bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 499: Development of a Novel Antithrombotic Therapy Targeting Platelet GPIbα: Assessment of Anfibatide In Vitro and in Phase I Clinical Trial
Authors: Hou Yan , Reheman Adili , Li Benjamin X , Dai Xiangrong , Yang Zhongqiang , Qian Fang , Zhang Guohui , Xu Zhengyu , Liu Jing , Liang Chaofan , Zhao Yanan , Shen Jie , Zhou Hui , Wang Yiming , Marshall Alexandra H , Lei Xi , Zarpellon Alessandro , Vanhoorelbeke Karen , Ruggeri Zaverio , Ni Heyu
Published: July 2021

Platelet adhesion and aggregation are critical for hemostasis and atherothrombosis. Interaction between the GPIb complex and VWF initiates platelet adhesion and contributes to complete vessel occlusion, particularly at sites of stenosis. However, no therapeutic targeting this pathway has been developed. Previously, we demonstrated in murine thrombosis models that Anfibatide, a snake venom-derived GPIbα antagonist, inhibited botrocetin-induced VWF-GPIbα binding and inhibited thrombus formation in vitro and in vivo, without significantly activating platelets or changing bleeding. We subsequently hypothesized that Anfibatide would be a safe and potent anti-thrombotic agent in humans.

We tested Anfibatide with human platelets in vitro and conducted the first anti-GPIbα phase I clinical trial with 94 healthy volunteers. In vitro, Anfibatide inhibited ristocetin-induced and low dose thrombin-induced human platelet aggregation. Surface plasmon resonance studies showed that Anfibatide binds to GPIbα and blocks binding of both VWF A1 domain and thrombin. In ex vivo perfusion chambers, Anfibatide strongly inhibited human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface, especially at high shear flow conditions. Importantly, Anfibatide effectively dissolved the preformed thrombi in perfusion chambers. Anfibatide incubation with human whole blood did not significantly change coagulation parameters measured by thromboelastography. After treating volunteers with Anfibatide, no serious adverse events, premature discontinuations due to adverse events, or deaths occurred during the study. Anfibatide bound to 95% of GPIb and inhibited 90% of platelet aggregation without prolonging activated partial thromboplastin time, prothrombin time, thrombin time, or bleeding time. There was no bleeding tendency, no alteration of platelet count, and no detectable anti-Anfibatide antibodies.

In conclusion, Anfibatide provides anti-thrombotic effects through inhibition of both GPIbα-VWF and GPIbα-thrombin interactions. These results demonstrate that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy.
Abstract 503: A Within-Subject Repeatability Coefficient Assessment of Snoring on Carotid Artery Velocities
Authors: McCormick Susan , Piwowarczyk Joanna , Lozanski Laurie , Kashtanjeva Besnike , Ilczyk Edyta , Skelly Christopher
Published: July 2021

Objectives:
Vascular surgeons use duplex ultrasound (US) to evaluate carotid stenoses as a means to determine stroke risk. An under-appreciated risk factor is Obstructive Sleep Apnea (OSA). Although the mechanism is unknown, preliminary studies suggest snoring may play a role. The goals were to determine 1) daily carotid artery velocity variability within subjects and 2) the effect of simulated snoring on carotid artery velocities.

Methods:
44 bilateral carotid artery US exams were performed on 11 subjects by 2 RVTs (4 exams/subject, 7 subjects/RVT A, 4 subjects/RVT B). Steps were taken to ensure measurements were made at identical sites under similar parameters for subsequent exams. PSV and EDV were measured at 8 locations at rest and during a simulated snore. For each location, limits of agreement was used to calculate PSV and EDV repeatability coefficients, which are the limits within which 95% of the differences will lie for two measurements made on the same subject. Repeatability coefficients were then used to determine significance for observed velocity changes in carotid arteries with and without stenosis during simulated snoring, at 3 locations. They were also used to assess velocity variability in CEA candidates. All US exams were performed in an IAC vascular testing accredited laboratory by RVTs.

Results:
Repeatability coefficients for RVT A ranged from 26 to 40cm/s for PSV and 11 to 20cm/s for EDV. For RVT B they ranged from 30 to 65 and 8 to 19cm/s for PSV and EDV, respectively. Maximum values occurred in the ICA ostia and ECA for RVT A, and in the ICA ostia and proximal for RVT B. In non-stenosed arteries, snoring most frequently caused a change in the PSV of the proximal ICA where it occurred in 17% of the arteries (4/24). However, the effect of snoring was greatest in stenosed arteries. In 75% of these arteries (6/8) there was a significant velocity change. 38% of proximal ICA PSVs and EDVs were affected.

Conclusion:
Repeatability coefficients can be used to determine significant changes in carotid artery velocities within diagnostic stenosis categories as measured by US. There is a possible connection between snoring, carotid velocities and stenosis which warrants further investigation to determine if it is part of the mechanism by which OSA increases stroke risk.
Abstract 509: Early Pathogenesis of Murine Aortic Valve Disease is Marked by Versican-Rich Proteoglycan Synthesis
Authors: Blaser Mark C , Kwon Jung-Woo , Wei Kuiru , Zhou Yu-Qing , Henkelman R M , Simmons Craig A
Published: July 2021

Background:
Aortic valve disease (AVD) is a cell-mediated pathology without pharmacotherapy whose early pathogenesis is poorly understood. In mice, the study of later-stage fibrotic & calcific AVD requires aggressive dietary/genetic induction unsuitable for the study of early AVD progression. Here, we examined the initial extracellular matrix & cellular changes induced in the mouse aortic valve by a mildly atherogenic diet.

Methods:
Male WT C57Bl/6J mice were fed control or high-fat (HF, BioServ F3282) diets for 4 or 16 months (n = 6-10 per group). Aortic valve function was assessed by echocardiography. Valve sections were (immuno)stained for Movat’s pentachrome, proteoglycans (PG), Sox9, & αSMA. Valve interstitial cells (VICs) from porcine aortic valves were cultured with 5 ng/ml TGF-β1 for 5 days. PG synthesis & mRNA levels were measured by g-HCL & qPCR, respectively.

