bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 337: VE-Cadherin Mechanotransduction Regulates Lung Endothelial Contractility and Barrier Integrity
Authors: Barry Adrienne K , Wang Ning , Leckband Deborah E
Published: July 2021

Dysfunctional regulation of endothelial adherens junctions is the direct underlying cause of vascular leak and pulmonary edema in acute lung injury. Indirect evidence suggests that cytoskeletal rearrangements are linked to adherens junction remodeling, but there is no clear demonstration that adherens junction proteins play an active role in the regulation of intracellular or extracellular mechanical tension. Here I investigate mechanotransduction (response to applied force) of VE-cadherin, the main structural protein at adherens junctions, and how this regulates global cell contractility and actin remodeling both locally and globally. Mechanical responses in human pulmonary artery endothelial cells were studied using magnetic twisting cytometry (MTC) experiments, in which shear stress was exerted on specific cell surface receptors by twisting magnetized beads bound to the cell surface. Remodeling of adherens junctions and F-actin in response to mechanical force was visualized by immunofluorescence. Confocal microscopy revealed force-actuated changes in both local and global cytoskeletal organization, as well as local rearrangements at both bead-cell and cell-cell junctions. This force-dependent remodeling correlated directly with the stiffening of VE-cadherin junctions with increasing applied stress. Treatment with cytoskeletal inhibitors significantly diminished this response. When a constant magnitude of shear stress was applied continuously over time, VE-cadherin junctions increased stiffness, suggesting active junction reinforcement. These data provide direct evidence that VE-cadherin complexes are tension sensors, and that associated mechanotransduction regulates global cell mechanics. Moreover, I present data showing that cadherin adhesions across the cell monolayer form a mechanically coupled network that regulates the endothelial barrier in response to force. This suggests a new mechanism regulating endothelial monolayer integrity.
Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice
Authors: Dayal Sanjana , Gu Sean X , Wilson Katinan M , Hutchins Ryan , Lentz Steven R
Published: July 2021

In vitro
studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis
in vivo
. We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that
Sod1-/-
mice formed stable occlusions significantly faster than
Sod1+/+
mice (P<0.05). In an inferior vena cava (IVC) stasis model
Sod1-
/- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs.
Sod1+/+
mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from
Sod1-/-
and
Sod1+/+
mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in
Sod1-/-
and
Sod1
+/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in
Sod1
-/- mice (P<0.05 vs.
Sod1
+/+ mice). Further,
in vivo
generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in
Sod1-/-
mice (P<0.05
vs. Sod1+/+
mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in
Sod1
-/- mice vs.
Sod1
+/+ mice, suggesting that altered generation of activated protein C in
Sod1-/-
mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.
Abstract 343: Endothelial Regulator of G-Protein Signaling-5 Maintains Endothelial Permeability and Controls Vascular Smooth Muscle Cell Migration in vitro
Authors: Loghmani Khouzani Houra , Hu Niandan , Ginski Greta , Chowdhury Arpita , Schmitto Jan , Martens Andreas , Theilmeier Gregor
Published: July 2021

GAP-inhibitor, RGS5 has been described as a VSMC marker and is associated with plaque stability. Its role in endothelial cells (EC) is unclear. We examined RGS5 expression patterns in human vasculature and hypothesized that loss of RGS5 alters EC biology and crosstalk with VSMC such, that it could promote atherogenesis.

Methods:
Human internal mammary artery (IMA), saphenous vein (SV) and failing myocardium (MYO) samples were stained for RGS5, αSMA and vWF. RGS5 or scrambled siRNA knocked down (RGS5-KD) HUVECs were used. oxLDL or TNFα induced THP1-adhesion to HUVECs was assessed. The 2h diffusion rate of Evans-blue bound to 0.1%-BSA/DPBS through a HUVEC monolayer was spectrophotometrically measured in the presence of PTX. HUVECs were also subjected to spheroid sprouting in collagen/methylcellulose. qRT-PCR using the Δ(ΔCT) method with HPRT as house-keeper was used to assess MMP-9, VEGF-A and caveolin-1 expression in transfected cells. To test RGS5-dependent paracrine effects of EC-secreted factors on VSMC, isotonic buffer was conditioned for 2h by transfected HUVECs (CB). CB was added to VSMC during a scratch assay.

Results:
RGS5 signal was enhanced in the endothelium of SV compared to IMA (63±9 vs 22±4 %vessel perimeter, p<0.05, n=4). Similar patterns were observed in myocardium. In IMA plaques RGS5 was prominently reduced in medial VSMC underlying and in the endothelium covering the plaque compared to non-diseased vessels. No effect on TNFα- or oxLDL-induced adhesion was observed in RGS5-KD HUVECs. HUVEC permeability increased in RGS5-KD cells (1 vs 2.5± 0.2 normalized OD, n=4, p<0.05), which was reduced by PTX (1.4±0.25). MMP-9 (1.0±0.03 vs 2.5±0.54, p<0.05, n=5), VEGF-A (1.0±0.02 vs 2.8±0.76, p<0.05, n=5) and Caveolin-1 (1.0±0.01 vs 1.6±0.17, n=3) expression (2-Δ (ΔCT)) were increased in RGS5-KD HUVECs. RGS5-KD increased the number of spheroid sprouts (2.1± 0.5 vs 3.5± 0.3,n=4, p<0.05). CB of RGS5-KD HUVECs increased VSMC migration (49±2.2 vs 67±2.6 %coverage, p<0.05, n=7).

