bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 367: Antibodies Against Oxidized LDL, ß2 Glycoprotein I and Chlamydia pneumoniae Heat Shock Protein 70 in Acute Coronary Atherothrombosis Development
Authors: Burazor Ivana , Vojdani Aristo
Published: March 2022

Background and aims:
Atherothrombosis is the major determinant of acute cardiovascular events, such as myocardial infarction. Inflammatory processes have been linked to all phases of atherogenesis. Data suggest that atherosclerosis also constitutes an autoimmune disease. We aimed to investigate the presence of anti oxidized LDL (ox-LDL) antibodies (IgG) and anti beta 2 glycoprotein I antibodies in acute coronary atherothrombosis development.

Methods:
The study included 206 participants of whom 106 were patients with acute coronary syndromes (ACS), (61.2 ± 3.21 years of age, 62% males) and 100 were age and sex matched controls with no known coronary artery disease. Patients with previous infection were excluded from the study. Blood was sampled, frozen and sent on dry ice to Immunosciences Lab. Inc (USA) for analyses. All traditional risk factors were noted. Anti ox-LDL antibodies (IgG), anti beta 2 glycoprotein I (IgG) antibodies were determined as well as anti Chlamydia pneumoniae heat shock protein (HSP) 70 (IgG) antibodies. Interleukin 6 and C- reactive protein were measured.

Results:
Our data showed significant prevalence of examined antibodies in patients with ACS: 30% of patients had anti Ox-LDL antibodies (IgG) detectable, 25% had anti beta glycoprotein I antibodies compared to 8% of controls. The total of 31% patients versus 13% of controls had anti Chlamydia pneumoniae HSP 70 (IgG) antibodies detectable, RR 2.36, (1.32-4.28 95% CI for RR, p=0.003) The levels of circulating antibodies were significantly higher in patients (p<0.001). Markers of inflammation differed significantly between the groups. Our data indicated linear correlation between examined antibodies and markers of inflammation.

In conclusion, the results of our study suggest that antibodies (IgG) against Ox- LDL, beta 2 glycoprotein I and Chlamydia pneumonia HSP 70 can be detected in patients with acute coronary atherothrombosis. The clinical relevance for circulating autoantibodies in cardiovascular outcomes is still debated. We believe that further research will help to assess if those autoantibodies could improve current cardiovascular risk stratification approaches and therapeutic algorithm, both in primary and secondary prevention.
Abstract 372: OxLDL-Induced Apoptotic Dendritic Cells as a Novel Therapy for Atherosclerosis
Authors: Frodermann Vanessa , van Puijvelde Gijs H , Wierts Laura , Lagraauw H Maxime , van Santbrink Peter J , Bot Ilze , Kuiper Johan , de Jager Saskia C
Published: March 2022

Introduction:
Tolerogenic dendritic cells (DCs) have been previously described to protect against atherosclerosis. A tolerogenic phenotype in DCs can be induced by apoptotic cell clearance and these DCs can prevent inflammatory responses. In this study, we took advantage of the fact that oxidized low-density lipoprotein (oxLDL) induces apoptosis in DCs. These oxLDL-induced apoptotic DCs (apop-DCs) enabled us to deliver an atherosclerosis-specific antigen to DCs in the context of regulatory signals.

Hypothesis:
We hypothesized that an adoptive transfer of apop-DCs would limit immune responses and thereby reduce atherosclerosis.

Methods and Results:
In vitro
DCs pre-exposed to apop-DCs showed significantly less pro-inflammatory cytokines IL-6 (2493±73 vs. 1500±95 pg/mL) and TNF-α (311±12 vs. 30±7 pg/mL), but significantly increased anti-inflammatory IL-10 production (12±12 vs. 106±11 pg /mL) in response to LPS, compared to control DCs. Moreover, these DCs resulted in a dramatic reduction of T cell proliferation, accompanied by an about 8-fold decrease of Th1 cells, while regulatory T cells (Tregs) were 4.5-fold increased.

Adoptive transfer of apop-DCs to LDLrKO mice prior to Western-type diet resulted in tolerogenic splenic DCs and increased circulating Tregs. Moreover, we determined a decreased proliferative capacity of splenic T cells and found less adventitial T cells in the aortic root. Serum IL-10 levels were significantly increased, while TNF- levels were reduced. Interestingly, we observed a 32% decrease in monocyte numbers, with 13% less Ly-6C
hi
and 14% less CCR2
+
monocytes, and a 48% decrease in serum CCL2 levels. This translated into significantly less lesional macrophages in the aortic root. The overall reduced inflammatory status upon apop-DC-treatment translated into a 40% reduction in aortic root lesion size.

Conclusions:
We are the first to show the potential of apop-DC-treatment for atherosclerosis. In the future treatment of patients with autologous oxLDL-induced apop-DCs may prove a safe and very efficient treatment of atherosclerosis and acute vascular syndromes.
Abstract 38: Identification of Thrombomodulin Binding Sites on Thrombin Activatable Fibrinolysis Inhibitor that Mediate Accelerated Activation by Thrombin
Authors: Marar Tanya , Boffa Michael B
Published: March 2022

Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM), or plasmin to generate the enzyme activated TAFI (TAFIa). TAFIa possesses basic carboxypeptidase activity which down-regulates fibrin clot lysis by removing carboxyl-terminal lysine residues from partially degraded fibrin thereby attenuating the positive feedback in the fibrinolytic cascade. Accumulating evidence from several studies suggests that TM and TAFI make direct contacts such that the enhancement of TAFI activation by TM is through binding at sites remote from the activation site (Arg92-Ala93). The elements of TAFI structure that allow accelerated activation of thrombin by TM are largely unknown. Therefore, alanine scanning mutagenesis of surface-exposed charged residues on TAFI was used to identify sites that mediate acceleration of activation by TM and which thus may indicate sites where TM binds to TAFI directly. The rates of activation by thrombin of the variants was measured in the presence or absence of TM. The variants R12A, E28A and R15A exhibited the lowest enhancement of catalytic efficiency in the presence of TM with decreases of 3.0-fold, 3.2-fold and 2.6-fold, respectively, compared to wild-type. The variants D75A/E77A/D78A, E106A, E112A/E116A, R12A/R15A and D54A/D56A showed about half the extent of rate enhancement of wild-type. On the other hand, the variants E99A and E116G exhibited approximately 2.0 to 2.5- fold increases in rate enhancement which may indicate increased binding between TAFI and TM. We determined the antifibrinolytic potential of each variant using an in vitro plasma clot lysis assay. The variants that showed reduced activation by thrombin/TM correspondingly showed impaired stimulation of antifibrinolytic potential by TM. Overall, the data indicate that the variants that showed the strongest TM dependence were focused around the activation peptide which would be near where the C-loop of EGF-3 of TM would contact TAFI. These residues contribute to the increased efficiency of TAFI activation by thrombin.
Abstract 394: Differential Effects of Platelet Inhibition on Angiotensin II-Induced Abdominal Aortic Aneurysm Rupture in Mice
Authors: Owens A Phillip , Boulaftali Yacine , Bergmeier Wolfgang , Mackman Nigel
Published: March 2022

Objective:
Currently, there is no proven medical approach to attenuate the expansion or rupture of abdominal aortic aneurysms (AAAs). It is estimated that >90% of AAA patients are on anti-platelet regiments without documented benefit or clinical evaluation. Therefore, we examined the effects of pharmaceutical blockade of platelet activation in either early or late-stage angiotensin II (AngII)-induced AAA formation.

Methods and Results:
Male
Ldlr
-/-
mice (8 weeks) fed a saturated fat and cholesterol-enriched diet were infused with AngII (1,000 ng/kg/min) via mini-osmotic pumps for 28 days. In one experiment, groups of mice received either aspirin (30 mg/L via drinking water [ASA]) or diet supplemented with the P2Y
12
inhibitor clopidogrel (50 mg/kg/day [Plavix]) 1 week prior to and throughout infusion (n > 15 all groups). All treatments were associated with a significant increase in aortic rupture-induced death versus placebo groups (P < 0.05; Plavix [58% vs. 0%]; and ASA [64% vs. 10%]). In a second experiment,
Ldlr
-/-
mice were similarly fed and infused for 28 days and then stratified, based on aortic lumen diameters, utilizing noninvasive ultrasonography into 4 different groups: ASA (30 mg/L via drinking water), ASA placebo control, Plavix (50 mg/kg/day via food), and Plavix placebo control. Mice were continuously infused with AngII for an additional 42 days. Ex vivo platelet reactivity to adenosine diphosphate and arachidonic acid was conducted throughout the experiment to confirm systemic drug delivery. All treatments were associated with a significant decrease in aortic rupture-induced death versus placebo groups (P < 0.05; [66% placebo vs. 8% ASA] and [58% placebo vs. 0% Plavix]. None of the treatments affected plasma cholesterol, lipoprotein distributions, or increases in systolic blood pressure in either study. In vivo imaging and histopathology demonstrated these anti-platelet agents reduced abdominal macrophage infiltration and matrix metalloproteinase activity in the intervention study.

Conclusion:
This study demonstrates different roles of platelets in the initiation and progression of AAA and warrants further investigation.
Abstract 396: High Doses of a Novel Niacin Analog Are Devoid of Clinically Relevant Adverse Events in Safety Trials in Healthy Volunteers
Authors: Benedict Claude , Neutal Joel , Kiristy Chris , Bachovchin William
Published: March 2022

Introduction:
High doses of niacin has beneficial effects on lipids and coronary outcomes. However, the use is limited due to side effects at higher doses, as shown by trials such as HPS2 THRIVE, including flushing, myopathy and increases in blood glucose. These dose limitations blunt the lipid responses and potential therapeutic effects of niacin in reducing coronary risk. ARI-3037MO is an orally active, novel niacin analog that induces beneficial changes in Low Density Lipoproteins (LDL), Triglycerides (TG) and in High Density Lipoproteins (HDL) in preclinical studies without evidence of flushing, liver toxicity or changes in blood glucose. Additionally, in a single ascending dose clinical trial in healthy volunteers, ARI-3037MO did not provoke flushing and produced significant changes in TGs and HDL at the highest dose.

Methods:
We evaluated the effect of repeat dosing high doses of ARI-3037MO on safety parameters in two separate studies in healthy volunteers. In the first placebo controlled trial, daily doses of 2g and 3.5g of ARI-3037MO were administered for 14 days to 9 subjects per group with 8 subjects receiving placebo. The 2g cohort was extended to 28 days of dosing. In the second trial, 7 subjects received 3g BID (total daily dose of 6g) administered for 24 days with 6 subjects receiving placebo.

Results:
In both studies, ARI-3037MO was well tolerated with no flushing or skin changes, no abnormalities in liver function tests or changes in blood glucose. There was also no effect on oral glucose tolerance test after 24 days of dosing with 3g BID. There was a trend toward lipid reduction in both of the studies, with the 3g BID dose showing a reduction of -19% in TGs, -7.4% in LDL-C, and a significant reduction of -8.7% in Apo B (p<0.05). Similarly, the 3g BID dose significantly increased HDL-C by 7.0% (p<0.05). In both studies, the effect of ARI-3037MO on lipids improved with the duration of dosing.

Conclusion:
The lack of niacin like adverse effects with very high doses of ARI-3037MO indicates that ARI-3037MO has a wide therapeutic index. This safety and tolerability profile with encouraging lipid effects suggest that ARI-3037MO represents a promising therapy for management of mixed hyperlipidemia, especially in the setting of metabolic syndromes and type II diabetes.
Abstract 404: The Effects of Resistance Training and Weight Loss on Arterial Stiffness in Older Adults
Authors: Jefferson Margie E
Published: March 2022

Aging and obesity are associated with increased arterial stiffness. While exercise and weight loss may have beneficial effects, the effect of resistance training (RT) on arterial stiffness is unclear, particularly in older adults.

