bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 342: Naturally Occurring Modified Forms of LDL
Authors: Orekhov Alexander N
Published: July 2021

Electronegative LDL, small,dense LDL, and desialylated LDL circulating in the blood of patients were obtained by different methods. Naturally, the question arises what are the similarities and differences between these forms of LDL modification. We believe that multiple modified LDL particle (small, dense, electronegative, desialylated, etc.) occurs in blood. Ex vivo experiments have revealed mechanisms of multiple modification of LDL in the blood. Fraction of native LDL was isolated from blood plasma of healthy subjects. Blood serum of patients with assessed atherosclerosis was also obtained. LDL and serum were mixed and incubated for various periods at 37°C. After 1 hour incubation of native LDL with atherosclerotic serum desialylated LDL appears. After 3 hours, LDL becomes able to cause accumulation of cholesterol in cultured cells. After 6 hours, LDL demonstrates reduction of neutral lipids and phospholipids as well as reduction in its size. After 36 hours, an increase in the electronegativity of the lipoprotein particle is detected. After 48-72 hours, loss of α-tocopherol, increase of susceptibility to oxidation, and accumulation of lipid peroxidation products in LDL are observed. Thus, multiple modification of LDL is a cascade of sequential changes in lipoprotein particle, namely: desialylation, loss of lipids, size reduction, increase of electronegative charge, lipid peroxidation in LDL. Desialylation of LDL particle is one of the first or primary events of atherogenic modification. We have established that the reason of LDL desialylation is trans-sialidase. We found trans-sialidase activity in the blood of patients with atherosclerosis and other cardiovascular diseases. We have shown that human neuraminidases 2 and 4 possess trans-sialidase activity. On the other hand, selective inhibitors of viral sialidases suppress trans-sialidase activity in the blood of atherosclerotic patients. Thus, trans-sialidase causing atherogenic desialylation of LDL may be of both endogenous and exogenous origin. Supported by Russian Ministry of Education and Science (Project RFMEFI61614X0010).
Abstract 386: Thrombin Inhibition by Dabigatran Reduces the Amount of Pro-Inflammatory CD11c-Positive Macrophages in Visceral Adipose Tissue of Low-Density Lipoprotein Receptor-Deficient Mice
Authors: Feldmann Kathrin , Grandoch Maria , Lehr Stefan , Fischer Jens W
Published: July 2021

Thrombin is involved in inflammatory processes. In obesity, chronic low-grade inflammation promotes the development of insulin resistance and diabetes mellitus type 2 (T2DM) which strongly drive atherosclerosis. Patients with T2DM exhibit increased circulating levels of tissue factor as well as thrombin-antithrombin complexes.

Aim of the present study was to analyse potential effects of thrombin inhibition by dabigatran on adipose tissue inflammation in a mouse-model of diet-induced obesity and atherosclerosis.

Female, 10 weeks old low-density lipoprotein receptor-deficient (LDLR-/-) mice received a Western-type diet containing 5 mg/g dabigatran or matching placebo for 20 weeks.

Flow cytometric analysis of the stromal vascular fraction revealed a reduction in pro-inflammatory macrophages (CD11b+F4/80+CD11c+) in visceral adipose tissue of dabigatran-treated animals (control 8.490 ± 1.996, dabigatran 3.606 ± 0.8884 CD11c+ macrophages/mg adipose tissue, n = 21), while the number of anti-inflammatory CD11c- macrophages was not changed. mRNA expression in the visceral adipose tissue as well as plasma concentrations of the pro-inflammatory cytokines interleukin (IL) 1 beta and IL 6 (quantitative real-time PCR (qPCR), multiplex immunoassay) were reduced in dabigatran-treated animals. No differences in body-weight gain and the amount of body fat (NMR measurement) were observed. Glucose tolerance and insulin resistance did not differ between groups, either. In vitro no direct influence of thrombin on macrophage polarization was detectable in bone marrow-derived macrophages (flow cytometry, qPCR). However, first results indicate that stimulation of the cells with conditioned medium from thrombin-treated, differentiated preadipocytes (3T3L1 cells) enhances M1 polarization of macrophages (flow cytometry).

In conclusion, thrombin inhibition by dabigatran promotes an anti-inflammatory phenotype in adipose tissue macrophages in a mouse model of diet-induced obesity and atherosclerosis, probably resulting from a changed expression profile of adipocyte cytokines. These effects might contribute to the known anti-atherosclerotic effects of thrombin inhibition in this model.
Abstract 387: Bioengineering L-605 Cobalt Chromium Cardiovascular Stent Biomaterial With Plasma Activated Coating, for Proactive Biocompatibility
Authors: Jeewandara Thamarasee , Wise Steven G , Michael Praveesuda L , Hung Juichien , Santos Miguel , Kondyurin Alexey , Weiss Anthony , Bilek Marcela M , Ng Martin K
Published: July 2021

Introduction:
Cobalt Chromium alloy L605 is an underlying biomaterial in new generation drug eluting stents (DES) and bare metal stents (BMS). Suboptimal biocompatibility of stents clinically manifest as thrombosis and restenosis. We optimized a plasma coating technology to activate an alloy L605 surface for enhanced biofunctionalization.

Objective:
Alloy L605 flat metal surface modified with plasma activated coating (PAC), covalently binds tropoelastin (TE) in its bioactive state.
In vitro
studies were conducted to assess biocompatibility of modified PAC biomaterial compared to the bare metal alloy L605.

