bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 246: Thoracic versus Abdominal Aortic Stiffness in Young and Old Non-Human Primates
Authors: Zhao Xin , Sun Zhe , Zhu Yi , McNulty Tara , Colman Ricki , Vatner Dorothy E , Meininger Gerald A , Vatner Stephen F
Published: March 2022

We demonstrated that thoracic aortic stiffness is increased in aging monkeys, with the mechanism involving increased vascular smooth muscle cell (VSMC) stiffness. In view of the importance of increased abdominal aortic stiffness to greater incidence of abdominal aortic aneurysms, the goal of this investigation was to determine differences in thoracic vs. abdominal aortic stiffness in young and old monkeys (fascicularis and rhesus). Most prior studies on aortic stiffness have been conducted in vitro or in anesthetized animals with indirect measurements of stiffness. A novel feature of the current investigation was to measure aortic diameter and pressure instantaneously and continuously in chronically instrumented, conscious young (n=25, 5-12yrs) and old male (n=21, >24yrs) monkeys, instrumented with aortic pressure gauges and ultrasonic aortic diameter crystals on the thoracic and abdominal aorta. The aortic stiffness index, β[[Unable to Display Character: ]][[Unable to Display Character: ]]calculated as a ratio of systolic and diastolic aortic pressures and diameters, was markedly greater, p<0.05, in abdominal, compared with thoracic, aorta in both young (33±5.0 in abdominal vs. 14±2.2 in thoracic) and old monkeys (57±6.3 in abdominal vs. 34±0.4 in thoracic). Although both thoracic and abdominal aortic stiffness were greater in old monkeys, p<0.05, surprisingly, abdominal aortic stiffness in young monkeys (33±5.0) was equal to thoracic aortic stiffness in old monkeys (34±0.4). Using atomic force microscopy, aortic VSMC stiffness was 2 fold greater, p<0.05, in old monkeys vs. young monkeys, but there were no differences in abdominal vs. thoracic aortic VSMC stiffness in either young or old monkeys. In summary, as expected, aging increased aortic stiffness, with the mechanism, in part, due to increased VSMC stiffness. However, aortic stiffness was increased more in the abdominal than thoracic aorta both in young and old monkeys, with the magnitude of the increase in the abdominal aorta of the young monkeys, similar to that which occurs with aging in the thoracic aorta. These findings point out the major differences in stiffness along the aorta and have relevance to the greater frequency of abdominal aortic aneurysms.
Abstract 247: Adipose-derived Stem Cells Suppress Aortic Inflammation and Aneurysm Expansion through Paracrine Mediated Mechanisms in an Elastase-induced Murine Model
Authors: Murphy Michael; P , Xie Jie , Jones Thomas , Feng Dongni , Cook Todd G , Babbey Clifford M , March Keith L
Published: March 2022

Introduction:
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 cells 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 fluorescent microscopy and confirmed by real-time PCR for human-specific Alu sequence.

Results:
ASC significantly suppressed AAA expansion at 14 days (n=10/group, p<0.01). At Day 1 there was a significant increase in Tregs 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 (insert CD16++?) 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). Hoescht 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 ASCs induced Treg, polarize macrophages to the M2 phenoype, 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 suppresses AAA formation by inducing anti-inflammatory immune cell phenotypes. 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 250: Oxidative Stress Mediated Increase of HMGB-1 and RAGE cCntributes to Ascending Aortic Aneurysm Development in vivo.
Authors: Ferrari Giovanni , Lai Eric K , Grau Juan B , Levy Robert J , Gorman Robert C , Gorman Joseph H , Bavaria Joseph E , Branchetti Emanuela
Published: March 2022

Background:
Ascending-Thoracic Aortic Aneurysm (TAA) is a life-threatening condition for which the only treatment available is the surgical replacement/repair of the dilated aorta. It is known that oxidative stress (ROS) plays an important role in aortic wall remodeling involved in TAA: ROS induces pathological changes in vascular smooth muscle cells (VSMC) and triggers a disorganized and excessive production of matrix components and mediators of elastolysis. We demonstrated that chronic infusion of Angiotensin II (AngII) induces TAA formation in mice with concomitant accumulation of both ROS and synthetic VSMC, similar to that seen in human aneurysmal tissues. We also demonstrated that two ROS triggered molecules, the Receptor for Advanced Glycation end product (RAGE) and its ligand HMGB-1/amphoterin, are highly expressed in dysfunctional human aortic tissue. Recently a few microRNAs (miRs) have been proposed to play a role in aneurysm formation. We found that miR-143 is significantly down-regulated in human TAA tissues. We hypothesized that miR-143 may play a role in TAA development by regulating RAGE/HMGB-1 expression.

Methods:
RAGE and HMGB-1 expression was tested in control and aneurysmal human ascending aorta. C57BL6/J mice were fed a hypercholesterolemic diet and infused with saline or Ang II (1000 ng/kg/min) for 28 days. MiR-143, HMGB-1 and RAGE expression was evaluated by Western blotting and qPCR in murine ascending aorta samples and in human aortic VSMC treated with AngII (100nM) for 4 and 24 hrs.

Results:
Human aneurysmal aorta shows down-regulation of miR-143 concomitant with up-regulation HMGB-1 and RAGE expression. AngII infusion induces TAA in mice together with up-regulation of HMGB-1 and RAGE and miR-143 down-regulation. AngII treatment of VSMC induces RAGE and HMGB-1 expression and down-regulation of miR-143. A miR target analysis, using bioinformatic miR-mRNA algorithm tools, identified HMGB-1 as a direct target of miR-143.

