bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 089: Role of LncRNA
Snhg18
in Diabetes Induced Macrophage Dysfunction and Atherosclerosis
Authors: Ganguly Rituparna , Reddy Marpadga A , Chen Zhuo , Amaram Vishnu , Bansal Anita , Lanting Linda , Natarajan Rama
Published: March 2019

Atherosclerotic vascular disease is the major cause of mortality in diabetic patients. Evidence from our laboratory and others showed that long non-coding RNAs (lncRNAs) play important roles in diabetic vascular complications. Here we investigated role of lncRNAs in hyperglycemia-induced macrophage dysfunction and accelerated atherosclerosis in diabetes. Hyperglycemia (~300-400 mg/dl) was induced in atherosclerosis-prone ApoE
-/-
mice by low dose Streptozotocin injections (50mg/kg/day i.p., 5 days) and were sacrificed at 20 weeks’ post diabetes. Aortic root F4/80 immunohistochemistry and morphometric analysis showed increased macrophage infiltration and enhanced atherosclerosis respectively in diabetic vs non-diabetic ApoE
-/-
mice. RNA-seq analysis identified ~45 up-regulated lncRNAs in bone marrow macrophages (BMM) from diabetic versus non-diabetic ApoE
-/-
mice. Among these, one of the robustly induced (~7.5 log
2
fold) lncRNA candidate was small nucleolar RNA host gene 18 (
Snhg18)
located on mouse chromosome 15. Gene expression analysis by RT-qPCR validated
Snhg18
upregulation in BMM from diabetic ApoE
-/-
mice and in peritoneal macrophages from type 2 diabetic db/db vs control db/+ mice. Moreover, diabetic-ApoE
-/-
aortic tissue also demonstrated increased expression of
Snhg18 as
compared to non-diabetic ApoE
-/-
s
.
Additionally,
Snhg18
was significantly increased in high glucose (HG) (25mM) treated RAW mouse macrophages
in vitro
. A human ortholog,
SNHG18
, located on chromosome 5 was also significantly increased in HG treated human THP1 monocytes. Cellular fractionation of RAW macrophages demonstrated that
Snhg18
was localized in both cytoplasmic and nuclear compartments. Overexpression of
Snhg18
in RAW macrophages significantly increased expression of proinflammatory (
Il1b
,
Il6
, and
Tnf)
and proatherogenic (
Thbs1
) genes but downregulated a nearby gene,
Semaphorin 5A
(
Sema5a).
These results suggest that diabetes induced lncRNA
Snhg18
upregulates macrophage inflammatory phenotype to modulate diabetes induced accelerated macrophage dysfunction and atherosclerosis development.
Abstract 090:
Slc44a2
Deficient Mice Exhibit Less Severity of Thrombosis in a Stenosis Model of Deep Vein Thrombosis
Authors: Maracle Chrissta X , Tilburg Julia , Zirka Gaia , van den Heijkant Lieke , Morange Pierre E , van Vlijmen Bart J , Thomas Grace M
Published: March 2019

Background/Objective:
Recent genome wide association studies identified
SLC44A2
as a novel susceptibility locus for venous thromboembolism (VTE), a region encoding the solute carrier family 44 member 2 protein (SLC44A2). Here we utilize
Slc44a2
deficient mice (KO) to determine the importance of SLC44A2 in thrombotic disease.

Methods:
Mice lacking
Slc44a2
were included in two models of venous thrombosis: 1) a spontaneous thrombosis model using siRNA targeting anti-coagulants
Serpinc1
and
Proc
and 2) a model of deep vein thrombosis (DVT) induced by flow restriction (stenosis) of the inferior vena cava (IVC).

Results:
In the model of spontaneous thrombosis,
Slc44a2
deficiency did not affect incidence as occurrence of thrombotic phenotype reached 100% in both wild type (WT; 12/12) and KO (12/12) mice 32 hours after siRNA injection. Platelet counts and fibrin deposition in the liver were also comparable. However, levels of circulating neutrophils were significantly higher (p=0.0017) in KO mice along with substantially lower amounts of plasma VWF antigen (p<0.0001). As these findings suggested a link between SLC44A2 and neutrophil function, a DVT model previously determined to be neutrophil dependent, was also employed. After 48 hours of IVC stenosis, 100% of the WT mice developed an occlusive thrombus (10/10) whereas 80% of the mice lacking
Slc44a2
did (12/15). Thrombus length was significantly reduced in KO animals (p=0.0184), as was thrombus weight (p=0.0413). Immunhistochemical staining of the thrombi revealed different repartitions in the “white” (platelet-rich) over the “red” (RBC-rich) parts of the thrombi depending on the mouse genotype. This observation suggests a different blood cell mobilization at the site of stenosis depending of the genotype. Plasma VWF levels were correlated with thrombus weight in WT (p=0.0175), but not in KO mice (p=0.3266), and had been previously confirmed to be lower in KO mice.

Conclusion:
These findings corroborate the original GWAS data, indicating a role for SLC44A2 in thrombotic disease. We have observed that SLC44A2 was not involved in a platelet-dependent model of thrombosis whereas it is involved in a thrombosis model where neutrophils and VWF have been shown to play a crucial role.
Abstract 091: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow
Authors: Zilberman-Rudenko Jevgenia , Reitsma Stephanie E , Puy Cristina , Smith Stephanie A , Wiesenekker Chantal P , Tucker Erik I , Travers Richard J , Maas Coen , Urbanus Rolf T , Gailani David , Schmaier Alvin H , Lupu Florea , Morrissey James H , Gruber Andras , McCarty Owen J
Published: March 2019

Background:
Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation
ex vivo
and
in vivo
.

