bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 24: Resolvin D2 Reduces Thrombus Burden and Attenuates Inflammatory Signaling Pathways in a Murine Model of Venous Thrombosis
Authors: Diaz Jose A , Shaydakov Maxim E , Chatterjee Anuran , Farris Diana M , Ballard-Lipka Nicole E , Hawley Angela E , Sigler Robert E , Henke Peter K , Myers Daniel D , Wakefield Thomas W , Conte Michael S
Published: March 2022

Background:
Specialized pro-resolving lipid mediators (SPM) derived from polyunsaturated fatty acids, such as Resolvin D2 (RvD2), play a critical role in the resolution of acute inflammation. In recent years, total organic synthesis of SPM have enabled investigation of their bioactivity in-vivo. It is known that the effects of RvD2 include limited neutrophil influx in-vivo, reduced neutrophil transmigration and counter-regulation of cytokines such as tumor necrosis factor-α TNF-α in mice. However, SPMs have not been studied in the context of experimental venous thrombosis (VT).

Methods:
Mice C57Bl/6, 10 week-old, 25grams. Groups: Control non-VT mice; VT-control-vehicle; VT-RvD2; Flow model: Electrolytic IVC model. Intra-peritoneal injection was used for RvD2 and vehicle administration 4 hours after surgery. Harvest was performed 2 days after surgery, acute VT. Thrombus weight, qRT-PCR for inflammatory cytokines panel and subsets of white blood cell (WBC) counts were performed at the site of thrombosis histologically.

Results (Figure 1):
We observed that RvD2 significantly reduced thrombus size when compared to vehicle controls (17.9mg±2.1mg vs. 4.5mg±0.8mg). The vein wall cytokine profile demonstrated a significant gene down-regulation of IL-6, CCL2, and TNF-a. In addition, the inflammatory cell counts showed reduction in all white cells subsets. No bleeding complications were observed.

Conclusions:
For the first time we have demonstrated that RvD2 reduces VT formation. The decreased thrombogenesis was accompanied by a decrease in WBC and proinflammatory cytokines. We demonstrated that RvD2 is a potent anti-inflammatory agent that reduced WBC and attenuated cytokines in the context of experimental VT. Further investigation of RvD2 in VT treatment and prophylaxis appears warranted.
Abstract 240: RhoGTPases and RNAi: A Large-scale Screen Using Ecis® to Measure Endothelial Integrity in Real-time
Authors: Amado-Azevedo Joana , de Menezes Rene , van Beusechem Victor , van Hinsbergh Victor , van Nieuw Amerongen Geerten P
Published: March 2022

Introduction:
Vascular leakage is a hallmark of many cardiovascular diseases and albeit its importance no specialized therapies are available to prevent or reduce it. RhoGTPases, their on/off switchers GEFs and GAPs and downstream effectors are known regulators of different cellular processes and studies have shown their positive and negative effects on the integrity of the endothelial barrier. Still, with data available on only a few family members, precise knowledge about this mechanism remains fragmentary. We hypothesize that there is a tight spatiotemporal regulation of a yet unknown combination of these regulatory proteins and that a systematic study of all members will provide more information on this mechanism. Using state-of-art RNAi and Electric Cell-substrate Impedance Sensing we measured the silencing effect of given genes on the endothelial barrier.

Methods:
A selective RNAi library of 270 siRNAs targeting RhoGTPases, GAPs, GEFs and Effectors was ordered. HUVECs of 12 donors were isolated, pooled and plated on 96 wells ECIS arrays. Before reaching confluence, cells were transfected and 72h later stimulated with 1U/ml of thrombin. Positive and negative controls were carefully chosen and three independent screens were carried out on different pools of cells. Trans endothelial electrical resistance was measured in real-time and recorded throughout the whole procedure.

Results:
Analysis of 3 critical time-points (72h post-transfection, direct thrombin response and recovery phase) confirmed already known key players such as Gef-H1, Larg or RhoA, which supports the reliability of this technique and its accuracy. However, it also revealed more than 10 new candidates involved in the regulation of the endothelial barrier. A few promoted a tighter endothelial barrier, some showed a protective effect upon thrombin challenge and others an intense disruptive response to the stimulus. Further characterization studies will be carried out and validation is ongoing.

Conclusions:
Using a novel screening approach we identified previously unknown candidate regulators involved with endothelial barrier formation, dysruption and restoration. These new molecular targets provide opportunities for future pharmacological intervention.
Abstract 243: Cyp2j2 Gene Delivery Inhibited Adventitial Remodeling via Attenuated Oxidative Stress
Authors: Xu Xizhen , Chen Jun Xiong , Wang Dao Wen
Published: March 2022

Vascular adventitial remodeling is an important pathologic process in multiple vascular diseases. Reactive oxygen species (ROS) are an important triggering factor involved in this process. Herein, we investigated whether EETs could prevent the adventitial remodeling via reducing oxidative stress induced by angiotensin II (Ang II). C57BL/6J mice were treated with Ang II by mini-pump to induce vascular remodeling after recombinant adeno-associated virus (rAAV)-mediated CYP2J2 overexpression. Results showed that Ang II treatment significantly induced vascular adventitial remodeling, while CYP2J2 overexpression markedly reduced adventitial hyperplasia, collagen accumulation, phenotype transformation and inward migration of AFs. The similar protective effects of soluble epoxide hydrolase inhibitors (sEHIs), TUPS, on aortic adventitial remodeling were observed as well. In vitro, CYP2J2, TUPS and 11,12-EET significantly prevented primary aortic adventitial fibroblasts (AoAFs) remodeling and oxidative stress induced by Ang II (100nmol/L) via regulating gp91phox, Cu/Zn-SOD and phosphorylation of p38MAPK and JNK. These results suggest that CYP2J2, TUPS and 11,12-EET attenuate vascular adventitial remodeling via direct effects on AoAFs, and the regulation of ROS level was involved in these favorable profiles. Exploitation of this beneficial activity may shed light on designing novel therapeutic approaches for remodeling in multiple kinds of vascular diseases.
Abstract 259: Aneurysm Development in Patients With a Bicuspid Aortic Valve Is Not Associated With Transforming Growth Factor-β Activation
Authors: Paloschi Valentina
Published: March 2022

Objective:
Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysms. Transforming growth factor-β (TGFβ) is a crucial factor of vascular remodeling, the impaired signaling of which can alter the structure and composition of the extracellular matrix. In this study, we analyzed the activity of TGFβ in aneurysmal and non-aneurysmal ascending aorta from BAV patients, using tricuspid aortic valve (TAV) patients as a reference group.

