bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 82: Angiogenesis Following Hind-Limb Ischemia in Mice Generates a Functionally Abnormal Microvasculature
Authors: Arpino John-Michael , Nong Zengxuan , Yin Hao , Li Fuyan , Ghonaim Nour W , Milkovich Stephanie , Ellis Christopher G , Pickering J Geoffrey
Published: March 2022

Extensive angiogenesis can occur in ischemic muscle. However, the extent to which newly formed microvessels regulate blood flow in ischemic and regenerating muscle remains unknown. To answer this, we developed an intravital video microscopy approach to red blood cell imaging in the extensor digitorum longus (EDL) muscle of mice following femoral artery excision. After surgery, blood flow ceased in the EDL microvasculature, followed by widespread muscle necrosis. However, 10 days later an extensive and highly branched network of flowing neovessels had regenerated. On day 14, branch pruning was evident, and vessel length density reached that of uninjured EDL. By day 28, the network grossly resembled that of the uninjured EDL, and complete arteriovenous units had developed. However, lumenal cross-sectional area of neocapillaries was 40% greater than uninjured capillaries (p<0.05). In addition, 47% of neoarterioles flowed directly into venules, bypassing capillaries. Mean red blood cell flow in neocapillaries was only 59% of uninjured capillary flow (p<0.05) and displayed reduced flow heterogeneity (p<0.05). To evaluate the functional responsiveness of the regenerated network, the EDL was subjected to local hypoxia (2% O
2
) via a polydimethylsiloxane (PDMS) membrane. Red blood cell flow in neocapillaries increased to only 66% of that in uninjured EDL capillaries (54.6±9.0 vs 82.6±7.4 μm/sec increase, p<0.05). Furthermore, the ability to sustain a hypoxic flow response was profoundly reduced in neocapillaries (p<0.05).

Conclusions:
Despite robust angiogenesis, the regenerated network in skeletal muscle is characterized by enlarged capillaries, slow red blood cell transit, non-functional flow units, and impaired flow responsiveness. These findings suggest that: 1) capillary density and bulk flow are inadequate indicators of functional angiogenesis; and 2) strategies beyond stimulating angiogenesis are needed to optimize flow to ischemic tissues.
Abstract 85: MicroRNA-210 Induces Atherosclerotic Plaque Stability Through Repression of Apoptotic Pathways in Vascular Smooth Muscle Cells
Authors: Jin Hong , Eken Suzanne , Chernogubova Ekaterina , Osterholm Cecilia , Perisic Ljubica , Razuvaev Anton , Gonzalez-Diez Maria , Roy Joy , Eriksson Per , Hedin Ulf , Maegdefessel Lars
Published: March 2022

Aim of the present study was to explore the role of miRNAs as potential regulators in patients with carotid artery stenosis and concordant vulnerable atherosclerotic plaques.

Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we were able to profile the regulation of miRNAs in patients with symptomatic vs. asymptomatic carotid stenosis undergoing carotid endarterectomy. A PCR-based miRNA array discovered 8 miRNAs (miRs-15b, -29c, -30c/d, -150, -191, -210 and -500) being substantially altered in symptomatic vs. asymptomatic patients. Next we investigated miRNA expression in 2 murine models of vascular injury and plaque vulnerability; balloon injury in Sprague-Dawley rats, and a newly-developed plaque rupture model in ApoE-/- mice, using incomplete ligation of the carotid artery with consecutive cuff injury to induce lesion rupture. In both models miR-210 was significantly repressed in response to vascular injury and plaque instability. In silico analysis revealed 48 previously validated targets of miR-210 in the vasculature. Functional miR-210 modulation analyses in both murine models revealed that the expression of three known tumorsuppressor genes (Aifm3, Apc, Casp8ap2) could be substantially altered through miR-210 modulation, affecting the proliferation rate of smooth muscle cells (SMCs) and plaque stability. By utilizing an in vitro cell streamer system, we subjected human carotid artery endothelial cells (hCAECs) and hCASMCs to laminar and oscillatory flow conditions. Interestingly, laminar flow induced miR-210 up-regulation, whereas turbulent flow suppressed miR-210 expression in both cell lines, suggesting that miR-210 repression in atherosclerotic plaques is a potential result of altered shear stress and accelerated flow patterns. Aifm3, Apc and Casp8ap2 were significantly up-regulated with disturbed flow.

The present study explores the role and therapeutic potential of miRNA regulation in patients with vulnerable atherosclerotic lesions, carotid artery stenosis and increased risk of stroke. We were able to identify miR-210 as a key modulator of pathologic processes in atherosclerosis-related vascular remodelling.
Abstract 87: KLF2 Regulates Aortic Aneurysm Morphology via Matrix Endopeptidase Regulation During Aneurysm Formation
Authors: Salmon Morgan , Pope Nicolas H , Davis John P , Johnston William F , Vandenbosch Matthew , Su Gang , Owens Gary K , Upchurch Gilbert R , Ailawadi Gorav
Published: March 2022

Objective:
Kruppel-like Factor 2(KLF2) is known to play an athero-protective role in the endothelium following statin treatment; however, its role in abdominal aortic aneurysm (AAA) formation remains unknown. We hypothesized that KLF2 is critical in aneurysm formation through IL-6 and matrix endopeptidase activation.

Methods:
8-12 wk male(n=6/group) C57/B6 mice underwent abdominal aortic elastase perfusion and were harvested at 3, 7, and 14 days for qPCR and immunohistochemistry(IHC) for KLF2. 8-12 wk male(n=6/group) MyH Cre+ KLF2 flx/flx (tamoxifen conditional smooth muscle(SMC) KLF2 KO) and MyH Cre+ KLF2 wt/wt mice underwent elastase perfusion following tamoxifen administration and harvested at 14 days. These mice were also bred to ApoE-/- and administered Angiotensin II for 28 days via osmotic pump(n=8/group). Separately, Angiotensin II treated KLF2 flx/wt ApoE-/- ERT Cre+ and KLF2 flx/wt ApoE-/- ERT Cre- mice(n=8/group) were also examined. Aortas were harvested at 14 and 28 days and evaluated by IHC. KLF2 was knocked down in vitro using siRNA in SMCs and macrophages and exposed to elastase, IL-1β or elastin degradation products(EDP).