Conclusion:
In conclusion, RGS5 levels could be involved in vascular differentiation. Its effects on permeability and VSMC migration point towards a prominent role in angiogenesis and atherogenesis. Atherosclerosis studies in RGS5-deficient animals are warranted.
Abstract 359: The T-Cell Negative Regulator, Lymphocyte Activation Gene-3, is Significantly Associated With SCARB1 Intronic Variant rs10846744 and Small HDL Particles
Authors: Golden Diana , Kolmakova Antonina , Rodriguez Annabelle
Published: July 2021

Objective:
We previously reported a significant association of SCARB1 intronic rs10846744 in reducing odds ratio for incident coronary heart disease (CHD) within participants of the Multi-Ethnic Study of Atherosclerosis (MESA), which was independent of lipid levels. We have now explored the underlying molecular mechanism for this clinical association.

Methods and Results:
Adults between 18-80 years of age with fasting HDL-C levels ≥ 60 mg/dl (HALP) donated fasting blood for rs10846744 genotyping, lipid/lipoprotein measurements, and EBV-B cell lymphocyte culture. Using RNA Seq, we found that lymphocytes isolated from HALP carriers homozygous for the risk C allele had significantly lower RNA expression of MHC class II (82-fold lower, p<0.05) and lymphocyte activation gene-3 (LAG3: a negative regulator of T cell activation) (5-fold lower, p<0.05) compared with carriers homozygous for the reference G allele. We next measured plasma soluble LAG3 (sLAG3) and observed that sLAG3 was significantly lower in carriers of the C allele (1722 ± 2983 pg/ml, 6-fold lower, p=0.009) compared with carriers of the G allele (11926 ± 1134). Given that traditional lipid levels did not attenuate the association of rs10846744 with CHD, we next determined whether sLAG3 associated with HDL subfractions as measured by NMR spectroscopy and 2D gel electrophoresis. As compared with GG carriers, CC carriers had significantly higher concentration of small HDL particles by NMR (24.8 ± 1.7 vs. 17.9 ± 0.8 μmol/L, p=0.0009, n=101) and by 2D gel electrophoresis (4.3 ± 0.5 vs. 2.0 ± 0.2, p=0.0002, n=63). For the entire cohort, sLAG2 was negatively associated with small HDL particles by NMR (p=0.02, n=98) and 2D gel electrophoresis (prebeta 2: p=0.04, n=59).

Conclusion:
This is the first observation that the SCARB1 intronic rs10846744 significantly associates with LAG3, and both are associated with higher concentrations of small HDL particles known to be linked to higher risk of CHD.
Abstract 368: Serum Matrix Metalloproteinases 1, 2 and 9, Tissue Inhibitor of Metalloproteinases-1 and Inflammatory Markers in Patients with Aortic Valve Sclerosis
Authors: Cossio-Aranda Jorge E , Springall Rashidi , Trevethan-Cravioto Sergio , Espinola-Zavaleta Nilda , Bojalil Rafael , Vallejo-Venegas Enrique , Verdejo-Paris Juan
Published: July 2021

Background:
Valvular Aortic Sclerosis (AVS) and Calcified Degenerative Valvular Aortic Stenosis (AS) are important risk factors for cardiovascular events. Matrix Metalloproteinases (MMPs) and other inflammatory markers might play an important role in the mechanisms underlying endothelial dysfunction described in AS. The participation of MMPs in AVS is poorly studied as of yet. We evaluated the concentrations of MMPs 1,2 and 9 and tissue-1 inhibitor (TIMP-1) and inflammatory markers in AVS patients.

Methods:
The research and the ethics committees of our institution approved the study. Samples were obtained from peripheral blood in a cohort of 107 subjects: 30 healthy blood donor controls, 47 with AS and 30 AVS. Serum concentrations of Interleukins (ILs) 1 and 6, Tumor Necrosis Factor (TNF-α) and MMPs 1, 2 and 9 and TIMP-1 were measured by quantitative immunoassay technique with commercial kits, while concentrations of C Reactive Protein (CRP) by nephelometry. In all patients endothelial function and carotid plaque, 256 slice coronary computed tomography and coronary calcium score were evaluated. A one way ANOVA nonparametric test (Kruskall-Wallis), Kaplan Meier and multivariate Cox regression model were performed.

Results:
Serum concentrations of CRP, ILs 6 and 10, MMPs-1, 2, 9 and TIMP-1 were higher in AVS and AS patients than in healthy controls (p<0.001), while no differences between the two patients groups were found. However, the MMP-9/TIMP-1 ratio was higher in patients with AVS than the rest of the groups (p<0.05). Endothelial dysfunction (73.9%), carotid plaque (34.2%), coronary plaque (39.7%) and coronary calcium score >400 (14%) was similar in both patients groups. During follow-up of 19.96±14.51 months, 50.7% of AVS and AS patients developed cardiovascular events. Type 2 diabetes was the stronger predictors for cardiovascular events in ASV (RR= 2.67, IC95% de 1.027 - 6.926).

Conclusions:
Our findings suggest an important inflammatory role of MMPs, TIMPs and cytokines in AVS disease, which could explain a higher prevalence of coronary artery disease in this patients.
Abstract 371: TNFα Drives Endothelial Dysfunction and Oxidative Stress via eNOS Uncoupling in Aortic Valve Disease
Authors: Farrar Emily J , Huntley Geoffrey , Pramil Varsha , Butcher Jonathan T
Published: July 2021

Objective:
Oxidative stress (OxS) is known to contribute to late stages of aortic valve disease (AVD), but its role in early-stage valve inflammation and as a driver of valve pathology has not been investigated. Here we show that inflammation drives increased OxS via eNOS uncoupling. Using an ex vivo model of AVD, we show that targeting eNOS uncoupling with tetrahydrobiopterin or superoxide dismutase can mitigate later-stage valve degeneration by blocking the downstream effects of eNOS uncoupling.

