bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 45: Endothelial Aryl Hydrocarbon Receptor Nucleatranslator (Arnt) is a Novel Regulator of Cardiac Endothelial Barrier Integrity in Diabetes
Authors: Knapp Maura , Zheng Mei , Sladojevic Nikola , Zhao Qiong , Birukov Konstaintin G , Liao James K , Wu Rongxue
Published: July 2021

Background:
Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes.

Methods and Results:
We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion.
In vitro
, trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway.

Conclusion:
ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.
Abstract 464: Cellular and Molecular Characterisation of Human Endothelial Progenitors in vivo Defined Based on Self-Renewal and Colony Forming Potential Identifies Notch Signaling as a Key Pathway
Authors: Patel Jatin , Shafiee Abbas , Wong Ho Yi , Alexis Josue , Khosrotehrani Kiarash
Published: July 2021

Background:
Although endothelial progenitors have long been described, there remains significant controversy around their identity
in vivo
. The endothelial colony forming (ECFC) assays suggested a hierarchy among endothelial cells
in vivo
. Our aim was to systematically test different endothelial cell populations sorted from the human placenta, a highly vascularised tissue, based on various cell surface markers for their ECFC potential.

Methods and Results:
Upon sorting based on key markers CD45, CD34 and CD34 it was easily established that most ECFC potential was concentrated in the CD45-CD34+ fraction. Among this population, single cell culture assays (>300 wells per cell type) were performed on sorted CD31neg, CD31int and CD31hi cells. Only the CD31int cells were able to grow high proliferative potential ECFC. CD31neg populations contained mesenchymal stem cells (MSC) whereas CD31hi cells only produced mature endothelial clusters. RNA sequencing of each fraction identified Notch signalling as a key driver of endothelial progenitors as opposed to MSC. When the CD31int population was further characterised it was found to be expressing VE-Cadherin as predicted by the RNAseq, however, was not in contact with the circulation as it did not stain for lectins injected intravenously. In accordance the self-renewing fraction of ECFC cultures
in vitro
was dependent on Notch signalling and controlled the expression of IL33 and CDKN1C (p57) to maintain progenitors in quiescence. This was validated
in vitro
and
in vivo
by performing shRNA and pharmacological inhibition of the different pathways.

Conclusion:
Our study uncovers a population of endothelial progenitors
in vivo
at the cellular and molecular level and identified a novel role for Notch signalling in maintaining progenitor self-renewal
in vivo
and
in vitro
.
Abstract 468: Smooth Muscle Cells Accelerate Endothelial Regeneration Through a Protein Kinase C-delta-Dependent Secretion of CXCR2 Ligands
Authors: Zhou Ting , Ren Jun , Parlato Matthew B , Binder Bernard Y , Phan Noel , Wang Qiwei , Stewart Danielle , Gupta Kartik , Feldman Conner , Si Yi , Liu Zhenjie , Sheibani Nader , Murphy William L , Liu Bo
Published: July 2021

Background:
Efficient regeneration of denuded endothelial cells (ECs) is an important process that counteracts the pro-stenotic activities. Unfortunately, this natural healing process is frequently compromised by disease conditions such as diabetes. Protein kinase C-delta (PKCδ) plays a complex role in the arterial injury response by regulating apoptosis of vascular smooth muscle cells (SMCs) as well as production of chemokines capable of attracting resident and circulating cells. In this study, we explored whether SMCs participate in endothelial repair through a PKCδ-dependent paracrine mechanism.

Methods and Results:
Following balloon injury to the rat carotid, SMC-specific gene transfer of
Prkcd
accelerated reendothelialization compared to the empty vector, reflected by a larger area excluded from Evans blue measured 14 days post injury (59.60±5.01% vs 25.38±7.52%). In contrast, SMC-specific knockdown of endogenous
Prkcd
delayed reendothelialization compared to the non-targeting shRNA control (41.31±6.54% vs 70.31±5.97%).
In vitro
, media conditioned by AdPKCδ-infected SMCs increased endothelial wound healing without affecting their proliferation and viability. In addition, SMCs in a PKCδ-dependent fashion attracted circulating angiogenic cells (CACs), a cell population that promotes neovascularization via production of angiogenic factors. A PCR-based array analysis identified
Cxcl1
and
Cxcl7
among others as PKCδ-mediated chemokines produced by SMCs. Blocking CXCL7 or CXCR2 significantly inhibited endothelial wound healing and CAC migration in response to AdPKCδ-infected SMC conditioned media.
In vivo
, PKCδ overexpression in SMCs following balloon injury increased CXCL7 production and stimulated CACs recruitment to injured arteries. Furthermore, insertion of a
Cxcl7
cDNA in the lentiviral vector that carries a
Prkcd
shRNA overcame the negative effects of
Prkcd
knockdown on reendothelialization.

