bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 54: Peptidylarginine Deiminase 4-dependent Generation of Neutrophil Extracellular Traps is Crucial for Deep Vein Thrombosis in Mice
Authors: Martinod Kimberly , Demers Melanie , Fuchs Tobias A , Wong Siu Ling , Brill Alexander , Gallant Maureen , Wang Yanming , Wagner Denisa D
Published: July 2021

Introduction

Histone hypercitrullination by the enzyme peptidylarginine deiminase 4 (PAD4) leads to nuclear chromatin decondensation that is needed for neutrophil extracellular trap (NET) formation. NETs consist of chromatin and granule proteins that are released into the extracellular environment. NETs were shown to be involved in thrombosis by promoting coagulation and platelet adhesion and were identified in the thrombus scaffold in animal models of deep vein thrombosis (DVT).

Objective

Whether NETs are involved in the pathogenesis of DVT or whether they are merely a consequence of neutrophil recruitment to the thrombus is unknown. We hypothesized that NET formation would be impaired in PAD4-deficient mice during deep vein thrombosis and that this may affect thrombus formation and/or stability.

Methods

PAD4-deficient mice are incapable of citrullinating histones and therefore fail to decondense chromatin during NETosis. We performed the inferior vena cava stenosis model of DVT in wild-type or PAD4-/- mice. Intravital microscopy was done to assess leukocyte vessel wall interaction in PAD4 deficiency.

Results

We induced NET formation in isolated peripheral blood mouse neutrophils with ionomycin and found that PAD4-/- neutrophils had a complete inability to produce NETs (WT, 20.65±2.61% NETs; PAD4-/-, not detected. n=4). Leukocyte-endothelial interactions in PAD4-/- mice were not impaired upon induction of systemic Weibel-Palade body release (WT, 55.2±11.8; PAD4-/-, 62.0±17.5 cells/min, n=5-6). In the DVT model, while a majority (9/10) of wild-type mice formed a thrombus 48 hours after stenosis, only 1 of 11 PAD4-/- mice formed a thrombus. Thrombus formation could be rescued by infusions of isolated WT bone marrow neutrophils into PAD4-/- mice, and extracellular H3Cit+ areas were seen within these thrombi. This data suggests that neutrophil PAD4 was essential for thrombus formation in deep veins.

Conclusion

NETs comprise a crucial part of the pathologic thrombus scaffold, and here we report that the lack of NETs inhibits pathological thrombosis. Chromatin decondensation initiated by PAD4 in neutrophils is a key player in the formation of deep vein thrombi and targeting neutrophil histone modification could be a new way to prevent DVT.
Abstract 540: Loss of Function Studies Highlight the Vasculoprotective Effect of A20 in Transplant Arteriosclerosis
Authors: Lee Andy , Peterson Clayton R , Moll Herwig , Csizmadia Eva , Daniels Kathleen , Notartomaso Ashley , Neidich Alon , Chien Victor , DaSilva Cleide G , Ferran Christiane
Published: July 2021

Transplant arteriosclerosis (TA) is an accelerated form of atherosclerosis largely driven by the immune response to the allograft. As for all atherosclerotic disease, inflammation is central to vascular remodeling associated with TA. We have shown that overexpression of the NF-κB inhibitory protein in totally mismatched (C57BL/6 to BALB/c) aortic to carotid vascular allografts protects from TA. A20 inhibits TA thanks to its anti-inflammatory and anti-apoptotic functions in endothelial cells (EC), anti-inflammatory and anti-proliferative functions in smooth muscles cells (SMC), pro-apoptotic function in neointimal SMC, and to favoring regulatory FOXP3+ over pathogenic Th1/Th17 T cells.

In this work, we analyzed the impact of A20 knockdown on TA by using aortic allografts from A20 heterozygous (A20-+/- HET) mice. Partial loss of A20 in aortic allografts caused significantly greater TA lesions, measured by intima over media ratios (I/M 1.79±0.18 in HET vs. 1.04±0.17 in wild type (WT) allografts; p<0.01). This was associated with higher inflammation as evaluated by VCAM-1 immunostaining and granulocyte infiltration, lower expression of the anti-inflammatory and tolerogenic cytokine IL-10, and greater incidence of thrombosis (3/11 vs. 0/10) in HET vs. WT allografts. These results are particularly relevant to newly discovered A20 single nucleotide polymorphisms (SNPs) that are less effective in inhibiting inflammation, and have been associated with increased risk of coronary artery disease in diabetic patients.

Based on these results, we conclude that A20 plays an important physiologic role in the vascular remodeling of TA. We propose that tailored A20-based vascular therapies could decrease incidence and severity of TA, and synergize with or minimize use of immunosuppression in solid organ transplantation.
Abstract 547: TGFß Response Coupled to Stress Signaling by TGRL Lipolysis in Vascular Endothelial Cells
Authors: Aung Hnin , Eiselein Larissa , Lamé Michael W , Ng Kit , Wilson Dennis W , Rutledge John C
Published: July 2021

