bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 503: Ixmyelocel-T, a Unique Mixed Cellular Therapy for the Treatment of Vascular Diseases Associated with Endothelial Dysfunction
Authors: Ledford Kelly J , Murphy Nikki , Zeigler Frank , Bartel Ronnda L
Published: July 2021

Ixmyelocel-T, an expanded autologous multicellular therapy cultured from bone marrow mononuclear cells (BMMNCs), is under investigation for the treatment of vascular diseases associated with endothelial dysfunction. This study examines the mechanisms by which ixmyelocel-T treatment may exert beneficial effects on the endothelium in comparison to BMMNCs.

To determine the effects on endothelial cells, ixmyelocel-T and BMMNCs were co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell inserts. Co-culture with ixmyelocel-T resulted in increased eNOS (1730±141, vs. 1371±135 pg/mL,
p
< 0.05) measured by ELISA, and nitric oxide (NO) production (1.97±0.2, vs. 1±0.1 relative fluorescence,
p
< 0.001) measured by DAF-2DA. Co-culture with BMMNCs did not have an effect on eNOS or NO in HUVECs. In TNFα stimulated HUVECs, ixmyelocel-T decreased the generation of reactive oxygen species (46±4 vs. 100±3 % of HUVEC,
p
< 0.01) measured with DCFH-DA and increased SOD activity (1.3±0.1, vs. 1±0.1 % of HUVEC,
p
< 0.05), whereas co-culture with BMMNCs didn’t have an effect on ROS or SOD in HUVECs. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T decreased apoptosis in TNFα treated HUVECs (0.78±0.02, vs. 1±0.05 relative to HUVEC,
p
< 0.001). Co-culture with BMMNCs had no effect on HUVEC apoptosis. Real time PCR analysis revealed that ixmyelocel-T decreased the expression of inflammatory markers (ICAM1, VCAM1, and MCP-1) in TNFα treated HUVECs, whereas treatment with BMMNCs had no effect. ELISA analysis indicated that ixmyelocel-T decreased MCP-1 (11983±5357 vs. 23312±11044 pg/mL,
p
< 0.05) and increased IL-10 secretion (61.3±11.2 vs. 1.2±0.5 pg/mL, p < 0.001), whereas treatment with BMMNCs had no effect.

Ixmyelocel-T stimulated NO production, reduced oxidative stress and inflammation, and prevented apoptosis in endothelial cells. BMMNCs did not exhibit similar results. This is most likely due to the anti-inflammatory cell phenotypes associated with ixmyelocel-T’s expansion process. This study indicates that ixmyelocel-T may be superior to BMMNCs in the treatment of diseases associated with endothelial dysfunction and vascular inflammation.
Abstract 507: Genetic Correlates of Cognitive Change in Patients Undergoing Carotid Interventions
Authors: Hitchner Elizabeth , Liao Phoebe , White Sharla , Rosen Allyson , Lane Barton , Zhou Wei
Published: July 2021

OBJECTIVES

Carotid interventions have been shown to decrease stroke risk and improve cerebral perfusion. However, nearly 40% of patients who undergo carotid revascularization procedures experience cognitive deterioration. We have demonstrated that subclinical microembolization is associated with memory decline, but whether genetic factors plays a role in post-procedural cognitive change is unclear. We herein seek to assess genetic determinants as potential risk factors for these procedures.

METHODS

Over one year period, patients undergoing carotid interventions at a single academic institution were recruited to participate. Patients underwent neuropsychological testing two weeks prior to and at one month following their procedure and MRI prior to and within 48 hours following their procedure. Saliva samples were collected for genetic testing and demographics were recorded. Logistic regressions were used to search for associations.

RESULTS

34 patients were included (18 CAS, 16 CEA); all were male with a mean age of 68. The majority of patients exhibit hypertension (94%) and have a history of smoking (76%). Other co-morbidities included diabetes (47%), obesity (35%), and CAD (44%). Consistent with previous findings, CAS was associated with higher incidence of microemboli (p=.0005) and with susceptibility to memory decline (p=.0085). Negative univariate associations were found for 5-HTT (serotonin transporter) short alleles with memory decline (p=.016) and brain-derived neurotrophic factor (BDNF) polymorphism with incidence of microemboli (p=.018). After adjusting for apolipoprotein E (ApoE) risk alleles, incidence of microemboli trended with memory decline (p=.099).

CONCLUSIONS

Despite a small number of patients, our study showed that genetic polymorphisms such as 5-HTT, BDNF, and ApoE may provide additional insight on post-operative changes in cognition. Further investigation of these polymorphisms is warranted to understand and potentially prevent cognitive deterioration following carotid revascularization procedures.
Abstract 518: Inhibition of Endoplasmic Reticulum Stress Reduces Aneurysm Induction in Different Aneuryms Models
Authors: Matrougui Khalid , Kassan Modar , Galan Maria , Pankey Edward , Kadowitz Philip , Sander Gary , Trebak Mohamed
Published: July 2021

