bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 134: Hypoxia in Coronary Plaque Enhance Thrombogenic Potential of Activated Macrophages
Authors: Maekawa Kazunari , Yamashita Atsushi , Nakamura Eriko , Asada Yujiro
Published: June 2021

Background:
Thrombogenic potential on disrupted atherosclerotic plaque is an important factor on the development of atherothrombosis, and inflammatory stimuli up-regulate thrombogenic factors. We have revealed that arterial glucose metabolism and hypoxia reflect the vascular wall thrombogenicity in rabbit. However, role of inflammation and/or hypoxia on the thrombogenicity in atherosclerotic plaque remain unclear. The present study aims to identify expression of hypoxic/metabolic markers and thrombogenic factors in human coronary plaques and also aims to evaluate the effect of of inflammatory and hypoxic stimuli on thrombogenicity in cultured macrophages.

Methods:
We immunohistochemically investigated numbers of inflammatory cells and microvessels, and expression of hexokinase (HK)-II, monocarboxylate transportor 4 (MCT4) (hypoxic/metabolic markers), and tissue factor (TF) in human vulnerable coronary plaques (n=22) and stable plaques (n=10). TF expression, procoagulant activity, and lactate production were assessed in THP-1 macrophages stimulated by tumor necrosis factor-α (10ng/mL) and interferon-γ (20ng/mL) under normoxic (21%O
2
) or hypoxic (1%O
2
) condition.

Results:
Numbers of macrophages, HK-II-, MCT4-, TF-immunopositive cells in the vulnerable plaques were significantly larger than those in the stable plaques. Many macrophages expressed HK-II, MCT4, and TF in the plaques. TF expression and procoagulant activity in the cultured macrophages were increased by the inflammatory stimuli, and were enhanced by hypoxia. The hypoxic condition also increased HK-II expression and lactate production, but the inflammatory stimuli did not affect them.

Conclusion:
Hypoxia in coronary plaque may enhance thrombogenic potential of macrophages activated by inflammatory stimuli.
Abstract 135: Endothelial Dysfunction in Acute Graft-versus-host Disease After Allogeneic Hematopoietic Cell Transplantation.
In vitro
Evidence of the Protective Effect of Defibrotide
Authors: Mir Enrique , Palomo Marta , Carreras Enric , Diaz-Ricart Maribel , Rovira Montse , Escolar Ginés
Published: June 2021

Acute Graft-Versus-Host Disease (aGVHD) is the most common early complication after allogeneic Hematopoietic Cell Transplantation (allo-HCT). We demonstrated endothelial dysfunction (ED) in association with allo-HCT. According to this data, aGVHD has been linked to an inflammatory process that may affect the endothelium.

To investigate the differential degree of endothelial damage in patients developing or not aGVHD, to identify potential biomarkers, and to explore the protective effect of defibrotide (DF) in this scenario. DF has orphan designation for GVHD prevention.

Patients blood samples were collected before allo-HCT, at day 0, and every week till day 28 after HCT. Plasma proteins (sTNFR1, sVCAM-1, VWF and ADAMTS-13) were measured as biomarkers of ED in individual samples from patients developing (GVHD, n=24), or not (NoGVHD, n=13), aGVHD. In
in vitro
assays, endothelial cells (EC) in culture were exposed to media containing pooled sera from patients to evaluate changes in the: a) expression of VCAM-1 and ICAM-1 on cell surfaces; b) presence of VWF on the extracellular matrix (ECM) and c) reactivity of the ECM towards platelets, under flow. The effect of DF was explored in the
in vitro
experiments by previous exposure of the EC (for 24h) followed by continuous incubation (100 μg/ml, added every 24h).

Levels of sTNFRI, sVCAM-1 and VWF in samples from group GVHD were significantly higher than in NoGVHD (increases of 100, 37 and 150% respectively, at diagnose, p<0.01). ADAMTS-13 activity and VWF levels were inversely related. In
in vitro
studies, cell surface expression of VCAM-1 and ICAM-1, presence of VWF and platelet adhesion on the ECM in response to GVHD samples were always superior (increases
vs
NoGVHD of 80, 40, 100 and 21%, respectively, at diagnose).
In vitro
exposure of EC to DF attenuated signs of endothelial injury reducing significantly (p<0.05) the expression of VCAM-1, ICAM-1 and VWF (reductions of 22, 30 and 30%, respectively) in the GVHD condition.

Our results demonstrate endothelial damage in association with aGVHD, as evidenced by elevated plasma levels of several biomarkers. The
in vitro
approach showed a marked proinflammatory and prothrombotic phenotype in association with aGVHD, which could be significantly prevented by defibrotide.
Abstract 147: Elevated Serum Resistin Levels are Associated with Angiographically Significant Coronary Artery Disease: A Meta-analysis.
Authors: Agasthi Pradyumna , Aloor Sivakanth , Annangi Srinadh , Sivakumar Kalaivani , Dammalapati Tirumala Rao , Sachdeva Rajesh
Published: June 2021

Background:
Resistin is a novel inflammatory cytokine secreted primarily by mononuclear inflammatory cells and adipocytes. It upregulates the expression of vascular adhesion molecules and stimulates smooth muscle cell proliferation, thereby contributing to atherogenesis in the vascular wall. We conducted a meta-analysis to evaluate the association between serum resistin levels and presence of angiographically significant coronary artery disease (CAD) in patients undergoing diagnostic coronary angiography (DCA).

Methods:
We searched MEDLINE, CINAHL and COCHRANE databases for studies reporting serum resistin levels in the patients with history of angina or positive stress test undergoing DCA. Patients with coronary stenosis > 50% in at least 1 coronary artery were considered to have significant CAD. They were included in the study group, and rest were included in control group. A calculation of weighted standardized mean difference (SMD) in serum resistin level between the CAD and control groups was conducted.