Conclusion:
Our data suggest that miR-143, RAGE and HMGB-1 may cooperate in the process of aortic remodeling behind TAA formation. Further studies, investigating the interplay between miR-143 and HMGB-1/RAGE activation, may lead to potential new pharamacological targets for the treatment of TAA.
Abstract 26: Who is Going to Stroke? Biomarkers for the Unstable Carotid Atheroma
Authors: Hedin Ulf , Matic Perisic Ljubica , Iglesias Maria Jesus , Lengquist Mariette , Razuvajev Anton , Roy Joy , Pontén Fredrik , Odeberg Jacob
Published: March 2022

Unstable carotid stenosis is an important source of extracranial atheroembolism and stroke. Current interventional therapy is based on surrogate markers for stroke risk and no precise diagnostic tool is available for identification of either individuals or lesions at risk. Thus, biomarkers for the unstable carotid atheroma may improve selection of patient for intervention.

Here, a high-throughput pipeline for identification of circulating biomarkers for unstable carotid atherosclerosis was developed. Carotid plaques were part of the Biobank for Karolinska Endarterectomies (BiKE) and obtained from patients treated for symptomatic (S) and asymptomatic (AS) carotid stenosis together with peripheral plasma and ‘local’ plasma (n = 47) sampled after clamping prior to arteriotomy. Screening of proteins was processed through Luminex based, suspension-bead assays using over 10,000 antibodies from the Human Protein Resource (HPR) and compared to peripheral plasma (n = 200 total). Plasma analysis identified 150 significantly dysregulated candidates based on three comparisons: proteins enriched in local plasma compared to peripheral plasma; in local plasma from S compared with AS patients; in peripheral plasma from S compared with AS patients. Pathway analysis revealed that detected proteins were related to ossification, lysosomes, magnesium transport, prenylation, collagens, lipid metabolism, and transcription factors. Identified candidate proteins were further filtered using public database mining, comparisons with transcriptomic data from BiKE, and qPCR/IHC in matched endarterectomies. Among the identified candidates, THEMIS2 and BLVRB were examples of proteins successfully validated as upregulated in both plaques and plasma from S patients and localized to CD68+ macrophages.

Extensive analysis of plasma and tissue samples from patients undergoing carotid surgery permitted identification of several potential biomarkers for unstable carotid atherosclerosis. Further validation in larger cohorts of unstable atherosclerosis is necessary in order to determine predictive power for the identification of individuals at risk as well as for the development of targeted bioimaging for lesion detection.
Abstract 265: Lipoprotein (a) Predicts Carotid Plaque, Extent of Coronary Artery Disease and Improves Discrimination and Risk Reclassification Beyond Conventional Risk Factors
Authors: Ooi Esther M , Barrett P Hugh R , Watts Gerald F , McQuillan Brendan M , Beilby John P , Thompson Peter L , Hung Joseph
Published: March 2022

Background and Aims:
Lipoprotein (a) [Lp(a)] has been identified as a risk factor for cardiovascular disease (CVD). We examined whether Lp(a) predicts subclinical atherosclerosis, extent of coronary artery disease and modifies discrimination and risk reclassification of CVD beyond conventional risk factors.

Methods:
Plasma Lp(a) and CVD risk factors were assessed in a random community-based cohort (Carotid Ultrasound Disease Assessment Study, CUDAS) and a cohort with premature angiographically defined coronary artery disease (CAD) (Carotid Ultrasound in Patients with Ischemic Heart Disease, CUPID). Carotid intima media thickness (IMT) and focal plaque were assessed by carotid B-mode ultrasound in both cohorts. The extent and severity of CAD was determined by the modified Gensini and Coronary Artery Stenosis ≥20% (CAGE) scores in CUPID.

Results:
In pooled analyses of 1414 individuals (830 men and 584 women) aged 20 to 70 years, Lp(a) was associated with risk of myocardial infarction and/or stroke [Odds Ratio (95% Confidence Intervals)][1.14 (1.05, 1.24)] and presence of focal plaque [1.08 (1.01, 1.16)], independent of age, gender and cardiometabolic risk factors (including body mass index, triglycerides, smoking, diabetes, hypertension, hypercholesterolemia and family history of CVD). Lp(a) was not associated with IMT. In CUPID, Lp(a) was an independent determinant of the Gensini score and in CUPID men, the CAGE ≥20% score (all P<0.01). The addition of Lp(a) to the basic model (age, gender and cardiometabolic risk factors) improved the C-statistics, integrated discrimination index and category-free net reclassification improvement for myocardial infarction and/or stroke.

Conclusions:
Lp(a) predicts risk of myocardial infarction and/or stroke, carotid plaque and extent of CAD. The addition of Lp(a) improves discrimination and risk reclassification of myocardial infarction and/or stroke beyond conventional risk factors.
Abstract 27: Therapeutic Targeting of Human Lipid Genes With in vivo CRISPR-Cas9 Genome Editing
Authors: Raghavan Avanthi , Chen Tao , Kuperwasser Nicolas , Ding Qiurong , Musunuru Kiran
Published: March 2022

