bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 191: Immobilized ApoA-I and High Density Lipoproteins are Anti-thrombotic and Inhibit Smooth Muscle Cell Attachment and Proliferation
Authors: Vanags Laura Z , Tan Joanne T , Michael Praveesuda S , Bilek Marcela M , Wise Steven G , Bursill Christina A
Published: July 2021

Aim:
The clinical efficacy of current endovascular stents is limited by thrombosis and restenosis. Plasma activated coated stainless steel (PAC) is able to covalently bind proteins to stainless steel (SS) surfaces in their bioactive state. High density lipoproteins (HDL) and its main apolipoprotein constituent apoA-I regulate key biological processes involved in restenosis and thrombosis, highlighting their potential for immobilization on PAC stent surfaces. We hypothesized that covalently bound apoA-I or reconstituted HDL (rHDL) to PAC will retain its biological properties and improve stent patency.

Methods and Results:
Covalent binding of 125I labelled apoA-I and rHDL was increased on PAC compared to SS (p<0.001). Thrombosis formation under static conditions was assessed by scanning electron microscopy. Thrombi were strikingly reduced on PAC and completely absent on PAC+apoA-I and PAC+rHDL, while extensive thrombi formed on all SS samples at all time points. Thrombus formation under flow conditions using a Chandler Loop (0.5U/ml heparin) showed PAC, PAC+apoA-I and PAC+rHDL had 98%, 97% and 94% lower thrombus weights, compared to SS samples, respectively (p<0.05). Lower heparin concentrations (0.3U/ml) revealed that PAC+apoA-I and PAC+rHDL were superior to PAC alone in reducing thrombus weights (98% and 95%, respectively, p<0.01). P-selectin protein expression was measured by ELISA in the plasma from the Chandler loop assay (0.3U/ml heparin). Plasma exposed to PAC+apoA-I had reduced p-selectin levels (32%) compared to the PAC control (p<0.05). Immobilized apoA-I and rHDL on PAC reduced smooth muscle cell (SMC) attachment by 70% and 80%, respectively, p<0.05. Conversely, endothelial cell (EC) attachment was increased by 36% on PAC+apoA-I (p<0.05). Immobilized PAC+apoA-I and PAC+rHDL also reduced TNF-α-induced SMC proliferation (26% and 20% respectively), compared to the PAC control (p<0.05).

Conclusion:
ApoA-I and rHDL covalently bind to PAC surfaces. Immobilized apoA-I and rHDL retain their anti-thrombotic properties, inhibit SMC attachment and proliferation, and increase EC attachment. This may represent a novel site-directed approach to increase stent patency.
Abstract 258: Significant Predictors of Survival Following Endovascular Abdominal Aortic Aneurysm Repair
Authors: Mousa Albeir Y , Broce Mike , Yacoub Michael , Bates Mark , AbuRahma Ali
Published: July 2021

Objective:
To identify significant predictors of survival after abdominal aortic aneurysm (AAA) repair.

Methods:
A retrospective review of the prospective endovascular aortic repair (EVAR) patient registry of the “Virginia’s group” maintained by the Society for Vascular Surgery Patient Safety Organization. Patients were classified into normal (eGFR ≥60 mL/min/1.73 m
2
), moderate chronic renal insufficiency (CRI; eGFR ≥30-59) or severe CRI (eGFR <30). Kaplan-Meier and Cox-regression were used to measure and determine the predictors of survival.

Results:
A total of 6410 EVAR patients were included with an age of 73.5 ± 8.4 years (majority were male 81.2%), with an average follow-up of 2.3 ± 2.0 years. Compared to patients with eGFR 30-59 and >60, those with the lowest eGFR <30 had the highest rate of post-op MI (3.8 vs. 2.2 and 0.8%;p<0.001) and stroke+MI+30-day mortality (3.8 vs. 2.4 and 0.8%; p<0.001). One year mortality was the highest in patients with the lowest eGFR (<30) (5.7 vs. 2.1 and 1.0%, respectively). Overall, 5-year survival was 80.6%. Significant predictors of decreased survival were, unstable angina (Hazard Ratio (HR), 2.5; P =0.007), age (by decade) (HR, 1.6; p<0.001, oxygen-dependent chronic obstructive pulmonary disease (HR, 3.3; P<0.001), eGFR <30 (HR, 2.4; P<0.001) and eGFR 30-59 (HR, 1.5; p<0.001), while pre-operative statin and aspirin use were associated with increased survival (HR, 0.79 and 0.78; both p=0.014, respectively).

Conclusion:
Reduced renal function at baseline was found to be associated with increased post operative cardiovascular events and long-term mortality following EVAR. Increased monitoring of patients with CRI seems warranted.
Abstract 261: Neutrophil Elastase Derived Fibrin Degradation Products are Increased in the Plasma of Patients with Abdominal Aortic Aneurysms and Correlate to the Intraluminal Thrombus Volume
Authors: Roy Joy , Silveira Angela , Liljeqvist Lindquist Moritz , Folkesson Maggie , Frebelius Siw , Hultgren Rebecka , Engstrom Jan , Swedenborg Jesper , Eriksson Per
Published: July 2021

Introduction:
Abdominal Aortic Aneurysms (AAA) often contain an intraluminal thrombus (ILT). AAA diameter and ILT volume are associated with growth of the aneurysm. Neutrophils, present in the ILT, contain elastase (NE). NE activity leads to production of fibrin degradation products (FDPs) with a specific epitope [[Unable to Display Character: –]] XDP. The present study evaluates NE-derived FDPs in aneurysm patients scheduled for elective aortic repair. The purpose of the study is to introduce an additional bio-marker for presence of AAA and possibly risk of rupture by measuring levels of NE derived FDPs in plasma of patients with AAA.

