bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 222: HDL-ApoA-I Exchange Rate is Inversely Correlated With Coronary Artery Disease Stability
Authors: Borja Mark S , He Yumin , Genest Jacques , Oda Michael N
Published: July 2021

Objective:
The exchange of apolipoprotein A-I between lipid-associated and lipid-free states is a key step in reverse cholesterol transport and is representative of HDL’s functional status. Reduced HDL-apoA-I exchange (HAE) is associated with the presence of cardiovascular disease. To build on this observation, we investigated the hypothesis that HAE in a coronary artery disease-identified patient decreases with increased coronary artery disease instability.

Method:
HAE was measured by electron paramagnetic resonance spectroscopy (EPR), wherein nitroxide-labeled lipid-free apoA-I is introduced into apolipoprotein B-depleted plasma, incubated at 37°C and measured. When added to plasma, the nitroxide-labeled apoA-I specifically interacts with HDL and displaces resident apoA-I. The EPR spectrum reports the population of lipid-bound, spin labeled apoA-I, which is directly proportional to the amount of resident apoA-I displaced. The relative level of apoA-I displaced is representative of the plasticity of HDL and its ability to make lipid-poor apoA-I available for ABCA1-mediated cholesterol efflux. We measured HAE in the plasma of three groups: stable coronary artery disease (n=22), 3 months following acute coronary syndrome (n=19), and a control group with no history of coronary artery disease (n=15).

Results:
HAE was significantly lower in both the stable coronary artery disease and acute coronary syndrome groups, compared to the control group (P<0.001 and, P<0.0001, respectively). Remarkably, the ACS subjects also had significantly lower HAE compared to those with stable CAD (P<0.01). By comparing HAE to apoA-I and HDL-C levels, we observed that stable CAD and ACS subjects have lower HAE per milligram/deciliter of apoA-I, consistent with a qualitative deficiency in their apoA-I.

Conclusions:
HAE activity is a by-product of apoA-I qualitative status, which is inversely correlated with coronary artery disease stability.
Abstract 225: Apolipoprotein A-IV Is a ß3 Integrin Ligand and an Endogenous Inhibitor of Platelets: Novel Mechanisms of Prevention and Treatment for Atherothrombosis
Authors: Xu Xiaohong , Ju Lining , Spring Christopher M , Yang Hong , Reheman Adili , Wang Yiming , Zhang Hailong , Yang Yan , Reddy Emily C , Song Jina , Zhu Guangheng , Lei Xi , Lang Sean , Chen Pingguo , Jin Joseph W , Connelly Phil , She YiMin , Cyr Terry , Zhu Cheng , Freedman John , Tso Patrick , Davidson Sean , Ni Heyu
Published: July 2021

Apolipoprotein A-IV (apoA-IV) is a lipid binding protein secreted by the intestine during dietary lipid absorption. Several clinical studies in different ethnic populations have demonstrated that apoA-IV levels inversely correlate with cardiovascular diseases. However, the roles of apoA-IV in platelet aggregation and atherothrombosis are completely unknown.

Here we isolated apoA-IV from plasma with beads coated with human platelet β3 integrin. Using a biomembrane force probe that detects single-molecule interactions, we clearly demonstrated that apoA-IV specifically binds to purified αIIbβ3 on beads and native αIIbβ3 on platelets or Chinese hamster ovary cells. ApoA-IV and αIIbβ3 interaction could be blocked by a monoclonal antibody against β
3
integrin, M1. Importantly, recombinant apoA-IV can competitively block fibrinogen-αIIbβ3 interaction, and specifically inhibited human and mouse platelet aggregation. Consistently, platelet aggregation was enhanced in mice lacking apoA-IV following stimulation with various agonists. In ex vivo perfusion chambers, apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV enhanced ex vivo thrombosis under both low and high shear stresses. Furthermore, in vivo intravital microscopy models revealed that FeCl
3
- and laser-induced thrombosis were enhanced in mice lacking apoA-IV, while transfusion of human or mouse recombinant apoA-IV significantly attenuated this process. To further identify the potential binding sites of apoA-IV for platelet αIIbβ3 integrin, we deleted apoA-IV N-terminal 38 amino acids and/or the C-terminal 41 amino acids. We observed that deletion of the N-terminus abrogated platelet-inhibitory function. Interestingly, we found that mutation of either of two highly conserved aspartic acid (D) residues at positions 5 and 13 abolished or reduced the inhibitory function of apoA-IV, suggesting that D5 and/or D13 participate in a direct protein-protein interaction between apoA-IV and αIIbβ3 integrin.

Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a novel link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases.
Abstract 274: Lipid Accumulation in CD11c-Expressing Intimal Myeloid Cells Induces Chemokine Production Required for Leukocyte Recruitment to Early Atherosclerotic Lesions
Authors: Siu Allan E , Jongstra-Bilen Jenny , Cybulsky Myron
Published: July 2021

Background:
In the early stages atherosclerosis myeloid cells accumulate through the recruitment of circulating monocytes and macrophage proliferation. However, the first foam cells appear in the artery wall prior to increased monocyte recruitment and macrophage proliferation. These initial foam cells are derived from myeloid cells residing in the normal intima, and express integrin alpha
X
(CD11c).

Rationale:
When CD11c
+
cells were depleted at the initiation of a cholesterol-rich diet (CRD) in
Ldlr
-/-
mice, lipid accumulation was reduced. We hypothesize that lipid accumulation in CD11c
+
intimal myeloid cells play an instrumental role in the recruitment of circulating monocytes to the artery wall.