Results:
At 4 months, valve function was unaffected by the HF diet (transvalvular velocity: 123.5 ± 8.5 vs. 128.2 ± 9.2 cm/s, p = 0.75), & no leaflets expressed αSMA. Distal tips of HF leaflets were thickened (84.4 ± 10.4 vs. 37.3 ± 2.7 μm) due to PG accumulation (11435 ± 1920 vs. 5448 ± 762 μm
2
, p < 0.01), not collagen deposition (1729 ± 815 vs. 1771 ± 663 μm
2
, p = 0.87). Versican was the only PG increased in HF leaflets (2.38-fold, p < 0.05), with no alterations in decorin or biglycan (p = 0.69 & 0.59). Chondrogenic processes were activated in HF leaflets, with a 3.42-fold increase in Sox9-positive leaflet area (p < 0.01). By 16 months, HF mice had elevated transvalvular velocities (169.7 ± 20.3 vs. 115.4 ± 3.6 cm/s, p < 0.05) & leaflets were αSMA-positive & myofibrogenic. Histology of 16-month mice for PGs & Sox9 is ongoing. Early PG expression patterns were recapitulated in culture: total PG production was increased 2.14-fold in TGF-β1-treated VICs (p < 0.05), while versican mRNA was solely elevated (1.53-fold, p < 0.05) & decorin & biglycan were decreased (-2.05-fold, p < 0.01 & -2.62-fold, p < 0.05 respectively).

Conclusions:
Diet-induced
de novo
PG synthesis is versican-rich, occurs along with increased Sox9 expression, & develops prior to myofibroblast activation, fibrosis, or deficits in valve function. These studies provide new insight into early AVD pathogenesis & the involvement of specific PGs therein.
Abstract 525: The Role of FHL2 in Mechanically Regulated Valve Interstitial Cell Osteogenic Differentiation
Authors: Lam Alan Y , Chen Jan-Hung , Simmons Craig A
Published: July 2021

Introduction:
Lesions in calcific aortic valve disease (CAVD) often include osteoblasts and occur preferentially in the natively stiffer fibrosa side of the valve, suggesting roles for osteogenesis and matrix stiffness in CAVD. Valve interstitial cells (VICs) have osteogenic potential that is modulated by matrix stiffness
in vitro
. Osteogenesis of mesenchymal stem cells is mediated by FHL2 and modulated by matrix stiffness via RhoA. However, the connection between FHL2 and RhoA and their roles in mechanically-regulated osteogenic differentiation of VICs and CAVD have not been established.

Hypothesis:
Matrix stiffness regulates RhoA-dependent FHL2 expression to direct VIC osteogenesis.

Methods/Results:
Primary porcine aortic VICs were grown on collagen-coated polyacrylamide gels of varying elastic moduli
in vitro
. With increasing substrate stiffness, VICs demonstrated increased RhoA activation by ELISA and increased FHL2 nuclear translocation by immunofluorescence and western blotting. Notably, significant increases were seen on 22 kPa substrates, a stiffness unique to the disease-prone fibrosa, compared to 11 kPa substrates (p<0.05). We investigated the regulatory relationship between RhoA and FHL2 in VICs by controlling RhoA levels with adenoviruses. With constitutively active RhoA, FHL2 nuclear translocation increased (p<0.06); in contrast, with dominant negative RhoA, FHL2 nuclear translocation decreased (p<0.05). The effect of RhoA levels on stiffness-dependent FHL2 activity was verified by knocking down RhoA levels pharmacologically (C3 toxin) which abrogated stiffness-dependent FHL2 nuclear translocation. Alkaline phosphatase activity as a marker of osteogenesis was highest on substrates with fibrosa-like stiffness and was reduced by FHL2 siRNA knockdown.

Conclusions:
Together, these results support the hypothesis that mechanically-regulated VIC osteogenic differentiation
in vitro
is mediated by FHL2 operating downstream of RhoA. New insights into the mechanotransduction mechanisms that regulate VIC osteogenesis may lead to novel strategies for treating CAVD.
Abstract 534: A Common Null Allele of LPA is Associated With Lp(a) Levels and Coronary Artery Disease Risk
Authors: Kyriakou Theodosios , Seedorf Udo , Goel Anuj , Hopewell Jemma C , Clarke Robert , Watkins Hugh , Farrall Martin
Published: July 2021

Objective:
Increased levels of Lp(a) lipoprotein are a highly heritable risk factor for coronary artery disease (CAD). The genetic determinants of Lp(a) levels are mainly due to genetic variation in the apolipoprotein(a) gene (LPA). We have tested the association of a null allele of LPA with Lp(a) levels and CAD risk in a large case-control cohort. We have also examined how null allele genotyping complements apolipoprotein(a) isoform typing to refine the relationship between LPA isoform size and circulating Lp(a) levels.

Approach and Results:
The LPA null allele (rs41272114) was genotyped in the PROCARDIS case-control cohort (4,073 CAD cases, 4,225 controls). Lp(a) lipoprotein levels were measured in 909 CAD cases and 922 controls; apolipoprotein(a) isoform size was estimated using SDS-agarose gel electrophoresis and a high-throughput qPCR based method.

Null carriers are common (null allele frequency 3%) and have significantly lower circulating Lp(a) levels (p=2.1x10-10) and reduced CAD risk (p=0.023) compared to non-carriers. An additive allelic model of apolipoprotein(a) isoform size, refined by null allele genotype and qPCR values, showed a sigmoid relationship with Lp(a) levels with baseline levels for longer isoform alleles and progressively higher levels of Lp(a) for shorter isoform alleles.

