bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 113: Screening of MicroRNAs Expressed in Isolated Cells of Human Abdominal Aortic Aneurysm for the Identification of Potential Biomarkers
Authors: Rafaelle Spear , Boytard Ludovic , Blervaque Renaud , Hot David , Vanhoutte Jonathan , Staels Bart , Chwastyniak Maggy , Amouyel Philippe , Haulon Stephan , Pinet Florence
Published: March 2022

Abdominal aortic aneurysm (AAA) is a vascular asymptomatic disease that is one of the leading causes of death in developed countries. Identifying biomarkers for AAA that can be detected easily in blood, before rupture could be useful.

The aim of this study was to investigate the potentiality of miRNAS as biomarkers of AAA by performing a profiling of miRNAs expressed in major cells present in the human AAA tissue.

The inflammatory cells as macrophages M1 and M2 and smooth muscle cells (SMC) were located by immunohistochemistry in 20 human AAA biopsies, showing a specific distribution towards the aneurysmal aortic wall. The cells were isolated by laser microdissection (LMD) from 20 human AAA biopsies obtained during surgical repair and control SMC from 14 healthy aortic biopsies harvested during organs multiretrieval. RNA extracted from 2 samples of each LMD isolated cells was screened on human miRNAs microarray. MiRNAs were selected with at least a 2-fold change and a detection value threshold corresponding to the value of miR-29b, described in experimental AAA models.

Out of the 850 human miRNAs tested for each sample, 408 were found to be present in AAA. Thirty miRNAs were common to each tested cells. Fifty-three miRNAs were found in SMC, of which 12 were specific to AAA compared to control aortas; 86 miRNAs were found in macrophages, of which 11 were specific to M1 macrophages, 37 to M2 macrophages and 38 common to both subtypes. Ten miRNAs were selected to be validated by quantitative RT-PCR in LMD isolated healthy SMC and aneurysmal SMC, M1 and M2 macrophages. We validated 4 miRNAs to be overexpressed in M1 macrophages and 1 overexpressed in M2 macrophages. Two miRs were validated to be less expressed in aneurysmal SMC compared to SMC from normal aortas. MiR-29b expression was specifically down-regulated in aneurysaml SMC compared to normal SMC.

In conclusion, the analysis of isolated cells allows to discriminate the miRNAs specifically expressed in inflammatory and vascular cells in AAA and to determine other miRNAs than those described in experimental AAA models as potential biomarkers of AAA.
Abstract 115: Adventitial Fibroblast Depletion of AT1a Receptors Attenuates AngII-Induced Ascending and Abdominal Aortic Aneurysms in Hyperlipidemic Mice
Authors: Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Cassis Lisa A , Daugherty Alan
Published: March 2022

Introduction and Objectives:
Infusion of angiotensin II (AngII) into mice induces aneurysm formation and accelerates atherosclerosis. These pathologies are ablated by whole body deficiency of angiotensin II type 1a (AT1a) receptors. It is currently unknown which cell type is stimulated by AngII through AT1a receptors to augment either pathology. Surprisingly, depletion of AT1a receptors in macrophages, endothelium, and smooth muscle has minimal effects. Advential fibroblasts have been implicated in vascular inflammation. The current study examined the effect of deletion of AT1a receptors in fibroblasts on AngII-induced pathologies.

Methods and Results:
LDL receptor -/- mice with fibroblast-specific deletion of AT1a receptors were produced by breeding hemizygous male mice expressing Cre under control of the S100A4 promoter to female AT1aR floxed mice. At 8 weeks of age, male, non-Cre littermates (N=13) and AT1a receptor x Cre-S100A4 (N=18) mice were fed a fat-enriched diet for 1 week before and during AngII infusion (1,000 ng/kg/min) for 28 days. AngII-infusion increased systolic blood pressures that were not influenced by fibroblast-specific deletion of AT1a receptors. AngII-induced expansion of the ascending aorta and abdominal aortic width were attenuated significantly in mice with fibroblast-specific AT1a receptor depletion (P=0.04 and P=0.043, respectively). In contrast, fibroblast-specific depletion of AT1a receptors had no effect on AngII-augmented atherosclerosis.

Conclusion:
Fibroblast-specific deficiency of AT1a receptors is the first cell type that has been identified to attenuate AngII-induced aortic aneurysms.
Abstract 118: Use of Computational Fluid Dynamics Studies in Predicting Aneurysmal Degeneration of Acute Type B Aortic Dissections
Authors: Shang Eric K , Nathan Derek P , Fairman Ronald M , Bavaria Joseph E , Gorman Robert C , Gorman Joseph H , Jackson Benjamin M
Published: March 2022

Objectives:
While uncomplicated acute type B aortic dissections are often medically managed with good outcomes, a subset develop subacute or chronic aneurysmal dilatation. We hypothesize that computational fluid dynamics (CFD) simulations may be useful in identifying patients at risk for this complication.

Methods:
Patients with acute type B dissection complicated by rapidly expanding aortic aneurysms (N=7) were compared to patients with stable aortic diameters (N=7). Three dimensional patient-specific dissection geometries were generated from CTA and used in CFD simulations of pulsatile blood flow. Hemodynamic parameters including false lumen flow and wall shear stress were compared.

Results:
Patients with rapid aneurysmal degeneration had a growth rate of 5.3 ± 2.7 mm/month compared to those with stable aortic diameters who had rates of 0.2 ± 0.02 mm/month. Groups did not differ in initial aortic diameter (36.1±2.9 vs 34.4±3.6 mm, P=0.122) or false lumen size (22.6±2.9 vs 20.2±4.5 mm, P=0.224). In patients with rapidly expanding aneurysms, a greater percentage of total flow passed through the false lumen (78.3±9.3 vs 56.3±11.8%, P=0.016). The time averaged wall shear stress on the aortic wall was also significantly higher (12.6±3.7 vs 7.4±2.8 Pa, P=0.028, see Figure).

