bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 79: Interleukin-19 Reduces Leukocyte-Endothelial Cell Interactions
Authors: England Ross N , Kelemen Sheri E , Preston Kyle J , Ellison Stephen P , Scalia Rosario , Autieri Michael V
Published: March 2022

Introduction

Vascular endothelial cells (EC) actively participate in the pathogenesis of atherosclerosis by increasing expression of cell adhesion molecules which tether and adhere to leukocytes. The goal of the present study was to test the hypothesis that interleukin-19 (IL-19), an anti-inflammatory Th2 cytokine, can decrease expression of EC adhesion molecules and subsequently decrease leukocyte-endothelial cell interactions in vitro and in vivo.

Methods and Results

Cultured human coronary artery EC were pre-treated for various times with IL-19 (100 ng/ml) followed by stimulation with tumor necrosis factor-α (TNF-α; 10 ng/ml). IL-19 significantly reduced TNF-α-driven mRNA and protein abundance of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (p<0.01). Surprisingly, IL-19 did not inhibit TNF-α-activation of the transcription factor NFκB. ICAM-1 and VCAM-1 transcript stability was significantly reduced in IL-19-treated EC (p<0.05). IL-19 treatment inhibited cytoplasmic localization of the mRNA stability factor HuR, and siRNA knock-down of HuR also decreased ICAM-1 and VCAM-1 transcript stability. IL-19 treatment of EC, but not treatment of monocytes, significantly reduced monocyte adhesion to EC monolayers (p<0.01). Leukocyte rolling and adhesion was quantitated in C57/B6 mice treated with a single systemic dose of IL-19 (10 ng/g) followed by TNF-α (400 ng) using intravital microscopy. IL-19 treatment resulted in a significant decrease (p<0.05) in TNF-α-driven leukocyte rolling and adhesion in vivo. Systemic administration of IL-19 (1.0ng/g/day) reduced CD68-positive cell infiltration into atherosclerotic plaque in LDLR
-/-
mice fed an atherogenic diet for 14 weeks (P<0.01).

Conclusion

These data are the first report that IL-19 can reduce leukocyte-endothelial cell interactions in vitro and in vivo, and suggest a reduction in HuR-mediated mRNA stability of ICAM-1 and VCAM-1 as a mechanism. These results suggest that IL-19 is an important negative regulator of vascular inflammatory diseases.
Abstract 83: Molecular Mechanism of Aortic Aneurysm Formation in Fibulin-4--Deficient Mice
Authors: Yamashiro Yoshito , Huang Jianbin , Mirzaei Hamid , Yanagisawa Hiromi
Published: March 2022

Background:
Fibulin-4 (Fbln4) is a secreted extracellular matrix protein, which is expressed in blood vessels and associated with microfibrils surrounding elastic fibers. Fbln4-null mice exhibit perinatal lethality with rupture of aortic aneurysm and a marked disruption of elastic fibers, and these phenotypes were recapitulated by smooth muscle cell (SMC)-specific deletion of Fbln4 in vivo (Fbln4SMKO). Severe aneurysms developed exclusively in the ascending aorta at postnatal day 90 (P90) and vascular SMCs were hyper-proliferative and failed to fully differentiate in the Fbln4SMKO aorta.

Objective:
To elucidate the molecular mechanism that initiates the aneurysmal change in Fbln4SMKO mice, we performed proteomics analysis of Fbln4-deficient (Fbln4-/-) SMCs and compared with wild-type SMCs.

Methods:
The early stage of ascending aneurysm was evaluated in Fbln4SMKO mice using microscopic methods and immunohistochemistry. The protein expression profiles were obtained from postnatal day (P) 7 wild-type and Fbln4
-/-
SMCs by two-dimensional fluorescence difference gel electrophoresis. Several differentially expressed proteins were identified by mass spectrometry and validated using qPCR and Western blot analysis.

Results:
Macroscopic observation revealed that the ascending aorta dilatation was not observed at P7 but became detectable at P14. Histologically, however, focal lesion of SMC proliferation was already noticeable in the sub-endothelial region at P7 and became evident at P14 with a thickened aortic media. Differential proteomic analysis showed that molecules involved in actin dynamics, including gelsolin, ezrin/radixin/moesin (ERM) protein, cofilin-2, and Rho GDI were upregulated in Fbln4
-/-
SMCs. In addition, phosphorylation of cofilin and ERM was increased in Fbln4
-/-
SMCs. Interestingly, SMC differentiation markers were normally expressed in Fbln4SMKO aortae until P7.

Conclusion:
Our study indicated that the phenotypic change of SMCs occurred after P7 in the Fbln4SMKO aorta and Fbln4 deficiency contributed to aneurysm formation by facilitating the alteration in actin dynamics. This information will shed light on the molecular mechanism of aneurysm initiation in the Fbln4 deficient SMCs.
Abstract 86: Apo(a) Inhibits Plasminogen-Dependent Abdominal Aortic Aneurysm Formation in Transgenic Mice
Authors: Huang Menggui , Gong Yanqing , Grondolsky Jessica , Zhao Nan , Hoover-Plow Jane L
Published: March 2022