Conclusions:
The LPA null allele (rs41272444) is associated with decreased circulating Lp(a) levels and decreased CAD risk. A joint genomic and isoform analysis revealed details of the relationship between apolipoprotein(a) isoform size and circulating Lp(a) level consistent with a threshold effect on lipoprotein secretion.
Abstract 547: PDE4 Inhibition Protects Against Neointimal Hyperplasia and Diminishes VCAM-1 Expression and Histone Methylation Through an Epac-Dependent Pathway
Authors: Kahles Florian , Lehrke Michael , Makowska Anna , Marx Judith , Bruemmer Dennis , Marx Nikolaus , Findeisen Hannes M
Published: July 2021

Introduction:
Upregulation of phosphodiesterase 4 (PDE4) activity causes cAMP-dependent vascular smooth muscle cell (VSMC) activation at sites of vascular inflammation and tissue remodeling.

Hypothesis:
PDE4 inhibition reduces VSMC activation and neointima formation following vascular injury.

Methods:
C57BL/6 mice (n=16) treated with roflumilast underwent guide wire-induced endothelial denudation injury of the femoral artery. Neointima formation was quantified after 4 weeks. In vitro, we analyzed the effects of roflumilast on VSMC proliferation and inflammation.

Results:
Roflumilast treatment attenuated neointima formation by 40% (17252 ± 2315 vs. 10119 ± 738 μm
2
, p=0,02) and femoral artery intima-media ratio by more than 50% (2,76 ± 0,32 vs. 1,35 ± 0,25 ,p=0,005). In vitro, roflumilast did not affect VSMC proliferation, but diminished TNF-α induced expression of the inflammatory marker vascular cell adhesion molecule 1 (VCAM-1) by 60% (p<0.05). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued in the presence of the Epac inhibitor ESI-09 and following siRNA mediated knockdown of Epac1. TNF-α induced NFκB p65 translocation and VCAM-1 promotor activity were not altered by roflumilast in VSMCs. However, Roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, while PKA activation showed no effect. In accordance with the reduction of VCAM-1 in vitro, roflumilast treated mice displayed decreased expression of VCAM-1 and the macrophage markers F4/80 and CD68 during neointima formation. Consistently, immunohistochemical analysis of the vessel wall showed reduced Mac2 staining, indicating decreased macrophage accumulation after vascular injury.

Conclusions:
PDE4 inhibtion reduces neointimal hyperplasia after vascular injury and regulates VCAM-1 through a novel Epac-dependent mechanism, which modulates specific histone methylation patterns. PDE4 inhibition might represent a novel approach for the treatment of vascular diseases, including atherosclerosis and in-stent restenosis.
Abstract 557: ACE2/Angiotensin-(1-7) Stimulates Vasoprotection-Relevant Functions of Human CD34
+
Cells
Authors: Singh Neha , Guo Lirong , Alberto Neville , Bartelmez Stephen B , Raizada Mohan K , Jarajapu Yagna P
Published: July 2021

Angiotensin converting enzyme 2 (ACE2) and angiotensin (Ang)-(1-7), key members of the vasoprotective axis of the renin-angiotensin system (RAS), have been shown to modulate hematopoietic functions of bone marrow-derived cells. This coupled with recent studies implicating the involvement of bone marrow stem/progenitor cells in the cardiovascular functions, we evaluated the expression of RAS components and functions of ACE2 and Ang-(1-7) in human CD34
+
cells, hematopoietic stem cells with vasoprotective functions.

Peripheral blood was obtained from healthy individuals and CD34
+
cells were isolated by immunomagnetic separation. Real-time PCR was carried out, and the effects of Ang II, Ang-(1-7), NorLeu
3
Ang-(1-7), and ACE2 activators, diminazene aceturate (DIZI) and a xanthenone derivative (XNT), were evaluated on migration, proliferation and adhesion of CD34
+
cells. Paracrine factors produced by CD34
+
cells were analyzed. CD34
+
cells showed the presence of mRNA transcripts of ACE, ACE2 and Mas1 receptor. In contrast, renin, angiotensinogen, and AT1 and AT2 receptors were not detectable. Treatment with 100nM Ang-(1-7) or 10nM NorLeu
3
Ang-(1-7) stimulated proliferation and migration of CD34
+
cells while adhesion of these cells to fibronectin was not affected. Ang-(1-7) treatment altered the paracrine profile of CD34
+
cells: increased

IL10, IL8, G-CSF, GM-CSF, IGFBP-3 and decorin (P<0.03), and decreased IGF1, thrombopoietin and TGFβ1 levels (P<0.05, n=5). In contrast, 100nM Ang II has no effects on proliferation and migration of CD34
+
cells. However, in the presence of Ang II, 100nM DIZI or 100nM XNT increased proliferation and migration of CD34
+
cells compared to the cells treated with Ang II alone (P<0.05, n=7). Treatment of total leucocytes with Ang II prior to the isolation of CD34
+
cells attenuated their migration to stromal-derived factor-1α and proliferation compared to the untreated (P<0.05, n=5), and enhanced their adhesion to fibronectin. These studies suggest that activation of endogenous ACE2 or treatment with Ang-(1-7) stimulates the functions of CD34
+
cells that are indicators of their cardiovascular protective functions while Ang II may attenuate these properties indirectly via acting on leucocytes.
Abstract 563: Mechanisms of VEGFR3-Dependent Pathological Lymphangiogenesis
Authors: Min Wang
Published: July 2021

