bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 644: CD36 is Essential to Increased Atherosclerosis Mediated by Oral Infection With Porphromonas gingivalis
Authors: Febbraio Maria , Brown Paul M
Published: July 2021

Epidemiological evidence strongly support a link between periodontal disease & cardiovascular disease, but the mechanism(s) remains poorly understood. Using the human periodontal disease associated bacteria, Porphyromonas gingivalis (Pg) as a model, we carried out studies in macrophages & low density lipoprotein receptor (LDLR) KO mice. Pg associated similarly with macrophages from wild type & CD36 KO mice, but there were differences in responses dependent on Toll-like receptor (TLR) 2. We observed decreased NFkB activation & IL1beta generation following Pg treatment in CD36 KO macrophages, despite similar levels of TLR2 expression. OxLDL strongly inhibited Pg mediated IL-1beta generation in a CD36 dependent manner. Macrophage foam cell formation as a result of incubation with oxLDL & PgLPS was increased in a CD36 dependent manner.

LDLR KO & CD36/LDLR double KO mice were orally infected with Pg & fed a Western diet (12 weeks). There was a significant increase in the cemento-enamel junction of molars of infected compared with uninfected mice, demonstrating the validity of the model. Histological analysis showed inflammatory cell infiltrates in gums of infected mice after 12 weeks, supporting a chronic inflammatory process. Differences in plasma parameters & weight gain did not necessarily track with atherosclerosis burden, however blood neutrophils & cytokines were increased in infected LDLR KO mice compared with all other groups. Infected LDLR KO mice had significantly increased atherosclerotic lesion burden compared with uninfected LDLR KO mice, and all of the increased lesion was CD36-dependent.

Our data suggest that atherosclerosis associated with periodontal disease is mediated by cellular inflammatory responses involving both CD36 & TLR2. Pg enhances oxLDL mediated foam cell formation in a CD36 dependent manner, and this may explain increased lesion burden. Generation of IL1beta, a key pro-atherogenic cytokine, is altered as a result of CD36 expression. Periodontal disease affects more than 20% of the population of the US/Canada, & is associated with increasing age, which is also a risk factor for atherosclerosis. Targeting CD36 may provide important supplemental therapy to current lipid lowering strategies to reduce atherosclerosis.
Abstract 65: ABCA1 Ligand Peptides That Stimulate Cellular Cholesterol Efflux With High Potency Increase Insulin Sensitivity in Metabolic and Genetic Mouse Models of Obesity and Type 2 Diabetes
Authors: Bielicki John K , Bittner Stefanie , Tabassum Juveria , Hafiane Anouar , Genest Jacques , Azhar Salman , Johansson Jan
Published: July 2021

Anti-diabetic activities of HDL apolipoprotein(apo)s have been described, complementing ability to reduce atherosclerosis. These effects have been attributed, in part, to apos stimulating cholesterol efflux from cells via ABCA1. This suggests apo mimetic peptides that promote ABCA1 cholesterol efflux may be useful to combat diabetes and its cardiovascular complications. Two such peptides (CS6253 and T6991-2) with optimized safety features (NOAEL = 500 mg/kg) and little TG elevating effects have been designed by us through structure-activity studies of a prototypic ABCA1 ligand peptide, ATI-5261, derived from the C-terminal (CT) domain of apoE. Glucose lowering and insulin sensitizing effects of these new peptides were presently evaluated in mouse models of obesity and diabetes. Peptides CS6253 and T6991-2 stimulated cholesterol efflux from macrophages via ABCA1 with high potency similar to the native apoE CT domain (Km= 0.33±0.14, 0.24±02, 0.21±0.02 μM). Administration of CS6253 at a dose of 30 mg/kg (SC) on alternate days for 6 weeks reduced atherosclerosis by 32% in apoE deficient mice fed high-fat, Western diet for 14 weeks (15±2 vs. 22±4% plaque lesions, CS6253 vs. control, p<0.01). T6991-2 administered (SC) at a dose of 10 mg/kg for 6 weeks in chow-fed ob/ob mice showed little effect on steady-state plasma glucose levels vs. controls (126±22 vs. 135±11 mg/dl, respectively); however, the levels of glucose were greatly attenuated with peptide treatment vs. controls in response to GTT (1.8±0.5 vs. 2.8±0.4 fold increases in plasma glucose at 60 min, respectively, p<0.01). Treatment of C57BL/6J mice fed high-fat diet (HFD) with T6991-2 (6 weeks, 30 mg/kg) also enhanced insulin sensitivity by 2.2±0.7 fold vs. controls (55±17 vs. 25±5% reduction in basal glucose levels, respectively, at 15 minutes post insulin, 0.75 Units/kg, p<0.01). The favorable anti-diabetic effects of T6991-2 were not due to changes in total body weight or β-cell function (i.e. unchanged plasma C-peptide levels). Our data indicate that single amphipathic α-helix peptides derived from apoE CT domain are sufficient to confer potent cellular cholesterol efflux and anti-diabetic activities, with therapeutic potential for diabetes and cardiovascular disease.
Abstract 650: Ceramide as a Mediator of Insulin Resistance--Associated Atherosclerosis
Authors: Kasumov Takhar , Li Ling , Previs Stephen , Gulshan Kailash , Willard Belinda , Smith Jonathan
Published: July 2021

Objectives:
Insulin resistance-induced hyperlipidemia and hyperlipoproteinemia are risk factors of atherosclerosis. Spingolipid ceramide is involved in insulin resistance and lipoprotein metabolism, and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD. Here we studied the role of spingolipids in insulin resistance associated atherosclerosis.