Approach and Results:
The response to exogenous TGFβ was analyzed with regard to gene expression in primary aortic smooth muscle cells that were isolated from 7 BAV and 5 TAV patients and in valve fibroblasts from 7 BAV and 8 TAV patients. The set of genes that were significantly changed by TGFβ (217 genes) was compared with gene expression profiles of the ascending aorta from BAV and TAV patients (139 arrays). By principle component analysis, based on the 217 genes, gene expression differed significantly in the intima/media region between aneurysmal BAV and TAV aortas, driven by the response in TAV patients. During the development of aneurysm, the levels of phosphorylated SMADs and the availability of free TGFβ were lower in BAV patients compared with TAV patients. Confocal microscopy analysis showed a higher co-localization of latency associated peptide and latent TGF-beta binding protein 3 in BAV aortas.

Conclusions:
Our findings suggest that TGFβ activation during aneurysm formation is muted in patients with BAV and, by interfering with repair processes, could explain the greater propensity of BAV to aortic complications.
Abstract 262: Association of Lipoprotein Subclasses with Arterial Stiffness in a High Risk Working Population: The Baptist Employee Healthy Heart Study (BEHHS)
Authors: Aziz Muhammad , Aneni Ehimen , Oni Ebenezer , Roberson Lara , Shaharyar Sameer , Ali Shozab S , Jamal Omar , Latif Muhammad A , Younus Adnan , Ahmad Rameez , Malik Rehan , Ogunmoroti Oluseye , Khan Sher A , Post Janisse , Campo Daniel D , Guzman Henry , Cury Ricardo , Agatston Arthur , Veledar Emir , Fialkow Jonathan , Feldman Theodore , Nasir Khurram
Published: March 2022

Introduction:
Arterial stiffness is an independent predictor of cardiovascular disease (CVD) morbidity and mortality, and has been shown to be of prognostic value in CVD. A risk factor independent association between arterial stiffness and traditional lipids has been described extensively but it is unclear whether an independent relationship exists with lipoprotein subclasses.

Methods:
The Baptist Employee Healthy Heart Study (BEHHS) is a lifestyle intervention study examining the effects of web-based programs on reducing CVD risk in high-risk persons. Participants had their brachial arterial augmentation index (AIx, a measure arterial stiffness) assessed using the EndoPAT 2000 device. The AIx is standardized to gender-matched non-selective populations. Cardio IQ™ Ion Mobility lipoprotein fractionation was utilized for advanced lipid subclass measurement.

Results:
The population consisted of 182 participants, (74% women, 49% Hispanic) with a mean age of 52 ± 9 years. There was a significant trend association between quartiles of AIx and total LDL, small LDL, small and large IDL, small, large and total HDL, and small VLDL. In multivariate analysis, only small and large IDL, total LDL and small VLDL showed independent significant association with AIx.

Conclusions:
Several lipoprotein subclasses demonstrate independent significant associations with arterial stiffness. Thus, a safe and relatively inexpensive blood test may be useful in identifying probable vascular disease in a relatively young high traditional CVD risk population. Longitudinal studies are required to fully understand the temporal relationship between the lipoprotein subclasses and arterial stiffness.
Abstract 281: Local MicroRNA Modulation Using a Novel Anti-mir-21-eluting Stent Effectively Prevents In-stent Restenosis
Authors: Chernogubova Ekaterina , Wang Dong , Deuse Tobias , Stubbendorff Mandy , Erben Reinhold G , Eken Suzanne M , Jin Hong , Heeger Christian , Behnisch Boris , Reichenpurner Hermann , Robbins Robert C , Spin Joshua M , Tsao Philip S , Schrepfer Sonja , Maegdefessel Lars
Published: March 2022

The major limitation of vascular angioplasty remains the occurrence of in-stent restenosis (ISR). The central role of miRNAs in the pathophysiology of the proliferative response in cardiovascular disease offers attractive concepts for possible therapeutic interventions, with systemic miRNA modulation however potentially causing substantial off-target effects. Aim of the current study was to test the feasibility of local miRNA modulation using drug eluted stent (DES) technology.

Utilizing human ISR tissue samples, as well as a humanized rodent model of balloon-injured human internal mammary arteries with subsequent stent implantation in RNU rats, we were able to identify several miRNAs dys-regulated, including the well-characterized miR-21. We inhibited miR-21 (with a FAM-tagged-LNA-anti-miR-21), using either systemic or local delivery via DES.

Systemic suppression of miR-21 showed a dose-dependent reduction of luminal obliteration, while not impeding vascular re-endothelialization. However, miR-21 expression in liver, heart, lung, and kidney appeared significantly reduced, with increased levels of serum creatinine, pointing towards the expected off-target effects of systemic miRNA modulation. Local mir-21 suppression, using the anti-miR-21-DES, effectively reduced myointimal hyperplasia and ISR compared to bare metal stents, while not exerting detectable off-target effects on any other organ we investigated. Additional in vitro experiments in cultured human coronary artery smooth muscle and endothelial cells further confirmed the ability of anti_miR-21 to limit SMC proliferation and migration by repressing mRNA and protein levels of its established target PTEN, while not affecting the proliferative response in the ECs.