Individuals with naturally occurring loss-of-function PCSK9, APOC3, and ANGPTL3 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels, reduced blood triglyceride levels, and/or protection against cardiovascular disease. We recently established that genome editing using a Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, delivered by adenovirus, can efficiently introduce loss-of-function mutations into the endogenous mouse Pcsk9 gene in vivo, resulting in near-complete knockout of Pcsk9 and a 35%-40% reduction in the blood cholesterol level. Thus, the in vivo genome-editing approach may have therapeutic potential for the prevention of cardiovascular disease in humans. To better evaluate the feasibility of the approach in humans, we are performing preclinical studies using a liver-humanized mouse model in which the endogenous mouse hepatocytes have been replaced with human-derived hepatocytes and in which the plasma lipoprotein profile better reflects human physiology, particularly with respect to LDL-C. One disadvantage of the S. pyogenes CRISPR-Cas9 system is that its Cas9 gene is too large to fit in adeno-associated virus (AAV) vectors, which offer significant therapeutic advantages over adenoviruses. We have adapted the smaller Staphylococcus aureus CRISPR-Cas9 system and demonstrated efficient genome editing of the human PCSK9, APOC3, and ANGPTL3 genes in vitro. Additionally, using a minimal liver-specific promoter and a novel human-hepatocyte-optimized AAV capsid protein, we are employing AAV vectors to knock out each of the three genes in both primary human hepatocytes and liver-humanized mice. This is allowing us to interrogate both the efficacy (lowering of LDL-C and/or triglycerides) and safety (degree of off-target mutagenesis) of genome editing in human hepatocytes in vitro and in vivo. These translational studies are providing critical information on the viability of an approach that could ultimately yield a one-shot, long-term therapy that permanently reduces a person’s blood LDL-C and triglyceride levels, thus serving as a “vaccination” against cardiovascular disease.
Abstract 270: IκB Kinase β Signaling in Adipose Progenitor Cells Promotes Obesity and Metabolic Disorders
Authors: Helsley Robert N , Sui Yipeng , Liu Zun , Park Se-Hyung , Zhou Changcheng
Published: March 2022

Objective:
Obesity is associated with a state of chronic low-grade inflammation that is a major contributor to diabetes and cardiovascular diseases. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been implicated in the pathogenesis of obesity-associated metabolic disorders. However, the role of IKKβ in adipose tissue development and metabolism remains elusive. We have recently revealed a potential role of IKKβ in the regulation of adipocyte differentiation. This study aims to investigate the impact of adipose progenitor cell IKKβ deficiency in diet-induced obesity and metabolic disorders.

Approach and Results:
To determine the role of IKKβ in adipose tissue development, we selectively deleted IKKβ in the white adipose lineage. This was attained by using a PDGFRβ-Cre/IKKβ-loxP system. Cell lineage analysis confirmed that PDGFRβ-Cre is active in primary adipose stromal vascular (SV) cells and PDGFRβ-positive SV cells had higher adipogenic potential than did PDGFRβ-negative cells. PDGFRβ-Cre-mediated IKKβ deletion diminished the ability of SV cells to differentiate into adipocytes. By analyzing mice that selectively lacked IKKβ in the white adipose lineage, we found that deficiency of IKKβ protected mice from high-fat diet-induced obesity. Moreover, IKKβ deficient mice had decreased plasma proinflammatory cytokine levels and enhanced insulin sensitivity. Mechanistically, IKKβ deficiency inhibited proteasome-mediated β-catenin ubiquitination and degradation in adipose SV cells, causing an accumulation of nuclear β-catenin protein levels and impaired adipocyte differentiation. Consistently, CRISPR-mediated IKKβ deletion in murine 3T3-L1 preadipocytes also increased nuclear β-catenin protein levels and blocked 3T3-L1 cell differentiation.

Conclusions:
Our results establish IKKβ as an important regulator of adipogenesis and adipose tissue development. Overnutrition-mediated IKKβ activation may serve as an initial signal that triggers adipose progenitor cell differentiation in response to consumption of a high-fat diet. Inhibition of IKKβ may present as a novel therapeutic approach to combat obesity and diabetes.
Abstract 276: Postprandial Effects on Monocyte Phenotype in Obese Humans With Metabolic Syndrome
Authors: Wu Huaizhu , Khan Ilvira , Pokharel Yashashwi , Dadu Razvan , Lewis Dorothy , Hoogeveen Ron , Ballantyne Christie
Published: March 2022

Monocytes are heterogeneous with 3 subsets in humans, identified by CD14 and CD16 levels and differing in numerous other markers. Under certain stimuli, monocytes undergo phenotypic changes, with accelerated trafficking into tissues, which play crucial roles in inflammation including atherogenesis. We used multicolor flow cytometry to examine postprandial effects of a high-fat meal, containing 910 kcal (53% from fat, 23% from saturated fat) and 535 mg cholesterol, on monocyte phenotype in 11 obese subjects with metabolic syndrome (MS) (BMI: 34.8 ± 2.2 kg/m2; fasting triglyceride [TG]: 217 ± 42 mg/dl) and 11 gender- and age-matched lean controls (BMI: 24.0 ± 0.7 kg/m2; fasting TG: 89 ± 10 mg/dl, P<0.01 vs obese group). Blood was taken after an overnight fast (T0) and at 3 (T3) and 5 (T5) hours after high-fat meal. At T0, obese subjects had higher counts of CD14dim/CD16+ nonclassical monocytes (ncM), but not CD14+/CD16- classical (cM) and CD14+/CD16+ intermediate monocytes (iM), than leans. Mean fluorescent intensity (MFI) of CD11c, CCR5 and HLA-DR, and percentages of TNFα+ and IL-1β+ cells were higher on/in iM and ncM than on/in cM (P<0.05) in each individual at T0, supporting inflammatory phenotype of iM and ncM. Compared to lean, obese subjects had higher levels of CD11c on iM and cM, CX3CR1 on iM and CCR5 and TNFα on/in cM and also higher proportion of Nile-red+ lipid-laden foamy monocytes at T0 (P<0.05), which correlated positively with fasting TG levels (P=0.02, r=0.53). After high-fat meal, both obese and lean groups had significant increases in TG levels (TG incremental area under the curve: 344 ± 52 in obese versus 225 ± 28 in leans, P=0.05). The proportions of foamy monocytes were significantly higher at T3 than T0 (P<0.05) in lean subjects and tended to be higher at T3 and T5 than T0 in obese subjects. Obese but not lean subjects had significant increases in CD11c MFI and percentages of IL-1β+ and TNFα+ cells in ncM at T3 compared to T0. In summary, in both obese and lean groups, iM and ncM had proinflammatory phenotype. Compared to leans, obese subjects with MS had increased lipid accumulation and enhanced inflammatory phenotype in monocyte subsets at baseline and showed further increases in inflammatory markers, particularly in ncM, after a high-fat meal.
Abstract 291: Multiple Mechanisms for the Inhibition of Intimal Hyperplasia by Herpes Simplex Virus Strain R7020
Authors: McCormick Sue , Skelly Christopher L
Published: March 2022

