bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 437: Endothelial Cell Gene Expression in Hypertensive Mice
Authors: Wilson Stephen J , Lowe Kate L , Bokoch Michael P , Srinivasan Yoga , Wu Roland S , Coughlin Shaun R
Published: July 2021

Hypertension is the major risk factor for premature cardiovascular disease, morbidity and mortality. It is the leading cause of death and disability globally, contributing to cardiovascular and renal diseases, including stroke, heart failure, coronary heart disease, and chronic kidney disease. We sought to determine the transcriptional changes that occur in cardiac endothelial cells secondary to hypertension, using ribosomal profiling of mice expressing the ribosomal protein Rpl22 tagged with the hemagglutinin (HA) epitope under control of Tie2-Cre (Tie2-Cre:RiboTag mice). Immunoprecipitation of HA-Rpl22 from heart tissue isolated from Tie2-Cre:RiboTag mice resulted in an approximate 10-fold enrichment of endothelial genes (Pecam-1, VE-cadherin) relative to genes highly expressed in cardiomyocytes (Tnnt2, Fabp3) as assessed by RT-PCR. Subsequently, Tie2-Cre:RiboTag mice were implanted with osmotic pumps delivering saline or angiotensin II (1000ng/kg/min) for 4 weeks. Angiotensin II infusion significantly increased blood pressure (p<0.0001, n=7-8) resulting in an increased heart to body weight ratio (6.07 ± 0.48 vs 8.14 ± 0.73; p<0.05, n=7-8). Transcriptomes from Tie2-Cre positive cells within the heart were isolated using Ribotag ribosomal immunoprecipitation and RNA-seq was performed. Five hundred and eighty seven differentially expressed genes were detected (fold change >1.5, p<0.05, n=3), representing pathways such as extracellular matrix metabolism, angiogenesis and fatty acid transport. Analysis of hypertension associated gene expression in endothelial cells across different organs may reveal the mechanism of hypertension-associated diseases.
Abstract 442: FOXO1/Notch Signaling Modulates Ambient Ultrafine Particle Impaired Vascular Repair
Authors: Baek Kyung In , Packard René R , Saffari Arian , Ma Zhao , Luu Anh P , Yen Hilary , Pieterson Andrew , Hsu Jeffrey J , Ren Bin , Sioutas Constantinos , Li Rongsong , Hsiai Tzung K
Published: July 2021

Introduction:
Exposure to ultrafine particles (UFP,
d
< 0.1 μm), redox-active components of particular matter (PM
2.5
), promotes endothelial dysfunction. Notch signaling in endothelial cells (EC) regulates differentiation and proliferation of vasculature. FOXO1 interacts with Notch signaling by enhancing assembly of activation complex during induction of Notch signaling. Whether UFP impair vascular repair by modulating FOXO1/Notch signaling axis remains elusive.

Hypothesis:
We hypothesized that UFP impairs vascular repair by attenuating Notch signaling via inhibition on FOXO1.

Methods and Results:
Control transgenic
Tg(fli1:gfp)
zebrafish embryos underwent tail amputation at 3 days post fertilization (dpf) developed complete vascular repair at 3 days post amputation (dpa), whereas exposure to UFP, or treatment with ADAM10 inhibitor to prevent Notch activation, or micro-injection of dominant negative(DN) Notch1b mRNA disrupted vascular network and impaired regeneration (*
P
< 0.05, n=20). By crossing the Notch reporter line
Tg(tp1:gfp)
with the
Tg(flk1:mCherry)
line, we demonstrated UFP inhibits endothelial Notch signaling on the amputated site at 3 dpa. Micro-injection of NICD mRNA only partially rescued endothelial Notch activity and impaired vascular repair in the presence of UFP (*
P
< 0.05, n=20). FOXO1 MO significantly inhibited Notch signaling, mimicking the UFP-impaired vascular repair. Injection of FOXO1 mRNA accentuated Notch activity and rescued UFP-impaired vascular repair. In human aortic endothelial cells, UFP suppressed FOXO1 expression and the co-localization with NICD, but not Master-Mind Like 1(MAML) or active NICD expression (*
P
< 0.05, n=3). As a corollary, UFP exposure induced dose and time-dependent reduction in Notch reporter activity, FOXO1 mRNA expression and the expression of Notch signaling related genes including the Notch ligand Dll4 and Notch target HES1. (*
P
< 0.05, n=3).

Conclusions:
In conclusion, UFP attenuated FOXO1/Notch cooperation to modulate Notch signaling and impaired vascular repair in embryonic zebrafish.
Abstract 443: Absence of Pro-Protein Convertase Subtilisin/Kexin 6 Increases Flow-Mediated Outward Remodeling in the Mouse Common Carotid Artery
Authors: Röhl Samuel , Arner Anders , Caidahl Kenneth , Hedin Ulf , Perisic Matic Ljubica , Razuvaev Anton
Published: July 2021

Objective:
Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines but their function in vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques, localization to smooth muscle cells in the fibrous cap and positive correlation to inflammation, extracellular matrix remodeling and cytokines. Here, our aim was to evaluate the effects of PCSK6 on flow-mediated vascular remodeling in mice using high-frequency ultrasound and myography.

Materials and Methods:
PCSK6 -/- and C57Bl/6J mice were compared in this study divided in baseline control and increased flow groups. Increased flow was created in the right common carotid artery (CCA) by ligation of the left CCA. All animals were subjected to high-frequency ultrasound examination prior to surgery, at 3 and 5 weeks after surgery. Upon euthanization 6 weeks post-surgery the right CCA was harvested for myography evaluation, subsequently fixed at optimal stretch and prepared for histological evaluation.