Conclusions:
Regeneration of denuded endothelium involves multiple cell types from the vascular wall as well as circulation. SMCs stimulate reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of ECs from uninjured endothelium and CACs from circulation.
Abstract 479: Smc Phenotype Switch Targeting Apoe is a Therapeutic Target in Popliteal Aneurysm
Authors: Busch Albert , Chernogubova Ekaterina , LI Yuhuang , Lorenz Udo , Roy Joy , Hultgren Rebecka , Maegdefessel Lars
Published: July 2021

Introduction:
Popliteal artery aneurysm (PAA) is an individually highly fatal disease, causing ischemia and eventual major amputation, mainly in 50-70 year old males. It is the most frequent peripheral artery aneurysm and correct treatment is challenging for clinicians, since both open and endovascular repair have only modest success rates, depending on the clinical presentation. In comparison to other aneurysm entities, little is known about its specific pathogenesis.

Material and Methods:
30 Human PAA and popliteal artery samples were analyzed by immunohistochemistry, mRNA and miRNA expression analysis and protein assays on the OLink platform in order to identify key features of the disease and crucial pathways involved. Furthermore, a novel murine model of PAA is characterized for future applications in basic research.

Results:
Vascular smooth muscle cells loose their contractile phenotype along with significant inflammatory and proteolytic changes in the vessel wall architecture. Extensive tissue remodeling with high cell turnover rates is a unique feature in comparison to AAA. This correlates with highly abundant expression of the apolipoproteins E and CI suggestive to have a pro-proliferative effect in peripheral vascular tissue. Additionally, array-based screening on gene and protein level revelaed the pentraxin-related protein 3 as a potential biomarker and soluble inducer of neutrophil homing. Other targets of interest in relation to ApoE were fatty acid binding protein 4 and Insulin growth facto binding protein 2. These findings are emphasized by a specific case of a solely mechanically induced PAA in a young male.

Conclusion:
Pathogenesis of PAA shows similar histologic features than AAA, yet differs in potential pathways involved. Increased cell turnover in the aneurysmal neck area suggests evaluation of alternative treatment strategies, targeting key processes in its pathogenesis.
Abstract 48: Role of Receptor for Advanced Glycation End Products (RAGE) in Regression of Diabetic Atherosclerosis
Authors: Senatus Laura M , Lopez-Diez Raquel , Liu Jianhua , Li Huilin , Daffu Gurdip , Li Qing , Rahman Karishma , Vengrenyuk Yuliya , Barrett Tessa , Friedman Richard , Ramasamy Ravichandran , Fisher Edward , Schmidt AnnMarie
Published: July 2021

Atherosclerosis is a chronic inflammatory disorder. Both progression and regression of atherosclerosis are adversely affected by diabetes. A key player in these processes is Receptor for Advanced Glycation End Products (RAGE). RAGE is a multiligand cell surface macromolecule, which binds ligands enriched in atherosclerotic plaques, such as advanced glycation endproducts (AGEs). RAGE is expressed on a wide array of cell types implicated in cardiovascular disease, such as endothelial cells, and inflammatory cells such as macrophages. The cytoplasmic domain of RAGE binds to the formin molecule DIAPH1 and DIAPH1 is required for RAGE ligands to activate cell signaling responses. RAGE acts as a key mediator of oxidative and inflammatory signaling pathways that are involved in atherosclerosis. We tested mechanisms of impaired regression of atherosclerosis in a murine model of aorta transplantation and found that deletion of
Ager
or
Diaph1
in diabetic mice recipients of
Ldlr
null mice atherosclerotic aortas accelerates atherosclerosis regression and significantly reduces the lesional macrophage content when compared to diabetic wild-type recipient mice. The antiatherosclerotic effects in diabetic
Ager
null mice and diabetic
Diaph1
null mice include reduced RAGE ligand AGEs in transplanted aortas, with reduced expression of a range of proatherogenic factors, including reactive oxygen species and inflammatory cytokines implicated in leukocyte recruitment and activation. We employed RNA sequencing to identify the key transcriptional events by which RAGE mediates its effects in donor or recipient macrophages in diabetic regressing plaques. Our results suggest that critical gene expression profiles, including those genes involved in inflammation, endothelial dysfunction, oxidative stress, monocyte/macrophage fate (recruitment, differentiation, proliferation), signal transduction and lipid metabolism, are beneficially modulated, at least in part, via
Ager
deletion in atherosclerosis regression. Taken together, these data increase our understanding of the role of RAGE in diabetic atherosclerosis, particularly in macrophages, and may provide avenues for therapeutic strategies to accelerate regression of atherosclerosis in diabetes.
Abstract 482: IDH2 Deficiency Induces Oxidative Stress and Vascular Inflammation
Authors: Kim Seonhee , Choi Su-jeong , Nagar Harsha , Piao Shuyu , Lee Ikjun , Kim Sungmin , Kim Cuk-Seong
Published: July 2021