Methods and Results:
We first observed evidence of eNOS uncoupling in the endothelium of calcified human aortic valve leaflets, which showed increased superoxide co-localized with cells positive for CD31 and VCAM-1, suggesting a connection between endothelial inflammatory activation and increased oxidative stress. We then showed that inflammatory cytokine TNF-α caused increased superoxide, free radicals, and hydrogen peroxide in aortic valve endothelial cells, as soon as 15 minutes after treatment. TNF-α did not cause increased oxidative stress in endothelial cells from the aorta, pointing to a valve-specific mechanism. TNF-α-driven oxidative stress decreased valve endothelial cell nitric oxide secretion, lowered eNOS and VE-cadherin protein levels, and increased expression of inflammatory adhesion molecules (VCAM1, ICAM1). Addition of eNOS co-factor tetrahydrobiopterin (BH4) mitigated the increases in OxS caused by TNF-α, demonstrating that eNOS uncoupling was the primary pathophysiological mechanism at play. Ex vivo, we used porcine aortic valve leaflets to screen the ability of BH4, catalase, and superoxide dismutase to protect against the downstream effects of eNOS uncoupling. We found that all three antioxidants mitigated increases in valve OxS, improved endothelial function, and protected against extracellular matrix remodeling. TNF-α+SOD also protected against increases in myofibroblastic protein α-smooth muscle actin and chronic elevation of VCAM1.

Conclusions:
These results present endothelial inflammatory oxidative stress as a new mechanism that connects early valve pathology with later stages of degeneration. Targeting these mechanisms via tailored antioxidant therapy could provide new avenues for treatment of CAVD.
Abstract 392: Warfarin Versus Aspirin in Patients with Systolic Heart Failure and Normal Sinus Rhythm: Insights From a Meta-Analysis
Authors: Shah Rachit , Mueller George , Patel Dhavalkumar , Molnar Janos , Patel Kalpesh , Jovin Ion
Published: July 2021

Background:
It is unknown whether warfarin or aspirin therapy is superior for the treatment of patients with systolic heart failure who are in sinus rhythm.

Methods:
We performed a systematic literature search for randomized trials comparing warfarin and aspirin in patients with systolic heart failure which provided the event rates for ischemic stroke, major hemorrhage and death in the two groups. Heterogeneity of the studies was analyzed by Q statistics. The studies were homogeneous for each outcome; therefore the fixed-effect model was used to compute the relative risk based on the number of events and total number of patients in each group. A two-sided alpha error of <0.05 was considered to be statistically significant (p<0.05).

Results:
We found 4 randomized clinical trials comparing warfarin and aspirin therapy in patients with systolic heart failure with a mean duration of follow up of 2.3 years enrolling a total of 3663 patients. The relative risk for ischemic stroke in patients treated with warfarin was 0.50 with 95% confidence interval (CI) of 0.33 - 0.75 (P= 0.001) while the relative risk for major hemorrhage was 1.94 with 95% CI of 1.40- 2.71 (P= 0.000) in comparison to the aspirin group. The relative risk of death was 1.01 with 95% CI of 0.89- 1.14 (P= 0.871) in the warfarin group compared to the aspirin group.

Conclusion:
Although warfarin therapy appears to reduce the risk of ischemic stroke in patients with systolic heart failure who are in sinus rhythm, the reduction comes at the cost of higher risk of bleeding and there is no evidence of an overall benefit on mortality.
Abstract 417: Structural Characterization of 20 kDa Fragments of Apolipoprotein B100
Authors: Reda Alexandra M , Tremblay Marie L , Rainey Jan K , McLeod Roger S
Published: July 2021

Apolipoprotein (apo) B100 is a 550 kDa hydrophobic glycoprotein that forms the structural backbone for the assembly of triglyceride-rich lipoproteins. ApoB100 circulates bound to the same lipoprotein particle on which it is synthesized (i.e. it is non-exchangeable) until the lipoprotein is cleared from the plasma via receptor-mediated uptake. The polypeptide length and aqueous insolubility are considerable obstacles to the determination of apoB100 structure. Based on primary structure algortihms, apoB100 is predicted to contain 5 domains, comprising an N-terminal globular mixed βα1 domain followed by alternating amphipathic β-strand and α-helix rich regions (N-βα1-β1-α2-β2-α3-C). Previous studies from our laboratory have shown that amino acids 1694-1880 (B1) of the β1 domain of apoB100 bind tightly to triglyceride droplets in vitro. We have expressed this 20 kDa protein fragment, and a second 20 kDa fragment B2 (amino acids 1881-2070) that lies at the junction between the β1 and α2 domains, in bacterial cell culture as hexahistidine-tagged fusion proteins. B1 was insoluble in aqueous solution, but soluble in the presence of 150 mM dodecylphosphocholine (DPC), whereas B2 was soluble in aqueous solution, with or without DPC. Circular dichroism (CD) spectroscopy of DPC-solubilized B1 and B2 fragments indicated an approximately equal proportion of α-helix, β-strand/β-turn and unordered structures in B1 (36%, 37% and 28%, respectively) vs. approximately 56% α-helix, 27% β-strand/β-turn and 17% unordered content for B2. Preliminary NMR spectroscopy of 13C/15N labeled B1 has been conducted. Estimates of protein secondary structure from backbone chemical shift assignments using the algorithm DANGLE (Dihedral ANgles from Global Likelihood Estimates) suggested significant α-helical character in B1. These studies suggest that the α-helical content may be underestimated by CD, and generation of a high-resolution structure of B1 is in progress. Our studies have provided the first atomic-level structural information for structural elements of apoB100 and suggest a central role for unique amphipathic structures in lipoprotein structure and function.
Abstract 421: TREM-Like Transcript 1 and Hyperlipidemia: A New Pandora's Box
Authors: Gonzalez Marieli , Morán Carlos , Vélez Javier J , Collado Carlos J , Cruz Nildris , Quidley José , Crespo María J , Chorna Natalia , Washington Anthony V
Published: July 2021