Aim:
Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner.

Methods/Results:
The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of coagulation using established and novel agents reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation.
In vivo
, FXII deficiency was protective against long-chain polyP occlusive lung thrombus formation in mice. Lastly, in a non-human primate model of sepsis, pretreatment of animals with an antibody blocking FXI activation by FXIIa (14E11) diminished LD
100
S. aureus
-induced platelet and fibrinogen consumption.

Conclusions:
This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could contribute to sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.
Abstract 125: Intensive Statin Therapy Negatively Modulates AdipoR Activity in the Monocyte-macrophage Lineage
in vivo
and
in vitro
Authors: Gasbarrino Karina , Zheng Huaien , Daskalopoulou Stella
Published: March 2019

Introduction:
Statins are widely used for primary and secondary prevention of cardiovascular disease and its complications through cholesterol lowering and anti-inflammatory effects. Adiponectin, an anti-inflammatory adipokine, acts via 2 receptors, AdipoR1 and AdipoR2, to exert protective effects on the vasculature. Recently, we demonstrated that AdipoR are highly expressed on macrophages and that decreased AdipoR2 activity is associated with atherosclerotic plaque instability. We aimed to investigate whether statins can modulate AdipoR activity in the monocyte-macrophage lineage.

Methods:
Blood monocytes were isolated from whole blood of patients (statin-naïve
vs
statin users) with severe carotid atherosclerosis by a Magnetic Cell-Sorting technique. AdipoR1 and AdipoR2 mRNA expression on circulating monocytes was determined using qRT-PCR.
In vitro
, THP-1 macrophages were treated for 24 or 72 hours with varying doses of atorvastatin or rosuvastatin (1μM, 10μM, 60μM) to determine the effect of statins on AdipoR activity (AMPK, PPAR-α) by qRT-PCR and Western Blot analyses. Macrophage cytokine secretion (IL-1β, IL-10, IL-6, TNF-α) was assessed by electrochemiluminescence.

Results:
AdipoR1 and AdipoR2 mRNA expression on circulating monocytes was significantly lower by 1.36- and 1.17-fold (P<0.05), respectively, in statin users (n=157) compared to statin-naïve patients (n=37). When analyzing by statin type and dose, only patients on high doses of atorvastatin (40-80 mg) or rosuvastatin (20-40 mg) had significantly lower AdipoR expression compared to statin-naïve patients (P<0.05). Statins effects on lowering AdipoR expression was only apparent in patients with unstable but not stable plaques.
In vitro
, higher doses and longer exposure of macrophages to atorvastatin or rosuvastatin resulted in a greater decrease in AdipoR mRNA expression and signalling through AMPK and PPAR-α, and greater secretion of pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α.

Conclusion:
In our study, intensive statin therapy resulted in negative modulation of AdipoR expression and function. Despite their known protective properties, statins may also elicit severe adverse effects, which are often associated with long-term treatment and high doses.
Abstract 127: Arterial Inflammation and Neointimal Hyperplasia Development in a Germ-free Mouse Model of Carotid Ligation
Authors: Wun Kelly , Theriault Betty , Pierre Joseph F , Leone Vanessa , Harris Katharine G , Chen Edmund B , Xiong Liqun , Jiang Qun , Eskandari Owen , Chang Eugene B , Ho Karen J
Published: March 2019

Backgound:
The microbiome plays a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia has been linked to inflammation, there has not been direct demonstration of the role of microbiota in this process. Germ-free (GF) mice raised in complete absence of microbes are an invaluable tool for studying causative links between commensal organisms and the host. We hypothesized GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally-raised (CONV-R) mice.

Methods:
Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers after 5 days. Systemic inflammation was assessed by multiplex immunoassays of serum. Identically treated CONV-R mice served as controls. GF and CONV-R outcomes were compared using standard statistical methods.

Results:
All GF mice remained sterile during the study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media area). Collagen content of neointimal lesions appeared qualitatively similar on Masson’s trichrome staining. Ki67 immunoreactivity was significantly reduced in GF carotid arteries at 5 days with increased arterial infiltration of neutrophils and M2 macrophages compared to CONV-R. GF mice also had significantly reduced serum IL-17 concentration and nearly undetected IL-10 at 5 days but not 28 days.

Conclusions:
GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which may be related to altered kinetics of wound healing and acute inflammation. Understanding how commensal microbes regulate arterial remodeling could provide new directions in pathophysiology of restenosis and offer novel strategies to reduce restenosis risk via microbiota manipulation.
Abstract 128: Disruption of Methylarginine Metabolism Impairs Vascular Homeostasis During Pregnancy
Authors: Georgopoulou Aikaterini , Johnson Mark , Noor Mohd N , Leiper James M
Published: March 2019