PURPOSE:
We examined the effects of RT with and without weight loss on arterial stiffness in 32 overweight and obese older adults (mean age = 68±3 yrs; BMI = 31.1±2.7 kg/m2, 56% female, 13% Black).

METHODS:
Participants were randomly assigned to either RT only (n=16) or RT + caloric restriction (RT+CR, n=16). Participants in each group underwent supervised moderate-intensity RT (i.e. 70% one-repetition maximum) 3 days per week for 20 weeks. Arterial stiffness was measured using tonometry, impedance cardiography and oscillometry to assess changes in brachial-ankle pulse wave velocity (baPWV), augmentation index (Alx), large and small artery elasticity and total arterial compliance.

RESULTS:
Overall baseline mean values for the arterial stiffness measures were as follows: total arterial compliance = 18.1±6.8 ml/mmHg, baPWV = 1521±277 cm/s, large artery elasticity = 14.6±5.4 ml/mmHg x 10, small artery elasticity = 4.3±2.2 ml/mmHg x 100, and AIx = 30.8±8.3%. As expected, body weight was significantly reduced in the RT+CR group (-6.0±1.2 kg, p<0.0001) compared to RT only (0.2±1.2 kg, p<0.0006 for comparison). In models adjusted for age, gender, and baseline arterial stiffness, we found that total arterial compliance decreased in the RT only group, but did not change in the RT+CR group (-2.4±1.2 ml/mmHg vs. 0.5±1.1 ml/mmHg respectively, p=0.09). On the other hand, both large artery elasticity (2.7±1.5 ml/mmHg x 10) and augmentation index (4.0±2.2%) increased in the RT+CR group, with no changes in the RT only group. No significant changes were observed for baPWV or small artery elasticity in either group. Overall, greater reductions in body weight were correlated with greater increases in large artery elasticity (r=-0.29, p=0.11), but no other arterial stiffness measures.

CONCLUSION:
Our data suggest that adding weight loss to a RT program may lead to greater increases in large artery elasticity in overweight and obese older adults. These changes could potentially contribute to improvements in cardiovascular health.
Abstract 407: The Association Between Diabetic Peripheral Neuropathy and Peripheral Artery Disease
Authors: Nichols Gregory A
Published: March 2022

Diabetic peripheral neuropathy (DPN) and peripheralartery disease (PAD) are common complications that occur more frequently in patients with type 2 diabetes than in the general population. Data on the association between these conditions is surprisingly scant. We searched the electronic medical records of 37,095 members of Kaiser Permanente Northwest who had type 2 diabetes identified prior to 2013 and had complete health plan eligibility in 2012 for physician-coded ICD-9-CM diagnoses of DPN or PAD in 2010-2012. We compared demographic (age, sex, diabetes duration) and clinical characteristics (BMI, HbA
1c
, blood pressure, lipids, and presence of ischemic heart disease [IHD], chronic kidney disease [CKD] and heart failure [HF]) associated with the presence of one or both of these conditions. To isolate the independent association between DPN and PAD, we constructed a multiple logistic regression model that controlled for risk factors for DPN and PAD. Of the 37,095 type 2 diabetes patients, 29% (n=10,636) had DPN and 7% (n=2,586) had PAD. Among patients with DPN, 15% had PAD compared with 4% without DPN (p<0.001). Nearly two-thirds of patients with PAD also had DPN, while about one-quarter of patients without PAD had DPN (62% vs. 26%, p<0.001). Characteristics of patients without PAD differed markedly depending on whether DPN was present. For example, mean age of patients with DPN but no PAD was 67.3 years, compare with 59.9 years among those without DPN or PAD (p<0.001), and those with DPN were two to three times more likely to have IHD, CKD or HF. Conversely, characteristics including age, sex and presence of comorbidities were similar among patients with PAD regardless of the presence of DPN. After controlling for the aforementioned common risk factors, presence of DPN more than doubled the probability of having PAD (OR 2.32, 95% CI 2.08-2.60). Our results demonstrate a strong association between DPN and PAD that remained even after controlling for a number of characteristics. Although our design did not evaluate the temporal relationship between these two conditions, older age and longer duration of diabetes among PAD patients suggests that DPN may often precede PAD. Research is needed to determine whether treating DPN can reduce the risk of PAD.
Abstract 461: Compositional Changes to High-Density Lipoproteins That Are Related to Increased Cardiocascular Disease Burden
Authors: McEneny Jane , McKavanagh Peter , York Edmund , Nadeem Nida , Lusk Lisa , Ball P , Trinick Tom , Stevenson Michael , Young Ian , Donnelly Patrick
Published: March 2022

Atherosclerosis is a vascular disease characterized by the build up of lipid derived plaques, and an individual’s risk of acute coronary syndrome is related to plaque burden and composition. Multiple risk markers for atherosclerosis and CVD act in a synergistic way through inflammatory pathways. One such inflammatory marker is serum amyloid A (SAA), which associates with HDL, negating its antiatherogeinc properties, which may have a causal role in atherosclerosis development.

This study examined if SAA was related to CVD burden, and if this influenced the antiatherogenic properties of HDL.

Subjects (n=240) referred to the rapid chest pain clinic at the Ulster Hospital UK, had atherosclerotic burden assessed by cardiac computerised tomography (CCT) and were classified as no CVD; mild CVD stenosis <50% and moderate/severe CVD stenosis >50%. HDL
2
and HDL
3
were isolated from serum by rapid ultracentrifugation. SAA was measured by an ELISA procedure, lipids by a colorimetric method and CETP activity by a fluorimetric assay.