Methods:
Low density lipoprotein receptors knockout (LDLR
-/-
) mice, a diet induced mouse model of insulin resistance and atherosclerosis, were fed a standard chow diet or a western diet (high fat with 0.2% cholesterol) for 12 weeks with or without myriocin, an inhibitor of
de novo
ceramide synthesis. A targeted lipidomics approach was used for hepatic and plasma profiling of ceramides. Metabolic
2
H
2
O-labeling technique was applied to quantify turnover rates of hepatic and plasma lipids and lipoproteins, including ApoB and ApoAI - the principle proteins of VLDL/LDL and HDL. Hepatic and intestinal expression of genes and proteins involved in insulin signaling, lipid and lipoprotein metabolism were characterized.

Results:
A western diet caused insulin resistance, increased hepatic and serum triglycerides and the altered distribution of hepatic ceramides, i.e. hepatic levels of long chain ceramides (C16 & C18), involved in apoptosis, were increased by 80% and 27%, respectively, while a very-long chain ceramide C24 was reduced more than twice. A western diet also induced dyslipidemia, hepatic oxidative stress, inflammation, apoptosis, mild fibrosis, and atherosclerosis. The plasma ratio of ApoB/ApoA1 was increased >2 fold which was due to increased production of ApoB. HDL turnover was not affected. Pharmacologic inhibition of sphingolipid biosynthesis with myriocin improved insulin sensitivity, hepatic steatosis, fibrosis, apoptosis, and prevented atherosclerosis. These changes were associated with decreased lipogenesis and ApoB production. In addition, myriocin significantly increased both plasma levels and flux of HDL.

Conclusions:
Modulation of sphingolipid metabolism may lead to the development of novel therapeutic strategies for the treatment of insulin resistance and associated atherosclerosis.
Abstract 670: A Metabolic Approach to Examining the Hexosamine Biosynthetic Pathway and Its Role in the Development of Diabetes-Associated Atherosclerosis
Authors: Petlura Christina , Walter Lisa , Werstuck Geoff
Published: July 2021

Introduction:
Diabetes is a disease affecting millions of people worldwide, and is a major independent risk factor for cardiovascular disease (CVD). Despite a vast amount of research, the molecular mechanisms that link diabetes to CVD are not well understood. Current evidence suggests that increased flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of hyperglycemia-associated diabetic complications. Our data suggest that increased HBP flux can induce vascular ER stress and accelerate atherogenesis in a mouse model. We hypothesized that this process can be attenuated by inhibiting the first and rate-limiting enzyme in the HBP - glutamine fructose-6-phosphate amidotransferase (GFAT) - using small molecules.

Methods and Results:
Using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) we have developed a methodology to monitor and quantify the levels of the end product of the HBP, uridine diphosphate
N
-acetylglucosamine (UDP-GlcNAc). Treatment of HepG2 cells with glucosamine (0.2-5 mM), or adenovirus-directed overexpression of GFAT caused a 3-7 fold increase in UDP-GlcNAc (
P<
0.001 &
P<
0.05, respectively). Inhibition of GFAT with three novel compounds - amrinone, lapachol or alloxan - decreased levels of UDP-GlcNAc by 1.5 (
P<
0.05), 3 (
P<
0.05) and 3.5-fold (
P<
0.001), respectively. Furthermore, we show that by modulating HBP flux, we can regulate ER stress levels in cultured HepG2 cells. The physiological relevance of this mechanism is supported by evidence of HBP augmentation in a hyperglycemic mouse model.

Conclusions:
These results support a role for the HBP in the development of atherosclerosis. Currently, MALDI imaging mass spectrometry is being performed on tissue sections to compare the levels of UDP-GlcNAc directly in hyperglycemic vs. normoglycemic mice. These studies may lead to the identification and validation of novel targets for the development of new pharmaceuticals to prevent diabetic atherosclerosis.
Abstract 7: A Subcutaneous, Potent and Durable RNAi Platform Targeting Metabolic Diseases, Genes PCSK9, ApoC3 and ANGPLT3
Authors: Fitzgerald Kevin , Borodovsky Anna , Querbes William , Sutherland Jessica , Hutabarat Renta , Milstein Stuart , Kuchimanchi Satya , Kallanthottathil Rajeev , Charisse Klaus , Yucias Kristina , Liebow Abigail , Sprague Andrew , Maier Martin , Kallend David , Horton Jay , Simon Amy
Published: July 2021

Aim:
There remains high unmet medical need for therapies to treat cardio/metabolic diseases. We validated in human trials, a platform for reducing the synthesis of genes expressed in the liver. The platform utilizes a GalNAc ligand attached to the 3’ end of the sense strand of an RNAi molecule to enable delivery specifically to the liver. Here we extend the platform to targets of interest in cardiovascular disease, including PCSK9, ANGPLT3 and ApoC3.

METHODS:
Chemically modified siRNAs were designed and were screened for potency
in vitro
. pM active siRNA molecules were developed targeting PCSK9, ANGPLT3 and ApoC3. The siRNAs were tested in either rodents or in non-human primates (NHPs) for activity.

RESULTS:
In NHPs a single dose of ALN-PCSsc at 6 mg/kg reduced PCSK9 levels up to 97% and LDL-C up to 67%. Moreover the nadir effect (without any rebound of LDL-C) lasted >30 days indicating that once a month or longer dosing frequency in clinic should be supported. Multidose studies in NHP at 2mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. The effects on both PCSK9 and LDL-C was also very durable with levels of LDL-C returning to baseline > 140 days post the last dose. Safety studies in rat at doses up to 225mg/kg (multi-dose) indicate that ALN-PCSsc is safe demonstrating a very wide therapeutic index. ALN-PCSsc was selected as a development candidate and is being advanced towards an IND

ALN-ANGsc (an siRNA targeting ANGPTL3) was tested in two models of hyperlipidemia, the OB/OB mouse and the hCETP-ApoB mouse. In the Ob/Ob model, treatment with ALN-ANGsc at 3mg/kg resulted in a significant lowering ANGPLT3 protein (>95%), total cholesterol(>60%), and triglycerides (>85%). Finally, we have developed a prototype molecule targeting Apoc3 with an ED90 for ApoC3 protein of <2.5mg/kg showing 50% lowering of triglycerides in and db/db mouse model of hypertriglyceridemia.

