bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 333: Hhcy Promotes Myocardial Infarction Induced Myocardiocyte Death via Mitochondria Mediated Inflammasome Activation
Authors: Zhou Jun , Chen Xiongwen , Tang Mingxin , Ai Xiaojie , Wang Hong , Yang Xiaofeng
Published: July 2021

Introduction:
Elevated level of serum homocysteine (Hcy) has been identified as a risk factor for accelerating progression of cardiovascular disease. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation by damaged mitochondria results in caspase-1 dependent inflammatory form of cell death.

Methods and Results:
AMI procedure was performed on hCBS/m
cbs
knockout mice at the age of 10weeks. Caspase-1 activity and myocardiocyte apoptosis were observed at the early stage of post-MI in severe HHcy mice (Hcy,120-220μM). Severe HHcy remarkably aggravated infarction size, cardiomyocyte area, and interstitial fibrosis 6 weeks after MI from 22.7 ± 4.3%, 340 ± 54μm
2
, 13.9 ± 1.9% in control mice (Hcy, 7-10μM) to 33.6 ± 6.5%, 485 ± 65μm
2
, 26 ± 4.5%, respectively, (
P
=0.035,
P
= 0.041,
P
=0.002). In the meantime, HHcy significantly increased LV cavity dilatation and dysfunction as compared with control mice by echocardiography (5.9 ± 0.2 vs. 4.2 ± 0.4mm,
P=
0.039; LV ejection fraction, 22.6 ± 3.9% vs. 36.8 ± 4.0%,
P
=0.011). Cultured neonatal mouse ventricular myocyte (NMVM) treated with the combination of DL-Hcy (500μM, 48h) and hypoxia (0.5% O2) showed that increased NLRP3 and caspase1 expression and activity, mitochondrial reactive oxygen species (mtROS) and mtDNA production, dissipation of mitochondrial membrane potential, and mitochondrial permeabilization. Moreover, synergetic effect of Hcy and hypoxia led to pronounced accumulation of damaged mitochondrial through suppressing mitophagy of damaged mitochondria. Caspase-1 activity and myocardiocyte death were lessened as NMVMs were administrated with mitochondria-targeted antioxidant Mito-temple and SOD2, whereas caspase-1 inhibition was not able to fully rescue damaged mitochondria.

Conclusion:
Acute myocardial infarction (AMI) initiates an intense inflammatory response in myocardium that promotes myocardiocyte death, ventricular remodeling, and cardiac dysfunction. Hcy accelerates cardiomyocyte death post-MI in part through mitochondrial-mediated NLRP3 inflammasome activation and inflammation, which contributes to adverse cardiac remodeling, ventricular dysfunction, and heart failure.
Abstract 336: Prevalence and Risk Factors for Idiopathic Venous Thromboembolism
Authors: Yau Patricia , Maldonado Thomas , Erb Juanita , Ranganath Neel , Sindet-Pederson Caroline , O’Reilly Deirdre , Rockman Caron , Berger Jeffrey S
Published: July 2021

Introduction:
Venous thromboembolism (VTE) is a major cause of morbidity and mortality in the United States. Despite medical advances in diagnostics and thromboprophylaxis, the incidence of VTE has not substantially decreased over time. Furthermore, many patients are diagnosed with VTE without an identifiable cause.

Objectives:
We sought to investigate the prevalence of idiopathic VTE and risk factors associated with this diagnosis.

Methods:
Patients diagnosed with deep vein thrombosis (DVT) or pulmonary embolism (PE) were enrolled into a prospective venous thromboembolic center (VTEC) registry at a large tertiary medical center from 7/2015-12/2015. VTE events were considered to be idiopathic if none of the following criteria were met: current pregnancy; current use of hormonal therapy or oral contraceptives, active malignancy; diagnosed thrombophilia; major surgery within 90 days; bed rest, restricted mobility, cast or mold, or serious trauma within 30 days. Patient demographics, comorbidities, social history, family history, laboratory and clinical data were analyzed and compared between groups. Multivariable logistic regression analysis was used to estimate odds of idiopathic VTE.

Results:
Of 223 patients with VTE, 93 (41.7%) were determined to be idiopathic. Patients with idiopathic and non-idiopathic VTE were of similar age, race/ethnicity, level of education, type of insurance, and type of VTE (DVT or PE). Patients with idiopathic VTE were more frequently female (68.8% vs. 46.4%; p < 0.01), more likely to have a history of COPD (8.6% vs. 1.1%, p = 0.03), hyperlipidemia (33.6% vs. 18.3%, p = 0.02), and less likely to be on a statin at the time of diagnosis (17.2% vs. 29.3%, p = 0.05) than non-idiopathic VTE. After multivariable adjustment, female sex (OR: 2.47, 95% CI 1.35-4.64) and history of varicose veins (OR 2.67, 95% CI 1.15-6.37) were significantly associated with the prevalence of idiopathic VTE.

Conclusions:
In a large tertiary medical center, idiopathic VTE occurred in more than 40% of all VTEs. Female sex and history of varicose veins were independently associated with having an idiopathic VTE. These data provide a basis for future investigation into this high-risk population and for further development of prediction models.
Abstract 34: Methotrexate Inhibits Vein Wall Scarring in Experimental Deep Vein Thrombosis: Implications for the Post-Thrombotic Syndrome
Authors: Wang Lang , Qi Guanming , Kessinger Chase W , Jhajj Harkamal S , Li Wenzhu , Henke Peter K , Jaffer Farouc A
Published: July 2021

Background:
Despite anticoagulant therapy, the post-thrombotic syndrome (PTS) remains a frequent, morbid complication of deep venous thrombosis (DVT). There are no effective clinical approaches beyond anticoagulation to prevent and mitigate PTS, which is driven by inflammation, impaired thrombus resolution, and vein wall scarring leading to valvular reflux. Methotrexate (MTX) is a FDA-approved antimetabolite drug that exerts multiple anti-inflammatory actions. Here we investigate the effects of MTX on vein wall scarring, a key mediator of PTS, in experimental murine DVT.