Conclusions:
Hemodynamic parameters derived from CFD simulations of acute type B aortic dissections were significantly different in dissections complicated by aneurysm formation. Thus, CFD may assist in predicting which patients may benefit from early stent grafting.
Abstract 12: Basigin Promotes Vascular Smooth Muscle Proliferation and Pulmonary Hypertension
Authors: Satoh Kimio , Suzuki Kota , Omura Junichi , Kikuchi Nobuhiro , Miyata Satoshi , Minami Tatsuro , Sugimura Koichiro , Aoki Tatsuo , Nochioka Kotaro , Tatebe Shunsuke , Miura Masanobu , Yamamoto Saori , Shimizu Toru , Yaoita Nobuhiro , Fukumoto Yoshihiro , Nakamura Kazufumi , Ito Hiroshi , Kadomatsu Kenji , Shimokawa Hiroaki
Published: March 2022

Background:
Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells (VSMC) by oxidative stress and promotes VSMC proliferation. However, the role of CyPA and its extracellular receptor Basigin (Bsg) in the pathogenesis of pulmonary hypertension (PH) remains to be elucidated. In this study, we tested our hypothesis that CyPA/Bsg signaling promotes the development of PH.

Methods and Results:
In the pulmonary arteries of PH patients, immunostaining revealed strong expression of CyPA and Bsg (n=5). In cultured human pulmonary arterial VSMC, hypoxia (O
2
2%) significantly increased CyPA secretion and Bsg expression compared with normoxic condition (O
2
21%) (n=4 per group). To determine the role of CyPA/Bsg signaling in PH development, CyPA
+/-
and Bsg
+/-
mice were exposed to hypoxia (O
2
10%) for 4 weeks. The pulmonary arteries (PA) of CyPA
+/-
and Bsg
+/-
mice exposed to normoxia did not differ in morphology compared with their littermate controls. In contrast, CyPA
+/-
(n=12) and Bsg
+/-
mice (n=15) exposed to hypoxia revealed significantly reduced right ventricular systolic pressure (RVSP), PA remodeling and RV hypertrophy compared with their littermate controls (all P<0.01). Importantly, after transplantation of bone marrow, the severity of PH was still exacerbated in Bsg
+/+
recipient mice compared with Bsg
+/-
recipient mice regardless of the source of bone marrow (Bsg
+/+
or Bsg
+/-
), suggesting the crucial role of Bsg in PA. To further evaluate the role of Bsg, we harvested VSMC from Bsg
+/+
and Bsg
+/−
mice. VSMC proliferation was significantly reduced in Bsg
+/−
compared with Bsg
+/+
in response to 2% FBS, suggesting the crucial role of Bsg in VSMC proliferation. Mechanistic studies demonstrated that Bsg
+/-
VSMC revealed reduced expression of oxidative stress genes, less secretion of cytokines/chemokines and growth factors. Finally, in the clinical study, plasma CyPA levels in PH patients were increased in accordance with the severity of pulmonary vascular resistance (P<0.001). Furthermore, event-free curve revealed that high plasma CyPA levels predicted poor outcome in PH patients (death or lung transplantation, P<0.001).

Conclusions:
These results indicate the crucial role of extracellular CyPA and vascular Bsg in the pathogenesis of PH.
Abstract 128: Angiotensin II Evoked Increases in Blood Pressure and Inflammatory Mediators in Adiponectin-Deficient Mice
Authors: Pamidimukkala Jayabala , O'Brien Alexis
Published: March 2022

Chronic low grade inflammation is considered a predisposing factor for vascular dysfunction and subsequent elevation of blood pressures. Adipose tissue is a well-known source of mediators that are proinflammatory. Adiponectin (APN) is one of the few proteins secreted by the adipose tissue that is known to have anti-inflammatory properties. APN deficiency has been shown to result in significant increases in the plasma levels of inflammatory mediators when provided with an appropriate stimulus. Our research question is does a proinflammatory milieu accelerate or potentiate the development of hypertension? Male wild type C57Bl/6J and adiponectin deficient (adipo-/-) mice (24-28 wks, 28-30g) were implanted with radiotelemeters (TA11PA-C10, DSI) for chronic measurement of blood pressures. The mice were allowed to recover from surgery and baseline blood pressures recorded for 7-10 days. These mice were then implanted subcutaneously with osmotic pumps containing Angiotensin II (ANGII, 800ng/kg/min) and blood pressures were recorded for another 14 days. Baseline blood pressures in the adiponectin deficient mice (105±2 mmHg, n=3) were significantly lower than that of wild type C57BL/6J mice (116±6, n=3). After 14 days, ANGII infusion increased the blood pressures to similar levels in adiponectin deficient( 144±10 mmHg) and the wild type mice (138±7 mmHg). In a separate group of mice, the effects of chronic ANGII infusion on plasma TNFalpha levels were evaluated using ELISA. After 14 days of infusion, plasma TNFalpha levels were similar in adiponectin deficient (5.30±0.97 pg/ml,n=3) and wild type (4.94±0.84 pg/ml, n=6) mice. The data suggest that adiponectin deficient mice were more susceptible to the prohypertensive effects of ANGII and the increases in blood pressures were not associated with significant differences in circulating inflammatory mediators such as TNFalpha.
Abstract 129: Predicting Patients Who Will Have Cognitive Decline After Carotid Endarterectomy or Stenting Using Structural Connectivity Graph Analysis
Authors: Soman Salil , Prasad Gautam , Hitchner Elizabeth , Rosen Allyson , Zhou Wei
Published: March 2022

Objectives:
Many CEA and CAS patients experience postoperative neurocognitive decline. We sought to apply structural connectivity metrics to identify patients at increased risk for postoperative decline based solely on preoperative imaging.

