apolipoprotein A-I Binding Protein (AIBP), an evolutionarily conserved intracellular and secreted protein, mediates cholesterol efflux from endothelial cells, which in turn disrupts lipid rafts and limits angiogenic signaling. Since lipid rafts are implicated in multiple cell signal cascades, to better understand the
in vivo
role of AIBP our laboratory has generated

Apoa1bp
-/-

mice. The

Apoa1bp
-/-

mice exhibit increased levels of inflammatory cytokines, and have an increased content of M1 macrophages in white adipose tissue in comparison to wild type mice when challenged with a high fat diet. Since AIBP accelerates cholesterol efflux from macrophages to HDL, and vascular lipid accumulation and inflammation are key factors in atherosclerosis, we hypothesized that AIBP is atheroprotective by suppressing macrophage lipid accumulation and inflammatory M1 macrophage polarization. Immunohistochemistry shows that AIBP is present in atherosclerotic lesion macrophages. However, elicited macrophages lacking AIBP expression do not exhibit any impairment in their ability to polarize to M1, suggesting that deficiency in secreted extracellular AIBP may be responsible for the M1 phenotype observed in

Apoa1bp
-/-

mice. Indeed, treating macrophages with recombinant AIBP prior to polarization resulted in suppression of M1 polarization. In a high-cholesterol diet feeding experiment,

Apoa1bp
-/-
Ldlr
-/-

mice had increased M1 macrophage content in their aorta and aortic root atherosclerotic lesions, as determined by FACS and immunohistochemistry, respectively. In conclusion, AIBP is an important negative regulator of macrophage polarization and lipid accumulation. A better understanding of AIBP’s regulatory functions in the context of atherosclerosis will provide new mechanistic insights and targeted therapies.
Abstract 27: Structure of Lipid-free and Lipid-bound Apolipoprotein A-I Determined by Stable Isotope-assisted Cross-linking and Small Angle X-ray Scattering
Authors: Melchior John T , Morris Jamie C , Walker Ryan G , Jones Martin K , Segrest Jere P , Thompson Thomas B , Davidson W. S
Published: June 2021

Apolipoprotein (apo)A-I, the major protein constituent of high density lipoproteins (HDL), acts as a conformationally dynamic scaffold on the surface of the particle. Evidence suggests apoA-I conformation dictates HDL functionality and capacity to interface with other HDL proteins; however, the details of apoA-I structure and HDL formation are not completely understood. X-ray crystal structures of truncated forms of apoA-I and its cousin, apoA-IV, show reciprocal domain swapping structures in the lipid-free state. Recently, we showed the truncated mutant of apoA-I is also highly dynamic in solution with a flexible N-terminus. The purpose of this study was to a) derive a structural model of wild-type (WT) apoA-I using the new in-solution truncation model as a template and b) determine the relationship between monomeric, and oligomeric forms of lipid-free WT apoA-I. Using a novel isotope labeling strategy and chemical cross-linking, we defined spatial relationships within and between different molecules of WT apoA-I. The cross-linking patterns revealed a high degree of structural similarity between WT apoA-I oligomers. We found that monomeric and dimeric WT apoA-I structures were similar, but not identical, to those predicted by the crystal and solution structures of truncated apoA-I. Using cross-linking data, we derived two interconverting structures each for monomeric WT apoA-I, both containing dynamic N- and C-terminal regions. Similar structures were generated for WT dimeric and trimeric species. Small angle X-ray scattering was used to derive molecular envelopes of the molecules and refine the arrangements to propose the most detailed molecular models of lipid-free WT apoA-I to date. The models were used in conjunction with the reported double-belt and trefoil structures reconstituted HDL to reveal evidence of a parsimonious relationship between lipid-free and lipid-bound WT apoA-I. We propose a structural framework of WT apoA-I self-association and lipidation, centric to the C-terminal region of the molecule, that forms a basis for understanding HDL particle formation at the cell surface.
Abstract 28: Adiponectin Stimulates Cholesterol Efflux Efficiently in Human THP-1 Macrophages and Modulates HDL-apoA-I Biogenesis
Authors: Gasbarrino Karina , Hafiane Anouar , Genest Jacques , Daskalopoulou Stella Styliani
Published: June 2021

Introduction:
Adiponectin (APN) is an anti-inflammatory and anti-atherogenic adipokine that is strongly correlated with circulating HDL levels. However, its role in macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. We examined the effect of APN on cholesterol efflux from human THP-1 macrophages, elucidated its kinetics, and investigated its role in HDL biogenesis.