Methods:
Stasis VT was established by inferior vena cava complete ligation in C57Bl6 mice (n=60) on day 0. MTX (0.7 mg/kg) or PBS was intraperitoneal injected daily from day 1 to sacrifice. Thrombus burden, vein wall scarring, inflammatory gene expression and macrophage content were assessed at multiple timepoints.

Results:
MTX decreased Carstairs’ and picrosirius red-assessed vein wall thickness by 36.52% on day 8 (n=6 for each group, p<0.05 vs PBS). Macrophage recruitment in the vein wall was inhibited by MTX at day 8 (p<0.05 vs. PBS). MTX further inhibited vein wall mRNA expression of inflammatory markers CD68 and cathepsin B (p<0.05 vs. PBS). At day 4, neutrophil infiltration in the vein wall was similar in both groups (p>0.05). Thrombus burden, weight, length and mass were similar for both groups on both day 4 (p>0.05) and day 8 (p>0.05).

Conclusions:
MTX attenuates vein wall scarring in association with the macrophage-mediated inflammatory response in VT, without impairing thrombus resolution. MTX administration following DVT may offer a new translatable approach to reduce the post-thrombotic syndrome.
Abstract 348: Lys 42, 43, 44 and Arg 12 of Thrombin Activable Fibrinolysis Inhibitor Comprise Thrombomodulin Binding Exosite Essential for Exerting Its Antifibrinolytic Activity
Authors: Kim Paul Y , Wu Chengliang , Stafford Alan R , Fredenburgh James C , Weitz Jeffrey I , Gils Ann , Declerck Paul J
Published: July 2021

The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin or plasmin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K
1/2
) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues eliminated TM binding, decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K
1/2
by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay. When thrombin or plasmin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1- and 4.0-fold compared with wild-type TAFI, respectively, suggesting that the mutation only impacted activation kinetics by thrombin-TM. Surface plasmon resonance data show that mutation of the four residues results in complete loss of binding, either in the presence or absence of thrombin. Together, our findings suggest that these four residues are critical for the interaction of TAFI with the thrombin-TM complex that modulates its antifibrinolytic activity.
Abstract 367: Extreme High-Density Lipoprotein Cholesterol Genetics: An Assortment of Large and Small Polygenic Effects
Authors: Dron Jacqueline S , Wang Jian , Low-Kam Cécile , Khetarpal Sumeet A , Robinson John F , McIntyre Adam D , Ban Matthew R , Cao Henian , Rhainds David , Dubé Marie-Pierre , Rader Daniel J , Lettre Guillaume , Tardif Jean-Claude , Hegele Robert A
Published: July 2021

Rationale:
Although HDL-C levels are known to have a complex genetic basis, most studies have focused solely on identifying rare variants with large phenotypic effects to explain extreme HDL-C phenotypes.

Objective:
Here we concurrently evaluate the contribution of both rare and common genetic variants, as well as large-scale copy number variations (CNVs), towards extreme HDL-C concentrations.

Methods:
In clinically ascertained patients with low (
N
=136) and high (
N
=119) HDL-C profiles, we applied our targeted next-generation sequencing panel (LipidSeq
TM
) to sequence genes involved in HDL metabolism, which were subsequently screened for rare variants and CNVs. We also developed a novel polygenic trait score (PTS) to assess patients’ genetic accumulations of common variants that have been shown by genome-wide association studies to associate primarily with HDL-C levels. Two additional cohorts of patients with extremely low and high HDL-C (total
N
=1,746 and
N
=1,139, respectively) were used for PTS validation.

Results:
In the discovery cohort, 32.4% of low HDL-C patients carried rare variants or CNVs in primary (
ABCA1
,
APOA1
,
LCAT
) and secondary (
LPL
,
LMF1
,
GPD1
,
APOE
) HDL-C–altering genes. Additionally, 13.4% of high HDL-C patients carried rare variants or CNVs in primary (
SCARB1
,
CETP
,
LIPC
,
LIPG
) and secondary (
APOC3
,
ANGPTL4
) HDL-C–altering genes. For polygenic effects, patients with abnormal HDL-C profiles but without rare variants or CNVs were ~2-fold more likely to have an extreme PTS compared to normolipidemic individuals, indicating an increased frequency of common HDL-C–associated variants in these patients. Similar results in the two validation cohorts demonstrate that this novel PTS successfully quantifies common variant accumulation, further characterizing the polygenic basis for extreme HDL-C phenotypes.

Conclusions:
Patients with extreme HDL-C levels have various combinations of rare variants, common variants, or CNVs driving their phenotypes. Fully characterizing the genetic basis of HDL-C levels must extend to encompass multiple types of genetic determinants—not just rare variants—to further our understanding of this complex, controversial quantitative trait.
Abstract 368: The GLP-1 Receptor Agonist Liraglutide Improves Autophagy Impairment in the Diabetic Heart
Authors: Bugyei-Twum Antoinette , Switzer Jennifer , Singh Krishna K , Connelly Kim A
Published: July 2021

Background:
Diabetes mellitus is a well-recognized risk factor for the development of heart failure. Although studies with the glucagon-like pepide-1 analog liraglutide—an approved treatment for type 2 diabetes—have demonstrated substantial cardioprotective effects of the drug in both human and experimental diabetes, the underlying mode of action/mechanism of liraglutide remains unclear. Here, we investigate the impact of liraglutide on autophagy—an evolutionary conserved mechanism thought to play an essential role in cell survival during stress—using a rodent model of type 2 diabetes, the Goto Kakizaki (GK) rat.