Results:
A literature search yielded 158 articles, but only 11 studies enrolling 3291 patients were included in the meta-analysis. The median age and body mass index (BMI) of the CAD group was 63 yrs (IQR 61-65) and 27 kg/m2 (IQR 25-29) versus 59 yrs (IQR 57-61) and 28 kg/m2 (IQR 26-29) in the control group. There were more females in the control group 48% (IQR 46-58) versus 27% (IQR 24-33) in CAD group. The unweighted median serum resistin levels in the CAD group were 5.7 ng/ml (IQR 4.1-16.1) vs 4.3 ng/ml (IQR 3.1-8.2) in the control group. The SMD of serum resistin level was 0.38 (95% CI 0.18- 0.57), P<0.001 comparing those in the CAD group and control group. None of the covariates (age, BMI and sex) accounted for statistical significance.

Conclusion:
An elevated serum resistin level is independently associated with the presence of angiographically significant CAD. Serum resistin levels can potentially be used as a biomarker for cardiac risk stratification.
Abstract 169: Hypertension Down Regulates Emilin1 in the Extracellular Matrix of Resistance Arteries in Humans and Mice, in Order to Increase the Myogenic Tone Through Overactive TGFβ, Thus Contributing to Blood Pressure Regulation
Authors: Carnevale Daniela , Perrotta Marialuisa , Fardella Stefania , Casaburo Manuel , Bizzotto Dario , Selvetella Giulio , Innocenzi Gualtiero , Bressan Giorgio , Lembo Giuseppe
Published: June 2021

Emilin1, a protein of the extracellular matrix of vessels, regulates TGFβ activity through proteolysis of the proTGFβ. Emilin1 KO mice, displaying increased TGFβ vascular activation, are hypertensive. As vascular Emilin1 is expressed from embryonic life to adulthood, we questioned whether the hypertensive phenotype results from a developmental defect or lack of a homeostatic role exerted in the adult. Thus, we inactivated Emilin1 in smooth muscle cells (VSMCs) of adult mice, with a tamoxifen inducible
Cre
-loxP, under the control of the SM myosin heavy chain promoter, finding that mice developed hypertension (HTN) as well as increased myogenic tone (MT) in resistance arteries. In order to evaluate the relevance of this mechanism in the human pathology, we enrolled 20 hypertensive (HT) and 20 normotensive (NT) patients, subjected to primary lumbar hernia neurosurgery, and dissected out resistance arteries from adipose tissue biopsies. We randomized vessels to receive vehicle, neutralizing antibody anti-TGFβ or non-relevant IgG and analyzed the MT. HT had a significantly increased MT, as compared to NT, rescued by the pretreatment with anti-TGFβ antibody. A pretreatment with the non-relevant IgG had no effect. Thus we hypothesized that Emilin1 could be down regulated by HTN to increase MT of resistances through overactive TGFβ, thus further sustaining high blood pressure (BP). In order to unravel this issue, we analyzed Emilin1 expression in the vessels walls of human arteries, finding a significant reduction in HT as compared to NT. We also evaluated Emilin1 expression in the resistance arteries of several murine models of HTN induced by: chronic AngII, DOCA-salt, L-NAME and obesity, finding that even in the experimental setting, HTN reduced Emilin1 in the vessel walls. Overall, we show that overactive TGFβ in VSMC increases the MT of resistance arteries, contributing to BP regulation. The data obtained in resistance arteries from HT and in the different murine models of HTN suggest that the down regulation of Emilin1 realized by HTN is a general mechanism to increase MT thus further sustaining high BP.
Abstract 17:
APOC3
A43T Variant Promotes ApoC-III Catabolism and Accelerates TG-rich Lipoprotein Clearance in Mice and Humans
Authors: Khetarpal Sumeet A , Millar John S , Varshini Amritha , Vitali Cecilia , Zeng Xuemei , Zanoni Paolo , Sun Zhiyuan , Nguyen David , McParland James T , McCoy Mary G , Natarajan Pradeep , Cuchel Marina , Mayne Leland , Englander S. W , Lund-Katz Sissel , Phillips Michael C , Yates Nathan A , Kathiresan Sekar , Rader Daniel J
Published: June 2021

Humans with loss-of-function (LoF) variants in
APOC3
, the gene encoding apolipoprotein C-III (apoC-III), have significantly reduced plasma triglycerides (TG) and protection from coronary disease. These findings suggest that apoC-III may be a viable therapeutic target for decreasing vascular risk through TG reduction, and that elucidation of the protective mechanism of
APOC3
LoF variants would inform such strategies. We report here the protective mechanism of the
APOC3
A43T missense variant, one of four recently identified CAD-protective variants. By genotyping >8,000 human participants with low TG, we identified 17
APOC3
A43T carriers and phenotyped 6 carriers and 54 matched controls. A43T heterozygotes demonstrate a significant reduction in apoC-III levels relative to non-carriers (50% reduction, P<0.05), resulting in decreased plasma TG (50% reduction, P<0.05). We generated viral vectors expressing WT or A43T apoC-III and expressed these in humanized mouse models to further explore the mechanism of reduced apoC-III levels due to the A43T variant. Mice expressing human CETP and the apoC-III A43T variant exhibit reduced plasma apoC-III (50% reduction, P<0.0001) despite equal hepatic expression and secretion relative to controls expressing WT human apoC-III. These mice also exhibit reduced plasma TG and VLDL-C, and increased HDL-C relative to WT-expressing mice, fully recapitulating the protective lipoprotein profile of the human A43T carriers. Radioisotope-labeled apoC-III turnover studies showed that the A43T mutation causes a >3-fold higher apoC-III clearance rate
in vivo
(P<0.0001) due to defective integration into lipoprotein particles and accelerated renal catabolism (40% increase, P<0.01). This results in increased lipoprotein lipase (LPL) activity (27% increase, P<0.01) and faster chylomicron-TG clearance (97% increase, P<0.01)
in vivo
. We are currently performing analogous studies of WT vs. A43T apoC-III turnover and VLDL clearance in human
APOC3
A43T carriers. Collectively, our results support the rationale for therapeutic efforts to target circulating apoC-III through disruption of its binding to lipoproteins, mirroring the genetics-driven approaches for targeting
PCSK9
that have recently yielded novel therapies.
Abstract 170: Development of a Novel Protein Nanobody Detection System for Serum Soluble Lectin-like Oxidised Low Density Lipoprotein Receptor-1
Authors: De Siqueira Jonathan , Seiler Caroline , Russell David A , Tomlinson Darren , Homer-Vanniasinkam Shervanthi , Ponnambalam Sreenivasan
Published: June 2021