Methods:
To assess the role if CD11c-expressing intimal myeloid cells in early atherosclerosis, CD11c
+
cells were deleted in
Ldlr
-/-
mice reconstituted with bone marrow from CD11c-DTR transgenic mice. Mice reconstituted with C57BL/6 bone marrow were used as controls. Depletion of CD11c
+
cells was maintained throughout a 10 or 21 days CRD by giving injections of diphtheria toxin every three days. Monocyte recruitment was assayed by pulse labeling and quantifying BrdU positive leukocytes in the aortic arch. Intimal gene expression of adhesion molecules and chemokines was carried out by real-time PCR. Functional characterization of intimal chemokine expression on the recruitment of circulating monocytes was evaluated by antibody blockade of chemokine receptor CCR5 as well as reconstituting
Ldlr
-/-
with CCL5 deficient bone marrow.

Results:
Recruitment of circulating monocytes was reduced in Ldlr-/- chimeras depleted of CD11c
+
cells. Correspondingly, intimal expression of chemokines CCL3, CCL4 and CCL5 were significantly blunted after 21 days of CRD in in
Ldlr
-/-
BM chimeras depleted of CD11c
+
cells. The recruitment of BrdU labeled circulating monocytes was reduced by antibody blockade of CCR5 - the receptor for CCL3, CCL4 and CCL5.

Conclusion:
Collectively these data suggest that lipid accumulation in resident CD11c
+
intimal myeloid cells promote the recruitment of circulating monocytes to nascent lesions through the expression of CCL3, CCL4 and CCL5.
Abstract 280: Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage
Authors: Yamashita Atsushi , Zhao Yan , Matsuura Yunosuke , Yamasaki Kazuaki , Moriguchi-Goto Sayaka , Iwakiri Takashi , Okuyama Nozomi , Koshimoto Chihiro , Kawai Keiichi , Tamaki Nagara , Zhao Songji , Kuge Yuji , Asada Yujiro
Published: July 2021

Aims:
Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.

Methods:
Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose (18F-FDG) and pimonidazole, a marker of hypoxia.

Results:
The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of 18F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p < 0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.

Conclusion:
Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.
Abstract 282: α5ß1 Integrin Signaling Through Focal Adhesion Kinase Mediates Oxidized LDL-Induced Endothelial Proinflammatory Gene Expression
Authors: Yurdagul Arif , Green Jonette , Albert Patrick , McInnis Marshall , Mazar Andrew , Orr Wayne
Published: July 2021

Endothelial cell activation drives early atherosclerotic plaque formation. Both fibronectin deposition and accumulation of oxidized LDL (oxLDL) occur early during atherogenesis and both are implicated in enhanced endothelial cell activation. However, interplay between these responses has not been established. We now show that oxLDL-induced NF-κB activation, proinflammatory gene expression, and monocyte binding is significantly enhanced when endothelial cells are attached to fibronectin compared to basement membrane proteins. This enhanced response does not result from altered oxLDL receptor expression, oxLDL uptake, or reactive oxygen species production, but instead results from oxLDL-induced activation of the fibronectin-binding integrin α5β1. Correspondingly, oxLDL activates focal adhesion kinase (FAK), the downstream signaling partner to integrin α5β1. We also found enhanced integrin α5 expression and active FAK in the endothelium overlying atherosclerotic plaques in both mice and humans. Preventing α5β1 signaling (blocking antibodies, knockout cells) inhibits oxLDL-induced NF-κB activation and VCAM-1 expression. Also, inhibiting FAK kinase activity utilizing a small molecule inhibitor (PF-573228), siRNA, and kinase dead fibroblasts reduces oxLDL endothelial cell activation. Furthermore, treatment with the α5β1 signaling inhibitor, ATN-161, significantly blunts atherosclerotic plaque development in ApoE deficient mice, characterized by reduced macrophage accumulation without affecting fibrous cap size. Therefore, our data suggest that α5β1-mediated crosstalk between fibronectin and oxidized LDL regulates inflammation in early atherogenesis.
Abstract 294: Differential Expression of Hedgehog/Notch and Transforming Growth Factor-ß in Human Abdominal Aortic Aneurysms
Authors: Doyle Adam , Redmond Eileen M , Cullen John P , Cahill Paul A , Morrow David
Published: July 2021

Objective:
The molecular mechanisms leading to the development of Adnominal Aortic Aneurysms (AAA) remain poorly understood. The aim of this study was to determine the expression of Sonic Hedgehog (SHh), Transforming Growth Factor Beta (TGF-β) and Notch signaling components in aneurysmal and non-aneurysmal aorta in vivo and demonstrate SHh control of Notch, TGF- β1 and SMC differentiation in vitro.

Methods:
Paired tissue samples were obtained from aneurysmal and non-aneurysmal (control) segments of the aortic wall of at least 8 patients with suitable anatomy undergoing open repair of infrarenal AAA. Protein and mRNA expression levels were determined by western blot analysis and quantitative Real-time PCR.

Results:
Aneurysm development resulted in a significant reduction in vascular smooth muscle (vSMC) differentiation gene protein and mRNA levels for α-actin and SMC22α, respectively. In parallel, significant reductions in Hh and Notch signaling component expression was observed in aneurysmal tissue when compared to control, concurrent with increased TGF-β1 expression. In vitro, Hh signaling inhibition with cyclopamine (40μmol/L) treatment for 24 h in human aortic smooth muscle cells (HASMC), resulted in decreased Hh/Notch signaling component and vSMC differentiation gene expression. Moreover, cyclopamine significantly increased TGF-β1 mRNA expression.