Endogenously produced asymmetrically methylated forms of L-arginine (ADMA) competitively inhibit all three isoforms of nitric oxide synthase enzymes and therefore have the potential to exert significant cardiovascular effects. Elevated circulating ADMA concentrations have been reported in a number of cardiovascular disease states, including preeclampsia, suggesting that impaired ADMA metabolism may contribute to pathology. In order to test the causal relationship between ADMA concentrations and hemodynamics in pregnancy we employed radiotelemetric measurement of hemodynamic function in mice with either global or fetal-specific deletion of the predominant ADMA metabolising enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1). Global deletion of DDAH1 in non-pregnant female mice caused an elevation in circulating ADMA concentrations (2.851±0.289 vs 1.216±0.072 μM p<0.01) and blood pressure (118±2.28 vs 110.36±0.82 mmHg p<0.05). During the first and second trimesters of pregnancy DDAH1
-/-
mice showed no further elevation in ADMA and normal pregnancy related changes in blood pressure were observed. However, during the third trimester ADMA levels in DDAH1
-/-
mice increased further and this was associated with increased concentrations of sEng and sFlt1 (2288±301.58 vs 1181.35 ± 161.83 pg/ml p<0.01 and 153.31 ± 8.57 vs 114.42 ± 8.79 ng/ml p<0.001) and an exaggerated hypertensive response (115.773 ± 3.7 vs 101.93 ± 3.27 mmHg p<0.05). Fetal-specific deletion of DDAH1 elevated fetal ADMA concentrations (4.97 ± 0.29 μM vs 1.7 ± 0.12 μM p<0.001) but did not impact on maternal ADMA concentrations or blood pressure during early pregnancy. However in the third trimester a significant elevation in maternal ADMA concentrations was detected (2.49 ± 0.17 vs 1.75 ± 0.13 μM p<0.01) and was associated with an elevation in maternal blood pressure. No changes in sEng or sFLt1 were detectable in fetal-specific DDAH1
-/-
mice. Our data indicate that elevated ADMA is sufficient to increase levels of sFLlt1 and sEng and disrupt maternal hemodynamics. The fetus is a significant source of ADMA in late pregnancy suggesting that dysfunctional fetal ADMA metabolism is sufficient to impair maternal hemodynamic function.
Abstract 132: Application of a Cryogel-Coated Prosthetic Vascular Graft Material for Delivery of Targeted Gene Therapies in a Rabbit Model
Authors: Huynh Cindy , Shih Ting , Contreras Mauricio , Mooney David , Pradhan-Nabzdyk Leena , LoGerfo Frank
Published: March 2019

Objectives:
Long-term success of prosthetic grafts (PG) in peripheral arterial disease is limited by development of anastomotic neointimal hyperplasia. We have constructed a bioactive prosthetic graft material (BPGM) capable of delivering biologic agents in vitro, and evaluate our BPGM in vivo with a rabbit carotid interposition bypass model.

Methods:
We synthesized our BPGM by cryopolymerization of RGD with methacrylated alginate and heparin, coating 1.5cm by 2mm electrospun PET (ePET) grafts, and dipcoating in fluorescent siRNA for 3 hours. Three rabbits received bare ePET and 3 received cryogel-coated ePET for a carotid interposition bypass (Figure 1). After 24 hours, bypass patency was assessed, and cell toxicity of the proximal anastomosis, mid-graft, and distal anastomosis examined with H&E staining. Confocal microscopy was used to visualize fluorescence, correlating with ability to deliver siRNA in vivo.

Results:
Graft patency was equal between groups, with no increased cell toxicity in rabbits receiving cryogel-coated ePET. Confocal microscopy demonstrated no difference in retained fluorescence between rabbits receiving cryogel-coated or bare ePET, and no increased transfection of cells at 24 hours (Figure 2).

Conclusion:
Creation of the optimal PG demands a material that is biocompatible, responsive, and nonthrombogenic. We have constructed a modified PG capable of in vitro delivery of targeted gene therapies, with comparable patency and biocompatibility in our large animal model. Additional optimization to achieve predictable and sustained release is needed to validate this as an effective and practical method to deliver biologic agents in vivo.
Abstract 137: Right Cerebral Insufficiency Associated with Right Aortic Arch with Mirror-Image Branching: a Case Study
Authors: Petit Joel , Kumar Senthil , Harrison Catherine , Scherf Aimee
Published: March 2019

We report a case of right cerebral insufficiency in a 65-year-old woman with right aortic arch with mirror-image branching. The patient was found to have intermittent occlusion at the origin of the right common carotid artery between the aortic arch and the sternoclavicular joint. Duplex showed intermittent bizarre carotid artery waveforms directly related to patient positioning. The patient underwent a transposition of the right common carotid to the right subclavian artery with subsequent resolution of symptoms. Right aortic arch with mirror-image branching can rarely present with right cerebral insufficiency due to intermittent mechanical occlusion of the right common carotid artery.
Abstract 148: Cardiac Myosin Promotes Thrombin Generation and Attenuates Tissue Plasminogen Activator-induced Plasma Clot Lysis
Authors: Zilberman-Rudenko Jevgenia , Deguchi Hiroshi , Orje Jennifer , Wyseure Tine , Mosnier Laurent O , McCarty Owen J , Ruggeri Zaverio M , Griffin John H
Published: March 2019