Introduction:
The Lectin-like Oxidised Low Density Lipoprotein (OxLDL) Receptor 1 (LOX-1) is a scavenger receptor found on vascular endothelial cells. LOX-1 is proteolysed at the cell surface and its soluble fragments (sLOX-1) are shed into the extracellular space. Clinical studies have demonstrated a link between serum sLOX-1 concentration and cardiovascular disease. Current technologies for the detection of sLOX-1 rely on costly monoclonal antibodies. Adhirons are antibody mimetics which can be directed against a wide range of molecules. We aimed to investigate whether Adhirons isolated against human sLOX-1 could be used to measure its concentration in solution.

Methods:
BL21 Star DE3
Escheriae Coli
underwent transformation with plasmids for 5 different cysteine-tagged Adhirons. Protein expression was induced and, following bacterial lysis, these were purified using nickel-agarose columns. The binding of Adhirons to the extracellular domain of immobilised, recombinant human LOX-1 was tested by modified direct enzyme linked immunosorbance assay (ELISA). Adhiron-based, sandwich ELISA and Chemiluminescence Enzyme Immunoassay (CLEIA) were developed to detect sLOX-1 in solution. The application of these techniques in the detection of LOX-1 in biological buffers was tested.

Results:
Direct ELISA demonstrated a limit of detection (LOD) of 5 nanograms per millilitre. A complimentary binding pair of Adhirons (H
1
capture, A
1
detection and
vice versa
) was identified. Colorimetry-based ELISA using these Adhirons detected sLOX-1 in phosphate buffered saline at an LOD of 150 nanograms per millilitre. CLEIA enabled a detection limit of 50 nanograms per millilitre, these results were reproduced in the presence of protein blockers (Bovine Serum Albumin, Casein).

Conclusion:
These experiments demonstrate proof of concept for the use of Adhirons as a viable platform for the detection of sLOX-1 in solution. Further refinement and optimisation is needed for the detection sLOX-1 levels in human blood samples (500 - 3000 picogram per millilitre concentration) in order to relate these to human disease.
Abstract 171: Urea Cycle Intermediates and Nitric Oxide Synthase Metabolites Decrease Whilst Asymmetric Dimethylarginine Remained Unchanged Following Cardiac Surgery
Authors: Garlick John , Wilson-O'Brien Amy , Kouremenos Konstantinos A , Williamson Nicholas , Wilson Andrew
Published: June 2021

Background:
Within the endothelium, Nitric Oxide Synthase (NOS) activity through the conversion of Arginine to Citrulline and Nitric Oxide (NO) is largely seen as beneficial. NO is a powerful bioactive molecule which elicits an antithrombotic and antioxidant effect preventing platelet aggregation and atherosclerosis. In immune cells NOS however is largely thought to be beneficial as a microbicidal agent involved in inflammatory responses. Systemic inflammation is also a common outcome in patients undergoing cardiac surgery and those receiving Cardiopulmonary Bypass (CPB) due to contact with foreign surfaces and surgical trauma. Through the Urea Cycle, Citrulline is converted back to Arginine via intermediate Ornithine. Asymmetric Dimethylmethylarginine (ADMA) is an inhibitor of all NOS isoforms, with circulating ADMA being an independent risk factor for mortality within ICU patients and is associated with an increased cardiovascular disease risk profile. Relatively recent studies have demonstrated the beneficial role of ADMA within diseases where iNOS production is overactive such as pulmonary fibrosis.

Hypothesis:
Cardiac surgery and CPB will alter NOS inhibitor ADMA and other metabolites .

Results:
Cardiac surgery patients (n=44) had arterial blood sampled over 4 time points: Morning of surgery (A), Pre (B) and post cardiac injury (C), and post-operative morning (D). Dimethylarginines (ADMA and SDMA) and Urea Cycle intermediates (Arginine, Citrulline and Ornithine) were measured via HPLC-MS/MS. Both Dimethylarginines are released as a function of proteolysis, ADMA remained unchanged over surgery (0.48±0.09μM Vs 0.46±0.08μM Vs 0.50±0.09μM Vs 0.43±0.07μM,
p<
0.05) whilst SDMA increased during surgery (B) from morning of the surgery (A) (0.73±0.21μM Vs 0.89±0.25μM;
p<0.01
) then decreasing post-surgery (D) (0.77±0.33μM) and Arginine (A; 111.83±53.74μM Vs B; 67.91±48.96μM;
p<0.01,
D; 60.41±34.26μM, A Vs D;
p<
0.001) and other metabolites steadily decreased.

