bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 8: Oxidized LDL Induces Macrophage Production of Prothrombotic Microparticles
Authors: Marchini Julio F , Manica Andre , Dutzmann Jochen , Folco Eduardo J , Weber Heinz , Libby Peter , Croce Kevin
Published: July 2021

Background:
Microparticles (MP) are submicron vesicles produced by activated or apoptotic vascular cells. Agonist proteins borne by MPs enables them to function as mediators of inflammation and thrombosis. Atherosclerotic plaques contain high levels of MPs, and plasma MP levels increase during acute coronary syndromes. The thrombotic consequences of plaque rupture may involve macrophage (MΦ) MPs. The activation pathways that promote MΦMP production remain poorly defined, and this study therefore tested the hypothesis that signals implicated in atherogenesis stimulates MΦMP production.

Methods and Results: We stimulated human primary MΦs with pro-inflammatory cytokines and atherogenic lipids, and assessed MΦMP production using light- and impedance-based flow cytometry. Oxidized LDL (oxLDL, 6-17 moles MDA/mg protein), but not IFN-γ, IL-1β, LPS or TNF, induced MΦMP production in a concentration- and time-dependent manner (293% increase, 16h peak, p<0.001). Non-oxidized LDL and minimally oxidized LDL components (POV-PC and PG-PC) did not augment MΦMP generation. oxLDL stimulation increased MΦMP tissue factor (TF) content by 78% (p<0.05), and oxLDL-induced MΦMP production correlated with activation of PI-3K/Akt signaling pathways. Inhibition of Akt signaling with the Akt inhibitor, KP372-1, reduced MΦMP release by 29% (p<0.01). Because statins inhibit inflammatory signaling that promotes MP production, we also examined the ability of mevastatin to reduce ox-LDL-stimulated MΦMP generation. Mevastatin treatment (10μM) reduced oxLDL-stimulated MΦMP production by 60% (p<0.01), MΦMP TF content by 43% (p<0.05), and attenuated oxLDL-stimulated Akt activation.

Conclusion:
oxLDL induces Akt-dependent production of prothrombotic MPs, a process inhibited by pretreatment with mevastatin. These findings link atherogenic signaling pathways, inflammation, and plaque thrombogenicity, and identify a novel potential mechanism for the LDL-independent antithrombotic effects of statins.
Abstract 95: A Conformable and Compliant Nanocomposite Polymer Stent-Graft for Endovascular Repair of Aortic Arch
Authors: Desai Mital , Ahmed Maqsood , Zhou Xiang , You Zhong , Seifalian Alexander M , Hamilton George
Published: July 2021

Objectives:
To develop new aortic stent-graft from polyhedral oligomeric silsesquioxane (POSS) and poly (carbonate-urea) urethane (PCU) nanocomposite polymer (NP) to achieve better conformability to arch anatomy and reduce aorta-prosthesis compliance mismatch.

Methods:
A constructed model of curved thoracic aorta from MRI was developed and anatomically accurate sutureless stent-graft was moulded using surface-modified Nitinol stents with composition of 55.8 wt.% Ni, diameter 0.62 mm and Af temperature less than 18 oC bonded to NP. An in vitro pulsatile flow phantom perfused NP stent-graft at physiological pulse pressure and flow. Diametrical compliance and stiffness index were calculated and comparison was made with ePTFE based in Gore TagTM thoracic stent-graft.

Results:
The NP stent-graft had uniform thickness of 150.7±6.6 μg and in its fully expanded shape, with diameter of 30 mm, matched curvature of aortic arch. Over temperature 37±1°C, mean pressure range 30-100 mm Hg, and pulse pressure 49±6 mm Hg, flow rate for NP stent-graft was 132±24 ml/min and ePTFE stent-graft was 140±25 ml/min (P=NS). Overall compliance of NP stent-graft was 3.3 ± 0.61%/mm Hg x 10-2 which was significantly better compared with ePTFE stent-graft (2.3±0.95 %/mm Hg x 10-2; P=0.0003). The ePTFE stent-graft was significantly stiffer with stiffness index β 92.7±46.1 compared to NP stent-graft (β 39.1±5.91; P<0.0001). The ePTFE stent-graft failed to exhibit anisotropic behaviour (i.e. visco-elasticity similar to natural vascular tissue) which was demonstrated by NP stent-graft.

Conclusion:
An innovative, curved, self-expanding and sutureless stent-graft is proposed for endovascular repair of thoracic aorta and aortic arch. The study confirmed the superior compliance, reduced elastic stiffness and visco-elasticity of POSS-PCU NP, which can help in improving device longevity by offering superior physiological and haemodynamic environment in vivo.
Abstract 101: Reconstituted High-density Lipoprotein - More Than the Sum of Its Parts.
Authors: Pirianov Grisha , Shoreim Azza , De Souza Ayesha , Torsney Evelyn , Cockerill Gillian
Published: July 2021

Rationale

Reconstituted high-density lipoprotein (rHDL, CSL-111) particles have been shown to reduce multiple organ failure following hypovolemic shock, perturb irritable bowel syndrome and, more recently, to inhibit aneurysm formation in animal models. To identify new endothelial targets of the protective effect we examined the differential changes that occur in the proteome of activated primary endothelial cells following treatment with rHDLs.

Methods

Primary human endothelial cells were isolated from human umbilical cords, grown to confluence and activated with TNFα (10ng/ml), following a 4 hr incubation half the cells received rHDLs (1mg/ml, ApoAI) and the remaining cells received carrier only (10% sucrose). Protein lysates were prepared following 20 hr incubation and differential protein expression compared by scanning DiGE-2dimentional PAGE. Protein identities were confirmed using LC/MS of tryptic digests of proteins recovered from preparatory gels. Western blotting confirmed differential expression and established whether the response required the whole particle or solely the protein component of rHDLs.

