bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 308: Cardiac Cycle Affects Ultrasound Measurements of Ascending Aortic Diameter in a Marfan Mouse Model
Authors: Chen Zheying , Sawada Hisashi , Rateri Debra , Daugherty Alan , Sheppard Mary
Published: July 2021

Objective:
Ultrasound measurements of aortic diameter are a common endpoint in preclinical studies. However, there is a lack of standardization in both image capture and analysis. For our study, we developed a standardized protocol for measuring ascending aortic diameter and examined effects of cardiac cycle in wild type and fibrillin-1 hypomorphic (FBN
mgR/mgR
) mice.

Methods and Results:
Twelve week old male and female FBN
mgR/mgR
mice were anesthetized and maintained at a heart rate of 450-550 beats per minute. Ultrasound images were captured using a Vevo 2100 system with a 40MHz tranducer. Images captured were standardized according to two anatomical landmarks: the innominate artery branchpoint and aortic valves. The largest luminal ascending aortic diameter between the sinotubular junction and the innominate artery were measured in mid-systole and end-diastole by two blinded, independent observers.

Aortic diameters were significantly different (p<0.05) when comparing systole and diastole within gender and genotype. Interestingly, wild-type male (n=4) and female (n=3) mice exhibited a 19% and 15% expansion of the ascending aorta respectively during systole compared to diastole. This difference was not recapitulated in either male (n=6) or female (n=5) FBN
mgR/mgR
mice (4% expansion in both; p<0.05 vs wild-type). Agreement between observers was excellent (R^2 = 0.99) but interobserver variability was a mean of .09 mm (%CV = 5%)

Conclusion:
As expected, there is a difference in aortic diameters between wild-type and FBN
mgR/mgR
mice. Luminal aortic diameters in FBN
mgR/mgR
vs wild-type mice of both genders are affected by cardiac cycle. Mid-systolic aortic expansion in wild-type vs FBN
mgR/mgR
mice were different. Error introduced by interobserver variability impacts ascending aortic measurements. Altogether, these phenomena may confound analyses of aortic dilation in FBN
mgR/mgR
mice, especially when studying interventions with modest effect sizes.
Abstract 32: Ly6C
Lo
Monocyte/Macrophages Are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis
Authors: Kimball Andrew S , Luke Cathy , Cai Qing , Obi Andrea , Jaffer Farouc , Gallagher Katherine , Henke Peter
Published: July 2021

Venous thrombosis (VT) results in vein wall injury by promoting inflammation and fibrosis leading to venous reflux, swelling, pain, and potentially, recurrent thrombosis. While prior work has shown that infiltrating leukocytes are important for VT resolution, as of yet, the precise roles of different leukocyte subsets are not well understood. Monocyte/macrophages (Mo/MΦs) are essential for the repair and resolution of tissue injury in other models, and come in inflammatory (Ly6C
Hi
) or pro-resolution (Ly6C
Lo
) subtypes. We hypothesized that infiltrating Mo/MΦs would be critical to VT resolution. In order to study this
in vivo
, we utilized a conditional macrophage depletion technique, using CD11b-DTR mice, to examine the effects of Mo/MΦs in a murine model of stasis VT by inferior vena cava ligation. Administration of 10ng/g diphtheria toxoid (DTx) every 48 hours by intra-peritoneal injection in CD11b-DTR mice resulted in an 89% and 55% decrease in circulating monocytes at 24hrs and 48hrs, respectively. When compared to saline controls, DTx injection had no effects on thrombogenic response or IVC thrombus cell populations in C57BL/6 control mice. At 8 days’ post-ligation, DTx treated CD11b-DTR mice had preferentially decreased vein wall-thrombus Ly6C
Lo
Mo/MΦs as compared with controls. DTx treated mice had significantly larger thrombi (1.7-fold) and less TGF-β, FSP-1, and plasminogen by western immunoblotting (all P-values ≤ 0.01). Consistent with a reduction in Ly6C
Lo
Mo/MΦs was a significant decrease in cellular TGF-β by intra-cellular flow cytometry. These findings suggest that Ly6C
Lo
Mo/MΦs are essential for normal VT resolution and may promote thrombus resolution via a plasminogen-mediated mechanism.
Abstract 330: Autophagy in Vascular Calcification
Authors: St. Hilaire Cynthia
Published: July 2021

Vascular calcification accompanies a variety of common cardiovascular-related diseases and correlates with premature death. Vascular calcification is a highly-regulated process but the precise mechanisms inducing this pathology are not fully understood, and currently no treatment exists that halts or reverses vascular calcification. We previously discovered the rare monogenetic disease Arterial Calcification due to Deficiency of CD73 (ACDC) which presents with vessel tortuosity and extensive calcifications in the medial layer of lower-extremity arteries. To study the mechanisms underlying this disease we previously created ACDC and Control patient iPSCs, and using a teratoma assay discovered that ACDC iPSC teratomas exhibit extensive calcifications, while Control iPSC teratomas do not. Drug screening identified that rapamycin inhibited calcification in this in vivo model. Using our in vitro calcification model with ACDC fibroblasts, we again found that rapamycin inhibited calcification, as well as expression and activity of the key enzyme involved in ectopic calcification, tissue non-specific alkaline phosphatase (TNAP). To corroborate these findings in vascular cells we used coronary artery smooth muscle cells (CASMCs) in our in vitro calcification assay. We found CASMCs calcify in vitro, and that both rapamycin treatment, and importantly, specific activation of autophagy, inhibited calcification in CASMCs. This suggests autophagy as a therapeutic target for the treatment of vascular calcification. Future work will identify how autophagy prevents calcification, and whether rapamycin, which induces autophagy, can be used as a therapy for medial calcification.
Abstract 331: Are Two Internal Thoracic Grafts Better Than One in Patients With Peripheral Vascular Disease?: Analysis of 858 Cases Between 1996- 2011
Authors: Teich Nadav , Mohr Refael , Pevni Demitry , Assaig Yael , Kramer Amir , Nesher Nahum , Paz Yosef , Ben-Gal Yanai
Published: July 2021