CONCLUSION:
We have developed a modular, robust and durable platform for the delivery of RNAi therapeutics to the liver. This platform is administered as a small volume subcutaneous dose and has a remarkable duration of effect in rodent NHP models. We have extended this platform to several targets of high interest includingPCSK9,ANGPLT3 and ApoC3.
Abstract 80: Modulating the Vascular Healing Response via SIRT1 Transgene Delivery
Authors: Jen Michele C , Chen Vincent Y , Shea Lonnie D , Ameer Guillermo A
Published: July 2021

Bypass graft procedures are commonly performed to treat narrowing or blocked vessels. However, damage to the vascular wall from the surgical intervention can lead to pro-inflammatory events and oxidative stress that promote intimal hyperplasia and may eventually lead to restenosis. Therefore, strategies that seek to reduce inflammation and oxidative stress during the initial stages of wound healing at the graft anastomoses are expected to inhibit intimal hyperplasia and reduce graft failure rates. Sirtuin-1 (Sirt1), an NAD+-dependent lysine deacetylase, protects against inflammation and oxidative stress by deacetylating the RelA/p65 subunit of NF-kB and FOXO transcription factors. We hypothesize that the localized overexpression of Sirt1 at the perivascular wall via lentivirus entrapped in a thermoresponsive gel referred to as nanonets will lead to an improved vascular healing response at the graft anastomoses and attenuate intimal hyperplasia via reduced tissue inflammation and oxidative stress. Human aortic adventitial fibroblasts (AoAFs) were transduced with lentiviruses encoding for Sirt1 and assessed for NF-kB activity, cytokine expression, senescence, and upregulation of antioxidant genes. To assess perivascular transgene delivery, nanonets containing lentiviruses encoding GFP were applied perivascularly around the aortas of rats. After one week of in vivo transduction, aortas were explanted and probed for GFP positive cells. Upon TNFα stimulation, NF-kB activity and cytokine expression levels were decreased when compared to non-transduced and empty vector-transduced AoAFs. Furthermore, Sirt1-overexpression prevented cellular senescence and protected against oxidative stress. Nanonets containing lentiviruses effectively transduced adventitial fibroblasts in vivo. In conclusion, Sirt1 overexpression in AoAFs may be protective against inflammation and oxidative stress.
Abstract 103: HDL Improves Cholesterol and Glucose Homeostasis and Reduces Atherosclerosis in TRAIL-/-ApoE-/- mice
Authors: Di Bartolo Belinda A , Cartland Sian P , Vellozzi Melissa , Rye Kerry-Anne , Kavurma Mary M
Published: July 2021

Background and Aims:
Atherosclerosis is characterized by the infiltration and accumulation of monocytes in the arterial wall, combined with elevated plasma low density lipoprotein (LDL) and triglycerides, and low levels of high density lipoprotein (HDL). Apolipoprotein A-I (apoA-I), the main HDL apolipoprotein, is not only athero-protective but can also increase insulin secretion from pancreatic β-cells. Our laboratory recently demonstrated that in response to a ‘western’ diet TNF-related apoptosis-inducing ligand (TRAIL)-deficiency accelerated plaque development in ApoE-/- mice, and promoted features of diabetes typical to those observed in humans. We sought to determine whether reconstituted HDL (rHDL) could improve TRAIL-dependent disease.

Methods and Results:
TRAIL-/-ApoE-/- and ApoE-/- mice on a ‘western’ diet for 12 w received 3 weekly infusions of either PBS (vehicle) or rHDL (20 mg/kg apoA-I, 1-palmitoyl-2-linoleoyl phosphatidylcholine) in the final 2 weeks of feeding prior to euthanasia. While no change in weight gain was observed, administration of rHDL significantly reduced plasma cholesterol, triglyceride and glucose levels, and improved glucose clearance and insulin sensitivity in TRAIL-/-ApoE-/- mice. Notably, rHDL treatment significantly attenuated atherosclerotic plaque size in brachiocephalic arteries of TRAIL-/-ApoE-/-, but not in ApoE-/- mice. rHDL-treated TRAIL-deficient mice also displayed elevated aortic mRNA expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. This was associated with significantly increased expression of the pan macrophage marker F4/80 and the M2 marker CD206, suggesting that the rHDL stimulated polarization towards an M2 phenotype. Immunohistological analysis of the rHDL-treated TRAIL-/-ApoE-/- pancreata revealed increased insulin expression and reduced macrophage infiltration relative to ApoE-/- pancreata.

