bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 326: MicroRNA-30c Reduces Hyperlipidemia and Atherosclerosis by Decreasing Lipid Synthesis and Lipoprotein Secretion
Authors: Soh James , Iqbal Jahangir , Quieroz Joyce , Fernandez-Hernando Carlos , Hussain M Mahmood
Published: July 2021

Hyperlipidemia is a risk factor for various cardiovascular and metabolic disorders. Overproduction of lipoproteins, a process critically dependent on microsomal triglyceride transfer protein (MTP), can contribute to hyperlipidemia. We show that microRNA-30c (miR-30c) interacts with the 3’-untranslated region of the MTP mRNA and induces degradation leading to reductions in apolipoprotein B secretion in cells. Further, miR-30c reduces hyperlipidemia and atherosclerosis in Western diet fed mice by decreasing lipid biosynthesis as well as assembly and secretion of triglyceride-rich apoB-containing lipoproteins without increasing either hepatic lipids or plasma transaminases. Therefore, miR-30c coordinately reduces lipid biosynthesis and lipoprotein secretion to control cellular and plasma lipid levels and might be useful in treating hyperlipidemia and associated disorders.
Abstract 332: Deletion of ADAMTS7 in Mice Reveals a Role in Vascular Smooth Muscle Cell Response to Injury
Authors: Bauer Robert C , Paschos Georgios K , Yu Christopher , Parmacek Michael S , Rader Daniel J , Reilly Muredach P
Published: July 2021

The zinc metalloprotease ADAMTS7 was recently shown by GWAS to be a locus on chr15 for human coronary artery disease (CAD). However, the molecular mechanisms tying ADAMTS7 function to atherosclerosis are unknown. We hypothesized that ADAMTS7 may promote atherosclerosis by facilitating vascular smooth muscle cell (VSMC) proliferation and migration in vivo. We obtained mice containing an exon-trapping cassette between exon 4 and 5 of Adamts7, leading to premature termination of the mRNA and a grossly truncated protein. These mice bred normally and did not display any obvious phenotype. The exon trapping cassette also contained a LacZ reporter construct, allowing for XGal staining to determine gene expression. We assessed Adamts7 gene expression via XGal staining in multiple tissues of interest, including the heart, lungs, liver, and spleen. We also confirmed KO of the gene message through TaqMan rtPCR in additional tissues. We performed wire injury and sham surgeries of femoral arteries in adult Adamts7+/+ and
-/-
mice (N=4 and 5, respectively) and investigated lesion formation at 28 days. Adamts7
-/-
mice showed reduced neointima formation (64%), intima-to-media ratio (47%) and percent stenosis (61%) as compared to wild type. By immunohistochemistry and Trichrome staining we observed trends toward reduced VSMC proliferation (Ki67), reduced collagen IV and fibronectin matrix staining, and less disruption of laminin in ADAMTS7
-/-
injured arteries. Finally, ADAMTS7 immunofluorescence of human primary aortic SMCs revealed colocalization to cortactin domains at the leading edge of migrating SMCs as well as to cytoskeletal filaments. In summary, Adamts7 deletion attenuates the VSMC and matrix responses to arterial injury suggesting a role for ADAMTS7 in VSMC migration in atherosclerotic lesion initiation and progression.
Abstract 339: Impaired Periaortic Adipocyte Differentiation in Angiotensin AT1 Receptor-Deficient Mice: Possible Role in Proinflammatory Phenotypic Modulation of Perivascular Adipose Tissue
Authors: Irie Daisuke , Yamada Hiroyuki , Kato Taku , Kawahito Hiroyuki , Ikeda Kouji , Okigaki Mitsuhiko , Matsubara Hiroaki
Published: July 2021

[BACKGROUND]
The angiotensin II type 1 (AT1) receptor in visceral white adipose tissue (WAT) is closely implicated in lipid metabolism and energy homeostasis. Recently, perivascular adipose tissue (PVAT) has been shown to play a crucial role in the development of atherosclerosis; however, the effects of AT1 on PVAT properties and their functional relevance in atherogenesis remain undefined.

[METHOD AND RESULT]
We examined the fat depot-specific difference of adipose tissue among epididymal WAT, PVAT surrounding thoracic aorta, and interscapular brown adipose tissue (BAT) in 8-week-old apoE deficient (apoE-/-) mice. The expression levels of brown adipocyte marker genes (UCP-1, PGC-1α, Elovl3, PPARα, and Cidea) were significantly higher in BAT and PVAT compared with WAT (P<0.01). White adipocyte marker genes (Igfbp3, DPT, Tcf21, and Hoxc9), which were hardly expressed in BAT, showed a moderate expression levels in PVAT, suggesting that PVAT has a strikingly different phenotype from the classical WAT and BAT. We next examined the properties of PVAT in 8-week-old apoE-/-/AT1 receptor deficient (Agtr1-/-) mice. After 4 weeks of western diet, the expression levels of adipocyte differentiation maker genes (PPARγ, FABP4, c/EBPα) were markedly increased in apoE
-/-
PVAT (P<0.05), which was completely diminished in apoE-/-/Agtr1
-/-
PVAT (P<0.01). To investigate the effect of AT1 on the periaortic adipocyte differentiation, we performed primary culture of preadipocyte from stromal vascular fraction in Agtr1
-/-
and Agtr1+/+ PVAT. The mRNA expressions of adipocyte differentiation marker genes (PPARγ, FABP4, and c/EBPα) were time-dependently increased in Agtr1+/+ adipocyte. In contrast, FABP4 and c/EBPα mRNA expressions were markedly inhibited in Agtr1
-/-
adipocyte, whereas PPARγ did not differ between the two groups during differentiation, suggesting that AT1 is essentially implicated in the terminal differentiation of periaortic adipocyte.

