bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 333: Vascular Type Ehlers Danlos Syndrome is Associated With Thrombocyte Dysfunction and Low Vitamin D Serum Concentration
Authors: Busch Albert ,   EDS Rsch Group, Maegdefessel Lars , Wildenauer René
Published: June 2021

Introduction:
The Ehlers-Danlos syndrome is a rare genetic disorder characterized by a heterogeneous mixture of orthopedic, visceral, ophthalmological and vascular affection based on an even broader variety of genetic subtypes. Besides disturbance in all-day life, vascular type EDS often results in life threatening spontaneous bleeding or aneurysm formation with high risk of rupture. Additional impairment of normal hemostasis, especially in emergency scenarios, is suspected.

Material and methods:
In collaboration with the German National EDS Initiative and funding by the Eva Luise and Horst Köhler Foundation for Rare Diseases, we set up a nationwide screening of blood coagulation tests in vascular type and mixture type EDS patients under normal conditions. Screening was made for blood count, bleeding time, factor XIII, fibrinogen, PFA100, Born aggregometry, ROTEM analysis, C-reactive protein and von Willebrand Factor activity.

Results:
The genetics of 25 vascular type EDS diagnosed patients in Germany are very heterogeneous, making results difficult to compare. Coagulation disorders of various kinds, however, do appear and affect every aspect of the coagulation cascade. Phenotypes worse within families with clear inheritance patterns suggesting a loss of function. Additionally Vitamin D3 shortage might impair bone mineralization worsening common orthopedic symptoms as well as vascular structural health.

Outlook:
Decision making for vascular surgery in EDS patients should be made very carefully. A complete coagulation workup together with a hematologist should precede surgery whenever possible. Our study will continue to enroll patients in order to generalize results and verify coagulation disorders.
Abstract 341: The Role of Nucleotidase in Arterial Thrombosis
Authors: Covarrubias Roman , Chepurko Elena , Novitskaya Tatiana , Dwyer Karen M , Robson Simon C , Cowan Peter J , Gumina Richard J
Published: June 2021

Objective:
To determine how leukocyte nucleotidase affects arterial thrombosis.

Approach and Results:
Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) is expressed on circulating cells, endothelium and smooth muscle cells where it hydrolyzes extracellular ATP or ADP to AMP. We have demonstrated that transgenic mice with a global overexpression of human CD39 (hCD39-Tg) are protected against ferric chloride-induced carotid artery thrombosis. Furthermore, transplant of hCD39-Tg bone marrow into WT recipient mice increases the time to thrombosis when compared to recipient mice (wild-type or hCD39-Tg) receiving wild-type bone marrow. Based upon these data and previously published work, we hypothesized that CD39 expression on leukocytes is responsible for the prolongation of the time to thrombosis measured in hCD39-Tg mice. To test this hypothesis, we first performed
ex vivo
mixing experiments. Addition of hCD39-Tg monocytes to WT blood inhibits the expression of activated glycoprotein IIb/IIIa on platelets in response to ADP as measured by FACS analysis (Baseline: 1224 ± 94.9 MFI
vs
hCD39-Tg monocytes: 663.5 ± 61.5 activated glycoprotein IIb/IIIa MFI: n=4; p< 0.001). Subsequently,
in vivo
we demonstrated that monocytes with increased CD39 contribute to extending the time to thrombosis. Clodronate liposome depletion of monocytes (WT: 69% decrease; hCD39-Tg: 63% decrease) resulted in a normalization of the time to thrombosis in hCD39-Tg mice (8.0 ± 1.07 minutes, n = 10) when compared to control loaded liposomes (120.0 ± 0.0, n = 14). No changes in the time to thrombosis were detected in wild-type mice treated with clodronate (8.6 ± 1.35 minutes, n = 8) or control liposomes (7.8 ± 0.80 minutes, n=8).

Conclusion:
Increased expression of CD39 on monocytes can inhibit platelet activation and extend the time to thrombosis following ferric chloride-induced carotid artery injury.
Abstract 347: Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease
Authors: Alicea Isamar , Gil de Lamadrid Daniel , Santiago Analaura , Cazares Joshua , Valentin Shirley , Rivera Alicia , Romero Jose R , Inostroza-Nieves Yaritza
Published: June 2021

Sickle Cell Disease (SCD) is characterized, in part, by vascular endothelial cell activation, increased oxidative stress and sickle cell adhesion. Excess levels of the vasoconstrictor, endothelin-1 (ET-1), activate endothelial cells, induce oxidative stress and inflammation in the vascular wall and regulate erythrocyte homeostasis, in part, via activation of protein disulfide isomerase (PDI). We measured circulating ET-1, TGFβ1 and PDI activity in two sickle transgenic knockout mouse models expressing human sickle hemoglobin (Hb), BERK and βS
Antilles
. We observed significant increases in all three markers (n=6; p<0.01) when compared to either C57BL/J6 mice or knockout mice expressing normal human Hb A. We then intraperitoneally injected BERK mice for 14 days with ET-1 receptor antagonists and observed significant reductions in these circulating markers (n=6; p<0.05). These studies are important as PDI is proposed to control hemostasis, thrombosis and is reported to regulate the major histocompatibility complex (MHC). In addition, activated endothelial cells express PDI and MHC. However, the relationship between ET-1 and MHC in SCD remains unclear. We characterized the role of ET-1 on MHC expression in the endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. Increased expression of MHC was significantly blocked by co-incubation of cells with 10μM BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies showed that ET-1 incubation produced an increase in Histone H3 acetylation of the promoter in MHC molecules (HLA-A2 4.6 ± 1.4 folds, HLA-DRB 6.0 ± 0.7 folds, p<0.01, n=3) in these cells; an event that was likewise blocked by BQ788. In addition, ET-1 stimulated recruitment of CIITA to the promoter of HLA-A2 (1.9 ± 0.7 folds, p<0.05, n=3) and HLA-DRB (3.0 ± 0.6 folds, p<0.01, n=3). These results implicate ET-1 as a novel regulator of MHC promoter activity and suggest that ET-1 receptor blockade represents an important therapeutic approach to improve both the inflammatory and vascular complications of SCD.
Abstract 35: DVT Inflammation Assessed by 18F-FDG-PET/CT Predicts Subsequent Vein Wall Scarring
Authors: Kessinger Chase W , Tawakol Ahmed , Wojtkiewicz Gregory R , Henke Peter K , Weissleder Ralph , Jaffer Farouc A
Published: June 2021