Objectives:
Bilateral internal thoracic artery( ITA) grafting is associated with improved survival. However, potential survival benefit of using two ITA`s in patients with peripheral vascular disease(PVD) is questionable due to their short life expectancy and the increased risk of sternal wound infection(SWI) compare to operations incorporating single ITA (SITA). The purpose of this study is to compare early and long-term outcome of bilateral ITA grafting(BITA) to that of SITA and vein grafts in PVD patients with multi-vessels coronary disease.

Methods:
Five hundred and thirty three PVD patients who underwent BITA between 1996 and 2011 were compared with 319 who underwent SITA .

Results:
SITA patients were more often female, more likely to have Diabetes, chronic obstructive lung disease, unstable angina, previous CABG, renal insufficiency, Cerebro- vascular disease and emergency operation .On the other hand congestive heart failure and triple vessel coronary disease were more prevalent among BITA patients. Operative mortality(3.5% vs. 4.1%, in the SITA and BITA respectively) and occurrences of SWI (6.6% vs 3.9%) and strokes(4.1% vs 6.8%) were not significantly different between groups . BITA patients did not have better Kaplan- Meier 10 year survival ( 52.1% vs.47.1%, p=0.145) and after propensity score matching(243 well matched pairs) BITA was not associated with better adjusted survival ( HR 1.108[95%CI 0.810-1.516] p=0.521)(cox model)

Conclusion:
This study does not support routine use of BITA in PVD patients .Earlier mortality from non-cardiac causes, reduces contribution of BITA and increases the influence of PVD and other co-morbidities on survival. Selective use of BITA in lower risk patients might un-mask the benefits of BITA
Abstract 337: Kidney Injury Affects Metabolism and Modification of Apolipoprotein A1 Promoting Renal Lymphangiogenesis
Authors: Zhong Jianyong , Yang Hai-Chun , Tsuchida Yohei , Matsusaka Taiji , Fogo Agnes , Linton MacRae , Davies Sean , Kon Valentina
Published: July 2021

Kidney disease causes ApoAI/HDL to lose their vasculoprotective effects. A likely mechanism for the protective loss is modification of ApoAI/HDL reactive carbonyls which are increased. Kidney injury increases reactive carbonyls and permits more ApoAI to escape into the glomerular ultrafiltrate. We examined how apoAI was metabolized in the injured proteinuric kidney. Nphs1-hCD25 mice (NEP25) expressing human CD25 in podocytes were injured by immunotoxin, LMB2. Six weeks after LMB2, we assessed urinary excretion and intrarenal localization of ApoAI and examined the renal lymphatic network with podoplanin. In vitro, we studied proximal tubular cells (PTC) and lymphatic endothelial cells (LEC) exposed to ApoAI or modified ApoAI (IsoLG-ApoAI). NEP25 mice developed glomerulosclerosis, increased urinary excretion of albumin and ApoAI with high molecular weight forms of ApoAI appearing in urine of injured mice assessed by western blot. Proteinuric kidneys had more ApoAI and VEGFC staining that localized to tubular epithelial cells and showed a dramatically more dense and complex lymphatic network than wild type mice. ApoAI localized to lymphatic vessels. Cultured PTC and LEC responded differently to normal and isolevuglandin (IsoLG)-modified ApoAI. In PTC, IsoLG-ApoAI promoted more ApoAI uptake and VEGFC secretion, a powerful lymphaniogenic stimulus. In LEC, IsoLG-ApoAI led to greater cell viability, migration and ROS production than normal ApoAI. Notably, supernatant of PTC exposed to ApoAI secreted a high molecular weight form of ApoAI. In conclusion, injury that disrupts the glomerular filtration barrier causing proteinuria also leads to a dramatic increase in filtered ApoAI followed by greater uptake by the proximal tubules, interstitium and lymphatic vessels. Proximal tubules can modify ApoAI and activate tubular secretion of VEGFC, which in turn promotes renal lymphangiogenesis and encourages delivery of kidney-modified ApoAI into circulation.
Abstract 347: Cigarette Smoking Associated Changes on Platelet Surface
Authors: Lowery Curtis L , Marsh James D , Kilic Fusun
Published: July 2021