Results:
Although lipids were similar in HDL
2
across the groups, lipids decreased in HDL
3
with increasing CVD burden. In addition, the concentration of SAA and the activity of CETP increased with increasing CVD burden (see table). Additionally, ordinal regression analysis illustrated that HDL
3
-SAA and HDL
2
-CETP where independently related to CVD burden (p=0.001).

Conclusions:
This study has shown that SAA was associated with increased CVD burden and that HDL had altered lipid composition and increased CETP activity. Thus, SAA and CETP may be useful biomarkers for the detection and assessment of CVD.

Funded by Heart-Research UK
Abstract 463: The Effect of Intrauterine Inflammation on the Early Development of Atherosclerosis
Authors: Nguyen Maria , Wallace Megan , Menheniott Trevelyan , Pepe Salvatore , Gaspari Tracey , Widdop Robert , Moss Timothy , Burgner David
Published: March 2022

Atherosclerosis is a chronic condition that may begin prior to birth, particularly in the presence of factors that act through inflammatory pathways. Inflammation may alter epigenetic marks, and changes in methylation are observed in atherosclerotic lesions. We hypothesised that intrauterine inflammation (chorioamnionitis, which complicates 1-4% of all births in the US) would alter development of early atherosclerosis. We report preliminary findings from a novel mouse model of chorioamnionitis and atherosclerosis.

We aim to investigate:

1. the effect of intrauterine inflammation on the development of early atherosclerosis in mice

2. molecular and epigenetic mechanisms

Methods:
Apolipoprotein E deficient pregnant mice (n=15-19 per group) received LPS (0.1 ng in 5 μl saline) or saline (5 μl) into each amniotic sac on gestational day 15. Offspring were weaned onto a high fat (21% v/v) diet at 4 weeks. Aortae, heart and plasma were collected from 6- and 12-week-old offspring. Aortae were analysed following Haematoxylin and Eosin (H&E) and Oil Red O staining. Heart and aortae cholesterol concentrations were quantified using colorimetric assay. Gene expression (Real-Time PCR) of candidate genes was measured.

Results:
Intra-amniotic LPS injection induced histological chorioamnionitis. In 6-week-old mice, intrauterine inflammation increased cholesterol concentrations in aorta and cardiac tissue (p<0.05), and increased aortic Dnmt1 mRNA levels (p=0.02; a key regulator of DNA methylation). No differences between LPS- and saline-treated animals were found in H&E stained aortic sinus. In 12-week-old mice, reduced mean intimal area (p=0.02) and mean intimal thickness index (p=0.02) were found in H&E stained brachiocephalic artery sections from LPS-treated offspring. No differences were found between groups in H&E stained aortic sinus or in an en face analysis of Oil Red O-stained aorta.

Conclusions:
Exposure to intrauterine inflammation has complex effects in this model. Contrary to our hypothesis, 12-week-old LPS-treated mice showed decreased intimal area with no other apparent histological differences, however early cholesterol and molecular biology changes exist. Differential epigenetic regulation may be an early underlying mechanism.
Abstract 468: Serum Amyloid A and Toll-Like Receptor 2 Activation Promote Dedifferentiation of Vascular Smooth Muscle Cells
Authors: Pessolano Lawrence G , Seidl Stephanie E , Rich Celeste B , Schreiber Barbara M
Published: March 2022

Smooth muscle cells (SMCs) regulate vascular tone, and during chronic inflammation associated with atherosclerosis, SMCs contribute to the disease process via de-differentiation from a contractile state. We study the impact of acute phase serum amyloid A (SAA), a cardiovascular risk marker that localizes to atherosclerotic plaques, on SMC function. The goal of this study was to define a role for SAA in SMC phenotypic expression. SMC marker expression was down-regulated by SAA, consistent with de-differentiation. Myocardin, a transcriptional co-activator of SMC marker gene expression, was also down-regulated by SAA, and its overexpression rescued the SAA-mediated repression of the smooth muscle α-actin and smooth muscle 22 α (SM22) promoters. SAA-mediated down-regulation of SM22 promoter activity was also rescued by expression of the myocardin family members, myocardin related transcription factor (MRTF)-A and MRTF-B. It was reported that SAA is a ligand for the Toll-like receptor (TLR)2, which has been implicated in atherosclerosis. Interestingly, FSL-1 and Pam3CSK4, known TLR2 ligands, down-regulated SM22 promoter activity, and the effects were rescued with myocardin overexpression. Moreover, knockdown of TLR2 using siRNA rescued the de-differentiated phenotype induced by SAA. In addition, SAA failed to induce de-differentiation in SMCs isolated from TLR2-/- mice. These data suggest that TLR2 activation, whether by SAA or by TLR2 ligands, promotes SMC de-differentiation characteristic of atherosclerosis.
Abstract 475: Circulating Immunoglobulins Are Not Associated With Intraplaque Mast Cell Number and Other Vulnerable Plaque Characteristics in Patients With Carotid Artery Stenosis
Authors: van der Velden Daniël , Willems Sanne , Quax P H , de Borst G J , de Vries J.P.P.M. , Moll F L , Kuiper J , Toes R E , de Jager S C , de Kleijn D P , Hoefer I E , Pasterkamp G , Bot I
Published: March 2022

Background:
Atherosclerosis is a chronic systemic inflammatory disease in which many immune cells participate. Recently we have shown that mast cell numbers are associated with atherosclerotic plaque vulnerability and interestingly with future cardiovascular events (S. Willems et.al. 2013). The endogenous trigger that activates mast cells within the lesion remains unidentified. We hypothesized that mast cells are activated via receptors for IgG and/or IgE.

In this study we aimed to establish whether specific immunoglobulin levels in plasma of atherosclerotic patients were related to intraplaque mast cell numbers. In addition, the levels were related to other vulnerable plaque characteristics and baseline clinical data.

