bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 130: Hypercholesterolemia Suppresses Carotid Artery Endothelial NOS3 Expression via Epigenetic Mechanisms
Authors: Sotolongo Alex , Jiang Yi-Zhou , Karanian John , Pritchard William , Davies Peter
Published: July 2021

Objective:
One of the first clinically detectable changes in the vasculature during atherogenesis is the accumulation of cholesterol within the vessel wall. Hypercholesterolemia is characterized by dysfunctional endothelial-dependent vessel relaxation and impaired NOS3 function. Since DNA methylation at gene promoter regions strongly suppresses gene expression, we postulated that high-fat/high-cholesterol diet suppresses endothelial NOS3 through promoter DNA methylation.

Methods:
Domestic male pigs were fed control diet (CD) or isocaloric high fat and high cholesterol diet (HC; 12% fat and 1.5% cholesterol) for 2, 4, 8 or 12 weeks prior to tissue collection. Furthermore, to determine the effects of risk factor withdrawal, an additional group of swine received HC for 12 weeks and then CD for 8 weeks; a control group received HC continuously for 20 weeks. Endothelial cells were harvested from common carotid aorta. In parallel in vitro studies, cultured human aortic endothelial cells (HAEC) were treated with human LDL, GW3956 (LXR agonist) and RG108 (DNA methyltransferase [DNMT] inhibitor). In cells from both sources, DNA methylation at the NOS3 promoter was measured using methylation specific pyro sequencing, and endothelial gene expression was measured using RT PCR.

Results:
HC diet increased plasma cholesterol level from 75 mg/dl on CD to a plateau of about 540 mg/dl within 2 weeks. Endothelial NOS3 expression was significantly reduced (71±9 % of CD) after 4 weeks of HC, a level sustained at subsequent time points. Withdrawal of HC for 8 weeks did not recover NOS3 expression. After 12-week HC, the NOS3 promoter was hypermethylated. Withdrawal of HC did not reverse NOS3 promoter methylation. In vitro treatment of HAEC with human LDL (200 mg/dl total cholesterol) or GW3956 (5μM) suppressed NOS3 mRNA to 50% and 30% respectively, suggesting that LXR/RXR is involved in suppression of NOS3. Nitric oxide production was consistently suppressed by GW3959. Both could be reversed through inhibition of DNMTs by RG108.

Conclusions:
DNA methylation and LXR/RXR pathway can mediate the HC-suppression of endothelial NOS3. The study identifies novel pharmaceutical targets in treating endothelial dysfunction. Crosstalk between these pathways is under investigation.
Abstract 134: Myeloid Disabled Homolog 2 (Dab2) Controls Liver Inflammation and Atherosclerosis
Authors: Adamson Samantha E , Marqueen Kathryn , Angdisen Jerry , Schulman Ira , Leitinger Norbert
Published: July 2021

Inflammatory (M1) macrophages contribute to the pathogenesis of atherosclerosis, but the intracellular mediators of macrophage polarization are not fully understood. We identified the adaptor protein
disabled homolog 2
(Dab2) as a novel regulator of phenotypic switching in macrophages.
Dab2
expression is upregulated in M2 and suppressed in M1 macrophages. Dab2 in macrophages dampens NF-κB signaling by binding to TRAF6. Genetic deletion of
Dab2
predisposes macrophages to adopt a proinflammatory M1 phenotype, and mice with myeloid cell-specific deletion of
Dab2
(

Dab2
fl/fl
LysM
Cre

mice) show increased inflammation upon challenge with lipopolysaccharide. To test the role of myeloid Dab2 in the pathogenesis of atherosclerosis we performed a bone marrow transplant of WT and Dab2 null bone marrow into LDLR
-/-
mice followed by western diet feeding for 9 or 20 weeks. Surprisingly, after 20 weeks of western diet feeding, Dab2-null chimeric mice had
decreased
lesion area by
en face
analysis of aortic arches and a trend toward decreased Oil red O positive area in aortic root lesions; however, aortic root lesions in Dab2 null-chimeric mice had increased macrophage content and caspase staining, indicative of lesion instability. Serum total cholesterol and triglyceride levels were significantly decreased in Dab2 null-chimeric mice after 20 weeks of western diet, and analysis of mRNA in liver tissue revealed increased levels of inflammatory cytokines. Histological analysis of livers of Dab2 null-chimeric mice showed large patches of cells that were positive for B220, a marker present on B cells and some dendritic-like cells. We conclude that myeloid Dab2 deficiency exacerbates hepatic immune responses which in turn control atherosclerotic lesion development.
Abstract 140: Nuclear Targeting Apelin Induces Phenotypic Transition of Vascular Smooth Muscle Cells
Authors: Bochaton-Piallat Marie-Luce , Chaabane Chiraz , Brun Cécile , König Stéphane , Audigier Yves , Baertschi Alex J
Published: July 2021

Apelin, and its receptor APJ, are a peptidic system playing a crucial role in vascular diseases. However, the role of apelin in atherogenesis and smooth muscle cell (SMC) proliferation remains unclear. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as being a marker of the R-SMCs in vitro and of intimal SMCs, both in pig and human.

The aim of our study was to investigate the effects of apelin on SMC phenotypic transition and S100A4 expression.

Apelin was highly expressed in R-SMCs including in their nucleus. P-SORT software analysis of preproapelin sequence suggested that N-terminal truncated apelin may target the nucleus, and we confirmed this in SMCs by overexpression of mutated preproapelin-His-tag. Transfection of mutated preproapelin-His-tag encoding plasmid in differentiated S-SMCs induced a transition towards a R-phenotype associated with increased proliferative activity, downregulation of SMC differentiation markers, and increased nuclear expression and release of S100A4. In contrast, transfection of S-SMCs with wild type preproapelin-His-tag encoding plasmid did not induce nuclear targeting of apelin or S100A4, and did not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB, known to induce a transition to the R-phenotype, yielded nuclear targeting of both apelin and S100A4. In vivo, Apelin was expressed in SMC nuclei of stent-induced intimal thickening while its expression in the media was mainly cytoplasmic.

