bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 51: The Role of CCL5 in T cell Recruitment to the Aorta
Authors: Li Jie , Ley Klaus
Published: March 2022

Introduction

T cell adaptive immunity is involved in the pathogenesis of atherosclerosis. Previously our lab found that antigen presenting cells (APCs) present antigens to effector T cells (Teff). This causes local T cell activation and cytokine production, which sustain local inflammation. However, the chemokine(s) that drive T cell recruitment into the atherosclerotic aorta are not known. By multi-color flow cytometry to analyze chemokine receptor expression on T cells from Apoe-/- aorta, we found that more than 60% of CD4+ T cells in Apoe-/- aorta express CCR5. I hypothesize that CCR5 plays an important role in T cell recruitment to atherosclerotic aortas.

Methods

T cells in the aorta of Apoe-/- mice that have been on western diet (WD) for > 3 months were analyzed by multi-color flow cytometry by staining T cell markers and chemokine receptors (CCR7, CCR5, CXCR3, CXCR6, CCR6). 400,000 Teff cells from CD45.1 Apoe-/- mice were sorted and incubated with explanted Apoe-/- aortas 12 to 18 hrs with media alone, CCL-5 (one of the ligands of CCR5) neutralizing antibody (0.5 ug/ml) or PTX (300ng/ml). Afterward, T cell infiltration into aorta was quantified by FACS. To monitor T cell-APC interaction, effector T cells from Apoe-/- mice were labeled with fluorescent dye SNARF before incubation with aortas from Apoe-/- CD11c-YFP+ mice in the above 3 conditions. Aortas were imaged by 2 photon imaging.

Results

There is a significant increase of CCR5+ T cells (63±4% of TCRβ+ T cells) in the aorta of Apoe-/- WD mice vs. Apoe-/- on chow diet (9±8%, p<0.05) or WT (13±8%, p<0.05) (n=3 in each group). CCL5 neutralizing antibody significantly decreased the number of T cells infiltrating into aorta from 997±74 (CD45.1 T cell count in the arota)(media alone) to 652±165 (P<0.05, media alone vs. α-CCL5), and PTX further decreased this number to 456±98 (p<0.05, media alone vs. PTX)(n=3 in each group). 2 photon imaging showed that T cells infiltrated into adventitia of Apoe-/- aorta and were co-localized with DCs in the aorta. Neutralizing CCL5 inhibited T cell infiltration and remaining T cells were localized mostly outside of the adventitia.

Conclusion

CCL5 plays a role in Teff recruitment into the aorta. This suggests that targeting CCR5 or its ligand CCL5 may be effective to treat atherosclerosis.
Abstract 511: Total Circulating Adiponectin Levels and its Receptors are Associated with Histological and Immunohistochemical Features of Carotid Plaque Instability
Authors: Gasbarrino Karina , Doonan Robert J. , Mantzoros Christos , Lai Chi , Veinot John , Daskalopoulou Stella S
Published: March 2022

Introduction.

Evidence shows that low plasma levels of adiponectin, a vasculoprotective adipokine, are associated with coronary artery disease and carotid-intima media thickness. The current study investigated for the first time the association between adiponectin and carotid plaque instability, namely the association between circulating adiponectin, expression of its receptors (AR1/AR2), and plaque instability as assessed by histology/immunohistochemistry.

Methods.

100 patients scheduled for carotid endarterectomy were recruited from the Royal Victoria Hospital, Montreal. Blood samples were collected to determine total adiponectin levels. Carotid plaque specimens were stained with hematoxylin and eosin and immunostained for CD3 (lymphocytes), CD68 (macrophages/foam cells), von Willebrand Factor (vWF, neovascularization), and AR1/AR2. A vascular pathologist categorized the plaques according to AHA classification and with a semi-quantitative scale of plaque instability. The intensity and percentage of macrophages/foam cells stained with AR1/AR2 were assessed using an optical microscope scoring method.

Results.

Patients with less infiltration of CD68+ inflammatory cells in their plaques had higher levels of total circulating adiponectin (12.26 + 6.74 μg/ml) when compared to patients with greater infiltration (8.99 + 6.97 μg/ml; p=0.010). The intensity of AR1 expression on macrophages was directly associated with overall grade of inflammation and neovascularization (p=0.025 and p=0.05 respectively). A trending inverse association was noted between the percentage of macrophages expressing AR1 and overall stability (p=0.084). Expression of vWF was inversely associated with the percentage of macrophages and foam cells expressing AR2 (p=0.033 and p=0.002, respectively), while increase cap infiltration was detected in plaques with a lower intensity of AR2 expression (p=0.013).

Conclusion.

Patients whose plaques were characterized as unstable were found to have lower plasma levels of total adiponectin, a higher expression of AR1 and a lower expression of AR2 in their plaques. These results suggest a potential role for adiponectin in the development of plaque instability, as well as differential roles for its two receptors.
Abstract 512: Angiotensin II Type 1 Receptor is Essential for the Initiation and Progression of Porcine Pancreatic Elastase-Induced Experimental Abdominal Aortic Aneurysms
Authors: Xuan Haojun , Xu Baohui , Wang Wei , Tanaka Hiroki , Glover Keith J , Fujimura Naoki , Iida Yasunori , Hu Xiaolei , Unno Naoki , Michie Sara A , Dalman Ronald L
Published: March 2022