Results:
KLF2 was elevated via qPCR and IHC 7 and 14 days following elastase-perfusion. 14 days following elastase perfusion, aortic dilation was significantly reduced in SMC specific KLF2 KO mice versus controls(49.5±5.76% vs. 99±5.72%, p<0.05) with reduced macrophage, T-cell, and neutrophil staining and greater elastin preservation and α-actin expression. In the Angiotensin II model, there was greater survival by Kaplan-Meier analysis in Klf2 flx/wt ERT Cre+ mice(Chi2=4.215, p=0.0453) and in SM-MHC Klf2 flx/flx mice(Chi2=4.342, p=0.0413) compared to their WT controls. In vitro studies demonstrated KLF2 siRNA transfection of SMCs during elastase exposure resulted in an attenuation of the matrix endopeptidase Astl1 and Ssc5d while knock-down of KLF2 in macrophages during IL-1β exposure resulted in an attenuation of IL-6 up-regulation. KLF2 binds astl1 and Ssc5d following elastase treatment in SMCs.

Conclusions:
Endogenous KLF2 activity is critical for AAA formation and SMCs appear to be an important source for KLF2. KLF2 could represent a putative therapeutic target to prevent aneurysm formation.
Abstract 88: Blockade of ETS-1 Attenuates EGF-Induced Collagen Loss in Carotid Plaques of Patients With Carotid Stenosis
Authors: Agrawal Devendra K , Rao Velidi H , Stoupa Samantha
Published: March 2022

Introduction:
Coronary thrombosis and rupture of plaques leading to stroke are major causes of morbidity and mortality. Here, we examined the role of v-ets erythroblastosis virus E26 oncogene homologue-1 (ETS-1) on the epidermal growth factor (EGF)-induced destabilization of collagen type I (Col-Iα1) and collagen type III (Col-IIIα1) involving matrix metalloproteinase-1 (MMP-1) and MMP-9 in isolated vascular smooth muscle cells (VSMCs) from the plaques of symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. To understand the molecular mechanisms underlying EGFR-regulated ETS-1, the signaling pathways directing ETS-1 production was investigated and examined if EGF-induced loss of fibrillar collagens in patients with carotid stenosis is regulated by ETS-1.

Methods:
VSMCs isolated from carotid plaques of both AS and S patients were treated with or without EGF. The mRNA transcripts and protein for ETS-1, MMP-9 and -1, EGFR, Col-I(α1) and Col-III(α1) were analyzed by qPCR and immunofluorescence, respectively.

Results:
Treatment of VSMCs with EGF significantly increased ETS-1 expression and immunoreactivity in S compared to AS. EGFR inhibitor (AG1478) decreased ETS-1 expression and upregulated Col-I(α1) and Col-III(α1) in EGF-treated VSMCs of both groups. In the transfection experiments, overexpression of ETS-1 in VSMCs increased mRNA transcripts of MMP-9 and MMP-1 and decreased the collagen mRNA transcripts. Knock-down of ETS-1 gene with either siRNA or transfection with dominant-negative form (ETS-DN) blocked EGF-induced MMP-1 and MMP-9 expression and increased mRNA transcripts for Col-I(α1) and Col-III(α1) in S compared to AS group. Inhibitors of p38MAPK (SB202190) and JNK-II (SP600125) decreased ETS-1, MMP-1 and MMP-9 transcripts and increased collagen transcripts in EGF-treated VSMCs, suggesting that p38MAPK and JNK pathways are involved in EGFR-induced collagen loss.

Conclusion:
These findings revealed a novel mechanism of ETS-1-regulated EGF-induced collagen loss in human carotid plaques and this could be a leading cause of plaque instability in patients with carotid stenosis. Selective blockade of ETS-1 and EGFR may be novel strategy and promising target for treating unstable and vulnerable plaques.
Abstract 112: Nitrated Apolipoprotein Ai/apolipoprotein Ai Ratio Is Increased in Diabetic Patients With Coronary Artery Disease
Authors: Chen Xueying , Bakillah Ahmed , Zhou Liye , Hoepfner Florian , Jacob Marrit , Pan Xiaoyue , Jiang Xian-Cheng , Lazar Jason , Schlitt Axel , Hussain M. Mahmood
Published: March 2022

Recent studies indicated that protective function of HDL may be more representative than its concentration. Apolipoprotein AI (apoAI), the major protein of HDL, is nitrosylated in vivo to nitrated apoAI (NT-apoAI) that might cause dysfunction. We measured plasma NT-apoAI and apoAI levels in 777 patients with coronary artery disease (CAD) by ELISA, and found that median NT-apoAI/apoAI ratio was significantly higher in diabetes mellitus (DM) (n=327) versus non-diabetic patients (n=450). Further analysis indicated that DM, thiobarbituric acid-reactive substances and C-reactive protein levels were independent predictors of higher NT-apoAI/apoAI ratio. There was negative correlation between NT-apoAI/apoAI and use of anti-platelet and lipid lowering drugs. The cholesterol efflux capacity of plasma from 67 individuals with differing NT-apoAI but similar apoAI levels from macrophages in vitro was negatively correlated with NT-apoAI/apoAI ratio. In conclusion, higher NT-apoAI/apoAI ratio is significantly associated with DM in this relatively large German cohort with CAD and may contribute to associated complications by reducing cholesterol efflux capacity.
Abstract 115: Microsomal Triglyceride Transfer Protein Is a Major Determinant of Plasma Ceramide And Sphingomyelin but Not of Hexosylceramide and Lactosylceramide
Authors: Iqbal Jahangir , Walsh Meghan T , Hammad Samar M , Cuchel Marina , Tarugi Patrizia , Hegele Robert A , Davidson Nicholas O , Rader Daniel J , Klein Richard L , Hussain M. Mahmood
Published: March 2022