This is the first experimental study to demonstrate the efficacy, feasibility and suitability of an anti-miRNA-eluting stent (anti-miR-21-DES) for the reduction of ISR through dominant inhibition of SMC proliferation.
Abstract 284: Testosterone Replacement Attenuates Hyperplasia Development in an Androgen Deficient Model of Vascular Injury Independent of Matrix Metalloproteinase Regulation
Authors: Freeman Brian M , Mountain Deidra J , Brock Timothy C , Chapman Jason R , Kirkpatrick Stacy S , Arnold Joshua D , Stevens Scott L , Goldman Mitchell H , Freeman Michael B , Klein Frederick A , Grandas Oscar H
Published: March 2022

Objectives:
Clinical and epidemiological studies have shown low testosterone (TST) is associated with increased risk of vascular disease, yet the molecular mechanisms remain unclear. Matrix metalloproteinases (MMPs) are implicated in dysfunctional remodeling. Our group has previously demonstrated an inverse relationship between TST levels and MMP activity in vitro. We have also demonstrated androgen deficiency (AD)-modulated inflammatory signaling does not influence systemic MMPs in vivo. Here we investigated the role of AD and TST replacement in MMP-modulated remodeling in response to injury.

Methods:
Sub-physiological and physiological TST replacement was administered via implanted pellets in aged orchiectomized rats (0.5-5mg, Table 1). Serum TST was determined by ELISA. At 14d TST replacement rats underwent balloon angioplasty of the left common carotid. Young intact (YI), aged intact (AI), and orchiectomized placebo (Plac) animals served as controls. Carotids were collected 14d post-injury for intima:media (I:M) and MMP quantification.

Results:
I:M was increased in Plac, sub-, and low-physiological TST animals compared to AI controls, and decreased with higher physiological TST (Fig 1). Injury-induced expression of MMPs involved in vascular remodeling was not significantly affected by TST status (Table 2).

Conclusions:
AD is associated with hyperplasia development in response to vascular injury while physiological TST replacement attenuated this effect. This attenuation occurs independent of MMP mechanisms known to be heavily involved in vascular remodeling. Future in vivo studies will examine molecular targets involved in AD and the effect of TST replacement in the acute inflammatory response to vascular injury. These studies are needed in determining if TST replacement therapy in AD men should be evaluated for attenuation of vascular pathogenesis.
Abstract 318: ApoE Modulates the Kinetics of HDL ApoA-I in Humans
Authors: Morton Allyson , Wang Liyun , Furtado Jeremy D , Sacks Frank M
Published: March 2022

Human HDL kinetic studies typically measure total apoA-I turnover, rather than partitioning by HDL size or apolipoprotein content. ApoE has potential roles in HDL metabolism by promoting enlargement and clearance. About 5-10% of plasma HDL apoA-I is associated with apoE. To determine whether apoE modulates the kinetics of apoA-I HDL, we compared the metabolism of apoA-I in HDL that has apoE with apoA-I in HDL that does not have apoE. We recruited 5 participants with low HDL-C (average 39 mg/dl, range 24-48 mg/dl) and BMI between 25-35 kg/m
2
. We gave the participants an intravenous bolus of d3-leucine, and collected blood at 14 time points to 70 hours. HDL was isolated from plasma by anti-apoA-I immunoaffinity chromatography, separated by anti-apoE chromatography into HDL that contain or do not contain apoE, and separated using ND-PAGE into 4 size fractions: alpha-1, alpha-2, alpha-3, and prebeta-1. ApoA-I was purified from the 8 HDL subtypes with SDS-PAGE, hydrolyzed into amino acids, and d3-leucine enrichment was measured by GC-MS. Pool size of apoA-I was determined from the protein bands, adjusted to plasma total apoA-I. We used SAAM-II modeling software to compute apoA-I fractional catabolic rates (FCR) and fluxes for each HDL subtype. The FCRs of apoA-I of HDL size fractions that contain apoE were about 2.5 to 5 times faster than that of apoA-I of the corresponding HDL size that does not contain apoE (table). Driven by the higher FCRs, a substantial amount of apoA-I flux in plasma occurs in apoE-containing HDL, varying from 20-40% among the 4 sizes. In summary, apoA-I HDL with apoE is an important, metabolically unique subspecies of HDL that could account for a substantial minority of total HDL flux in plasma.
Abstract 328: ABCG1 Has a Critical Role in Pulmonary Type 2 Cells and Surfactant Lipid Metabolism
Authors: de Aguiar Vallim Thomas Q , Goulbourne Christopher N , Cheng Joan , Han Tieyan , Frank Joy , Ford David A , Baldan Angel , Tarling Elizabeth
Published: March 2022

Pulmonary alveolar proteinosis (PAP) is a rare disease caused by the accumulation of surfactant proteins and lipids in the pulmonary alveoli resulting in respiratory distress. For almost all reported cases the underlying cause is unknown. Synthesis and secretion of surfactant in the lung is restricted to epithelial type 2 pneumocytes (T2 cells). However, subsequent clearance of surfactant from the hypophase is dependent upon both T2 cells and macrophages. Both T2 cells and macrophages express high levels of the sterol-transporter ATP Binding Cassette transporter G1 (ABCG1). Further, Abcg1-/- mice exhibit a severe pulmonary lipidosis, characterized by elevated levels of multiple lipid species and the presence of lipid-loaded macrophages and T2 cells containing abnormal lamellar bodies. Recent advances have implicated a role for specific granulocyte-macrophage colony stimulating factor (GM-CSF) autoantibodies and the innate immune system in the pathogenesis of PAP. We have recently demonstrated that the lungs of Abcg1-/- mice accumulate specific oxidized phospholipids and that this is associated with a local increase in innate immune natural antibody-secreting B-1 B cells and both local and circulating natural antibodies. Here we show that the lungs of mice containing ABCG1-deficient T2 (T2KO) cells and wild-type macrophages contain elevated levels of both phospholipids and esterified cholesterol. In addition, the T2KO cells in these mice accumulated lamellae bodies, unesterified cholesterol and exhibited abnormal gene expression. Collectively, our results suggest that ABCG1 is critical for normal surfactant metabolism and that the pulmonary expansion of B-1 B cells and the subsequent increase in natural antibodies in Abcg1-/- mice may provide a novel model in which to study the role of the innate immune system in PAP.
Abstract 329: CRISPR/Cas-mediated Gene Editing in Human iPSC-derived Macrophage Reveals Lysosomal Acid Lipase Functions in Human Macrophages
Authors: Zhang Hanrui , Li Wenjun , Hinkle Christine , Zhang Xuan , Bauer Robert , Tohyama Junichiro , Patel Kevin , Trindade Kevin , Lin Jennie , Millar John , Billheimer Jeffrey , Yang Wenli , Morrisey Edward , Phillips Michael C , Rader Daniel J , Reilly Muredach P
Published: March 2022