Thirty percent of all deaths from coronary heart disease each year in the US are attributable to cigarette smoking. The acute effects of smoking on the cardiovascular systems are associated with arterial thrombosis. Cigarette smoking produces central nervous system-mediated activation of the sympathetic nervous system, which stimulates secretion of pro-thrombotic molecules, serotonin (5-HT) and catecholamines into the blood at supraphysiological levels. 5-HT and catecholamines activate their platelet-specific receptors in an additive manner to change the platelet biology and physiology. Our studies of blood samples from smokers (collected just after smoking) showed several-fold elevation in plasma 5-HT and catecholamines, and platelet aggregation. To explore smoking-associated changes on platelets, the plasma membrane glycans and proteins were eluted and analyzed in MALDI-MS and LC-MS, respectively. The surface glycans of nonsmokers’ platelets were high-mannose-type but those of smokers’ platelets were on smokers’, sialylated N-glycans. Removing the N-glycan counteracted smoking-mediated platelet aggregation, suggesting that N-glycans play a role in adhesiveness of the platelet surface. MS analysis of the membrane proteins identified enzymes and proteins exclusively on smokers’ platelet plasma membrane. These included enzymes involved in glycan biosynthesis and proteins important in membrane trafficking of secretory vesicles. We hypothesize that, upon smoking, glycosylation enzymes normally sequestered in intracellularly, are translocated to the plasma membrane to induce changes in surface glycans. Interestingly, we showed that treating nonsmokers’ platelets with 5-HT and catecholamines predisposed them to a prothrombotic state. Furthermore, pharmacological blockade of receptors for 5-HT or catecholamines counteracted the 5-HT-catecholamine-mediated aggregation and alterations in level and composition of surface glycans. Achieving our proposed aims potentially will provide novel biomarkers and pharmacological targets for preventing and treating smoking-associated thrombosis.
Abstract 36: Factor XII RNAi-based Therapeutic as a Prophylactic Anti-thrombotic Therapy
Authors: Melquist Stacey , Pei Tao , Hamilton Holly , Chu Qili , Schienebeck Casi , Chapman Christine , Hagen Collin , Walters Megan , Milarch Tracie , Trilling Zach , Andersen Aaron , Christensen Gary , Casper Jeff , Klas Crystal , Hegge Julia , Li Zhen , Kanner Steven
Published: July 2021

Introduction:
Current anticoagulants effectively prevent thromboembolic (TBE) events, but unmet medical need remains due to elevated risk of major bleeding events, particularly where Factor Xa inhibitors are contraindicated. Factor XII (F12) zymogen initiates the intrinsic coagulation pathway through both autocleavage and Factor XI cleavage. F12 deficient mice show protection from induced thrombosis without increased bleeding risk. F12 deficiency in humans is not associated with increased bleeding risk, suggesting F12 is not essential for hemostasis.

Methods:
Hepatocyte-targeted RNA interference (RNAi) triggers for reducing liver F12 expression were developed for subcutaneous (SQ) administration. Specific rodent/human/non-human primate (NHP) cross-reactive RNAi triggers were designed
in silico
and screened for knockdown activity
in vitro
and in wild-type mice. Structure activity relationship (SAR) studies of active RNAi triggers identified optimal modifications for lead identification. Candidate RNAi triggers were evaluated for activity and safety in NHPs. In addition, candidate RNAi triggers were evaluated for activity in two rodent models of thrombosis and bleeding risk.

Results:
F12 RNAi triggers exhibited significant and sustained knockdown of serum F12 levels in both mice and NHPs. SAR studies enabled identification of a candidate lead molecule that demonstrated >95% knockdown after a single 1 mg/kg dose in mice. Evaluation of two SQ doses of this RNAi trigger in NHPs demonstrated >93% knockdown of serum F12 levels at nadir with durable knockdown of >90% for over 5 weeks after the final dose. Concomitant increases in coagulation biomarker aPTT were observed, consistent with functional F12 depletion. Both arteriovenous shunt studies in rats and FeCl
3
-induced TBE studies in mice exhibited prevention of clot formation after F12 knockdown. Importantly, F12 knockdown did not increase bleeding times in explicit hemostasis studies in mice.

Conclusions:
Development of a SQ-administered F12-specific RNAi trigger offers potential for a novel, infrequently dosed prophylactic treatment for TBE without increased bleeding risk. Administration by medical personnel would eliminate adherence concerns inherent to current therapies.
Abstract 365: Inhibiting Hmg Coa Reductase Reduces Palmitate-Induced Inflammation in Adipocytes
Authors: Pathak Asha K , Han Chang Yeop , Omer Mohamed , Wang Shari , Chait Alan
Published: July 2021

Adipose tissue inflammation associates with insulin resistance and increased cardiovascular disease risk. We previously observed that 3T3-L1 adipocytes exposed to palmitate become inflamed and demonstrate increased plasma membrane cholesterol and lipid raft content. It is known that palmitate induces translocation of NAPH oxidase and toll-like receptor 4 into lipid rafts, increasing adipocyte inflammation. However, it is unclear (1) how palmitate alters plasma membrane cholesterol content; and (2) whether increased cholesterol content in the plasma membrane is related to adipocyte inflammation induced by palmitate exposure. We hypothesize that mechanisms involved in increasing plasma membrane cholesterol content after palmitate treatment could be related to cholesterol synthesis and/or ER stress, and that increased cholesterol in lipid rafts is essential for induction of inflammation in adipocytes. To test these hypotheses, differentiated murine 3T3-L1 adipocytes were exposed to palmitate for 24 hours, with and without pre-treatment with HMG-CoA reductase inhibitors (statins) or HDL. RT-PCR was used to evaluate gene expression of inflammation (
Saa3
,
Ccl2
), ER stress (
Bip
,
Chop
), and HMG-CoA reductase (
Hmgcr
). Cholera toxin subunit β staining and flow cytometry were used to evaluate plasma membrane lipid raft content. In differentiated adipocytes, palmitate-induced inflammation neither increased expression of ER stress genes nor HMG-CoA reductase gene expression. However, treatment with 3 different statins (simvastatin, lovastatin, atorvastatin) significantly reduced palmitate-induced adipocyte inflammation as indicated by decreased gene expression of
Saa3
and
Ccl2
(
P
<0.05). A similar effect was seen with pre-treatment with HDL. Lipid raft content induced by palmitate was decreased by HMG-CoA reductase inhibitors (difference in mean fluorescence intensity
P
<0.05) and also by pre-treatment with HDL. These findings indicate that ER stress was not involved in increased plasma membrane cholesterol after palmitate-induced inflammation in adipocytes. However, regulating cholesterol content in lipid rafts plays an important role in adipocyte inflammation induced by palmitate.
Abstract 370: Contrast-Induced Nephropathy and Long-Term Outcomes in Patients With Diabetes Undergoing Coronary and Peripheral Angiography and Intervention
Authors: Hopkinson Dennis A , Powell Samuel P , Chawla Raveen , Bottinor Wendy , Turlington Jeremy S , Patel Kalpesh , Danyi Peter , Jovin Ion S
Published: July 2021

Background:
Patients with diabetes may be at increased risk of contrast-induced nephropathy (CIN) when undergoing coronary and/or peripheral angiography or intervention but there is little data on long-term outcomes. We examined the relationship between diabetes, CIN and long-term outcomes in patients undergoing coronary and/or peripheral angiography and intervention.