Conclusions:
This is the first demonstration of rHDL improving atherosclerosis and rescuing features of diabetes which are dependent on TRAIL. Understanding how rHDL may be beneficial in TRAIL-dependent disease could identify new strategies in the treatment of atherosclerosis and associated diabetic complications.
Abstract 12: EMMPRIN-Targeted Magnetic Nanoparticles for in vivo Visualization and Regression of Acute Myocardial Infarction in a Model of Acute Coronary Artery Occlusion
Authors: Cuadrado Irene , Garcia Miguel Maria Jose , Herruzo Irene , Turpin Mari Carmen , Martin Ana , Reventun Paula , Saura Marta , Zamorano Jose-Luis , Zaragoza Carlos
Published: July 2021

Extracellular matrix metalloproteinase inducer EMMPRIN, is highly expressed in patients with acute myocardial infarction (AMI), and induces activation of several matrix metalloproteinases (MMPs), including MMP-9 and MMP-13. To prevent Extracellular matrix degradation and cardiac cell death we targeted EMMPRIN with paramagnetic/fluorescent micellar nanoparticles with an EMMPRIN binding peptide AP9 conjugated (NAP9), or an AP9 scramble peptide as a negative control (NAPSC). NAP9 binds to endogenous EMMPRIN as detected by confocal microscopy of cardiac myocytes and macrophages incubated with NAP and NAPSC in vitro, and in vivo in mouse hearts subjected to left anterior descending coronary artery occlusion (IV injection 50mγ/Kg NAP9 or NAP9SC). Administration of NAP9 at the same time or 1 hour after AMI reduced infarct size over a 20% respect to untreated and NAPSC injected mice, recovered left ventricle ejection fraction (LVEF) similar to healthy controls, and reduced EMMPRIN downstream MMP9 expression. In magnetic resonance scans of mouse hearts 2 days after AMI and injected with NAP9, we detected a significant gadolinium enhancement in the left ventricle respect to non-injected mice and to mice injected with NAPSC. Late gadolinium enhancement assays exhibited NAP9-mediated left ventricle signal enhancement as early as 30 minutes after nanoprobe injection, in which a close correlation between the MRI signal enhancement and left ventricle infarct size was detected. Taken together, these results point EMMPRIN targeted nanoprobes as a new tool for the treatment of AMI.
Abstract 155: MicroRNA-146a Suppression of NF-κB-driven Monocyte/Macrophage Activation and Atherosclerosis is Regulated by Cellular ApoE Expression
Authors: Li Kang , Ching Daniel , Luk Fu Sang , Raffai Robert L
Published: July 2021

Apolipoprotein E (apoE) exerts anti-inflammatory properties that protect against atherosclerosis and other inflammatory diseases. However, the mechanisms by which apoE suppresses the activation of leukocytes remain incompletely understood. An assessment of apoE expression levels among major leukocyte subsets in wild-type mice revealed that only macrophages and monocytes express apoE abundantly. An absence of Apoe expression in mouse macrophages and monocytes resulted in enhanced NF-κB signaling and an exaggerated inflammatory response upon stimulation with lipopolysaccharide. This correlated with reduced levels of microRNA-146a, a critical negative regulator of NF-κB signaling. Restoration of apoE expression in Apoe-/- macrophages and monocytes enhanced miR-146a levels, while silencing apoE expression in wild-type mouse monocytes reduced miR-146a levels. Mechanistically, apoE increased the expression of transcription factor PU.1, which raised levels of pri-miR-146 transcripts, demonstrating that apoE exerts a transcriptional control over miR-146a. In vivo, even a small amount of apoE expression in macrophages and monocytes of hypomorphic apoE mice led to increased miR-146a levels, which inhibited macrophage proinflammatory responses, Ly-6Chigh monocytosis and atherosclerosis in the settings of hyperlipidemia. Accordingly, cellular enrichment of miR-146a through the systemic delivery of miR-146a mimics emulsified in lipid microparticles infused into apoE deficient mice attenuated systemic inflammation and atherosclerosis in the absence of plasma lipid reduction. Together, our data demonstrate that apoE suppresses NF-κB-mediated inflammation and atherosclerosis by enhancing miR-146a levels in monocytes and macrophages. Ongoing studies are exploring the impact of apoE on the cellular release of endogenous miR-146a in exosomes and high density lipoproteins from macrophages that can be communicated to cells in the circulation and artery to suppress inflammation and atherosclerosis.
Abstract 172: Product of Lipid Oxidation During Inflammation is a New Potent Ligand for β2 Integrin-mediated Macrophage Migration
Authors: Yakubenko Valentin , Brown Kathleen , Tischenko Miraslava , West Xiaoxia , Podrez Evgeny , Byzova Tatiana
Published: July 2021

2-(ω-carboxyethyl)pyrrole (CEP) and ethylpyrrole (EP) derivatives of proteins are formed through adduction of end products of docosahexaenoate (DHA) oxidation[[Unable to Display Character: ]] with the ε-amino groups of protein lysyl residues, followed by cyclization and dehydration. Our previous studies demonstrated that EP-modified proteins are present in healthy tissue, while CEP-modified proteins are detected in the tissue during inflammation including inflamed peritoneal wall and atherosclerotic lesions.

Potential role of CEP in inflammatory response was evaluated by testing leukocyte recruitment during thioglycollate-induced peritoneal inflammation. We found that injection of anti-CEP antibody dramatically reduced the accumulation of macrophages in the peritoneal cavity at 72 hours, while the earlier accumulation of neutrophils was not affected. Migration of neutrophils was a prerequisite for CEP production in the peritoneum. In vitro adhesion assay revealed strong binding of peritoneal macrophages to CEP, while adhesion to EP was not detected. Moreover, CEP presence in 3D fibrin gel significantly increased migration of macrophages toward MCP-1 gradient. We have previously demonstrated that CEP is ligand for CD36 and TLR2. However, the adhesion of TLR2/CD36-double deficient macrophages to CEP was similar to wild type cells; suggesting that other receptors are involved in CEP-mediated macrophage adhesion/migration. Since αDβ2 and αMβ2 integrins are major macrophage adhesion receptors we tested their ability to bind CEP. Isolated αM and αD I-domains, which are critical for integrin ligand recognition, showed a concentration-dependent binding to immobilized CEP, but not to EP in the biacore assay. Moreover, αDβ2 and αMβ2-transfected but not control HEK293 cells strongly adhered to CEP. This adhesion was significantly inhibited by anti-CEP, anti-β2 and anti-αM (or αD) antibodies, but not by anti-β1 antibody.