Methods:
Thirty-two weeks old male GK rats and sex/age-matched Wistar controls were treated with liraglutide (0.2 mg/kg/day) or PBS twice daily for 8 consecutive weeks. At 40-weeks of age, cardiac structure/function were assessed by echocardiography and LV tissue samples were collected to assess the expression of inducers/markers of autophagy (mTOR, phospho-mTOR, LC3-I/II, p62, and Beclin-1).

Results:
Autophagy was inhibited in the heart of diabetic GK animals when compared to Wistar controls, as confirmed by a significant increase in mTOR expression/activation—a negative regulator of autophagy. This was further confirmed by an observed decrease in the LC3 II/I ratio in diabetic GK animals when compared to controls (p<0.05). P62 expression levels, however, were found to be significantly upregulated in GK animals with respect to controls (p<0.01); suggesting an impairment or defect in basal autophagy in diabetic GK animals. Liraglutide-treatment—which was associated with an improvement in both HbA1C level and LV hypertrophy in GK animals—resulted a significant reduction in mTOR expression/activation in GK animals when compared to their untreated counterparts. This improvement in autophagy status was further corroborated by an observed increase in LC3 II/I ratio and Beclin-1, as well as a reduction in p62 expression levels.

Conclusion:
Overall, our data suggests that the cardioprotective effects of liraglutide may stem from its ability to activate autophagy in the diabetic heart and improve autophagy impairment in the setting of type 2 diabetes.
Abstract 369: Naringenin Supplementation to a Chow Diet Reduces Plasma Lipids and Adiposity, and Suppresses Rer In

Ldlr
-/-

Mice Fed a Chow Diet
Authors: Burke Amy C , Telford Dawn E , Sutherland Brian G , Edwards Jane Y , Huff Murray W
Published: July 2021

Previously, we have shown that intervention by the addition of the citrus flavonoid naringenin to a chow diet enhances the reversal of diet-induced metabolic dysregulation, obesity, and atherosclerosis. However, the metabolic effects of naringenin in the absence of obesity and metabolic dysregulation are unknown. In the present study, we assessed the effect of naringenin supplementation to a chow diet on plasma lipids, adiposity, respiratory exchange ratio (RER), ambulatory activity and tissue lipolysis. For 8 weeks,

Ldlr
-/-

mice were fed an isoflavone-free chow diet supplemented with or without 3% naringenin. Over 8 weeks, there was no difference in caloric intake between the two groups. Naringenin supplementation reduced plasma VLDL-cholesterol (C) (-46%;
P
<0.05), VLDL-triglycerides (-43%;
P
<0.05), and LDL-C (-27%;
P
<0.05) compared to mice consuming chow alone. Chow-fed mice maintained body weight, whereas mice fed chow with naringenin were ~1.4 g lighter (
P
<0.05) with significantly reduced adiposity (-48%;
P
<0.05). Histological analysis of epididymal white adipose tissue showed naringenin supplementation reduced adipocyte size and number. Between 6 and 8 weeks of diet, mice were assessed in metabolic cages. Naringenin supplementation had no effect on food intake, ambulatory activity or energy expenditure during both the light and dark cycles. Consistently, naringenin-treated mice had significantly lower RER compared to mice fed chow alone (0.97 vs 0.99;
P
<0.05). This difference was driven by a significant suppression in RER during the light cycle (0.96 vs 1.00;
P
<0.05), but not the dark cycle (0.97 vs 0.98
N.S
), suggesting an enhanced starvation response. Triglyceride lipolysis was highest in white adipose tissue, followed by liver and muscle. Naringenin supplementation to chow increased the lipolytic rate in adipose, but not in muscle or liver, suggesting reduced adiposity was related to increased expression of ATGL or HSL. In conclusion, compared to chow alone, naringenin supplementation reduced plasma lipids and decreased body weight
via
increased adipose tissue lipolysis and suppressed RER, with no change in energy expenditure.
Abstract 374: Interleukin-19: A Novel Pro-Angiogenic and Anti-Inflammatory Adipokine
Authors: Vrakas Christine , Keleman Sheri E , Scalia Rosario , Autieri Michael V
Published: July 2021

Uncontrolled inflammation leads to many of the chronic diseases associated with obesity. Due to a lack of oxygen in the tissue, expanding adipose tissue becomes hypoxic and pro-inflammatory. Adipocytes release pro-angiogenic factors in an effort to restore blood flow to the tissue. Presently, little is known about the potential for endogenously expressed anti-inflammatory cytokines to attenuate inflammation and also provide pro-angiogenic effects. IL-19 is uniquely anti-inflammatory, pro-angiogenic and is both expressed by and targets various cells types. IL-19 expression in adipocytes and stromal vascular cells is increased in visceral compared to subcutaneous fat, and is also increased in visceral fat on high fat diet (HFD) compared to normal chow diet. There is no known mechanism to explain the role of IL-19 in adipose tissue expansion, and we hypothesized that IL-19 may have pro-angiogenic and anti-inflammatory properties in expanding adipose tissue. We have identified a gene regulatory factor, Interleukin Enhancer-Binding Factor 3 (ILF3) that is induced in adipocytes and stromal vascular cells by HFD and IL-19 treatment. We found that both IL-19 and VEGF induce ILF3 expression in cultured human endothelial cells (hECs). Proliferation is significantly reduced when ILF3 is knocked down using siRNA in hECs. Furthermore, when ILF3 is knocked down and hECs are stimulated with VEGF several angiogenic cytokines are also decreased. Through immunohistochemistry we found that ILF3 translocates from the nucleus to the cytoplasm in visceral fat of C57BL/6 mice fed a HFD, and remains in the nucleus when fed a normal chow diet. In summary IL-19 may be a unique HFD responsive adipokine functioning to reduce inflammation and increase angiogenesis in expanding adipose tissue. The angiogenic function of IL-19 may work through induction of the gene regulatory factor, ILF3.
Abstract 376: Nedd4 Regulated Bk Channels in Diabetes Mellitus
Authors: ZHANG DAI-MIN , Chen Shao-liang , Zhu Yanrong , Ye Peng
Published: July 2021