Materials and Methods:
42 male patients, undergoing aortic repair for AAA were included. As controls, we collected blood samples from 42 men who attended an AAA screening program but had no AAAs on ultrasound. Computed Tomography (CT) images were available for 34 AAA patients and analyzed using A4 Clinics software (VASCOPS, Austria). Patient demographics, maximum diameter, aortic volume and ILT volume were recorded. Peak wall stress (PWS), peak wall rupture index (PWRI) and mean ILT stress were estimated by Finite Element Analysis using the A4 Clinics software. Plasma levels of elastase digests of cross-linked fibrin (E-XDP) were determined with a sandwich ELISA.

Results:
E-XDP levels were higher in AAA patients than in age-matched controls (8.5 vs 1.2 U/ml, p<0.0001). E-XDP levels correlated with ILT volume (r = 0.64, p<0.0001), aortic volume (r = 0.64, p<0.0001) and maximum diameter (r = 0.59, p=0.0003). AAA patients with other concomitant peripheral aneurysms had higher E-XDP levels than those with only an AAA (13.6 vs 6.8 U/ml, p=0.028). PWS, PWRI and bleeding signs in the thrombus did not significantly affect E-XDP levels. Interestingly, the mean ILT stress correlated significantly to E-XDP levels (r= 0.45, p=0.008).

Conclusions:
The study shows that it is feasible to measure E-XDP levels in plasma of patients with AAA and that E-XDP correlates with ILT volume and mean ILT stress. These results support the notion that the resident neutrophils in the ILT can actively lyse fibrin in the ILT, which may decrease ILT strength. E-XDP holds potential as a biomarker of the ILT in AAA patients and needs to be further investigated in AAA rupture risk assessment.
Abstract 273: HDL Particle Concentration Inversely Associates with Incident Metabolic Syndrome in the Multiethnic Dallas Heart Study
Authors: Mani Preethi , Neeland Ian J , McGuire Darren K , Ayers Colby , Khera Amit , Rohatgi Anand
Published: July 2021

Objective:
Metabolic syndrome (MetS) increases atherosclerotic cardiovascular disease (ASCVD) risk. Low HDL cholesterol (HDL-C) is a diagnostic criterion of MetS and a major ASCVD risk factor. HDL particle concentration (HDL-P) associates with incident ASCVD independent of HDL-C, but its association with incident MetS has not been studied. We hypothesized that HDL-P would be inversely associated with incident metabolic syndrome independent of HDL-C and other recognized risk factors.

Methods:
HDL-P was measured by NMR and visceral fat by MRI in participants of the Dallas Heart Study, a probability-based population sample of adults age 30-65. Participants with prevalent MetS, DM, CVD, cirrhosis, cancer, HIV, or renal failure were excluded. Incident MetS as defined by NCEP ATPIII criteria was determined in all participants after median follow-up period of 9.4 years.

Results:
Among a cohort of 1120 participants without DM or MetS at baseline (57% women, 45% Black, mean age 43), 22.8% had incident MetS at follow-up. HDL-P and HDL-C were modestly correlated (r=0.54, p<0.0001). The lowest quartile of HDL-P was associated with younger age, men, Hispanic ethnicity, lower total, HDL, and LDL cholesterol levels and particle sizes, and less reported alcohol intake. Participants in the lowest sex and race stratified quartile of HDL-P had the highest incidence of MetS (Figure). In models adjusted for traditional risk factors, HDL-C, visceral fat, HOMA-IR, and hs-CRP, the lowest quartile of HDL-P was associated with 65% increased risk of incident MetS (Figure).

Conclusion:
HDL-P is independently associated with incident MetS after adjustment for HDL-C, adiposity, inflammation, and markers of insulin sensitivity. Further studies are warranted to validate these findings and elucidate the mechanisms underpinning this association.
Abstract 282: Biomarkers of Muscle Damage in Patients with Peripheral Artery Disease
Authors: Cluff Kim , Mehraein Hootan , Howard Kaitlyn , Koutakis Panagiotis , Casale George P , Pipinos Iraklis I , Subbiah Jeyamkondan
Published: July 2021

Introduction:
Peripheral artery disease (PAD), a manifestation of systemic atherosclerosis (fat, plaque deposition and hardening of arterial walls), characterized by blockages of the arteries supplying the legs affects approximately 8 million lives in the United States. The classic symptom of PAD is intermittent claudication (IC), de[[Unable to Display Character: ﬁ]]ned as walking-induced calf pain and gait dysfunction relieved by rest. At the level of the skeletal muscle ischemic injury is manifested as a gradual and characteristic degradation of muscle histology. This injury includes altered metabolic processes, damaged organelles, and compromised bioenergetics in the affected muscle. In this study, we evaluated the hypothesis that Fourier Transform Infrared (FTIR) spectroscopy of human biopsy samples (gastrocnemius muscle) can be used to identify biochemical alterations in PAD muscle and characterize severity of muscle damage.

Method:
FTIR spectral profiles were collected from muscle biopsies of the gastrocnemius from 13 patients consisting of 4 controls, 5 claudicating patients, and 4 critical limb ischemia (CLI) patients. Statistical analysis of the data included an analysis of variance, and partial least squares regression (PLRS) to identify significant differences in spectral peaks and correlate them with clinical diagnosis.

Results:
When comparing spectral peaks between controls, claudicants, and CLI patients, significant differences (p<.05) were found in the fingerprint region at spectral peaks between wavenumbers 1200-1250 cm-1. These spectral peaks have been attributed to alterations in protein content, lipids, and DNA or phospholipid groups. FTIR spectral biomarkers, of the muscle, also correlated (r=0.91) with clinical diagnosis of PAD.