Loss-of-function mutations of lysosomal acid lipase (
LIPA
or LAL) are the cause of the rare lysosomal disorders, Wolman disease and Cholesteryl Ester Storage Disease (CESD), which manifest with hepatomegaly, hyperlipidemia and premature atherosclerosis due to lysosomal cholesteryl ester (CE) accumulation mainly in hepatocytes and macrophages. Surprisingly, recent genome-wide association studies (GWAS) have implicated
LIPA
alleles, which associate with higher
LIPA
mRNA, also associate with increased risk of coronary artery disease (CAD). To date, the mechanistic insights into the role of human
LIPA
were largely derived from skin fibroblasts of CESD patients. Here we use CRISPR/Cas techniques to knock-out LIPA in human iPSC, and then differentiate to macrophage (IPSDM) to explore human macrophage
LIPA
phenotypes.
LIPA
mRNA was markedly induced by ~20 fold upon iPSC to IPSDM differentiation, was comparable between IPSDM and peripheral blood mononuclear cells (PBMC)-derived macrophages (HMDM, n=9) and was ~7 fold higher than in skin fibroblasts. Knock-out
LIPA
(

LIPA
-/-

) IPSDM had barely detectable
LIPA
mRNA. Control and

LIPA
-/-

IPSDM were loaded with [
3
H]-cholesteryl oleate (CO)-labeled acetylated-LDL ([
3
H]CO-ac-LDL) followed by efflux to apolipoprotein-AI (apoA-I) in the presence of ACAT inhibitor (n=5). Efflux of [
3
H]-cholesterol was abolished in

LIPA
-/-

IPSDM with massive lysosomal [
3
H]-CE accumulation, indicating deficiency in LIPA-mediated lysosomal CE hydrolysis. In non-lipid loading state,

LIPA
-/-

IPSDM had high levels of CE mass compared with minute amounts in control IPSDM (11.34±0.49 vs. 2.42±0.44 ug/mg protein, n=3). Yet, with ac-LDL loading, CE mass was similarly increased in both control and

LIPA
-/-

IPSDM (22.34±1.70 vs. 21.96±2.18 ug/mg).

LIPA
-/-

also did not impact IPSDM lysosomal apoB degradation or [
3
H]-labeled free cholesterol efflux to apoA-I (4h and 20h) with ac-LDL loading. In conclusion,

LIPA
-/-

IPSDM reveals macrophage-specific hallmarks of human
LIPA
LOF. CRISPR/Cas and IPSDM provide critical tools in assessing the impact of human
LIPA
genetic variation in diseases including the impact of GWAS CAD alleles in macrophage-specific functions in atherosclerosis.
Abstract 333: Protective Role of Gut Microbiota
Akkermansia municiphila
Against Development of Atherosclerosis in Mice
Authors: LI Jin , Woo Connie , Xu Aimin
Published: March 2022

Gut microbiota is involved in both onset and progression of obesity. Obesity is associated with several cardiovascular diseases, among which atherosclerosis is the leading cause of morbidity and mortality. However, the role of gut microbiota in obesity-related cardiovascular complication remains to be explored. We found that the fecal amount of Akkermansia municiphila (Akk), a beneficial bacterium, decreased in apolipoprotein E-deficient (apoE -/-) mice fed a Western diet. In this study, we investigated whether Akk may exert beneficial role against the development of atherosclerosis.

ApoE -/- mice were oral gavaged with either PBS or Akk daily along with feeding a Western diet for 8 weeks. Atherosclerotic lesions in these mice were analyzed by H&E staining and Oil Red O staining. Local and systemic expressions of inflammatory molecules including MCP-1, ICAM, VCAM, IL-1beta and TNF-alpha were evaluated.

Western diet substantially altered gut microbiota pattern in apoE -/- mice. However, treatment of Akk, without changing the gut microbiota composition, significantly reduced atherosclerotic lesion. Although decreased body weight and fat mass were observed in Akk treatment group, the changing levels were physiologically insignificant. Serum total cholesterol, triglyceride, HDL and LDL levels were not affected by Akk treatment. However, local expression levels of MCP-1, ICAM, VCAM and TNF-alpha along with macrophage infiltration at the lesions were diminished by Akk. Furthermore, Akk treatment ameliorated systemic inflammatory status by decreasing circulating MCP-1 and IL1-beta levels. It appeared that Akk treatment was able to decrease gut permeability by inducing the expression levels of tight junction proteins, ZO-1 and occludin. Akk-induced reduction of gut permeability led to elimination of bacterial products penetration and serum LPS level was decreased. We speculated that the protective effect of Akk against atherosclerosis was mediated by decreasing gut permeability and LPS level in circulation.

Collectively, our results demonstrated that Akk can decrease atherosclerotic lesions by ameliorating local and systemic inflammation. Probiotic Akkermansia municiphila may be a potential therapeutic target for atherosclerosis.
Abstract 343: Association Between Electrocardiographic Parameters and Coronary Atherosclerotic Burden in Symptomatic Patients Without History of Coronary Artery Disease
Authors: Adigun Rosalyn , Chaikriangkrai Kongkiat , Sunkara Anusha , Cherry Mariyam , Chang Su Min
Published: March 2022

Introduction:
Coronary artery calcium score (CACS) and the electrocardiogram (ECG) are tests available in the evaluation and risk stratification of coronary heart disease risk. The aim of this study is to evaluate the association between conduction abnormalities detected by ECG and coronary atherosclerotic burden determined by CACS in symptomatic patients with no history of coronary artery disease (CAD).

