bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 329: Mechanism by Which Anacetrapib Lowers Plasma Lipoprotein (a) Concentration
Authors: Thomas Tiffany A , Zhou Haihong , Roddy Thomas , Previs Stephen , Lassman Michael , Hubbard Brian , Jumes Patricia , Wagner John A , Gutstein David E , Ramakrishnan Rajasekhar , Marcovina Santica M , Rader Daniel J , Ginsberg Henry , Millar John , Reyes-Soffer Gissette
Published: July 2021

Objective:
Anacetrapib, a CETP inhibitor, was previously shown to decrease plasma lipoprotein (a) [Lp(a)] levels by 35-40% in subjects also taking a statin. Thus, anacetrapib is an efficacious Lp(a)-lowering agent. The goal of this study was to define the mechanism by which anacetrapib lowers plasma Lp(a) levels.

Methods:
39 moderately hyperlipidemic volunteers were enrolled in a fixed-sequence study, in which 75% were on atorvastatin 20mg/day, plus placebo for four weeks (period 1), and then atorvastatin plus anacetrapib (100 mg/day) for 8 weeks (period 2). The other 25% of the subjects received double placebo for four weeks, and then placebo plus anacetrapib for 8 weeks. Turnover studies using D3-leucine were performed at the end of each period. The present analysis utilized samples from a subset of subjects (n=12) who had plasma Lp(a) levels greater than 10 nM at the end of period 1 and had a greater than 10% reduction in Lp(a) by the end of period 2. The fractional synthetic rate (FSR:equal to fractional catabolic rate at steady state) of mature Lp(a), isolated from a D:1.019-1.21 g/ml density interval, was determined from the enrichment of a leucine-containing peptide specific to apo(a). The production rate (PR) of mature Lp(a) was calculated from the FSR and the Lp(a) pool size. To date, we have calculated the FSR and PR in 4 participants.

Results:
Baseline Lp(a) mean levels were 45.7 ± 6.3nM in the entire group and 56.5 ± 33.6nM in the 12 qualifying subjects. Anacetrapib lowered Lp(a) by 43 ± 22% in the 12 subjects and 21 ±12% in the 4 subjects with turnover data. In these 4 subjects, the reduction in mature Lp(a) was associated with a 24% reduction in FSR and a 41% reduction in PR. Lp(a) kinetics analyses of the remaining 8 subjects are in progress.

Conclusion:
These preliminary results suggest that anacetrapib decreases Lp(a) levels by significantly decreasing the production of mature Lp(a). Additional analyses are planned to determine if the reduced production of Lp(a) results from decreased entry of Lp(a) into plasma or reduced conversion of a precursor form to the mature Lp(a).
Abstract 333: CD36 Mediates Cholesterol Uptake via Na/K-ATPase/lyn Signaling in Macrophages
Authors: Chen Yiliang , Kennedy David , Li Zhichuan , Xie Zijian , Silverstein Roy L
Published: July 2021

Atherosclerosis, the leading cause of death in the developed countries, is characterized by macrophage foam cell formation. We previously showed that CD36, a scavenger receptor highly expressed in macrophages, mediates oxidized-LDL uptake, contributes to intracellular cholesterol accumulation and foam cell formation, and regulates macrophage migration and pro-inflammatory signaling. Consistently,
cd36
deletion in mice protects from diet-induced atherosclerosis. Mechanistically, we discovered a novel signaling pathway, in which oxidized LDL (oxLDL) binding to CD36 activates Lyn kinase and initiates a cascade that is necessary for the pro-atherogenic cellular phenotype. How CD36 regulates Lyn kinase remains undefined. Since we previously showed that the Na/K-ATPase (NKA) regulates Src family kinases, including Lyn, we
hypothesized
that CD36 regulates Lyn kinase via an interaction with NKA. We used co-immunoprecipitation, FRET, and a novel cross linking assay to demonstrate that CD36 physically associates with NKA on the macrophage surface. Using a Lyn kinase activity assay, we showed that the interaction regulates Lyn kinase activity in response to oxLDL in macrophages. Moreover, a newly developed peptide inhibitor specifically blocked Lyn activation in response to oxLDL and attenuated oxLDL-stimulated cholesterol uptake (135.6±3.4 μM cholesterol/mg protein after 24 hours vs 173.8±7.7 μM cholesterol/mg protein in vehicle treated cells; p=0.0005; n=6). Taken together, we
conclude
that CD36 signals through NKA to regulate Lyn kinase activity in macrophages, which may be a molecular mechanism underlying cholesterol overloading and foam cell formation.
Abstract 336: Angiotensinogen Gene Polymorphism Confers a Multiple Risk Factor for Atherosclerosis and Its Susceptibility Traits
Authors: Dzimiri Nduna , Elhawari Samar , Al-Najai Mohammed , Muiya Paul , Gueco Daisy , Vigilla Mary Grace , Andres Editha , Mazher Nejat , Alshahid Maie , Meyer Brian F
Published: July 2021