Conclusions:
These findings indicate that the regulation of circulating ADMA compared to SDMA is homeostatic even within cardiac surgery. NOS substrate Arginine and Urea Cycle intermediates are consumed which may be predictive of post-operative morbidity.
Abstract 174: Periadventitial or Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury
Authors: Helkin Alex , Bruch David , Wilson David R , Bader Rebecca , Maier Kristopher G , Gahtan Vivian
Published: June 2021

Introduction:
Oral statin administration reduces intimal hyperplasia (IH) after arterial injury by only ~25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid tagged polysialic acid-polycaprolactone micelles)
in vitro
on vascular smooth muscle cell (VSMC) migration and
in vivo
after arterial injury. We hypothesized both free and micelles containing statins would inhibit VSMC chemotaxis and local statin treatment would reduce IH in Sprague-Dawley (SD) rats following carotid balloon injury greater than oral delivery.

Methods:
VSMCs pretreated with free simvastatin (1 μM, 20 min or 20 hrs) or simvastatin-loaded micelles underwent chemotaxis to thrombospondin-1 (20 μg/mL) or serum-free media using a modified Boyden chamber. Next, SD rats (13-15 wks) who underwent balloon injury of the common carotid artery (5 atm/5 min) received statin therapy – either intraluminal simvastatin loaded micelles (50 μM, pressurized for 5 min) prior to injury or periadventitial pluronic gel (50 μM simvastatin) following injury. Arteries were harvested 14 days postoperatively. Morphometric analysis was performed using the intimal/media area ratio defined as intima/(intima + media). Findings were compared to our historic animals which received oral simvastatin (10 mg/kg/day) or controls without statin therapy. Statistical analysis was by ANOVA and
p
<0.05 was significant.

Results:
Simvastatin loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%,
p
<0.05). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively,
p
<0.05). However, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03,
p
>0.05).

Conclusions:
Intraluminally and periadventially delivered statins were effective in reducing IH. The efficacy of single dose, locally delivered statins alone may lead to novel treatment modalities for the prevention of IH. The different routes of administration allow for treatment at the time of both open and endovascular procedures, without the need for systemic therapy.
Abstract 178: Angiogenesis is Triggered by Nutrient Deprivation via Gcn2/atf4-dependent Regulation of Vegf and H2s Production
Authors: Longchamp Alban , Mirabella Teodelinda , Hine Christopher , Brace Lear E. , Knudsen Nelson , Treviño-Villarreal J. Humberto , Mejia Pedro , Tao Ming , Sharma Gaurav , Wang Rui , Corpataux Jean-Marc , Haefliger Jacques-Antoine , Ahn Kyo Han , Lee Chih-Hao , Chen Christopher , Ozaki Charles Keith , Mitchell James R.
Published: June 2021

Objective:
Angiogenesis is crucial to maintain tissue homeostasis under nutrient and oxygen deprivation (ischemia). Although considerable evidence supports that angiogenesis is regulated by hypoxia-HIF1α induction of vascular endothelial growth factor (VEGF), the role of nutrient deprivation in angiogenesis is poorly defined.

Approach and Results:
We report that nutrient deprivation in the form of dietary sulfur amino acid restriction (Methionine/cysteine Restriction; MR) promotes VEGF expression and functional growth of new capillaries in skeletal muscle of mice (Fig.1 A, B). This occurred independently of hypoxia or HIF1α, but instead required the amino acid-sensing eIF2α kinase GCN2 and the transcription factor ATF4 (Fig. 1C). In addition to increased VEGF, nutrient deprivation boosted production of the pro-angiogenic gas hydrogen sulfide (H
2
S) via increased GCN2/ATF4-dependent expression of the H
2
S-generating enzyme cystathionine-gamma-lyase (CGL). The genetic requirement for CGL in angiogenesis triggered by nutrient deprivation, exercise or local VEGF overexpression, as well as the ability of local CGL overexpression to promote angiogenesis in vivo, revealed the critical importance of CGL-derived H
2
S in angiogenesis (Fig. 1D). Finally, plasma H
2
S was reduced in patients with vascular disease (versus non-diseased age-matched controls), and correlated with 2-year survival following vascular surgery (Fig.1 E, F).

Conclusions:
These data reveal a nutrient-sensing pathway targetable by diet as a previously unrecognized central regulator of VEGF expression and angiogenesis independent of canonical hypoxic signaling. This discovery points to novel dietary interventions and GCN2/ATF4/CGL/H
2
S-based strategies to manipulate angiogenesis.

Figure 1
Abstract 185: Androgen Deficiency-induced Hyperplasia Development is Attenuated by Physiological Testosterone Replacement Independent of Metabolite Dihydrotestosterone
Authors: Univers Junior , Freeman Brian M , Mountain Deidra J , Kirkpatrick Stacy S , Arnold Joshua D , Goldman Mitchell H , Klein Frederick A , McNally Michael M , Stevens Scott L , Terry Paul D , Freeman Michael B , Grandas Oscar H
Published: June 2021

Objectives:
Androgen deficiency (AD) is associated with increased risk of cardio- and peripheral vascular disease, yet the underlying biochemical mechanisms remain unclear. Systemically testosterone (TST) is enzymatically reduced to its more potent metabolite dihydrotestosterone (DHT) or is aromatized to estradiol, which differentially stimulate androgen and estrogen receptor-mediated pathways, respectively. We have previously demonstrated an inverse relationship between TST levels and the cellular processes of intimal hyperplasia (IH) in vitro. Here we investigated TST and DHT replacement in the attenuation of IH in an in vivo model of AD.