Methods:
This is a cross-sectional study of 843 consecutive patients without history of CAD who presented with chest pain to our emergency department. All patients were evaluated with ECG and CACS by MDCT. The cohort was categorized into 4 groups: CACS 0, 1-100, 101-400, and >400. PR prolongation was defined as PR interval >200 ms. QTc prolongation was defined as normal (<420 ms), mild (420 [[Unable to Display Character: –]] 440ms) and moderate-to-severe (>440ms). Other ECG parameters evaluated include bundle branch blocks (BBB), ST segment changes, and T wave abnormalities.

Results:
The cohort had a mean age of 54 ± 13 years and 44% were male. Median Framingham 10-year risk for cardiovascular disease was 5% (Interquartile range; IQR 1% - 16%). 59% of the cohort had CACS 0; 22.7% (CACS 1-100), 9.5% (CACs 101-400) and 8.8% (CACS >400). PR prolongation was present in 5% of the cohort. 66% had normal QTc and 34% had prolonged QTc - mild (180), moderate-to-severe (105). In multivariate analysis adjusted for Framingham risk, PR prolongation was independently associated with presence (OR 3.22; 95%CI 1.08, 1.12; p<0.001) and severity (CACS>400) of coronary calcification (OR 2.51; 95%CI 1.10, 5.75; p=0.03). Similarly, QTc prolongation was independently associated with presence (mild: OR 1.01; 95%CI 0.70, 1.46; p=0.96, moderate-to-severe: OR 1.64; 95%CI 1.05, 2.56; p=0.03) and severity (CACS>100) of coronary calcification (mild: OR 1.66; 95%CI 1.10, 2.53; p=0.02, moderate-to-severe: OR 2.05; 95%CI 1.26, 3.35; p=0.004). BBB (3.6%), ST segment changes (11.9%), and T wave abnormalities (22.7%) were not associated with the presence or extent of coronary calcification.

Conclusion:
In patients without history of CAD, PR and QTc prolongation were independently associated with presence and severity of coronary atherosclerotic burden detected by CACS, while BBB, ST segment and T wave changes were not.
Abstract 349: Sgk1 Is a Pivotal Player in Vascular Inflammation and Atherogenesis
Authors: Borst Oliver
Published: March 2022

Objective:
Atherosclerosis, an inflammatory disease of arterial vessel wall, requires migration and MMP-9-dependent invasion of macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor NF-κB, which is regulated by IκB and thus IκB-kinase IKK. Regulators of NF-κB include SGK1. The present study explored involvement of SGK1 in vascular inflammation and atherogenesis.

Approach and Results:
Gene-targeted ApoE-deficient mice without (apoe-/-sgk1+/+) or with (apoe-/-sgk1-/-) additional SGK1 knockout received 16-weeks cholesterol-rich diet. Atherosclerotic lesions in aorta and carotid artery, vascular CD45+ leukocyte infiltration, Mac-3+ macrophage infiltration, VSMC content, MMP-2 and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe-/-sgk1-/-mice. Migration of SGK1-deficient CD11b+F4/80+ macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1-/- than in sgk1+/+ macrophages and in control plasmid- or inactive K127NSGK1-transfected than in constitutively active S422DSGK1-transfected THP-1 cells. Plaques of apoe-/-sgk1-/- mice displayed reduced macrophage number and macrophage MMP-9 content. In THP-1 cells MMP-inhibitor GM6001 abrogated S422DSGK1-induced MMP-9 production and invasion. MMP-9 transcript levels were significantly reduced in sgk1-/- macrophages and strongly upregulated in S422DSGK1-transfected THP-1 cells compared to control plasmid- or K127NSGK1-transfected THP-1 cells. Phosphorylation of IKK and IκB and nuclear translocation of p50 were significantly lower in sgk1-/- macrophages than in sgk1+/+ macrophages and significantly higher in S422DSGK1-transfected THP-1 cells than in control plasmid- or K127NSGK1-transfected THP-1 cells. Treatment of S422DSGK1-transfected THP-1 cells with IKK-inhibitor BMS-345541 abolished S422DSGK1-induced increase of MMP-9 transcription and gelatinase activity.

Conclusions:
SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of macrophage migration and MMP-9 transcription via regulation of NF-κB.
Abstract 360: Reduction of Atherosclerotic Lesions by the Chemotherapeutic Agent Carmustine Associated to a Lipid Nanoemulsion
Authors: Maranhao Raul C , Daminelli Elaine N , Martinelli Ana Elisa M
Published: March 2022

After injection in bloodstream, a lipid nanoemulsion (LDE) resembling low-density lipoprotein (LDL) concentrate in atherosclerotic lesions of cholesterol-fed rabbits. Here, rabbits with atherosclerosis were treated with carmustine, an antiproliferative agent used in cancer chemotherapy, associated to LDE to investigate the effects on the lesions. Eighteen male New Zealand rabbits were fed a 1% cholesterol diet for 8 weeks. After 4 weeks nine animals were treated intravenous saline solution and nine with intravenous LDE-carmustine (4 mg/kg, weekly for 4 weeks). LDE-carmustine inhibited atherosclerotic lesions by 90%, compared to controls. LDE-carmustine reduced presence of macrophages, smooth muscle cells, and regulatory T-cells in the arterial intima as well as the expression of matrix metallopeptidase-9, interleukin-1β and TNF-α and lipoprotein receptors (LDL receptor, LDL-related protein-1 and scavenger receptor class B member 1). In conclusion, LDE-carmustine treatment resulted in marked reduction of lesion area, invasion by macrophages and intimal smooth muscle cells and pro-inflammatory factors. Therefore, this new formulation shows great potential for therapy of atherosclerotic cardiovascular disease.
Abstract 375: Alkaline Phosphatase-driven Vascular Calcification Interacts with Hypercholesterolemia and Leads to Severe Coronary Atherosclerosis and Heart Failure in Mice
Authors: Savinov Alexei Y , Yadav Manisha C , Millán José Luis , Savinova Olga V
Published: March 2022

Objective:
Asymptomatic calcification of coronary arteries can independently predict future coronary heart disease. It is not yet clear, however, whether the temporal presence of calcification in coronary arteries is pathogenic. We have recently demonstrated that upregulation of tissue-nonspecific alkaline phosphatase (TNAP) in vascular smooth muscle cells
in vivo
is sufficient to induce medial calcification. The purpose of this study was to test the hypothesis that TNAP-driven intimal calcification can promote coronary atherosclerosis in a mouse model.