[CONCLUSION]
Our findings demonstrate that AT1 regulates the expression levels of late stage of adipocyte-differentiation marker genes in PVAT, suggesting that AT1-mediated modulation of periaortic adipocyte differentiation could be a novel therapeutic target for the prevention of atherosclerosis.
Abstract 34: Apolipoprotein A-I Increases Insulin Secretion from β-cells via a Protein Kinase A Dependent Mechanism
Authors: Cochran Blake J , Rye Kerry-Anne
Published: July 2021

Introduction:
The progression to hyperglycaemia in type 2 diabetes is marked by β-cell insulin secretory dysfunction and cell loss. We have previously demonstrated that apolipoprotein (apo) A-I, the major protein constituent of high density lipoproteins (HDL) increases insulin expression and secretion from β-cells. Clinical data also suggests that pharmacological elevation of HDL levels is associated with improved glycemic control in patients with type 2 diabetes. With the current interest in HDL raising therapeutics, defining the mechanism by which apoA-I acts on insulin secretion is of importance.

Objective:
To elucidate the cell signalling events responsible for increasing insulin secretion from pancreatic β-cells treated with lipid-free apoA-I.

Methods:
Ins-1E (rat insulinoma) cells were pre-treated for 30 min with the Protein kinase A (PKA) specific inhibitor H89 (20 μM), soluble and transmembrane adenyl cyclase specific inhibitors (KH7, 30 μM and 2’5’ dideoxyadenosine, 50 μM, respectively) or vehicle control, then incubated for 1 h with lipid-free apoA-I (final concentration 1 mg/mL) under both basal (2.8 mM) and high (25 mM) glucose conditions. The insulin concentration in the culture supernatants was determined by radioimmunoassay and the cells were either lysed for protein analysis by western blotting or treated with 0.1 M HCl for determining cAMP by enzyme immunoassay.

Results:
Incubation of Ins-1E cells with apoA-I increased insulin secretion up to 3-fold. This increase was no longer apparent when the cells were pre-treated with H89. Incubation with apoA-I increased cAMP accumulation in Ins-1E cells 2.5-fold. This increase was totally inhibited when the cells were pre-incubated with 2’5’ dideoxyadenosine but not by KH7, indicating that transmembrane adenyl cyclase(s) are responsible for this response. ApoA-I also activated the small GTPase Cdc42, which may link cell surface apoA-I receptors with transmembrane adenyl cyclases.

Conclusion:
ApoA-I increases insulin secretion from pancreatic β-cells via a PKA-dependent mechanism involving transmembrane, but not soluble, adenyl cyclases and possibly Cdc42. This provides a possible explanation of the clinical observations that increased HDL may be beneficial in type 2 diabetes.
Abstract 348: Serial MRI Differentiates the Natural History of Stable and Vulnerable Plaques in a Rabbit Aorta
Authors: Pham Tuan A , Hua Ning , Phinikaridou Alkystis , Giordano Nick , Hamilton James A
Published: July 2021

Non-invasive monitoring of human atherosclerotic plaque progression would be extremely valuable for determining the characteristics of vulnerable plaques before they rupture and for evaluating therapeutic interventions. We have shown that MRI is a reliable method for differentiating stable and vulnerable aortic atherosclerotic plaques in a rabbit model of atherothrombosis. In this study, we imaged rabbits at 4 time points (baseline, 1, 2 and 3 months) to monitor the progression of plaques and investigate when stable and vulnerable plaques can be differentiated. MR images and histology of two plaques in the same rabbit aorta are compared below. The top row of black blood (BB) T1 images (with fat suppression) show no significant changes in the lumen or the vessel wall area, and no thrombus in the stable plaque after triggering to induce plaque rupture. The histology showed an intact fibrous cap, overlying diffuse lipids. In comparison, there is a marked increase in vessel wall area in the vulnerable plaque (from 4.1 to 15 mm2 over 3 months). The second row (T1BB FS with Gd) shows some uptake of Gd into the stable plaque and surrounding tissue, but the vulnerable plaque showed rapidly increased Gd uptake between months 2 and 3. The vulnerable plaque also showed a large luminal thrombus in the MR images of the live rabbit and in the corresponding histology slice. In addition, the vulnerable plaque showed excessive outward remodeling at 2 months (data not shown). Our study shows a progression of vulnerable features, clearly evident at 2 months, in the plaque that disrupted. We are currently investigating whether therapies initiated at 2 months will reduce the progression of plaque vulnerability.
Abstract 37: Metabolomic Signatures of Metabolic Risk Factors for Atherosclerotic Cardiovascular Disease
Authors: Yin Xiaoyan , Hwang Shih-Jen , Adourian Aram , Courchesne Paul , Gordon Neal , Fox Caroline , O’Donnell Christopher J , Subramanian Subha , Juhasz Peter , Muntendam Pieter , Larson Martin G , Levy Daniel
Published: July 2021

Background:
Multiple cardiovascular disease (CVD) risk factors cluster in the same individuals and their concurrence is used to diagnose metabolic syndrome (MetSyn), which carries substantial risk for CVD. We hypothesized that MetSyn is associated with multiple metabolomic derangements.