Methods:
Under an IRB approved protocol 28 patients underwent presurgical T1 structural and 30 direction diffusion tensor imaging (DTI) MRI and neuropsychological tests before and 1 month after surgery. Patients with decline showed decreased performance on the Rey-AVLT on 1 month follow up. The T1 images were processed using FreeSurfer 5.3, with resulting segmentations reviewed and edited as needed under neuroradiologist supervision. Whole brain tractography was performed using Diffusion Toolkit and visually inspected. Connectivity matrices were then generated, and graph metrics were computed using the Brain Connectivity Toolbox.

Results:
Controlling for age, classifiers using the graph analysis metrics “weighted optimal community structure” & “binary component sizes” were able to identify patients that would experience cognitive decline with 81% sensitivity 83% and specificity (p<.05, false discovery rate .05). These two measures were computed at 10 proportion edge thresholds from .1 to 1 at intervals of .1 in weighted and binary networks respectively.

Conclusions:
Applying preoperative structural connectivity analysis in CEA and CAS patients may identify patients at increased risk for postoperative cognitive decline, and in so doing may help risk stratify patients and guide them to preventive interventions.
Abstract 133: Prognostic Utility of the GDF-15/FSTL-1 Axis for Adverse Cardiac and Limb Events in Patients With Peripheral Artery Disease
Authors: Johnston-Cox Hillary A , Widera Christian , LeLeiko Rebecca M , Eberhardt Robert T , Holbrook Monika , Palmisano Joseph N , Gokce Noyan , Wang Na , Sharmeen Farhana , Farber Alik , Pande Ashvin N , Wollert Kai C , Vita Joseph A , Hamburg Naomi M
Published: March 2022

Background:
Peripheral artery disease (PAD) confers greater vulnerability to cardiovascular events. Inflammatory activation and tissue ischemia induce expression of a vasoprotective cytokine network including GDF15 and FSTL1. We sought to evaluate the clinical utility of novel vascular stress biomarkers for risk stratification in PAD.

Methods and Results:
We followed 245 PAD patients prospectively over two years for adverse cardiac events (n=40, myocardial infarction, unstable angina, coronary revascularization, transient ischemic event, stroke, congestive heart failure, cardiac death) and adverse limb events (n=52, worsening claudication, peripheral revascularization, critical limb ischemia, amputation, graft or stent failure). At baseline, we measured serum GDF15 and FSTL-1 and disease severity by the ankle-brachial index (ABI). In survival analyses, higher GDF15 was associated with both adverse cardiac and limb events (P<0.0001, P<0.01). FSTL-1 was associated with adverse limb (P=0.03) but not cardiac events. In multivariable models adjusting for relevant covariates including ABI, higher GDF15 remained associated with higher risk of limb events (HR 1.83, 95% CI 1.10, 3.06, P=0.02) and cardiac events (HR 1.92, 95% CI 1.09, 3.32, P=0.02). There was a trend for an association of higher FSTL1 with limb events (HR 2.34, 95% CI 0.93, 5.75, P=0.07). Addition of GDF15 to clinical variables led to an increase in the c statistic (P<0.05) and net reclassification improvement (P<0.01) for both cardiac and limb events.

Conclusions:
Our findings show that vascular stress markers involving the GDF15-FSTL1 axis provide additional prognostic value over clinical factors and disease severity for predicting adverse cardiac and limb events in patients with PAD.
Abstract 148: Critical Role of Toll-Like Receptor 4 in Ischemia-Induced Skeletal Muscle Damage
Authors: Navi Ali , Abraham David , Shi-wen Xu , Hamilton George , Baker Daryll , Tsui Janice
Published: March 2022

Objectives:
Inflammation contributes to skeletal muscle damage in patients with critical limb ischaemia (CLI), in whom patient oriented functional outcomes are poor. Previously, we showed that TLR4, a pattern recognition receptor, is upregulated and activated in skeletal muscle of patients with CLI. This study aimed to investigate whether the TLR4 pathway contributes to skeletal muscle damage in CLI, using in vitro and in vivo models.

Methods:
Human myoblasts, isolated from skeletal muscle biopsies, were cultured to myotubes and exposed to simulated ischaemia, with or without TLR4 inhibitor pre-treatment. Western blot analyses of P-NFkB (signal-related kinase) and cleaved caspase-3 were carried out on cell lysates to assess downstream signalling activity and apoptosis respectively. Further, hind-limb ischaemia by excision of femoral artery was induced in 12-week old TLR4-/- and wild-type (WT) mice with and without administration of the TLR4 antagonist, LPS-RS (n=18 per group). Systemic levels of IL6 & TNF-α, tissue perfusion, inflammatory damage and apoptosis within the ischaemic muscle were assessed at days 3, 7 and 21 by ELISA, laser Doppler, H&E staining and Fluorescent IHC-P, respectively.

Results:
Simulated ischaemia in cultured human myotubes was associated with upregulation of TLR4, P-NFkB and cleaved caspase-3. Pre-treatment with TLR4 inhibitor reduced P-NFkB and cleaved caspase-3 expression (P<0.05). In in vivo studies, TLR4-/- mice and mice given LPS-RS exhibited reduced systemic IL6 & TNFα levels (P<0.05) following hind-limb ischaemia. This was associated with improved tissue perfusion recovery (P<0.05), reduced inflammatory cell infiltration and diminished apoptosis (P<0.05) in the ischaemic limb as compared to untreated WT mice at days 3, 7 and 21.

Conclusion:
TLR4 inhibition prior to ischaemia in human myotubes in vitro, was associated with inhibition of the signalling pathway and reduced ischaemia-induced apoptosis. Further, endogenous deletion and exogenous inhibition of TLR4 in vivo were associated with reduced systemic IL6 & TNF-α levels and skeletal muscle damage following hind-limb ischaemia. These findings suggest that TLR4 plays a key role in ischaemia-induced skeletal muscle damage.
Abstract 151: ZBTB46 is a Shear Stress Sensitive Transcription Factor in Endothelial Cells
Authors: Rezvan Amir , Sun He Ying , Jo Hanjoong
Published: March 2022

Background:
ZBTB46, also known as zDC or BTBD4, is a member of the BTB-ZF protein family which comprises a diverse group of transcription factors. Recently, ZBTB46 was identified as a transcription repressor specific to classical dendritic cells (cDCs) among immune cells and responsible for keeping cDCs in a quiescent state. ZBTB46 down-regulation by TLR signaling leads to cDC activation. Interestingly, endothelial cells also express ZBTB46, and may be viewed as part of the immune system; expressing functional TLRs and interacting with other immune cells. However, the role of ZBTB46 in endothelial cells has not been studied. Endothelial cell activation in areas of disturbed flow is a critical step in initiation of atherosclerosis, leading to heart attacks and stroke.