Results:
The vascular circumference at optimal stretch in myography was strongly correlated (Pearson r=0.74, p<0.001) and in agreement with the diastolic circumference measured by high-frequency ultrasound in examined animals. A significant increase in diastolic circumference was seen at 3 and 5 weeks after surgery in PCSK6 -/- mice with increased flow compared to PCSK6 -/- control group (1.6±0.15 mm vs 1.4±0.08 mm, p<0.05 and 1.7±0.09 mm vs 1.4±0.12 mm, p<0.01). Myography revealed a significant increase in circumference at optimal stretch (1.7±0.21 mm vs 1.4±0.08 mm, p<0.05) in PCSK6 -/- mice subjected to increased flow compared to PCSK6 -/- control group. A significant flow-mediated increase in medial contractility was identified (0.68±0.14 mN/mm vs 0.45±0.11 mN/mm, p<0.05) in C57Bl/6J mice compared to C57Bl/6J control where as an absence of flow-mediated increase in medial contractility was seen in PCSK6 -/- mice.

Conclusion:
Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow.
Abstract 444: A Six-Microrna Panel Identified as a Potential Biomarker for Early-Stage Atherosclerotic Lesions
Authors: Karere Genesio , Glenn Jeremy , Galindo Samuel , Garcia Roy , Chevalier Frederic , Dick Edward , Cox Laura
Published: July 2021

Introduction:
Atherosclerosis is a progressive disease, and it’s the common cause of cardiovascular disease (CVD). CVD is the leading cause of morbidity and mortality in the US. It is important to detect early-stage asymptomatic atherosclerosis prior to progression to plaques. However, it is not feasible to obtain target tissues from humans with early-stage atherosclerosis. We tested the hypothesis that a panel of microRNAs (miRNAs) expressed in atherosclerotic lesions and expressed in plasma of the same baboons are potential biomarkers indicative of initiation and progression of atherosclerosis.

Methods and Results:
We used small RNA-Seq to identify miRNA expression profiles in atherosclerotic lesions and in plasma of baboons. We challenged adult baboons (n=24) with a high cholesterol, high fat (HCHF) diet for two years. After the diet challenge, common iliac arteries were harvested, fixed in 10% buffered formalin and stained with Sudan IV. We observed interesting fatty streak lesion variations, including early stage (EGES), flat (F) and raised (R) lesions, corresponding, respectively, to AHA lesion types I, II and V. We identified 45 miRNAs differentially expressed in fatty streak lesions (ES vs F=0; ES vs R =36; F vs R=9), and 43 miRNAs in plasma of the same animals (ES vs F=0; ES vs R =21; F vs R=22). Further, we observed that miR-30-5p, miR-340-5p, miR-548-5p and let-7-3p were expressed in flat lesions as well as in plasma, whereas miR-30-5p and miR-21-5p were expressed in raised lesions and in plasma. FDR < 0.05.

Conclusions:
We conclude that a panel of six miRNAs differentially expressed in fatty streak lesions and differentially expressed in plasma of the same animals is a potential biomarker indicative of initiation and progression of atherosclerosis in baboons. Future studies will focus on translating baboon findings to humans.
Abstract 445: Venous Adventitial Cells May Protect Against Human Vein Graft Stenosis
Authors: Sobel Michael , Chen Lihua , Tang Gale , Kenagy Richard D
Published: July 2021

Introduction:
A p27
Kip1
single nucleotide polymorphism (SNP) is associated with vein graft failure. The protective genotype (AA) is associated with slower growth of human saphenous vein adventitial cells, but not of smooth muscle cells (SMCs). We investigated the influence of patient clinical characteristics on the migration of adventitial cells and SMCs from vein tissue and the interaction between adventitial cells and SMCs on cell growth.

Methods and Results:
Tissue explants of the adventitia and intima/media were prepared from samples of vein used for leg bypass, and the number of cells emerging from explants was counted over time (15 replicates/vein, 43 veins). Correlating migration with clinical characteristics of the patient, the degree of leg ischemia was an important factor. Migration of
adventitial
cells was slower with more severe ischemia (AAI<0.6; N=12) compared to milder ischemia (AAI≥0.6; N= 31): 37.0 ± 4.9 vs 53.8 ± 7.7 cells/explant day 7; mean ± SEM, P<.03). Migration of
SMCs
from the intima/media explants showed no influence of ischemia (19.1 ± 3.0 vs 24.1 ± 6.1; P=.12). The p27 SNP genotype, diabetes, smoking, and wounds/infection bore no correlation with migration. To test cell interactions, adventitial cells and SMCs from 11 veins were seeded in triplicate on opposite sides of 0.4 um Transwell filters that prevent cell migration (6 AA and 5 CC p27 SNP genotypes), such that the same or different cells were opposed. Cells were counted for a Day4/Day1 proliferation ratio. Compared to AA SMCs, AA adventitial cells significantly inhibited AA SMC growth (10.9 ± 2.5% inhibition; P<.01). In contrast, CC adventitial cells did not significantly inhibit CC SMC growth (4.1 ± 3.5% inhibition, P=.20).

Conclusions:
Migration of adventitial cells, but not SMCs, from vein tissue is inhibited by increased ischemia. This suggests that adventitial cells may be beneficial, since more severe ischemia has been associated with poorer graft patency. Supporting this, adventitial cells with the protective AA genotype (but not the CC genotype) inhibit the growth of SMCs. These data suggest that adventitial cells and their in situ precursors may inhibit intimal hyperplasia and graft failure by inhibiting SMC proliferation in a p27 SNP genotype-dependent manner.
Abstract 446: Cellular Uptake of Glycol Chitosan Nanoparticles in Idiopathic Pulmonary Fibrosis Fibroblasts in Response to the Collagen-Rich Microenvironment
Authors: Nho Richard , Yhee Ji-Young
Published: July 2021