Methods and Results:
We studied 4070 consecutive, predominantly (98%) male patients undergoing coronary and peripheral angiography and intervention and assessed the association between diabetes, CIN and long-term outcomes including renal dysfunction at 3 months, the need for dialysis and mortality. The mean age of the patients was 66.6 years. Approximately two fifths of the patients (n=1671, 41.05%) were diabetic. Patients with diabetes were the same age but had higher baseline creatinine compared to the patients without diabetes. CIN occurred in 70 (4.19%) diabetic patients and in 64 (2.67%) patients without diabetes at 72 hours after the procedure (odds ratio [OR] 1.59; 95% confidence interval [CI] 1.13 - 2.25; P=0.008). At 3 months, renal dysfunction was seen in 179 (10.71%) diabetic patients versus 174 (7.25%) of the non-diabetic group (OR 1.53, CI 1.23 - 1.91; P=0.0001). After a follow-up of 5 years, 31 (1.86 %) patients with diabetes had developed end-stage renal disease and were started on dialysis versus 13 (0.54 %) of the non-diabetic group (OR 3.47, CI 1.81 - 6.65; P<0.0001). 478 (28.61 %) patients of the diabetic group had died versus 479 (19.97 %) of the non-diabetic group (OR 1.61, CI 1.39 - 1.86; P<0.0001). On multivariate analysis, after adjustment for age, comorbidities, medical therapy and baseline creatinine, the presence of diabetes was significantly associated with CIN (OR 1.50, CI 1.06 - 2.43: p=0.02) and was significantly associated with the incidence of end stage renal disease requiring dialysis (OR 3.64, CI 2.07-10.04; P<0.0001) and with mortality at 5 years (OR 1.58, CI 1.42-2.03, P<0.0001).

Conclusion:
In this cohort of patients undergoing coronary and/or peripheral angiography and intervention diabetes was associated with CIN, with end-stage renal disease and the need for hemodialysis and was associated with an increased mortality.
Abstract 372: PPARα/γ Agonist Tesaglitazar Reduces Inflammatory Macrophage Numbers And Induces Adipose Ucp-1 Expression In Diabetic Ob/ob Mice
Authors: Osinski Victoria , Harms Matthew , Boucher Jeremie , McNamara Coleen
Published: July 2021

Rationale:
PPARα/γ agonist tesaglitazar effectively improves insulin sensitivity and dyslipidemia in diabetic mice and human subjects. The cellular and molecular mechanisms whereby this occurs remain unclear. Given the established expression of PPARs in macrophages and
in vitro
findings demonstrating attenuated inflammation with PPAR agonism, we hypothesized that tesaglitazar attenuates macrophage-mediated inflammation within the adipose to improve metabolic outcomes in diabetic mice.

Methods & Results:
To validate that tesaglitazar reduces diabetic symptoms, we treated Ob/ob mice with tesaglitazar or vehicle control for one week and observed significant reductions in fasting blood glucose, insulin, and triglyceride levels with drug treatment when compared to vehicle treatment. Using flow cytometry, we determined that treatment with tesaglitazar results in reduced numbers of total macrophages (CD45+CD11b+F4/80+) in the epididymal and subcutaneous adipose depots. Furthermore, this reduction in macrophages numbers is primarily due to reduced numbers within the M1, or pro-inflammatory, compartment (CD11b+F4/80+CD11c+CD206-) of the macrophage population. Given the established role of inflammation in inhibiting adipocyte Ucp-1 expression, we measured Ucp-1 expression levels in epididymal adipocytes and observed a significant induction of Ucp-1 mRNA expression with tesaglitazar treatment.

Conclusion:
As anticipated, treatment with tesaglitazar improves insulin sensitivity and dyslipidemia in Ob/ob mice. Interestingly, tesaglitazar treatment also reduced the number of inflammatory macrophages in adipose and induced adipocyte Ucp-1 mRNA expression. Taken together, our findings suggest that tesaglitazar has anti-inflammatory effects indicated by reduced numbers of pro-inflammatory macrophages and increased thermogenic capacity as seen by increased Ucp-1 expression.
Abstract 378: Red Blood Cells From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Through Up-Regulation of Arginase I
Authors: Zhou Zhichao , Mahdi Ali , Tratsiakovich Yahor , Kövamees Oskar , Yang Jiangning , Pernow John
Published: July 2021