Abdominal Aortic Aneurysm (AAA) is a lethal disorder found primarily in adults over 65. Unlike coronary heart disease, the incidence of AAA is rapidly increasing, and currently there is no nonsurgical treatment. AAA is a chronic inflammatory disease that includes early leukocyte infiltration, destruction of the elastic lamina and extracellular matrix, expansion of the aorta, and eventually fatal aorta rupture. Components of the plasminogen (Plg) and MMP pathways that mediate leukocyte infiltration are prominent in AAA. The goal of this study is to investigate regulators of these pathways in AAA, namely the inhibition Plg by apo(a). In AAA patients, the association of elevated Lp(a) and progression of AAA is controversial. Apolipoprotein(a) [apo(a)], a component of lipoprotein(a) [Lp(a)], has a high homology to Plg, and interferes with Plg functions in vitro. In our previous studies apo(a), independent of Plg, inhibited neutrophil recruitment in a peritonitis model of inflammation, and in Plg deficient mice macrophage infiltration was impaired in CaCl2 induced AAA formation. Our hypothesis is that apo(a) inhibits AAA formation by reducing neutrophil or macrophage infiltration by interfering with Plg. To test our hypothesis, the CaCl2 induced AAA model was performed in 5 strains of mice: WT; Plg-/-; apo(a)tg; apo(a)tg:Plg-/-; and Lp(a)tg mice (6-14 mice analyzed per genotype). Three weeks after injury, the WT aorta diameter increased from 0.54 to 0.88mm (62%). The diameter changed in WT 0.31±0.11mm, apo(a)tg 0.06±0.02mm, apo(a):Plg
-/-
0.39±0.07mm and Lp(a) 0.08±0.07mm. The dilation was significantly lower in apo(a) and Lp(a) mice compared to WT (P<0.001). The H&E, Trichrome and EVG stain showed elevated leukocyte infiltration in WT and apo(a):Plg
-/-
injured aorta, accompanied by thining of the media layer and elastic lamellae fragmentation. In contrast, the aorta of apo(a) mice remained preserved. Neutrophils and macrophages were decreased in apo(a) mice compared to WT(P<0.05). In apo(a):Plg
-/-
mice, only neutrophil infiltration was decreased not macrophages. These results suggest that inhibition of neutrophils in apo(a) mice is independent of Plg, but reduced macrophage infiltration in apo(a) mice is Plg-dependent during AAA formation.
Abstract 107: Impaired Lipid Metabolism and Enhanced Atherosclerosis in Clock Mutant Mice
Authors: Pan Xiaoyue , Jiang Xian-Cheng , Hussain M. Mahmood
Published: March 2022

Clock is a key transcription factor that controls circadian regulation. Here, we show that dominant-negative Clock mutant protein (Clock
Δ19/Δ19
) enhances hyperlipidemia and atherosclerosis in

Apoe
-/-

mice.

Clk
[[Unable to Display Character: ∆]]19/[[Unable to Display Character: ∆]]19
Apoe
-/-

mice display hypercholesterolemia, show enhanced cholesterol absorption and express more NPC1L1, ACAT2 and MTP in the intestine. Macrophages from

Clk
[[Unable to Display Character: ∆]]19/[[Unable to Display Character: ∆]]19
Apoe
-/-

mice express high levels of scavenger receptors and take up more modified lipoproteins compared to

Apoe
-/-

mice; but they express low levels of ABCA1 and are defective in cholesterol efflux. Molecular studies reveal that Clock regulates ABCA1 expression in macrophages by modulating USF2 expression. Thus, Clock
Δ19/Δ19
protein enhances atherosclerosis by increasing intestinal cholesterol absorption by enterocytes, augmenting uptake of modified lipoproteins by macrophages and reducing cholesterol efflux from macrophages. These studies establish that circadian Clock plays a pivotal role in the regulation of cholesterol metabolism and atherosclerosis.
Abstract 11: T Cell Functions in a Novel Antigen-specific Experimentally-induced Model of Atherosclerosis
Authors: Tse Harley Y , Welch Kathryn , Van Dusen Hillary , Zhao Xiaoqing , Thummel Ryan , Eitzman Daniel T , Tse Kevin Y , Ley Klaus , Shaw Michael K
Published: March 2022

Introduction

While it is realized that inflammation plays a key role in the development of atherosclerosis, the underlying mechanism of how T cells, directly or indirectly, trigger and propagate the disease process, is not forthcoming. This is because there is no knowledge of what constitutes an atherogenic T cell and how to isolate it. New and innovative ideas are needed.

Hypothesis

We reason that T cells are antigen specific and identification of the atherogenic antigenic epitopes of an autoantigen should lead us to identification of the atherogenic T cells themselves.

Methods

Since the T cell receptor only recognizes a short peptide of the antigen in association with major histocompatibility complex (MHC) proteins, our focus was directed towards elucidating the peptides that form a ligand with MHC class II proteins and bind to atherogenic T cells. It has been known that oxidized low-density lipoprotein, ApoB100, heat-shock protein 60 and β2 glycoprotein I are the major atherogenic antigens. Because different MHC haplotypes have different binding motifs of peptide sequences and most atherosclerosis experiments are carried out in the H-2
b
ApoE
-/-
mice, we searched for H-2
b
binding motifs along the entire amino acid sequence of murine ApoB100 according to the prediction model of Liu et al.
ApoE

-/-

mice fed Western diet (WD) for 8 weeks were immunized with the identified peptides in CFA. Mice were boosted in 2 weeks and 3 weeks later were sacrificed and the aortic trees were isolated. The extent of atherosclerosis was assessed via en face staining of the aortic samples with Oil Red O.

Results

We identified eighteen such peptides, six of which were selected for synthesis. Two of the peptides, peptide6 (P6) and peptide3 (P3) induced strong proliferative responses in immunized mice, but only P6 was atherogenic in enhancing development of atherosclerosis in WD-fed and P6-immunized mice. The proliferative T cells were of the CD4 type, producing IL-17, IFNγ as well as IL-10 and IL-13. T cell clones were derived from P6-primed mice and adoptive transfer of these clones into WD-fed recipients also showed enhanced atherosclerosis.