Abdominal aorta aneurysm (AAA) is a major cause of mortality in men. The role of endoplasmic reticulum (ER) stress in the pathogenesis of AAA is unknown. Human AAA specimens from diabetic patients were found to have increased expression of ER stress markers. The inhibition of ER stress signaling with (taurine-conjugated ursodeoxycholic acid, Tudca) administration reduces the development of aneurysm in Ang-II infused and calcium chloride (CaCl2)-induced mouse models. Type 2 diabetic (db-/db-) and control (db-/db+) mice were infused with angiotensin II (Ang-II, 1000ng/kg/min) and treated with or without the ER stress inhibitor Tudca for 4 weeks. Mice infused with Ang-II displayed an increase in blood pressure, plasma cholesterol and CRP levels, which were reduced after ER stress inhibition. Human AAA and Ang-II-induced AAA are characterized by an increase in lumen, cross sectional area and were associated with increased macrophage infiltration, expression of the ER stress marker CHOP and elastic fiber disruption, and these indicators of aneurysm disease were reduced by Tudca treatment. To determine the effect of ER stress inhibition on aneurysm formation in a model that did not exhibit blood pressure and metabolic changes, we exposed the adventitial surface of carotid arteries of db-/db- and db-/db+ mice to CaCl2 and then treated the mice with or without Tudca. Our results indicate that in the CaCl2 induced carotid artery aneurysm model that treatment with Tudca significantly reduced carotid artery aneurysm formation. These results provide evidence in support of a role for that ER stress activation in the induction of aneurysm in type 2 diabetes.
Abstract 52: Statin Therapy Accelerates Venous Thrombus Resolution: Assessment In Stasis And Chemical Injury Induced Thrombosis
Authors: Kessinger Chase W , Kim Jin Won , Thompson Brian , Sillesen Martin , Hara Tetsuya , McCarthy Jason R , Lin Charles P , Henke Peter K , Jaffer Farouc A
Published: July 2021

Introduction

In deep vein thrombosis (DVT), both the induced inflammation response and fibrinolytic capacity are critical in resolving experimental DVT. Unresolved, obstructing thrombi can lead to the burdensome and costly sequelae of the post-thrombotic syndrome. Recently, statins have shown to reduce the incidence of DVT (JUPITER trial). In this study, we investigated the in vivo effects of statin therapy on resolution of already formed DVT using serial intravital microscopy (IVM) imaging and thrombus mass measurements.

Methods

DVT was induced in C57Bl/6J male mice (N=190) by topical ferric chloride of the femoral vein (chemical injury) or ligation of the IVC (stasis). One day after DVT induction, mice started treatment of atorvastatin (1.14mg/kg), mevastatin (10 mg/kg) or PBS by once daily oral gavage. Serial IVM was preformed at days 2, 4 and 6 utilizing FITC-dextran (ex/em 490/520nm), CLIO-AF555 (ex/em 555/565nm), and MMPSense680 (ex/em 680/700nm) imaging agents for thrombus architecture, macrophage content and MMP activity measurements. Thrombus masses were collected at days 4, 7, 10 in stasis DVT mice and combination statin/enoxaparin (10mg/kg/d, s.c.) were also compared to statin therapy. Platelet activation and coagulation were analyzed using aggregometry and thromboelastography on whole-blood.

Results

We found that statin therapy reduced DVT burden and inflammation in both chemical- and stasis-induced established DVT up to 10 days compared to control (p<0.05). Serial IVM imaging of statin animals showed an increase in DVT resolution from day 2 to 4 (Δlength -21± 6.0 vs. control -7.1± 5.6%, p<0.01), and decreased inflammation compared to controls (p<0.001). Inflammation was also found to predict the extent of DVT resolution(r=0.84). Statin therapy also reduced thrombus plasminogen activator inhibitor-1, tissue factor, and Mac-3 levels, as well as reducing platelet activation and clot strength(p<0.05).

Conclusion

Statin therapy accelerates DVT resolution despite reducing thrombus related inflammation. The net reduction of thrombus burden tracked with statin-mediated favorable effects on fibrinolysis, coagulation, and platelet function. These results support statins as a translatable therapy to improve the resolution of DVT.
Abstract 536: Metformin Accumulation and Accentuated Ampk Phosphorylation Contribute to Sustained Inhibition of Agonist-Induced Contractility in Endothelium-Denuded Rat Aorta
Authors: Pyla Rajkumar , Osman Islam , Hansen Paul , Fagan Susan C , Segar Lakshman
Published: July 2021

Objective

Metformin, a widely used antidiabetic drug, has been shown to exert protective effects in the vasculature by improving endothelium-dependent vasodilation. However, the beneficial effect of metformin toward smooth muscle contraction and the molecular events involved in this process remain unclear. Using endothelium-denuded aorta, the present study has therefore examined the extent of metformin accumulation and the likely changes in AMP-activated protein kinase phosphorylation during smooth muscle contraction induced by G-protein coupled receptor agonists, including phenylephrine (PE) and serotonin (5-HT).

Methods

Endothelium-denuded rat aortic ring preparations were mounted in organ baths for isometric tension measurements. The contractile responses to cumulative concentrations of PE or 5-HT were determined before and after pretreatments with metformin or AICAR (a direct AMPK activator). The changes in AMPK phosphorylation were determined by immunoblot analysis. Metformin accumulation was determined by LC-MS/MS.

Results

Concentration dependency studies revealed that pretreatment with 3 mM metformin resulted in significant inhibition of PE- or 5-HT-induced contractility (p < 0.05). In parallel, pretreatment with 1 mM AICAR led to a similar inhibition of agonist-induced contractility (p < 0.05). Notably, the inhibitory effects of metformin on smooth muscle contractility persisted even after the washout of this drug. In contrast, washout of AICAR restored the contractile responses to PE or 5-HT. Pretreatment of aortic rings with 40 μM compound C, an AMPK inhibitor, resulted in significant reversal of metformin and AICAR-mediated decrease in agonist-induced contractility (p < 0.05). An association between metformin/AICAR treatment and AMPK phosphorylation was confirmed by immunoblot analysis. Importantly, metformin treatment resulted in its significant accumulation in the aortic tissue as revealed by LC-MS/MS analysis.