Big conductance calcium activated potassium(BK) channel plays a critical role in pathophysiological regulation of vascular function. Recent studies indicated that the expression reduction of BK channels in high glucose condition exacerbated vessel dilation, and led to coronary artery diseases, while BK channel expression was reserved in A-kinase anchoring protein(AKAP) knockout mice at same condition. Here, We are to investigate heterologous co-expression of Nedd4 ligase, ubiquitin protein ligase, and KCa1.1 in HEK293 cells. The result shown that co-expression reduced BK current density without modulation of kinetic properties as measured by path clamp techniques. Modulation of current density was dependent on ligase activity and was lost in AKAP knockout mice with diabetes mellitus. Taken together, our data disclose a novel mechanism of KCa1.1 channel regulation that NEDD4 decreased BK channels expression in diabetes mellitus depending on AKAP signal complexity. These findings provide a new insight into potential therapeutic target in vascular diseases, especially in diabetes mellitus.This work was supported by the National Natural Science Foundation of China(Grant No. 8137034)
Abstract 38: Neutrophil-Derived S100a8 and S100a9 Promotes Reticulated Thrombocytosis and Atherogenesis in Diabetes
Authors: Nagareddy Prabhakara R , Kraakman Michael J , Lee Man Kit Sam , Al-Sharea Annas , Dragoljevic Dragana , Berger Jeffery , Goldberg Ira , Murphy Andrew
Published: July 2021

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. It remains unknown if hyperglycemia promotes platelet production and whether this contributes to enhanced atherothrombosis. Here we show that in response to hyperglycemia, neutrophil-derived S100A8/A9 interacts with the receptor for advanced glycated end-products (RAGE) on hepatic Kupffer cells resulting in increased production of interleukin-6 (IL-6), a pleiotropic cytokine implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor, c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose co-transporter 2 inhibitor (dapagliflozin), depleting neutrophils/Kupffer cells or inhibiting S100A8/A9 binding to RAGE (paquinimod) all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we show that patients with type 2 diabetes have reticulated thrombocytosis, which correlates with glycated hemoglobin, and also have increased plasma S100A8/A9 levels. These studies provide novel insights into the mechanisms that regulate platelet production and may help to develop strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.
Abstract 384: Low Carbohydrate High Protein (LCHP) Diets are Atherogenic by Supplying Excess Amino Acids to Alter the Macrophage mTORC1-Autophagy Signaling Axis
Authors: Zhang Xiangyu , Sergin Ismail , Evans Trent , Vélez Astrid R , Razani Babak
Published: July 2021

Low carbohydrate high protein (LCHP) diets are commonly used in weight loss programs. However, the overall health benefits of such regimens are controversial with recent studies even suggesting an increased cardiovascular risk in certain populations. A few reports in animal models corroborate these concerns demonstrating increased LCHP-induced atherosclerosis. Interestingly, the downstream sequelae of such diets on tissues and cellular signaling are largely inferred with relevant mechanisms undefined. We first confirmed in the ApoE-null mouse model that LCHP diets are indeed atherogenic with the development of complex lesions. Using mass spectrometry, we find high protein feeding not only increases serum amino acid levels but increases amino acid load to tissues including the spleen and aorta with resultant activation of the mTORC1 signaling pathway particularly in macrophages. The involvement of mTORC1 is clearly causal as the atherogenic effect of LCHP-feeding is abrogated in macrophage-specific Raptor-null mice. Further mechanistic evaluation of the effects of amino acids on macrophages reveals dichotomous roles on a predominant mTORC1 target, autophagy. Certain amino acids such as Leucine potently activate mTORC1 via recruitment to lysosome and in turn suppress autophagy via ULK1 phosphorylation, whereas others such as glutamine act indirectly by downregulating the transcription of autophagy chaperones including p62/SQSTM1. This combined suppressive effect on autophagy leads to macrophage inflammasome activation and IL-1β release, accumulation of deleterious protein aggregates, and increased cell death. The in vivo relevance of this LCHP-amino acid-mTORC1-autophagy axis is supported by 1) the absence of increased atherosclerosis in macrophage autophagy-deficient (ATG5-/-) mice fed a LCHP diet, and 2) the absence of reduced atherosclerosis in mice dually deficient in macrophage mTORC1 and autophagy (Raptor/ATG5-/-). Our data provide the first mechanistic details of the deleterious effects of high protein diets on macrophages and the development of atherosclerosis. Incorporation of these concepts in clinical studies will be important to define the vascular effects of dietary protein.
Abstract 39: Myeloid Klf2 Regulates Arterial and Venous Thrombosis
Authors: Nayak Lalitha , Lapping Stephanie , Vasudevan Neelakantan , Schmaier Alvin H , Mackman Nigel , Jain Mukesh K
Published: July 2021