While it is known that high blood cholesterol levels are associated with increased risk and poor outcomes during cardiovascular disease (CVD), all of the mechanisms that regulate cholesterol levels remain unclear. Recent studies on the Triggering Receptor Expressed in Myeloid (TREM) like transcript (TLT)-1 null mice demonstrate changes in cholesterol regulation. TLT-1 was identified as a platelet specific immunoreceptor prompting our studies on TLT-1’s potential involvement in CVD. We crossed our treml1-/- mice on to the apoe-/- background and placed them on high fat diet (HFD). To our surprise, the treml1-/- /apoe-/- (DKO) mice had total cholesterol levels 1.5 fold higher compared to apoE controls. These changes can be seen in treml1-/- mice that are not on the apoE background as well. There are no cholesterol differences in mice not on HFD, demonstrating that these changes are diet-induced. These differences led us to investigate, “why the treml1-/- mouse has higher cholesterol?” Serum chemistry panels reveal high triglycerides, increased glucose levels, and potential liver damage in the treml1-/- mice. Histological evaluation of liver sections revealed large lipid-filled vacuoles and ballooning of DKO and treml1-/- hepatocytes suggesting direct liver involvement in regulating the aforementioned cholesterol levels. What brings these findings together is that we have identified an alternate transcript of TLT-1, TLT-1s, associated with mitochondrial fractions of hepatocytes. Immunohistochemistry of liver sections demonstrate a unique TLT-1s expression in the perivascular hepatocytes of the central vein. Preliminary metabolic analysis of mice livers confirm the hyperlipidemia and demonstrate significant changes in protein metabolism as well as Krebs cycle intermediates. Interestingly, treml1-/- mice have smaller lesions in the aortic sinus which may be explained by less reactive treml1-/- platelets (Gonzalez et al.). Our data suggests that treml1 gene products may affect energy metabolism and mutations in TLT-1 may affect energy use in platelets. This uncanny hypothesis potentially explains how we could have high cholesterol levels, with smaller atherosclerotic lesions and less reactive platelets in the face of hyperlipidemia.
Abstract 435: Brown Adipose Tissue Activation in the Postprandial State Reflects on Plasma Lipoproteins and Immune Cell Response in Humans
Authors: Mellerowicz Alexandra , Ericsson Madelene , Hedlund Mattias , Nilsson Stefan K
Published: July 2021

Background:
Brown adipose tissue (BAT) has a unique to ability to use excess energy for heat production. It is therefore an attractive target organ for counteracting obesity and related metabolic diseases where overfeeding is an underlying cause. BAT has in murine models been shown to clear postprandial lipids quickly. The postprandial response is associated to systemic inflammatory alterations and an increased lipid pressure possibly driving atherosclerosis development. We hypothesized that BAT activation would affect postprandial lipid clearance and that this would reflect in an altered immune cell response.

Methods:
Young male volunteers were subject to an oral fat tolerance test at two separate occasions during both cold stimulation and in thermoneutral control conditions. Body temperature and EMG activity was monitored and energy expenditure (EE) was measured. Blood samples were taken at baseline and every 30 min for 2 h. Plasma lipids and the immune cell response.

Results:
Cold stimulation during OFTT resulted in a 19,4 % higher EE compared to warm conditions (P=0,007). Surprisingly, no changes in plasma TG were observed. A 2-fold elevation in free fatty acids (FFA) was seen in cold which also correlated positively with EE (P=0,008). Total plasma cholesterol increased compared to warm conditions by 0,56 mmol/L (P=0,050). LDL-c and HDL-c were increased in cold (0,20 mmol/L difference P=0,048 and 0,16 mmol/L P=0,002) whereas remnant-c was unaltered between the two thermal conditions. White blood cell count (WBC) after OFTT was significantly increased in cold (P = 0,018) by 0,29 х 109/L.

Discussion:
BAT activation in the postprandial state results in increased HDL-c, possibly indicating increased vascular lipolysis and associated pre-β HDL particle formation. Increased VLDL production due to elevated FFA levels in the cold state and might explain why plasma TG is unaltered and also why LDL-c remains at a higher concentration in the cold.

Conclusions:
BAT might be an attractive target for obesity treatment but potentially displays pro-atherogenic properties that must be addressed in longitudinal studies.
Abstract 444: Angiotensinogen Inhibition Reduces Atherosclerosis and Body Weight Gain Induced by High-Carbohydrate Diet
Authors: Balakrishnan Anju , Howatt Deborah A , Wu Congqing , Mullick Adam E , Graham Mark J , Crooke Rosanne M , Cassis Lisa A , Lu Hong , Daugherty Alan
Published: July 2021

Background and Objective:
Angiotensinogen (AGT) is the unique precursor of the renin angiotensin system. Our previous studies demonstrated that inhibition of AGT markedly reduced development of atherosclerosis and ablated body weight gain induced by a saturated fat-enriched diet. In addition to high fat intake, high carbohydrate consumption is an important component of contemporary diets. The purpose of this study was to determine whether AGT inhibition prevented atherosclerosis and obesity in LDL receptor -/- mice fed a high carbohydrate diet.