Conclusions

Our identification of a T cell epitope of ApoB100 allows cloning of a population of peptide-specific atherogenic T cells.
Abstract 113: Streptococcal Serum Opacity Factor Activity Diverts Human High Density Lipoprotein (HDL)-Cholesteryl Esters (CE) to the LDL-Receptor Pathway in Human Huh7 Hepatocytes
Authors: Fields David W , Gillard Baiba K , Courtney Harry S , Gotto Antonio M , Pownall Henry J
Published: March 2022

Plasma concentrations of HDL-cholesterol (C) are negatively correlated with atherosclerotic cardiovascular disease. Nevertheless, current evidence suggests that this correlation is not axiomatic and that some forms of HDL are dysfunctional and atherogenic. Moreover, clinical trials of interventions that raise HDL-C have not been uniformly successful suggesting that mechanisms by which HDL-C is increased determine its anti atherogenic potency. Additionally, mice overexpressing the HDL receptor, SR-BI, have lower plasma HDL-C concentrations and less atherosclerosis. Thus, new strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. Serum opacity factor (SOF) disrupts HDL and forms three products_lipid-free apo A-I, a CE-depleted remnant neo HDL, and a cholesteryl ester-rich microemulsion (CERM) containing apo E and the CE of up to ~400,000 HDL particles. Hepatic CE uptake in Huh7 and HepG2 cells is faster when delivered by CERM than from the parent HDL, and cleared
in vitro
, in part, via the LDL-receptor (LDLR). We investigated the therapeutic potential of SOF in promoting RCT by comparing the uptake of HDL-, LDL- and CERM-CE in the final RCT steps_hepatocyte uptake, CE metabolism to cholesterol and bile salts, and their secretion and transfer to bile. HDL and LDL with [
14
C]CE were labeled with [
14
C]CE by incubation with lipoprotein deficient serum and the lipoproteins recovered by ultracentrifugation. CERM-labeled with [
14
C]CE was obtained from SOF activity vs. HDL labeled with [
14
C]CE. Incorporation efficiencies for HDL and LDL were 67% and 52% while [
14
C]CE specific activities were 3.93 x 10
-4
and 3.04 x 10
-4
mCi/mg CE, respectively. [
14
C]Metabolites from the hepatic uptake of HDL, LDL, and CERM were quantified by a two-solvent TLC system that resolves CE, C, and the major bile salts. The [
14
C]CE of all three particles was taken up by cells and converted to free cholesterol with half-times of 2.7, 3.8, and 3.7 h respectively from HDL, CERM and LDL. Rates of bile salt synthesis and secretion are currently being investigated. These data show that SOF diverts potentially toxic HDL-CE to the hepatic LDLR pathway where it is hydrolyzed.
Abstract 114: Correlation of Specific HDL Subspecies with Arterial Stiffness in Youth with Type 2 Diabetes
Authors: Gordon Scott M , Lu Jason , Dolan Lawrence , Urbina Elaine , Shah Amy , Davidson W. Sean
Published: March 2022

Human epidemiological studies demonstrate that increased HDL cholesterol (HDL-C) concentrations are associated with a decreased risk of cardiovascular disease. However, recent drug therapies designed to raise plasma HDL-C concentrations have failed to reduce cardiovascular events. These findings suggest that the cardio-protective effects of HDL may lie in specific HDL subspecies and not in the HDL-C number. Thus, we sought to define the composition of HDL in adolescents with T2D and determine whether specific HDL subspecies are associated with early markers of arterial disease.

Youth with T2D (n=10) and two control groups lean (n=9) and obese (n=11) adolescents were recruited in a cross sectional study. Plasma was separated into 18 fractions using gel filtration chromatography. Lipid associated proteins were then isolated and identified using mass spectrometry. Concurrently, arterial thickness and stiffness measures including common/bulb/internal carotid intima media thickness (IMT) and pulse wave velocity (PWV) were measured.

Compared to lean and obese controls, youth with T2D exhibited decreased phospholipid content in fractions containing large HDL subspecies that was inversely associated with pulse wave velocity (p<0.001). No association between HDL-C and pulse wave velocity was seen. Proteomic analysis of the HDL sized fractions demonstrated decreases in 17 of 45 identified proteins in the T2D group compared to lean youth. The most striking protein decreases occurred in apolipoprotein (apo) A-1, apoC-I, apoE and paraoxonase-1 (p<0.05) in fractions associated with large HDL particles.

Our data demonstrate early changes in the lipid and protein compositions of specific HDL subspecies in adolescents with T2D that are related to early markers of arterial disease. These findings suggest that analyzing the composition of HDL, rather than HDL-C, may be useful in assessing cardiovascular risk in this population.
Abstract 117: Does Lipoprotein (a) Impact the Classification of Intermediate to High Risk Patients using Reynold’s Risk Score
Authors: Low Li Shien , Gout Marcel , Siracusa Mary , Zenebe Wondwossen , Parikh Devang , Kazlauskaite Rasa , Lundberg Gina , Doukky Rami , Braun Lynne , Volgman Annabelle
Published: March 2022

Introduction

Elevated lipoprotein (a) (Lpa) has been linked repeatedly with cardiovascular disease (CVD) events. Two models of the Reynold’s Risk Score (RRS) A & B were validated to be effective predictors of CVD events. Lpa was included in RRS A but was excluded in RRS B. We sought to determine the proportion of patients (pts) that are reclassified into a different risk category utilizing RRS A instead of the simplified RRS B.

Methods

Female pts with Lpa and data for RRS seen from 2006-12 at the Heart Centers for Women at RUMC and Emory Healthcare were included. Charts were reviewed for pt characteristics and biomarkers using Berkeley Heart Lab including: Total, LDL, and HDL cholesterol, triglycerides, ApoB, Lpa, HS-CRP, HgB A
1
c and history of hypertension, diabetes, current smoking, family history and blood pressure. HDL was substituted for ApoA
1
in all cases in RRS A. RRS A and B were calculated. The proportions of pts within different risk strata were compared between the two models.