Vascular remodeling, i.e., the growth and/or enlargement of both blood and lymphatic vessels, is tightly associated with inflammatory diseases, such as chronic airway inflammation, rheumatoid arthritis, inflammatory bowl disease, and chronic inflammatory skin psoriasis. The lymphatic system collects extravasated fluid, macromolecules, and immune cells from tissues and returns them to the blood circulation. While VEGFR2 and its ligand VEGF-A functions as master regulators of vasculogenesis and angiogenesis, VEGFR3 is critical for lymphangiogenesis. These conclusions are based on observations in genetically modified mice for VEGFR3 and its ligands. VEGFR3 is expressed on the blood and lymphatic endothelium during development, and is restricted to the lymphatics in the adult. Extensive studies have thus far focused on the role of VEGFR3 in lymphangiogenesis during development, however the function, regulation and intracellular mediators of the VEGFR3-dependent pathways in pathologic formation of lymphatic vessels during diseases remain poorly characterized. Our recent studies have identified two critical intracellular mediators, AIP1 and Bmx, in regulating VEGFR3 expression and activity. Genetic deletion of AIP1, a Ras-GAP protein, delays developmental lymphangiogenesis in neonatal skin and mesentery, and mounts weaker VEGF-C-induced cornea lymphangiogenesis. Deletion of Bmx, a non-receptor tyrosine kinase, has no effect on developmental lymphangiogenesis but blunts pathological lymphangiogenesis in mouse models. Mechanistically, AIP1 and Bmx sequentially regulate different steps during the VEGFR3 activation. AIP1 constitutively binds to VEGFR3 and enhances VEGFR3 endocytosis and stability. Bmx is not expressed in developmental lymphatics but is induced in response to pathological stimuli such as ischemia, and associates with an active form of VEGFR3 to regulate its downstream signaling without an effect on receptor phosphorylation. I will report the most recent progress as to how AIP1 and Bmx coordinately regulate VEGFR3-dependent lymphangiogenesis.
Abstract 565: LPA-PKD-1-HDAC7/NCoR1-FoxO1 Signaling Axis Regulates Endothelial Cell CD36 Transcription and Stimulates Arteriogenic Responses
Authors: Ren Bin , Ramakrishnan Devi P , Walcott Brian , Chen Yiliang , Best Brad , Storz Peter , Silverstein Roy L
Published: July 2021

Lysophosphatidic acid (LPA), a bioactive signaling phospholipid, down-regulates CD36 expression in microvascular endothelial cells (MVECs) via protein kinase PKD-1 signaling, thereby abolishing endothelial cell responses to its antiangiogenic ligand thrombospondin-1. However, little is known regarding mechanisms by which MVEC-specific CD36 transcription is regulated. We describe that in MVECs LPA represses CD36 transcription by activating a PKD-1 signaling that induces formation of a HDAC7/NCoR1/FoxO1 complex in the nucleus. Promoter analysis first identified FoxO1 as a transcription factor responsible for the CD36 transcription, which was confirmed by a chromatin-immunoprecipitation assay. Using a combination of PKD-1 gene transduction with co-immmunoprecipitation assay, we showed an increased interaction of HDAC7/NCoR1 with FoxO1 in response to LPA. However, HDAC7 and FoxO1 interaction was attenuated with PKD-1 silencing. Furthermore, based on results from an angiogenesis profiling with real time qPCR, doxycycline inducible constitutively active PKD-1 plasmids were transduced into tumor associated endothelial cells using a Lentiviral system to induce the PKD-1 expression. The results showed that turning off CD36 transcription reprograms by PKD-1 signaling was accompanied by an induced expression of ephrin B2 and activation of MAPK/ERK1/2 signaling, which are two critical “molecular signatures” involved in arteriogenesis. Moreover, three dimensional spheroid assay, a modified Boyden Chamber assay and
in vivo
Matrigel assay revealed that turning off CD36 transcription promoted angiogenesis
in vitro
and
in vivo
in a PKD-1-dependent manner. Immunofluorescence microscopy also showed the presence of this signaling pathway in the vasculature of Lewis lung carcinomas grown in
cd36
deficient mice. In summary, our data suggest that a LPA-PKD-1-HDAC7/NCoR1-FoxO1 signaling axis is critical for transcriptional regulation of CD36 and mediates silencing of this antiangiogenic switch. This subsequently results in MVEC reprogramming for proangiogenic and arteriogenic responses. Therefore, targeting this signaling cascade could be a novel approach for malignant tumors, cardiovascular ischemia and other thrombotic diseases.
Abstract 582: Inflammatory Cytokines Reduce Thrombin Activatable Fibrinolysis Inhibitor Protein Levels in HepG2 Cells via TTP-Mediated Destabilization of TAFI mRNA
Authors: Novakovic Dragana , Boffa Michael B
Published: July 2021

Thrombin activatable fibrinolysis inhibitor (TAFI) is the zymogen form of a basic carboxypeptidase (TAFIa) with both anti-fibrinolytic and anti-inflammatory properties. The role of TAFI in inflammatory disease is multifaceted and involves both recognition of specific pro-inflammatory substrates by TAFIa as well as regulation of TAFI gene expression by inflammatory mediators. In this study we addressed the hypothesis that decreased TAFI levels observed in inflammation are due to destabilization of its mRNA via the A/U-rich responsive element binding protein tristetraprolin (TTP). TAFI protein levels were measured in conditioned medium of HepG2 cells with recently developed assay specific for TAFIa. Treatment of cells with pro-inflammatory cytokines TNFα, IL-6 in combination with IL-1β, or with bacterial lipopolysaccharide (LPS) decreased TAFI protein levels by approximately 2-fold over 24 to 48 hours of treatment. Conversely, treatment of HepG2 cells with the anti-inflammatory cytokine IL-10 increased TAFI protein levels by 2-fold at both 24 and 48 hour time points. We employed luciferase reporter gene studies using human TAFI promoter constructs and found no change in promoter activity with these treatments. We then hypothesized that changes in mRNA stability may be involved through binding of TTP to the TAFI 3’-UTR, as we recently characterized the role of TTP in mediating TAFI mRNA stability. Using constructs expressing β-globin fusion mRNAs containing the TAFI 3’-UTR, we found that TNFα, IL-6, IL-1β, and LPS reduce TAFI mRNA half-life by 30%. This effect appears to be dependent on TTP binding, since these cytokines did not alter the stability of a fusion transcript lacking the TTP binding site. IL-10 caused an increase in fusion transcript stability by 38%, an effect that was also observed when the TTP binding site was mutated. Using reporter constructs expressing fusion mRNA’s containing the luciferase coding region and the TAFI 3’-UTR, we found that translation rate remained unaffected with both pro- and anti-inflammatory treatments. In conclusion, destabilization of TAFI mRNA appears to be the main mechanism behind decreased TAFI protein levels observed in the presence of pro-inflammatory mediators.
Abstract 584: ApoE3[K146N/R147W] Acts as a Dominant Negative ApoE Form that Prevents Remnant Clearance and Inhibits the Biogenesis of HDL
Authors: Fotakis Panagiotis , Kardassis Dimitris , Zannis Vassilis
Published: July 2021