Objective:
While venous thrombosis (VT)-induced inflammation facilitates thrombus resolution, inflammation causes vein wall scarring (VWS). Recently, statins have shown to improve VT resolution and reduce VT inflammatory components. In this study, we hypothesized that early VT inflammation detected by 18F-FDG positron emission tomography/computed tomography (PET/CT) could predict subsequent late stage VWS, and would be attenuated by statin therapy.

Methods:
Stasis VT was induced in 8-12 week old male C57BL/6 mice (n=31) in either the right jugular vein (n=13) or inferior vena cava (IVC,n=18). Animals in the IVC VT cohort were randomized to statin (n=8) or control (n=10) treatment. Statin, rosuvastatin (5mg/kg), was administered by oral gavage, daily starting 24 hours prior to VT induction; control mice received saline. All mice underwent survival FDG-PET/CT venography imaging on day 2. FDG inflammation signals (standard uptake value=SUV) were measured in the thrombosed vein and compared to the sham-operated venous segments or treatment control. On day 14, mice were sacrificed and VT tissue was resected. Picrosirius red staining allowed measurement of collagen and vein wall thickness in VT sections.

Results:
FDG-PET/CT at day 2 revealed increased inflammation signal activity in jugular VT (SUV 1.43 ± 0.3 VT vs. 0.81 ± 0.3 contralateral vein, p<0.0001). Statin-treated mice showed a trend of decreased inflammation signal at day 2 in the IVC VT models (SUV 1.02 ± 0.1 statin VT vs. 1.42 ± 0.2 control VT, p=0.07). Day 14 histological analysis revealed significantly reduced vein wall injury in statin-treated animals (thickness, 32±9.4 μm statin; vs. 56.2±14.7 μm control, p=0.02). Day 2 FDG-PET inflammation in VT correlated positively with the magnitude of day 14 VWS (jugular VT, Spearman r=0.62, p=0.02; IVC VT r=0.74, p<0.001, respectively).

Conclusions:
Quantitative FDG-PET/CT imaging demonstrates that early in vivo VT inflammation predicts subsequent VWS, a driver of post-thrombotic syndrome (PTS). The overall findings strengthen: (i) the link between inflammation and PTS; (ii) the translational potential of FDG-PET inflammation to predict VWS and PTS; and (iii) the concept that statins and other anti-inflammatory therapies could reduce VWS and PTS.
Abstract 354: Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation into Immunogenic Dendritic Cells
Authors: Loperena Roxana , Chen Wei , Kirabo Annet , Harrison David G
Published: June 2021

Fatty acid oxidation leads to formation of highly reactive isoketals that adduct to protein lysines. We have shown that dendritic cells (DCs) from hypertensive mice accumulate isoketal-adducted proteins and promote T cell activation. In hypertension, the endothelium is activated to produce reactive oxygen species and to express adhesion molecules and chemokines that attract inflammatory cells. Human monocytes that traverse the endothelium differentiate into monocyte-derived DCs upon exposure to a pro-inflammatory state. We hypothesized that human endothelial cells exposed to mechanical stretch promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive cyclical stretch (10%) using the Flexcell
®
Tension System for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and monocyte chemokine CCL4 and the p22

phox

subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocyte in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also had an accumulation of isoketal-adducted proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively,
p
= 0.0001) as evidenced by intracellular staining and flow cytometry. Monocytes co-cultured with 10%-stretched HAECs induced a 1,500-fold increase in CD4
+
T cell proliferation and a 1,300-fold increase in CD8
+
T cells proliferation as monitored by CFSE compared to 5% stretch controls. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, prevented both CD4
+
and CD8
+
T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote their differentiation into immunogenic DCs potentially via the JAK/STAT3 pathway. These findings give insight into a new mechanism of lymphocyte activation in the vascular endothelium during hypertension.
Abstract 368: Novel Role of Cofilin in Retinal Neovascularization
Authors: Kumar Raj , Janjanam Jagadish , Singh Nikhlesh K , Rao Gadiparthi N
Published: June 2021

Pak1 plays an important role in several cellular processes including cell migration, but its role in pathological angiogenesis is not known. Here we have determined its role in pathological retinal angiogenesis using Oxygen Induced Retinopathy (OIR) model. VEGFA induced Pak1 and its effector cofilin phosphorylation in time-dependent as well as p38β-dependent manner in HRMVECs. Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVECs F-actin stress fiber formation, migration, proliferation, sprouting, and tube formation. In accordance with these observations, hypoxia induced Pak1 and Cofilin phosphorylation with p38β being downstream to Pak1 and upstream to cofilin. Furthermore, Pak1 deficiency abolished hypoxia-induced p38β and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, siRNA-mediated downregulation of p38β or cofilin levels in WT mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that, while p38β-Pak1-cofilin axis is required for HRMVECs migration, proliferation, sprouting and tubulogenesis, Pak1-p38β-cofilin signaling is essential for hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization.
Abstract 374: Inhibition of Mitochondrial CaMKII Reduces Vascular Smooth Muscle Migration and Neointima Formation and Halts Mitochondrial Mobility
Authors: Nguyen Emily , Koval Olha , Grumbach Isabella
Published: June 2021