Our results suggest that nuclear targeting of apelin in SMCs acts on S100A4 expression and release, cell proliferation and differentiation. The pathophysiological consequences of this retargeting could be instrumental in the understanding of artherosclerosis.
Abstract 156: Development of a New Bioactivatable Fluorescent Probe for the Study of the Proteolytic Activities Degrading Apolipoprotein A-I in vitro and in vivo
Authors: Maafi Foued , Li Baoqiang , Gebhard Catherine , Brodeur Mathieu , Villeneuve Louis , Lesage Frédéric , Rhainds David , Rhéaume Eric , Tardif Jean Claude
Published: July 2021

Introduction and Objective:
The possible benefits of high-density lipoproteins (HDL) against atherosclerosis have been largely attributed to its major protein component, apolipoprotein A-I (apoA-I). However, apoA-I can be degraded by diverse processes, including proteases localized in atherosclerotic plaques, which could reduce the effectiveness of HDL-based therapies. Here we describe the development of a new bioactivatable near-infrared apoA-I-Cy5.5 fluorescent probe and its initial use in the assessment of proteolytic activities that degrade apoA-I in vitro, in vivo and ex vivo.

Methods and Results:
Fluorescence emission of our probe is quenched by saturation of Cy5.5 fluorophore molecules on the full-length apoA-I protein. In vitro proteolysis of the apoA-I probe showed up to 11-fold increase of near-infrared fluorescence (n=5, P ≤ 0.05). Using this apoA-I-Cy5.5 probe, we were able to quantify proteolytic activities from a wide range of proteases targeting serine (chymase), cysteine (cathepsin S) and metalloproteases (MMP-12). Also, we detected activation of the apoA-I-Cy5.5 probe on aortic cryosections from Ldlr-/--Tg for human apoB atherosclerotic (ATX) mice using an in situ zymography assay and observed that broad-spectrum protease inhibitors protect the probe from protease activities, as shown by decreased fluorescence compared to conditions without protease inhibitors (-54%, n= 6 per group, P ≤ 0.001). In vivo, using a combined Fluorescence Molecular Tomography-Magnetic Resonance imaging system, the injected probe exhibited a trend for increased fluorescence in the aorta when infused in ATX mice compared to C57BL/6J wild-type mice. Ex vivo imaging of these aortas showed a 10-fold increase of fluorescence in ATX (n=5) mice compared to CTL (n=3) mice (P ≤ 0.05).

Conclusion:
Given the potential importance of HDL functionality in the assessment of cardiovascular risk, this novel protease-activatable apoA-I probe may help to improve HDL-based therapies through better characterization of the alterations of functionality of apoA-I or lipid-poor HDL particles in different pathophysiological settings.
Abstract 158: PPAP2B Expression Regulates the Development of Atherosclerosis
Authors: Mueller Paul , Yang Liping , Morris Andrew , Smyth Susan
Published: July 2021

Coronary artery disease (CAD) is the leading cause of death in both men and women worldwide. The bioactive lipid lysophosphatidic acid (LPA) accumulates in human atheroma compared to levels in healthy control tissue. LPA can be degraded by the membrane protein lipid phosphate phosphatase 3 (LPP3), encoded by the gene
PPAP2B.
Noncoding polymorphisms in
PPAP2B
associate with CAD risk, and gene expression analysis indicates that individuals possessing the risk allele exhibit lower levels of
PPAP2B
mRNA in leukocytes and vascular cells. We hypothesize that decreased LPP3, as a result of low
PPAP2B
expression, accelerates experimental atherosclerosis. Murine models of tissue specific LPP3 knockdown were generated using the Cre-Lox system driven by either the MX-1 promoter (MX-1Δ) or the smooth muscle cell specific SM22 promoter (SM22Δ) in mice on the atherogenic LDLr -/- background. Following 12 weeks on Western diet, knockdown of LPP3 in both the MX-1Δ and the SM22Δ mice resulted in significantly more atherosclerosis by
en face
analysis compared to LPP3-fl/fl littermate controls. LPA content determined using LC/MS/MS tended to be higher in the proximal aortas of MX-1Δ and SM22Δ knockdown mice compared to controls. Significantly higher macrophage gene expression was observed in the MX-1Δ mice while IL-6 levels were significantly higher in the SM22Δ mice compared to controls. Finally, targeting LPP3 expression in the MX-1Δ, but not the SM22Δ, mice resulted in significantly higher circulating levels of LPA compared to controls. These results are consistent with accelerated atherosclerosis in LPP3 knockdown mice and suggest a protective role for LPP3 in CAD. Increased vascular LPA content, as a consequence of decreased LPP3 expression, may promote the infiltration of monocyte/macrophages into lesions as well as exacerbate inflammation to accelerate the development of CAD. Our findings provide mechanistic insight into the genome wide association studies that linked genetic variation in PPAP2B with risk of CAD and focus attention on the LPA/ LPP3 signaling nexus as a novel therapeutic strategy to prevent atherosclerosis.
Abstract 162: Detection of N-alpha-acetyltransferase Activity Towards Endogenous Inhibitor of Nitric Oxide Synthase Asymmetric Dimethylarginine in the Liver and Kidneys
Authors: Rodionov Roman N , Burdin Dmitrii V , Martens-Lobenhoffer Jens , Brilloff Silke , Jarzebska Natalia , Demyanov Anton V , Weiss Norbert , Bode-Böger Stefanie
Published: July 2021

Background:
Endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been proposed as a risk factor and mediator of cardiovascular diseases. ADMA can undergo hydrolysis by dimethylarginine dimethylaminohydrolase or be processed through an alternative pathway by alanine:glyoxylate aminotransferase 2. We and other have also shown that ADMA can be N-acetylated to form asymmetrical Nα-acetyldimethylarginine (Ac-ADMA). The goal of the study was to characterize this novel pathway in vivo and identify the organs, which are responsible for the ADMA-acetylating activity.

