bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 303: CD44 Activation Protects Human Sclerotic-derived Valvular Interstitial Cells from Calcification
Authors: Poggio Paolo , Branchetti Emanuela , Sainger Rachana , Grau Juan , Lai Eric , Ferrari Giavanni 
Published: July 2021

Introduction.
The activation of Valvular Interstitial Cells (VIC) towards an osteoblast-like phenotype is a cellular hallmark of pathological progression towards Aortic Stenosis (AS). In recent years several clinical trials have failed to halt or revert the progression of this prevalent disease. The ability to prevent end-stage AS requires the understanding of the molecular events associated with the early phase of valve degeneration, a condition known as Aortic Sclerosis (ASc). In the last few years the transmembrane receptor CD44 has been studied as a putative molecule for cardiovascular drug therapy. We reported that the functional interaction between CD44 and one of its ligand, Osteopontin (OPN), protects vascular smooth muscle cells from calcification. More recently, we demonstrated that sclerotic tissues show increased expression of Bone Morphogenetic Protein 4 (BMP4) and BMP4 directly stimulates osteoblast-like transdifferentiation and calcification of VICs. Therefore we hypothesized a direct role of CD44 activation in protecting human Aortic Sclerosis-derived VICs from calcification.

Methods.
Human VICs from Control, ASc, and AS (n=5 each group) were isolated. Histological, cellular and molecular analysis, and in situ Proximity Ligation Assay were used to investigate the role of CD44 and OPN in VIC calcification. BMP4 treatments were used to promote VIC activation. Osteoblast-like transdifferentiation was analyzed using Alkaline Phosphatase (ALP) expression.

Results.
CD44 and OPN, as well as their functional binding, were increased in sclerotic and stenotic tissues compared to healthy controls in vitro and ex vivo. CD44-OPN binding prevented in vitro calcification induced by inorganic phosphate on human ASc-derived VICs. A neutralizing antibody against CD44, under BMP4 treatments, promoted calcium deposition along with increased expression of OPN and ALP.

Conclusion.
Our results generate an important insight into the molecular mechanism of VIC calcification. We proved that CD44-OPN direct interaction inhibits calcification of Aortic Sclerosis-derived VICs, suggesting that CD44 activation could have a protective role against VIC osteoblast-like transdifferentiation and calcification in the early stage of the disease.
Abstract 307: Deficiency of Lim-only Protein Fhl2 Accelerates Neointima Formation After Carotid Artery Ligation
Authors: Kurakula Kondababu , de Waard Vivian , van Tiel Claudia M , de Vries Carlie J 
Published: July 2021

Rationale
The LIM-only protein FHL2, also known as DRAL or SLIM3, has a regulatory function in many physiological processes and is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells, but not in macrophages. FHL2 regulates SMC phenotype, but its function in vascular injury is unknown.

Objective
To assess the role of FHL2 in SMC-rich lesion formation after vascular injury and to elucidate the underlying mechanism.

Methods & Results
Cultured aortic SMCs from FHL2-KO mice showed increased migration and increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. In agreement with this, overexpression of FHL2 in SMCs reduced CyclinD1 expression. In addition, FHL2-KO SMCs showed enhanced expression of pro-inflammatory cytokines in a NFkB dependent manner. Consistent with these findings, NFkB activity is higher in FHL2-KO SMCs. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice developed accelerated lesion formation compared with wild-type (WT)-mice. Furthermore, FHL2-KO mice displayed high number of macrophages in lesions and enhanced expression of RANTES and stromal derived factor-1α (SDF-1α) in ligated carotid arteries. SDF-1α expression was also increased in plasma of FHL2-KO mice. Finally, FHL2-KO mice showed enhanced Ki67 expression in lesions compared with wild-type (WT)-mice.

Conclusion
FHL2 deficiency in mice results in an exacerbated neointima formation by enhanced proliferation and migration of SMCs, possibly regulated via ERK1/2 and NFkB pathway.
Abstract 326: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix Stores and Induces Vascular Permeability in vivo
Authors: Hendel Alon , Granville David J 
Published: July 2021

Introduction
The formation of unstable and leaky neovessels underlies the pathogenesis of a large number of chronic inflammatory diseases. Granzyme B (GZMB) is a serine protease that is expressed and released by a variety of immune cells and accumulates in the extracellular matrix (ECM) during chronic inflammation where it cleaves a number of ECM proteins, including fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN in both normal and diseased tissue. We hypothesize that GZMB cleavage of FN will release VEGF from its extracellular stores and promote vascular permeability as a mechanism that contributes to neovessel leakage during chronic inflammation.