Results

Of 604 women, 502 of them age 45 to 80 years old were included in this analysis, because RRS has been validated only in this age group. The table summarizes that a high proportion of women were reclassified into different risk strata using RRS B score.

Discussion

The addition of Lpa reclassifies up to 81% of pts’ risk categories. Given that model A was the better fitting model, we propose the incorporation of Lpa in women whose risk is > 5% as determined by the simplified RRS B. Treatment implications may impact cardiovascular outcomes.
Abstract 12: Targeted Deletion of I?B kinase ß Protects Mice from Obesity and Atherosclerosis
Authors: Sui Yipeng , Park Se Hyung , Helsley Robert N , Rios-Pilier Jennifer , Zhou Changcheng
Published: March 2022

Vascular inflammation plays an essential role in the initiation and progression of atherosclerosis. IκB kinase β (IKKβ), a central coordinator of inflammatory responses through activation of NF-κB, has been implicated in vascular pathologies, but its cell-specific role in atherogenesis remains elusive. Here we show that IKKβ functions in smooth muscle cells to regulate vascular inflammatory responses and atherosclerosis development. Targeted deletion of IKKβ in smooth muscles cells protected LDL receptor-deficient mice from vascular inflammation and atherosclerosis induced by high-fat feeding. Unexpectedly, IKKβ-deficient mice were also protected against diet-induced obesity and associated metabolic disorders. Cell lineage analysis revealed that adipose mesenchymal stem cells express multiple smooth muscle cell markers. Ablation of IKKβ inhibited adipose stromal vascular cell differentiation, leading to a decreased number of mature adipocytes and an accumulation of adipocyte progenitor cells in the white adipose tissue. Furthermore, short hairpin RNA-mediated reduction of IKKβ expression or pharmacological inhibition of IKKβ activity in preadipocytes reduced the expression of adipogenic genes and diminished the ability of these cells to differentiate in vitro. These results demonstrate a pivot role of IKKβ in linking vascular inflammation with adipose development and atherosclerosis.
Abstract 129: Investigation of Atherosclerosis in Association with Arthritic Inflammation
Authors: Bäcklund Alexandra , Ria Massimiliano , Chernogubova Ekaterina , Ketelhuth Daniel FJ , Holmdahl Rikard , Hansson Göran K. , Hamsten Anders
Published: March 2022

Background and Aims

It is well established that patients with Rheumatoid Arthritis (RA) have a higher risk of developing coronary artery disease (CAD). However, it is unclear how the inflammatory process affects atherosclerosis and what specific factors are involved. In this study, we investigated the effect of the pro-atherogenic factor hyperlipidaemia on arthritis incidence and severity. Simultaneously, we have investigated the mutation of the neutrophil cytosolic factor 1 (Ncf-1), a known genetic factor that promotes arthritis susceptibility, and MHCII H-2q on atherosclerosis development.

Results

We observed that diet-induced hyperlipidaemia prior to induction of collagen induced arthritis (CIA) protected mice against the disease while genetically hyperlipidemic without the use of diet, Ldlr-/- x human ApoB100 transgenic (Ldlr-/-hApoBtg), were equally susceptible to CIA compared to their heterozygous littermates. Next, we investigated Ncf-1 mutation on Ldlr-/-hApoBtg mice. Surprisingly, despite the increased inflammatory/arthritic phenotype induced by Ncf-1 mutation, no difference in the atherosclerotic lesion size was observed.

Conclusions

Our data shows that hyperlipidaemia-induced by diet have substantial effects on arthritis susceptibility, which in turn differs from hyperlipidaemia acquired on birth due to genetic alterations. By developing a model carrying pro-arthritogenic factors, MHCII H-2q and Ncf-1 mutation, as well as the pro-atherosclerotic factors, Ldlr-/- and hApoBtg, we have established a new model where both arthritis and atherosclerosis are present. These mice will be used to further characterize the impact of systemic inflammation in the form of arthritis in the atherosclerotic process and vice versa.
Abstract 130: Platelet Rap1 Signaling, Mediated by CalDAG-GEFI and P2Y12, Contributes to Atherosclerotic Lesion Development in Mice
Authors: Boulaftali Yacine , Owens A.Phillip , Beale Ashley , Piatt Raymond , Andre Patrick , Conley Pamela B. , Mackman Nigel , Bergmeier Wolfgang
Published: March 2022

Background and Objective

The development and progression of atherosclerotic lesions is mediated by various blood cell types, including platelets and neutrophils. We recently identified CalDAG-GEFI (CDGI) as a critical component of integrin-mediated adhesion of platelets and neutrophils at sites of vascular injury. In platelets, signaling by CDGI mediates the early activation of Rap1, while receptor for ADP, P2Y12, the target of the clinically used drug Plavix, is required for sustained Rap1 activation and thrombus stability. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr-/-) mice lacking CDGI and/or P2Y12 in hematopoietic cells.

Methods and Results

Ldlr-/- mice were irradiated and reconstituted with bone-marrow from wild-type (WT), CalDAG-GEFI-/- (CdgI), P2y12-/-, or CdgI-/- P2y12-/- (DKO) mice. Reconstituted mice were fed a fat and cholesterol enriched diet (21% fat, 0.2% cholesterol) for 12 weeks, ad libitum. Atherosclerotic lesions in the aortic sinus of Ldlr-/-;CdgI-/- chimeras were 42% smaller than those in Ldlr-/-;WT controls (0.18 ± 0.02 mm2 vs 0.31 ± 0.05 mm2, respectively; n = 13 each group, p < 0.001). Lesions in Ldlr-/-;P2y12-/- chimeras were also significantly smaller compared to controls (0.22 ± 0.10 mm2; n = 13, p < 0.05). Lesions in Ldlr-/-;DKO were reduced by 48% compared to WT controls (0.16 ± 0.02 mm2; n = 13, p < 0.001 ), but they were not statistically different from Ldlr-/-;CdgI-/- chimeras. Platelet adhesion and activation on collagen under flow was markedly impaired in Ldlr-/-;CdgI-/- and Ldlr-/-;DKO blood, but only partially impaired in Ldlr-/-;P2y12-/- blood. Importantly, firm neutrophil adhesion to collagen-bound platelets was significantly reduced in Ldlr-/-;CdgI-/- and Ldlr-/-;DKO blood, but not in Ldlr-/-;P2y12-/- blood. Total cholesterol and triglyceride levels were similar among groups.