Methods:
APN dose-dependent (0.1 to 60 μM) and time-dependent (0.5 to 24 hours) cholesterol efflux studies were performed in
3
[H]-cholesterol labeled human THP-1 macrophages in the presence of apoA-I. Following efflux studies, the HDL fractions within media were concentrated (10kDa cut-off filter) and subjected to analytical FPLC and 2D-PAGGE technique to reveal HDL species.

Results:
APN stimulated ABCA1-mediated cholesterol efflux in a dose-dependent and time-dependent manner. Kinetics analysis revealed that increased molar doses of APN and apoA-I had similar Km efficiency of cholesterol efflux but greater velocity (
Km
=3.24±0.71 μM,
Vmax
=4.90±0.07 efflux/6h) when compared to apoA-I alone (
Km
=3.33±0.57 μM,
Vmax
=3.83±0.24 efflux/6h). Importantly, once APN was tested against a fixed dose of apoA-I (10 μg/mL), it promoted cholesterol efflux with
Km
= 0.17±0.06 μM. This was associated with a 75.7% decrease in intracellular free cholesterol in THP-1 cells in the presence of APN and apoA-I when compared to apoA-I alone (P<0.01). APN alone had no effect on the level of residual efflux (reached a level of 1%). The FPLC cholesterol profiles demonstrated that in the presence of APN and apoA-I there was increased lipidated nascent HDL (nHDL) during the process of cholesterol efflux, compared to apoA-I alone. This was associated with increased size of nHDL-apoA-1 pre-β and α species via 2D-PAGGE analyses. By immunoblotting for apoA-I and APN, APN oligomers exhibited a molecular weight range of 9 to 20 nm, appearing within the size range of nHDL-apoA-I.

Conclusion:
In addition to promoting macrophage cholesterol efflux
in vitro
, APN can modulate HDL-apoA-I biogenesis, by increasing the generation of nHDL particles. These findings suggest that APN may be of potential therapeutic value in the modulation of HDL’s protective role in atherosclerosis.
Abstract 303: Asthma Associates With Human Abdominal Aortic Aneurysm and Rupture
Authors: Liu Conglin , Wemmelund Holger , Lindholt Jes S , Levy Bruce D , Libby Peter , Shi Guo-Ping
Published: June 2021

Objective:
Both asthma and abdominal aortic aneurysms (AAA) involve inflammation. It remains unknown whether these diseases interact.

Methods and Results:
Databases analyzed included Danish National Registry of Patients, a population-based nationwide case-control study included all patients with ruptured AAA and age- and sex-matched AAA controls without rupture in Denmark from 1996 to 2012; Viborg vascular trial, subgroup study of participants from the population-based randomized Viborg vascular screening trial. Patients with asthma were categorized by hospital diagnosis, bronchodilator use, and the recorded use of other anti-asthma prescription medications. Logistic regression models were fitted to determine whether asthma associated with the risk of ruptured AAA in Danish National Registry of Patients and an independent risk of having an AAA at screening in the Viborg vascular trial. From the Danish National Registry of Patients study, asthma diagnosed <1 year or 6 months before the index date increased the risk of AAA rupture before (odds ratio [OR]=1.60-2.12) and after (OR=1.51-2.06) adjusting for AAA comorbidities. Use of bronchodilators elevated the risk of AAA rupture from ever use to within 90 days from the index date, before (OR=1.10-1.37) and after (OR=1.10-1.31) adjustment. Patients prescribed anti-asthma drugs also showed an increased risk of rupture before (OR=1.12-1.79) and after (OR=1.09-1.48) the same adjustment. In Viborg vascular trial, anti-asthmatic medication use associated with increased risk of AAA before (OR=1.45) or after adjustment for smoking (OR=1.45) or other risk factors (OR=1.46).