Hypothesis:
ZBTB46 is expressed in quiescent endothelial cells under laminar shear conditions and is down-regulated by low and oscillatory shear stress patterns as seen in areas of disturbed flow, leading to endothelial cell activation.

Methods:
We used the mouse partial carotid ligation model to assess ZBTB46 shear responsiveness
in vivo
and a cone and plate cell culture model to assess shear responsiveness
in vitro
. We measured endothelial cell mRNA using qPCR and protein via western blot. We used immunofluorescence to show localization of ZBTB46 in the arterial wall.

Results:
We showed that ZBTB46 is expressed in both human and mouse endothelial cells by mRNA and protein measurements. We also showed that ZBTB46 is down-regulated under low and oscillatory shear conditions after 48 hours both
in vivo
(Figure 1-A) and
in vitro
(Figure 1-B). Further functional studies including monocyte adhesion and migration assays as well as mechanistic studies assessing upstream and downstream pathways are currently underway.
Abstract 161: Differential Effects of Short- and Long-Term Increase in Endothelial ROS on Coronary Vascular Function
Authors: Shafique Ehtesham , Choy Wing , Liu Youhong , Feng Jun , Sellke Frank , Abid Ruhul
Published: March 2022

Introduction:
Increase in reactive oxygen species (ROS) is often associated with vascular pathophysiological conditions. Our recent findings demonstrated that transient increase in ROS improved coronary endothelial function. However, long-term effects of endothelium-specific increase in ROS on endothelial function are not known.

Hypothesis:
The short-term (four to eight weeks)
versus
the long-term (16 to 20 weeks) increase in NADPH oxidase-derived ROS will have differential effects on vascular endothelium.

Methods:
Our binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) induces 1.8±0.42-fold increase in NADPH oxidase-derived ROS in endothelium. Using these animals, we have examined the effects of short-term
vs.
the long-term effects of ROS on endothelial cell (EC) signaling and coronary endothelial function.

Results:
We demonstrate that whereas EC-dependent coronary vasodilation was significantly increased after short-term increase in ROS, coronary vasodilation was drastically reduced after long-term increase in EC-ROS in Tet-OFF Nox2 mice compared to their Tet-ON (control) littermates. Using EC isolated from mouse heart, we demonstrate that, whereas Nox2-derived both short-term and long-term increase in ROS increased activation of AMPK and eNOS, and nitric oxide (NO) synthesis, long-term increase in ROS abrogated these positive effects due to increased peroxynitrite and nitrotyrosylation of MnSOD. We also show that long-term ROS increase resulted in abrogation of EC proliferation and reduction in EC mitochondrial content compared to short-term ROS exposure.

Conclusion:
This study demonstrates that whereas short-term increase in ROS is beneficial for EC function, long-term increase in ROS results in loss of EC mitochondria and inhibition of endothelial function.
Abstract 178: Cilostazol Attenuated Inflammation and Matrix Metalloproteases Induced by TNF-α
Authors: Umebayashi Ryoko , Uchida Haruhito A , Makino Hirofumi
Published: March 2022

Objective:
Previously, we demonstrated that cilostazol, a phosphodiesterase-3 inhibitor, attenuated AngII-induced abdominal aortic aneurysms (AAAs) in male apolipoprotein E (apoE) deficient mice. However, the mechanism of this protective effect is unknown. The purpose of this study was to elucidate the effect of cilostazol on inflammation and MMPs in several cell types present in AAAs.

Methods and Results:
First, aortic endothelial cells (ECs) were isolated from male apoE deficient mice. After confluence from 3 to 5 passages, ECs were incubated with saline, TNF-alpha, or TNF-alpha/cilostazol for 24 hours. Many inflammatory genes, such as MCP-1, IL-1beta, COX-2, ICAM-1 and VCAM-1, were increased by TNF-alpha, but the enhancements of them were attenuated by co-treatment of cilostazol. Western blot revealed that cilostazol attenuated TNF-alpha-induced ICAM-1 protein expression. Second, aortic vascular smooth muscle cells (VSMCs) were isolated from male apoE deficient mice. Cultured VSMCs were incubated with saline, TNF-alpha, or TNF-alpha/cilostazol for 24 hours. Similar to ECs, cilostazol decreased gene expression of inflammation such as MCP-1, COX-2, iNOS, OPN, and gene expression of MMP-2 and -9 that were induced by TNF-alpha. Third, we harvested peritoneal macrophages from male apoE deficient mice. 24 hours incubation with TNF-alpha induced MMP-2 and MMP-9 gene expression. Co-treatment with cilostazol attenuated gene expression of these proteases.