Conclusion

In conclusion, metformin accumulation contributes to sustained inhibition of arterial smooth muscle contractility
via
activation of AMPK. The present findings suggest that metformin has the potential to ameliorate chronic hypertension in patients with type 2 diabetes.
Abstract 567: Causal Assessment of Uric Acid and Risk of Myocardial Infarction, a Mendelian Randomization Study
Authors: Keenan Tanya , Ho Weang K , Voight Benjamin F , Rasheed Asif , Mucksavage Megan L , Reilly Muredach , Danesh John , Rader Daniel J , Saleheen Danish
Published: July 2021

Introduction

Prospective studies suggest that high serum uric acid (UA) levels are associated with increased risk of myocardial infarction (MI), but causality of this association remains uncertain. Mendelian randomization studies assess causality by using genetic variants as unbiased proxies for circulating biomarkers. We tested the hypothesis that genetically raised UA levels are causally related to elevated MI risk.

Methods

All reported UA associated genetic variants were assessed for pleiotropy in public genome-wide association databases and in the Pakistan Risk of Myocardial Infarction Study (PROMIS), in which we measured >25 atherosclerosis-related biomarkers in >10,000 people. Genetic variants lacking pleiotropic associations (p<0.01) were evaluated for association with MI in 7031 cases and 6360 controls in PROMIS.

Results

Among the individuals studied (mean age 54 ± 10 years; 81% male), 1316 had UA measurements with mean UA 5.71 ± 1.54 mg/dl. Of the 36 genetic variants associated with UA at genome-wide significance (p <5x10-8), 5 were independent and not associated with any factors investigated. For each variant, genetically raised UA was not associated with MI risk in a concordant direction (Table). A score-based instrumental variable analysis found no causal relationship between UA levels and MI (OR 0.91, 95% CI 0.74 - 1.12, p = 0.39). This analysis will be repeated with CARDIoGRAM data in time for presentation.

Conclusions

(1) Genetically raised UA levels were not associated with elevated MI risk, and (2) the collection of UA genetic instruments did not reject the null hypothesis of no causal relationship. We suggest that UA levels are not causally related to MI risk.
Abstract 72: Vascular and Cardiac Effects of Inducible Nitric Oxide Synthase in Hyperhomocysteinemic Mice
Authors: Dayal Sanjana
Published: July 2021

Background

Diet-induced hyperhomocysteinemia (HHcy) produces endothelial and cardiac dysfunction and promotes thrombosis through a mechanism proposed to involve oxidative stress. Inducible nitric oxide synthase (iNOS) is upregulated in HHcy and can generate oxidants such as superoxide. We tested the hypothesis that iNOS mediates the adverse oxidative vascular, thrombotic, and cardiac effects of HHcy.

Methods

Mice deficient in iNOS (
Nos2-/
-) and their wild-type (WT) littermates were fed either a control (C) diet or a high methionine/low folate (HM/LF) diet for 3-6 months. Vasomotor function was assessed in cerebral arterioles in response to the endothelium-dependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Thrombotic occlusion of the carotid artery was measured in response to photochemical injury (rose bengal). Regional myocardial injury was measured after 30 minutes of ischemia and 2 hours of reperfusion of the left coronary artery.

Results

Nos2
-/- and WT mice fed the HM/LF diet developed similar levels of HHcy, with a 2-fold increase in plasma total homocysteine (P<0.001 vs. C diet). As expected, WT mice with HHcy exhibited endothelial dysfunction, with impaired dilatation to acetylcholine but not nitroprusside, and enhanced susceptibility to thrombosis, with shortened times to occlusion of the carotid artery (P<0.05 vs. C diet). Interestingly, deficiency of iNOS resulted in decreased, rather than increased dilatation responses to acetylcholine (P<0.05 vs. WT mice).
Nos2
-/- mice also exhibited shortened times to thrombotic occlusion in mice fed the C diet (P<0.05 vs. WT mice) and failed to protect from endothelial dysfunction or accelerated thrombosis in mice fed the HM/LF diet. Deficiency of iNOS did not alter myocardial infarct size in mice fed the C diet but significantly increased infarct size and cardiac superoxide production in mice fed the HM/LF diet (P<0.05 vs. WT mice).

Conclusions

These findings suggest that endogenous iNOS protects from, rather than exacerbates, endothelial dysfunction, thrombosis, and myocardial ischemia-reperfusion injury. In the setting of HHcy, iNOS appears to function to blunt cardiac oxidative stress rather than as a source of superoxide.
Abstract 79: Examining the Role of Endoplasmic Reticulum Stress Induced Glycogen Synthase Kinase-3a/ß in Accelerated Atherosclerosis
Authors: McAlpine Cameron , Werstuck Geoff
Published: July 2021

Introduction

Cardiovascular disease accounts for a third of all deaths in the western world, however our understanding of the molecular mechanisms of atherosclerosis development is incomplete. Our previous findings have suggested that endoplasmic reticulum (ER) stress plays a causative role in atherogenesis. The objective of this study is to investigate how ER stress promotes atherosclerosis with a specific focus on ER stress-induced Glycogen Synthase Kinase (GSK)-3α and β.

Methods and Results

Established mouse models of accelerated atherosclerosis including hyperglycemia, hyperhomocysteinemia and relative dyslipidemia in female ApoE-/- mice were examined. A subset of mice from each group was supplemented with valproate (625 mg/kg), a compound with GSK3α/β inhibitory properties. Hyperglycemia, hyperhomocysteinemia and high fat diet elevated ER stress levels in multiple tissues, enhanced GSK3β activity, increased atherosclerotic plaque size, and induced hepatic steatosis. Valproate-supplementation significantly attenuated GSK3 activation, aortic plaque development and hepatic steatosis without altering ER stress levels. ER stress and valproate appears to modulate GSK3 activity by altering its phosphorylation status.