Methods and Results:
Eight week old male LDL receptor -/- mice were randomized to receive either control or AGT antisense oligonucleotides (ASO; 50 mg/kg/week intraperitoneal injection; N = 10 mice/group). Feeding a high carbohydrate diet was initiated after 6 weeks of ASO injection, and maintained for 12 weeks with continuous ASO injection. AGT-ASO administration profoundly reduced plasma AGT concentrations (Control vs AGT ASO: 3582 ± 117 vs 227 ± 15 ng/ml; P<0.001). High carbohydrate diet feeding resulted in profound increases of plasma cholesterol concentrations in both groups. Atherosclerotic lesions in aortic arches were measured using an en face method after termination. AGT inhibition led to significant decreases (P<0.001) in percentage lesion areas of aortic arches. Prior to high carbohydrate diet feeding, there was no difference of body weight between the two groups (Control vs AGT ASO: 26.6 ± 0.6 vs 25.9 ± 0.7 g; P=0.4). Although high carbohydrate diet only increased body weight modestly, mice injected with AGT ASO had less increases of body weight compared to mice injected with control ASO (Control vs AGT ASO: 28.5 ± 0.6 vs 26.8 ± 0.6 g; P<0.05). Echo MRI analyses demonstrated that the lower body weight in mice administered AGT ASO was attributed to less fat mass gain, whereas lean mass was comparable between the two groups. Additionally, AGT inhibition resulted in lower blood hemoglobin A1c (5.5 ± 0.1 vs 5.1 ± 0.1%; P=0.005).

Conclusions:
AGT inhibition reduces high carbohydrate diet-induced atherosclerosis and body weight gain.
Abstract 457: Sulforaphane at Physiological Concentrations Inhibits TNF-α-Induced Monocyte Adhesion to Human Vascular Endothelial Cells and Improves Vascular Inflammation in Mice Through a Nuclear Factor-κB--Mediated Mechanism
Authors: Nallasamy Palanisamy , Anandh Babu Pon Velayutham , Shah Halley , Brooke Elizabeth A S , Zhu Hong , Zhen Wei , Liu Dongmin , Li Yunbo , Jia Zhenquan
Published: July 2021

Introduction:
TNF-α, plays an important role in endothelial dysfunction and is involved in the pathogenesis of atherosclerosis.

Aim:
We investigated the protective effect of cruciferous vegetable phytochemical sulforaphane at physiological concentrations on TNF-α-induced vascular inflammation.

Methods:
Human umbilical vascular endothelial cells (HUVEC) were pretreated with sulforaphane (0.5 - 8 μM) before addition of TNF-α for 6 h. Cell adhesion was measured with calcein-AM labeled human monocytes. In animal study, ten weeks old male C57BL/6 mice were fed a diet containing 0 or 300 ppm sulforaphane for 2 weeks followed by TNF-α administration. The chemokines and adhesion molecules in the serum were measured using ELISA Kit. VCAM-1 and F4/80 expressions in mice aorta were performed using immunohistochemistry.

Results:
Sulforaphane at physiological concentrations (0.5 - 2 μM) significantly inhibited TNF-α-induced adhesion of monocytes to HUVECs and suppressed TNF-α-induced production of monocyte chemoattractant protein -1 (MCP-1) and interleukin-8 (IL-8). Furthermore, sulforaphane inhibited TNF-α-induced NF-κB transcriptional activity, IκBα degradation and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that sulforaphane can inhibit endothelial inflammation by suppressing NF-κB signaling. In animal study, dietary sulforaphane (300 ppm in the diet) significantly suppressed TNF-α-induced increase in circulating chemokines and adhesion molecules in C57BL/6 mice. Sulforaphane treatment also reduced the presence of VCAM-1 and monocytes-derived F4/80-positive macrophages in the aorta of TNF-α treated mice. Histology showed that sulforaphane treatment significantly prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers' delicate organization as shown by Verhoeff-van Gieson staining.

Conclusion:
sulforaphane at physiological concentrations protects against TNF-α-induced vascular endothelial inflammation, in both in vitro and in vivo models. This anti-inflammatory effect of sulforaphane may be, at least in part, associated with interfering with the NF-κB pathway.
Abstract 46: Differential Gene and Transcription Factor Binding Site Expression Between the Intima and Media Drives Intimal Hyperplasia
Authors: DeSart Kenneth , Jiang Zhihua , Tao Ming , O'Malley Kerri , Berceli Scott
Published: July 2021

Background:
Vein grafts (VG) fail due to rapid intimal expansion in the weeks to months following implantation. Histologically, this results in a metabolically active intima juxtaposed to a quiescent medial layer. In this study, we leverage this dichotomy to identify the key genes that control intimal growth and the critical transcription factors (TF) that drive their activation.

Methods:
Microsurgical techniques were used to separate the intima and media of rabbit carotid VGs harvested at 1 (n=4), 3 (n=6), and 6 (n=4) months (m) following implantation. Each layer was independently analyzed for differential mRNA expression using a whole-genome microarray. rVista and TRANSFAC were used to identify TF binding sites within 5000 bp of differentially expressed genes.

Results:
Early growth of the intima was driven by an enhanced proliferative response and an accumulation of cell mass (Fig). During this early hyperplastic phase, substantial differences in intimal and medial gene expression were observed (197 genes at 1m and 346 at 3m, p<0.001) that resolved by 6m (8 genes). Genes involved in cell cycle regulation and cellular movement dominated the 1 and 3m differences (Fig). TF binding site analysis of up-regulated intimal genes at 1 month revealed enrichment of ETS and HOX, while CREB and bHLH-ZIP were the most over-represented among up-regulated 3m intimal genes. In the medial layer, RXR and zinc finger TF binding sites were most prominent among up-regulated genes at 1 and 3m, respectively.