Experimental, clinical and pathological studies have indicated an important link between inflammation and thrombosis. However, the precise mechanisms regulating myeloid cell function and the importance of various myeloid lineages involved in thrombosis remain incompletely defined. Published work from our group shows that a systemic deficiency of KLF2 renders animals susceptible to thrombosis. Using conditional knockouts, we examined the role of myeloid KLF2 in thrombosis. Carotid thrombosis assay (Rose Bengal model) show a robust reduction in time to occlusion in MY-K2-KO mice. No difference was noted in complete blood counts and coagulation assays between MY-K2-KO and control mice. Adoptive transfer of KLF2-KO neutrophils significantly shortened the time to occlusive thrombosis in control mice compared with the occlusion time in control mice given control neutrophils. No change in thrombosis time was noted in MY-K2-KO mice transfused with either control or KLF2-KO neutrophils. Neutrophil depletion with Ly6G antibody reversed the prothrombotic phenotype in MY-K2-KO mice and prolonged thrombosis time while no change was noted in thrombosis time in control mice following neutropenia. MY-K2-KO mice also developed significantly larger venous clot burden as compared to controls (complete ligation of inferior vena cava model). KLF2 deleted neutrophils demonstrated significantly increased tissue factor (TF) expression and activity. KLF2 deleted monocytes did not reveal change in TF expression as compared to controls. MY-K2-KO neutrophils also demonstrated increased generation of neutrophil extracellular traps and increased expression of neutrophil elastase (NE) and myeloperoxidase (MPO) as compared to controls. Other enzymes reported as important to the generation of NETs were not altered. TF, NE and MPO promoter-luciferase reporter assays demonstrated that KLF2 overexpression significantly abrogates p65-induced promoter activity. Collectively these studies identify neutrophil KLF2 as an important regulator of both arterial and venous thrombosis and illuminate the molecular mechanisms involved suggesting that modulation of neutrophil KLF2 may alter thrombosis.
Abstract 393: Pancreatic Beta Cell Export of miR-375 to High-Density Lipoproteins is Inversely Associated With Insulin Secretion
Authors: Sedgeman Leslie R , Sheng Quanhu , Guo Yan , Vickers Kasey C
Published: July 2021

microRNAs (miRNAs) are critical regulators of glucose metabolism and contribute to the pathogenesis of Type 2 Diabetes (T2D). Recently, we reported that high-density lipoproteins (HDL) transport and deliver functional miRNAs to recipient cells. Here, we report that miR-375 is decreased on HDL in two models of chronic hyperglycemia -- T2D human subjects and Zucker Diabetic Fatty (ZDF) rats. Since miR-375 expression in the islets is 10X greater than in other organs, we tested whether pancreatic beta cells have the ability to export miR-375 to HDL through
in vitro
export assays, incubating HDL with INS1 beta cells or primary human islets. Indeed, we found miR-375 to be readily exported to HDL from INS1 cells and primary islets
in vitro
. To determine if cholesterol transporters contribute to HDL-miR-375 export from beta cells, Abca1, Abcg1 and Scarb1 (SR-BI) were inhibited using siRNAs; however, we found that knockdown of each of these transporters failed to affect the beta cell’s ability to export miR-375 to HDL. Nonetheless, enhancing insulin secretion with tolbutamide resulted in the suppression of HDL-miR-375 export, suggesting that miRNA export and insulin secretion are inversely regulated. To determine the roles of Argonaute (Ago) family proteins in HDL-miRNA export, INS1 cells were transfected with siRNAs against
Eif2c1-4
to knockdown Ago1-4. We found HDL-miR-375 export to be suppressed when Ago1, but not Ago2-4, were inhibited, suggesting that miRNA export is downstream of miRNA processing by Ago1. We are currently investigating the relationship between HDL-miR-375 export, insulin secretion, and miRNA processing in pancreatic beta cells to elucidate the mechanism(s) controlling HDL-miR-375 export. Collectively, results suggest that a large fraction of HDL-miRNAs originate from pancreatic beta cells and HDL-miRNAs are exported independent of cholesterol transporters.
Abstract 396: Acrolein Modification Weakens the Antimicrobial Activity of Apolipoprotein A1
Authors: Weers Paul , Hong Hea J
Published: July 2021

Human apolipoprotein A-I (apoA-I) has been show to exhibit antimicrobial activity by neutralizing lipopolysaccharides and destabilizing inner membranes of gram-negative bacteria. Previous studies showed that acrolein, a highly reactive αβ unsaturated aldehyde generated in cigarette smoking, modifies ε-amino side chains of lysine residues in apoA-I. The current study investigated the effect of acrolein exposure on the structure and antimicrobial activity of apoA-I. Incubation of apoA-I with acrolein using a 1:20 molar ratio, acrolein modification was evident by the appearance of apoA-I oligomers due to intermolecular crosslinking. Increase of the acrolein to protein ratio resulted in heavily cross-linked apoA-I, with protein bands appearing at 63, 98, and 126 kDa. The presence of acrolein-modified lysines in the oligomers was verified through Western blot analysis using mab5F6 antibody that specifically detects acrolein modified lysine residues in proteins. The structural changes of modified apoA-I was analyzed using circular dichroism. The α-helical content of acrolein-modified apoA-I was not significantly different from the unmodified protein. However, the midpoint of guanidine-induced denaturation increased from 0.97 to 1.50 M guanidine upon modification, indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the protein fold was altered due to cross-linking. To measure the effect of acrolein modification on the interaction with bacterial membranes, binding experiments were performed with phosphatidylglycerol entrapped with calcein. This showed that the percentage of calcein released by apoA-I decreased from 87.5 ± 2.3 % to 4.7 ± 0.13 % when the protein was modified by acrolein. Thus acrolein-modified apoA-I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interactions with anionic phospholipid vesicles. In addition, binding of apoA-I to lipopolysaccharides was significantly weaker when the protein was modified by acrolein. These results suggest that apoA-I modification by acrolein results in a protein with a decreased ability to bind to bacterial membranes and is thus less potent as an antimicrobial protein.
Abstract 4: Deletion of Macrophage Low-density Lipoprotein Receptor-related Protein 1 (LRP1) Accelerates Atherosclerosis Regression by Increasing CCR7-dependent Egress of Plaque Macrophages
Authors: Mueller Paul , Zhu Lin , Giunzioni Illaria , Tavori Hagai , Stafford John M , Linton MacRae , Fazio Sergio
Published: July 2021