We previously showed that increased arginase activity is a key mechanism for endothelial dysfunction in patients with type 2 diabetes mellitus (T2DM) thereby arginase inhibition improves endothelial function. Recently, we demonstrated a crucial role of red blood cells (RBCs) in control of cardiac function via an arginase-dependent regulation of nitric oxide export from RBCs, suggesting a direct interaction of RBCs with cardiovascular function. Considering an increase in arginase activity in T2DM, we hypothesized that RBCs induce endothelial dysfunction in T2DM via up-regulated arginase I. Healthy rat aortas were incubated with RBCs from patients with T2DM (T2DM-RBCs) and age-matched healthy subjects (H-RBCs) for 18 h in the absence and presence of the arginase inhibition or scavenging of reactive oxygen/nitrogen species (ROS/RNS). Following the incubation, endothelium-dependent and -independent relaxations (EDR and EIR) were determined using wire myograph. Human internal mammary arteries (IMAs) obtained from non-diabetic patients who underwent cardiac surgery were also incubated with RBCs for functional evaluation. Arginase activity and protein expression were determined in RBCs. EDR was impaired in vessels incubated with T2DM-RBCs (Emax: 43.2±3.0% in aortas, n=8; 32.3±2.7% in IMAs, n=3) but not H-RBCs (Emax: 74.3±3.4% in aortas; 71.5±5.1% in IMAs) in comparison with buffer (Emax: 74.4±2.3% in aortas; 73.1±5.0% in IMAs; P<0.01 vs. T2DM-RBCs). EIR was not affected by T2DM-RBCs. The impairment in EDR in rat aortas was fully reversed by inhibition of arginase, ROS and RNS in RBCs. Arginase activity was significantly elevated in T2DM-RBCs. The increased arginase activity was attributed to arginase I, as there was increased arginase I expression in RBCs, whereas no arginase II expression was detected. Moreover, high glucose and RNS stimulation increased arginase activity in H-RBCs, while ROS/RNS scavenging decreased arginase activity in T2DM-RBCs. This study demonstrates a novel mechanism behind endothelial dysfunction that T2DM-RBCs induce endothelial dysfunction via ROS/RNS-dependent up-regulation of arginase I. Targeting arginase I in RBCs may serve as a novel therapeutic tool for treatment of endothelial dysfunction in T2DM.
Abstract 387: HDL-apolipoprotein A-I Exchange Occurs in the Absence of HDL Particle Remodeling
Authors: Borja Mark S , Phelps Armen , Oda Michael N
Published: July 2021

Objective:
Apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states. This is a key event in reverse cholesterol transport, necessary for
de novo
HDL biogenesis. However, the mechanism of HDL-apoA-I exchange (HAE) is poorly understood. In this study, we test whether HDL remodeling is obligatory to HAE or an independent process.

Method and Results:
Recombinant human apoA-I modified with single cysteine mutations were expressed in bacteria and labeled with Alexa 488 and Alexa 647 fluorophores. ApoA-I
Alexa647
was used to synthesize rHDL particles of defined sizes with phosphatidyl choline (POPC) and cholesterol. Reconstituted HDL of 17.0, 12.2, 9.6, 8.4 and 7.8 nm were obtained. These particles were incubated with excess lipid-free apoA-I
Alexa488
to initiate HAE, which was quantified using non-denaturing gradient gel electrophoresis to separate HDL particles from lipid-free apoA-I. Gels were imaged using FITC and Cy5 filters. The extent of apoA-I
Alexa488
binding and apoA-I
Alexa647
released from HDL was determined by densitometry. HAE occurred without significant changes in rHDL particle size for all subclasses of rHDL. HAE in the forward (lipid-free to bound) direction did not exhibit size dependence, but HAE in the reverse (lipid-bound to free) direction appeared to be size-dependent, with the 8.4 nm rHDL displaying 2-fold higher apoA-I
Alexa647
dissociation compared to the 17.0 nm particle.

Conclusions:
The bidirectional rate of HAE can be measured simultaneously using fluorescent apoA-I probes. Reconstituted HDL particles predominantly retained their size, indicating that apoA-I exchange occurs without remodeling of HDL particles. This result has significant implications as to the possible molecular processes driving apoA-I exchange with HDL.
Abstract 389: The Role of Amadori-Glycation in High Density Lipoprotein Dysfunction and Oxidative Stress in Patients With Diabetes
Authors: Kasumov Takhar , Golizeh Makan , Lee Kwangwon , Ilchenko Serguei , Wang Shuhui , Smith Jonathan , Kashyap Sangeeta
Published: July 2021

Objectives:
Diabetes is associated with HDL dysfunction and oxidative stress. We recently demonstrated that HDL from patients with T2D has reduced anti-oxidant activity and it is pro-inflammatory. Here we used the
2
H
2
O-based metabolic labeling approach to test the hypothesis that hyperglycemia-induced glycation contributes to HDL dysfunction and oxidative stress in diet-controlled patients with T2D.

Methods:
HDL from patients with T2D and age- and BMI-matched healthy controls (n=7/group) was isolated and it’s anti-oxidant and cholesterol efflux properties were quantified. ApoAI cross-linking and HDL particle size distribution were anlyzed. HDL proteome composition and post-translational modification of proteins were quantified by shot-gun proteomics aproach. Metabolic
2
H
2
O-labeling coupled with high resolution mass spectrometry was applied to quantify HDL turnover and HDL proteome dynamics.

Results:
Despite significant differences in fasting blood glucose, insulin, insulin resistance and HbA1c, patients with T2D and controls had similar lipid (triglycerides, total cholesterol, and HDL cholesterol) and lipoprotein (apoAI and apoB100) profile. HDL from patients with T2D displayed increased levels of cross-linked apoAI and lipid-poor pre-β1 HDL partcicles. ApoB-depleted serum from T2D patients had reduced PON1 activity and macrophage-cholesterol efflux capacity, and increased levels of lipid peroxidation products measured as TBARS (all
P<0.05
). HDL from patients with T2D was enriched with Amadori glycated apoAI and transferrin (Tf), the key HDL proteins involved in cholesterol and iron transport. The extent of glycations were associated with increased degradation of apoAI and Tf. ApoAI glycation was inversely correlated with ABCA1-induced cholesterol efflux activity of HDL (r=-0.52, P<0.005), while Tf glycation had positive association with the TBARS levels (r=0.40, P<0.05). Our
in vivo
HDL flux study demonstrated that glycated apoAI and Tf species were degraded 3 and 10 fold faster than the respective non-modified native proteins.