Conclusion:
These results suggest that SHh/Notch and TGFβ signaling are differentially regulated in aneurysmal tissue, compared with non-aneurysmal tissue. Changes in these signaling pathways and the resulting changes in vSMC content may play a causative role in the development of AAA.
Abstract 313: A Novel Catheter-Based Radionuclide Imaging System to Characterize Atherosclerotic Plaque
Authors: Zaman Raiyan , Kosuge Hisanori , Carpenter Colin , Pratx Guillem , Sun Conroy , Xing Lei , McConnell Michael
Published: July 2021

Objectives:
Atherosclerosis underlies coronary artery diseases—the leading cause of death in the United States and worldwide. In this study, we developed a novel catheter-based radionuclide imaging (CRI) system to image
18
F-fluorodeoxyglucose (
18
F-FDG), a radionuclide, a marker of vascular inflammation, in murine carotid arteries and characterized the system for spatial resolution from multiple scintillating materials.

Methods:
The catheter system includes 35 mm and 8 mm fixed focal length lenses, which are subsequently connected to a CMOS camera and fiber holder. The distal ferrule of an image bundle is terminated with a wide-angle lens. The novelty of this system is a scintillating balloon with a crystal tip in the front of the wide angle lens to image light from the decay of
18
F-FDG emission signal. The scintillating balloon is fabricated from 1mL of silicone RTV catalyst mixed with 1 mL base and 50 mg/mL calcium fluoride doped with Europium (CaF
2
:Eu). To identify the optimal scintillating materials with respect to resolution, we calculated modulation transfer function (MTF) of Yttrium Aluminum Garnet doped with Cerium (YAG:Ce), anthracene, and CaF
2
:Eu phosphors using a thin line optical phantom (Fig. 1a-1b). Macrophage-rich FVB murine atherosclerotic carotid plaque model (n = 4) was used in
ex vivo
experiments. Confirmatory imaging was also performed by an external optical imaging system (IVIS-200).

Results:
Analysis of the different phosphors (Fig 1b) showed that CaF
2
:Eu enabled the best resolution of 1.2μm. The CRI system visualized
18
F-FDG in atherosclerotic plaques (Fig. 1d). The ligated left carotid (LR) artery exhibited 4.9х higher
18
F-FDG signal intensity compared to the non-ligated right carotid (negative control) artery (2.6х10
4
±1.4х10
3
vs. 5.4х10
3
±1.3х10
3
, A.U., p = 0.008) and confirmed with IVIS-200 (Fig. 1d).

Conclusions:
This CRI system enables high-resolution and sensitive detection of
18
F-FDG uptake by murine atherosclerotic plaques.
Abstract 318: Determinants of Red Blood Cell Omega-3-Fatty Acid Content in Patients With Peripheral Arterial Disease
Authors: Nosova Emily V , Owens Christopher D , Chong Karen C , Alley Hugh F , Conte Michael S , Grenon S Marlene
Published: July 2021

Objectives:
Despite available medical therapies, patients with peripheral arterial disease (PAD) remain at high risk for cardiovascular events. n-3 polyunsaturated fatty acids (PUFAs), derived from marine sources, have been shown to improve cardiovascular mortality. The omega-3 index (O3I), a proportion of the n-3 PUFAs eicosapentanoic acid and docosahexanoic acid in the red blood cell membrane, correlates with cardiovascular risk. We sought to determine the clinical factors associated with the O3I in patients with PAD.

Methods:
This cross-sectional study included 111 patients, who had an ankle-brachial index of < 0.9 associated with claudication symptoms. We used linear regression to determine the association between demographic and lifestyle factors and the O3I.

Results:
The mean age of the cohort was 69 ± 8 years, 37% had diabetes mellitus (Hemoglobin A1c: 7 ± 1%), and 94% had smoked. The mean O3I was 5 ± 2%. Table 1 shows the variables associated with O3I in regression analyses. In a multivariate model, an independent association was maintained with increasing age, BMI, and a history of smoking and fish oil intake.

Conclusions:
In a cohort of patients with PAD, older age, elevated BMI, and prior fish oil supplementation predicted a higher O3I. A history of smoking correlated with a lower O3I. Our findings suggest that targeting nutrition and tobacco use with therapeutic interventions has potential to reduce cardiovascular risk among PAD patients.
Abstract 328: Capillary Formation and Autologous Regulation of Androgen Receptor Expression in Endothelial Progenitor Cells by DHT: Potential Implications for EPC-Mediated Cardiovascular Repair in Men
Authors: Plutino Yuliya , Barchiesi Federica , Simigdala Nikiana , Imthurn Bruno , Dubey Raghvendra
Published: July 2021

Therapeutic use of Endothelial Progenitor Cells (EPCs) is known to repair and protect against cardiovascular damage. Since androgens are associated with cardiovascular protection in men, we investigated the mechanism(s) by which androgens (dihydrotestosterone; DHT) can modulate androgen receptor (AR) dependent EPC activity.

Tube/capillary formation and androgen receptor expression by cultured EPCs was assessed using 2D-cultures/co-cultures, western blotting and RT-PCR.