Intimal hyperplasia (IH) is the primary cause of vein bypass graft failure. Smooth muscle cells (SMCs) are a key element of IH as they phenotypically switch from a contractile to a synthetic state which may lead to IH. R7020 is an engineered strain of Herpes Simplex Virus-1 which inhibits IH in animal models. Although it has many characteristics which make it a strong candidate for use as a prophylaxis agent how it inhibits IH is not well understood. IH is multifaceted and R7020 infects a wide range of cell types regulating their cellular functions and pathways. Therefore we hypothesized that R7020 inhibits IH by altering multiple cellular functions and cell types. The objective of this study was to identify mechanisms used by R7020 to function in blood vessels that may contribute to its inhibition of IH. In this study the cytopathic effect of R7020 on SMCs was determined in vitro and in a rabbit IH model. The effects of R7020 on SMC viability and the adhesion of peripheral blood mononuclear cells (PBMCs) to SMCs were quantified in the absence and presence of TNF-α. Microarray analysis was used to identify genes whose expression was altered by R7020 in vivo. The cytopathic effects which include filopodia positive for R7020 tegument proteins extending from cell to cell and the formation of syncytia suggest that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid. The directionality of the filopodia in vivo suggests that the virus preferentially travels from the media towards the intima targeting the SMCs that would lead to IH. The formation of syncytia would also inhibit SMC proliferation as cells that form syncytia do not proliferate. In addition, R7020 induced the fusion of PBMCs with syncytia which may limit the effect of macrophages on IH. Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH. Microarray analysis detected a significant change in the expression of 58 genes in R7020 infected arteries relative to non-infected at 24 h. The encoded proteins are associated with the immune system, cell death, proliferation/motility and vascular tone. Thus this study identifies multiple mechanisms which R7020 may be using to inhibit IH.
Abstract 3: Cholesterol Crystals Produced by Endothelial Cells During Hyperlipidemia Initiate Atherogenesis
Authors: Baumer Yvonne , McCurdy Sara , Weatherby-Carvalho Tina , Boisvert William A
Published: March 2022

Atherosclerosis is caused by prolonged dyslipidemia and chronic inflammation. Although much is known about advanced stages of atherosclerosis, events leading to its initiation are poorly understood. Cholesterol crystals (CC) have been associated with advanced plaque, but their origin and potential role in the initiation of atherosclerosis and early plaque development are unknown. Using polarized light, scanning and transmission electron microscopy we show for the first time that CC are present in the very early plaque induced after only 1 week of high fat diet in ldlr-null mice. We determined that the source of CC is the endothelial monolayer by treatment of human aortic endothelial cells with LDL for 5 days. Other cell types like monocytes, fibroblasts and human umbilical vein endothelial cells failed to form CC upon LDL, AcLDL or oxLDL treatment. To identify in vitro formed CC we used polarized light, transmission electron and confocal microscopy. Furthermore, we identified that CC are generated within autophagolysosomes of the endothelium. The formation of CC by endothelial cells is accompanied with changes in intracellular cAMP levels. The consequences of early subendothelial CC formation are incomplete junction formation with subsequent impaired endothelial barrier integrity, processes known to be involved in atherosclerosis initiation and progression. Interestingly, increase of intracellular cAMP inhibited endothelial CC production in vitro and in vivo. By performing western Blot, flow cytometry and various microscopy techniques we could show that the inhibition of CC-formation as the results of cAMP increase is accompanied by a decrease autophagolysosomes as the CC producing organelles. We could furthermore demonstrate that this protective effect of cAMP could be inhibited by additional application of an eNOS inhibitor.

In summary, we could demonstrate that CC are present in very early stages of atherosclerosis. These early subendothelial CC depositions are produced by autophagolysosomes within the endothelial monolayer in a cAMP/eNOS dependent manner. With these findings we might have found the so far missing link to initiation of plaque development in atherosclerosis.
Abstract 306: A Protective Role of IL-1 beta in Abdominal Aortic Aneurysms
Authors: Ruhlman Melissa , Carson Jeffrey , Xiong Wanfen , Dale Matthew , Meisinger Trevor , Baxter B T
Published: March 2022

Objectives:
Abdominal aortic aneurysm (AAA) is a chronic disease characterized by persistent inflammation maintained by an array of inflammatory cells and mediators. Interleukin 1 beta (IL-1β) has largely been considered a pro-inflammatory cytokine but, interestingly, our previous studies of human AAAs showed that activated T-cells from patients with AAAs expressed lower levels of IL-1β compared to matched control patients without AAAs. This led us to hypothesize that IL-1β could have a protective role in preventing the development of AAA.

Methods:
Abdominal aortic aneurysms were induced in IL-1 receptor KO (IL-1R
-/-
) and control mice using the CaCl
2
murine model of AAA. Mice were sacrificed six weeks after aneurysm induction. Aortic diameters were measured and compared at the time of aneurysm induction and sacrifice. Aortic samples were further evaluated histologically using connective tissue staining to assess the aortic structure in both groups.