Conclusion

Our findings reveal a critical role for CDGI and P2Y12, and Rap1-dependent platelet activation, in promoting atherosclerotic lesion development in hypercholesterolemic mice. Further studies are required to determine if the observed protection in Ldlr-/-;CdgI-/- chimeras is due to impaired neutrophil function.
Abstract 131: Alphavbeta3 Integrins Mediate Shear Stress-Induced Inflammation and Early Atherogenesis
Authors: Chen Jie , Green Jonette , McInnis Marshall , Traylor James G. , Mazar Andrew P. , Orr A. Wayne
Published: March 2022

Rational

The frictional shear stress generated by blood flow activates integrins on the endothelial cell surface, and signaling downstream of newly activated integrins regulates several flow-induced responses including inflammation and permeability. Signaling through transitional matrix binding integrins (α5β1, αvβ3 and αvβ5) drives shear stress-induced NF-κB activation and proinflammatory gene expression; however the specific contribution of each of these integrins remains unclear.

Objective

To address which transitional matrix-binding integrin mediates shear stress-induced endothelial cell.

Methods and Results

We now show that the small molecule αv integrin inhibitor S247, but not the α5 inhibitor ATN-161, abolished flow-induced proinflammatory signaling (PAK, NF-κB). Blocking αv expression using siRNA similarly prevents flow-induced activation of PAK and NF-κB, whereas siRNA-mediated knock-down of α5 did not. Furthermore, αv and β3 knockdown but not α5 and β5 knockdown inhibited flow-induced ICAM-1 and VCAM-1 expression (both mRNA and protein) following either short term (5 hr) laminar and long term oscillatory (24 hr) shear stress, indicating αvβ3 is the mediator for flow induced endothelial cell inflammation. In contrast, αv knockdown did not affect TNFα-induced ICAM-1 and VCAM-1 expression, suggesting the anti-inflammatory effect of αv inhibition is specific to shear stress. Integrin inhibition did not affect all shear stress responses, as neither α5 nor αv siRNA affected shear stress-induced extracellular-signal regulated kinase (ERK1/2) activation. Lastly, inhibiting αv integrins using the small molecule inhibitor S247 reduces atherosclerotic plaque formation in ApoE knockout mice.

Conclusion

Taken together, these studies suggest that αvβ3 integrins mediate shear stress-induced proinflammatory responses associated with the transitional matrix
in vitro
and early atherosclerotic plaque formation
in vivo
.
Abstract 136: Quercetin Intake With Exercise Modulate Adipose Response in Mice Fed Atherogenic Diet
Authors: Garelnabi Mahdi , Mahini Halleh , Massie Melissa , Cook Carla , Santanam Nalini
Published: March 2022

Adipose tissue is shown to influence atherosclerotic plaque formation and contribute to cardiovascular disease. Recent studies have focused on the potential role of adipose tissue physiopathology on several metabolic diseases.

Aims and Objectives

In the current study we hypothesized that the combined action of quercetin and exercise will promote the releases of adipose anti-atherogenic metabolites and contribute to the reduction of the plaque formation.

Study Design

C57BL6 LDL -/- mice fed atherogenic diet (40) was divided into four groups (10 each). These groups are as follows: Control mice, left untreated; control quercetin group, orally supplied with 100 μg/day of quercetin without exercising; exercise group without quercetin, and exercise group with quercetin supplements. The exercise groups were run on a treadmill for 30 minutes, 15m/m/ 5 days/week for 30 days. All animals were on atherogenic diet. At the end of the month of treatment, mice were sacrificed, and adipose tissue gene expressions for selected genes were analyzed.

Results and Conclusion

We previously reported that mice on quercetin and exercise have significant (P<0.001) drop in body weigh accompanied with a 78% drop in the atherosclerotic plaque development in exercising groups. In the current abstract we investigated whether the drop in weight is mediated through an adipose tissue based mechanism. The adipose gene expression studies have shown significant (P<0.0001) up regulation of PPARg in exercised mice compared to the control and quercetin fed group, and PPARg also increased with exercise and quercetin intake although these changes were not significant. Adiponectin on the other hand elevated with exercise or quercetin intake alone and there was no appreciable changes with the combination of exercise and quercetin intake compared to control. Exercise; quercetin or the combination increased SREBP-1; however, interestingly quercetin intake with exercise significantly increased leptin up regulation. Circulating leptin levels are known to correlate with white adipose tissue mass. This finding brings an amazing perspective to the role of antioxidants taken with exercise which need further investigation to establish its mechanism(s) that modulate adipose metabolism.
Abstract 138: Treatment of Atherosclerosis by Systemic Protease Inhibition
Authors: Hu Jie Hong , Touch Phanith , Zhang Jingwan , Liu Shihui , Lund Ida K , Høyer-Hansen Gunilla , Dichek David A
Published: March 2022

Background

Elevated activity of urokinase plasminogen activator (uPA) and matrix metalloproteases (MMPs) in human arteries is associated with accelerated atherosclerosis (athero), aneurysms, and plaque rupture.
Apoe
-null mice with macrophage-targeted uPA overexpression (SR-uPA mice) have elevated vascular uPA and MMP activity, accelerated athero, dilated aortic roots, and increased histologic features of plaque rupture. SR-uPA mice are therefore a useful experimental setting for beginning to investigate whether inhibition of uPA or MMPs can prevent athero progression, aortic dilation, and plaque rupture.