Angiotensin (Ang) II type 1 receptor (AT1) activity is critical for experimental abdominal aortic aneurysm (AAA) formation in response to Ang infusion in hyperlipidemic mice. The role of AT1 activity in exogenous Ang II-independent, experimental AAA is unknown. Genetic and pharmacological inhibition strategies were used to isolate the significance of AT1 function in porcine pancreatic elastase (PPE)-induced AAAs in normolipidemic C57BL/6 mice. AAA progression was monitored via serial in-vivo transabdominal ultrasound measurements at 40 mHz. Aortic diameters enlarged remarkably from day 3 to day 14 in control mice following PPE infusion. Progression was abrogated, however, following either targeted AT1a receptor deletion, or treatment with the AT1 inhibitor telmisartan, beginning day 3 prior to PPE infusion. Additionally, telmisartan treatment initiated 4 days following PPE-infusion led to stabilization /regression of existing AAAs. Histologically, AT1a deficiency or telmisartan treatment resulted in preserved medial smooth muscle cellularity and elastic fibers, and attenuated mural angiogenesis and leukocyte infiltration. Telmisartan treatment did not affect PPARγ-regulated gene expression in aneurysmal aortae, however, and the PPARγ antagonist (GW9662) did not influence telmisartan-induced AAA suppression. In conclusion, AT1 activity is required for the initiation and progression of experimental AAAs, independent of the specific methodology used to induce aneurysm formation. Strategies targeting AT1 activity may prove effective in limiting human AAA progression.
Abstract 524: Myxomaviral Anti-angiogenesis Protein Serp-1 Inhibits Pancreatic Cancer Growth in Mice
Authors: Zheng Donghang , Chen Hao , Bartee Mee , Davids Jennifer , Lucas Alexandra
Published: March 2022

Modification of the tumor microenvironment by inflammatory cells represents a newly recognized driving force in cancer with a critical role in tumor invasion, growth, angiogenesis, and metastasis. Increased serine proteases in the thrombolytic cascade, specifically urokinase-type plasminogen activator and its receptor, correlate with inflammatory cell invasion, angiogenesis, cancer growth and invasion. Inflammation in pancreatic cancer is linked with myeloid-derived suppressor cell activity and cancer progression. Myxomavirus is a complex DNA virus encoding highly potent immune modulators such as Serp-1, which inhibits uPA, tPA, plasmin and coagulation factor X. Serp-1 has also been demonstrated to inhibit angiogenesis in a chicken chorioallantoic membrane (CAM) model. To explore the potential use of anti-inflammatory, anti-angiogenesis proteins that target thrombotic and thrombolytic as well as inflammatory pathways, for cancer treatment, severe combined immunodeficient (SCID) mice engrafted with human cancer cells were treated with Serp-1 and neuroserpin, a selective mammalian inhibitor of thrombolytic proteases or control saline. Proteins were tested for efficacy in preventing human pancreatic and breast cancer cell growth in NOD/SCID mice (n=67 total) at 4 weeks after implantation. Serp-1 and neuroserpin inhibited pancreatic cancer Hs766t (n=27; P=0.03 and P=0.01, respectively), but not breast cancer MDA231 growth (n=11) when compared to saline controls (N =5). Serpin inhibition of pancreatic tumor growth was associated with decreased MDSC as shown by flow cytometry analysis of splenocyte (P=0.009). Serpin-mediated anti-inflammatory activity in pancreatic tumor growth was also demonstrated by reduced macrophage infiltration in tumors (P=0.001). Furthermore, RT-PCR array analysis revealed a five-fold decrease in the expression of inducible nitric oxide synthase (iNOS), which has been shown to play important role in tumor angiogenesis.

Conclusions

Anti-inflammatory protease inhibitors, virus-derived Serp-1 and mammalian neuroserpin, may provide new treatment approaches by targeting the angiogenesis and inflammatory responses that support cancer cell growth.
Abstract 526: Angiogenesis and Kallikrein-kinin System Activation after Exercise Training in Rats with Sustained Adrenergic Hyperactivity
Authors: Santana Eduardo T , Serra Andrey J , Manchini Martha T , Antonio Ednei L , Tucci Paulo J , Silva Jose A
Published: March 2022

Several mechanisms are involved to exercise-related cardioprotection.

Purpose

Kallikrein-kinin system may contribute to this effect inducing endothelial vascular growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression.

Methods

In a model of β-adrenergic hyperactivity, male Wistar rats were assigned into four groups: sedentary non-treated (Con); sedentary isoproterenol-treated (Iso); exercised only (Ex); and exercised isoproterenol-treated (Iso+Ex). Left ventricles were processed for Real time PCR and Western blot analysis in order to quantify the expression of kinins and angiogenic molecules.

Results

In Iso+Ex, a significant increase of tissue kallikrein gene and protein expression was observed (p<0.01). In the same group, an augmented eNOS (p<0.05), VEGF and VEGF receptor mRNA expression (p<0.01) was observed. The expression of angiogenic IL-1 and Il-8 were increased after exercise (p<0.01). Expression of integrin linked kinase (ILK), another angiogenesis promoter, also augmented after training (p<0.01).

Conclusion

Our results represent the first demonstration that exercise increases the tissue kallikrein gene and protein expression in rat myocardium after β-adrenergic hyperactivity. Together with the observed augmented eNOS and VEGF and angiogenesis-related molecules mRNA expression, a feasibly pathway might be drawn to understand the kallikrein-kinin system protective role to avoid the abnormalities developed due excessive catecholaminergic activation as observed in cardiac failure. Our data suggest a strong evidence of beneficial exercise action, particularly related to angiogenesis, in response of myocardium towards β-adrenergic hyperactivity.
Abstract 529: A Chloride Intracellular Channel-4 (Clic4) and VEGF-A Signaling Pathway Regulates Maturation of the Collateral Circulation
Authors: Lucitti Jennifer L , Faber James E
Published: March 2022

The density of native (pre-existing) cerebral collaterals in healthy tissues varies widely and is a major determinant of the wide variation in infarct volume in ischemic stroke. Collateral number in adults has been shown to be dependent on 3 processes: formation in the embryo, maturation in the perinate (i.e. slight pruning, increase in diameterand muralization) and maintenance thereafter. However, the mechanisms underlying these processes are not well understood. We previously found that collaterogenesis is led by a unique “arteriolar” angiogenic sprouting-like mechanism involving the VEGF-Notch signaling pathway. We also observed that mice lacking chloride intracellular protein 4 (Clic4), a protein which appears to reside upstream of VEGF-A in hypoxia-induced signaling, had fewer pial collaterals on postnatal day (P)1 that underwent greater pruning by P21, to yield lower collateral number and more severe stroke in adults. Therefore, herein we hypothesized that Clic4 is required for both formation and maturation of the collateral circulation. Wildtype and