Methods and Results:
The initial water contact angle (Kruss contact angle analyser, DS10) of PAC was 44.4
0
±3.40 and correlating surface energy computed with the Owens-Wendt-Rabel-Ström kinetic model was 62.35±2.4 mJ/m
2
. Thrombogenicity studies with heparinised whole blood (0.5 U/mL, 37
0
C), in a modified chandler loop showed significantly less clot formation at 60 mins for PAC, PAC+TE Vs. L605 surfaces; 2.2±1.01 mg, 3.6±0.85 mg and 10.3±3.26 mg respectively (P<0.05). Static blood assays with platelet rich plasma (PRP) on PAC and PAC+TE surfaces were compared to L605 (figure 1). Samples incubated with tropoelastin and treated with SDS-Elisa (90
0
C, 10 mins), to assess covalent binding showed higher absorbance for PAC vs. L605. Robust adhesion of PAC polymer to L605 was quantified as surface element percentage with energy dispersive X-ray spectroscopy (2 kV, Xray 2000 cps). Future studies will be conducted on an alloy L605 coronary stent platform.

Conclusion:
The plasma coating is hydrophilic and hemocompatible. The PAC technology will be translated to a commercially available alloy L-605 coronary stent platform to establish a pre-clinical model of hemocompatibility
in vitro,
as an alternative to animal model testing. Early events of coagulation and inflammation will be determined via biological evaluation assays of medical devices (ISO 10993-4).
Abstract 388: Body Mass Index Predicts Major leeding Risks in Patients on Warfarin
Authors: Ogunsua Adedotun A , Touray Sunkaru , Liu Justin K , Escobar Jorge , Ip Tiffany , Gore Joel
Published: July 2021

Background:
Despite the lack of an optimum dosing strategy in obese patients, warfarin remains the most commonly used anticoagulant. Body mass index (BMI) > 30 has been linked to increased time to obtain a therapeutic institutionalized normalized ratio (INR) on initiation of warfarin as well as higher maintenance dose. Obese patients have higher dosage requirements, however, few studies have examined the relationship between warfarin and bleeding events in these patients.

Aim:
We examined the performance of BMI in predicting the incidence of bleeding at an anticoagulation clinic (ACC) over a one year period.

Methods:
863 patients followed in an ACC were evaluated for bleeds according to BMI over a one year period. BMI was defined as weight (kg)/height (m2). CHADS2VASC scores were calculated. Major (gastrointestinal, intracerebral and retroperitoneal hemorrhage) and minor bleeding events (epistaxis, hematuria, vaginal and skin bleeds) were ascertained.

Results:
71 ( 8.2%) of the 863 patients had a bleeding event, the mean age of the cohort was 69.5 years, and 44% were females. BMI categories were normal weight (21%), overweight (38%), obese class I, (21%), II (9 %), and III (11.3%) respectively. The prevalence of major and minor bleeding events were 35.2% and 64.8% respectively. In univariate analyses, hazard ratio for major bleeding risks increased with higher obesity categories (HR 1.30, 1.85, and 1.93 for class I,II, III respectively). In multivariable adjusted model, obesity (BMI>30) significantly increased the risk of major bleeds. (HR 1.84, P<.001) (Table 1).

Conclusion:
Bleeding risk is higher in obese compared to normal weight individuals who are on warfarin. Risk is higher with increasing BMI. This result suggests that BMI plays a role in bleeding events in patients on warfarin. Future studies are needed to understand the mechanism by which obesity increases bleeding risk for patients on warfarin and whether similar risk exist for the novel oral anticoagulants.
Abstract 395: Elevated Von Willebran Factor in an in vitro Model of Fabry Disease
Authors: Kang Justin J , Kaissarian Nayiri M , Shu Liming , Desch Karl C , Bodary Peter F , Shayman James A
Published: July 2021

Fabry disease is an X-linked disorder caused by a defect in the gene encoding the lysosomal hydrolase, alpha-galactosidase A (GLA). The loss in GLA function causes an age-dependent accumulation of globotriaosylceramide in the endothelial cells, which results in decreased nitric oxide bioavailability and premature mortality in the affected patients. Based on this, we hypothesized that GLA deficiency promotes endothelial inflammation. We observed a significant age-dependent increase in the plasma von Willebrand Factor (vWF) in Fabry mice compared to age-matched wild type mice (p=0.046 at 2 months and p=0.0004 at 17 months). To evaluate the molecular mechanisms of increased vWF in Fabry disease, small interfering RNA (siRNA) technique was used to knock-down GLA in EA.hy926 cells. Our initial studies with this cell line demonstrated that thrombin (2U/mL) induced a time-dependent vWF secretion into the cell culture medium with an 80-fold increase in the level of vWF after 90 min stimulation (p=0.001 compared to basal level; n=3 replicates per group), measured by a human antibody against vWF and AlphaLISA. Next, the siRNA studies revealed a greater than 95% knockdown of GLA protein (via western blotting) in the GLA-siRNA group compared to the control group with no effect in the scrambled (SCR) sequence-siRNA group (CON vs. GLA vs. SCR arbitrary units: 1.0±0.03 vs. 0.01±0.01 vs. 1.1±0.08, p<0.001; GLA vs. SCR). Importantly, no difference in GAPDH protein was observed between groups. To determine the direct effect of GLA deficiency on vWF, the confluent cells in each group were incubated in serum-free medium overnight. Consistent with the hypothesis, we observed approximately 60% increase in vWF in the cell culture medium in the GLA siRNA group compared with the untreated and scrambled-sequence control groups. (CON vs. GLA vs. SCR arbitrary units: 38.5±4.7 vs. 61.3±1.1 vs. 31.4±3.8, p=0.02; GLA vs. SCR; n=3 replicates per group).