Background:
Restenosis after angioplasty for coronary vascular disease remains a critical problem in cardiovascular medicine. Vascular smooth muscle cell (VSMC) migration and proliferation cause restenosis through neointima formation. Mitochondrial motility is likely necessary for cell proliferation and migration, and is inhibited in microdomains with increased Ca
2+
. The Ca
2+
/calmodulin-dependent kinase II (CaMKII) in mitochondria (mtCaMKII) is proposed to control mitochondrial matrix Ca
2+
uptake through mitochondrial Ca
2+
uniporter (MCU). Thus, we hypothesized that blocking mtCaMKII decreases VSMC migration and neointima formation by decreasing mitochondrial motility.

Methods:
mtCaMKII was inhibited by expression of the mitochondria-targeted CaMKII inhibitor peptide (CaMKIIN) in a novel transgenic mouse model in smooth muscle only (SM-mtCaMKIIN) or delivered by adenoviral transduction (Ad-mtCaMKIIN).

Results:
In our models, mtCaMKIIN was detected selectively in mitochondria of VSMC. mtCaMKIIN significantly reduced mitochondrial Ca
2+
current and Ca
2+
content compared to WT in vivo and in vitro. SM-mtCaMKIIN mice showed significantly reduced neointimal area 28 days after endothelial injury (n=8, p<0.05) and fewer proliferating neointimal cells by PCNA staining. In vitro, Ad-mtCaMKIIN mildly reduced VSMC proliferation and mitochondrial ROS production without altering maximal respiration after PDGF treatment. Ad-mtCaMKIIN abolished VSMC migration, as did mitoTEMPO and MCU inhibitor Ru360. Ad-mtCaMKIIN blocked mitochondrial mobility towards the leading edge, while relocation of mitochondria was seen in WT cells 6 h after PDGF treatment. Mitochondrial redistribution was also inhibited by Ru360, but not by mitoTEMPO or cytoplasmic CaMKII inhibition. Mitochondrial fission promotes cell migration. Accordingly, PDGF increased mitochondrial particles in WT VSMC, while mitochondria in Ad-mtCaMKIIN cells were fragmented and unresponsive to PDGF treatment.

Conclusions:
mtCaMKIIN prevents mitochondrial distribution to the leading edge and reduces VSMC migration and neointima formation. These data suggest mitochondrial Ca
2+
regulation plays an important role in VSMC migration by altering mitochondrial location.
Abstract 378: Cytochrome b5 Reductase 3 Regulates Vascular Smooth Muscle Phenotypic Switching
Authors: Vijayvargiya Amogha , Nguyen Anh , Miller Megan P , Hahn Scott , Straub Adam C
Published: June 2021

Contractile vascular smooth muscle cells (VSMC) play a key role in the regulation of arterial blood vessel tone and cardiovascular health. However, in many vascular diseases, VSMCs undergo a phenotypic switch from a contractile state to a synthetic phenotype, where loss of the contractile markers myosin heavy chain 11 (Myh11), smooth muscle alpha actin (ACTA2) and transgelin (SM22) are observed and proliferation is increased. Recent evidence from our lab demonstrates that cytochrome b5 reductase 3 (Cyb5R3) regulates the redox state of soluble guanylate cyclase to control cGMP levels in VSMCs. Because cGMP modulates protein kinase G activity, a critical kinase that maintains VSMCs in a contractile state, we tested the hypothesis that Cyb5R3 is critical for maintenance of the contractile phenotype. To test this hypothesis, we transduced primary rat aortic smooth muscle cells with non-targeting (NT) or Cyb5R3 shRNA followed by serum starvation for 24, 48, and 72 hours to induce phenotypic switching. After each time point, mRNA measurements of Cyb5R3, Myh11, ACTA2, and SM22 were conducted using RT-PCR. In NT shRNA transduced VSMCs, we observed a significant increase in Cyb5R3, Myh11, ACTA2, and SM22 mRNA, but not in Cyb5R3 knockdown VSMCs. Next, we conducted proliferation studies by serum starving NT shRNA and Cyb5R3 shRNA treated VSMCs for 24 hours followed by stimulation of platelet growth factor BB (PDGF-BB, 40 ng/mL). After 24 hours of PDGF-BB treatment, Cyb5R3 deficient cells showed augmented proliferation compared to control cells measured by
3
H-thymidine incorporation. Together, our data suggest that Cyb5R3 is essential for VSMC phenotypic switching and proliferation, which may unravel a new therapeutic target for treating individuals with cardiovascular disease.
Abstract 380: Notch4 Uncouples Endothelial Nitric Oxide Synthase Leading to Arteriovenous Malformations
Authors: Huang Lawrence , Nystoriak Matthew A , Youn Ji Youn , Navedo Manuel F , Cai Hua , Wang Rong A
Published: June 2021