In summary, we found that CEP-modified proteins drive the migration of macrophages during inflammation by engaging macrophage integrins αMβ2 and αDβ2. Hence, regulation of CEP-mediated adhesion/migration can control the accumulation of macrophages within the site of inflammation and may regulate the development of chronic inflammatory deseases.
Abstract 208: The Interaction of Neutrophils and Interferon Gamma Promotes Post-infarct Survival in a Mouse Model of Myocardial Infarction
Authors: Finger Stefanie , Knorr Maike , Kossmann Sabine , Schoenfelder Tanja , Karbach Susanne , Muenzel Thomas , Wenzel Philip
Published: July 2021

Background:
Myelomonocytic cells are involved in both the initial injury phase and the healing process of myocardial infarction (MI). However, the exact interaction of inflammatory myeloid cells and prominent cytokines remains only partly understood.

Objective:
The goal of the study was to investigate the either cardioprotective or in part adverse role of lysozyme M positive (LysM+) and granulocyte-receptor 1 positive (Gr-1+) immune cells on cardiac injury and healing in a murine model of MI.

Methods and results:
MI was induced in 8 to 12 week-old male mice (C57BL/6 background) by ligation of the left anterior descending (LAD) coronary artery. Compared to LysMCre controls, LysM+ cell depleted LysMiDTR transgenic mice (depletion 3d prior MI by diphtheria toxin application, 25 ng/g body weight) showed a decreased influx of CD45.2+/CD3-/CD11b+/Gr-1high neutrophils into infarcted myocardium 1d post MI (measured by flow cytometric analysis). Additionally cardiac mRNA expression levels of inflammatory cytokines like INFγ and TNFα were decreased 7d post MI. To more specifically assess the role of neutrophils, we depleted C57BL/6 mice with a monoclonal anti-Gr-1 antibody and found increased mortality early after MI as well as a decrease in INFγ mRNA expression 1d and 7d post MI. MCP-1 (CCL2) and CCR2 mRNA were reduced 3d after MI according to decreased amount of CD11b+/Ly6G-/Ly6Chigh inflammatory monocytes in the infarcted myocardium of anti-Gr-1 treated mice. LAD ligated INFγ-/- mice displayed a significantly reduced survival, worsening of left ventricular function and an impaired inflammatory cell infiltration compared to C57BL/6 controls.

Conclusion:
We provide evidence that neutrophils and INFγ play an essential role in survival and cardiac remodeling following MI. Our data indicate that neutrophils are required for monocyte chemotaxis. We conclude, that strategies to combat the inflammatory injury in MI must consider a potentially beneficial effect of early neutrophil influx into infarcted myocardium.
Abstract 23: Immunosuppression with FTY720 Reverses Cardiac Dysfunction to Prevent Chronic Heart Failure in Mice that Survive Diet-induced Myocardial Infarction
Authors: Luk Fu Sang , Kim Roy Y , Li Kang , Ching Daniel , Joshi Sunil , Honbo Norman , Imhof Isabella , Zhu Bo-qing , Lovett David , Karliner Joel S , Raffai Robert L
Published: July 2021

We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in the HypoE/SR-BI-/- mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study we tested whether FTY720 could also reverse cardiac dysfunction in mice that survive MI and subsequently develop chronic heart failure (CHF). HypoE/SR-BI-/- mice were bred to Mx1-Cre mice and offspring were fed a high fat diet (HFD) for 21 days to provoke hyperlipidemia, coronary atherosclerosis and recurrent MIs. HypoE/SR-BI-/-Mx1-Cre mice were subsequently given oral FTY720 in drinking water or not. Hyperlipidemia was permanently reversed by inducible Cre-mediated gene repair of the HypoE allele (also known as the Apoeh/h allele) that rapidly restores normal apoE expression in all tissues and by switching mice to a normal chow diet. In cohorts of mice that survived this period of HFD, left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among HypoE/SR-BI-/-Mx1-Cre mice six weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks after cessation of HFD. Reversal of heart failure did not result from reduced atherosclerosis as the burden of both aortic and coronary atherosclerosis increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In the heart, FTY720 led to reduced expression of MMP-2 along with the expression of genes involved in Type I innate inflammation that we have recently demonstrated as major contributors to heart failure. Our data demonstrate the benefit of immunosuppression with FTY720 post MI to prevent progressive pathological remodeling of the heart, which leads to CHF.
Abstract 230: Protein Farnesylation Inhibitor Tipifarnib Prevents Development of Chronic Hypoxia-induced Pulmonary Hypertension
Authors: Duluc Lucie , Ahmetaj-Shala Blerina , Mitchell Jane , Abdul-Salam Vahitha B , Edwards Robert , Aldabbous Lulwah , Iannone Lucio , Dubois Olivier D , Storck Elisabeth M , Tate Edward W , Zhao Lan , Wilkins Martin , Wojciak-Stothard Beata
Published: July 2021

Study objectives:
Small GTPase RhoB contributes to pulmonary vascular remodelling in pulmonary hypertension. Farnesylation of RhoB augments cell proliferation while geranylgeranylation of RhoB is believed to be pro-apoptotic. We hypothesized that farnesyltransferase inhibitors may prevent vascular pathology in pulmonary hypertension. We studied the effects of farnesyltransferase inhibitor Tipifarnib on pulmonary vascular remodelling and vasoreactivity in chronically hypoxic pulmonary hypertensive mice and cultured human pulmonary artery endothelial cells (HPAECs). Chemical proteomics was used to characterise Tipifarnib-induced changes in protein prenylation while label- free quantitative proteomics was used to characterise changes induced by overexpression of RhoB prenylation mutants: farnesylated-only or geranlygeranylated-only RhoB in HPAECs. The effects of Tipifarnib on Ras and Rho GTPases expression and activity, filamentous and globular actin levels and eNOS pathway, were also studied.