Isocitrate dehydrogenase 2 (IDH2) plays an essential role protecting cells against oxidative stress-induced damage. A deficiency in IDH2 leads to mitochondrial dysfunction and the production of reactive oxygen species (ROS) in cardiomyocytes and endothelial cells. However, the physiological function of IDH2 in vascular system is mostly unknown In this study, we investigated whether IDH2 knockdown causes mitochondrial dysfunction and vascular inflammation
in vitro
and
in vivo
. IDH2 knockdown decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III, which lead to increased mitochondrial superoxide. In addition, the levels of fission and fusion proteins (Mfn-1, OPA-1, and Drp-1) were significantly altered and MnSOD expression also was decreased by IDH2 knockdown. Furthermore, knockdown of IDH2 decreased eNOS phosphorylation and nitric oxide (NO) concentration in endothelial cells. Interestingly, treatment with Mito-TEMPO, a mitochondrial-specific superoxide scavenger, recovered mitochondrial fission-fusion imbalance and blunted mitochondrial superoxide production, and reduced the IDH2 knockdown-induced decrease in MnSOD expression, eNOS phosphorylation and NO production in endothelial cells. Endothelium-dependent vasorelaxation was impaired, and the concentration of bioavailable NO decreased in the aortic ring in IDH2 knockout mice. These findings suggest that IDH2 deficiency induces endothelial dysfunction through the induction of dynamic mitochondrial changes and impairment in vascular function. Key words: IDH2, mitochondria, endothelial cells, ROS
Abstract 486: A Novel Synthetic Glycolipid TLR4 Antagonist (FP7) Negatively Regulates in vitro and in vivo Hematopoietic and Non-Hematopoietic Vascular Toll Like Receptor 4 Signalling
Authors: Palmer Charys , Peri Francesco , Neumann Frank , Pirianov Grisha
Published: July 2021

Objectives:
The toll-like receptors (TLRs), including TLR4, have been shown to play a crucial role in vascular inflammatory based diseases. The main goal of this study was to determine the potential of FP7, a synthetic glycolipid active as TLR4 antagonist, to modulate hematopoietic and non-hematopoietic vascular TLR4 proinflammatory signalling.

Methods:
Human umbilical vein endothelial cells (HUVEC), THP-1 monocytes, THP-1-derived macrophages and Angiotensin II-infused Apo E deficient mice were
in vitro
and
in vivo
models respectively. Western blotting, antibody array and ELISA approaches were used to explore the effect FP7 on TLR4 functional activity on two levels: modulation of TLR4-induced mitogen activated protein kinases (MAPK) and p65 NF-kB activity and expression of TLR4 dependent proinflammatory proteins in response to liposaccharide (LPS) and ligands of sterile inflammation; minimally oxidized LDL (moxLDL) and small fragments of hyaluronan (sfHA).

Results:
Following activation of TLR4 by ligands of non-sterile and sterile inflammation,
in vitro/in vivo
data revealed that FP7 inhibited MAPK and p65 NF-kB phosphorylation associated with down regulation of specific TLR4 dependent proinflammatory proteins proteins, such as MCP-1, ICAM-1, RANTES, MIP-1 gamma, KC etc. In addition to inhibition of LPS-induced TLR4 signalling, FP7 negatively regulated TLR4 activation in response to ligands of sterile inflammation; moxLDL and sfHA (
in vitro
) and Angiotensin II infusion (
in vivo
).

Conclusions:
These results demonstrate the ability of FP7 to negatively regulate in vitro and in vivo hematopoietic and non-hematopoietic vascular TLR4 signalling, suggesting the potential therapeutic use of this novel TLR4 antagonist for pharmacological intervention of vascular inflammatory based diseases such as atherosclerosis and aneurysm.
Abstract 496: Genetic Neutrophil Deficiency Ameliorates Cerebral Ischemia-Reperfusion Injury
Authors: Frieler Ryan A , Chung Yutein , Song Jianrui , Vigil Thomas M , Mortensen Richard M
Published: July 2021

Background:
Neutrophils respond rapidly to cerebral ischemia and are thought to contribute to inflammation-mediated injury during stroke. Neutralizing antibodies and inhibition of neutrophil chemotactic molecules can be protective during models of stroke, but many of these techniques have the potential to result in cross-reactivity and non-specificity with other immune cell types. Using myeloid Mcl1 knockout mice as a model of genetic neutrophil deficiency, we investigated the contribution of neutrophils to stroke pathophysiology.

Methods:
Myeloid Mcl1 knockout mice were subjected to transient 90-min middle cerebral artery occlusion and infarct size was assessed by MRI after 24 hours reperfusion. Immune cell mobilization and infiltration was assessed by flow cytometry after 24 hours reperfusion.

Results:
We found that myeloid Mcl1 knockout mice had significantly reduced infarct size when compared to heterozygous and wild type control mice (MyMcl1
+/+
: 78.0 mm
3
; MyMcl1
+/-
: 83.4 mm
3
; MyMcl1
-/-
: 55.1 mm
3
). This was accompanied by a nearly complete absence of neutrophils in the ischemic hemisphere of myeloid Mcl1 knockout mice. Although myeloid Mcl1 knockout mice were protected from cerebral infarction, no significant differences in the expression of inflammatory genes were detected. Inhibition of neutrophil chemotaxis using CXCR2 pepducin treatment partially reduced neutrophil mobilization and recruitment to the brain after stroke, but did not reduce infarct size 24 hours after transient MCA occlusion.

Conclusions:
These data confirm that neutrophils have an important role in infarct development during stroke pathophysiology and suggest that complete deficiency, but not partial inhibition, is necessary to prevent neutrophil-mediated injury during stroke.
Abstract 50: TLR2 Plays A Key Role in Platelet Hyperreactivity and Accelerated Thrombosis Associated With Hyperlipidemia
Authors: Biswas Sudipta , Zimman Alejandro , Gao Detao , Byzova Tatiana , Podrez Eugene
Published: July 2021

Introduction:
Platelet hyperreactivity, common in many pathophysiological conditions, is associated with increased atherothrombotic risk. Platelet hyperreactivity, specifically in dyslipidemia, has been mechanistically linked to accumulation in circulation of specific oxidized phospholipids (oxPC
CD36
), ligands for pattern-recognition receptor CD36. Although significant progress in understanding of CD36 mediated signaling in platelets has been made in recent years, many details of the molecular mechanism are still missing.

