bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 260: BAFF 60mer is Critical for B Cell Activation and BAFF Depletion Suppresses AAA Formation
Authors: Serbulea Vlad , Spinosa Michael , Montgomery William , Sahu Srabani , Srikakulapu Prasad , McNamara Coleen A , Upchurch Gilbert R , Ailawadi Gorav , Leitinger Norbert , Meher Akshaya K
Published: March 2019

Marginal zone and follicular B cells together constitute the B2 cell population, which is known to promote cardiovascular diseases by secretion of pathogenic antibodies. However, it is not completely understood how B2 cells are activated. Here, we tested the hypothesis that B cell activating factor (BAFF) activates B2 cells and promote abdominal aortic aneurysm (AAA) formation. Since BAFF can either exist as a 3mer or multimerize to a highly active 60mer, we further examined if the 60mer is critical for B2 cell-mediated pathogenicity. Anti-BAFF antibody (Ab) Sandy-2 was injected to C57BL/6 male mice at 1 mg/kg once in every 14 days. AAA was induced by topical elastase model after 14 days of Sandy-2 injection. Native PAGE and ELISA methods were used to determine binding of Abs to recombinant BAFF 3mer and 60mer. For
in vitro
experiments, B cells were isolated from murine spleens. Activation of B cells was examined by Western blotting and RNA sequencing and by surface expression of CD23 and MHC II by flow cytometry. Metabolic reprogramming of B cells by BAFF was determined by extracellular flux analysis using a Seahorse XF24 Flux Analyzer. Sandy-2 bound to both 3mer and 60mer, resulting in suppressed AAA formation (n=8, p<0.05) with (1) marked depletion of B2 cells, transitional 2, germinal center, plasma and memory B cells, but not transitional 1 and B1 cells, (2) a lower level of IgG1 and IgG2, and (3) a lack of immunoglobulin deposition in AAA sections.
In vitro
, the 60mer, but not the 3mer, significantly activated both NF-kB1 and -kB2 signaling, and induced expression of B2 cell activation markers and anti-apoptotic genes in B cells. Inhibitors of NF-kB signaling decreased B cell activation in response to 60mer. The 60mer treatment significantly increased mitochondrial respiration and glycolysis in B cells, supporting an activated status. An antibody against multimerization site of BAFF (anti-multiBAFF) significantly suppressed B cell activation relative to a control Ab, in a neutrophil:B cell co-culture model. The effect of the anti-multiBAFF Ab on AAA formation is currently being tested in our laboratory. Altogether, our results suggest a critical role for BAFF 60mer in skewing B cells to an activated B2 cell phenotype, supporting a pathogenic role of B2 cells in AAA.
Abstract 262: Circulating Exosomes from PAD Patients Modulate Vascular Repair and Inflammation
Authors: Sorrentino Thomas A , Duong Phat , Bouchareychas Laura , Sansbury Brian E , Mitchell Pete , Chen Mian , Chung Allen , Schaller Melinda S , Spite Matthew , Raffai Robert L , Conte Michael S
Published: March 2019

Objectives:
Peripheral arterial disease (PAD) is a chronic disease characterized by inflammation. Recent work suggests that circulating exosomes may contribute to vascular injury and remodeling. We hypothesize that exosomes from PAD subjects negatively modulate vascular repair via miRNA and bioactive lipid mediators (LM).

Methods:
Exosomes (particle size 30-100nm) were isolated from plasma of healthy (n=6) and PAD (n=6) subjects. Exosome miRNA was isolated and assessed by qPCR. Targeted metabolo-lipidomics was performed by liquid-chromatography-tandem mass spectrometry. VSMC and EC migration were assessed via scratch assay. Monocyte-derived macrophage gene expression after exposure to exosomes was assessed via RT-qPCR.

Results:
Compared to healthy subjects, exosomes from PAD subjects contained lower levels of pro-angiogenic miR-126 and miR-210 (25.2±6.4 vs 8.3±1.7, p<0.05 and 0.29±0.07 vs 0.08±0.02, p<0.05, respectively). Exosomes contained arachidonic acid, eicosapentaenoic acid and docosapentanoic acid, as well as both pro-inflammatory and pro-resolving bioactive LMs and their pathway markers, including prostaglandins, leukotrienes, lipoxins, resolvins (D- and E-series) and maresins. By principle component analysis, exosome LM profiles differed significantly between healthy and PAD subjects. Exosomes from PAD subjects increased VSMC migration (1.5±.09-fold vs 1.0±.09-fold wound closure, p<0.005) and decreased EC migration (1.5±.04-fold vs. 1.8±.06-fold wound closure, p<0.005) compared to healthy controls. Both PAD and healthy exosomes increased MDM expression of pro-inflammatory genes TNF-α and MCP-1.

Conclusion:
Plasma-derived exosomes from PAD patients contain an altered profile of vascular-active miRNA and LMs and confer effects on VSMCs and ECs that may impair vessel remodeling. We describe the first known evidence that plasma exosomes contain pro-resolving LMs. Collectively these data suggest that circulating exosome-based signaling may modulate vascular inflammation and repair in PAD patients.
Abstract 267: Testosterone to Estradiol Ratio Reflects Systemic and Plaque Inflammation and Predicts Future Cardiovascular Events in Men With Severe Atherosclerosis
Authors: van Koeverden Ian D , de Bakker Marie , de Borst Gert J , Pasterkamp Gerard , den Ruijter Hester
Published: March 2019

Objective:
The effects of testosterone on cardiovascular disease (CVD) as reported in literature have been ambiguous. Recently, the interplay between testosterone and estradiol as assessed by testosterone/estradiol (T/E2) ratio was suggested to be better informative on the normal physiological balance. Considering the role in CVD, we hypothesized that a low T/E2 ratio in men with CVD is associated with increased inflammation, a more unstable plaque and a worse cardiovascular outcome.