Angiotensinogen (AGT) constitutes a pivotal component of the renin-angiotensin system which controls the systemic blood pressure and several other cardiovascular functions. In order to evaluate the role of the
AGT
gene polymorphism in atherosclerosis (CAD) and its risk factors, we first sequenced the gene in 200 individuals in search of informative SNPs. Of the >100 SNPs identified, we proceeded to evaluate the association of 8 variants in 3232 CAD cases versus 2292 angiographed controls by the Applied Biosystems real-time PCR system. Of the studied variants, the recessive mode of inheritance (TT) of rs3789679C>T [Odds ratio(95% Confidence Interval) = 1.23(1.05-1.45); p=0.012]; TT of rs699C>T [1.21(1.11-1.32); p<0.0001] as well as the dominant models (AG+GG) of rs5051A>G [1.22(1.10-1.37; p<0.0001) and AG+GG of rs2067853A>G [1.23(1.07-140);p=0.003] conferred risk for CAD. Interestingly, rs699 (p<0.0001), rs5051 (p=0.003) and rs2067853 (p=0.001) were equally implicated in hypertension, while the rs699 was further associated with hypercholesterolaemia (p=0.002) and hypertriglyceridaemia (p<0.0001), and the rs5051 was similarly associated with type 2 diabetes mellitus (T2DM) (p=0.011). One 8-mer haplotype from the 8 studied SNPs ACGGATAT (χ
2
=9.83; p=0.0017) and several of its variants, including the 7-mer ACGGATA (χ
2
=9.40; p=0.0022), 6-mer GGATAT (χ
2
=11.54; p=0.0007) and 5-mer GATAT (χ
2
=10.89; p=0.001) conferred significant risk for CAD. Furthermore, three 8-mer haploptypes GTGGGTGG (χ
2
=6.96; p=0.0083), ACGGGTAT (χ
2
=4.70; p=0.030) and GTAGGCGG (χ
2
=5.69; p=0.017) were associated with HTN. Similar trends were also observed for T2DM, obesity and hyperlipidaemia. These observations show that the
AGT
gene polymorphism confers a common risk for atherosclerosis and its major susceptibility traits.
Abstract 341: Akt1 Deletion Promotes Survival in a Model of Spontaneous Myocardial Infarction and Atherosclerosis
Authors: Kerr Bethany , Ma Lining , West Xiaoxia , Malinin Nikolay , Weber Malory , Ding Liang , Podrez Eugene , Byzova Tatiana
Published: July 2021

The role of Akt1 kinase in cardiac dysfunction and atherosclerosis is hotly contested. Many models use transient Akt1 activation or deletion to study its function in cardiac diseases. The effects of Akt1 knockout were tested in mouse models dependent on short-term Western diets to induce atherosclerotic lesions; however, in these models, the complete cascade ending in infarction does not occur. In contrast, the ApoE
-/-
/SR-BI
-/-
double knockout (DKO) mouse represents a model of spontaneous myocardial infarction resulting from severe dyslipidemia and atherosclerosis leading to death between 5-7 weeks of age. In DKO mice and humans, dyslipidemia and inflamed arterial occlusions lead to plaque rupture and myocardial infarction. Importantly, Akt1 activation is dramatically elevated in the myocardium and endothelium of DKO vs. WT mice. To assess the role of chronic Akt1 activation in atherosclerosis, we generated ApoE
-/-
/SR-BI
-/-
/Akt1
-/-
triple knockout (TKO) mice. We found that TKO mice exhibited decreased cardiac dysfunction and hypertrophy resulting in longer lifespan vs. DKO. In addition, TKO mice have diminished endothelial VCAM expression, decreased atherosclerotic lesions
in vivo
, and reduced foam cell formation
in vitro
. ROS production, which is regulated by Akt1 signaling and contributes to atherosclerosis during dyslipidemia, was reduced in TKO hearts, plasma, endothelial cells, and macrophages. This led to reduced proatherogenic lipid oxidation product accumulation in TKO vs. DKO. Moreover, Akt1 deletion in TKO mice decreased levels of CD36, the main oxidized lipid receptor, in hearts, on endothelial cells, and on macrophages. Thus, during dyslipidemia, chronic Akt1 activation results in elevated oxidative stress, generation of proatherogenic lipid oxidation products, and expression of CD36 (a known atherothrombotic regulator) resulting in enhanced atherosclerosis and spontaneous myocardial infarction. Inhibition of Akt1 by genetic deletion results in partial rescue of mice leading to decreased atherosclerosis, improved cardiac function, and elongated lifespan. Continued study of chronic Akt1 activation and its effects on atherosclerosis will enable the development of additional targets for heart disease therapeutics.
Abstract 342: Ultrasound-Enhanced Release of Bevacizumab from Echogenic Liposomes for Therapeutic Inhibition of Atherogenesis
Authors: Klegerman Melvin E , Zou Yuejiao , Britton George L , Naji Ali K , Huang Shao-Ling , McPherson David D
Published: July 2021

Introduction.
Bevacizumab (BEV) is a humanized monoclonal antibody to vascular endothelial growth factor (VEGF) that can ameliorate atheroma progression, the mechanism of which is believed to be through inhibition of neovascularization. Systemically, BEV has multiple adverse effects, which may be diminished in a controlled release delivery vehicle.

Hypothesis.
The purpose of this study was to test the hypothesis that BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US).

Methods.
BEV-ELIP were prepared by a rehydration/loading-freeze/thaw-centrifugation-lyophilization method. BEV-ELIP samples were resuspended in 50ml of porcine plasma and divided into two portions, one of which was subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 minutes. After various time intervals, aliquots were fractionated by centrifugation and assayed for immunoreactive BEV with a direct enzyme-linked immunosorbent assay (ELISA). We considered immunoreactive BEV in washed pellet suspensions to represent ELIP surface-exposed VEGF-binding sites. To assure that BEV was accurately measured in plasma, we repeated the release protocol with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method; BEV was measured by quantitation of fluorescence emission using a SpectraMax M5 microplate reader.

Results.
BEV-ELIP encapsulation efficiency was 43.2 ± 9.7% (SD, n = 5); 34.1 ± 29.3% of the encapsulated BEV was spontaneously released within the first 30 minutes. US caused an additional 4.1% of BEV to be released from the surface exposed compartment. Fluorescence measurements indicated that 43.2 ± 6.2% (n = 3) of encapsulated FITC-BEV-ELIP was spontaneously released in 60 minutes and that US caused an additional 5.7% to be released in that period, but, overall, US caused an additional 100 μg of BEV to be released or exposed per aliquot.