Results:
At the time of sacrifice, IL-1R
-/-
mice demonstrated a 73.9 ± 3.3% increase in aortic diameter compared to a 25.9 ± 5.8% increase in wild type control mice (p<0.05). On histology, IL-1R
-/-
mice had more severe disruption of the aortic architecture compared to control mice. These data suggest that in the absence of IL-1β signaling, mice are more susceptible to aneurysms and develop more severe pathology.

Conclusion:
The current mouse data, in combination with our previous human studies, suggest a protective role for IL-1β in abdominal aortic aneurysm. These results are unexpected given that related pro-inflammatory cytokines TNF-α and IFN-γ both promote aneurysm formation.
Abstract 34: Elevated Plasma CETP Activity and Hyper-procoagulant R451Q-CETP Mutation Linked to Venous Thrombosis Risk
Authors: Deguchi Hiroshi , Xu Xiao , Elias Darlene J , Griffin John H
Published: March 2022

Cholesteryl ester transfer protein (CETP) is an important lipid transfer factor in plasma that enhances prothrombinase activity in purified reaction mixtures. To test the hypothesis that CETP activity is linked to venous thrombosis (VTE), we measured CETP lipid transfer activity in plasmas of 49 male VTE patients (age < 55 yrs old) and matched controls. CETP activity levels were significantly higher for VTE patients than controls (p= 0.0008) (see Figure panel A). Elevated CETP activity (> 90th percentile of controls) was significantly associated with VTE risk (odds ratio= 4.1 (95%CI, 1.4-13)). A subset of patients carrying CETP mutations A373P and R451Q, which were also linked to VTE risk, showed significantly higher plasma CETP activity compared with non-carriers (110 % (105-115 %) vs 80 % (66-92 %) (p=0.0004)). To test the hypothesis that these mutations enhance procoagulant activity of CETP, we made recombinant CETPs in a stable HEK293 cell expression system and measured the procoagulant activity of recombinant wt-CETP, A373P-CETP, and R451Q-CETP that were added to reaction mixtures containing factors Xa, Va, prothrombin and lipids. Quite remarkably, the R451Q-CETP mutant had five-fold higher thrombin generation activity compared to wt-CETP or A373P-CETP (see Figure panel B). In summary, our data show that (1) elevated CETP lipid transfer activity in plasma was associated with the risk of VTE and (2) recombinant R451Q-CETP which is linked to VTE has much higher procoagulant activity than wt-CETP. These clinical and basic findings indicate that the lipid transfer activity of CETP may contribute to risk for thrombosis and that further studies of this hypothesis are merited.
Abstract 355: Dyslipidemia, Obesity, Insulin Resistance and Atherosclerosis in LDL-Receptor Deficient Rats
Authors: Hamed-Berair Rihab E , Lorkiewicz Pawel , Sithu Srinivas D , Riggs Krista A , Malovichko Marina , Winner Millicent G , Agarwal Abhinav , Riggs Daniel W , Bhatnagar Aruni , Srivastava Sanjay
Published: March 2022

The LDL-receptor (LDLR) plays a major role in removing circulating LDL. Deletion of the receptor in mice results in profound dyslipidemia and atherosclerotic lesion formation. We established a new LDLR deficient (KO) rat model of atherosclerosis. When maintained on normal chow (NC), the KO rats displayed a 2.5-fold increase in plasma total cholesterol and 1.5 to 2.0- fold increase in triglycerides levels than corresponding WT rats. Lipidomics analysis of the plasma showed that deficiency of LDLR significantly (P<0.05) affected the abundance of 65 lipids. On normal chow, KO rats gained more weight and were more glucose intolerant than WT rats. Plasma leptin levels were higher and adiponectin levels were lower in KO than WT rats. Untargeted metabolomics analysis of polar extracts of plasma revealed that deficiency of LDLR differentially regulated the abundance of 20 metabolites (P<0.05). Measurement of circulating micro RNAs showed differential regulation of 35 micro RNAs, several of which are associated with obesity, insulin resistance and dyslipidemia. On Western diet (WD), the KO rats displayed exaggerated obesity and age-dependent increase in glucose intolerance. Their plasma levels of adiponectin and leptin were lower in comparison with WT rats. Lipidomics and metabolomics analyses of plasma showed differential regulation of 53 lipids (P<0.05) and 9 polar metabolites (P<0.05) as compared with WT rats maintained on WD. No appreciable aortic lesions were observed in KO rats fed NC for 64 weeks or WD for 16 weeks; however after 34-52 weeks of Western diet, the KO rats developed exuberant atherosclerotic lesions in the aortic arch and throughout the abdominal aorta. Several microRNAs associated with atherosclerosis, insulin resistance and obesity were also differentially regulated in the KO rats maintained on WD. These data suggest that LDLR-KO rat may be a useful model for studying obesity, insulin resistance, and atherosclerosis; and a panel of signature plasma lipids, polar metabolites and circulating microRNAs may serve as biomarkers of atherosclerosis and its risk factors.
Abstract 357: Intimal Smooth Muscle Cells Are Vascular Tissue Specific Innate Immune Effector Cell
Authors: Jiang Xintong , Hansson Göran K , Yan Zhongqun
Published: March 2022

Intimal smooth muscle cells (SMC) represent the largest cell population in atherosclerotic plaques and restenotic lesions. These cells have multiple functions that differ from those of SMC of tunica media. Compared with medial SMC, intimal SMC are hyperresponsive to a large array of endogenous and exogenous inflammatory stimuli. However, the underlying molecular basis is elusive.