In conclusion, the data provide evidence of endothelial activation in the setting of GLA deficiency, demonstrated by the robust elevation of vWF mediated by GLA gene disruption. Future study is required to further understand the mechanism of the elevated vWF and decreased nitric oxide bioavailability in Fabry disease.
Abstract 396: Nanoscale Extracellular Matrix Alters Endothelial Function Under Disturbed Flow
Authors: Nakayama Karina H , Narayanan Vinay , Lai Edwina S , Ostrowski Maggie A , Fuller Gerald G , Dunn Alex R , Huang Ngan F
Published: July 2021

With 25 million Americans suffering from at least one clinical manifestation of atherosclerosis, there is a pressing need to better understand the biological basis for the development of atherosclerosis. Endothelial cells (EC) that form the inner lining of blood vessels are primarily responsible for resisting atherosclerotic lesion formation. Although hemodynamic shear stress has been thought to be the dominant modulator of EC function, spatial patterning cues from nanoscale extracellular matrices (ECMs) that underlie the endothelium may play an equally important role. Therefore, the goal of this work was to examine how modulation of EC alignment using ECM nanopatterning cues modulates the formation of atherosclerosis. We hypothesized that ECs are more responsive to aligned nanopatterning cues than to disturbed flow, and can resist functional changes associated with atherosclerosis in the presence of disturbed flow. ECs were cultured on fibrillar scaffolds with nanotopography to either induce longitudinal alignment of ECs to mimic the morphology of healthy ECs, or to cause ECs to orient randomly (control) to mimic pathological ECs. ECs were then exposure to disturbed flow for 24 hours. Time-lapse microscopy and F-actin staining were used to quantify migration velocities and cellular orientation. ECs on aligned nanofibrillar scaffolds were induced to align along the direction of the fibrils, even after exposure to disturbed flow, and demonstrated a 2-fold decrease in migrational velocities, compared to randomly oriented ECs. This data suggests that nanoscale cues from aligned nanofibrillar scaffolds direct cell morphology and motility. Functional effects of nanofibrillar organization to resist inflammation, revealed a 2-fold decrease in monocyte adhesion on aligned ECs compared to randomly-oriented ECs, suggesting that ECs on aligned nanofibrillar scaffolds were less prone to atherosclerosis. These results were validated by analysis of inflammatory and shear-mediated markers (NFKB, ICAM, KLF2). This work reveals fundamental insights into the role of nanoscale ECM cues on early events of atherosclerosis, and has important translational potential in the generation of aligned nanofibrillar vascular grafts that resist lesion formation.
Abstract 407: Use of a Previously Validated Blood-based Test Demonstrates Increased Diagnostic Accuracy as Measured by AUC over Usual Care in the Evaluation of Obstructive Coronary Artery Disease in Males and Females.
Authors: Rhees Brian , Johnson Andrea , Lansky Alexandra , McPherson John , Budoff Matthew , Honigberg Robert , Monane Mark , Wingrove James A
Published: July 2021

Background:
Current evaluation of stable, non-acute patients presenting with symptoms suggestive of obstructive coronary artery disease (CAD) is costly and often exposes patients to radiation and contrast-dye side effects. These risks are coupled with relative poor diagnostic accuracy, as consistently demonstrated by low yields at invasive coronary angiography. We hypothesized that the use of a previously validated blood-based test incorporating age, sex and whole-blood gene expression, in conjunction with a clinician’s clinical assessment, may improve on usual care methods for the evaluation of these patients.

Methods and Results:
This analysis includes evaluable data from two prospective multicenter clinical studies [[Unable to Display Character: –]] PREDICT (NCT005617, N=523) and COMPASS (NCT1117506, N=431) where patients were assessed for both pre-test CAD risk according to Diamond-Forrester (D-F) criteria as well as for obstructive CAD using either invasive coronary angiography or cardiac computed tomography angiography (CCTA). All patients in COMPASS were also assessed by myocardial perfusion imaging (MPI); a subset of N=307 subjects were assessed by MPI in PREDICT. Previously, we demonstrated diagnostic superiority, as measured by AUC, for a score combining patient age, sex and whole-blood gene expression (ASGES), in a combined set of men and women from both PREDICT (ASGES = 70%, D-F = 66%, MPI = 54%) and COMPASS (ASGES = 79%, D-F = 69%, MPI = 59%). In this expanded analysis, we report results stratified by sex and demonstrate superiority of the ASGES, as measured by AUC, to MPI for males and females in PREDICT (male ASGES = 66%, MPI = 55%; female ASGES = 65%, MPI = 48%) and in COMPASS (male ASGES = 73%, MPI=60%; female ASGES 73%, MPI = 55% respectively). In addition, we demonstrate that ASGES improves CAD risk classification when compared to D-F criteria in females in both PREDICT (ASGES = 65%, D-F = 51%) and COMPASS (ASGES = 73%, D-F = 58%).

Conclusions:
We demonstrate that use of a gender-specific, blood-based test incorporating age, sex, and gene expression provides better diagnostic accuracy for patients considered for referral to cardiology and advanced cardiac testing, when compared to usual care methods of D-F type risk classification and MPI.
Abstract 423: CC-Chemokine Class Inhibition Attenuates Inflammatory Induced Pathological Angiogenesis Whilst Preserving Ischaemia Driven Physiological Angiogenesis
Authors: Ridiandries Anisyah , Tan Joanne T , Prosser Hamish C , Hawkins Clare , Bursill Christina A
Published: July 2021

Introduction:
Angiogenesis is critical for survival and in the regenerative response to tissue hypoxia. An imbalance in its regulation causes excessive angiogenesis, exacerbating inflammatory diseases such as cancer and atherosclerosis. Increasing evidence suggests the CC-chemokine class promote inflammatory-driven angiogenesis but, in contrast, have no role in hypoxia-driven angiogenesis. Inhibition of the CC-chemokine class may therefore regulate angiogenesis differently depending on the pathophysiological context.