Approach:
Testosterone and estradiol concentrations were determined in blood samples of 709 male carotid endarterectomy patients included in the Athero-Express Biobank Study. T/E2 ratio was associated with baseline characteristics, atherosclerotic plaque specimens, inflammatory biomarkers and three-year follow-up information.

Results:
Patients with low T/E2 ratio had more unfavorable inflammatory profiles compared to patients with high T/E2 as observed by higher levels of C-reactive protein (CRP) (3.08 μg/mL vs. 1.21 μg/mL (p<0.001)) and higher leukocyte counts (8.95*109/L vs. 7.84*109/L (p<0.001)) in blood. In atherosclerotic plaques, a negative association between T/E2 ratio and number of neutrophils (B=-0.56(p=0.010)), smooth muscle cells (B=-0.049(p=0.046)), interleukin-6 (IL-6) (B=-0.15 p=0.009) and Il-6receptor (B=-0.13 p=0.024) was found. Decreased T/E2 ratio showed an overall trend towards histological features that represent the vulnerability of atherosclerotic lesions. Furthermore, in multivariate cox regression analysis, low T/E2 ratio was independently associated with an increased risk for major cardiovascular events (MACE) during three-year follow-up (HR 1.77 (95%CI: 1.08 - 2.90 p=0.023)).

Conclusion:
In male patients with manifest atherosclerotic disease, low T/E2 ratio was associated with increased systemic inflammation, increased inflammatory plaque proteins and an increased risk of future major cardiovascular events as compared to men with normal T/E2 ratio. Normalization of T/E2 ratio may be a useful tool for the secondary prevention of CVD in men.
Abstract 268: Macrophage Depletion Attenuated Renal Injury and Fibrosis in Angiotensin II Hypertensive Mice
Authors: Huang Lei , Wang Aimei , Li Weihong , Liu Chang , Liu Yueyang , Yang Zhihnag , Zhou Ming-Sheng
Published: March 2019

Monocyte/macrophage recruitment is closely associated with the degree of hypertensive renal injury. Here we investigated the direct role of macrophage using Liposome encapsulated clodronate (LEC) to deplete monocyte/macrophage in Ang II-induced hypertensive renal injury. C57BL/6 mice were treated with a pressor dose of angiotensin II (Ang, 1.4 mg/kg/day by mini-pump) plus LEC or the PBS-liposome for two weeks. Ang II mice developed hypertension (186 ± 5 vs. 110 ± 4 mmHg in control, p<0.05) , albuminuria (33.9 ± 4.9 vs. 83.6 ± 2.3 μg/mg creatinine in control, p<0.05), glomerulosclerosis and renal fibrosis. LEC reduced Ang II-induced albuminuria (59.6 ± 1.9 μg/mg creatinine, p<0.05) and protected against renal structural injury with a mild reduction in systolic blood pressure (153 ± 3 mmHg, p<0.05). Ang II significantly increased renal macrophage infiltration (MOMA2
+
cells) and the protein expression of renal tumor necrosis factor α and interleukin β1, which were significantly reduced in Ang II mice treated with LEC. Ang II increased renal oxidative stress (as demonstrated by increased oxidative fluorescence densities, NADPH oxidative activity and the protein expression of NADPH oxidase subunits gp91phox and p22phox) and the expression of profibrotic factors transform growth factor β1 and fibronectin. Ang II also inhibited the phosphorylation of endothelial nitric oxide synthesis (eNOS, ser1177). LEC revised most of the changes in the Ang II-induced molecules as mentioned above. Our results suggest that renal macrophage is a main mediator to contribute to Ang II-induced hypertensive renal injury and fibrosis, the underlying mechanisms may involve reduction in macrophage-drove renal inflammation and restoration of the balance between renal oxidative stress and eNOS. These findings may lead us to develop a novel therapy directed at targeting the infiltration of macrophages or the macrophages-derived cytokines for treatment of hypertensive renal diseases.
Abstract 276: Excessive Plasmin Activity Compromises Hepatic Sinusoidal Vascular Integrity After Acetaminophen Overdose
Authors: Gao Siqi , Silasi-Mansat Robert , Behar Mandi , Lupu Florea , Griffin Courtney
Published: March 2019

The serine protease plasmin degrades extracellular matrix (ECM) components both through direct cleavage and indirectly through activation of matrix metalloproteinases. We previously reported that excessive plasmin activity and subsequent ECM degradation causes hepatic sinusoidal fragility and lethal hemorrhage in developing embryos. We now report that excessive plasmin activity in a murine acetaminophen (APAP) overdose model likewise compromises postnatal hepatic sinusoidal vascular integrity. We found that hepatic plasmin activity is upregulated significantly at 6 hr after APAP overdose. This plasmin upregulation precedes both degradation of the ECM component fibronectin around liver vasculature and bleeding from centrilobular sinusoids. Importantly, administration of the pharmacological plasmin inhibitor tranexamic acid or genetic reduction of plasminogen, the circulating zymogen of plasmin, ameliorates APAP-induced hepatic fibronectin degradation and sinusoidal bleeding. In conclusion, these studies demonstrate that reduction of plasmin stabilizes hepatic sinusoidal vascular integrity after APAP overdose.
Abstract 283: Fenofibrate Induces Endothelial Cell Tubule Formation Independent of Phospholipogenesis
Authors: Saffaf Omar , Belaygorod Larisa , Saffaf Khalid , Semenkovich Clay F , Zayed Mohamed
Published: March 2019