Results:
Serum levels of total IgG, total IgE and oxLDL specific IgG were determined in 135 patients that underwent carotid endarterectomy. The studied patient population with a mean age of 67 and a male prevalence (71%) reflects a relatively typical population of patients with vascular occlusive diseases. The majority of patients were symptomatic (74%) as illustrated by the incidence of amaurosis fugax, a TIA or a stroke.

No associations were observed between either the serum immunoglobulin levels or the total mast cell numbers in atherosclerotic plaques (total IgG vs. total mast cells r= -0,038, p=0.664) (tIgE vs total mast cells r=-0.038, p=0.664) (oxLDL-IgG vs total mast cells r=0.137 p=0.114). Furthermore, no associations were found between immunoglobulin levels and the following plaque characteristics: lipid core size, degree of calcification, number of macrophages or smooth muscle cells, amount of collagen and number of microvessels.

Interestingly, we observed a negative association between statin use and total IgE (97.5 [43.1-276.8] vs. 157.3 [75.2-545.4] ng/mL, p=0.012) and statin use and oxLDL-IgG (288 [224-406] vs. 399 [282-584] mU/mL, p=0.004).

Conclusions:
In patients suffering from carotid artery disease, total IgG or IgE and oxLDL-IgG levels do not associate with plaque mast cell numbers or vulnerable plaque histopathological characteristics These data may indicate that other endogenous factors activate mast cells within the atherosclerotic plaque.
Abstract 488: Remote Ischemic Preconditioning Attenuates Renal Ischemia-Reperfusion Injury in a Model of Thoracoabdominal Aortic Aneurysm Open Repair
Authors: Lazaris Andreas , Athanasiadis Dimitrios , Kapelouzou Alkistis , Katsimpoulas Michail , Balafas Evaggelos , Vasdekis Spyros , Kostakis Alkiviadis , Liakakos Theodoros
Published: March 2022

Introduction:
Temporary renal ischemia consists a significant factor that increases morbidity and mortality after thoracoabdominal aortic aneurysm (TAAA) open repair. Various measures have been described to protect against renal ischemia-reperfusion (IR) injury. Remote ischemic preconditioning (RIPC) has been considered a means of decreasing IR injury in various tissues. Aim of the study was to identify the effect of RIPC in reducing renal IR injury in an animal model of TAAA open repair.

Material and methods:
An animal based experimental study was designed. Twenty male swine were divided in 4 groups: Sham (control) group, IR group, RIPC I group, RIPC II group. Animals underwent a 30 minutes visceral ischemia after a concomitant subphrenic and infrarenal aortic clamping (IR group). In RIPC groups, two types of 15 minutes of lower body brief ischemia followed by 15 minutes of reperfusion, preceded the visceral ischemia (15 minutes ischemia and 15 minutes reperfusion in RIPC I group, and 3 cycles of 5 minutes ischemia followed by 5 minutes of reperfusion in RIPC II group). Elective renal vein blood was retrieved from the renal vein in specific time intervals of reperfusion, ranging from 30 minutes to 24 hours post-ischemia. Using ELISA techniques, serum was examined for various parameters indicating IR injury (MDA), inflammation and renal tissue damage (CRP, TNFa, Troponin I), as well as renal function (Urea, Creatinine, Cystatin C, NGAL).

Results:
All examined parameters were found to be less impaired in animals of RIPC groups compared to IR group, at a significant statistical level.

Discussion:
Blood data demonstrate a beneficial effect in reducing IR renal injury induced in a way similar to the one during a TAAA open repair, when RIPC has been preceded the IR injury.
Abstract 493: Ultrasound-Based Image Registration Algorithm for Measuring Regional Wall Strain Changes Leading to Abdominal Aortic Aneurysm Rupture
Authors: Richards Michael S , Couper Nathan , Mix Doran , Chandra Ankur
Published: March 2022

OBJECTIVES:
Size-based assessments of AAA do not accurately identify rupture risk. AAA rupture potential is related to wall failure which is dependent on material properties and compliance. Our hypothesis is that transcutaneous ultrasound (US) regional strain measurements can be used to quantify changes in aortic wall mechanical properties indicative of an increase in rupture risk.

METHODS:
A patient-specific AAA cryogel phantom was fabricated and mechanical compliance and tested on an in vitro hemodynamic simulator. A Sonix-Touch system imaged the phantom through the maximum axial diameter under physiologic, pulsatile conditions with increasing mean arterial pressure (MAP) of 95, 120, 140, 160, and 180 until rupture (200mmHg). An US image registration algorithm was used to measure the circumferential wall strain from systole to diastole.

RESULTS:
The spatial mean of the circumferential wall strain (%) in the known location of rupture (solid box) decreased from 2.7, 2.0, 2.4, 1.7, 1.6, and 1.2 for MAP of 95, 120, 141, 160, 180 and 200 mm Hg (rupture) respectively. For comparison the mean strain levels in the adjacent wall of the aneurysm (dashed) were 3.4, 3.4, 3.0, 3.2, 2.1 and 1.8 for the same MAP. The mean diameter of the AAA increased from 5.48cm, 5.83cm, 6.13cm, 6.34cm, 6.54cm, and 6.69cm (rupture) over the range of MAP.

CONCLUSIONS:
At the location of eventual rupture, the mean regional strain decreased as the diameter of the phantom enlarged and progressed to rupture relative to other regions of the wall. This suggests the possibility of applying regional AAA wall strain as a more accurate method of predicting AAA rupture risk.
Abstract 500: Resting State Functional MRI Predicts Lesions When Diffusion-Weighted Imaging Fails in Ischemic Stroke
Authors: Kasam Mallikarjun
Published: March 2022

Purpose:
DWI is the current standard to identify and manage stroke. We assessed the hypothesis that resting state fMRI may detect the infarcts in patients with acute ischemic stroke more effectively than DWI.