Aim:
Using the broad-spectrum CC-chemokine inhibitor ‘35K’, compare the role of the CC-chemokine class in inflammatory versus ischaemia driven angiogenesis.

Results:
Adenoviruses expressing GFP (AdGFP) or 35K (Ad35K) were injected into mice 3 days prior surgery. Using the murine femoral cuff model of inflammatory angiogenesis, we found a significant decrease in adventitial capillaries (39.7%) and arterioles (75.5%) in the Ad35K group compared to the AdGFP controls, p<0.05. Ad35K mice also had significantly less macrophages (CD68, 32.5%). In contrast, 35K had no effect on markers of neovascularisation including: blood flow recovery and hindlimb capillary density, in the murine hind limb ischaemia model. Consistent with our in vivo findings, in vitro angiogenic assays with endothelial cells, revealed that 35K protein had inhibitory effects on inflammation-induced tubule formation (95.8%), proliferation (88.3%) and migration (15.8%), p<0.05. In hypoxia the inhibitory effects of 35K were more modest for tubule formation (45.2%) and proliferation (43.8%), and migration was completely preserved. Under inflammatory conditions 35K inhibited VEGF (53.4%) and HIF-1α (87.3%) cell protein levels, yet in hypoxia 35K had no effect on VEGF and had a more modest inhibitory effect on HIF-1α (65.4%).

Conclusion:
Broad-spectrum CC-chemokine inhibition by 35K inhibits inflammation-induced angiogenesis, whilst preserving ischaemia-driven angiogenesis in vitro and in vivo. CC-Chemokine inhibition may be therapeutic strategy to reduce pathological angiogenic diseases without the severe side effects of current therapies caused by complete inhibition in both contexts.
Abstract 440: Increased Migration and Proliferation of Human Venous Valve Wall Smooth Muscle Cells are Mediated by FGF2
Authors: Kikuchi Shinsuke , Chen Lihua , Wight Thomas N , Azuma Nobuyoshi , Sobel Michael , Sasajima Tadahiro , Clowes Alexander W , Kenagy Richard D
Published: July 2021

Introduction:
Lesions causing coronary and peripheral vein graft failure often occur at valves. To test the hypothesis that intrinsic differences in cells of the valve wall contribute to graft failure we measured cell migration from tissue explants and migration and proliferation of cultured cells from valve and non-valve sections of human saphenous vein (HSV).

Methods:
After removal of endothelium, HSVs were dissected into an outer wall with adventitia and some media and an inner wall with intima and some media. Cell migration from explants and migration (microchemotaxis chambers) and proliferation of cultured cells at passage 6 were measured.

Results:
Cells migrated faster from valve compared to non-valve tissue of the inner wall (Figure A; P<0.01, N=24 veins), but not the outer wall (not shown). PDGF-BB- or serum-mediated migration of valve inner wall smooth muscle cells (VSMC) was greater than with non-valve SMCs (NVSMC; P<0.05, N=6 pairs, 6.5 ± 1.2 vs. 4.2 ± 0.8 fold of control for PDGF-BB and 4.7 ± 0.6 vs. 3.4 ± 0.4 for serum). VSMC also proliferated faster than NVSMC after stimulation with PDGF-BB plus 2% serum (P<0.01, N=6 pairs, 3.3 ± 0.4 vs. 2.2 ± 0.2 fold of control). Finally, a blocking antibody to FGF2 had no effect on PDGF-mediated migration or proliferation of NVSMC (migration: IgG 2.2 ± 0.3 vs anti-FGF2 2.0 ± 0.4, N=6; proliferation: IgG 2.6 ± 0.1 vs anti-FGF2 2.5 ± 0.2, N=7), but largely blocked the enhanced migration and proliferation of VSMC (migration: Figure B; P<0.01, N=6; proliferation: control IgG 3.4 ± 0.3 vs. Anti-FGF2 Ab 2.7 ± 0.3 fold; P<0.01, N=7).

Conclusion:
The increased migration and proliferation of VSMC compared to NVSMC is largely mediated by FGF2.
Abstract 445: Beraprost, a Prostacyclin Analogue, Inhibits Human Vascular Smooth Muscle Cell Migration via Activation of Exchange Protein Activated by cAMP (Epac)
Authors: McKean Jenny S , Shewan Derryck , Gibson George , Nixon Graeme F
Published: July 2021

Following vein grafting, endothelial damage initiates vascular smooth muscle cell (VSMC) migration to the intima leading to neointimal hyperplasia and restenosis. Inhibition of VSMC migration is therefore a therapeutic target to prevent restenosis. Previous studies have shown that cyclic adenosine monophosphate (cAMP) inhibits VSMC migration although the downstream mediators are unknown. This suggests that cyclic AMP-producing agonists, such as the prostacyclin analogue beraprost which binds to prostanoid receptors, could be used as a potential drug against neointimal formation. We hypothesised that prostacyclin analogues inhibit VSMC migration by activating specific cAMP downstream mediators. The aims of this project were to examine the role of cAMP mediators protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) in this inhibitory effect. Saphenous veins were obtained from patients undergoing coronary artery bypass. VSMCs were primary cultured and migration measured using a chemotaxis chamber. Cell migration stimulated by PDGF was significantly inhibited by 68 ± 5% (n=6) in the presence of a therapeutically relevant concentration of beraprost (1 nM). To examine potential mechanisms of this inhibition, selective activators of PKA and Epac were used. Both Epac and PKA activators significantly inhibited migration towards PDGF. However, fluorescence resonance energy transfer using cells transfected with specific probes revealed that 1 nM beraprost only activates Epac and not PKA in VSMCs (n=4). The downstream mechanisms were further investigated. Pulldown assays demonstrated that beraprost-mediated stimulation of Epac produces subsequent activation of the monomeric G-protein Rap1 and ultimately inhibits RhoA-induced cytoskeleton rearrangement required for migration (n=3). Using siRNA to knockdown Rap1, the inhibition of PDGF-induced migration by beraprost in VSMC cells was shown to be dependent on the Rap1 pathway (n=3). In conclusion, the prostacyclin analogue beraprost inhibits VSMC migration via activation of Epac/Rap1. This may provide a new therapeutic target to prevent neointimal hyperplasia.
Abstract 453: Hydrogen Sulfide Increases Endothelial Permeability through Sulfane Sulfur Modification of Occludin
Authors: Yuan Shuai , Yurdagul Arif , Orr A. Wayne , Kevil Christopher G.
Published: July 2021