Hypothesis:
CD36 is known to cooperate with other receptors in cells such as macrophages during inflammation and apoptosis. Moreover, we have recently reported that platelets can also respond to oxidation specific epitopes via Toll Like Receptors. So, we hypothesized that TLRs may have a role in oxPC
CD36
induced platelet activation.

Methods and Results:
In this study, using
in vitro
approaches as well as mice with genetic deletion of MyD88 or TLRs, we demonstrate that TLR2 is required for platelet activation by oxPC
CD36
. oxPC
CD36
induce formation of CD36/TLR2/TLR6 complex in platelets and downstream signaling via TIRAP-MyD88-IRAK1/4-TRAF6, which leads to platelet integrin activation via the SFK-Syk-PLCγ2 pathway. Studies in hyperlipidemic

ApoE
-/-

mice have demonstrated that TLR2 deficiency abolishes platelet hyperreactivity and accelerated thrombosis induced by hyperlipidemia. At the same time, no effect of TLR2 deficiency on platelet activation nor thrombosis was observed in normolipidemic mice.

Conclusions:
Taken together, our studies reveal that TLR2 plays a key role in platelet hyperreactivity and the prothrombotic state in the settings of hyperlipidemia by sensing a wide range of endogenous lipid-peroxidation ligands and activating innate immune signaling cascade in platelets.
Abstract 506: Intramural Structural Changes in Human Femoropopliteal Arteries With Age
Authors: Poulson William , Rogers Alexis , Batra Rishi , Deegan Paul , Kamenskiy Alexey , MacTaggart Jason
Published: July 2021

Introduction:
Femoropopliteal artery (FPA) disease is common and therapeutic interventions and reconstructions leave significant room for improvement. Models of arterial growth and remodeling can help better understand the pathophysiology of FPA disease, but they require detailed data on arterial structural composition to produce accurate predictions. The goal of this work was to quantify intramural collagen and elastin in human FPAs and determine how these constituents change with age.

Methods:
FPAs were obtained from 32 tissue donors without hallmarks of peripheral artery disease (average age 50±18 years, age range 15-72 years). Longitudinal pre-stretch, wall thickness and lumen diameter were used to calculate
in situ
volume of tissue in a 1cm-long arterial segment. Arteries were fixed in formalin and sectioned in both transverse and longitudinal directions. Elastin and collagen contents were quantified with image analysis using Verhoeff-Van Gieson and Masson’s Trichrome stained slides scanned at 10x.

Results:
Ageing was associated with an increase in
in situ
tissue volume of 31mm
3
per decade of life, and increases in the overall wall and tunica media thicknesses of 94μm and 19μm per decade of life respectively (p=0.01). Volume fraction of elastin did not change with age (p=0.49) and remained at 4.8±1.6%, although elastic fibers did become more fragmented (p<0.01). Amorphous medial collagen increased with age from occupying 35±11% of the media at ages younger than 30 years, to 54±9% of the media at ages older than 60 years. This translated into an increase of 4.1% in medial collagen per decade of life (p<0.01). Volume fraction of fibrillar adventitial collagen did not change with age (p=0.09) and remained at 34±7%.

Conclusions:
In human FPAs, arterial walls thicken and collagen content increases with age. Though total elastin content remains stable, elastic fiber architecture deteriorates with aging. Quantification of intramural structural changes in human FPAs across age groups can be instrumental for developing models of arterial growth and remodeling that can help better understand the pathophysiology of peripheral artery disease.
Abstract 524: Identification of Roles for the Rho-Specific Guanine Nucleotide Dissociation Inhibitor (RhoGDI) Ly-Gdi in Platelet Function
Authors: Ngo Anh T , Thierheimer Marisa L , Babur Özgün , Rocheleau Anne D , Nan Xiaolin , McCarty Owen J , Aslan Joseph
Published: July 2021

Introduction:
Upon activation, platelets undergo specific morphological alterations critical to hemostatic plug and thrombus formation via actin cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in regulating platelet function.

Methods and Hypothesis:
Through an approach combining pharmacology, cell biology and systems biology methods we assessed the hypothesis that RhoGDI proteins regulate Rho GTPase-driven platelet functions downstream of platelet integrin and glycoprotein receptors.

Results:
We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular staining and super resolution microscopy assays find that Ly-GDI displays an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI in platelets, as Ly-GDI is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Notably, inhibition of PKC diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42.

Conclusion:
In conclusion, our results support roles for Ly-GDI as a localized regulator of Rho GTPases in platelets and link PKC and Rho GTPase signaling systems to platelet function.
Abstract 527: The Functional Significance of the Adenosine A
2b
Receptor in the Internal Mammary Artery
Authors: Archacki Stephen R , Wang Qing K
Published: July 2021

Objective:
The endothelium is the initial target that leads to cardiovascular disease. Knowing that the internal mammary arteries (IMA) are resistant to the development of atherosclerosis, which contrasts with coronary arteries (Cor) which are athero-prone, we hypothesize that genes over-expressed in the endothelial cells (ECs) of between these two arteries will identify genes that resist atherosclerosis.