Methods:
As part of the SABRe CVD initiative, a multi-project investigation of biomarkers of CVD and its risk factors, we designed a 2x2x2 factorial study of MetSyn risk factors that included 650 individuals from the Framingham Heart Study (out of a total N of ∼3200) assigned to 8 unique groups of approximately 81 individuals each, sampled from high vs. low strata of BMI, lipids, and glucose. We conducted gas chromatography-mass spectroscopy (GC/MS) on plasma samples from 650 eligible individuals. General linear modeling was used to identify biomarkers that differed across all 8 groups or differed in their main effects on individual risk factors.

Results:
Characteristics of the study sample (mean±SD), according to group assignment, are presented in the Table. GC/MS characterized 149 metabolites; of these 18 differed across all groups at P<5x10
-8
and 36 differed at P<0.00001. The top 3 most highly significant metabolites across all groups were glucose (P=2x10
-40
), glutamic acid (P=4x10
-26
), and sphingomyelins (lowest P=8x10
-25
). The top 3 most highly significant main effects of metabolites for BMI were: glutamic acid (P=2x10
-18
), sitosterol (P=2x10
-10
), and uric acid (P=3x10
-10
), for dyslipidemia: sphingomyelins (lowest P=1x10
-27
), glutamic acid (P=5x10
-20
), and lactic acid (P=7x10
-12
), and for dysglycemia: glucose (P=1x10
-42
), fructose (P=3x10
-7
), and 2-hydroxybutanoic acid (P=6x10
-7
).

Conclusions:
Metabolomic profiling identified multiple biomarker signatures of MetSyn and its major metabolic risk factors. These novel findings warrant external replication. Understanding the pathways represented by our results may help to unravel the molecular derangements contributing to MetSyn and its constituent risk factors. This knowledge may identify therapeutic targets for the prevention and treatment of CVD.
Abstract 373: Cigarette Smoke Produces Phenotypic Modulation of Cerebral Vascular Smooth Muscle Cells: Role of KLF4 and Epigenetic Control
Authors: Ali Muhammad S , Jabbour Pascal M , Tjoumakaris Stavropoula I , Gonzalez L Fernando , Rosenwasser Robert H , Koch Walter J , Dumont Aaron S
Published: July 2021

Objectives:
Cigarette smoke is one of the most important environmental factors associated with cerebral aneurysm formation and progression. Cigarette smoke causes phenotypic modulation of Cerebral Vascular Smooth Muscle Cells (VSMCs) which is considered an important underlying mechanism in cerebral aneurysm formation/progression. We studied epigenetic changes caused by Cigarette Smoke Extract (CSE) in VSMC differentiation marker genes.

Methods:
Rat cerebral VSMCs were treated with CSE at 40 ug/ml (optimal dosage based on preliminary data) dissolved in HEPES buffer (biological activity comparable to soluble components of cigarette smoke in humans) for 2 hours. Cells at 80-90% confluence were fixed with formaldehyde. DNA was sheared using sonication. Chromatin immune-precipitation was performed using following antibodies: 1) anti-KLF4 (Krupple Like Factor 4), 2) anti-HDAC2 (Histone Deacetylase 2), 3) anti-H3K9Ac (Histone 3 Lysine 9 Acetylation), 4) anti-H3K27triMe (Histone 3 Lysine 27 Tri-Methylation) and 5) anti-H4Ac (Histone 4-Acetylation). qPCR was performed with primers specific to CArG containing promoter regions of differentiation marker genes {(alpha-Actin and Myosin Heavy Chain (MHC)} and Myocardin. Results were normalized against input DNA. As part of in vivo experiments Pluronic Gel (40% w/v) containing CSE at 0.8 mg/ml was applied to rat carotid vessels for 6-8 hours. Vessels were harvested and frozen in liquid nitrogen and chromatin immune-precipitation was performed.

Results:
CSE stimulation promoted a non-differentiated phenotype of cerebral VSMCs. We demonstrated a marked increase in percent enrichment of alpha-actin, MHC and Myocardin promoters with KLF4, HDAC2, and H3K27triMe antibodies and decreased enrichment with H3K9Ac antibody. Although H4Ac antibody showed decreased enrichment at the alpha-Actin promoter, this was not observed for MHC and Myocardin promoters. This was consistent for both in vivo and in vitro studies.

Conclusions:
We previously demonstrated that CSE decreases expression of cerebral VSMCs marker genes and SRF co-activator, Myocardin and increases expression of transcription factor, KLF4. Further it promotes a pro-inflammatory phenotype. Decreased expression of the above mentioned genes is a result of direct binding of KLF4 to the CArG containing promoter region. Further, KLF4 recruits HDAC2 which deacetylases H3K9 and H4 causing DNA compaction thereby repressing transcription. Our results provide insight into the underlying molecular mechanisms involved in CSE-induced VSMC phenotypic modulation and provide future potential therapeutic targets applicable to cerebral aneurysms.
Abstract 380: Favorable Effects of Flavonoids on Endothelial Function and Arterial Stiffness in Healthy Smokers
Authors: Kokkou Eleni , Siasos Gerasimos , Tousoulis Dimitrios , Oikonomou Evangelos , Kollia Marilena , Kioufis Stamatios , Gouliopoulos Nikolaos , Verveniotis Aleksis , Athanasiou Dimitrios , Papaeleutheriou Dimitrios , Papavasiliou Athanasios G , Stefanadis Christodoulos
Published: July 2021

Background:
Smoking is associated with impaired vascular function. Consumption of concord grape juice (CGJ), a rich source of flavonoids, has previously been shown to have antithrombotic and antioxidant effects and moreover can modify cardiovascular risk factors. In the present study we assessed the hypothesis that CGJ can improve endothelial function and arterial stiffness in healthy smokers.