Hypothesis

Treatment with a murine monoclonal antibody inhibiting mouse uPA (mU1), or a limited-spectrum synthetic MMP inhibitor (XL784) will reduce athero in SR-uPA mice.

Methods

SR-uPA mice were fed a high-fat diet for 10 wk beginning at 5 - 6 wk of age. Fat-fed mice were injected with mU1 or control antibody twice per wk (for 10 wk), or were given either XL784 (125, 250, or 500 mg/kg) or vehicle by daily oral gavage. Mice were then anesthetized, formalin-perfused, and athero was measured in isolated aortic roots and pinned aortas. Groups were compared by t-test and ANOVA.

Results

None of the treatments altered peripheral blood monocyte counts or plasma cholesterol. mU1 reduced aortic root intimal lesion area by 20% (
P
< 0.05) and aortic root circumference by 12% (
P
= 0.01). All 3 doses of XL784 significantly decreased oil-red O-positive lesion area (36 - 42%;
P
< 0.05 for all). All XL784 doses also reduced aortic root intimal lesion area (22 - 29%;
P
< 0.05 for low dose only). The highest XL784 dose also reduced total macrophage-positive lesion area (33%;
P
< 0.05). All XL784 doses yielded trends towards decreased aortic root circumference (5 - 10%). Neither mU1 nor XL784 significantly altered percent aortic surface lesion coverage.

Conclusions

In SR-uPA mice, pharmacologic inhibition of either uPA or selected MMPs significantly decreased aortic root athero and limited aortic dilation. Differential effects of both agents on aortic root versus surface athero suggest prevention of athero progression rather than initiation. These approaches to protease inhibition show promise for preventing the progression and complications of established athero.
Abstract 145: Development of Radiolabeled Liposomes for Atherosclerotic Plaque Imaging
Authors: Ogawa Mikako , Umeda Izumi O , Kosugi Mutsumi , Kawai Ayumi , Magata Yasuhiro
Published: March 2022

Objectives

Molecular imaging enables non-invasive
in
vivo
detection of vulnerable plaques. Macrophage infiltration is characteristics for atherosclerotic vulnerable plaques, and many apoptotic cells are seen in this region. Also, it is known that macrophages recognize phosphatidylserine (PS) that is exposed on the cell surface during apoptosis to phagocytize these cells. In this study, we prepared radiolabeled phosphatidylserine liposomes for detection of vulnerable plaques.

Methods

PS liposomes and control liposomes (phosphatidylcholine (PC) liposomes) were prepared by lipid film hydration. The prepared sizes were 100 nm and 200 nm (PS100, PS200, PC100, and PC200 liposomes). The liposomes were radiolabeled by encapsulating
111
In-NTA using active-loading method. The
111
In-liposomes were incubated with mouse peritoneum macrophages for 2hr and uptake level was investigated. For biodistribution study,
111
In-liposomes were intravenously injected to ddY mice. Also, the
111
In-liposomes were injected to apoE -/- mice, and the aortas were harvested for autoradiography and Oil-Red O staining. For SPECT imaging, WHHL rabbits were used.

Results

The
in
vitro
uptake levels to macrophages were 60.5, 14.7, 32.0, 14.4 %dose/mg protein, for
111
In-PS100,
111
In-PC100,
111
In-PS200,
111
In-PC200, respectively. The liver uptake was high in all liposomes, and blood clearance was faster in PS liposomes than PC liposomes. The
in
vivo
distributions of
111
In-labeled PS liposomes to atherosclerotic regions were well matched with Oil-Red O staining in apoE -/- mouse. Target-to-non-target ratio was highest in
111
In-PS200. The aorta was successfully visualized by SPECT at 48hr after the
111
In-labeled PS liposome injection.

Conclusions

By
in
vitro
uptake study, it is revealed that macrophage targeting was accomplished by PS. Also, atherosclerotic region was successfully detected by
111
In-PS200 in apoE-/- mice and WHHL rabbits
in
vivo
.
Abstract 147: Coordinate Regulation of Chemo-attractive And -repulsive Guidance Cues Instructs Leukocyte-endothelial Interactions in Atherosclerosis
Authors: Ramkhelawon Bhama , van Gils Janine , Fernandes Luciana , Stewart Merran , Guo Liang , Menezes Gustavo , Cara Denise , Chow Camille , Kinane Bernard , Fisher Edward A , Balcells Mercedes , Alvarez-Leite Jacqueline , Moore Kathryn J
Published: March 2022

Leukocyte influx into the arterial wall plays a critical role in the development and progression of atherosclerosis. Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues plays a role in regulating leukocyte trafficking into the vascular wall during atherogenesis. Gene expression profiling revealed that members of the Netrin, Semaphorin and Ephrin family of guidance molecules are differentially regulated in athero-prone and athero-protected regions of the aortae of Ldlr-/- mice. Netrin-1 and Semaphorin 3A are highly expressed in the outer curvature of the aorta, and potently inhibit chemokine-directed migration of monocytes in vitro. Endothelial expression of these negative guidance cues is down-regulated by pro-atherogenic factors associated with monocyte entry into the vessel wall, including oscillatory shear stress and pro-inflammatory cytokines. Furthermore, using intravital microscopy we show that in vivo delivery of Netrin-1- or Semaphorin3A-blocking peptides increases leukocyte adhesion to the endothelium ~2-fold compared to the control peptides. Conversely, we show that a guidance cue that is highly expressed in atheroprone regions of the aorta, EphrinB2, is up-regulated in endothelium cells under pro-atherosclerotic flow conditions (eg. oscillatory shear stress, pro-inflammatory cytokines) and functions as a monocyte chemoattractant. These data suggest a new paradigm for leukocyte-endothelial interactions in which the coordinate regulation of chemo-attractive and -repulsive guidance cues regulates leukocyte trafficking in health and disease.
Abstract 148: AGES, Receptor for Advanced Glycation End Products (RAGE), Reverse Transmigration of Macrophages & Polarization
Authors: Senatus Laura M , Shen Xiaoping , Zou Yu Shan , Li Huilin , Daffu Gurdip , Rosario Rosa , Schmidt Ann Marie
Published: March 2022