Methods:
TNAP was overexpressed in endothelial cells in wild type mice and in mice harboring “wicked high cholesterol” (WHC) mutation in the low density lipoprotein receptor. Hypercholesterolemia was induced in WHC mice by feeding an atherogenic diet. Physiological, histological, and biochemical data were collected longitudinally.

Results:
In the absence of hypercholesterolemia, pan-endothelial overexpression of TNAP induced systemic generalized arterial calcification with normal calcium and phosphate metabolism, but with elevated blood pressure and physiological left ventricular (LV) hypertrophy. Calcific nodules first appear in the arterial intima and expand into the arterial media. Combined with the WHC mutation and 6-8 weeks on atherogenic diet, TNAP-driven arterial calcification led to severe atherosclerosis with 100% morbidity characterized by occlusive coronary artery disease (histologically), pathological cardiac hypertrophy with LV dilation, and reduced cardiac ejection fraction (EF).

Conclusions:
Arterial calcification promotes lipid deposition and is pathogenic when cholesterol is elevated due to genetic and dietary causes.
Abstract 376: Traf-stop-hdl-nanoparticles Reduce Atherosclerosis
Authors: Seijkens Tom T , Zarzycka Barbara , Gijbels Marion J , de Winther Menno P , Weber Christian , Nicolaes Gerry A , Mulder Willem J , Lutgens Esther
Published: March 2022

Inhibition of the co-stimulatory CD40-CD40L receptor/ligand dyad drastically reduces atherosclerosis. However, its long-term blockage results in immune suppression. Inhibition of the CD40-CD40L dyad further downstream in the signaling pathway is therefore required. The interaction between CD40 and its signaling intermediate TNF receptor associated factor 6 (TRAF6) plays a pivotal role in atherosclerosis. To identify drug like molecules that inhibit the CD40-TRAF6 interaction, an
in silico
structure-based virtual ligand screening approach was used. Several small molecule inhibitors (SMI) blocking CD40-TRAF6 interactions were identified. Surface plasmon resonance experiments confirmed direct binding of the compounds to the TRAF6 C-domain. Two of these SMIs, the TRAF-STOPs, reduced atherosclerosis by >40% in
Apoe-/-
mice, when they were treated from 12-18 wks of age, by hampering monocyte and neutrophil recruitment. In accordance, expression of chemokines and cytokines was remarkably reduced in compound treated macrophages.

Interestingly, when the TRAF-STOPs were administered to mice with existing atherosclerosis (from 22-30 wks of age), TRAF-STOPS were able to halt atherosclerosis, resulting in a >45% decrease in atherosclerotic plaque area.

However, these SMIs had a low solubility, and had a half-life of only 8 hrs, and had to be injected daily. To improve the therapeutic applicability of our TRAF-STOPs, TRAF-STOP 6877002 was packed in HDL-based nanoparticles, and administered twice a week for 6 wks (wk 12-18) to
ApoE-/-
mice. The HDL-TRAF-STOP nanoparticles preferentially homed to macrophages, and the expression level in plaque macrophages was high. HDL-TRAF-STOP nanoparticle treatment reduced atherosclerosis by 42.6%.

These newly developed, nanoparticle based CD40-TRAF6 inhibiting SMIs (TRAF-STOPs) are a promising lead for the development of therapeutics for the treatment of atherosclerosis.
Abstract 384: Association between Renin Angiotensin System Gene Polymorphisms and Severity of Coronary Artery Disease and Restenosis after Coronary Stenting
Authors: Zhu Min , Yang Minjun , Jiang Jianjun , Zhu Huanhuan , Chen Xiaofeng
Published: March 2022

Background and Objective:
Coronary artery disease (CAD) represents the most important cause of sudden cardiac death. Percutaneous coronary intervention (PCI) is an effective technique for coronary revascularization but a considerable number of patients develop restenosis after stenting. Renin angiotensin system (RAS) plays an important role in restenosis by promoting neo-intimal hyperplasia. This study evaluateed the polymorphisms of the genes encoding key RAS components including angiotensinogen (AGT), angiotensin converting enzyme (ACE) and angiotensin type 1a (AT1a) receptor in relation to severity of coronary artery disease and in-stent restenosis after coronary stenting.

Methods and Results:
Five hundred and twenty six patients who underwent angiography due to suspected CAD were prospectively recruited to this study. Five single nucleotide polymorphisms (M235T, G217A, G152A, G-6A, A-20C) of AGT gene, ACE insertion/deletion (I/D) and AT1a A1166C polymorphisms were genotyped from genomic DNA with direct sequencing. Severity of coronary atherosclerosis was assessed by angiographic Gensini score. A subsequent coronary angiography was performed 6-9 months later for suspected restenosis for patients (N=273) who underwent coronary stent implantation. In-stent restenosis was evaluated by means of quantitative angiography. Forty-five patients (16.5%) revealed in-stent restenosis. Haplotypes were constructed after linkage disequilibrium analysis. In a multivariate analysis of angiographic restenosis, there was no significant association of the polymorphisms of AGT(M235T, G217A, G152A, G-6A, A-20C, ACE I/D, and AT1a A1166C with the occurrence of in-stent restenosis (P>0.05). However, in the CAD group, frequency of ACE D allele was significantly higher in patients with angiographically defined multi-vessel disease compared to patients with single vessel CAD ( p = 0.013). Additionally, a significant association was found between the frequency of D allele and severity of CAD assessed by the Gensini score (P=0.028).