Recently we discovered that skeletal muscle myosin, which is in the same family as cardiac myosin, exerts prothrombotic effects by binding factor Xa and enhancing prothrombin activation in the presence of factor Va. Thus, we tested the influence of cardiac myosin on thrombus formation and fibrinolysis. Studies of the effects of cardiac myosin on thrombogenesis
ex vivo
using fresh human flowing blood showed that perfusion of blood over cardiac myosin-coated surfaces at 300 s
-1
shear rate caused extensive fibrin deposition. Addition of cardiac myosin to blood also promoted the thrombotic responses of human blood flowing over collagen-coated surfaces, evidence of myosin’s thrombogenicity. Further studies showed that cardiac myosin enhanced thrombin generation in whole blood, platelet rich plasma and platelet poor plasma, indicating that myosin promotes thrombin generation in plasma primarily independently of platelets or other blood cell components. In a purified system composed of factor Xa, factor Va, prothrombin and calcium ions, cardiac myosin greatly enhanced prothrombinase activity. Experiments using Gla-domainless factor Xa showed that the Gla domain of factor Xa was not required for cardiac myosin’s prothrombinase enhancement in contrast to phospholipid-enhanced prothrombinase activity which requires that Gla domain. In studies of tissue plasminogen activator (tPA)-induced plasma clot lysis, increasing concentrations of cardiac myosin attenuated tPA-mediated clot lysis. The ability of cardiac myosin to inhibit tPA-induced plasma clot lysis was ablated in the presence of the carboxypeptidase inhibitor from potatoes, an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI). Clot lysis assays using TAFI-deficient plasma confirmed the requirement for TAFI for the antifibrinolytic action of cardiac myosin. We hypothesize that cardiac myosin-dependent thrombin generation increases TAFI activation and subsequent inhibition of clot lysis. In summary, here we show that cardiac myosin is both procoagulant and anti-fibrinolytic due to its ability to bind factor Xa and strongly promote thrombin generation. This raises new questions about potential procoagulant functions for cardiac myosin in coronary health and disease.
Abstract 168: Response Gene to Complement 32 Suppresses Adipose Tissue Thermogenic Genes through Inhibiting Beta3 Adrenergic Receptor mTORC1 Signaling
Authors: Chen Sisi , Mei Xiaohan , Yin Amelia , Yin Hang , Cui Xiao-Bing , Chen Shi-You
Published: March 2019

Our previous study have shown that response gene to complement 32 (RGC-32) deficiency (Rgc32-/-) protects mice from diet-induced obesity and increases thermogenic gene expression in adipose tissues. However, the underlying mechanisms by which RGC-32 regulates thermogenic gene expression remain to be determined. In the present study, we found that RGC-32 expression in white adipose tissue (WAT) was suppressed during cold exposure-induced WAT browning. Rgc32-/- significantly increased thermogenic gene expression in the differentiated stromal vascular fraction (SVF) of inguinal WAT (iWAT). Interestingly, Rgc32-/- and cold exposure regulated a common set of genes in iWAT as shown by RNA sequencing data. Pathway enrichment analyses showed that RGC-32 deficiency downregulated PI3K/Akt signaling-related genes. Consistently, Akt phosphorylation was also decreased in Rgc32-/- iWAT, which led to an increase in β3-adrenergic receptor expression and subsequent activation of mTORC1. β3-adrenergic receptor antagonist SR 59230A and mTORC1 inhibitor rapamycin blocked the Rgc32 deficiency-induced thermogenic gene expression in iWAT both in vitro and in vivo. These results indicate that RGC-32 suppressed iWAT thermogenic gene expression through down-regulation of β3-AR expression and mTORC1 activity via a PI3K/Akt-dependent mechanism.
Abstract 181: The Use of Recombinant Human Lipoprotein Lipase as a Treatment for Hypertriglyceridemia
Authors: Wolska Anna , Francone Omar L , Amar Marcelo J , Pryor Milton , Remaley Alan T
Published: March 2019

Pancreatitis affects 270,000 number of people a year in the US and has a mortality rate of approximately 5%. Hypertriglyceridemia is a well established risk factor for acute pancreatitis, accounting for 10% of cases. To investigate recombinant human lipoprotein lipase (rhLPL) as a potential therapy for pancreatitis caused by hypertriglyceridemia, we examined effect of rhLPL produced in CHO cells on lipoproteins in human plasma and hypertriglyceridemic mice. IV injection of 500 μL of 20% Intralipid into mice increased plasma TG levels over 200-fold to about 6000 mg/dL at 15 min. When mice were co-injected with rhLPL (2.5 μg/g BW; IV), TG levels reached 2200 mg/dL and by 3 h were completely normal unlike control animals, which took 24 hours. Plasma TGs were decreased by 81% and 63% compared to control mice when rhLPL was administered IV at a dose 2 μg/g BW and 1 μg/g BW consecutively for mice treated IP with 1 mL of 20% Intralipid. The effect of rhLPL was sustained for 6 hours for high dose rhLPL. Enhanced lipolysis from rhLPL treatment resulted in a 2-fold increase of plasma FFA, but the albumin binding capacity for FFA did not appear to be exceeded because all FFA were bound to either lipoproteins or albumin. Moreover, no pathological changes were observed in the pancreas of treated mice, as assessed by histology. Amylase levels in rhLPL treated mice given Intralipid at 6 h were increased only 1.4-fold, whereas in control mice 2.8-fold. Addition of rhLPL in vitro (2 μg per 250 μL of human plasma) resulted in a significant decrease in lipoprotein particle numbers of all subpopulations of VLDL, small LDL, and small HDL, whereas a significant increase was observed for large LDL and large HDL particle number. The sizes of VLDL shifted towards smaller particles and the opposite was observed for LDL and HDL particles. Overall, there was a 57% reduction of TGs in plasma treated with rhLPL. In summary, rhLPL at relatively low doses accelerates the clearance of TG-rich lipoproteins in mice after a fat load. It does so without causing accumulation of toxic levels of FFA in plasma and reduces hypertriglyceridemia induced markers of pancreatitis. Furthermore, observed changes in human lipoprotein subpopulation distribution after rhLPL treatment are positive in terms of atherosclerosis risk.
Abstract 183: Association of LDL Particles With Cerebral Amyloidosis
Authors: Parekh Trusha , King Sarah M , Reed Bruce , Chui Helena C , Krauss Ronald M , Yassine Hussein
Published: March 2019