Results:
We infused ADMA in mice for three days. Control mice received saline infusion. Half of the mice underwent bilateral nephrectomy 24 hours before completion of the infusion, the rest had a sham surgery. ADMA infusion resulted in a 3.5 fold rise in plasma Ac-ADMA levels in the sham-operated mice (1.35 ± 0.45 nmol/L vs. 4.77 ± 0.88 nmol/L). In nephrectomized mice plasma Ac-ADMA levels were markedly increased even after saline infusion (45.08 ± 9.25 nmol/L) and further raised 5-fold after ADMA infusion (229.65 ± 91.30 nmol/L). We assessed tissue distribution of Ac-ADMA in mice and found the highest levels in pancreas, small intestine, liver and kidney. We incubated the lysates of those organs with ADMA for 24 hours and estimated production of Ac-ADMA. Only liver and kidney lysates showed significant increase in Ac-ADMA levels upon addition of ADMA (from 0.03 ± 0.01 nmol/μg protein to 0.18 ± 0.06 nmol/μg protein and from 0.06 ± 0.01 nmol/μg protein to 0.11 ± 0.02 nmol/μg protein).

Conclusions:
We showed that overload of ADMA leads to increase in plasma Ac-ADMA levels. This observation is consistent with the hypothesis that Ac-ADMA is formed from ADMA in vivo. The striking rise in plasma Ac-ADMA concentrations after bilateral nephrectomy suggests that Ac-ADMA is predominantly eliminated via the kidneys. We demonstrated that liver and kidney are the organs, in which acetylation of ADMA takes place. The high levels of Ac-ADMA in pancreas and intestine may be due to accumulation of Ac-ADMA in those organs. In the next step we want to identify the enzyme responsible for acetylation of ADMA and explore the clinical implications of this novel pathway of ADMA metabolism.
Abstract 166: Activation of Integrin α5 Mediated by Flow Requires Its Translocation to Membrane Lipid Raft in Vascular Endothelial Cells
Authors: Sun Xiaoli , Zhu Yi
Published: July 2021

Rationale:
Local flow patterns determine the uneven distribution of atherosclerotic lesions. Sensing mechanical forces, membrane lipid raft and integrin are crucial for shear stress-regulated endothelial function.

Objective:
In this study, we investigate the role of lipid raft and integrin α5 in regulating the inflammatory response in endothelial cells (ECs) under the atheroprone flow.

Methods and Results:
Lipid raft proteins were isolated from ECs exposed to pulsatile shear stress (PS) or oscillatory shear stress (OS) and then analyzed by quantitative proteomics. Among 396 proteins redistributed in lipid raft, integrin α5 was most significantly elevated in lipid raft under OS. Moreover, OS increased the level of activated integrin α5, which was exclusively located in lipid raft. Disruption of F-actin-based cytoskeleton and knockdown of caveolin-1 prevented the OS-induced integrin α5 translocation and activation, suggesting significant roles of the cytoskeleton and caveolin-1 in integrin α5 translocation. Furthermore, OS activation of integrin α5 induced the NLRP3 inflammasome, leading to increased inflammatory responses. In vivo, activation of integrin α5 and increased expression of adhesion molecules were seen in the atheroprone areas of LDLR-/- mice. Knockdown of integrin α5 greatly attenuated EC dysfunction in partially ligated carotid arteries in LDLR-/-mice. Consistently, genetic ablation of integrin α5 in α5-/- murine embryonic fibroblasts and α5+/- mice caused a reduction of inflammation under the atheroprone flow.

Conclusions:
Atheroprone flow activates integrin α5 through lipid raft anchoring with ensuing induction of NLRP3 inflammasome in ECs. These findings reveal a novel mechanotransduction mechanism by which atheroprone flow causes endothelial dysfunction.
Abstract 169: Impaired Shear Stress-induced Endothelial Nitric Oxide Production in High Glucose Condition is Restored by Inhibiton via NADPH Consumption by Polyol Pathway Activation
Authors: Umemoto Tomio , Kuroki Masatoshi , Ueba Hiroto , Kawakami Masanobu , Fujita Hideo , Momomura Shin-ichi
Published: July 2021

Endothelial dysfunction leading to cardiovascular disease risk involves a decrease in nitric oxide (NO) production. In physiological conditions shear stress is a potent stimulation of endothelium-derived NO production and flow mediated NO production is regulated by the activation of endothelial NO synthase (eNOS). In endothelial cells, eNOS, aldose reductase (AR), a rate limiting enzyme of polyol pathway, and glutathione reductase (GR) share a NADPH as an obligate cofactor. In diabetec condition intracellular polyol pathway is activated and this may decrease shear stress-induced endothelial NO production and increase intracellular oxidative stress via inhibition of eNOS and GR by NADPH consumption. Therefore we investigated whethter AR inhibitor epalrestat improved endothelial NO production under high glucose condition to elucidate the mechanism of endothelial dysfunction in diabetes.

We incubated human umbilical vein endothelial cells (HUVECs) in normal (5mM) and high (30mM) glucose condition for 72 hours, with or without epralrestat, or 100U/ml superoxide dismutase (SOD), respectively. After exchange of medium for Krebs’ buffer, HUVECs were exposed to 12dyne/cm2 steady laminar fluid shear stress for 5 minutes. NO release from HUVECs was measured as NO2 using a NOx analyzing HPLC system by Griess reaction. Next we harvested the cells in lysis buffer and analyzed phosphorylation of Akt (shear induced intracellular signal transduction) and eNOS by western blotting, and measured intracellular 8-OHdG and ratio of NADPH/NADP.