Treatment with DHT (100nM) induced capillary/tube formation by EPCs (≈179±3%; p<.05 vs control) and these effects were attenuated by AR antagonist flutamide and AR siRNA. DHT (10-100nM) up-regulated AR protein expression and this effect was abrogated by AR antagonist flutamide (1μM), AR silencing siRNA and cycloheximide (protein synthesis inhibitor). Compared to AR protein, DHT did not induce AR mRNA expression and DHT-induced AR expression was not blocked by the transcription inhibitor actinomycin-D (10ng/ml). Treatment with proteasome inhibitor MG132 (100nM) mimicked the effects of DHT on AR protein expression, moreover, when combined with DHT, the stimulatory effects on AR expression were additive. To ascertain the role of protein stabilization in mediating AR up-regulation in EPCs, the effects of MG132 and DHT on Raf-1, which is known to undergo proteasomal degradation, were assessed. Treatment with MG132, but not DHT, increased Raf-1 expression in EPCs. Moreover, the stimulatory effects of DHT on capillary formation were enhanced in presence of MG132. These findings suggest that DHT up-regulates AR expression and function in EPCs via protein stabilization, however, participation of other pathways cannot be ruled out.

In EPCs, DHT induces capillary formation via AR and regulates AR expression in an autologous fashion. Since, androgens induce endothelial growth, and most patients receiving EPCs for cardiovascular repair are older, the low endogenous levels of androgens would decrease the potential of stem cell mediated tissue repair. More importantly, pretreatment of patients or priming of EPCs with androgens/DHT, as well as co-administration of DHT with EPCs may potentiate ARs and EPC mediate cardiovascular tissue repair in men.
Abstract 340: Psychological Stress Impairs Both Endothelial Function and Insulin Sensitivity via Activation of Inflammation and ER Stress Pathways Mediated by Corticotropin-Releasing Hormone
Authors: Chung Ick-Mo , Kim Hee Young , Lee Youn Jung
Published: July 2021

Background:
Psychological stress may contribute to atherogenesis, although the underlying mechanism is poorly understood yet. We studied the role of corticotropin-releasing hormone (CRH) in the pathogenesis of stress-induced endothelial dysfunction and insulin resistance with specific focus on inflammation and ER stress pathways.

Methods and Results:
Immobilization stress (IS) was applied (2 hr/d) for 14 d to male 8-wk OLETF rats, whereas control groups comprised of rats without IS (n≥7 for each). 14d-IS decreased acetylcholine (Ach)-induced NO production, measured by direct NO electrochemical microsensor, from the aorta along with decreased expression of eNOS mRNA compared with control group. 14d-IS did not significantly change plasma glucose, but increased plasma insulin (342.5±6.3 vs. 496.0±51.6 pg/ml, p<0.05) and HOMA-IR (3.1±0.2 vs.4.7±0.5 mmol/LxμIU/mL, p<0.05) compared with control group. IS increased phosphorylation of both IRS1 serine and ERK, and increased expression of ER stress marker BiP. IS also increased translocation of NF-kB subunit p65 into nucleal fraction, and increased mRNA expression of both TNF-α and MCP-1 in the liver. Real-time RT-qPCR study showed that 1d IS increased expression of CRH mRNA and urocortin 2 mRNA in the hypothalamus by 2.5 folds and expression of CRH receptor type 2 mRNA in the aorta by 7.8 folds compared with control group. In cultured monocyte U937 cells, CRH induced translocation of NF-kB subunit p65 into nuclear fraction, and increased expression of BiP, phosphorylated inositol requiring enzyme 1, and MCP-1 mRNA. In cultured HUVECs, CRH pretreatment inhibited insulin-stimulated eNOS phosphorylation at Ser
1177
.

Conclusions:
Psychological stress impairs both endothelial function and insulin sensitivity probably via activation of NF-kB-mediated inflammation and ER stress pathways by CRH.
Abstract 348: α1a-Adrenoceptor Genetic Variant-Triggered Hyperproliferation Is Mediated Through Aberrant Interaction With Spinophilin in Smooth Muscle Cells
Authors: Babaeva Ekaterina , Gradinaru Irina , Schwinn Debra A , Oganesian Anush
Published: July 2021