Elevation of circulating triglyceride-rich lipoproteins (TGRL) in the postprandial state is associated with increased endothelial cell inflammation and dysfunction potentially contributing to atherosclerosis. Previous studies from our laboratory showed that lipolysis products of TGRL induced endothelial cell barrier permeability, up-regulation of cytokines and adhesion molecules, and apoptosis. Gene array studies implicated the Stress Associated Protein Kinase and c-Jun N-terminal kinase (SAPK/JNK) pathway and identified the transcription factor activating transcription factor 3 (ATF3) as highly induced in this process. Additional studies demonstrated activation of second messengers for transforming growth factor beta (TGFβ) signaling (SMAD2/SMAD4) in response to lipolysis product treatment. In the present study, we asked whether TGFβ signaling was implicated in the induction of apoptosis by lipolysis products and whether there was a linkage between TGFβ receptor activation and pro-inflammatory responses. siRNA knockdown of ATF3 transcription abrogated both IL-8 and E-selectin up-regulation in lipolysis product-treated human aortic endothelial cells (HAEC). In addition, ATF3 knockdown prevented activation of caspase 3/7 and induction of p53, markers of apoptosis. Inhibition of the TGFβ Type I activin receptor-like kinase (ALK) receptors (ALKs 4, 5 and 7) by SB 431542 (10 μM) also inhibited inflammatory up-regulation, ATF3 induction and apoptosis. Both ATF3 knockdown and TGFβ receptor blockade prevented c-Jun phosphorylation, a co-activating transcription factor with ATF3 of AP-1 transcription sites. Our findings implicate TGFβ signaling in the inflammatory and apoptotic responses to TGRL lipolysis and suggest a complex interaction between TGFβ receptors and the SAPK subset of MAP Kinases.
Abstract 56: Macrophage Reverse Cholesterol Transport in Mice Relies on The Lymphatic Vasculature
Authors: Martel Catherine , Li Wenjun , Fulp Brian , Platt Andrew , Gautier Emmanuel L , Westerterp Marit , Bittman Robert , Tall Alan R , Chen Shu-Hsia , Chen Shu-Hsia , Thomas Michael J , Kreisel Daniel , Swartz Melody A , Sorci-Thomas Mary G , Randolph Gwendalyn J
Published: July 2021

Reverse cholesterol transport (RCT) refers to mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used two models of disrupted lymphatic drainage from skin -one surgical and the other genetic- to quantitatively track RCT following injection of [3H]cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analogue (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aorta of donor atherosclerotic apoE-/- mice with [2H]6-labeled cholesterol and surgically transplanting these aortas into recipient apoE-/- mice that were treated with anti-VEGFR3 mAb to block lymphatic regrowth or with control mAb to allow such regrowth. [2H]-Cholesterol was retained in aortas of anti-VEGFR3 treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. Therefore, supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.
Abstract 565: Nuclear Receptor NR4A1 Represses SDF1a expression in macrophages and Reduces Atherosclerosis.
Authors: Hamers Anouk A
Published: July 2021

Rationale

NR4A1 is a nuclear receptor, also known as Nur77 and is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. NR4A1-knockout (NR4A1
-/-
) mice lack Ly6C
-
monocytes, but have normal LyC6
+
numbers. Macrophages play a critical role in atherosclerosis and we have demonstrated that NR4A1 has an anti-inflammatory function in THP-1 macrophages. The aim of the current study is to assess the function of this receptor in myeloid cells in atherosclerosis.

Objective

This study aims to delineate the function of Nuclear Receptor NR4A1 in macrophages in atherosclerosis.

Methods and results

Bone marrow-derived macrophages (BMM) from wild-type and NR4A1
-/-
mice were cultured and classically activated with LPS or alternatively activated with IL4. NR4A1
-/-
BMM exhibit changed expression of M2-specific markers and a pro-inflammatory polarization in response to LPS with enhanced expression of IL12, IFNγ, and SDF1α. Nitric oxide synthesis is also strongly induced in (non)-stimulated NR4A1
-/-
BMM. SDF1α is a potent chemotactic factor for B cells and monocytes. The chemoattractive activity of NR4A1
-/-
BMM is abolished by SDF1α inhibiting antibodies as well as by overexpression of NR4A1 in the NR4A1-deficient cells. Luciferase experiments show that NR4A1 binds directly to several response elements present in the mouse and human SDF1α promoter. A ChIP assay confirms these results. The effect of bone marrow-specific NR4A1-deficiency on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr
-/-
) mice. Ldlr
-/-
mice with a NR4A1
-/-
bone marrow develop 2.1-fold larger lesions than wild type bone marrow transplanted mice; containing more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF1α expression is also higher in these lesions, which may explain the observed aggravation of lesion formation, since SDF1α is not only a chemoattractant for monocytes but also enhances smooth muscle cell proliferation.

Conclusion

In macrophages the nuclear receptor NR4A1 has an anti-inflammatory function, represses SDF1α expression and, most importantly, bone-marrow transplantation studies in Ldlr
-/-
mice revealed that NR4A1 inhibits atherosclerosis.
Abstract 66: SR-BI Mediates Cholesteryl Ester (CE) Sorting in Tetraspanin Microdomains with CD81 Facilitating CE Nano-endocytosis and CE Transport to Lipid Droplets
Authors: Bocharov Alexander V , Vishnyakova Tatyana G , Baranova Irina N , Lizunov Vladimir A , Zimmerberg Joshua , Eggerman Thomas L , Patterson Amy P
Published: July 2021

SR-BI functions as a neutral lipid transporter facilitating cholesteryl ester (CE) sorting, internalization and accumulation in LDs. The mechanism of CE transport remains obscure. We hypothesized that HDL lipid transport requires CD81 as a co-receptor and tetraspanin enriched domains (TED) for CE sorting, similar to hepatitis C uptake.