Conclusion:
Genetic polymorphisms of RAS are not associated with in-stent restenosis after coronary stenting. However, the presence of the ACE I/D polymorphism is potentially associated with the severity of CAD.
Abstract 394: Murine Cre/flox Studies Indicate Yolk Sac Derived Macrophages Are the Cellular Source of FXIIIA
Authors: Beckers Cora M , Griffin Kathryn J , Simpson Kingsley J , Brown Jane M , Cordell Paul A , Smith Kerrie A , Drinkhill Mark J , Kearney Mark T , Vacher Jean J , Pease Richard J , Grant Peter J
Published: March 2022

Fibrin clot cross-linking FXIIIA has a plasma and cellular pool. It is present in platelets, macrophages and connective tissues. However, it is unknown how FXIIIA is secreted and which cell type maintains the plasma pool, although the platelet is considered the main candidate. To address this we have induced lineage specific deletion in mice floxed in coding exon 7 of the FXIIIA gene. Mice homozygous for the floxed gene and transgenic for Pf4cre-recombinase (thrombopoietic deletion), CD11bcre or LysMcre (myeloid deletions) were generated. A FXIIIA KO was obtained using a CMV-driven cre. Plasma and platelet FXIIIA protein & activity and FXIIIA mRNA in brain, heart and aorta were determined and compared to WT, FXIIIA floxed and thrombocytopenic Mpl (thrombopoietin receptor) KO mice.

FXIIIA KO and Pf4FXIIIA KO mice lack platelet FXIIIA protein & activity, whereas both are normal in the Mpl KO. pFXIIIA is absent in the FXIIIA KO and decreased in Pf4FXIIIA KO (by 85%), CD11bFXIIIA KO (40%) and LysMFXIIIA KO (60%) whereas in WT, FXIIIA floxed and Mpl KO pFXIIIA was unaltered. Mpl KO platelet size as determined by FACS was also unaltered. These data confirm that platelets are not the source of pFXIIIA. These data also demonstrate that the pFXIIIA secreting cells are Pf4 dependent & Mpl independent: a combination not seen in BM-derived cells. Thus, we are looking for a macrophage-like cell outside of the BM.

Resident macrophages in the brain are yolk sac-derived and uniquely, not replaced from BM under normal conditions. In young uninjured brain FXIIIA mRNA levels were knocked down by 96.8% in Pf4FXIIIA KO and by 37.8% in the Mpl KO, confirming, for the first time, the existence of a Pf4 dependent Mpl independent cell from the yolk sac.

In heart cell fractions, the smallest Thy1+ fraction (3% of total) contained the largest FXIII-A mRNA pool, which decreases in Pf4cre & CD11bcre-Flox mice, further supporting involvement of yolk sac-derived macrophages.

Further study of FXIIIA mRNA expression in Pf4 dependent, Mpl independent cells in the cardiovascular system demonstrated that the pFXIIIA profile was most closely mirrored by the reduction in arterial wall FXIIIA mRNA suggesting that yolk sac derived arterial wall resident macrophages are the cellular source of FXIIIA.
Abstract 398: Inhibition of Tissue Factor Reduces Thrombosis and Inflammation Without Increased Bleeding in a Mouse Model of Ischemic Stroke
Authors: Wang Shaobin , Reeves Brandi , Kirchhofer Daniel , Pawlinski Rafal
Published: March 2022

Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, is highly expressed by astrocytes surrounding blood vessels in the central nervous system. Using mouse models of hemorrhagic and ischemic stroke, we previously demonstrated that astrocyte TF plays a critical role in brain hemostasis while also contributing to neuronal damage. Importantly, using mice with different levels of astrocyte TF expression, we determined that an astrocyte TF activity around 5% of the wild type level was sufficient to limit bleeding in hemorrhagic stroke and at the same time was low enough to reduce infarct size and behavioral deficits in ischemic stroke, without increased hemorrhagic transformation. Therefore, we hypothesized that pharmacologic inhibition of TF may improve outcomes after ischemic stroke and have a wide safety margin.

To test this hypothesis, C56Bl/6J mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hour followed by 24 hours of reperfusion. Inhibitory rat anti- mouse TF antibody (1H1) and control IgG were administered as a bolus intraperitoneal injection immediately after ischemia. Inhibition of TF dose-dependently reduced brain infarct size in female mice (36.6±1.4% of total brain volume in IgG group [n=19] vs 28.3 ± 1.7% in 25mg/kg 1H1 group [n=9, p<0.01] and 18.5 ±2.3% in 75mg/kg 1H1 group [n=8, p<0.001]; mean±SEM). Importantly, 1H1 did not exacerbate hemorrhagic transformation during the reperfusion phase. Furthermore, TF inhibition significantly reduced microvascular thrombosis (analyzed by fibrin staining), expression of inflammatory markers in the injured brain (IL-6, MCP-1 and KC levels- analyzed by ELISA) and attenuated behavioral deficit after ischemic stroke. Subjecting male mice to the same MCAO condition resulted in partial lethality in IgG treated group (4 out of 6 mice died with 24 hours) which was prevented by 1H1 (25mg/kg) treatment (0 out of 5, Log-rank test p=0.031).