Conclusion:
Corresponding to the most active phase of intimal growth, smooth muscle cells demonstrated marked up-regulation of an array of genes that modulate cell proliferation and migration, with these genes under the control of a discrete set of TFs. While previous attempts at modifying TF binding (via an E2F decoy) in the initial days following VG implantation have been unsuccessful, manipulation of a broader set of TFs in the time frame of weeks to months may prove a more promising strategy.
Abstract 465: Deciphering the Genetic Regulation of
ADAMTS7
, a Novel Locus for Coronary Artery Disease
Authors: Ou Kristy , Bauer Robert C , Zhang Xuan , Cui Jian , Rader Daniel J , Reilly Muredach P
Published: July 2021

Recent genome-wide association studies (GWAS) have identified an association between the
ADAMTS7
locus and coronary artery disease (CAD) in humans. While ADAMTS7 is proposed to play a role in vascular smooth muscle cell (VSMC) migration and neointimal formation, the molecular regulation of human
ADAMTS7
gene expression has not yet been explored. We assessed
ADAMTS7
expression levels in primary mouse aortic smooth muscle cells (mAoSMC) as well as primary human coronary and human pulmonary artery smooth muscle cells (hCASMC, hPASMC) in response to treatment with various stimulatory agents. No differences in
ADAMTS7
expression were observed upon treatment with PDGF, angiotensin II, and nicotine in any of the cell lines tested. However, TNFα upregulated
ADAMTS7
by 4-fold in mAoSMC but not in human VSMCs while H
2
O
2
upregulated
ADAMTS7
by 4-fold in hCASMC but not mAoSMCs. No agents modulated expression in hPASMC. Basal levels of
ADAMTS7
varied among different VSMC lines; hCASMC had 2-fold greater levels of
ADAMTS7
when compared to hPASMC and hAoSMC. These data demonstrate that
ADAMTS7
is differentially regulated not only across different species, but also among different VSMC types, underscoring the complex genetic regulation of
ADAMTS7
. In an attempt to elucidate important regulatory genomic regions controlling
ADAMTS7
expression, we utilized data from the ENCODE project to identify regions surrounding
ADAMTS7
that are enriched for regulatory domains, e.g. DNase hypsersensitivity (DHS) and H3K27 acetylation, in relevant cell types. We then overlayed data from CARDIoGRAM and C4D CAD GWAS to look for CAD-associated SNPs in these potential enhancers. Ultimately we selected 5 regions of interest (e.g., rs5029904 lies in region with increased H3K27 acetylation in ENCODE layered 7 cell lines, DHS peaks in serum fed and starved hAoSMCs, binding peaks for transcription factors via ENCODE ChIP-Seq experiments) for further studies in luciferase reporter assays multiple cell types to look for tissue specific expression mediated by these genomic regions. GWAS variants in these domains may affect the transcriptional regulation of
ADAMTS7
, thus providing insight into the role for ADAMTS7 in human atherosclerosis.
Abstract 481: Downregulation of MicroRNA-143 Induces Proximal Aorta Dilatation in C57BL6/J Mice
Authors: Branchetti Emanuela , Rylski Bartosz , Poggio Paolo , Lai Eric K , Gorman Robert C , Gorman Joseph H , Bavaria Joseph E , Ferrari Giovanni
Published: July 2021

Background:
Thoracic Aortic Aneurysm (TAA) is a silent life-threatening condition which leads to death if not surgically treated. TAA development is secondary to the remodeling of the aortic wall structure due to pathological changes in both vascular smooth muscle cells (VSMC) and extracellular components. The phenotypic switch of VSMC from a contractile to a pathological synthetic state by inducing excessive production of matrix components is associated with the aberrant remodeling of the aorta. MicroRNA (miR)-143 and -145 are critical in controlling VSMC phenotype and are highly expressed in the aorta. In investigating the molecular mechanisms controlling VSMC phenotype we found that accumulation of reactive oxygen species (ROS) linearly correlates with synthetic VSMC accumulation as well as dilatation of the ascending aorta in humans. Furthermore ROS contribute to the onset of TAA in a murine model based on Angiotensin II (AngII) infusion. Interestingly we found that miR-143 is down regulated in patients with aneurysmal aorta. We hypothesized that miR-143 may be regulated by ROS and its down regulation could contribute to TAA development.

Methods:
MiR expression, MMP-2, MMP-9, and VSMC markers of phenotype (Vimentin, Elk-1, Myocardin) were tested in human isolated aortic VSMC treated with ROS. C57BL6/J mice were subjected to AngII for 28 days in the presence or absence of LNA-anti-miR-143. Two-dimensional M Mode echocardiography was performed to measure aortic diameter.

Results:
ROS treatment of human VSMC induces a dose dependent down regulation of miR-143 and -145 together with up regulation of MMP-2, MMP-9 and VSMC synthetic markers Vimentin and Elk-1. Both miR-143/145 are down regulated in the aneurysmal proximal aorta of C57BL6/J mice treated with AngII. Injection of LNA-anti-miR-143 alone induces dilatation of the proximal aorta but has no effect on AngII induced TAA development.

Conclusion:
ROS induced down regulation of miR-143 is involved in the pathological remodeling of the proximal aorta leading to TAA. Understanding the mechanism behind ROS-mediated regulation of miR-143 may unveil new targets for the development of therapeutic strategies against TAA.
Abstract 515: Effects of the Glucagon-Like Peptide-1 Analog Exendin-4 on Reendothelialization and Intimal Hyperplasia Formation in an Animal Model of Vascular Injury
Authors: Eriksson Linnea , Saxelin Robert , Röhl Samuel , Roy Joy , Caidahl Kenneth , Nyström Thomas , Hedin Ulf , Sjöholm Åke , Razuvaev Anton
Published: July 2021

Background and aims:
Diabetic patients have an increased risk of late stent thrombosis formation and restenosis, after angioplasty. It is hypothesized that delayed re-endothelization is a major underlying problem for late-stent thrombosis and also that rapid re-endothelization is essential for preventing restenosis. Exendin-4 is a stable GLP-1 receptor agonist, which is applied in clinical treatment of diabetes. Beside its insulinotropic action, it may also exert direct beneficial effects on endothelial function. We previously reported that exendin-4 is able to activate the GLP-1 receptor on human coronary artery endothelial cells, leading to increased cell proliferation and decreased apoptosis. Therefore, our aim was to study if exendin-4 can influence endothelialization and decrease neointima formation after vascular injury.

