bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 209: An XX Sex Chromosome Complement Promotes the Development of Obesity, Hypercholesterolemia and Atherosclerosis in Male and Female

Ldlr
-/-

Mice Fed a Western Diet
Authors: Alsiraj Yasir , Thatcher Sean , M. Ali Heba , Temel Ryan , Daugherty Alan , Cassis Lisa
Published: July 2021

Background:
Underlying mechanisms contributing to sexual dimorphism of cardiovascular diseases are not well understood. Sex hormones are primary contributors to sexual dimorphism of cardiovascular diseases. By comparison, little is known regarding the contribution of genes on sex chromosomes (XX and XY) to sexual dimorphism of cardiovascular diseases, even though the X chromosome contains around 5% of the human genome. In this study, we hypothesized that genes on sex chromosomes influence the development of obesity, hypercholesterolemia and atherosclerosis.

Methods and Results:
Transgenic male mice with deletion of
Sry
from the Y-chromosome expressing
Sry
on autosomes (8-12 weeks of age) were bred to female

Ldlr
-/-

mice to generate female and male mice with an XX or an XY sex chromosome complement (FXX, FXY, MXX, MXY). Mice were fed a Western diet (Teklad TD88137) for 3 months. XX mice exhibited increased body weight compared to mice with an XY sex chromosome complement, regardless of gonadal sex (FXX, 41.2 ± 2.4; FXY, 31.7 ± 2.5 g; P<0.05; MXX, 51.5 ± 1.2; MXY, 41.7 ± 1.8 g; P<0.05). Moreover, XX mice had increased serum cholesterol concentrations, regardless of gonadal sex (FXX, 2501 ± 192; FXY, 890 ± 141 mg/dl; P<0.05; MXX, 3814 ± 344; MXY, 1297 ± 385 mg/dl; P<0.05). Elevations in serum lipids were manifest as increased VLDL and LDL-cholesterol. The extent of atherosclerosis in aortic arch was significantly increased in XX compared to XY mice (XXF, 37 ± 2.1; XYF, 20 ± 3.2; XXM, 38 ± 3.6; XYM, 24 ± 3.6 % lesion surface area; P<0.05). In the aortic sinus, atherosclerotic lesion surface area was significantly increased in XX mice, regardless of gonadal sex (FXX, 60.4 x 10
4
± 3.6 x 10
4
; FXY, 32.4 x 10
4
± 3.8 x 10
4
μm
2
; P<0.05; MXX, 67.1 x 10
4
± 9.6 x 10
4
; MXY, 36.2 x 10
4
± 3.7 x 10
4
μm
2
; P<0.05).

Conclusion:
Results demonstrate that an XX sex chromosome complement promotes diet-induced obesity, hypercholesterolemia and atherosclerosis regardless of gonadal sex. Future studies will identify the role of genes on the X or Y chromosome as mechanisms for these effects.
Abstract 21: Endothelial Epsins Promote LPS-Induced Inflammation During Septic Shock
Authors: Chen Hong
Published: July 2021

Introduction:
Sepsis is caused by a deleterious host response to infection, which is primarily responsible for further injury of host tissue and cause of organ dysfunction. Despite significant progress, the pathophysiology of sepsis and the underlying regulatory mechanisms are still not fully understood. We have established that endothelial epsins play a pivotal role in mediating internalization and degradation of Thrombomodulin after LPS challenge.

Hypothesis:
Given LPS triggers “cytokine storm” that causes hyper-permeability in the endothelium of lungs and excessive inflammation, we assessed the hypothesis that epsins play a role in promoting endothelial permeability and augmenting inflammation.

Methods and Results:
Using innovative tissue-specific inducible epsins double knock out animal models, we investigated the role for epsins during sepsis. We administered lethal dose of LPS into endothelial-specific inducible epsins mutant mice, myeloid cell-specific epsins mutant mice, and platelet-specific epsins mutant mice (n>10). We uncover a potent protective role for endothelial epsins deficiency against the development of LPS-induced sepsis, whereas deletion of epsins in myeloid cells offers 40% ~ 50% of protection, and loss of epsins in platelets exhibits no protection. We further show that endothelial epsin-deficiency upregulates Thrombomodulin surface protein expression by preventing its internalization and subsequent degradation induced by LPS exposure. Sustained surface Thrombomodulin activity subsequently impaired the heightened Tissue Factor expression and activation that usually occurs in response to LPS. Given LPS challenge mimics chronic inflammatory conditions, we show endothelial epsin-deficiency downregulates LPS-induced proinflammatory cytokine production and suppresses endothelial hyper-permeability in lungs assessed by ELSA and Evans Blue perfusion, respectively.

Conclusions:
Endothelial epsins depletion inhibits septic shock after LPS challenge by protecting Thrombomodulin against internalization and degradation, blocking proinflammatory cytokine production and inhibiting endothelial leakage in the lungs, highlighting the therapeutic potential for targeting epsins during sepsis.
Abstract 212: Endothelium-Specific Deletion of Epsins Attenuates Atherosclerosis in ApoE-deficient Mouse Model Through Stabilization of IP3 Receptor 1 and Maintaining ER Homeostasis
Authors: Dong Yunzhou , Brophy Megan L , Wen Aiyun , Yu Lili , Wu Hao , Song Kai , Rahman Ashiqur , Wong Scott , Kwak Sukyoung , Krygier Kenneth , Chen Hong
Published: July 2021

Background:
Atherosclerosis is caused in part by endothelial dysfunction due to ER stress. Understanding the mechanisms that cause ER stress and endothelial dysfunction are essential in establishing new therapies to treat atherosclerosis. Epsins are endocytic adaptor proteins that mediate the internalization of plasma membrane receptors. However, whether epsins affect atherosclerosis remains unknown. To this end, we assessed the hypothesis that endothelial epsins play a critical role in regulating atherosclerosis.