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the presence of persistent fibrotic fibroblasts which have apoptosis-resistant properties in response to cell death inducing conditions including type I collagen rich matrix. Although selective targeting of fibrotic fibroblasts is a feasible concept for the effective therapy of IPF, no reliable drug carriers for targeting IPF fibroblasts have been reported. To address this limitation, biodegradable glycol chitosan nanoparticles (CNPs) were utilized as a carrier system for selective targeting of IPF patients-derived fibroblasts. Primary human lung fibroblasts from non-IPF and IPF patients (n = 6/group) were treated with various doses (50–450 μg/ml) of CNPs, and the subcellular localization of CNPs was examined as a function of time (3, 6, and 30 h) with a confocal microscope. CNPs were found in the cytoplasm of fibroblasts at 30 h post-treatment on tissue culture plates in the absence of collagen while the intracellular CNPs were clearly observed at 3 h post-treatment. Importantly, the cellular uptake of the CNPs was significantly increased at 3, 6, and 30 h post-treatment, when control and IPF fibroblasts were cultured on collagen, suggesting that fibroblast and collagen interaction promotes the CNP’s cellular internalization process. Clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis were then suppressed by chlorpromazine (30 μM), genistein (100 μM), or amiloride (50 μM), respectively to elucidate the underlying mechanism of cellular uptake of CNPs in fibroblasts on the collagen matrix. Amiloride pre-treatment remarkably reduced the cellular internalization of CNPs in fibroblasts cultured on the collagen matrix. Furthermore, additional results also showed that sodium-hydrogen exchanger 1 (NHE-1) and PI3K play important roles in the increased cellular uptake of CNPs in the lung fibroblasts on the collagen matrix, suggesting that the subcellular localization of CNPs is largely due to the macropinocytosis. We propose that our approach can be a promising tactic for targeted delivery of therapeutics to the fibrotic fibroblasts residing in collagen matrix, which may lead to the improved treatment outcomes of this deadly disease.
Abstract 457: Selective Epidermal Growth Factor Receptor Inhibition in Cd4+ T Cells Induces Anergy and Limits Atherosclerosis Development
Authors: Zeboudj Lynda , Maître Mikael , Guyonnet Lea , Laurans Ludivine , Lemarie Jeremie , Giraud Andreas , Bourcier Simon , Nour-Eldine Wared , Tedgui Alain , Mallat Ziad , Tharaux Pierre-Louis , Ait-Oufella Hafid
Published: July 2021

Background:
Several Epidermal Growth Factor receptor (EGFR) inhibitors have been successfully developed for the treatment of cancer. They inhibit tumor cell survival, proliferation and migration. EGFR is also expressed by leukocytes, but little is known about its role in the modulation of the immune response. We aimed to determine whether EGF-R expressed on CD4
+
T cells is functional, and to address the consequences of EGF-R inhibition in atherosclerosis, a T cell-mediated chronic inflammatory disease of the vascular wall.

Method and Results:
Mouse CD4
+
T cells expressed Egfr, and the EGFR tyrosine kinase inhibitor AG-1478 blocked
in vitro
T cell proliferation and Th1/Th2 cytokine production.
In vivo
, treatment of
Ldlr

-/-

mice with the Egfr inhibitor Erlotinib for 8 weeks induced T cell anergy, reduced T cell infiltration within atherosclerotic lesions and protected against atherosclerosis. Selective deletion of Egfr in CD4
+
T cells resulted in decreased T cell proliferation and activation both
in vitro
and
in vivo
, as well as reduced IFN-γ, IL-17A, IL-4 and IL-10 production. Atherosclerotic lesion size was reduced by 2-fold in irradiated
Ldlr

-/-

mice reconstituted with bone marrow from
Cd4Cre Egfr

lox/lox

mouse, compared to
Cd4Cre Egfr

+/+

chimeric mice, after 4, 6 and 12 weeks of high fat diet, associated with marked reduction in T cell infiltration in atherosclerotic plaques. Finally, human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both
in vivo
and
in vitro
.

Conclusion:
EGFR is expressed by human and mouse CD4
+
T cells. EGFR pharmacological inhibition or genetic invalidation induced T cell anergy
in vitro
and
in vivo
, and reduced atherosclerosis development. Our results suggest that targeting EGFR may be a novel strategy to combat atherosclerosis.
Abstract 458: Role of Colony Stimulating Factor 1 Receptor in Graft Vascular Disease
Authors: Almonte Vanessa , Wunimenghe Oriyanhan , Riascos-Bernal Dario F , Chinnasamy Prameladevi , Jayakumar Smitha , Sibinga Nicholas E
Published: July 2021

Heart and kidney transplants are effective treatments for end-stage organ failure, but their long-term success is limited by graft vascular disease (GVD), the leading cause of solid organ transplant failure. This process manifests as concentric thickening of vessel walls due to neointimal hyperplasia in the donor organ, characterized by expansion of cells, notably smooth muscle-like cells (SMLCs) and macrophages (MPs), which accumulate, proliferate, and eventually occlude the lumen of arteries. Our lab recently reported that loss of colony stimulating factor-1 (CSF1) expression in either donor or recipient mice limits GVD, and showed that SMLCs isolated from neointimal lesions express high levels of CSF1 and its receptor, CSF1R. While CSF1-mediated activation of CSF1R has been studied extensively in MP biology, its role in SMLCs and GVD has not been well characterized. We hypothesize that CSF1R activation in neointimal MPs and SMCs occurs after organ transplantation and promotes the development of GVD.