Introduction:
The K146N/R147W substitutions in human apolipoproteinE3 (apoE3) have been associated with a dominant form of type III hyperlipoproteinemia which is expressed at an early age.

Methods:
The effects of the K146N,R147W substitutions on the lipid and lipoprotein profiles and the HDL phenotypes were studied by adenovirus mediated gene transfer of the full length and a truncated apoE3[K146N/R147W]-202 mutant using different mouse models.

Results:
A low dose of adenovirus expressing the apoE3[K146N/R147W] mutant in apoE deficient or in apoA-I x apoE double deficient mice exacerbated the hypercholesterolemia and increased greatly plasma apoE and triglycerides levels. In apoE deficient mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and resulted in the accumulation of discoidal apoE containing HDL particles in plasma. Similar doses of WT apoE3 cleared the cholesterol of apoE deficient mice without induction of hypertriglyceridemia and promoted formation of spherical HDL particles. A unique feature of the truncated apoE3[K146N/R147W]-202 mutant is that it prevented the induction of hypertriglyceridemia but did not correct the hypercholesterolemia. Other apoE-202 truncated mutants tested did not induce hypertriglyceridemia but corrected hypercholesterolemia. Infection of apoE deficient mice with adenoviruses expressing the apoE3[K146N/R147W] and lipoprotein lipase corrected the hypertriglyceridemia but did not prevent the formation of discoidal HDL particles. Infection of apoE deficient mice with adenoviruses expressing the apoE3[K146N/R147W] and lecithin:cholesterol (LCAT) acyltransferase corrected hypertriglyceridemia, normalized the plasma cholesteryl ester to total cholesterol ratio and generated spherical HDL particles. The combined data indicate that the K146N/R147W substitutions in the full length and the truncated apoE3[K146N/R147W] mutant prevent receptor mediated remnant clearance.

Conclusion:
The lipid/lipoprotein and HDL abnormalities observed in the different mouse models suggest that the apoE3[K146N/R147W] mutant acts a dominant negative ligand that exacerbates the dyslipidemia and also affects the activity of LCAT and thus inhibits the maturation of HDL.
Abstract 591: Genetic Risk Score Does Not Explain Hypercholesterolemic Phenotype in Children with Hypercholesterolemia of Unknown Genetic Origin
Authors: Sjouke Barbara , Fouchier Sigrid W , Wiegman Albert , Kastelein John J , Hovingh G Kees
Published: July 2021

Background:
Autosomal Dominant Hypercholesterolemia (ADH) is caused by mutations in
LDLR
,
APOB
or
PCSK9
. In our previous study, we identified a causative mutation in these genes in 255 (95%) out of 269 children with clinically defined ADH (vd Graaf Circulation 2011). In the current study we addressed whether these 14 ADH children were characterized by a polygenetic form of dyslipidemia, defined by the presence of an excessive number of LDL-C raising SNPs (gene risk score (GRS)).

Methods:
Upon reassessment of the molecular diagnosis and clinical phenotype in the 14 ADH children we excluded: 2 patients in whom a pathogenic ADH mutation was identified by re-sequencing, one patient in whom a secondary cause of hypercholesterolemia became evident, 2 patients in whom hypercholesterolemia was not identified anymore, and one patient who was lost to follow-up. In total, 8 index patients and their 24 first degree relatives (median[IQR] = 3[2-4] relatives/index case) were included. We genotyped all individuals for 15 SNPS with an effect on LDL-C levels (Teslovich Nature 2010) and created a GRS.

Results:
The GRS was 16.1±2.2 in the indices and 16.2±2.2 in their relatives, irrespective of their LDL-C levels (p=0.928) The GRSs did not differ between individuals with LDL-C levels above the 90
th
percentile (n=21) and those with LDL-C levels below the 90
th
percentile (n=11) (GRS 16.4±2.1 vs.15.7±2.3; p=0.399 and effect size LDL-C 1.15±0.15 vs. 1.18±0.20 mmol/L; p=0.616).

Conclusions:
In children with a definite clinical diagnosis of ADH, absence of mutations in known genes is very rare. In these individuals, we did not find a suggestion of a polygenetic cause for the phenotype, suggesting that other, as of yet unidentified ((epi)genetic) mechanisms might constitute the underlying cause of hypercholesterolemia in these patients. Further research is carried out to elucidate this mechanism.
Abstract 594: Macrophage Proteomics Identifies Mechanistic Links Between Type 2 Diabetes and Atherosclerosis
Authors: Coats Brittney , Schoenfelt Kelly , Becker Lev
Published: July 2021

Obesity and Type 2 Diabetes (T2Ds) are associated with increased risk of atherosclerosis and CHD. However, the mechanism(s) linking these diseases are incompletely understood. Macrophages play essential roles in the pathogenesis of both disorders, suggesting that changes in macrophage biology link obesity/T2D and atherosclerosis. We previously showed that the atherogenic actions of cholesterol-loaded macrophages (foam cells) are an emergent property of a dysregulated sterol responsive protein network secreted by macrophages (MSRN). Thus, we hypothesized that obesity/T2Ds may impact atherogenesis by similarly disrupting the MSRN.