Cerebral amyloidosis is a condition in which β-Amyloid (Aβ) proteins are deposited in the cerebral cortex and is a predictor of Alzheimer’s disease (AD). In the Aging Brain Study, we reported an association between LDL cholesterol and cerebral amyloidosis assessed using PET PiB imaging. LDL comprises multiple species of varying size, density and protein composition, including very large LDL-I which is enriched in ApoE and can bind to ApoE receptors. In this study, LDL particle fractions were measured in plasma samples of 58 participants (40 women and 18 men) of the Aging Brain study. Cerebral amyloidosis was assessed using Pittsburgh Compound B index-Positron Emission Tomography (PiB-PET) imaging. LDL subfractions were analyzed by the method of ion mobility. The subjects were divided into three groups based on PiB tertiles. Compared to the first tertile, total plasma cholesterol as well as LDL cholesterol were greater in patients in the second and third PiB index tertiles (p values = 0.05 and 0.03 respectively). Amongst the LDL subfractions, levels of LDL-I as well as very small LDL-IVa particles were significantly greater in the second and third PiB index tertiles and independent of LDL cholesterol levels (p values = 0.03 and 0.04 respectively). A significant inverse association was also observed between LDL-I and hippocampal volumes (r=-0.33, p=0.02). We suggest that LDL-I level may be a mechanistic biomarker for extent of cerebral amyloidosis. Lipoprotein receptors, particularly LRP-1, participate in Aβ clearance from the brain and its hepatic degradation. One potential mechanism for our findings is competition between plasma-derived LDL-I and brain Aβ that may retard Aβ clearance and degradation.
Abstract 190: Mir-33 Inhibition Alters Monocyte/macrophage Kinetic Processes to Promote Atherosclerotic Plaque Regression
Authors: Afonso Milessa S , Sharma Monika , van Solingen Coen , Koelwyn Graeme J , Schlegel P. Martin , Ouimet Mireille , Beckett Lauren , Babunovic Susan , Rahman Karishma , Fisher Edward A , Moore Kathryn J
Published: March 2019

Previous studies have identified microRNA-33 (miR-33) as a key regulator of metabolic gene pathways involved in cholesterol and fatty acid homeostasis. Inhibition of miR-33 promotes the regression of atherosclerosis, in part, by enhancing plasma HDL and reverse cholesterol transport levels. However, it has become clear that miR-33 inhibition can also reduce plaque inflammation, independent of its effects on cholesterol homeostasis, yet the mechanisms of these actions are still not well understood. To investigate how miR-33 inhibition alters monocyte/macrophage dynamics during atherosclerosis regression, we fed
Ldlr
-/-
mice a western diet for 14 weeks to establish plaques, after which mice were switched to chow diet and treated with anti-miR-33 or control anti-miR oligonucleotides for 4 weeks. As we reported previously, anti-miR-33 treatment

increased plasma levels of HDL cholesterol by 30%, and concurrently reduced plaque size, macrophage content and necrotic area, compared to control anti-miR treatment. Analysis of the monocyte/macrophage kinetic processes contributing to macrophage burden in plaques revealed that anti-miR-33 treatment normalized the monocytosis associated with hypercholesterolemia. Mice treated with anti-miR33 had 40% fewer circulating monocytes than control anti-miR treated mice, and this was associated with a reduction of splenic common myeloid progenitor (CMP) cells. Surprisingly, monocyte tracking assays

also revealed an increase in Ly6C
hi
monocyte recruitment into plaques of anti-miR-33 treated mice. Although somewhat counterintuitive, these data are consistent with recent findings that Ly6C
hi
monocytes are required for atherosclerosis regression and are a source of tissue reparative M2 macrophages. Indeed, we find that M2 macrophages, as well as atheroprotective regulatory T cells are enriched in plaques of anti-miR-33 treated mice. Collectively, our results provide insight into the mechanisms underlying anti-miR-33’s atheroprotective actions, which include both anti-inflammatory and cholesterol homeostatic effects.
Abstract 192: Relation Between Poor Sleep Quality and Hypercholesteremia
Authors: Ramirez Francisco E , Nedley Neil
Published: March 2019

Background:
Problems with sleep are linked with higher risk of cardiovascular pathologies. We explore the effect that problems with sleep may have on hypercholesterolemia as well as mental health on participants of an 8-week lifestyle health program.

Methods:
The focus of the program is educational. Those who chose to participate met once a week for 8 weeks for a 2 hour program. It consisted of a 45 minute DVD presentation and a facilitated group discussion. The Depression and Anxiety Assessment Test (DAAT) was administered at baseline and completion. It assessed depression level based on DSM-5 [The Diagnostic and Statistical Manual of Mental Disorders Volume 5] criteria, demographics, sleep quality and inquires about cholesterol levels. The depression was classified according to DSM-5 into 4 categories as none (0-6), mild (7-10), moderate (11-19) or severe (20 or more). They were taught various healthy lifestyle habits. Sleep quality was self reported and was divided between bad quality and good quality the last two weeks.

Results:
Of n=5997 participants that finished the program, n=5380 knew their cholesterol levels and were used in this study. Mean age was 52.3 (SD 15.1), n=4209 (70%) females. At baseline participants that reported bad sleep were n=1124, that group baseline mean depression was 14.8 (SD 7.2), n=313 (27.8%) had hypercholesterolemia. At the end of the 8- week program their mean depression was 8.1 (SD 6.3), the change was significant with a paired t-test t(1123)=7.761 p<.001. By the end also 58.1% reported improvement in sleep quality. An improvement in cholesterol was reported in 11.8% of this group at the end of the program. At baseline those with good sleep quality were n=4256, their baseline mean depression was 11.2 (SD 7.4), n=1006 (23.6%) had hypercholesterolemia. Their end mean depression was 5.9 (SD 5.7), paired t-test reported significant change t(4255)=55.03 p<.001. An improvement in their cholesterol was reported in 26.3% of this group at the end of the program.