Sphingolipids are present in cell membranes and in plasma lipoproteins. Mechanisms of sphingolipid transport to plasma lipoproteins are unknown. We found that plasma levels of ceramide and sphingomyelin were significantly lower in homozygous abetalipoproteinemia subjects defective in microsomal triglyceride transfer protein (MTP) function compared with heterozygotes, and in mice deficient in both intestinal and hepatic MTP compared with control mice. In contrast, plasma concentrations of hexosylceramide, lactosylceramide and different sphingosines were not affected by MTP deficiency. MTP deficiency had no effect on ceramide and sphingomyelin synthesis, but reduced their secretion. MTP was found to transfer ceramide and sphingomyelin. These studies suggest that MTP is a critical determinant of plasma ceramide and sphingomyelins, but not of glycosylceramide and sphingosine. The MTP-dependent transport processes to carry ceramide and sphingomyelin might have evolved to protect against toxicity associated with cellular ceramide accretions and to supply sphingomyelin to other tissues to maintain membrane integrity and to control cellular differentiation and proliferation.
Abstract 118: Phospholipid Transfer Protein, High Density Lipoprotein Particle Concentration and Cardiovascular Disease Risk
Authors: Vaisar Tomas , Hutchins Patrick , Tang Chongren , Prazen Bryan , Zhao Xue-Qiao , Heinecke Jay W
Published: March 2022

High density lipoprotein (HDL) plays a key role in reverse cholesterol transport from arteries and protects against development of atherosclerosis by removing cholesterol from lipid loaded macrophages in atherosclerotic lesions. Low levels of HDL-cholesterol (HDL-C) strongly associate with increased risk of cardiovascular disease (CVD) in clinical and epidemiological studies. However, recent trials with drugs that elevate HDL-C failed to reduce CVD events. In contrast recent studies strongly suggested that HDL sterol efflux capacity-its ability to remove cholesterol from macrophages-predict incident cardiovascular disease risk. Moreover, CVD risk strongly associates with low levels of HDL particle concentration (HDL-P). However, the relations between HDL efflux capacity, HDL particle concentration and HDL composition are not well understood. Using proteomics analysis, we have previously identified over 50 proteins on HDL. Here we investigated the relationship of HDL composition, and HDL-P in two independent populations of people with and without CVD (Study 1, n=40, and Study 2, n=65). Using quantitative targeted multiplex MS/MS analysis quantifying majority (40) of HDL associated proteins, we found that phospholipid transfer protein (PLTP) and clusterin (CLU or ApoJ) strongly correlated in HDL (r = 0.9, P < 0.001) indicating that the two proteins likely coexist in a single population of HDL particles. Furthermore, abundance of these two proteins correlated strongly with the concentration of large HDL particles (PLTP r = 0.58; CLU r = 0.61, both P < 0.001) and negatively with the concentration of small HDL particles (both PLTP and CLU r = -0.49, P < 0.001) as determined by calibrated ion mobility analysis. Moreover, both PLTP and CLU were significantly decreased in CVD subjects, and both predicted CVD status after adjustment for age and sex (PLTP, OR 0.53, 95% CI, 0.28-0.94, P = 0.02). Collectively, this data supports the hypothesis that PLTP and CLU play important roles in determining HDL-P and that these are interrelated factors determining HDL’s anti-atherogenic properties.
Abstract 119: Identifying and Quantifying Eight ApoA-I HDL Subspecies by a Novel Modified Sandwich ELISA
Authors: Yamamoto Rain , Furtado Jeremy D , Sacks Frank M
Published: March 2022

HDL-cholesterol is a well-established independent risk factor used in CVD risk stratification. However, the cardioprotective effect of HDL-cholesterol is challenged by genetic studies and pharmaceutical trials, which do not support the causal relationship between increased HDL-cholesterol levels and CVD. Previously, our lab found that HDL can be classified into subspecies based on the presence of apoC-III, where for example 7% of the total plasma apoA-I in HDL has apoC-III. We also showed that the apoA-I concentration of HDL that has apoC-III predicts increased risk of CHD, whereas that of HDL that does not have apoC-III predicts decreased risk of CHD. We hypothesized that some of the many other proteins that are located in HDL are present in apoA-I HDL subspecies.

We developed a novel sandwich ELISA to measure apoA-I in HDL that is associated with a specific protein of interest, and applied it to apoA-II, apoC-III, and apoE, which define known subspecies of apoA-I HDL. We also applied the ELISA to apoC-I, apoC-II, and plasminogen, to determine whether their association with HDL constitutes a subspecies of apoA-I HDL, and in what concentrations they exist in HDL. We confirmed the existence of all the apoA-I lipoprotein subtypes using pooled plasma samples collected from healthy men and women (age: 40±14 (mean±SD), triglyceride: 116±63 mg/dl, LDL-cholesterol: 118±28 mg/dl, HDL-cholesterol: 45±10 mg/dl). The concentration (percentage) of total plasma apoA-I in HDL associated with apoA-II was 103±11 mg/dl (71±12%), with apoC-I was 23±4 mg/dl (16±2%), with apoC-II was 4±1 mg/dl (2±1%), with apoC-III was 9±3 mg/dl (6±1%), with apoE was 8±5 mg/dl (5±3 %) and with plasminogen was 4±1 mg/dl (3±1%). The results for apoA-II, apoC-III, and apoE are consistent with previously reported values. In conclusion, we established a convenient sandwich ELISA, which demonstrated the existence of new apoA-I HDL subspecies that are defined by presence of apoC-I, apoC-II, and plasminogen, as well as known subspecies of apoA-I HDL; and we quantified the concentration of such subspecies. Future studies will investigate these abundant HDL subtypes in relation to CVD risk.
Abstract 120: Inhibitory Effects of Cholesterol on Lysophosphatidylcholine Metabolism in Human Monocytes
Authors: Fernandez-Ruiz Irene , Aluganti Narasimhulu Chandrakala , Sengupta Bhaswati , Parthasarathy Sampath
Published: March 2022

Background and aims:
In hypercholesterolemic conditions blood cells are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PtdCho). While the metabolism of lyso-PtdCho by monocytes has been studied, the effects of high cholesterol, which complexes with lyso-PtdCho, are not well understood. In this study we used a novel assay developed in our laboratory, based on the ability of lyso-PtdCho to solubilize and deliver cholesterol into the cells, to study whether cholesterol affects the metabolism of lyso-PtdCho.