Methods:
We studied the effect of a 2 weeks oral treatment with 7cc/Kg/day of CGJ (Welch’s 100% Concord Grape) in 20 healthy smokers (aged 26±5y) on two occasions (day 0: baseline and day 14). The study was carried out on two separate arms, one with CGJ and one with placebo, according to a randomized, placebo-controlled, double-blind, cross-over design. Measurements were carried out before (pSm) and immediately after (Sm0) cigarette smoking. Endothelial function was evaluated by flow-mediated dilation (FMD) of the brachial artery. Carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness and augmentation index (AIx) as a measure of arterial wave reflections.

Results:
At baseline measurements, compared to pSm, cigarette smoking decreased FMD values (8.58±3.17% vs.5.65±1.98%, p<0.001) and moreover caused an increase in AIx (4.19±9.13% vs. 6.26±9.81%, p=0.013) and PWV (6.02±0.67m/sec vs. 6.21±0.68m/sec, p<0.046). Treatment with CGJ, improved pSm values of: FMD (7.87±2.79% vs. 9.43±2.62%, p=0.024), PWV (6.11±0.58m/sec vs. 5.70±0.6m/sec, p=0.013) and AIx (3.03±7.70% vs. -0.59±8.56%, p=0.016), while there was no statistically significant difference with placebo administration. Finally, compared with placebo, at day 14, treatment with CGJ, blunted the acute smoking-induced increase in PWV (placebo: from 5.67±0.64m/sec Psm to 6.00±0.78m/sec Sm0), (CGJ: from 6.00±0.73m/sec Psm to 6.23±0.66m/sec Sm0) (p=0.012).

Conclusion:
Concord grape juice contains specific flavonoids that may improve endothelium-dependent vasodilation and vascular elastic properties of the arterial tree in healthy smokers. Improved endothelial function and decreased arterial stiffness is a potential mechanism by which flavonoids may prevent cardiovascular events.
Abstract 381: Favorable Effects of Habitual Physical Activity on Vascular Function: Ikaria Study
Authors: Oikonomou Evangelos , Chrysohoou Christina , Siasos Gerasimos , Tousoulis Dimitrios , Zaromitidou Marina , Gialafos Elias , Zisimos Konstantinos , Marinos George , Kioufis Stamatios , Miliou Antigoni , Pitsavos Christos , Papavasiliou Athanasios G , Stefanadis Christodoulos
Published: July 2021

Background:
Physical activity (PA) has substantial vascular and cardiac health benefits and can ameliorate cardiac risk. Measurement of endothelial function is well validated in large population studies as strong predictor of adverse cardiovascular outcomes. In the present study we evaluated the effect of habitual physical activity on vascular function in residents of Ikaria Island. The inhabitants of this island show increased longevity and high prevalence of healthy aging.

Methods:
The study was conducted on a subgroup population of IKARIA study consisted of 327 subjects (155 men) aged 40-91 years, permanent inhabitants of Ikaria Island. Endothelial function was evaluated by ultrasound measurement of flow-mediated-dilatation (FMD). We evaluated PA using the shortened version of the self-reported International Physical Activity Questionnaire (IPAQ). Overall the study sample was divided in three groups according to the categorical score achieved in IPAQ questionnaire: low PA (n=75), moderate PA (n=200) and vigorous PA (n=48). Subjects in the low PA group were recorded as physical inactive and the rest as physical active.

Results:
Mean FMD (5.79±3.19%) was inversely associated with age (r=-0.242, p<0.001). Inactive subjects had significantly lower FMD compared to active subjects (4.95±3.02% vs. 6.06±3.23%, p=0.008). Multiple linear regression analysis after adjustment for several known confounders (age, diabetes mellitus, hypertension, smoking, hypercholesterolemia, cardiovascular disease, body mass index) revealed significant differences in FMD values between moderate vs. low PA group [b=1.24, 95%CI: (0.28, 2.20), p=0.011] whereas a trend was evident between vigorous and low PA group [b=1.11, 95%CI: (-0.16, 2.38), p=0.089]. Moreover subjects walking for more than 10 minutes less than two times weakly had significantly lower FMD compared with subjects walking three to five days weakly or almost every day (5.13±2.82% vs. 6.95±3.84% vs. 6.00±3.23%, p=0.005).

Conclusions:
The present study revealed another cardioprotective mechanism of habitual PA through beneficial modification of endothelium function.
Abstract 384: Interleukin-6 Mediates the Altered Platelet Function Associated with Experimental Colonic Inflammation
Authors: Yan Li-Sue S
Published: July 2021