To examine the mechanism linking ER stress to the activation of pro-atherogenic pathways, cultured THP1-derived macrophages were treated with the ER stress inducing agents, glucosamine (5mM), thapsigargin (1.5μM) or palmitate (800μM). GSK3α/β activity was inhibited with the specific inhibitor CT99021 (4μM). In macrophages, ER stress upregulated the mRNA expression of genes controlling lipid biosynthesis while GSK3α/β inhibition attenuated lipid synthesis. The expression of downstream targets of the PERK ER stress signaling pathway, CHOP and ATF4, were downregulated by GSK3α/β inhibition. Further, PERK-/- mouse embryonic fibroblasts (MEFs) had significantly attenuated lipid accumulation while expression of constitutively active GSK3β-S9A restored the ability of PERK deficient cells to accumulate lipids.

Conclusions

These findings support the a common pathway that links cardiovascular risk factors to the activationof pro-atherogenic pathways by a mechanism involving ER stress-induced GSK3α/β.
Abstract 81: Pharmacological Inhibition of Calpain Attenuates Adipose Tissue Apoptosis, Macrophage Accumulation and Inflammation in Diet-induced Obese Mice
Authors: Subramanian Venkateswaran , Balakrishnan Anju , Howatt Deborah A , Moorleghen Jessica J , Katz Wendy S
Published: July 2021

Background and Objective

Obesity is associated with low-grade chronic inflammation and apoptosis that contributes to development of insulin resistance and other metabolic complications. Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not defined. Calpains are calcium-dependent neutral cysteine proteases that are essential for multiple cellular functions, such as cytoskeletal remodeling and apoptotic cell death. Recent studies have demonstrated that activated calpain promotes adipocyte differentiation in vitro, and enhances macrophage recruitment during nephropathy. However, the functional role of calpain activation in adipose tissue macrophage accumulation and obesity remains to be elucidated. The purpose of this study was to define whether pharmacological inhibition of calpain influences diet-induced obesity and adipose tissue macrophage accumulation in mice.

Methods and Results

Male C57BL/6 mice (8 weeks old; n=10 per group) were fed either low (10% kcal) or high (60% kcal) fat diet for 12 weeks. Calpeptin, a calpain inhibitor (2.5 mg/kg/day) or vehicle (DMSO) was administered daily by osmotic mini-pumps for 12 weeks. Calpeptin administration did not influence high fat diet induced body weight and fat mass gain throughout the study. Calpain inhibition had no effect on glucose and insulin tolerance in obese mice. However, calpain inhibition highly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as evident by TUNEL, Picro Sirius and CD68 immunostaining. Real-time PCR analysis showed that calpain inhibition significantly suppressed inflammatory cytokines (TNFα, IL-6, MCP-1, F4/80) expression in adipose tissue (P<0.05 vs vehicle). In addition, Oil Red O staining revealed that calpain inhibition also suppressed accumulation of hepatic fat.

Conclusion

Pharmacological inhibition of calpain attenuated macrophage accumulation, adipocyte apoptosis, fibrosis and inflammation in diet-induced obese mice without influencing body weight gain and insulin tolerance.
Abstract 106: Prevention of Abdominal Aortic Aneurysm by Anti-MiRNA-712 or Anti-miR-205 in Angiotensin II--Infused Mice
Authors: Kim Chanwoo , Kumar Sandeep , Son Dong Ju , Jang In-Hwan , Jo Hanjoong
Published: July 2021

Abdominal aortic aneurysm (AAA) is characterized by weakening of the vessel wall, followed by progressive expansion of the diseased aortic segment. MicroRNAs (miRNAs) have emerged as key regulator of gene expression in the cardiovascular diseases and may play a key role in therapeutically targeting AAA development. Although,vascular wall degradation by matrix metalloproteases (MMPs) is the key mechanism in AAA development, their targeting through miRNAs have never been studied. We identified microRNA-712 (miR-712) as a novel Angiotensin II(AngII)-sensitive miRNA which is upregulated in the abdominal aortic endothelium of AngII-infused mice. Mechanistically, we identified that miR-712 directly regulates two key endogenous inhibitors of MMP: tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion inducing cysteine-rich protein with kazal motifs (RECK). Furthermore, inhibition of miR-712 by subcutaneous injection of anti-miR-712 significantly decreased MMP activity in the AngII-infused abdominal aorta wall, prevented the dilatation of aortae and significantly reduced AAA incidence from 80% (8/10) to 20% (2/10), compared to its mismatched control in ApoE
-/-
mice. Interestingly, based on the seeding sequence, we identified miR-205 as the human homolog of miR-712. miR-205 was also upregulated by AngII treatment and like miR-712 regulated MMPs activity via TIMP3 and RECK. Moreover, inhibition of miR-205 dramatically inhibits AngII-induced AAA development. We also found that miR-205 was significantly upregulated in the aortic sections of AAA patients in comparison to the healthy controls. Our findings demonstrate that AngII-sensitive miRNAs, miR-712 and miR-205, regulate MMP activity through TIMP3 and RECK and play important role in the pathogenesis of AAA. These results suggest that targeting these miRNAs using their inhibitors may hold promise as a therapeutic strategy to prevent the development of AAA.
Abstract 13: Suppression of Atherosclerosis by CD39
Authors: Kanthi Yogendra , Hyman Matthew , Liao Hui , Baek Amy , Visovatti Scott , Sutton Nadia , Takabe Wakako , Ni Chih-Wen , Jo Hanjoong , Pinsky David
Published: July 2021

Atherosclerotic plaque rupture and thrombosis remains the leading cause of death in the United States. We investigated the role of CD39, a potent ecto-enzymatic regulator of platelet activation and leukocyte trafficking, in atherosclerosis. We generated mice deficient in CD39 on a hyperlipidemic,
apoE
-/-
background and noted a two-fold higher plaque burden in when compared to

apoE
-/-

controls (
P
=0.003). We noted higher levels of circulating markers of platelet activation, soluble P-selectin (39%) and RANTES (60%), in the CD39-deficient mice (
P
=0.003 and
P
=0.015, respectively, n=7-11). CD39-haploinsufficient mice had 1.8-fold greater enhanced platelet reactivity in response to ADP compared to controls (
P
=0.03, n=3-8). Macrophages from CD39-deficient mice had significantly higher lipoprotein uptake
in vitro
. Correspondingly, CD39 overexpression in RAW cells inhibited scavenger receptor expression and lipoprotein uptake.