Activation of α1-Adrenergic Receptors (α1ARs), members of the G protein-coupled receptor (GPCR) superfamily, in response to stimulation of the sympathetic nervous system by catecholamines plays a major role in regulating cardiovascular (CV) function and blood pressure (BP). Among three α1AR subtypes (α1a,α1b,α1d) expressed in vasculature α1aARs predominate in human vascular smooth muscle cells (SMC), particularly in resistant vessels most involved in BP control. Polymorphisms in the α1aAR gene are also related to hypertension (HTN), one of the major CV risk factors. Recently, we discovered a novel, unique mechanism of hypertension triggered by naturally occurring human α1aAR-G247R (247R) genetic variant, identified in the 3rd intracellular loop (3iL) of the receptor in hypertensive patient. In fibroblasts, 247R signals via constitutive active coupling to the βarrestin1/MMP7/EGFR pathway. Here we report that 247R expression in SMC triggers constitutive, Gq- and MMP/EGFR/ERK-dependent hyperproliferation. Agonist (phenylephrine) treatment of cells inhibits hyperproliferation and induces hypertrophy and α1aAR inverse agonist prazosin inhibits hyperproliferation and hypertrophy indicating Gq-dependent pathway. Expression of 247R in SMC also triggers upregulation of spinophilin (SPL), a ubiquitous multi-domain scaffold protein that binds to 3iL of several GPCRs and competes with βarrestins for 3iL binding. We hypothesized that SPL mediates constitutive signaling of 247R and examined whether SPL directly interacts with α1aAR-WT or 247R. Our preliminary results demonstrate a distinct interaction of α1aAR-WT and 247R with SPL, although the SPL-α1AR-WT interaction appears to be strongest. SPL 1-480aa fragment demonstrates stronger interaction with α1aAR-WT indicating that this domain is responsible for interaction with 3iL. Interestingly the SPL 480-817 fragment has stronger interaction with 247R. Thus, expression of the naturally occurring human α1aAR genetic variant in vascular cells activates distinct signaling pathways leading to aberrant hyperproliferation and hypertrophy, and may eventually result in hypertension and other CV diseases suggesting a possible novel mechanism underlying some forms of human hypertension.
Abstract 35: Plasma Fibronectin is a Vital Supportive Factor in Hemostasis and a Unique Self-Limiting Regulator in Thrombosis
Authors: Wang Yiming , Reheman Adili , Spring Christopher , Kalantari Jalil , Marshall Alexandra , Wolberg Alisa , Gross Peter , Weitz Jeffrey , Rand Margaret , Freedman John , Mosher Deane , Ni Heyu
Published: July 2021

The role of plasma fibronectin (pFn) in thrombosis/hemostasis is controversial. We previously demonstrated that pFn supports thrombosis in pFn-/- mice. Interestingly, depletion of pFn in fibrinogen (Fg)/VWF-/- mice resulted in enhanced thrombus formation, revealing a functional switch of pFn in the absence of Fg/VWF. However, the mechanism controlling this switch is unknown. Furthermore, the hemostatic function of pFn is unclear.

To address these questions, we established Fg/pFn-/- and VWF/pFn-/- mice in addition to triple deficient mice (Fg/VWF/pFn-/-, TKO). TKO mice had significantly higher mortality (23.1% vs. 9.1%) and longer tail bleeding time than their pFn+ Fg/VWF-/- littermates. Autopsy revealed severe subcutaneous or abdominal bleeding. pFn depletion in Fg-/- mice also increased mortality from 5.5% to 25.6% and prolonged bleeding times; the latter effect was reversed with pFn infusion. Using intravital microscopy, we found that pFn deposition at the site of injury preceded significant platelet accumulation, suggesting that pFn is an efficient and rapid hemostatic factor. Based on thromboelastography, fibrin clots from pFn+ mice were significantly stronger than those from pFn-/- mice. Consistent with this, inferior vena cava occlusion was significantly delayed in pFn-/- mice. Under confocal microscopy, fluorescently-labeled pFn was actively recruited into the fibrin network in mouse and human plasma. Under electron microscopy, physiological levels of pFn doubled the diameter of fibrin fibers, suggesting that pFn contributes to lateral aggregation of fibrin protofibrils. Interestingly, pFn enhances platelet aggregation when linked with fibrin and inhibits aggregation when fibrin is absent. pFn therefore gradually switches from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium.

Our data established pFn as an important supportive factor in hemostasis and a unique self-limiting regulator of thrombosis, suggesting the therapeutic potential of pFn transfusion in controlling bleeding complications, particularly for those receiving antithrombotic therapy for heart attack and stroke.
Abstract 353: Triciribine, a Selective Akt Inhibitor, Inhibits Myofibroblast Differentiation and Ameliorates Pulmonary Vascular Remodeling in Pulmonary Hypertension
Authors: Somanath Payaningal R , Abdalla Maha
Published: July 2021

Objectives:
Idiopathic pulmonary fibrosis (IPF) is an incurable, chronic and progressive disease with severely poor prognosis and often leads to pulmonary hypertension (PH). Persistent myofibroblast (MF) differentiation in response to TGFβ, marked by de novo expression of αSMA stress fibers, is the central orchestrator of tissue fibrosis and vasculopathy. Our previous studies indicated the integral role of Akt in myofibroblast differentiation and fibrosis. Here, we investigated the efficacy of Triciribine (TCBN), a selective Akt inhibitor, currently in clinical trials for cancer therapy, as a potential therapeutic option for IPF and PH.

Methods:
WT and Akt1 null mice, as well as those treated with TCBN were subjected to 3 week hypoxia along with normoxia control, and the lungs were used for imaging and western analysis to study the biochemical, structural and vascular changes in the lungs. Cutting edge techniques such as microfil-cast based vascular imaging, confocal imaging and analysis of extracellular matrix protein turnover was performed for the data collection and analysis.

Results:
Under hypoxia, WT mice exhibited robust vascular remodeling leading to thickening of the artery wall, reduction in lumen size and loss of capillaries in the lungs. This effect was significantly less in Akt1 null mice. Treatment with TCBN significantly ameliorated vascular remodeling in mice due to hypoxia, and resulted in normalization of the vasculature with increased lumen size, reduced wall thickness and increased number of capillary branches as compared to untreated controls. TCBN also affected expression of a number of different extracellular matrix proteins and matrix metalloproteases in addition to changes in the expression of contractile proteins alpha-actin, myocardin and serum response factor in the adventitial fibroblasts.