Objective:
Hydrogen sulfide (H2S) is an endogenous signaling molecule which exists in three biological pools, namely free H2S, sulfane sulfur and acid labile sulfur. H2S also regulates protein function by protein sulfuration. Fine tuning endothelial permeability is crucial for vascular functions. Therefore, we sought to determine how sulfane sulfur and sulfuration of cell junction proteins alters endothelial permeability.

Methods:
Endothelial permeability was evaluated by albumin leakage or transendothelial electrical resistance (TEER) in transwell inserts. Exogenous H2S was studied by treating human umbilical vein endothelial cells (HUVECs) with H2S donors including sodium sulfide (Na2S), diallyl trisulfide (DATS). To investigate the effect of endogenous H2S, endothelial cells were isolated from aorta (MAECs) of wild type and CSE knockout mice. Biological pools of H2S were measured using monobromobimane followed by detection of sulfide-dibimane with reverse phase HPLC. Protein sulfhydration is determined by 2-methylsulfonyl benzothiazole (MSBT)/cyanoacetate tag-switch assay.

Results:
DATS treatment elevated sulfane sulfur and induced rapid albumin leakage through HUVEC monolayer. Comparatively, Na2S increased permeability only at later time points and to a lesser extent. Total sulfide and sulfane sulfur were reduced in CSE deficient MAECs. Moreover, MAECs lacking CSE showed enhanced barrier function compared to wild-type MAECs, characterized by decreased albumin leakage and increased of TEER. The tag-switch assay showed increased sulfuration of occludin in HUVECs. In conclusion, H2S bioavailability regulates endothelial solute permeability.
Abstract 470: Statin Treatment in Patients with Acute Myocardial Infarction According to Left Ventricular Systolic Function
Authors: Kim Sang-Hyun , Kim Hack-Lyoung , Im Moon-Sun , Seo Jae-Bin , Chung Woo-Young , Zo Joo-Hee , Kim Myung-A , Lee Hyang-Lim
Published: July 2021

Aim:
Statin decreases low density lipoprotein cholesterol level and prevents atherosclerosis. Although statin may be beneficial for patients with acute myocardial infarction (AMI) due to its direct anti-atherosclerotic effect, statin is not usually prescribed to all patients with AMI. This study was performed to identify different determinants of statin treatment and its prognostic impacts in patients with AMI according to their baseline LV systolic function.

Methods:
12,988 patients with AMI were enrolled into nationwide registry database study. Patients were stratified into 2 groups according to their LV ejection fraction (LVEF): LVEF < 40% vs ≥ 40%, and determinant factors for statin treatment and MACE were identified in each group. Major adverse cardiovascular events (MACE) within 12 months of AMI including death, nonfatal MI and revascularization were assessed.

Results:
1,962 patients (15.1%) with LVEF < 40% were older and had more un¬favorable cardiovascular risk factors than those with LVEF ≥ 40%. Statin treatment rate was significantly lower in group with LVEF < 40% than those with LVEF ≥ 40 % (70.5% vs 76.2%, p=0.001). Mean LDL-C level of patients with LVEF < 40% was lower than those with LVEF ≥ 40 %. MACE rate in patients with LVEF < 40% was higher than those with LVEF ≥ 40% (26.8% vs 11.4%, P < 0.001). Independent predictors for 12-month MACE in patients with LVEF ≥ 40% were history of previous myocardial infarction, high Killip stage, three vessel disease, and lower renal function, which are already known risk factors.

Conclusions:
The patients with LVEF < 40% were less treated with statin and showed lower LDL-C level and higher MACE rate than those with LVEF ≥ 40 %. Statin treatment may be considered in patients with AMI and LVEF < 40%.
Abstract 471: Impact of Enrichment Strategies for Future Clinical Trials Assessing PCSK-9 Inhibitors Efficacy in Primary Prevention Settings
Authors: Nasir Khurram , Veledar Emir
Published: July 2021

Introduction:
To date multiple ongoing clinical trials are testing impact of human monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9) in high risk patients with established CVD already on statins. It is of interest to explore the added value of these therapies in individuals free of established CVD. The aim of this study is to assess the implications of population baseline risk considered for clinical trials, in evaluating impact of PCSK9 inhibitors in primary prevention settings.

Methods:
We simulated scenarios among primary prevention cohorts recruited with 5 year CVD event rates ranging from 5-25%. In the control group, 30% relative risk reduction (RRR) with highest tolerated statin dose was considered. The NNT and drug cost with additional PSCK9 inhibitor therapy to prevent one cardiac event were calculated across varying (20-40%) RRR thresholds. The annual cost of PCSK9 inhibitors was estimated in range of $3000-$7,000. We considered ≤ $0.5 million as upper limit for willingness to pay to prevent 1 event in 5 years.

Results:
The table highlights: a) absolute event rates anticipated in the controls as well with additional PCSK9 inhibition; b) the calculated NNT; and c) costs to prevent one cardiac event across the spectrum of baseline CVD risk levels. Among those with estimated 5 year absolute risk of 10%, the threshold for willingness to pay was achieved provided PCSK9 inhibition achieved 40% RRR with annual cost of $3000. Among individuals with >20% absolute 5 year baseline risk, the threshold for willingness to pay was realized even with 20% RRR in all scenarios if annual cost was limited to $3000.