Altered fluid mechanics contribute to atherosclerosis, with non-laminar shear stress enhancing regional plaque formation as seen in arterial bifurcations. We examined coronal sections of aortas from
apoE
−/−
mice and observed that CD39 is poorly expressed in the endothelium in regions of turbulent blood flow, where plaque develops, supporting our hypothesis that endothelial CD39 can be induced by fluid phase shear forces. HUVEC treated with physiologic laminar shear stress (LS) (15 dynes/cm
2
) had a 5.9-fold increase in CD39 protein (
P
=0.004, N=3-7) and a concordant increase in nucleotidase activity (
P
=0.03 N=3) compared to static controls (SS). We identified Krüppel like factor 2 (KLF2) as an upstream candidate for transcriptional regulation of CD39 induction by fluid shear forces. Silencing KLF2
in vitro
led to a 55% decrease in CD39 mRNA induction with LS vs SS controls (
P
=0.002, N=3-4). Chromatin immunoprecipitation revealed that KLF2 binds to the CD39 (
P
=0.01, N=3) and this binding was further enhanced under laminar shear stress (
P
=0.0007, N=3). These data show that CD39, an anti-thrombotic, anti-inflammatory enzyme is a critical regulator of atherosclerosis by modulating platelet, macrophage and endothelial function and mechanistically identify KLF2 as a direct, upstream regulator of CD39 expression.
Abstract 134: Short-Term, High-Dose Fish Oil Supplementation Increases the Production of Downstream n-3 Fatty Acid Metabolites in Patients With Peripheral Artery Disease
Authors: Grenon Marlene , Owens Christopher , Alley Hugh , Chong Karen , Yen Priscilla , Boscardin John , Spite Matthew , Conte Michael S
Published: July 2021

OBJECTIVES:
Patients with peripheral artery disease (PAD) experience significant morbidity and mortality, at least partially related to vascular inflammation and endothelial dysfunction. The OMEGA-PAD I Trial (NCT01310270), a randomized, double-blinded, placebo-controlled trial addressed the hypothesis that short-duration, high-dose n-3 polyunsaturated fatty acids (n-3 PUFA) oral supplementation improves endothelial function (EF) and inflammation in subjects with PAD.

METHODS:
Eighty patients with stable, mild-severe claudication and ABI<0.9 received 4.1gm of fish oil (FISH) vs placebo capsules (CTL) for 1 month. The primary endpoint was EF as measured by brachial artery flow-mediated vasodilation (FMD). Secondary endpoints included biomarkers of inflammation, generation of n-3 fatty acid-derived lipid metabolites, lipid profile and walking impairment questionnaires.

RESULTS:
The FISH and CTL group were no different with regards to age, baseline EF, inflammation and lipid profiles. Following treatment, there was a significant reduction in triglycerides (-34 ± 46, p=0.0001) and an improvement in HDL (+2 ± 6, p=0.03) in the FISH group. These changes were accompanied by an increase in the omega-3 index of 4 ± 1% (p<0.00001). We observed a significant increase in the production of downstream metabolites of n-3 fatty acids including 18-, 15- and 5-hydroxy eicosapentaenoic acids and 4-hydroxy docosahexaenoic acid in the FISH group. n-3 PUFA led to a significant improvement in FMD in the FISH group (+0.7 ± 4.0%, p=0.04) and a non-significant improvement in the CTL (+0.6 ± 2.5, p=0.18) group. There were no significant differences between groups in pro-inflammatory markers or walking parameters post-treatment.

CONCLUSIONS:
High-dose, short-duration n-3 PUFA supplementation significantly improves the metabolo-lipidomic profile of patients with PAD. Longer studies are needed to assess the effects of n-3 PUFA on inflammation, vascular function, and clinical endpoints in patients with established PAD and to determine whether generation of n-3 fatty acid-derived bioactive lipid mediators is related to clinical outcomes.
Abstract 135: Should We Use Radial Artery in Patients With Peripheral Vascular Disease Undergoing Myocardial Revascularization?
Authors: Nesher Nahum , Mohr Rafael , Teich Nadav , Kramer Amir , Pevni Demitri , Paz Yosef , Benjamin Medalion , Ben-Gal Yanai
Published: July 2021

Objective:
Bilateral Internal Mammary (IMA) grafting is associated with improved survival. However, many surgeons are reluctant to use this revascularization technique in patients with peripheral vascular disease (PVD) due to their short life expectancy and the potential increased risk of sternitis.. The composite T graft with radial artery attached end-to-side to the left IMA provides arterial revascularization without increased risk of sternitis. The purpose of this study is to compare early and long-term outcome of these two revascularization strategies, in patients with PVD.

Methods:
Between 1999 and 2008, 462 consecutive patients with PVD (340 bilateral IMA and 122 single IMA and radial artery) underwent arterial myocardial revascularization.

Results:
Occurrence of female sex (35.2% versus 20.9%, radial group versus bilateral IMA, respectively), patients older than 70 (86% versus 59.7%), diabetes (52.52% versus 36.5%), emergency operation (26.2% versus 16.2%) and COPD (23.8% versus 9.4%) was higher in the radial group. The radial group also had a higher logistic Euroscore (25.4 versus 16.49). On the other hand recent MI (22.6% versus 13.1%) was more prevalent in the bilateral IMA group. Operative mortality of the two groups was similar (8.2% versus 6.5% for the radial and bilateral IMA, respectively). Occurrence of sternal infection was also similar (3.3% versus 0.9%). Median follow-up was 7.8 years. Kaplan Meier 10 year survival (47.1% versus 56.7%,), as well as Cox adjusted survival (p= 0.741) of the two groups were similar.