Conclusions.
We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody. These results warrant investigation of the formulation’s anti-angiogenic efficacy in vitro and in vivo.
Abstract 344: Bcl-x Inactivation in Macrophages Accelerates Progression of Advanced Atherosclerotic Lesions in Apoe
-/-
Mice
Authors: Lesnik Philippe , Deswaerte Virginie , Gautier Emmanuel L , Saint-Charles Flora , Pirault John , Rucker Edmund B , Beliard Sophie , Chapman John , Jessup Wendy , Huby Thierry , Shearn Andrew I
Published: July 2021

Background:
Bcl-x is the most abundantly expressed member of the Bcl-2 gene family in macrophages but its role in macrophage apoptosis during atherogenesis is unknown.

Methods and results:
We previously reported dual pro- and anti-atherogenic effects of macrophage survival in early versus advanced atherosclerotic lesions respectively, potentially reflecting growing impairment of efferocytosis during plaque progression. Here, we specifically inactivated Bcl-x in macrophages and evaluated its impact on atherosclerotic lesion formation in Apoe
-/-
mice at various stages of the disease. Bcl-x deficiency in macrophages increased susceptibility to apoptosis, resulting in the depletion of tissue macrophages in vivo, including its major pool, Küppfer cells in the liver. We also observed increased cholesterol levels, that was however not associated with any acceleration of early atherosclerotic plaque progression. This observation, suggests that the atheroprotective effect of macrophage apoptosis at that stage of disease was counterbalanced by enhanced cholesterol levels. Bcl-x KOmac/Apoe
-/-
mice exhibited significantly larger advanced lesions than control mice. These lesions showed vulnerable traits. Such enhanced lesion size may occur as a result not only of apoptotic cell accumulation but also of elevated cholesterol levels.

Conclusions:
Modulation of macrophage resistance to apoptosis through targeted deletion of Bcl-x has a major impact on the entire macrophage cell population in the body, including Küpffer cells. Macrophage survival may, therefore, not only influence atherosclerotic plaque development and vulnerability but also cholesterol metabolism.
Abstract 346: Change in Alcohol Consumption Status Associated with Changes in Fibrinogen Levels over 13 Years of Follow-up: The Coronary Artery Risk Development in Young Adults Study
Authors: Okwuosa Tochi M , Klein Oana , Chan Cheeling , Schreiner Pamela , Liu Kiang , Green David
Published: July 2021

Introduction:
Moderate alcohol consumption is inversely related to coronary/cardiovascular disease (CVD) risk and mortality. Conversely, fibrinogen has been positively associated with atherosclerosis and thrombosis. No studies to date have examined longitudinal associations between alcohol consumption and blood fibrinogen levels.

Methods:
We included 2548 CARDIA participants (55% women, 43% black) with fibrinogen and alcohol use data at year 7 (1992-93; ages 25-37) and year 20 examinations (2005-06). Fibrinogen was measured using automated nephelometry. Alcohol use was self-reported and categorized as current, former or never drinkers at each exam. ANCOVA models were used to relate changes in alcohol use to changes in mean fibrinogen levels stratified by sex and race, after controlling for year 7 fibrinogen. Subsequent models also adjusted for conventional cardiovascular risk factors (Figure).

Results:
Over 13 years, mean fibrinogen increased by 71mg/dL vs. 70mg/dL (p-difference=NS) in black men (BM) vs. white men (WM), and 78mg/dL vs. 68mg/dL (p-difference<0.05) in black women (BW) vs. white women (WW), respectively. The Pearson’s correlation coefficients between years 7 and 20 fibrinogen were 0.60 (BM), 0.47 (WM), 0.54 (BW) and 0.53 (WW) (each p<0.001). Compared with never-drinkers, becoming or staying a drinker was associated with a smaller longitudinal increase in fibrinogen for BM, BW and WW after multivariable adjustment (Figure). Conversely, WM who stayed drinkers had a larger increase in fibrinogen (p<0.01). Relative to never-drinkers, fibrinogen increased more among BM (p<0.05), WM (p<0.001) and WW (p=NS) who were no longer alcohol users by year 20.

Conclusions:
In this young cohort, we observed an inverse longitudinal relationship between alcohol consumption and fibrinogen in BM, BW and WW. These results provide a novel insight into the mechanism for established protective effect of moderate alcohol intake on CVD outcomes.
Abstract 355: β-Adrenergic Stimulation Impact on Cerebrovascular Contractility Through Actin Cytoskeleton Disorganization in Rabbits
Authors: Kim Nari , Kim Hyoung Kyu , Heo Hye-Jin , Ko Kyung Soo , Rhee Byoung Doo , Han Jin
Published: July 2021

Beta-adrenoceptor stimulations have been well-known to initiate cardiac hypertrophy accompanied by extracellular remodeling that predisposes heart failure and cardiac dysarrhythmia. However, less is known concerning cerebrovascular dysfunction induced by beta-adrenergic stimulation. We investigated whether cerebrovascular functions and related biochemical signal pathways were altered during beta-adrenergic stimulations in rabbits.

New Zealand white rabbits have been injected with isoproterenol (ISO, 0.3 mg/kg/day) for 7 days. We have studied the alteration of protein expression in cerebral arteries (CAs) using 2DE proteomics and western blot analysis and further analyzed those data using bioinformatics software. Reactive oxygen species generation and followed DNA damage were assessed deteriorative effect of ISO on CAs. Vasocontractility response to angiotensin II (Ang II) and related biochemical signal pathways were assessed to evaluate functional alteration of CAs.

Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins, (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin) and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1) in ISO-stimulated CAs. As a cause of dysregulation of actin cytoskeleton organization, we found decreased level of RhoA/ROCK1, major regulators of actin cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca
2+
efflux and contraction response to Ang II and high K
+
in ISO-stimulated CAs.