To test this hypothesis, we isolated peritoneal macrophages (PMΦ) from C57BL/6 mice fed a chow or obesogenic diet and interrogated their secreted proteomes using mass spectrometry. Based on stringent statistical analysis (FDR<5%), we identified 36 proteins that were differentially expressed in media conditioned by macrophages from obese relative to lean mice. Unsupervised gene set enrichment analysis demonstrated that obesity-induced changes to the PMΦ proteome significantly targeted the MSRN. Importantly, this phenomenon could not be explained by cholesterol accumulation or inflammation, since PMΦ’s from obese mice did not accumulate cholesterol and pro-inflammatory cytokine expression was not induced in PMΦ’s from obese or atherogenic mice.

Our previous studies showed that treating hypercholesterolemic
Ldlr
-/- mice with TZD (insulin sensitizer) could normalize MSRN protein expression
in vivo
, suggesting that insulin resistance is a key regulator of the network. To test this hypothesis, we placed C57BL/6 mice on an obesogenic diet for 7 weeks and treated the mice with TZD for 2 weeks. TZD therapy had no effect on body weight, but normalized fasting glucose levels. Moreover, PMΦ’s isolated from TZD-treated mice, demonstrated normalized protein expression patterns. Collectively, our studies suggest that insulin resistance in T2Ds reproduces macrophage protein dysfunction observed in atherogenic models, and provides a plausible mechanism by which T2Ds may act to increase risk of atherosclerosis.
Abstract 616: Interleukin 22 Downregulates ABCG1 and Impairs Cholesterol Efflux in Macrophages
Authors: Hofmann Bowman Marion , Chellan Bijoy , Yan Ling , Sonntag Timothy , Reardon Catherine
Published: July 2021

IL22 belongs to the IL10 cytokine family and is expressed by T helper cells. IL22 functions on epithelial cells and has been shown to improve epithelial barrier function in inflammatory bowel disease, asthma, and psoriasis; autoimmune diseases associated with elevated serum IL22. Patients with psoriasis have increased coronary artery disease and it was previously shown that macrophages from patients with psoriasis have impaired cholesterol efflux. The function of IL-22 on macrophage cholesterol metabolism is not known.

Methods:
ABCA1, ABCG1 and CD36 mRNA and protein expression, cholesterol uptake and efflux were studied in murine macrophages and human THP-1 macrophages. C57BL6/J mice with transgenic expression of hS100A12 and hS100A8/9 in myeloid cells were generated by using a bacterial artificial chromosome (hBAC/S100 mice). hBAC/S100 and WT littermate mice were breed into mice lacking the receptor for advanced glycation endproducts, RAGE.

Results:
Peritoneal macrophages from hBAC/S100 mice have reduced ABCG1 mRNA and protein expression, increased cholesterol uptake, and reduced cholesterol efflux compared to WT. This was abolished in hBAC/S100 mice lacking RAGE, the receptor for S100/calgranulin. Recombinant S100A12 or S100A8 protein (2.5 μg/ml) had no effect on ABCG1 expression in WT peritoneal macrophages or human THP-1 cells, suggesting other systemic intermediary products in hBAC/S100 mice. Serum IL22 and mRNA in splenic T cells were significantly increased in hBAC/S100 mice, and this was abolished in hBAC/S100 mice lacking RAGE. Moreover, r S100A12 increased IL22 mRNA by 2-fold in cultured human THP-1. Importantly, THP-1 macrophages treated with r IL22 (100 ng/ml) had reduced expression of ABCG1 and impaired cholesterol efflux to mouse serum, but not to Apoa1. Up regulation of ABCG1 and ABCA1 in response to LXR agonist TO901317 in THP-1 cells abolished the detrimental effects of IL22 on cholesterol efflux.

Conclusion:
S100/calgranulin induces IL22 in a RAGE dependent manner. IL22 down regulates ABCG1 and impairs cholesterol efflux in macrophages. This raises the hypothesis that IL22-mediated down regulation of cellular cholesterol efflux may be linked to improved epithelial barrier function, but may also augment atherosclerosis.
Abstract 631: Lipid Composition of Gold Nanoparticle-Templated High-Density Lipoprotein Analogs Dictates Cholesterol Efflux Function
Authors: Mutharasan R Kannan , Singh Amritha T , McMahon Kaylin M , Thaxton C Shad
Published: July 2021

Background:
Reverse cholesterol transport, the process by which cholesterol is effluxed from cells to high-density lipoproteins (HDL) and is delivered to the liver for clearance, is a promising pathway to augment for treatment of atherosclerosis. Though structure-function relationships for nascent, discoidal HDL and cholesterol efflux have been well studied, how the lipid composition of spherical HDL species - which varies in pathophysiological conditions - impacts their ability to mediate cholesterol efflux has not been investigated.

Methods and Results:
Spherical gold nanoparticles (5 nm) were used to synthesize spherical HDL analogs (HDL-NP) by adding ApoAI protein, and various lipids. With this strategy a panel of HDL-NP varying in lipid content was generated. HDL-NP designs tested include: dipalmitylphosphatidylcholine (DPPC, saturated fatty acid), dioleoylphosphatidylcholine (DOPC, unsaturated fatty acid), sphingomyelin, lysophosphatidylcholine (LPC), and mixtures thereof. All of these species are found in natural HDL. After characterizing protein and lipid stoichiometry of the purified HDL-NP, these HDL-NP designs were tested in the cellular reverse cholesterol transport assay using J774 mouse macrophages. These studies demonstrate that all HDL-NP designs mediate more efflux than equimolar amounts of ApoAI protein control, and further demonstrate that HDL-NP designs incorporating unsaturated phospholipid (DOPC), sphingomyelin, and LPC - each of which can increase disorder in the lipid membrane and thus give rise to opportunity for cholesterol to intercalate and bind - enhance cholesterol efflux compared to saturated phospholipid (DPPC) design.