Methods:
In THP-1 monocytes
14
C-lyso-PtdCho metabolism was analyzed by TLC and cytotoxicity by WST-1 assay.

Results:
Monocytes incubated with lyso-PtdCho alone metabolize the incorporated lyso-PtdCho by 4h mainly into phosphatidylcholine, with a small percentage into sphingomyelin and phosphocholine. The latter could represent PLase C reaction that would generate mono- and diacyl-glycerols, important signaling molecules. However in cells incubated with cholesterol or cholesteryl ester (CE)/lyso-PtdCho micelles, at 4h the radioactivity remained mainly in the lyso-PtdCho fraction (p<0.01 and p<0.001). Lyso-PtdCho decreased monocyte viability at concentrations over 30μM, but cholesterol and CE inhibited the lyso-PtdCho-induced cytotoxicity (p<0.001). Lyso-PtdCho and CE/lyso-PtdCho, but not cholesterol/lyso-PtdCho, increased the gene expression of the pro-inflammatory chemokine MCP-1 (p<0.05). Peripheral blood monocytes from LDL-R -/- mice on a high fat diet had higher levels of CE than monocytes from mice in a normal diet (n=10, p<0.05). Monocyte CE content in these mice positively correlated with plasma LDL/HDL ratio (p<0.05, R
2
=0.65).

Conclusions:
We show that cholesterol and CE inhibit the cytotoxic effects of lyso-PtdCho in monocytes, but only cholesterol counteracted the pro-inflammatory action of lyso-PtdCho. Since lyso-PtdCho concentrations in plasma are increased in hypercholesterolemic conditions, and its content is increased during the oxidation of LDL, the current studies might indicate that cholesterol and lyso-Ptdcho could influence each other’s metabolism. This is important as lyso-PtdCho has been suggested to influence plaque vulnerability.
Abstract 123: The Immunoproteasome Does Not Influence Macrophage Differentiation but Survival of Differentiated Macrophages
Authors: Wang Feilong , Nesbitt Lisa , Lerman Amir , Herrmann Joerg
Published: March 2022

Background:
Atherosclerosis is an inflammatory disease with T cells and macrophages as the leading plaque cell populations. The immunoproteasome (IP) influences T cell survival and differentiation but its impact on macrophage survival and differentiation remains unknown, which was the objective of this study.

Methods:
Murine monocyte/macrophage Raw264.7 cells were stimulated by IFN-gamma (20 ng/mL) plus LPS (10pg/mL) or IL-4 (20 ng/mL) for 18 hours to induce M1 and M2 macrophages, and these differentiated macrophages were then re-exposed to IFN- gamma and IL-4 for another 24 hours, without and with IP subunit beta 5i inhibition by siRNA or Onx0914. Nitric oxide synthase and arginase activity served as differentiation markers (analyzed by enzymatic assays) as did the cytokine/chemokine profile (analyzed by antibody arrays). Cell viability was tested by an XTT assay and apoptosis by TUNEL FACS.

Results:
IP beta5i knockout/inhibition did not influence M1 and M2 macrophage differentiation. Also, no significant differences in apoptosis were observed between RAW264.7 cells treated with and without beta 5i inhibitor during macrophage differentiation. With re-exposure to IFN- gamma and IL-4, however, there was more apoptosis and less survival of differentiated M1 macrophages with beta5i inhibition. Conversely, beta 5i inhibition achieved less apoptosis and more survival of differentiated M2 macrophages when switching IL-4- to IFN-gamma-stimulating conditions.

Conclusions:
IP inhibition does not influence macrophage differentiation but survival of differentiated macrophages of potential significance for the atherosclerotic disease process.
Abstract 128: Recombinant Lecithin Cholesterol Acyltransferase Generates Mature High Density Lipoprotein with Increased Capacity to Efflux Cholesterol via ABCG1 in Cynomolgus Monkeys
Authors: Peach Matthew , Chen Michelle M , Cai Feng , Peng Joanna Z , Tang Chongren , Hale Clarence , Everds Nancy , Fitzpatrick V. D , Shaywitz Adam J , Zhou Mingyue , Xu Ren
Published: March 2022

Introduction:
Lecithin cholesterol acyltransferase (LCAT) esterifies free cholesterol to cholesteryl esters on the HDL particle and is considered a key component of reverse cholesterol transport. We have developed a recombinant human LCAT fusion protein with high LCAT activity and characterized the effects of this molecule on lipoprotein metabolism and cholesterol efflux in cynomolgus monkeys.

Methods:
Cynomolgus monkeys received a single injection of rhLCAT at 0, 0.5, 1, or 5 mpk. Blood samples were drawn for analysis at pre-dose and up to 5 days after administration. HDL-C and apoAI were measured using standard clinical analyzers, apoE was measured by ELISA, and rhLCAT was measured by MSD immunoassay. ApoAI containing HDL subfractions were determined by two-dimensional non-denaturing gradient gel electrophoresis as described in the literature. Cholesterol efflux was measured ex vivo from ABCA1, ABCG1, or SRB1 transfected BHK cells, or J774 mouse macrophages using apoB depleted serum.

Results:
After administration, serum rhLCAT concentrations increased dose proportionately. HDL-C increased rapidly, beginning within the first 6 hours after dosing, and was dose dependent with maximum increases of 87% observed in the 1 and 5 mpk dose groups. ApoAI concentrations increased by as much as 15% and 21% in the 0.5 and 1 mpk groups, respectively, but decreased in the 5 mpk group by as much as 23%. The ratio of HDL-C to apoAI increased dose dependently indicating the formation of HDL particles containing increasingly more cholesterol. 2D gels confirmed the formation of large, primarily α-1, HDL particles. ApoE on HDL particles increased dose dependently with large increases observed following the 1 mpk (169%) and 5 mpk (910%) doses. Changes in the HDL particle were accompanied by changes in the ability of HDL to efflux cholesterol from cells. ABCG1 mediated efflux increased by as much as 21% and 24% in the 1 and 5 mpk dose groups, respectively. Conversely, efflux via ABCA1, SRB1, and from J774 cells was reduced dose dependently.