Methods:
We studied 10 patients within 7 days of the acute onset of stroke patients retrospectively with an approved Institutional Review Board. All patients underwent multimodal MR imaging, including DWI and resting state fMRI sequences in the using a GE 3T MRI scanner. High resolution T2-weighted images were also acquired. The resting state fMRIs were processed using a published method by a single rater with extensive experience in neuroimaging data processing to avoid inter-rater variability. DWI data was analyzed using Analyze 11.0 software.

Results:
fMRI provides more accurate information on the irreversible status of ischemic brain tissue during an acute stroke compared with diffusion weighted imaging. Similar results were observed across all the 4 patients studied.

Results:
fMRI provides more accurate information on the irreversible status of ischemic brain tissue during an acute stroke compared with diffusion weighted imaging. Similar results were observed across all the 4 patients studied.

Conclusion:
The results of this project have direct clinical implications. fMRI show areas of damage when DWI has normalized after an acute ischemic stroke. DWI can normalize after showing an area of restricted diffusion in areas of the brain that is irreversibly injured. Therefore, fMRI reliably indicates damaged brain tissue in the setting of acute ischemic stroke where DWI is unable to detect. Such information is critical to make treatment decisions when considering which acute stroke patients should undergo reperfusion therapies. fMRI provides complimentary information to CT/MRI perfusion and has the potential to obviate the need of CT/MRI perfusion which are invasive, time and resource intensive. Our study is limited by their qualitative nature and small sample size.
Abstract 505: Young Adults With Juvenile Dermatomyositis Have Endothelial Dysfunction: Studies of Brachial Artery Reactivity and Oxidized Lipids
Authors: Pachman Lauren M , McMahon Maureen , Morgan Gabrielle A , Korcarz Claudia E , Stein James H , Amorouso Maria , Huang Ching C
Published: March 2022

Objective:
Older patients with Juvenile Dermatomyositis (JDM) have increased carotid wall thickness and decreased nailfold capillary end row loops (ERL). This study tested young adults with JDM and decreased ERL for brachial artery reactivity (BAR) and HDL antioxidant function.

Methods:
20 adults, 14 F, mean age 21.8± 4.2 yrs; mean age at JDM onset 7.6 ± 3.7 years, mean duration of untreated disease (DUD) at diagnosis 5.4 ± 4.8 months, and mean total duration of disease 14.2 ±3.9 years. JDM adults had a mean Total Disease Activity Score (DAS) 1.9 ± 2.5 and were medication free. They were matched by age, race, sex and BMI with 20 healthy controls (mean age 23.8 ± 4.2 years). All subjects were tested for: BAR, ERL number, height and weight. The antioxidant function of HDL was evaluated by the change in fluorescence intensity caused by oxidation of dichlorfluorescein-diacetate by oxidized LDL ±HDL was measured (mean HDL function in healthy controls ranges from 0.44 - 0.66).For BAR, individuals were fasting and rested supine for 10 minutes before ultrasound imaging. Brachial artery diameter was measured at rest and one minute after 4.5 minutes of supra-systolic forearm occlusion. BAR was expressed as the percent diameter change from baseline in response to increased sheer stress [(post-deflation - baseline diameter)/baseline diameter] x100.

Results:
The # of ERL were significantly lower in the JDM group, 6.35 ± 1.29 vs the controls, 7.4±0.58, p=0.003. The TNF-α -308A allele in the JDM group was 40% vs. 15% in controls. JDM were shorter than their controls, for JDM women, 159.7 ±8.8cm vs. 165.6 ±5.4cm, p=0.048, and for JDM men, 172.7±3.6cm compared to controls, 181.5 ±4.6cm, p=0.0046, perhaps a consequence of steroid administration. BAR in JDM was 3.35±3.06%, and 5.22±3.07% in normal controls (p=0.068). After adjusting for height, age, sex, and BA diameter, the BAR in JDM adults was significantly lower than controls by 2.31% (p=0.04). There were no associations of BAR with ERL, DUD, or DAS or oxidized lipids.

Conclusion:
BAR is decreased in young adults off medication for JDM, indicating endothelial cell dysfunction. Larger studies in young adults with JDM are needed to validate this finding
Abstract 506: Nanocarrier-Targeted Mesenchymal Stem Cell for Angiogenesis and Wound Healing
Authors: Wang Bo , Li Yan , Kovalski Leticia , Tian Runxia , Liu Zhao-Jun , Velazquez Omaida
Published: March 2022

Objectives:
Wound healing and angiogenesis are impaired in patients with diabetes and peripheral vascular disease. We developed a novel treatment by systemic administration of mesenchymal stem cells (MSC) coated with nanocarrier composed of adhesion molecule -nanoparticle complex (AMNC). AMNC-MSC significantly enhanced wound healing and angiogenesis, in wild type and diabetic mice. AMNC-MSC targeted homing is specific to the injured tissue and superior to BSA (control)-NC coating or uncoated MSC. We now investigate the biosafety profile of the AMNC-MSC.

Method:
1x 10
6
HUVEC were coated with AMNC. Cytotoxicity of AMNC was evaluated by trypan blue. Cell apoptosis was analyzed by flow cytometry using FITC-Annexin-V and propidium iodide. 12-14 week-old C57BL6 mice (N=15) underwent 6mm full thickness dorsal dermal wounding and then assigned to 3 IV treatment groups: (i) 1x 10
6
AMNC-MSC, (ii) 1x 10
6
MSC and (iii) equivalent volume saline (No Treatment (NT); (n=5/group). Day 8 post-wounding and treatment, comprehensive metabolic panel, complete blood count and urinalysis were obtained.

Result:
AMNC-MSC had no cytotoxicity to HUVEC. Blood tests and urinalysis revealed that the hematological, hepatic, renal and pancreatic functions were normal in all groups. Hyperglycemia was observed among all groups at comparable level (p=0.136), likely a stress response. All other blood/urine parameters were within normal, in all groups.