Results:
Oral administration of Tipifarnib significantly attenuated chronic hypoxia-induced pulmonary hypertension in mice. This protective effect was associated with a marked improvement of endothelium-dependent vasodilatation, reduction in pulmonary vascular muscularization and a decrease in the right ventricular systolic pressure. Tipifarnib reduced farnesylation and increased geranyl-geranylation of several proteins, including Rho GTPases and Ras and reduced their activity in vitro and in vivo. Tipifarnib-induced changes in cultured cells were associated with a decrease in cell proliferation, decreased polymerization of actin and increased eNOS mRNA and protein levels. Proteomic analysis of HPAECs overexpressing prenylation mutants of RhoB suggests that protective effects of Tipifarnib may result, at least in part, from inhibition of RhoB farnesylation.

Conclusions:
We demonstrate protective effects of farnesyltransferase inhibitor Tipifarnib in chronic hypoxia-induced pulmonary hypertension. The effects of Tipifarnib may result from inhibition of small GTPases activity and increase in NO signalling.
Abstract 25: Critical Limb Ischemia Progression is Associated with an Inflammatory Profile
Authors: Gremmels Hendrik , Teraa Martin , Fledderus Joost O , de Jong Olivier G , van der Graaf Yolanda , Moll Frans L , Verhaar Marianne C
Published: July 2021

Background:
Patients with Critical Limb Ischemia (CLI) have a high risk of amputations and mortality. We hypothesize that inflammation is involved in atherosclerotic disease progression. In this study we investigate whether levels of plasma cytokines are associated with disease progression in CLI.

Methods:
Data were collected from a randomized controlled trial cohort investigating cell therapy for CLI (the JUVENTAS study) from 2006 to 2012. The primary outcome measures were major amputation and mortality at 6 months; secondary outcomes included Ankle/Brachial Index (ABI) and Transcutaneous O2 Pressure. Plasma was collected at inclusion in the study and a panel of cytokines consisting of GROa, HGF, LIF, SCF, SCGFb, SDF1a, TRAIL, IL-6, IL-8, FGFb, GCSF, GMCSF, IP10, MCP1, PDGFbb, RANTES, TNFa and VEGF was measured and evaluated for predictive power.

Results:
Data on 108 patients was collected with a follow-up of 6 months. Patients who underwent a major outcome had significantly higher levels of IL-6, IL-8 and GROa (p=0.0004, 0.006 and 0.009 resp.). Univariate Kaplan-Meier analysis showed that amputation-free survival was 94.4% in the lowest tertile, 74% in the middle tertile and 64% in the highest tertile of plasma IL-6 levels (p=0.009, Fig A). Adjustment for potential confounders in multivariate Cox proportional hazards models showed that IL-6 remained independent predictor of major outcome. The ROC of a model using Il-6 as single predictor for major outcomes is 0.73 (Fig. B)

Conclusion:
Plasma levels of inflammatory cytokines, in particular IL-6 are associated with disease progression in CLI and can serve as an independent predictor of amputation-free survival.
Abstract 267: CD93: A Novel Myocardial Infarction- Associated Protein with Glucose Regulatory Properties in Humans and Mice
Authors: Bäcklund Alexandra , Strawbridge Rona , Jin Hong Jin , Tsikrika Panagiota , Silveira Angela , Ketelhuth Daniel F , Eriksson Per ,   IMPROVE Study Group, Maegdefessel Lars , Hamsten Anders
Published: July 2021

Introduction:
Coronary artery disease (CAD) is a complex disorder involving metabolic, and inflammatory mechanisms. Previously, we have shown that plasma levels of soluble CD93 (sCD93) are inversely associated with CAD events in women. Yet, little is known about the role of sCD93 in disease progression or its association with metabolic pathways.

Hypothesis:
In this study we asked whether plasma levels of sCD93 could be associated with type 2 diabetes (T2D), a major risk factor for CAD. In parallel, we investigated CD93-mediated effects in metabolism and inflammation using a newly established mouse model.

Methods:
Soluble CD93 was measured by Mesoscale® in plasma of subjects from the IMPROVE cohort, (3371 subjects). IMPROVE is a prospective, multicentre, longitudinal, observational study with subjects presenting at least 3 established CVD risk factors but no clinical coronary artery disease at baseline. T2D was defined clinically or by blood fasting glucose ≥7mmol/L. Detailed ultrasound carotid intima-media thickness (cIMT) phenotypes at baseline, 15 and 30 months were assessed.

In parallel, cd93 heterozygous deficient mice were established by KOMP repository using C57Bl/6N ES cells (CD93low). These mice were then used in a T2D model by giving a high fat diet (21% fat 0.2% cholesterol) or crossed into C57Bl6/N.ApoE deficient mice in order to establish a murine atherosclerosis model.

Results:
Soluble CD93 levels presented significantly lower in subjects with T2D compared to those without (n= 901 vs 2470; mean ± sd = 156.6±40.0ng/ml vs 164.1±44.8ng/ml, respectively; p<0.0001). However, no association between sCD93 cIMT was observed. In line with this, analysis of ApoE-/-CD93low revealed no changes in atherosclerotic lesion development compared to ApoE-/-CD93 sufficient littermates. Nevertheless, in consensus with the clinical findings, CD93low mice showed impaired glucose clearance and reduced insulin sensitivity compared to littermate controls in the T2D model.