Patients with inflammatory bowel disease (IBD) are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have also revealed alterations in platelet activation, enhanced platelet-leukocyte interaction, and elevated plasma levels of prothrombotic cytokines, such as IL-6. This study was directed towards determining whether: 1) experimental colitis, induced by 6 days of dextran sodium sulfate (DSS) ingestion, is associated with platelet activation and the formation of platelet-leukocyte aggregates (PLAs), 2) IL-6 deficiency alters these responses to DSS colitis, and 3) the platelet abnormalities observed in DSS mice can be recapitulated by chronic infusion of murine recombinant IL-6. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood. Platelets were identified by characteristic light scattering and membrane expression of CD41. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin (with JON/A antibody). The combination of CD41, CD45.2, Gr-1, F4/80 and isotype control antibodies were used to detect and quantify aggregates of leukocytes, neutrophils and monocytes with platelets in control, wild type (WT) colitic, IL-6
-/-
colitic, and WT mice implanted with IL-6 loaded Alzet osmotic minipumps (for 6 days). Our results indicate that DSS colitis is associated with increased numbers of activated platelets and the formation of aggregates of leukocytes (PLA), neutrophils (PNA) and monocytes (PMA) with platelets. These platelet responses to experimental colitis were largely undetected in IL-6
-/-
mice. Chronic infusion (at a rate that yielded plasma IL-6 levels similar to those detected in DSS colitic mice) of IL-6 recapitulated the increased platelet activation and formation of PLA, PNA, and PMA observed in DSS-colitic mice. Collectively, these findings show that the altered platelet function detected in human IBD can be reproduced in an animal model of colonic inflammation and that interleukin-6 plays a critical role in the genesis of these platelet abnormalities in the setting of experimental IBD.
Abstract 411: An Anion-Exchange Chromatography Isolated Subfraction of Mouse Apolipoprotein A-I Is Unable to Activate Cellular Cholesterol Release from Mouse Peritoneal Macrophage Foam Cells
Authors: Okumura-Noji Kuniko , Cavigiolio Giorgio , Huang Rong , Davidson W Sean , Akita Nobukatsu , Okuhira Kei-ichiro , Yokoyama Shinji , Tsujita Maki
Published: July 2021

Aim:
Human and mouse plasma apoA-I separate on anion exchange chromatography (6M urea) in distinct subfractions. To investigate the nature of this diversity, we analyzed the protein subfractions by IEF and Urea-PAGE, and compared their ability to generate HDL in vitro.

Methods:
Human and mouse plasma HDLs were delipidated then applied to an DE52 ion-exchange column and eluted by 30-60mM Tris-HCl, pH7.80 containing 6M urea. Mouse, human, and recombinant apoA-Is were analyzed by IEF (Ettan IPGphor3) followed by CBB protein staining. Urea-PAGE analysis was performed with 14% acrylamide gel in 6M urea buffer. ApoA-Is abilities to efflux cholesterol from mouse peritoneal foam cells and to shift the size of the ACAT-accessible cholesterol pool were determined.

Results:
Human plasma and recombinant human apoA-I showed two apoA-I subtypes by IEF and Urea-PAGE analysis; whereas HDL from C57BL/6 and 129SV mice contained three apoA-Is at pI=5.37, 5.31, 5.27 (mP3-1, mP2, mP3-3), and pI=5.44, 5.37, 5.32, respectively. Subfractions mP2 and mP3-3 were isolated on a DE52 column and, despite their difference in pI, were able to mediate cellular cholesterol efflux at levels similar to human plasma apoA-Is. Subfraction mP3-1, with the highest pI, eluted between mP2 and mP3-3 on DE-52 column. Interestingly, for both mouse strains, mP3-1 showed very poor cholesterol efflux and the incorporation of 14C-oleic acid to cellular CE was significantly higher when compared to mP2 and mP3-3.

Conclusion:
We found that differences in the pI of anion-exchange chromatography apoA-I subfractions did not directly affect HDL generation except for mouse subtype mP3-1. mP3-1 binds strongly to the anionic DE52 resin, probably by hydrophobic interaction, and is unable to mediate cellular cholesterol efflux. Further investigation of the nature of the differences in these apoA-I subtypes will provide insights on factors that may be implicated in the biogenesis of HDL in vivo.
Abstract 428: Progression Patterns of Carotid Atherosclerotic Plaques with and Without Intraplaque Hemorrhage Monitored for 6 Months: A Multicenter Magnetic Resonance Imaging Study
Authors: Sun Jie , Balu Niranjan , Xu Dongxiang , Hippe Daniel , Hatsukami Thomas , Yuan Chun
Published: July 2021

Background:
Carotid magnetic resonance imaging (MRI) is emerging as an effective tool for monitoring plaque progression noninvasively. However, data from multi-center studies are scarce. Intraplaque hemorrhage (IPH) has been shown to promote plaque growth. In this multi-center study over 6 months, we sought to study plaque progression in subjects with and without IPH.

Methods:
As the placebo arm of a multi-center clinical trial, 59 subjects with 16-79% carotid stenosis by ultrasound and a lipid core (LC) on baseline MRI completed a follow-up MRI at the end of a 6-month period. Thirty-nine (66.1%) were on concurrent rosuvastatin therapy. Lumen, wall and LC volumes, and IPH presence were measured on both scans blinded to time sequence at a core lab. Plaque progression was calculated as annualized changes within the common coverage between scans using the carotid bifurcation as a landmark.

Results:
IPH was present on 14 slices from 3 (5.1%) subjects at baseline. Subjects without IPH (n=56) showed a significant reduction in LC volume (p=0.031), a trend towards reduction in wall volume (p=0.215) and no apparent change in lumen volume (p=0.910). Although all subjects with IPH were on rosuvastatin during the study period, compared to subjects without IPH, they had increased LC (62.9±46.2 mm3/year vs. -8.8±29.9 mm3/year, p<0.001) and wall (100.1±78.1 mm3/year vs. -12.7±75.8 mm3/year, p=0.015) volumes, but decreased lumen volume (-93.4±114.7 mm3/year vs. 0.90±59.7 mm3/year, p=0.014).