Conclusions:
Our results demonstrate the crucial role of Akt as an integral mediator of MF differentiation in IPF and PH. Further, TCBN could potentially be a therapeutic option for IPF and PH.
Abstract 355: NOX Deficiency Alters Endothelial Response to Blood Flow--Induced Shear Stress
Authors: Winkel Leah C , Fontijn Ruud D , Musters Rene J , Wentzel Jolanda J , Horrevoets Anton J , van der Heiden Kim
Published: July 2021

Increased levels of Reactive oxygen species (ROS) lead to vascular complications like atherosclerosis. Important producers of ROS are the family of NADPH oxidases (NOXes). NOX subtypes have recently been shown to be responsive to blood flow-induced shear stress. Shear stress is also responsible for the focal nature of atherosclerosis by regulating expression of the athero-protective transcription factors KLF2 and Nrf2. These transcription factors inhibit pro-inflammatory and promote antioxidant gene expression. High, pulsatile blood flow in straight vessel segments induces KLF2 and Nrf2. In contrast, disturbed flow at vessel bends and bifurcations inhibits them, thus rendering the vessel wall susceptible to systemic risk factors for vascular disease. Several studies indicate that ROS might influence KLF2/Nrf2 expression and/or activation. In this study we set out to determine the interrelationship between shear stress-induced NOX subtypes and KLF-2/Nrf2 activation.

HUVEC were exposed to 1.5 Pascal pulsatile shear stress (athero-protective) for 6 days in IBIDI flow chambers and compared to static conditions (athero-prone). NOX 1 and NOX 5 were expressed at undetectable levels for PCR analyses in static HUVEC. NOX 2 and NOX 4 were expressed in static HUVEC. Upon exposure to shear stress, NOX 2 expression decreased to undetectable levels while NOX 4 was reduced by 50%. This shift in NOX 2/NOX 4 balance resulted in a ROS shift from O
2-
to H
2
O
2
, as determined by DHE and CMDCF staining. Subsequently, we silenced NOX 2 and 4 by transducing HUVEC with lenti-viral shRNAs, using non-targeting shRNA and non-transduced cells as controls. Silencing did not affect static or shear stress-induced KLF2 and Nrf2 expression, activation, or target gene expression. However, we did observe an Nrf2-independent effect of NOX silencing on antioxidant expression, which is currently under further investigation.

Our preliminary results show a shift in NOX/ROS balance under shear stress, without affecting KLF2 or Nrf2. Together with the anti-oxidant activities of KLF2 and Nrf2, this shift in oxidant balance might be causative in the quiescent, anti-atherogenic effect of high, pulsatile shear stress on ECs.
Abstract 362: Genomewide Assessment of Posttranscriptional Events Orchestrated by Quaking that Determine Monocyte Fate
Authors: van der Veer Eric P
Published: July 2021

The post-transcriptional events that enable monocytes to home to sites of vascular injury, and subsequently differentiate into macrophages within atherosclerotic lesions are poorly understood. We discovered that the mRNA and protein levels of the RNA-binding protein Quaking (QKI) are augmented in CD14+ myeloid cells extracted from advanced human atherosclerotic plaques (4.2-fold upregulated vs. early lesions; p<0.01). This prompted us to investigate how the RNA-binding properties of QKI could influence monocyte fate.

Our approach was to examine, at a genome-wide level, how altering the expression levels of QKI could impact pre-mRNA splicing, expression, and localization upon differentiation of human monocytes into Mf. For this, we employed both array-based gene expression analyses as well as next-generation sequencing techniques (RNA-seq) of THP-1 monocytic cells as well as CD14+ monocytes derived from the peripheral blood of a unique, QKI haploinsufficient subject (with sibling control). Despite low expression levels of QKI in monocytes, the abrogation of QKI in these cells perturbed cellular adhesion and the ensuing establishment of the cytoskeletal architecture. Interestingly, our investigation of post-transcriptional events that are associated with the conversion of the monocyte to a macrophage, uncovered: 1) 536 alternative splicing events (p<0.05) that are directly mediated by binding of QKI proximal to the splice site (QBS)(Fig. 1A); and 2) 1214 differentially expressed genes (minimally +/- 1.5-fold; p<0.05) that indicate that QKI modulates monocyte activation and differentiation by regulating inflammation, cell growth and survival, RNA editing and lipid metabolism via activation of the LXR/RXR pathway (Fig. 1B).

Collectively, our data illustrate the post-transcriptional events that drive monocyte to macrophage differentiation, and identify the RNA-binding protein QKI as an orchestrator of this inflammatory response.
Abstract 365: Microparticle-Induced Activation of the Vascular Endothelium Requires Caveolin-1/Caveolae
Authors: Andrews Allison M , Rizzo Victor
Published: July 2021