Methods and Results:
In an ApoE-deficient murine model, endothelial-specific double knockout mice (EC-iDKO/ApoE-/-) significantly attenuated atherosclerotic lesion and macrophage infiltration in the aortic root and arch compared to ApoE-/- mice fed Western diet for ten weeks. To elucidate underlying mechanisms, mass spectrometry analysis identified IP3R1 as one of major interactors of epsins upon exposure to oxLDL. We show that epsins bind IP3R1 and facilitate its degradation in the endothelium. Downregulation of IP3R1 by atherosclerosis promoting agents augmented ER stress in primary mouse aortic endothelial cells. In line with these findings from mouse studies, in human atherosclerotic lesion, IP3R1 is dramatically downregulated and decrease in IP3R1 expression is associated with increased ER stress. Conversely, loss of epsins stabilized IP3R1, attenuated ER stress, reduced expression of P-selectin, adhesion molecules, and chemoattractant MCP-1 in primary mouse aortic endothelial cells. Molecular mapping analysis revealed that epsin’s UIM domain is critical in facilitating the degradation of IP3R1. Delivery of a synthetic UIM peptide conjugated with atheroma-targeting Lyp-1 peptide significantly attenuated atherosclerosis in ApoE-/- animal models by stabilizing IP3R1 and maintaining ER homeostasis. Importantly, atherosclerotic lesion was restored in EC-iDKO/ApoE-/- mice by breeding them onto an endothelial-specific IP3R1 heterozygous background, suggesting that endothelial epsins promote atheroma progression in part by mediating IP3R1 degradation.

Conclusions:
Our data identified a novel mechanism of epsins in regulating atherosclerosis and provide a potential target to treat atherosclerosis.
Abstract 214: Immune Response Biomarkers Characterize Endothelial Injury and Predict Acute Coronary Syndrome
Authors: Harrington Douglas S , Simonini Americo
Published: July 2021

Acute Coronary Syndromes (ACS) are caused by free-radical mediated endothelial injury. Soft unstable coronary artery wall lesions form that can rupture causing thrombosis and obstruction. Three software systems (Akaike, Bayesian, Drop-in Deviance) tested 45 biomarkers and global risk factors to predict risk of ACS. A novel 9 protein algorithm was developed in 3 unique longitudinal, outcome-based studies and independently verified in MESA (Multiethnic Study of Atherosclerosis). The proteins selected include CTACK (Cutaneous T-Cell Attracting Chemokine)-homing of memory t-cells to tissue injury site; EOTAXIN (Eosinophil Chemotactic Protein)-attracts eosinophils to endothelial injury facilitating fibrin removal; MCP-3 (Monocyte Chemotactic Protein 3)-attracts monocytes and modulates activity; IL-16 (cytokine)-chemoattractant and modulator of T cell activation; sFAS (soluble FAS Ligand)-suppresses apoptosis; FAS Ligand (transmembrane protein tumor necrosis factor family member) induces apoptosis; HGF (Hepatocyte Growth Factor)-acts on epithelial cells and endothelial cells for angiogenesis, tumorogenesis, and tissue regeneration/repair; HDL (High-density Lipoprotein)-reverse cholesterol transport and inhibits oxidation, inflammation, endothelial activation, coagulation, and platelet aggregation; HbA1c (Hemoglobin A1c)-three-month average plasma glucose concentration. We conclude that the selection of biomarkers associated with endothelial damage and immune system response correlates with risk of ACS and reflects the underlying pathophysiology. Further studies are warranted.
Abstract 224: TRAF-STOP-RHDL-Nanoparticles Reduce Atherosclerosis
Authors: van Tiel Claudia M , Seijkens Tom T , Kusters Pascal J , Zarzycka Barbara , Soehnlein Oliver , Gijbels Marion J , Fay Francois , Bleijlevens Boris , Gerdes Norbert , de Winther Menno P , Nicholaes Gerry A , Mulder Willem J , Weber Christian , Lutgens Esther
Published: July 2021

Background:
Inhibition of the costimulatory CD40-CD40L receptor/ligand dyad drastically reduces atherosclerosis. However, its long-term blockage can result in immune suppression. We recently identified small molecule inhibitors that block the interaction between CD40 and TNF Receptor Associated Factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. We investigated the potential of the TRAF-STOPs to treat atherosclerosis.

Results:
Treatment of ApoE-/- mice with either TRAF-STOP 6877002 or 6860766 reduced both initial and established atherosclerosis and induced a stable plaque phenotype with increased collagen and VSMC content, decreased lipid core, and a decrease in macrophage number. There were no signs of immune suppression or toxicity. In vitro experiments showed that the TRAF-STOPs reduced inflammation in macrophages, but not in T- or B cells, endothelial cells or vascular smooth muscle cells. Intravital microscopy demonstrated that the TRAF-STOPs reduced monocyte recruitment to the plaque. The CD36-mediated uptake of ox-LDL by macrophages and foam cell formation was also inhibited by TRAF-STOPs. Transcriptomics analysis and Ingenuity pathway analysis of TRAF-STOP-treated bone marrow-derived macrophages revealed that the top ranking canonical pathways for both TRAF-STOPs involved pro-inflammatory immune responses and cholesterol biosynthesis. 6877002 also affected cell cycle regulation. Surface plasmon resonance experiments and mutation studies demonstrated that 6877002 and 6860766 had a different interaction site within the TRAF6 C-domain, which explained the additional effect of 6877002. To target TRAF-STOPs specifically to macrophages, 6877002 was incorporated into rHDL nanoparticles. Flowcytometry and fluorescent microscopy demonstrated accumulation of rHDL-6877002 in plaque macrophages after a single dosis. Six weeks of rHDL-6877002 treatment reduced atherosclerosis in ApoE-/- mice.