Cellular migration is fundamental processes linked to diverse pathological states including atherosclerosis and diabetes. RAGE is a multiligand cell surface macromolecule, which binds ligands such as advanced glycation endproducts (AGEs) in diseased settings such as atherosclerosis. We tested the hypothesis that RAGE contributes to impaired regression of diabetic atherosclerosis by suppression of reverse transmigration of macrophages in an AGE-enriched environment. We performed our studies in vitro, a reverse transmigration assay, using primary murine aortic endothelial cells from wild-type C57BL6 mice and wild-type or RAGE deficient primary bone marrow derived macrophages (BMDMs). Our data is shown in the table.

We evaluate if the expression of RAGE modulates influx and/or egress of macrophages from the atherosclerotic lesions in the non-diabetic and diabetic states. We used a monocyte bead-tracking model in an aorta transplantation study with LDL-/-diabetic mice fed high-fat diet as a baseline (donors), and non-diabetic RAGE-/- or WT mice as recipient. Migration patterns evaluated by labeled monocytes, showed a decrease in bead number in RKO recipients (23±0.05 p<0.05) compared to WT (30±0.05 p<0.05) indicating emigration of monocyte- derived cells from the plaque in the diabetic state.

We also tested if RAGE plays roles in macrophage polarization. AGEs-RAGE axis increases M1 polarization (MCP 1, TNF alpha, iNos, p<0.05) and suppresses M2 polarization (Arg1, IL-10, p<0.05) in a manner prevented by deletion of RAGE in BMDMs. Our studies suggest AGEs-RAGE axis action delays reverse transmigration of macrophages and augments pro-inflammatory gene expression.
Abstract 151: Thrombosis Prone Plaques Exhibit Inflammation Prior to Rupture: Intravascular Detection via Macrophage-targeted Optical Imaging
Authors: Stein Ashley F , Hara Tetsuya , McCarthy Jason R , Mauskapf Adam , Ntziachristos Vasilis , Jaffer Farouc A
Published: March 2022

Introduction

Inflammation modulates atherogenesis and the destabilization of plaques. However, the role of macrophages in plaque rupture is incompletely understood. Here we investigated the in vivo spatial distribution of plaque macrophages prior to triggered plaque rupture, using intravascular molecular imaging and a near-infrared fluorescence (NIRF) macrophage targeted agent, CLIO-CyAm7. We hypothesized that CLIO-CyAm7 would illuminate macrophages in vivo and preferentially localize to plaques that are vulnerable to plaque rupture.

Methods

Atherosclerosis was induced in rabbits (n=14) using a 12-week hyperlipidemic diet with alternating 1% high cholesterol and normal chow, concomitant with aortic balloon injury at 2 weeks. Rabbits were injected with 2.5mg/kg (n=8) or 5mg/kg (n=6) of CLIO-CyAm7 at 24 or 48 hours, respectively, prior to thrombosis triggering using Russell’s Viper Venom and histamine over 48 hours. The 2.5mg/kg subgroup was imaged in vivo using NIRF imaging and intravascular ultrasound (IVUS), immediately prior to thrombosis triggering. After sacrifice, ex vivo imaging, fluorescence microscopy (FM), Carstairs and Ram-11 immunostaining were performed.

Results

FM identified significantly higher CLIO-CyAm7 signal in areas of atherosclerosis compared to normal segments of the aorta (2.5mg/kg and 5.0mg/kg, p=0.03 and p=0.03). This signal was primarily localized at the intimal-luminal border of atheroma, with some penetration in the media and adventitia. CLIO-CyAm7 colocalized with a subset of superficial macrophages. Carstairs staining identified fibrin-rich thrombi, indicating plaque rupture in 6 of 14 rabbits (43%). There was increased CLIO-CyAm7 in areas underlying thrombus and particularly at the shoulder of ruptured plaques. Plaque macrophages were detectable via
in vivo
intravascular NIRF imaging. Areas of atherosclerosis, determined by IVUS, showed significantly higher NIRF peak signal-to-noise ratio than normal segments of the aorta (40.2±16.9 and 5.9±1.9, p=0.02).

Conclusion

Macrophages are consistently present in plaques that undergo triggered rupture, and can be detected via CLIO-CyAm7 intravascular NIRF molecular imaging. In vivo macrophage imaging may identify thrombosis-prone plaques.
Abstract 16: Low Doses of Ionizing Radiation Promote Therapeutic Revascularization in Ischemic Tissues
Authors: Ministro Augusto , Nunes Raquel J , Oliveira Paula , Rocha André , Grillo Isabel , Dinis da Gama A. , Constantino Susana
Published: March 2022

Lower limb ischemia is a major health problem. Because of the absence of effective treatment in the advanced stages of the disease, amputation is undertaken, even though it is associated to morbidity and mortality. Therefore, novel therapeutic approaches are necessary, aiming at the formation of stable, well-organized and functional blood vessels in ischemic tissues.