Analyzing the phenotype of smooth muscle cells (SMC) isolated from balloon injury induced neointima of rat carotid artery, we found that unlike SMC of tunica media, the SMC of the intima exhibit an innate immune phenotype, characterized by constitutively expressing a broad panel of pathogen pattern recognition receptors (PRR) seen typically in macrophages. These PRR confer on intimal SMC an ability to react to an extensive list of microbial components by producing inflammatory mediators including nitric oxide, cytokines and chemokines. Additionally, intimal SMC produce the leukocyte chemokine CCL-5 (or RANTES) in the absence of exogenous stimuli, implying an intrinsic innate immune activity. The master program that determines the phenotype of intimal SMC remains to be defined but may involve a number of transcription pathways. These observations suggest that intimal SMC are vascular tissue specific innate immune effector cells with functions similar to those of macrophages.
Abstract 359: CD4+ Natural Killer T Cells Promote Atherosclerosis via Cytotoxic Mechanism
Authors: Li Yi , To Kelly , Kanellakis Peter , Hosseini Hamid , Deswaerte Virginie , Tipping Peter , Smyth Mark , Toh Ban-Hock , Bobik Alex , Kyaw Tin
Published: March 2022

CD4
+
NKT cells are atherogenic lymphocytes, but the mechanisms by which they promote atherosclerosis are not known. Here we investigated the role of other lymphocytes and NKT cell-derived cytokines and cytotoxins in NKT cell’s atherogenicity. First, CD4
+
NKT cells were adoptively transferred into ApoE
-/-
mice; deficient in T, B cell and NKT cells (ApoE
-/-
Rag2
-/-
) and in T, B, NKT and NK cells (ApoE
-/-
Rag2
-/-
γc
-/-
). At the end of 8 week high fat diet, CD4
+
NKT cells augmented aortic root atherosclerosis assessed by total intimal lesion area, lipid content and macrophage accumulation in both ApoE
-/-
Rag2
-/-
mice and ApoE
-/-
Rag2
-/-
γC
-/-
mice. These data indicate that CD4
+
NKT cells can exert atherogenic effects in the absence of other lymphocytes. As NKT cells secrete cytokines and cytotoxins, we next investigated the role of NKT cell-derived cytokines and cytotoxins in atherosclerosis development. CD4
+
NKT cells from mice deficient of IFN-γ, IL-4 and IL-21 cytokines and perforin and granzyme B cytotoxins were transferred into NKT cell-deficient ApoE
-/-
Jα18
-/-
mice; controls were ApoE
-/-
Jα18
-/-
mice that received PBS or wildtype NKT cells. At completion of 8 week high fat diet, wildtype CD4
+
NKT cells and those deficient in IL-4, IFN-γ or IL-21 increased total intimal lesion area by ~65%, ~95%, ~80% and ~70% compared to vehicle control mice respectively. In contrast CD4
+
NKT cells deficient in perforin or granzyme B failed to augment lesion size, lipid content and macrophage accumulation. Apoptotic cells, necrotic cores and proinflammatory VCAM-1 and MCP-1 were reduced in mice receiving perforin-deficient NKT cells. Our data suggest that CD4
+
NKT cells require perforin and granzyme-B for atherosclerosis development, thereby increasing apoptotic and necrotic cells in lesions that in turn augments inflammation. Targeting NKT cell apoptotic cell mediators may be useful in attenuating atherosclerosis.
Abstract 372: Tumor Necrosis Factor-α Regulates Triggering Receptor Expressed on Myeloid Cells-1-dependent Matrix Metalloproteinases in the Carotid Plaques of Symptomatic Patients with Carotid Stenosis
Authors: Rao Velidi H , Stoupa Samantha , Rai Vikrant , Agrawal Devendra K
Published: March 2022

Aims:
Unstable atherosclerotic plaques in carotid artery are characterized by rupture of their fibrous cap, leading to transient ischemic attack or stroke. However, the underlying mechanisms are unclear. Our aim was to investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on plaque stability involving matrix metalloproteinases (MMPs) and examine the effect of inflammatory cytokine TNF-α in symptomatic (S) compared to asymptomatic (AS) patients with carotid stenosis.

Methods and Results:
The mRNA transcripts for TREM-1, MMPs (MMP-1, MMP-3, and MMP-9) and collagen type I and III were analyzed by qPCR and immunofluorescence, respectively in whole plaque and plaque smooth muscle cells (pSMCs) isolated from carotid endarterectomy tissues of patients with carotid stenosis. The mRNA transcripts of TREM-1, MMP-1 and MMP-9 were increased while Col I and Col III mRNA transcripts were decreased in pSMCs of S compared to AS patients. Stimulation of pSMCs with TNF-α further increased the mRNA transcripts of TREM-1 and MMPs without any additional effect on Col I (α1) or Col III (α1) mRNA levels. The inhibitors of NF-kB (BAY11-7085), JNK (SP600125) and PI3K (LY294002) signaling pathways attenuated the effect of TNF-α. Modulation of TREM-1 in TNF-α-treated pSMCs by the administration of TREM-1 decoy receptor rTREM-1/Fc, monoclonal antibodies, or siRNA to TREM-1 attenuated the expression of MMPs and Col I (α1) and Col III (α1) in both S and AS groups.

Conclusion:
Increased expression of TREM-1 in pSMCs of symptomatic than in asymptomatic patients suggest a potential role of TREM-1 in plaque destabilization. This is further supported by the pronounced effect of inflammatory cytokine, TNF-α, to increase of mRNA transcripts for TREM-1 and MMPs. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.
Abstract 40: Increased Expression of Transforming Growth Factor-Beta 1 in the Gastrocnemius of Patients with Peripheral Artery Disease is a Localized Response in the Ischemic Muscle
Authors: Ha Duy M , Carpenter Lauren A , Casale George P , Kim Julian K , Swanson Stanley A , Koutakis Panagiotis , Papoutsi Evlampia , Pipinos Iraklis I
Published: March 2022

Introduction:
Patients with peripheral artery disease (PAD) develop a myopathy in their ischemic lower extremities, which is characterized by myofiber degeneration and myofibrosis. We previously showed that increased expression of transforming growth factor-beta 1 (TGF-β1), which stimulates collagen deposition, is associated with increased fibrosis in PAD gastrocnemius. Expression was localized to the microvasculature, suggesting that elevated TGF-β1 may be a systemic condition rather than a local response in PAD patients.