Methods:
Sub- to high physiologic levels of TST or DHT was administered via pellet implants in aged orchiectomized rats (0.5-5mg). Young intact (YI), aged intact (AI), and orchiectomized placebo (Plac) rats served as controls. After 14d hormone replacement rats underwent balloon angioplasty of the left common carotid. 14d post-injury animals were euthanized, systemic hormone levels were determined by ELISA and comparative weight analysis of androgen sensitive organs (Table 1), and carotid intima:media (I:M) was quantified.

Results:
I:M was decreased in AI animals and with higher physiological TST replacement compared to YI controls (Fig 1). I:M was higher in Plac, sub- and low-physiological TST animals and at all DHT levels.

Conclusions:
Aging and the normal reduction of TST was protective against IH when compared to young animals. However, pathological AD and sub-physiological hormone replacement increased IH. While physiological TST replacement attenuated this effect, equivalent DHT replacement was not protective, but instead exacerbated the hyperplastic response. Future studies will investigate if the protective effect of physiological TST replacement could be via its conversion to estradiol and downstream estrogen receptor signaling and if estrogen therapy attenuates IH in AD males.
Abstract 2: Loss of
Rictor
in Macrophages Suppresses Their Viability and Reduces Atherosclerosis in LDLR Null Mice
Authors: Babaev Vladimir R , Ding Lei , Zhang Youmin , May James M , Linton MacRae F
Published: June 2021

The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that plays a central role in the regulation of cell viability, growth and metabolism. mTOR complex 2 (mTORC2) directly activates phosphorylation of Akt at S
473
, promoting pro-survival signaling. Rictor is an essential component of mTORC2, and genetic loss of
Rictor
inactivates the complex. To examine whether macrophage mTORC2 signaling has an impact on atherosclerosis, we transplanted male
Ldlr
null mice with bone marrow isolated from male mice with myeloid-specific
Rictor
deletion (
Rictor
-/-
, n=9) and control marrow from
Rictor
flox-flox mice (
Rictor
flox/flox
; n=10). Compared to control mice reconstituted with
Rictor
flox/flox
cells, the recipients of
Rictor
-/-
bone marrow cells exhibited dramatic changes in blood cells including lower levels of white blood cells, B-cells, T-cells and monocytes but had similar levels of neutrophils. After 8 weeks of the Western diet, both groups of recipients had similar levels of body weight, blood glucose, plasma total cholesterol and triglycerides. However,
Rictor
-/-
→
Ldlr
-/-
mice developed smaller atherosclerotic lesions in the proximal and distal aorta (46 and 40% reduction, respectively). These lesions contained less macrophage area and more apoptotic macrophages than lesions of control
Rictor
flox/flox
→
Ldlr
-/-
mice. Importantly, blood monocytes and peritoneal macrophages isolated from
Rictor
-/-
→
Ldlr
-/-
mice were more sensitive to apoptotic stimuli compared to control
Rictor
flox/flox
cells. In response to LPS,
Rictor
-/-
macrophages exhibited the M1 phenotype with high levels of pro-inflammatory gene expression. Both
Rictor
-/-
blood monocytes and macrophages had lower levels of
Il10
gene expression than
Rictor
flox/flox
cells. Thus, loss of
Rictor
and, consequently, mTORC2 in monocyte/macrophages significantly compromises their survival, and this markedly diminishes early atherosclerosis in
Ldlr
-/-
mice. Our results indicate that mTORC2 is a key signaling regulator of macrophage survival and inflammatory responses and promote atherosclerosis.
Abstract 202: MiR-1200 Differentially Modulates Plasma LDL and HDL-cholesterol Levels to Reduce Hyperlipidemia and Atherosclerosis in Mice
Authors: Zhou Liye , Irani Sara , Hussain Mahmood
Published: June 2021

High LDL and low HDL are risk factors for heart disease. Drugs that can lower LDL and increase HDL might be ideal for the treatment of cardiovascular diseases. We performed a high throughput screening of human microRNA (miR) mimic library to identify miRs that regulate apoB (LDL scaffolding protein) and apoAI (major HDL protein) secretion in human hepatoma Huh-7 cells. MiR-1200 potently decreased apoB and increased apoAI secretion. We found that seed sequence of miR-1200 interacts with the 3’-untranslated region of apoB mRNA to enhance posttranscriptional degradation and reduce apoB secretion. In contrast, miR-1200 increased apoAI protein and mRNA levels by increasing transcription. Mechanistic studies revealed that miR-1200 reduced the expression of B-Cell Lymphoma 11B (BCL11B), a repressor of apoAI transcription, to increase apoAI expression.
In vivo
studies showed that overexpression of miR-1200 significantly reduced LDL-cholesterol and increased HDL-cholesterol levels without causing hepatic steatosis in Western diet fed C57Bl6J mice. Additionally, miR-1200 reduced atherosclerosis in Western diet fed

Apoe
-/-

mice. Physiologic studies showed that miR-1200 reduced VLDL production. Further, HDL from miR-1200 injected mice showed increased cholesterol efflux capacity from lipid loaded macrophages suggesting increases in functionally adept HDL. In short, we have identified a novel miR-1200 that reduces dyslipidemia by reducing apoB-containing lipoproteins and increasing functional HDL and suggest that it can be useful in treating hyperlipidemia and atherosclerosis.
Abstract 203: Regulation of Lipid Phosphate Phosphatase 3 in Cardiac Ischemia/Reperfusion Injury and Hypertrophy
Authors: Chandra Mini , Fox Jonathan , Orr Wayne , Kevil Christopher , Miriyala Sumitra , Panchatcharam Manikandan
Published: June 2021