Methods and Results:
Microarray analysis showed over 1,000 differentially expressed in the ECs of IMA vs Cor. The most statistically significant different gene was the adenosine A
2B
receptor. This indicates the A
2B
receptor may be involved in a resistance to atherosclerosis. Western blot analysis showed higher A
2B
expression in the IMA than in coronary arteries with or without disease from proteins harvested from these human arteries and ECs. Overexpression of A
2B
in ECs blunted: monocyte adhesion, cell adhesion molecule expression, migration, and the transendothelial migration of monocytes-- processes directly associated with the development of atherosclerosis. Knockdown of A
2B
expression by siRNA promoted these processes.

Conclusions:
ECs derived from the IMA and Cor are distinctly different in gene expression, which may be responsible for their differential sensitivities for atherosclerosis. This study defined how the A
2B
receptor may act as an atherosclerotic-resistance gene, which blunted monocyte adhesion and cell adhesion molecule expression, EC migration and retarded the transendothelial migration of monocytes.
Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis
Authors: Adili Reheman , Mast Katherine , Holinstat Michael
Published: July 2021

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis
ex vivo
, supporting a key role for 12-LOX in regulation of
in vivo
thrombosis. While pharmacologically targeting 12-LOX
in vivo
has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic
in vivo
was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation
ex vivo
in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis.
Ex vivo
flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin.
In vivo
, PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl
3
-induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX
-/-
mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX
-/-
mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity
in vivo
and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.
Abstract 547: Glycocylated Hemoglobin Targets Among Diabetics and Cardiovascular Disease: Do We Need to Change Focus?
Authors: Oni Ebenezer T , Buddhavarapu Siva , Nangrani, Kunal , Patel Ricky , Madubum Chinelo , Chand Raja , Sam Tanyka
Published: July 2021

Background:
Diabetes is an established risk factor for cardiovascular disease (CVD). Glycosylated hemoglobin (HbA1C) targets are surrogate markers to monitor disease progression and guide treatment. While HbA1C targets are relevant in ascertaining glycemic control, the relationship of the established cutoffs and cardiovascular disease risk are not clear.

Methods:
We conducted a step wise logistic regression from a cohort of 1,065 diabetic patients attending the Ambulatory Care Clinic at The Brooklyn Hospital Center between 2012- 2014. CAD was defined as a diagnosis of coronary artery disease in the health records.

Results:
Twelve percent were females, 9% of the individuals achieved an HbA1C target ≤ 7%., and 12% had a history of CAD. Thirty-eight percent were taking metformin, 18% were on insulin. Only 7% of individuals with CAD had their A1C at target. Both a linear regression of HbA1C and logistic regression of HbA1C > 7% did not show any significant association with CAD, See table 1.

Conclusion:
This cross-sectional analysis of diabetic patients on treatment did not show an association between glycosylated hemoglobin and CAD. It is therefore imperative for future studies to evaluate the relationship between HbA1C and cardiovascular outcomes and ascertain the usefulness of this marker to guide CVD treatment strategies in diabetics.

Model 1-unadusted, Model 2- adjusted for BMI, Model 3- adjusted for BMI, LDL, HDL, gender, Metformin use, insulin use.
Abstract 549: Visceral Adipose Tissue Associates With Coronary Plaque Burden Beyond Cardiovascular Risk Factors in Psoriasis
Authors: Rivers Joshua P , Dey Amit K , Chung Jonathan H , Rana Anshuma , Chaturvedi Abhishek , Rodante Justin A , Lerman Joseph B , Playford Martin P , Yao Jianhua , Chen Marcus Y , Bluemke David A , Powell-Wiley Tiffany M , Mehta Nehal N
Published: July 2021

Background:
Psoriasis (PSO), a chronic inflammatory disease associated with increased cardiovascular (CV) risk, provides a reliable human model to study inflammatory atherogenesis. PSO has been known to be associated with cardiometabolic dysfunction including adipose tissue dysfunction. Recently, visceral adiposity (VAT) was shown to be associated with increased CV events, but whether VAT is associated with subclinical atherosclerosis as assessed by coronary plaque burden has not been characterized.

Hypothesis:
We hypothesized that VAT volume by CT is associated with total burden (TB) and more specifically with non-calcified burden (NCB) by CCTA.

Methods:
Consecutive PSO patients (N=68) underwent CT scans to measure abdominal adiposity. VAT volume was quantified from the level of the diaphragm to the pubic symphysis and reported in volume. Coronary plaque characterization was performed by CCTA (Toshiba 30 slice) via QAngio CT software (Medis, The Netherlands). The relationship of VAT with TB and NCB was analyzed using unadjusted and adjusted multivariable regression models (STATA 12).

Results:
The cohort was middle-aged, predominantly male, at low CV risk by FRS with mild to moderate PSO by skin disease severity (Table 1). VAT volume associated with both TB (beta coefficient= 0.49, p-value <0.001) and NCB (beta coefficient= 0.51, p-value <0.001). This relationship remained significant after adjustment for cardiovascular risk factors for TB (beta coefficient= 0.28, p-value = 0.004) and NCB (beta coefficient = 0.34, p-value <0.001).