We suggested that ISO stimulations reduced RhoA/ROCK activities and modified key actin based cytoskeletal and antioxidative proteins in CAs, which consequently lead to impair cerebrovascular contractility response to Ang II in rabbits.
Abstract 363: Lim Domain Only 4 Is a Shear-Sensitive Protein, Playing a Critical Role in Endothelial Inflammation and Atherosclerosis
Authors: Takabe Wakako , Ni Chih-Wen , Son Dong Ju , Alberts-Grill Noah , Jo Hanjoong
Published: July 2021

Recently, we have shown that disturbed flow, characterized by low and oscillatory shear stress, caused by a partial ligation of mouse left carotid artery (LCA) rapidly induces atherosclerosis. Using the partial ligation model and genome-wide microarray study with aortic endothelial RNAs obtained directly from the flow-disturbed carotid arteries, we previously identified mechanosensitive genes in mouse endothelial RNA including
LIM domain only 4
(
lmo4
). Here we report that LMO4 is a shear-sensitive protein that regulates endothelial inflammation.
Lmo4
was up-regulated by disturbed flow in mouse LCA compared to the contralateral right CA (RCA) exposed to stable flow. At protein levels, LMO4 expression was significantly higher not only in LCA in our surgical model but also in the lesser curvature (flow-disturbed and athero-prone region of mouse aortic arch) compared to the greater curvature (stable-flow and ather-protected region). In addition, immunohistochemical staining of LMO4 in human coronary arteries revealed that its expression is detectable only in intimal endothelial cells, but not in medial cells. While LMO4 is known as a potential oncogene and associated with growth, migration and invasion of breast cancer cells, its role in cardiovascular system is not known to our knowledge. We tested a hypothesis that LMO4 is a mechanosensitive gene and plays a critical role in regulation of endothelial cell biology. LMO4 protein expression was robustly induced by oscillatory shear stress (OS) compared to laminar shear (LS) in human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with siRNA against LMO4 significantly inhibited OS-induced inflammation and migration, but not apoptosis and cell cycle progression. Further, LMO4 siRNA treatment significantly blunted expression of VCAM-1 and interleukin-8 induced by OS in endothelial cells. These results suggest that LMO4 is a shear-induced gene that plays a critical role in OS-induced endothelial inflammation and migration, and potentially in atherosclerosis.
Abstract 435: Increased Serum Level of Cholesterol Decreases 3-Month Mortality After Acute Ischemic Stroke
Authors: Kim Dohoung , Kim Chi K , Jang Hyunduk , Kim Jun Y , Ryu Wi-Sun , Lee Seung-Hoon
Published: July 2021

Introduction:
Recently, it was reported that obesity, one of the risk factor for cardiovascular diseases, paradoxically decreased mortality in patients with coronary heart disease. Nutritional status associated with obesity may play a role on this paradoxical effect, and cholesterol is also an indicator of nutritional status. Although cholesterol is a well-known risk factor for ischemic stroke, according to prognostic effect of cholesterol on ischemic stroke few studies have been done. We investigated whether cholesterol levels after acute ischemic stroke are associated with early-mortality.

Methods:
We prospectively included patients with acute ischemic stroke admitted consecutively to our hospital, from October 2002 to October 2008. A total of 2,313 patients were analyzed for this study. Cholesterol levels were measured from fasting blood samples which were drawn within 24 hours of admission. For the analyses of mortality, the patients were divided by baseline measurements into quartiles of cholesterol levels (5.23 mmol/L). Patients were followed for 3-months from enrollment into the study, and mortality data were collected.

Results:
The three-month mortality rate in the highest quartile of cholesterol was significantly lower than in the lowest quartile (2.1% versus 6.4%, p<0.05). After adjusting possible confounders including age, gender, hypertension, diabetes, smoking, stroke severity and subtypes, and statin use with the Cox proportional hazard models, adjusted hazard ratio of the highest quartile for all-cause death was 0.49 (95% confidence interval 0.25-0.95).

Conclusion:
Our study demonstrated that increased serum level of cholesterol paradoxically decreases three-month mortality after acute ischemic stroke.
Abstract 496: Competition Between Filamin and Kindlin-2 Regulates Integrin Activation
Authors: Das Mitali , Ithychanda Sujay , Bledzka Kamila , Qin Jun , Plow Edward F
Published: July 2021

Cell migration and adhesion during hemostasis, angiogenesis and inflammation are dynamically regulated by integrin heterodimeric adhesion receptors. Their interactions with cytosolic proteins, filamin (FLN), talin (TLN) and Kindlin (Kn2) enable them to convey intracellular signals (inside-out-signaling) to the external environment by engaging extracellular matrix ligands. While TLN and Kn2 activate integrins, FLN inhibits cell migration. TLN and Kn2 bind to membrane-proximal and -distal NPxY motifs of β integrin cytoplasmic tails (CTs), respectively, and an integrin binding site for FLN resides in between these two sequences. Competition between TLN and FLN regulates integrin activation, but it is unknown if FLN and Kn2 compete and regulate integrin inside-out signaling. This competition was tested using αIIbβ3 (platelet-specific) and β7 (lymphocyte-specific; strong FLN binder) integrins in multiple cell types. siRNA depletion of FLNA in K562 cells stably expressing αIIbβ3 integrin (K562-αIIbβ3) significantly enhanced PAC-1 (specific for activated αIIbβ3) binding compared to control siRNA, demonstrating its effect on β3 activation. In pulldown assays using GST-β3 CT, Kn2 bound β3 in CHO lysates transfected with Kn2, either alone or with FLN repeat 21; however, FLN binding to β3 CT was observed only when FLN repeat 21 was expressed alone. Under similar conditions using GST-β7 CT, FLN-β7 interaction was not perturbed by Kn2. This was more pronounced in endothelial cell lysates where GST-β7 CT bound endogenous FLNA but not Kn2. Weak talin-β7 CT binding in this assay was noted. Moreover, in K562-αIIbβ3 cells, exogenous Kn2 overcame the suppressive effect of FLN on αIIbβ3 activation. Overall, our data shows that FLN inhibits β3 integrin function, and competition between FLN and Kn2 can indeed regulate integrin activation.
Abstract 521: The Thrombotic Potential of the Endothelium Is Regulated by Hemodynamic Flow
Authors: Simmers Michael B , Mackman Nigel , Blackman Brett R , Wamhoff Brian R
Published: July 2021

BACKGROUND:
Vascular wall mechanisms underlying human arterial and venous thrombosis are still evolving. TNFα and other cytokines have been shown to be potent mediators of endothelial cell-derived tissue factor in vitro, which mediates fibrin deposition in the vascular wall. Circulating levels of TNFα detected in humans with cardiovascular disease are approximately 0.05 ng/ml.