Conclusions:
TRAF-STOP 6877002 and 6860766 can overcome the current limitations of long-term CD40 and CD40L inhibition and nanoparticle-mediated delivery TRAF-STOP to plaque macrophages may become a future therapy for atherosclerosis.
Abstract 226: C-type Lectin Receptor Clec9a on Cd8a+ Dendritic Cells Promotes the Development of Atherosclerosis in Mice
Authors: Yacine Haddad , Lahoutte Charlotte , Clément Marc , Laurans Ludivine , Sarvenaz.Metghalchi Sarvenaz.Metghalchi , Zeboudj Lynda , Giraud Andreas , Loyer Xavier , Vandestienne Marie , Wain-Hobson Julien , Esposito Bruno , Potteaux Stephane , Ait-Oufella Hafid , Reis e Sousa Caetano , Tedgui Alain , Mallat Ziad , Taleb Soraya
Published: July 2021

Necrotic core formation during the development of atherosclerosis is associated with a chronic inflammatory response and promotes accelerated plaque development and instability. We hypothesized that sensing of necrotic cells by CLEC9A, a C-type lectin receptors selectively expressed by the CD8α
+
subset of dendritic cells (CD8α
+
DCs), plays a determinant role in the inflammatory response of atherosclerosis.

Reconstitution of lethally-irradiated Ldlr-/- with bone marrow from CLEC9A-/- mice significantly reduced atherosclerotic lesion size in aortic root after 5 weeks of high fat diet (HFD) (-45%, p=0,0059) and after 7 weeks of HFD (-40%, p=0,0017), as compared to mice transplanted with wild-type bone marrow-derived cells. However, no effect of CLEC9A was observed after 13 weeks of HFD (p=0,4996), suggesting early effect of CLEC9A on atherosclerosis development.

The same phenotype was observed in 20-week-old Apoe-/-CLEC9A-/- compared to Apoe-/- mice put on chow diet (-50%, p=0,0022).

Interestingly, an increase of IL-10 expression (+60%, p=0,0093) was observed in spleens of mice deficient for CLEC9A. Furthermore, the beneficial effect observed in CLEC9A-/- was abolished in CLEC9A-/-IL-10-/- compared to IL-10-/- (p=0,4452). Moreover, a specific deletion of Clec9a in CD8α
+
DC cells significantly increases Il10 expression, reduces macrophage and T cell contents within the lesions, and significantly limits the development of atherosclerosis. In conclusion, our results identify a new role of Clec9a in regulating vascular inflammation and atherosclerosis development and potentially identify a new target for disease modulation.
Abstract 227: Megalin Regulates Angiotensinogen and Contributes to Atherosclerosis
Authors: Ye Feiming , Howatt Deborah , Mullick Adam , Grahan Mark , Balakrishnan Anju , Vander Kooi Craig , Danser A.H. Jan , Wang Jian-An , Daugherty Alan , Lu Hong
Published: July 2021

Objective:
Angiotensinogen (AGT) is the only substrate of the renin-angiotensin system to generate angiotensin peptides, including angiotensin II (AngII), a critical contributor to atherosclerosis. AGT interacts with megalin in proximal convoluted tubules of kidney. The purpose of this study was to determine effects of megalin on AGT, AngII, and atherosclerosis in mice.

Methods and Results:
Male C57BL/6 mice were injected subcutaneously with vehicle (PBS) or megalin second generation antisense oligonucleotides (ASO) for 5 weeks. Inhibition of megalin was confirmed by more than 80% reduction of megalin mRNA in kidney. Urine was collected using metabolic cages after 5 weeks of vehicle or ASO injections. At termination, blood was collected with EDTA and protease inhibitor cocktail to measure plasma concentrations of renin and AngII, respectively. Urine AGT and renin concentrations were profoundly increased, accompanied by reduction of renal, but not plasma, AngII production. To determine whether megalin inhibition influences atherosclerosis, male LDL receptor -/- mice were injected with vehicle, control ASO, or megalin ASO for 13 weeks. Western diet was started 1 week after the first injection and continued for 12 weeks. Atherosclerosis was quantified by en face analysis of the aortic intimal surface from the ascending aorta to 3 mm proximal to the left subclavian artery branch. Megalin ASO administration led to more than 70% reductions of megalin mRNA in kidney. Consistent with the effects in C57BL/6 mice, AGT concentrations were significantly higher in urine (Vehicle, control ASO and megalin ASO groups: 85 ± 10, 109 ± 71, and 4616 ± 637 ng/ml, respectively; P<0.001 by one way ANOVA with Holm-Sidak method). Plasma total cholesterol concentrations did not differ among groups. Megalin inhibition reduced atherosclerotic lesion area compared to the other two groups (Percent lesion area in Vehicle, control ASO and megalin ASO groups: 23 ± 2, 20 ± 2, 12 ± 1 %, respectively; P < 0.001 by one way ANOVA with Holm-Sidak method).

Conclusions:
Inhibition of megalin increased urine AGT and renin, reduced renal AngII concentrations, and diminished hypercholesterolemia-induced atherosclerosis in mice.
Abstract 228: The Establishment of a Novel Atherosclerosis Regression Model in Mice
Authors: Tang Chaojun , Ren Lijie , Liu Yifei , Wang Yinyan , Cao Yiren , Wang Lei , Wu Fan , You Tao , ZHU Li
Published: July 2021

Background:
Inhibition of plaque progression by various anti-atherosclerotic drugs is a canonical aspect underlying their therapeutic efficacy. Although existed animal models of plaque regression are available for drug evaluation, they are either highly invasive or time-consuming. We therefore aimed to establish a practical method to study the mechanism of atherosclerosis regression based on hemodynamic regulation of atherosclerosis.