Conclusion:
Levels of sCD93 were inversely correlated with T2D in a CAD high-risk cohort, furthermore the mouse T2D models indicates CD93 as a glucose-regulating molecule. However, it remains to be determined how CD93 is involved in glucose regulation.
Abstract 269: Exendin-4 Prevents Glucose-induced Secretion of Tissue Transglutaminase-2 from Cardiac Microvascular Endothelial Cells: Potential Role in the Prevention of Matrix Remodeling in Diabetes
Authors: Shihab Ali S , Richardson Vanitra A , Dokken Betsy B
Published: July 2021

Diabetes causes endothelial dysfunction, which is the initial trigger for vascular complications in diabetic patients. Hyperglycemia initiates a cascade of events that alters protein expression and secretion by endothelial cells. Tissue transglutaminase-2 (tTG2) is an enzyme that under physiologic conditions is sequestered inside the endothelial cell, but under pathologic conditions causing decreased bioavailability of nitric oxide, tTG2 is secreted, activated, and catalyzes irreversible crosslinking of proteins in the extracellular matrix (ECM). Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist, used in the treatment of type 2 diabetes, which has vasculo-protective effects. We hypothesized that hyperglycemic stress would induce secretion of tTG2, and that this effect would be attenuated by Ex-4. Mouse cardiac microvascular endothelial cells (MCECs) were serum-starved and exposed to control (5.5 mM glucose) or hyperglycemic (25 mM glucose) conditions, with or without Ex-4 (10 nM) x 72 hrs. Proteins from conditioned media were isolated, trypsinized, and analyzed using LC-MS/MS (LTQ Orbitrap Velos). Immunoblots from cell homogenate were probed for tTG protein expression. Conditioned media from MCECs exposed to high-glucose but not Ex-4 contained tTG2, which was absent in media from cells exposed to high-glucose and Ex-4, as well as in media from control cells, suggesting that Ex-4 prevented the secretion of tTG2 induced by hyperglycemic stress. Protein expression in cell lysate was not different. These findings may have important implications for the etiology of diabetic vascular complications, and for the role of Ex-4 to prevent the pathologic ECM remodeling associated with diabetic vasculopathy. Further studies are ongoing to determine the mechanisms of glucose-induced secretion of tTG2, as well as the mechanisms by which Ex-4 prevents this effect.
Abstract 286: The Role of Gut Microbiota in Neointimal Hyperplasia After Vascular Injury
Authors: Ho Karen J , Xiong Liqun , Hubert Nathaniel , Nadimpalli Anuradha , Chang Eugene B , Kibbe Melina R
Published: July 2021

Introduction:
There is increasing evidence that the gut microbiome regulates susceptibility to certain diseases through systemic effects of microbe-derived metabolites. Sodium butyrate is a short chain fatty acid that is produced by microbial fermentation of dietary fiber and has known anti-proliferative and anti-migratory effects on vascular smooth muscle cells (VSMC). We hypothesized that perturbation of the gut microbiome with antibiotics would alter systemic serum butyrate concentration and impact neointimal hyperplasia after vascular injury.

Methods:
10-wk-old male Lewis Inbred rats were treated with vancomycin (“vanco”) in the drinking water (0.5mg/mL) ± sodium butyrate (“buty”, 0.5mg/mL) for 4 wks prior to undergoing left carotid angioplasty. Serum butyrate concentration was assessed by gas chromatography. Gut microbial composition was assessed by 16S rRNA gene surveys of fecal samples. VSMC were treated with butyrate (0-5mM) and assessed for cell proliferation using cell counting, cell migration using a transwell assay, and cell cycle progression using FACS.

Results:
Post-angioplasty carotid arteries from vanco-treated rats developed 38% more neointima than controls (0.032±0.004mm2 vs. 0.044±0.003 mm2, P=0.02), but vanco+buty treatment prevented this increase in intimal area (0.035±0.004 mm2, P=.62 vs. control). Analysis of gut microbial communities revealed unique shifts in bacterial clustering by treatment group, which correlated with changes in serum butyrate levels, with the lowest butyrate level detected in vanco-treated rats (0.54±0.1 μmol/mL control, 0.017±0.1 μmol/mL vanco, 0.45±0.1 μmol/mL vanco+buty, P=.008). In vitro, butyrate treatment inhibited VSMC proliferation at 24-48 hrs in a dose-dependent manner, which correlated with induction of G0/G1 cell cycle arrest (P=.001) and a reduction in chemotaxis (P=.03).

Conclusions:
Oral vancomycin treatment induced a shift in the gut microbial community that was associated with decreased serum butyrate levels and increased neointimal hyperplasia, both of which were reversed by oral butyrate supplementation. These data demonstrate proof-of-concept that there is a correlation between gut microbial dysbiosis and susceptibility to neointimal hyperplasia.
Abstract 290: Lower Limb Revascularization of Patients with Peripheral Artery Disease Reduces Human Enterovirus Infection of the Gastrocnemius
Authors: Kim Julian , Casale George , Zhu Zhen , Swanson Stanley , Koutakis Panagiotis , Pipinos Iraklis
Published: July 2021

Introduction:
Peripheral artery disease (PAD), caused by obstruction of arteries supplying the lower extremities, is characterized by leg muscle degeneration. Recently, we showed that infectious human enterovirus (HEV), which has been implicated in other myodegenerative diseases, is present in ischemic muscle of PAD patients and that viral copy numbers correlate with disease severity.

Hypothesis:
We tested the hypothesis that prevalence of infectious HEV and viral copy number in the gastrocnemius of patients with PAD decrease in response to revascularization.

Methods:
Gastrocnemius biopsies were collected from controls (N=14) and from PAD patients (N=17), before and after revascularization. Biopsies were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity and for viral sub-genotype.