Clic4
-/-

mice formed similar numbers of collaterals through embryonic day (E)18.5. Thus, collaterogenesis does not require Clic4. However, perinatal pruning was strongly augmented in

Clic4
-/-

mice, resulting in reduced collateral number and larger infarct volume in adults after MCA occlusion. Pial diameter was also reduced, consistent with our previous findings. Overexpression of VEGF-A in

Clic4
-/-

mice by crossing them to

Vegf
hi/+

mice did not affect collaterogenesis but inhibited the excessive perinatal pruning, resulting in adults with the abundant collaterals and small infarct volume seen in wildtype mice. We conclude that Clic4 is not required for embryonic collaterogenesis but is essential for perinatal stabilization of nascent collaterals through a mechanism involving VEGF signaling.
Abstract 53: Noninvasive Detection of Inflammation of Murine Stasis Deep Venous Thrombosis (DVT) Using Integrated 18F-FDG PET/CT
Authors: Hara Tetsuya , Truelove Jessica , Jokivarsi Kimmo , Brownell Anna-Liisa , Wojtkiewicz Gregory R , Weissleder Ralph , Tawakol Ahmed , Jaffer Farouc A
Published: March 2022

Background

Noninvasive imaging of inflammation in DVT could offer new insights into the resolution of deep venous thrombosis (DVT) in living subjects. While a few clinical studies have reported incidental uptake of 18F-FDG PET in a subgroup of DVTs, limited information exists regarding the cellular mechanisms and time dependence of FDG uptake in DVT. Here we investigated whether FDG-PET could enhance murine stasis-induced DVT in acute to subacute DVT, and assessed the inflammatory cell infiltrate within DVT.

Methods

C57BL/6 mice (n=6) underwent ligation of the jugular vein to induce stasis DVT. At DVT timepoints from day 2 to day 7, 18F-FDG PET (25-35 μCi/g) and contrast CT venography was performed. After PET/CT imaging, animals were sacrificed and veins were resected. Gamma-counting and histological analyses of thrombus neutrophils and macrophages were then performed.

Results

PET/CT images demonstrated the FDG signal accumulated in thrombosed jugular veins in acute DVT, and significantly more than in contra-lateral sham veins (SUV = 1.27±0.11 vs. 0.79±0.069, SUVmax = 1.45±0.090 vs. 1.02±0.056, p<0.05, respectively). The %IDGT was significantly higher in thrombosed veins (7.21±0.17% vs. 1.54±0.47% sham vein, p<0.05). The % positive area of neutrophil in thrombus was 25 times higher than macrophages (14.1±2.9% vs. 0.58±0.091%, p<0.0001) in earlier-stage (day2-4) DVT.

Conclusions

Noninvasive FDG-PET/CT can visualize inflammation in experimental DVT. Thrombus neutrophils appear to be the cellular source underlying accumulated FDG signal in earlier-stage DVT. FDG-PET may enable assessment of thrombus age and provide new insights into the resolution of DVT.
Abstract 533: Micro RNA-based Strategy to Preserve Endothelial Function After Angioplasty
Authors: Santulli Gaetano , Wronska Anetta , Marx Steven , Kitajewski Jan , Marks Andrew , Totary-Jain Hana
Published: March 2022

Coronary artery disease is currently a leading cause of death worldwide. Despite all the benefits of drug-eluting stents (DES), concerns have been raised over their long-term safety, with particular reference to stent thrombosis due to delayed endothelial cell (EC) coverage. Here we describe a method that exploits the endogenous miRNAs to specifically inhibit vascular smooth muscle cell (VSMC) proliferation, the major cause of restenosis, without affecting re-endothelialization. By inserting four target sequences of the EC specific mir-126 into the 3’UTR of p27 expressing adenoviruses (p27-126TS), we were able to specifically inhibit VSMC proliferation and migration while EC were able to proliferate, migrate and form capillary-like networks. Balloon injured rat carotid artery infected with p27-126TS viruses exhibited complete inhibition of restenosis with complete re-endothelialization after two weeks (Fig. A). Moreover, hypercoagulability assessed by measuring plasma levels of D-dimer in the serum of rats treated with the p27-126TS was similar to the non-injured control (Fig. B). Finally, the vasodilatative response to acetylcholine of balloon injured carotid arteries treated with p27-126TS adenovirus were comparable to those of the control-noninjured vessel (Fig. C). This discovery may ultimately lead to a safer therapy than the non-selective DES and may diminish the need for prolonged dual antiplatelet therapy following percutaneous coronary intervention.
Abstract 542: Dedicator of Cytokinesis 2 (DOCK2) Promotes Vascular Lesion Formation through Stimulation of Smooth Muscle Cell Proliferation and Migration
Authors: Guo Xia , Wang Jia-Ning , Cui Xiao-Bing , Chen Shi-You
Published: March 2022