Conclusion:
In summary, these results demonstrate that lipid content of HDL-NP - analogs of spherical HDL - dictates cholesterol efflux function, a finding which sheds light on the functional importance of lipid content variation seen in mature, spherical HDL species.
Abstract 632: Lipoprotein Lipase Activators: A New Potent Drug Class for Treatment of Dyslipidemia
Authors: Ericsson Madelene , Larsson Mikael , Caraballo Remi , Enquist Per-Anders , Elofsson Mikael , Willems van Dijk Ko , Rensen Patrick C , Olivecrona Gunilla , Nilsson Stefan K
Published: July 2021

Aim:
Elevated plasma TG and low HDL-cholesterol are part of CVD residual risk. Lipoprotein lipase (LPL) is the central enzyme controlling plasma TG hydrolysis and affecting de novo HDL formation. Thus LPL is an attractive target for correcting dyslipidemia and reduction of CVD residual risk. We have identified a novel first-in-class small molecule LPL activator - LP071. The objective with this study was to characterize the plasma lipid metabolism in LPL activator treated mice.

Methods:
ApoE3L:CETP mice were treated week with LP071. Plasma lipid profiles were analyzed using FPLC fractioning. LPL activity was investigated systemically and in specific tissue. Using triton WR1339 blockade chylomicron and VLDL secretion were assessed. Functional lipid clearance test were performed by oral fat tolerance tests.

Results:
Fasting plasma TG levels decreased with 97 % in apoE3L:CETP mice treated with LP071; HDL-c were increased by 116 % and (V)LDL-c were decreased with 91 %. Plasma free glycerol and NEFA were significantly lowered, 26 % and 22 % respectively. After an oral lipid load the plasma lipid response was significantly blunted in LP071 treated mice compared to controls. LPL activity in subcutaneous adipose tissue was increased 1500 % and in BAT LPL was increased by 100% at 3 h after a lipid gavage in WT mice treated with LP071.

Conclusions:
LP071 is a potent compound that can improve plasma lipid levels to a less atherogenic profile. The LP-class LPL activators can potentially help battle CVD residual risk in the future.
Abstract 647: Hyperglycemia Increases Diet-Induced Coronary Artery Disease and Myocardial Infarction in SRBI Knockout ApoE-Hypomorphic Mice
Authors: Gonzalez-Jara Leticia A , Werstuck Geoff , Trigatti Bernardo
Published: July 2021

Background:
Type I diabetes is characterized by high levels of glucose and free fatty acids in plasma. Several epidemiological studies have established diabetes as an independent risk factor for the development of cardiovascular disease. Diabetic animal models have shown increased development of aortic atherosclerosis, however the impact on coronary artery (CA) disease is less clear. Conventional mouse models of atherosclerosis do not reproducibly develop CA atherosclerosis. The HDL receptor, SRBI, has been associated with protection against atherosclerosis. SRBI knockout (KO) ApoE-hypomorphic mice exhibit increased aortic sinus atherosclerosis and diet-induced occlusive CA atherosclerosis when compared to SRBI+/+ ApoE-hypomorphic mice. We assessed the hypothesis that diabetes will increase the development of CA and aortic atherosclerosis and myocardial infarction in SRBI KO ApoE-hypomorphic mice.

Methods:
Hyperglycemia was induced by multiple low-dose i.p. injections of streptozotocin (40 mg/kg body weight). Controls received vehicle. Mice were fed a regular chow diet for 11 weeks (n=6-7) or a high-fat/high-cholesterol diet for 4 weeks (n=14-15). CA and aortic atherosclerosis and cardiac fibrosis were evaluated.

Results:
Diabetic SRBI KO ApoE-hypomorphic mice maintained on normal chow diet exhibited small plaques in the aortic sinus and the burden of atherosclerosis in CA was low. No fibrosis in hearts was detected. When mice were challenged with a high-fat/high-cholesterol diet, survival was significantly reduced in the diabetic group (p<0.01 vs control). High-fat/high-cholesterol diet fed diabetic mice exhibited significantly larger plaques in the aortic sinus as well (p<0.05 vs control). CA atherosclerosis burden was increased (p<0.001 vs control) along with higher levels of cardiac fibrosis (p<0.01 vs control).

Conclusion:
Hyperglycemia significantly increased the burden of aortic and CA atherosclerosis and cardiac fibrosis in SRBI KO ApoE-hypomorphic mice fed a high-fat/high-cholesterol diet.
Abstract 677: The Role of Discoidin Domain Receptor 1 in Vascular Calcification in Atherosclerosis and Diabetes
Authors: Lino Marsel , Bendeck Michelle P
Published: July 2021

Background:
Atherosclerosis and diabetes share many common pathogenic mechanisms. Vascular calcification is a common and severe complication in patients with atherosclerosis and type-2 diabetes (T2D), and occurs when vascular smooth muscle cells (VSMCs) trans-differentiate into osteoblast-like cells, in a process driven by runt-related transcription factor-2 (RUNX2). Our laboratory has recently discovered that Discoidin Domain Receptor-1 (DDR1) deletion reduces vascular calcification
in vivo
in

Ldlr
-/-

mice. Additionally, we have shown that RUNX2 activity is reduced in

Ddr1
-/-

VSMCs. However, little is known about the mechanism by which DDR1 mediates calcification and RUNX2 activity.

Rationale:
The insulin signaling pathway plays an important role in T2D and VSMC function. It has recently been discovered that PI3K binds to the DDR1 tyrosine kinase domain upon DDR1 activation.

Hypothesis:
DDR1 promotes vascular calcification by inducing RUNX2 activity via the PI3K/Akt signaling pathway in T2D.