Conclusions:
Recent active asthma increased risk of AAA and ruptured AAA. These findings document and furnish novel links between airway disease and AAA, 2 common diseases that share inflammatory aspects.
Abstract 310: High Frequency Ultrasound Reveals Dobutamine Increases Murine Aortic Strain
Authors: Adelsperger Amelia R , Castle Paige , Greve Joan M , Goergen Craig J
Published: June 2021

Dobutamine increases heart rate and cardiac output, similar to the effects the sympathetic nervous system has on the body during exercise. This makes dobutamine an excellent surrogate for exercise, and it is often administered during stress tests. The purpose of this study was to quantify changes in arterial strain using
in vivo
high frequency ultrasound in order to better understand the effects of dobutamine on vascular dynamics. Here, C57BL/6 mice were infused with 40 μg/min/kg of dobutamine at a flow rate of 0.002 mL/min via a tail vein catheter (n=3; 19 weeks old, 31 ± 2.0 g). Ultrasound images of the suprarenal aorta, infrarenal aorta, iliac arteries, and carotid artery were acquired before the infusion of dobutamine (baseline) and again after the heart rate had plateaued during the infusion using B- and M-Modes images in both short and long axis. These mice showed an average heart rate increase of 76±18.7 bpm roughly 20 minutes after the start of the dobutamine infusion. Green-Lagrangian circumferential cyclic strain, calculated from arterial diameters in systole (maximum, dashed) and diastole (minimum, solid), increased throughout the vasculature after infusion. The suprarenal aorta increased from 22.5±1.3% to 41.9±2.4%, while the infrarenal aorta increased from 21.2±2.5% to 38.0±1.7%. The iliac artery increased from 22.4±2.9% to 42.0±2.7%, and the carotid artery increased from 26.0±2.3% to 43.5±5.2%. The average strain for all four locations increased from 23.0±2.1% (A) to 41.4±2.4% (B), representing a 79.6% increase from baseline. These results suggest that high frequency ultrasound is capable of capturing rapid changes in vascular dynamics due to dobutamine infusion. The effects of dobutamine on these healthy vessels can be used as a baseline for future studies that investigate murine disease models including advanced atherosclerosis and abdominal aortic aneurysms.
Abstract 321: Prasugrel, a Platelet P2y
12
Receptor Antagonist, Improves Abnormal Gait in a Novel Murine Model of Thrombotic Hind Limb Ischaemia
Authors: Ohno Kousaku , Tomizawa Atsuyuki , Mizuno Makoto , Jakubowski Joseph A , Sugidachi Atsuhiro
Published: June 2021

Background:
The efficacy of P2Y
12
inhibition for the prevention of cardiovascular events in patients with peripheral arterial disease (PAD) has been established. However the therapeutic effects on ischaemic limb manifestations are less clear. Accordingly, we developed a novel murine model of thrombotic hind limb ischaemia to reflect that found in patients with PAD exhibiting ischaemic limb symptoms. We further investigated the effects of P2Y
12
inhibition by prasugrel in this model.

Methods and Results:
Thrombus formation induced by application of ferric chloride to the femoral artery resulted in a significant reduction in blood flow in the injured limb. In gait analysis of the ischaemic limb using the CatWalk system, maximum contact area and stance phase duration were reduced and swing phase duration increased in this model. Blood flow reduction and gait abnormalities gradually recovered over 21 days to levels present before arterial injury. Compared to wild-type (WT) mice, significant increases in blood flow and improvement in gait were observed in P2Y
12
-deficient mice. In addition, daily oral administration of prasugrel (3 mg/kg/day) to WT mice resulted in significant inhibition of blood flow reduction and gait abnormalities to levels found in P2Y
12
deficient mice.