The objective of this study is to determine the role of dedicator of cytokinesis 2 (DOCK2) in vascular lesion formation after experimental angioplasty. By using a rat carotid artery balloon-injury model, we demonstrate that neointima formation is closely associated with DOCK2 expression. DOCK2 is not expressed in normal arteries. However, its expression was observed in the developing neointima and media after injury as shown by immunohistochemistry staining, qPCR and Western blot analyses. shRNA knockdown of DOCK2 via adenovirus (Ad)-mediated gene delivery dramatically inhibited the neointimal formation by 65% as compared to those treated with control shRNA or saline. Similar results were obtained when intima/media area ratios were compared between DOCK2 knockdown and control groups. Loss of function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that DOCK2 is essential for both the proliferation and migration of RASMCs. PDGF-BB induced DOCK2 expression in RASMCs. shRNA knockdown of DOCK2 significantly inhibited PDGF-BB-induced proliferation and migration of RASMCs. DOCK2 appeared to stimulate RASMC migration via inducing focal adhesion contact and stress fiber formation as shown by the altered expression of vinculin and F-actin, respectively. Taken together, our studies demonstrate that DOCK2 plays an important role in vascular lesion formation following vascular injury. Therefore, targeting DOCK2 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.
Abstract 551: The Dual Effect of Cigarette Smoke on Macrophage Abca1-dependent Cholesterol Efflux and Protease Production Leads to Decreased Collagen and Increased Necrosis in Plaques of ApoE Deficient Mice
Authors: Sklepkiewicz Piotr L , Lemaitre Vincent , Zelonina Tina , Westerterp Marit , Tall Alan , D’Armiento Jeanine
Published: March 2022

Epidemiological evidence links smoking to an increase in the incidence and severity of cardiovascular disease. However, the molecular mechanisms responsible for susceptibility are unknown. The accumulation of lipids in macrophages induces an inflammatory response, with secretion of cytokines and proteases, and leads to necrosis and loss of collagen content within atherosclerotic lesions. Therefore, the effect of cigarette smoke on lipids in the atherosclerotic process and consequences was examined. Initial studies revealed that cigarette smoke attenuated cholesterol efflux. Subsequently, the end result of cigarette smoke exposure on atherosclerotic lesions in ApoE deficient mice demonstrated a significant increase in necrosis along with a dramatic decrease in the collagen content of lesions in ApoE deficient mice exposed to smoke for 20 weeks. Cigarette smoke extract (CSE) impaired the cholesterol efflux ability for ApoAI and correlated with ABCA1 loss and increased MMPs (9, 12, 13, in human THP-1 cells additionally MMP1 is increased), TLR4/Myd88 and TNFα expression in mouse bone marrow derived macrophages (BMdM) in vitro and in vivo in peritoneal macrophages elicited after 5 days of smoke exposure. Furthermore, by increasing ABCA1 expression with LXR agonist treatment the CSE-induced up regulation of MMPs (MMP-9 and 13) and TNFα was inhibited signifying a correlation between the cigarette smoke induced loss of ABC transporters and MMP induction. Moreover, BMDMs isolated from Cre-LysM-ABCA1 deficient mice displayed an increase in mRNA expression of MMPs (9, 12, 13) at a basal level, which was appreciably augmented with CSE as compared to control macrophages. The LXR induced decrease in MMPs and TNFα expression as well as decreased MMP-9 activity in macrophages was confirmed to be ABCA1-dependent since ABCA1 KO macrophages exposed to CSE did not exhibit this response. These findings establish that the effect of cigarette smoke on macrophage ABCA1-dependent cholesterol efflux impairment leads to both an increase in proteases and inflammatory cytokines and this combination of events is likely synergistic in ultimately generating a more vulnerable plaque through the increase in plaque necrosis and a decrease in collagen content.
Abstract 552: The Immunosuppressant FTY720 Increases Longevity and Prevents Chronic Heart Failure in HypoE Mice Deficient in the SR-BI Receptor that Survive Myocardial Infarction Caused by Diet-induced Coronary Atherosclerosis
Authors: Kim Roy Y , Wang Guanying , Imhof Isabella , Honbo Norman , Luk Fu Sang , Li Kang , Kumar Nikit , Zhu Bo-qing , Eberle Delphine , Karliner Joel S , Raffai Robert L
Published: March 2022

FTY720 is an analogue of sphingosine-1-phosphate, which is cardioprotective during acute injury. Whether FTY720 affords cardioprotection and prevents chronic heart failure (CHF) after myocardial infarction (MI) is unknown. Initially we added FTY720 (0.3mg/kg/day) to the drinking water of C57BL/6J mice. After ex vivo cardiac I/R injury these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Next, we fed FTY720 to a previously described mouse model of diet-induced coronary atherosclerosis, MI and premature death. This model was developed by breeding our hypomorphic apoE mice, also referred to as “HypoE” mice, which express sub-physiological apoE in all tissues due to Apoeh/h alleles, to mice deficient in the SR-BI receptor (Apoeh/h/SRB1-/- mice). These mice were given a high fat diet (HFD) for 4 weeks and treated or not with oral FTY720 (0.05mg/kg/day). This therapy sharply reduced mortality (p<0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia or atherosclerosis. Rather, FTY720 produced immunosuppression by reducing T and B cells and increasing the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen and lymph nodes. Importantly, FTY720-treated mice exhibited increased TGF-β and reduced IFN-γ expression in the heart. Cardiac expression of CD4 was increased and correlated with molecules involved in natural Treg activity including TGF-β and GITR. Lastly, Apoeh/h/SRB1-/- mice were bred to Mx1-Cre mice. Apoeh/h/SRB1-/-Mx1-Cre mice were given HFD for 3.5 weeks and hyperlipidemia was reversed by inducible Cre mediated gene repair of the HypoE allele using pIpC injections. The mice were then treated or not with oral FTY720 (0.05mg/kg/day). Fifteen weeks after lipid lowering, sham-treated mice had developed profound CHF while in mice receiving FTY720 cardiac function returned to normal. We conclude that long-term FTY720-treatment enhances LV function and increases longevity in mice with CHF and prevents CHF in mice that survive MI. These benefits resulted not from atheroprotection but from profound systemic immunosuppression resulting in reduced cardiac inflammation.
Abstract 556: Significant Interaction of Smoking and Metabolic Syndrome for Subclinical Vascular Inflammation
Authors: Jamal Omar , Ali Shozab S , Blaha Michael J , Shaharyar Sameer , Agatston Arthur A , Blumenthal Roger S , Santos Raul D , Nasir Khurram
Published: March 2022

BACKGROUND

Emerging data suggest an additive interaction of current smoking and metabolic syndrome (MS) on cardiovascular risk. In this study we aim to assess if this increased risk can be explained by an increased risk of subclinical vascular inflammation.