In high glucose condition NO2 was decreased and 8-OHdG increased compared to low glucose. NO2 was restored and 8-OHdG was reduced by epalrestat significantly (p<0.01, p<0.05, respectively, vs. high glucose condition). In SOD-treated HUVECs, NO2 was not restored (n.s. vs. high glucose condition) despite of complete reduction of 8-OHdG (p<0.01). Both Akt and eNOS phosphorylation by shear stress was affected neither by high glucose, epalrestat nor SOD. Intracellular NADPH/NADP ratio was decreased in high glucose condition, but this reduction was restored by epalrestat.

These results showed that polyol pathway activation plays a key role in endothelial NO production under high glucose condition via a cofactor NADPH.
Abstract 187: Paraoxonase-2 Regulates Blood Coagulation through Endothelial Redox-Signaling and Inflammation
Authors: Ebert Julia , Wilgenbus Petra , Horke Sven
Published: July 2021

Background:
Enhanced coagulation increases the risk for cardiovascular diseases (CVDs) such as stroke, myocardial infarction or atherosclerosis. Increased oxidative stress and inflammation, predominantly in the vascular wall and platelets, are important underlying mechanisms of a pro-coagulant state. Paraoxonase-2 (PON2), an anti-oxidative protein with anti-inflammatory properties, has an emergent role in CVDs, as it counter-acts atherosclerosis. Previous studies revealed enhanced atherosclerosis in PON2-/- mice and diminished PON2 expression in human atherosclerotic endothelium. We hypothesized that PON2 affects coagulation by controlling redox-mediated inflammatory processes provoking atherosclerosis.

Methods and Results:
In several coagulation tests, PON2-/- mice showed significantly shortened clotting times (8.58±0.09 sec) compared to WT (9.32±0.16 sec prothrombin time; p<0.001; n=42). Confocal microscopy, flow cytometry and gene expression studies revealed enhanced vascular oxidative stress (p<0.001; n=6) and a pro-inflammatory endothelium in PON2-/- mice. In line with this, the endogenous interleukin-6 plasma level was increased compared to WT (2-fold) as disclosed by cytokine profiling. Additionally, plasmatic coagulation factors VIII, IX and XI displayed significantly elevated activities in PON2-/- mice (p<0.05; n=10). Further, PON2-/- platelets showed a pro-coagulant activity, due to increased endogenous thrombin potentials triggered by an enhanced phosphatidylserine plasma membrane exposure (p<0.01; n=7). To locate PON2’s dominant effect to either endothelial cells, plasma or platelets, we established bone marrow chimeras and transgenic mice with exclusively endothelial and hematopoietic PON2 expression (Tie2cre-PON2-/-). Coagulation time analyses revealed that the pro-coagulant effect was attenuated in WT chimera with PON2-/- bone marrow (9.22±0.15 sec; n=12) and in Tie2cre-PON2-/- (10.02±0.3 sec; n=23), indicating that much of the effects originates from PON2 functions in the endothelium.

Conclusion:
We found that PON2 regulates specific pathways of blood coagulation based on a redox-mediated endothelial-dependent modulation of important players in inflammation and hemostasis.
Abstract 188: Selective Inhibition of Vascular Smooth Muscle Cell Migration by Targeting Plasminogen Activator Inhibitor-1
Authors: Goyal Neha , Weng Zhen , Fish Philip , Strawn Tammy , Myears Samantha , Ji Yan , Wu Jianbo , Fay William P
Published: July 2021

Introduction:
Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of mammalian plasminogen activators and an important regulator of cell migration. We have shown that tiplaxtinin, a small molecule, specific inhibitor of PAI-1, inhibits intimal hyperplasia in a murine vein graft model. However, little is known about the effects of pharmacological inhibition of PAI-1 on vascular cell migration under physiologically relevant conditions.

Methods:
We studied the effects of tiplaxtinin on migration of smooth muscle cells (SMCs) and endothelial cells (ECs).

Results:
Tiplaxtinin significantly inhibited migration of murine SMCs through 3-dimensional (3-D) collagen matrix in a concentration-dependent manner. Tiplaxtinin did not inhibit SMC proliferation, and it did not inhibit migration of PAI-1-deficient SMCs, suggesting that tiplaxtinin’s effect on SMCs was non-toxic and PAI-1-dependent. The anti-migratory effect of tiplaxtinin on SMCs was preserved in collagen 3-D matrix containing vitronectin and other extracellular matrix molecules, further supporting the physiological significance of the effect. In contrast to SMCs, tiplaxtinin did not inhibit migration of human aortic ECs in vitro or murine ECs in vivo, the latter assessed in a murine carotid injury model. To study the basis for the differential effect of tiplaxtinin on SMCs vs. ECs, we compared expression of LDL receptor-related protein 1 (LRP1), a motogenic receptor for PAI-1, between cell types by RT-PCR and found that LRP1 gene expression was significantly lower in ECs than in SMCs. Furthermore, recombinant PAI-1 stimulated the migration of wild-type mouse embryonic fibroblasts (MEFs), but not LRP1-deficient MEFs.