Fenofibrate, a proliferator-activated receptor (PPAR)α agonist, is the only oral medication demonstrated to prevent lower extremity amputations in diabetic patients. Phosphatidylcholines, generated by choline-ethanolamine phosphotransferase 1 (CEPT1) via the Kennedy Pathway, also induce PPARα activation, but their metabolism is altered in the setting of diabetes. It is unknown whether CEPT1 is essential for fenofibrate-mediated endothelial cell (EC) function. To evaluate this, we generated a murine model for conditional knockdown of
Cept1
in the endothelium (
Cept1EC
KO). Heart ECs (MHECs) were harvested from 6wk old
Cept1EC
KO and wildtype (WT) littermates, and cultured
in vitro
on growth factor-reduced Matrigel. Cultures were then supplemented with VEGF (50ng/mL), bFGF (50ng/mL), and fenofibrate (25uM), then assessed longitudinally at 0, 4, and 6 hours. We observed that compared to WT,
Cept1EC
KO MHECs had significantly less tubule formation (p < 0.0001). VEGF and bFGF failed to rescue
Cept1EC
KO MHECs, but demonstrated a robust agonist response in WT MHECs (bFGF: p=0.003; VEGF: p=0.0002). Interestingly, fenofibrate demonstrated complete rescue of
Cept1EC
KO MHECs at 4 and 6 hours of culture. This finding demonstrates that fenofibrate restores EC function even in the setting of impaired phospholipid biosynthesis. This observation may partially explain how fenofibrate confers added benefits in subjects with diabetic and peripheral arterial disease. Future work will further elucidate this mechanism of action in diabetic subjects.
Abstract 286: Role of Glutathione in the Regulation of VEGFR2 in Human Aortic Endothelial Cells
Authors: Prasai Priya K , Pattillo Christopher B
Published: March 2019

Tissue ischemia due to blood vessel occlusion favors new blood vessel formation or angiogenesis. Although hypoxia is one of the important regulators of angiogenesis, angiogenesis is a rare event in many cardiovascular diseases (CVD). This decreased angiogenesis could be attributed to many factors including altered glutathione (GSH) redox state. Previously, our laboratory has shown an intricate relationship between decreases in GSH in ischemic tissues and angiogenesis. Therefore, we hypothesized that altered GSH redox state regulates angiogenesis by affecting the vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR2) pathway. We used human aortic endothelial cells (HAEC) and treated with diamide (DA), an oxidant that oxidizes GSH to GSSG, thereby altering GSH redox state. DA treatment decreased GSH and increased GSSG concentrations resulting in decreased GSH/GSSG. Using western blotting, we found that decreased GSH/GSSG activates VEGFR2. To identify the mechanism of VEGFR2 activation after DA treatment, we measured VEGF release into the media using ELISA. There was no difference in extracellular VEGF between the groups suggesting that DA activates VEGFR2 ligand-independently. We then investigated the role of protein tyrosine phosphatases (PTP) using a pan-PTP inhibitor, sodium orthovanadate, which did not alter VEGFR2 activation after DA treatment suggesting no role for PTPs in this pathway. We also studied the role of intracellular H
2
O
2
in DA-induced activation using PEG-catalase to quench H
2
O
2
and then treated with DA. PEG-catalase pretreatment did not affect VEGFR2 activation showing H
2
O
2
is not involved in DA-induced VEGFR2 activation. In order to validate the DA-induced receptor activation, we inhibited glutathione reductase using 2-AAPA to increase intracellular GSSG levels. 2-AAPA treatment alone activated VEGFR2, confirming receptor activation after altering GSH/GSSG. Lastly, altering GSH/GSSG decreased the proliferation of HAECs. Our results elucidate a novel role of glutathione especially GSSG in ligand-independent VEGFR2 activation and subsequent inhibition of cell proliferation. Based on our data, GSSG could be a possible mechanism for decreased angiogenesis in CVD.
Abstract 304: Differential Effects of LRP1 in AngII-induced Ascending Aortic PathologiesBetween Male and Female Mice: Lack of Association With Elastin Fragmentation
Authors: Wright Bradley C , Sawada Hisashi , Moorleghen Jessica J , Howatt Deborah A , Rateri Debra L , Majesky Mark W , Daugherty Alan
Published: March 2019

Objective:
Low-density lipoprotein receptor-related protein 1 (LRP1), a transmembrane protein, is important in maintaining elastin fiber integrity of the aortic wall. Smooth muscle cells (SMCs) of the ascending aorta are composed of the inner medial layers from the cardiac neural crest and the outer layers from the second heart field (SHF). LRP1 depletion in SMCs of male mice augments angiotensin II (AngII)-induced ascending aortic dilation and rupture attributed specifically to SMCs of SHF origin. The purpose of this study was to determine whether depletion of LRP1 in SHF-derived SMCs (SHF-SMC LRP1) has differential effects on AngII-induced aortic dilation, rupture, and elastin fragmentation between male and female mice.

Methods and Results:
Female LRP1 floxed mice were bred to male LRP1 floxed mice with Mef2c-Cre transgene to generate SHF-SMC LRP1 +/+ and -/- mice. Male and female mice at 12-14 weeks of age were infused with either saline or AngII (1,000 ng/kg/min) for 28 days (n = 12-31). Ascending aortic diameter (AoD) was measured by ultrasound, rupture was determined by necropsy, and elastin fragmentation was assessed by Movat’s staining. In male mice of both genotypes, AoD and elastin breaks were increased by AngII infusion, and AoD was positively correlated with elastin fragmentation (r
2
= 0.48, p < 0.001). SHF-SMC LRP1 deletion augmented aortic dilation, rupture rate, and elastin fragmentation in AngII-infused male mice. In females, AngII infusion increased AoD in SHF-SMC LRP1 -/- mice, but not in their wild type controls. AngII-induced elastin fragmentation did not differ between SHF-SMC LRP1 +/+ and -/- female mice, and no correlation between AoD and elastin fragmentation was detected (r
2
= 0.01, p = 0.88). Despite the extent of AngII-induced elastin fragmentation that was equivalent to that in males, aortic rupture is lower in female than in male LRP1 deleted mice (9 vs 38%, p = 0.02).