Hypothesis:
We hypothesize that increased expression of TGF-β1 in PAD gastrocnemius is a localized response in the ischemic muscle, which contributes to the myofibrosis of PAD. To test this hypothesis, we evaluated TGF-β1 expression and collagen deposition in the gastrocnemius of the diseased and contralateral legs of patients with unilateral PAD.

Methods:
Gastrocnemius biopsies were collected from both limbs of PAD patients with unilateral disease (n=10). Muscle homogenates were analyzed by qPCR for TGF-β1 transcripts expressed as fold change in relation to myosin heavy chain. TGF-β1 in paraffin-embedded gastrocnemius sections was labeled for fluorescence analysis and expression measured as grey scale units (gsu) by quantitative fluorescence microscopy. Sections were stained by Masson Trichrome and collagen density was measured by spectral analysis.

Results:
TGF-β1 transcripts were increased in the diseased compared to contralateral muscle (mean +/- s.e.; 4.65 +/- 0.95 vs. 0.61 +/- 0.15; p = 0.007) as was TGF-β1 protein expression (919 +/- 169 gsu vs. 593 gsu +/- 115; p = 0.013). Increased TGF-β1 expression was accompanied by increased collagen deposition in the diseased versus contralateral muscle (2162 gsu +/- 99 vs. 1739 gsu +/- 73; p = 0.007).

Conclusions:
TGF-β1 expression in PAD gastrocnemius does not represent a systemic condition that may interact with local ischemic events and thereby contribute to PAD myopathy. Instead, it is an integral and localized response in ischemic muscle, which stimulates fibrosis in the diseased muscle. Consequently, successful intervention in the fibrotic response of PAD muscle may be dependent on addressing tissue hypoxia or metabolic shifts associated with ischemia.
Abstract 403: E-selectin Transfer by Recombinant Adeno-associated Virus Improves Angiogenesis in a Murine Model of Hindlimb Ischemia
Authors: Horkan Davis , Li Yan , Liu Zhao-Jun , Velazquez Omaida
Published: March 2022

Novel cell-based therapies, including optimization of the wound microenvironment via chemical or genetic means, hold promise to revolutionize the treatment of chronic limb ischemia and ischemic wounds.

We examined the hypothesis that increasing expression in ischemic tissue of the endothelial adhesion molecule E-selectin by adeno-associated viral transduction would promote angiogenesis in a murine model of hindlimb ischemia.

12-week-old female C57BL/6 mice (N=10) underwent unilateral femoral artery ligation. After ligation, each mouse was injected in the ipsilateral semimebranosus muscle with 1x108 viral particles of either adeno-associated virus recombinant with human E-selectin gene (AAV/E-selectin) or adeno-associated virus recombinant with green fluorescent protein gene as control (AAV/GFP) suspended in phosphate-buffered saline (N=5/group).

Ischemia was confirmed by laser Doppler perfusion imaging on post-ligation day one. Mice were sacrificed on post-ligation day seven and semimembranosus muscle harvested from the ligated limb. Muscle was fixed, embedded, and sectioned in 5 μm intervals.

To assess the efficacy of viral transduction, we performed immunohistochemistry (IHC) of each section from the AAV/GFP group with anti-GFP antibody and from the AAV/E-selectin group with anti-human E-selectin antibody.

Angiogenesis was evaluated by capillary density with IHC of all slides for endothelial cell marker VEGFR2 with anti-VEGFR2 antibody.

Relative fluorescence index per high-powered field (RFI) was recorded for each slide. E-selectin RFI was 4 times greater, VEGFR2 RFI was 2 times greater, and E-selectin/VEGFR2 RFI ratio was 2 times greater in AAV/E-selectin treated tissue than in control.

In conclusion, this study demonstrates the AAV/E-selectin vector as an effective means to upregulate E-selectin expression in ischemic muscle and supports E-selectin upregulation as a possible means to improve ischemia-induced angiogenesis.
Abstract 405: Functional Characterization of Coronary Artery Disease (CAD) Associated SNPs at the ADAMTS7 Locus
Authors: Ou Kristy , Nuernberg Sylvia , Bauer Robert C , Rader Daniel J , Reilly Muredach P
Published: March 2022