Results

Of the proteins identified by LC/MS which were differentially expressed in response to rHDLs, Perilipin 3 (PLPN3, 1.3 fold, p<0.003); Growth factor receptor-bound protein 2 (GRB2, 2.5 fold, p<0.003) and Vimentin (1.6 fold, p<0.007) were induced, whilst sinexin 6 (SNX6, 1.2 fold p< 0.007); Glutathione S Transferase-1 (GST-1, 1.6 fold, p<0.02) and Crystallin-zeta like (CrystZL, 1.2 fold, p<0.004) were reduced. We found that the entire lipoprotein particle was necessary for the induction of PLN3 and reduction of SNX6. Concentration of rHDL < 100μg/ml were able to modulate the maximal effect observed. However, both rHDL and lipid free ApoAI were able to induce cytokine-inhibited expression of GRB2 (rHDL>ApoAI).

Conclusion

The PC component of rHDL is necessary for an acute effect on proteins involved in endosomal trafficking. Further studies are needed to understand the role this could play in the protective effect of HDL therapy.
Abstract 109: Preliminary Structural Into the Sterol Regulatory Element-Binding Protein (SREBP) Interaction With SREBP Cleavage-Activating Protein (SCAP)
Authors: Ziarek Joshua J , Wagner Gerhard
Published: July 2021

Maintenance of cholesterol and fatty acid homeostasis is critical for membrane architecture, protein localization, and protein trafficking. Dysregulation can lead to severe health concerns including obesity, diabetes, heart attack and stroke. Vital to the management of lipid molecules are the sterol regulatory element binding protein (SREBP) family of transcription factors, which transcribe more than 30 genes controlling lipid homeostasis. The activation of SREBP is tightly regulated through association with SREBP cleavage-activating protein (SCAP). When sterol levels are high, SCAP maintains SREBP in the endoplasmic reticulum. As sterol concentrations are reduced, SCAP escorts SREBP to the Golgi apparatus where it is activated. Liver-specific knockout of SCAP results in a 70-80% decrease in lipid biosynthesis and germline knockout is hypothesized to be embryonic lethal. Nonetheless, how SCAP recognizes and restricts the location of SREBP is relatively unknown. Structural insight to this protein-protein interface may yield future drug targets; however, their large molecular mass makes them challenging. We hypothesize both CTDs can be functionally produced in vitro to investigate the complex affinity, stoichiometry, and architecture. So far, we have recombinantly produced milligram quantities of both proteins. Circular dichroism and 1D NMR spectroscopy support in vitro SCAP refolding into a β-propeller fold. In contrast to previously published suggestions of a seven-bladed propeller motif, our sequence analysis and homology modeling support an eight-bladed architecture. Both X-ray crystallography and NMR are currently being pursued to elucidate the SCAP structure. In conclusion, recombinant expression of SCAP and SREBP is feasible and future research will use a combined biochemical and cell-based approach to determine the complex affinity, dynamics, and binding interface.
Abstract 118: The Effect of CETP Inhibition With Anacetrapib on the Metabolism of PCSK9
Authors: Millar John S , Reyes-Soffer Gissette , Ginsberg Henry N , Rafeek Hashmi , Lassman Michael , Wong Allen , Jumes Patricia , Wagner John A , Gutstein David E , Rader Daniel J
Published: July 2021

Introduction

Inhibition of CETP activity with anacetrapib has been associated with a significant reduction in low density lipoprotein (LDL)-cholesterol (C) levels both as a monotherapy and in combination with a statin. We recently reported that this reduction in LDL-C is due to a significant increase in the fractional clearance rate (FCR) of LDL apoB. One potential mechanism by which anacetrapib could influence LDL apoB clearance is through effects on PCSK9 metabolism. We conducted kinetic studies on PCSK9 to determine if a change in the metabolism of PCSK9 is responsible for the reduction in LDL-C levels following treatment with anacetrapib.

Methods

Moderately hyperlipidemic volunteers (n=39) were enrolled in a fixed-sequence study: 75% were on atorvastatin (20mg/day) plus placebo and 25% were on double placebo for four weeks. Following this baseline period subjects added anacetrapib (100 mg/day) to their background treatment for 8 weeks. Studies using D3 leucine tracer were performed at the end of each treatment period to determine the kinetics PCSK9.

Results

Statin treated subjects had lower LDL-C levels (84 vs 118 mg/dL) and higher PCSK9 levels (234 vs. 208 ng/mL) at baseline than subjects on placebo. The difference in PCSK9 was due to a higher PCSK9 production rate (PR) (19.7 vs. 16.7 ug/kg/day) but similar PCSK9 FCR (1.87 vs. 1.78 pools/day). Adding anacetrapib to background treatment was associated with a significant reduction in LDL cholesterol (~ -35%) and apoB levels (~ 20%) in both panels. Anacetrapib had no significant effect on PCSK9 levels in either of the background treatment groups. Consistent with the lack of change in PCSK9 levels, there was no effect of anacetrapib on the PR (18.1 vs. 19.3 ug/kg/day) or FCR of PCSK9 (1.82 vs. 1.93 pools/day).