Notch is distinctively expressed in arterial but not venous endothelial cells. Notch signaling regulates arteriovenous (AV) specification, and mutations in Notch signaling lead to AV malformation (AVM) in mice. AVMs are characterized by abnormal AV shunts that displace capillaries. Mechanisms underlying AVM pathogenesis remain poorly understood, hindering therapeutic development. We reported that endothelial expression of constitutively active Notch4 (Notch4*) in mice initiates AVMs
de novo
through enlargement of microvessels without an increase in endothelial cell number or proliferation. Here, we hypothesized that Notch4* disrupts endothelial nitric oxide synthase (eNOS) signaling and vascular tone, thereby permitting vessel enlargement and AV shunting. We show that arteries isolated from Notch4* mutant mice exhibited decreased arterial tone compared to controls, suggesting that Notch4* impaired vascular tone. Administering the NOS inhibitor N
G
-nitro-L-arginine abolished Notch4*-mediated vascular tone impairment. Deletion of the eNOS gene and administration of the NOS inhibitor N
G
-nitro-L-arginine in the Notch4* mutant mice attenuated Notch4*-induced AVM initiation, measured by decreased AV shunt diameter, delayed AV shunting, reduced hemorrhage, pathological lesions, and improved survival, suggesting that eNOS is essential for Notch4* action. In addition, uncoupled eNOS-derived superoxide production was elevated in the Notch4* mice. Our results show that inhibition of eNOS signaling attenuates Notch4*-mediated AVM formation. Furthermore, Notch4* impairs eNOS activity, leading to superoxide production, which results in arterial dysfunction and AV shunt formation.
Abstract 390: Human Aortic Valve Interstitial Cells Exhibit Decreased Cholesterol Efflux Associated with Hypo- α1-nascent HDL Particles
Authors: Hafiane Anouar , Schwertani Adel , Genest Jacques
Published: June 2021

Objectives:
High plasma levels of lipoprotein (a) (Lp(a)) are associated with increased risk of aortic valve stenosis. The purpose of this study was to determine ability of human aortic valve interstitial cells (HAVICs) to generate HDL particles from plasma of patients with high Lp(a), and understand the mechanism involved.

Methods:
HAVICs were isolated from consented participants and cultured in DMEM, 10% FBS, 5% glucose. ABCA1 proteins expression was tested in HAVICs lyses after detection by polyclonal anti-ABCA1 antibody. Cholesterol efflux was determined in HAVICs against HDL isolated by PEG from patients with aortic valve stenosis exhibiting mild or severe plasma Lp(a), compared to normolipidemic controls. FPLC and 2D-PAGGE were used to illustrate HDL profiles from these patients after incubation with HAVICs.

Results:
Our data showed abundant expression of ABCA1, and a significant decrease in HAVICs cholesterol efflux (5%, for 8h) in response to HDL from plasma of patients with high Lp(a) and aortic valve stenosis (mild 16±0.01%cpm, severe 23.4±0.21%cpm) compared to normolipidemic HDL (29.57cpm; P<0.01). This was consistent with FPLC profiles indicating a similar reduction in in cholesterol within nascent HDL (nHDL) when compared to nHDL generated from HDL isolated from plasma of normal controls. Importantly, we noticed a generation of smaller population of HDL particles formed following cholesterol efflux. Plasma from patient with aortic valve stenosis showed a distinct decrease in preβ- and α1-migrating HDL, but no in other α-migrating HDL particles, when compared to plasma from normolipidemic controls. We also observed a defect in α1-HDL in the nHDL generated from patient with high Lp(a) and aortic valve stenosis compared to nHDL from plasma of normal controls.

Conclusion:
These findings demonstrate a defect in HAVICs ABCA1 cholesterol removal associated a loss of α1 species of nHDL in patient with high Lp(a) and aortic valve stenosis. Therefore, the HAVICs cholesterol efflux defect associated with the elevated plasma Lp(a) may explain the pathological changes associated aortic valve calcification.
Abstract 400: Determining the Contribution of Endothelial Lipase-mediated Lipolysis to Brain Phospholipid Metabolism
Authors: Vitali Cecilia , Khetarpal Sumeet A , Billheimer Jeffrey T , Subbaiah Papasani V , Rader Daniel J
Published: June 2021

Polyunsaturated fatty acids (PUFA) are essential constituents of the cell membrane. Brain PUFA content is critical for synaptic function and neuroinflammation and PUFA deficiency may underlie several neurological disorders. Prior work in murine models has showed that the predominant source of central nervous system (CNS) PUFAs is 2-acyl-lysophophatidylcholine (2-lysoPC), a class of phospholipids (PLs) circulating on blood lipoproteins that is formed from the enzymatic activity of phospholipases. Endothelial lipase (EL) is a phospholipase that is a critical determinant of HDL metabolism in humans and the predominant generator of 2-lysoPC on HDL particles. While the contribution of EL to circulating HDL metabolism is well-established, its contribution to CNS fatty acid availability and brain function has not been explored. In order to investigate the physiological contribution of EL to brain FA availability in vivo, we studied PL-derived PUFA uptake in mice with EL deficiency. We adopted a method for measuring EL activity using a fluorescent synthetic HDL particle containing sn2-TopFluor-PC, a fluorescent precursor of 2-lysoPC and demonstrated its utility in vitro. Next, we tested the clearance of this synthetic HDL-derived 2-lysoPC in C57BL/6 (WT) vs EL KO mice (n=12/group). Circulating plasma 2-lysoPC clearance was significantly impaired in in EL KO mice (-32%, p=0.024) and was accompanied by a slower remodeling of the HDL particles. Measurement of labeled PL content in tissues showed a significant reduction in hepatic uptake in KO mice (22% decrease, p=0.002). No significant differences were observed in total brain PL uptake, but the distribution of PL uptake differed between WT and KOs. Ongoing studies are evaluating the physiological consequences of reduced circulating 2-lysoPC clearance, reduced hepatic uptake, and altered distribution of brain PL uptake in EL KO mice. Collectively, our data demonstrate that EL is a key physiological regulator of systemic PL catabolism.
Abstract 406: Achievement of Treatment Target in Korean Patients With Familial Hypercholesterolemia
Authors: Lee Chan Joo , Oh Jaewon , Kim Jung Sun , Lee Sang-Hak
Published: June 2021

Background:
In many cases of familial hypercholesterolemia (FH), there remains difficulty in achievement of treatment target. However, despite growing attention to FH, data on the treatment and its results in these patients are very limited.