Conclusion:
Future discussion for clinical trials assessing efficacy of PCSK9 inhibitors in reducing CVD events in primary prevention settings must focus on enrichment strategies for recruiting a ‘high risk’ population. These strategies will likely facilitate acceptable NNT and public willingness to pay for these emerging novel therapies.
Abstract 488: Dipeptidyl Peptidase-4 Links Metabolic Regulation With Innate Immune Signaling
Authors: Zhong Jixin , Rao Xiaoquan , Oghumu Steve , Deiuliis Jeffrey , Satoskar Abhay , Frieman Matthew , Rajagopalan Sanjay
Published: July 2021

Objective:
Dipeptidyl peptidase-4 (DPP4) is well known for its ability to modulate post-prandial blood glucose by inactivation of incretin hormones. DPP4 is highly expressed on innate and adaptive immune cell populations and is recognized to be regulated upon inflammatory activation. We investigated the role of myeloid DPP4 in post-prandial glucose regulation and a role for TLR4-MyD88 in humans and mice.

Methods and Results:
We evaluated DPP4 expression on circulating immune cells in patients with atherosclerosis (ATH, n=20) and compared them with healthy controls without atherosclerosis using flow cytometry. ATH patients had increased DPP4 expression on peripheral blood monocytes (4.22 ± 0.44 vs. 5.76 ± 0.44, in control vs. ATH, p=0.037) as well as CD4+ T cells (27.46 ± 2.67 vs. 48.06 ± 1.96, control vs. ATH, p<0.0001). More importantly, DPP4 expression on T cells and monocytes correlated with plasma non HDL-Cholesterol level (R2=0.153, p=0.036 and R2=0.244, 0.012 respectively). Stimulation with LPS increased DPP4 expression on human peripheral blood monocytes by ~4-fold. DPP4 up-regulation by LPS was abrogated by Toll-like receptor-4 (TLR4) knockdown using siRNA. Bone marrow monocytes deficient for MyD88, an adaptor protein for TLR signaling, displayed reduced expression of DPP4. Consistent with these findings, MyD88-/- mice also showed improved post-prandial glycemia in response to oral glucose challenge, a phenotype seen in DPP4-/- mice.

Conclusions:
Our results suggest a previously unrecognized role for TLR4-MyD88 as a critical regulator of DPP4 expression and post-prandial glucose control.
Abstract 492: Atherogenic Dyslipidemia in Patients with Transient Ischemic Attack
Authors: Choi Hye-Yeon
Published: July 2021

Background:
The risk of recurrent stroke following transient ischemic attack (TIA) is reported up to 15% at 90 days after the attack. Urgent assessment and combined use of preventive treatments in appropriate patients can reduce this risk significantly. The atherogenic dyslipidemia in patients with TIA may be associated with intracranial artery stenosis and higher risk of early recurrent stroke. This study was aimed to investigate if dyslipidemia was related to atherosclerotic vascular lesions in the TIA patients.

Methods:
Patients with TIA who admitted to single hospital were included. TIA was defined as an acute loss of focal cerebral function, lasting fewer than 24 hours. If brain magnetic resonance imaging scans showed acute infarction, the patient was judged to have a TIA with a new lesion; if not, the event was classified as TIA without a new lesion. Radiologic findings were reviewed retrospectively. Association between presence of atherosclerotic vascular lesions and lipid profile were examined. Atherogenic dyslipidemia was defined as high density lipoprotein (HDL) cholesterol ≤ 40 mg/dL and triglycerides ≥ 150 mg/dL. Level of non-HDL cholesterol was calculated as total cholesterol concentration minus HDL cholesterol concentration. Presence of classic risk factors of atherosclerosis and vascular disease were also reviewed.

Results:
Among 117 TIA patients, 106 patients who had available results were included to the analysis. Mean age was 64 years (± 13), and 48 were men (42.1%). Atherosclerotic vascular lesion was shown in 56 patients (52.8%). Presence of atherosclerotic vascular lesions was associated with age, diabetes, low density lipoprotein (LDL) cholesterol concentrations, and level of CRP. Atherogenic dyslipidemia was not related to vascular lesions. Among 106 patients, only 18 patients (17.0%) were diagnosed as TIA with a new lesion. Presence of acute cerebral infarction was associated with presence of atherosclerosis. Diabetes and current smoking were associated with new ischemic lesion related TIA.

Conclusions:
In TIA patients, concentrations of LDL cholesterol were related atherosclerotic vascular lesion which is known to be associated with recurrent stroke after TIA. It might be helpful to screen high risk TIA patient.
Abstract 510: Effect of Repetitive Intra-Arterial Infusion of Bone Marrow Mononuclear Cells in Patients With No-Option Limb Ischemia: The Randomized, Double-Blind, Placebo-Controlled JUVENTAS Trial
Authors: Teraa Martin , Sprengers Ralf W , Schutgens Roger E , Slaper-Cortenbach Ineke C , van der Graaf Yolanda , Algra Ale , van der Tweel Ingeborg , Doevendans Pieter A , Mali Willem P , Moll Frans L , Verhaar Marianne C
Published: July 2021

Background:
Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering clinical trials suggest that bone marrow-derived cell therapy enhances neovascularization, improves tissue perfusion, and prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of bone marrow mononuclear cells (BMMNCs) in patients with severe, nonrevascularizable limb ischemia can prevent major amputation.