METHODS:
Human endothelial cells were co-cultured with or without smooth muscle cells in the presence or absence of human-derived, region-specific hemodynamics. Endothelial cells were exposed to TNFα at various concentrations and incubated in human, platelet-free-plasma supplemented by Alexa 488-labeled fibrinogen. Conversion of A488-fibrinogen to A488-fibrin and deposition on the endothelium was quantified by confocal microscopy.

RESULTS:
In static cultures, samples treated with 1ng/ml TNFα demonstrated minimal fibrin deposition whereas samples treated with 10ng/ml or 20ng/ml exhibited dense fibrin networks (1.17e5 vs. 2.69e7 vs. 3.61e7 mean fluorescence intensity, respectively). Fibrin deposition was tissue factor-dependent. In contrast, endothelial/smooth muscle cell co-cultures primed with inflammatory-prone hemodynamics derived from the internal carotid sinus and 0.05ng/ml TNFα deposited a dense fibrin network (1.9e7 mean fluorescence intensity). Identical experiments in which only endothelial cells were cultured yielded similar results demonstrating the result is hemodynamic-specific and not a consequence of the presence of smooth muscle cells.

CONCLUSIONS:
Hemodynamic priming of endothelial cells in vitro shifts the dose-dependent fibrin deposition response to TNFα into a more human physiological range, two orders of magnitude below that required to induce fibrin deposition in static endothelial cell cultures. Similar approaches can be used to investigate the differential mechanisms underlying vascular wall-mediated venous and arterial thrombosis.
Abstract 58: Adipose-Specific Deletion of ARV1 Results in Decreased HDL Cholesterol, Reduced White Adipose Tissue Mass and Improved Glucose Tolerance in Mice
Authors: Lagor William R , Tong Fumin , Fields David W , Lin Wen , Billheimer Jeffrey T , Ahima Rexford S , Sturley Stephen L , Rader Daniel J
Published: July 2021

ACAT related enzyme 2 required for viability 1 (ARV1) was identified as a gene required for viability in yeast in the absence of cholesterol esterification. ARV1 encodes a putative lipid transporter believed to be important in trafficking of lipids from the ER to the Golgi. ARV1 deficient yeast exhibit profound alterations in cholesterol, phospholipid, and sphingolipid metabolism, accompanied by a constitutively activated unfolded protein response and impaired GPI anchor synthesis. To study the role of ARV1 in mammalian lipid metabolism, we have generated mice with an adipose specific deletion of ARV1 using Cre/loxP technology with Cre expression driven by the Ap2 promoter. ARV1 adipose specific knockout (ASKO) mice exhibited significant reductions in plasma total cholesterol (↓21%, p < 0.05), HDL cholesterol (↓25%, p < 0.01), and phospholipid (↓17.6%, p < 0.05) levels, while fasting triglyceride levels were unaffected. ARV1 ASKO mice also had substantial reductions in epididymal adipose (WT: 0.41 +/- 0.07 g vs. KO: 0.10 +/- 0.07 g, p = 0.0002) and subcutaneous adipose tissue mass (WT: 0.32 +/- 0.03 g vs. KO: 0.11 +/- 0.08 g; p= 0.0002) on a chow diet. In contrast to nearly every other lipodystrophic mouse model, the reduced fat mass in these animals was paradoxically accompanied by improved glucose tolerance (WT AUC: 32,055 vs. KO AUC: 21,470 mg/dl*minutes, p<0.05). These data identify mammalian ARV1 as an important player in adipose tissue biology, and support an important role for adipose tissue in circulating lipoprotein metabolism.
Abstract 6: ADAMTS13 Reduces VWF-Mediated Acute Myocardial Ischemia/Reperfusion Injury in Mice
Authors: Gandhi Chintan , Lentz Steven , Chauhan Anil
Published: July 2021

Background and objective:
ADAMTS13 cleaves von Willebrand factor (VWF), a large multimeric protein that plays an important role in thrombus formation by binding to platelets following vascular injury. Epidemiological studies implicate elevated VWF levels and reduced ADAMTS13 activity in plasma as risk factors for myocardial infarction, but it remains unknown whether the ADAMTS13/VWF axis contributes to myocardial infarction pathogenesis. We tested the hypothesis that ADAMTS13 reduces VWF-mediated myocardial ischemia/reperfusion injury in mice.

Methods:
Myocardial infarction was induced in male mice (8-10 weeks of age) by ligating the left anterior descending coronary artery for 30 minutes followed by 23.5 hours of reperfusion. The extent of myocardium damage was evaluated by measuring infarct size (%) in 2 mm serial sections stained with 2% triphenyl-2, 3, 4-tetrazolium-chloride.

Results:
Adamts13
-/-
mice had significantly larger infarcts (mean ± SEM: 21.4 ± 1.3%,
P
<0.05) than WT mice (16.9 ± 1.2%) after myocardial ischemia/reperfusion injury.
Adamts13+/-
mice, which have a 50% reduction in ADAMTS13 activity, had similar sized infarcts (16.6 ± 1.3%) compared to those in WT mice. Because VWF remains the only known substrate of ADAMTS13 in multiple experimental models, we hypothesized that ADAMTS13 reduces myocardial injury through its proteolytic effect on hyper adhesive ULVWF and /or VWF. To test this hypothesis, we used a VWF-blocking antibody. Interestingly, WT mice treated with the VWF-blocking antibody showed a marked reduction in infarct size (7.9 ± 0.6%,
P
< 0.001) compared with WT mice treated with control Ig (17.5 ± 1.5 %). Finally,
Adamts13
-/-
mice treated with the VWF-blocking antibody had infarct sizes (8.0 ± 1.5%) that were similar to those WT mice treated with VWF-blocking antibody, demonstrating that increased infarct size in the
Adamts13
-/-
mice in this acute myocardial ischemia/reperfusion injury model is VWF-dependent.