Methods:
From nine sites in Korea, 122 consecutive unrelated men and women who were diagnosed with heterozygous FH by Simon Broome criteria were initially enrolled. Atorvastatin 20 mg or similar-potency drugs were prescribed and the dose was escalated every 2 months (for the first 6 months) or 6 months (thereafter) if needed. Forty one subjects were dropped and 81 subjects who underwent regular laboratory check-up were finally analyzed. The primary evaluation points were achievement rates of low-density lipoprotein cholesterol (LDL-C) <70 mg/dL, LDL-C<100 mg/dL, and LDL-C down to 50% of baseline levels at 12 month. The secondary evaluation point was % change of LDL-C at 12 month.

Results:
Patients’ mean age was 53 years and 59.3% were males. 21.0% were definite type FH and 28.4% had coronary artery disease (CAD). The mean total cholesterol and LDL-C were 319 mg/dL and 232 mg/dL, respectively. At 12 month, 7.4% received atorvastatin 10mg or similar, 21.0% received atorvastatin 20mg or similar, 16.0% received atorvastatin 40mg or similar, 4.9% received atorvastatin 80mg or similar, and 49.4% received atorvastatin (mean 57 mg) or similar plus ezetimibe 10mg. The mean follow-up total cholesterol and LDL-C were 201 mg/dL and 124 mg/dL, respectively. The mean % change of LDL-C was -45.6%. The achievement rates of LDL-C<70 mg/dL, <100 mg/dL, and LDL-C down to 50% of baseline were 1%, 21%, and 44%, respectively. The achievement rates were not significantly different between the patients without or with CAD (1.8%, 26.3%, 47.4% vs. 0%, 8.7%, 34.8%, respectively, all p values > 0.05).

Conclusions:
The achievement rate of treatment target in FH was low in Korea even after maximum tolerable dose of lipid lowering drugs. Improvement of awareness on this issue and more aggressive treatment are needed for this population.
Abstract 412: Are Body Mass Index and Serum Low Density Lipoproteins Cholesterol Increase Risk for Thromboembolic Events and Give Additive Information with CHADS2 Score in Nonvalvular Atrial Fibrillation Patients?
Authors: Harbi Hassan M , Alroshodi Abdulaziz , Alzahrani Mashari , Alfhaid Abdullah , Attamimi Mashary , Alqarni Khaled , Alshehri Fahad , Alzead Ahmed , Almutairi Ghazi , Alghasab Naif , Alrashed Abdulaziz
Published: June 2021

Introduction:
Atrial fibrillation (AF) is associated with atrial thrombus formation and peripheral embolization, which leads to ischemic stroke or systemic thromboembolism. The CHADS2 score is tool for estimating risk of stroke in nonvalvular atrial fibrillation (NVAF) patients. Obesity and Low density lipoprotein (LDL) are modifiable risk factors pose a great risk for stroke and venous thromboembolism. However, data on their implication in identify thromboembolic events in patients with NVAF are quite limited.

Objective:
To determine whether body mass index (BMI) and Serum LDL would be give incremental information with CHADS2 scheme in predicting thromboembolic event in NVAF Saudi patients.

Methodology:
The study consisted of 541 patients with AF seen in our institution from 2008 to 2013 were identified in database, 175 were NVAF. Thromboembolic end points were defined as ischemic stroke and systemic embolism. During follow- up period of 730 days. BMI, LDL and CHADS2 components associated with thromboembolic end points identified by Cox regression analysis.

Results:
Of 175 patients with NVAF, 26 (14.9 %) patients were identified to have thromboembolic events. Majority of them had stroke. Age (>or =75 years) and Peripheral vascular disease were significant factors for thromboembolic events. Thromboembolic risk in patients had previous stroke or TIA was 38.5% (P value = .000). Events rate of thromboembolic increased when CHADS2 score increased. No statistically significant associations were observed with BMI and thromboembolic risk. Study showed LDL associated with an increased risk of thromboembolic events (P value = .009) despite 61.5% of patients were in desirable level of LDL. Using a Combination of serum LDL and CHADS
2
score give additional implication on thromboembolism risk assessment (p value =.005) of NVAF patient.

Conclusion:
Study demonstrated LDL associated with thromboembolic risk among NVAF Saudi patients. So keeping this association in consideration during thromboembolism risk assessment In addition to the conventional CHADS2 scheme is recommended. Further study is needed to search for modifiable risk factors that are associated with increased risk thromboembolism in NVAF patient.
Abstract 413: CD34+CD31+ Endothelial Cells From Human Obese Subcutaneous but Not Omental Fat Show Adipogenic Potential
Authors: James Ashley , Haynes Bronson , Dobrian Anca
Published: June 2021