Conclusion

These results indicate that in our study population the increase in LDL FCR observed in response to anacetrapib treatment is not likely to be related to changes in the metabolism of PCSK9.
Abstract 134: Kidney and Blood Pressure Abnormalities, and Cardiac Enlargement Accompany Moderate Coronary Artery Atherosclerosis in Western Diet Fed SR-BI/LDLR Double Knockout Mice
Authors: Fuller Mark T , Trigatti Bernardo
Published: July 2021

Mice lacking the HDL receptor, SR-BI, are susceptible to spontaneous or diet-inducible occlusive coronary artery (CA) atherosclerosis and myocardial infarction (MI) in the context of absence, or reduced expression of apoE, respectively. Our lab has generated mice lacking both SR-BI and the LDL receptor (dKO mice). When fed diets that are very high in cholesterol, dKO mice develop rapid CA atherosclerosis and large transmural MIs, and exhibit significantly reduced survival. However, when fed a western type diet (WTD) with only moderately high cholesterol (22% fat, 0.125% cholesterol), dKO mice do not appear to exhibit reduced survival, and develop only mild CA atherosclerosis with little to no cardiac fibrosis. However they have significantly larger hearts than LDLR single KO (sKO) controls fed the same diet for the same period of time. We therefore examined additional factors that may account for the cardiac enlargement observed in these mice such as blood pressure (BP) and kidney pathology. 10 week old dKO mice and sKO controls were fed a WTD for 10 weeks; BP was measured before feeding, and periodically throughout feeding. Mice were euthanized after 10 weeks and blood and tissues were collected for analysis.

DKO mice had reduced cholesterol associated with VLDL and LDL sized lipoproteins compared to sKO control mice after 10 weeks of WTD feeding. As expected, heart weights were significantly increased in dKO mice compared to sKO controls. Additionally, dKO mice had significantly lower body weights compared to sKO controls treated equivalently. Before the onset of feeding, there was no significant difference in BP between dKO and sKO mice. After 1 week of WTD feeding, dKO mice had significantly increased BP compared to both pre-diet levels and to sKO controls fed the same diet for the same timeframe. However, BP in dKO mice dropped significantly after 9 weeks of WTD compared to pre-diet measurements, while BP in sKO mice was constant throughout the study. Oil red O staining of kidney sections revealed striking lipid deposition and enlargement of glomeruli in kidneys from dKO mice, this was not observed in sKO control mice.
Abstract 149: Pluripotency Factors Krüppel-Like Factor 4 and Octamer-binding Transcription Factor 4 Alter Indices of Plaque Stability in Long Term Western Diet Fed Mice by Regulating Smooth Muscle Cell Phenotypes
Authors: Shankman Laura S , Cherepanova Olga A , Gomez Delphine , Owens Gary K
Published: July 2021

The bulk of life threatening thrombotic events have been associated with disruption of the fibrous cap, an atheroprotective layer of smooth muscle α-actin positive (ACTA2+) cells that form around the plaque, and the presence of a large foam cell-laden necrotic core within the plaque. Despite the overwhelming research demonstrating that ACTA2+ cells are beneficial for plaque stability, and cells positive for macrophage-markers are detrimental, there are major ambiguities regarding the origins of these cells, and their role in lesion stability. To clearly define the contribution of smooth muscle cells (SMCs) within atherosclerotic lesions, we generated SMC specific lineage tracing
Apoe-/-
mice containing a SM myosin heavy chain (
Myh11
) tamoxifen-inducible cre-recombinase gene and a floxed STOP ROSA eYFP gene (
Myh11 YFP ApoE-/-
mice) thus allowing activation of eYFP exclusively in fully differentiated SMCs before the onset of atherosclerosis and subsequent determination of the fate of these cells and their progeny irrespective of continued expression of MYH11 or other SMC marker genes.

Remarkably, our results reveal that 86% of SMCs cannot be identified using traditional SMC markers, such as ACTA2, and 23% of presumed macrophages (LGALS3+ cells) are derived from SMC origins. The last finding was confirmed in human coronary atheromas using the ISH-PLA approach.

SMC specific knockout (KO) of the pluripotency factor
Klf4
in
Myh11 YFP ApoE-/-
mice did not alter the frequency of phenotypically modulated (ACTA2-eYFP+) SMCs within atherosclerotic lesions of mice fed a high fat diet for 18 weeks, however, decreased the number of ACTA2-eYFP+ SMCs that expressed LGALS3, and increased several indices of plaque stability, suggesting a detrimental role for KLF4 in SMCs within atherosclerotic lesions. Conversely, SMC specific
Oct4
KO resulted in a dramatic reduction in the number of ACTA2-eYFP+ SMCs within the lesion with marked decreases in indices of plaque stability. In summary results show that the majority of SMC-derived cells within advanced atherosclerotic lesions cannot be identified using conventional SMC marker genes, and that phenotypic switching of SMC during atherogenesis is differentially regulated by the pluripotency factors KLF4 and OCT4.
Abstract 17: The Association Between Erythrocyte n-3 Polyunsaturated Fatty Acids (n-3 PUFAs) Content and Inflammation in Male Patients With Peripheral Artery Disease (PAD)
Authors: Grenon Marlene , Owens Christopher D , Alley Hugh , Chong Karen , Vittinghoff Eric , Harris William , Hughes-Fulford Millie , Rapp Joseph , Conte Michael
Published: July 2021

Objectives

Dietary intake of n-3 PUFAs has been associated with cardiovascular disease, though the relationship to PAD is unclear. PAD patients have an increased burden of systemic inflammation. In a cross-sectional PAD cohort study, we evaluated the relationship between n-3 PUFAs content of red blood cells (omega-3 index) and biomarkers of systemic inflammation.

Methods

This was a prospective cohort study of patients (n=83) presenting to vascular surgery clinic for evaluation of PAD. The omega-3 index was measured using gas chromatography and inflammatory markers (hsCRP, IL-6, TNF-α and ICAM-1) via ELISA kits. We used linear regression to evaluate the independent association between the omega-3 index and inflammation.

Results

70 patients had PAD while 13 were found to have a normal ankle-brachial index (ABI). Mean (±SD) age was 67 ± 7 years. Mean ABI was 0.85 ± 0.23. The omega-3 index decreased across defined AHA hsCRP categories (Figure 1, p=0.04). One percentage point decrease in the omega-3 index was associated with increases in CRP (14%, 95% CI 0, 25, p=0.04), IL6 (8%, 95% CI 1, 15, p=0.02) and possibly ICAM-1 (4%, 95% CI -1, 12, p=0.13), but not TNF-α. After adjusting for age, race, HDL, smoking status, ABI and the body-mass index, the omega-3 index remained significantly (negatively) associated with systemic inflammation as measured by hsCRP in a male population at risk or suffering from PAD (p=0.05).