Recently, we found that low doses of ionizing radiation (IR) promote angiogenesis and we assessed the hypothesis that they may be therapeutically useful in vascular occlusive disease.

By using a mouse model of hind limb ischemia we quantified the blood flow expressed as a ratio of ischemic to non-ischemic limb perfusion in irradiated and unirradiated mice at different times and our results suggest that, at 15 days post-ischemia, low-dose IR significantly increases perfusion recovery (p=0.00075; n=6 mice per group). Moreover, a statistically significant increase in the capillary density was noted in ischemic irradiated muscles when compared to ischemic unirradiated ones (p=0.00009; n=6 mice per group). On the contrary, no difference was observed when comparing non-ischemic muscles irradiated or not. These results are representative of at least four independent experiments. From an
in vitro
microarray data obtained in our lab, we selected the genes whose expression is significantly altered by low doses of IR and that represent the best candidates for a pro-angiogenic response. Their expression was evaluated in endothelial cells isolated from the gastrocnemius muscles from irradiated and unirradiated mice. A laser capture microdissection microscope was used followed by RNA extraction and cDNA synthesis. Several pro-angiogenic genes were preamplified before quantitative RT-PCR analyses. Our results suggest that low-dose IR induces the expression of VERGR1, VEGFR2, FGF2, TGFB2 and ANG2 in endothelial cells isolated from ischemic muscles.

In conclusion, our data have the potential to propose a new strategy for neovascularization using low doses of IR. The use of a hind limb ischemia model in larger animals will be fundamental to firmly establish the benefits and exclude any harmful effects, before considering low-dose IR as a valid tool to be used in contemporary human medicine.
Abstract 162: Exposure of Human Macrophages to Tobacco Smoke Induces Hmgb1 Release on Microvesicles That Cause Monocyte Recruitment and Impairment of Insulin Signaling in Adipocytes
Authors: Chen Yan , Li Guangping , Liu Yanxia , Williams Kevin , Liu Ming-Lin
Published: March 2022

Background_

Smokers show increased prevalence of insulin resistance and type 2 diabetes. The underlying mechanisms, however, remain poorly understood. We recently implicated a role for HMGB1, a damage-associated molecular pattern previously shown to be released as a soluble molecule. We reported that exposure of human macrophages to tobacco smoke extract (TSE) provokes the release of HMGB1 in two forms: as a soluble molecule and on membrane microvesicles (MV). The HMGB1 on TSE-induced MVs mediates crucial crosstalk with adipocytes, e.g., these particles stimulate MCP1 secretion by adipocytes and the recruitment of monocytes.

Aims_

To explore the mechanisms by which HMGB1 is released on MVs, and the harmful effects of the TSE-MVs on macrophage-adipocyte crosstalk.

Methods/Results_

To examine how HMGB1 is released on MVs, we found that exposure of human macrophages to TSE upregulated HMGB1 expression, induced its gradually translocation from the nucleus to the cytosol and plasma membrane, in a time-dependent manner, and then the release of HMGB1 both as a soluble molecule and on TSE-MVs. Inhibition of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), by using CRM1 inhibitor III, attenuated the translocation of HMGB1 from the nucleus and its export on microvesicles. To examine biological effects, we found that treatment of adipocytes with TSE-MVs inhibited insulin-induced phosphorylation of IRS1 (Y612) and Akt (S473). Co-incubation of adipocytes with TSE-MVs and human monocytes produced more severe inhibition of insulin signaling than either TSE-MVs or monocytes did alone. Most importantly, pretreatment of macrophages with CRM1 inhibitor III, before exposure to TSE, attenuated the ability of TSE-MVs released by these cells to impair insulin-induced phosphorylation of Akt (S473) in adipocytes.

Conclusion_

Our results indicate that exposure of human macrophages to smoke upregulates HMGB1 expression and causes its release on microvesicles, which mediate harmful crosstalk from macrophages to adipocytes, causing the MCP1 secretion by adipocytes, further recruitment of monocyte/macrophages, and impairments in insulin signaling that may contribute to insulin resistance and the development of T2DM in tobacco smoke exposure.
Abstract 166: Omega3-Rich Diet Modulates Leptin through Lipid Sensing G-Protein Coupled Receptors in Catalase Transgenic Mice
Authors: Santanam Nalini , Massie Melissa , Selbe Myriaha , Efaw Logan , Crain Courtney , Cook Carla , Tamski Holly
Published: March 2022

Diet induced obesity is a major risk factor for cardiometabolic diseases. Oxidative stress plays an important etiological role in these diseases. Hence, we hypothesized that increased antioxidant expression will prevent obesogenic events. We tested our hypothesis by feeding normal chow (nc), high fat diet (HF) and omega-3 rich diet (OM3) to C57Bl/6J mice or human catalase transgenic mice-[Tg(CAT)
+/0
] (antioxidant enzyme that detoxifies hydrogen peroxide) (n=8) for 6 weeks. Our results indicated no difference in food consumption among the groups, but a significant increase in both body weight and adipose weight on HF fed diet, but not with OM3 diet in Tg(CAT)
+/0
mice compared to C57Bl/6J mice. There was a 140-fold induction in
leptin
mRNA expression in the adipose tissue even at basal levels in Tg(CAT)
+/0
mice compared to C57Bl/6J, which was further increased to 170-fold after HF diet. The diet rich in OM3 brought the
leptin
levels down by <80%. The fluctuations in leptin levels in Tg(CAT)
+/0
mice seems to be regulated by changes in lipid sensing PPARγ and G-protein coupled receptors (GPR120, GPR41/43). Our results suggest that increase in antioxidant enzyme catalase modulates leptin pathway. This modulation can be regulated by omega-3 rich diet. The physiological significance of these findings in relation to obesity will be the focus of our future studies.