Conclusion:
Our results indicate that cilostazol exerts anti-inflammatory effect on selected cell types that promote arterial vascular wall inflammation. This effect might, in part, contribute to attenuated AngII-induced AAAs in male apoE deficient mice.
Abstract 181: VAMP8 Mediates Endothelial Granule Exocytosis
Authors: Zhu Qiuyu , Bao Clare , Ture Sara , Ferlito Marcella , Morrell Craig N , Yamakuchi Munekazu , Lowenstein Charles J
Published: March 2022

Endothelial cells maintain the integrity of the vasculature. In response to injury, endothelial cells undergo exocytosis, releasing various compounds into the blood stream by vesicle fusion with the plasma membrane. Endothelial exocytosis provokes inflammation and thrombosis as one of the earliest responses to vascular injury. Enhanced understanding of these events may lead to novel treatments for vascular diseases such as atherosclerosis, stroke, myocardial infarction, and thrombosis. However, the exocytic machinery that regulates granule secretion from endothelial cells is not completely defined. We tested the hypothesis that specific proteins in the soluble N-ethylmaleimide sensitive factor receptor attachment protein (SNARE) superfamily are associated with the endothelial-specific secretory organelles, Weibel-Palade bodies (WPBs), and mediates endothelial exocytosis. We found that human endothelial cells express members of the SNARE superfamily, including vesicle associated membrane protein 3 (VAMP3), VAMP8, syntaxin 4 (STX4), and synaptosomal associated protein 23 (SNAP23). Out of all these proteins, we found that only VAMP8 co-localizes with WPBs containing von Willebrand Factor (VWF), the major component of endothelial granules. Furthermore, VAMP8 also interacts with STX4, another SNARE component of the exocytic machinery. To explore the role of VAMP8 in regulating endothelial granule exocytosis, we knocked down VAMP8 expression with siRNA in endothelial cells. Decreasing VAMP8 expression impairs exocytosis; conversely, rescue of VAMP8 expression after VAMP8 knockdown restores exocytosis to normal level. Mice lacking VAMP8 have decreased endothelial exocytosis, as measured by decreased plasma VWF levels and decreased leukocyte rolling along vessels. In conclusion, our data suggest that VAMP8 plays a critical role in mediating endothelial exocytosis and may be a potential therapeutic target for vascular inflammatory and thrombotic diseases.
Abstract 187: Prevention of Circulating Progenitor Surge After Vascular Injury Effectively Reduces Restenosis
Authors: Tillman Bryan W , Kelly Jeremy , Richards Tara D , Chen Alex F , Donnenberg Albert D , Donnenberg Vera
Published: March 2022

Background:
Restenosis is an aberrant healing response that leads to failure of half of all vascular interventions and circulating progenitor cells have been implicated in restenotic pathology. Based on our previous findings of a surge specific to macrovascular injury, we hypothesized that a surge among progenitors and vascular injury signaling would be evident in the murine model and that an approach to prevent this surge would reduce restenosis.

Methods:
C57BL6 mice underwent injury of the femoral artery following either a depleting antibody to the progenitor marker sca-1 or a control antibody (n=8 per group). A third group had a non-vascular surgery (n=5). Antibody was administered pre-op and then until day 9. Blood and bone marrow were collected on days 1, 3, and 9 and analyzed by flow cytometry for progenitor markers CD34 and c-kit. Plasma was analyzed for the vascular injury signal, stromal cell-derived factor 1/CXCL12 (sdf1alpha). Vessel explants were imaged with lectin microscopy. Finally, animals treated with either sca-1 depleting antibody (n=4) or an isotype control (n=3) underwent vessel explant at 28 days and a lumen ratio was calculated.

Results:
Microscopy confirmed injury of endothelial monolayer and serologically, wire injury increased sdf1alpha by over 3 fold. Injury of the femoral artery resulted in a surge of CD34+/c-kit+ progenitors by over 2.2 fold relative to non-operative controls with a peak on day 1. Furthermore, this surge was prevented by treatment with a sca-1 antibody (P= 0.004). At 28 days, progenitor depletion increased the lumen from 20% among isotype treated controls to 79% among animals treated with direct progenitor depletion (P=0.04).

Conclusions:
It has not been previously demonstrated whether changes among progenitor cells impact restenotic pathology. Our findings suggest that a surge occurs in both a key vascular injury cytokine and among circulating progenitor cells after macrovascular injury. We have further demonstrated the efficacy of a depleting sca-1 antibody to mitigate progenitor surge and most importantly to reduce restenosis in a significant fashion. Future studies of strategies to block circulating progenitor cells may provide new therapies for this prevalent life and limb threatening disease.
Abstract 19: CD39 Expression on Circulating Blood Components Prolongs the Time to Ferric Chloride--Induced Carotid Artery Thrombosis in Mice
Authors: Reynolds Adam , Huttinger Zachary M , Aurand William L , Huttinger Ryan M , Stanfill Katerine , Wheeler Debra G , Novitskaya Tatiana , Robson Simon C , Dwyer Karen M , Dwyer Peter J , Gumina Richard J
Published: March 2022

Objective:
To determine the effect of increased CD39 expression on circulating blood components on
in vivo
arterial thrombosis in mice.

Approach and Results:
We have previously demonstrated that increased global expression of hCD39 conveys an anti-thrombotic phenotype in mice suggesting a pivotal role for CD39 in modulating the balance between a pro-thrombotic and anti-thrombotic milieu. We hypothesized the expression of CD39 on circulating blood components can regulate occlusive thrombus formation. Bone marrow transplantations were performed (WT to WT; WT to hCD39-Tg; hCD39-Tg to hCD39-Tg; hCD39-Tg to WT) to determine the contribution of CD39 expressed on circulating blood cells and platelets on occlusive arterial thrombus formation in mice. Complete blood counts and fluorescence-activated cell sorting (FACS) analyses were performed on whole blood obtained from mice 60 days after transplantation of bone marrow. No significant differences in the circulating levels of white blood cells, red blood cells, and platelets were observed between the groups. FACS analysis confirmed that the circulating blood components were reconstituted from the transplanted bone marrow. Carotid artery thrombosis was induced by ferric chloride (FeCl
3
) application and the time to thrombotic occlusion (blood flow of 0 ml/min) was measured. The operator was blinded to the animal genotype and treatment during all experiments. Transplantation of CD39-Tg bone marrow into WT mice resulted in a marked resistance to thrombosis (CD39-Tg to WT: 315.7.00 ± 54.6 min; WT to WT: 7.58 ± 0.89 min;
P
<0.001). CD39-Tg mice that received WT bone marrow demonstrated a significantly lower time to vessel occlusion than CD39-Tg mice that received CD39-Tg bone marrow (WT to CD39-Tg: 17.15 ± 6 min; CD39-Tg to CD39-Tg 414.3 ± 61.95 min;
P
<0.001).