Methods:
A carotid plaque regression model in mice was developed based on the previously described model. Briefly, a single slipknot was applied to all three branches of the left carotid artery (LCA) in ApoE
-/-
mice. After 2 weeks on HFD, the blood flow in LCA was reconstructed by slipknot release. Mice were housed for additional 4 weeks. Carotid ultrasonography was used to verify successful induction of disturbed flow and the reconstruction of the blood flow. Atherosclerotic plaques were stained with Sudan IV. The infiltration of smooth muscle cells and macrophages were determined by immunofluorescence staining.

Results:
Histology studies demonstrated similar cellular components and lipid deposition between the d-flow induced plaques in the LCA in WT mice and atherosclerotic plaques in aorta of ApoE
-/-
mice on HFD. However, a significantly higher proportion of SMC (n=5, p<0.0001) and lower macrophage infiltration (n=6, p=0.0056) were observed in the induced carotid plaques compared to the aortic plaques. After the release of the knots, an abrupt increase in flow velocity indicated a successful restoration of blood flow from the LCA to its branches. At this point, the restored flow in ligated LCA was still lower compared to the untied right carotid artery (RCA). After the procedure, mice were given chew diet for additional 4 weeks and blood flow was re-accessed using carotid ultrasonography. Results showed no significant difference of blood flow between LCA and RCA, indicating nearly complete regression of carotid atherosclerosis.

Conclusion:
We have established a highly reproducible atherosclerosis regression model with less invasiveness and genetic independence that supplies a practical tool for evaluation of anti-atherosclerotic drugs and for the pathophysiology of atherosclerosis regression.
Abstract 229: Mucosal Immunity Shapes the Lipoprotein Small-RNA Fingerprint from Commensal, Dietary and Environmental Microbiota
Authors: Allen Ryan M , Ramirez-Solano Marisol A , Zhu Wanying , Zhao Shilin , Sheng Quanhu , Vickers Kasey , Linton MacRae
Published: July 2021

Cardiovascular disease (CVD) is a leading cause of mortality in developed countries and is a frequent comorbidity of numerous metabolic and inflammatory diseases that demands more effective therapies. Dyslipidemias are a classical risk factor for CVD, but emerging alternative functions of lipoproteins have implicated them in novel narratives for the pathophysiology of many diseases that warrant further study. Our lab has identified functional, intercellular gene regulatory networks mediated by extracellular transport of microRNAs (miRNA) by lipoproteins. Here, we quantified the landscape of small RNAs (sRNA) on human and animal lipoproteins and discovered that most lipoprotein-sRNAs are derived from microorganisms of multiple kingdoms, primarily bacteria. Based on these observations, our over-arching hypothesis is that lipoprotein-sRNA signatures are shaped by the interface of host tissues with resident, environmental and dietary microbiota, and likely participate in unique gene regulation networks that contribute to complex (patho)physiological traits. To investigate this hypothesis, we developed a sRNA-sequencing analysis pipeline that identifies and quantifies both host and non-host sRNAs. Using this bioinformatic tool, we identified and validated a number of lipoprotein-sRNAs derived from bacteria that are similar in size to miRNAs with identical seed regions, termed Doppelganger (Dopl)-miRNAs. We hypothesized that mucosal immunity contributes to non-host sRNAs on lipoproteins, as mucosal linings are a primary interface between host tissues and microorganisms. To this end, we investigated the lipoprotein-sRNAs of mice lacking the polymeric immunoglobulin receptor (pIgR
-/-
), which is devoid of polymeric IgA and IgM at mucosal linings and models human respiratory and intestinal diseases. We report a global increase in lipoprotein-miRNAs juxtaposed with a profound decrease in bacterial sRNA and Dopl-miRNAs from specific taxa. This work unfurls novel links between microbiota, mucosal immunity, and lipoprotein-sRNA gene networks and emphasizes the potential for novel nucleic-acid based therapeutics.
Abstract 23: Identification of NCF4 as a Novel Regulator in Arterial Remodeling and Advanced Atherosclerosis
Authors: Bäcklund Alexandra , Gertow Karl , Jin Hong , Olsson Lina , Gonzalez-Diez Maria , Sabater-Lleal Maria , Strawbridge Rona J , Sennblad Bengt , Öhrvik John , Zabaneh Delilah , Shah Sonia , Veglia Fabrizio , Folkersen Lasse , Gigante Bruna , Leander Karin , Vikström Max , Silveira Angela , Colombo Gualtiero I , Gabrielsen Anders , Hedin Ulf , Franco-Cereceda Anders , Paulsson-Berne Gabrielle , Nyyssönen Kristiina , Tuomainen Tomi-pekka , Savonen Kai , Rauramaa Rainer , Smit Andries J , Giral Philippe , Mannarino Elmo , van der Laan Sander W , Pasterkamp Gerard , Hansson Göran K , Eriksson Per , Baldassarre Damiano , Tremoli Elena , de Faire Ulf , Humphries Steve E , Hamsten Anders , Holmdahl Rikard , Maegdefessel Lars
Published: July 2021

Arterial wall remodeling is a central multifactorial process in the development and progression of cardiovascular diseases. We employed an approach aimed at observing genetic variants associated with the progression of carotid intima-media thickness (cIMT) in order to identify novel pathways effecting vessel remodeling. This was achieved by conducting gene-centric analysis of 400,000 variants in 3,042 subjects with repeated cIMT measurements.