Conclusion:
This multi-center study over 6 months demonstrated significant regression in LC volume in plaques without IPH. By contrast, plaques with IPH may be poorly controlled despite statin therapy.
Abstract 458: Omega-3 Supplementation Does Not Affect Inflammatory Gene Expression in the Small Intestine of Patients with Type 2 Diabetes
Authors: Labonté Marie-ève , Couture Patrick , Tremblay André J , Hogue Jean-Charles , Lemelin Valéry , Lamarche Benoît
Published: July 2021

Recent evidence suggests that diet-induced inflammation in the small intestine is linked to obesity and insulin resistance. Long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to have anti-inflammatory effects by down-regulating inflammatory gene expression in adipocytes and mononuclear cells. However, the extent to which EPA and DHA may exert their anti-inflammatory effects by down-regulating inflammation in the gut is unknown. The objective of the study was to investigate the impact of EPA+DHA supplementation on the expression of inflammatory genes in the small intestine of patients with type 2 diabetes. A total of 12 men with type 2 diabetes were recruited in this placebo-controlled randomized crossover study. After a 4-week run-in period, patients received in random sequence 5 g/d of fish oil providing 3 g of EPA+DHA or placebo (corn and soybean oil) for 8 weeks, each separated by a 12-week washout period. Gene expression was assessed by real-time PCR in duodenal biopsy samples obtained in the fasted state at the end of each treatment phase. Intestinal mRNA expression levels for interleukin(IL)-6 and tumor-necrosis factor(TNF)-α were hardly detectable after either treatment (< 100 copies/10^5 copies of the reference gene ATP synthase O subunit, ATP5o). Intestinal mRNA expression of IL-18 and of the transcription factor STAT3 (signal transducer and activator of transcription 3) was higher (> 5000 copies/10^5 copies ATP5o) but still relatively low and EPA+DHA supplementation had no impact on any of these levels (P ≥ 0.73 between treatments). Plasma C-reactive protein (CRP) concentrations after supplementation with EPA+DHA (5.2 ± 4.5 mg/L) were not significantly different than values measured after placebo (8.0 ± 10.8 mg/L, P = 0.2). In conclusion, these data suggest that gene expression of pro-inflammatory cytokines and STAT3 in duodenal cells is low in patients with type 2 diabetes and not affected by EPA+DHA supplementation.
Abstract 471: Nuclear Receptor Nur77 Reduces Atherosclerosis
Authors: Hamers Anouk A , de Winther Menno P , Gijbels Marion J , de Waard Vivian , de Vries Carlie J
Published: July 2021

Rationale:
Nuclear receptor Nur77, also known as NR4A1, TR3 or NGFI-B, is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. Macrophages play a critical role in atherosclerosis and the function of Nur77 in lesion macrophages has not yet been investigated.

Objective:
This study aims to delineate the function of Nur77 in macrophages and to assess the effect of bone marrow-specific deficiency of Nur77 on atherosclerosis.

Methods and results:
We investigated Nur77 in macrophage polarization using bone marrow-derived macrophages (BMM) from wild-type and Nur77-knockout (Nur77
-/-
)-mice (n=10). Nur77
-/-
BMM exhibit changed expression of M2-specific markers and an inflammatory M1-phenotype with enhanced expression of IL12, IFNγ, and SDF-1α and increased NO synthesis in (non)-stimulated Nur77
-/-
BMM cells. SDF-1α expression in non-stimulated Nur77
-/-
BMM is repressed by Nur77 and the chemoattractive activity of Nur77
-/-
BMM is abolished by SDF-1α inhibiting antibodies. Furthermore, Nur77
-/-
mice show enhanced thioglycollate-elicited migration of macrophages and B-cells. The effect of bone marrow-specific deficiency of Nur77 on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr
-/-
) mice. Ldlr
-/-
mice with a Nur77
-/-
-deficient bone marrow transplant develop 2.1-fold larger atherosclerotic lesions than wild-type bone marrow transplanted mice. These lesions contain more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF-1α expression is higher in lesions of Nur77
-/-
-transplanted mice, which may explain the observed aggravation of lesion formation.

Conclusions:
In conclusion, in bone-marrow derived cells the nuclear receptor Nur77 has an anti-inflammatory function, represses SDF-1α expression and inhibits atherosclerosis.
Abstract 495: High-Density Lipoprotein-Mediated Modulation of Autoimmunity in Normolipidemic Lupus-Prone Mice
Authors: Black Leland L , Yu Shaohua , Srivastava Roshni , Tsimikas Sotirios , Anantharamaiah G M , Kabarowski Janusz H
Published: July 2021

The notion that quantitative and/or qualitative modulation of High-Density Lipoprotein (HDL) can protect against autoimmunity and the development of Systemic Lupus Erythematosus (SLE) is supported by demonstration of its anti-inflammatory and immunosuppressive properties in hypercholesterolemic rodent models lacking Apolipoprotein A-I (Apo A-I) or treated with 4F Apo A-I mimetic peptide. However, it is not known whether similar effects of Apo A-I (HDL) deficiency or 4F treatment are manifested in the absence of hypercholesterolemia, or whether raising HDL elicits opposite effects to those caused by Apo A-I deficiency. It also remains to be determined whether beneficial effects of 4F also include mitigation of SLE-associated HDL dysfunction (depressed HDL levels and PON-1 activity) that we previously showed was also inherent in normolipidemic SLE-prone
gld
mice. We transferred SLE from SLE-prone Sle123 mice into congenic wild-type, Apo A-I knockout or Apo A-I transgenic mice by bone marrow transplantation. Transgenic Apo A-I overexpression resulted in significant improvement in several Lupus phenotypes, including lower anti-DNA auto-antibodies, reduced
in situ
T lymphocyte proliferation, decreased activated CD4
+
T lymphocytes and CD4
+
effector/memory cells, and improved kidney pathology. Surprisingly, Apo A-I deficiency had no significant influence on the above Lupus phenotypes and, as with Apo A-I overexpression, did not affect CD4
+
FoxP3
+
regulatory T cell numbers. Feeding
gld
mice diet supplemented with 4F peptide (∼6mg/Kg body weight: synthesized from D-amino acids to make it orally bio-available) did not reduce auto-antibodies or modify any of the Lupus phenotypes above other than kidney pathology, which was significantly improved. Interestingly, this beneficial effect of 4F was mediated in the absence of any significant improvement in HDL levels or PON-1 activity. Considering that HDL deficiency did not modulate major Lupus phenotypes in the absence of hypercholesterolemia, we conclude that supra-physiologically high levels of Apo A-I (HDL) may be required to have broad therapeutic benefit, while Apo A-I mimetic peptide treatment may be a more clinically viable strategy to treat glomerulonephritis in SLE.
Abstract 50: Regulation of Peripheral Tissue Inflammation Links Autoimmunity and Atherosclerosis
Authors: Brzoza-Lewis Kristina L , Zabalawi Manal , Wilhelm Ashley J , Owen John S , Fulp Brian , Gerelus Mark , Ferguson Daniel , Thomas Michael J , Sorci-Thomas Mary
Published: July 2021