Conclusions:
Directly quantified VAT directly associated with TB and NCB independent of cardiovascular risk factors. These findings suggest that adipose tissue dysfunction may in part contribute to the high CV events observed in psoriasis and support efforts to provide weight control as a strategy to reduce CV disease in psoriasis.
Abstract 566: MicroRNA-146a Deficiency Prevents PCSK9 Gain-of-Function Mutation-induced Hypercholesterolemia in Mice
Authors: Mohammadmoradi Shayan , Javidan Aida , Jiang Weihua , Moorleghen Jessica , Subramanian Venkateswaran
Published: July 2021

Background and Objective:
Mimetic mediated activation of microRNA 146a (miR-146a) reduces atherosclerosis via suppression of nuclear factor-κB-driven inflammation in mice. The purpose of this study was to determine whether miR-146a influences plasma cholesterol in hypercholesterolemic mice.

Methods and Results:
To induce hypercholesterolemia, female C57BL/6 miR-146a WT (n=8) and miR-146a KO (n=8) mice were injected intraperitoneally with an adeno-associated viral vector (AAV) expressing the proprotein convertase subtilisin/kexin type 9 (PSCK9 D377Y) gain-of-function mutant at a dose of 3 x 10
10
genomic copies/mouse. After infection, mice were fed a Western diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for sixteen weeks, and plasma PCSK9 and total cholesterol concentrations were monitored monthly using an enzymatic assay. Plasma PCSK9 concentrations were profoundly increased 4 weeks post injection (Baseline: WT - 179 ± 12 vs KO - 207 ± 12; Week 4: WT - 1700 ± 148 vs KO - 2689 ± 305 ng/ml) and remained significantly high during 16 weeks (WT - 882 ± 142 vs KO - 718 ± 109 ng/ml; p<0.05 vs baseline) of Western diet feeding. Consistent with increased plasma PCSK9 concentrations, plasma cholesterol concentrations were increased in both groups of mice. Interestingly, miR-146a KO group mice showed less significant increase in plasma cholesterol compared to WT group (Baseline: WT - 88 ± 3 vs KO - 83 ± 3; Week 4: WT - 328 ± 25 vs KO - 195 ± 18 mg/dl) irrespective of the comparable plasma PCSK9 concentrations. Also, lipoprotein distribution analysis with size exclusion gel chromatography revealed that miR-146a KO mice showed a strong reduction in high density lipoprotein (HDL) particles while very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles were not affected.

Conclusion:
Our findings suggests that miR146a plays a critical role in the regulation of HDL particles in PCSK9 gain-of-function mutant-induced hypercholesterolemia in mice. Future studies will identify gene targets influenced by miR-146a in regulating HDL-cholesterol in hypercholesterolemic mice.
Abstract 572: The Effect of Inflammation and Soluble Epoxide Hydrolase Inhibition on Fatty Acid Epoxide Incorporation Into VLDL
Authors: Walker Rachel E , Savinova Olga V , Pedersen Theresa L , Newman John W , Shearer Gregory C
Published: July 2021

Objective:
We have previously observed fatty acid epoxides, a class of potent anti-inflammatory oxylipins, in circulating VLDL. The source of these epoxides is unknown. Cytochrome P450 (CYP450) produces them via oxygenation of polyunsaturated fatty acids (PUFAs), and soluble epoxide hydrolase (sEH) converts them to diols. Our objectives were 1) to investigate if incorporation of epoxides into VLDL occurs via hepatic VLDL synthesis and 2) to determine if incorporation is modulated by inflammation or by inhibition of hepatic sEH.

Approach and Results:
A 2х2 factorial design was used for treatment assignment. Livers were isolated from rats treated with pro-inflammatory lipopolysaccharide (LPS, 10 mg/kg ip) or saline. AUDA, an inhibitor of sEH (10 μM), was included or excluded in the perfusate (Control, N=3; LPS, N=4; AUDA, N=4; LPS+AUDA, N=4). Livers were perfused for 180 minutes. VLDL was isolated by ultra-centrifugation, then analyzed by LC-MS/MS for oxylipin content. Analyzed epoxides and diols were derived from alpha-linolenic acid (ALA), linoleic acid (LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Two-way ANOVA’s were used with triglyceride concentration as a covariate. Concentrations (nM) are reported as mean [95% CI]. DHA-derived epoxides increased with AUDA treatment (3.91 [3.01, 5.07]) compared to livers without AUDA (2.06 [1.58, 2.67]) (p=0.004), but other epoxides were unchanged by AUDA. EPA and ALA-derived epoxides decreased with LPS treatment (0.32 [0.22, 0.47]; 2.44 [2.07, 2.87]) compared to animals without LPS (0.73 [0.46, 1.16]; 3.28 [2.71, 3.96]) (p=0.01; 0.02). AA and DHA-derived diols decreased with LPS treatment (1.01 [0.82, 1.25]; 0.21 [0.17, 0.26]) compared to animals without LPS (1.46 [1.15, 1.86]; 0.31 [0.24, 0.39]) (p=0.03; 0.03).