To test this idea, carotid arteries from 8-12 week old C57/B6J male mice were transplanted orthotopically into female or male mice. At day 30 post transplantation, sex-mismatched transplants developed significant neointimal lesions not seen in sex-matched controls. Neointimal, adventitial, and to a lesser extent medial cells were positive for CSF1R and CD68, which were scarcely detected in control untransplanted arteries. Cells in the media of transplanted vessels co-stained for smooth muscle alpha actin (SMA) and calponin. SMA-positive cells were found in neointimal lesions, with few cells co-expressing calponin. Proliferation, assessed by Phospho-histone H3 staining, was evident in cells of uncertain origin in the media and neointima. In conclusion, H-Y antigen-driven histoincompatibility in this mouse transplant model yielded vascular lesions that resemble GVD, with significant neointima formation, preservation of medial cells, and evidence of CSF1R expression and of cell proliferation. Future studies will focus on lineage tracing of smooth muscle and myeloid cells to evaluate neointimal cell origins, plus genetic depletion of CSF1R in these cell lineages to determine the requirement for CSF1R expression in the development of GVD.
Abstract 460: Intra- and Inter-Day Reproducibility of Low-Flow Mediated Constriction Response in Young Adults
Authors: Harbin Michelle M , Ostrem Joseph D , Evanoff Nicholas G , Kelly Aaron S , Dengel Donald R
Published: July 2021

Purpose:
When assessing vasomotor endothelial function of the brachial artery by reactive hyperemia, blood flow is stopped and creates a period of low-flow mediated constriction (L-FMC). Little is known about how this parameter influences flow-mediated vasodilation (FMD). The purpose of this study was to better understand this relationship and to determine the intra- and inter-day reproducibility of brachial L-FMC in healthy adults.

Methods:
Brachial L-FMC and FMD were measured on 26 healthy, young adults (13 males, 13 females; 24.6 ± 2.7 years). Each subject had two assessments conducted on two separate visits, separated by a minimum of seven days. Brachial artery pre-occlusion baseline diameter was imaged during rest and prior to cuff-occlusion. Continuous imaging of the artery was captured during the last 20 seconds of cuff-occlusion to 180 seconds post-cuff release. An L-FMC was considered present if the relative change from pre-occlusion baseline to L-FMC artery diameter was less than -0.1%. Peak FMD was measured as the greatest 10-second average in brachial artery diameter following occlusion compared to pre-occlusion baseline.

Results:
Overall, there was a strong, positive correlation between increased brachial L-FMC and blunted FMD (visit 1 test 1: r=0.758,
p
<0.001; visit 1 test 2: r=0.706,
p
<0.001; visit 2 test 1: r=0.836,
p
<0.001; visit 2 test 2: 0.857,
p
<0.001). The reproducibility of intra- and inter-day L-FMC diameter were ICC = 0.627, CV = 54.4% and ICC = 0.734, CV = 43.5%, respectively.

Conclusion:
The results of the present study suggest that the degree of vasoconstriction to low-flow conditions influences the subsequent maximal dilation during reactive hyperemia. However, L-FMC in young adults is variable as evidenced by the weak inter- and intra-day reproducibility of the measure. Further research is needed to study brachial L-FMC reproducibility among varying subject populations and the implications L-FMC has on the interpretation of FMD results.
Abstract 462: Expression, Phosphorylation and Interaction Partners of the Transcription Factor Grainyhead-like 3 in the Endothelium
Authors: Kohlgrüber Stefanie , Dyballa-Rukes Nadine , Metzger Sabine , Nguyen Anh , Garmey Jim , McNamara Coleen , Haendeler Judith , Altschmied Joachim
Published: July 2021

The transcription factor Grainyhead-like 3 (GRHL3) regulates apoptosis, migration and NO-bioavailability and thus, critical functions of endothelial cell, which are impaired in many cardiovascular diseases. However, due to the lack of a good antibody, all experiments concerning the regulation of GRHL3 itself were performed on the RNA level. After establishing a new antibody, we analyzed the GRHL3 protein in aortic sections of ApoE-deficient mice fed a high fat diet, a model for atherosclerosis. The diet resulted in a reduction of GRHL3 levels in the endothelium, which was corroborated
ex vivo
in endothelial cells treated with LDL. This simulated high fat diet also led to a decrease in endothelial NO-synthase. As the activity of transcription factors is regulated by post-translational modifications and protein-protein interactions, we analyzed the phosphorylation of GRHL3 and identified potential interaction partners. Using a combination of immunoprecipitation and -blotting we demonstrated for the first time that GRHL3 is phosphorylated on tyrosine residues. Furthermore, this phosphorylation was NO-inducible and Src-kinase-dependent. After characterization of the modified residues, we will assess their relevance by determining the impact of phospho-mimetic and non-phosphorylatable mutants on functional parameters of endothelial cells. To identify potential interaction partners of GRHL3 we immunoprecipitated the protein and analyzed the co-precipitated proteins by mass spectrometry. We identified the DBHS-proteins NONO and SFPQ, which have been implicated in the regulation of transcription and alternative splicing. The interaction with these two proteins was validated by co-immunoprecipitation. As a next step, we will overexpress and downregulate these proteins in endothelial cells to evaluate their cross-talk with GRHL3. Taken together our findings demonstrate (i) a downregulation of GRHL3 in a disease setting, (ii) a Src-kinase dependent, NO-inducible phosphorylation and (iii) an interaction with other gene-regulatory proteins. The analysis of the functional consequences of these different aspects of GRHL3 regulation will further shed light on the GRHL3 network in the endothelium and thus, its functions in the vasculature.
Abstract 463: The Key Regulatory Elements for SM22 Transcription
Authors: Yang Maozhou , Warming Soren , Shen Jianbin , Jiang Hong , Li Hui J , Krawetz Steve , Li Li
Published: July 2021