Conclusion:
FTIR spectroscopy was able to characterize the secondary effects of PAD on the gastrocnemius by identifying unique biochemical signatures of diseased PAD skeletal muscle. These signatures can discriminate control from PAD muscle and correlate with the clinical presentation of the PAD patient. FTIR spectroscopy provides novel spectral biomarkers that may complement existing diagnosis and treatment monitoring methods for PAD.
Abstract 30: Apolipoprotein CIII Sialylation is a Critical Determinant of Liver Triglyceride Metabolism
Authors: Yassine Hussein N , Ramrakhiani Ambika , Parekh Aarushi , Walker Ryan , Goran Michael , Trenchevska Olgica , Nedelkov Dobrin , Nelson Randall , Koska Juraj , Reaven Peter D , Yen Frances T
Published: July 2021

Apolipoprotein (apo) CIII inhibits lipoprotein lipase (LPL) activity and remnant particle uptake in the liver by the lipolysis stimulated receptor (LSR); and therefore modulates liver and systemic lipid metabolism. Although post-translational modifications of apo CIII lead to asialylated, mono- or di-sialylated variants, the biological relevance of these sialylations is not known. Our objectives were to determine the association of apo C-III sialylation with plasma triglycerides and liver fat content in vivo, and to evaluate the effect of apo CIII sialylation on lipid uptake in liver cells or LPL activity in vitro. In 209 obese non-diabetic adolescent Hispanic participants, apo CIII variants in plasma were measured using mass spectrometric immunoassay. Increased plasma apo C-III sialylation (di- to mono-sialylated apo CIII ratio) was associated with lower triglyceride levels (r=-0.43, p<0.001) and liver fat (by MRI, r=-0.27, p<0.001) independent of total apo CIII concentrations. Higher plasma concentrations of the mono-, but not the di-sialylated variant, were associated with higher triglycerides concentrations (r=0.53, p<0.001). In HepG2 liver cells, immunoprecipitated apo CIII from VLDL of participants in the upper quartile of plasma apo CIII sialylation less effectively inhibited LSR-mediated VLDL uptake compared to apo CIII from those in the lower quartile of apo CIII sialylation (28% difference, p=0.02). This difference in inhibition was abolished by removal of sialic acid with neuraminidase. Apo CIII isolated from VLDL of participants with different level of apo CIII sialylation showed similar capacity to inhibit fatty acid release by LPL of control VLDL. VLDL particles with higher apo CIII sialylation also demonstrated a two-fold increase in ApoE. We conclude that a greater apo CIII sialylation together with particle enrichment in apo E allow for more efficient VLDL liver uptake and less liver fat accumulation. The measurement of plasma apo CIII sialylation may be a useful index of triglyceride metabolism and risk of fatty liver.
Abstract 305: Development of an Antidote-Controlled RNA Probe for Molecular Thrombi Imaging
Authors: Steen-Burrell Kady-Ann , Powell Gray Bethany , Sullenger Bruce
Published: July 2021

Vascular thrombosis is a major underlying factor in many cardiovascular diseases worldwide and is also a major post-surgical complication. Early detection and treatment of thrombi improves outcomes for patients since the responsiveness of thrombi to fibrinolytic treatment decreases with thrombus age. The serine protease thrombin plays a central role in thrombogenesis and remains associated with the thrombus thereby facilitating further activation of coagulation factors and platelets after initial clot formation. Therefore, targeting and imaging clot-bound thrombin may provide a way of distinguishing newly formed thrombi from older, constituted ones. The goal of this study was to determine whether a thrombin-binding RNA aptamer conjugated to a near-infrared fluorophore could detect clot-bound thrombin and if a complementary antidote oligonucleotide could remove clot-bound aptamer thereby creating a subtraction image. The thrombin-binding aptamer conjugate, Tog25t-AF680, was assessed for its in vitro binding capability to newly formed human plasma clots using fluorescence reflectance imaging. In comparison to a labeled non-binding control RNA, we observed a greater than 6-fold increase in near-infrared fluorescence (NIRF) target-to-background ratio (TBR) with Tog25t-AF680. Upon treatment with the antidote oligonucleotide (AO4), we observed an approximately 57% reduction in fluorescence signal after 10 minutes of incubation. Our results suggest that thrombin is a suitable target for imaging newly formed clots and that a RNA aptamer conjugate binds to clot-bound thrombin with high specificity. This thrombin-targeting near-infrared probe has the potential to be used as a diagnostic tool for arterial and venous thrombosis and may further advance our understanding of the role of thrombin in vivo.
Abstract 309: Twelve Year Followup for Managing Coronary Artery Diease Using a Nutrigenomics Based Diet and Supplement Program With Quarterly Assessment of Biomarkers
Authors: Gundry Steven R
Published: July 2021

Introduction:
Coronary Artery Disease (CAD) is thought to be progressive; standard protocols call for a low fat/low cholesterol diet, exercise, and lipid lowering agents in an effort to slow the onset of recurrent MI’s, stents, CABG’s, stroke, or death. This results in an approximate 30-40% new event rate in 5 yrs.

Methods:
Based upon our experience using a Nutrigenomic-based, Lectin-limited diet to prevent/reverse Metabolic Syndrome and CAD, we have enrolled and followed 978 pts (aged 42-89 yrs) with known CAD, defined as previous MI, stent, CABG, or positive stress test/angiogram, positive Corus score greater than 30, into a diet and supplement based, physician coached program, which emphasizes large amts of leafy green vegetables, olive oil, radical reduction of grain, legumes, nightshades, and fruits; and 4 oz amts of animal proteins, emphasizing shellfish, wild fish, and grass fed meats, while avoiding commercial poultry (Matrix Protocol). All Apo E 4 genotypes ate large amts of shellfish and avoided animal fats and cheeses. All pts were instructed to take 2-4,000 mg of high DHA fish oil, 200mg of Grape Seed Extract, and 50 mg of Pycnogenol per day. Supplements were individualized based on results of Advanced Cardiovascular Risk Markers, which were sent to three core labs, (Berkeley Heart Labs, and Singulex, Alameda, CA, Health Diagnostics Labs, Richmond,VA) q 3 months and followed to measure compliance and to change supplement/eating regimens.

Results:
Pts have been followed for 1.5 to 12 years (mean 9 yrs). While enrolled, 13/978 pts (1.3%) have received a new stent, two that were predicted by a rising Lp-PLA2, two required CABG, based on a rising Corus score, despite HDL’s of 110-120 mg/dl. There have been no MI’s, unstable angina. One pt underwent carotid endarterectomy ; one pt suffered a CVA and died, while in atrial fibrillation, A second pt expired from a ruptured cerebral berry aneurysm. Total CV events over 12 years is 16/978 (1.6%).