Utilizing BODIPY-CE labeled recombinant HDL (rHDL/BP-CE) based on either human apoA-I or an apoA-I mimetic peptide, L-37pA, and confocal and TIRF microscopy, we found that after initially binding to SR-BI at the cellular filopodia and lamellipodia, HDL CE was laterally transported to the bases of filopodia, accumulating in 1-2 μm plasma membrane microdomains. While CE was rapidly internalized, neither apoA-I, L37pA nor SR-BI was, suggesting that these microdomains are an important part of the CE sorting mechanism. Internalized CE was transported in 50-100 nm CE-containing intracellular nano-vesicles. In a process of multiple collisions potentially involving transient nano-vesicle/LD fusion, BODIPY-CE was transferred to LDs. To identify CE-sorting microdomains, SR-BI expressing HeLa cells were transfected with GFP-CD81 or GFP-CD82. After a 1 h incubation with rHDL/BP-CE, CE was co-localized with both tetraspanin proteins demonstrating that these are TED (the observation was confirmed by anti-CD81 and -CD-82 antibody immunostaining). Following a 2 h chase, only GFP-CD81, not CD82, was found internalized and co-localized with CE transporting nano-vesicles. CE transport was not affected by inhibitors of de novo neutral lipid synthesis such as the ACSL-3 inhibitor, triacsin c, ACAT inhibitor, Sandoz 58-035 or ATGL inhibitor, A926500, indicating that CE was directly transported through a novel nano-endocytosis mechanism involving CD81 and not requiring CE hydrolysis and de novo synthesis.

We conclude that TED function as CE sorting sites where CD81 helps to form nano-vesicles delivering CE to LDs. Further understanding of their function will help develop novel anti-atherogenic compounds accelerating reverse cholesterol transport.
Abstract 75: Somatic Overexpression and Knockdown in Mice to Identify Causal Genes Underlying 26 Lipid-associated Loci
Authors: Pashos Evanthia , Stylianou Ioannis M , Marchadier Dawn , Picataggi Antonino , Redon Valeska S , Lukmanova Daniya , Xu Yuxin , Frank-Kamenetsky Maria , Barnes Carmen , Ruda Vera , Fitzgerald Kevin , Kathiresan Sekar , Rader Daniel J
Published: July 2021

Genome-wide association studies (GWAS) have identified 95 loci in the human genome that harbor common variants associated with plasma lipid traits. Of the 95 loci, 17 harbor genes known to cause monogenic lipid disorders and collectively a third of them contain genes with characterized roles in lipid metabolism. Therefore in the majority of loci the causal genes are unknown. We selected 32 genes, not previously implicated in lipid metabolism and representing a total of 26 loci, to test for their ability to modify plasma lipid concentrations upon somatic overexpression in vivo. We utilized adeno-associated virus serotype 8 (AAV8) to overexpress the selected genes specifically in the livers of both C57BL/6 mice and in an appropriate humanized mouse model (either mice expressing human apolipoprotein A-I for HDL loci or Apobec1-knockout, Ldlr haploinsufficient mice expressing human apolipoprotein B-100 for triglyceride and LDL loci). Approximately half of the genes tested reproducibly affected plasma lipids. For 13 of the interrogated loci the lipid-associated variants also correlated with expression variations of the respective genes in liver (liver expression quantitative trait loci-eQTLs). We demonstrate a causal role for 7 of these 13 genes. The overexpression of these 7 genes not only affected the predicted lipid class, but additionally exerted its effect in the predicted direction in 6 of 7 cases (Tmem57, Slc39a8, Ppp1r3b, Vkorc1, Tbkbp1 and Ube2l3). Additionally for a subset of the examined genes we proceeded to develop small interfering RNA (siRNA) nanoparticles that were particularly targeted to the liver. We were able to obtain robust knockdown for a significant number of genes and, in several cases, observe reciprocal effects on plasma lipids from our overexpression and knockdown studies. This work has identified several novel lipid regulators, whose further investigation can uncover novel mechanisms and pathways controlling plasma lipids.
Abstract 108: Diagnostic and Prognostic Biomarker Potential of miR-24 in Abdominal Aortic Aneurysm Disease and Rupture
Authors: Chernogubova Ekaterina , Eken Suzanne , Spin Joshua , Jin Hong , Raaz Uwe , Roy Joy , Eriksson Per , Tsao Philip , Maegdefessel Lars
Published: July 2021

MicroRNAs (miRNAs) have lately received much attention regarding their suitability and feasibility as biomarkers for cardiovascular diseases. Aim of this current study was to explore the diagnostic and prognostic value of detecting circulating levels of miRNAs in abdominal aortic aneurysms (AAAs).

Using a PCR-based array, we profiled the 168 most abundant blood miRNAs in 20 patient plasma samples with AAA disease, undergoing surgical repair of their enlarged aorta vs. 20 samples from a matched screening cohort without aneurysm. We were able to identify a total number of 12 miRNAs that were significantly altered in diseased patient samples as compared to controls. Next we investigated these 12 miRNAs in plasma from ApoE-/- mice with angiotensinII (AngII)-infusion induced AAAs, in order to determine the potential prognostic value of miRNAs being released into circulation. Indeed we were able to detect that the expression of 4 out of the 12 miRNAs (miRs-126 and -668 both increased; miRs-24 and -210 both decreased), was substantially altered in plasma samples drawn from AAA mice immediately before rupture occurred between days 10 and 14 after AngII pump implantation compared to mice with AAA that did not rupture for the remainder of study (28 days), as well as saline-infused controls. The expression of miR-24 was furthermore significantly different in plasma samples from human patients with acutely ruptured AAAs (n=7) compared to patients with non-ruptured AAAs (AA diameter between 55-78 mm; n=7) undergoing surgical repair, as well as un-diseased controls (n=7). Using the AngII model, as well as the elastase infusion model, we were able to show that overexpression of miR-24 protects from aneurysm progression as well as aortic rupture (in the AngII model) by repressing the expression of its target gene chitinase3-like1 (Chi3l1), which regulates macrophage survival and cytokine release in expanding murine AAAs.