Conclusion:
In male mice, AngII infusion promotes dilation and rupture of ascending aorta that is augmented in SHF-SMC LRP1-/- mice and positively associated with elastin fragmentation. However, AngII only promotes ascending aortic dilation in female mice with SHF-SMC LRP1 deletion, despite elastin fragmentation being equivalent between SHF-SMC LRP1 +/+ and -/- mice.
Abstract 310: Retrograde Hemorrhage and Ischemic Injury after REBOA in a Porcine Model of Uncontrolled Aortic Injury
Authors: Go Catherine , Thirumala Partha , Kuhn Jenna , Chen Yanfei , Chun Youngjae , Tillman Bryan
Published: March 2019

Introduction:
Resuscitative Balloon Occlusion of the Aorta (REBOA) has gained popularity as a less invasive approach to temporize traumatic noncompressible hemorrhage, yet mortality remains over 70%. Although associated injuries may account for some deaths, contributions from ischemia and ongoing retrograde bleeding are also likely, with REBOA occlusion frequently in Zone 1 (descending thoracic aorta) and often greater than 20 minutes. This study examined retrograde blood loss and ischemic injury after REBOA in a porcine model of aortic injury.

Methods:
Six anesthetized swine with invasive hemodynamic and neurophysiologic monitoring (Muscle Evoked Potentials and Somatosensory Evoked Potentials) underwent 8 Fr femoral access and Zone 1 positioning of a REBOA balloon prior to aortic injury. The thoracic aorta was injured with a 22 Fr dilator, followed by aortography and immediate REBOA inflation proximal to the injury. Profound deterioration of the first three animals with one hour of REBOA prompted the next three animals to undergo only 30 minutes of REBOA. Blood loss was recovered with a cellsaver. Animals underwent permanent stent repair of the aortic injury and resuscitation with the intent to recover.

Results:
Despite proximal hemorrhage control documented angiographically, blood loss from retrograde bleeding was substantial averaging 3.7 L and 3.5 L for the 30- and 60-minute groups, respectively. After balloon inflation, mean pressure fell an average of 62 mmHg within 20 minutes (p < 0.001), while cardiac output decreased 20-40%. In the lower extremities, Neuromonitoring revealed ischemic loss of motor signals at a mean of 27 minutes. Even after resuscitation with blood, bicarbonate, saline and pressors, all six animals arrested shortly after balloon deflation, amidst falling bicarbonate (p less than 0.001), and rising lactate (p less than 0.01) relative to baseline.

Conclusions:
Retrograde hemorrhage is an underappreciated event during REBOA control of aortic injuries, that may contribute to spinal cord ischemia, tissue ischemia and death. This study suggests that improved outcomes for noncompressible hemorrhage will require balance of competing goals of hemorrhage control and distal perfusion.
Abstract 312: Upregulation of Notch3 Contributes to Aneurysm Formation in a Murine Model of Marfan Syndrome
Authors: Fitzgerald Matthew , Meisinger Trevor , Batra Rishi , Liu Zhibo , Baxter B. Timothy , Xiong Wanfen
Published: March 2019

Notch3 functions as a receptor for membrane-bound ligands (Jag1, DLL1) in signaling pathways for cell-cell communication, gene regulation and control of cell differentiation during embryonic and adult vascular development. However, the role of Notch3 in Marfan Syndrome (MFS), which the main cardiovascular complication is thoracic aneurysms that results in high mortality, has yet to be fully elucidated. Previously we have shown that during aneurysm development, aortic smooth muscle cells (SMCs) in mgR mice, a murine model of MFS, display premature switch from a synthetic to a more contractile phenotype. Therefore we
hypothesized
that Notch3 could be playing a key role in this aberrant differentiation and aneurysm formation.

To test this hypothesis we examined Notch3 mRNA and protein levels in the aortic tissue and SMCs from 1, 4, and 8 weeks-old mgR mice and wild type controls by real time-PCR, Western blot, and immunohistochemistry. Aortic histological changes were examined by Verhoeff-Van Gieson (VVG) elastin staining. In addition, mice were treated with DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a known γ-secretase inhibitor. The effects of notch signaling inhibition on aneurysm rupture were evaluated.

We found that aortic mRNA and protein levels of Notch3 in mgR mice was upregulated ~2 fold by 1 week, and 4-fold at 4 and 8 weeks compared to WT littermate controls. The expression levels of upstream and downstream targets of Notch3, Jagged1 and p21, respectively, were significantly increased in the aorta of mgR mice compared to wild type controls. DAPT treatment prolonged the median lifespan of mgR mice from 16 weeks (untreated) to 32 weeks (DAPT treated). The results from VVG staining of aortic tissue showed that DAPT treatment inhibited aortic elastic degradation in mgR mice.

Taken together, our findings indicate that Notch3 is upregulated in the early aortic development and continues to increase during the course of the aneurysm formation in mgR mice. The increase in Notch3 expression may contribute to aneurysm development in MFS. This study lays the groundwork to further examine Notch3 as a novel potential target for treatment.
Abstract 317: Inflammasome Inhibitor MCC950 Prevents the Development of Sporadic Thoracic and Abdominal Aortic Aneurysms and Dissections in Mice
Authors: Ren Pingping , Appel Richard , Zhang Lin , Luo Wei , Zhang Chen , Wang Yidan , Guo Jiao , Robertson Avril A. B. , Cooper Matthew A. , Coselli Joseph S. , Shen Ying H. , LeMaire Scott A.
Published: March 2019

Background:
Aortic aneurysms and dissections (AAD) are common interrelated cardiovascular diseases that cause more than 10,000 deaths in the United States each year and are a leading cause of death in people 55 years of age or older. Currently, surgery is the only effective treatment; thus, there is a critical need to develop pharmacological agents that can prevent the initiation or progression of AAD. Increasing evidence suggests that the NLRP3 inflammasome plays a critical role in aortic destruction. In this study, we aimed to test the therapeutic potential of MCC950, a potent and specific NLRP3 inhibitor, for preventing AAD.