Rs16997464 on chr22 intergenic between neutrophil cytosolic factor-4 (
NCF4)
and colony stimulating factor 2 receptor beta (
CSF2RB
) was associated with cIMT progression

at array-wide significance (p <4.5x10
-7
). The potential causative genes within this locus were investigated using a human vascular and non-vascular tissue biobank. Expression of 9 genes near rs16997464, were analyzed with the most significant association being with
NCF4
in aortic adventitia. The effect of the variant on the function of the
NCF4
gene product was further analyzed by comparing the oxidative burst capacity of neutrophils from subjects with different rs16997464 genotypes. We observed that neutrophils homozygous for the minor T allele, associated with slower cIMT progression, produced more extracellular ROS than neutrophils homozygous for the G allele, indicating a functional effect of rs16997464 on the
NCF4
gene product p40
phox
, a component of the NADPH oxidase 2 complex (NOX2).

In parallel, we investigated if the chr22 locus also influenced the cellular composition of the atherosclerotic plaque, by utilizing data from the Athero-Express Biobank. Here we found that the minor T allele associated with a higher smooth muscle cell (SMC) content in the plaque. Finally, using a partial ligation model in mice where
ncf4
is mutated, resulting in a reduced but not absent NOX2-associated ROS formation, we observed a reduced neointima formation in the
ncf4
-mutated strain compared with wild-type littermates.

Thus, this study identified rs16997464 in the
NCF4-CSF2RB
locus as a novel genetic determinant of cIMT progression, and provides evidence suggesting that
NCF4
is involved in SMC proliferation and alteration of vessel wall pathophysiology.
Abstract 238: Sub-Endothelial Matrix Targeted Liposomal Nanoparticles For Vascular Therapeutics
Authors: Cudnik Jonathan S , West Phillip C , Fisher Richard K , Mattern-Schain Sam , Best Michael , Kirkpatrick Stacy S , Freeman Michael B , Grandas Oscar H , Mountain Deidra J
Published: July 2021

Objectives:
Vascular intervention results in intimal denudation, exposure of the sub-endothelial matrix, and subsequent intimal hyperplasia (IH), under the control of numerous remodeling mechanisms. Reduction of IH-induced restenosis may be achieved by manipulation of these remodeling pathways through targeted molecular inhibition. Spatially-controlled nanoparticles designed to co-localize to exposed sub-endothelial matrices could provide an optimal delivery system for targeted vascular therapeutics. To his end, we aimed to develop the framework for a surface-modified liposomal drug delivery platform designed to preferentially bind collagen type IV.

Methods:
Non-targeted control liposomes (NTL) were formed with bulk DOPC-PEG, 30% cholesterol, and 0.1 mol% Rhodamine-DOPE. DSPE-PEG-DBCO lipids were conjugated to peptides previously shown to bind collagen IV via copper-free click chemistry and inserted at 5 mol% to form collagen-targeting liposomes (CTL), either at hydration (PreCTL) or by post-insertion via micellar transfer (PostCTL). Peptide conjugation was confirmed by MALDI-TOF, and liposomes were characterized by DLS and electrophoretic mobility. Liposome binding was assayed on collagen IV matrices dried at 3ug/cm
2
and quantified by fluorescence at 0-24hr static 37°C incubation.

Results:
All liposome formulations exhibited a narrow size distribution (~100nm) and neutral-low positive charge. CTLs demonstrated a significant increase in binding vs. NTLs (Fig1).

Conclusions:
CTLs demonstrated significant affinity for collagen IV binding in a static environment compared with NTLs. Future studies aim to optimize the binding capacity of CTLs via further lipid modifications and under flow conditions mimicking vessel wall hemodynamics. Considering the efficacy demonstrated here, CTLs show promise as the framework for a spatially-controlled drug delivery platform for future application in targeted vascular therapeutics.
Abstract 240: TGFβ-Activated Kinase 1 is Required for Arteriovenous Fistula Maturation
Authors: Hu Haidi , Hashimoto Takuya , Bai Hualong , Guo Jianming , Isaji Toshihiko , Yatsula Bogdan , Santana Jeans M , Kudze Tambudzai , Dardik Alan
Published: July 2021

Objectives:
Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to have high rates of primary failure to mature. Transforming growth factor-β-activated kinase 1 (TAK1) plays important roles in regulation of extracellular matrix (ECM) production and deposition as well as inflammatory signaling and prevention of apoptosis; however the function of TAK1 in mechanotransduction in response to hemodynamic changes such as occur during AVF maturation is not well understood. Since deposition of ECM is critical to AVF maturation, we hypothesized that TAK1 is a critical regulator of AVF maturation.

Methods:
Aortocaval fistulae were created via needle puncture in wild-type (WT) C57BL/6J mice. AVF diameter was serially assessed weekly by duplex ultrasound; AVF were harvested at days 7 or 21 for histological analysis using computerized morphometry, as well as qPCR and Western blot. Some mice were treated with either the TAK1 inhibitor 5Z-7-oxozeaenol (OZ, 0.5mg/kg/day, 7 days) or Lentiviral TAK1 ShRNA (1x 10
8
pfu/ml; adventitial transduction). Mouse lung endothelial cells (MLECs) were exposed to laminar shear stress (3 or 20 dyne/cm
2
, 1h); TAK1 signaling was detected by Western blot and immunofluorescence.

Results:
TAK1 mRNA expression was increased at days 7 and 21 in AVF (2.0-fold; day 21; p<0.05) compared with sham controls. In AVF treated with OZ there was reduced fistula diameter (p=0.0059) and wall thickness (p=0.031) at day 21, as well as reduced collagen and fibronectin deposition (p<0.05), compared to controls. TAK1 knockdown with ShRNA also showed reduced fistula diameter at day 21 (p<0.05), compared to controls. MLECs exposed to arterial magnitudes of laminar shear stress showed increased TAK1 phosphorylation and downstream signaling (p<0.05) compared to MLECs exposed to venous shear stress or static conditions, which was reduced in cells either pretreated with OZ (p<0.0001) or transfected with TAK1 ShRNA (p<0.05).