The present study explores the biomarker potential of miRNAs being released into circulation during initiation, propagation, and ultimately rupture of AAA disease in mice and humans. The identification of miR-24 potentially offers prognostic value to determine which patients present with increased risk of rapid AAA expansion and subsequent rupture.
Abstract 116: Simvastatin Affects Monocyte Adhesion and Infiltrative Activity in Patients With Abdominal Aortic Aneurysms
Authors: Samadzadeh Kiana M , Nguyen Anthony , Chun Kevin C , Lee Eugene S
Published: July 2021

Purpose:
The pleiotropic effects of statin drugs on reducing inflammation have been well regarded in decreasing AAA expansion. We hypothesize that increased monocyte activity plays a central role in AAA formation and expansion. This study examines whether statins can prevent monocyte cell adhesion, transmigration, and matrix metalloproteinase (MMP) and inhibitor (TIMP) concentrations in AAA patients compared to non-AAA patients.

Methods:
Peripheral blood was collected for monocyte and serum isolation from control (n=4) and AAA (n=8) patients. Monocyte adhesion and transmigration were assessed under untreated, statin treated, and statin + mevalonate (statin inhibitor) treated conditions in vitro. Luminex assays determined MMP and TIMP concentrations from cell culture and patient serum.

Results:
Untreated AAA patient monocytes showed higher levels of adhesion (p=0.05) and transmigration (p=0.04) compared to control subjects (Figure 1A & 1B). Statin treatment caused a decrease in AAA monocyte adherence to the endothelium (p=0.03) and high concentrations of mevalonate reversed statin treatment effects (p=0.04) (Figure 1A). A similar trend was noted in monocyte transmigration (Figure 1B). Higher concentrations of MMP-9 were found in AAA patient serum compared to controls (p=0.01) (Figure 1C). TIMP-4 concentration were decreased in AAA patients compared to controls (p=0.02) (Figure 1D).

Conclusions:
Statins reduce monocyte interaction with the endothelium in vitro, leading to decreased levels of MMP-9 and increased levels of TIMP-4, implying a possible mechanism by which statins reduce AAA expansion.
Abstract 122: STAT3 Mediates Toll-Like Receptor 4--Dependent Abdominal Aortic Aneurysm Formation in Angiotensin II--Treated ApoE-/- Mice
Authors: Qin Zhexue , Park Ho-Jin , Beasley Debbie , Galper Jonas , Zhang Yali
Published: July 2021

Abdominal aortic aneurysm (AAAs) is a life threatening disease and currently the only treatment is surgical or endovascular repair. Using an animal model for AngiotensinII (AngII) mediated AAA formation in the ApoE-/- mouse, we demonstrate that AAA formation is dependent on the activation of the JAK/STAT pathway. STAT3 activity was increased in the abdominal aortas of Ang II treated ApoE-/- mice. Treatment of ApoE-/- mice with AngII and the p-STAT3 inhibitor S3I-201 decreased the incidence of AAA formation and aortic diameter by greater than 50% compared to placebo. While AngII treatment increased activity of MMP2 and MMP9 in aortas of ApoE-/- mice, S3I-201 treatment inhibited p-STAT3 stimulation and decreased MMP2 and MMP9 activity to levels similar to those in control aortas. The increase in p-STAT3 levels and the secretion of MMP2 and MMP9 in AngII treated bone marrow derived macrophages (BDMs) from ApoE-/- mice was inhibited by pretreatment with S3I-201. Interestingly, AngII stimulated TLR4 expression in AAAs and in BDMs from ApoE-/- mice. Based on these findings we determined the role of TLR4 on AAA formation and p-STAT3 stimulation in ApoE-/-TLR4-/- mice. The incidence of AAA formation, increase in aortic diameter and MMP2 and MMP9 activity were markedly decreased in AngII treated ApoE-/-TLR4-/- mice compared to ApoE-/- controls. Importantly, AngII stimulation of p-STAT3 was also decreased in ApoE-/-TLR4-/- mice compared to control. Similar effects on AngII stimulated p-STAT3 and activity of MMP2 and MMP9 were demonstrated in BMDs from ApoE-/-TLR4-/- mice. Treatment of ApoE-/- mice with the TLR4 inhibitor Eritoran (Eisai, Inc) also decreased the incidence and severity of AAAs, MMPs secretion and p-STAT3 activity. Finally, we determined that S3I-201 decreased AngII stimulated TLR4 expression in AngII treated ApoE-/- mice and in BDM from ApoE-/- mice compared to placebo. These data supported the conclusion that AngII stimulation of AAAs is mediated via a TLR4 dependent activation of p-STAT3 and that both p-STAT3 and TLR4 might be potential therapeutic targets for the treatment of AAAs.
Abstract 136: Stent-Supported Prosthetic Vein Valve
Authors: Ruddy Jean Marie , Tanner Daniel , Bernhard Kathleen , Brewster Luke P , Ku David N
Published: July 2021

Background:
In chronic venous insufficiency, incompetent valves prevent effective blood return and lead to varicose veins, lower extremity edema, and stasis ulcers. Several surgical interventions have been described, including valve transplantation, but these extensive procedures are only moderately effective and require inpatient hospitalization. Valve replacement technology continues to evolve and this project reports the design, verification, and pre-clinical validation of a new prosthetic vein valve.

Methods:
A venous valve has been constructed from a hydrogel and bare-metal stent that has the natural open configuration. Design verification was performed by testing competency, flow resistance, mechanical fatigue, and in vitro thrombogenicity. CFD analysis was used to quantify the shear rates within the valve that may induce platelet aggregation. A large animal study has been initiated to evaluate in vivo performance by open implantation into ovine jugular vein followed by periodic venography. Animals were not heparinized during implantation but were maintained on aspirin in the post-operative period.

Results:
The valve presents little resistance, allowed physiologic flow rates 400-600mL/min, and tolerated up to 2800mL/min. Preclinical testing shows the valve is also competent under physiologic pressures of 35-50mmHg and computed shear stress rates were maintained below 4000s-1. Fatigue testing up to 500,000 cycles did not cause valve damage. Antegrade venography confirmed patency of the venous valve continuing beyond 4 weeks in sheep. Placement in sheep would be much better with catheter placement using endosurgical techniques.