Methods and Results:
The Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe, nonrevascularizable limb ischemia. Patients were randomly assigned to repetitive (3 times; 3-week interval) intraarterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, ie, major amputation at 6 months, with major amputation rates of 19% in the BMMNC versus 13% in the placebo group (relative risk, 1.46; 95% confidence interval, 0.62-3.42). The safety outcome (all-cause mortality, occurrence of malignancy, or hospitalization due to infection) was not significantly different between the groups (relative risk, 1.46; 95% confidence interval, 0.63-3.38), neither was all-cause mortality at 6 months with 5% versus 6% (relative risk, 0.78; 95% confidence interval, 0.22-2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen pressure improved during follow-up, but there were no significant differences between the groups.

Conclusions:
Repetitive intra-arterial infusion of autologous BMMNCs into the common femoral artery did not reduce major amputation rates in patients with severe, nonrevascularizable limb ischemia in comparison with placebo. The general improvement in secondary outcomes during follow-up in both the BMMNC and the placebo group, as well, underlines the essential role for placebo-controlled design of future trials.

Clinical Trial Registration:
URL: http://www.clinicaltrials.gov. Unique identifier: NCT00371371.
Abstract 556: Targeted Screening Reveals microRNAs of Therapeutic Interest in Radiotherapy-Induced Vascular Disease
Authors: Eken Suzanne M , Christersdottir Tinna , Pirault John , Jin Hong , Chernogubova Ekaterina , Li Yuhuang , Korzunowicz Greg , Winter Hanna , Hansson Göran K , Halle Martin T , Maegdefessel Lars
Published: July 2021

Radiotherapy is an established therapeutic method in many different cancer types. The success of modern cancer treatment raises a new problem, namely radiotherapy-induced vascular disease (vRTx) or vasculopathy, which typically emerges in irradiated areas of the heart, neck and brain and manifests itself as coronary disease, heart failure or carotid stenosis. A chronic inflammatory response likely underlies vRTx. Like in atherosclerosis, complex biological processes such as vascular cell proliferation, remodeling, oxidative stress, and tumor growth factor β (TGF-β)-regulated nuclear factor kappa B (NF-κB) activation are involved. These processes are tightly regulated by a multitude of factors, which separately are difficult to influence.

A set of miRNAs known to orchestrate these processes in other disease contexts likely also play a role in vRTx, and might be modifiable in order to treat or prevent RTx-induced vascular disease. We selected miR-29b, miR-125a, miR-126, miR-143, miR-145, miR-146a, miR-155, miR-221, miR-222, and miR-503 and determined their potential contribution to vRTx. Utilizing unique biobank material from microvascular free tissue transfer reconstructions, where irradiated (R) vascular tissue (branches from external carotid arteries and internal jugular veins) can be compared with non-irradiated control tissue (NR) within the same patient (n=10), we could precisely determine which miRNAs become deregulated after irradiation. Currently we are using a mouse model of local irradiation for further analysis and assessment of miRNA modulation effects.
Apoe
-/-
mice are irradiated in the mediastinal area; control littermates were sham irradiated. We checked the expression of the 10 pre-selected miRNAs in R
vs.
NR vascular tissue (ascending and thoracic aorta).

Of the 10 miRNAs, three (miR-29b up-; miR-143, miR-145 downregulated) were significantly differentially regulated between R and NR human arteries. miR-146a trended upward, but not significantly. Ongoing studies are aimed at histological analysis of mouse R
vs.
NR tissue, functional modulation of deregulated miRNAs
in vivo
to assess vRTx outcome, and
in vitro
assessment of miRNA expression differences in different human vascular cell lines exposed to radiation.
Abstract 56: Platelet Specific Knockout of Glucose Transporter 3 Leads to Altered Metabolism and Decreased Platelet Activation
Authors: Fidler Trevor P , Middleton Elizabeth , Rowley Jesse W , Boudreau Luc , Campbell Robert A , Souvenir Rhonda , Boilard Eric , Weyrich Andrew S , Abel E. D
Published: July 2021

Patients with diabetes display increased thrombosis and platelet activation. In these disorders, the systemic milieu is characterized by multiple metabolic changes including increased glucose concentrations. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. Therefore we hypothesized that decreasing platelet glucose uptake would limit glycolysis thereby decreasing energy production and platelet reactivity. Platelets import glucose via two glucose transporters GLUT1 and GLUT3. GLUT1 is expressed on the plasma membrane and GLUT3 is expressed predominantly on alpha granule membranes (85%) and to a lesser extent on the plasma membrane (15%). To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout (KO) of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. Platelet glycogen content and glycolytic intermediates were significantly reduced in GLUT3 KO platelets compared to controls, and following mitochondrial uncoupling exhibited reduced glycolysis rates. Interestingly, under these conditions, mitochondrial maximal respiration was increased two-fold, with no change in mitochondrial density, or citric acid cycle intermediates.
In vitro
, GLUT3 deficient platelets display a 90% reduction of spreading on fibrinogen and collagen matrixes and significant reductions in CD62p surface translocation and GPIIbIIIa activation following stimulation with multiple agonists. Additionally makers of alpha granule release were significantly reduced.
In vivo
analysis of GLUT3 KO mice using a 10% ferric chloride model of arterial thrombosis and a tail-bleed model indicated no alteration in thrombosis between littermate controls and knockouts. However in a KBx/N model of rheumatoid arthritis GLUT3 KO mice exhibited significantly reduced disease severity. Together, these data indicate that GLUT3-mediated glucose uptake is essential for platelet activation, spreading and alpha granule release. GLUT3 modulates mechanisms that promote rheumatoid arthritis but not those that regulate in vivo thrombus formation.
Abstract 575: NEDDylation Promotes Endothelial Dysfunction: A Role for HDAC2
Authors: Pandey Deepesh , Hori Daijiro , Kim Jae H , Bergman Yehudit , Berkowitz Dan E , Romer Lewis H
Published: July 2021