Conclusion:
In patients with PVD long-term outcome of arterial revascularization with left IMA and radial artery is similar to that of bilateral IMA. Therefore we recommend the use of single IMA and radial artery if double IMA's cannot be used safely.
Abstract 137: The Response of an in vitro Cynomolgus Macaque System to Statin Treatments Mirrors the Human in vitro System and Clinical Outcomes
Authors: Simmers Michael , Feaver Ryan , Turk Jim , Cole Banumathi , Collado M Sol , Berzin Erica , Lawson Mark , Mackey Aaron , Manka David , Wamhoff Brian , Qualls Charles W , Blackman Brett
Published: July 2021

Introduction:
There is an urgent unmet need to improve the predictive value of animal and in vitro systems for drug development. The purpose of this study was to develop an in vitro vascular system that reflects the in vivo biology of the cynomolgus macaque (cyno).

Methods:
We co-cultured endothelial (EC) and smooth muscle cells (SMC) from cyno arteries and exposed them to physiologically relevant hemodynamics. Cyno velocity profiles were obtained from arterial regions prone to atherosclerosis (atheroprone) or healthy regions. Velocity waveforms were applied to the co-culture along with oxidized LDL from cyno or humans, and TNFα, which are pathogenic in humans and mimic advanced inflammatory conditions (AIC). Using RNA sequencing, we quantified the response of the system to 4 different statin treatments (atorvastatin, simvastatin, cerivastatin, rosuvastatin) under AIC. Using computational analyses of differentially expressed genes (DEGs), we characterized the response of our system by identifying functional biological themes that were associated with each statin treatment.

Results:
We found that AIC in the cyno system dramatically increased inflammatory DEGs and decreased vascular health DEGs. Cerivastatin elicited the most DEGs in both the cyno and human vascular system while the response to rosuvastatin did not reach statistical significance for any genes. Statin treatment decreased inflammatory cytokine gene expression, but not adhesion molecule expression under AIC. Except for rosuvastatin, all statins tested significantly increased the expression of genes that promote vascular health, while suppressing the expression of inflammatory genes. Biological themes were regulated by statin treatment in both the human and cyno vascular systems including: cholesterol biosynthesis, thrombosis, ethanol degradation, cell cycle regulation and notch signaling. Finally, we discovered functional themes related to glycogen metabolism, which may be relevant as a potential mechanism driving the risk of hyperglycemia with statin treatment.

Conclusions:
The cyno vascular system described here mirrors many of the well-known phenotypes of the human vascular system, and should provide a valuable predictive tool for in vivo studies.
Abstract 144: Bone-Marrow Chimeras Demonstrate that the Epigenetic Signature in the Bone Marrow Myeloid Cells Influences the Peripheral Wound M1-Dominant Macrophage Phenotype
Authors: Gallagher Katherine A , Joshi Amrita , Hogikyan Emily , Coleman Dawn , Carson William , Kunkel Steven
Published: July 2021

Introduction:
Diabetic wounds are characterized by a chronic inflammatory state that is maintained by overexpression of pro-inflammatory cytokines generated by macrophages. In normal wound healing, macrophages mobilized from the circulation initially exhibit an M1 phenotype and secrete pro-inflammatory mediators (i.e., IL-12) followed by a prolonged M2 anti-inflammatory phase. In type 2 diabetes (T2D), this M1 to M2 switch appears to be markedly attenuated. We have previously demonstrated that post-translational histone methylation changes in BM cells are maintained in peripheral macrophages and promote an M1 dominant phenotype in T2D wounds that result in impaired wound healing. We hypothesize that chimeric mice reconstituted with bone marrow from our T2D murine model will demonstrate delayed wound healing and expression of this epigenetic signature in peripheral wound macrophages.

Methods:
BM chimeras were created using GFP+ mice on a C57BL/6 background. GFP+ mice were fed a high-fat diet (HFD, 60% fat) for 14 weeks and BM from these mice and normal diet (ND, 12% fat) mice was transferred into irradiated recipients. Change in wound area compared to initial wound size using Image J software (NIH) at day 3 post-wounding. Levels of the demethylase enzyme, Jmjd3, in macrophages (CD11b
+
) MACS isolated from wounds at day 3 were quantified by RT-PCR in ND→ND and HFD→ND GFP
+
chimeric mice. (N=8) Data are expressed as mean ± S.E.

Results:
Peripheral blood analysis was performed weekly and at 8 weeks confirmed 96.5% donor chimerism. Wound healing was significantly delayed in HFD→ND GFP
+
chimeric mice compared to controls and analysis of wound tissue macropahges in mice at day 3 post-wounding demonstrated increased Jmjd3 and IL-12 production.