Conclusion:
These findings reveal a new role for anti-thrombotic enzyme ADAMTS13 in reducing VWF-mediated myocardial ischemia/reperfusion injury.
Abstract 78: Chemokine-Driven β2 Integrin Adhesion Influences Macrophage Cytokine Gene Expression and Atherosclerotic Lesion Composition Through an Induced Rac2-Myosin IIa Interaction
Authors: Morrison Alan R , Ike Kenneth , Yarovinsky Timur , Bender Jeffrey
Published: July 2021

Chemokine-driven β2 integrin adhesion facilitates the activation and subendothelial migration of monocyte/macrophages in both acute and chronic vascular inflammatory processes. We have shown that engagement of β2 integrin contributes to stabilization of labile mRNAs encoding inflammatory cytokines (TNF-α, GM-CSF, IL-1), through translocation of the RNA binding protein HuR. These changes in cytokine gene expression influence monocyte/macrophage effector function, skewing the delicate balance between physiologic and pathologic responses. Using bone marrow-derived macrophages from wild-type and gene-deleted mice, we demonstrate that the hematopoietic cell-specific low molecular weight G protein, Rac2, is activated (GTP-bound) by β2 integrin engagement and required for effective integrin-induced nuclear-to-cytosolic HuR translocation. Moreover, this process depends on both the isoprenylation state of Rac2 and its interactions with Myosin IIA, an atypical myosin component of the cytoskeletal network. The HMG CoA reductase inhibitor atorvastatin leads to loss of Rac2 isoprenylation and consequent reduction in HuR translocation. The Myosin IIA-specific inhibitor, blebbistatin, disrupts the induced Rac2-myosin association, reduces translocation of HuR, and inhibits stabilization of cytokine mRNA. In Apo-E deficient mice, gene deletion of Rac2 influences atherosclerotic plaque composition, with more heavily calcified plaques identified by
ex vivo
microCT angiography. This provides evidence that Rac2’s effect on macrophage effector function influences consequential vascular pathology. In summary, chemokine-driven integrin engagement leads to isoprenylation-dependent activation of Rac2, resulting in translocation of HuR through an induced Rac2-Myosin IIA complex. This constitutes an important molecular switch in the activation of monocyte/macrophage inflammatory cascades, prolonging cytokine production and influencing the composition within vascular pathologies such as atherosclerosis. Targeting this newly identified signaling pathway has potential to yield a novel class of molecular therapeutics directed at influencing the course of vascular disease.
Abstract 85: Atherosclerosis Induces Thoracic and Abdominal Aortic Disease in Preaneurysmal Transgenic Fibulin-4 Mice
Authors: Ramnath Natasja , Van der Heiden Kim , Speelman Lambert , Ridwan Yanto , Van Heijningen Paula , Vermeij Marcel , Kanaar Roland , Rouwet Ellen , Essers Jeroen
Published: July 2021

Extracellular matrix degradation in the aortic wall plays an important role in the formation and progression of aneurysms. In mice, a deficiency of the extracellular matrix protein fibulin-4, induces matrix metalloproteinases (MMP) upregulation and transforming growth factor-β dysregulation. This results in the formation of aneurysms in the thoracic aorta of homozygous Fibulin-4
R/R
mice. In humans, aneurysms usually develop in the abdominal aorta during aging and are most often associated with atherosclerosis. To investigate the molecular mechanisms underlying the interaction of aneurysmatic and atherosclerotic aortic disease we developed a new mouse model.

Fibulin-4 knockdown (Fibulin-4
+/R
) mice, which display mild extracellular matrix changes, were crossbred with atherosclerotic ApoE
-/-
mice. The double mutant mice were fed a high fat diet for 10 or 20 weeks starting at 9 weeks after birth. Thoracic and abdominal aortic architecture were assessed
in vivo
using high-resolution ultrasound. MMP activity in the aorta was determined using fluorescent imaging with protease-activatable near-infrared fluorescence probes. Following sacrifice, plaque burden was evaluated through quantitative oil-red-o staining and histological analysis.

Already after 10 weeks of high fat diet, ApoE
-/-
Fibulin-4
+/R
mice displayed increased matrix metalloproteinase activity in the abdominal aorta and increased plaque formation at the level of the visceral arteries as compared with ApoE
-/-
Fibulin-4
+/+
mice, with further increases observed after 20 weeks of high fat diet. Surprisingly, after 20 weeks high fat diet the aortic diameter increased up to 60% in the aortic arch and 20% in the abdominal aorta in the ApoE
-/-
Fibulin-4
+/R
mice as compared with ApoE
-/-
Fibulin-4
+/=
mice.

This novel mutant mouse model is the first viable murine model which mimics the clinical situation of coexistent aneurysm formation and atherogenesis. It provides the opportunity to unravel the biological processes underlying aortic wall degeneration and to identify markers that elucidate key events in the early stages of the pathogenic sequence that culminates in an aneurysm. We identified that partial degradation of the extracellular matrix under the influence of atherosclerosis leads to aortic dilatation. In addition, we show that protease-activatable fluorescent sensors are promising new tools to monitor non-invasively the enhanced matrix metalloproteinases activity in the aortic wall that precedes aortic wall degeneration.
Abstract 87: Sterol-Mediated Regulation of γδ T Lymphocytes
Authors: Cheng Hsin-Yuan , Hedrick Catherine
Published: July 2021

Objective:
gammadelta (γδ) T lymphocytes, which bridge innate and adaptive immunity, may be differentially regulated by cholesterol and play distinct roles from the conventional alphabeta (αβ) subset in the development of atherosclerosis. γδ T cells represent a small subset of total T cells (∼ 3-5% of CD3+ cells). These cells express unique γδ T cell receptors, and recognize lipid and peptide antigens without processing and presentation by MHC molecules. γδ T cells react much faster toward pathogen invasion than αβ T cells, and thus are considered one of the first lines of immune defense.