Gene transcription is controlled by an array of transcriptional regulatory elements. SM22 gene regulation has been widely used to characterize the molecular mechanisms of smooth muscle cell (SMC) phenotypic modulation during cardiovascular development and in vascular diseases. Our previous studies show that the proximal CArG box (CArGnear) in the SM22 promoter is required for its transcription in arterial smooth muscle cells (SMC). However, the role of the CArG box in visceral SMCs has not yet been explored. Here we aim to determine the role of CArG boxes in regulating S
M22
transcription in both vascular and visceral SMCs. Using bacterial chromosome (BAC) recombineering, we knock-in a
lacZ
reporter into a
SM22
BAC to trace SM22 transcriptional activities in transgenic mice. Anatomic/histology analyses show that the
lacZ
expression patterns in the BAC transgenic mice recapitulate that of the endogenous
SM22
transcription during embryogenesis and in adult
.
Similar to the endogenous
SM22
regulation, the expression of
lacZ
is highly sensitive to vascular remodeling: carotid injury abolishes lacZ expression in the arterial wall. Using seamless BAC recombineering mutagenesis, we generate mutations in the proximal and/or distal CArG box in the SM22-lacZ-BAC. Consistent with our previous results, we find that mutating the CArGnear box disrupts the
lacZ
expression in the aorta; this mutation also drastically reduces its expression in visceral SMCs including stomach, uterus and bladder. Interestingly, mutating the distal CArG (CArGfar) box does not affect the lacZ expression in arterial, venous and visceral SMCs. Mutating both CArG boxes nearly abolishes
lacZ
expression in all SMCs. This study provides evidence supporting the generation of SM22 knockout mice by mutating the CArG boxes in the SM22 promoter using the CRISPR technology.
Abstract 47: FXR1 Regulates Inflammatory MRNA Stability and VSMC Proliferation by Modulation of HuR Activity
Authors: Herman Allison , England Ross , Haines Dale , Kelemen Sheri , Ray Mitali , Autieri Michael V
Published: July 2021

Vascular smooth muscle cells (VSMC) play a critical role in the etiology and progression of many vascular diseases including atherosclerosis and restenosis. Our laboratory has found that one anti-inflammatory interleukin, IL-19, is atheroprotective and can decrease vascular inflammation by reduction in mRNA stability of inflammatory transcripts by reduction of activity of HuR, an mRNA stability protein. HuR translocates from the nucleus to the cytoplasm where it recognizes AU-rich elements present almost exclusively in the 3’UTR of pro-inflammatory genes. Proteins and pathways which limit HuR translocation are understudied, but may reduce inflammatory mRNA stability. Using MASS SPEC to identify HuR-interacting proteins under different inflammatory conditions, we identified one protein, Fragile X-related protein (FXR1), which interacts with HuR in inflammatory, but not basal conditions, a novel interaction. FXR1 mRNA expression is enhanced in muscle cells, but nothing has been reported on expression of FXR1 in VSMC or function for FXR1 in vascular disease. The FXR1 promoter contains multiple cholesterol-response elements, and in this study we demonstrate that FXR1 expression is increased in injured arteries and TNFα and oxLDL stimulated human VSMC, but also by IL-19. RNA EMSA demonstrates that FXR1 directly interacts with ARE in 3’UTR. SiRNA knock down of FXR1 in VSMC increases stability of inflammatory mRNA and protein abundance as well as VSMC proliferation, while overexpression of FXR1 reduces both their abundance and stability in addition to reducing proliferation. Since FXR1 appears to be a novel repressor of inflammatory proteins, and is also induced by IL-19, our overall hypothesis is that FXR1 expression and HuR interaction is an inflammation responsive, counter-regulatory mechanism to reduce abundance of pro-inflammatory proteins and therefore reduce inflammation.
Abstract 473: Dissecting Aortic Aneurym in Angiotensin II - Infused Mice: The Mechanisms Behind Lesion Variability
Authors: Trachet Bram , Piersigilli Alessandra , Aslanidou Lydia , Fraga-Silva Rodrigo A , Sordet-Dessimoz Jessica , Villanueva-Perez Pablo , Stampanoni Marco F , Stergiopulos Nikolaos , Segers Patrick
Published: July 2021

Angiotensin II-infused mice develop dissecting aneurysms, characterized by intramural rather than intraluminal thrombus, suprarenal rather than infrarenal lesions, medial dissections rather than circumferential elastin degradation and a variability in lesion shape that remained as yet unexplained. In order to provide insight into these intriguing lesions we scanned murine aortas at baseline and after 10, 18 and 29 days of Angiotensin II infusion, both in vivo (ultrasound, micro-CT) and ex vivo (phase-contrast X-ray tomographic microscopy). Dissecting aneurysms of varying severity occurred in 31/34 mice. All of these were characterized by a medial tear near the ostium of thoracic and abdominal aortic side branches, with a predilection for the left and ventral aspects of the ostium of the celiac artery. In 25/31 animals an intramural hematoma was formed. Fully ruptured branch ostia occurred significantly more often in the supraceliac aorta, affecting in particular the left suprarenal artery (the first branch cranial to the celiac artery, 23/25). Animals with a thoracic tear (6/31) had significantly larger intramural hematoma than animals with an abdominal tear (p<0.05), and the length of the hematoma correlated to the number of ruptured side branches (r
2
=0.78). In 11 mice a parallel false channel was formed. The volume of free-flowing intramural blood in the false channel was significantly larger for left than for ventral tears, but was not related to the length of the tear. Our data suggest that (i) medial tears are the primary event in dissecting AAA formation, (ii) an intramural hematoma is formed if the adventitia covering the medial tear dissects and leads to the accumulation of intramural blood from ruptured side branches, (iii) adventitial dissection and hematoma formation progress in the direction of least resistance/smallest side branches (i.e. cranial of the celiac artery) and (iv) a false channel is formed if the radial expansion of the adventitia due to blood flowing out of the medial tear acts in the same (leftward) direction as the expansion due to a ruptured left suprarenal artery. We conclude that Ang II-infused mice can be a valuable model to study the under-researched role of side branches in the formation and progression of aortic dissections.
Abstract 474: Early Events in Dissecting Aneurysms Induced by Angiotensin-II Infusion: The Geometry-Driven Aspect of a Multifaceted Problem
Authors: Aslanidou Lydia , Trachet Bram , Ferraro Mauro , Piersigilli Alessandra , Fraga-Silva Rodrigo , Sordet-Dessimoz Jessica , Villanueva-Perez Pablo , Stampanoni Marco , Segers Patrick , Stergiopulos Nikolaos
Published: July 2021