Generation of reactive oxygen species (ROS) has been implicated in myocardial infarction (MI), stroke and sudden cardiac death. Mitochondrial respiration is a major source of ROS production and lipids regulate mitochondrial oxidative metabolism and homeostasis through effects on mitochondrial fusion and fission and on the activity of mitochondrial membrane proteins. Lipid phosphate phosphatases (LPPs) control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. These include phosphatidic acid (PA), and lysophosphatidic acid (LPA). Oxidative stress was identified to transactivate microRNA-92a, which is a negative regulator of LPP3. We found that LPP3 expression was markedly down regulated in ischemic regions after ischemia/reperfusion (I/R) injury. We observed a similar trend in the myocardium from patients with acute MI at 24h. Our in vitro studies indicate that overexpression of LPP3 protects the cardiomyocyte against ROS-induced cardiac injury and reduction of LPP3 by conditional specific cardiac knockout of the LPP3 gene in mice increases cardiac dysfunction and mortality. These mice are viable and fertile but showed increased mortality ~8 months (Fig1). Blood pressure was similar in LPP3
fl/fl
(96 ± 9 mmHg; n = 19) and Myh6- LPP3
Δ
mice (92 ± 7 mmHg; n = 19), although heart rates were significantly higher in Myh6- LPP3
Δ
3 month old mice (642 ± 21 bpm, compared to LPP3
fl/fl
with 600± 17 bpm; P<0.001). Knockdown of LPP3 enhanced cardiomyocyte hypertrophy induced by LPA based on analysis of sarcomere organization, cell surface area, levels of fetal genes ANP and BNP, and ANF release from nuclei, which are hallmarks of cardiomyocyte hypertrophy, indicating that LPP3 negatively regulates cardiomyocyte hypertrophy induced by LPA.
Abstract 22: Absence of Circadian Gene Bmal in Macrophage Enhances Atherosclerosis
Authors: Tian Xiao Yu , Huang Yuhong , Wong Wing Tak , Chawla Ajay , Huang Yu
Published: June 2021

Backgrounds:
Peripheral cell-intrinsic clock is present in all types of cells and is important for synchronization of physiological function in response to changes of environment. Bmal (encoded by Arntl gene) is a transcription factor that forms complex with Clock and induces rhythmic transcription. Peripheral clock has been demonstrated in monocyte to regulate inflammatory responses. In this regards, we hypothesize that disruption of circadian rhythm in myeloid cells enhances atherosclerosis in Apoe
-/-
mice.

Methods and Results:
Apoe
-/-
mice housed under weekly switch between regular (ZT12-ZT0: dark) and inverted (ZT12-ZT0: light) ligh/dark cycle developed more atherosclerotic lesion than Apoe-/- mice housed under regular L/D cycle. Myeloid cell-specific Bmal knockout mice (

Arntl
LoxP/LoxP
Lyz2
Cre

) as Bmal
MKO
and Bmal wild type (

Arntl
LoxP/

LoxP
) as Bmal
MWT
were crossbred with Apoe-/- to generate Apoe-Bmal
MKO
and Apoe-Bmal
MWT
mice. Mice with disruption of myeloid circadian rhythm (Apoe-Bmal
MKO
) on Western diet showed enhanced atherosclerotic plaque formation by en face oil red o staining, compared with their WT control. Flow cytometry showed more infiltrating macrophages within the plaque of Apoe-Bmal
MKO
mice, accompanied with upregulation of proinflammatory genes Tnf, Il1b, Nos2, S100a8; chemokines and adhesion molecules Vcam1, Icam1, Sele, Ccl2, Ccl8; and downregulation of anti-inflammatory genes. Aortic root staining showed more macrophages by CD68 staining, more CD11c
+
pro-inflammatory macrophages, and more Ly6c
+
infiltrating monocyte-derived macrophages in Apoe-Bmal
MKO
mice. In vitro culture of bone marrow derived macrophage from Apoe-Bmal
MKO
and Apoe-Bmal
MWT
mice demonstrated that deletion of myeloid Bmal impaired rhythmic transcription of Ccl2 and Ccl8, causing higher expressions. Meanwhile, lipid profile and endothelial function were unaltered in Apoe-Bmal
MKO
compared with Apoe-Bmal
MWT
mice.

Conclusions:
Our data showed disruption of myeloid circadian rhythm increased inflammation and enhanced atherosclerosis
Abstract 226: Mechanism of LCAT Activation by Compound A
Authors: Freeman Lita A , Demosky Stephen J , Konaklieva Monika , Kuskovsky Rostislav , Gordon Scott M , Ossoli Alice F , Vaisman Boris L , Shamburek Robert D , Aponte Angel , Gucek Marjan , Tesmer John J , Levine Rodney L , Remaley Alan T
Published: June 2021

Background:
Lecithin:cholesterol acyltransferase (LCAT) catalyzes cholesteryl ester (CE) production from free cholesterol (FC) and phosphatidylcholine (lecithin), promoting HDL formation.

Objective:
To investigate activation of LCAT by Compound A (Amgen), a previously described small-molecule activator of LCAT, with the ultimate goal of developing novel LCAT activators for therapeutic use.

Methods:
LCAT activity in plasma from Familial LCAT Deficiency (FLD) patients with different mutations was quantitated by TLC before and after addition of Compound A. HEK293 cells were transiently transfected with plasmids containing wild-type (WT) or mutant LCAT cDNA. Media was then incubated with either vehicle or Compound A and LCAT activity was quantitated using a novel plate assay utilizing Methylumbelliferyl Palmitate as a substrate.

Results:
Compound A increased LCAT activity for a subset of FLD mutations to a level above which renal disease may occur. Mutations of Cys31 in vitro strongly affected basal LCAT activity as well as activation by Compound A. Charged residues at position 31 profoundly decreased activity whereas bulky hydrophobic groups increased LCAT activity up to 3-fold (p < 0.005, all). Mass spectrometry of WT LCAT incubated with Compound A revealed a +103.017 m/z adduct to the tryptic peptide containing Cys31, indicative of a cyanopyrazine adduct to LCAT Cys31. Molecular modeling identified potential binding sites of Compound A to LCAT.