Materials and Methods:
Balloon injury of the left common carotid artery (LCCA) was performed on Sprague-Dawley rats. Rats were randomized into two groups and treated for four weeks with exendin-4 (1 nmol/day) or saline. Intimal hyperplasia and endothelialization was monitored non-invasively by high frequency ultrasound and upon sacrifice with Evans blue, respectively. The LCCA was then sectioned for subsequent morphometric and immunohistochemical analyses. To further investigate if and how smooth muscle cells (SMCs) are directly affected by exendin-4 treatment, we studied proliferation and apoptosis of SMCs
in vitro
.

Results and conclusions:
Our findings show that exendin-4 selectively reduces SMC proliferation and intimal hyperplasia through activation of cAMP signaling and endothelial nitric oxide synthase without influencing re-endothelialization. Treatment with exendin-4 did however improve arterial wall elasticity. Together, these effects of exendin-4 are highly desirable and may lead to improved outcome for patients undergoing vascular interventions
Abstract 536: RNA-Seq Identifies Differential Expression of Platelet Transcripts Between People With Hyper- and Hypoaggregation to Collagen
Authors: Qayyum Rehan , Faraday Nauder , Becker Diane M , Kral Brian G , Becker Lewis C
Published: July 2021

Background:
Platelet aggregation plays an important role in the pathogenesis of myocardial infarction, and high platelet aggregability is associated with increased risk of events. We hypothesized that inter-individual variability in platelet function is associated with differences in the mRNA transcripts found in platelets from hyper-aggregators and hypo-aggregators.

Methods:
Eight African American (discovery cohort) and four European American (validation cohort) males with family history of early onset coronary artery disease were enrolled. Individuals were divided into hyper- and hypo-aggregators based on maximal aggregation to collagen using whole blood impedance aggregometry. RNA was extracted from leukocyte-depleted platelet-rich plasma and approximately 70 million 100bp paired-end reads per sample were obtained using an Illumina HiSeq 2500. RNA-seq analysis was performed using Bowtie/TopHat/Cufflinks software pipeline. Genes with expression ≥ 1 fragments per kilobase of transcript per million reads (FPKM) were considered to be expressed. On log2 scale, ≥ 2-fold (discovery cohort) and ≥ 1-fold (replication cohort) change in gene expression were considered to be significantly differentially expressed.

Results:
Among the 9,373 expressed genes, the most highly expressed nonubiquitous genes included GP1BB, GP9, PPBP, PF4, & TUBB1, confirming our methods. Hyper-aggregability was associated with up-regulation of 239 genes and down-regulation of 29 genes in the discovery cohort (44 and 2 replicated in validation cohort respectively). Pathway analysis found that genes related to sphingosine-1P3 (such as RHOA [ras homolog family member A], GNAZ [G protein, alpha z polypeptide], VEGFA [vascular endothelial growth factor A]) and platelet aggregation pathways (such as GP9 [platelet glycoprotein IX], TLN1 [talin 1], ITGB3 [platelet glycoprotein IIIa]) were up-regulated in hyper-aggregators.

Conclusion:
RNA-seq and pathway analyses found that hyper- and hypo-aggregators have significant differences in gene expression. Increased expression of sphingosine-1P3 and platelet aggregation pathway genes in hyper-aggregators highlights the relationship of signal transduction with increased platelet aggregation.
Abstract 543: Regulation of Atherosclerosis by Myeloid-Derived Suppressor Cells
Authors: Foks Amanda C , van Puijvelde Gijs H , Frodermann Vanessa , van der Heiden Thomas , Bot Ilze , Kuiper Johan
Published: July 2021

Objective:
Restoration of immune homeostasis in atherosclerosis represents the ultimate goal of an immune-based therapy. Myeloid-derived suppressor cells (MDSCs) are a population of immature myeloid cells that potently suppress immune responses in various pathological settings, via multiple mechanisms, including inhibition of T cell responses. They express the myeloid markers CD11b and Gr-1 and can accumulate in various lymphoid and non-lymphoid tissues. In the present study, we determined the role of MDSCs in atherosclerosis by an adoptive transfer of CD11b
+
Gr-1
+
cells into LDLr
-/-
mice fed a Western-type diet.

Methods and Results:
We isolated CD11b
+
Gr-1
+
cells from the bone marrow of LDLr
-/-
mice fed a Western-type diet for 2 weeks with magnetic beads and found that they strongly suppressed αCD3/CD28-induced splenocyte proliferation in an IFN-γ and iNOS-dependent manner. Subsequently, we adoptively transferred MDSCs into LDLr
-/-
mice fed a Western-type diet for 6 weeks, which resulted in a 35% reduction in atherosclerotic lesion formation in the aortic root. MDSC treatment reduced splenic Th1 and Th17 cells with 50% and diminished B cells, in particular circulating B2 cells, and concomitantly impaired their proliferative capacity.

Conclusions:
Our data prove that MDSCs could represent a novel cell-based immune-therapy to dampen pro-atherogenic immune responses and thereby reduce atherosclerosis.
Abstract 60: B Cell--Specific Deletion of Id3 is Atheroprotective and Enhances B1b B Cell Numbers and Antibody Production
Authors: Morris-Rosenfeld Sam , Perry Heather M , McNamara Coleen A
Published: July 2021

Introduction:
B cells have been shown to have subset specific functions in atherosclerosis. B1a B cells, which contribute to innate immunity, are atheroprotective due to the spontaneous generation of protective natural antibodies. B1b B cells, the sister population to B1a B cells, also produce natural antibodies and unlike B1a B cells, have memory and inducible properties. B1b B cells have not been studied in atherosclerosis. Our lab has demonstrated that the helix-loop-helix transcription factor Id3 is important for B cell mediated atheroprotection. However, how Id3 in B cells regulates atheroprotection is incompletely understood.