Conclusion:
The group with bad sleep quality seem to have more hypercholesterolemia and more severe depression. The 8-week program was safe and was associated with at least some degree of improvement in the vast majority.
Abstract 195: Therapeutic Silencing of FSP27 Attenuates the Progression of Atherosclerosis in LDL-Receptor Deficient Mice
Authors: Rajamoorthi Ananthi , Lee Richard G , Baldan Angel
Published: March 2019

Obesity, hepatosteatosis, and hypertriglyceridemia are components of the metabolic syndrome and independent risk factors for cardiovascular disease. The lipid droplet-associated protein CIDEC (cell death-inducing DFFA-like effector C), known in mice as FSP27 (fat-specific protein 27), plays a key role in maintaining triacylglyceride (TAG) homeostasis in adipose tissue and liver, and controls circulating TAG levels in mice. Importantly, mutations and SNPs in
CIDEC
are associated to dyslipidemia and altered metabolic function in humans. Here we tested whether systemic silencing of
Fsp27
using antisense oligonucleotides (ASOs) was atheroprotective in LDL receptor knock-out (
Ldlr
-/-
) mice. Animals were fed a high-fat, high-cholesterol diet for 12 weeks while simultaneously dosed with saline, ASO-ctrl, or ASO-Fsp27. Data show that, compared to control treatments, silencing
Fsp
27
significantly reduced body weight gain and visceral adiposity, prevented diet-induced hypertriglyceridemia, and reduced atherosclerotic lesion size both in
en face
aortas and in the aortic root. Our findings suggest that therapeutic silencing of
Fsp27
with ASOs may be beneficial in the prevention and management of atherogenic disease in patients with metabolic syndrome.
Abstract 200: Behavior Modification and Hypothalamic Appetite Regulation in a Stress-less Mouse Model
Authors: Santanam Nalini , Seidler Abbagael , Amos Debbie , Grover Lawrence
Published: March 2019

Obesity is a major crisis in the Appalachian region. Being obese not only increases risk to heart disease and diabetes, but also promotes behavior modifications. Behavior changes such as excess food consumption, stress, depression is very commonly observed among obese individuals. We hypothesized, lower redox stress by modulating appetite regulation will decrease obesity associated behavioral stress. Behavior changes and appetite regulation were determined in C57Bl6 mice (control) and Bob-Cat mice that express high antioxidant catalase in an obese (Ob/Ob) background (stress-less mouse) (n=6-8) fed either normal chow (NC) or high-fat (HF-45% lard-TD06415) diet for eight weeks. Weekly body weights were monitored. Fat mass changes were determined using ECHO-MRI. Comprehensive lab animal monitoring system (CLAMS) was used to determine energy expenditure. Motor and anxiety-like behavior was tested using Open Field (locomotor behavior) and Rota-Rod (motor coordination and strength) testing systems at baseline and 8 weeks. Changes in appetite regulating genes in the hypothalamus were determined using real time PCR. The C57Bl6 mice on the NC stayed longer on the Rota-Rod as the weeks progressed compared to the mice on HF, where the effect was more dependent on the trials (learning) rather on endurance. The Open Field test showed that the C57Bl6 mice on HF diet were more anxious (spent more time in the edges rather than center) compared to the mice on NC. Catalase overexpression lowered levels of anxiety but had less endurance levels in the HF fed mice. CLAMS showed heat production was higher in stress-less mice vs C57Bl6. Catalase overexpression showed an induction in anorexigenic POMC, and decrease in orexigenic Npy vs C57Bl6. Redox regulation in the stress-less mice modulated both hypothalamic appetite regulation and behavior changes associated with obesity. These findings suggest that redox modulation, may be regulating metabolic pathways that lower obesity and improve the overall behavioral and nutritive state.
Abstract 201: Non-invasive, Mechanical Treatment for Atherosclerosis
Authors: Urso Justin , Narasimhulu Chandrakala , Parthasarathy Sampath , Kassab Alain , Vasu Subith
Published: March 2019

Background:
Atherosclerosis is the foremost cause of cardiovascular diseases, and results in millions of deaths worldwide. All current treatments for atherosclerosis involve, at the least, minimally invasive surgery. The treatment procedures result in a necessary recovery time, have a risk of complication, and do not reverse plaque buildup, but rather offset the symptoms. Preventative measures depend on a reliance on pharmaceuticals to slow the growth of atherosclerotic regions.

Methods:
Lipoproteins were isolated from human plasma, acetylation of lipoproteins was performed. RAW 264.7 cells were incubated with Ac-LDL for 24hrs. After 24hrs of incubation, cells were treated with ultrasonic waves (Vevo 3100) followed by 2hrs incubation at 37°C. RNA was isolated, cDNA was synthesized and quantitative RT-PCR was performed to detect mRNA levels of cholesterol transport as well as pro-inflammatory gene expressions. Foam cells were stained with Oil Red O after the ultrasonic wave treatment.

Results:
Reduced Oil Red O staining was observed in sound waves treated foam cells as compared to untreated cells. Gene analysis showed the increased cholesterol transport genes such as ABCA1 and ABCG1, and reduced pro-inflammatory gene expressions were observed.