Methods/Results:
In a sporadic AAD model induced by a high-fat diet and angiotensin II infusion,, aortic challenge induced significant aortic enlargement and the development of a range of AAD manifestations, including aneurysm without dissection, dissection, and rupture. Importantly, MCC950 treatment significantly inhibited aortic enlargement and the development of AAD, particularly in the ascending and suprarenal aorta. The overall incidence of AAD decreased from 86% in the challenged control group to 40% in the challenged MCC950 treatment group (
P
<0.001). The incidence of ascending AAD decreased from 56% in the challenged control group to 18% in the challenged MCC950 treatment group (
P
=0.0001), and the incidence of suprarenal AAD decreased from 60% in the challenged control group to 20% in the challenged MCC950 treatment group (
P
<0.001). The reduction in AAD incidence was similar in male and female mice. Furthermore, aortas from the MCC950 group showed reductions in inflammasome activation, macrophage infiltration, MMP-9 activity, and elastic fiber destruction. Likewise, in the cultured macrophages, MCC950 treatment inhibited H
2
O
2
-induced activation of the NLRP3 inflammasome, IL-1β secretion, and MMP-9 activation.

Conclusion:
The NLRP3 inhibitor MCC950 is effective at preventing inflammasome activation, aortic destruction, inflammation, and the development of AAD in a mouse model of sporadic disease. Further studies are needed to determine whether MCC950 has similar effects in other AAD models.
Abstract 321: The Aortic Luminal Area is a Potential Marker of Increased Rupture Risk in AbdominalAorticAneurysms
Authors: Siika Antti , Lindquist Liljeqvist Moritz , Hultgren Rebecka , Gasser Christian , Roy Joy
Published: March 2019

Introduction:
Diameter is currently the only factor used to estimate rupture risk of abdominal aortic aneurysms (AAAs). Many large AAAs, however, do not rupture, and a significant portion of small AAAs do. Our aim was to investigate if simple two-dimensional geometric measurements can improve rupture risk prediction in AAAs, and relate these measurements to biomechanical determinants of AAAs.

Methods:
Thirty patients with ruptured AAAs (mean age was 77 ± 5 years and 23 were male) and 60 patients (mean age 60 ± 8 years, and 46 were male) with asymptomatic AAAs were included. At the location of the maximal diameter, the diameter, the luminal area and the vessel area were measured. Finite element analysis was used to compute 3D-geometric and biomechanical parameters of the asymptomatic AAAs, using A4 Clinics Software (VASCOPS, Austria). An automatic matching function was used to construct diameter-matched groups.

Results:
Analysis of all stable AAAs (n=60) and ruptured AAAs (n=30) showed that ruptured AAAs had a significantly larger diameter, 77 ± 15 mm vs. 62 ± 13 mm (p<0.01) and significantly larger luminal area 2281 ± 1964 mm
2
vs. 1059 ± 674 mm
2
(p<0.01). In order to control for diameter as a confounder, two diameter-matched groups, one with ruptured AAAs (n=28) and one with stable AAAs (n=15) were formed (74 ± 12 mm vs 73 ± 11, p = .67). Diameter-matched ruptured AAAs had a larger luminal area (1954 ± 1254 mm
2
vs. 1120 ± 623 mm
2
, p = .02) and a lower relative ILT area (55 ± 24 % vs 68 ± 24%, p= .03). In multivariate regression of 60 asymptomatic AAAs, including the maximal diameter, the luminal area explained the largest amount of variance in the biomechanical rupture risk parameters, followed by the ILT-area.

Conclusions:
We demonstrate that the luminal area is increased in ruptured AAAs compared to stable AAAs. Further, we show that this finding may in part be explained by a correlation between luminal area and biomechanical rupture risk parameters.
Abstract 329: Systems-based Target Discovery Reveals PPARa as a Key Metabolic Regulator for Macrophage Activation in Vein Graft Disease
Authors: Decano Julius , Zhang Hengmin , Singh Sasha , Mlynarchik Andrew , Mojcher Alexander , Wang Jianguo , Lee Lang Ho , Halu Arda , Higashi Hideyuki , Mattson Peter , Qiao Jiao , Choi Jung , Creager Michael , Libby Peter , Anderson Daniel G , Ozaki C. Keith , Aikawa Elena , Aikawa Masanori
Published: March 2019

Objectives:
Vein graft (VG) failure rates after surgical bypass for PAD remain high, which can cause limb loss. Yet, the underlying mechanisms for VG lesion development remain obscure. We took a systems approach to explore key pathways in a mouse model.

Approach and Results:
We implanted the inferior vena cava into the left carotid artery of fat-fed Ldlr-/- mice and monitored lesion development up to 4 weeks post-surgery. Unbiased proteomic profiling followed by hierarchical clustering showed distinct clusters. One protein cluster is decreased in vein grafts with overt lesions (poor outcome) relative to the group of non-diseased veins devoid of lesions and vein grafts with minimal lesion (favorable outcome). Conversely, this can be viewed as increased in the “favorable” outcome group. Using the “favorable outcome” predominant protein cluster, network analysis and pathway enrichment identified PPARα pathway as a top-ranked key hub node with high betweeness centrality measure. This implies that PPARα may play a key regulatory role in limiting lesion severity. We examined whether network-based prediction of PPARα can also serve as a therapeutic target to attenuate the development of experimental VG lesions. A blinded
in vivo
loss-of-function study using PPARα siRNA encapsulated in macrophage-targeted lipid nanoparticles (LNP) demonstrated higher plaque volume as seen by 3D ultrasound and histologic morphometry in the test group (n=7) versus the control group (n=8). In contrast, a randomized drug study on another set of VG mice using the new PPARα-selective agonist pemafibrate demonstrated less plaque lesions and macrophage accumulation in the drug group (n=11) compared to the control group (n=11). In vitro validation studies (alongside loss-of-function) including high-throughput qPCR, single cell qPCR, CyTOF, ELISA, FACS, and metabolic assays all suggested that pemafibrate-induced PPARα activation promotes a phenotype shift of activated macrophages into a less inflammatory state through metabolic reprogramming.