Hypothesis:
Loss of Id3 specifically in B cells regulates an atheroprotective subset.

Methods:
To determine the impact of loss of Id3 specifically in B cells, we generated B cell specific Id3 knockout mice on the
Apoe
-/-
background (Id3
fl/fl
Apoe
-/-
CD19
cre/+
). To measure atherosclerosis, 8 week old male Id3
fl/fl
Apoe
-/-
CD19
cre/+
mice were fed Western diet for 16 weeks (n=7). These mice developed significantly less atherosclerosis in the aortic sinus compared to litter matched wildtype controls (Id3
fl/fl
Apoe
-/-
CD19
+/+
, n=10) suggesting loss of Id3 in B cells was atheroprotective. Additionally, immunohistochemistry of atherosclerotic plaques demonstrated decreased intraplaque macrophage accumulation and fewer apoptotic bodies compared to wildtype. Immunophenotyping of Id3
fl/fl
Apoe
-/-
CD19
cre/+
mice by flow cytometry revealed a specific increase of B1b B cells in the peritoneal cavity, spleen, and in circulation compared to wildtype (n=12-19). Additionally, 8 week old Id3
fl/fl
Apoe
-/-
CD19
cre/+
mice (n=8) were shown by ELISA to have significantly greater circulating IgM and, after 16 weeks of Western diet feeding (n=10), significantly greater circulating natural antibodies compared to wildtype (n= 9 & 11 respectively).

Conclusion:
B cell-specific loss of Id3 enhances the B1b B cell pool, natural antibody production, and atheroprotection suggesting B1b B cells are important in defending against atherosclerosis in an antibody dependent manner.
Abstract 612: Effect of Bariatric Surgery on HDL-Associated MicroRNAs
Authors: Tabet Fatiha , Vickers Kasey C , Cuesta Torres Luisa F , Ammori Basil , Yadav Rahul , Hama Salam , Liu Yifen , Schofield Jonathan , Barter Philip , Soran Handrean , Rye Kerry-Anne
Published: July 2021

Background and aims:
We have recently demonstrated that high-density lipoproteins (HDL) transport microRNAs (miRNAs) in the plasma. Here we aim to investigate how HDL-associated miRNAs are regulated in morbidly obese subjects and more importantly, how weight loss after bariatric surgery affected HDL-miRNAs in these subjects.

Methods:
Plasma HDL were isolated from morbidly obese subjects (n=23) before, 6 months and 12 months after bariatric surgery by immunoprecipitation using goat anti-human apoA-I microbeads. We also isolated HDL from 12 normal subjects using the same methods. miRNAs were extracted from the isolated HDL and miR-223, miR-24 and miR-126 levels were determined by TaqMan miRNA assays.

Results:
miR-223, miR-24 and miR-126 were present on HDL from obese subjects before and after bariatric surgery. Before surgery, levels of miR-223, miR-24 and miR-126 on HDL from morbidly obese subjects were significantly higher than the levels on HDL from normal subjects. At 12 months after bariatric surgery, miR-223, miR-24 and miR-126 levels on HDL from obese subjects were increased by 2.9-fold, 2.3-fold and 2.7-fold (p<0.05 all), respectively, compared to before surgery.

Conclusion:
HDL-associated miRNAs are differentially regulated in healthy versus morbidly obese subjects. miR-223, miR-24 and miR-126 are sensitive to weight loss induced by bariatric surgery.
Abstract 639: CD36 Recruits Na/K-ATPase/Lyn Complex to Mediate Proatherogenic Phenotypes in Macrophages
Authors: Chen Yiliang , Kennedy David , Ramakrishnan Devi Prasadh , Wenxin Huang , Li Zhichuan , Xie Zijian , Siverstein Roy L
Published: July 2021

Atherosclerosis is characterized by accumulation of macrophage foam cells in the arterial wall. We previously showed that CD36, a scavenger receptor highly expressed in macrophages, mediates oxidized-LDL (oxLDL) uptake, contributes to intracellular cholesterol accumulation and foam cell formation, and regulates macrophage migration and pro-inflammatory signaling. Genetic deletion of cd36 in mice is protective against diet-induced atherosclerosis. Mechanistically, we discovered that binding of oxLDL to CD36 activates Lyn kinase and initiates a cascade that is necessary for the pro-atherogenic cellular phenotype. Nevertheless, how CD36 regulates Lyn kinase remains undefined. We previously showed that Na/K-ATPase (NKA) regulates Src family kinases, including Lyn and we now hypothesized that CD36 regulates Lyn kinase via an interaction with NKA. We used co-immunoprecipitation and a novel immunofluorescence-based cell surface cross linking assay to demonstrate that CD36 physically associates with NKA on the macrophage surface. In a NKA α1 subunit heterozygous null mouse model in which ~60% of macrophage NKA expression is downregulated, we demonstrated that recruitment and activation of Lyn and its downstream signaling events in response to oxLDL were abolished. Functionally, we showed that NKA haploinsufficiency significantly blunted oxLDL uptake, foam cell formation and macrophage migration under atherogenic conditions. Importantly NKA α1 heterozygous null mice when bred into an apoe null background developed less atherosclerosis (26.7% lesion area in NKA control mice v.s. 13.4% lesion area in NKA heterozygous null mice) assessed by en face oil red O staining of aortae after 12 weeks on high fat diet. We conclude that by controlling Lyn kinase activity NKA critically regulates oxLDL/CD36 induced pro-atherogenic signaling.