Conclusions:
In conclusion, acute femoral artery thrombosis resulted in hind limb ischaemia and moderate gait abnormalities in mice. In addition, the present study suggests a therapeutic role for P2Y
12
antagonism in reducing the manifestations of limb ischaemia in PAD.
Abstract 324: Ankle Brachial Index as a Predictor for Abnormal Cardiovascular Stress Test and Cardiovascular Outcomes
Authors: Shakoor Muhammad T , Mohani Amir , Maqsood Khawar , Lotfi Amir
Published: June 2021

Background:
The combination of ankle brachial index (ABI) and other risk stratification tools has been evaluated previously. However baseline ABI in patients admitted for chest pain prior to stress testing to predict increased probability of abnormal stress test and MACE (major cardiovascular adverse events) has not been studied.

Methods:
Patients (n= 342) admitted for chest pain with normal or unchanged electrocardiogram and negative troponin T who were referred for myocardial perfusion study were enrolled in this prospective observational study and followed for one year. The first part of the study was to assess if an abnormal ABI increases the probability of an abnormal stress test and the second part was to measure one-year outcome for MACE (Angina, MI, CHF, TIA/Stroke and deaths) based on their stress test and ABI results. Multivariable logistic regression modeling is used to interpret the data.

Results:
In our study population 83% of the patients had Framingham risk score less than 20. Twenty-nine percent of the patients with normal ABI and 40% of the patients with abnormal ABI had abnormal stress test. The difference is statistically insignificant. After adjusting for different variables abnormal ABI was found to have stronger correlation with MACE (Odds ratio 2.9, p 0.01, CI 1.2 - 6.7) as compared to abnormal stress test (Odds ratio 0.92, p 0.84 CI 0.3 - 2.1).

Conclusion:
In our study cohort an abnormal ABI, irrespective of the stress test results, was an independent predicator of MACE at one year. We can conclude that there is very weak temporal relationship between abnormal ABI and abnormal stress test in low risk population and asymptomatic peripheral vascular disease is a strong independent predictor of MACE, even in this low risk population. We can also conclude that ABI during stress testing can be used for risk stratification. Future studies may need more power in this low risk population.
Abstract 336: Metabolites Reflecting Fresh Venous Thrombus: Lactic Acid and Guanine Enhance Whole Blood Coagulation
Authors: Yamashita Atsushi , Sugita Chihiro , Moriguchi-Goto Sayaka , Nakamura Eriko , Maekawa Kazunari , Asada Yujiro
Published: June 2021

Background:
Thrombus formation is a multicellular dynamic process involving platelets, leukocytes, and erythrocytes. Recent studies demonstrated that microemvironment affects cellular metabolism and that metabolites can alter the cellular function. The present study aims to identify metabolites reflecting fresh venous thrombus and their role on thrombus formation in rabbit.

Methods:
We performed metabolomic analysis of rabbit venous blood and jugular venous thrombus 4 hours after endothelial denudation and blood stasis using capillary electrophoresis-time of flight mass spectrometry. Effects of the altered metabolites on blood coagulation and platelet aggregation were assessed with rotation thromboelastometry and platelet aggregometer.

Results:
The metabolomics analysis identified 226 metabolites (133 cationic and 93 anionic metabolites) in the venous blood and thrombus. The levels of 7 or 4 of them were significantly more (thrombus/ blood ratio >5) or less (thrombus/ blood ratio <1/2) in thrombus than those in blood, and the metabolites included glycolysis, nucleotides, and redox-related metabolites. Three metabolites were detectable only in the blood or venous thrombus. Among the metabolites (thrombus/ blood ratio >5), lactic acid and guanine dose-dependently enhanced whole blood clotting with thromboelastometry. Adenosine monophosphate inhibited collagen-induced platelet aggregation.