Conclusion:
Increased expression of CD39 on circulating bone-marrow-derived blood components markedly prolongs the time to carotid artery occlusion following ferric-chloride-induced injury in mice.
Abstract 190: Classic Lipid and Novel Inflammatory Phenotypes in an Induced Pluripotent Stem Cell--Derived Macrophage Model of Tangier Disease
Authors: Zhang Hanrui , Shah Rhia , Bermingham Kate , Hinkle Christine , Rodrigues Amrith , Cuchel Marina , Liu Ying , Yang Wenli , VanDorn Daniel , Chou Stella , Morrisey Edward E , Rader Daniel J , Reilly Muredach P
Published: March 2022

Tangier disease (TD) is an autosomal recessive disorder caused by mutations in the ATP-binding cassette transporter A1 (ABCA1). These result in a greatly reduced ability to transport cholesterol out of cells, leading to the accumulation of cholesterol in macrophages and many other body tissues expressing ABCA1. We recruited two TD individuals, TD1, compound heterozygote at S2046R/K531N, and TD2, homozygous for the E1005X/E1005X truncation mutation, as well as heterozygote/non-affected family members, and healthy controls. We reprogrammed PBMC of TD patients and controls to pluripotency by Sendai viral transduction with Oct3/4, Sox2, Klf4 and c-Myc. TD-iPSC expressed pluripotency markers including SSEA-3, SSEA-4, TRA-1.60, and TRA-1.81, and maintained a normal karyotype. TD iPSC differentiated efficiently to macrophages. iPSC-derived macrophages (IPSDM) characteristics paralleled those of primary PBMC-derived macrophages (HMDM) at morphological, phenotypic and transcriptomic levels. TD-IPSDM and HMDM showed no cholesterol efflux to apolipoprotein A-I (apoA-I) and impaired efflux to HDL
3
. Treatment of TD cells with LXR agonists, which upregulate ABCA1 expression, failed to enhance cholesterol efflux to apoA-I in TD-IPSDM and HMDM consistent with the absence of functional ABCA1. In both IPSDM and HMDM, the heterozygote ABCA1 mutation carrier had an intermediate defect in cholesterol efflux and a partial response to the LXR agonist consistent with the presence of one functional allele. Relative to control-IPSDM, TD-IPSDM also showed a higher cholesterol ester/total cholesterol (CE/TC) ratio upon acetylated-LDL loading. Compared with control-IPSDM, TD-IPSDM showed enhanced phagocytosis of zymosan particles and greater inflammatory response to ATP treatment in lipopolysaccharide (LPS)-primed IPSDM, evidenced by markedly elevated gene expression of IL-1beta, IL-6, IL-8 and CCL5, but not TNF-alpha. These observations provide further support for the utility of IPSDM in defining more subtle macrophage phenotypes that are less obvious manifestations of Mendelian disorders. We conclude that macrophages derived from TD IPS can effectively recapitulate pathologic hallmarks of the disease.
Abstract 197: p300-Based Acetylation Signaling Regulates Cytoskeletal Reorganization and Procoagulant Phenotype of Platelets
Authors: Aslan Joseph E , RIgg Rachel , Loren Cassandra P , Itakura Asako , Baker Sandra M , Pang Jiaqing , McCarty Owen J
Published: March 2022

Introduction:
Reversible acetylation signaling catalyzed by acetyltransferases and deacetylases regulates multiple cellular events. We recently showed a role for deacetylation signaling in platelet function via the actions of the deacetylase enzyme HDAC6. Here we investigate the role of acetylation mediated by the p300 class of acetyltransferase proteins in platelet function.

Methods:
To test the

hypothesis

that p300 acetylation signaling controls cytoskeletal remodeling and the exposure of phosphatidylserine (PS) in platelets, we used cell biological and hematological methods, including enzymatic assays, microscopy, cell sorting, aggregometry and
in vitro
assays of thrombus formation.

Results:
Platelets express high levels of p300 protein. Upon stimulation with the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP), total platelet acetyltransferase enzyme activity increases. Pre-treatment of platelets with pharmacological inhibitors of p300 blocks agonist-induced acetyltransferase activity and prevents the acetylation of p300 substrate proteins. Inhibition of p300 blocked the deacetylation of HDAC6 and tubulin and disrupted the platelet tubulin cytoskeleton. p300 inhibition also increased basal platelet PS exposure via a caspase-dependent apoptotic pathway. On a functional level, p300 inhibition prevented platelet shape change, aggregation and spreading while increasing the procoagulant activity of platelets in whole blood.

Conclusions:
These results suggest that p300-based acetylation signaling regulates separable aspects of platelet function. p300 plays a role in the regulation of the platelet cytoskeleton as well as PS exposure to effect platelet spreading, aggregation, thrombus formation as well as platelet procoagulant activities. Future studies of the platelet acetylome will aim to identify p300 target substrates and specify roles for p300 in in the context of platelet activation, thrombus formation and pathologies of the cardiovascular system.

Acknowledgements:
This work is funded by the American Heart Association (13POST13730003 to J.E.A. and 13EIA12630000 to O.J.T.M.) and the National Institutes of Health (R01HL101972 to O.J.T.M.).
Abstract 212: Genetically Modified Mesenchymal Stem Cells Help Reduce Inflammation and Glucose-Mediated Insulin Response in Obese Diabetic Mouse Models
Authors: Sen Sabyasachi , Yadava Nagendra
Published: March 2022

MSCs are multipotent cells that differentiate into fat, muscle, bone and cartilage. High Glucose (HG) exposure of bone marrow derived MSCs leads to: increased ROS accumulation both in cytosol and mitochondria ( analysed by FACS using Mitosox Red dye) and increased intra-cellular lipid droplet accumulation. We hypothesized that intra-cellular antioxidant up-regulation specifically mitochondrial (super-oxide dismutase-2), may help reduce superoxide accumulation in mitochondria which will subsequently help reduce fat droplet accumulation and associated cellular inflammation.