Conclusion:
Systems-based target discovery on VG demonstrates that PPARα agonism reduces lesion development and inflammatory burden. Targeting this pathway might furnish a novel therapeutic option to prevent VG lesion development.
Abstract 345: Combining Morphological and Mechanical Risk Factors May Improve Carotid Plaque Progression Prediction: an Magnetic Resonance Image-Based Follow-Up Study
Authors: Tang Dalin , Wang Qingyu , Canton Gador , Hatsukami Thomas S , Billiar Kristen L , Wu Zheyang , Yuan Chun
Published: March 2019

It has been hypothesized that combining morphological and mechanical risk factors may improve plaque progression prediction. In this paper, plaque burden (PB), cap thickness (CT), lipid percent (LP), plaque wall stress (PWS), plaque wall strain (PWSn) and their combinations were used as predictors to identify the best predictor(s) for plaque progression measured by plaque area increase (PAI) from baseline (T1) and follow-up (T2).

In vivo magnetic resonance image (MRI) carotid plaque data were acquired from 8 patients (5 m, mean age 71) with follow-up (18 months) with informed consent obtained. We built 3D thin-layer models for the 41 matched slices to obtain plaque stress/strain data using patient-specific material properties determined from Cine MRI and patient arm blood pressure. The mean values of the 5 predictors from all lumen points of each slice were obtained for analysis. For each predictor Y, a threshold value Yc was determined so that optimal agreement rate was obtained (agreement cases are either {(Y>Yc) and (PAI >0)} or {(Y≤Yc) and (PAI ≤0)}). For PWS and PWSn, we used (either {(Y0)} or {(Y≥Yc) and (PAI ≤0)}). Two combinations of predictors

COM1=a1*PB+a2*CT+a3*LP,

COM2=b1*PB+b2*CT+b3*LP+b4*PWS+b5*PWSn

were considered, where COM1 combines PB with morphological features, and COM2 combines all 5 predictors. The coefficients in these formulas were determined by using iterative linearly fitting method and initial guess of threshold values from single predictor results.

The optimal threshold values for the 5 single predictors and 2 combination predictors are given in Table 1. PWSn was the best single predictor and COM2 was the best combination predictor.

The threshold value-based method is simple and easy for clinical implementation. Our preliminary results indicate that combining PB, CT, LP, PWS, and PWSn could improve the agreement rate by nearly 10%, compared to using PB alone. Large-scale patient studies are needed to confirm our findings.
Abstract 347: Significant Association Between the Qualifying Neurologic Event and the In-hospital Risk of Stroke or Death Following Carotid Endarterectomy and Carotid Artery Stenting
Authors: Tsantilas Pavlos , Knappich Christoph , Schmid Sofie , Kallmayer Michael , Breitkreuz Thorben , Zimmermann Alexander , Kuehnl Andreas , Eckstein Hans-Henning
Published: March 2019

Objectives:
The purpose of this observational study was to analyze the association between the initial neurological status and the risk of any in-hospital stroke or death in patients treated with carotid endarterectomy (CEA) or carotid artery stenting (CAS) under routine conditions in Germany.

Methods:
Secondary data analysis based on the German statutory quality assurance database for carotid procedures between 2009-2014. The primary outcome was any periprocedural stroke or all-cause death until discharge. To analyze the association between initial neurological status and outcome, a multilevel multivariable regression analyses adjusting for confounders was performed.

Results:
From a total of 182,033 patients documented between 2009 and 2014, 144,347 patients treated with CEA and 14,794 patients treated with CAS were included in the analysis. In total, there 68% were men and the mean age of the cohort was 70.5±9.1 years. The risk of any in-hospital stroke or death in patients treated with CEA was 2.0% (n=2923/144,347). The raw risk of any in-hospital stroke or death was 1.4% in asymptomatic patients and 3.0% in symptomatic patients treated with CEA. Within the group of symptomatic patients, risk of any in-hospital stroke or death after CEA increased from 1.2% (amaurosis fugax, AFX), 2.3% (TIA), 2.8% (minor stroke), 4.4% (major stroke), 4.8% (crescendo TIA, cTIA) to 9.0% (stroke in evolution, SIE). The risk of any in-hospital stroke or death in patients treated with CAS was 3.6% (n=538/14,794). The raw risk of any in-hospital stroke or death was 1.7% in asymptomatic patients and 6.1% in symptomatic patients treated with CAS. Within the group of symptomatic patients, risk of any in-hospital stroke or death increased from 1.0% (AFX), 4.1% (TIA), 4.1% (minor stroke), 5.4% (major stroke), 5.2% (cTIA) to 11.7% (SIE). Regression analysis revealed that the severity of initial neurologic symptoms was associated with an increased risk of any in-hospital stroke or death in both patients treated for CEA and CAS.