Methods
GZMB-mediated VEGF release from either FN coated wells or endogenously produce endothelial cell (EC) matrix was measured by VEGF ELISA. VEGF-release supernatants were used to treat EC and VEGF receptor 2 (VEGFR2) activation was evaluated by immunoblotting for phosphorylated VEGFR2. Evan’s blue was injected intravenously to CD1 mice followed by ear injection of either mouse GZMB, saline control, GZMB + neutralizing mouse VEGF antibody or GZMB+ IgG control (n=5 for each experimental group). Vascular leakage was evaluated by Evan’s blue dye extraction.

Results
GZMB effectively releases VEGF from both FN and from EC matrix, while inhibition of GZMB prevented VEGF release. GZMB-mediated VEGF release resulted in significant activation of VEGFR2 in EC monolayer signified by increased VEGFR2 phosphorylation. GZMB ear injection resulted in a significant increase in vascular permeability in vivo. Importantly, co-injection of GZMB and neutralizing mouse VEGF antibody significantly reduced vascular leakage compared to co-injection of GZMB and matching IgG control.

Conclusions and Impact
GZMB increases VEGF bioavailability by releasing it from the ECM leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.
Abstract 328: Viral Anti-Atherogenic Proteins Alter Monocyte and Neutrophil Invasion in Mice and Bag3 Gene Expression
Authors: Davids Jennifer , Saikaly Sami , Lucas Alexandra 
Published: July 2021

Aim
Atherosclerosis is characterized by chronic inflammation and cell death (apoptosis). Serine protease inhibitors, or serpins, regulate inflammatory, thrombotic and apoptotic pathways. Poxviruses encode cross-class serpins that prevent host cell apoptosis. Serp-2, from Myxoma, reduces plaque, inflammation and apoptosis in animal models; CrmA, from Vaccinia, does not. Both serpins target Caspase 1 and Granzyme B, but the reasons for the differing effects in vivo are unknown. In prior research three Myxoma viral proteins, including Serp-2, reduced monocyte invasion, plaque growth, and aneurysm formation in ApoE-/- mice after angioplasty. These same proteins alter expression of a shared cohort of 48 apoptosis-related genes in human monocytes treated with camptothecin. This study assesses the effects of Serp-2 and CrmA on apoptotic gene expression in a mouse peritoneal inflammation model.

Methods
Mouse strains deficient for Granzyme B (GzmB, N=13) and Caspase 1 (Casp1, N=15) were compared to their respective background mice (C57Bl/6 and Nod, N=15 each) 18 hours after treatment with PMA and either Serp-2 or CrmA. RNA was isolated and analyzed by RT-PCR and normalized to GAPDH, then to the PMA-only treated control.

Results
Compared to human monocytes, mouse peritoneal exudates from knockout mice displayed differential alteration of BCL2-associated athanogene 3 (BAG3) in response to treatment with Serp-2 or CrmA. Serp-2 treatment reduced expression levels compared to CrmA treatment in GzmB (p=0.0267) and C57Bl/6 mice (p=0.0280). Casp1-/- mice treated with Serp-2 downregulate BAG3, expressing 9.7-fold less than Nod mice (p=0.0006), but CrmA had no significant effect in either strain. An associated differential migration of Ly6Chi and Ly6Ghi cells was also discovered in knockout mice with serpin treatments.

Discussion
One candidate gene found in human monocytes, BAG3, has reduced gene expression after Serp-2 treatment in mouse peritoneal exudates but increased by CrmA. BAG3 is known to alter cell migration and apoptosis, important to atherosclerotic progression. BAG3 is regulated by 3 myxomaviral proteins in human and mouse cells, underscoring a potential role as a lynchpin in viral protein anti-inflammatory and anti-apoptotic pathways.
Abstract 354: Prevalence and Predictors of Non Alcoholic Fatty Liver Disease Among Normal Weight Non Diabetic Individuals
Authors: Sinha Pragya , Oni Ebenezer T , Jamal Omar , Blaha Michael J , Agatston Arthur A , Blumenthal Roger S , Nasir Khurram , Santos Raul 
Published: July 2021

BACKGROUND
Nonalcoholic fatty liver disease(NAFLD) is generally considered a close manifestation of obesity and metabolic abnormalities. In the past, autopsy studies have also demonstrated NAFLD in 15% of non-obese subjects. Increasingly, NAFLD is considered an early manifestation of CAD. However, there is limited data on the prevalence and predictors of NAFLD in normal weight (NW) individuals.

METHODS
We evaluated 6,385 healthy non diabetic Brazilian subjects (43±10 years, 79% males) without clinical coronary heart disease between November 2008 and July 2010. NAFLD was diagnosed by ultrasound.