Conclusions:
TAK1 regulates ECM production during AVF maturation. Strategies to alter TAK1 function in vivo may be a novel therapeutic approach to improve AVF maturation.
Abstract 257: Activation of the Renin-Angiotensin II-Aldosterone-System Leads to Increase in Protein Disulfide Isomerase
Authors: Lopez Pablo , Inostroza-Nieves Yaritza , Rosado Jamie , Rivera-Gonzalez Lorena , Lozano Alexandra , Rivera Alicia , Pulido-Perez Patricia , Cazares Joshua , Torres-Rasgado Enrique , Nishiyama Akira , Pérez-Fuentes Ricardo , Ortiz Rudy M , Romero Jose R
Published: July 2021

Activation of the Renin-Angiotensin-Aldosterone-System (RAAS) is proposed to play a role in the development of insulin resistance and type II diabetes. Angiotensin II (AngII) is a principal effector molecule that binds AT1 receptors (AT1R) in various tissues. Recent data shows that activated endothelial cells secrete protein disulfide isomerase (PDI), a multifunctional protein that has been shown to be critical to the initiation and regulation of thrombus formation. However, the effects of RAAS on PDI regulation are unknown. We hypothesized that RAAS activation would lead to increased PDI levels thus contributing to inflammation. First, we studied the in vitro effects of AngII on EA.hy926 human endothelial cells and measured PDI activity. Our results show that AngII increased PDI activity; an event that was blocked by preincubation with 0.5 M losartan, an AT1R antagonist (ARB) (P<0.05, n=6). We then studied the in vivo effects of exogenous AngII infusion in Sprague–Dawley rats under the following conditions: (1) control; (2) AngII infused; (3) AngII + ARB. In these rats, AngII infusion led to significant increases in plasma PDI levels that were partially prevented by ARB treatment (P<0.05, n>5). We then studied the obese Otsuka Long Evans Tokushima Fatty (OLETF) (n = 6/group) rats, a model of naturally increased AngII and RAAS mediated insulin resistance and hypertension. OLETF and their lean strain controls were randomly assigned to the following groups: (1) untreated Long Evans Tokushima Otsuka (lean, control); (2) untreated OLETF; (3) OLETF + ARB. Our results show that OLETF rats had increased insulin resistance and significantly greater circulating PDI activity than control rats (P<0.05) that was likewise blocked by ARB treatment (P<0.05). To assess the relevance to humans of these findings we measured circulating PDI levels in patients with type 2 diabetes. In our cohort, PDI activity was significantly greater in patients with type 2 diabetes than non diabetics (P<0.001, n=134). Our data suggest that RAAS activation represents a novel mechanism for PDI secretion. Thus we posit that PDI may contribute to the deleterious effects of RAAS-mediated vascular disease.
Abstract 258: Mir-155 Mitigates Acute Oscillatory Shear Stress (OSS)-Induced Vascular Inflammation and Barrier Dysfunction Through Down Regulation of the AT1R-ETS1 Pathway
Authors: Mohamed Islam , Thomas Sheena , Sutliff Roy , Willet Nick , Searles Charles
Published: July 2021

Introduction:
Shear stress forces play an integral role in dictating the vascular wall response to changes in blood flow, induction of pro-inflammatory response and hence development of atherosclerosis. Previously, our group and others have identified an inverse relationship between microRNA-155 (miR-155) and AT1R expression and/or activity in atheroprone areas of chronic low magnitude oscillatory shear stress (OSS) in vasculature and in-vitro.

Hypothesis:
we hypothesized that acute induction of OSS mediates vascular inflammation and dysfunction, via activation of the AT1R-ETS1 pathway and dysregulation of miR-155.

Methods:
12-week old C57B/6J mice were subjected to abdominal aortic coarctation (AAC), a unique model of acute induction of acute OSS, for 3 days. Downstream segments of acute OSS were compared to upstream unidirectional shear stress (USS) segments of the thoracic aorta using RT-PCR, western blot analysis and unpaired student t-test.

Results:
Acute OSS resulted in vascular inflammation evidenced by upregulation of the AT1R-ETS1 pathway and several of its downstream targets including phosphorylated ERK2, MCP-1 and VCAM-1 in OSS segments compared with USS. This was associated with loss of EC barrier function as evaluated by extravasation of Evans-blue dye assay along with increased expression of MMP3 and MMP9 in in OSS segments compared with USS. However, vascular miR-155 expression was also higher in OSS segments compared with USS (n=6-12, P<0.05). Nevertheless, tail vein injections of miR-155 overexpressing lentivirus particles after AAC resulted in further upregulation of miR-155 expression and inverse downregulation of the AT1R-ETS1 pro-inflammatory pathway and MMPs 3 and 9 expression in OSS segments compared with USS versus scramble control (n=5-6, P<0.05).