Discussion:
Epigenetic changes in BM cells appear to be maintained in peripheral wound macrophages and contribute to increased M1 macrophages and impaired wound healing in T2D wounds.
Abstract 155: Sustained CD4+ T-Cell Activation Promotes Transplant-Associated Recipient Cardiovascular Disease
Authors: Zhou Jing , Qin Lingfeng , Yi Tai , Ali Rahmat , Li Qingle , Jiao Yang , Li Guangxin , Tobiasova Zuzana , Huang Yan , Zhang Jiasheng , Sadeghi Mehran M , Giordano Frank J , Pober Jordan S , Tellides George
Published: July 2021

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in transplant patients. The mechanisms underlying increased recipient CVD are poorly understood. We have established an animal model of transplant-associated recipient CVD by combining murine models of atherosclerosis (apolipoprotein E-deficient recipients, ApoE
-/-
) and of transplant rejection (heterotopic cardiac transplantation). Mice were placed on a normal chow diet for 3 months after transplantation and then analyzed. ApoE
-/-
recipients of rejecting allografts developed more severe aortic atherosclerosis compared to syngeneic controls, most strikingly across a major histocompatibility complex (MHC) class II antigen barrier (Fig. A). Atherosclerotic lesions were associated with increased CD4
+
T cell infiltration and sustained CD4
+
T cell activation as assessed by flow cytometry. Total cholesterol and triglyceride levels were unchanged. Cardiac function and aortic distention by echocardiography demonstrated significant impairment in recipients of MHC class II mismatched grafts (Fig. B). In addition, quantitative assessment of proinflammatory cytokines demonstrated significantly elevated plasma levels of interferon (IFN)-γ, the signature Th1 cytokine, as well as IFN-γ inducer cytokine IL-12 in mice with MHC class II mismatched grafts. Our study provides an experimental model of transplant-associated recipient CVD, reveals the critical role of sustained CD4
+
T cell activation by donor-recipient immunological crosstalk, and has the potential for identifying novel therapeutic interventions for transplant-associated recipient CVD.
Abstract 156: Macrophage Low-Density Receptor--Related Protein 1 Deficiency Facilitates Atherosclerosis Regression in Hyperlipidemic Mice
Authors: Zhu Lin , Yancey Patricia G , Ding Lei , Blakemore John L , Zhang Youmin , Linton Macrae F , Fazio Sergio
Published: July 2021

Atherosclerosis regression is characterized by egress of macrophages out of the artery wall. We have previously shown that macrophages lacking low-density receptor related protein 1 (MFLRP1-/-) are pro-inflammatory and lead to increased lesion formation in apoE-/- mice. To study the role of macrophage inflammation during atherosclerosis regression, bone marrow from four different types of mice (wild-type, MFLRP1-/-, apoE-/- and apoE-/-/ MFLRP1-/-) was transplanted into apoE-/- recipient mice who had been fed a Western-type diet for 12 weeks. ApoE-/- recipient mice transplanted with apoE-/- bone marrow were sacrificed 2 weeks post-BMT for determination of baseline aortic atherosclerosis. After 8 weeks on chow diet, cholesterol levels were normalized in mice reconstituted with wild-type (WT) and MFLRP1-/- bone marrow (157± 36 mg/dl and 136 ± 33 mg/dl, respectively), and significantly lowered in mice with apoE-/- (302±33 mg/dl) or apoE-/-/ MFLRP1-/- (294±52 mg/dl) macrophages compared to baseline (387±34 mg/dl). Total atherosclerotic lesion area in the aortic root decreased by 15% in mice receiving WT macrophages, and decreased by an additional 10% in mice transplanted with LRP1 deficient macrophages (p<0.05 compared to WT). Similarly, mice reconstituted with apoE-/-/ MFLRP1-/- bone marrow had 15% (p < 0.01) smaller lesion size than mice receiving apoE-/- marrow. The lesion area positive for CD68 was significantly smaller in MFLRP1-/- mice compared to WT mice, and in apoE-/-/ MFLRP1-/- mice compared to apoE-/- mice. The ratio of necrotic to total lesion area was significantly lowered by WT and LRP1-/- macrophages, and was also reduced in recipients of apoE-/-/MFLRP1-/- compared to apoE-/- bone marrow. Here we demonstrate that absence of LRP1 in macrophages, which is known to cause pro-inflammatory changes, promotes atherosclerosis regression. Our study supports the novel idea that pro-inflammatory macrophages efficiently egress from the plaque in a regressive environment caused by switching from a high-fat to a chow diet. This observation sets the stage for a change in paradigm on how to target inflammation for prevention of atherosclerotic cardiovascular events.
Abstract 171: Role of α-Actinin-4-Dependent Endothelial Cell Stiffness in Neutrophil Transmigration
Authors: Hordijk Peter , Schaefer Antje , te Riet Joost , Ritz Katje , Hoogenboezem Mark , Anthony Eloise , Mul Erik , de Vries Carlie , Daemen Mat , Figdor Carl , van Buul Jaap
Published: July 2021

Inflammation is causally linked to many chronic human disorders and constitutes a growing problem in the ageing population. The inflammatory process is driven by interactions of activated leukocytes with the endothelial lining of blood vessels. This requires binding of leukocyte β2-integrins to endothelial ICAM-1 (InterCellular Adhesion Molecule-1), which allows leukocyte adhesion, spreading, crawling and transendothelial migration (TEM). Integrin binding induces ICAM-1 clustering and its consequent association to F-actin which enforces leukocyte adhesion. Here, we analyzed the molecular basis of this positive feedback loop.

We show that ICAM-1 clustering promotes its binding to F-actin through distinct complexes with FilaminB, Cortactin and α-Actinin-4. We found that α-Actinin-4 regulates endothelial cell peripheral stiffness, which is sensed by adherent neutrophils and promotes adhesion, spreading, crawling and TEM. Conversely, increasing endothelial cell stiffness stimulates the ICAM-1-α-Actinin-4 interaction. Finally, we found that the endothelial lining of atherosclerotic plaques, which is characterized by increased stiffness and leukocyte infiltration, shows increased expression of α-Actinin-4. These results identify α-Actinin-4-regulated endothelial cell stiffness as a novel pro-inflammatory event that promotes ICAM-1-mediated leukocyte adhesion and TEM.
Abstract 174: Lacking Gata2 in Endothelial Cells Induces Apoptosis During Vascular Development
Authors: Ni Chih-Wen , Smith Tom , Lawson Nathan D
Published: July 2021