Previous publications from our laboratory show that cholesterol-fed LDLr-/-, apoA-I
-/-
(DKO) mice develop accelerated atherosclerosis, as well as, a severe autoimmune disorder not seen in diet-fed LDLr
-/-
mice (SKO). This phenotype is characterized by the expansion of cholesterol loaded T cells within lymphoid organs such as skin draining lymph nodes and spleen as well as the manifestation of aberrant T cell responses. In order to examine the link between the progression of atherosclerosis and the onset of autoimmunity we examined the type and amount of immune cell infiltration in both the aorta and the skin of DKO and SKO mice fed an atherogenic diet for 12 weeks. In a subset of mice the immunomodulator, Fingolimod (FTY720), a sphingosine analogue was administered along with the diet. After 12 weeks, plaque burden in the aortic root was measured as both neutral lipid accumulation, and as the type and number of immune cells present. As expected, diet alone increased both lipid and immune cell infiltration in the aortic root, as well as in the skin of DKO compared to SKO mice. Interestingly, FTY-diet DKO mice showed a sharp reduction in both lipid accumulation as well as CD4+ cell infiltration in the aortic root. This decrease in aortic lipid deposition in FTY-diet DKO was well below that of any of the other groups, including diet-only SKO mice, while FTY-diet SKO were not different than diet-only SKO mice. These data suggest that heightened aortic inflammation observed in response to dietary cholesterol in the absence of apoA-I, renders the DKO aorta more susceptible to the immunomodulatory effects of FTY. Quite unexpectedly, the skin of FTY-diet DKO mice continued to show massive cholesterol accumulation and inflammation with a preservation of subcutaneous fat, in light of the dramatic reduction in aortic infiltrates observed. When DKO mice lacking T and B cells (Rag1-/-, LDLr-/-, apoA-I-/-) were studied, massive skin cholesterol accumulation was still observed and TKO mouse dermis showed infiltrates largely composed of neutrophils and monocytes, suggesting that dermal cholesterol imbalance, in the absence of apoA-I, was the basis for disease initiation in the skin. Overall, these studies illustrate the vastly different roles of apoA-I and immunomodulatory drugs in skin versus aorta cholesterol balance.
Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region
Authors: Hirbawi Jamila , Bledzka Kamila , Ma Yan Qing , Qin Jun , Plow Edward F
Published: July 2021

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α
IIb
β
3
that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W
679
V
680
, resulted in a 50% reduction in co-activator activity and a single point mutation of Y
673
A also led to a 50% loss of function. A combination mutant consisting of the W
679
V
680
deletion and the Y
673
point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β
3
CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β
3
CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K
D
values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.
Abstract 525: Reduced Ability of Endothelial Progenitor Cells to Inhibit Smooth Muscle Cell Proliferation and Migration Compared to Aortic Endothelial Cells Under Flow
Authors: Sprague Eugene A , Bailey Steven R
Published: July 2021

Early endothelial restoration at sites of vascular intervention is one of the recognized important potential treatment strategies that can be exploited to limit arterial restenosis associated with intimal smooth muscle cell hyperplasia. In recent years, much attention has been focused on the potential of circulating bone marrow derived cells named endothelial progenitor cells (EPC) to differentiate into mature endothelial cells upon attachment at injured vascular sites and, thereby, potentially limit intimal hyperplasia. Our objective was to compare the influence of isolated porcine blood EPC and aortic endothelial cells (HAEC) on the proliferation and migration of underlying aortic smooth muscle (SMC) cells using a novel arterial wall model system exposed to arterial flow patterns. An in vitro arterial wall model co-culture system was constructed as the bottom plate within a parallel plate flow chamber. A 2mm thick collagen gel containing porcine SMC was overlayed by a porous (5μm) polyester mesh. After 2 days, either porcine aortic EC or CD34+ EPC or no cells (control) were seeded on the surface and allowed to attain confluence. These constructs were then placed into a flow chamber and exposed to arterial flow (12 dynes/cm
2
) for 48h using circulating culture medium. After the flow regimen, SMC that had migrated through the mesh were immunologically identified and counted. The mesh was removed and the percent proliferating cells in the underlying gel were identified by PCNA staining.EC coverage resulted in an almost 7-fold inhibition of SMC migration compared to control (127±33 vs 840±193 SMC/cm
2
) and more than 4- fold more than observed with EPC coverage (553±7 SMC/cm
2
). Similarly, EC coverage resulted in reducing SMC proliferation to 3.2% compared to 8.5% in EPC covered SMC and 14% PCNA positive SMC in the control underlying SMC. These result indicate that blood-derived CD34+ EPC are less effective in limiting adjacent SMC proliferation and migration than native arterial endothelial cells. This may have important implications for strategies designed to employ or attract these cells as a means to limit restenosis at stented interventional sites.
Abstract 547: 7-Ketocholesterol-Mediated Alterations in Vascular Function: A Link Between Vascular and Metabolic Complications of Chronic Particulate Matter Exposure
Authors: Rao Xiaoquan , Maiseye Andrew , Zhong Jixin , Gopalakrishnan Bhavani , Ying Zhekang , Liu Cuiqing , Guschina Lyubov , Villamena Frederick , Harkema Jack , Sun Qinghua , Rajagopalan Sanjay
Published: July 2021