Conclusions:
Despite the early upregulation of the shear-sensitive miR-155, our data suggest that it could serve as a negative feedback regulator to acute OSS-induced upregulation of the pro-inflammatory AT1R-ETS1 pathway. Further studies are in progress to evaluate the effect of exogenous miR-155 on OSS-induced oxidative stress and vascular dysfunction, which can serve as the basis for developing novel miRNA-based therapeutic modalities.
Abstract 26: Identification of a Novel Gene, DENND5B, Associated With Plasma High Density Lipoprotein Levels and Intestinal Fat Absorption
Authors: Gordon Scott M , Yang Zhihong , Pryor Milton , Mendelson Michael , Levy Daniel , Remaley Alan T
Published: July 2021

In an effort to identify novel high density lipoprotein (HDL) receptors expressed by the liver, a bacteriophage library expressing liver cDNA fragments was used to screen for clones that bind to HDL. The gene DENND5B showed significant enrichment for HDL binding over 3 cycles of biopanning, with negative selection against low density lipoprotein binding. Although little is known about the recently described DENN-domain containing family of proteins, some members act as guanine nucleotide exchange factors involved in the activation of Rab proteins and membrane trafficking. To examine the influence of DENND5B on HDL physiology, a DENND5B knockout mouse was generated using a custom zinc finger nuclease. DENND5B
-/-
mice had decreased plasma total cholesterol (-30.9%, p < 0.0001) and phosphatidylcholine (-31.4%, p < 0.0001). When plasma was analyzed by size-exclusion chromatography, the lipid effects were attributed entirely to decreased HDL. When fed western diet for 4 months, DENND5B
-/-
mice showed significantly smaller increases in plasma lipids and body weight compared to wild type mice. After sacrifice, DENND5B
-/-
mice were found to have a distended small intestine that was whitish in color. To evaluate fat absorption, mice were given an oral gavage of vegetable oil (10 uL/gram body weight) and plasma lipids were measured at 0, 2, and 4 hours post-gavage. DENND5B
-/-
mice had significantly reduced plasma triglyceride (p < 0.05) and free fatty acids (p < 0.01) in response to oil gavage compared to wild type mice. To identify a possible role for this gene in human lipoprotein metabolism, we examined gene expression data from 5,458 participants in the Framingham Heart Study. Consistent with the mouse data, a significant negative relationship exists between DENND5B expression and plasma HDL-C (p = 4.82 x 10
-5
). In summary, using a phage display screening approach, we have identified a novel gene associated with circulating HDL levels in mice and humans. Additionally, in a homozygous knockout mouse model, a defect in intestinal fat absorption is present. Future studies will be aimed at evaluating the influence of this gene on HDL function, fat absorption, and atherosclerotic disease burden.
Abstract 260: Monocyte to Macrophage Conversion is Guided by Isoform Switching of the Actin Capping Protein Y-Adducin
Authors: Prins Jurriën , de Bruin Ruben G , van Zonneveld Anton Jan , van der Veer Eric P
Published: July 2021

Background:
Modulation of cellular function necessitates a versatile utilization of the transcriptome, thereby altering the proteome. These functional adaptations are post-transcriptionally guided by RNA-binding proteins (RBP), which confer cells with the capacity to rapidly respond to various stimuli and stressors. We recently discovered a pivotal role for the RBP Quaking (QKI) in directing the conversion of monocytes into macrophages, with in particular alternative splicing of pre-mRNAs critically impacting the acquisition of a pro-inflammatory phenotype. Computational analysis indicated that this differentiation process was dependent on dynamic mobilization of the actin cytoskeletal network, which prompted us to focus our attention on an exclusion event of exon 13 at the C-terminus of the actin-capping protein gamma-Adducin (ADD3). At present, the functional consequences of ADD3 isoform switching are unknown.

Methods and Results:
Stimulation of primary human monocytes with GM-CSF initially led to a decrease in ADD3 mRNA expression while ADD3 exon 13 usage was preserved. However, mRNA expression was restored upon differentiation towards the pro-inflammatory macrophage phenotype (t=3 days), while exon 13 inclusion strikingly dropped from 47.2% ± 10.5 to 7.7% ± 2.6 (p=0.0387, n=3). Having previously shown that QKI protein expression increases upon conversion to a macrophage, we sought to pinpoint whether QKI specifically regulates this ADD3 splicing event. Therefore, we mutated the QKI binding site (ACUAA → ACGAA) proximal to exon 13 using an ADD3 minigene. These studies revealed that disrupting the capacity for QKI to bind at this intronic sequence almost completely abolished exon 13 splicing (ACGAA: 94.9% ± 0.0025 inclusion vs. ACUAA: 2.0% ± 0.0009 inclusion; p<0.0001, n=3). Using predictive bioinformatics tools, exclusion of exon 13 was found to induce conformational changes in ADD3 protein structure. We subsequently utilized atomic force microscopy to study actomyosin dynamics and cell stiffness, pressure and tension in monocytes overexpressing the distinct ADD3 isoforms.

Conclusions:
QKI-mediated splicing of ADD3 triggers an isoform switch that impacts monocyte and macrophage function by altering the actin cytoskeleton.
Abstract 27: ApoA-I Improves Lymphatic Function Through a Platelet-Dependent Mechanism in Atherosclerotic Mice
Authors: Milasan Andreea , Dallaire François , Jean Gabriel , Tardif Jean-Claude , Merhi Yahye , Sorci-Thomas Mary , Martel Catherine
Published: July 2021

Rationale:
Lymphatic vessels (LVs) are now recognized as prerequisite players in the modulation of cholesterol removal from the artery wall in experimental conditions of plaque regression, and a particular attention has been brought on the role of the collecting LVs in early atherosclerosis-related lymphatic dysfunction. Whereas recent findings revealed that apoA-I restores the neovascularization capacity of the lymphatic system during tumor necrosis factor-induced inflammation, the effect of apoA-I on collecting LV function during atherosclerosis has not been tested.

Objective:
In the present study, we address whether and how apoA-I can enhance collecting LV function in atherosclerosis-associated lymphatic dysfunction.