Altogether, our data suggest that the overabundance of astrocyte TF expression resulted from selective pressure to limit intracerebral hemorrhage after traumatic brain injury, but in the modern era poses the additional risk of increased injury during ischemic stroke.
Abstract 410: Human SR-BI/SR-BII Overexpression Enhances LPS-induced Liver and Kidney Inflammation and Acute Organ Injury
Authors: Bocharov Alexander V , Baranova Irina N , Souza Ana C , Vishnyakova Tatyana G , Remaley Alan T , Yuen Peter S , Star Robert A , Patterson Amy P , Eggerman Thomas L
Published: March 2022

SR-BI and its splice variant SR-BII are HDL receptors that recognize bacteria and other pathogens. It has been reported that the SR-BI/II knockout mouse is more sensitive to endotoxin-induced inflammation and sepsis. Since the SR-BI/II KO model demonstrates a number of immunity-related deficiencies and metabolic alterations, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with LIV11-directed liver/kidney expression of hSR-BI or hSR-BII. 6 hours after intraperitoneal LPS injection transgenic hSR-BII and hSR-BI mice demonstrated markedly higher serum levels of pro-inflammatory cytokines/chemokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared to WT mice. In line with the pro-inflammatory status of hSR-BI and hSR-BII transgenic mice, considerably (3-6-fold) higher expression of iNOS was detected in liver and kidney of these animals, while serum NO levels were similar in all groups of mice. Despite lower levels of plasma HDL, LPS-induced corticosterone was only moderately reduced (by 30%) in plasma of both groups of transgenic mice when compared to WT animals. Histological examination of tissues from LPS-challenged mice revealed hepatic and renal injury in hSR-BII transgenic mice, while no statistically significant changes in damage score were observed between the hSR-BI transgenic and WT mice. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, could substantially increase LPS-triggered inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs that are susceptible to LPS toxicity during sepsis.
Abstract 412: Radio Protective P105 Deficiency Aggravates Vein Graft Disease And Lesion Instability via Increased Inflammatory Responses
Authors: de Vries Margreet R , Wezel Anouk , Peters Erna H , Karper Jacco C , Kuiper Johan , Bot Ilze , Quax Paul H
Published: March 2022

Aim:
Vein grafts are often used to bypass atherosclerotic lesions; however, patency rates are troublesome due to the development of vein graft disease. Deficiency of toll like receptor (TLR)4, a key initiator of inflammatory signaling, results in reduced vein graft disease. As TLR4 signaling is regulated by the accessory molecule RadioProtective 105 (RP105), we aimed to investigate the effects of RP105 on vein graft disease.

Methods and results:
Vein graft surgery was performed on Rp105-/- mice (n=13) and C57BL/6 mice (n=11), as well as on LDLr-/-/Rp105-/- mice (n=11) and LDLr-/- mice (n=11) fed a western type diet, 28 days later lesion size and composition was analysed. A 90% increase in vein graft lesion size was observed in RP105-/- mice compared to controls. Lesion size however, did not differ between LDLr-/-/RP105-/- mice and LDLr-/- mice, but interestingly, a significant increase in the number of unstable lesions and intraplaque hemorrhage upon RP105 deficiency was observed. In both experimental setups, an increase in lesional macrophages was seen. Peritoneal Rp105-/- macrophages showed an increase in proliferation. Whereas, Rp105-/-smooth muscle cells and bone marrow derived mast cells secreted increased levels of the monocyte chemoattractant CCL2. In both the Rp105-/- and LDLr-/-/Rp105-/- vein grafts the amount of lesional CCL2 was significantly increased, as well as the number of activated perivascular mast cells.

Conclusions:
Together, these data indicate that RP105 has a protective role in vein graft disease by dampening the inflammatory effect, since RP105 deficiency results in an increased inflammatory response and exacerbated CCL2 production by both mast cells and smooth muscle cells.
Abstract 414: Key Role of STAT4 Deficiency in the Hematopoietic Compartment on Insulin Resistance and Adipose Tissue Inflammation
Authors: Dobrian Anca D , Ma Kaiwen , Glenn Lindsey M , Hatcher Margaret A , Haynes Bronson A , Lehrer Eric J , Nalder Jerry L
Published: March 2022

Adipose tissue inflammation is a hallmark of obesity and contributes to insulin resistance (IR). Recent data from our lab showed that STAT4 global deficiency reduces inflammation and improves insulin sensitivity in obese mice. STAT4 is expressed in adipocytes and cells of hematopoietic lineage. The objective of this study was to determine the contribution of selective STAT4 deficiency in adipocyte vs hematopoietic cells to IR and adipose tissue inflammation. For this purpose we sub-lethally irradiated

Stat-4
-/-
C57Bl6

mice and reconstituted them with bone marrow cells (BMC) from

Stat-4
+/+
C57Bl6

congenic donors to restore STAT4 expression in BM- derived cells (STAT4-/- recipients). These mice were compared to irradiated
C57Bl6 wild-type
mice reconstituted with

Stat-4
-/-
C57Bl6

BMCs to produce STAT4 deletion selectively in BM-derived cells (STAT4+/+ recipients). Mice received a HFD (60%kcal fat) for 12 weeks (n=7 mice/group). Body weights, plasma cholesterol, triglycerides and free fatty acids were not different between the STAT4+/+ and STAT4-/- recipients. However the STAT4-/- recipients had significantly increased fasted plasma glucose. Congruent with this data the IP-ITT and GTT showed significantly higher area under the curve for the STAT4-/- recipients indicating increased glucose intolerance and insulin resistance compared to STAT4+/+ recipients. Adipose tissue immune cell composition was determined by flow cytometry. The total number of CD45+ lymphocytes was significantly higher in the STAT4-/- recipients compared to STAT4+/+ recipients. Also, the numbers of CD3+CD4+ and CD3+CD8+ cells were significantly higher in the STAT4-/- recipient group. Adipocyte size was significantly higher and the number of crown-like structures was significantly increased in the STAT4 -/- recipients indicating adipocyte hypertrophy and increased macrophage infiltration. Following in vivo insulin stimulation, activation of IR and IRS-1 was increased in liver and muscle in the STAT4+/+ recipient group suggesting improved insulin signaling in the mice that received STAT4-/- BMCs. These studies provide clear evidence for the importance of STAT4 in hematopoietic cells in regulating IR and adipose tissue inflammation in obesity.