We exposed hMSCs to HG (25mM) and normal glucose (NG, 5.5 mM) and interrogated mitochondrial superoxide accumulation and cellular oxygen consumption rate (OCR using Seahorse) and interrogated response to mitochondrial complex I-IV 4 substrate addition. We used AdSOD2 to up-regulate SOD2 prior to HG exposure. HG exposure increased TNF α (Tumor Necrosis Factor, 4 fold) and IL6 (interleukin6, 6-fold) mRNA and increased lipid accumulation (2.5 fold) and led to impaired OCR on SeaHorse and impaired Mitochondrial Complex 1.

With prior SOD2 upregulation in MSCs, TNF α and IL6 mRNA expression was reduced in HG and improved Complex-1 function.

Next we delivered the SOD2 upregulated MSC intra-peritoneally to obese diabetic (db/db) mice. We confirmed homing-in of eGFP labeled MSC, delivered IP, to different fat pockets. The mice that received MSC-SOD2 (experimental) showed reduced adipocyte and systemic inflammation (IL6 and TNF alpha levels) compared the mice that received MSC-Null (control). Interestingly the experimental group showed consistent reduction in fat mass (Echo-MRI), local adipocyte inflammation, systemic inflammation in serum and marked improvement in glucose tolerance tests (GTT).

We conclude that local delivery of mitochondrial superoxide dismutase using stem cells as a gene delivery vehicle reduces inflammation and glucose tolerance tests in vivo. We plan to carry out further glucose clamp studies to consolidate our findings. In conclusion local rather than systemic up-regulation of a mitochondrial anti-oxidant helps in reducing local and systemic inflammation, total fat mass and improves glucose mediated insulin response in obese diabetic mouse models.
Abstract 217: Cardiac Dysfunction in Offspring of Obese Multiparous Females
Authors: Rebholz Sandra , Jones Thomas , Cash James , Wortman Matthew , Tso Patrick , Woollett Laura
Published: March 2022

Emerging evidence indicates that maternal events prior to birth have a powerful impact on the risk for age-related diseases. Indeed, mothers who are obese during pregnancy confer an increased risk on their offspring for developmental programming of metabolic diseases. An often-overlooked risk factor for female obesity, and thus developmental programming, is multiparity. We have developed a novel mouse model in which multiparous mice become obese and develop severe inflammation − mirroring the pathology of obese multiparous humans. We have previously reported that the adult offspring of multiparous mice exhibit increased adiposity and a reduction in expression levels of genes associated with energy expenditure when fed low fat diets, despite similar food consumption. Recent studies show that these defects are associated with reduced energy expenditure and reduced mitochondrial function in hepatocytes of these mice compared to adult offspring of primiparous mice. Since a change in mitochondrial function in cardiomyocytes can lead to abnormal heart function, we fed male offspring a high fat diet (41% calories; milk fat) to stress the metabolic system. All mice accrued the same amount of fat on the high fat diets. We then and measured cardiac function using an echocardiogram. We found that there were significant differences in the ejection time, left ventricle mass, fractional shortening, and importantly in stroke volume and cardiac output of male offspring from females in their 1
st
vs 4
th
(multiparous) pregnancy. No differences in heart rate and aorta diameter were observed. These data suggest that the offspring of obese multiparous females are more prone to develop reduced cardiac function on a high fat diet, possibly indicating that maternal environment may be a novel risk factor for heart disease.
Abstract 218: Contributions of Microangiopathy to the Pathogenesis of Coronary Macroangiopathy of Nonobese Diabetes
Authors: Yamamoto Hiroaki
Published: March 2022

Background:
Visceral obesity and metabolic syndrome have been proposed as major coronary risk factors, however non-obese diabetes patients have also significant coronary atherosclerosis.

Purpose:
To elucidate the difference of prognosis between obese diabetes and non-obese diabetes.

Methods:
Up to December 2003, 606 patients who were initially treated with PCI, were recruited as cohort, and followed up for mean 10.3 years. Patients were divided into four groups according to diabetes and obesity. Endpoints are occurrence of new coronary lesions.

Results:
Non-obese diabetes had more frequently new coronary lesions than obese diabetic patients, indicating the incidence rate at 10 years was 56% and 31%, respectively (p=0.0017). At baseline, frequency of chronic kidney disease (CKD) was almost equal in these two groups, however, during follow-up, percentage of death associated with renal failure significantly increased up to 21.4% in non-obese diabetes, whereas 4.4% in obese diabetes. This increase is coincidence with occurrence of new coronary lesions, indicating diabetic microangiopathy is strong promoting factor in pathogenesis of coronary atherosclerosis in non-obese diabetes.

Conclusion:
In lean diabetes, microangiopathy especially nephropathy are frequently occurred, and this may contribute to progression of coronary atherosclerosis via the same mechanisms such as oxidative stress and glycation endproducts, or indirectly via secondary hypertension. these intriguing interaction of microangiopathy and macroangiopathy make unique coronary anatomy observed in diabetes.
Abstract 221: PDGF-Mediated Activation of CREB in Vascular Smooth Muscle Cells Alters Cell Cycling via Rb, p27kip1 and Fox01
Authors: Molnar Peter , Perrault Raissa , Zahradka Peter
Published: March 2022

Introduction:
Vascular injury initiates signals which lead to the secretion of humoral factors and ultimately to vessel repair. PDGF is one such factor that promotes conversion of smooth muscle cells (SMC) from their contractile state to the synthetic phenotype, which is characterized by the ability to migrate and proliferate. If not properly controlled, such changes can lead to neointimal hyperplasia or restenosis. As CREB has been shown to participate in vascular remodeling, it is plausible that it is an important player in PDGF-mediated phenotypic switch.