Conclusions:
This is the first study that demonstrates that ultrasonic wave treatment could be an effective non/minimally-invasive treatment for atherosclerosis. This novel treatment has the capability to reverse plaque buildup naturally, without the need for surgery, and without the use of pharmaceuticals.
Abstract 203: Glyoxalase 1 Overexpression on Endothelial Progenitor Cell Therapy Accelerates Diabetic Wound Healing
Authors: Li Hainan , O'Meara Megan , Zhang Xiang , Wang Yihan , Wang Jie-Mei
Published: March 2019

Introduction:
Vascular progenitor cells (VPCs) represent the repair and regenerative potential of vasculature. VPC function has been found severely impaired in diabetes with mechanisms not fully understood. We previously demonstrated that inositol requiring-enzyme 1α (IRE1α) activity is significantly attenuated in diabetic VPCs, contributing to impaired angiogenesis
in vitro
. Methylglyoxal (MGO) is one of the highly reactive species generated in hyperglycemic environment. We hypothesize that MGO scavenger glyoxalase 1 (GLO1) reverses VPC dysfunction through augmenting of IRE1α, resulting in improved diabetic wound healing.

Methods and Results:
VPCs were isolated and cultured from the bone marrow of adult male db/db type-2 diabetic and their healthy control db/+ mice. MGO (10μM, 24h) induced immediate and severe VPC dysfunction, including impaired tube formation (Matrigel tube formation assay) and proliferation (MTT assay), increased apoptosis (Caspase 3/7 activity), which was rescued by adenovirus-mediated GLO1 over-expression (50MOI, 48h prior to MGO exposure). IRE1α expression (by Western Blot) in VPCs was decreased upon MGO exposure, but reversed by GLO1 over-expression. IRE1α RNase activity was impaired by direct MGO modification
in vitro
. Cell therapies using db/db VPCs with GLO1 over-expression significantly improved diabetic wound healing
in vivo
(6-mm full-thickness excisional wound) through facilitating angiogenesis (detected by wound capillary density), compared with diabetic control cell therapy.

Conclusions:
Our data suggest that GLO1 can improve VPC function in diabetes and facilitate angiogenesis by rescuing compromised efficacy of diabetic cell therapies. This is at least partly through augmenting IRE1α expression and activity. We believe that our data will provide useful information for developing novel therapeutic approaches to rescue progenitor/stem cell-mediated angiogenesis and tissue repair in diabetes.
Abstract 214: Functional Analysis of the Low Density Lipoprotein Receptor Using Saturation Mutagenesis Strategies
Authors: Chadwick Alexandra C , Evitt Niklaus H , Musunuru Kiran
Published: March 2019

Low density lipoprotein receptor (LDLR) genetic mutations are well-documented to cause familial hypercholesterolemia (FH) in patients. These mutations affect a broad range of LDLR functions, including LDL binding and receptor trafficking, and five classes exist to describe their functional consequences. Although over 1,000 LDLR mutants have been identified, an additional 15,000+ LDLR mutants are possible by only a single amino acid substitution in the ~160kDa protein. Since cataloging mutants would undoubtedly be of therapeutic benefit, we are systematically classifying the effect of every single amino acid substitution on LDLR function using saturation mutagenesis. LDLR was knocked out of HEK293 cells using standard CRISPR/Cas9 practices and decreased protein levels were assessed by flow cytometry and immunoblot staining. In order to introduce each individual mutation into the genomic DNA sequence for high-throughput, functional analyses, the PiggyBac transposon system was used to insert a serine-integrase attachment site into a single allele in the
LDLR
locus, whereby efficient knock in of LDLR mutants from a pooled cDNA library can be achieved. Mutant LDLR libraries containing ~800 single amino acid substitutions were generated using commercial oligo pools and Gibson cloning. Of particular therapeutic interest is the identification of mutations that alter LDL’s ability to bind to the receptor. In our first set of experiments, fluorescently labeled LDL was incubated with cells containing pooled single amino acid substitutions in LDLR, and cells were sorted using flow cytometry. Sorted groups ranging from fluorescent negative to positive were sent for next generation sequencing in order to identify severe vs benign mutants on receptor function. As some of these mutations may decrease LDL binding activity due to inhibited receptor trafficking, we also stained pooled cells with an antibody directed against LDLR, sorted by flow cytometry and analyzed using next generation sequencing with similar strategies. Ultimately, cataloging LDLR genetic mutations should be of great therapeutic benefit, as it will allow better diagnosis and treatment of patients suffering from FH.
Abstract 216: Modeling Statin Hepatotoxicity with Acute Liver Specific Deletion of HmgCoA Reductase
Authors: De Giorgi Marco , Jarrett Kelsey E , Burton Jason C , Doerfler Alexandria M , Hurley Ayrea , Hsu Rachel H , Lagor William R
Published: March 2019