METHODS

We evaluated 6,464 healthy non-diabetic Brazilian subjects (43±10 years, 79% males). Participants were divided into 4 subgroups by MS and smoking status. High-sensitivity C reactive protein (hs-CRP) was measured to assess degree of underlying inflammation. We excluded 945

(14%) individuals with missing hs-CRP levels.

RESULTS

Overall 1,062/5,519 (19%) had hs-CRP≥3 mg/dl. The figures show prevalence of high CRP across smoking and MS status. As compared with never smokers without MS, in multivariate

analysis the adjusted OR’s for elevated hs-CRP were 1.28 (95% CI: 0.98-1.67), 2.09 (95% CI: 1.76-2.48), and 4.24 (95% CI:2.95-6.08) for smokers without MS, nonsmokers with MS, and current

smokers with MS. The interaction for MS and smoking with high hs-CRP was statistically significant(p=0.05) The risk of hsCRP≥3 with smoking was 1.58 (1.00-2.52) fold vs smokers among non MS

individuals.

CONCLUSION

The current study further demonstrates a synergistic interaction of current smoking and MS for vascular inflammation, highlighting those with MS are at higher risk and needed to be

aggressively targeted for smoking cessation.
Abstract 559: Systemic Oxidative Stress is Associated with Myocardial Perfusion in Whites but not in Blacks: The Bogalusa Heart Study
Authors: Fernández Alonso Camilo , Li Shengxu , Blandon Roberto , Srinivasan Sathanur R , Chen Wei , Berenson Gerald S
Published: March 2022

Central and peripheral vascular damage by oxidative stress-mediated mechanisms has been described in male and female patients with inflammatory pathologies. However, information is scant on the association between oxidative stress and myocardial perfusion in biracial (black-white) asymptomatic young adults.

We assessed the hypothesis that the association between oxidative stress and myocardial perfusion differs among biracial (black-white) asymptomatic young adults.

As a part of the Bogalusa Heart Study, systemic oxidative stress (in terms of urinary creatinine-indexed levels of 8-epi-PGF2alfa -isoprostane-) and non-invasive myocardial perfusion (in terms or Subendocardial Viability Ratio -SEVR) were assessed in 605 asymptomatic individuals aged 29-51 years (mean 43.4; 68.7% white and 54.4% female), along with evaluation of traditional cardiovascular risk factors. The race-specific association of urinary creatinine -indexed 8-epi-PGF2alfa with SEVR was tested through multivariable- adjusted linear regression analyses.

SEVR levels were significantly higher in white and male participants (p<0.001), whereas creatinine-indexed 8-epi-PGF2alfa levels were lower; when compared to their counterparts. In race-specific multivariable-adjusted linear regression analyses, controlling for age, sex, body mass index, smoking, hypertensive and diabetic status; along with traditional cardiovascular risk factors, a significant inverse association was observed between creatinine-indexed 8-epi-PGF2alfa and SEVR in white (B= -0.246, p<0.001) but not in black participants (B= 0.007, p=0.93).

In conclusion, the described observations help support the hypothesis that the impact of oxidative stress on myocardial perfusion (coronary microcirculation) is different among race (black-white) groups. Further, this may aid to emphasize and enhance race-specific risk factor screening and disease evaluation.
Abstract 564: Influence of Coronary Artery Disease-Associated Genetic Variants on Risk of Venous Thromboembolism
Authors: Bruzelius Maria , Strawbridge Rona J , Tregouet David A , Bergendal Annica , Silveira Angela , Gertow Karl , Sundström Anders , Kieler Helle , Syvänen Ann-Christine , Morange Pierre-Emmanuel , Odeberg Jacob , Hamsten Anders
Published: March 2022

Objective

To determine whether genetic variation robustly associated with coronary artery disease (CAD) also influences risk of venous thromboembolism (VTE) in a well-defined, female case-control study (n=2753) from Sweden (the ThromboEmbolism Hormone Study (TEHS)).

Approach and Results

Single nucleotide polymorphisms associated with CAD in genome-wide association studies were genotyped in TEHS. Association with VTE was assessed by logistic regression (Bonferroni-corrected p-values). A strong association was found between
rs579459
in the
ABO
locus and VTE risk (p=3.8×10
13
). Adjustment for known VTE-associated variants within the
ABO
,
F5
,
F2
,
F11
and
FGG
loci, along with age and body-mass index, identified a further suggestive association for rs1333049 in the
ANRIL
locus (p=0.003). However, replication analysis in 3 French cohorts (total n=5503) failed to confirm the
ANRIL
association with VTE, but indicated that the effect direction is consistent across cohorts. Furthermore, marked variation was found in minor allele and minor allele frequency of the
ANRIL
variant in a survey of studies conducted across Europe.