Conclusions:
We conclude that simple Nutrigenomic-based dietary interventions, emphasizing lectin avoidance, with compliance and supplement choices based upon q 3 month assessment of biomarkers, represents a quantum leap forward in preventing/modifying Cardiovascular events in known CAD patients.
Abstract 321: Lymphatic derived HDL Is an Effective Donor for the Trans-Intestinal Cholesterol Efflux Pathway That Is Impaired During Insulin Resistance
Authors: Mangat Rabban , Borthwick Faye , Vine Donna F , Proctor Spencer D
Published: July 2021

Introduction:
Emerging evidence shows that the proximal small intestine secretes cholesterol into the intestinal lumen via the trans-intestinal cholesterol excretion (TICE) pathway. It is thought TICE can contribute up to 30-40% of fecal neutral sterol excretion. Plasma apoB containing lipoproteins are known to donate cholesterol to the TICE pathway whilst conflicting evidence exists on the role for plasma HDL. Due to the anatomical proximity of lymphatic vessels to the basolateral membrane of enterocytes, it may serve as a candidate donor to TICE, but to date this has not been tested.

Objective:
To determine if HDL derived from mesenteric lymph can act as cholesterol donor for TICE in JCR:LA cp rats as a model of insulin resistance (IR).

Methods:
Mesenteric lymph was collected following intralipid infusion via lymphatic cannulation from control rats. Lymph HDL was isolated using ultra-density centrifugation and labeled with H3 cholesterol. Jejunal explants were obtained from control and IR rats (as a model of reduced TICE) fed chow or a high fat/cholesterol diet. TICE was measured with Ussing Chambers as appearance of H3-cholesterol labeled lymph HDL using micelles as acceptors.

Results:
Relative to free cholesterol [FC; used as marker of non-specific lipid permeability], lymph derived HDL TICE was 77% higher in control tissue (n=5, p<0.05) under chow fed conditions, suggestive of an effective donor for TICE. Lymph HDL TICE was also reduced (89%, p<0.05) in IR rats compared to control. Furthermore SR-B1 mRNA was reduced (-65%) in enterocytes from IR rats compared to control, and may explain reduced TICE by lymph HDL in IR. Under conditions of high fat/cholesterol fed diet, TICE from FC [and mannitol as a marker of paracellular transport] was increased in both control and IR rats, suggesting an elevated non-specific permeability of lipids by the basolateral membrane.

Conclusions:
Consistent with evidence that the lymphatics have a role in reverse cholesterol transport, this data supports that lymph HDL is an effective donor for TICE possibly by the SR-B1 pathway. While we have shown that the lymph HDL TICE pathway may be impaired during insulin resistance, a high fat/cholesterol diet may exacerbate lipid permeability via non-specific efflux pathways.
Abstract 334: The LXRβ Selective Agonist, VTP-38443, Significantly Decreases Plaque Cholesterol Ester Content and Inflammation in a Murine Model of Accelerated Atherosclerosis
Authors: McGeehan Gerard M , Lala Deepak S , Zhao Yi , Noto Paul B , Zhuang Linghang , Claremon David A , Meng Shi , Bukhtiyarov Yuri , Gregg Richard R
Published: July 2021

The ligand-activated transcription factors LXRα and LXRβ are important regulators of cholesterol metabolism and inflammation. Activation of LXR was previously shown to inhibit atherosclerosis in animal models through activation of reverse cholesterol transport (RCT) and suppression of vascular inflammation. VTP-38443 is a potent selective and orally bioavailable modulator of LXRβ that is being pursued for the prevention of subsequent vascular events following an acute coronary syndrome (ACS) episode. VTP-38443 is a potent binder (Ki=12 nM) and activator (EC50=17 nM) of LXRβ. It is ~20x selective for LXRβ versus LXRβ in both binding and activation. In primary human cells, it induces the expression of the cholesterol efflux pumps ABCA1 and ABCG1, markers of RCT, and promotes cholesterol efflux from human fibroblasts at low concentrations (EC50 = 14 nM). Orally dosed VTP-38443 demonstrates robust induction of ABCA1 and ABCG1 in mice (ED50 < 0.3 mg/kg) and primates (ED50 < 0.1 mg/kg). Based on this robust activity, VTP-38443 was tested in the apoE -/- carotid artery ligated mouse model, an accelerated atherosclerosis model. ApoE-/- mice (9 week) were fed a Western diet for 2 weeks at which point the left common carotid artery was ligated. Mice remained on the Western diet for 2 weeks post-surgery and were dosed BID with VTP-38443 (0.05, 0.2 and 1 mg/kg/dose) or a non-selective LXR agonist, TO90137 (20 mg/kg/day) during this time. Plasma cholesterol and TGs were measured as were cholesterol esters and FDG-6-phosphate, a biomarker of plaque inflammation, in the carotid plaques. VTP-38443 gave a dose-dependent reduction in plasma cholesterol (35% at 1 mg/kg) and a modest dose-dependent increase in plasma TGs ranging from 5% to 45% of the TG increase seen with TO90137. Plaque analysis showed a highly significant decrease in carotid cholesterol ester content at all doses, achieving >90% reduction at the highest dose. In addition, there was a significant reduction (~40-50%) in FDG-6 phosphate at all doses of VTP-38443, indicating a decrease in vascular inflammation. These data indicate that LXRβ activation may decrease plaque cholesterol content and reduce plaque inflammation in ACS.
Abstract 37: A Multi-locus Genetic Risk Score for Abdominal Aortic Aneurysm
Authors: Ye Zi , Austion Erin , Schaid Daniel J , Kullo Iftikhar J
Published: July 2021

Background:
Genome-wide association studies (GWAS) have identified several common variants associated with abdominal aortic aneurysm (AAA). We investigated whether a multi-locus genetic risk score (GRS) for AAA was associated with presence and progression of AAA in a case-control study.