Hmg-CoA Reductase (Hmgcr) catalyzes the conversion of Hmg-CoA to mevalonate, which is the rate-limiting step in the cholesterol biosynthetic pathway. Hmgcr is the target of statins, which are the front line therapy for hypercholesterolemia. Statin-responsiveness varies greatly between individuals, and liver toxicity and transaminase elevations are observed in 1-3% of patients. In addition to cholesterol,
Hmgcr
activity is also required for the synthesis of heme A, dolichol, ubiquinone (Coenzyme Q), farnesylated and geranylgeranylated proteins, isopentenyl tRNA, and vitamin K2. Surprisingly, very little is known about the effects of statins on these non-sterol isoprenoids
in vivo
, which may be essential for hepatocyte viability and function. The goal of this project is to investigate the effects of
Hmgcr
inhibition in the liver, in order to better understand the physiological regulation and function of the mevalonate pathway. We have developed a new inducible genetic model of
Hmgcr
deletion, which enables us acutely inhibit the mevalonate pathway in the liver, thus avoiding well-known compensatory responses to statins in rodents.
Hmgcr
floxed mice were injected with liver-specific AAV-Cre recombinase (AAV-Cre) and followed for 1, 2, 4 and 8 weeks. We observed massive apoptosis followed by a burst of hepatocyte proliferation at 2 weeks post-injection. We also observed compensatory upregulation of genes in the mevalonate pathway, likely a response to sterol depletion. Hepatocytes from AAV-Cre mice had expanded and swollen endoplasmic reticulum (ER), and upregulation of Chop and Bax, suggesting ER stress-induced apoptosis. Genetic analysis of AAV-Cre treated livers showed progressive loss of the null allele over time, indicating a selective advantage for hepatocytes that escaped complete
Hmgcr
deletion. The injection of a five-fold higher dose of AAV-Cre was acutely lethal in less than 2 weeks, confirming that liver Hmgcr activity is essential for survival. This new model will be used to identify the essential mevalonate-derived products in the liver, and the molecular pathways underlying statin hepatotoxicity and responsiveness.
Abstract 222: Translocator Protein as Novel Marker for Early Atherosclerosis
Authors: Kopecky Chantal M , Ryder William J , Szajer Jeremy , Parmar Arvind , Pandzic Elvis , Rye Kerry A , Cochran Blake J
Published: March 2019

Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. Here, we aimed to determine if (i) the TSPO radioligand,
18
F-PBR111, can identify fatty streak formation that precedes atherosclerotic lesion development and (ii) TSPO expression can be used to identify distinct macrophage populations during different stages of lesion progression. Atherosclerosis-prone apoE
-/-
mice were maintained on a high-fat diet for 3, 9 and 12 weeks. C57BL6 mice maintained on chow diet served as controls. Mice were anesthetized, injected with
18
F-PBR111 (8-18 MBq, 0.1 mL, 0.2 nM) via the lateral tail vein and imaged using an Inveon PET/CT Scanner over 60 min. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, fluorescently labelled anti-TSPO antibody and the macrophage markers F4/80 and CD11b. Tissues were visualized on a Lightsheet Z.1 microscope and analysed with 3D imaging software. A 3-fold increase in the uptake of
18
F-PBR111 was observed by PET/CT in the aortas of atherosclerotic mice relative to controls. Increased
18
F-PBR111 uptake was observed in the aortic arch and thoracic aorta, corresponding with sites of atherosclerotic lesion development. Light sheet microscopy revealed that TSPO expression increased correspondingly to atherosclerosis progression. The expression pattern of macrophage populations in early lesions differed from that in mature plaques. While F4/80-positive macrophages were evident uniformly throughout the aortas, CD11b-positive macrophages were increased in atherosclerotic areas. In conclusion, imaging TSPO expression is a new approach for studying atherosclerotic lesion progression and is associated with specific inflammatory cell infiltration. The TSPO ligand
18
F-PBR111 is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.
Abstract 225: Electronic Cigarettes Induced Exacerbation of Atherosclerosis: Contribution of MicroRNA-21
Authors: Malovichko Marina V , Wickramasinghe Nalinie S , Kalani Anuradha , Sithu Srinivas D , Gordon Terry , Chen Lung-Chi , Bhatnagar Aruni , O’Toole Tim E , Conklin Daniel J , Srivastava Sanjay
Published: March 2019

Smoking is the foremost cause of preventable disease and pre-mature deaths. Although the rates of smoking have declined in the recent past, advent of electronic cigarettes (e-cig) pose new challenges. E-cig are extremely popular in youth and usage of e-cig has increased by 9-fold in the youth between 2011 and 2015. E-cig are battery-operated devices used for the delivery of aerosolized nicotine. Unlike conventional cigarettes, e-cig have lower levels of carcinogens. However, aerosolization of e-liquids generates reactive chemicals such as acrolein, which has adverse effects on cardiovascular health. To examine the effect of e-cig on atherosclerosis we exposed 8-week old apoE-KO mice (maintained on Western diet) to filtered air or e-cig (36 mg/ml nicotine aerosolized in glycerol/propylene glycol, 50/50, v/v) for 12 weeks (3 h/day; 7 days a week). Our data show that exposure to e-cig increased the aortic lesion area by 24% (P<0.05), without affecting plasma cholesterol (e-cig 1088±61 vs air 1102±61 mg/dL) and triglyceride (e-cig 95±9 vs air 77±5 mg/dL). Exposure to e-cig also increased the expression of micro RNA-21 (miR-21) in the aorta by 26% (P<0.05). In situ hybridization assay showed that miR-21 co-localizes with macrophages in atherosclerotic plaques.
In vitro
, e-cig constituents acrolein and nicotine induced the expression of miR-21 by 1.8-2.0 fold, in bone marrow derived macrophages. Similarly, 4,hydroxynonenal (HNE) generated by acrolein and nicotine induced reactive oxygen species (ROS) formation by 1.3-fold, and increased the expression of miR-21 in macrophages by 2-fold. Exposure to e-cig also activated matrix metalloproteinases (MMPs) in atherosclerotic plaques. Acrolein activated MMPs
ex vivo
in atherosclerotic plaques, and increased the secretion of MMP-9 from macrophages. Acrolein-induced ROS formation and MMP-9 secretion was prevented by free radical scavengers. Moreover, we also observed that HNE-induced macrophage apoptosis was significantly increased by miR-21 deficiency. This process was mediated by the activation of both, intrinsic and extrinsic pathways of apoptosis. Collectively, these data suggest miR-21 prevents e-cig-induced atherogenesis by inhibiting inflammatory and apoptotic pathways in macrophages.