Conclusions:
Our findings yield important mechanistic insight into LCAT activation that can be used to design novel LCAT activators for therapeutic use.
Abstract 230: Lipoprotein X Causes Renal Disease in LCAT Deficiency
Authors: Neufeld Edward B , Ossoli Alice , Thacker Seth G , Vaisman Boris , Pryor Milton , Freeman Lita A , Brantner Christine A , Baranova Irina , Francone Nicolás O , Demosky Stephen J , Vitali Cecilia , Locatelli Monica , Abbate Mauro , Zoja Carlamaria , Franceschini Guido , Axley Milton J , Karathanasis Sotirios K , Calabresi Laura , Remaley Alan T
Published: June 2021

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, and chemical properties, to wild-type and
Lcat
-/-
mice. Our
in vitro
and
in vivo
studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in
Lcat
-/-
mice, which have low HDL but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of
Lcat
-/-
mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive
in vivo
EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes, where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA
2
and induced podocyte secretion of pro-inflammatory IL-6
in vitro
and renal Cxl10 expression in
Lcat
-/-
mice. In conclusion, LpX is a nephrotoxic particle that in the absence of LCAT induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
Abstract 241: O-GlcNAcylation of Runx2 in Nuclear Matrix Targeting Signal Modulates Vascular Calcification
Authors: Byon Chang Hyun , Heath Jack , Mao Xia , Sun Yong , Chen Yabing
Published: June 2021

Vascular calcification is prevalent in patients with atherosclerosis, diabetes, and chronic kidney diseases and increases the mortality and morbidity of those patients. Osteogenic differentiation of vascular smooth muscle cells (VSMC) has been recognized as a key feature of the calcification process. Using smooth muscle-specific deletion mouse model, we have demonstrated an essential role of SMC-derived Runx2 in regulating vascular calcification in atherosclerosis in vivo. The present study further defined Runx2 regulation and its functional domains that control osteogenesis of VSMC. Using a serial of Runx2 truncation mutants, we located the domain between amino acids 391 and 432 as responsible region for Runx2 osteogenic function in VSMC. This region contains the nuclear matrix targeting signal, which has been shown to interact with Smad proteins. Accordingly, we determined the contribution of Smad proteins in Runx2-regulated VSMC calcification. By knockdown individual Smad in VSMC, we demonstrated that Smads 1/5/8, but not Smads 2/3, were required for Runx2 osteogenic function and VSMC calcification. In addition, we found the osteogenic function of Runx2 was abolished by inhibition of protein O-GlcNAcylation, a unique posttranslational modification that we have recently reported to be critical in regulating VSMC calcification. Runx2 O-GlcNAc modification was further demonstrated by immunoprecipitation. Based on bioinformatics analysis, we found several putative O-GlcNAcylation sites within Runx2 osteogenic domain. Using site-directed mutagenesis, we demonstrated that mutation on S385, S387, T404, T406, T412, S413, or T427 decreased Runx2 O-GlcNAcylation and Runx2 binding to Smads 1/5/8, and thus decreasing Runx2 activity and VSMC calcification. In summary, we have identified the Runx2 osteogenic functional domain in VSMC; and demonstrated that O-GlcNAc modification of Runx2 in its osteogenic functional domain is critical for Runx2-regualted VSMC calcification. Our studies provide molecular insights into the regulation and function of Runx2 in VSMC calcification, which may shed lights on novel targets that are amenable to drug discovery for vascular calcification.
Abstract 243: Regression of Carotid Intima-media Thickness and Improvement of Lipid Profile by the Addition of High-purity Eicosapentaenoic Acid (Icosapent Ethyl) to Statins in a Morbidly Obese Patient with Type II Diabetes Mellitus
Authors: Reddy Kota J , Chowdhury Sumita
Published: June 2021

Objective:
The AHA scientific statement on obesity identifies obesity as an important independent metabolic/genetic risk factor for ASCVD. For patients at high risk of ASCVD and with less-than-anticipated therapeutic response to statin therapy, the 2013 ACC/AHA cholesterol guideline recommends increasing statin intensity, or if on maximally tolerated statin intensity, considering non-statin therapy. The 2013 ACC/AHA cholesterol guideline evidence statements also describe the JELIS trial, in which 1.8 g/day of EPA added to statin therapy led to a 19% relative risk reduction in major coronary events in Japanese patients with hypercholesterolemia. Additionally, in the CHERRY trial of high-risk cardiac patients treated with moderate- to high-intensity statin therapy, the addition of 1.8 g/day of EPA resulted in significantly greater prevalence of coronary plaque regression compared to statin therapy alone (50% vs 24%; p<0.001). Further, in patients with DM, ASCVD is the primary cause of death.

Methods:
We reviewed the impact of initiating a prescription high-purity EPA drug, icosapent ethyl (2 g/day), in a morbidly obese (485 lb) 51-year-old African-American male patient with hypertension, DM, and ASCVD (evidenced by high CIMT) while on maximally tolerated statin therapy. Diet, exercise, and medications were stable for the duration of this report. Lipids, A1C, HOMA-IR, and CIMT were evaluated before and 4 months after initiating icosapent ethyl.

Results:
See table.