Conclusions:
Tiplaxtinin, a pharmacological inhibitor of PAI-1, inhibits SMC migration under physiological conditions, while having no inhibitory effect on EC migration. The differential effect of PAI-1 inhibition on SMCs vs. ECs appears to be mediated by LRP1 and may be of clinical significance, as it is advantageous to prevent intimal hyperplasia by inhibiting SMC migration without inhibiting EC migration, which is key to preserving an intact, anti-thrombotic vascular endothelium.
Abstract 196: Modulation of Inflammatory Macrophage Phenotype Through Alternative Splicing
Authors: Lin Jennie , Zhang Hanrui , Xue Chenyi , Qu Liming , Li Mingyao , Reilly Muredach
Published: July 2021

How alternative splicing (AS) regulates macrophage activation to the pro-inflammatory M1 phenotype, which is causally implicated in cardiometabolic diseases, remains unknown. Because AS is a major mechanism by which cell-specific functions are derived from the genome, we hypothesized that it modulates inflammatory macrophage activation. In an unbiased approach to detect AS, we performed deep RNA-seq (~130 million reads/sample) on non-activated M0, M1-activated (18 hours LPS / IFN-γ), and anti-inflammatory M2-activated (18 hours IL-4) macrophages of isogenic primary human monocyte derived macrophages from six individuals. At false detection rate (FDR)-adjusted P-value < 0.01, 1790 and 2072 genes were differentially expressed between M0 and M1, and M0 and M2 macrophages, respectively. Using DDS-Seq, a computational tool detecting AS using all rather than junction reads alone, 505 AS events for M1 and 511 events for M2 activation were detected at FDR P < 0.01. These events included cassette exons, alternative 3’ / 5’ splice sites, and tandem 3’ untranslated regions. Gene ontology analysis (Ingenuity, Qiagen) revealed differential enrichment for causal networks between M1 and M2 activation states (Table 1). AS genes in M1 activation, including PCR-validated ARRB1 and MAP4K4, were enriched for macrophage-specific and immune functions (Table 2). Also, 14 splicing factors, including CELF1 (3.68-fold increase, FDR P < 0.001), were upregulated between M0 and M1. 65% siRNA CELF1 knockdown decreased M1 marker CCL5 mRNA expression 48.5% compared to non-targeting control (P = 0.002). These findings suggest that during macrophage activation AS occurs in unique phenotype-specific patterns that modulate inflammatory phenotype.
Abstract 198: Familial Hypercholesterolemia and Intra-Cranial Aneurysm: A Novel Phenotypic and Genetic Association
Authors: Pandey Arvind K , Becker Jason R
Published: July 2021

Background:
Autosomal dominant familial hypercholesterolemia (FH) is characterized by elevated levels of low density lipoprotein (LDL) cholesterol. Mutations in LDLR, APOB, and PCSK9 genes are established causes of this condition. However, in up to 20-40% of cases, the genetic cause has not been defined. FH is associated with coronary ectasias, but extra-cardiac vascular malformations have not been described.

Methods:
A subject with hypercholesterolemia was evaluated, and a three generation pedigree was constructed. Whole exome sequencing (WES) was performed (Illumina HiSeq 2500, 100x avg coverage) and reads were aligned to a human reference genome (hg19) using BWA. Variants were called with GATK Unified Genotyper and annotated using SeattleSeq.

Results:
Pedigree analysis suggested an autosomal dominant mode of inheritance of the hypercholesterolemia trait (Fig 1). Multiple family members also had intracranial aneurysms. Targeted analysis of the classical genes associated with FH (LDLR, APOB, and PCSK9) did not identify any missense, nonsense, or splice site variants with a minor allele frequency (MAF) < 0.01. Expanded analysis of the entire exome yielded rare missense variants (MAF < 0.001) in 845 genes and nonsense variants in 22 genes.

Conclusions:
Utilizing WES we rapidly screened an individual for pathogenic DNA variants in genes previously associated with FH. The genetic factor underlying our patient’s FH appears to be distinct from the known causes of FH. Genetic analysis of additional family members will be necessary to define the novel genetic cause of FH in this pedigree and explore the relationship of intracranial aneurysms with FH.
Abstract 210: Biosynthesis of D-series Resolvin by Isolated Vascular Cells and Tissues
Authors: Komshian Sevan R , Chatterjee Anuran , Wu Bian , Mottola Giorgio , Chen Mian , Conte Michael S
Published: July 2021

Introduction:
Resolvin-D1 (RvD1) and other specialized pro-resolving lipid mediators (SPM) are synthesized in-vivo from docosahexaenoic acid (DHA) through transcellular pathways involving leukocytes. We investigated if vascular tissues, in the absence of inflammatory cells, can contribute to the local production of SPM.

Methods:
Primary cultures of human saphenous vein endothelial (EC) and smooth muscle (SMC) cells were supplemented with DHA in cell culture media (10% serum) for 4h-24h. Freshly harvested rabbit aorta was incubated intact or following gentle EC denudation in medium with or without DHA for 48h. RvD1 levels were quantified by ELISA, and lipoxygenase (LO) expression by western blotting.

Results:
In the absence of DHA supplementation, EC and SMC produced undetectable levels of RvD1. DHA treatment produced a dose and time-dependent increase in RvD1 production by EC and SMC (10.1 ±1.0 pg, 7.4 ±0.2 pg respectively; 1000nM DHA; 24h; Fig A, B). 5-LO expression was demonstrated in both cell types, however DHA induced increased 5-LO expression in EC (Fig C) but not in SMC. DHA-treated intact rabbit aorta segments produced 0.24±0.05 pg RvD1/mg tissue versus 0.13±0.01 pg RvD1/mg tissue in media alone. Moreover, EC-denuded aortas produced significantly less RvD1 (Fig D).

Conclusions:
Human vascular cells and rabbit vascular tissue can biosynthesize RvD1 de novo from its precursor DHA, signifying a potentially important local source of SPM in the vasculature.
Abstract 213: Myofilament Ca2+ Sensitization and Site-specific Phosphorylation of Contractile Proteins Following Myocardial Infarction: A Novel Role for Mast Cells
Authors: Ngkelo Anta , Kirk Jonathan , Richart Adele , Bonnin Philippe , Vilar Jose , Heymes Christophe , Rodewald Hans-Reimer , Kass David , Silvestre Jean-Sebastien
Published: July 2021

Inflammatory cells orchestrate post-ischemic cardiac remodeling after myocardial infarction (MI). Studies in Kit mutant mice suggest key roles for mast cells in post-ischemic tissue remodeling. However, Kit mutations affect multiple cell types of both immune and non-immune origin. The aim of this study was to address the impact of mast cells on cardiac function following MI, using selectively mast cell-deficient mice (Cpa3Cre/+ mice).