Conclusions:
The administration of rhLCAT in cynos resulted in the formation of large HDL particles that were enriched in cholesterol and ApoE. These larger particles showed increased capacity to efflux cholesterol via the ABCG1 transporter.
Abstract 138: Cigarette Smoke and Monocytes Induce Endothelial Dysfunction
Authors: Castiglioni Silvia , Barbieri Silvia S , Corsini Alberto , Bellosta Stefano
Published: March 2022

Cigarette smoke induces endothelial cell (EC) dysfunction. Circulating monocytes (HM) adhere to a damaged endothelium and migrate into the intima, differentiate to macrophages and contribute to atherosclerotic plaque growth. We demonstrated that HM exposed to cigarette smoke condensate (CSC) increase the transmigration of HM through EC. Thus, we hypothesized that CSC may affect HM/EC interaction. We analyzed the effects on EC of CSC (30 μg/ml) alone and/or of medium conditioned by HM incubated without (CM) or with CSC (CM/CSC). CSC, CM or CM/CSC did not affect EC vitality (MTT assay). CM and CM/CSC increased the expression (measured by RT-PCR) of adhesion molecule ICAM1 (36-fold and 90-fold, p<0.01 vs control or CM, respectively). The effect on VCAM1 was even more dramatic (63-fold vs control for CM, p<0.01 and 725-fold vs control, p<0.01 with CM/CSC). In addition, CM and CM/CSC enhanced the expression of matrix metalloproteinase (MMP)-3 (+60% vs control, p<0.01; +200% vs control, p<0.01, respectively) and MMP-9 (+70% vs control, p<0.01; +140% vs control, p<0.01, respectively). MMP-1 and MMP-2 were not affected. Then, we used the iCELLigence System to analyze EC biological status (cell number, adhesion, viability, and morphology). Only the addition of CM/CSC to confluent EC monolayer dramatically modified the pattern of the electric flow, indicative of modifications in EC morphology. Indeed, we observed an altered actin cytoskeleton organization by immunofluorescence studies performed with FITC-phalloidin. In particular, CM/CSC caused the shrinking of the EC cytoplasm by about 50% (p<0.01 vs control or CM). Moreover, CM/CSC altered sub-cellular localization of beta-catenin promoting its trafficking from membrane into the cytosol and the nucleus. In contrast, EC morphology was not affected by exposure to CSC or CM. Finally, CM and CM/CSC reduced (by 40% and 60%, p<0.05, respectively) the expression of the small signaling G protein Rac 1 GTPase involved in cytoskeletal remodeling. In conclusion, these results indicate that monocytes, in the presence of cigarette smoke, secrete some soluble factors that affect endothelium morphology, thus increasing its permeability and facilitating monocytes transmigration through the endothelial monolayer.
Abstract 148:
Stat2
Deficiency Does Not Protect From Atherosclerosis in
Ldlr
Knockout Mice Fed a Western Diet
Authors: Gupta Rajat , Schindler Christian , Wu Huaizhu , Ballantyne Christie , Lagor William R
Published: March 2022

Signal Transducer and Activator of Transcription (
Stat2
) is a transcription factor that plays a critical role in type I interferon (IFN) signaling. Recent evidence supports a role for the type I IFN pathway in atherosclerosis, since recombinant IFN alpha and IFN beta both promote atherosclerosis in mice. We tested the hypothesis that constitutive type I IFN signaling through
STAT2
may promote atherosclerotic lesion formation. Male
Ldlr
KO and
Stat2/Ldlr
DKO mice on a C57BL6/J background were fed a western diet for 16 weeks. Total cholesterol levels were indistinguishable at baseline (
Ldlr
KO: 232 ± 24 vs.
Stat2/Ldlr
DKO: 239 ± 36 mg/dl, p=0.39), but significantly higher in the
Stat2/Ldlr
DKO mice after 16 weeks of western diet (
Ldlr
KO: 856 ± 203 Vs
Stat2/Ldlr
DKO: 1015 ± 176 mg/dl, p=0.004). Despite consuming less food than
Ldlr
KO controls, the
Stat2/Ldlr
DKO animals weighed significantly more than controls and had greater fat mass in the inguinal subcutaneous and perigonadal depots respectively (
Ldlr
KO: 0.45±0.23 Vs
Stat2/Ldlr
DKO: 0.69 ± 0.27 g, p=0.003 and
Ldlr
KO: 0.89 ± 0.50 Vs
Stat2/Ldlr
DKO: 1.39 ± 0.56 g, p=0.003). Surprisingly, no significant differences were observed in percent lesional area of aortae (
Ldlr
KO: 9.7% ± 5.6% Vs
Stat2/Ldlr
DKO: 12.5% ± 6.8%, p=0.156). In conclusion, STAT2 does not appear to have a major impact on atherosclerosis in the absence of exogenous type I IFNs or other agonism of the pathway.
Abstract 149: Relationship between Epicardial Fat Thickness and Underlying Coronary Atherosclerotic Plaque Size and Burden
Authors: Coura Laura M , Gutierrez Paulo S
Published: March 2022

A paracrine effect of epicardial fat in underlying coronary atherosclerosis has been proposed, due to the inflammatory role of adipose tissue. Most studies evaluate globally fat (by magnetic resonance) and atherosclerosis (by computerized tomography). We analyzed microscopically, segment by segment, the potential local influence of fat in atherosclerotic plaque size and burden. Thickness of epicardial fat, coronary lumen area, and area delimited by the internal elastic lamina were measured with an image analysis system coupled to a light microscope, and plaque size (area delimited by the internal elastic lamina less lumen area) and burden (plaque area/ area delimited by the internal elastic lamina) were calculated in 192 coronary artery segments from 9 hearts from necropsies of patients that died with coronary atherosclerosis. To compare epicardial and myocardial faces of the arteries, the measures were made in each half; total amounts were also calculated. The relationships between epicardial fat and underlying atherosclerosis, both considering epicardial half and the whole segment, and between total fat and the total arterial segment, were tested by Pearson’s test. Plaque size and burden in each half were compared by paired Wilcoxon signed rank test. We considered correlation strength as
r
=0.10 to 0.29- weak,
r
= 0.30 to 0.49- medium, and
r
= 0.50 to 1.0- strong correlation, and results significant if
P
< or = 0.05.