Conclusion:
This new prosthetic valve design meets physiologic and hemodynamic criteria for clinical use and patency has been maintained at the 4 week time point. Early occlusions may be prevented with operative anticoagulation
Abstract 154: Stress-Induced Loss of Nuclear HMGB1 From Ischemic Muscle Is Modulated by Autophagy and Accompanies Myoblast Differentiation
Authors: Xu Jun , Cui Xiangdong , Hong Guiying , Tzeng Edith , Sachdev Ulka
Published: July 2021

Introduction:
The nuclear protein HMGB1 can initiate innate immunity as well as sustain autophagy during times of cellular stress. We hypothesize that autophagy, a mechanism of cellular preservation, is a critical process in skeletal muscle biology during ischemia in patients with PAD. We further hypothesize that HMGB1 mobilization is associated with critical myoblast functions such as differentiation, and that this process may be modulated by autophagy.

Methods:
Human C2C12 myoblasts were cultured on laminin and exposed to hypoxia (1% O2) and serum depletion. Cells were also cultured with 2% serum to induce differentiation over 72hr. C57B6 mice were injected with 3-methyladenine (3MA), an autophagy inhibitor, before femoral artery ligation (FAL) and muscles were harvested after 24hr. Cell fractions from the cultured cells and muscles were probed for HMGB1. Immunoblots were analyzed using Image J.

Results:
Hypoxia and serum depletion induced complete loss of nuclear HMGB1 after 48hr. In myoblasts cultured in differentiation conditions, there was a 3 fold relative loss of nuclear HMGB1 compared with controls (p=0.014, t-test;N=3). While ischemia resulted in loss of nuclear HMGB1 in vivo, modulation of autophagic signaling with 3MA resulted in the retention of nuclear HMGB1 24 hours after FAL (Figure 1).

Conclusion:
Loss of nuclear HMGB1 by stressed myoblasts during differentiation suggests that this danger signal may be important in the regulation of muscle repair after injury. Additionally, our data indicate that autophagy may play a role in controlling the mobilization of myocyte HMGB1, affecting its inflammatory and reparative functions in ischemic muscle.
Abstract 16: FDG-PET/CT Enables the Detection of Recurrent, Same-Site Deep Vein Thrombosis by Illuminating Recently Formed Neutrophil-Rich Thrombus
Authors: Hara Tetsuya , Truelove Jessica , Tawakol Ahmed , Wojtkiewicz Gregory , Kessinger Chase W , Hucker Willam J , MacNabb Megan H , Henke Peter K , Weissleder Ralph , Jaffer Farouc A
Published: July 2021

Background:
Specific detection of recurrent deep vein thrombosis (DVT) is a challenging clinical problem. As DVT formation and resolution are inflammatory processes, we experimentally investigated whether noninvasive 18F-fluorodeoxyglucose (FDG)-PET imaging could identify inflamed, recently formed thrombi and thereby improve the diagnosis of recurrent DVT.

Methods:
We established a stasis-induced DVT model in murine jugular veins and also a novel model of recurrent stasis DVT in mice. C57BL/6 mice (n = 30) underwent ligation of the jugular vein to induce stasis DVT. FDG-PET/CT was performed at DVT timepoints of day 2, 4, 7, 14, or 2+16 (same-site recurrent day 2 DVT overlying a 16 days old DVT). Antibody-based neutrophil depletion was performed in a subset of mice prior to DVT formation and FDG-PET/CT. In a clinical study, 38 patients with lower extremity DVT or controls undergoing FDG-PET were retrospectively analyzed.

Results:
Stasis DVT demonstrated that the highest FDG signal occurred day 2, followed by a time-dependent decrease (p<0.05). Histological analyses demonstrated that thrombus neutrophils (p<0.01), but not macrophages, correlated with thrombus PET signal intensity. Neutrophil depletion decreased FDG signals in day 2 DVT compared to that of control mice (p<0.05). Recurrent DVT demonstrated significantly higher FDG uptake than organized day 14 DVT (p<0.05). The FDG DVT signal in patients also exhibited a time-dependent decrease (p<0.05). Recurrent day 2 DVT showed neutrophil-dominant infiltration pattern with few macrophages, similar to standalone day 2 DVT.

Conclusions:
Noninvasive FDG-PET/CT identifies neutrophil-dependent thrombus inflammation in murine DVT, and demonstrates a time-dependent signal in both murine and clinical DVT. FDG-PET/CT may offer a molecular imaging strategy to accurately diagnose recurrent DVT.
Abstract 165: cAMP-Phosphodiesterase 1C Regulates Neointimal Hyperplasia by Controlling Platelet-Derived Growth Factor Receptor ß Degradation via the Endosome-Lysosome Pathway
Authors: Cai Yujun , Knight Peter A , Nagel David J , Yan Chen
Published: July 2021