In obesity, excess lipid storage and reduced adipogenesis causes adipocytes to undergo hypertrophy leading to inflammation and insulin resistance. Increasing adipogenic potential may reduce lipid overload and ectopic lipid accumulation. In mice, the vascular niche is a source of adipocyte progenitors and a subset of endothelial cells (ECs) can trans-differentiate to functional adipocytes. It is unknown if a similar process occurs in humans. Our hypothesis is that pro-inflammatory cytokines contribute to de-differentiation and support induction of a pro-adipogenic program in a subset of CD31+CD34+ EC in human fat. The aim of this study is to determine if CD34+ CD31+ ECs isolated from human obese fat can be trans-differentiated to adipocytes and if this process is depot-specific. CD31+CD34+ ECs isolated from subcutaneous (SC) or omental (OM) fat of obese subjects undergoing bariatric surgery (BAMVECs) were treated with 2.5ng/mL of IFNγ and TNFα for 24 hrs then cultured in adipocyte induction medium (AIM), containing insulin and rosiglitazone, for up to 21 days. Cells without cytokine treatment and/or grown in media without induction were used as controls. Lipid accumulation was quantified with AdipoRed. OM BAMVECs showed no lipid accumulation in AIM alone (RFU ratio 1.1 vs control) nor with prior cytokine treatment (RFU ratio 1.6 vs control). However, SC BAMVECs showed lipid accumulation in AIM alone (RFU ratio 2.7 vs control) and with a trend to increased lipid accumulation with prior cytokine treatment (RFU ratio 4.1 vs control). By RT-PCR, SC BAMVECs pre-treated with cytokines had a 6-fold increase in pre-adipocyte commitment marker zfp423 and a 106,000 increase in adiponectin vs controls. The increases vs controls in AIM alone were 2.6x for zfp423 and 21,700x for adiponectin. We conclude that SC but not OM BAMVECs can trans-differentiate to adipocytes as shown by lipid droplet accumulation and increased expression of adipocyte markers. Cytokine stimulation trended to increase EC plasticity. This indicates that CD34+ CD31+ ECs within the SC depot, when stimulated, may play a protective role by supplementing the adipocyte pools thus increasing growth and function of adipose tissue and potentially reducing ectopic fat storage.
Abstract 417: Control of Adipose Tissue Macrophage Metabolism by Oxidized Phospholipids
Authors: Serbulea Vlad , Meher Akshaya K , Adamson Samantha , Leitinger Norbert
Published: June 2021

Our group has previously shown that oxidized phospholipids (OxPL) induce a unique macrophage (MΦ) phenotype known as “Mox,” distinct from pro- and anti-inflammatory M1 and M2 phenotypes. We have also shown that Mox make up 30% of the MΦs found in atherosclerotic lesions. OxPL induce TLR2-dependent inflammatory gene expression in MΦs. TLR2 activation by peptidoglycan was shown to induce accumulation of ceramides, which can alter bioenergetics by inhibiting the mitochondria. Recently, it has been shown that spleen tyrosine kinase (Syk) is phosphorylated upon TLR2 agonist Pam3CSK stimulation in MΦs. The effect of OxPL on bioenergetics has never before been studied. Here we test the hypothesis that OxPL change MΦ bioenergetics and inflammatory capacity via a TLR2-Syk-Ceramide pathway. Using flow cytometry, we found that in mice fed a high-fat diet, more than 20% of all adipose tissue MΦs can be described as Mox. Concomitantly, using immunohistochemistry and liquid-chromatography mass spectrometry, we measured increased levels of OxPL in the stromal vascular fraction of obese murine adipose tissue, as compared to lean controls. We treated bone marrow-derived MΦs (BMDMs) from WT, TLR2-KO, and Syk-KO mice with OxPL and measured bioenergetics using a Seahorse Flux Analyzer. Our results show that OxPL decrease oxygen consumption rate (OCR), a measure of oxidative phosphorylation, and decrease extracellular acidification rate (ECAR), a measure of glycolytic capacity. These MΦs can be described as quiescent, but they still engage in low-level cytokine production. Moreover, OxPL result in the accumulation of ceramides, as quantified by mass spectrometry. Finally, using Syk-KO MΦs and Syk inhibitors, we show that OxPL-induced inflammatory gene expression and ceramide accumulation are dependent on Syk. In summary, our results demonstrate that OxPL accumulate in obese adipose tissue and induce a change in the inflammatory and metabolic profiles of MΦs, involving a TLR2-ceramide-Syk dependent pathway. These results suggest that OxPL are triggers of adipose tissue inflammation and subsequent development of insulin resistance. Furthermore, we identify Syk as a therapeutic target for inhibiting diet-induced adipose tissue inflammation and insulin resistance.
Abstract 419: Metabocin
TM
Increases Lipolytic Pathways to Counter High Fat Diet-Induced Non-Alcoholic Fatty Liver Disease
Authors: Thyagarajan Baskaran , Cisneros Sara , Cook Ross , Baskaran Padmamalini
Published: June 2021

Non-alcoholic fatty liver disease (NAFDLD) is a comorbidity of high fat diet-induced obesity. NAFLD leads to steatohepatitis and hepatic steatosis, which progress into cirrhosis and liver cancer. Currently, there is no single strategy to counter NAFLD. In our effort to understand the role of transient receptor potential vanilloid receptor in the pathophysiology of high fat diet-induced, non-alcoholic fatty liver disease, we discovered that mammalian liver expresses transient receptor potential vanilloid 1 protein and that Metabocin
TM
{(
E
)-
N
-[(4-hydroxy-3-methoxyphenyl) methyl]-8-methylnon-6-enamide; also know as capsaicin; an agonist of transient receptor potential vanilloid 1 channel protein} significantly prevented mice from diet-induced NAFLD. Feeding high fat diet (60% calories from fat) for 32 weeks, from the age of 6 weeks until 38 weeks, caused hypertension, hyperglycemia, glucose intolerance and hyper triglyceridemia in the wild type mice and Metabocin
TM
prevented these effects. Metabocin
TM
markedly prevented hepatic steatosis and decreased lipid accumulation in the liver. Also, Metabocin
TM
significantly increased the expression of lipolytic PPAR alpha, PPAR gamma co activator 1 alpha, sirtuin-1 and forkhead box protein 01, while suppressed the expression of lipogenic stearoyl CoA desaturase 1. Further, Metabocin
TM
increased liver lipolysis, facilitated the activation of sirtuin-1 via Ca
2+
/Calmodulin dependent protein kinase II/AMPK-dependent mechanism and induced an interaction between sirtuin-1 and PPAR alpha. Metabocin
TM
also increased the expression of lipin-1 (a transcriptional regulator of PPAR alpha) and mitochondrial UCP-1. Our data provide evidence for the emergence of Metabocin
TM
as a novel drug molecule to antagonize NAFLD and its associated pathologies.
Abstract 42: Genetic Ablation of TMEM16F Exhibits Strain-specific Lethality in Mice
Authors: Kretz Colin A , Gilbert Gary , Ginsburg David
Published: June 2021

Background:
Scott Syndrome is a rare bleeding disorder characterized by a defect in platelet phosphatidylserine (PS) exposure. The syndrome has recently been linked to mutations in TMEM16F.