Methods and Results:
A 6-week systemic treatment with lipid-free apoA-I enhanced lymphatic transport and abrogated collecting lymphatic vessel permeability in atherosclerotic Ldlr
–/–
mice when compared to control. As injection of apoA-I has been shown to protect wild-type mice against flow restriction-induced thrombosis, and that platelets are identified as key elements in the maintenance of lymphatic vessel integrity via their interaction with lymphatic endothelial cells (LECs), we have tested whether the effects of apoA-I could be mediated through a platelet-dependent mechanism. Our
in vivo
results show that apoA-I kinetics in lymph reflected that of blood.
Ex vivo
experiments performed with washed platelets isolated from mouse blood reveal that apoA-I decreased thrombin-induced but not podoplanin-induced platelet aggregation. Whereas this result suggests that apoA-I limits platelet thrombotic potential in blood but not in lymph, we demonstrate that treatment of human LECs with apoA-I increases the adhesion of bridge-like platelets on human LECs.

Conclusions:
Our results suggest that apoA-I can mediate beneficial effects on lymphatic function by promoting platelet adhesion to the lymphatic endothelium and consequently restore collecting LV integrity. Altogether, we bring forward a new pleiotropic role for apoA-I in lymphatic function and unveil new potential therapeutic targets for the prevention and treatment of atherosclerosis.
Abstract 3: Cellular Contributions of the Circadian Clock in Atherogenesis
Authors: Li Xueling , Ruan Ling , Bentley Austin , Haigh Stephen , Huo Yuqing , Singla Bhupesh , Csanyi Gabor , Stepp David , Fulton David , Rudic R. Daniel
Published: July 2021

Atherosclerosis is a leading cause of death despite the improvements in lipid and blood pressure control. The circadian clock, a molecular network of genes and proteins that controls 24-hour timing, has emerged to have a surprising role in the control of metabolic and vascular function. Herein we examined the impact of circadian rhythm dysfunction in atherogenesis by implementation of vascular transplant and PCSK9 based approaches to induce accelerated lesion development in mice. We find that atherogenesis is exacerbated in Bmal1-KO aortic grafts immersed in the hypercholesterolemic milieu of ApoE
-/-
mice. To assess if atherosclerosis was ‘circadian rhythm dependent’ we subjected wild-type mice to a shortened light cycle (4L/4D) and induced atherosclerosis by intravenous injection of a human PCSK-9 adeno associated virus. Atherosclerosis in the jet-lagged PCSK-9 mice was robustly increased relative to the atherosclerosis observed in WT mice on a normal light cycle (12L/12D), providing further evidence that circadian rhythm and the circadian clock contribute to atherosclerosis. However, atherosclerosis is a complex disease that is the net result of interplay between intrinsic (vascular cells) and extrinsic mechanisms (metabolism, blood pressure, and hormones) and the importance of clock function in individual cell types is poorly understood. We found that deletion or silencing of key circadian transcription factors resulted in an enhanced inflammatory and pro-oxidant phenotype with diminished NO production and greater lipid uptake in both macrophages and endothelial cells. Loss of circadian function in smooth muscle cells similarly resulted in enhanced production of reactive oxygen species and greater cell proliferation. Surprising, the silencing of Bmal2 in endothelial cells resulted in greater lipid uptake in oxLDL treated HAEC as well as increased expression of markers of autophagy, suggesting that Bmal2 may orchestrate numerous output functions in different cell types. In conclusion, we find that the circadian clock and circadian rhythm have a profound impact on atherosclerosis, to influence vascular cell inflammatory and lipid uptake responses, and identify an unexpectedly prominent role for the side-partner of Bmal1, Bmal2.
Abstract 304: Association Between Plaque Morphology and Time Interval After the Neurologic Index Event in Patients With Symptomatic Carotid Stenosis
Authors: Tsantilas Pavlos , Kuehnl Andreas , Pelisek Jaroslav , Maegdefessel Lars , Wendorff Carina , Wendorff Heiko , Schmid Sofie , Zimmermann Alexander , Eckstein Hans-Henning
Published: July 2021

Objectives:
Instable plaques are more common in patients with symptomatic carotid stenosis compared to asymptomatic patients. Clinically symptomatic patients are at high risk for a recurrent stroke in the first days after a neurologic index event. Histopathologic plaque stabilisation or remodelling mechanisms of symptomatic plaques are unclear. Therefore, our study aimed to find changes of plaque morphology in dependence of time interval between neurologic index event and plaque removal.

Methods:
Plaques of patients that were removed from surgically treated patients with symptomatic carotid stenosis between 2004 and 2016 were included. Histological analyses of those carotid plaques were performed to assess the type of plaque (American Heart Association classification), the stability of plaque (thickness of the fibrous cap </>200μm), the extent of calcification, inflammation, neovascularization and the deposition of collagen and elastin. Statistical analysis was applied in form of an ordinal regression analysis, adjusted for common risk factors of atherosclerosis.

Results:
Out of 348 included plaques, the patients’ median age was 71 (Q1-Q3, 65 - 77) years and 69% were male. Median time interval between index event and plaque removal was 10 days (Q1-Q3, 4-28 days). Most common index event was a transient ischemic attack with 37% (128 of 348), followed by stroke in 28% (97 of 348), amaurosis fugax in 22% (76 of 348) and instable symptoms (crescendo transient ischemic attack, stroke in evolution) in 12% (43 of 348), respectively. The ordinal regression analysis revealed, that the time interval as continuous independent variable had no significant influence on plaque type, plaque stability, extent of calcification, inflammation or neovascularization and the deposition of collagen and elastin.

Conclusions:
The examined plaque morphology features of patients with symptomatic carotid stenosis showed no differences in relation to the time interval between neurologic index event and plaque removal. To find potential symptomatic plaque remodelling mechanisms, currently ongoing molecular and histomorphological analysis aim at identifying novel markers of apoptosis and cell fate-driven mechanisms in fibrous cap-enriched vascular smooth cells.