Conclusion:
The glycolysis, nucleotides, and redox-related metabolites may reflect fresh venous thrombus, and lactic acid and guanine may enhance blood coagulation in venous thrombus formation. The metabolic change could provide new insight into the process of venous thrombus formation.
Abstract 340: Recombinant Soluble Apyrase Inhibits Vascular Smooth Muscle Cell Migration
Authors: Adeola Oluwaseun , Ji Yan , Fish Phillip , Strawn Tammy , Weisman Gary A , Chan Ridong , Fay William
Published: June 2021

Background:
Purinergic receptor activation by extracellular nucleotides is involved in thrombosis and neointimal hyperplasia that accompany atherosclerosis and postangioplasty restenosis. Human apyrases [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases)] are membrane bound enzymes that hydrolyze extracellular nucleotides, thereby inhibiting purinergic receptor activation. CD39, the first identified human apyrase, is constitutively expressed on endothelial cell (EC) and vascular smooth muscle cell (VSMC) surfaces. APT102, a recombinant soluble form of CD39L3, has been shown to reduce platelet activation through its ADPase activity, but its effects on VSMC and EC function are yet to be established. We tested the hypothesis that APT102 will inhibit migration of VSMCs and ECs.

Methods:
We studied cell migration using a modified Boyden chamber assay in which 5x10
4
cells suspended in 0.2% FBS/DMEMF12 were added to the upper chamber of transwells separated from the lower chamber medium by a microporous membrane through which VSMCs and ECs can migrate. APT102 (100 nM) or vehicle control was added to the upper chamber; lower chamber contained 2.5% FBS/DMEMF12 and either ATP (10 μM) or vehicle control. Transwells were incubated at 37
0
C for 6 h, after which cells that migrated through pores and adhered to the lower chamber side of the membrane were fixed, stained and counted.

Results:
ATP (10μM) significantly enhanced migration of both VSMCs and ECs. APT102 significantly inhibited VSMC migration and completely abrogated the pro-migratory effect of ATP. In contrast, APT102 had no inhibitory effect on EC migration, either spontaneous or ATP-enhanced.

Conclusion:
APT102 inhibits VSMC but not EC migration. These results suggest that pharmacological targeting of extracellular nucleotides may provide a safe and effective therapeutic strategy to inhibit neointimal hyperplasia and restenosis after angioplasty, without delaying endothelial cell recovery, which is a significant limitation of drug-eluting stents. Further studies are needed to clarify the mechanism(s) underlying the differential effect of extracellular nucleotide degradation by APT102 on VSMC and EC migration.
Abstract 344: Kruppel-like Factor 15: A Critical Transcriptional Regulator of Hypoxia Induced Endothelial Arginase 2
Authors: Pandey Deepesh , Berkowitz Dan , Romer Lew
Published: June 2021

Objective:
Kruppel Like Factor 15 has recently been shown to be critical for activation of proinflammatory processes in vascular smooth muscle and atherogenesis. Although KLF15 is abundantly expressed in vascular endothelium there is a significant lack of knowledge regarding the role of KLF15 in the regulation of vascular endothelial function. Here we tested the hypothesis that a KLF15 is a critical regulator of Arg2 transcription in response to hypoxia.

Approach and Results:
Adenoviral mediated augmented expression of KLF15 in human pulmonary microvascular endothelial cells (HPMEC) and in rat pulmonary arteries decreased Arg2 mRNA, protein, and activity. HPMECs exposed to 48 hours of hypoxia exhibited a robust increase in Arg2 protein and mRNA expression and a reciprocal decrease in KLF15 protein levels that remain sustained after 24 hours of reoxygenation. Further, decreased KLF15 protein levels observed in hypoxia exposed HPMEC occurs through augmented conjugation of Ubiquitin to KLF15 that results in enhanced clearance of KLF15 via proteasomal degradation. Chromatin immunoprecipitation indicated direct binding of KLF15 to the Arg2 promoter, which was relieved when HPMEC were exposed to hypoxia, Finally, overexpression of KLF15 reversed hypoxia-induced augmentation of arginase activity and decrements in Nitric Oxide production in HPMEC, and also reversed hypoxia-induced endothelial dysfunction in isolated mice aortic and rat pulmonary artery rings.

Conclusions:
KLF15 is a critical regulator and repressor of endothelial Arg2 expression, and thereby, NO and pulmonary endothelial function. Overexpression or activation of KLF15 may represent novel therapeutic strategies for pulmonary hypertension.