Conclusion:
AMNC-MSC has no observed cytotoxicity to human endothelial cells,
in vitro
. Intravenous administration of AMNC-MSC does not cause metabolic or hematologic toxicity, in mice. We demonstrate initial biosafety of novel nanocarrier-targeted MSC therapy which holds promise in pro-angiogenic and pro-healing regenerative medicine.
Abstract 51: Sortilin 1 Promotes Matrix Vesicle-Mediated Vascular Microcalcification via Rab11 Trafficking and C-Terminal Phosphorylation
Authors: Goettsch Claudia , Hutcheson Joshua D , Aikawa Masanori , Singh Sasha , Libby Peter , Aikawa Elena
Published: March 2022

Background:
Arterial microcalcification predicts cardiovascular events and contributes to plaque rupture. We recently demonstrated that matrix vesicles (MVs) participate in the formation of microcalcifications. Yet, the precise mechanisms by which MVs regulate calcification in atheroma remain unknown. We hypothesized that sortilin 1, a protein associated with arterial calcification in GWAS, promotes calcification via its phosphorylation and recruitment to MVs.

Results:
We detected sortilin 1 in calcified MVs of human plaques using transmission electron microscopy-based immunogold labeling. Sortilin 1 loading into MVs increased in sortilin 1-overexpressing smooth muscle cells (SMCs), and rose further in MVs derived from calcifying SMCs. Nanoparticle tracking analysis indicated no alteration in MV release. Silencing sortilin 1 reduced tissue non-specific alkaline phosphatase (TNAP) activity within MVs (-45%; p<0.01), whereas sortilin 1 overexpression promoted TNAP activity within MVs twofold (p<0.05). Silencing Rab11 reduced sortilin 1 protein (-44%, p<0.01) as well as TNAP activity (-34%, p<0.01) and mineralization (-36%, p<0.01). Further, silencing Rab11 abolished the induction of TNAP activity by sortilin 1 overexpression. We used a mass spectrometry-based strategy to monitor sortilin 1 post-translational modifications in calcifying SMCs and identified a calcification-dependent phosphorylation within the C-terminal domain. Mutations on Ser825 and Ser819, which prevented sortilin 1 phosphorylation, 1) abolished SMC TNAP activity (-43%; p<0.05) and calcification (-72%; p<0.05), 2) mitigated TNAP activity (-36%; p<0.05) and hydroxyapatite content (-34%; p<0.05) in MVs, and 3) prevented loading of sortilin 1 into MVs. In contrast, mutations on Ser825 and Ser819 that mimic a constitutive active phosphorylation enhanced TNAP activity without affecting sortilin 1 expression.

Conclusion:
Sortilin 1 promotes MV-mediated vascular microcalcification via Rab11 trafficking and C-terminal phosphorylation. Our data provide mechanistic insight into the correlation between sortilin 1 and calcification and identifies sortilin 1 as a potential therapeutic target to modulate the formation of microcalcifications.
Abstract 570: Aptamers Against Factor Xa or Thrombin Synergize With Catalytic Site Inhibitors of FXa or Thrombin, Respectively, to Effectively Anticoagulate Blood in an Ex Vivo Oxygenator Circuit
Authors: Gunaratne Ruwan , Frederiksen James , Lohrmann Jens , Bompiani Kristin M , Smock-Woodruff Rebecca , Sullenger Bruce A
Published: March 2022

Introduction:
The RNA aptamers 11F7t and R9D-14T, which inhibit Factor (F)Xa and thrombin respectively, function as reversible anticoagulants but achieve less intense anticoagulation than unfractionated heparin (UFH). Neither aptamer inhibits the catalytic site of its target; 11F7t binds a FXa exosite in a manner that impedes FVa binding, while R9D-14T binds thrombin exosite I, thereby obstructing fibrinogen binding. However, 11F7t synergizes with a FXa catalytic site inhibitor and R9D-14T synergizes with a thrombin catalytic site inhibitor; each combination prevents clot formation for >180 minutes (min) in a whole blood thromboelastography (TEG) assay, thereby replicating the effect of UFH (5U/mL).

Aims:
To determine whether 11F7t plus a FXa catalytic site inhibitor, or R9D-14T plus a thrombin catalytic site inhibitor, can also maintain the fluidity of blood circulated continuously for 120 min within an
ex vivo
membrane oxygenator circuit.

Methods:
Human whole blood anticoagulated with (A) 11F7t (2μM) + edoxaban (2μM), (B) 11F7t (2μM) + apixaban (2μM), (C) 11F7t (2μM) + fondaparinux (2μM), (D) R9D-14T (10μM) + argatroban (10μM), or (E) UFH (5U/ml) was circulated continuously within the oxygenator circuit at 33°C for 120 min at a flow rate of 50 mL/min. Activated Clotting Time (ACT) was assessed at 0, 5, 60, and 120 min after circulation was initiated, and post-circulation scanning electron microscopy (SEM) images of the oxygenator membranes were obtained.

Results:
11F7t (2μM) or R9D-14T (10μM) alone failed to prevent visible clot formation in the circuit. In contrast, strategies (A) through (E) each prevented visible clot formation for 120 min. At 120 min, ACTs (mean ± SE) were, respectively, 565.8 ± 34.7, 446.5 ± 7.4, 387.6 ± 1.6, 903.4 ± 19.0, and 411.4 ± 16.5 seconds. Post-circulation SEM images of oxygenator membranes were similar for all five strategies and revealed minimal fibrinous and cellular debris.