Conclusions

CAD-associated genetic variants in the
ABO
and
ANRIL
loci appear to influence risk of VTE. Differences in cohort characteristics, including allele frequencies, may account for the failure to replicate the association with rs1333049 in the French cohorts.
Abstract 568: Identification of Novel Pre-translational Regulatory Mechanisms for NF-?B Activation
Authors: Huang Xiao , Gong Ren , Li Xinyuan , Virtue Anthony , Yang Fan , Yang Irene H , Tran Anh H , Yang Xiao-feng , Wang Hong
Published: March 2022

NF-κB controlled transcriptional regulation plays a central role in inflammatory and immune responses. Currently understanding about NF-κB activation mechanism emphasizes IκB-tethered complex inactivation in the cytoplasm. In the case of NF-κB activation, IκB phosphorylation leads to its degradation followed by NF-κB relocating to the nuclear and transactivation of NF-κB targeted genes. Pre-translational mechanism mediated NF-κB activation remains poorly understood. In this study, we studied NF-κB pre-translational regulation by performed a series of data-base mining using experimental data-based NCBI Unigene EST profile database, Gene Expression Omnibus (GEO) database, Transcription Element Search System (TESS) database, AceView database, Epigenomics databases, and TargetScan software. We reported the following findings: 1) NF-κB family/signaling genes are differentially expressed in 20 human and 19 mouse tissues; 2) Heart and vessels are the inflammation privilege tissues and less easy to be inflamed because lacking of NF-κB signaling key molecular expression; 3) NF-κB activation is readily induced by cardiovascular disease risk factors oxidized-LDL and pro-inflammatory cytokines in endothelial cells; 4) Transcription factors C/EBPs and NF-κB have higher binding site frequencies in the promoters of Pro-inflammatory cytokine-induced NF-κB genes; 5) Most NF-κB signaling genes have multiple alternative promoters and alternatively spliced isoforms; 6) NF-κB family genes have DNA methylation characteristics and may be subjected to epigenetic regulation; 7) 27 out of 38 NF-κB signaling gene can be regulated by microRNAs, with the binding qualities to mRNA targets being equivalent to what have been approved experimentally. Our findings provide important insight in the mechanism of NF-κB activation, which may contribute to cardiovascular disease, inflammatory diseases and immunological disorders.
Abstract 61: LXR Agonist Treatment of Nonhuman Primates Increases LDL Cholesterol due to Decreased Hepatic LDL Receptor Expression
Authors: Temel Ryan E , Hong Cynthia , McDaniel Allison L , Marshall Stephanie M , Horton Jay D , Tontonoz Peter
Published: March 2022

Reverse cholesterol transport (RCT) is a process whereby cholesterol from foam cells in atherosclerotic plaques is removed by HDL, transported to the liver, secreted into bile, and excreted in the feces. Treatment of mice with LXR agonist can significantly increase RCT and inhibit atherosclerosis. Therefore, it would be anticipated that LXR agonists would also increase RCT and be anti-atherogenic in humans. However, cynomolgus monkeys treated with the LXR agonist GW3965 had significant increases in LDLc, which could be due to elevated hepatic expression of the inducible degrader of the LDL receptor (IDOL). IDOL is an E3 ubiquitin ligase that promotes the ubiquitination of the LDL receptor (LDLR) on its cytoplasmic domain, thereby targeting it for degradation. We recently discovered that GW3965 treatment of mice caused an ~3-fold increase in IDOL expression in the small intestine and consequently increased the turnover of the LDLR. In livers of GW3965 treated mice, LDLR protein was unchanged and IDOL expression was only increased by ~20%. However, treatment of human hepatoma cells with GW3965 increased IDOL 7-fold resulting in very low levels of LDLR protein expression. Therefore, we hypothesized that in humans and nonhuman primates, LXR agonist treatment would increase LDLc by inducing IDOL-dependent degradation of hepatic LDLR. After treating cynomolgus monkeys with GW3965 for 7 days, we found that HDLc was increased by ~20% while LDLc was increased by ~100%. The increase in LDLc was accompanied by a dramatic increase in plasma apoB100 and apoE concentration and a significant decrease in hepatic LDLR protein. We are currently determining the potential role of hepatic IDOL expression on these effects. We also found that unlike mice, monkeys treated with GW3965 did not display an increase in hepatic lipids, biliary cholesterol, and fecal neutral sterol excretion. These findings show that LXR activation has very different effects on lipoprotein metabolism and RCT in monkeys compared to mice. Our data suggest that LXR agonists are likely to exert both pro- and anti-atherosclerotic effects in primates and that these complex effects must be considered in the development of LXR agonists as drugs.
Abstract 62: The Effect of ETC-1002 on Low Density Lipoprotein-Cholesterol and Additional Risk Factors in Patients With Type 2 Diabetes
Authors: Rosenberg Noah , MacDougall Diane , Hanselman Jeffrey C , McBride Scott , Strange Poul , Gutierrez Maria , Margulies Janice , Newton Roger
Published: March 2022

Background

Patients with type 2 diabetes (T2DM) are at high risk for cardiovascular (CV) disease and over half do not achieve LDL-C goal. Statins lower CV risk by reducing LDL-C and inflammation, but have been associated with worsening glycemic control and new onset diabetes, resulting in class label warnings for all statins. ETC-1002 is an oral investigational drug in Phase 2 development to treat dyslipidemia and other cardio-metabolic risk factors through dual adenosine monophosphate-activated protein kinase activation and adenosine triphosphate-citrate lyase inhibition.

Methods

Sixty T2DM patients received either ETC-1002 (80 mg for 2 weeks followed by 120 mg for 2 weeks) or placebo (PBO) for 4 weeks following a 4 week T2DM drug washout and 1 week PBO run-in.

Results

ETC-1002 lowered LDL-C by 43% (PBO corrected mean -39%; p<.0001) and non-HDL-C by 32% (PBO corrected mean -31%; p<.0001). Median reductions in high sensitivity C-reactive protein (hsCRP) were 41% and 11% for ETC-1002 and PBO (p=.0011). Post hoc analyses showed additional benefits of ETC-1002. An LDL-C goal of <100 mg/dL was achieved by 88% of ETC-1002 (21/24) and 4% of PBO subjects (1/24) not at baseline goal (p<.0001 versus PBO). Reductions in post-prandial hyperglycemia following standard meal challenge were observed which were significant in a subgroup of patients with BMI ≥ 30 kg/m
2
(p=.0246 versus placebo). Using continuous glucose monitoring, ETC-1002 treatment resulted in significant peak glycemic reduction between hours 6-12 and a trend between hours 12-18 and 18-24. A weight loss trend was observed with ETC-1002. In patients with 24 hour diastolic ambulatory blood pressure (DBP) > 80 mmHg, a 7 mmHg reduction versus PBO was observed (p=.0474). ETC-1002 was safe and well tolerated with no drop-outs, dose limiting side effects or serious adverse events, and no liver function tests >2 times upper limit normal.