Methods:
The study comprised of 1098 patients with AAA (74±8 years, 83% men) and 6538 controls (67±10 years, 58% men) enrolled in the Mayo Vascular Disease Biorepository. AAA was defined as a transverse diameter of abdominal aorta ≥ 3.0 cm or history of AAA repair. Controls were participants without known AAA. We found five single nucleotide polymorphisms (SNPs) previously shown to be associated with AAA at GWAS significance (P≤10^-8) in NHGRI GWAS catalog and PubMed. A GRS for AAA for each individual was calculated from four SNPs (rs2383107, rs7025486, rs599839, rs1466535) that were replicated in our cohort, by summing the number of risk alleles for each SNP weighted by their estimated effect sizes in GWAS catalog or published largest meta-analysis.

Results:
GRS was associated with presence of AAA: odds ratio (OR) (95% confidence interval): 1.06 (1.03-1.08). The association remained significant after adjustment for age, sex, cardiovascular risk factors, and atherosclerotic cardiovascular diseases: adjusted OR: 1.05 (1.03-1.08). Further adjustment for each SNP did not attenuate association of GRS with presence of AAA (All P<0.001). GRS was not associated with family history of aortic aneurysm (P=0.4). Adding GRS to conventional risk factors improved net reclassification index by 16% (P<0.001).In a subset of patients with AAA who had sequential imaging studies (n=628), GRS was associated with AAA growth rate ≥ 1.75 mm/year (median of the cohort) after adjustment for baseline AAA size: adjusted OR: 1.07 (1.00-1.14). No conventional risk factors were associated with AAA growth. Patients with GRS > 5.24 (median of the cohort) had 1.31 times higher odds of having AAA and 1.64 times higher odds of having AAA growth rate ≥1.75 mm/year compared to those with GRS ≤ 5.24 (both P≤0.005).

Conclusions:
A multi-locus GRS was associated with presence of AAA and aneurysm growth, suggesting genetic predisposition to disease initiation and progression.
Abstract 402: Plasminogen Inhibits Sdf-1-induced Cxcr4 Internalization In Bone Marrow-derived Mononuclear Cells
Authors: Han Hongyu , Zhang Lindi , Fan Yi , Gong Yanqing
Published: July 2021

In multiple pathological settings including stokes and myocardial infarction (MI), stem cells are mobilized from the bone marrow to injury site to promote tissue repair. However, naturally recruited stem cells are not sufficient for heart function recovery. How to enhance stem cell mobilization remains challenge but may lead to develop novel strategy for stem cell therapies. Our previous studies have shown that Plasminogen (Plg) enhances stem cell mobilization and contributes to cardiac repair after MI. Mechanism studies reveal that Plg regulates CXCR4 expression, the receptor of SDF-1, a major chemoattractant for stem cells, during stem cell mobilization. Using bone marrow mononuclear cells, our data show that Plg is able to increase CXCR4 expression in a dose-dependent manner. Moreover, Plg decreases CXCR4 internalization induced by SDF-1. SDF-1 (50nM) induces a rapid internalization of CXCR4 on BMNC indicated as much lower CXCR4 expression compared with control (0.9 ± 0.5% vs 10.5 ± 2.6%) in cell surface. However, Plg treatment (100nM) decreases CXCR4 internalization, indicated as significantly higher expression (5.5 ± 0.9%) of CXCR4 on the cell surface. Immunofluorescent staining results show that CXCR4 translocates into cytoplasma after SDF-1 treatment. Plg rescued the CXCR4 translocation induced by SDF-1 and CXCR4 is mainly expressed on the cell surface as shown in the figure, confirming that Plg may prolong CXCR4 expression through inhibiting CXCR4 internalization by SDF-1. These data has indicated a novel mechanism for Plg-regulated CXCR4 expression and may contribute stem cell mobilization from bone marrow to the circulation during MI injury.
Abstract 413: Designed Ankyrin Repeat Proteins Against Activation Specific Binding Sites of The Leukocyte Integrin αmβ2: Novel Strategies in Vascular Inflammation
Authors: Diehl Philipp , Siegel Patrick , Holien Jessica , Bassler Nicole , Flierl Ulrike , Tonnar Xavier , Olivier Christoph , Anto Michel Nathaly , Wolf Dennis , Zirlik Andreas , Steiner Daniel , Bode Christoph , Moser Martin , Parker Michael , Peter Karlheinz
Published: July 2021

Introduction:
Vascular inflammation is the underlying condition of several cardiovascular diseases and is mainly mediated by activated leukocytes. The leukocyte integrin αMβ2 with its activation specific epitope (I domain) is strongly involved in leukocyte adhesion to endothelial cells and thus represents an interesting therapeutic target. Designed Ankyrin Repeat Proteins (DARPins) are a novel class of linear, thermostable, highly specific recombinant binding proteins that overcome several limitations of immunoglobulins.

Hypothesis:
DARPins selected against the mouse I domain (mId) of αMβ2 bind specifically to activated leukocytes and can be used as a novel diagnostic tool as well as a therapeutic, anti-inflammatory agent.

Methods:
Using phage display, binding proteins were selected against recombinant I domain. Specific binding behavior to only activated leukocytes was assessed in FACS. Docking studies were used to define specific interaction sites of selected DARPins with the I domain. Therapeutic, anti-inflammatory effects of anti-mId DARPins was assessed in a sepsis mouse model.

Results:
DARPins selected against the I domain bind in FACS specifically to activated monocytes (activated vs. non-activated 61±4 % vs. 19±6 %, p<0.05). Docking studies revealed amino acid positions responsible for the specific binding behavior. Mutagenesis of these residues showed significantly reduced binding of the mutated DARPin using FACS analysis (anti-mId DARPin vs. mutated anti-mId DARPin 61±4 vs. 29±7, p<0.05) proving that binding of the wild type DARPin to its target is specific. Furthermore, anti- I domain DARPins showed anti-inflammatory effects in a mouse sepsis model (peritoneal cells: anti-mId DARPin vs control: 2,049±189 103/ml vs. 3,382±213 103/ml, p<0.01).