Microparticles (MPs) are small membrane fragments shed from normal as well as activated, apoptotic or injured cells. Recent findings show a strong correlation between elevated levels of circulating MPs and several cardiovascular disease states including hypertension and atherosclerosis. Moreover, emerging evidence implicates MPs as a causal and/or contributing factor in altering normal vascular cell phenotype through initiation of proinflammatory signal transduction events and paracrine delivery of proteins, mRNA and miRNA. However, little is known regarding the mechanism by which MPs influence these events. Caveolae are important membrane microdomains, which function as centers of signal transduction and endocytosis. A number of known MP signaling events are associated with caveolae-mediated pathways, yet the relationship between caveolae and MPs has not been established. Here, we tested the concept that MP induced signaling in endothelial cells (ECs) depends on caveolae. MPs were generated from mouse lung ECs exposed to TNF-α for 24 hrs. MPs were isolated using low and high-speed centrifugation, resuspended and then exposed to ECs. Consistent with previous reports, MPs activated ECs as evidenced by upregulation of intracellular adhesion molecule (ICAM) expression. ICAM upregulation was associated with activation of NF-κB, Poly [ADP-ribose] polymerase 1 (PARP1) and epidermal growth factor receptor (EGFR). This response was absent in ECs lacking cavolin-1. To test whether caveolae-mediated endocytosis is a feature of the proinflammatory response, ECs were pretreated with dynasore, a dynamin2 inhibitor. Similar to observations in cells lacking caveolin-1, inhibition of endocytosis significantly attenuated MPs effects including activation of EGFR, NF-κB and PARP1 and ICAM expression. Last, we found that the EGFR localized in caveolae indicating that compartmentation of proximal signaling components in caveolae is essential for induction of an endothelial pro-inflammatory phenotype by MPs.
Abstract 366: Inhibition of Interleukin-1ß by a Monoclonal Antibody Therapy Reduces Vascular Calcification in Ldlr-/- Mice
Authors: Awan Zuhier , Denis Maxime , Roubtsova Anna , Gram Hermann , Seidah Nabil G , Genest Jacques
Published: July 2021

Objective:
Given the link between cholesterol and activation of inflammation via interleukin-1β, we thus tested the effects of IL-1β inhibition on atherosclerotic calcification in mice.

Methods and Results:
A mouse monoclonal antibody (mAB) against IL-1β or placebo was administered subcutaneously to Ldlr-/- and Tg(Pcsk9) models fed a Western diet. Drug level, anthropometric, lipid and glucose profiles were determined. PCSK9, SAA1 and cytokine expressions were measured by ELISA. Aortic calcification was determined by micro-CT and X-Ray densitometry and aortic flow velocity was assessed by ultrasound. Circulating levels of IL-1β in Ldlr-/- mice were significant twice that observed in Tg(Pcsk9) mice. Both mAb and placebo treated mice did not differ in their growth, lipid, glucose profiles and other cytokines while plasma SAA1 levels were lower in mAb-treated mice. Calcifications were significantly diminished in mAb-treatment Ldlr-/- mice (a reduction of 75% by X-ray and 96% by micro-CT) and reduced insignificantly in mAb-treatment Tg(Pcsk9) mice, whereas aortic flow velocity was unchanged in both models.

Conclusions:
Herein we demonstrate that aortic calcifications can be inhibited by IL-1β mAb in LDL-receptor deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease.
Abstract 384: The Prothrombotic Profile of BCR-ABL Inhibitors Ponatinib, Nilotinib and Imatinib
Authors: Loren Cassandra P , Rigg Rachel A , Aslan Joseph E , Healy Laura D , Gruber Andras , McCarty Owen J
Published: July 2021

Background:
Thrombotic complications observed in patients treated with the tyrosine kinase inhibitor ponatinib resulted in the temporary suspension of the drug by the FDA in late 2013. Ponatinib is a pan-BCR-ABL inhibitor designed for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Ponatinib has been shown to inhibit a subset of the class III/IV family of receptor tyrosine kinases. The pathogenic mechanism underlying the prothrombotic phenotype associated with ponatinib remains ill-defined.

Hypothesis:
We tested the hypothesis that BCR-ABL inhibitors regulate platelet activation, spreading, and aggregation.

Methods & Results:
Our results show that treatment of platelets with ponatinib (1 μM) abrogated platelet spreading on fibrinogen (50 μg/ml) or collagen (100 μg/ml). Both nilotinib and imatinib inhibited platelet spreading, although not to the same extent as ponatinib at equimolar concentrations. We next examined the effects of BCR-ABL inhibitors on platelet activation and aggregation in response to the GPVI-agonist, CRP, which mediates platelet activation in a receptor tyrosine kinase-dependent manner. Intriguingly, our results show that ponatinib abrogated platelet aggregation in response to CRP (1 μg/ml), whereas equimolar concentrations of nilotinib or imatinib had minimal effects on CRP-induced platelet aggregation.

Conclusions:
While tyrosine kinase inhibitors are generally associated with bleeding diathesis, thrombotic complications have been observed in patients treated with ponatinib. Our results indicate that ponatinib, nilotinib, and imatinib inhibit platelet spreading and aggregation, with ponatinib having the most significant effect, suggesting that ponatinib may act as a platelet antagonist. Our future work will be focused on identifying platelet proteins affected by the BCR-ABL inhibitors as well as the pathogenic mechanisms underlying the prothrombotic phenotype associated with ponatinib.
Abstract 401: Altered Bile Acid Profiles With Colesevelam Treatment Are Associated With Improved Glycemic Control in Type 2 Diabetes
Authors: Beysen Carine , Chan Miram , Tsang Ellen , Fessler Chancy , Czerwieniec Gregg , Hellerstein Marc , Turner Scott
Published: July 2021