RESULTS

Overall 39% (n=2494) individuals (41±9 years, 62% males) had normal weight (BMI<25 kg/m
2
& normal waist circumference). The prevalence of NAFLD in this group was 11% compared to 43% in the overweight (BMI 25-29.9) and 79% in the obese (BMI≥30), respectively (p<0.0001). As shown in the table below, increasing age, male gender and metabolic risk factors (MRF) remained significant predictors of NAFLD in NW individuals in univariate and multivariate analyses. As compared to those with no known MRF, the adjusted odds ratio for presence of NAFLD with increasing risk factors (1,2 and 3) were 1.56 (1.13-2.14), 3.93 (2.75-5.75) and 3.81 (1.92-7.59) respectively.

CONCLUSION
Approximately 1/10th of normal weight non-diabetic individuals had NAFLD in our cohort. High triglyceride and low HDL were independent predictors of NAFLD. Increasing metabolic risk factor burden can be considered for further testing to identify those at risk for NAFLD in this group.
Abstract 366: Biglycan Deficiency Does Not Affect Angiotensin Ii-induced Atherosclerosis but Promotes Aortic Aneurysms Formation
Authors: Tang Tao , Thompson Joel C , Wilson Patriticia G , Yoder Meghan H , Tannock Lisa R 
Published: July 2021

Background
Proteoglycans play a critical role in the development of atherosclerosis due to their ability to bind and retain atherogenic lipoproteins. Of all the vascular proteoglycans, biglycan has been shown to be the one most closely associated with apolipoprotein B. Our previous studies showed that angII increases vascular biglycan content and predisposes to diet-induced atherosclerosis in Ldlr null mice. The purpose of this study was to determine whether biglycan deficiency protected against angII induced atherosclerosis in vivo.

Methods and Results
Bgn KO or WT mice, crossed to Ldlr null (C57B/6 background), were infused with angII (1000 ng/kg/min) or saline for 28 days followed by 6-week western diet feeding. Bgn KO mice showed no difference in atherosclerotic lesion area at either aortic sinus or en face surface. Unexpectedly, Bgn KO mice exhibited a striking mortality (77% for males and 48% for females) due to aortic rupture upon angII infusion. Thus, a lower dose of angII was then infused to mice to study atherosclerosis. There was no difference in lesion area between Bgn KO or WT mice under angII (500 ng/kg/min) infusion followed by 6-week western diet feeding or under one-year normal chow feeding without angII infusion. However, angII (500 ng/kg/min) still induced greater aortic rupture in Bgn KO mice (30% for males and 13% for females) than in WT mice (0% for both gender). Besides rupture, 7 Bgn KO mice out of 35 also developed aneurysm at mid-thoracic aorta whereas only 1 Bgn WT mouse out of 25 developed abdominal aneurysm after angII (500 ng/kg/min) infusion. Therefore, our study demonstrated that biglycan deficiency did not affect atherosclerotic lesion development, but induced a striking aneurysm phenotype upon angII infusion.
Abstract 370: Oxidized Low-density Lipoprotein is Related to Carotid Atherosclerosis in Spinal Cord Injury Subjects
Authors: Schreiber Roberto , Paim Layde R , Matos-Souza José R , Silva Anselmo A , Campos Luis F , de Azevedo Eliza R , Alonso Karina , de Rossi Guilherme , Bellinazzi Vera R , Gorla José I , Cliquet Alberto , Nadruz Wilson 
Published: July 2021

Objective
Physical activity is associated with improved carotid atherosclerosis in spinal cord injury (SCI) subjects independent of variation in hemodynamic, metabolic and inflammatory variables. The present study investigated the role of oxidized low-density lipoprotein (Ox-LDL), matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in this regard.

Design and Methods
We studied 40 men with chronic (>1 year of injury) SCI and no voluntary motor activity [17 sedentary (47% tetraplegic) and 23 athletes (52% tetraplegic)] by clinical, anthropometric, laboratory (glucose, lipid profile and C-reactive protein), hemodynamic (blood pressure and cardiac output) and carotid ultrasound analysis. All enrolled subjects were normotensive, non-diabetics, non-smokers and normolipemic. Plasmatic Ox-LDL, MMP2, MMP8, MMP9, TIMP1 and TIMP2 were determined by ELISA. Data are presented as mean±SEM. A p-value <0.05 was considered significant.