Conclusion

In patients with T2DM and dyslipidemia, ETC-1002 produced statin-like reduction in LDL-C, robust reduction in hsCRP and was associated with modest to significant improvement of blood pressure, glucose control and weight loss. The novel mechanism of action of ETC-1002 may provide unique benefit to these patients.
Abstract 67: The Novel Role of the Matricellular Protein Cyr61 in the Extracellular Matrix Bridging LPA and Integrin Signaling Pathways Leading to Smooth Muscle Cell Migration
Authors: Zhang Fuqiang , Wu Daniel D , Hao Feng , Xu Kan , Xu Xuemin , Cui Mei-Zhen
Published: March 2022

Lysophosphatidic acid (LPA), a potent bioactive lipid, markedly induces smooth muscle cell (SMC) migration. We identified a novel function of the de novo synthesized matricellular protein Cyr61, which bridges LPA and integrin signaling pathways leading to cell migration. LPA transiently and markedly induced Cyr61 mRNA expression and the temporal and spatial expression of Cyr61 proteins in SMCs. The de novo synthesized Cyr61 proteins promptly accumulate in the Golgi apparatus and then translocate to the extracellular matrix. Using primary SMCs from LPA receptor knockout mice, we identified that LPA1 receptor is required for LPA-induced Cyr61 expression. Neutralization or knockdown of Cyr61 protein expression blocked LPA-induced cell migration, indicating the novel regulatory role of the induced matricellular protein Cyr61 in LPA-induced cell migration. LPA induces the activation of intracellular focal adhesion kinase (FAK) in SMCs. Interestingly, 1) knockdown of Cyr61 blocked LPA-induced cellular FAK activation and cell migration, and 2) knockdown of the expression of integrins alpha6, beta1 and beta3 prevented LPA or Cyr61-induced FAK activation and cell migration. These data reveal for the first time that a novel LPA/Cyr61 pathway controls cell migration and that the matricellular protein Cyr61 bridges LPA signaling and integrin signaling, which, in turn, activates FAK leading to cell migration.
Abstract 68: The WWOX Gene Modulates HDL Metabolism and Lipoprotein Gene Expression
Authors: Iatan Iulia , Y. Choi Hong , Ruel Isabelle , Reddy MV Prasad Linga , Kil Hyunsuk , Lee Jaeho , Odeh Mohammad Abu , Salah Zaidoun , Abu-Remaileh Muhannad , Weissglas-Volkov Daphna , Nikkola Elina , Civelek Mete , Awan Zuhier , Croce Carlo M , Aqeilan Rami I , Pajukanta Päivi , Aldaz Marcelo C , Genest Jacques
Published: March 2022

Low plasma HDL cholesterol (HDL-C) constitutes a major risk factor for coronary artery disease. We previously identified a region-wide association between low HDL-C and the WW domain-containing oxidoreductase (
WWOX
) gene in dyslipidemic families and low HDL-C cases and controls (rs2548861, P=6.9x10
-7
). Here, through a combination of
in vivo
functional studies, gene microarray and next generation resequencing, we investigated the role of WWOX in lipoprotein and HDL metabolism. Using total

Wwox
-/-

and
Wwox
liver-specific (

hep
-/-

) mice, we found decreased apoA-I and ABCA1 protein levels in hepatic tissues. Analyses of lipoprotein profiles in

Wwox
-/-

littermates also showed marked reductions in serum HDL-C and apoA-I levels, with no effect in

Wwox hep
-/-

mice. A sex-specific difference was however observed in female
Wwox

hep
-/-

mice, showing a significant increase in plasma triglycerides levels. We further obtained evidence of a gender-specific effect by gene microarray analyses in

Wwox hep
-/-

hepatocytes, where it was identified that
WWOX
disruption alters primarily lipid metabolic pathways in females. We observed a significant reduction (P<0.05,
Abstract 80: Loss of Id3 in B Cells Leads to Increased Adipose Tissue T15-IgM and Protects Against Diet-Induced Glucose Intolerance
Authors: Harmon Daniel B , Oldham Stephanie N , McNamara Coleen A
Published: March 2022

Background

B1 B cells [[Unable to Display Character: –]] the major producers of anti-inflammatory natural T15-IgM antibodies that promote tissue homeostasis [[Unable to Display Character: –]] have been identified in adipose tissue, but their role in adipose tissue response to high-fat diet (HFD)-induced obesity is unclear. Our group and others have shown that the helix-loop-helix protein Id3 is a key regulator of B cell function in disease; mice null for Id3 have increased IgM levels and are protected from HFD-induced obesity. In this study, we test the hypothesis that B cell-specific Id3 deletion protects against the consequences of HFD-induced obesity through increased local production of natural IgM antibodies.

Methods and Results

C57Bl/6 floxed Id3 (Id3fl/fl) and CD19cre/+ mice were crossed to create an Id3fl/fl CD19cre/+ mouse. While these mice had similar body and adipose depot mass as littermate controls after 12 weeks of HFD, they had significantly improved glucose clearance (2hr blood glucose: 314 ± 33 vs. 420 ± 38 mg/dl; n=7-11; p<0.05). At baseline, these mice were found to have significantly more B1b B cell than controls in the peritoneal fluid (3.2E5 ± 0.4E5 vs. 0.9E5 ± 0.4E5 cells; n=5-8; p<0.01), epididymal adipose tissue (4072 ± 923 vs. 1149 ± 652 cells/g fat; n=5-8; p<0.05), and omental adipose tissue. There were no differences in B1a or B2 cells in any compartment, and the mice had similar T cell and macrophage numbers in epididymal adipose tissue. ELISA analysis on whole epididymal adipose tissue revealed significantly higher levels of both total IgM (0.64 ± 0.06 vs. 0.24 ± 0.01 ng/ug protein; n=3-7; p<0.001) and T15-IgM (7.6 ± 1.5 vs. 2.8 ± 0.4 U/ng protein; n=3-7; p<0.05) in mice null for Id3 in B cells compared to controls. No differences were observed in the serum. In addition, omental fat from Id3fl/fl CD19cre/+ mice cultured
ex vivo
secreted significantly more T15-IgM than omental fat from littermate controls (40.0 ± 5.9 vs. 17.5 ± 4.2 U/mg fat; n=3-4; p<0.01).