While research on dissecting aneurysms in Angiotensin-II infused mice spans more than a decade, the temporal sequence of initial events still remains unclear. Recent findings in our group suggested that focal medial tears at the vicinity of suprarenal side branches are the primary event in disease formation. In this study we used a combined experimental-computational approach to investigate the hypothesis that initial events of dissecting AAAs originate at branching sites along the aorta. Male apolipoprotein-deficient mice were infused with Angiotensin-II (n=11) and saline 0.9% (n=6) for 3 days and scanned with contrast-enhanced microCT prior to sacrifice. One animal presented an in-vivo rupture during the microCT scan, and was rescanned after 2.5 hours to observe real-time morphological changes. In all other animals, the excised aortic tissue was imaged with Phase Contrast X-Ray Tomographic Microscopy (PCXTM) at 6.5um isotropic resolution. An automatic morphing code was developed to map the ex-vivo geometry onto the in vivo geometry, and a finite element simulation yielded a stress distribution that represents an estimation of the wall tension, not only due to the pressurization, but also due to the local stretch field. We found that the nanoparticulate microCT contrast agent had infiltrated the aortic wall in 11/11 Ang-II infused animals, while no infiltration was observed in 6/6 control mice. The infiltration affected at least one pair of intercostal arteries in 11/11 mice, and in 9/11 mice the coeliac region was also affected. Image-guided histology allowed us to determine the circumferential distribution of microlesions at branching sites, including disruption of elastin fibers, apoptotic cell appearance, subintimal leukocyte infiltration and intramural hematomas. In the animal whose aorta had ruptured during the in vivo scan, the initial hematoma had originated around 3 pairs of intercostal arteries and quickly propagated afterwards. Mouse-specific finite element simulations revealed a co-location of computed peak stresses at the vessel wall and histologically identified vascular damage. We conclude that the aortic geometry, and side branches in particular, play a pivotal role in the onset of dissecting AAA.
Abstract 475: Novel Gene Therapy Facilitates Critical Limb Ischemia Reperfusion in Mouse Hindlimb Gangrene Model
Authors: Parikh Punam P , Lassance-Soares Roberta , Regueiro Manuela , Shao Hongwei , Li Yan , Liu Zhao-Jun , Velazquez Omaida C
Published: July 2021

Lack of a reliable animal hindlimb gangrene model limits molecular investigation of critical limb ischemia. We sought to develop a mouse hindlimb gangrene model, used to assess the efficacy of novel gene therapy. We hypothesized that priming ischemic hindlimb tissue with E-selectin/adeno-associated virus (AAV) enhances therapeutic angiogenesis and attenuates gangrene.

We tested two methods to induce hindlimb gangrene. First, FVB mice underwent femoral artery ligation (FAL) to achieve critical limb ischemia. Second, FVB underwent combined FAL and administration of NG-nitro-L-arginine methyl ester (L-NAME), nitric oxide synthase inhibitor, which further reduces hindlimb perfusion. Prior to FAL and L-NAME use, gangrene-induced mice were intramuscularly administered E-selectin/AAV (treatment) or LacZ/AAV (control) to the hindlimb. Gangrene was assessed with a standardized ischemia score ranging from 0 (no gangrene) to 11 (forefoot gangrene), recorded on postoperative day (POD)’s 2,7,14. Hindlimb reperfusion using Laser doppler imaging was quantified by mean perfusion of ligated:non-ligated limb on same POD’s. Live animal Dil perfusion plus laser scanning confocal microscopy quantified limb neovascularization.

Most FVB did not develop gangrene with FAL-only (n=2/8, 25% gangrene incidence). Combined FAL and L-NAME consistently induced hindlimb gangrene (n=14/14, 100% gangrene incidence). Laser doppler imaging score on POD 7 for E-selectin/AAV (n=7) and LacZ/AAV (n=7) was 0.41 vs 0.27 (P=0.071) and on POD 14 was 0.54 vs 0.29 (P=0.017). Dil perfused ligated hindlimb in E-selectin/AAV and LacZ/AAV revealed significantly higher mean neovascularization intensity score of 44 vs 21 (P=0.037). Dil perfused non-ligated limb in respective mice demonstrated mean intensity score of 50 vs 25 (P=0.006). Mean limb ischemia score on POD 2,7,14 for E-selectin/AAV and LacZ/AAV was 1.9, 2.9, 3.7 vs 2.7, 3.9, 5.3 (P=0.104).

We developed a highly reliable mouse hindlimb gangrene model where E-selectin-based novel gene therapy improved limb reperfusion and neovascuclarization in critical limb ischemia. This hindlimb gangrene model can be used to further understand Redox pathways contributing to gangrene, facilitating future translational studies.
Abstract 481: Pharmacological Inhibition of Calpain Attenuates Mineralocorticoid Receptor Agonist and High Salt-Induced Abdominal Aortic Aneurysm Incidence and Aortic Rupture in Mice
Authors: Javidan Aida , Jiang Weihua , Subramanian Venkateswaran
Published: July 2021

Background and Objective:
Recently, we demonstrated that pharmacological and genetic inhibition of calpains, a class of calcium-activated neutral cysteine proteases, attenuated angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) in mice. A newly identified non-invasive model of aneurysm has been developed in mice using deoxycorticosterone acetate (DOCA) coated pellets and oral administration of high salt. It is interesting to note that in this model, systemic administration of renin angiotensin inhibitors had no effect on aneurysm formation. The purpose of this study was to test whether pharmacological inhibition of calpain would influence AAA formation induced by mineralocorticoid receptor agonism and high salt in mice.