In response to myocardial infarction, mast cells progenitors’ numbers (Lin-CD45+CD34+FcγRII/IIIhighβ7+) increased in the white adipose tissue (day 3) and in the heart (day 5) and mature mast cells (CD117+FcεRI+Sca1+) peaked at day 7 in the cardiac tissue. To assess the functional effect of this mast cell infiltration in the heart, cardiac function 14 days following MI was assessed in wild-type (WT) and Cpa3Cre/+ mice. There was a significant decrease of the left ventricular shortening fraction in Cpa3Cre/+ compared to WT mice (17.27±1.9 % vs 28.15±1.7, p<0.01) that was not due to adverse cardiac remodeling; with no significant difference on levels of fibrosis, cardiomyocyte apoptosis, infarct size or neovascularization. By contrast, we observed a significant reduction in cardiomyocyte contractility (both cell shortening and ±dL/dt) in Cpa3Cre/+ mice compared to WT mice 14 days post MI. In addition, Ca2+ sensitivity (EC50) declined in Cpa3Cre/+ -derived skinned myocytes (p<0.01 vs WT) and correlated with increased PKA-dependent phosphorylation of both cardiac troponin (cTn)I (Ser22/23) and myosin-binding protein-C (MyBPC) (Ser273) (p<0.05).

This study identifies a novel function of mast cells on modulating cardiomyocyte contractility via alteration of PKA-regulated Ca2+ handling and myofilament protein phosphorylation. Identification of the mast cell-dependent signaling pathway that targets cardiac performance will provide a new platform of regulators with potential cardioprotective properties.
Abstract 217: Quaking Post-Transcriptionally Guides Monocyte Adhesion and Differentiation into the Pro-Inflammatory Macrophage
Authors: de Bruin Ruben G , Shiue Lily , Djarmshi Anjana , de Boer Hetty C , Leung Wai Yi , van Gils Janine M , Prins Jurrien , Duijs Jacques M , van der Zande Patrick H , Rabelink Ton J , Jukema Wouter J , van Esch Hilde , Kazan Hilal , Biessen Erik A , Ares Manuel , van Zonneveld Anton Jan , van der Veer Eric P
Published: July 2021

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce new transcription necessary for macrophage identity, but post-transcriptional control of human macrophage differentiation is less well understood. Here, we detail our discovery that levels of the RNA-binding protein Quaking (QKI) are low in monocytes of early atherosclerotic lesions, but abundant in macrophages of advanced plaques. Specific depletion of QKI protein impaired monocyte adhesion, migration and differentiation into macrophages, and lesion formation. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal developmental changes in RNA levels and alternative splicing of RNA transcripts enriched in QKI-bound sequence elements. The importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in post-transcriptionally guiding macrophage identity and function. These studies implicate QKI as a novel target for therapeutic intervention in inflammatory diseases.
Abstract 233: Intra-arterial Uridine 5’-triphosphate (UTP) Increases Macrophage/Monocyte Recruitment in the Developing Collateral After Femoral Artery Ligation
Authors: McEnaney Ryan M , Pennetti Sean M , Tzeng Edith
Published: July 2021

Objectives:
Arteriogenesis is a process by which small resistance vessels mature into large caliber collateral arteries in response to arterial occlusive disease. Inflammatory cells of monocyte/macrophage lineage are necessary to promote vascular remodeling. We have previously reported that mice lacking the P2Y2 receptor, a purinergic receptor whose agonists include ATP and UTP, exhibited impaired collateral development and persistently reduced perfusion when subjected to femoral artery ligation (FAL). Thus, we hypothesize that purinergic signaling via the P2Y2 receptor mediates inflammatory cell recruitment during arteriogenesis.

Methods:
C57Bl6 mice underwent FAL and intra-arterial infusion of 200uL of PBS or 100uM UTP in PBS (N=3 each group). Hindlimb perfusion was assessed with laser Doppler imaging at day 7. In a separate experiment, P2Y2 -/- mice underwent femoral artery ligation alongside C57Bl/6 (wild type receptor) mice without UTP infusion. Thigh adductor muscles were fixed, sectioned, and immunostaining was performed for inflammatory cells (anti-CD45) and macrophages/monocytes (anti-F4/80). Two collateral vessels were analyzed per animal, and positive cells were quantified per high-power field.

Results:
UTP treated mice showed a trend toward increased peri-collateral CD45 positive cells at 12hr, and significantly increased F4/80 positive cells at day 3 (18.3+/-3.3 vs 11.5+/-1.3, p=0.044). This corresponded to improved perfusion at 7 days in UTP treated mice (58.5%+/-3.9 vs 36.0%+/-2.8, p=0.026). P2Y2 -/- mice recruited fewer F4/80 positive cells to the collateral vessels at day 3 when compared to the wild type (1.8 ± 1.3 vs 11.8 ± 2.9, p=.012).

Conclusion:
UTP treatment increases macrophage recruitment to the immature collateral vessel wall and improves perfusion recovery following FAL. In the absence of P2Y2 receptor, macrophage recruitment was significantly inhibited. These findings suggest that purinergic signaling via the P2Y2 receptor is an important mediator of vascular inflammation which is required for collateral growth. Ongoing studies will focus on the cellular mechanisms involved and whether enhanced collateral maturation and limb perfusion can be driven by the pharmacologic administration of nucleotides.
Abstract 236: Vascular Smooth Muscle Cells Stimulate Re-endothelialization Through Protein Kinase C-delta-dependent Release Of CXCL7 and Recruitment of Circulating Angiogenic Cells
Authors: Ren Jun , Wang Qiwei , Parlato Matthew , Morgan Stephanie , Giles Jasmine , Greenberg Jason , Murphy William , Liu Bo
Published: July 2021

Objective:
In response of injury, vascular smooth muscle cells (VSMCs) undergo proliferation, migration, as well as apoptosis. We have previously shown that gene transfer of a pro-apoptotic mediator protein kinase C-delta (PKCδ) reduces intimal hyperplasia. Subsequently, we showed that VSMCs are a rich source of chemokines. In this study, we explored whether VSMCs may play a role in regulation of re-endothelialization through PKCδ-dependent release of chemokines.