Conclusions:
Adding high-purity prescription EPA treatment (icosapent ethyl) to a maximally tolerated statin dose in a morbidly obese African-American patient with DM led to substantial regression of carotid intima-media thickness and to improvements in measured lipid parameters and two laboratory measures of DM. The clinical implications of adding icosapent ethyl to statin therapy is being evaluated in the ongoing cardiovascular outcomes trial, REDUCE-IT.
Abstract 250: Kidney and Liver are the Main Organs of Alanine: Glyoxylate Aminotransferase 2 Expression in Humans
Authors: Jarzebska Natalia , Georgi Sophia , Jabs Normund , Brilloff Silke , Maas Renke , Rodionov Roman N , Zietz Christian , Weiss Norbert
Published: June 2021

Background:
Numerous epidemiological studies have demonstrated an association between elevated levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) with cardiovascular diseases. ADMA can be hydrolysed by dimethylarginine dimethylaminohydrolase (DDAH). It can also be metabolized through a much less explored alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2), which converts ADMA to α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). It has been shown in previous Northern Blot and in-situ RNA-hybridisation experiments that the kidney is the main organ of
Agxt2
expression in rats, while RT-PCR and Western Blot studies suggested that
Agxt2
is expressed in the mouse kidney and liver at comparable levels. The goal of the current study was to analyse the expression of the enzyme in human tissues.

Methods and Results:
We performed immunohistochemical staining using two rabbit polyclonal anti-AGXT2 antibodies in both frozen and paraformaldehyde-fixed samples from a normal tissue bank. We saw the strongest expression of
AGXT2
using both of the antibodies in the proximal convoluted tubule of the kidney and in the liver with a homogenous pattern throughout all the samples. We also observed weak staining in the skeletal and heart muscle. Aorta, small intestine and lungs were negative for
AGXT2
expression. We also confirmed our immunohistochemistry data by RT-PCR.

Conclusions:
Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We are currently designing experiments to show the direct localization of AGXT2 in a human cell.
Abstract 263: Role of Micro RNA-21 in Atherosclerosis
Authors: Hamed-Berair Rihab E , Sithu Srinivas D , Wickramasinghe Nalinie , Shah Jasmit , Agawral Abhinav , Winner Millicent G , Wysoczynski Marcin , Bhatnagar Aruni , Li Yong , Srivastava Sanjay
Published: June 2021

Micro RNAs (miR) are short non-coding RNAs that regulate several genes under pathophysiological conditions. Accumulating evidence suggest the involvement of miR in atherogenesis. However, limited information is available about atherogenic miR and the underling mechanisms by which miR affect atherogenesis. Our data shows that 12 weeks of western diet (WD) in LDL receptor-knockout (LDLR-KO) mice upregulated 99 and downregulated 50 miR in the aorta. Among the 41 differentially expressed miR associated with macrophage inflammation and apoptosis, expression of micro RNA-21 (miR-21) was increased by 1.4-fold (P<0.05). WD also increased the expression of miR-21 by 1.5-fold in bone marrow derived macrophages (BMDM).
In vitro
, LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in BMDM. Basally, miR-21 deficient BMDM showed increased secretion of IL-6, IL-9 and CXCL-2,-3,-4, and -10 (P<0.05)); and increased early and late apoptosis (2-3-fold, P<0.05). We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with LPS significantly increased the activation of NF-κB and enhanced the secretion of several pro-inflammatory cytokines including TNFα, IL-6, IL-12 and CXCL-2 (2-10 fold; P<0.05); interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86). Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase -3, -7 and -9. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis, at least in parts, by preventing macrophage apoptosis and inflammation.
Abstract 267: Identification of Proteolytic Substrates for ADAMTS7 Using Terminal Amine Isotope Labeling of Substrates
Authors: Zhang Xuan , Klein Theo , Cui Jian , Ou Kristy , Hinkle Christine , Li Wenjun , Overall Christopher M , Reilly Muredach P
Published: June 2021

ADAMTS7
, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, has been recently identified as a novel genetic locus for coronary artery disease. Although a handful of ADAMTS7 substrates have been identified through traditional yeast two-hybrid screening and standard proteomic profiling, the molecular mechanisms of ADAMTS7 contribution to atherosclerosis have not been fully explored in the context of its proteolytic action. Here we utilized terminal amine isotope labeling of substrates (TAILS), a cutting-edge proteomic approach to enrich N-terminal peptides, enable systemic characterization of proteolytic events and substrate identification for ADAMTS7. Briefly, primary vascular smooth muscle cells (SMC) from ADAMTS7 WT and KO mice were treated with TNFα to stimulated endogenous ADAMTS7 expression, followed by TAILS using dimethyl labeling in cellular proteome and secretome. In 3 biological replicates, we identified 1,720 and 1,616 quantified N-terminal peptides in SMC proteome and secretome, which composed of 415 and 122 unique N-termini of mature protein as well as 1,185 and 1,161 cleaved neo-N-termini driven by proteolytic processing. As expected, most of acetylated peptides in SMC proteome and secretome are natural N-termini of mature proteins (82% and 77%, respectively) while free N-terminal peptides are remarkably enriched with cleaved neo-N termini (85% and 96%, respectively). Importantly, 106 and 86 neo-N-termini (corresponding to 65 and 54 unique proteins) were enriched in ADAMTS7 WT SMC proteome and secretome respectively (WT/KO isotope ratio >2), suggesting that these proteins underwent elevated proteolysis in ADAMTS WT SMCs and thus are proteolytic substrate candidates for ADAMTS7. Interestingly, gene pathway analysis indicates that these proteins are enriched with genes involved in proteinaceous extracellular matrix and extracellular region, such as vimentin, fibulin and MMPs. Biochemistry studies are ongoing to validate these ADAMTS7 substrate candidates. In summary, our study presents a systemic profiling on proteolytic events in SMC cellular proteome and secretome and identifies candidate ADAMTS7 substrates relevant to smooth muscle function and vascular biology.