Conclusions:
HDL dysfunction and oxidative stress in T2D is related to glycation-induced instability of HDL proteins, including apoAI and Tf.
Abstract 397: Aldehyde-Modified Hdl Has CD36-dependent Pro-Atherogenic Effects in Macrophages
Authors: Holme Rebecca L , Chadwick Alexandra C , Chen Yiliang , Silverstein Roy L , Sahoo Daisy
Published: July 2021

The role of high density lipoproteins (HDL) in protecting against cardiovascular disease is compromised when HDL undergoes modification during conditions of oxidative stress; however, the mechanisms underlying these changes in HDL function are not well defined. Reactive aldehydes such as acrolein (a major component in cigarette smoke) or major products of lipid peroxidation such as 4-hydroxynonenal (HNE) or malondialdehyde (MDA) are known to oxidize HDL in cardiovascular disease. To test the hypothesis that modification of HDL with aldehydes impairs HDL’s athero-protective functions in macrophages, we first measured the ability of modified HDL to protect against foam cell formation. Cholesterol-loaded peritoneal macrophages isolated from wild-type C57Bl/J mice were incubated with native HDL, acrolein-modified HDL (acro-HDL), HNE-modified HDL (HNE-HDL) or MDA-modified HDL (MDA-HDL) for 24 h. Contrary to native HDL, oxidized forms of HDL were unable to prevent foam cell formation as shown by increased Oil red-O staining. Next, using a Boyden chamber assay, we demonstrated that acro- and MDA-HDL had impaired abilities to promote macrophage migration (64% and 67% of native HDL cell migration, respectively). Finally, using a secreted alkaline phosphatase reporter THP-1 cell-based assay, we determined that acro-HDL promotes activation of the pro-inflammatory NFkappaB pathway. Interestingly, immunoblot and quantitative RT-PCR analyses revealed that incubation of macrophages with acro- and MDA-HDL leads to increased expression of the pro-atherogenic receptor, cluster of differentiation 36 (CD36). Therefore, we repeated the foam cell formation and migration experiments using similar ligands, but this time, in CD36-null peritoneal macrophages. We found that both of these functions were dependent on CD36; however, the extent of the functional changes varied based on the type of oxidative modification present on HDL. In conclusion, modification of HDL with reactive aldehydes generates a particle that has pro-atherogenic effects in macrophages, many of which are dependent on CD36.
Abstract 404: Induction of MiR133a Expression By IL-19 Targets LDLRAP1 and Reduces Oxidized LDL Uptake in VSMC
Authors: Gabunia Khatuna , Herman Allison , Ray Mitali , Kelemen Sheri , England Ross , DeLaCadena Raul , Foster William , Elliott Katherine , Eguchi Satoru , Autieri Michael
Published: July 2021

Introduction:
The transformation of vascular smooth muscle cells (VSMC) into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. We tested the hypothesis that one mechanism was reduction in VSMC foam cell formation.

Methods and Results:
In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we show that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. We also show that IL-19 reduces lipid accumulation in VSMC, as well as LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients.

Summary and conclusions:
miR133a targets LDLRAP1 3’UTR and reduces its expression. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.
Abstract 412: Apelin-13 Enhances Atherosclerotic Plaque Stability in Apoe-Deficient Mice
Authors: Fraga-Silva Rodrigo A , Seeman Hugo , Montecucco Fabrizio , da Silva Analina R , Burger Fabienne , Costa-Fraga Fabiana P , Anguenot Léa , Mach François , dos Santos Robson A , da Silva Rafaela F , Stergiopulos Nikolaos
Published: July 2021

Introduction:
Atherosclerosis remains one of the main cause of death worldwide and substantial efforts have been made to identify novel approaches to improve the management of this disorder. Apelin is an endogenous peptidergic family with essential role on the cardiovascular hemostasis and pathologies. Recent studies pointed out a fundamental contribution of Apelin system on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define the beneficial or deleterious role of Apelin in atherosclerosis.

Objective:
To better understand the role of Apelin system on atherosclerosis, we aimed to investigate the actions of Apelin-13 treatment on atherosclerotic plaques composition, focusing on features of plaque vulnerability.

Methods:
Apolipoprotein E gene-deleted mice (n=40) were fed with western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposed the vessel to distinct patterns of shear stress, resulting in plaque formation with different composition. The mice were treated with Apelin-13 (2 mg/Kg/day) or vehicle for the last 3 weeks of experimental period.

Results:
Apelin-13 treatment did not change atherosclerotic plaque size in the aorta, neither altered the lipid content of low shear stress and oscillatory shear stress-induced plaques in the carotid. However, Apelin-13 remarkably ameliorated plaque stability by increasing intraplaque collagen content, which was associated with a reduction of MMP-9 expression. Furthermore, Apelin decreased cell infiltration (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, Apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acids serum levels, while HDL, triglycerides serum levels were not significantly changed.