Methods and Results:
To determine whether calpain inhibition influenced DOCA and high salt-induced AAAs, 9 month old male C57BL/6 mice were administered calpain inhibitor, BDA-410 (30 mg/kg/day) or vehicle (n=10 per group) by oral gavage 1 week before DOCA pellet implantation and throughout the subsequent 21 days. Mice were implanted with DOCA pellets (50mg/ mouse for 21 days) and maintained on high salt (0.9% NaCl and 0.2% KCl) water. Calpain inhibition significantly suppressed AAA incidence (defined as increase in aortic diameter or AAA rupture; Vehicle = 60% vs BDA = 20%; P<0.05) compared to the vehicle group. Calpain inhibition also significantly protected DOCA + high salt-induced aortic rupture (Vehicle = 6/10 vs BDA = 1/10; P<0.05) in mice.

Conclusion:
These data demonstrate that calpain activation plays a critical role in not only AngII-induced, but also aldosterone pathways accelerated AAA formation in mice.
Abstract 484: Multimerization of BAFF Regulates B Cell Function and Growth of Aortic Aneurysms
Authors: Serbulea Vlad , Jakobs Philipp , Sahu Srabani , Srikakulapu Prasad , McNamara Coleen A , Ailawadi Gorav , Upchurch Gilbert R , Leitinger Norbert , Meher Akshaya K
Published: July 2021

B cell activating factor (BAFF) regulates differentiation and survival of B cells by binding to the surface receptors BAFF receptor (BR3), transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA). During differentiation, intracellular metabolic reprogramming is crucial, such as, naïve B cells are metabolically quiescent, whereas, antibody producing plasma cells are metabolically active. We have reported that depletion of B cells protects mice from abdominal aortic aneurysm (AA), however it is not clear how B cells promote AA growth. BAFF exists as a 3mer (binds only to BR3) or as a 60mer (binds to BR3, TACI and BCMA). Therefore, we hypothesize that BAFF multimerization regulates the immune and metabolic phenotype of B cells by binding to BAFF receptors and modulate AA growth. Immunohistology was performed on AA tissues collected from patients undergoing open AA repair. Experimental AA was induced by elastase perfusion of abdominal aorta or angiotensin II infusion (1000 ng/kg/min) method in 8 weeks old male C57BL/6 or apolipoprotein E knockout mice, respectively. Western blotting, flow cytometry and Seahorse extracellular flux assays were used to determine immune and metabolic changes in B cells in response to recombinant BAFF 3mer and 60mer. BR3+ B cells were detected in the milieu of BAFF in human AAs. Mouse AAs demonstrated significant infiltration (>50/section) of CD138+ plasma B cells, but few (4-10/section) CD20+ B cells. In vitro, BAFF 3mer induced canonical NF-kB, whereas, 60mer induced both canonical and non-canonical NF-kB signaling. Moreover, the 3mer significantly decreased mitochondrial density, oxygen consumption rate, and surface expression of IgD and IgM indicating a metabolically quiescent state of B cells. However, these parameters were significantly increased by the 60mer similar to plasma cells. Anti-BR3 IgG1, but not a control IgG1 antibody decreased BAFF 60mer-induced oxygen consumption rate by 50%. In a pilot study (n=10/group), anti-BR3 IgG1, but not the control IgG1 aggravated angiotensin II-induced AA growth. Altogether, our results suggest that BAFF 3mer and 60mer oppositely regulate immune and metabolic phenotype of B cells and inhibition of BAFF-BR3 signaling is detrimental for AA growth.
Abstract 485: On the Relative Effectiveness of Machine Learning and Statistical Methods in Predicting Abdominal Aortic Aneurysm Rupture in the Asian Population
Authors: Canchi Tejas , Patnaik Sourav S , Ng E Y , Srinivasan Dinesh K , Narayanan Sriram , Finol Ender A
Published: July 2021

Background:
Abdominal Aortic Aneurysms (AAA) have a high risk of mortality if they rupture, making it one of the leading causes of death in elderly men. Hence, a better prediction method is necessary to identify high rupture risk patients.

Methods:
Retrospective data (clinical and AAA geometric parameters) were collected of 114 Asian AAA patients from an IRB approved database. We applied a novel machine learning algorithm (MLA) to predict AAA rupture by generating a decision tree of rupture risk predictors. For comparative analysis, a binary logistic regression (backward stepwise Wald) was also performed. The rupture status was the dependent variable for both methods; each method provided the highest weighted risk factors and effectiveness of classification as assessed by Receiver Operating Characteristic (ROC) curves.

Results:
For the MLA, the most significant risk factors were DAAA (maximum diameter) (> 57 mm), presence of hypertension, D4 (diameter at the iliac bifurcation) (> 23.7 mm), age (> 79 years), and D3 (right common iliac artery diameter) (> 14 mm), as seen in Fig. 1a. Similarly, the backward stepwise logistic regression analysis showed a strong association for rupture with DAAA (O.R. 2.635), L1 (distance between the lowest renal artery and the iliac bifurcation) (O.R. 1.658), D3 (O.R. 1.876), NL (AAA neck length) (O.R. 0.294), and age (O.R. 0.681). The MLA exhibited a lower classification accuracy (80.7% vs. 99.2%) and area under the curve (ROC analysis) (56.3% vs. 98.2%) when compared to the regression analysis, as seen in Fig. 1b.

Conclusions:
This is the first known report of predictive rupture risk in the Asian AAA patient population using MLA and statistical methods. Although the MLA did not provide improved classification accuracy compared to a standard statistical method, the risk factors obtained from both methods were similar. Future studies will aim at improving the prediction ability of MLA in a racially diverse AAA patient population.
Abstract 491: Racial Disparities in the Trends of Acute Myocardial Infarction Outcomes Among Medicaid Patients, 2007-2011
Authors: Adegbala Oluwole M , Akinjero Akintunde , Alliu Samson , Adejumo Adeyinka C , Akintoye Emmanuel , Otuada David , Ajayi Tokunbo , Onyeakusi Nnaemeka , Edo-Osagie Eseosa , Durojaiye Modupeoluwa , Lichstein Edgar , Akinyemiju Tomi
Published: July 2021

Background:
Although, in-hospital mortality from acute myocardial infarction (AMI) have declined in the United States recently, there is a gap in knowledge regarding racial differences in this trend. We sought to evaluate the effect of race on the trends in outcomes after Acute Myocardial Infarction among Medicaid patients in a nationwide cohort from 2007-2011

Methods:
We extracted data from the Nationwide Inpatient Sample (NIS) for all hospitalizations between 2007 and 2011 for Medicaid patients aged 45 years or older with principal diagnosis of AMI using ICD-9-CM codes. Primary outcome of this study was all cause in-hospital mortality. We then stratified hospitalizations by racial groups; Whites, African Americans and Hispanics, and assessed the time trends of in-hospital mortality before and after multivariate analysis.