Methods and Results:
We constructed an adenovirus that expresses PKCδ under VSMC-specific SM22 promoter (AdPKCδ) and use it to drive PKCδ expression in VSMCs of injured rat carotid arteries. Compared to the empty vector (AdNull), AdPKCδ accelerated re-endothelialization, reflected by a larger area that was excluded of Evan’s blue (25.38±7.52% v.s 59.60±5.01%). Media conditioned by PKCδhigh VSMCs had no measurable effects on endothelial cell functions, including BrdU incorporation, transwell migration, scratch wound healing, and tube formation. Interestingly, PKCδhigh VSMCs conditioned media attracted circulating angiogenic cells (CACs), a population of cells that promote neovascularization via production of angiogenic factors. Using a PCR array analysis, we identified a group of PKCδ upregulated chemokines in VSMCs, including MCP-1, CXCL1, and CXCL7. Neutralizing CXCL7, but not MCP-1 or CXCL1, significantly blocked PKCδhigh VSMC conditioned media induced migration of CACs. In vivo, we observed more CD133+ (a marker of CACs) cells lined up to the lumen of PKCδhigh expressing vessels, while the number of CD133+ cells was comparable in the blood of AdPKCδ treated rats and AdNull treated rats. Immunofluorescence staining revealed that delivery of PKCδ gene increased CXCL7 expression in media VSMCs.

Conclusions:
Our data suggest that VSMCs stimulate re-endothelialization through PKCδ-dependent release of CXCL7 and recruitment of CACs.
Abstract 239: Olfactomedin 2 Regulates Vascular Smooth Muscle Cell Phenotypic Modulation by Mediating the Interaction Between Runx2 and SRF
Authors: Shi Ning , Cui Xiao-Bing , Chen Shi-You
Published: July 2021

Olfactomedin 2 (Olfm2) is a novel regulator for vascular smooth muscle cell (SMC) differentiation, but it is unclear whether Olfm2 is also involved in SMC phenotypic modulation, an important process associated with vascular injury. In this study, we found that Olfm2 was induced during PDGF-BB-induced SMC phenotypic modulation. Olfm2 knockdown attenuated PDGF-BB-induced suppression of SM marker genes including SM myosin heavy chain and SM22α, and also inhibited PDGF-BB-stimulated SMC proliferation and migration. On the other hand, Olfm2 overexpression down-regulated SM markers gene expression, and promoted SMC proliferation marker PCNA expression. Moreover, PDGF-BB slightly induced expression of Runx2, which interfered with the formation of SRF/myocardin ternary complex, but dramatically enhanced SRF-Runx2 interaction, suggesting that certain factors mediate SRF-Runx2 interaction. Indeed, Olfm2 physically interacted with both SRF and Runx2. Blockade of Olfm2 inhibited SRF association with Runx2, leading to increased association between SRF and myocardin, which in turn activated the transcription of SM markers, whereas overexpression of Olfm2 promoted SRF binding to Runx2. These results demonstrated that Olfm2 mediates the interaction between SRF and Runx2, contributing to SMC phenotypic modulation.
Abstract 241: Endothelial Function is Preserved with Endoscopic Vein Harvest for Lower Extremity Bypass
Authors: Wheeler Adam R , Kendrick Daniel E , Allemang Matthew T , Gosling Andre F , Nagavalli Anil , Kim Ann H , Hausladen Alfred , Kashyap Vikram S
Published: July 2021

Objective:
Endoscopic vein harvest for lower extremity arterial bypass technique has been questioned due to concern for endothelial damage during procurement. We sought to compare NO mediated endothelial dependent relaxation (EDR) in vein segments harvested with open surgical (OH) versus endoscopic (EH) techniques.

Methods:
Saphenous vein segments were harvested for lower extremity bypass. 3-4mm vein rings were mounted on force transducers. Segments were mounted in 37° oxygenated Krebs solution and maximally contracted using KCl. NE was used to achieve submaximal contraction. EDR was determined using increasing concentrations of bradykinin (BDK). Endothelial independent relaxation was confirmed using sodium nitroprusside. Two-way ANOVA was used to analyze differences between harvest techniques across BDK concentration. Student t-test was used to examine nitrite levels in each cohort.

Results:
Vein segments harvested from patients (n=13) led to 28 rings (11 rings; 5 patients EH v. 17;8 OH). Both cohorts achieved moderate relaxation to maximal BDK concentration, [10
-6
M]; (49.5% EH vs. 40.55%, OH, P = .270). Analysis by two way ANOVA for mean % relaxation for BDK concentration [10
-11
- 10
-6
M] showed improved EDR in EH samples compared to OH (P =.029). Mean nitrite tissue bath concentration measurements post-BDK were 279 nM (EH) v. 194 nM (OH) (P = .264). Histology and IHC confirmed intact endothelium by morphometric analysis and CD31 staining.

Conclusion:
Endothelial function is preserved when utilizing endoscopic harvesting techniques. The advantages of minimally invasive vein procurement for lower extremity bypass can be obtained without concern for damaging venous endothelium.
Abstract 244: Arterial Calcification Decreases Functional Recovery in a Murine Model of Hindlimb Ischemia.
Authors: Zettervall Sara L , Monk Stephanie , Wang Xue-Lin , Lin Tonghui , Guzman Raul J
Published: July 2021

Objectives:
Clinical studies have demonstrated associations between arterial calcification and adverse outcomes among patients with peripheral arterial disease (PAD). The causal relationship between calcification and the pathophysiology of ischemia, however, remains unclear. This study aims to evaluate the effects of vitamin D3-induced arterial calcification on functional recovery and resolution of ischemia in the rat hindlimb model.