Tmem16f
-/-

mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding. We now report analysis of an additional gene targeted
Tmem16f
allele generated in C57BL/6 ES cells.

Results:
JM8 ES cell were obtained from EUCOMM, and successful Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120

Tmem16f
+/gt

(+/gt) intercross progeny identified no surviving

Tmem16f
gt/gt

(gt/gt) mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryo’s exhibiting no morphological abnormalities on gross or routine histologic examination. Though complete deficiency of TMEM16F is lethal in the C57BL/6J genetic background between E17.5 and birth, an F2 intercross of +/gt mice outcrossed one generation to 129x1SvJ resulted in gt/gt mice surviving to weaning, though at reduced numbers (6/75 total progeny compared to ~19 expected, p <0.002). Progeny testing of surviving gt/gt mice suggest a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3.

Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05).

Conclusion:
These data suggest the existence of a viability-determining genetic modifier of TMEM16F in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover novel functions for TMEM16F and the regulation of hemostatic function.
Abstract 424: Transintestinal Cholesterol Excretion Can Drive Massive Cholesterol Elimination in Mice
Authors: De Boer Jan F , Schonewille Marleen , Boesjes Marije , Wolters Henk , Bloks Vincent W , Bos Trijnie , Van Dijk Theo H , Jurdzinski Angelika , Boverhof Renze , Van Deursen Jan M , Oude Elferink Ronald P , Moschetta Antonio , Kremoser Claus , Verkade Henkjan J , Kuipers Folkert , Groen Albert K
Published: June 2021

High plasma cholesterol levels increase the risk of cardiovascular disease (CVD). Transintestinal Cholesterol Excretion (TICE) is a recently emerged pathway of cholesterol removal and has the potential to lower plasma cholesterol levels and confer protection against CVD. Under control conditions, TICE accounts for about 30% of fecal cholesterol loss in mice. Using a panel of knock-out and transgenic mice as well as pharmacological manipulations we show that in mice TICE is regulated by intestinal activation of the Farnesoid X Receptor (FXR), via its target Fibroblast Growth Factor 15/19 (FGF15/19). Activation of FXR by the agonist PX20606 (PX) resulted in a >10-fold increased fecal cholesterol excretion as well as TICE and 40% reduced plasma cholesterol levels. The induction of fecal cholesterol excretion and TICE was absent in PX-treated intestine-specific FXR-null mice but was regained when those mice were co-treated with FGF19. Moreover, FGF19 treatment alone was sufficient to induce fecal cholesterol loss to a similar extend as was observed in PX-treated wild-type mice. PX treatment resulted in an increased muricholate/cholate-ratio and thereby induced a more hydrophilic bile salt pool. Not surprisingly, cholesterol absorption was reduced in PX-treated mice. However, experiments in which mice were co-treated with PX and the cholesterol absorption inhibitor ezetimibe revealed that the stimulating effect of PX on fecal neutral sterol excretion and TICE were completely independent of differences in cholesterol absorption. Of note, treatment of mice with a combination of PX and ezetimibe stimulated fecal cholesterol loss and TICE so strongly that those mice lost about 60% of their entire estimated body cholesterol content each day. The stimulation of fecal cholesterol loss and TICE by PX and PX/ezetimibe treatment was severely blunted in Abcg8-KO mice and this could not be restored by hepatic reintroduction of Abcg8, indicating a decisive role for intestinal ABCG5/G8 in PX-induced fecal cholesterol loss and TICE. Our data strongly suggest that hydrophilic bile acids stimulate ABCG5/G8 activity in the intestine, leading to an increased flux of cholesterol from the body into the intestinal lumen that is subsequently excreted with the feces.
Abstract 428: Lipoprotein(a) Targeting With RNAi Delivery Platforms in Transgenic Mice and Cynomolgus Monkeys
Authors: Melquist Stacey , Wakefield Darren , Hamilton Holly , Chapman Christine , Grondolsky Jessica , Almeida Aaron , Almeida Lauren , Hegge Julia , Chu Qili , Trubetskoy Vladimir , Hoover-Plow Jane , Rozema David , Lewis David , Kanner Steven
Published: June 2021

Background:
Meta-analyses of clinical studies have demonstrated that high levels of lipoprotein(a), known as Lp(a), correlate with residual risk of cardiovascular disease (CVD) while Mendelian genetic studies indicate that Lp(a) is an independent cause of CVD. Lp(a) is a complex lipoprotein particle similar in structure to LDL (consisting of cholesterol, triglycerides, phospholipids and apoB-100), but contains the genetically variable liver-expressed protein apo(a). Lp(a) may contribute to atherosclerosis by targeting oxidized phospholipids to vascular injury sites, promoting both inflammation and accumulation of lipids.

Methods:
RNA interference (RNAi) based approaches for reducing liver apo(a) expression were developed using both a targeted intravenous platform called Dynamic Polyconjugates (DPC)
TM
and a subcutaneous (SQ) format. The DPC platform comprises an RNAi trigger conjugated to cholesterol, and a hepatocyte-targeted, membrane active peptide to promote endosomal release of the RNAi trigger. The SQ platform is an RNAi trigger targeted to liver by N-acetyl galactosamine in a proprietary format. RNAi activity was assessed in two different humanized transgenic mice and cynomolgus monkeys.