Neointimal hyperplasia is associated with the development of diverse vascular diseases such as atherosclerosis, vein bypass graft disease and restenosis after percutaneous coronary interventions. Vascular injury-mediated neointimal hyperplasia involves the phenotypic modulation, migration and proliferation of vascular smooth muscle cells (VSMCs). Cyclic nucleotide is vital in regulating VSMC migration and proliferation, which are controlled by cyclic nucleotide phosphodiesterase (PDE) isozymes. To identify the regulation and function of PDEs in VSMC pathogenesis of vascular diseases, we performed systematic screening for all 22 known PDE genes in normal contractile VSMCs and proliferating synthetic VSMCs. Interestingly, we observed that expression of PDE1C was very low in normal contractile VSMCs but drastically elevated in synthetic VSMCs in vitro as well as in vivo in various mouse models of vascular injuries, including carotid artery ligation, femoral artery wire injury and vein bypass graft. Consistently, PDE1C is also highly induced in VSMCs in neointimal lesion of human coronary artery. More importantly, injury-induced neointimal formation was significantly attenuated in pde1c-deficient mice or by perivascular administration of PDE1 inhibitor. Furthermore, PDE1 inhibition also reduced the spontaneous vascular remodeling of human saphenous vein explant in an ex vivo culture model. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of PDGF receptor beta (PDGFR-β). We found that PDE1C knockdown or inhibition markedly decreased the levels of PDGFR-β protein but not mRNA, which was blocked by endosome and lysosome inhibitors. Furthermore, PDE1C inhibition is capable of promoting PDGFR-β internalization and attenuating PDGFR-β membrane accumulation. Finally, we found that PDE1C co-localized with PDGFR-β on cell membrane, which was able to regulate PDGFR-β protein degradation through modulating a transmembrane adenylyl cyclase-cAMP-PKA signaling. Taken together, our present data demonstrated that PDE1C plays a critical role in regulating VSMC proliferation and neointimal hyperplasia, in part, through promoting endosome/lysosome dependent PDGFR-β protein degradation.
Abstract 167: A Cell-Autonomous Role for Endothelial GTP Cyclohydrolase 1 and Tetrahydrobiopterin in Blood Pressure Regulation
Authors: Chuaiphichai Surawee , McNeill Eileen , Douglas Gillian , Crabtree Mark J , Bendall Jennifer K , Hale Ashley B , Alp Nicholas J , Channon Keith M
Published: July 2021

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and nitric oxide (NO) generation. Augmentation of BH4 levels can prevent eNOS uncoupling and improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de-novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of
GCH1,
encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo.

Mice with a floxed
GCH1
allele (

GCH1
fl/fl

) were crossed with Tie2cre mice to delete
GCH1
in endothelial cells.

GCH1
fl/fl

Tie2cre mice demonstrated virtually absent NO bioactivity and significantly greater O
2
•

-

production.

GCH1
fl/fl

Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in

GCH1
fl/fl

Tie2cre aortas were in part mediated by NOS-derived hydrogen peroxide (H
2
O
2
), which mediated vasodilatation through soluble guanylate cyclase.
Ex vivo
supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H
2
O
2
production in

GCH1
fl/fl

Tie2cre aortas.

GCH1
fl/fl

Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalised by NOS inhibitor, L-NAME. Taken together, these studies reveal an endothelial cell-autonomous requirement for
GCH1
and BH4 in regulation of vascular tone and blood pressure, and identify endothelial cell BH4 as a pivotal regulator of NO vs. H
2
O
2
as alternative eNOS-derived endothelial derived relaxing factors.
Abstract 169: Smad3 Drives Cross-Talk Between TGFß and Wnt/ß-Catenin Signaling Pathways in Smooth Muscle Cells
Authors: DiRenzo Daniel M , Shi Xu Dong , Guo Lian-Wang , Kent K Craig
Published: July 2021

Restenosis (neo-intimal hyperplasia) occurs in approximately 25-50% of patients undergoing arterial interventions, primarily due to the proliferation and migration of arterial smooth muscle cells (SMCs) into the peri-luminal area. Recently, Wnt/β-catenin signaling has been shown to promote SMC proliferation and enhance neo-intimal hyperplasia but its mechanism of activation is unclear. Interestingly, Wnt/β-catenin has been shown to be activated by TGFβ in mesenchymal stem cells and fibroblasts. We have shown that TGFβ and its downstream signaling protein, Smad3, are upregulated following vascular injury and that Smad3 overexpressing SMCs display enhanced proliferation, migration, and neo-intimal hyperplasia.
These results led us to hypothesize that TGFβ, through Smad3, activates Wnt/β-catenin to regulate SMC behavior following arterial injury
.

In primary rat SMCs, TGFβ (5ng/mL) led to β-catenin activation and relocalization from the plasma membrane to the cytoplasm / nucleus within 24 hours. Furthermore, qRT-PCR results demonstrated that expression of Wnt11 (22 fold) and Wnt9a (3.9 fold) were significantly upregulated after 24 hours of TGFβ stimulation (p<0.05, n=3). In addition, 24 hours of TGFβ stimulation in SMCs overexpressing Smad3 (TGFβ/Smad3) further enhanced the gene expression of Wnt11 (>300 fold) and Wnt9a (14 fold) and also stimulated significant increases in Wnt2b (41 fold), Wnt5a (2.9 fold), and Wnt4 (3.2 fold) (p<0.05, n=3) as measured by qRT-PCR. Western blot results demonstrated that the combined TGFβ/Smad3 stimulation increased β-catenin protein levels, suggesting that TGFβ activates canonical Wnt signaling leading to stabilization of β-catenin protein. In normal rat carotid arteries, β-catenin protein was undetectable via immunohistochemistry but could be seen in SMCs of the vessel media at 3 days post-balloon angioplasty and in neo-intimal cells at 7 and 14 days. Smad3 was also expressed in neo-intimal cells at 7 and 14 days post-angioplasty suggesting that TGFβ, through Smad3, is responsible for Wnt/β-Catenin activation during vascular injury. In conclusion, this work describes a novel cross-talk in SMCs between TGFβ and Wnt signaling which may provide a viable target for future anti-restenotic treatments.
Abstract 173: MicroRNA-365 Inhibits the Proliferation of Vascular Smooth Muscle Cells by Targeting Cyclin D1
Authors: Kim Myung-Hyun , Ham Onju , Lee Se-Yeon , Choi Eunmi , Lee Chang Yeon , Park Jun-Hee , Lee Jiyun , Seo Hyang-Hee , Seung Minji , Choi Eunhyun , Min Pil-Ki , Hwang Ki-Chul , Kwon Hyuck Moon
Published: July 2021

Background:
Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miRNAs) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in cardiovascular disease. However, the roles and mechanisms of miRNAs in VSMCs and neointimal formation are far from being fully understood.