Results:
Correlation coefficients:
epicardial face fat thickness
- 1) with plaque area at epicardial half -
r
= 0.22,
P
<0.01; 2) with plaque burden at epicardial half-
r
= 0.18,
P
=0.02; 3) with total plaque area-
r
= 0.19,
P
<0.01; 4) with total plaque burden-
r
= 0.10,
P
=0.18;
total epicardial fat thickness
- 1) with total plaque area -
r
= 0.22, p<0.01; 2) with total plaque burden-
r
= 0.06,
P
=0.39.

Comparison between halves:
area size
- mean 1.20 mm at epicardial face and 1.5 mm at myocardial face,
P
=0.96;
plaque burden
- mean 33.7% at epicardial face and 45.7% at myocardial face,
P
=0.98.

Conclusion:
the correlations were weak and/or non-significant, and the atherosclerotic process involves equally both halves of the coronary arteries; thus our data indicate that there is no significant effect of fat tissue in underlying coronary atherosclerosis.
Abstract 173: Upregulation of Sema7A Induced by Disturbed Flow Contributes to the Development of Atherosclerosis
Authors: Yang Fei , Hu Shuhong , Tang Chaojun , Zhu Li
Published: March 2022

Background:
Atherosclerosis preferentially develops in areas of arteries with arches and branches, where the flow is disturbed (D-flow). D-flow upregulates proatherogenic genes and promotes the dysfunctional endothelial cell phenotype. Semaphorin 7A (Sema7A), a membrane-associated glycosylphosphatidylinositol (GPI)-linked cell-surface glycoprotein, was recently found to be expressed in the endothelial cells and played important roles in several pathophysiological processes. We hypothesize that Sema7A is involved in the proatherogenic process induced by D-flow. Methods and Results: We performed partial carotid ligation (PCL) on C57BL/6 mice by ligation of the external carotid artery (ECA), internal carotid artery (ICA), and ophthalmic artery (OA) of the left carotid artery (LCA), leaving the superficial temporal artery (STA) untied. The ultrasound image was used to monitor blood flow. Successful partial ligation was obtained as blood flow velocity was reduced by 70% (n=3) and D-flow was generated in the LCA while the blood flow in RCA remained normal. After 48h ligation, mice were sacrificed and total RNA was isolated from carotid arteries for the analysis of Sema7A gene expression by real-time PCR. Results showed that Sema7A expression in the LCA was 2.6-fold higher (P=0.0237, n=3) in D-flow condition compared to RCA. To ask whether Sema7A is involved in D-flow-induced atherosclerosis, we generated Sema7A-/-ApoE-/- mice and Sema7A+/+ApoE-/- mice, and performed PCL at 2 weeks on high fat diet. The results showed that atherosclerotic lesion size in the LCA under disturbed condition was decreased by 50.74% (P=0.0072) in ApoE-/-Sema7A-/- mice (0.2411±0.05265%, n=6) compared to Sema7A+/+ApoE-/- control mice (0.4894 ± 0.05186%, n=6). Conclusion: Our data showed that D-flow promotes the upregulation of Sema7A gene expression in endothelial cells. Deletion of Sema7A attenuates atherosclerotic plaque formation induced by D-flow.
Abstract 174: MicroRNA-216a Induces a Premature Senescent-Like Phenotype and Regulates Angiogenic Activity in Human Vascular Endothelial Cells
Authors: Yang Shujun , Mi Xuenan , Wang Yuxuan , Chen Yu , Hui Rutai , Zhang Weili
Published: March 2022

Background:
Among the age-associated functional and structural changes, of particular note is cellular senescence of endothelial cells, which plays a key role in endothelial dysfunction and atherogenesis. But the molecular basis of which is not fully defined. We have identified that a particular microRNA,
miR-216a
, was up-regulated in senescent endothelial cells, here, the present study aimed to determine the role of
miR-216a
in regulating premature senescence and angiogenic activity of endothelial cells.

Methods and results:
Human umbilical vein endothelial cells (HUVECs) were cultured and population-doubling levels (PDLs) were calculated during passages. Briefly, PDLs was computed as Log2 (Ch/Cs), where Ch was the number of viable cells at harvest and Cs was the number of cells seeded. PDL8 and PDL44 were respectively identified as young and senescent HUVECs, with increased expression of p53 and p21 and shortened telomere length in PDL44. We found that
miR-216a
expression is up-regulated by 64% in PDL44 (
P
<0.05). Next, we examined the effect of
miR-216a
on senescence-associated β-galactosidase (SA-β-gal) activity, a characteristic of senescence-related growth arrest. In PDL8 with stable
miR-216a
lentiviral transfection, we found that
miR216a
overexpression increased the percentage of SA-β-gal[[Unable to Display Character: –]]positive cells by 1.8-fold compared with control at about 15 days. We also found that telomerase activity was dependent on modulation of
miR-216a
levels, which confirms a crucial role for
miR-216a
in HUVEC senescence. To further analyze the effect of
miR-216a
on endothelial functions, we tested the cell proliferation, migration, adhesion, and tube-formation abilities. The results showed that
miR-216a
overexpression accelerated the decline of endothelial functions at about 15 days, which led to a significant inhibition of endothelial proliferation and migration by 15% (
P
<0.001) and 8% (
P
=0.001) and increased adhesion capability of THP-1 cells to HUVECs by 1.9-folds (
P
=0.001). There was no significant impairment of tube formation in the Matrigel assay.

Conclusions:
Our data indicated that
miR-216a
can promote the premature endothelial senescence and may serve as a target in regulating endothelial dysfunction associated with atherosclerosis.
Abstract 180: Risks and Predictors of Ischemic/bleeding Complications in Cardiac Patients Undergoing Elective Non-cardiac Surgery - An Analysis of the Prague-14 Study
Authors: Motovska Zuzana , Dusek Ladislav , Ondrakova Martina , Knot Jiri , Havluj Lukas , Robert Gurlich , Bartoska Radek , Dupa Valer , Bittner Lukas , Widimsky Petr
Published: March 2022

Objective:
To assess risks and predictors of ischemic/bleeding complications in consecutive patients with at least one cardiovascular disease undergoing elective major non-cardiac surgery.