Conclusions

In a contemporary cohort of patients with PAD, the omega-3 index was negatively associated with biomarkers of inflammation. Our findings suggest a rationale for future studies of dietary manipulation of omega-3 index to reduce inflammation in patients with PAD.
Abstract 174: miR30c Regulates Plasminogen Activator Inhibitor-1 In Platelet
Authors: Luo Mao , Wan Qin , Liu Fei , Li Rong , Xia Jiyi , Ren Meiping , Chen Ni , Strawn Tammy , Fay William P , Wu Jianbo
Published: July 2021

Objective.

PAI-1 mRNA and protein have been detected in human platelets. Recently some miRNAs have been found in human platelets, which are involved in the regulation of genes and the protein synthesis. However, little is known about the physiological roles of individual miRNAs in platelets. In this study, we investigated whether miR30c can regulate platelet-derived plasminogen activator inhibitor-1(PAI-1).

Methods and Results.

Expression of miR-30c, PAI-1, miR-21 and its targeted gene TIMP1 were found in healthy human leukocyte-depleted platelets (LDPs) by real time PCR. In luciferase reporter gene assay, miR-30c targets the 3’ untranslated region (3’ UTR) of PAI-1 mRNA through a miR-30c binding site. Transfection of miR-30c mimic into MEG-01, a megakaryoblastic cell line, significantly reduced PAI-1 protein level compared with negative control. Inhibition of miR-30c by transfecting miR-30c inhibitor significantly increased PAI-1 protein level. Furthermore, miR-21 expression was significantly down-regulated after transfecting with miR-30c mimic in PAI-1-/- mice LDPs, conversely, the expression of its target gene TIMP1 was significantly up-regulated after transfecting with miR-30c mimic in PAI-1-/- mice LDPs.

Conclusion.

These results provide a novel regulatory mechanism of miR30c- regulated PAI-1 protein through its influence on the downstream miR21 and its target gene TIMP1 expression in platelet, suggesting that miR-30c might be a potential new strategy for anti-thrombosis.
Abstract 181: BET Bromodomain Inhibition Suppresses Endothelial Inflammation and Atherosclerosis
Authors: Brown Jonathan , Duan Qiong , Griffin Gabriel , Paranal Ronald , Bair Steven , Newton Gail , Lichtman Andrew , Kung Andrew , Luscinskas Francis , Yang Tianlun , Croce Kevin , Bradner James , Plutzky Jorge
Published: July 2021

Introduction

The BET bromodomain-containing family of proteins (BRD2, BRD3, BRD4) are epigenetic readers that coactivate transcription. Recent evidence indicates that BETs promote carcinogenesis and inflammation in sepsis, while BET bromodomain inhibitors are promising anti-cancer therapies. However, the role of chromatin remodeling in atherosclerosis in general and through BETs in particular remains unknown.

Hypothesis

We hypothesized that BET bromodomain-containing proteins coactivate proinflammatory responses in the vasculature with functional effects that promote atherogenesis.

Methods and Results

BET bromodomain inhibition, achieved with the highly selective, small-molecule inhibitor JQ1 significantly reduced early atherosclerosis (12 weeks) in cholesterol-fed, LDL receptor-null mice. In pursuing mechanisms for this effect, we identified BET protein expression in mouse and human endothelial cells (ECs) as well as endothelium from human atherosclerotic plaque. Treating human umbilical vein endothelial cells (HUVECs) with either JQ1 or siRNA to BRD2 or BRD4 potently suppresses TNFα-induced expression of adhesion molecules (SELE, VCAM1) and chemokines (CCL2, CXCL8). In chromatin immunoprecipation studies, TNFα stimulation of ECs recruited BETs to adhesion molecule and chemokine promoters coincident with RNA polymerase II and cyclin T1 localization, without altering NF-κB recruitment. In functional studies, JQ1 suppressed 1) monocyte adhesion to TNFα-activated HUVECs, 2) leukocyte rolling on cremaster post-capillary venules (intravital microscopy); 3) leukocyte transmigration (parallel-plate flow chamber); and 4) monocyte recruitment in thioglycolate-induced peritonitis
in vivo
.

Conclusions

BET bromodomain-containing proteins are novel determinants of pro-inflammatory transcription in the endothelium. Targeting chromatin by BET bromodomain inhibition may be a therapeutic strategy to limit atherosclerosis and other disorders involving endothelial inflammation.
Abstract 182: Quantitative Proteomics Reveals Dynamic Changes of Plasma Proteome in APOE-Deficient Mice at Four Phases of Atherosclerosis
Authors: Jing Linhong , Jin Zhu-qiu , Xie Wei , Zhang Shuaipeng J
Published: July 2021

Introduction

To understand the mechanism for atherosclerotic disease at molecular level, we analyzed the dynamic changes of plasma proteome in mice at four different disease stages vs. control based on hypothesis that a panel of plasma proteins might associate with the progression of atherosclerosis.

Methods

Male APOE knockout (-/-) mice were fed with high fat diet and euthanized at 8, 12, 18 or 24 weeks of age, representing the early, intermediate, moderate and later stages of disease. Age-matched male wild type mice fed with high fat diet served as controls. At the time of euthanasia, aorta and plasma were collected. A mass spectrometry-based proteomics approach was employed to simultaneously quantify proteins in eight pooled samples.