Methods:
αβ and γδ T lymphocytes from mice were evaluated for their lipid content, gene expression, proliferation, apoptosis, and activation.

Results and conclusion:
Nile Red staining revealed higher intracellular lipid content in γδ T cells, comparing to the αβ subset (1.3 folds of αβ, p<0.05; n=5). We therefore hypothesize that the basal higher sterol content elicits the turnover and activation of γδ T lymphocytes for rapid response. We found that genes involved in cholesterol esterification (ACAT1 and ACAT2) and efflux (ABCA1) are upregulated in γδ cells compared to conventional αβ T cells. PCSK9, which downregulates LDLR posttranslationally, is elevated in γδ T cells, suggesting cholesterol metabolism is differentially regulated in the two subsets.

BrdU incorporation and annexin V staining revealed significantly higher proliferative (3.4 folds of αβ, p<0.001; n=8) and apoptotic (34.9 folds of αβ, p<0.001; n=6) rates of γδ T cells in vivo, suggesting a higher turnover rate of the γδ subset. Staining for ganglioside GM1 showed elevated lipid raft levels in γδ T cells (1.9 folds of αβ, p<0.001; n=8), implying enhanced TCR signaling and activation. Notably, the percentages of total (2.3 folds of αβ, p<0.01; n=3) and pro-inflammatory cytokine IL-17- producing (3.9 folds of αβ, p<0.01; n=4-5) γδ T cells increase in ApoE
-/-
mice fed a high cholesterol, high fat diet, implying a high sterol/high fat environment may promote γδ T cells activation in vivo. Taken together, our data suggest γδ T cells are more sensitive to cholesterol, and a high sterol environment may enhance γδ T cell turnover and activation in vivo.
Abstract 96: Aberrations in the Sonic Hedgehog/Notch Signaling Pathway in Abdominal Aortic Aneurysms
Authors: Doyle Adam J , Gillespie David L , Redmond Eileen M , Knight Peter , Kumar Neil G , Hislop Sean J , Roztocil Elisa , Chandra Ankur , Cullen John P , Morrow David J
Published: July 2021

Objectives:
The molecular mechanisms leading to the development of Adnominal Aortic Aneurysms (AAA) remain poorly understood. Vascular Smooth Muscle Cells (VSMCs) are fundamental to maintaining a healthy arterial wall and changes in VSMC phenotype may be pivotal to aneurysm development. We have recently determined a role for a Hedgehog (Hh)/Notch signaling cascade in regulating adult VSMC phenotype. The aim of this study was to investigate Hh/Notch signaling components in aneurysmal and non-aneurysmal aorta. As crosstalk between Notch and Transforming Growth Factor Beta (TGFβ) has been postulated we also investigated expression of this growth factor.

Methods:
Tissue samples were obtained from aneurysmal and non-aneurysmal segments of the aortic wall of at least 5 patients with suitable anatomy undergoing open repair of infrarenal AAA. All samples analyzed were paired from the same patient with aneurysmal and non-aneurysmal specimens. Protein and mRNA expression levels were determined by western blot analysis and quantitative Real-time PCR respectively.

Results:
SHh, Notch 1 and 3 IC protein expression was decreased by at least 50% in aneurysmal tissue when compared to non-aneurysmal tissue. In addition, SHh mRNA expression was also decreased by 65%, while there was a decrease in Dll4, Notch 1 and Notch 3 by 66%, 57% and 54% respectively. In contrast, aneurysmal tissue had significantly increased expression of TGFβ and MMP9. TGFB protein and mRNA expression was significantly increased by 5.45±2.13 and 2.5±.2 fold respectively in aneurysmal tissue when compared to non-aneurysmal tissue. Furthermore, MMP9 mRNA expression was significantly increased by 4.7±1.6 fold. In parallel experiments, SMC alpha actin protein expression was significantly decreased by 90% in aneurysmal tissue when compared to non-aneurysmal tissue.

Conclusion:
These results suggest that SHH/Notch and TGFβ signaling is altered in aneurysmal tissue, compared with non-aneurysmal tissue. Changes in these signaling pathways and resulting changes in VSMC phenotype may play a role in the development of AAA.
Abstract 106: Transintestinal Cholesterol Excretion and Macrophage Reverse Cholesterol Transport are not Stimulated in Hepatic ABCG8 Knockdown Mice Treated with an LXR Agonist
Authors: McDaniel Allison L , Temel Ryan E , Brown J M , Lee Richard G , Graham Mark J , Crooke Roseanne M
Published: July 2021