Methods:
Arterial calcification was induced in 14 Sprague-Dawley rats using Vitamin D3. A control group of 14 rats received vehicle only. After 2 weeks to allow for calcification, unilateral hindlimb ischemia was induced by ligation and excision of the superficial femoral artery. Functional status and ischemia status were evaluated weekly for 1 month in a blinded manner.

Results:
28 rats were assessed at all designated time periods. Arterial calcification was significantly greater in Vitamin D3-treated animals than in controls. Vitamin D3-treated rats had significantly worse total ischemia scores at each time point compared with controls. (Figure 1). All rats in the control group regained normal functional status and ischemia scores by week 4. Functional and ischemia scores in vitamin D3-treated animals did not return to baseline by the conclusion of the study.

Conclusions:
Vitamin D3 treatment significantly reduces functional recovery in a rat model of hindlimb ischemia. Future research aimed at understanding the relationship between vitamin D3 treatment, arterial calcification, and lower extremity ischemia may help to improve outcomes in patients with PAD.
Abstract 248: Adipose Stromal Cells Mitigate Excessive Aortic Inflammation and Aortic Aneurysm Expansion through Paracrine Factors in an Elastase-induced Murine Abdominal Aortic Aneurysm Model
Authors: Xie Jie , Jones Thomas J , Feng Dongni , Cook Todd G , Babbey Clifford M , March Keith L , Murphy Michael P
Published: July 2021

Objective:
Abdominal aortic aneurysm (AAA) formation is characterized by activation of innate and adaptive immune responses that initiate aortic wall degradation. We hypothesize that human adipose stromal cells (ASC) favorably modulate these immune responses through paracrine mechanisms and attenuate AAA expansion in a murine elastase model.

Methods:
AAA was induced in C57BL/6 mice by topical treatment of the abdominal aorta with elastase. Human ASC or saline was injected i.v. within 4 hours of elastase treatment. The aortic diameter was measured by ultrasound and video micrometry. Monocytes/macrophages and lymphocyte sub-populations were quantified by flow cytometry. Regulatory T cells (Treg) and M2 macrophages were quantified by immunohistochemical (IHC) staining. ASC were labeled with Hoechst and their distributions in the lung and aorta were imaged by fluorescent microscopy and confirmed by real-time PCR for human-specific Alu sequence.

Results:
ASC significantly suppressed AAA expansion at 14 days (percent change from baseline: 64.1±4.2% (saline) vs. 24.6±6.2% (ASC), n=10/group, p<0.01). At Day 1 there was a significant increase in Treg in ASC treated mice (n=5/group, p<0.01) with a concurrent decrease of CD4+CD28-, CD8+CD28- T cells, and neutrophils (n=5/group, p<0.05). At Day 7, activated monocytes (CD115+CXCR1-LY6C+) and total aortic tissue resident macrophages were decreased (n=10/group, p<0.05) with a significant polarization to the tissue reparative M2 phenotype (n=5/group, p<0.05). Hoechst labeled ASC were detected in the lung without engraftment in the aorta at 24 hrs and were cleared at 96 hrs. Transmembrane co-culture of human peripheral blood mononuclear cells with ASC induced Treg, polarized macrophages to the M2 phenotype, inhibited neutrophil transmigration, and suppressed lymphocyte proliferation, as further supporting the mechanism of paracrine immunomodulation in the in vivo model of AAA.

Conclusion:
Systemic injection of ASC significantly induced anti-inflammatory immune cell phenotypes and suppressed AAA formation. These effects are mediated by release of paracrine factors and suggest that further characterization of the ASC secretome may lead to more potent recombinant protein therapies for AAA.
Abstract 257: Pilot Study of the Safety and Feasibility of the Treatment of Paclitaxel Associated to a Cholesterol-Rich Nanoemulsion in Patients With Aortic Atherosclerotic Disease
Authors: Maranhao Raul C , Shiozaki Afonso A , Senra Tiago , Morikawa Aleksandra T , Deus Debora F , Paladino-Filho Antonio T , Pinto Ibraim M
Published: July 2021

Previously, we showed that the toxicity of chemotherapeutic agents used in cancer treatment can be drastically reduced by association to nanoemulsions (LDE) that mimic the lipid composition of low-density lipoprotein (LDL), without loss of pharmacological action. Injected into the circulation, LDE concentrates associated drugs, such as the anti-proliferative agent paclitaxel, in neoplastic tissues and in atherosclerotic lesions. In rabbits with atherosclerosis lesions were reduced by 65% by LDE-paclitaxel treatment. Tolerability and safety of high dose LDE-paclitaxel was shown in several patients with advanced cancers. Based on those findings, this pilot study was designed to test, in 9 aged patients with extensive aortic atherosclerosis, LDE-paclitaxel at 175 mg/m2 body surface dose (I.V., 3/3 weeks for 6 cycles). All were under statin treatment that was not discontinued during the 18-week treatment period. No observable clinical or laboratorial toxicity was observed. One death occurred but was unrelated with treatment toxicity. Images acquired by Multiple Detector Computer Tomography Angiography (64-slice scanner) taken before and at 2-3 month after the treatment end showed that the mean volume of the aortic artery wall was clearly reduced in 4 of the 8 patients, while in 3 it remained unchanged and in one it clearly increased. Therefore, the treatment was tolerable for patients with cardiovascular disease and these results encourage large, placebo-controlled clinical studies to ascertain the ability of LDE-paclitaxel to reduce atherosclerotic lesions.