Hypothesis and aim:
We assessed the hypothesis that a relationship exists between CREB and PDGF-dependent proliferation of coronary artery SMCs. We also aimed to identify the pathways both upstream and downstream of CREB activation.

Methods:
Vascular SMCs from porcine explants and Western blotting were used to monitor protein levels and changes in phosphorylation resulting from various treatments. Multiple cell growth methods were applied to study cell proliferation. A dominant-negative CREB mutant and constitutive expression of p27kip1 were used to assess the involvement of these proteins in mediating the actions of PDGF.

Results:
Phosphorylation of CREB by PDGF was dependent on both Src and PI3 kinase, and partially dependent on MAPK. Dominant-negative CREB decreased PDGF-dependent cell proliferation by approximately 75%, and both PCNA expression and Ser-780 phosphorylation of Rb were inhibited. While no change in either cyclin D or cdk4 levels was observed, PCNA and Rb mediate the signals transduced through CREB. These cell cycle proteins likely controlled via p27kip1 expression in response to CREB-dependent Fox01 acetylation.

Conclusion:
CREB phosphorylation is required for SMC proliferation in response to PDGF. Additionally, p27kip1 mediates the actions of CREB in SMCs, likely as a result of changes in Fox01 activity.
Abstract 229: Development of a Conditional Knockout of Cholesterol Synthesis in the Mouse
Authors: Weerasekera Ranjuna , McKenzie Alison , Emmer Joseph , Cai Yanhong , Patel Kriya , Tyshynsky Roman , Patel Shailendra B
Published: March 2022

Dhcr24 (3β-hydroxysteroid-Δ24 reductase) is responsible for reducing the C24-C25 double bond in sterol intermediates of cholesterol and this step is necessary to synthesize cholesterol. In humans, mutations in the Dhcr24 gene cause desmosterolosis (OMIM #602398), characterized by severe developmental abnormalities and elevated levels of desmosterol in plasma and tissue. The first case was reported in 1998 when a premature infant died shortly after birth, had elevated levels of desmosterol and phenotypic abnormalities. Although initially reported as a ‘cholesterol-free mouse’ in Science, the global Dhcr24 knockout mouse shows neonatal lethality within 10h of birth, a finding reproduced in many different laboratories, despite sharing the original knockout mouse.

We now report a conditional knockout model using the LoxP-cre deletion system and show proof-of-principle by studying effects Dhcr24 deletion in the liver.
Abstract 250: Palmitic Interesterified Fat Induces Atherosclerosis and Inflammatory Cytokine Secretion in LDL Receptor Knockout Mice
Authors: Afonso Milessa S , Lavrador Maria Silvia F , Koike Marcia , Bombo Renata P , Nunes Valeria S , Catanozi Sergio , Castilho Gabriela , Passarelli Marisa , Nakandakare Edna R , Lottenberg Ana Maria
Published: March 2022

Rationale:
Interesterified fat was developed to substitute trans fat, once several epidemiological and clinical studies demonstrated its harmful effects on cardiovascular risk. However, the effects of these fats have not yet been completely elucidated. Thus, the aim of this work was to evaluate the effect of interesterified fats containing stearic and palmitic fatty acids on atherosclerosis development and cytokines secretion from peritoneal macrophages isolated from LDLr knockout mice.

Protocol:
Weaning male LDLr-KO mice were randomly divided into six groups (n=15-20) fed during 16 weeks on a high fat diet (40% of energy as fat) enriched with polyunsaturated (PUFA), TRANS, palmitic (Palm), palmitic interesterified (Palm Inter), stearic (Stear) or stearic interesterified (Stear Inter). Analyses: total plasma cholesterol and triglyceride concentrations, aortic macrophage infiltration (CD68 staining) and atherosclerosis (oil red O), and inflammatory biomarkers secretion were determined.

Results:
Dietary intake was similar in all groups. Interesterification failed to alter plasma cholesterol concentrations: Palm vs Palm inter (499.2±22.5 vs 526.8±20.7, p>0.05); Stear vs Stear inter (342.6±21.8 vs 363.9±22.5, p>0.05). Similar results were observed for triglycerides: Palm vs Palm inter (309±49 vs 303±33, p>0.05); Stear vs Stear inter (239±28 vs 237±27, p>0.05). As expected, TRANS induced the largest lesion area. However, Palm Inter induced the highest lesion area and macrophage infiltration as compared to Palm, Stear, Stear Inter and PUFA groups. Concerning pro inflammatory cytokines secretion, harmful effects of Palm inter were also observed for IL-6, IL-1β and MCP-1.

Conclusion:
Interesterified fats containing palmitic acid increased pro inflammatory cytokines secretion that contributed to aortic macrophage infiltration and atherosclerosis development.
Abstract 255: Association of 24-Hour Heart Rate and Rate Pressure Product With Atherosclerosis Risk Factors in Nonhypertensive Individuals
Authors: Cheng Cynthia , Daskalakis Constantine
Published: March 2022

Heart rate (HR) is an established marker of sympathetic nervous system (SNS) activity and an independent predictor of cardiovascular morbidity and mortality. However, the exact mechanism by which the SNS mediates its deleterious effects remains unknown. The main objective of this study was to determine if 24-hour mean HR and heart rate pressure product (HR24 and RPP24) obtained using ambulatory blood pressure (BP) monitoring are associated with atherosclerosis risk factors in young-middle aged non-hypertensive patients in the preclinical phase of disease.

Data were obtained from 186 non-hypertensive individuals aged 18-45. HR24 and RPP24 were significantly associated with known atherosclerosis risk factors (elevated SBP/DBP (p< 0.001), endothelial dysfunction (p< 0.05), insulin resistance (p< 0.001), and inflammatory adipokines: CRP (p< 0.001), IL-6 (p< 0.001), and PAI-1 (p< 0.001). These data suggest that elevated HR and RPP may be potential markers of elevated risk for future atherosclerotic events in these asymptomatic individuals, prior to the development of clinically defined hypertension.