Methods & Results:
In this study, cell cycle specific cyclin D1 was found to be a potential target of miR-365 by direct binding. Through an in vitro experiment, we showed that exogenous miR-365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR-365 was observed to block G1/S transition in platelet-derived growth factor (PDGF)-induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF, angiotensin II (Ang II), and serum, led to the downregulation of miR-365 expression levels. The expression of miR-365 was confirmed in balloon injured carotid arteries. Taken together, our results suggest an anti-proliferative role for miR-365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1.

Conclusions:
Therefore, miR-365 may influence neointimal formation in atherosclerosis patients.
Abstract 182: Increased Adipose-Derived Mesenchymal Stem Cells Counts and Pro-B-Type Natriuretic Peptide in Patients With Critical Limb Ischemia
Authors: Farres Houssam , Sutton Caroline , Zubair Abba , Dortch John D , Hakaim Albert
Published: July 2021

Mesenchymal stem cells (MSCs) have been shown to improve regeneration of injured tissues in vivo. Several in vitro studies and animal models have demonstrated improvement in MSCs paracrine effects under hypoxic conditions. Moreover, several studies suggested that the pro B-type natriuretic peptide (pro-BNP) could be involved in the stimulation of postischemic vascular regeneration. The purpose of this study was to investigate the effect of critical limb ischemia, in a human model, on in-situ adipose derived mesenchymal stem cells (ADMSCs) and to determine whether serum levels of N-terminal pro-BNP correlate with ADMSCs counts and associated paracrine effects.

Lipoaspirate samples of ≥ 10mL were collected from ischemic limbs (ischemic group) and compared to control (without ischemia). MSCs were characterized by frequency, viability, differentiation potential, cytokines expression, and cell surface markers. Serum NT pro-BNP was measured as well.

MSCs counts were 9-to-10-fold higher in patients with ischemic limbs (mean 7952 MSC/mL ± 542) than controls (mean 790 MSC/mL ± 65). Pro-BNP levels (1878-4757 pg/mL) were approximately 8-to-26-fold higher than in age- and sex-matched controls. Furthermore, there were positive correlations between pro-BNP levels and MSCs counts in the ischemic group.

In conclusion, patients with critical limb ischemia (CLI) have higher levels of pro-BNP and MSCs counts than controls. Increased levels of pro-BNP and MSCs counts can be considered humoral and cellular surrogates of ischemia and hypoxia in patients with CLI. This supports recent studies that suggest that the increase production of peripheral BNP may be a stem cells-mediated response to stimulate angiogenesis in the ischemic skeletal muscles.
Abstract 2: Elucidation of the Mechanism of Apolipoprotein(a)/Lipoprotein(a)-Induced Interleukin 8 Gene Expression: Insight into Potential Receptors and Signaling Pathways and the Role of Oxidized Phospholipids
Authors: Scipione Corey A , Sayegh Sera , Boffa Michael B , Koschinsky Marlys L
Published: July 2021

Elevated plasma Lipoprotein(a) (Lp(a)) levels are a risk factor for a variety of atherosclerotic disorders, although the exact role(s) of Lp(a) in atherogenesis remains poorly understood. It is clear, however, that the unique pathogenic effects attributed to Lp(a) are mediated by its apolipoprotein(a) moiety. Over the past decade, a growing body of evidence suggests that the harmful effects of apo(a) involve stimulation of inflammatory responses such as monocyte chemotaxis. It was previously reported that Lp(a), through apo(a), induces IL-8 gene expression by macrophages. The kringle V domain of apo(a) was implicated in this effect, along with oxidized phospholipids potentially present in this domain. In the current study, we found that a recombinant apo(a) species containing 17 kringle-IV domians (17K) elicited a dose-dependant increase in IL-8 mRNA abundance and protein secretion in THP-1 macrophages. This effect was significantly reduced by mutation of the strong lysine binding site (sLBS) in kringle IV type 10, but not by treatment with lysine analogue ε-ACA. Notably, this mutation eliminates the EO6-reactive oxidized phsopholipid in apo(a). Removal of kringle V did not reduce the ability of apo(a) to enhance IL-8 expression. Therefore, to examine the potential role of the modification of apo(a) by oxidized phospholipids, we used an siRNA knockdown approach to screen candidate receptors. We found that CD36 and TLR2, but not TLR4, are required for apo(a)-induced IL-8 expression. Specific MAPK pathway inhibitors were used to investigate the mechanism of IL-8 induction by apo(a): inhibitors of JNK and ERK1/2 abolished the effect of apo(a) whereas an inhibitor of p38 had no effect. Accordingly, apo(a) also stimulated an increase in JNK phosphorylation. To assess the role of downstream transcription factors, luciferase reporter gene experiments were conducted using the IL-8 promoter. Apo(a) induced expression of this reporter construct, as expected, and this induction was eliminated by mutation of known binding sites for NFκB or AP-1. Our results provide tantalizing mechanistic links between apo(a) lysine binding function, oxidative modification, and stimulation of intracellular signaling pathways through inflammation-linked receptors.
Abstract 208: Improved Vasodilatory Function in Ins2Akita Type 1 Diabetic Mice with Daily Intake of Oral L-Arginine in Combination with Salsalate
Authors: Krishnan Manikcam , Janardhanan Preethi , Gaudette Colleen , Reddick Robert , Van Haperen Rien , Natarajan Mohan , Mohan Sumathy
Published: July 2021

Endothelial dysfunction due to deficient nitric oxide (NO) generation cause progressive vascular complications in diabetes. Our earlier findings revealed that Inhibitor kappa B kinase beta (IKK-beta) -an upstream regulator of an inflammatory mediator, nuclear factor kappa-B (NF-kB), competitively inhibits endothelial nitric oxide synthase (eNOS), the chief source of NO in blood vessels. Therefore, we investigated the influence of an inhibitor of IKK beta, salsalate in augmenting eNOS activity. The co-operative effect of L-arginine supplementation (substrate of eNOS) with salsalate intake in enhancing eNOS activity has been evaluated.