Conclusions:
Treatment with LPS and AUDA have significant effects on incorporation of epoxides and diols into VLDL, supporting hepatic incorporation controlled by inflammation. Inflammation decreased select EPA- and ALA-derived epoxides. In contrast, sEH inhibition increased only DHA-derived epoxides. Surprisingly, in VLDL only epoxides derived from omega-3 fatty acids were affected by either inflammation or inhibition of sEH.
Abstract 615: Transactivation of EGFR by TGFβ Induces Hypertension by Increasing Arteriolar Myogenic Response
Authors: Carnevale Daniela , Casaburo Manuel , Fardella Stefania , Bizzotto Dario , Da Ros Francesco , Cifelli Giuseppe , Innocenzi Gualtiero , Bressan Giorgio , Lembo Giuseppe
Published: July 2021

Myogenic tone (MT) is an intrinsic property of arteriolar smooth muscle cells (SMCs) that contract when intraluminal pressure is increased. MT is a relevant physiological mechanism to maintain stable blood flow through important organs like kidney and brain, thus preventing organ damage that could derive from increase in blood pressure (BP). On the other hand, enhancement of the basal MT could raise BP by increasing systemic vascular resistance, thus further contributing to hypertension. Several studies in animal models suggest that primary dysfunctions in SMCs can directly cause abnormalities in BP. However, in hypertensive animal models investigated so far, the increment of MT is accompanied by a stronger response to G protein coupled receptor (GPCR) agonists, thus making it difficult to distinguish whether increased vascular resistance and BP are primary effects of a more robust MT or the consequence of increased reactivity to agonists. Here we show an increased MT in mice deficient of
Emilin1
, an extracellular inhibitor of TGFβ signaling, displaying a phenotype of spontaneous hypertension. We also found that the higher TGFβ signaling in
Emilin1
deficient SMCs stimulates heparin binding epidermal growth factor (HB-EGF) expression and subsequent transactivation of the EGF receptor. When step-increases in intraluminal pressure are applied to mesenteric resistance arteries (MRA), the combined stimulation of mechanosensor and EGF transactivation results into activation of transient receptor potential classical type 6 (TRPC6) and melastatin type 4 (TRPM4) channels, stimulation of voltage dependent calcium channels (VDCC). To put our data into translational perspective, we measured MT on resistance arteries isolated from hypertensive patients and untreated normotensives, finding increased MT and TGFβ signaling in the former group. In addition, by using an antibody neutralizing EGFR signaling we found a normalization of the increased MT, thus confirming the relevance of TGFβ-EGFR pathway in humans. Overall our results suggest that primary increase of MT induced by TGFβ-EGFR transactivation can cause hypertension and that higher TGFβ-EGFR signaling and MT are common alterations of resistance arteries of hypertensive patients.
Abstract 625: Modest Elevations of Extracellular Sodium Activate Dendritic Cells and Contribute to Hypertension
Authors: Barbaro Natalia , Foss Jason , Dikalov Sergey , Harrison David , Kirabo Annet
Published: July 2021

Recently, it has become apparent that sodium can accumulate in the interstitium of hypertensive humans and animals, and such high salt concentrations drive immune cells toward a pro-inflammatory phenotype through poorly defined mechanisms. We hypothesized that dendritic cells (DCs) are activated by modest elevations of extracellular sodium via formation of IsoLGs leading to hypertension. Isolated mouse splenic DCs were cultured in media with either normal salt (NS, 150 mM NaCl), high salt (HS, 190 mM NaCl) or HS plus the gp91ds-tat peptide which inhibits assembly of NADPH oxidase for 24 hours. DCs exposed to HS demonstrated a marked increase in superoxide production compared to NS (146.3±9.5 vs. 100.0±5.0 % control, p<0.001). This was NADPH oxidase dependent because incubation with the gp91ds-tat peptide prevented this effect. Using flow cytometry, we found that DCs cultured in HS had a significantly higher expression of CD86 and more IsoLG-protein adducts than those cultured in NS. These effects were not mimicked by an equiosmolar concentration of mannitol. Western blot analysis of protein extracts from DCs indicated HS markedly increased phosphorylation of the NADPH oxidase subunit p47
phox
and association of p47
phox
with gp91
phox
. Importantly, we found that the salt-activated DCs promote production of cytokines interferon gamma and interleukin 17 by primed T cells. To determine whether these salt-activated DCs promote hypertension, mice received adoptive transfer of splenic DCs that were cultured for 24 hours in either NS (n = 6); HS (n = 5); or HS plus an IsoLG scavenger (n = 6). Mice were then implanted with radiotelemeters to measure mean arterial pressure (MAP) and infused with a subpressor dose (140 ng/kg/min) of angiotensin II (AngII) for two weeks. This caused no increase in MAP in mice that received NS DCs (+0.8 +/- 3.3 mmHg), whereas MAP increased significantly with AngII infusion in mice that received HS DCs (+13.4 +/- 5 mmHg). This pro-hypertensive effect of salt on DCs was attenuated by scavenging of IsoLGs (-3.3 +/- 4.4 mmHg) during salt exposure. These findings provide a mechanistic link between salt, inflammation and hypertension involving increased oxidative stress and IsoLG production in DCs.
Abstract 637: Role of Nuclear Smooth Muscle Alpha-Actin in the Differentiation of Vascular Smooth Muscle Cells
Authors: Ma Shuangtao , Milewicz Dianna M
Published: July 2021