Results
Sedentary SCI subjects and SCI athletes presented similar clinical, anthropometric, laboratory and hemodynamic features. Carotid intima-media thickness (IMT) of SCI athletes was lower than that of SCI sedentary individuals (0.54±0.01 vs 0.69±0.02 mm; p<0.001). SCI athletes presented lower Ox-LDL (38.4±2.1 vs 54.1±4.1 U/L; p<0.001) levels in comparison with SCI sedentary individuals. Conversely, MMPs and TIMPs levels as were as MMP8/TIMP1, MMP9/TIMP1 and MMP2/TIMP2 ratios were similar between the SCI groups. Bivariate correlation analysis including all SCI subjects showed that carotid IMT correlated with Ox-LDL (r=0.54; p<0.001), but not with any MMPs or MMP/TIMP ratios. Further linear regression analysis adjusted for the presence or not of physical activity showed that Ox-LDL was still associated with carotid IMT (beta=0.293±0.141; p=0.044) in the whole sample.

Conclusions
Plasmatic Ox-LDL levels are associated with increased carotid IMT in SCI subjects and are lower in physically active SCI individuals than in sedentary SCI ones. These findings may provide a potential mechanism by which physical activity attenuates SCI-induced atherosclerosis.
Abstract 38: Endoplasmic Reticulum Stress Pathway-mediated Apoptosis is Involved in Ang II Induced Abdominal Aortic Aneurysm
Authors: Qin Yanwen , Cao Xu , Liu Ou , Li Huihua , Zhang Hongjia , Qin Yongfen , Tang Chaoshu , Du Jie 
Published: July 2021

Backgroud—
Abdominal aortic aneurysms (AAA) represent a unique and dramatic example of vessel wall remodeling characterized by degeneration of the elastic media. Apoptosis of vascular smooth muscle cells plays an important role in the pathogenesis of AAA. However, the potential mechanism remains poorly understood. Endoplasmic reticulum stress (ER stress)-induced apoptosis has been proved to be one of the important pathogenesis of cardiovascular disease. However, whether ER stress is involved in AAA is still not known. We assessed the hypothesis that ER-associated apoptosis is involved in Angiotensin II (Ang II)-induced AAA in apolipoprotein E-deficient mice.

Methods and Results—

Mice were infused with Ang II (1000 ng/kg per minute) with or without ER stress inhibitor (taurine-conjugated ursodeoxycholic acid) for 4 weeks. Mice infused with Ang II displayed an increase in aortic diameter. Detection of apoptosis was performed with the TUNEL assay. We performed Western blot and Real-time PCR to analyze indicators of ER molecule chaperone and ER-associated apoptosis. Glucose Regulated Proteins 78 and 94 (GRP78/BiP and GRP94), the ER chaperone, were up-regulated significantly in AAA compared to control. Furthermore, the hallmarks of ER-associated apoptosis, C/EBP homologous protein (CHOP), caspase-12 and PERK-eIF2-ATF4 signaling pathway were found to have activated in the AAA. The inhibition of ER stress significantly decreased maximal aortic diameter by 31% and abdominal aortic weight by 35% (
P
<0.05, respectively). ER stress inhibitor also reduced GRP 78, CHOP and caspase-12 expression (
P
<0.05, respectively). Taken together, these results suggested that apoptosis induced by ER stress may contribute to the development of AAA.

Conclusions—
ER stress response is involved in the pathogenesis of Ang II induced AAA in apolipoprotein E-deficient mice. ER stress inhibition attenuates AAA formation during Ang II infusion in apolipoprotein E-deficient mice. Therefore, ER stress could be a potential target for AAA.
Abstract 384: Association of Insulin Resistance With Ambulatory Blood Pressure in Normotensive and Prehypertensive Patients
Authors: Cheng Cynthia , Daskalakis Constantine , Falkner Bonita 
Published: July 2021

Introduction
Hypertension and insulin resistance are well established risk factors for atherosclerosis. Accumulated evidence also suggests that insulin resistance plays a contributory role in the development of essential hypertension. Previous studies have focused on patients with established hypertension, using clinic blood pressure (BP) measurements. Ambulatory Blood Pressure Monitoring (ABPM) records BP at 20-30 minute intervals over 24 hours, providing an average BP exposure over time. We tested the hypothesis that insulin resistance and blood pressure are associated in normotensive and prehypertensive patients, using ABPM to quantify average BP.

Methods
Data were collected for 140 subjects (87 female, 53 male; 117 non-black, 23 black). In addition to clinic BP, ABPM was used to obtain average SBP and DBP over 24 hours. Insulin resistance (IR) was quantified using the QUantitative Insulin sensitivity ChecK Index (QUICKI), calculated from fasting glucose and insulin levels with the formula QUICKI = 1/(log [Fasting insulin in mU/l] + log [Fasting plasma glucose in mg/dl]). Multiple linear regression was used to assess IR as a predictor of ABPM-derived BP measures, controlling for BMI, gender, age, and race.