Methods:
Subgroup analysis of the PRAGUE-14 study was performed. This prospective study analyzed the impact of antithrombotic therapy interruption on outcomes in non-selected surgical patients with known cardiovascular disease. Logistic regression models were applied to assess the association between potential predictors and selected binary coded end-points (perioperative ischemia, bleeding and hospital mortality).

Results:
Study population consisted of 1200 patients, 742 of them underwent elective surgery; age (median) 72.0, 66.2% males, CAD in 396, stroke in 70, atrial fibrillation in 209, venous thromboembolism (VTE) in 55, valvular heart disease in 108, artificial valve in 18, cardiomyopathy in 13.

Preoperatively, 62.0% patients were receiving betablockers, 55.1% ACE inhibitors, 32.9% statins. 84.6% of patients were treated with antithrombotic therapy i.e. 439 aspirin, 31 DAPT, 192 oral anticoagulants. Prophylaxis of VTE in perioperative period was applied in 93.5%.

In-hospital ischemic complications occurred in 3% (vs 9.8% in acutely operated patients, p<0.001), bleeding complications in 10.90% (vs 12.0%, n.s.), and both of them in 0.7% (vs. 4.2%, p<0.001). Mortality of patients, who underwent planned surgery, was 2.2% (vs. 6.8% in acute surgery, p<0.001).

Multivariate linear regression identified that pre-procedural anemia and history of (coronary or non-coronary) stent implantation were significantly related to ischemic complications. Periprocedural antithrombotic management has no impact on ischemic complications. Termination of warfarin less than 4 days before surgery was significantly related to the risk of bleeding.

Conclusions:
Risk of perioperative ischemic complications is significantly lower in cardiac patients who are undergoing non-cardiac surgery electively (in comparison to acutely), and this risk is significantly related to a history of preprocedural anemia and history of stent implantation. For prevention from periprocedural bleeding, warfarin should be discontinued more than 4 days before surgery
Abstract 192: Neurotrophin 3 Mediates the Fibrogenic Response to Pro-inflammatory Stimulation in Human Aortic Valve Interstitial Cells
Authors: Yao Qingzhou , Song Rui , Ao Lihua , Yu Xiyong , Fullerton David , Meng Xianzhong
Published: March 2022

Background:
Calcific aortic valve disease is characterized by inflammation, fibrosis and calci[[Unable to Display Character: ﬁ]]cation. Aortic valve interstitial cell (AVIC) proliferation and over-production of extracellular matrix (ECM) proteins result in valvular fibrosis and thickening, and an inflammatory milieu in valvular tissue is believed to promote aortic valve fibrosis and calcification. However, the factors that mediate AVIC fibrogenic response remain to be identified. Recently, we observed that AVICs of aortic valves affected by calcific disease express higher levels of neurotrophin 3 (NT3). It is unknown whether NT3 induces the fibrogenic response in AVICs and whether this growth factor plays a role in valvular fibrogenic response to pro-inflammatory stimulation. Hypotheses: NT3 is a pro-fibrogenic factor in the aortic valve and mediates the fibrogenic response to pro-inflammatory stimulation in human AVICs. Methods and Results: Stimulation of human AVICs with lipopolysaccharide (LPS, 0.20 μg/ml) induced proliferation and up-regulated production of MMP-9 and collagen III. TLR4-dependent NT3 over-production was observed in cells exposed to LPS and was attenuated by inhibition of the NF-κB, ERK1/2 or p38 MAPK pathway. Neutralization of NT3 or blocking the Trk receptors (neurotrophin receptors) with K252a suppressed LPS-induced fibrogenic response and reduced collagen deposition. Treatment of human AVICs with recombinant NT3 (0.10 μg/ml) also induced proliferation accompanied by up-regulated production of MMP-9 and collagen III. NT3-induced fibrogenic response was preceded by Akt phosphorylation and cyclin D1 up-regulation mediated by the Trk receptors. Inhibition of the Trk receptors, Akt or cyclin abrogated NT3-induced fibrogenic response. Conclusions: This study provides the first evidence that NT3 is capable of inducing human AVIC proliferation and ECM protein production via the Trk-Akt-cyclin D1 axis and plays an important role in mediating the TLR4-evoked fibrogenic response in human AVICs. Thus, NT3 is a pro-fibrogenic mediator in aortic valves. Up-regulation of AVIC production of NT3 by pro-inflammatory stimuli may have a mechanistic role in aortic valve fibrosis associated with calcific aortic valve disease.
Abstract 204: Conjugated Linoleic Acid Induces an Atheroprotective Macrophage MΦ2 Phenotype and Limits Foam Cell Formation
Authors: de Gaetano Monica , Alghamdi Kawthar , Marcone Simone , Belton Orina
Published: March 2022

Background:
Atherosclerosis, the underlying cause of heart attack and stroke, is a progresive dyslipidemic and inflammatory disease where monocyte-derived macrophage cells play a pivotal role. Although most of the mechanisms that contribute to the progression of atherosclerosis have been identified, there is limited information on those governing regression. Conjugated linoleic acid (CLA) is a group of isomers of linoleic acid that differ in the position and/or geometry of their double bonds. We have previously shown that a specific CLA blend (80:20 cis-9,trans-11:trans-10,cis-12-CLA) induces regression of pre-established atherosclerosis in vivo, via modulation of monocyte/macrophage function. However, the exact mechanisms through which CLA mediates this effect remain to be elucidated.

Methods:
Here, we address if CLA primes monocytes towards an anti-inflammatory MΦ2 macrophage and examine the effect of individual CLA isomers and the atheroprotective blend on monocyte-macrophage differentiation cytokine generation, foam cell formation and cholesterol metabolism in human peripheral blood monocyte (HPBMC)-derived macrophages.