Results:
The overall mortality from AMI among Medicaid patients declined during the study period (8.80% in 2007 to 7.46% in 2011). In the adjusted models, compared to 2007, in-hospital mortality from AMI for Medicaid patients decreased across the 3 racial groups; Whites (aOR= 0.88, CI=0.70-0.99), African Americans (aOR=0.76, CI=0.57-1.01), Hispanics (aOR=0.87, CI=0.66-1.25). While the length of hospital stay declined significantly among African American and Hispanic with 2 days and 1.76 days decline respectively, the length of stay remained unchanged for Whites. There was non-significant increase in the incidence of stroke across the various racial groups; Whites (aOR= 1.23, CI=0.90 -1.69), African Americans (aOR=1.10, CI=0.73 -1.64), Hispanics (aOR=1.03, CI=0.68-1.55) when compared to 2007.

Conclusion:
In this study, we found that in-hospital mortality from AMI among Medicaid patients have declined across the racial groups. However, while the length of stay following AMI declined for African Americans and Hispanics with Medicaid insurance, it has remained unchanged for Whites. Future studies are necessary to identify determinants of these significant racial disparities in outcomes for AMI.
Abstract 498:
In vivo
Assessment of Murine Valvular and Vascular Calcification Using
18
F Sodium Fluoride Micro Positron-Emission Tomography and Computed Tomography
Authors: Hsu Jeffrey J , Wu Alexander , Lu Jinxiu , Umar Soban , Demer Linda L , Tintut Yin
Published: July 2021

Calcific aortic valvular and vascular disease (CAVVD) is associated with increased morbidity and mortality. In addition to the commonly practiced computed tomography (CT) to evaluate for the presence of CAVVD, recent work has demonstrated the use of
18
F-sodium fluoride positron-emission tomography (
18
F-PET) to assess both valvular and vascular calcification in humans. In this pilot study, we combined
18
F μPET and μCT to assess for aortic valvular and aortic arch calcification
in vivo
. Aged

Apoe
-/-

mice (n=5) were injected with ~200 μCi
18
F-sodium fluoride and, one hour later, were imaged with fused μPET-μCT (Figure A,C). Intravenous contrast was used for the μCT studies to assist with anatomic localization (Figure B,D). To assess valvular hemodynamics, direct cardiac catheterization was performed on the mice to determine the peak-to-peak pressure gradient (PPG) across the aortic valve, between the left ventricle and aorta (Figure E,F). All mice were found to have aortic arch calcification present on both μPET and μCT imaging. In mice with aortic valve calcification specifically identified on μPET-μCT (Figure A-D, red arrows), there was increased
18
F uptake in the heart and aorta (58.8 ± 7.7 %ID/cc) compared to the mouse without aortic valve calcification (16.2 %ID/cc). Additionally, in the mice with aortic valve calcification, the mean transvalvular PPG was 9.7 ± 2.5 mmHg, and in the mouse without valvular calcification, the PPG was 3.3 mmHg. Alizarin red staining of histological sections from the aortic valves and aortic roots from these mice was performed to assess for the presence of calcium mineral. In conclusion, these findings suggest that the use of
18
F μPET-μCT in small animals provides a method to determine the presence of CAVVD
in vivo
. Future studies will determine whether changes in
18
F μPET-μCT signal reliably correlate with meaningful changes in CAVVD.
Abstract 502: Effect of Intensive Versus Standard Blood Pressure Control on Stroke and Serious Adverse Event: A Bivariate Analysis on the Net Clinical Benefit
Authors: Marszalek Jolanta , Mehrsefat Sara , Chi Gerald
Published: July 2021

Background:
Intensive blood pressure (BP) control is associated with a reduced risk of stroke but a greater risk of serious adverse event (SAE), thereby offsetting the benefit-to-risk ratio of this strategy.

Methods:
Using a previously described method of bivariate analysis, efficacy and safety outcomes were collectively analyzed as net clinical benefit (NCB) based on data from three randomized controlled trials comparing intensive versus standard BP control among 17,115 patients (ACCORD, SPS3, and SPRINT trials). Bivariate risk difference was assessed by the minimum distance from NCB curve to 95% CI of risk difference in all-cause stroke and SAE.

Results:
Compared to standard BP target, intensive strategies reduced all-cause stroke by 0.70% (95% CI: 0.19% to 1.21%; P = 0.007) and increased SAE by 1.85% (95% CI: 1.35% to 2.34%; P < 0.0001). With a non-inferiority margin of 25%, standard strategy was preferable with respect to NCB in diabetics (bivariate risk difference: 1.29% [0.40% to 2.31%]) and in patients without diabetes or prior stroke (1.55% [0.76% to 2.37%]). Among patients with stroke history, intensive strategy failed to achieve non-inferiority in NCB (-0.18% [-1.01% to 0.83%]).

Conclusions:
Bivariate analysis is a novel method that allows simultaneous display and assessment of efficacy and safety outcomes. Among patients with cardiovascular risk factors, intensive BP control failed to demonstrate a favorable risk-benefit profile against standard BP control with respect to the tradeoff between stroke and SAE.

Figure 1.
Net clinical benefit of intensive vs standard BP control

Figure 2.
Forest plot of bivariate risk difference in stroke and SAE