Conclusion:
The anticoagulant synergy achieved by combining aptamer 11F7t or aptamer R9D-14T with a FXa or thrombin catalytic site inhibitor, respectively, can maintain the fluidity of blood circulated continuously within an
ex vivo
oxygenator circuit. Such synergy may prove useful in clinical settings that require intense anticoagulation.
Abstract 580: Lung Cancer Chemotherapy Agents Increase Procoagulant Activity via Protein Disulfide Isomerase-Dependent Tissue Factor Decryption
Authors: Lysov Zakhar , Swystun Laura , Kuruvilla Sara , Arnold Andrew , Liaw Patricia , Novakovic Dragana , Boffa Michael B
Published: March 2022

Background:
Lung cancer patients undergoing chemotherapy have an elevated risk for thrombosis. However, the mechanisms by which chemotherapy agents increase the risk for thrombosis remains unclear.

Objectives:
To determine the mechanism(s) by which lung cancer chemotherapy agents cisplatin, carboplatin, gemcitabine, and paclitaxel elicit increased tissue factor (TF) activity on endothelial cells, A549 cells, and monocytes.

Methods:
TF activity, TF antigen, and phosphatidylserine exposure were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Cell surface protein disulfide isomerase (PDI) and cell surface free-thiol levels were measured on HUVEC and A549 non-small cell lung carcinoma cells.

Results:
Treatment of HUVECs, A549 cells, and monocytes with lung cancer chemotherapy significantly increased cell surface TF activity. However, elevated TF antigen levels were observed only on cisplatin- and gemcitabine-treated monocytes. Cell surface levels of phosphatidylserine were increased on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Chemotherapy also resulted in increased cell surface levels of PDI and reduced cell surface free thiol levels. Glutathione treatment and PDI inhibition, but not phosphatidylserine inhibition attenuated TF activity. Furthermore, increased TF activity was reversed by reducing cysteines with dithiothreitol.

Conclusions:
These studies are the first to demonstrate that lung cancer chemotherapy agents increase procoagulant activity on endothelial cells, blood monocytes, and A549 cells by TF decryption through a disulfide bond formation in a PDI-dependent mechanism.
Abstract 586: Ordered Opening of the 4-Helix Bundle in the LDL-Receptor Binding Domain of Human Apolipoprotein E3 as Revealed by Fluorescence Polarization Analysis
Authors: Narayanaswami Vasanthy , Hernandez Roy , Beck Wendy , Tran Tuyen
Published: March 2022

Apolipoprotein E3 (apoE3) is an anti-atherogenic protein that helps maintain triglycerides and cholesterol levels in the plasma. It is responsible for binding and cellular uptake of plasma lipoproteins via the low-density lipoprotein receptor (LDLr) family of proteins. It is a highly alpha-helical protein that can exist in lipid-free and lipid-bound states, and undergoes a dramatic conformational re-organization when transitioning between the two states. The objective of this study is to understand the mechanism of chemical-induced unfolding of the 4-helix bundle N-terminal (NT) domain housing the LDLr binding sites. The rationale is that the ease of unfolding is a reflection of ease of helix bundle opening during lipid interaction. We tested the hypothesis that the 4-helix bundle undergoes a concerted opening to reveal the hydrophobic interior, analogous to opening an umbrella. Using X-ray and NMR structure guided rational approach, single cysteine-containing constructs were designed; a fluorescent probe was attached to the free -SH groups to serve as a spectroscopic reporter about the microenvironment and mobility of selected segments of the protein: the 4 main helices observed in the X-ray structure (H1 to H4) and the relatively flexible helixes flanking the helix bundle as observed in the NMR structure. We induced unfolding by chemical denaturation and monitored changes in protein secondary structure and tertiary conformation by circular dichroism and fluorescence spectroscopy, respectively. The overall global was relatively unaltered in the labeled variants; however, fluorescence emission and polarization studies revealed that during unfolding, the microenvironment of the probe on H3 and H4 is more polar and that these helices become more mobile at lower denaturant concentrations, compared to probes on H1 and H2. The data suggest that the 4-helix bundle undergoes a sequential unfolding pattern of the main helices in the following order: helix 4/helix 3, helix 2 and lastly helix 1. Our results offer insights into the mechanism of lipid and lipoprotein binding of apoE3 NT domain, with the segment towards the C-terminal end likely triggering lipid interaction.
Abstract 587: Dynamics of the Apolipoproteome: Turnover of HDL- and LDL-Associated Apolipoproteins by LC/MS/MS
Authors: Turner Scott , Holmes William , Czerwieniec Gregg , Tsang Ellen , Dalidd Marcy , Chen Miriam , Hellerstein Marc , Beysen Carine
Published: March 2022

The production rate and fractional catabolic rate of apoB and apoAI have been measured using stable isotope infusions of 13C or 2H leucine for many years and has led to our improved understanding of the pathophysiology of dyslipidemia and atherosclerosis. Modern tandem mass spectrometry methods have demonstrated the highly complex complement of proteins that associate with HDL and LDL and alterations in these proteins may impact lipoprotein function and cardiovascular risk. Very little is known about the turnover of the proteins that comprise the apolipoproteome. To study the fesability of measuring the turnover of multiple apolipoproteins associated with HDL and LDL we performed 20 hour IV infusions of U-13C labeled leucine in 20 healthy subjects. VLDL, LDL and HDL were isolated by ultracentrifugation and digested with trypsin. We analyzed peptides containing one leucine with analysis of multiple transitions using an Agilent triple quad. The enrichment of HDL and LDL peptides from multiple apolipoproteins were determined. The fractional synthesis of each protein was calculated by comparison of the average peptide enrichment for each protein to the average enrichment in VLDL apoB after 20 hours, at which time the VLDL apoB is fully turned over and its enrichment reflects the hepatic leucine precursor pool enrichment. Mean and SD values for HDL associated proteins are presented in table 1. The range of protein turnover rates on HDL confirms the dynamic state of the particle components. Fractional synthesis was similar for the majority of proteins on HDL and LDL with the notable exception of apoL1 and apoM. Using these methods, the regulation of the apolipoprotein composition of HDL and LDL can now be investigated.