Results:
cis-9,trans-11-CLA and the atheroprotective 80:20 CLA blend regulates expression of pro-inflammatory mediators and modulates the inflammatory cytokine profile of macrophages and foam cells. In addition, cis-9,trans-11-CLA and CLA blend primes HPBMCs towards an anti-inflammatory MΦ2 phenotype, characterised by increased scavenger receptor (CD36) and efflux protein (ABCA-1) expression. Furthermore, this altered macrophage phenotype impacts on foam cell formation, inhibiting ox-LDL accumulation and promoting cholesterol efflux via both PPARγ and LXRα dependent pathways.

Conclusion:
The data increases the understanding of the pathways regulated by CLA in atheroprotection, namely, inhibiting the progressive acquisition of a pro-inflammatory macrophage phenotype.
Abstract 206: Endothelium-derived 5-methoxytryptophan Protects Endothelial VE-cadherin and Barrier Function by Inhibiting p38 Mitogen-activated Protein Kinase Activation
Authors: Chu Ling-yun , Cheng Huei-Hsuan , Wu Kenneth K
Published: March 2022

VE-cadherin at adherens junction is important in maintaining the integrity of endothelium. Inflammatory cytokines and growth factors such as TNFα and VEGF have been reported to suppress endothelial VE-cadherin level and increase endothelial permeability. 5-methoxytryptophan was first identified by our group as an endogenous factor produced by human fibroblasts and endothelial cells to control the expression of cyclooxygenase-2 and proinflammatory cytokines. Since endothelial cells are the major source of 5-methoxytryptophan production, we assessed the hypothesis that 5-methoxytryptophan plays a role in maintaining vascular integrity. In this study, we determined the influence of proinflammatory mediators on 5-methoxytryptophan production in human umbilical vein endothelial cells and assessed the relation between 5-methoxytryptophan production and VE-cadherin and endothelial permeability. TNFα, IL-1β, lipopolysaccharide (LPS) as well as VEGF reduced VE-cadherin and increased endothelial permeability. They did not affect cell viability as evaluated by MTT assay but reduced 5-methoxytryptophan production by >70% compared to vehicle control. Addition of synthetic 5-methoxytryptophan rescued VE-cadherin and prevented endothelial leakage. TNFα, IL-1β, LPS or VEGF activated p38 mitogen-activated protein kinase (MAPK) as analyzed by phosphorylated p38. 5-methoxytryptophan pretreatment abrogated p38 MAPK activation accompanied by preservation of VE-cadherin and maintenance of barrier function. In conclusion, our findings show for the first time that endothelium-derived 5-methoxytryptophan protects VE-cadherin and endothelial barrier function from damage by endotoxins and cytokines via inhibition of p38 MAPK activation.
Abstract 223: Inhibition of STAT3 Phosphorylation by Sulforaphane Reduces Adhesion Molecule Expression in Vascular Endothelial Cell
Authors: Ahn Hee Y , Cho Young S , Kim Chan H
Published: March 2022

This study explored the mechanism(s) by which sulforaphane inhibits intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressions stimulated with lipopolysaccharide (LPS) in human umbilical vein endothelial cell (HUVEC).

The protein levels of ICAM-1 and VCAM-1 were investigated by primary antibodies of anti-human ICAM-1, anti-human VCAM-1. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) were investigated by phospho-STAT3 antibody. The STAT3-specific small interfering RNA (siRNA) was used to knockdown the expression of STAT3. THP-1 monocyte adhesion photograph images were obtained using a fluorescence microscope. Sulforaphane prevented LPS-mediated increase of ICAM-1 and VCAM-1 expression. Sulforaphane prevented LPS-mediated increase of STAT3 phosphorylation. AG490, Janus kinase (JAK)-2 inhibitor and Stattic, STAT3 inhibitor, reduced LPS-induced the expressions of ICAM-1 and VCAM-1, respectively. STAT3 small interfering RNA (siRNA) treatment reduced ICAM-1 and VCAM-1 expression stimulated with LPS. Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC. These data provide a novel mechanism where sulforaphane decreases an expression of ICAM-1 and VCAM-1 onto vascular wall through an inhibition of STAT3 phosphorylation.
Abstract 245: Prevention of Glyceryl Trinitrate Tolerance by Flavonoids
Authors: Zhang Lu , Leung Susan W , Man Ricky Y
Published: March 2022

Background and propose:
The anti-ischemic effects of glyceryl trinitrate (GTN) are rapidly blunted after continuous application. This phenomenon of GTN tolerance may be the result of an increased oxidative stress and/or a reduced bioactivation of GTN by the enzyme aldehyde dehydrogenase (ALDH). The polyphenolic compounds flavonoids are reported to potentiate vascular relaxation and inhibit oxidation. Therefore, in the present study, we assessed the hypothesis that flavonoids can prevent GTN tolerance. Whether or not this effect of flavonoids is related to their anti-oxidative property or modulation of the activity of ALDH was also examined.

Experimental approach:
Isolated rat aortic rings were incubated with modified Krebs-Henseleit solution containing GTN (30 μM) in organ chamber at 37°C for 1 hour to induce tolerance. Flavonoids (10 μM of apigenin, luteolin, kaempferol, quercetin, genistin, genistein, narigenin, catechin or epicatechin) or the vehicle (1 mM dimethyl sulfoxide) were added to the aortic rings 30 minutes before GTN treatment. Those rings without incubation with GTN were served as the respective control groups. After removing the treatment solution, rings were contracted with phenylephrine (1 μM) followed by cumulative addition of GTN (0.1 nM to 30 μM). GTN-induced relaxation was calculated as change in isometric tension of aortic rings from the contraction induced by phenylephrine. Vascular ALDH activity was assayed by spectrophotometry as the rate of NAD+ consumption.

Key results:
GTN-induced relaxation in rat aortic rings was reduced by prior incubation with GTN; this reduction was prevented by apigenin (10 μM), which did not affect the relaxations in the group without prior treatment with GTN. Apigenin (10 μM) also increased the vascular ALDH activity which was reduced by prior GTN incubation.

Conclusion:
Apigenin appears to have the potential to prevent the development of GTN tolerance, likely by restoring the bioactivation of GTN by ALDH.