Conclusions

Loss of Id3 expression in B cells leads to elevated B1b B cells and increased local T15-IgM production in omental adipose tissue, along with improved glucose tolerance in mice fed a HFD, suggesting that T15-IgM may protect against the metabolic dysfunction caused by obesity.
Abstract 105: Evidence that Serum Amyloid A Mediates Inflammation and Matrix Degradation in Abdominal Aortas of Angiotensin II--Infused ApoE-/- Mice
Authors: Ji Ailing , Haggerty Christopher M , Wroblewski Joanne M , Shridas Preetha , Fornwalt Brandon K , De Beer Maria C , De Beer Frederick C , Webb Nancy R
Published: March 2022

Objectives:
Infusion of angiotensin II (AngII) into apoE
-/-
mice results in abdominal aortic aneurysms (AAA) that exhibit features of human AAA, including medial degradation, inflammation, and rupture. We determined that apoE
-/-
mice deficient in acute phase serum amyloid A (SAA) are protected from AngII-induced AAA (unpublished data). SAA is thought to orchestrate the inflammatory response to tissue injury by stimulating cytokine induction, inflammatory cell recruitment, and extracellular matrix turnover. In this study we performed
in vitro
and
in vivo
studies to test the hypothesis that SAA promotes AAA by augmenting inflammation and matrix degradation in abdominal aortas of AngII-infused mice.

Methods and Results:
Male apoE
-/-
mice were infused with 1000 ng/kg/min AngII or saline for 10 days. Abdominal aortas, defined as the 10.4mm region of the aorta immediately above the right superior renal vessel, were analyzed
in vivo
using a 7T ClinScan MRI and
ex vivo
by immunohistochemistry and
in situ
zymography. Vessel wall distensibility was markedly lower in the AngII-infused mouse abdominal aorta (0.54%/mm Hg) compared to saline control (1.07%/mm Hg). Sections of AAA corresponding to regions of lowest distensibility were distinguished by breaks in the elastin lamina that co-localized with intense matrix metalloproteinase (MMP) activity and prominent macrophage and SAA immunoreactivity. In more distensible regions of AAA, there was no evidence of elastin breaks, and MMP activity and macrophage and SAA immunoreactivity were less pronounced. Primary mouse aortic smooth muscle cells (VSMC) were cultured for 3 days and then treated for 8 hours in the presence or absence of 50μg/ml purified mouse SAA. Results from RT-PCR arrays indicated that SAA altered the expression of genes involved in inflammation (52 out 84 analyzed) and extracellular matrix/cell adhesion (24 out of 84 analyzed). Conventional RT-PCR confirmed that SAA induces the expression of Ccl2, MMP9 and MMP13 in mouse aortic VSMCs.

Conclusions:
SAA stimulates chemokine and MMP expression in VSMC that may lead to macrophage infiltration, medial degradation, and decreased arterial distensibility in AngII-induced AAA. SAA is potentially a useful biomarker and therapeutic target for human AAA.
Abstract 107: Finite Element Models With Patient-Specific Wall Strength Estimations Improve Growth Predictions of Abdominal Aortic Aneurysms
Authors: Lindquist Liljeqvist Moritz , Hultgren Rebecka , Gasser Christian , Roy Joy
Published: March 2022

OBJECTIVES:
Abdominal aortic aneurysms (AAA) grow discontinuously and there is no reliable method to predict growth rate specifically. If AAA growth rate could be predicted, surveillance intervals and elective surgery could be planned in a safe and cost-efficient manner. In this study, we used finite element modeling (FEM) with estimations of patient specific factors including wall strength and wall thickness to find a biomechanical parameter that predicted AAA growth.

METHODS:
41 patients (9 women, 32 men) who had undergone two computed tomography angiographies (CT-A) within 9-18 months were included. Two FEM were made per CT-A; one (standard) with and one (matched) without modeling influence of patient specific age, sex, mean arterial pressure and family history. Annual growth of diameter and volume were measured. The baseline AAA volume, diameter, intraluminal thrombus (ILT) volume, mean wall stress (MWS), mean ILT stress (MIS), peak wall stress (PWS) and peak wall rupture risk (PWRR = maximal wall stress/wall strength ratio) were estimated. Linear and non-linear correlations between growth and biomechanical parameters were tested with Pearson product-moment and Spearman’s rank correlation coefficients, respectively.

RESULTS:
Standard baseline PWRR correlated with diameter growth (r=0.32 P=0.040). Baseline AAA volume (r=0.57 P=0.0001), diameter (r=0.53 P = 0.0003), ILT volume (r=0.50, P=0.0009), standard (r=0.44 P=0.0087) and matched (r=0.32 P=0,043) PWRR and matched MIS (r=0.39 P=0.013) correlated with volume growth. PWS and MWS did not correlate with AAA growth.

CONCLUSIONS:
PWRR, based on patient specific wall stress and strength estimations, can predict both diameter and volume growth in our small sample. As this parameter previously has been shown to predict rupture, it is promising as a future clinical predictor for AAA progression and outcome. Our results also suggest a role for the ILT in AAA expansion since both the mean stress and volume of the ILT correlated with AAA volume growth.