Conclusion:
Periprocedural risk for any stroke or death did not significantly differ between asymptomatic patients and patients with AFX but between asymptomatic patients and patients with TIA, stroke, cTIA or SIE.
Abstract 362: Plasminogen Activator Inhibitor 1 Forms a Complex With FXIa on the Surface of Endothelial Cells, Blocking Its Activity and Inducing the Clearance, Internalization and Degradation of FXIa
Authors: Puy Cristina , Ngo Anh , Gailani David , Gruber Andras , McCarty Owen J
Published: March 2019

Background:
The activation of coagulation factor (F) XI by activated coagulation FXII (FXIIa) is a prothrombotic process. The endothelium is known to play an antithrombotic role by preventing thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes regulation of activated FXI (FXIa) activity.

Hypothesis:
Endothelial cells (ECs) express anticoagulant properties that block the procoagulant activity of FXIa.

Methods:
We used a chromogenic assay to measure FXIa, kallikrein or FXIIa activity on cultured human umbilical vein ECs (HUVECs). To detect FXIa-inhibitor complexes in either the supernatant (SN) or cell lysate (CL), HUVECs were exposed to FXIa for 2 hrs at 4°C, then washed and lysed, followed by immunoprecipitation and western blot (WB) using an anti-FXIa mAb to the catalytic domain. To assess FXIa binding and internalization, HUVECs were exposed to FXIa for 2 hrs a 4C, washed and incubated at 37C for 5 min to 2 hrs, followed by fixation, and immunostaining with an anti-FXI antibody.

Results:
HUVECs block the activity of FXIa but not kallikrein or FXIIa activity. The catalytic domain of FXIa (30 kDa) formed a covalent bond with one inhibitor generating one band at 80 kDa. The formation of FXIa-inhibitor complexes appeared in the SN when ECs were transferred to 37C. Mass spectrometry analysis of the 80kDa band revealed that FXIa forms a complex with plasminogen activator inhibitor 1 (PAI-1). WB for PAI-1 from the SN and CL detected a band at 80 kDa, while the expression of PAI-1 on the HUVEC surface decreased after incubation with FXIa. FXIa bound to the EC surface at 4C. After 5 mins at 37C, FXIa was internalized and localized beneath the plasma membrane, and after 30 mins to 2 hr at 37C, FXIa accumulated in the early and late endosomes and lysosomes. The serine protease inhibitor PPACK blocked FXIa-PAI-1 complex formation and internalization of FXIa. A blocking anti-PAI-1 antibody increased the cleavage of the chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation on ECs in recalcified plasma.

Summary:
FXIa forms a complex with PAI-1 on the surface of ECs blocking its activity. Inhibition of FXIa on the endothelium may support internalization and clearance of FXIa from the circulation.
Abstract 363: Platelet Mass Retraction is a Negative Feedback Regulator of Thrombin Generation and Activity
Authors: Stalker Timothy J , Nettey Leonard , Matzko Chelsea , Poventud-Fuentes Izmarie , Sinno Talid , Brass Lawrence F , Tomaiuolo Maurizio
Published: March 2019

Endogenous anticoagulants, including antithrombin III, tissue factor pathway inhibitor, and activated protein C (aPC) provide a biochemical means to inhibit thrombin generation and/or activity. In addition, we have previously shown that platelet mass retraction prevents the escape and exchange of solutes, thereby providing a biophysical mechanism to limit thrombin generation and activity. The relative contribution of each of these biochemical and biophysical mechanisms to the termination of thrombin activity during the hemostatic response
in vivo
remains poorly understood. Here, using computational simulations coupled with
in vivo
experimental models, we tested the hypotheses that 1) platelet mass retraction itself is physiologically relevant to inhibit thrombin generation; and 2) that given the spatial localization of protein C activation on the endothelial cell surface, aPC mediated thrombin inhibition has negligible effects on the hemostatic response. Our computational model has several innovative aspects, including 3D representations of the vessel, extravascular space, and injury, blood flow, and a simplified but anatomically correct model of coagulation. Our simulations show that tissue factor localization combined with flow exiting the injury limit thrombin generation/activity to the extravascular space. Our experiments using a mouse jugular puncture injury model show rich deposition of fibrin in the extravascular space and little to no fibrin in the luminal side. Additionally, our simulations illustrate how platelet mass retraction inhibits thrombin generation by decreasing the delivery of substrates, such as prothrombin and factor X, and at the same time it limits thrombin activity by constraining its movement. These effects are mediated by physical forces only and are independent of the biochemical inhibitory pathways. Our
in vivo
experiments using aPC inhibitory antibodies show that lack of aPC mediated thrombin inhibition has negligible effects on the hemostatic response. In conclusion, this study suggests that platelet mass retraction is an underappreciated mechanism that negatively regulates thrombin generation, while aPC mediated inhibition of thrombin generation plays a minor role in hemostasis.
Abstract 375: Interaction of Plasma Autotaxin and Extracellular Vesicles in Rheumatoid Disease Patients Induces Circulating Cell Activation
Authors: Duchez Anne-Claire , Hasse Stephan , Fortin Paul R , Boilard Eric , Bourgoin Sylvain G
Published: March 2019

Background:
the frequency and severity of cardiovascular disease (CVD) is higher in patients suffering from Rhumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) than healthy individuals, likely due to the direct impact of systemic inflammation on the atherosclerotic plaque. Several factors can contribute to cardiovascular damage, including pro-inflammatory lipid mediators such as lysophosphatidic acid (LPA) produced by a lysophospholipase enzyme named autotaxin (ATX). Extracellular vesicles (EVs) are also abundant in blood from patient developing atherosclerotic plaque, and recently EVs from arthritic synovial fluid were found to interact with phospholipase and release inflammatory lipids.

Objective:
we therefore sought to understand how EVs and ATX may cooperate in the context of rheumatoid disease to promote inflammation.