Results

Image analysis of lesions in aortas (stained with Sudan IV) from 40 APOE -/- mice (10 mice at each time point) demonstrated the development of four different phases of disease (Fig.). No lesion was observed in the control mice. We have identified 57 dysregulated proteins (e.g., fibrinogen beta and gamma chains, complement C3, C4B, C9, apolipoproteins, etc.) at four disease stages. These protein’s expression levels, biological functions (e.g. immune modulation and inflammation), and pathways (e.g., complement and cogulation cascades) provide a better understanding of the basic molecular processes in atherosclerosis.

Conclusions

1) Proteomics analysis of plasma proteome changes at different stages of APOE-deficient mice facilitated the mechanistic studies of atherosclerosis. 2) A panel of plasma proteins was identified as potential “signatures” of atherosclerosis which may help early diagnosis and therapeutic intervention.
Abstract 19: Prognostic Value of Plasma Choline and Betaine Depend on the Intestinal Microflora-generated Metabolite Trimethylamine N-oxide
Authors: Wang Zeneng , Tang W. H. Wilson , Fu Xiaoming , Britt Earl B , Wu Yuping , Koeth Robert A , Levison Bruce , Hazen Stanley L
Published: July 2021

Background

Recent metabolomics and animal model studies show trimethylamine-N-oxide (TMAO), an intestinal microbiota-dependent metabolite formed from dietary phosphatidylcholine (PC), is linked with coronary artery disease (CAD) pathogenesis. Prognostic determinants of other PC metabolites recently associated with prevalent CAD risk, such as choline and betaine, have not been described.

Methods

We examined the relationship between fasting plasma choline, betaine, and TMAO and future risk of major adverse cardiac events (MACE=death, myocardial infraction, stroke) over 3-year follow-up in 3,916 subjects undergoing elective coronary angiography. All analytes were quantified by stable isotope dilution LC/MS/MS.

Results

In our study cohort (mean age 63±11 years, 64% male), median [IQR] TMAO, choline, and betaine levels were 3.7 [2.4-6.2] μM, 9.8 [7.9-12.2] μM, and 41.1 [32.5-52.1] μM, respectively. Modest but statistically significant correlations were noted between TMAO and choline (r=0.33, p<0.001) and between TMAO and betaine (r=0.09, p<0.001). Higher plasma TMAO, choline and betaine levels were associated with a 2.7-fold, 1.9-fold, and 1.4-fold increased risk of MACE, respectively (Quartiles 4 vs 1: p<0.01, each). Following adjustments for traditional risk factors and hsCRP, elevated TMAO (Quartiles 4 vs 1: Hazard ratio, 1.97 [95% CI 1.50-2.60], p<0.01), choline (HR 1.34 [95% CI 1.03-1.74], p<0.05) and betaine levels (HR 1.33 [95% CI 1.03-1.73], p<0.05) each predicted increased MACE risk, but only TMAO predicted MACE risk when all 3 metabolites were included in the model (HR, 1.73 [95% CI 1.3-2.31], p<0.01). Choline and betaine only predicted increased MACE risk in those with elevated TMAO levels.

Conclusion

Elevated plasma levels of TMAO, and to a lesser extent choline and betaine, are associated with incident MACE risk. However, high choline and betaine levels are only associated with higher risk of future MACE with concomitant increase in TMAO. Collectively, these findings further support the atherogenic associations between intestinal-microflora dependent metabolism of PC and atherosclerosis in humans.
Abstract 205: Aging and Exercise Training Alter Coronary Microvascular Function: Role of the Adiponectin/AMPKinase Axis
Authors: Chen Bei , Kang Lori , Dougherty Patrick , Dominguez Catherine , Tsuda Shigeharu , Stabley John , Muller-Delp Judy
Published: July 2021

Our lab has demonstrated age-related coronary microvascular dysfunction can be reversed by aerobic exercise training; however, the mechanisms that underlie age- and exercise training-induced adaptations of the coronary microvasculature remain to be identified. The adipokine, adiponectin, possesses cardioprotective properties, including anti-atherosclerotic and anti-inflammatory effects in the vascular wall, and is known to inhibit vascular smooth muscle (VSM) proliferation in large arteries. Circulating adiponectin declines with age and increases with regular exercise training. Therefore, we hypothesized that alterations in signaling through the adiponectin/AMPKinase axis contribute to age- and exercise training-induced adaptations of the coronary microvasculature, particularly in VSM. Young (4 mos) and old (22 mos) male Fischer 344 rats were randomly assigned to either a sedentary or an exercise training group. Circulating adiponectin levels were measured by ELISA. Coronary arterioles from the left ventricular (LV) free wall were isolated for assessment of contractile function and VSM proliferation, and levels of contractile and proliferative proteins. Circulating adiponectin levels decreased with age and increased after 10 wks of exercise training in both young and old rats. Age impaired contractile responses of coronary arterioles, and increased proliferation of arteriolar VSM. Age reduced levels of AMPKinase and contractile proteins, smooth muscle myosin 1 (SM1) and smooth muscle myosin 2 (SM2), and increased total levels and phosphorylation of ribosomal protein S6 (rpS6), a downstream effector of mammalian target of rapamycin (mTOR). Exercise training reversed age-induced contractile dysfunction and attenuated VSM proliferation in coronary arterioles. Exercise training restored contractile function and reduced proliferation in coronary arterioles from old rats. Exercise training also returned AMPKinase, SM1, rpS6 and phosphorylated rpS6 levels in arterioles of old rats to those found in arterioles of young rats. These data indicate that changes in adiponectin/AMPKinase axis contribute to age- and exercise training-induced alterations of coronary microvascular phenotype and function.
Abstract 234: Cigarette Smoke Induction of Mmp9 in Aortic Vascular Smooth Muscle Cells is Mediated by the Jak/stat Pathway
Authors: Ghosh Abhijit , Pechota Angela , Coleman Dawn , Upchurch Gilbert R , Eliason Jonathan L
Published: July 2021

Introduction

Cigarette smoke has a strong correlation with abdominal aortic aneurysm (AAA) formation and is characterized by increased levels of MMP9 in the aorta. We hypothesized that smoke-induced MMP9 regulation is mediated by the Jak/Stat pathway thereby contributing to AAA formation.