Although the function of the cytoplasmic smooth muscle (SM) α-actin in contractile units has been well characterized in vascular SMCs, whether the SM α-actin is present in the nucleus and its nuclear function are yet to be determined. Our previous study discovered the mutations in
ACTA2
, which encodes SM α-actin, predispose to both thoracic aortic disease and occlusive vascular diseases (stroke and coronary artery disease). We hypothesize that nuclear SM α-actin is critical for differentiation of SMCs. The cytoplasmic and nuclear fractions of mouse SMCs were isolated and the SM α-actin was detected in both cytosol and nucleus by immunoblotting. Moreover, both cytoplasmic and nuclear SM α-actin were dramatically upregulated during the differentiation of SMCs stimulated with TGF-β. Two-dimensional gel electrophoresis, which can separate actin isoforms based on distinct isoelectric points, was used to show that the ratio of SM α-actin to β-actin was increased in nuclear fraction when compared to cytoplasmic fraction with differentiation of SMCs, driven by serum starvation and TGF-β treatment. Furthermore, differentiation was associated with a significantly higher nuclear to cytoplasmic ratio of SM α-actin, indicating SM α-actin accumulates in the nucleus during differentiation. Co-immunoprecipitation experiments identified that nuclear SM α-actin is a component of the chromatin remodeling complex, Ino80, and the immunofluorescence confirmed co-localization of SM α-actin and Ino80 in the nucleus. To investigate the transcriptional function of nuclear SM α-actin, we performed the chromatin immunoprecipitation quantitative PCR, which revealed that the SM α-actin, but not β-actin, accumulated to the promoter region of SMC differentiation markers, including
Myh11
,
Cnn1
, and
Tagln
, during the differentiation of SMC. As expected, the
Acta2
mutant SMCs (pArg149Cys) disrupt the differentiation of SMCs based on decreased contractile protein levels and increased proliferation. Furthermore, specific
Acta2
missense mutations disrupt the ability for SM α-actin to localize to the nucleus. Taken together, our study found the SM α-actin concentrates into the nucleus of SMCs and may play a role in chromatin remodeling required for differentiation of SMCs.
Abstract 643: CX
3
CR1 Identifies Adventitial Macrophage Progenitor Cells (AMPCS), a Local Source of Self-Renewing Macrophages in Postnatal Murine Arteries
Authors: Toledo Deborah F , Williamson Anna , Schwarz Nisha , Fernando Sanuja , Di Bartolo Belinda , Nicholls Stephen J , Psaltis Peter J
Published: July 2021

Background:
Macrophages are integral to vascular biology and disease. Although traditional paradigms assert that vascular macrophages originate from circulating monocytes, recent data suggest that some are seeded in utero from CX
3
CR1
+
progenitors, and are maintained postnatally by local self-renewal. We previously identified a novel population of locally-maintained adventitial macrophage progenitor cells (AMPCs) as a source of self-renewing macrophages in adult mouse arteries.

Hypothesis:
AMPCs express CX
3
CR1 and do not derive from definitive hematopoiesis.

Methods:
Single-cell disaggregates were prepared from postnatal murine aortas. AMPC content was assessed by macrophage colony-forming unit (CFU-M) assays. Flow cytometry together with fluorescence activated cell sorting (FACS) were used to investigate the immunophenotypic profile of AMPCs and fate-mapping to assess their origins.

Results:
CFU-M prevalence in C57BL/6J aortic cells was highest in neonatal mice (~100 per 10
5
cells), and diminished progressively with age (~55/10
5
at 3w, ~15/10
5
at 12w, ~5/10
5
at 52w, n>4, P<0.01). Secondary replating of single cells from aortic CFU-M revealed striking self-renewal capacity, with 1 in 10 cells forming new CFU-M (n=8). Undifferentiated CFU-M displayed >95% expression of the stem cell markers Sca-1 and c-Kit, and high levels of CX
3
CR1, but did not acquire the monocyte/macrophage markers CD11b or F4/80 until treated with macrophage-colony stimulating factor (n=6). Mice deficient in CX
3
CR1 (Cx
3
cr1
GFP/GFP
) produced fewer CFU-M than heterozygous (Cx
3
cr1
+/GFP
) littermates (8.7±0.7 vs 15.3±0.7 per 10
5
, P<0.001, n>7). FACS selection confirmed that CFU-M forming AMPCs were exclusively contained within a CX
3
CR1
+
subpopulation that did not express either CD11b or F4/80 (n=3). Finally, CFU-M analysis from Flt3
Cre
xRosa
mT/mG
mice demonstrated that AMPCs arise from a FLT3
-ve
source, indicating that their origins are independent of definitive hematopoiesis (n=4).

Conclusion:
Clonogenic, self-renewing murine AMPCs express CX
3
CR1 but not the monocyte/macrophage markers CD11b and F4/80. The high prevalence of AMPCs in neonatal aorta is consistent with prenatal seeding from CX
3
CR1
+
progenitors, independent of definitive hematopoiesis.