Conclusions:
DARPins selected against the I domain of αMβ2 bind specifically to activated leukocytes and inhibit leukocyte function as a new class of anti-inflammatory agents under in vivo conditions.
Abstract 415: Resolvin D1 Limits 5-lipoxygenase Nuclear Localization and Leukotriene B4 Synthesis by Inhibiting a Calcium-activated Kinase Pathway
Authors: Fredman Gabrielle
Published: July 2021

Introduction:
Imbalances between pro-inflammatory and pro-resolving mediators can lead to chronic inflammatory diseases, such as atherosclerosis. Hypothesis: The balance of arachidonic acid (AA)-derived mediators in leukocytes is thought to be achieved through intracellular localization of 5-lipoxygenase (5-LOX): nuclear 5-LOX favors the biosynthesis of pro-inflammatory leukotriene B
4
(LTB
4
), while, in theory, cytoplasmic 5-LOX could favor the biosynthesis of pro-resolving lipoxin A
4
(LXA
4
). This balance is shifted in favor of LXA
4
by resolvin D1 (RvD1), a specialized pro-resolving mediator (SPM) derived from docosahexaenoic acid (DHA), but the mechanism is not known. Hence we hypothesized that RvD1 regulates 5-LOX localization in macrophages.

Methods/Results:
Here we report a new pathway through which RvD1 promotes nuclear exclusion of 5-LOX and thereby suppresses LTB
4
and enhances LXA
4
in macrophages. RvD1, by activating its receptor FPR2/ALX, suppresses cytosolic calcium and decreases activation of the calcium-sensitive kinase CaMKII. CaMKII inhibition suppresses activation P38 and MAPKAPK2 kinases, which reduces Ser271 phosphorylation of 5-LOX and shifts 5-LOX from the nucleus to the cytoplasm. As such, RvD1’s ability to decrease nuclear 5-LOX and the LTB
4
:LXA
4
ratio
in vitro
and
in vivo
was mimicked by macrophages lacking CaMKII or expressing S271A-5-LOX. Importantly, nuclear localization of 5-LOX has been reported in unstable advanced human atherosclerotic lesions, hence strategies to reverse this process are of interest. In this regard, RvD1 treatment of
Ldlr
-/-
mice reduced macrophage 5-LOX localization and stabilized advanced plaques.

Conclusions:
Knowledge of this mechanism may provide new strategies for promoting inflammation resolution in chronic inflammatory diseases, like atherosclerosis.
Abstract 426: Abdominal Aorta Dilatation and Aneurysm in Kawasaki Disease Vasculitis Mouse Model: Role of IL-1 Signaling
Authors: Wakita Daiko , Kurashima Yosuke , Lee Youngho , Shimada Kenichi , Chen Shuang , Crother Timothy R , Lehman Thomas J , Fishbein Michael C , Arditi Moshe
Published: July 2021

Background:
Kawasaki disease (KD) is the most common cause of acquired cardiac disease among US children. KD causes coronary artery aneurysms in up to 25% of untreated patients, and less frequently aneurysms in other systemic arteries including the abdominal aorta.

Objective:
To evaluate the development of abdominal aorta dilatation and aneurysm in KD mouse model and investigate the role of IL-1 signaling.

Methods and Results:
We investigated the incidence and progression of abdominal aorta aneurysm (AAA) and dilatation in the Lactobacillus casei cell wall extract (LCWE)-induced KD mouse model at 1, 2, 5 wks. Over 80% of the mice developed significant dilation of abdominal aorta at 1 wk with progressively greater dilatation at 5 wks, with greater severity in males. KD mice showed fusiform and saccular AAA, which were always below the renal artery. Immunohistochemistry showed significant intimal proliferation, massive myofibroblastic proliferation that breaks the elastin layer, infiltration of large numbers of neutrophils and macrophages into the media and adventitia. IL-1R- or IL-1beta-deficient mice were completely protected from the KD associated abdominal aorta dilatation and AAA. Active form Caspase-1 was detected at infiltrated macrophages and the Caspase-1-deficient mice showed significant reduction of AAA formation. Blockade of IL-1/IL-1R signaling with IL-1R antagonist (Anakinra), or neutralizing antibody against IL-1α or IL-1β significantly prevented the AAA in the KD mice.

Conclusions:
We report a new model of AAA and aortic dilatation in the LCWE-induced KD mouse model. These studies suggest that in children with KD the incidence of abdominal aortic dilatation and AAA maybe higher than currently appreciated, thus requiring prospective studies to determine the frequency of these vascular complications. Our findings also demonstrate that IL-1 plays an important role in development of LCWE-induced abdominal aortic lesions and blockade of IL-1 signaling may be a promising therapeutic target not only for KD vasculitis and coronary arteritis, but also for abdominal aorta dilatation and AAA associated with the disease.
Abstract 430: A Novel Tram Stent Method in the Treatment of Coronary Bifurcation Lesions
Authors: Arokiaraj Mark
Published: July 2021

Aims:
A novel stent was designed for the treatment of coronary bifurcation lesion, and it was investigated for its performance by finite element analysis. This study was performed in search of a novel method of treatment of bifurcation lesion with provisional stenting.

Methods:
A coronary bifurcation model was created with the proximal vessel of 3.2 mm diameter, and the distal vessel after the side branch (2.3 mm) was 2.7 mm. A novel stent was designed with connections that had a profile of a tram. Laser cutting and shape setting of the stent was performed, and thereafter it was crimped and deployed over a balloon. The contact pressure, stresses on the arterial wall, stresses on the stent, the maximal principal log strain of the main artery and the side-branch were studied. Also multiple designs were tested in the tram area to suite the standard crimping. The study was performed in Abaqus. A firm lesion model with a stenosis of about 75% was then created with a discrete lesion in the sidebranch and the main vessel. The accompanying figure shows the model used for evaluation.

Results:
The stresses on the main branch and the distal branch were minimally increased after deployment of this stent. The side branch was preserved, and the stresses on the side branch were lesser; and at the confluence of bifurcation on either side of the side branch origin the Von Mises stresses were marginally increased as shown in figure. However, the stresses at the bifurcation and in the main branch were significantly lesser than the stresses by the existing techniques used in the treatment of bifurcation lesions.