Bile acid sequestrants (BAS) such as colesevelam are effective therapies for lowering LDL-cholesterol and improving glycemic control in individuals with type 2 diabetes. It is however not clear if changes in glucose metabolism with BAS treatment are regulated via changes in whole body BA metabolism. We performed a post-hoc analysis of circulating bile acid levels to investigate whether changes in glucose and lipid parameters are associated with alterations in plasma bile acids (BA) concentrations with colesevelam treatment. Subjects with type 2 diabetes (n=49) treated with diet and exercise, sulfonylurea, metformin or a combination thereof, were treated with 3.75 g/day colesevelam for 12 weeks. Fasting bile acid concentrations were measured by LC/MS and glucose and lipid kinetics were measured using stable isotope techniques at baseline and post-treatment. Colesevelam treatment reduced fasting LDL-cholesterol and increased HDL-cholesterol and triglyceride concentrations. No changes were seen in fasting total cholesterol concentrations and de novo lipogenesis (DNL). Beta-cell function (HOMA-B), glycolytic disposal of oral glucose and fasting plasma glucose clearance improved significantly. Colesevelam treatment resulted in a smaller (-40% from baseline) and more hydrophilic fasting plasma BA pool. All BA’s measured except for cholic acid, glycocholic acid and taurocholic acid decreased with treatment. Changes in total BA concentrations were inversely correlated with changes in A1C and a positive correlation was seen with fasting insulin concentrations. Changes in BA concentrations did not correlated with changes in lipid parameters. These data suggest that altered bile acids levels may contribute to improved glycemic control with colesevelam treatment in subjects with type 2 diabetes.
Abstract 409: Is Accelerated Atherosclerosis a Microvascular Complication of Diabetes?
Authors: Stoute Heidi K , Venegas-Pino Daniel , Veerman Kaley J , Werstuck Geoff H
Published: July 2021

Introduction:
The complications of diabetes have traditionally been categorized as micro- or macrovasculature in nature. Individuals with diabetes are at an increased risk of macrovascular complications, including cardiovascular disease and stroke, and also suffer from microvascular disorders including retinopathy, nephropathy and neuropathy. There is increasing evidence these micro- and macrovascular conditions may be linked. Our objective is to determine if direct effects of hyperglycemia on a specific microvascular bed, the vasa vasorum, promotes diabetes-associated accelerated atherosclerosis.

Methods and Results:
The effects of hyperglycemia on retinal and vasa vasorum neovascularization and aortic atherogenesis were examined in streptozotocin-injected apolipoprotein-E deficient (ApoE-/-) mice and normoglycemic ApoE-/- controls. Microvessel densities of the retina and vasa vasorum were quantified at 5, 10, 15 and 20 weeks of age. Atherosclerotic lesion volume in the ascending aorta was determined at the same time points. The expression levels of pro-angiogenic factors, VEGF and VEFGR2, were also determined. Data from normoglycemic ApoE-deficient mice indicate that there is an expansion in vasa vasorum density as the atherosclerotic lesion area increases. In contrast, hyperglycemic ApoE-/- mice have significantly larger atherosclerotic lesions (2 fold, P<0..05) but have no detectible neovascularization of the vasa vasorum A deficiency in VEGF expression in the artery walls of the hyperglycemic mice may explain lack of neovascularization.

Conclusions:
These findings indicate that the microvessel structure of the vasa vasorum is altered by hyperglycaemia. The development and progression of atherosclerosis in hyperglycemic ApoE-deficient mice directly correlates with, and may be influenced by, microvascular changes.
Abstract 418: Bifunctional Role of Autophagy in Intracellular ApoB Degradation and Lipid Droplet Mobilization
Authors: Christian Patricia , Zamani Mostafa , Qui Wei , Zhang Rianna , Adeli Khosrow
Published: July 2021

Introduction:
As apolipoprotein B (apoB) is translated and translocated into the ER, lipids from cytoplasmic lipid droplets (LDs) are added to promote folding and to initiate very-low-density-lipoprotein (VLDL) assembly. However, without sufficient lipid availability, apoB is misfolded and subject to proteasomal degradation. Evidence now shows that apoB can also be degraded through autophagy under certain conditions, and that LDs are also subject to autophagic degradation, a process referred to as lipophagy. We postulate that apoB autophagy and LD lipophagy integrate to regulate hepatic lipid export and VLDL production.

Methods/Results:
Studies were conducted in vitro using the human hepatoma cell line, HepG2 and ex vivo using primary Syrian Golden hamster hepatocytes. Cells were fat loaded with/without 0.4 mM OA for 4 hours and simultaneously treated with an autophagy inhibitor 3-methyadenine (3-MA; 100uM) or an autophagy inducer, Torin 1 (250nM). HepG2 cells were transfected with 10 nM of atg12 siRNA, an essential autophagy related gene, for a total of 72 hours. In freshly isolated primary hamster hepatocytes, inhibition of autophagosome formation, through treatment with 3-MA, significantly increased cellular levels of newly synthesized apoB, without a significant increase in apoB secreted into the media. Interestingly, treating these cells with Torin 1 to promote autophagy also significantly increased apoB recovery. However, modulation of autophagy activity also affected the average number of LDs per cell, indicating that lipophagy activity had also been modified, potentially affecting VLDL formation. Conversely, while inhibition and induction of autophagy in HepG2 cells, a human hepatoma cell line, reduced and increased apoB co-localization with autophagosomes respectively, siRNA knockdown of atg12 as well as 3-MA treatment decreased apoB recovery. However, this did not appear to be due to reduction in LD breakdown through autophagy. The data obtained with primary hamster hepatocytes suggest that autophagy may play a dual role in VLDL assembly in vivo by regulating both degradation of apoB and lipidation of VLDL particles through mobilization of lipid from LDs. Defects in these pathways can induce hepatic LD accumulation and steatosis.