Results
In the entire cohort using ABPM, there was a significant association of IR with DBP24 (p=0.012) but not SBP24 (p=0.11). The association between IR and clinic SBP was marginally significant (p=0.09); DBP NS (p=0.33). After stratifying by gender, IR was significantly associated with SBP24, Daytime SBP (p=0.064, 0.044), DBP24 and Daytime DBP (p=0.02 and p=0.015) in males; no association was found in females.

Conclusions
This study demonstrates a significant association of IR with average 24-hour DBP in males and females combined. Stratified by gender, our finding using ABPM of a significant association of IR with SBP in males but not in females supports previous findings using clinic BP in the NHANES cohort. In conclusion, these data demonstrate the presence of IR in prehypertension, and lend support to the theory that IR may contribute to later development of sustained hypertension, particularly in males. Additionally, our study highlights the importance of further research using ABPM for a more precise estimate of average BP level.
Abstract 387: HDL Structure and Function in Individuals with Extreme High HDL-C Who Have Coronary Artery Disease Compared with Those Without CAD
Authors: Agarwala Anandita P , Billheimer Jeffrey , Rodrigues Amrith , Risman Marjorie , Cuchel Marina , Rader Daniel 
Published: July 2021

Plasma levels of high-density lipoprotein cholesterol (HDL-C) are strongly inversely associated with coronary artery disease (CAD) in epidemiologic studies, and high HDL-C is generally associated with apparent ‘protection’ from CAD. We have been recruiting individuals with high HDL-C for about 15 years, and while most have no CAD, a minority has premature CAD, a paradoxical phenotype. We hypothesize that such individuals may have HDL with altered structure and/ or function, and are systematically comparing these individuals (cases) to older individuals with extreme high HDL-C without CAD (controls) and a healthy control group with normal HDL-C levels.
We identified 60 subjects with HDL-C above the 90th percentile, premature CAD, and no other major risk factors for coronary disease. We selected 2 controls per case, each matched for age, race, gender, and HDL level. Demographic information and lipid profile (mean ± SD) of the study groups are shown below. Controls are well matched to the cases.
Studies are well underway to assess HDL size distribution by NMR, HDL composition, total and ABCA1-specific cholesterol efflux capacity, lecithin-cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) activity in cases and controls. We will also compare to a group of healthy controls with normal HDL-C levels. We expect that the findings from this study will provide insight into the etiology of CAD in this paradoxical phenotype.
Abstract 406: In Vivo Measurement of Cholesterol Deposition in Peripheral Macrophages
Authors: Weibel Ginny , Rothblat George H , Shivers Debra , Billheimer Jeffrey T , Wilson Aisha 
Published: July 2021

Cholesterol accumulation in macrophages contained in peripheral tissue is an initial step in the initiation and progression of atherosclerosis. Due to the difficulty in harvesting atherosclerotic macrophages and the complexity of atherosclerotic plaques, it is difficult to obtain reliable measurements of cholesterol mass contained in these cells in vivo. We describe a method for measuring cholesterol accumulation in vivo in peripheral macrophages and demonstrate rapid cholesterol deposition in macrophages when transplanted in a mouse with an atherosclerotic genetic background.
Previously, we described a novel method for measuring in vivo cholesterol mass changes in macrophages by entrapping macrophages from donor mice into porous hollow fibers and then implanting the fibers into the peritoneal cavity of a recipient mouse. This procedure allows for the recovery of the cells after recovery of the fibers. We now show that subcutaneous implantation of macrophage-containing fibers results in cholesterol mass changes when the fibers are implanted for 24h. Using this system, we implanted fibers containing wild type peritoneal macrophages subcutaneously into mice with three different genetic backgrounds: 1) wild type, 2) LDLr apoBec dKO and 3) LDLr apoBec dKO/human apoB background. Serum cholesterol levels for the LDLr apoBec dKO and LDLr apoBec dKO/human apoB was 484±101 and 917±12mg/dl respectively, whereas serum cholesterol content of in wild-type controls was 108±6mg/dl. Significant cholesterol deposition in the macrophages was detected as early as 4h after implantation. Following 24h of implantation there was a 3-fold increase in cholesterol mass in the macrophages implanted in the LDLr apoBec dKO, and a five-fold increase in cholesterol content in the macrophages implanted in the LDLr apoBec dKO/human apoB mice. This increase in cell cholesterol mass was a result of expansion of both the unesterified and esterified cholesterol pools, resulting in a 50% / 50% distribution after 24h.