Conclusion:
Apelin-13 treatment for 3 weeks did not alter the lesion size, but significantly enhances the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of Apelin system enhances plaque stability.
Abstract 413: Lipoprotein SCARB1 Intronic Variant Associated With Increased Coronary Heart Disease Risk Affects Cardio-Protective Gene Networks
Authors: Golden Diana , Mehta Sameet , Kolmakova Antonina , Rodriguez Annabelle
Published: July 2021

Introduction:
We previously reported a common intronic
SCARB1
(12q24.31) variant, rs10846744, located in an enhancer region, to be significantly associated with coronary artery disease (CAD) in the Multi-Ethnic Study of Atherosclerosis (MESA). RNA-Seq showed expression of the immune checkpoint inhibitor lymphocyte activation gene 3 (
LAG3
, 12p13.31), to be 5-fold lower in carriers of the risk allele, while low plasma LAG3 protein levels were also significantly associated with increased CAD risk in MESA, after multivariate regression analysis.

Hypothesis:
That the
SCARB1
rs10846744 variant disrupts long-range transcriptional regulation of
LAG3
disturbing cardio-protective and anti-inflammatory gene networks to promote CAD.

Methods and Results:
Using functional genomics (HiC global chromatin capture, ChIP-Seq and RNA-Seq) in reference and risk EBV-transformed B lymphocytes to assess 3D chromatin architecture and gene-gene interactions at a 2.5kb resolution, we did not observe direct chromatin contacts between
SCARB1
and
LAG3.
In the reference allele, an enhancer-rich intermediate contact (12q13.13) was found containing genes associated with cholesterol (
SOAT2
) and NR2F2 signaling (
RARG
). This same 12q13.13 region was in direct contact with 22q12.3 (
APOLI)
, an apoprotein associated with HDL and innate immunity. Micro-looping within the rs10846744 12q24.31 region showed direct contacts with other enhancers (
NCOR2
) and cardiovascular loci (
TMEM132B
), while
LAG3
micro-looping on 12p13.31 was associated with immune regulatory networks (
CD4
). Loci associated with viral infection, cytokine production, heart failure and autoimmunity were also identified. NR2F2 disrupted contacts in the risk allele, implicating NR2F2 as a dysfunctional rs10846744 transcriptional repressor altering gene networks. The risk allele included contacts near
PCSK9
,
VLDLR
and 2q33.1, a CAD locus.

Conclusion:
Functional genomics of the
SCARB1
rs10846744 enhancer region identified a number of intra- and inter-chromosomal chromatin contacts in reference cells that were markedly disrupted in risk cells. Perturbing NR2F2 and/or genes disrupted in the
SCARB1
-NR2F2 immuno-cardiovascular axis may protect against CAD in the rs10846744 risk population.
Abstract 427: Histopathological and Molecular Effects of SIRT3 Deletion in Advanced Calcific Aortic Valve Disease
Authors: Roos Carolyn M , Hagler Michael A , Verzosa Grace C , Zhang Bin , Fujimoto Hirokazu , Miller Jordan D
Published: July 2021

Increasing age is the greatest risk factor for development and progression of calcific aortic valve disease (CAVD), and mitochondrial dysfunction has been implicated in the pathogenesis of cardiovascular calcification. Previous findings by our group suggested a significant reduction in expression of multiple sirtuin (SIRT) isoforms in normocholesterolemic aged mouse aortic valves, however, it is unclear if losses in SIRT3—a major mitochondrial SIRT isoform—modulates progression of aortic valve calcification. Therefore, we hypothesized that loss of SIRT3 in a hypercholesterolemic mouse model of CAVD will result in augmented calcium burden in aortic valve, increased osteogenic signaling, and impaired aortic valve function. To test this we used mice that were Ldlr
-/-
/ApoB
100/100
mice that were either wild-type (LA-SIRT3
+/+
) or null for SIRT3 (LA-SIRT3
-/-
) and fed a western diet (TD88137) for 12 months. Alizarin red was used to quantitate calcium burden, qRT-PCR was used to measure changes in mRNA levels, and high-resolution echocardiography was used to assess aortic valve function (cusp separation distance). In line with our hypothesis, we observed a substantial increase in calcium burden in LA-SIRT3
-/-
mice compared to their LA-SIRT3
+/+
littermates(11.7±4.0, 4.8±1.8; respectively). Interestingly, expression of Runx2 and osterix—classic markers of osteogenic differentiation—were decreased in LA-SIRT3
-/-
mice compared to their LA-SIRT3
+/+
littermates. Expression of Msx2, were markedly increased in aortic valve tissue from LA-SIRT3-deficient mice. Despite these histological and molecular changes, SIRT3 deletion did not alter cusp separation distance (LA-SIRT3
+/+
= 0.81±0.04; LA-SIRT3
-/-
= 0.81±0.02). Collectively, this data suggest losses in SIRT3 can contribute to accelerated valve calcification through unclear molecular mechanisms, but these changes are not sufficient to drive reductions in cusp separation distance. Additional experiments delineating the histopathological and molecular sequelae of SIRT3 deletion will be critical to understanding its role in the pathogenesis of CAVD.
Abstract 431: Nlrp3 Inflammasome Inhibits Cholesterol Efflux in Macrophages by Downregulating Gpr109a Expression. A Novel Proatherogenic Mechanism
Authors: Tumurkhuu Gantsetseg , Dagvadorj Jargalsaikhan , Zhang Wenxuan , Crother Timothy , Shimada Kenichi , Arditi Moshe , Chen Shuang
Published: July 2021

Recent studies suggest that Nlrp3 inflammasome activation plays a critical role in the development of atherosclerosis and
Chlamydia pneumoniae (Cpn
) infection has been shown to accelerate atherogenesis. Herein, we asked whether Cpn infection induced acceleration is via Nlrp3 inflammasome activation in hypercholestrolemia mouse model.

Nlrp3
-/-
Ldlr
-/-

,

Casp1
-/-
Ldlr
-/-

and

Ldlr
-/-

mice were infected intranasally with
Cpn
followed by western diet (WD) for 16 weeks.