Conclusions:
There is a potential for a novel Tram-stent method in the treatment of coronary bifurcation lesions.
Abstract 473: Development of High-throughput ABCA1, ABCG1 and SR-BI Mediated HDL Efflux Assays
Authors: Storey Carl , Tang Chongren
Published: July 2021

Studies have shown an inverse association between HDL-efflux capacity and cardiovascular disease (CVD). The efflux of cholesterol from cells to HDL can occur by several mechanisms, including unmediated aqueous diffusion and ABCA1, ABCG1 and SR-BI specifically mediated process. We developed a high throughput assay to measure pathway specific HDL efflux capacity using BHK cells expressing inducible ABCA1, ABCG1, or SR-BI in 96-well plate format. We found that ABCA1-mediated cholesterol efflux in BHK cells correlated significantly with that of J774 macrophages when apoA-I, HDL or 2% apoB-depleted serum were used as acceptors (R
2
=0.98 to 0.99, p<0.0001). Compared with J774 macrophages, ABCA1-mediated efflux using BHK cells is more sensitive, consistent and has better reproducibility (Intro-assay CV=3.0%, R
2
=0.99; Inter assay CV=3.2%, R
2
=0.98). The consistence and reproducibility for ABCG1 and SR-BI mediated cholesterol efflux were also excellent as judged by intro-assay and inter-assay CV and R
2
. When pathway specific assay was employed in a high-throughput format to measure HDL efflux capacity among subjects with or without CVD, we found that ABCA1-mediated cholesterol efflux was significantly lower in CVD subjects when compared with that of control (p<0.01), while ABCG1 or SR-BI mediated cholesterol efflux was not significantly different among the two groups. ABCG1-mediated cholesterol efflux was significantly associated with HDL-C (R
2=
0.65, p<0.01), but ABCA1 or SR-BI mediated cholesterol efflux was not correlated with HDL-C. Those studies demonstrate that BHK cells with inducible ABCA1, ABCG1, or SR-BI expression provide a sensitive, reproducible high throughput efflux assay that can be used to screen large number of serums in mechanistic studies or drug discovery program.
Abstract 504: Cystatin-C Risk-Stratifies Patients for Acute Kidney Injury and 1-Year Major Vascular Events Following Contrast -Enhanced CT Imaging in the Emergency Care Setting
Authors: Mitchell Alice M , Kline Jeffrey A , Williams Roxanne Y , Basile David P , Teague Shawn D , Molitoris Bruce A
Published: July 2021

Background:
Despite poor sensitivity in acutely ill patients, serum creatinine (and estimated glomerular filtration rate [eGFR
SCR
]) remains the sole means of risk-stratifying patients for acute kidney injury (AKI) prior to contrast-enhanced CT imaging (CECT).

Hypothesis:
We hypothesized that an acute phase marker of renal dysfunction, cystatin-C (expressed as eGFR
CYS
), would more accurately predict contrast-induced nephropathy (CIN) than eGFR
SCR
. Given the risk of arterial vascular events subsequent to AKI, we also evaluated eGFR
CYS
in risk-stratifying patients for major adverse events (MAE) within 1 year of CECT.

Methods:
We followed 462 consecutive adults, without end-stage renal disease, undergoing CECT (any indication) in the outpatient, emergency care setting for CIN and 1-year MAE: death, renal failure, myocardial infarction, stroke, and/or peripheral vascular event requiring intervention (blinded, adjudicated outcome). We excluded patients with life-threatening CECT indications and collected serum for eGFR
SCR
and eGFR
CYS
prior to CECT. Predictive accuracy was defined as the area under the receiver operating characteristic curve (AUROC) and likelihood ratios (LR+ and LR-). A threshold of ≤60 ml/min/m
2
defined an abnormal eGFR
SCR
or eGFR
CYS
.

Results:
CIN occurred in 14% and a MAE in 17% (low observer variability, κ>0.9) of our heterogeneous population: mean age 50 yrs (±16 yrs), 51% discharged after CECT, 16% with diabetes mellitus (DM), and only 16% with eGFR
SCR
≤60ml/min/m
2
. CIN was associated with 1-year MAE: RR 2.4 (1.5-4.0) after adjusting for age and existing co-morbidities (active malignancy, CHF, DM, and CAD). The AUROC, LR+ and LR- for eGFR
SCR
were 0.55 (0.47-0.63), 0.9 (0.4-2.1) and 1.0 (0.9-1.1). In comparison, the AUROC, LR+, and LR- for eGFR
CYS
were 0.79 (0.62-0.96), 5.5 (3.9-7.6) and 0.43 (0.31-0.57), respectively. The MAE rate did not differ in patients with normal (13%) or abnormal (15%, p=0.5) pre-CECT eGFR
SCR
. Whereas, an abnormal eGFR
SCR
was associated with a 29% (p<0.01) increase in MAE.

Conclusions:
In patients undergoing CECT in the outpatient setting, eGFR
CYS
more accurately predicted CIN and more effectively risk-stratified patients for 1-year MAE than eGFR
SCR
. These findings warrant prospective validation.
Abstract 505: Effect of Angiotensin II Infusion on Renal TNFα and IL6 mRNA Expression in Male Adiponectin Deficient Mice
Authors: Pamidimukkala Jayabala , Wang Xia , Yu Esther , Phan Joy
Published: July 2021