We previously showed that mice lacking macrophage LDL receptor-related protein 1 (LRP1) undergo accelerated lesion formation due to increased apoptosis, decreased efferocytosis, and enhanced macrophage transformation into the inflammatory M1 phenotype.
In vitro,
LRP1-deficient macrophages (MΦLRP1
-/-
) show enhanced plasticity with exaggerated polarization towards either the inflammatory M1- or the anti-inflammatory M2-phenotype depending on the stimulant (LPS or IL-4, respectively). During atherosclerosis regression, the M2:M1 macrophage ratio increases as lesion M1 macrophages egress and inflammation resolves. Thus, we hypothesize that atherosclerosis regression is accelerated in MΦLRP1
-/-
mice via enhanced macrophage M2 polarization and CCR7-dependent M1 macrophage egress. ApoE-/- mice on high fat diet for 12 weeks were reconstituted with bone marrow from wildtype (WT) or MΦLRP1
-/-
mice and then placed on chow diet for 8 weeks. In this model, apoE is reintroduced into circulation to correct the hyperlipidemia and induce regression of atherosclerotic lesions. A cohort of apoE
-/-
mice reconstituted with apoE
-/-
bone marrow served as baseline controls. Lesions in both WT and MΦLRP1
-/-
mice regressed relative to controls (11% and 22%, respectively; p<0.05), but MΦLRP1
-/-
lesions were 13% smaller than those of WT mice (p<0.05). LRP1 deletion increased M2 transformation of macrophages and a higher M2:M1 macrophage ratio (p<0.01) in the plaque. MΦLRP1
-/-
lesions contained 36% fewer M1 macrophages compared to WT (p<0.01).
In vivo
studies of reverse cholesterol transport (RCT) revealed that MΦLRP1
-/-
have a 1.4-fold higher RCT compared to WT mice (p<0.01). MΦLRP1
-/-
lesions contained 2.5-fold more CCR7
+
macrophages relative to WT lesions (p<0.01), and in our
in vivo
egress assay 4.6-fold more CCR7
+
macrophages were found in mediastinal lymph nodes.
In vitro
, M1-differentiated MΦLRP1
-/-
macrophages expressed 1.6-fold higher
Ccr7
mRNA compared to WT controls (p<0.01). Thus, the absence of macrophage LRP1 accelerates atherosclerosis regression due to enhanced transformation of macrophages into an anti-inflammatory M2 phenotype, increased cholesterol efflux, and increased CCR7-driven egress of M1 macrophages from lesions.
Abstract 407: High Density Lipoprotein Mediated Protection of Macrophages Against Apoptosis Requires Scavenger Receptor Class B Type 1 Activity and Sphingosine-1-Phosphate
Authors: Chathely Kevin , González Leticia , Trigatti Bernardo
Published: July 2021

Background/Objectives:
Prevention of macrophage apoptosis in advanced atherosclerotic lesions can help stop atherosclerosis progression to vulnerable plaques. High density lipoprotein (HDL) can protect macrophages from apoptosis that has been induced by a variety of agents. We hypothesize that this is the consequence of the sphingolipid, sphingosine-1-phosphate (S1P), specifically carried by HDL, and transferred to S1P receptor 1 (S1PR1) on the cells via the HDL receptor, scavenger receptor class B type 1 (SR-B1).

Methods:
Apoptosis was induced in murine peritoneal macrophages from wild type and different knockout mice with the ER stress inducing agent tunicamycin. Apoptosis was then observed and detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling through fluorescent microscopy. All experiments were conducted with an n of 3 or 4.

Results:
Treatment of cells with HDL protected them against tunicamycin induced apoptosis. In contrast, pre-treatment of HDL with S1P lyase, which irreversibly cleaves S1P, eliminated the ability of HDL to protect macrophages. Furthermore, HDL-dependent protection of macrophages against apoptosis required both the HDL receptor SRB1 and the S1PR1. Inhibitor of SRB1’s lipid transport activity also prevented HDL dependant protection against apoptosis.