Methods

Aqueous extract of cigarette smoke (AEC) was applied to male rat aortic vascular smooth muscle cells (RASMC) for 24 hours (h) in serum free medium (SFM), using SFM alone for controls. Media were collected for MMP2 and MMP9 zymography and proteins extracted from cells for phospho-Stat3 (pStat3), total Stat3 (T-Stat3), phospho-Jak2 (pJak2) and total Jak2 (T-Jak2) western blots. RASMC were transfected by siRNAs for Jak2 and Stat3 before AEC treatment to evaluate induction of MMPs. Coimmunoprecipitation and immunofluorescence microscopy investigated complex formation and cellular distribution of Jak2 and Stat3 following AEC treatment.

Results

MMP9 was increased in the medium of the AEC treated RASMC (P=0.005) and pro-MMP2 slightly elevated (P=0.055) at 24h compared to controls. By western blot protein extracts from AEC treated RASMC showed up regulation of pStat3, pJak2 and T-Jak2 and stable T-Stat3 compared with controls. siRNA transfection of RASMC for Jak2, Stat3 and a combination of Jak2 and Stat3 reduced MMP9 (P<0.005) and pro-MMP2 (P<0.05) in medium of AEC treated RASMC compared with AEC-treated cells transfected with control siRNA. Coimmunoprecipitation of total proteins from RASMC with total Jak2 antibody revealed increased levels of pStat3 in the AEC treated cells compared to controls, while separation of nuclear and cytoplasmic components followed by coimmunoprecipitation revealed more abundant Jak2/T-Stat3 complexes in the nuclear fraction than the cytoplasm following AEC treatment. Immunofluorescence microscopy revealed increased presence of pJak2, T-Jak2, pStat3 and T-Stat3 in the cytoplasm and nucleus of the AEC treated cells compared to the control cells.

Conclusion

This study suggests that cigarette smoke may result in AAA formation through Jak/Stat-mediated MMP9 production. siRNA inhibition of the Jak/Stat pathway greatly reduced AEC-induced MMP9 by RASMC and suggests a potential therapeutic target for the treatment of AAA.
Abstract 235: Macrophage Depletion Reduces Angiotensin Ii-induced Aortic Inflammation but Not Experimental Aneurysm Formation
Authors: Glover Keith J , Xu Baohui , Xuan Haojun , Tanaka Hiroki , Nagai Taku , Fujimura Naoki , Iida Yasunori , Wang Wei , Furusho Yuko , Miyata Masaaki , Michie Sara A , Matsuyama Takami , Dalman Ronald L
Published: July 2021

Massive mural macrophage infiltration is observed in experimental and clinical abdominal aortic aneurysm (AAA) disease. However, causational relationships, if any, between macrophage infiltration and aneurysm pathogenesis remain uncertain. This study examined the consequences of total and activated macrophage depletion on aneurysm formation and progression in the angiotensin II infusion, apolipoprotein E-deficient (Ang II/Apo E
-/-
) AAA model. Mice were evaluated via serial
in vivo
aortic ultrasonography for 28 days during the Ang II infusion, and histology at sacrifice. In CD11b-diphtheria toxin (DT) receptor transgenic (DT
+
) Ang II/ApoE
-/-
mice, DT injection failed to prevent aneurysm formation despite successful macrophage depletion. AAAs developed in 40% of DT
+
Ang II/ApoE
-/-
mice, a rate similar to littermate control, DT
-
Ang II/ApoE
-/-
receiving Ang II and DT. Similar results were observed when Ang II/Apo E
-/-
mice were treated with folate receptor (FR) β immunotoxin (to deplete activated macrophages characterized by FRβ expression) compared with control toxin. Histologically, DT treatment in DT
+
Ang II/ApoE
-/-
mice reduced aortic CD68
+
mural macrophages by 77% and 60% in mice with or without aneurysms, respectively. Similarly, activated macrophage depletion via FRβ immunotoxin resulted in a 33% and 50% reduction in mural CD68
+
macrophages in mice with and without aneurysms, respectively. In conclusion, neither the total or activated mural macrophages appear to mediate aneurysmal aortic enlargement in the Ang II/ApoE
-/-
AAA model.
Abstract 237: MicroRNA patterns in Abdominal Aortic Aneurysm Appear to Reflect Adventitial Inflammation
Authors: Jones Gregory T , Phillips Victoria L , van Rij Andre A
Published: July 2021

MicroRNAs (miRNA) have been shown to play an important role in vascular biology. In this study we examined the miRNA profile within human abdominal aortic tissue and plasma to identify differential expression patterns between aneurysm cases and controls.

Whole miRNome profiles were assessed using array-based assays (Affymetrix miRNA 2.0) in 27 AAA and 14 control aortic biopsies. Results were validated in 89 AAA and 21 controls using real-time PCR (Taqman-based) assays. A subset of case and control aortic samples were also microdissected. Separate intima/medial and adventitial miRNome profiles were compared with matching total wall. In addition, whole miRNome profiles of EDTA plasma from 22 AAA and 20 age and gender matched controls were examined and validated by RT-PCR in 62 AAA and 62 control plasmas.

In aortic tissue, 18 miRNA were highly significantly (p<1x10-4, q<1x10-3) differentially expressed in AAA versus controls. Variable principal component analysis indicated that these miRNA were expressed in three distinct clusters. MiRNA within each cluster were subsequently validated by RT-PCR. The strongest validated associations were miR-146a (p<0.0004), mir-150 (p<0.001), miR-223 (p<0.0001), and mir-140-3p (p=0.006). Aortic wall sub-layer analysis indicated that total wall miRNA profiles reflected adventitial related miRNA in AAA and intima/medial related miRNA in controls.