Results:
In Lp(a) transgenic mice expressing human apo(a) and apoB-100 genes, treatment with a single DPC dose reduced serum Lp(a) by >98% at nadir and >90% for >5 weeks. A single DPC injection in cynomolgus monkeys resulted in >95% reduction in endogenous Lp(a) particles at 5 weeks post injection, without significant changes in toxicity biomarkers. Treatment with a SQ RNAi trigger reduced serum apo(a) in transgenic mice by >90% at nadir, while that in monkeys reached 64% reduction of Lp(a). Additional modifications to the RNAi trigger improved both depth and duration of knockdown in transgenic mice.

Conclusion:
Long-duration reductions in circulating apo(a) and Lp(a) have been shown with hepatocyte-directed delivery of apo(a)-specific RNAi triggers in mice and monkeys. This represents an intriguing mechanism to evaluate in humans for reducing residual risk of CVD in LDL-controlled individuals with elevated Lp(a).
Abstract 429: Structural Stability of Streptococcal Serum Opacity Factor
Authors: Rosales Corina , Gillard Baiba K , Yelamanchili Dedipya , Gotto Antonio M , Pownall Henry J
Published: June 2021

Despite its putative cardioprotective qualities, raising plasma high density lipoprotein-cholesterol (HDL-C) levels via pharmacologic means has failed to add protection against atherosclerosis, particularly when used as co-therapy with a statin. Two scenarios argue against the raising-plasma-HDL-is-better hypothesis and suggest that enhancing the final RCT step, hepatic cholesterol disposal, is a better cardioprotective strategy. First, probucol prevents CVD events and increases survival in humans and reduces CVD in SR-BI/apo E DKO mice. Despite its anti atherogenic properties, probucol lowers plasma HDL-C levels. Second, SR-BI over expressing vs. WT mice have lower plasma HDL-C but less atherosclerosis whereas the converse is true in SR-BI KO mice, suggesting that increasing HDL-C disposal is a rational cardioprotective strategy. One possible agent for therapeutic development is streptococcal serum opacity factor (SOF), a 100 kDa protein that clouds human serum via a novel HDL-targeting mechanism. SOF diverts HDL-CE to the LDL receptor and to bile acid secretion in vitro and in vivo, increases plasma HDL-C clearance in mice in an apo E-, LDLR-dependent mechanism thereby increasing hepatic CE uptake and reducing plasma cholesterol levels. SOF is active at 10
-14
M. Given its novel mechanism and potent reduction of plasma cholesterol levels in mice, we studied the structure and stability of SOF using its activity as a marker of its integrity versus several physicochemical challenges—extremes of pH, the denaturant, guanidinium chloride, heat, and ionic strength. SOF was highly resistant to all of these challenges. SOF has only one cysteine so it cannot be stabilized by internal disulfide bonds. Thus, SOF is an unusually stable protein that undergoes reversible unfolding-folding when challenged with a variety of physicochemical perturbants. These studies have helped us identify optimal conditions for crystallizing SOF for X-ray structure analysis.
Abstract 430: Efficacy of ApoC-II Mimetic Peptide in Novel Apoc2 Mutant Mice With Hypertriglyceridemia and Impaired Insulin Sensitivity
Authors: Sakurai Toshihiro , Sakurai Akiko , Vaisman Boris L , Amar Marcelo J , Liu Chengyu , Gordon Scott M , Drake Steven K , Pryor Milton , Sampson Maureen L , Yang Ling , Freeman Lita A , Noguchi Audrey , Remaley Alan T
Published: June 2021

Apolipoprotein C-II (apoC-II) is a cofactor for activating lipoprotein lipase, which hydrolyzes triglycerides (TGs). ApoC-II deficiency causes hypertriglyceridemia in humans. Here, we created Apoc2 mutant mice by using zinc finger nucleases and characterized the mice on lipid and glucose metabolism. We also investigated the efficacy of C-II-a, an apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice had apoC-II with an uncleaved signal peptide that strongly binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had elevated plasma TG (757.5 ± 281.2 mg/dL) derived from very-low density lipoprotein and chylomicron remnants (40-200 nm) and low HDL cholesterol (31.4 ± 14.7 mg/dL) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dL; HDL cholesterol, 55.9 ± 14.3 mg/dL). The homozygous mice had significantly increased body weight and % fat mass (36.4 g; 26.0% at 6-mon-old) compared to wild-type mice (29.6 g; 17.5%). Less movement and much heat (Energy expenditure) in homozygous mice was observed by metabolic cage. Both Glucose and insulin tolerance tests also revealed insulin resistance in homozygous mice. Intravenous injection of C-II-a (0.2, 1, and 5 μmol/kg body weight) decreased plasma TG in a dose-dependent manner, with a maximum reduction of 90% at 30 minutes after the high dose. TG did not return to baseline until 2 days later. Subcutaneous or intramuscular injections also had similar effects. Plasma half-life of C-II-a was 1.33 ± 0.72 hours, suggesting that C-II-a only acutely supports activation of lipolysis, and the sustained TG reduction is due to the relatively slow synthesis of TG-rich lipoproteins. Additionally, insulin tolerance test in both wild-type and homozygous mice showed improvement tendency of insulin sensitivity in intraperitoneal injections of C-II-a (1 μmol/kg) five times for 9 days. In conclusion, we found a novel mouse model of apoC-II deficiency with insulin resistance and obesity as well as hypertriglyceridemia and also revealed apoC-II mimetic peptide can ameliorate hypertriglyceridemia and insulin resistance in the mice, could be a potential new therapy for apoC-II deficiency and insulin resistance.