Results:
HEV RNA was detected in biopsies of 65% (11/17) of PAD patients and in none of the controls. After revascularization, the gastrocnemius of 5 patients who were HEV positive before surgery no longer harbored detectable virus. The prevalence of HEV infection was reduced from 65% (11/17 patients) before surgery to 35% (6/17 patients) after surgery. Positive-strand viral RNA copy numbers were decreased significantly after revascularization. Mean positive-strand copy number (average 10
2.61
copies/mg muscle wet weight) in post-surgical biopsies that were HEV-positive were lower (p<0.001) compared to mean copy number (average 10
5.46
copies/mg muscle wet weight) in pre-surgical biopsies that were HEV-positive. Copy numbers of negative-strand (template) viral RNA were also decreased after revascularization. Viral replication was confirmed by detection of negative-strand viral RNA in all specimens positive for HEV. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein. By sequence analysis, HEV in PAD muscle exhibited 97% homology with Coxsackievirus B1.

Conclusion:
Our data introduce HEV infection as a potential mechanism for degeneration of ischemic muscle in PAD patients and demonstrate that revascularization of PAD patients reduces both prevalence of HEV infection and viral copy number in the gastrocnemius.
Abstract 296: Turbulence in Saphenous Vein Bypass Grafts by Duplex Ultrasound Imaging
Authors: Koons Katelyn C , Sehgal Chandra M , Shang Eric K , Foley Paul J , Etkin Yana , Davis Julia T , Fairman Ronald M , Jackson Benjamin M
Published: July 2021

Objectives:
Turbulent flow distal to stenoses in saphenous vein bypass grafts (SVBG) may influence vein graft remodeling including intimal hyperplasia. This study aims to develop and validate ultrasound indices for turbulence intensity that may be useful in predicting stenosis, occlusion, and remodeling in SVBG.

Methods:
25 patients with leg SVBG’s underwent regularly scheduled duplex ultrasounds and simultaneous measurement of turbulence indices, using a Philips iU22 ultrasound module and 9-3 MHz linear transducer. 14 patients had significant stenosis and downstream turbulence. In each of these 14 patients, color Doppler at maximal stenosis was used to determine the color scale to avoid aliasing. Then color Doppler video loops were recorded downstream of the stenosis. Color Doppler videos were also recorded in a normal region absent of stenosis for comparison. Custom image analysis algorithms were used to determine percent area reverse flow (%ARF), reverse mean velocity (RMV), and turbulent flow volume (TFV = %ARF х RMV) as surrogates for turbulence intensity.

Results:
Peak %ARF was 20±21 in normal segments and 56±27 in turbulent segments (P=.0002). Peak RMV was 7.7±7.6 cm/s in normal segments and 16.7±7.7 cm/s in turbulent segments (P=.01). Peak TFV was 1.3±1.3 m/s in normal segments and 6.5±3.4 m/s in turbulent segments (P=.0001), and correlated with upstream peak systolic velocity (see figure).

Conclusions:
Surrogates for SVBG turbulence can be detected and quantified using duplex ultrasound. These measures correlate with percent stenosis, and may predict progressive stenosis and remodeling; turbulence imaging may also be useful in situations where a stenosis is inadequately imaged on ultrasound or its hemodynamic significance is uncertain. A larger study with appropriate follow-up to determine clinical and ultrasound outcomes in these patients’ SVBGs is underway.
Abstract 337: Berberine Attenuates Hyperlipidemia in Mice by a Novel Underlying Mechanism Involving Trib1
Authors: Singh Amar B , Liu Jingwen
Published: July 2021

Recent genome wide association studies have identified tribbles homolog 1 (Trib1) as a risk factor in Myocardial Infarction. Trib1 has been shown to regulate VLDL production and hepatic lipogenesis in mice. Liver-specific overexpression of Trib1 reduced hepatic VLDL production and knockdown of Trib1 elevated plasma TG and cholesterol due to the increased VLDL production. However, currently little is known about the regulation of endogenous Trib1 expression and the relationship with plasma lipid homeostasis. In this study, we demonstrate that treating hyperlipidemic mice with 200 mg/kg of berberine (BBR), a natural lipid lowering compounds for 2 weeks reduced plasma total cholesterol by 24% and VLDL-cholesterol by 73% as compared to control mice. Hepatic gene expression analysis revealed that LDLR mRNA levels were not changed by BBR treatment. However, Trib1 mRNA levels were 70% (p<0.01) higher in BBR-treated group than that of control group. The elevation of hepatic Trib1 mRNA levels was also observed in BBR-treated mice fed a regular diet. To further characterize the LDLR-independent mechanism responsible for plasma TG lowering, BBR (150 mg/kg) was orally administered to LDLR knockout mice and we observed a 47% (p<0.05) reduction of serum TG levels after 6-days of BBR treatment while serum TC levels were not reduced. Importantly, the reduction of serum TG in LDLR KO mice by BBR treatment was accompanied by approximately 2-fold (p<0.05) increase in hepatic Trib1 mRNA levels in BBR-treated group as compared to control group. Furthermore, upregulation of Trib1 mRNA expression by BBR was inversely associated with the expression of key lipogenic genes (ACC1, SCD1, C/EBPα, GPAT1, MLXIPL and DGAT1) and measures of lipogenesis. By using HepG2 in vitro model system, we show BBR treatment also significantly increases Trib1 mRNA expression and reduces mRNA and protein expression of lipogenic genes. Taken together, our studies demonstrate for the first time that hepatic expression of Trib1 is upregulated by the natural lipid lowering compound BBR and that Trib1 plays an important role in the hypolipidemic effect of BBR independent of LDLR.