BACKGROUND:
Both human and animal studies have suggested a compelling link between inhaled particulate matter <2.5nm (PM2.5) exposure and acceleration of atherosclerosis. In this study, we assessed the hypothesis that 7-Ketocholesterol (7KC), a major redox product of cholesterol may mediate adverse vascular and metabolic effects of PM2.5 exposure.

METHODS:
Apolipoprotein E
-/-
(apoE-/-) mice were exposed to concentrated ambient PM2.5 or filtered air (FA) for 6 hours per day, 5 days per week for a total of 6 months in a versatile aerosol concentrator exposure system. Vasomotor tone changes were assessed in and atherosclerosis lesion quantification performed. 7KC and total cholesterol levels were determined by LC-MS in various tissues including aorta and 7KC accumulation and reverse transport studies in cultured murine macrophages subjected to PM2.5 exposure. eNOS activation and NO generation in response to 7KC exposure was assessed in cultured endothelial cells.

RESULTS:
PM2.5 increased aortic plaque (1.4-fold, p<0.05 vs. FA; n=7). Relaxation to the endothelium-dependent agonist acetylcholine was attenuated (Rmax, 74.86±6.2 vs. 94.55±7.29%, p<0.05 for PM2.5 vs. FA; n=7). 7KC levels in the aorta of PM2.5-exposed mice was significantly higher than that of FA (1251±453 vs. 8588±2088 ng/g tissue, p<0.05 PM2.5 vs. FA; n=5) while levels in liver were 1-fold higher (p=0.06; n=5). 7KC reduced eNOS activation in cultured endothelial cells through regulation of 1177, 495 and 113 phosphorylation sites of eNOS in a time and dose dependent manner and reduced NO generation when assessed by EPR. Enhanced uptake of 7KC was demonstrated in cultured macrophages after PM2.5 pre-exposure (56.2±0.3 vs. 47.8± 0.9 % p =0.01 for PM2.5 vs. control; n=3) while reverse transport of 7-KC did not differ in the presence of HDL (50 μg/ml). CD36 expression inside macrophages increased after PM2.5 exposure (1.5-fold, p<0.01; n=5) suggesting internalization of 7-KC via CD36 dependent mechanisms.

CONCLUSIONS:
Chronic PM2.5 exposure enhances 7KC levels paralleled by alterations in endothelial function through eNOS dependent pathways. These findings suggest novel secondary mediators by which air-pollution may modulate adverse vascular effects.
Abstract 76: Preoperative Genome-wide Differences in Inflammatory Gene Expression Predict Success Versus Failure in Lower Extremity Vein Bypass
Authors: O’Malley Kerri A , Novelo LG Leon , Lopez M Cecilia , DeSart Kenneth , Butler Khayree , Restrepo Christian , Moldawer Lyle L , Berceli Scott A , Baker Henry V , Casella George , Nelson Peter R
Published: July 2021

Introduction:
Vein bypass grafting is standard surgical therapy in the treatment for peripheral arterial disease (PAD), especially for those with limb threatening ischemia. However, the durability of vein grafts remains problematic with recent studies reporting disappointing 1-year primary patency rates as low as 61%. We hypothesized that an individual’s systemic inflammatory state, characterized by genome-wide inflammatory gene expression, would be predictive of ultimate clinical success or failure of these grafts.

Methods:
41 patients undergoing vein bypass grafting for symptomatic PAD (85% critical limb ischemia; 25% claudication) had complete pre-operative gene expression data for analysis and one-year follow-up. There were 16 failures (39%) by one-year. Gene expression was analyzed for the entire genome using the novel Affymetrix GGH2 6.9 million feature oligonucleotide array system. Expression levels for the 35,123 gene transcripts and initial unsupervised and supervised (success/failure outcome) clustering was performed using BRB array tools. To then determine the additional predictive influence of specific genes or groups of genes, a novel objective Bayes method for dichotomous outcomes using stochastic search algorithms and probit regression models was employed. A series of increasingly predictive candidate models of gene combinations were derived. Clinical parameters including Rutherford disease severity score at presentation, vein conduit (GSV vs. composite vein), diabetes, smoking, statin use, and antiplatelet/anticoagulant use were standardized in the predictive model.

Results:
A random search through the space of possible models including the six clinical covariates and different combinations of gene products was executed, resulting in a list of 20 models ranked based on their posterior probabilities. The additional predictive power of the top gene products are listed in Table 1 and genes with the highest marginal posterior probabilities are listed in Table 2. This modeling approach suggests that the genes identified, independent of any relationship functionally, exhibit class prediction potential for bypass outcome.

Conclusions:
Using both a powerful and novel gene array system and a novel probit model selection method, we were able to identify a small number of gene clusters with predictive capability for bypass success or failure. This may both be prognostic and feasible for use in clinical practice as a point-of-care tool moving forward. Validation of these models is currently underway.