Both in vitro cell cultures and diabetic/transgenic animal models were used. Human aortic endothelial cells incubated with 25 mM glucose with or without L-arginine (2mM), the cofactor sepiapterin (100uM) and salsalate(50uM) were examined for NO production. Combined L-arginine and salsalate treatment enhanced NO production, phospho eNOS expression and eNOS-dimer vs monomer ratio. For in vivo assays, diabetic Ins2Akita mice were fed with L-arginine and salsalate for 15 weeks. Aortae of these mice exhibited increased phospho eNOS expression and increased eNOS dimer/monomer ratio. Arterial ring assays showed improved acetylcholine mediated vasodialation. Transgenic-Akita mice that overexpress human eNOS in endothelial cells (Tie-2eNOS-Ins2 Akita) generated in-house showed improved vasodialation.

In conclusion, enhancing eNOS activity in diabetic mice either by genetic modification as seen in Tie-2 eNOS-Akita mice or by nutritional approach (supplementation of L-arginine with simultaneous inhibition of IKK beta) restores normal vasodialatory function.
Abstract 209: Dimethylarginine Dimethylaminohydrolase 1 and Insulin Resistance
Authors: Piper Sophie E , Leiper James M
Published: July 2021

Type 2 diabetes is a prevalent metabolic condition and is the result of an impaired response to insulin. Insulin resistance and type 2 diabetes are clearly associated with obesity and the secondary cardiovascular complications of this condition are serious and life threatening.

Asymemetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases and increased levels are seen in multiple pathologies. Increased plasma levels of ADMA have been associated with patients with type 2 diabetes, insulin resistance and obesity, although a causal link between ADMA and diabetes has not been established. Dimethylarginine dimethylaminohydrolase (DDAH) is the enzyme that catalyses the metabolism of ADMA. There are two isoforms of the enzyme which are both involved in the control of ADMA and NO.

The interplay of insulin with NO release is well established but the initial causes for the onset of insulin resistance are not well defined. Elevated levels of ADMA are linked to insulin resistance and transgenic mice that over-express ddah1 show increased insulin sensitivity. Of note is that metformin, an insulin sensitising drug that is widely used in the treatment of insulin resistance, reduces plasma glucose and ADMA concentrations.

In order to elucidate the physiological role of DDAH1 in glucose homeostasis we investigated the glucose handling in a ddah1 global knockout model.

Intra-peritoneal glucose tolerance tests in ddah1 global knockout mice demonstrate insulin resistance. Baseline plasma glucose levels were 25% higher in ddah1 knockouts and peak levels were 53% higher in ddah1 knockouts. The kinetics of plasma glucose accumulation and clearance in ddah1 knockout mice suggests dysfunction in both the liver and skeletal muscle. On a normal chow diet, hepatocyte specific ddah1 knockout mice and skeletal muscle specific ddah1 knockout mice show no insulin resistance. On a high fat diet however the hepatocyte specific ddah1 knockout mice show significant insulin resistance and lower metabolic rate than their fat fed wild-type counterparts.

These studies demonstrate for the first time a causal link between ADMA accumulation and insulin resistance. Furthermore these data establish DDAH1 activity is a significant regulator of insulin resistance.
Abstract 220: Reduction in Atherosclerosis Plaque by Intravenous Injections of Engineered Bone Marrow Stem Cells With Low Notch Activity in Apo E-/- Mice
Authors: Liu Zhao-Jun , Tan Yurong , Li Yan , Wang Bo , Tian Runxia , Goldschmidt-Clermont Pascal J , Seo David , Velazquez Omaida C
Published: July 2021

Objectives:
We test a novel paradigm in the pathogenesis of atherosclerosis as an imbalance in the pro-repair function of circulating bone marrow-derived stem cells (BMDSC) that can be modulated by Notch activity.

Methods:
Significance Analysis of Microarray (SAM) and PCRArray/immunoblotting were used to study Notch activity in empirically-derived repair-competent versus repair-incompetent BMDSC (RC-BMDSC vs. RI-BMDSC). In parallel experiments, BMDSC with low-Notch activity were generated by transducing cells with dominant-negative Master-mind like 1 (DN-MAML1)/lentivirus (Notch-inhibited). Mock/lentivirus transduced cells were used as control. 5 x 10^6 Lin- BMDSC were infused bi-weekly for 4 consecutive weeks i.v. into Apo E -/- mice under high fat diet (HFD); (N=5/group). Atherosclerosis plaque burden was assessed using Oil Red O staining and blinded computerized quantitative measurements.

Results:
Of 289 genes with differential expression in RC-BMDSC vs. RI-BMDSC, by SAM, Notch1 expression was significantly elevated in RI-BMDSC compared to RC-BMDSC, p<0.0009. Notch activity was higher in BMDSC (EPC and MSC) from ApoE-/- mice before initiation of HFD (Notch target genes, Hes-1 and Hey-1, with 6.6- and 8.2-fold higher expression, respectively (p<0.05)). Compared to mice infused with control Lin-BMDSC, ApoE-/- mice treated with DN-MAML1/Lin-BMDSC had significantly decreased aortic plaque burden (p<0.05).

Conclusions:
For the first time we demonstrate a direct correlation between Notch activity and pro-repair function in BMDSC that modulates atherosclerosis plaque burden. Lower Notch activity in BMDSC appears to confer a pro-repair or repair-competent BMDSC phenotype. These novel findings provide rationale for further studies into clinically-relevant cell-based therapy for atherosclerosis.