Conclusions:
These results suggest that the HDL mediated protection of macrophages against apoptosis may involve SRB1 mediated delivery of S1P from HDL to the S1PR1. Understanding the mechanisms by which HDL elicits atheroprotective signalling in macrophages will provide insight into new targets for therapeutic intervention.
Abstract 409: Androgen Inhibits Key Atherosclerotic Processes by Directly Activating
ADTRP
Transcription
Authors: Chen Qiuyun , Luo Chunyan , Leineweber Kirsten , Tang Bo , Zhang Weiyi , Li Sisi , Pook Elisabeth , Cheung Shing-Hu , Bechem Martin , Hu Jing-Shan , Laux Volker , Wang Qing
Published: July 2021

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the
ADTRP
gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of
ADTRP
was regulated by androgen, but the molecular mechanism is unknown. Here, we tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating
ADTRP
expression. Luciferase assays with an
ADTRP
promoter luciferase reporter revealed that androgen regulated
ADTRP
transcription. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE) located at +324bp from the
ADTRP
transcription start site. The ARE is required for the transcriptional activation of
ADTRP
. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was reduced by
ADTRP
siRNA and enhanced by overexpression of
ADTRP
. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of
ADTRP
expression.
Abstract 415: Repetitive Surgical Procedures in Vascular Patients Are Associated With Perioperative Cardiac Events
Authors: Janssen Henrike , Felgner Larissa , Gillmann Hans-Jörg , Schrimpf Claudia , Rustum Saad , Lichtinghagen Ralf , Teebken Omke , Theilmeier Gregor , Larmann Jan
Published: July 2021

Introduction:
Vascular surgery patients are at risk of myocardial injury after non-cardiac surgery (MINS) associated with perioperative mortality. Underlying mechanisms are largely unknown. In animal models surgery combined with blood loss promotes atherosclerotic lesion progression and plaque destabilization. It is unknown whether repetitive surgery contributes to cardiac risk in patients.

Hypothesis:
Repetitive surgical procedures in vascular surgery patients are associated with increased MINS rates.

Methods:
With IRB approval, after obtaining informed consent, pre- and day-1 post-operative (OP) plasma samples were collected from 663 patients undergoing elective aortic-, peripheral vascular or carotid surgery. High-sensitive cardiac troponin T (hs-cTNT) (Roche) was measured pre- and post-OP. Additional 3
rd
generation cTnT or hs-cTNT measurements were prompted on clinical suspicion for acute coronary syndrome. MINS was defined as any new (delta ≥50%) hs-cTNT ≥50 ng/L or 3
rd
gen. cTnT >0.03 ng/mL. Data are presented as median (inter quartile range (IQR)) and were compared using Wilcoxon matched-pairs signed tank test. Incidence of the combined endpoint between 1
st
and 2
nd
surgeries was compared using one-sided chi square test. p<0.05 was considered significant.

Results:
We identified 40 patients with two repetitive surgical procedures. For 37 patients pre- and post-OP blood samples were available. Median time between surgeries was 53 (43.5-208) days. There were no statistical differences in pre-OP medication or risk factors. Pre-OP hs-cTNT values were slightly higher prior to the 2
nd
procedure (1
st
vs. 2
nd
procedure, 11.2 (6.6-19.9) vs 12.6 (7.9-31.3) ng/l, n=37, p<0.05). One patient experienced MINS after the 1
st
surgery. Five patients reached the endpoint after the 2
nd
procedure (1
st
vs 2
nd
procedure, n=37, p<0.05).

Conclusions:
In vascular patients repetitive surgery is associated with myocardial injury. The underlying mechanisms need to be examined in more detail. However, clinicians should recognize the elevated cardiovascular risk associated with repetitive operations. Among other things, prophylactic strategies for prevention of perioperative cardiac events should focus on patients undergoing repetitive operations.
Abstract 43: Cholesterol Efflux Pathways Suppress Inflammasome Activation in Mice and Humans
Authors: Westerterp Marit , Fotakis Panagiotis , Ouimet Mireille , Bochem Andrea E , Zhang Hanrui , Molusky Matthew M , Wang Wei , Abramowicz Sandra , Wang Nan , Welch Carrie L , Reilly Muredach P , Stroes Erik S , Moore Kathryn J , Tall Alan R
Published: July 2021

Plasma high-density-lipoprotein (HDL) has several anti-atherogenic properties, including its key role in functioning as acceptor for ATP-binding cassette A1 and G1 (ABCA1 and ABCG1) mediated cholesterol efflux. We have shown previously that macrophage
Abca1/g1
deficiency accelerates atherosclerosis, by enhancing foam cell formation and inflammatory cytokine expression in atherosclerotic plaques. Macrophage cholesterol accumulation activates the inflammasome, leading to caspase-1 cleavage, required for IL-1β and IL-18 secretion. Several studies have suggested that inflammasome activation accelerates atherogenesis.

We hypothesized that macrophage
Abca1/g1
deficiency activates the inflammasome. In

Ldlr
-/-

mice fed a Western type diet (WTD), macrophage
Abca1/g1
deficiency increased IL-1β and IL-18 plasma levels (2-fold;
P
<0.001), and induced caspase-1 cleavage. Deficiency of the inflammasome components
Nlrp3
or
caspase-1
in macrophage
Abca1/g1
knockouts reversed the increase in plasma IL-18 levels (
P
<0.001), indicating these changes were inflammasome dependent. We found that macrophage
Abca1/g1
deficiency induced caspase-1 cleavage in splenic CD115
+
monocytes and CD11b
+
macrophages. While mitochondrial ROS production or lysosomal function were not affected, macrophage
Abca1/g1
deficiency led to an increased splenic population of monocytes (2.5-fold;
P
<0.01). Monocytes secrete ATP, and as a result, ATP secretion from total splenic cells was increased (2.5-fold;
P
<0.01), likely contributing to inflammasome activation.
Caspase-1
deficiency decreased atherosclerosis in macrophage
Abca1/g1
deficient