Ldlr
-/-

mice
infected with Cpn
infection had markedly increased lesion size in the aortic sinus and aorta
en face
compared to WD only group. Casp1 activation in lesion macrophages in

Ldlr
-/-

mice was also increased in
Cpn
group
vs
controls.

Nlrp3
-/-
Ldlr
-/-

or

Casp1
-/-
Ldlr
-/-

mice with and without
Cpn
resulted in significantly smaller of plaques in aortic root and aorta compare to

Ldlr
-/-

mice. However, no difference was observed between
Cpn
infected and uninfected groups in the double knockout animals. Furthermore, foam cell formation of

Nlrp3
-/-
,

and
Casp1
-/-
peritoneal macrophages after treatment with OxLDL and
Cpn
was significantly reduced when compared with WT cells. Interestingly, expression levels of the cholesterol efflux transporter, ATP-binding cassette A1 (ABCA1), was increased by RT PCR and western analysis in the KO macrophages. Further investigations found that the niacin receptor Gpr109a, a known positive regulator of ABCA1, was upregulated in Nlrp3 KO macrophages during foam cell formation. Hydroxy-butyrate, an activating ligand of GPR109, was produced by macrophages after
Cpn
infection indicating a feedback loop. Intact IL-1 signaling suppressed Gpr109a expression suggesting a pathway by which the inflammasome and IL-1β could enhance foam cell formation. In aortic root lesions, macrophage expression of Gpr109a was increased in

Nlrp3
-/-
Ldlr
- -

mice compare with

Ldlr
-/-

mice on WD and infected with
Cpn
. In conclusion, the activation of the NLRP3 inflammasome negatively regulate Gpr109a receptor and its downstream cholesterol efflux transporter ABCA1, which leads to more foam cell formation and acceleration of atherosclerosis in

Ldlr
-/-

mice.

This work was supported by the National Institutes of Health grant HL111483 (to S. Chen)
Abstract 433: Map Kinase Phosphatase-5 Deficiency in Macrophages Protects Against Atherogenesis
Authors: Zhang Xinbo , Zhang Cheng , Tao Bo , Zhang Lei , Bennett Anton M , Yu Jun
Published: July 2021

Objective:
To determine the role of macrophage MAP Kinase Phosphatase-5 (MKP-5) in the pathogenesis of atherosclerosis.

Approach and Results:
Lethally irradiated LDLR
-/-
mice were transplanted with wild-type (WT) or MKP-5 deficient (MKP-5
-/-
) bone marrow and subjected to high-fat feeding. Mice transplanted with MKP-5
-/-
bone marrow developed smaller atherosclerotic lesions accompanied by decreased lipid deposition and macrophage content compared to WT. Lack of MKP-5 in macrophages led to decreased plasma levels of interleukin-1α (IL-1α) and IL-7, elevated anti-inflammatory cytokines IL-1 receptor antagonist (IL-1rn) and IL-4. Mechanistically, lack of MKP-5 in macrophages inhibited ox-LDL-induced foam cell formation through enhanced cholesterol efflux mediated by increased expression of ATP-binding cassette transporters ABCA1 and ABCG1.

Conclusions:
These data suggest that the macrophage MKP-5 deficiency reduces atherosclerosis progression and foam cell formation through amelioration of cholesterol efflux and inhibition of inflammation.
Abstract 439: High Dimensional Single-Cell Immune Contexture of Human Atherosclerotic Plaques and Blood
Authors: Rahman Adeeb , Chudnovskiy Aleksey , Amir El-ad David , Kim-Schulze Seunghee , Li Jennifer R , Pina Christian , Moss Noah , Remark Romain , Gnjatic Sacha , Faries Peter , Merad Miriam , Giannarelli Chiara
Published: July 2021

Atherosclerosis is a disease characterized by immune infiltration of the arterial wall in response to tissue damage and systemic inflammation. In the era of precision medicine, is essential to gain insights on immune contexture of atherosclerotic tissue taking into account disease-specific cell variation in patients. We applied high-dimensional technologies for the analysis of multiple parameters at the single-cell level in clinical samples of patients undergoing carotid endatherectomy (CEA, n=15). Using time-of-flight mass-cytometry (CyTOF), we simultaneously analyzed 32 parameters at the single-cell level in peripheral blood mononuclear cells (PBMCs) and atherosclerotic-tissue associated immune cells of the same patient. Using viSNE, we mapped single-cell heterogeneity into two dimensions to discriminate PBMCs and tissue-associated CD45+ immune cells. Next, we employed Phenograph to cluster cells into phenotypically related populations, which were annotated based on canonical marker expression patterns. We identified several major immune subsets including two subsets of macrophages (CD163
low
and CD163
high
), monocytes, dendritic cells (DCs), B and T cells. The most prevalent CD45+ cells identified in atherosclerotic tissue were CD4
+
(25.8%) and CD8
+
(25.2%) T cells, macrophages (12.8%), monocytes (7.7%) and B (2.1%) cells. Using a regression analysis similar to that employed by CITRUS, we determined that macrophages and a subset of CD8 T cells characterized by low expression of CD127 were selectively enriched in tissue vs. blood. Multiplexed immunohistochemistry confirmed that T cells comprised a major portion of the CD45+ cells in atherosclerotic tissue, even more abundant than macrophages. This study of deep phenotyping across-atherosclerotic tissue and blood demonstrate a significant T cell tissue infiltration of a specific subset of CD8 T cells. This suggests that adaptive T cell immunity plays a critical role in advanced atherosclerosis. The extension of this systems biology analysis pipeline to larger datasets can improve our understanding of the core mechanisms of chronic inflammation in atherosclerosis.