Gata2 is a transcription factor implicated in hematopoietic development. Previously, we utilized zinc-finger nucleases to generate a zebrafish mutant in gata2a, which reveals a novel role for this gene in vascular development. In particular, gata2a mutant embryos exhibit defects in blood flow circulation due to the formation of shunts within trunk blood vessels, although artery and vein identity appears normal. Here, we show that endothelial cells in gata2a mutant embryos specifically undergo apoptosis as revealed by positive staining of activated caspase-3 at 30hpf but not 24hpf. This endothelial apoptosis can be rescued by re-constitution of gata2a or overexpression of a well-known anti-apoptotic protein, bcl2l1. Interestingly, knocking-down p53 has no effect on the rescue of apoptosis in gata2a mutants, suggesting a p53 independent pathway of apoptosis. Furthermore, we find that mouse retinal vascular endothelial cells lacking gata2 also appear to undergo apoptosis, which prevent angiogenic sprouting from the superficial to media layer of retina vasculature. These findings reveal a new role of gata2 in vascular development whereby gata2 retains an anti-apoptotic function in endothelial cells.
Abstract 200: Development and Proof of Concept Testing of an Ex Vivo Perfusion Chamber for Studying Platelet Adhesion to Coronary Stents Under Physiological Conditions
Authors: Strawn Tammy , Kelly Hayden , Jones John E , Chen Meng , Fay William P
Published: July 2021

Background:
Current-generation drug-eluting stents unblock atherosclerotic arteries, inhibit restenosis, but also render the artery prone to thrombosis by inhibiting endothelial cell function. Hence, there is strong clinical interest in developing stents that inhibit restenosis, yet are less thrombogenic. Goals of this study were to 1) develop an
ex vivo
method to measure platelet adhesion and aggregation on coronary stents under clinically-relevant conditions, and 2) test the hypothesis that treating metal stents with trimethylsilane (TMS) through a novel glow discharge process that coats stents with a nanoscale layer of hydrophobic plasma coating, with or without additional post-deposition glow discharge treatment with NH
3
and O
2
gas, generating elemental nitrogen and oxygen on the stent surface, inhibits platelet adhesion to stents.

Methods:
Bare-metal stents (BMS) made of 316L stainless steel was deployed in Masterflex tubing by inflating an over-the-wire coronary balloon within the stent. Tubing containing stents was filled with porcine indium-labeled platelets in anticoagulated whole blood and the ends were connected to create a closed circuit, which was connected to a peristaltic pump to generate unidirectional flow with shear stress of 67 s
-1
, similar to that present in human coronary arteries. After 30 min of continuous flow at 37°C the tubing was disconnected and extensively rinsed to remove blood. Platelet adhesion to stents was measured with a gamma counter and by scanning electron microscopy (SEM).

Results:
Platelet adhesion to metal stents treated with TMS followed by NH
3
and O
2
(DC-TMS+NH
3
/O
2
) was 56±8.2% reduced compared to BMS (n=12; P=0.003), whereas TMS treatment alone did not significantly inhibit platelet adhesion. SEM confirmed significantly decreased platelet deposition on DC-TMS+ NH
3
/O
2
stents vs. BMS.

Conclusions:
A novel glow discharge surface modification involving TMS, NH
3
, and O
2
gas generates stents with a bioactive surface that resists platelet adhesion under physiological conditions. Our
ex vivo
perfusion circuit facilitates rapid and precise comparison of stent thrombogenicity under physiological conditions, which should prove useful to other investigators developing coronary stents for clinical use.
Abstract 205: Protective Role for B-1 B Cells and IgM Antibodies in Obesity
Authors: Harmon Daniel , Oldham Stephanie N , McSkimming Chantel C , Perry Heather M , Kirby Jennifer L , Kaplan Jennifer L , Hallowell Peter , Schirmer Bruce , Tsimikas Sotirios , Taylor Angela , McNamara Coleen
Published: July 2021

Innate B-1 B cells can protect against inflammatory disease through production of natural IgM antibodies, but little is known regarding their role in obesity-induced metabolic dysfunction. In this study, we explore the role murine B-1 B cells play in regulating diet-induced glucose intolerance. In addition, we examine bariatric surgery samples for the presence of B cells in human adipose tissue and circulating natural IgM antibodies.

We show that mice with increased visceral adipose tissue B-1b B cells due to B cell specific deletion of
Id3
(Id3
BcellKO
) have attenuated high-fat diet-induced glucose intolerance compared to littermate controls. Omental visceral adipose tissue from Id3
BcellKO
mice had enhanced local natural IgM antibody secretion (49.0 ± 5.9 vs. 17.5 ± 4.2 U/mg fat), and demonstrated attenuated HFD-induced secretion of TNFa (2.5 ± 0.3 vs. 6.7 ± 1.3 pg/mg fat) and IFNg (0.10 ± 0.02 vs. 0.48 ± 0.14 pg/mg fat). Adoptive transfer of B-1b B cells null for
Id3
protected against diet-induced glucose intolerance in

Rag1
-/-

hosts, while B-1b B cells unable to secrete IgM had no effect. Additional studies in humans undergoing bariatric surgery showed CD20+CD27+CD43+ B cells, previously shown to have B-1-like characteristics, within omental adipose tissue. A correlation was found between their presence and serum natural IgM levels (r=0.52). In addition, natural IgM levels were inversely associated with the inflammatory chemokine, MCP-1 (r=-0.21). Finally, IgM, but not IgG, natural antibodies were inversely associated with insulin resistance (r=0.32).

Together, our findings provide the first evidence that B-1b B cells protect against diet-induced glucose intolerance in an IgM-dependent manner in mice, and suggest that anti-inflammatory natural IgM antibodies may modulate the inflammatory and metabolic consequences of obesity in humans.