Adiponectin is a well-known adipokine with insulin sensitizing and anti-inflammatory actions. Low circulating levels of adiponectin are associated with a proinflammatory milieu leading to the development of cardiovascular and metabolic disorders. We have previously shown that male adiponectin deficient (adipo-/-) mice have lower baseline blood pressures but are more susceptible to prohypertensive effect of Angiotensin II (ANGII). In addition to being a vasoconstrictor, ANGII is also known to stimulate proinflammatory cytokine expression in cardiovascular & renal tissue. However, low dose infusion of ANGII for 14 days did not increase plasma TNFα or IL6 appreciably in the adipo -/- mice. The present study evaluates the expression of these proinflammatory cytokines in renal tissue. Briefly, male wild type C57Bl/6J and adiponectin deficient (adipo-/-) mice (24-28wk, ~30g) were implanted with osmotic pumps containing Angiotensin II (ANGII, 800ng/Kg body weight/min) or Saline. At the end of 14 days infusion, the mice were euthanized to obtain blood and tissue samples. The kidneys were snap frozen in liquid nitrogen and the total RNA was extracted using RNA mini kit (Qiagen). Quantitative real-time PCR was performed with Eppendorf Realplex 4 mastercycler and SYBR Green ROX Mastermix. TNFα mRNA expression normalized to GAPDH was similar in adipo-/- and C57Bl/6J mice. TNFα mRNA expression levels were not increased by ANGII infusion. IL6 expression was undetectable in all groups (n=3 animals/group). We also carried out additional microarray analysis of markers of endothelial activation and adhesion molecules. Preliminary data show that ANGII increases expression of E-Selectin, VCAM1, Collagen1a1 and eNOS by two fold in the C57BL/6J mice. The saline treated adipo-/- had lower expression of eNOS, VCAM-1 and Collagen1a1. Interestingly, ANGII infusion increased expression of E-Selectin, VCAM1 and Collagen 1a1 in these mice but eNOS expression was unchanged. The data suggest that chronic ANGII infusion did not change renal expression of cytokines appreciably but downstream markers of inflammation and fibrosis were increased.
Abstract 559: FTY720 Protects Against Diet Induced Coronary Artery Disease and Myocardial Infarction in Diabetic SR-B1-/- ApoE-Hypomorphic Mice
Authors: Gonzalez-Jara Leticia A , Werstuck Geoff , Trigatti Bernardo
Published: July 2021

Background:
Epidemiological studies have established diabetes as a risk factor for the development of cardiovascular diseases. Increased development of aortic atherosclerosis has been shown in diabetic animal models, but the effect on coronary artery (CA) disease is less clear. Conventional mouse models of atherosclerosis do not reproducibly develop CA atherosclerosis. SR-B1-/- apoE-hypomorphic mice develop increased aortic sinus atherosclerosis and diet-induced CA atherosclerosis when compared to SR-B1+/+ apoE-hypomorphic mice. FTY720, a sphingosine-1-phosphate analog, has been shown to play a protective role in both atherosclerosis and diabetes. We assessed the hypothesis that FTY720 supplementation will decrease the development of CA and aortic atherosclerosis and myocardial infarction in diabetic SR-B1-/- apoE-hypomorphic mice fed an atherogenic diet.

Methods:
Diabetes was induced by multiple low-dose i.p. injections of streptozotocin (40 mg/kg body weight). Controls received vehicle. Mice were fed a high-fat/high-cholesterol diet for 4 weeks (n=14-15). FTY720 was administered on the drinking water (0.0036 g/l, n=14-15). CA and aortic atherosclerosis and cardiac fibrosis were evaluated.

Results:
Diabetic SR-B1-/- apoE-hypomorphic mice fed a high-fat/high-cholesterol diet presented reduced survival (p<0.01) and significantly larger plaques in the aortic sinus (p<0.05) compared to control. CA atherosclerosis burden (p<0.001) and levels of cardiac fibrosis (p<0.01) were increased as well. FTY720 supplementation significantly protected diabetic mice against aortic and CA atherosclerosis (p<0.05) and myocardial fibrosis (p<0.05).

Conclusion:
FTY720 significantly reduced the burden of aortic and CA atherosclerosis and cardiac fibrosis in diabetic SR-B1-/- apoE-hypomorphic mice challenged with a high-fat/high-cholesterol diet.
Abstract 569: Lymphatic Function Is Impaired Before Atherosclerosis Onset in a Model of Atherosclerosis-prone Mice
Authors: Milasan Andreea , Clavet-Lanthier Marie-Elaine , Mayer Gaetan , Martel Catherine
Published: July 2021

Introduction:
Macrophages and cholesterol are the two main constituents driving the inflammatory response that characterizes atherosclerosis. In a recent study, the lymphatic system has been identified as a novel prerequisite player in the removal of cholesterol out of the atherosclerotic lesion. It has been shown that without a functional lymphatic network, cholesterol gets trapped in the artery wall. The lymphatic vessels are composed of two main components, namely the absorptive capillaries, responsible for the uptake of the cells, molecules and fluid, and the collecting vessels, characterized by pumping units (lymphangions) that are propelling the lymphatic content toward the blood circulation in a unidirectional manner. The relative roles of the lymphatic capillaries and collectors in the context of atherosclerotic disease are still unclear.

Methods and results:
Lymphatic function has been evaluated in 3-month old atherosclerosis-prone (LDLR-/-; hApoB100+/+, also called ATX mice), LDLR-/-, atherosclerosis-protected (PCSK9-/- mice, deficient in a convertase that induces the degradation of the LDL receptor) and wild-type (WT) mice. Our preliminary data show that, like LDLR-/- mice, pre-atherosclerotic ATX mice exhibit impaired lymphatic cellular transport. Immunohistochemistry and immunofluorescence imaging portrays a relatively normal number of sprouting and diameter of lymphatic vessel capillaries in the adventitial layer of the aortic sinus and in the skin dermis of ATX mice compared to WT or PCSK9-/- animals.

Conclusions:
Our preliminary results suggest that lymphatic transport is impaired even before the onset of atherosclerosis, and that i) the LDLR is associated with lymphatic vessel function, and that ii) the collecting lymphatic vessels are most likely responsible for the impairment in lymphatic dysfunction in LDLR-/- and ATX mice. These preliminary results suggest that characterizing the functional pumping capacity of the collecting vessels would be a prerequisite in understanding the interplay between atherosclerosis progression and lymphatic transport. We hope that in the long run we will be able to identify new therapeutic targets to enhance lymphatic transport and ultimately limit atherosclerosis progression.