Ldlr
-/-

mice fed WTD for 8 weeks (225822 vs 138606 μm
2
;
P
<0.05). Of therapeutic interest, one injection of reconstituted HDL (100 mg/kg) in macrophage
Abca1/g1
knockouts decreased plasma IL-18 levels (
P
<0.05). Tangier disease patients, with a homozygous loss-of-function for ABCA1, showed increased IL-1β and IL-18 plasma levels (3-fold;
P
<0.001), suggesting that cholesterol efflux pathways also suppress inflammasome activation in humans. These findings suggest that macrophage cholesterol efflux pathways suppress inflammasome activation, possibly contributing to the anti-atherogenic effects of HDL treatment.
Abstract 434: The Majority of Outpatients With Severe Hypercholesterolemia at an Academic Tertiary Centre Have Not Been Screened for FH
Authors: Ajufo Ezim , Ueda Masako , Hossain Erik , Sikora Tracey , Damrauer Scott M , deGoma Emil M , Dilzell Kristen , DerOhannessian Stephanie , Cuchel Marina , Rader Daniel J
Published: July 2021

Background:
National and International guidelines recommend, as a minimum, that individuals with LDL-C ≥190mg/dL are asked about a family history of ASCVD and hypercholesterolemia to screen for familial hypercholesterolemia (FH), a monogenic condition that carries considerable ASCVD risk. However, FH is grossly underdiagnosed and undertreated in the US. One reason for this may be failure to screen severely hypercholesterolemic individuals for FH.

Hypothesis:
We evaluated the hypothesis that poor identification of FH might be due to inadequate screening for FH among individuals with severe hypercholesterolemia.

Methods:
An EHR query was used to identify active adult patients in the University of Pennsylvania outpatient EHR database (N=310 802) with LDL-C≥220mg/dL, excluding secondary causes of hypercholesterolemia. The EHR was then systematically reviewed for structured notation of family history information.

Result:
The query identified 3,475 individuals with severely elevated LDL-C. Among them, only 47.9% (1666) had family history data relating to ASCVD (968), hypercholesterolemia (336) or both (362) in the EHR. The history was positive in 94.2% (1569) of these cases. Overall, patients with LDL-C ≥220mg/dL were more likely to be screened (OR 1.57; 95% CI 1.47-1.68) than those with LDL <220mg/dL. Within the former group, the odds of being screened were higher in Caucasians (OR 1.52; 95% CI 1.33-1.75), with more severe LDL-C elevation or a history of ASCVD (OR 1.36; 95% CI 1.10 - 1.67). Conversely, the presence of diabetes (OR 0.73; 95% CI 0.61-0.86) or hypertension (OR 0.82; 95% CI 0.72-0.94), made screening less likely to occur. Interestingly, the setting of clinical care was also important; individuals seen in secondary care (OR 1.68; 95% CI 1.41-2.26), general (OR 1.69, 95% CI 1.47-1.95) or preventative cardiology (OR 1.82; 95% CI 1.47-2.25) were more likely to be screened. Finally, statin prescription was more common in those screened (OR 1.39; 95% CI 1.16-1.88), but this did not affect LDL-C goal achievement.

Conclusion:
At a large academic centre, the majority of outpatients with severe hypercholesterolemia had no record of being screened for FH. This report sheds light on factors that might be relevant to the underdiagnosis of FH in the US.
Abstract 438: Macrophages in a BAPN/AT2 Induced Model of Murine Aneurysm are Predominantly Lyve-1 and Tim-4 Negative
Authors: Byrne John S , Besla Rickvinder , Li Angela , Al-chami Edouard , Legg Karen , Chen Emily , Ouzounian Maral , Lindsay Thomas , Robbins Clint
Published: July 2021

Macrophages are key effector cells in aneurysm progression. Aneurysm macrophages may derive from monocyte recruitment and turnover of resident cells. We tested the hypothesis that aneurysm macrophages have a non-resident (lyve-1/tim-4 negative) phenotype.

C57/Bl6 mice were administered beta-aminopropriononitrile (BAPN) and angiotensin-2 (AT2). At four weeks, whole aortas were excised, photomicrographed, and single cell suspensions created for immunophenotyping with flow cytometry. Results were compared to wild-type controls.

BAPN/AT2 causes aortic dilatation (p<0.01) with maximal aneurysmal degeneration at the suprarenal aorta (wild-type control aortic diameter 0.93±0.03mm, n=8; BAPN/AT2 2.00±0.10mm, n=23; p<0.0001). BAPN/AT2 significantly increased aortic cd45+ myeloid-lymphoid cells and cd11b+ f4/80+ macrophages (p<0.02). Maximal aneurysm diameter correlated positively with aortic cell numbers of cd45+ myeloid-lymphoid cells (R=0.8168, p<0.001) and macrophages (R=0.5977, p=0.02). In wild-type, aortic macrophages are predominantly lyve-1 positive (67±3%, n=10). Whilst overall macrophage numbers are increased in aneurysm, the percentage of lyve-1 positive macrophages is significantly reduced (40±3%, n=18; p<0.001). The number of lyve-1 negative macrophages (R=0.6875, p=0.02), correlated with maximal suprarenal aortic diameter. In controls and aneurysm, lyve-1 positive, but not lyve-1 negative, macrophages are also tim-4 positive.

In a BAPN/AT2 murine aneurysm model, aortic cd45+ myeloid-lymphoid cells and macrophages are increased and cell counts correlate with maximal aortic dilatation. Wild-type murine aortic macrophages are predominantly lyve-1/tim4 positive, with a significant relative increase in lyve-1/tim4 negative macrophages in aneurysm, suggesting lyve-1/tim-4 may indicate a resident macrophage phenotype.