Transintestinal cholesterol excretion (TICE) is a recently discovered pathway by which cholesterol travels from plasma to the small intestine for direct excretion into the feces. Hallmarks of animal models with TICE include severely diminished biliary cholesterol secretion but near normal levels of hepatic cholesterol and fecal neutral sterol excretion. Using an ATP binding cassette transporter G8 (ABCG8) antisense oligonucleotide (ASO) to knock down ABCG8 specifically in liver (G8
HKD
), we created a novel mouse model with significantly decreased biliary cholesterol excretion but a 658% increase in hepatic cholesterol accumulation and a 78% reduction in fecal neutral sterol excretion, indicating a dysfunction in the TICE pathway. LXR agonists have previously been shown to stimulate the TICE pathway. In order to more definitively prove the TICE pathway was disfunctional in G8
HKD
mice, we treated wild type (WT) and G8
HKD
mice with the LXR agonist T0901317 and measured markers of TICE stimulation. As expected, in WT mice, T0901317 doubled biliary cholesterol concentrations. A similar effect was seen in G8
HKD
mice treated with T0901317, but biliary cholesterol concentrations remained significantly less than their WT counterparts. These levels of biliary cholesterol closely mirrored hepatic ABCG8 mRNA expression. T0901317 stimulated fecal neutral sterol excretion by >1000% in wild type mice but only by 190% in G8
HKD
mice. These data indicate that TICE is disfunctional in G8
HDK
mice since the pathway was not stimulated to the same extent in WT and G8
HKD
mice by an LXR agonist. Some controversy remains over whether the TICE pathway transports macrophage derived cholesterol. In order to address this issue, we performed a macrophage RCT assay on WT and TICE disfunctional G8
HKD
mice. T0901317 stimulated macrophage RCT (fecal neutral sterol 3H dpm) by >2300% in wild type mice but only by 370% in G8
HKD
mice. T0901317 increased fecal acidic sterol 3H count by 65-75% in both wild type and G8
HKD
mice. These results indicate that macrophage RCT is impaired when the TICE pathway is decreased. In sum, our data shows that hepatic ABCG8 plays a key role in the TICE pathway and that impairing the TICE pathway through hepatic ABCG8 knockdown causes decreased macrophage RCT.
Abstract 127: The Mediterranean Diet With and Without Weight Loss Improves Several Features of Low-Density Lipoprotein Metabolism in Men With Metabolic Syndrome
Authors: Richard Caroline , Couture Patrick , Desroches Sophie , Lamarche Benoît
Published: July 2021

Objective

To assess the impact of a Mediterranean type diet (MedDiet), without weight loss (-WL) and with weight loss (+WL), on features of LDL and LDL- apolipoprotein (apo) B metabolism in men with metabolic syndrome (MetS).

Methods and Results

The diet of 19 men with MetS (NCEP-ATP III, age 24-62 years) was first standardized to a typical North American control diet that they consumed for 5 weeks under isoenergetic feeding conditions (all foods and beverages provided). Subjects were then fed a MedDiet for 5 weeks also under isoenergetic conditions (MedDiet-WL), after which they underwent a 20-week weight loss period in free-living conditions (average weight loss -10.2 ± 2.9% body weight, P<0.01). This was followed by consumption of the MedDiet (5 weeks) under isoenergetic conditions (MedDiet+WL). Features of LDL and apo B kinetics were assessed at the end of the three controlled feeding periods. MedDiet-WL reduced VLDL-apoCIII (-41.5%), cholesteryl ester transfer protein (-7.0%) and hepatic lipase concentrations (-7.1%), reduced the proportion of small LDL (LDL<255 å, -11.7%) and increased LDL peak particle diameter (LDL-PPD, +0.7%) as well as the proportion of medium size LDL (LDL255-260 å, +11.1%) vs. control diet (all P<0.05). MedDiet-WL also decreased LDL-apoB pool size (PS, -14.2%) and increased the fractional catabolic rate (FCR) of LDL-apoB (+30.4%) vs. the control diet (both P<0.05), with no change in LDL-apoB production rate (PR). MedDiet+WL was associated with further increase in LDL-PPD (0.2%) and further reduction in VLDL-apoCIII concentrations (-28.7%) compared with MedDiet-WL (both P<0.05) but had no further impact on LDL-apoB PS and on LDL-apoB FCR.

Conclusion

Data from this controlled feeding study suggest that consumption of MedDiet, even in the absence of weight loss, decreases LDL-C concentrations and LDL-apoB pool size primarily by enhancing the clearance of LDL particles in men with MetS. This is accompanied by a reduction in the proportion of small dense LDL and an increase in LDL particle size. Combining weight loss to MedDiet has no additional impact on LDL-apoB pool size and catabolism but may have further beneficial effects on the LDL particle size phenotype in men with MetS.
Abstract 163: The Prevalence of the Metabolically Healthy Obese Phenotype in an Aging Population and its Association with Subclinical Cardiovascular Disease: The Brazilian Study on Healthy Aging
Authors: Roberson Lara L , Siddiqui Shozab A , Blaha Michael J , Agatston Arthur A , Blumenthal Roger S , Santos Raul D , Nasir Khurram , Sposito Andrei C
Published: July 2021

BACKGROUND

Obese and overweight individuals have been shown to be at higher risk of CVD events than normal weight individuals. Current literature has elucidated a new phenotype, Metabolically Healthy Obese (MHO), with risks of CVD similar to that of normal weight individuals. Few studies have examined the MHO phenotype in an aging population, especially in association with subclinical cardiovascular disease.

METHODS

The cross sectional study population consisted of 208 individuals (79% Female), age 80 and older (mean age 84±4, range 80-102). Anthropometrics & biochemical parameters were measured. The Adult Treatment Panel definition of metabolic syndrome (MetS), excluding waist circumference, criteria was used to define metabolically healthy (<3 MetS components) versus unhealthy. A combination of BMI and waist circumference were used to define normal weight and overweight/obese. Multidetector-row cardiac CT for coronary artery calcium score (CACS) was used to detect subclinical atherosclerosis. High reactive C reactive protein (hs-CRP) was measured to assess degree of underlying inflammation.

RESULTS

The prevalence of MHO defined by BMI≥25 kg/m2 &/or waist circumference >88cm in women, >102cm in men & having 3mg/dl, Uric Acid >6 mg/dl (p=NS). Gender, total cholesterol, HDL, LDL, triglycerides, and SBP was significantly associated with MHO (p<0.05).

CONCLUSIONS

Our results suggest that the MHO phenotype is still seen in octogenarians, but at lower rates than in the general population suggesting MHO may not simply be an intermediary stage, driven by length of spent in the obese state. Those with this phenotype tended to have lower triglycerides, higher HDL, and lower body fat % than their metabolically at risk obese counterparts (p<0.05), however, degree of subclinical CVD was not different. Further studies are needed to explore the related risk of CVD among MHO octogenarians.