Emerging evidence strongly supports a role for HDAC2 in the transcriptional regulation of endothelial genes and vascular function. We have recently demonstrated that HDAC2 reciprocally regulates the transcription of Arginase2, which is itself a critical modulator of endothelial function via eNOS. Moreover HDAC2 levels are decreased in response to the atherogenic stimulus OxLDL via a mechanism that is apparently dependent upon proteasomal degradation. NEDDylation is a post-translational protein modification that is tightly linked to ubiquitination and thereby protein degradation. We propose that changes in NEDDylation may modulate vascular endothelial function in part through alterations in the proteasomal degradation of HDAC2. In HAEC, OxLDL exposure augmented global protein NEDDylation. Pre-incubation of mouse aortic rings with the NEDDylation activating enzyme inhibitor, MLN4924, prevented OxLDL-induced endothelial dysfunction. In HAEC, MLN enhanced HDAC2 abundance, decreased expression and activity of Arg2, and blocked OxLDL-mediated reduction of HDAC2. Additionally, HDAC2 was shown to be a substrate for NEDD8 conjugation and this interaction was potentiated by OxLDL. Further, HDAC2 levels were reciprocally regulated by ectopic expression of NEDD8 and the de-NEDDylating enzyme SENP8. Our findings indicate that the observed improvement in endothelial dysfunction with inhibition of NEDDylation activating enzyme is likely due to an HDAC2-dependent decrease in Arginase2. NEDDylation activating enzyme may therefore be a novel target in endothelial dysfunction and atherogenesis.
Abstract 577: Medical Marijuana Use: Effect on Lipid Metabolism and Atherosclerosis
Authors: Ponce Orellana Carolina , Roldan Paola , Sibbitt Wilmer , Qualls Clifford , Roldan Carlos
Published: July 2021

Background:
Dyslipidemia and aortic (Ao) and carotid (Ca) atherosclerosis are common in systemic lupus erythematosus (SLE) and are associated with increased morbidity and mortality. Medical use of Marijuana is common in SLE. Marijuana use has been associated with stroke and myocardial infarction in isolated case reports and limited and retrospective series. In contrast, Marijuana in atherogenic animal models has resulted in favorable lipid metabolism and inhibition of atherosclerosis. However, there are limited data on the effect of medical Marijuana on the lipid profile and atherosclerosis in humans.

Methods:
76 SLE patients (69 women, age 38 ± 12 years) underwent laboratory evaluations including lipid profile [total cholesterol, LDL, HDL, and triglycerides levels in mg/dL] and Ca ultrasonography and transesophageal echocardiography for assessment of Ca and Ao atherosclerosis, respectively. Atherosclerosis was defined as intima-media thickening (mean intima-media thickness in 26 healthy controls+2SD) or plaques (as >50% focal intima-media thickening as compared with surrounding walls). Fifteen (20%) patients were regular users of medical Marijuana (≥3 times per week for ≥1 year).

Results:
As shown in Table 1, Marijuana users versus non-users had lower total and LDL cholesterol (both p ≤0.006) and triglycerides (p=0.13). Also, Ao and Ca intima media thickness, intima-media thickening, or plaques were similar in users and non-users.

Conclusions:
Medical Marijuana use in SLE patients may not have a detrimental effect on lipid metabolism or atherogenesis. These findings are preliminary and need confirmation in a larger controlled study.
Abstract 580: Regulation of Inflammatory Gene Expression by Angiotensin II-induced KLF4 in Vascular Smooth Muscle Cells
Authors: Jin Wen , Reddy Marpadga A , Chen Zhuo , Das Sadhan , Lanting Linda , Zhang Lingxiao , Wang Mei , Natarajan Rama
Published: July 2021

Angiotensin II (Ang II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a critical role in the pathogenesis of Cardiovascular Diseases (CVDs). However, the role of Ang II-induced transcription factors in the diverse effects of Ang II remains unclear. We profiled Ang II induced gene expression by microarray analysis of RNA isolated from Ang II-treated and control VSMC. Our results identified numerous differentially regulated genes including several key transcription factors in Ang II-stimulated VSMC compared with controls. Ingenuity Pathway Analysis indicated that Ang II-regulated genes are involved in VSMC dysfunction highly relevant to CVDs. We validated the expression of several genes by RT-qPCR and further characterized the functions of the most differentially regulated gene, KLF4, known to regulate growth factor induced VSMC phenotypic switching. We demonstrated that Ang II induced the expression of KLF4 in cultured VSMC
in vitro
, in mice aortas cultured
ex vivo
, and in aortas isolated from Ang II-infused mice
in vivo
. Ang II-induced KLF4 expression was inhibited by Losartan, demonstrating regulation via the AT1 receptor. Transfection experiments using WT and mutant KLF4 promoter constructs revealed the key role of
cis
-elements with consensus binding sites for p53, SP1 and YY1 in Ang II-induced KLF4 promoter activation. Next, we performed gene expression profiling by Affymetrix gene arrays after siRNA mediated KLF4 knockdown in VSMC. The differentially expressed genes were subsequently analyzed by DAVID to obtain enriched biological processes and potential pathways relevant to cardiovascular functions. Results showed that KLF4 knockdown upregulated the expression of several genes related to cell proliferation and hypertrophy. Interestingly, KLF4 knockdown also enhanced the expression of multiple pro-inflammatory genes including IL-6 and downregulated several anti-inflammatory genes including Thrombomodulin, suggesting an anti-inflammatory role for KLF4 in VSMC. Together, these results suggest that KLF4 may act as a novel molecular brake to modulate Ang II actions that, when disrupted, can further augment Ang II mediated VSMC dysfunction associated with vascular diseases.