Results:
we observed that plasma from RA and SLE patients, analysed by ELISA, contained high concentrations of ATX compared to the plasma from healthy people (pvalue =0.0001). The plasma of these patients, analysed by nanoparticle flow cytometry, also contained high concentration of EVs and these EVs are decorated by ATX, on their surface. As the presence of EVs indicated cell activation due to different endogenous stimuli such as LPA, we found in vitro that different species of LPA activate circulating blood cell to produce EVs (observed by flow cytometry and electronic microscopy), suggesting that LPA serves as an activating signal for EV production. In summary, our results show that ATX bind EVs and produces LPA that activates circulating cells in RA and SLE patients. ATX and LPA could serve as biomarkers and therapeutic target in accelerated CVD in patients with rheumatoid diseases.
Abstract 384: Differential Effects of Niacin and Omega-3 Fatty Acids on HDL-apolipoprotein A-I Exhange and Cholesterol Efflux Capacity in Subjects with Metabolic Syndrome
Authors: Borja Mark S , Hammerson Bradley , Tang Chongren , Savinova Olga V , Harris William S , Shearer Gregory C , Oda Michael N
Published: March 2019

Objective:
Niacin and omega-3 fatty acids are two therapeutic agents that have been extensively studied for their ability to reduce cardiovascular disease risk, but their effectiveness has more recently been called into question. In this study, we investigate whether these agents alone and in combination alter HDL function, in particular, HDL-apolipoprotein A-I exchange (HAE), a measure of HDL dynamics, and serum cholesterol efflux capacity (CEC).

Approach:
Fifty-six subjects with metabolic syndrome (MetSyn) were recruited to a double-blind trial and randomized to 16 weeks of treatment with dual placebo, extended release niacin (ERN, 2g/day), prescription omega-3 ethyl esters (P-OM3, 4g/day), or combination. HDL function was assessed at baseline and following 16 weeks of treatment by measuring HAE, macrophage CEC, and ABCA1-specific CEC.

Results:
Compared to placebo, ERN and P-OM3 alone significantly increased HAE by 15.1 [8.2, 22.0] (p<0.0001) and 12.4% [5.9, 18.9] (
P
<0.0001) respectively while the combination therapy increased HAE by 10.0% [3.2, 16.8] (
P
=0.002). When evaluated by HAE:apoA-I ratio (a measure of apoA-I specific activity), ERN increased apoA-I specific activity by 20.1% [4.8, 36.9] (
P
=0.008), P-OM3 by 30.1% [14.4, 45.9] (
P<
0.0001), however with combination there was no increase, 9% [-6.6, 26.6] (
P
=0.34). Triglyceride-adjusted macrophage CEC showed marginally significant increases with P-OM3 therapy (
P
=0.05). No therapy significantly improved ABCA1-specific CEC.

Conclusions:
Much of the effect of ERN on HDL function can be attributed to this therapy raising apoA-I levels, but P-OM3 raises HDL function by independent means, increasing apoA-I specific activity. Future investigation is needed to determine whether interaction between ERN and P-OM3 therapies in combination reduces their overall effectiveness.
Abstract 388: High-density Lipoprotein Cholesterol Efflux Capacity is not associated with Atherosclerosis and Cardiovascular Events: the CODAM Study
Authors: Josefs Tatjana , Wouters Kristiaan , Tietge Uwe J , Dullaart Robin P , van der Kallen Carla J , Stehouwer Coen D , Schalkwijk Casper G , Goldberg Ira J , Fisher Edward A , van Greevenbroek Marleen M
Published: March 2019

Cholesterol Efflux Capacity (CEC) is considered to be a key atheroprotective property of high-density lipoproteins (HDL). However, its role in atherosclerosis risk is still controversial. In this study, we have analyzed the relationship between HDL CEC and atherosclerosis surrogates (carotid intima media thickness (cIMT), endothelial dysfunction (EnD); Z-Scores) in a well-characterized clinical population in the Cohort of Diabetes and Atherosclerosis Maastricht (CODAM) study, which consists of 574 individuals (age 59.6±0.3yrs, 61.3% men, 24.4% T2DM). Multiple linear regression analyses were performed to identify the associations in baseline data between HDL CEC (%), plasma HDL cholesterol (HDL-C, mmol/L), HDL size (nm), apoprotein A1 (ApoA1, g/L), and HDL Particle Concentration (HDL-P, nmol/L (LN), by NMR) with cIMT and EnD. Logistic regression analyses were done to identify the association of the above-mentioned HDL characteristics and CV events (CVE; N=499, 80 CVE cases). Analyses were adjusted for lifestyle, medication and metabolic confounders.

The results show that HDL-C (ß=-0.65, 95%CI -0.91; -0.42), HDL size (ß=-0.57, 95%CI -0.98; -0.15), apoA1 (ß=-0.65, 96%CI -1.01; -0.29), and HDL-P (ß=-0.77, 95%CI -1.47; -0.11) were inversely associated with EnD (N=532), whereas HDL CEC was not (ß=-0.03, 95%CI=-0.24;0.24). Interestingly, preliminary analyses show that these associations are lost in diabetic subjects (N=131), but not in non-diabetics (N=401). An inverse association also exists between HDL-C, HDL size, apoA1, and HDL-P with cIMT (N=494), which was not statistically significant likely due to variability and relative insensitivity of cIMT measurements. Further, a 1 unit increase of HDL-C was associated with ~4fold lower prevalence of CVE (OR=0.25, 95%CI 0.08;0.83), and for HDL-P, with ~20fold less CVE (OR=0.05, 95%CI 0.00;0.70), but no association was found between HDL CEC and CVE (OR=1.26, 95%CI 0.41;3.84).

Our results show that certain HDL-related parameters have atheroprotective associations, but HDL-CEC is not one of them. This result agrees with, but differs from, some other studies, and might reflect population differences in the relationships between measurements of HDL lipids/particle number, and HDL CEC.