In plasma, 12 miRNA were initially identified (including three which were also observed in tissue) however only miR-151-5p (p=0.005) was successfully validated by RT-PCR.

The general pattern of AAA associated miRNA expression indicated regulation of genes known to be involved with B-cell and T-cell function. This was also consistent with the concentration of these miRNA within the AAA adventitia, as these cells are key regulators of the vascular (adventitial) inflammation associated with AAA.

In conclusion, microRNAs appear to have significant potential as AAA biomarkers. However, further work is required to determine the specificity of such associations to account for potential confounders, including as concurrent arterial disease.
Abstract 240: Serum Cholesterol and Angiotensin-Induced Abdominal Aortic Aneurysm in Hypercholesterolemic Mice
Authors: Prins Petra A , Hill Michael , Airey David , Nwosu Sam , Perati Prudhvidhar R , Abramo Jason M , Kon Valentina , Fazio Sergio , Sampson Uchechukwu
Published: July 2021

Background

Although hyperlipidemia is known to augment the incidence of abdominal aortic aneurysms (AAA) in the AngII-induced model of apolipoprotein E
-/-
mice, its relationship to AAA size is unknown. Therefore, we evaluated the relationship between total cholesterol concentration (TC) and change (delta) in aortic diameter.

Methods

TC was measured in 36 male mice that underwent a 4-week infusion period with saline (n=9) or AngII (1500 ng/kg/min; n=27), along with serial measurements of pulse rate (PR), and pulse (PP), mean arterial (MAP), systolic (SBP) and diastolic (DBP) pressure. A linear mixed effect model was used to assess the relationship between all hemodynamic parameters and delta. Nonparametric and linear regression methods were used to evaluate TC in relation to delta.

Results

TC did not differ between AngII and control mice (Figure, bottom left) (p=0.18). The burden of atherosclerosis was greater among AngII-exposed mice versus control, but did not differ by presence or size of AAA (Figure, bottom right). None of the hemodynamic parameters were predictive of delta (SBP, p = 0.66; DBP, p = 0.66; MAP, p = 0.55; PP, p = 0.66; and PR, p = 0.39). Mean TC was higher among mice with large versus small AAA (552.6 vs. 393.5 mg/ ml, p<0.05; Figure, top right). The nonparametric smoothing line (Figure, top left) suggests a first order relationship between delta and TC (p for trend < 0.001). AngII (ß = 0.48, p < 0.001) and TC (ß = 0.0015, p = 0.003) were independent predictors in the linear model for delta.

Conclusions

Our findings suggest that TC is incrementally associated with AAA size. These findings may have potential clinical relevance for risk assessment in AAA patients.

Figure
Abstract 246: Echo Particle Image Velocimetry Generates Wall Shear Stress Biomarkers with Higher Accuracy than Doppler: Clinical Studies on 24 Healthy and 12 Tia Subjects
Authors: Gurung Arati , Mazzaro Luciano , Gates Philip E , Strain David , Shore Angela C , Shandas Robin
Published: July 2021

Wall shear stress (WSS) is an important modulator of vascular disease. We assessed the hypothesis that WSS profiles derived from Echo Particle Image Velocimetry (EchoPIV) are characteristic of vascular conditions such as TIA with superior accuracy to Doppler. We obtained ultrasound images of CCA from 24 healthy and 12 TIA subjects. Doppler provided centerline peak velocity and WSS estimate based on parabolic velocity distribution. Velocity profiles measured by EchoPIV varied from patient to patient and within the cardiac cycle. Curve-fitting to a power law function with an exponent term (n-value) showed that in healthy carotid arteries, a blunt velocity profile is present during systole with an average n-value of 5, whereas during diastole a more parabolic profile with an n-value of 2 is present. Therefore, assuming a fully parabolic velocity distribution for WSS estimation, the basis for Doppler velocimetry, introduces a mean error of 48% during diastole and 98% during systole. EchoPIV offers characterization of WSS profiles; time waveform analysis revealed smoother and fewer number of peaks per cycle for healthy and increased flow pulsatality (reverberations) for TIA (Figure). Echo PIV generated means for phase averaged peak velocity (27.95 vs 16.13 cm/s), WSS (6.45 vs 4.65 Dynes/cm2) and flow rate (0.3179 vs 0.2215 L/min) with standard deviations 3.66 vs 2.35, 2.8 vs 1.74 and 0.0723 vs 0.0744 were significantly different between healthy and TIA subjects respectively (all p < 0.01). Direct measurement of the velocity profile using Echo PIV provides superior estimates of WSS in carotid vessels. TIA populations have compromised hemodynamic variables compared to healthy subjects.
Abstract 254: TSP-2 siRNA Coating of Prosthetic Arterial Graft Material to Reduce Intimal Hyperplasia in a Rat Model
Authors: Pratt Wande B , Contreras Mauricio A , Pradhan-Nabzdyk Leena , LoGerfo Frank W
Published: July 2021

Introduction

Intimal hyperplasia represents a complex pathologic vascular response to injury. Our group previously revealed that TSP-2 was associated with this response, and demonstrated that intraluminal delivery of TSP-2 siRNA can suppress the intimal hyperplastic response in an animal model. Furthermore, we have shown that siRNA can be incorporated into prosthetic graft material using a dip-coating technique. We sought to determine whether this method could be employed for siRNA delivery in order to attenuate the response to arterial injury, and to reduce neointima formation in an in vivo model.