Methods/Results:
To study the role of DDR1 in vascular calcification in T2D,

Ldlr
-/-

(SKO) and

Ldlr-/-; Ddr1
-/-

(DKO) mice were placed on a modified Western diet (40% fat, 43% carbohydrates, 0.5% cholesterol) for 12 weeks. Oil Red-O staining of the descending aorta showed reduced plaque burden in DKO mice (9.1±2.7% vs 4.4±2.9% surface area; p<0.05). Measurements of vascular calcification are ongoing. To determine the mechanism by which DDR1 modulates VSMC calcification, primary VSMCs collected from

Ddr1
+/+

and

DDR1
-/-

mice were cultured in osteogenic media for 12 days to induce calcification. Proliferation and calcification were significantly reduced in

DDR1
-/-

VSMCs. Additionally,

DDR1
-/-

VSMCs showed significantly reduced p-Akt levels when stimulated with insulin.
Significance:
Current treatments for vascular calcification are non-specific and often pose a risk to bone health. This is the first study to test the role of DDR1 in vascular calcification in an animal model of T2D, which will provide novel insight into the mechanism of vascular calcification and uncover new potential therapeutic approaches.
Abstract 8: Effect of Intensive Lipid Therapy on Plaque Lipid Depletion as Assessed by Magnetic Resonance Imaging
Authors: Zhao Xue-Qiao , Hulphers Elizabeth , Iquith Daniel , Bai Hua , Shadzi Peymon , Contreras Andrea , Neradilek Moni , Gill Edward , Colletti Patrick , Hatsukami Tom , Yan Chun
Published: July 2021

Background:
Previously we demonstrated plaque lipid depletion by carotid MRI during lipid lowering. Effect of different lipid therapy on plaque lipid content was investigated.

Methods:
Subjects (n=182) with coronary or carotid artery disease, apoB ≥120 mg/dl, and lipid treatment history <1 year were randomized to: (1) single therapy (Rx) - atorvastatin (Atorva) (10-80 mg/day); or (2) double Rx - Atorva plus extended release-niacin (ERN) (2 g/day); or (3) triple Rx - Atorva, ERN plus colesevelam (3.8 g/day) with appropriate placebos for 2 years. These Rx lowered treatment-group average LDL-C by 38%, 29%, 45% and triglycerides by 17%, 22%, 39%, and raised HDL-C by 7%, 24%, 30%. Of the 182 subjects who underwent high-resolution, multi-contrast bilateral carotid MRI scans at baseline, 46 were identified with lipid-rich necrotic core (LRNC) and completed 2-year follow-up scans by July 2013. Cross-sectional images (10 slices on average) with 2 mm thickness were obtained and centered at the carotid bifurcation. All images were analyzed for quantifications of wall area and plaque composition while blinded to therapy, lab results and clinical course, using the previously published MRI criteria. LRNC-volume (LRNC-V) and %LRNC (LRNC area/wall area) were compared pre- and post-therapy in each of the 3 Rx groups using Mann-Whitney test.

Results:
Overall, the 46 subjects with measurable LRNC at baseline had a significant plaque lipid content reduction. LRNC-V decreased from 58.8±9.0 to 39.7±10.2 mm3 (p<0.001) and %LRNC from 13.3±1.0 to 8.1±1.2 (p<0.001) after 2 years of Rx. Single Rx (n=17) showed non-statistical significant reductions in LRNC-V (78.0±20.7 vs. 69.8±25.3 mm3, p=0.2) and %LRNC (14.3±1.9 vs. 11.2±2.8, p=0.2). However, double Rx (n=16) had significant reductions in LRNC-V (51.9±10.3 vs. 21.4±6.0 mm3, p<0.001) and %LRNC (13.3±1.5 vs. 6.1±1.0, p=0.001). Triple Rx (n=13) also showed significant reductions in LRNC-V (42.3±9.8 vs. 23.1±6.9 mm3, p=0.005) and %LRNC (12.1±1.9 vs. 6.6±1.9, p=0.002).

Conclusions:
Intensive combination lipid therapy of atorvastatin plus niacin or atorvastatin, niacin and colesevelam significantly depletes carotid plaque lipid content as assessed by MRI over 2 years.
Abstract 81: Novel Adult Endothelial Hierarchy Defined by Endovascular Progenitor Reexpression of SOX18
Authors: Patel Jatin , Seppanen Elke J , Rodero Mathieu P , Roy Edwige , Fisk Nicholas M , Yoder Mervin C , Francois Mathias , Khosrotehrani Kiarash
Published: July 2021

Introduction:
During adult life, formation of new blood vessels in response to insults or physiological signals is thought almost exclusively to result from the sprouting and extension of existing vessels defined as angiogenesis. An alternate proposal for de novo vascularization relates to endothelial progenitors and their ability to assemble the intimal layers of mature vessels. However, the cell lineage and possible hierarchy of such precursors remains controversial. We here aimed to define endothelial progenitors in vivo in situations of neo-vessel formation in adult mice.

Methods and Results:
Based on protein expression levels of markers CD31 and VEGFR2 using flow cytometry, three populations of endothelial cells could be identified among VECAD+CD34+CD45- cells in a variety of angiogenic situations such as wound healing, tumours, placental development, or in subcutaneous Matrigel plugs. Lineage tracing based on sox18 re-expression using a sox18CreER/Rosa-YFP reporter strategy demonstrated that the CD31loVEGFR2- endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2- transit amplifying and a definitive differentiated CD31hiVEGFR2hi population. Loss of SOX18, in a dominant-negative mouse model, did not affect levels of EVP populations in vivo, but depleted both the intermediate and definitive populations. Only the sorted CD31loVEGFR2- as opposed to the other two endothelial populations could form colonies in vitro and engraft in vivo in a Matrigel plug assay.

Conclusions:
These results define and characterize an in vivo endothelial hierarchy and suggest a prominent role for vessel resident EVP populations in situations of neo-vessel formation. This data opens the medium for new perspectives in modulating blood vessel formation.