SNPs in the genomic region containing the gene
ADAMTS7,
a gene that codes for a metalloproteinase involved in vascular smooth muscle cell migration, are significantly associated with CAD in humans. Recently, our lab demonstrated that knock out of
Adamts7
in mice is protective against atherosclerosis, suggesting that
ADAMTS7
is involved in the pathogenesis of cardiovascular disease. Several of the top CAD-associated SNPs (p<5e-8) identified by CARDIoGRAMplusC4D consortium lie in putative enhancer regions of
ADAMTS7
.. We investigated the functionality of CAD associated SNPs that overlap 5 active regulatory elements as predicted by vascular smooth muscle cell (VSMC) ChIP-seq signals, ENCODE histone methylation peaks, and aortic smooth muscle cell DNase hypersensitivity peaks. Several regions containing association SNPs showed enhancer activity in minimal promoter luciferase reporter assays. Region 2 showed an 8.2 (p<0.01) and a 32.4 (p<0.005) fold increase in luciferase activity in HEK293T and A7r5 rat aortic smooth muscle cell lines, respectively, when normalized to empty vector control, while Region 5 demonstrated a 4.9 (p<0.01) and 1.8 (p<0.01) normalized fold increase in 293T and A7r5 cells, respectively. Both of these regions contain binding motifs for TCF21, a transcription factor involved in the development of the human coronary vasculature that is also a GWAS locus for CAD, that is disrupted by genotype of the CAD-associated SNP. We performed electrophoretic mobility shift assays (EMSAs) using purified TCF21 protein and showed altered binding in Region 5 based on disease associated SNP variation. Additionally, EMSAs using human coronary artery smooth muscle cell (hCASMC) nuclear extract detected a band shift demonstrating protein binding at both predicted enhancer regions 2 and 5. However, we were not able to detect a significant difference in protein binding between the major and minor allele, suggesting transcription factors other than TCF21 may be regulating these regions. More work is being done using Capture-C technology to interrogate physical interactions between these regions and the promoter of
ADAMTS7
, but our current data suggest that top CAD-associated SNPs alter regulatory activity at the
ADAMTS7
locus.
Abstract 411: Decreased Circulating and Neutrophil Mediated Vegf Release in Stable Long-term Cardiac Transplant Recipients
Authors: Chaar Diana , Vitiello Damien , Neagoe Paul-Eduard , Ducharme Anique , Carrier Michel , Pelletier Guy , Racine Normand , Lizkowski Mark , Sirois Martin G , White Michel
Published: March 2022

Background:
A chronic state of inflammation may contribute to the long-term cardiac and vascular complications following cardiac transplantation (CTx). However, the assessment of neutrophil pro- or anti-inflammatory response has not been investigated in long-term CTx recipients. Herein, we measured the circulating levels of pro-inflammatory (interleukin-8; IL-8), anti-inflammatory (interleukin 1 receptor antagonist; IL-1RA) and a pro-angiogenic and inflammatory cytokine (vascular endothelial growth factor; VEGF), concomitantly with their release from neutrophils of CTx recipients and as compared to healthy volunteers.

Methods:
Eighteen CTx recipients aged 49.6 ± 3.1 years being transplanted for 145 ± 20 months were aged-matched to 20 healthy control (HC) subjects. Seven (39%) patients exhibited coronary allograft vasculopathy (all CAV1). VEGF, IL-8 and IL-1ra plasmatic levels were measured in resting state. Circulating neutrophils were isolated, purified and stimulated by vehicle (PBS), N-Formyl-Met-Leu-Phe (fMLP, 10-7 M), bacterial lipopolysaccharide (LPS, 1 μg/ml), or tumor necrosis factor alpha (TNF-α, 10 ng/ml).

Results:
Compared with HC, CTx recipients exhibited a decrease (-80%) in circulating levels of VEGF (225 ± 42 (HC) versus 44 ± 10 pg/ml (CTx); (p < 0.001). There were no differences in the levels of IL-8 and IL-1ra. Under basal or stimulated conditions, neutrophils from CTx patients exhibited a significant decrease on their capacity to release VEGF, IL-8 and IL-1ra upon stimulation.

Conclusions:
Long-term CTx recipients exhibit a marked reduction in the circulating levels of VEGF, as well as neutrophil-mediated release of VEGF, IL-8 and IL-1ra. The mechanisms and physiological impacts of these findings in relationship with various severities of CAV deserve additional investigations.
Abstract 42: Platelet Polyphosphate Accelerates the Inhibition of Tissue Factor Pathway Inhibitor by Factor XIa.
Authors: Puy Cristina , Gruber András , Tucker Erik I , Gailani David , Smith Stephanie A , Choi Sharon H , Morrissey James H , McCarty Owen J
Published: March 2022

Introduction:
Our group has recently identified that alpha tissue factor pathway inhibitor (TFPIα) can be neutralized by activated factor XI (FXIa), accelerating plasma clotting time and fibrin generation, although this reaction was slow and required relatively high concentrations of FXIa. Activated platelets secrete the anionic polymer polyphosphate (polyP), which potently accelerate the activation of FXI by thrombin and the activation of FV by FXIa. PolyP is also able to inhibit the anticoagulant effect of TFPIα in plasma but the mechanism for this reaction is still uncertain. We assessed the hypothesis that polyP of the size secreted by activated human platelets is cofactor for the inhibition of TFPIα by FXIa.

Methods:
To measure the binding of polyP to TFPI, biotinylated-polyP was added to TFPIα-coated wells. Binding was detected with HRP-streptavidin. Activated FX (FXa) inhibition by TFPIα was measured in the FXa-initiated clotting time of FX-depleted plasma. TFPIα inhibition of FXa generation by the tissue factor (TF)-FVIIa complex was measuring using a FXa chromogenic substrate. TFPIα was pretreated with FXIa in the absence or presence of polyP

Results:
We found that polyP was able to bind to TFPIα in a concentration-dependent manner. In the absence of polyP, the pretreatment of TFPIα (5 nM) with 1 nM FXIa for 1 hour abrogated the anticoagulant effect of TFPIα in the FXa-initiated clotting time of FX-depleted plasma. In the presence of polyP (10 μM), the pretreatment of TFPIα (5 nM) with 0.25 nM FXIa for 30 min was enough to completely abrogate the anticoagulant effect of TFPIα in plasma. Also, the presence of polyP potently accelerated the effect of FXIa to inhibit the capacity of TFPIα to block the generation of FX by the TF-FVIIa complex.

Conclusion:
Our study provides a novel molecular link between hemostatic activation of platelets and FXI. The results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPIα by FXIa, which is significantly enhanced by the release and presence of activated platelet-derived polyphosphates.