Methods

Carotid artery endothelial cell denudation was performed on male Wistar rats using a 2 Fr Fogarty balloon catheter. ePET graft material was dip-coated in several siRNA solutions (i.e. saline, TSP-2 siRNA, PEI-complexed TSP-2 siRNA) for 50 minutes. The graft was cut longitudinally, and then wrapped circumferentially around the denuded carotid artery. Arterial segments were then isolated at 21 days. TSP-2 gene expression was measured by qRT-PCR, and intimal hyperplasia was assessed by quantitative morphometry.

Results

Balloon denudation increased TSP-2 gene expression by 10-fold, when compared to non-denuded arteries after 21 days (p = .01). Fluorophore-conjugated siRNA confirmed transmural delivery following denudation. When compared to saline treated animals, TSP-2 siRNA reduced TSP-2 mRNA expression by 70% (p = .001). The addition of the transfection reagent PEI to TSP-2 siRNA treated grafts, however, did not significantly augment gene silencing. Morphometric analysis of arterial cross sections demonstrated significantly less neointima formation–as measured by intima-media (I/M) total area ratio–among grafts dip-coated with unmodified and PEI-treated TSP-2 siRNA (I/M: 0.2 and 0.4, respectively) compared to saline grafts (I/M: 1.0, p < .001).

Conclusions

In the rat carotid artery balloon injury model, dip-coating of prosthetic graft material with subsequent perivascular delivery of TSP-2 siRNA is an effective method to achieve transmural transfection and consistent gene silencing, while significantly suppressing the intimal hyperplastic response following arterial injury.
Abstract 284: microRNAs are Novel Plasma Biomarkers for Diagnosis and Prognosis of Abdominal Aortic Aneurysm Disease
Authors: Chernogubova Ekaterina , Eken Suzanne M , Jin Hong , Roy Joy , Hamsten Anders , Eriksson Per , Tsao Philip S , Maegdefessel Lars
Published: July 2021

MicroRNAs (miRNAs) have been identified as transcriptional and posttranscriptional inhibitors of gene expression, thought to “fine tune” the translational output of their target mRNAs. Recently, they have received much attention regarding their suitability as biomarkers for disease. Our goal was to explore the diagnostic and prognostic value of miRNAs in abdominal aortic aneurysms (AAAs), a disease for which currently no established biomarker exists.

Using a PCR-based array platform, we profiled the 168 most abundant blood miRNAs in 20 patient plasma samples with AAA disease, undergoing surgical repair of their enlarged aorta vs. 20 samples from an age, risk factor, and medication matched control group without aneurysm. We were able to identify a total number of 12 miRNAs being significantly altered in diseased patient samples as compared to controls. We further investigated these 12 miRNAs in plasma (as well as in aortic tissue) from apoE-/- mice with angiotensinII (AngII)-infusion induced AAAs, enabling us to discover a potential prognostic value of miRNAs being released into circulation. Indeed we were able to detect that the expression of 4 out of the 12 miRNAs (miRs-126 and -668 both increased; miRs-24 and -210 both decreased), was substantially modified in plasma samples drawn from mice with AAA immediately before rupture occurred between days 10 and 14 after AngII pump implantation compared to mice with AAA that did not rupture for the remainder of study (28 days), as well as saline-infused controls. Importantly, the expression of miRs-24 and -126 appeared also significantly different in plasma samples from patients with ruptured AAAs (n=7) compared to patients with non-ruptured AAAs (abdominal aortic diameter between 55-78 mm; n=7) and un-diseased controls (n=7).

The present study explores the diagnostic and prognostic biomarker potential of miRNAs being released into circulation during initiation, propagation, and ultimately rupture of AAA disease in mice and humans. The identification of miRs-24, -126, -210, and -663 potentially offers great prognostic value to determine which patients present with an increased risk of AAA rupture.
Abstract 293: Embryological Origin of Human Smooth Muscle Cells Influences Their Ability to Support Vasculogenesis.
Authors: Low Lucinda , Cheung Christine , Bennett Martin R , Sinha Sanjay
Published: July 2021

Lineage-tracking studies in avian and mouse embryos have revealed that SMCs in different regions of the vasculature are derived from different embryological origins. We have developed an in vitro protocol to differentiate human embryonic stem cells (hESCs) into SMCs through different embryonic lineages, namely neuroectoderm, lateral plate mesoderm (LM) and paraxial mesoderm. As mural cells are thought to augment the formation of functional blood vessels during revascularisation, this study aims to determine whether embryologically-distinct SMCs differ in their ability to support vasculogenesis. We assessed the hypothesis that LM-SMCs are superior in supporting endothelial network formation in vitro.

Using an mStrawberry-expressing hESC line, we derived the three origin-specific SMCs and co-cultured them with GFP-expressing HUVECs in a 3D in vitro matrigel assay. Endothelial network formation was assessed using real-time confocal imaging. Quantitative analysis revealed that LM-SMCs alone had a supportive effect on network formation and survival, with an increase in HUVEC network area after 4 days (3.23-fold increase ± 1.04-fold vs HUVECs alone; p<0.05; n=3) and 8 days (6.38-fold increase ± 0.39-fold vs HUVECs alone; p<0.001; n=3). In addition, LM-SMCs appear to facilitate more complex endothelial networks, with narrower cords and more branch points, compared with the other SMC types and HUVECs alone. To identify whether the LM-SMC-specific influence on network formation and survival was a paracrine effect, the three SMC types were tested in a paracrine 3D in vitro assay, where they shared media with HUVECs seeded in an adjacent well. Preliminary results reveal that the supportive effect of LM-SMCs is, at least partly, paracrine, with an increase in HUVEC network area after 4 days (7.08-fold increase vs HUVECs alone; n=1) and 8 days (21.39-fold increase vs HUVECs alone; n=1). Currently, we are exploring possible soluble factors that may be responsible and investigating this effect in an in vivo matrigel assay. In conclusion, the embryological origin of SMCs influences their functional ability to support vasculogenesis. This research will provide insight into which SMC type will be most effective in future revascularisation therapy.